HK1219430B - Biocompatible hydrogel polymer matrix for delivery of cells - Google Patents

Description

Biocompatible hydrogel polymer matrices for cell delivery

Cross-references

This application claims priority to U.S. Provisional Application No. 61/785,477, filed March 14, 2013, which is incorporated herein by reference.

Background Art

Cell-based therapy is an important option for the treatment of clinical indications including diseases, tissue damage, neurological disorders, blood disorders, cancers, developmental defects, trauma and plastic surgery disorders. Many cell-based therapies are target-specific, wherein cells are directly administered to the target site. When cells are not properly retained at the target site after administration, there is a simultaneous loss of cells that can be used for the intended treatment and an increased risk of cell differentiation at other sites. When cells are administered to the target site without adequate protection, the cells may undergo physicochemical changes such as overgrowth, necrosis, apoptosis or aging. When the administered cells are physicochemically altered or not retained at the desired target site, the therapeutic efficacy is weakened.

Summary of the Invention

In one aspect, the present invention provides a fully synthetic, polyglycol-based biocompatible hydrogel polymer matrix comprising a fully synthetic, polyglycol-based biocompatible hydrogel polymer comprising at least one first monomeric unit bound to at least one second monomeric unit via at least one amide bond, thioester bond, or thioether bond, wherein the polymer forms a matrix that encapsulates at least one cell and a culture medium that supports the growth of the at least one cell. In certain embodiments, the fully synthetic, polyglycol-based biocompatible hydrogel polymer matrix provides controlled release of the at least one cell to the target site in the animal body when implanted into the target site in the animal body. In some embodiments, the at least one first monomeric unit is PEG-based and fully synthetic, and the at least one second monomer is PEG-based and fully synthetic. In certain embodiments, the cell is selected from a mammalian cell, an insect cell, a protozoan cell, a bacterial cell, a viral cell, or a fungal cell. In some embodiments, the mammalian cell is a stem cell. In certain embodiments, the culture medium comprises a growth factor.

In another aspect, the present invention provides a biocompatible hydrogel polymer matrix based on polyglycol, comprising at least one first monomeric unit bound to at least one second monomeric unit by at least one amide bond, thioester bond, or thioether bond; culture medium; and at least one cell. In certain embodiments, the biocompatible hydrogel polymer matrix based on polyglycol is completely synthetic. In certain embodiments, the biocompatible hydrogel polymer matrix based on polyglycol is based on polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG), or a copolymer thereof. In some embodiments, the biocompatible hydrogel polymer matrix based on polyglycol is completely synthetic and based on PEG. In certain embodiments, the at least one first monomeric unit is based on PEG and completely synthetic, and wherein the at least one second monomeric unit is based on PEG and completely synthetic. In certain embodiments of the biocompatible hydrogel polymer based on polyglycol, the biocompatible hydrogel polymer matrix based on polyglycol encapsulates the cells. In some embodiments of polyglycol-based biocompatible hydrogel polymers, the polyglycol-based biocompatible hydrogel polymer supports the viability and growth of cells on the surface of the polymer. In certain embodiments of polyglycol-based biocompatible hydrogel polymers, the polyglycol-based biocompatible hydrogel polymer supports the viability and growth of cells within the polyglycol-based biocompatible hydrogel polymer matrix. In some embodiments, the cells are microorganisms. In certain embodiments, the cells are selected from mammalian cells, insect cells, protozoan cells, bacterial cells, viral cells, or fungal cells. In one embodiment, the mammalian cells are stem cells.

In some embodiments of the biocompatible hydrogel polymer matrix based on polyglycol, the culture medium comprises a growth medium. In certain embodiments, the culture medium comprises a growth factor. In some embodiments, the biocompatible hydrogel polymer matrix based on polyglycol releases the cells at a target site in the human body. In certain embodiments, the at least one cell survives for at least one hour in the biocompatible hydrogel polymer matrix based on polyglycol. In some embodiments, the at least one cell survives for 5 days in the biocompatible hydrogel polymer matrix based on polyglycol. In certain embodiments, the at least one cell proliferates and grows in the biocompatible hydrogel polymer matrix based on polyglycol. In certain embodiments, the controlled release of the at least one cell to the target site of the animal body comprises diffusion of the at least one cell from the biocompatible hydrogel polymer matrix based on polyglycol. In some embodiments, the controlled release of the at least one cell to the target site of the animal body is carried out at least in part by degradation and bioabsorption of the biocompatible hydrogel polymer matrix based on polyglycol.

In certain embodiments of the biocompatible hydrogel polymer matrix based on polyglycol, the first monomer unit is derived from multi-arm-(5-50k)-SH, multi-arm-(5-50k)-NH2 or multi-arm-(5-50k)-AA monomer. In some embodiments, the first monomer unit is a diol, trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol or a polyglycerol derivative. In certain embodiments, multi-arm is selected from 2 arms, 3 arms, 4 arms, 6 arms and 8 arms. In some embodiments, the first monomer unit comprises one or more polyethylene glycol moieties. In certain embodiments, the first unit monomer is derived from 4-arm-5k-SH, 4-arm-2k-NH2, 4-arm-5k-NH2, 8-arm-20k-NH2, 4-arm-20k-AA or 8-arm-20k-AA monomer.

In some embodiments of the biocompatible hydrogel polymer matrix based on polyglycol, the second monomeric unit is derived from a multi-arm-(5-50k)-SG, a multi-arm-(5-50k)-SGA, or a multi-arm-(5-50k)-SS monomer. In certain embodiments, the second monomeric unit is a glycol, trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol, or a polyglycerol derivative. In some embodiments, the multi-arm is selected from 2 arms, 3 arms, 4 arms, 6 arms, and 8 arms. In certain embodiments, the second monomeric unit comprises one or more polyethylene glycol moieties. In some embodiments, the second monomeric unit is derived from a 4-arm-10k-SG, an 8-arm-15k-SG, a 4-arm-20k-SGA, or a 4-arm-20k-SS monomer.

On the other hand, there is provided herein a biocompatible preformulation based on polyglycol, comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, at least one cell and culture medium, wherein the biocompatible preformulation based on polyglycol is at least partially polymerized and/or gelled in the presence of water to form a biocompatible hydrogel polymer matrix based on polyglycol that encapsulates the cells. In certain embodiments, the biocompatible preformulation is completely synthetic. In some embodiments, the culture medium supports the growth of the at least one cell. In certain embodiments, the biocompatible preformulation is based on polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG) or its copolymer. In some embodiments, the biocompatible preformulation is based on PEG. In certain embodiments, the biocompatible preformulation is completely synthetic and based on PEG.

In certain embodiments of the biocompatible preformulations based on polyglycol, the cell is a microorganism. In some embodiments, the cell is a mammalian cell, an insect cell, a protozoan cell, a bacterial cell, a viral cell, or a fungal cell. In certain embodiments, the mammalian cell is a stem cell.

In some embodiments of the polyalkylene glycol-based biocompatible preformulation, the culture medium comprises a growth medium. In certain embodiments, the culture medium comprises growth factors.

In some embodiments of the biocompatible preformulation based on polyglycol, the nucleophilic group includes sulfhydryl or amino group.In certain embodiments, the electrophilic group includes epoxide, N- succinimidyl succinate, N- succinimidyl glutarate, N- succinimidyl succinamide or N- succinimidyl glutaramide.In certain embodiments, the first compound and the second compound include one or more polyglycol moieties.In some embodiments, the first compound is selected from multi-arm -(5-50k)-SH, multi-arm -(5-50k)-NH2 and multi-arm -(5-50k)-AA, and the second compound is selected from multi-arm -(5-50k)-SG, multi-arm -(5-50k)-SGA and multi-arm -(5-50k)-SS.In certain embodiments, the first compound and the second compound are independently glycol, trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol or polyglycerol derivatives. In some embodiments, the multi-arm is independently selected from 2 arms, 3 arms, 4 arms, 6 arms, and 8 arms.

In certain embodiments of the polyalkylene glycol-based biocompatible preformulation, the first compound is selected from 4-arm-5k-SH, 4-arm-2k-NH2, 4-arm-5k-NH2, 8-arm-20k-NH2, 4-arm-20k-AA, and 8-arm-20k-AA, and the second compound is selected from 4-arm-10k-SG, 8-arm-15k-SG, 4-arm-20k-SGA, and 4-arm-20k-SS. In a specific embodiment, the first compound is 8-arm-20k-NH2 and/or 8-arm-20k-AA, and the second compound is 4-arm-20k-SGA.

In some embodiments of the polyglycol-based biocompatible preformulation, the at least one first compound is a polyglycol-based, fully synthetic biocompatible compound comprising one or more nucleophilic groups, and the at least one second compound is a polyglycol-based, fully synthetic biocompatible compound comprising one or more electrophilic groups. In certain embodiments of the polyglycol-based biocompatible preformulation, the at least one first compound is a PEG-based, fully synthetic biocompatible compound comprising one or more nucleophilic groups, and the at least one second compound is a PEG-based, fully synthetic biocompatible compound comprising one or more electrophilic groups.

In certain embodiments of the polyglycol-based biocompatible preformulation, the polyglycol-based biocompatible preformulation gels to form a polyglycol-based biocompatible hydrogel polymer matrix in about 20 seconds to 10 minutes. In some embodiments, the polyglycol-based biocompatible hydrogel polymer matrix gels at a predetermined time.

In certain embodiments of the polyglycol-based biocompatible preformulation, the first compound and the second compound do not react with the cells during the formation of the polyglycol-based biocompatible hydrogel polymer matrix. In some embodiments, the cells remain unchanged after the polyglycol-based biocompatible hydrogel polymer matrix is formed. In certain embodiments, the viability of the cells is not affected by the formation of the polyglycol-based biocompatible hydrogel polymer matrix. In some embodiments, the viability of the cells is not affected by the formation of the polyglycol-based biocompatible hydrogel polymer matrix. In certain embodiments, the physicochemical properties of the cell wall are not affected by the formation of the polyglycol-based biocompatible hydrogel polymer matrix.

In certain embodiments, the polyglycol-based biocompatible hydrogel polymer matrix is bioabsorbable. In some embodiments, the polyglycol-based biocompatible hydrogel polymer matrix is bioabsorbed within about 1 to 70 days. In other embodiments, the polyglycol-based biocompatible hydrogel polymer matrix is bioabsorbed within about 14 to 180 days. In certain embodiments, the polyglycol-based biocompatible hydrogel polymer matrix is substantially non-bioabsorbable.

In some embodiments, the cells are released from the biocompatible hydrogel polymer matrix based on polyglycol by diffusion, degradation of the biocompatible hydrogel polymer matrix based on polyglycol, or any combination thereof. In certain embodiments, the cells are initially released from the biocompatible hydrogel polymer matrix based on polyglycol by diffusion and subsequently released by degradation of the biocompatible hydrogel polymer matrix based on polyglycol. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix based on polyglycol within 180 days. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix based on polyglycol within 14 days. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix based on polyglycol within 24 hours. In other embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix based on polyglycol within one hour. In certain embodiments, the release of the cells is substantially inhibited until the time when the biocompatible hydrogel polymer matrix based on polyglycol begins to degrade. In some embodiments, the polyglycol-based biocompatible hydrogel polymer matrix has a pore size that is small enough to substantially inhibit the release of the cells before the time when the polyglycol-based biocompatible hydrogel polymer matrix begins to degrade. In certain embodiments, at least a portion of the cells are released before the time when the polyglycol-based biocompatible hydrogel polymer matrix begins to degrade. In other embodiments, the polyglycol-based biocompatible hydrogel polymer matrix has a pore size that is large enough to allow at least a portion of the cells to be released before the time when the polyglycol-based biocompatible hydrogel polymer matrix begins to degrade.

In certain embodiments, the biocompatible polyglycol-based hydrogel polymer matrix minimizes degradation or denaturation of the cells. In some embodiments, the cells are protected from the enzymes and pH conditions of the gastrointestinal tract. In certain embodiments, the cells remain viable after release from the biocompatible polyglycol-based hydrogel polymer matrix.

In a further aspect, provided herein are methods for treating a disease or condition by administering a biocompatible polyglycol-based preformulation, wherein the preformulation comprises at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, at least one cell, and culture medium, wherein the biocompatible polyglycol-based preformulation at least partially polymerizes and/or gels in the presence of water to form a biocompatible polyglycol-based hydrogel polymer matrix that encapsulates the cells. In another aspect, provided herein are methods for treating a disease or condition by administering a biocompatible polyglycol-based polymer matrix, wherein the polymer matrix comprises at least one first monomeric unit bound to at least one second monomeric unit via at least one amide bond, thioester bond, or thioether bond; culture medium; and at least one cell.

In another aspect, provided herein is a polyglycol-based biocompatible hydrogel polymer matrix comprising at least one first monomeric unit bound to at least one second monomeric unit via at least one amide bond, thioester bond, or thioether bond, and a culture medium. In a further aspect, provided herein is a polyglycol-based biocompatible hydrogel polymer matrix comprising at least one first monomeric unit bound to at least one second monomeric unit via at least one amide bond, thioester bond, or thioether bond, and at least one cell.

In a further aspect, provided herein are polyglycol-based biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, and at least one cell, wherein the polyglycol-based biocompatible preformulation at least partially polymerizes and/or gels in the presence of water to form a polyglycol-based biocompatible hydrogel polymer matrix that encapsulates the cells. In another aspect, provided herein are polyglycol-based biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, and culture medium, wherein the polyglycol-based biocompatible preformulation at least partially polymerizes and/or gels in the presence of water to form a polyglycol-based biocompatible hydrogel polymer matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of the invention which illustrates illustrative embodiments utilizing the principles of the invention and the accompanying drawings, in which:

Figure 1 shows the effect of addition of degradable acetate amine 8-Arm-20k-AA or 4-Arm-20k-AA on degradation time. Degradation occurred at 37°C in phosphate-buffered saline (PBS).

FIG2 shows the effect of polymer concentration on degradation time for 75% amine acetate formulations and 100% amine acetate formulations.

FIG3 shows the effect of polymer exposure to air as percent water weight loss over time.

A sample graph generated by the Texture Analyzer Exponent software running the hardness test is shown in Figure 4. The peak force was recorded as the polymer hardness, which represents the point when the probe had reached the target penetration depth of 4 mm.

Figure 5 shows a graph of a sample generated by the Texture Analyzer Exponent software running an elastic modulus under compression test. The modulus was calculated from the initial slope of the curve up to 10% of the maximum compressive stress.

Fig. 6 shows an exemplary graph generated by the Texture Analyzer Exponent software running the adhesion test. A contact force of 100.0g was applied for 10 seconds. Adhesion was measured as the peak force after the probe was lifted from the sample. Adhesion energy or adhesion work was calculated as the area under the curve representing adhesion (points 1 to 2). Stringiness was defined as the distance the probe traveled when affecting adhesion (points 1 and 2).

Figure 7 shows a graph of hardness plotted as a percentage versus degradation time for the following polymer formulation: 4.8% solution of 8-Arm-20k-AA/8-Arm-20k-NH2 (70/30) and 4-Arm-20k-SGA containing 0.3% HPMC. Error bars represent the standard deviation of three samples. The degradation time of the polymer was 18 days.

Figure 8 shows the cumulative % elution of chlorhexidine.

Figure 9 shows the cumulative % elution of triamcinolone for the polymer at 60, 90 and 240 days.

Figure 10 shows the cumulative % elution of methylprednisolone for the short degradation time version of the Depo-Medrol loaded hydrogel polymer.

Figure 11 shows the cumulative % elution of methylprednisolone for the long degradation time version of the Depo-Medrol loaded polymer.

FIG12 shows the effect of solid phosphate powder concentration on polymer gel time (A) and solution pH (B).

FIG13 shows the effect of sterilization on the gel time of polymers at different concentrations of (A) and (B).

FIG14 shows the storage stability of the kit at 5°C, 20°C and 37°C.

DETAILED DESCRIPTION

Cell therapy is used for many clinical indications at multiple target sites and through several cell delivery modes. Cells are guided to the target site by local or systemic administration. Important examples of cell therapy include stem cell transplantation, cell differentiation and damaged tissue replacement, whereby the target tissue has improved function. Cell-based therapies can be applied to damaged tissues after events such as myocardial infarction, infection or cancer treatment. During and after the application of cell-based therapies to the target site, the therapeutic cells may not be adequately protected. Unprotected cells may undergo apoptosis, necrosis, overgrowth or aging; thus reducing the efficacy of the treatment. Cell-based therapies delivered in a suitable environment enable cells to survive. In some cases, cells delivered in a suitable environment proliferate, differentiate and integrate with the target tissue. Cell-based therapies retained at the target site in a suitable environment enable appropriate cell functionality at the target site.

Systemic cell-based therapy exposes many areas of the body to the administered cells in addition to the target site. The diffusion of stem cells away from the target site increases the risk of undesirable cell differentiation and subsequent complications. Importantly, the loss of cell retention at the target site increases the amount of administered cells required for therapeutic efficacy. Local cell delivery directly to the target site limits the exposure of administered cells to the area surrounding the target site. Local cell delivery and cell retention enable the administration of controlled therapeutic doses. In some cases, the therapeutic dose is controlled by the extended release of cells. In some cases, because the dose can be increased while there is less concern about adverse side effects, local cell delivery therapy is more effective. In further cases, the extended release of cells also reduces the number of doses required during treatment.

Biocompatible preformulations for forming a biocompatible hydrogel polymer matrix enable cells to be directly administered and retained at the target site. The biocompatible preformulation is at least partially polymerized and/or gelled to form a biocompatible hydrogel polymer matrix. The biocompatible hydrogel polymer matrix contains at least one cell. In some embodiments, the biocompatible hydrogel matrix comprises a biocompatible hydrogel scaffold. After the polymer matrix or preformulation is applied to the target site, the biocompatible hydrogel polymer matrix provides structural and nutritional support for the cells. In some embodiments, after the polymer matrix or preformulation is applied to the target site, the biocompatible hydrogel scaffold provides structural and nutritional support for the cells. In certain embodiments, after the polymer matrix or preformulation is applied to the target site, the biocompatible hydrogel polymer matrix provides structural and nutritional support for the cells. The biocompatible hydrogel polymer matrix enables cells to remain at the target site for a predetermined amount of time. In certain embodiments, the biocompatible hydrogel polymer matrix provides a protective and nutrient-rich environment suitable for cell survival, growth, or proliferation. In certain embodiments, the biocompatible hydrogel polymer matrix provides a protective and nutrient-rich environment suitable for cell survival, proliferation, differentiation, and tissue integration. The biocompatible preformulations used to form the biocompatible hydrogel polymer matrix also enable controlled release of cells at the target site. In certain embodiments, the controlled release of cells at the target site is carried out by diffusion, degradation of the biocompatible hydrogel polymer matrix, or any combination thereof. In some cases, the hydrogel polymer matrix is biodegradable. In some cases, cell delivery, retention, and controlled release using the biocompatible hydrogel polymer matrix minimize cell overgrowth, aging, apoptosis, and necrosis. In some cases, the biocompatible hydrogel polymer matrix protects cells from the enzymes and pH conditions of the gastrointestinal tract. In some cases, the polymer matrix is configured to maintain the physicochemical properties of the cells during administration, retention, degradation of the biocompatible hydrogel polymer matrix, or release of the cells to the target site. In some cases, the cells remain alive during and after the polymerization of the biocompatible hydrogel polymer matrix. In some cases, the cells remain viable when added to a biocompatible hydrogel polymer matrix that has already polymerized and/or gelled. In some cases, the cells remain viable during administration. In some cases, the cells remain viable after delivery to a target site. In some cases, the cells remain viable during release from the biocompatible hydrogel polymer matrix. In some cases, the cells remain viable during degradation of the biocompatible hydrogel polymer matrix.

The biocompatible hydrogel polymer matrix enables delivery of cells to a target site, where the cells are ultimately released from the polymer matrix by diffusion, polymer matrix degradation, or any combination thereof. In some cases, the polymer matrix degradation time is controlled by varying the composition of biocompatible preformulation components that allow for proper application and placement of the biocompatible hydrogel polymer matrix. In some cases, the polymer matrix degradation time is controlled by varying the pH of the preformulation that allows for proper application and placement of the biocompatible hydrogel polymer matrix. In some cases, the polymer matrix degradation time is controlled by varying the concentration of biocompatible preformulation components that allow for proper application and placement of the biocompatible hydrogel polymer matrix. In some embodiments, cells are released from the biocompatible hydrogel polymer matrix in a precise and consistent manner. In some cases, the biocompatible hydrogel polymer matrix is bioabsorbed over a defined period of time. In some embodiments, the biocompatible hydrogel polymer matrix provides sustained release of cells at the target site. In certain embodiments, sustained and controlled release reduces systemic exposure to the cells. In certain embodiments, controlled release allows the cells to remain at the target site. In some cases, cells are released from the biocompatible hydrogel polymer matrix over an extended period of time. In some cases, the delivery of cells in the biocompatible hydrogel polymer matrix provides a depot (e.g., under the skin) of cells, wherein the depot releases cells over an extended period of time (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 3 weeks, 4 weeks). In some cases, the biocompatible hydrogel polymer matrix releases cells after a delay as a delayed burst.

The biocompatible hydrogel polymer matrix comprising at least one cell can be used as a liquid biocompatible preformulation to start, and it uses minimally invasive technology to be delivered to the target site. The initial liquid state enables the preparation to be delivered to the target site (for example, for the lung, for the chest cavity, for the thoracic cavity, for the abdominal cavity, for the laparoscope, for the bladder, for the cystoscope, for the joint space, for the arthroscope, etc.) by the small catheter guided by endoscope or other image-guided techniques. Once entering the body, the liquid preparation is polymerized into the biocompatible hydrogel polymer matrix. In some cases, the biocompatible hydrogel polymer matrix adheres to tissue, and at least one cell remains on the target site. In some cases, the biocompatible hydrogel polymer matrix is delivered to the target site after polymerization. In some cases, the composition of the biocompatible preformulation components of the suitable application and placement of the biocompatible hydrogel polymer matrix is controlled polymerization time by changing. The gelation of control allows the use of the biocompatible hydrogel polymer matrix to directly deliver at least one cell to the affected target tissue, thus minimizing systemic exposure. In some embodiments, the biocompatible hydrogel polymer matrix can be polymerized in vitro. In certain embodiments, exposure to the cells is limited to tissues surrounding the target site. In some embodiments, the patient is not systemically exposed to the cell therapy. In certain embodiments, the biocompatible preformulation allows the cells to remain viable during and after polymerization. In some embodiments, after polymerization and/or gel formation, the cells are combined with the biocompatible hydrogel polymer matrix. In some embodiments, after delivery to the target site, the biocompatible hydrogel polymer matrix is further polymerized and/or gelled.

Cells can also be applied directly to the wound or surgical site via a biocompatible hydrogel polymer matrix. A biocompatible preformulation can form a biocompatible hydrogel polymer matrix that is easily applied to the wound or surgical site and the surrounding skin. The biocompatible hydrogel polymer matrix enables cells to be applied directly to the wound or surgical site. The biocompatible preformulation can be polymerized and/or gelled before or after being applied to the wound or surgical site. In some cases, once the biocompatible preformulation is applied, for example, by spraying it onto the wound or surgical site in liquid form, the biocompatible preformulation quickly gels and forms a solid biocompatible hydrogel polymer matrix layer on the wound or surgical site. The biocompatible hydrogel polymer matrix seals the wound or surgical site and also adheres to the surrounding skin. The biocompatible hydrogel polymer matrix layer on the wound or surgical site acts as a barrier to prevent the wound or surgical site from becoming infected. In some cases, the biocompatible hydrogel polymer matrix layer in contact with the skin makes the skin surface sticky, thereby making the bandage adhere more effectively to the skin. Most importantly, the biocompatible hydrogel polymer matrix is non-toxic. After healing has begun, the biocompatible hydrogel polymer matrix dissolves and is absorbed without producing toxic by-products. In some embodiments, after applying stem cells with the biocompatible hydrogel polymer matrix, the wound or surgical site heals by the formation of a graft. In certain embodiments, the biocompatible preformulation is applied to the wound or surgical site without cell loss of viability. In certain embodiments, the biocompatible hydrogel polymer matrix keeps the wound or surgical site sealed for 24-48 hours and protects it from infection, which avoids repeated visits to the hospital and therefore saves costs. In certain embodiments, exposure to the cells is limited to the tissue surrounding the target site. In some embodiments, the patient is not systemically exposed to the cell therapy.

In some embodiments, the biocompatible hydrogel polymer matrix is also loaded with one or more additional components, such as buffer or therapeutic agent. The physical and chemical properties of the biocompatible hydrogel polymer matrix are such that multiple cell types and additional components can be used together with the biocompatible preformulations that form the biocompatible hydrogel polymer matrix. In some embodiments, the additional components enhance the viability and functionality of the cell. In some embodiments, the additional components include activating factors. In some embodiments, the activating factors include growth factors for cell growth stimulation and proliferation.

Exemplary biocompatible hydrogel components

Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, at least one cell, and optionally additional components. An exemplary additional component is a culture medium. In certain embodiments, the culture medium is a buffer. In certain embodiments, the culture medium contains nutrients for at least one cell. In certain embodiments, at least one cell is a stem cell. In certain embodiments, at least one first compound is formulated in a buffer. In certain embodiments, at least one second compound is formulated in a buffer. In certain embodiments, at least one cell is formulated in a buffer. In certain embodiments, at least one biocompatible preformulation component is a solid. In certain embodiments, all components of the biocompatible preformulation are solid. In certain embodiments, at least one biocompatible preformulation component is a liquid. In certain embodiments, all components of the biocompatible preformulation are liquids. In certain embodiments, the biocompatible preformulation components form a biocompatible hydrogel polymer matrix at the target site by mixing at least one first compound, at least one second compound, at least one cell, and optional additional components in the presence of water and delivering the mixture to the target site so that the biocompatible hydrogel polymer matrix at least partially polymerizes and/or gels at the target site. In certain embodiments, the biocompatible preformulation forms a biocompatible hydrogel polymer matrix at the target site by mixing at least one first compound, at least one second compound, and at least one cell in the presence of water and delivering the mixture to the target site so that the biocompatible hydrogel polymer matrix at least partially polymerizes and/or gels at the target site. In certain embodiments, optional additional components, such as buffers, are added after the formulation is combined. In certain embodiments, the biocompatible preformulation forms a biocompatible hydrogel polymer matrix before being applied to the target site by mixing at least one first compound, at least one second compound, at least one cell, and optional additional components in the presence of water and delivering the mixture to the target site so that the biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled before being applied to the target site. In certain embodiments, the biocompatible preformulation forms a biocompatible hydrogel polymer matrix before being applied to the target site by mixing at least one first compound, at least one second compound, and at least one cell in the presence of water and delivering the mixture to the target site so that the biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled before being applied to the target site. In certain embodiments, optional additional components, such as buffer, are added after the formulation is combined. In certain embodiments, the biocompatible preformulation is biodegradable. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold. In certain embodiments, the biocompatible hydrogel scaffold comprises the at least one first compound and at least one second compound. In certain embodiments, the biocompatible hydrogel scaffold comprises at least one first compound, at least one second compound, and a buffer. In certain embodiments, the biocompatible hydrogel scaffold is fully synthetic.

Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, a buffer, and optional additional components. An exemplary additional component is at least one cell. In certain embodiments, the cell is a stem cell. In certain embodiments, the buffer is a culture medium. In certain embodiments, the culture medium provides nutrients to the cells. In certain embodiments, the at least one first compound is formulated in a buffer. In certain embodiments, the at least one second compound is formulated in a buffer. In certain embodiments, at least one biocompatible preformulation component is a solid. In certain embodiments, all biocompatible preformulation components are solid. In certain embodiments, at least one biocompatible preformulation component is a liquid. In certain embodiments, all biocompatible preformulation components are liquid. In certain embodiments, the biocompatible preformulation forms a biocompatible hydrogel polymer matrix at the target site by mixing the at least one first compound, the at least one second compound, the buffer, and the optional additional components in the presence of water and delivering the mixture to the target site, thereby causing the biocompatible hydrogel polymer matrix to at least partially polymerize and/or gel at the target site. In certain embodiments, the biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled at the target site by mixing at least one first compound, at least one second compound, and a buffer in the presence of water and delivering the mixture to the target site. In certain embodiments, optional additional components, such as cells, are added after the formulation is combined. In certain embodiments, the biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled before being applied to the target site by mixing at least one first compound, at least one second compound, a buffer, and optional additional components in the presence of water and delivering the mixture to the target site. The biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled before being applied to the target site. The biocompatible preformulation forms a biocompatible hydrogel polymer matrix before being applied to the target site. In certain embodiments, the biocompatible hydrogel polymer matrix is at least partially polymerized and/or gelled before being applied to the target site by mixing at least one first compound, at least one second compound, and a buffer in the presence of water and delivering the mixture to the target site. The biocompatible preformulation forms a biocompatible hydrogel polymer matrix before being applied to the target site. In certain embodiments, optional additional components, such as cells, are added after the formulation is combined. In certain embodiments, the biocompatible preformulation is biodegradable. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold. In certain embodiments, the biocompatible hydrogel scaffold comprises at least one first compound, at least one second compound, and a buffer. In certain embodiments, the biocompatible hydrogel scaffold is fully synthetic.

In some embodiments, the biocompatible preformulation compound comprises monomers that are polymerized into polymers. In some embodiments, the biocompatible preformulation monomers are polymerized to form a biocompatible hydrogel polymer matrix. In some embodiments, the polymer is a biocompatible hydrogel polymer matrix. In some embodiments, the polymer is a biocompatible hydrogel scaffold. In some embodiments, the biocompatible preformulation compound gels to form a biocompatible hydrogel polymer matrix. In some embodiments, the biocompatible preformulation compound gels to form a biocompatible hydrogel scaffold. In some embodiments, the biocompatible preformulation compound polymerizes and gels to form a biocompatible hydrogel polymer matrix. In some embodiments, the biocompatible preformulation compound polymerizes and gels to form a biocompatible hydrogel polymer scaffold. In some embodiments, the biocompatible hydrogel polymer matrix is further polymerized after the hydrogel polymer matrix is formed. In some embodiments, the biocompatible hydrogel polymer matrix gels after the hydrogel polymer matrix is formed. In some embodiments, the biocompatible hydrogel polymer matrix is further polymerized and gelled after the hydrogel polymer matrix is formed.

In some embodiments, the first or second compound comprising more than one nucleophilic or electrophilic group is based on diol. In some embodiments, the compound based on diol includes ethylene glycol, propylene glycol, butylene glycol, alkyl glycols of various chain lengths and any combination or copolymer thereof. In some embodiments, the compound based on diol is a compound based on polyglycol. In some embodiments, the compound based on polyglycol includes but is not limited to polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG) and polyglycol copolymers. In some embodiments, the compound based on diol includes polyethylene glycol, polypropylene glycol, polybutylene glycol, polyalkyl glycols of various chain lengths and any combination or copolymer thereof. In some embodiments, the compound based on diol is completely synthesized. In some embodiments, the compound based on polyglycol is completely synthesized.

In some embodiments, the first or second compound comprising more than one nucleophilic or electrophilic group is a polyol derivative. In certain embodiments, the first or second compound is a dendritic polyol derivative. In some embodiments, the first or second compound is a glycol, trimethylolpropane, glycerol, diglycerol, pentaerythritol (pentaerythritiol), sorbitol, hexaglycerol, tripentaerythritol or a polyglycerol derivative. In certain embodiments, the first or second compound is a glycol, trimethylolpropane, pentaerythritol, hexaglycerol or a tripentaerythritol derivative. In some embodiments, the first or second compound is trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol or a polyglycerol derivative. In some embodiments, the first or second compound is pentaerythritol, dipentaerythritol or a tripentaerythritol derivative. In certain embodiments, the first or second compound is a hexaglycerol (2-ethyl-2-(hydroxymethyl)-1,3-propanediol, trimethylolpropane) derivative. In some embodiments, the first or second compound is a sorbitol derivative.In certain embodiments, the first or second compound is a glycol, propylene glycol, glycerol, diglycerol, or polyglycerol derivative.

In some embodiments, the first and/or second compound comprises a polyethylene glycol (PEG) chain comprising 1 to 200 ethylene glycol subunits. In certain embodiments, the first and/or second compound may further comprise a polypropylene glycol (PPG) chain comprising 1 to 200 propylene glycol subunits. The PEG or PPG chain extending from the polyol is the "arm" connecting the polyol core to the nucleophilic or electrophilic group.

Exemplary nucleophilic monomers

The biocompatible preformulation comprises at least one first compound comprising more than one nucleophilic group. In some embodiments, the first compound is a monomer configured to form a polymer matrix by reacting a nucleophilic group in the first compound with an electrophilic group in the second compound. In some embodiments, the first compound monomer is completely synthetic. In some embodiments, the nucleophilic group is a hydroxyl, sulfhydryl, or amino group. In preferred embodiments, the nucleophilic group is a sulfhydryl or amino group. In some embodiments, at least one first compound is diol-based. In some embodiments, diol-based compounds include ethylene glycol, propylene glycol, butylene glycol, alkyl glycols of various chain lengths, and any combination or copolymer thereof. In some embodiments, diol-based compounds are polyglycol-based compounds. In some embodiments, polyglycol-based compounds include but are not limited to polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG), and polyglycol copolymers. In some embodiments, diol-based compounds include polyethylene glycol, polypropylene glycol, polybutylene glycol, polyalkyl glycols of various chain lengths, and any combination or copolymer thereof. In some embodiments, diol-based compounds are completely synthetic. In some embodiments, the polyglycol-based compounds are entirely synthetic.

In certain embodiments, the nucleophilic group is connected to the polyol derivative by a suitable linker. Suitable linkers include but are not limited to esters (for example, acetate) or ethers. In some cases, the monomer comprising an ester linker is more sensitive to biodegradation. The example of the linker containing the nucleophilic group includes but is not limited to thioglycolate, aminoacetate (glycine) and other amino acid esters (for example, alanine, beta-alanine, lysine, ornithine), 3-mercaptopropionic acid ester, ethylamine ether or propylamine ether. In some embodiments, the polyol core derivative is attached to polyethylene glycol or polypropylene glycol subunits, and the subunits are connected to the linker comprising the nucleophilic group. The molecular weight of the first compound (nucleophilic monomer) is approximately 500 to 40000. In certain embodiments, the molecular weight of the first compound (nucleophilic monomer) is about 100, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 12000, about 15000, about 20000, about 25000, about 30000, about 35000, about 40000, about 50000, about 60000, about 70000, about 80000, about 90000, or about 100000. In some embodiments, the molecular weight of the first compound is between about 500 and 2000. In certain embodiments, the molecular weight of the first compound is between about 15000 and about 40000. In some embodiments, the first compound is water soluble.

In some embodiments, the first compound is multi-arm-(5k-50k)-polyol derivative, which includes polyglycol subunits and more than two nucleophilic groups. Multi-arm refers to the number of the polyglycol subunits connected to the polyol core, and these polyglycol subunits connect the nucleophilic group to the polyol core. In some embodiments, multi-arm is 3 arms, 4 arms, 6 arms, 8 arms, 10 arms, 12 arms. In some embodiments, multi-arm is 4 arms or 8 arms. In some embodiments, the first compound is multi-arm-(5k-50k)-NH2, multi-arm-(5k-50k)-AA or a combination thereof. In certain embodiments, the first compound is 4 arms-(5k-50k)-NH2, 4 arms-(5k-50k)-AA, 8 arms-(5k-50k)-NH2 and 8 arms-(5k-50k)-AA or a combination thereof. In some embodiments, the polyol derivative is diol, trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol, or a polyglycerol derivative.

Examples of structures of monomers containing more than one nucleophilic group with a trimethylolpropane or pentaerythritol core polyol are shown below. The compounds shown have a thiol or amine electrophilic group connected to PEG subunits of varying lengths via acetate, propionate, or ether linkers (e.g., the following structures: ETTMP (A; n = 1), 4-arm-PEG-NH2 (B; n = 1), and 4-arm-PEG-AA (C; n = 1)). Monomers with other polyol cores were constructed in a similar manner.

A: (n=0 to 6)

B: (n=0 to 6)

C: (n=1-6)

Suitable first compounds containing a nucleophilic group (for use in amine-ester chemistry) include, but are not limited to, pentaerythritol polyethylene glycol amine (4-arm-PEG-NH2) (molecular weight selected from about 5,000 to about 40,000, e.g., 5,000, 10,000, or 20,000), pentaerythritol polyethylene glycol aminoacetate (4-arm-PEG-AA) (molecular weight selected from about 5,000 to about 40,000, e.g., 5,000, 10,000, or 20,000), hexaglycerol polyethylene glycol amine (8-arm-PEG-NH2) (molecular weight selected from about 5,000 to about 40,000, e.g., 10,000, 20,000, or 40,000), or tripentaerythritol polyethylene glycol amine (8-arm (TP)-PEG-NH2) (molecular weight selected from about 5,000 to about 40,000, e.g., 10,000, 20,000, or 40,000). In such compounds, the 4 (or 8) arm-PEG-AA contains an ester (or acetate) group, whereas the 4 (or 8) arm-PEG-NH2 monomer does not contain an ester (or acetate) group.

Other suitable first compounds containing a nucleophilic group (for use in thiol-ester chemistry) include, but are not limited to, ethylene glycol dimercaptoacetate, trimethylolpropane trimercaptoacetate, pentaerythritol tetramercaptoacetate, ethylene glycol di-3-mercaptopropionate, trimethylolpropane tris-3-mercaptopropionate, pentaerythritol tetra-3-mercaptopropionate, polyol-3-mercaptopropionate, polyester-3-mercaptopropionate, propylene glycol 3-mercaptopropionate, ethoxylated trimethylolpropane tris-3-mercaptopropionate, and ethoxylated trimethylolpropane tris-3-mercaptopropionate.

Exemplary electrophilic monomers

The biocompatible preformulation comprises at least one second compound comprising more than one electrophilic group. In some embodiments, the second compound is a monomer configured to form a polymer matrix by reacting the electrophilic group in the second compound with the nucleophilic group in the first compound. In some embodiments, the second compound monomer is completely synthesized. In some embodiments, the electrophilic group is an epoxide, a maleimide, a succinimide group, or an α-β unsaturated ester. In preferred embodiments, the electrophilic group is an epoxide or a succinimide group. In some embodiments, at least one second compound is based on a diol. In some embodiments, diol-based compounds include ethylene glycol, propylene glycol, butylene glycol, alkyl glycols of various chain lengths, and any combination or copolymer thereof. In some embodiments, diol-based compounds are compounds based on polyglycols. In some embodiments, polyglycol-based compounds include but are not limited to polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG), and polyglycol copolymers. In some embodiments, the diol-based compound includes polyethylene glycol, polypropylene glycol, polybutylene glycol, polyalkylene glycols of various chain lengths, and any combination or copolymer thereof. In some embodiments, the diol-based compound is completely synthetic. In some embodiments, the polyglycol-based compound is completely synthetic.

In certain embodiments, the electrophilic group is connected to the polyol derivative by a suitable linker. Suitable linkers include but are not limited to esters, amides or ethers. In some cases, the monomer comprising an ester linker is more sensitive to biodegradation. The example of the linker comprising an electrophilic group includes but is not limited to succinimidyl succinate, succinimidyl glutarate, succinimidyl succinamide, succinimidyl glutaramide or glycidyl ether. In some embodiments, the polyol core derivative is attached to polyethylene glycol or polypropylene glycol subunits, and these subunits are connected to the linker comprising the electrophilic group. The molecular weight of the second compound (electrophilic monomer) is approximately 500 to 40000. In certain embodiments, the second compound (electrophilic monomer) has a molecular weight of about 100, about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 12000, about 15000, about 20000, about 25000, about 30000, about 35000, about 40000, about 50000, about 60000, about 70000, about 80000, about 90000, or about 100000. In some embodiments, the molecular weight of the second compound is between about 500 and 2000. In certain embodiments, the molecular weight of the second compound is between about 15000 and about 40000. In some embodiments, the second compound is water soluble.

In some embodiments, the second compound is a multi-arm-(5k-50k)-polyol derivative, which includes a polyglycol subunit and more than two electrophilic groups. Multi-arm refers to the number of the polyglycol subunits connected to the polyol core, and these polyglycol subunits connect the nucleophilic group to the polyol core. In some embodiments, multi-arm is 3 arms, 4 arms, 6 arms, 8 arms, 10 arms, 12 arms or any combination thereof. In some embodiments, multi-arm is 4 arms or 8 arms. In certain embodiments, the second compound is multi-arm-(5k-50k)-SG, multi-arm-(5k-50k)-SGA, multi-arm-(5-50k)-SS, multi-arm-(5-50k)-SSA and combinations thereof. In certain embodiments, the second compound is selected from 4-arm-(5-50k)-SG, 4-arm-(5-50k)-SGA, 4-arm-(5-50k)-SS, 8-arm-(5-50k)-SG, 8-arm-(5-50k)-SGA and 8-arm-(5-50k)-SS and combinations thereof. In some embodiments, the polyol derivative is a diol, trimethylolpropane, glycerol, diglycerol, pentaerythritol, sorbitol, hexaglycerol, tripentaerythritol or a polyglycerol derivative.

Examples of structures of monomers with a pentaerythritol core polyol containing more than one electrophilic group are shown below. The compounds shown have a succinimidyl electrophilic group, a glutarate or glutaramide linker, and PEG subunits of varying lengths (e.g., the following structures: 4-arm-PEG-SG (D; n=3) and 4-arm-PEG-SGA (E; n=3)). Monomers using other polyol cores or different linkers (e.g., succinate (SS) or succinamide (SSA)) are constructed in a similar manner.

D: (n=1 to 6)

E: (n=1 to 6)

Suitable second compounds comprising an electrophilic group include, but are not limited to, pentaerythritol polyethylene glycol maleimide (4-arm-PEG-MAL) (molecular weight selected from about 5,000 to about 40,000, e.g., 10,000 or 20,000), pentaerythritol polyethylene glycol succinimidyl succinate (4-arm-PEG-SS) (molecular weight selected from about 5,000 to about 40,000, e.g., 10,000 or 20,000), pentaerythritol polyethylene glycol succinimidyl glutarate (4-arm-PEG-SG ... from about 5,000 to about 40,000, for example, 10,000 or 20,000), pentaerythritol polyethylene glycol succinimidyl glutaramide (4-arm-PEG-SGA) (molecular weight selected from about 5,000 to about 40,000, for example, 10,000 or 20,000), hexaglycerol polyethylene glycol succinimidyl succinate (8-arm-PEG-SS) (molecular weight selected from about 5,000 to about 40,000, for example, 10,000 or 20,000), hexaglycerol polyethylene glycol succinimidyl glutarate (8-arm-PEG-SS) (molecular weight selected from about 5,000 to about 40,000, for example, 10,000 or 20,000), EG-SG) (molecular weight selected from about 5000 to about 40000, for example, 10000, 15000, 20000 or 40000), hexaglycerol polyethylene glycol succinimidyl glutaramide (8-arm-PEG-SGA) (molecular weight selected from about 5000 to about 40000, for example, 10000, 15000, 20000 or 40000), tripentaerythritol polyethylene glycol succinimidyl succinate (8-arm (TP)-PEG-SS) (molecular weight selected from about 5000 to about 40000, for example, 0, for example, 10,000 or 20,000), tripentaerythritol polyethylene glycol succinimidyl glutarate (8-arm (TP)-PEG-SG) (molecular weight selected from about 5,000 to about 40,000, for example, 10,000, 15,000, 20,000 or 40,000), or tripentaerythritol polyethylene glycol succinimidyl glutarate (8-arm (TP)-PEG-SGA) (molecular weight selected from about 5,000 to about 40,000, for example, 10,000, 15,000, 20,000 or 40,000). The 4 (or 8) arm-PEG-SG monomer contains an ester group, while the 4 (or 8) arm-PEG-SGA monomer does not contain an ester group.

Other suitable second compounds comprising an electrophilic group are sorbitol polyglycidyl ethers, including but not limited to sorbitol polyglycidyl ether, sorbitol polyglycidyl ether, sorbitol polyglycidyl ether, sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, glycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, sorbitol polyglycidyl ether,

Formation of biocompatible hydrogel polymer matrix

Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, at least one cell, and optional additional components. An exemplary additional component is a culture medium. In certain embodiments, the culture medium is a buffer. In certain embodiments, the culture medium is a nutrient-rich culture medium. In certain embodiments, the cells are stem cells. The biocompatible preformulation undergoes polymerization and/or gelation to form a biocompatible hydrogel polymer matrix. In certain embodiments, the biocompatible hydrogel polymer matrix is biodegradable. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold.

Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, a culture medium, and optionally additional components. An exemplary additional component is at least one cell. In certain embodiments, the cell is a stem cell. In certain embodiments, the culture medium is a buffer. In certain embodiments, the culture medium is a nutrient-rich culture medium. The biocompatible preformulation undergoes polymerization and/or gelation to form a biocompatible hydrogel polymer matrix. In certain embodiments, the biocompatible hydrogel polymer matrix is biodegradable. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold.

In certain embodiments, the preformulation is safely polymerized in a target site in or on a mammalian body, for example, at a wound site, a surgical site, or a joint. In certain embodiments, the biocompatible hydrogel polymer matrix forms a wound patch, a suture, or a joint spacer. In some embodiments, the first compound and the second compound are monomers that form a polymer matrix by reacting the nucleophilic group in the first compound with the electrophilic group in the second compound. In certain embodiments, the monomer is polymerized at a predetermined time. In some embodiments, the monomer is polymerized under mild and near-neutral pH conditions. In certain embodiments, the biocompatible hydrogel polymer matrix does not change volume after gelation.

In some embodiments, the first compound reacts with the second compound to form an amide, thioester, or thioether bond. When a thiol nucleophile reacts with a succinimidyl electrophile, a thioester is formed. When an amino nucleophile reacts with a succinimidyl electrophile, an amide is formed.

In some embodiments, one or more first compounds comprising an amino group react with one or more second compounds comprising a succinimidyl ester group to form first and second monomeric units connected by an amide bond. In certain embodiments, one or more first compounds comprising a thiol group react with one or more second compounds comprising a succinimidyl ester group to form first and second monomeric units connected by a thioester bond. In some embodiments, one or more first compounds comprising an amino group react with one or more second compounds comprising an epoxide group to form first and second monomeric units connected by an amine bond. In certain embodiments, one or more first compounds comprising a thiol group react with one or more second compounds comprising an epoxide group to form first and second monomeric units connected by a thioether bond.

In some embodiments, the first compound is mixed with a different first compound prior to being added to the one or more second compounds. In other embodiments, the second compound is mixed with a different second compound prior to being added to the one or more first compounds. In certain embodiments, the properties of the biocompatible preformulation and the biocompatible hydrogel polymer matrix are controlled by the properties of the at least one first monomer mixture and the at least one second monomer mixture.

In some embodiments, one first compound is used in the biocompatible hydrogel polymer matrix. In certain embodiments, two different first compounds are mixed and used in the biocompatible hydrogel polymer matrix. In some embodiments, three different first compounds are mixed and used in the biocompatible hydrogel polymer matrix. In certain embodiments, four or more different first compounds are mixed and used in the biocompatible hydrogel polymer matrix.

In some embodiments, one second compound is used in the biocompatible hydrogel polymer matrix. In certain embodiments, two different second compounds are mixed and used in the biocompatible hydrogel polymer matrix. In some embodiments, three different second compounds are mixed and used in the biocompatible hydrogel polymer matrix. In certain embodiments, four or more different second compounds are mixed and used in the biocompatible hydrogel polymer matrix.

In some embodiments, a first compound comprising an ether bond connected to a nucleophilic group is mixed with a different first compound comprising an ester bond connected to a nucleophilic group. This enables the concentration of ester groups in the resulting biocompatible hydrogel polymer matrix to be controlled. In certain embodiments, a second compound comprising an ester bond connected to an electrophilic group is mixed with a different second compound comprising an ether bond connected to an electrophilic group. In some embodiments, a second compound comprising an ester bond connected to an electrophilic group is mixed with a different second compound comprising an amide bond connected to an electrophilic group. In certain embodiments, a second compound comprising an amide bond connected to an electrophilic group is mixed with a different second compound comprising an ether bond connected to an electrophilic group.

In some embodiments, a first compound comprising an aminoacetate (e.g., glycine-derived) nucleophile is mixed with a different first compound comprising an amine nucleophile (e.g., ethylamine ether) at a specified molar ratio (x/y). In certain embodiments, the molar ratio (x/y) is 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or 95/5. In certain embodiments, a first compound comprising an aminoacetate (e.g., glycine-derived) nucleophile is mixed with a different first compound comprising an amine nucleophile (e.g., ethylamine ether) at a specified weight ratio (x/y). In certain embodiments, the weight ratio (x/y) is 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or 95/5. In certain embodiments, the mixture of two first compounds is mixed with one or more second compounds in a molar amount equal to the sum of x and y.

In some embodiments, a first compound comprising more than one nucleophilic group is premixed with at least one cell in the presence of water. In some embodiments, a first compound comprising more than one nucleophilic group is premixed with cells in the absence of water. Once premixing is complete, a second compound comprising more than one electrophilic group is added to the premix in the presence of water to form a biocompatible hydrogel polymer matrix. Shortly after final mixing, the biocompatible hydrogel polymer matrix is delivered to the target site. In certain embodiments, an optional additional component is added to the premix, the second compound, or the mixture just before the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, after the biocompatible hydrogel polymer matrix mixture is delivered to the target site, an optional additional component is added to the premix, the second compound, or the mixture. In some embodiments, the additional component is a buffer. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled before being delivered to the target site. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled at the target site.

In some embodiments, a first compound comprising more than one nucleophilic group is premixed with a buffer in the presence of water. In some embodiments, a first compound comprising more than one nucleophilic group is premixed with a buffer in the absence of water. Once premixing is complete, a second compound comprising more than one electrophilic group is added to the premix in the presence of water to form a biocompatible hydrogel polymer matrix. Shortly after final mixing, the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, just before the biocompatible hydrogel polymer matrix mixture is delivered to the target site, optional additional components are added to the premix, the second compound, or the mixture. In certain embodiments, after the biocompatible hydrogel polymer matrix mixture is delivered to the target site, optional additional components are added to the premix, the second compound, or the mixture. In some embodiments, the additional component is at least one cell. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled before delivery to the target site. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled at the target site.

In other embodiments, a second compound comprising more than one electrophilic group is premixed with at least one cell in the presence of water. In other embodiments, a second compound comprising more than one electrophilic group is premixed with cells in the absence of water. Once premixing is complete, a first compound comprising more than one nucleophilic group is added to the premix to form a biocompatible hydrogel polymer matrix. Shortly after final mixing, the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, an optional component is added to the premix, the first compound, or the mixture just before the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, after the biocompatible hydrogel polymer matrix mixture is delivered to the target site, an optional additional component is added to the premix, the first compound, or the mixture. In some embodiments, the additional component is a buffer. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled before delivery to the target site. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled at the target site.

In other embodiments, the second compound comprising more than one electrophilic group is premixed with a buffer in the presence of water. In other embodiments, the second compound comprising more than one electrophilic group is premixed with a buffer in the absence of water. Once premixing is complete, the first compound comprising more than one nucleophilic group is added to the premix to form a biocompatible hydrogel polymer matrix. Shortly after final mixing, the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, an optional component is added to the premix, the first compound, or the mixture just before the biocompatible hydrogel polymer matrix mixture is delivered to the target site. In certain embodiments, after the biocompatible hydrogel polymer matrix mixture is delivered to the target site, an optional additional component is added to the premix, the first compound, or the mixture. In some embodiments, the additional component is at least one cell. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled before delivery to the target site. In some embodiments, the biocompatible hydrogel polymer matrix is polymerized and/or gelled at the target site.

In some embodiments, a first compound comprising more than one nucleophilic group, a second compound comprising more than one electrophilic group, and at least one cell are mixed together in the presence of water to form a biocompatible hydrogel polymer matrix. In some embodiments, a first compound comprising more than one nucleophilic group, a second compound comprising more than one electrophilic group, and a buffer are mixed together in the presence of water to form a biocompatible hydrogel polymer matrix. In some embodiments, a first compound comprising more than one nucleophilic group, a second compound comprising more than one electrophilic group, at least one cell, and a buffer are mixed together in the presence of water to form a biocompatible hydrogel polymer matrix. In certain embodiments, the first compound comprising more than one nucleophilic group, the second compound comprising more than one electrophilic group, and/or the cell are diluted in an aqueous buffer having a pH range of about 5.0 to about 9.5, wherein a single dilution or pure monomer is mixed and forms a biocompatible hydrogel polymer matrix. In some embodiments, the aqueous buffer is in a pH range of about 6.0 to about 8.5. In certain embodiments, the aqueous buffer is in a pH range of about 8. In certain embodiments, the aqueous buffer is a culture medium. In certain embodiments, the culture medium is a nutrient-rich culture medium.

In certain embodiments, the concentration of the monomer in the aqueous buffer is about 1% to about 100%. In some embodiments, dilution is adopted to regulate the viscosity of the monomer diluent. In certain embodiments, the concentration of the monomer in the aqueous buffer is about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100%.

In some embodiments, the electrophilic and nucleophilic monomers are mixed in a ratio such that there is a slight excess of the electrophilic group in the mixture. In certain embodiments, the excess is about 10%, about 5%, about 2%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or less than 0.1%.

In certain embodiments, the gelation time or curing time of the biocompatible hydrogel polymer matrix is controlled by selecting the first and second compounds. In some embodiments, the concentration of the nucleophilic or electrophilic groups in the first or second compound affects the gelation time of the biocompatible preformulation. In certain embodiments, temperature affects the gelation time of the biocompatible preformulation. In some embodiments, the type of aqueous buffer affects the gelation time of the biocompatible preformulation. In some embodiments, the aqueous buffer is culture medium. In certain embodiments, the concentration of the aqueous buffer affects the gelation time of the biocompatible preformulation. In some embodiments, the nucleophilicity and/or electrophilicity of the nucleophilic and electrophilic groups of the monomer affect the gelation time of the biocompatible preformulation. In some embodiments, the cell type affects the gelation time of the biocompatible preformulation. In some embodiments, the cell concentration affects the gelation time of the biocompatible preformulation.

In some embodiments, the gel time or curing time of the biocompatible hydrogel polymer matrix is controlled by the pH of the aqueous buffer. In certain embodiments, the gel time is about 20 seconds to 10 minutes. In some embodiments, the gel time is less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 4.8 minutes, less than 4.6 minutes, less than 4.4 minutes, less than 4.2 minutes, less than 4.0 minutes, less than 3.8 minutes, less than 3.6 minutes, less than 3.4 minutes, less than 3.2 minutes, less than 3.0 minutes, less than 2.8 minutes, less than 2.6 minutes, less than 2.4 minutes, less than 2.2 minutes, less than 2.0 minutes, less than 1.8 minutes, less than 1.6 minutes, less than 1.4 minutes, less than 1.2 minutes, less than 1.0 minute, less than 0.8 minute, less than 0.6 minute or less than 0.4 minute. In certain embodiments, the pH of the aqueous buffer is about 5 to about 9.5. In some embodiments, the pH of the aqueous buffer is about 7.0 to about 9.5. In specific embodiments, the pH of the aqueous buffer is about 8. In some embodiments, the pH of the aqueous buffer is about 5, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 9.0, or about 9.5.

In certain embodiments, the gelation time or the solidification time of biocompatibility preformulation are controlled by the type of aqueous buffer. In some embodiments, aqueous buffer is a physiologically acceptable buffer. In certain embodiments, aqueous buffer includes but is not limited to, aqueous saline solution, phosphate buffered saline, borate buffered saline, each component is dissolved in the borate and phosphate buffer combination in each buffer, N-2-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid (HEPES), 3-(N-morpholino) propanesulfonic acid (MOPS), 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid (TES), 3-[N-tris(hydroxymethyl)ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid (EPPS), tris[hydroxymethyl]-aminomethane (THAM) and tris[hydroxymethyl]methylaminomethane (TRIS). In some embodiments, thiol-ester chemical reaction (for example, ETTMP nucleophile and SGA or SG electrophile) is carried out in borate buffer. In certain embodiments, the amine-ester chemical reaction (NH2 or AA nucleophile and SGA or SG electrophile) is carried out in a phosphate buffer. In some embodiments, the aqueous buffer is a culture medium. In certain embodiments, the culture medium includes but is not limited to DMEM, IMDM, AlgiMatrix ^™ , fetal bovine serum, RPMI, SensiCell ^™ , GlutaMAX ^™ , FluoroBrite ^™ , LB, M9 Minimal, Terrific Broth, 2YXT, MagicMedia ^™ , ImMedia ^™ , SOC, YPD, CSM, YNB, Grace Insect Media (Grace's InsectMedia), 199/109 and HamF10/HamF12. In certain embodiments, the cell culture medium can be serum-free. In certain embodiments, the culture medium can include additives. In some embodiments, culture medium additives include but are not limited to antibiotics, vitamins, proteins, inhibitors, small molecules, minerals, inorganic salts, nitrogen, growth factors, amino acids, serum, carbohydrates, lipids, hormones and glucose. In some embodiments, growth factors include but are not limited to EGF, bFGF, FGF, ECGF, IGF-1, PDGF, NGF, TGF-α and TGF-β. In certain embodiments, the culture medium may not be aqueous. In certain embodiments, non-aqueous culture medium includes but is not limited to frozen cell storage, lyophilized culture medium and agar.

In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold. In certain embodiments, the biocompatible hydrogel scaffold comprises a preformulation of at least one first compound and a preformulation of at least one second compound. In certain embodiments, the biocompatible hydrogel scaffold comprises a buffer. In certain embodiments, the biocompatible hydrogel scaffold is fully synthetic. In certain embodiments, the biocompatible hydrogel scaffold provides an environment suitable for sustained cell viability and/or growth.

In certain embodiments, during the formation of the biocompatible hydrogel polymer matrix, the first compound and the second compound do not react with the cell. In some embodiments, the cell remains unchanged after the polymerization of the first and second compounds (i.e., monomers). In certain embodiments, the cell does not change the property of the biocompatible hydrogel polymer matrix. In some embodiments, the physicochemical properties of the cell and the biocompatible hydrogel polymer matrix preparation are not affected by monomer polymerization. In certain embodiments, the biocompatible hydrogel polymer matrix is used to deliver cells to minimize degradation or degeneration of the cell. In some cases, the physicochemical properties of the cell are not affected by the delivery or release of the cell to the target site.

In some embodiments, the biocompatible hydrogel polymer matrix formulation further comprises a contrast agent for visualizing the biocompatible hydrogel polymer matrix formulation and locating tumors using, for example, X-ray, fluoroscopy, or computed tomography (CT) imaging. In certain embodiments, the contrast agent enables visualization of bioabsorption of the biocompatible hydrogel polymer matrix. In some embodiments, the contrast agent is a radiopaque material. In certain embodiments, the radiopaque material is selected from, but not limited to, sodium iodide, potassium iodide, and barium sulfate, or tantalum and similar commercially available compounds or combinations thereof. In other embodiments, the biocompatible hydrogel polymer matrix further comprises a pharmaceutically acceptable dye.

In some embodiments, the biocompatible hydrogel polymer matrix formulation further comprises a viscosity enhancer. Examples of viscosity enhancers include, but are not limited to, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, polyvinyl cellulose, and polyvinyl pyrrolidone.

Areas treated – target areas

In certain embodiments, the target site is internal to a mammal. In certain embodiments, the target site is internal to a human. In certain embodiments, the target site is on a human body. In certain embodiments, the target site is accessible by surgery. In certain embodiments, the target site is accessible by minimally invasive surgery. In certain embodiments, the target site is accessible by an endoscopic device. In certain embodiments, the target site is a wound on the skin of a mammal. In other embodiments, the target site is in a joint or on a bone of a mammal. In certain embodiments, the target site is a surgical site in a mammal.

In some embodiments, the biocompatible preformulation or biocompatible hydrogel polymer matrix is used as a sealant or adhesive. In certain embodiments, the biocompatible preformulation or biocompatible hydrogel polymer matrix is used to seal a wound on a mammal. In other embodiments, the biocompatible preformulation or biocompatible hydrogel polymer matrix is used to fill a body cavity, for example, in a joint space, to form a gel pad. In other embodiments, the biocompatible preformulation or biocompatible hydrogel polymer matrix is used as a carrier for delivering cells to a target site.

In some embodiments, the biocompatible hydrogel polymer matrix formulation is polymerized ex vivo. In certain embodiments, the ex vivo polymerized biocompatible hydrogel polymer matrix formulation is delivered via conventional routes of administration (e.g., oral, implantable, or rectal). In other embodiments, the ex vivo polymerized biocompatible hydrogel polymer matrix formulation is delivered to the target site during surgery.

Delivery of biocompatible hydrogel formulations to target sites

In some embodiments, the biocompatible preformulation is delivered to the target site as a biocompatible preformulation via a catheter or needle to form a biocompatible hydrogel polymer matrix at the target site. In other embodiments, the biocompatible preformulation is delivered to the target site in or on a mammal using a syringe and needle. In some embodiments, the biocompatible preformulation is delivered to the target site using a delivery device. In some embodiments, the biocompatible preformulation is delivered to the target site such that the biocompatible preformulation substantially covers the target site. In certain embodiments, the biocompatible preformulation substantially covers the exposed portion of the diseased tissue. In some embodiments, the biocompatible preformulation is not intended to spread to any other location. In some embodiments, the biocompatible preformulation substantially covers the diseased tissue and does not significantly cover healthy tissue. In certain embodiments, the biocompatible hydrogel polymer matrix does not significantly cover healthy tissue. In some embodiments, the biocompatible preformulation gels at the target site and completely covers the diseased tissue. In some embodiments, the biocompatible hydrogel polymer matrix adheres to the tissue. In some embodiments, the biocompatible hydrogel polymer matrix mixture gels at the target site after delivery, thereby covering the target site. In some embodiments, the biocompatible hydrogel polymer matrix mixture gels at the target site before delivery.

In some embodiments, the gel time of a biocompatible preformulation is set based on a physician's preference for delivering the biocompatible preformulation mixture to the target site. In most cases, the physician delivers the biocompatible preformulation mixture to the target site within 15 to 30 seconds. In certain embodiments, the gel time is approximately 20 seconds to 10 minutes. In some embodiments, the gel time or curing time of the biocompatible preformulation is controlled by the pH of the aqueous buffer. In certain embodiments, the gel time or curing time of the biocompatible preformulation is controlled by selecting the first and second compounds. In some embodiments, the concentration of the nucleophilic or electrophilic groups in the first or second compound affects the gel time of the biocompatible preformulation. In some embodiments, the cell concentration affects the gel time of the biocompatible preformulation. In some embodiments, the cell type affects the gel time of the biocompatible preformulation. In some embodiments, optional additional components affect the gel time of the biocompatible preformulation.

In some embodiments, the solidification of the biocompatible hydrogel polymer matrix is verified after administration. In certain embodiments, in vivo verification is carried out at the delivery site. In other embodiments, ex vivo verification is carried out. In some embodiments, the solidification of the biocompatible hydrogel polymer matrix is verified by optical fiber vision of an endoscopic device. In certain embodiments, the solidification of the biocompatible hydrogel polymer matrix comprising a radiopaque material is verified using X-ray, fluoroscopy or computed tomography (CT) imaging. The lack of flow in the biocompatible hydrogel polymer matrix indicates that the biocompatible hydrogel polymer matrix has gelled and the biocompatible hydrogel has fully solidified. In further embodiments, the solidification of the biocompatible hydrogel polymer matrix is verified by assessing the residues in the delivery device (e.g., residues in the catheter of a bronchoscope or other endoscopic device, or residues in the syringe for delivering the biocompatible hydrogel polymer matrix). In other embodiments, the solidification of the biocompatible hydrogel polymer matrix is verified by depositing a small sample (e.g., about 1 mL) on a piece of paper or in a small container and then assessing the flow properties after exceeding the gel time.

In some embodiments, the biocompatible preformulation delivers at least one cell to the target site. In some embodiments, the biocompatible preformulation delivers nutrients to at least one cell located at the target site. In some embodiments, the biocompatible preformulation provides structural support for at least one cell located at the target site. In some embodiments, the biocompatible preformulation delivers at least one cell and at least one buffer to the target site. In some embodiments, the biocompatible hydrogel polymer matrix delivers at least one cell to the target site. In some embodiments, the biocompatible hydrogel polymer matrix delivers nutrients to at least one cell located at the target site. In some embodiments, the biocompatible hydrogel polymer matrix provides structural support for at least one cell located at the target site. In some embodiments, the biocompatible hydrogel polymer matrix delivers at least one cell to the target site.

Bioabsorption of biocompatible hydrogel polymer matrices

In some embodiments, the biocompatible hydrogel polymer matrix is a bioabsorbable polymer. In certain embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed within about 5 to 30 days. In some embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed within about 30 to 180 days. In some embodiments, the biocompatible hydrogel polymer is bioabsorbed within about 1 to 70 days. In preferred embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed within about 14 to 180 days. In some embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed within about 365 days, 180 days, about 150 days, about 120 days, about 90 days, about 80 days, about 70 days, about 60 days, about 50 days, about 40 days, about 35 days, about 30 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In certain embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed in less than 365 days, 180 days, less than 150 days, less than 120 days, less than 90 days, less than 80 days, less than 70 days, less than 60 days, less than 50 days, less than 40 days, less than 35 days, less than 30 days, less than 28 days, less than 21 days, less than 14 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, the biocompatible hydrogel polymer matrix is bioabsorbed in more than 365 days, more than 180 days, more than 150 days, more than 120 days, more than 90 days, more than 80 days, more than 70 days, more than 60 days, more than 50 days, more than 40 days, more than 35 days, more than 30 days, more than 28 days, more than 21 days, more than 14 days, more than 10 days, more than 7 days, more than 6 days, more than 5 days, more than 4 days, more than 3 days, more than 2 days, or more than 1 day. In some embodiments, the biocompatible hydrogel polymer matrix is substantially non-bioabsorbable.

The biocompatible hydrogel polymer matrix is slowly bioabsorbed, dissolved, and/or excreted. In some cases, the rate of bioabsorption is controlled by the number of ester groups in the biocompatible and/or biodegradable hydrogel polymer matrix. In other cases, the higher the concentration of ester units in the biocompatible hydrogel polymer matrix, the longer its in vivo lifespan. In further cases, the electron density at the carbonyl group of the ester unit controls the in vivo lifespan of the biocompatible hydrogel polymer matrix. In some cases, a biocompatible hydrogel polymer matrix without ester groups is substantially non-biodegradable. In other cases, the molecular weight of the first and second compounds controls the in vivo lifespan of the biocompatible hydrogel polymer matrix. In further cases, the number of ester groups per gram of polymer matrix controls the in vivo lifespan of the biocompatible hydrogel polymer matrix.

In some cases, the lifetime of a biocompatible hydrogel polymer matrix can be estimated using a model that controls the temperature and pH at physiological levels while exposing the biocompatible hydrogel polymer matrix to a buffer solution. In some cases, the biodegradation of the biocompatible hydrogel polymer matrix is substantially non-enzymatic.

In some embodiments, the choice of reaction conditions determines the degradation time of the biocompatible hydrogel polymer matrix. In certain embodiments, the concentration of the monomers of the first compound and the second compound determines the degradation time of the resulting biocompatible hydrogel polymer matrix. In some cases, a higher monomer concentration results in a higher degree of crosslinking in the resulting biocompatible hydrogel polymer matrix. In some cases, more crosslinking results in a slower degradation of the biocompatible hydrogel polymer matrix. In certain embodiments, the temperature determines the degradation time of the resulting biocompatible hydrogel polymer matrix. In some cases, a higher monomer concentration results in a higher degree of crosslinking in the resulting biocompatible hydrogel polymer matrix.

In certain embodiments, the composition of the linker in the first and/or second compound affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In some embodiments, the more ester groups present in the biocompatible hydrogel polymer matrix, the faster the biocompatible hydrogel polymer matrix degrades. In certain embodiments, the higher the concentration of mercaptopropionate (ETTMP), acetamide (AA), glutarate, or succinate (SG or SS) monomers, the faster the degradation rate.

In certain embodiments, the composition of the cells affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In certain embodiments, the concentration of the cells affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In certain embodiments, the composition of the buffer affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In certain embodiments, the concentration of the buffer affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In certain embodiments, the pH of the buffer affects the degradation rate of the resulting biocompatible hydrogel polymer matrix. In certain embodiments, the composition of the optional additional components affects the degradation rate of the resulting biocompatible hydrogel polymer matrix.

Preformulations and hydrogel matrices for cell delivery in disease treatment

Treating tendon injuries by stem cells requires controlled delivery and release of cells at the target area. For example, bone marrow mesenchymal stem cells (MSCs) have a beneficial effect on the healing of equine tendon injuries. Existing methods inject MSCs in autologous bone marrow aspirates in large quantities (10-20 million cells), but due to systemic clearance, less than 25% of MSCs remain in the damaged area after 24 hours. The retention of cells at the delivery site can promote cell implantation into the tissue, thereby resulting in an increased amount of available MSCs to contribute to tissue healing. The biocompatible preformulations and hydrogel polymer matrix described herein are configured to deliver cells, such as MSCs in a flexible, injectable and absorbable gel that is clinically tolerated and compatible with cell survival and growth. In some embodiments, the biocompatible preformulations and hydrogel polymer matrix described herein provide improved cell viability compared to cells injected without the use of a biocompatible preformulation. In certain embodiments, the biocompatible hydrogel polymer matrix acts as a scaffold for supporting the growth of cells loaded on or within the hydrogel polymer matrix.

In some embodiments, the biocompatible preformulations or hydrogel polymer matrix described herein are delivered to a target site on or in a mammal. In certain embodiments, the biocompatible preformulations or hydrogel polymer matrix are delivered to a target site in a joint. In some embodiments, the biocompatible preformulation forms a biocompatible hydrogel polymer matrix within the joint. In certain embodiments, the biocompatible preformulation forms a viscous biocompatible hydrogel polymer matrix to seal a wound on or in an animal. In some embodiments, the biocompatible preformulation forms a suture. In certain embodiments, a wound patch, a joint spacer, or a suture is at least partially gelled at a target site in or on a mammal. In some embodiments, a wound patch, a joint spacer, or a suture is at least partially polymerized at a target site. In some embodiments, a wound patch, a joint spacer, or a suture is at least partially adhered to the target site.

In certain embodiments, the biocompatible preformulation is used as a "liquid suture" or drug delivery platform to deliver drugs directly to a target site in or on a mammal. In some embodiments, the target site is a joint, a wound, or a surgical site. In some embodiments, the spreadability, viscosity, optical clarity, and adhesive properties of the biocompatible preformulation or hydrogel polymer matrix are optimized to produce an ideal material for use as a liquid suture for disease treatment. In certain embodiments, the gel time is controlled to be from 50 seconds to 15 minutes.

In some embodiments, a biocompatible preformulation or hydrogel polymer matrix comprising at least one cell is delivered to a target site in a mammal. In some embodiments, the biocompatible preformulation or hydrogel polymer matrix is configured to deliver cells to damaged tissue to treat a disease or injury. In some embodiments, the disease includes but is not limited to cancer, diabetes, Alzheimer's disease, Parkinson's disease, Huntington's disease, and celiac disease. In some embodiments, the injury is caused by heart failure, muscle injury, brain injury, or a neurological disorder. In some embodiments, the injury is a spinal cord injury. In some embodiments, the delivered cells are configured to treat an orthopedic disease or injury. In some embodiments, the delivered cells are configured to repair tendons, joints, bone defects, muscles, or nerves.

Control of cell release rate

In some embodiments, the biocompatible hydrogel polymer matrix slowly delivers at least one cell to the target site by diffusion and/or osmosis over a period of several hours to several days. In certain embodiments, the cells are delivered directly to the target site. In some embodiments, the procedure of delivering the biocompatible hydrogel polymer matrix containing cells to the target site is repeated several times if necessary. In other embodiments, the cells are released from the biocompatible hydrogel polymer matrix by biodegradation of the biocompatible hydrogel polymer matrix. In some embodiments, the cells are released by a combination of diffusion, osmosis and/or biocompatible hydrogel degradation mechanisms. In certain embodiments, the release curve of the cells released from the biocompatible hydrogel polymer matrix is unimodal. In some embodiments, the release curve of the cells released from the biocompatible hydrogel polymer matrix is bimodal. In certain embodiments, the release curve of the cells released from the biocompatible hydrogel polymer matrix is multimodal.

In some embodiments, the cells are released from the biocompatible hydrogel polymer matrix by diffusion or osmosis. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix within 180 days. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix within 14 days. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix within 24 hours. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix within one hour. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix within about 180 days, about 150 days, about 120 days, about 90 days, about 80 days, about 70 days, about 60 days, about 50 days, about 40 days, about 35 days, about 30 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 0.5 days, about 6 hours, about 4 hours, about 2 hours, or about 1 hour. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix in more than 180 days, more than 150 days, more than 120 days, more than 90 days, more than 80 days, more than 70 days, more than 60 days, more than 50 days, more than 40 days, more than 35 days, more than 30 days, more than 28 days, more than 21 days, more than 14 days, more than 10 days, more than 7 days, more than 6 days, more than 5 days, more than 4 days, more than 3 days, more than 2 days, more than 1 day, more than 0.5 day, more than 6 hours, more than 4 hours, more than 2 hours, or more than 1 hour. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix in less than 180 days, less than 150 days, less than 120 days, less than 90 days, less than 80 days, less than 70 days, less than 60 days, less than 50 days, less than 40 days, less than 35 days, less than 30 days, less than 28 days, less than 21 days, less than 14 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 0.5 day, less than 6 hours, less than 4 hours, less than 2 hours, or less than 1 hour. In some embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix in about one day to about fourteen days. In certain embodiments, the cells are substantially released from the biocompatible hydrogel polymer matrix in about one day to about 70 days.

In some embodiments, the release of cells from the biocompatible hydrogel polymer matrix is controlled by the composition of the biocompatible hydrogel polymer matrix. In certain embodiments, cells are released when the biocompatible hydrogel polymer matrix begins to degrade. In some embodiments, the pore size of the biocompatible hydrogel polymer matrix is small enough to prevent the early release of cells (i.e., release before the biocompatible hydrogel polymer matrix degrades). In certain embodiments, the pore size of the biocompatible hydrogel polymer matrix is large enough to allow the early release of cells.

In some embodiments, large PEG groups in the monomers result in large pores in the resulting biocompatible hydrogel polymer matrix, thereby allowing elution of large cells. In certain embodiments, large molecular weight monomers result in a biocompatible hydrogel polymer matrix with large pores. In some embodiments, a large monomer molecular weight of about 10 kDa results in a biocompatible hydrogel polymer matrix with large pores. In certain embodiments, a large monomer molecular weight of about 20 kDa results in a biocompatible hydrogel polymer matrix with large pores.

In some embodiments, the small PEG groups in the monomer result in a small pore size in the resulting biocompatible hydrogel polymer matrix, thereby limiting the elution of small (and large) cells. In certain embodiments, monomers of small molecular weight result in a biocompatible hydrogel polymer matrix with a small pore size. In some embodiments, a small monomer molecular weight of about 5kDa results in a biocompatible hydrogel polymer matrix with a small pore size. In certain embodiments, a small monomer molecular weight of about 10kDa in an 8-arm monomer results in a biocompatible hydrogel polymer matrix with a small pore size. In some embodiments, the elution of cells by the small pore size restriction is limited.

Exemplary cells

In some embodiments, the biocompatible hydrogel polymer matrix comprises at least one cell. In some embodiments, the biocompatible hydrogel polymer matrix is delivered together with the cell. Examples of cells include, but are not limited to, mammalian, insect, protozoan, bacterial, viral, or fungal cells. In some embodiments, the cell can be genetically engineered. In some embodiments, the cell can be a vaccine.

In certain embodiments, the cell is a mammalian cell. Examples of mammalian cells include, but are not limited to, humans, mice, hamsters, rats, dogs, and primate cells. Examples of human cells include, but are not limited to, embryonic, adult, bone marrow stroma, embryonic germline, fetus, oligopotent progenitor cells, somatic cells, and induced pluripotent cells. Mammalian cells include, but are not limited to, established or developed cell lines. Examples of cell lines include, but are not limited to, HEK-293, CHO, 293-T, A2780, BHK-21, BCP-1, DU145, H1299, HeLa, High-Five, HUVEC, MCF-7, and RBL. Mammalian cells include, but are not limited to, stem cells. Examples of stem cells include, but are not limited to, adult, embryonic, hematopoietic, embryonic, mesenchymal, multipotent, neural, pluripotent, totipotent, umbilical cord, and unipotent cells.

In certain embodiments, the cell is an insect cell. In certain embodiments, the insect cell is genetically engineered. In certain embodiments, the insect cell is non-infectious. Examples of insect cells include, but are not limited to, Spodoptera frugiperda, Drosophila, and Trichoplusia ni cells.

In certain embodiments, the cell is a protozoan cell. In certain embodiments, the protozoan cell is genetically engineered. In certain embodiments, the protozoan cell is a vaccine. In certain embodiments, the protozoan cell is non-infectious. Examples of protozoan cells include, but are not limited to, Giardia lamblia, Entamoeba histolytica, Plasmodium knowlesi, and Balantidium coli cells.

In certain embodiments, the cell is a bacterial cell. In certain embodiments, the bacterial cell is genetically engineered. In certain embodiments, the bacterial cell is a vaccine. In certain embodiments, the bacterial cell is non-infectious. Examples of bacterial cells include, but are not limited to, Acetobacter aurantius, Agrobacterium radiobacter, Anaplasma phagocytophilum, Azorhizobium caulinodans, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, pertussis), Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Helicobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittac, Clostridium botulinum botulinum), Clostridium difficile, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis pertussis), Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma hominis hominis), Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia rickettsii, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi typhi), Salmonella typhimurium, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pseudotuberculosis.

In certain embodiments, the cell is a viral cell. In certain embodiments, the viral cell is genetically engineered. In certain embodiments, the cell is a vaccine. In certain embodiments, the cell is non-infectious. In certain embodiments, the viral cell is a bacteriophage. Examples of viral cells include, but are not limited to, adenoviruses, herpes viruses, pox viruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, retroviruses, and hepadnaviruses.

In certain embodiments, the cell is a fungal cell. In certain embodiments, the fungal cell is genetically engineered. In certain embodiments, the fungal cell is non-infectious. Examples of fungal cells include, but are not limited to, Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Yarrowia lipolytica, Saccharomyces exiguus, and Pichia pastoris.

Exemplary culture media

In some embodiments, the biocompatible hydrogel polymer matrix comprises a buffer or culture medium. In some embodiments, the biocompatible hydrogel polymer matrix comprises a buffer and at least one cell. In some embodiments, the culture medium is a buffer. In some embodiments, the culture medium comprises a growth medium. In some embodiments, the culture medium is nutrient-rich. In certain embodiments, the culture medium provides nutrients sufficient to support cell viability, growth, and/or proliferation. In certain embodiments, culture medium includes but is not limited to DMEM, IMDM, AlgiMatrix ^™ , fetal bovine serum, RPMI, SensiCell ^™ , GlutaMAX ^™ , FluoroBrite ^™ , LB, M9Minimal, Terrific Broth, 2YXT, MagicMedia ^™ , ImMedia ^™ , SOC, YPD, CSM, YNB, Grace Insect Media (Grace's Insect Media), 199/109, and HamF10/HamF12. In certain embodiments, the cell culture medium can be serum-free. In certain embodiments, the culture medium includes additives. In some embodiments, culture medium additives include but are not limited to antibiotics, vitamins, proteins, inhibitors, small molecules, minerals, inorganic salts, nitrogen, growth factors, amino acids, serum, carbohydrates, lipids, hormones and glucose. In some embodiments, growth factors include but are not limited to EGF, bFGF, FGF, ECGF, IGF-1, PDGF, NGF, TGF-α and TGF-β. In certain embodiments, this culture medium may not be aqueous. In certain embodiments, non-aqueous culture medium includes but is not limited to frozen cell storage, lyophilized culture medium and agar.

Exemplary combinations

In some embodiments, one or more optional components may be introduced into the biocompatible hydrogel polymer matrix formulation. Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, at least one cell, and optional additional components. An exemplary additional component is a buffer. In certain embodiments, the cell is a stem cell. In certain embodiments, the additional component is a culture medium. In certain embodiments, the culture medium is nutrient-rich. A biocompatible hydrogel polymer matrix is formed by mixing the first compound, the second compound, and at least one cell in the presence of water; wherein the biocompatible hydrogel polymer matrix gels at the target site. In some embodiments, a buffer or other additional components may be added to the preformulation mixture before or after the biocompatible hydrogel polymer matrix is formed. In some embodiments, the first and second compounds do not react with the at least one cell during formation of the biocompatible hydrogel polymer matrix. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold. In certain embodiments, the biocompatible hydrogel scaffold comprises the at least one first compound and the at least one second compound. In certain embodiments, the biocompatible hydrogel scaffold comprises a buffer.In certain embodiments, the biocompatible hydrogel scaffold is fully synthetic.

Provided herein are biocompatible preformulations comprising at least one first compound comprising more than one nucleophilic group, at least one second compound comprising more than one electrophilic group, a buffer, and optionally additional components. An exemplary additional component is at least one cell. In certain embodiments, the cell is a stem cell. In certain embodiments, the buffer is a culture medium. In certain embodiments, the culture medium is nutrient-rich. A biocompatible hydrogel polymer matrix is formed by mixing the first compound, the second compound, and the buffer in the presence of water; wherein the biocompatible hydrogel polymer matrix gels at the target site. In some embodiments, the at least one cell or other additional component can be added to the mixture before or after the biocompatible hydrogel polymer matrix is formed. In some embodiments, the first compound and the second compound do not react with the at least one cell during the formation of the biocompatible hydrogel polymer matrix. In certain embodiments, the biocompatible hydrogel polymer matrix comprises a biocompatible hydrogel scaffold. In certain embodiments, the biocompatible hydrogel scaffold comprises the at least one first compound, the at least one second compound, and the buffer. In certain embodiments, the biocompatible hydrogel scaffold is completely synthetic.

In certain embodiments, the biocompatible preformulation or biocompatible hydrogel polymer matrix comprises at least one additional component. Additional components include, but are not limited to, proteins, biomolecules, growth factors, anesthetics, antimicrobials, antivirals, immunosuppressants, anti-inflammatory agents, antiproliferative agents, anti-angiogenic agents, and hormones.

In some embodiments, the biocompatible hydrogel polymer matrix or biocompatible preformulation further comprises a visualization agent for visualizing the placement of the biocompatible hydrogel polymer matrix at the target site. The visualization agent helps visualize placement using minimally invasive delivery, for example, using an endoscopic device. In certain embodiments, the visualization agent is a dye. In specific embodiments, the visualization agent is a colorant.

In some embodiments, the biocompatible hydrogel polymer matrix formulation further comprises a contrast agent for visualizing the biocompatible hydrogel formulation and locating tumors using, for example, X-ray, fluoroscopy, or computed tomography (CT) imaging. In certain embodiments, the contrast agent is radiopaque. In some embodiments, the radiopaque material is selected from sodium iodide, potassium iodide, barium sulfate, or tantalum and similar commercially available compounds or combinations thereof.

Example

The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The following are general characteristics of the biocompatible preformulation and biocompatible hydrogel polymer matrix that are biocompatible.

The following are some of the properties of the adhesive biocompatible hydrogel polymer matrix.

The biocompatible preformulation chemical components used to form the biocompatible hydrogel polymer matrix are listed in Table 1. The biocompatible preformulation components will be referred to by their abbreviations. Several USP grade viscosity enhancers were purchased from Sigma-Aldrich and stored at 25°C. They include methylcellulose (10-25 MPA.S), abbreviated as MC; hypromellose (hydroxypropyl methylcellulose 2910), abbreviated as HPMC; and povidone K-30 (polyvinyl pyrrolidone), abbreviated as PVP.

Store the biocompatible preformulation components at 5°C and allow them to warm to room temperature before use, which typically takes 30 minutes. After use, purge the contents with _N2 for approximately 30 seconds before sealing with parafilm and returning to 5°C. Alternatively, store the biocompatible preformulation components at -20°C and allow them to warm to room temperature under an inert gas flow before use, which typically takes 30 minutes. Purge the biocompatible preformulation components with inert gas for at least 30 seconds before returning to -20°C.

0.15 M phosphate buffer was prepared by dissolving 9.00 g (0.075 mol) of NaH ₂ PO ₄ in 500 mL of distilled water at 25° C. with magnetic stirring. The pH was then adjusted to 7.99 by dropwise addition of 50% aqueous NaOH. Several other phosphate buffers were prepared in a similar manner: 0.10 M phosphate buffer at pH 9, 0.10 M phosphate buffer at pH 7.80, 0.10 M phosphate buffer at pH 7.72, 0.10 M phosphate buffer at pH 7.46, 0.15 M phosphate buffer at pH 7.94, 0.15 M phosphate buffer at pH 7.90, 0.4 M phosphate buffer at pH 9, and 0.05 M phosphate buffer at pH 7.40.

Prepare sterile 0.10M phosphate buffer at pH 7.58 containing 0.30% HPMC for use in the kit. First, dissolve 1.417g of HPMC in 471mL of 0.10M phosphate buffer at pH 7.58 by vigorous shaking. Allow the viscous solution to clarify overnight. Filter the solution through a 0.22μm filter (Corning #431097) by applying a slight vacuum. The viscosity of the resulting solution was 8.48cSt+/-0.06 at 20°C.

A sterile 0.10M phosphate buffer with a pH of 7.58 containing 0.3% HPMC was prepared. First, 0.10M phosphate buffer was prepared by dissolving 5.999g (0.05mol) _{of NaH2PO4} in 500mL of distilled water at 20°C with magnetic stirring. Then, a 50% aqueous NaOH solution was added dropwise to adjust the pH to 7.58. Then, 1.5g of HPMC was dissolved in 500mL of the above buffer by vigorous shaking. The viscous solution was allowed to clarify overnight. The solution was filtered through a 0.22μm filter (Corning#431097) by applying a slight vacuum. The viscosity of the resulting solution was measured by the procedure described in the viscosity measurement section and was measured to be 8.48cSt+/-0.06 at 20°C.

Phosphate buffered saline (PBS) was prepared by dissolving two PBS tablets (Sigma Chemical, P4417) in 400 mL of distilled water with vigorous shaking at 25° C. The solution had the following composition and pH: 0.01 M phosphate, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.46.

A 0.058 M phosphate buffer solution was prepared by dissolving 3.45 g (0.029 mol) of NaH ₂ PO ₄ in 500 mL of distilled water at 25° C. with magnetic stirring, and then the pH was adjusted to 7.97 by adding 50% aqueous NaOH solution dropwise.

0.05 M borate buffer was prepared by dissolving 9.53 g (0.025 mol) of Na ₂ B ₄ O ₇ ·10H ₂ O in 500 mL of distilled water under magnetic stirring at 25° C. 6.0 N HCl was then added dropwise to adjust the pH to 7.93 or 8.35.

The antiseptic liquid component was prepared in a similar manner using a commercial 2% chlorhexidine solution. 0.3 g of HPMC was dissolved in 100 mL of the 2% chlorhexidine solution. The viscous solution was allowed to settle overnight at 5°C. The resulting clear blue solution had the following composition: 2% chlorhexidine, 0.3% HPMC, and an unknown amount of non-toxic blue dye and detergent.

The other liquid components were prepared in a similar manner by simply dissolving the appropriate amount of the desired additives into the solution. For example, a preservative liquid component containing 1% denatonium benzoate (a bittering agent) was prepared by dissolving 2 g of denatonium benzoate in 200 mL of a 2% chlorhexidine solution.

Alternatively, commercially available drug solutions are used as the liquid components. For example, saline solution, Kenalog-10 (10 mg/mL triamcinolone acetonide solution), and Depo-Medrol (40 mg/mL methylprednisolone acetate) are used.

The amine or thiol component (usually within the range of 0.1 mmol arm equivalent) is added to a 50 mL centrifuge tube. A volume of reaction buffer is added to the tube via a pipette to give a final concentration of solids in the solution of approximately 5%. The mixture is vortexed slightly to dissolve the solids before adding an appropriate amount of ester or epoxide. Immediately after the addition of the ester or epoxide, the entire solution is shaken for 10 seconds and then allowed to stand.

Table 1. Components used in biocompatible preformulations.

In all cases, gel time was measured from the time the ester or epoxide was added until the solution gelled. The gel point was recorded by pipetting 1 mL of the reaction mixture and observing the dropwise viscosity increase. Polymer degradation was performed by adding 5 to 10 mL of phosphate-buffered saline to approximately 5 g of material in a 50 mL centrifuge tube and incubating the mixture at 37°C. Degradation time was measured from the time the phosphate buffer was added until the polymer completely dissolved into the solution.

Example 1: Preparation of Biocompatible Hydrogel Polymer Matrix (Amine-Ester Chemistry)

8-Arm-20K-NH2 solution was prepared in a Falcon tube by dissolving about 0.13g of solid monomer in about 2.5mL of sodium phosphate buffer (buffer pH 7.36). The mixture was shaken at ambient temperature for about 10 seconds until complete dissolution was achieved. The Falcon tube was allowed to stand at ambient temperature. In another Falcon tube, 0.10g of 8-Arm-15K-SG was dissolved in the same phosphate buffer as above. The mixture was shaken for about 10 seconds until all the powder was dissolved. The 8-Arm-15K-SG solution was immediately poured into the 8-Arm-20K-NH2 solution and a timer was started. The mixture was shaken, mixed for about 10 seconds, and 1mL of the mixture solution was aspirated with a mechanical high-precision pipette. The gel time of 1mL of liquid was collected, and then the remaining liquid was verified to be unable to flow. The gel time data of the preparation was recorded and the gel time data was about 90 seconds.

Example 2: Preparation of Biocompatible Hydrogel Polymer Matrix (Amine-Ester Chemistry)

An amine solution was prepared in a Falcon tube by dissolving approximately 0.4 g of solid 4-arm-20k-AA and approximately 0.2 g of solid 8-arm-20k-NH2 in approximately 18 mL of sodium phosphate buffer (buffer pH 7.36). The mixture was shaken at ambient temperature for approximately 10 seconds until complete dissolution was achieved. The Falcon tube was allowed to stand at ambient temperature. To this solution was added 0.3 g of 8-arm-15K-SG. The mixture was shaken and mixed for approximately 10 seconds until all the powder was dissolved. 1 mL of the mixture was aspirated using a mechanical high-precision pipette. The gel time of the formulation was collected using the above method. The gel time was approximately 90 seconds.

Example 3: Preparation of Biocompatible Hydrogel Polymer Matrix (Thiol-Ester Chemistry)

An ETTMP-1300 solution was prepared in a Falcon tube by dissolving approximately 0.04 g of monomer in approximately 5 mL of sodium borate buffer (buffer pH 8.35). The mixture was shaken at ambient temperature for approximately 10 seconds until complete dissolution was achieved. The Falcon tube was allowed to stand at ambient temperature. To this solution was added 0.20 g of 8-Arm-15K-SG. The mixture was shaken for approximately 10 seconds until the powder dissolved. 1 mL of the mixture was aspirated using a mechanical high-precision pipette. The gel time was found to be approximately 70 seconds.

Example 4: Preparation of Biocompatible Hydrogel Polymer Matrix (Thiol-Epoxide Chemistry)

An ETTMP-1300 solution was prepared in a Falcon tube by dissolving approximately 0.04 g of monomer in approximately 5 mL of sodium borate buffer (buffer pH 8.35). The mixture was shaken at ambient temperature for approximately 10 seconds until complete dissolution was achieved. The Falcon tube was allowed to stand at ambient temperature. 0.10 g of EJ-190 was added to the solution. The mixture was shaken for approximately 10 seconds until complete dissolution was achieved. 1 mL of the mixture was aspirated using a mechanical high-precision pipette. The gel time was found to be approximately 6 minutes.

Example 5: In vitro bioabsorption test

Prepare a 0.10 molar buffer solution of pH 7.40 with deionized water. Transfer a portion (50 mL) of this solution to a Falcon tube. Prepare the sample polymer in a 20cc syringe. After solidification, cut 2-4 mm thick slices from the polymer block and place them in a Falcon tube. Prepare a circulating water bath and maintain it at 37°C. Place the Falcon tube with the polymer in a water bath and start timing. Monitor and record the dissolution of the polymer. Depending on the type of sample polymer, the dissolution time range is 1-90 days.

Example 6: Gelation and Degradation Times of Amine-Ester Polymers

The amines studied were 8-arm-20k-NH2 and 4-arm-5k-NH2. The details of the formulation and the material properties are shown in Table 2. For 8-arm-20k-NH2, a phosphate buffer containing 0.058 M phosphate and a pH of 7.97 was found to be necessary to obtain an acceptable gel time of approximately 100 seconds. The use of 0.05 M phosphate buffer at a pH of 7.41 resulted in a more than 2-fold increase in gel time (270 seconds).

For 8-Arm-20k-NH2, the ratio of 4-Arm-10k-SS to 4-Arm-20k-SGA was varied between 50:50 and 90:10. While gelation times remained consistent, degradation times shifted significantly around a ratio of 80:20. For formulations with ratios of 75:25 and 50:50, degradation times soared to one month and longer. Using smaller amounts of 4-Arm-20k-SGA (80:20, 85:15, and 90:10) resulted in degradation times of less than 7 days.

For comparison, 4-arm-5k-NH2 was used in a formulation with an 80:20 ratio of 4-arm-10k-SS to 4-arm-20k-SGA. As expected, the degradation time remained consistent, indicating that the degradation mechanism was unaffected by the change in amine. However, the gelation time increased by 60 seconds, likely reflecting the relative accessibility of reactive groups in the high-molecular-weight 8-arm amine and the low-molecular-weight 4-arm amine.

Table 2. Gelation and degradation times for different 4-arm-10k-SS/4-arm-20k-SGA ratios using 8-arm-15k-SG esters.

Example 7: Gelation and degradation time of thiol-ester polymers

The thiols studied were 4-arm-5k-SH and ETTMP-1300. The details and material properties of the formulation are shown in Table 3. It was found that a 0.05M borate buffer at pH 7.93 produced a gel time of approximately 120 seconds. Increasing the amount of 4-arm-20k-SGA in the formulation increased the gel time to 190 seconds (4-arm-10k-SS to 4-arm-20k-SGA ratio of 25:75), up to 390 seconds (4-arm-10k-SS to 4-arm-20k-SGA ratio of 0:100). Using a 0.05M borate buffer at pH 8.35 resulted in a gel time of 65 seconds, a decrease of approximately two-fold. Therefore, the gel time can be adjusted by simply adjusting the pH of the reaction buffer.

The ratio of 4-arm-10k-SS to 4-arm-20k-SGA was varied between 0:100 and 100:0. In all cases, the degradation time did not change significantly, typically 3 to 5 days. It is likely that degradation occurs via an alternative pathway.

Table 3. Gelation and degradation times for different 4-arm-10k-SS/4-arm-20k-SGA ratios using 4-arm-5k-SH and ETTMP-1300 thiol.

Example 8: Gelation and Degradation Times of Amine-Ester and Thiol-Ester Polymers

The ester 4Ar-10k-SG was used to study amines (4Ar-5k-NH2) and thiols (4Ar-5k-SH). The details of the formulation and material properties are shown in Table 4. When using the amine, 0.058M phosphate buffer at pH 7.97 produced a gel time of 150 seconds. When using the thiol, 0.05M borate buffer at pH 8.35 produced a gel time of 75 seconds.

As expected based on the lack of degradable groups, the amine-based polymers did not appear to show signs of degradation. However, the thiol-based polymers degraded within 5 days. This suggests that degradation occurs via an alternative pathway, as observed in the thiol formulations containing 4-Arm-10k-SS and 4-Arm-20k-SGA (described above).

Table 4. Gelation and degradation times of amine and thiol biocompatible preformulations containing 4-arm-10k-SG

Example 9: Gelation and degradation time of thiol-sorbitol polyglycidyl ether polymer

For ETTMP-1300, conditions such as high pH (10), high solution concentration (50%), or high borate concentration (0.16 M) were necessary to gel the mixture. Gelation times ranged from approximately 30 minutes to several hours. The conditions investigated included: pH 7 to 12; solution concentrations from 5% to 50%; borate concentrations from 0.05 M to 0.16 M; and thiol to epoxide ratios from 1:2 to 2:1.

The high pH required for the reaction to occur may lead to degradation of the thiol. Therefore, a polymer containing EJ-190 and 4-Arm-5k-SH was prepared. A 13% solution formulation exhibited a gel time of 230 seconds at pH 9 to 10. The degradation time was 32 days. At a lower pH of approximately 8, the mixture exhibited gel times in the 1 to 2 hour range.

Example 10: General Procedure for Preparing Polymerizable Biocompatible Preformulations

Several representative viscous formulations are listed in Table 5, along with specific reaction details for preparing polymerizable biocompatible preformulations. A biocompatible hydrogel polymer is prepared by first dissolving the amine component in phosphate buffer or the thiol component in borate buffer. The appropriate amount of the ester component is then added, and the entire solution is vigorously mixed for 10 to 20 seconds. The gelation time is measured from the time the ester is added until the solution gels.

Table 5. (A) Summary of reaction details for several representative viscous formulations without viscosity enhancers; (B) More detailed listing of selected reaction details including molar numbers (degradation times were measured in phosphate buffered saline (PBS) at 37°C).

(A)

(B)

Table 6. Gel time of 8-arm-20k-NH2/4-arm-20k-SGA (1/1) viscous polymer containing HPMC as viscosity enhancer with different buffers and concentrations.

The gelation time ranged from 60 to 300 seconds and was found to be easily adjustable by adjusting the pH of the reaction buffer, the concentration of the buffer, or the concentration of the polymer. An example of gelation time control for a single formulation is shown in Table 6, where the gelation time of the 8-arm-20k-NH2/4-arm-20k-SGA (1/1) polymer was varied between 1.5 and 15.5 minutes.

In some cases, the stickiness of the polymers results from a mismatch in the molar equivalents of the components. A variety of sticky materials were formed using a combination of a 4- or 8-arm amine with a molecular weight of 2,000 to 20,000 and a 4- or 8-arm ester with a molecular weight of 10,000 to 20,000. It was found that the 4-arm ester formed a stickier material than the 8-arm ester. For the amine component, it was found that a smaller molecular weight resulted in a stickier material and a higher molar ratio of amine to ester.

A mismatch (amine to ester molar ratio) of at least 3 is required to qualitatively induce tack. More preferably, a ratio of about 5 produces the desired level of tack combined with polymer strength. Polymers with amine to ester molar ratios greater than 5 can also be formed, but some reaction conditions, such as polymer concentration, may need to be adjusted to achieve reasonable gel times. In addition, the use of viscosity-enhancing solutions has been found to improve the polymer by increasing its strength and elasticity, thereby allowing for higher amine to ester molar ratios (Example 11; Table 9).

The resulting material is typically transparent and elastic. Adhesion is qualitatively detected by touch. Thus, the adhesive material adheres to a person's finger or other surface and remains in place until removed. Degradation times range from 1 to 53 days. In some cases, polymer properties such as gelation and degradation times, pore size, and swelling can be optimized for different applications without losing adhesive properties.

Example 11: General procedure for preparing solutions with enhanced viscosity

Polymer solutions with enhanced viscosity were prepared by adding viscosity enhancers to the reaction buffer. Table 9B lists the viscosity enhancers studied, including observations on the properties of the polymers formed. Stock solutions of the reaction buffer were prepared using varying concentrations of methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), or polyvinylpyrrolidone (PVP). As an example, a 2% (w/w) HPMC solution in the buffer was prepared by adding 0.2 g of HPMC to 9.8 mL of 0.10 M phosphate buffer at pH 7.80 and subsequently shaking vigorously. The solution was allowed to stand overnight. Buffer solutions with HPMC concentrations ranging from 0.01% to 2.0% were prepared in a similar manner. Buffer solutions with PVP concentrations ranging from 5% to 20% and MC concentrations ranging from 1.0% to 2.0% were also prepared by similar methods.

The polymer was formed using the same method as described above in the general procedure for preparing viscous materials (Example 10). A typical procedure involves first dissolving the amine component in phosphate buffer containing the desired concentration of viscosity enhancer. An appropriate amount of the ester component is then added and the entire solution is vigorously mixed for 10 to 20 seconds. The gel time is measured from the time the ester is added until the solution gels.

Several representative formulations are listed in Tables 7 and 8 along with specific reaction details. The molar equivalent percentage of the degradable amine acetate component is represented by the ratio specified in parentheses. For example, a formulation with 75% degradable amine would be written as 8Arm-20k-AA/8Arm-20k-NH2 (75/25). The polymer was prepared as follows: the amine component of the formulation was first dissolved in phosphate buffer. The appropriate amount of the ester component of the formulation was then added, and the entire solution was vigorously mixed for 10 to 20 seconds. The gel time was measured from the time the ester was added until the solution gelled.

The gelation time depends on several factors: pH, buffer concentration, polymer concentration, temperature, and the biocompatible preformulation monomer used. Previous experiments have shown that once the components are in solution (which typically takes up to 10 seconds), the degree of mixing has little effect on the gelation time. The effect of adding biocompatible preformulation monomers on the buffer pH was measured. For the 8-arm-20k-NH2 and 4-arm-20k-SGA formulations, the buffer pH dropped slightly from 7.42 to 7.36 after adding the biocompatible preformulation monomers. For the 8-arm-20k-AA/8-arm-20k-NH2 (70/30) and 4-arm-20k-SGA formulations, the buffer pH dropped from 7.4 to 7.29 after adding the biocompatible preformulation monomers. It was found that the additional reduction in pH was due to the acidic residues in the degradable amine acetate. The same pH drop was observed for the 4-arm-20k-AA amine. In some cases, quality control specifications for the pH of the amine acetate solution may be required to improve the consistency of the degradable formulation.

The effect of the reaction buffer pH on the gelation time was measured. The gelation time increased in an approximately linear manner with increasing hydronium ion concentration. More generally, the gelation time decreased with increasing buffer pH. In addition, the effect of the phosphate concentration of the reaction buffer on the gelation time was determined. The gelation time decreased with increasing phosphate concentration. Furthermore, the effect of polymer concentration on the gelation time was studied. The gelation time decreased significantly with increasing polymer concentration. At low polymer concentrations, where the gelation time was greater than 5 minutes, the ester hydrolysis reaction began to compete with polymer formation. The effect of temperature on the gelation time appeared to follow the Arrhenius equation. The gelation time is directly related to the degree of reactivity of the polymer solution, so this behavior is not unusual.

The rheology of the polymer during gelation was determined as a function of time relative to the gel point. With 100% representing the gel point and 50% representing half the time before the gel point, the viscosity of the reaction solution remained relatively constant until approximately 80% of the gel point. After this point, the viscosity increased dramatically, indicating the formation of a solid gel.

The gel time stability of a single formulation using the same batch of biocompatible preformulation monomers was measured over the course of approximately one year. The biocompatible preformulation monomers were processed according to the standard protocol outlined above. The gel time remained relatively stable; some changes in the reaction buffer may result in differences in gel time.

Table 7. (A) Summary of reaction details for several representative viscous formulations; (B) More detailed listing of selected reaction details including molar numbers (degradation times measured in phosphate buffered saline (PBS) at 37°C).

(A)

(B)

Table 8. (A) Summary of reaction details for several representative viscous formulations; (B) More detailed listing of selected reaction details including molar numbers (degradation times measured in phosphate buffered saline (PBS) at 37°C)

(A)

(B)

Cytotoxicity and hemolysis assessment

Several polymer samples were sent to NAMSA for cytotoxicity and hemolysis evaluation. Cytotoxic effects were evaluated according to ISO 10993-5 guidelines. Hemolysis was evaluated according to a procedure based on ASTM F756 and ISO 10993-4.

A 4.8% solution of the polymers 8-arm-20k-NH2 and 4-arm-20k-SGA containing 0.3% HPMC was found to be non-cytotoxic and non-hemolytic. A 4.8% solution of the polymers 8-arm-20k-AA/8-arm-20k-NH2 (70/30) and 4-arm-20k-SGA containing 0.3% HPMC was found to be non-cytotoxic and non-hemolytic. In addition, a formulation containing 4-arm-20kAA and 8-arm-15k-SG was also non-cytotoxic and non-hemolytic.

Gelation and degradation time measurements

In all cases, gelation time was measured from the time the ester was added until the solution gelled. The gel point was recorded by pipetting 1 mL of the reaction mixture and observing the viscosity increase dropwise until the mixture stopped flowing. Polymer degradation was performed by adding 1 to 10 mL of phosphate-buffered saline per 1 g of material in a 50 mL centrifuge tube and incubating the mixture at 37°C. The temperature was maintained using a digital water bath. Degradation time was measured from the time the phosphate buffer was added until the polymer completely dissolved in the solution.

The effects of reaction buffer pH, phosphate concentration, polymer concentration, and reaction temperature on gelation time were characterized. The buffer pH was varied between 7.2 and 8.0 by dropwise addition of 50% aqueous NaOH or 6.0 N HCl. Phosphate concentrations of 0.01 M, 0.02 M, and 0.05 M were prepared and adjusted to pH 7.4. Polymer concentrations ranging from 2% to 20% solutions were studied. Reaction temperatures of 5, 20, and 37°C were tested by maintaining the monomer, buffer, and reaction mixture at appropriate temperatures. The 5°C environment was provided by a refrigerator, while the 37°C temperature was maintained by a water bath. The measured room temperature was 20°C.

The effects of degradation buffer pH and the ratio of degradable amines in the polymer formulation on degradation time were investigated. The degradation buffer pH was varied between 7.2 and 9.0 by dropwise addition of 50% aqueous NaOH or 6.0 N HCl. The degradable amine components studied were either 4-arm-20k-AA or 8-arm-20k-AA, and the percentage of degradable amine relative to non-degradable amine varied between 50% and 100%.

Degradation times are primarily dependent on buffer pH, temperature, and the biocompatible preformulation monomers used. Degradation occurs primarily through hydrolysis of ester bonds; in biological systems, enzymatic pathways may also play a role. Figure 1 compares the degradation times of formulations containing varying amounts of 4-arm-20k-AA and 8-arm-20k-AA. In general, increasing the amount of degradable amine acetate relative to the non-degradable amine decreases degradation time. Additionally, in some cases, 8-arm-20k-AA exhibits longer degradation times per molar equivalent than 4-arm-20k-AA, which becomes particularly pronounced when the percentage of amine acetate decreases below 70%.

The effect of buffer pH on degradation time was studied. A pH range of 7.2 to 9.0 was studied. Generally, a high pH environment resulted in greatly accelerated degradation. For example, increasing the pH from approximately 7.4 to 7.7 reduced the degradation time by approximately half.

The degradation time of different acetamide formulations was evaluated. The formulation containing 70% acetamide had a degradation time of approximately 14 days, while the formulation containing 62.5% acetamide had a degradation time of approximately 180 days.

Figure 2 shows the effect of polymer concentration on the degradation time of different acetamide formulations, where increasing polymer concentration slightly increases the degradation time (75% acetamide formulation). The effect is less pronounced for the 100% acetamide formulation, where the rate of ester hydrolysis is more pronounced.

It has also been found that the monomers used in the formulation play a role in the degradation pattern of the polymer. For the 8-arm-20k-AA/8-arm-20k-NH2 (70/30) and 4-arm-20k-SGA polymers, degradation occurs uniformly throughout the material, resulting in a "smooth" degradation process. The polymer absorbs water and swells slightly in the first few days. Then, the polymer gradually becomes softer but still maintains its shape. Finally, the polymer loses its shape and becomes a highly viscous fluid.

When the amount of degradable amine is reduced, a fragmentation degradation process is observed, and non-degradable regions may appear in the polymer. For example, the 4-arm-20k-AA/8-arm-20k-NH2 (70/30) and 4-arm-20k-SGA formulations degrade into several large fragments. For applications where the polymer is subjected to significant forces, fragmentation can also occur as the polymer becomes softer and weaker over time.

Polymer concentration

More dilute polymer solutions can be used with minimal changes in mechanical properties. Polymer concentrations of 3.0%, 3.5%, and 4.0% were studied for a formulation of 8-Arm-20k-AA-20k/8-Arm-20k-NH2 (75/25) with 4-Arm-20k-SGA and 0.3% HPMC. Gel time steadily increased with decreasing polymer concentration. Hardness decreased slightly with decreasing polymer concentration. The polymer's adhesive properties remained essentially unchanged. Elastic modulus decreased slightly with decreasing polymer concentration.

Table 9 (A) Reaction details for specific viscous formulations; (B) Formulation results for specific viscous formulations with various viscosity enhancing agents (biocompatible hydrogel surface diffusion assay performed at an angle of approximately 30° on a hydrophilic, biocompatible hydrogel surface containing 97.5% water; a drop of polymer solution from a 22 gauge needle was applied to the surface prior to gelation); (C) Clarity of solutions containing various viscosity enhancing agents as measured by % transmittance at 650 nm.

(A)

(B)

The hydrogel surface spreading test categories are: 1) no spreading, a compact droplet remains in place; 2) slight spreading, the droplet slowly drips; 3) severe spreading, the droplet completely wets the surface. Water belongs to category 3.

(C)

sample % transmission at 650nm 0.10 M phosphate buffer, pH 7.80 100.0% 10% PVP 99.9% 1.5% HPMC 95.7% 1.0% HPMC 96.8% 0.5% HPMC 99.1% 0.1% HPMC 99.6%

Methylcellulose (MC) was found to perform similarly to hypromellose (HPMC), providing workable viscous solutions in a concentration range of 0 to 2% (w/w). However, HPMC was more soluble than MC, and HPMC solutions had greater optical clarity; thus, HPMC was favored. Povidone (PVP) readily dissolved in buffer but provided only minimal viscosity enhancement even at 20% (w/w). Higher molecular weight grades of PVP could be used but have not been investigated.

In most cases, the polymer remains unchanged after adding low concentrations of HPMC or PVP. However, at approximately 0.3% HPMC, the polymer undergoes a noticeable change, characterized by increased elasticity, as evidenced by the material's ability to extend beyond its normal limits without breaking. Above 1.5% HPMC, the polymer becomes slightly softer and exhibits lower elasticity. The gel time also remains within 10 seconds of the gel time of the formulation without adhesive. In the case of PVP, the polymer undergoes significant changes above 10% PVP. The polymer becomes more opaque, while its elasticity and adhesiveness increase significantly. At 15% to 20% PVP, the polymer becomes similar to a viscous material, but with improved mechanical strength. The gel time also increases by approximately 20 seconds relative to the formulation without adhesive. Therefore, adding lower concentrations of PVP or HPMC to the polymer solution may be beneficial in improving the polymer's elasticity and lubricity.

The results of the biocompatible hydrogel surface diffusion test showed that most formulations belonged to category 2.

Based on these observations, a formulation using 0.3% HPMC was selected for further evaluation. Above 1.0% HPMC, the solution became significantly more difficult to mix, and monomer dissolution became an issue. At 0.5% HPMC and above, bubble formation during mixing became noticeable. Furthermore, the solution was not easily filtered through a 0.5 μm syringe filter to remove bubbles. However, the 0.3% HPMC solution was easily filtered even after moderate mixing, yielding a bubble-free, optically clear polymer.

Viscosity measurement

The viscosity of the obtained buffer solutions was measured using a Cannon-Fenske viscometer tube of appropriate size from Ace Glass. The viscometer sizes used ranged from 25 to 300. The selected solutions were measured in triplicate at 20°C and 37°C. The results are shown in Table 9B. To calculate the approximate dynamic viscosity, it was assumed that all buffer solutions had the same density as water.

To characterize the rheology of the polymer during gelation, a Size 300 viscometer was used with a formulation designed to gel after approximately 15 minutes. The formulation used consisted of a 2.5% solution of 8-Arm-20k-NH2 and 4-Arm-20k-SGA esters and 0.3% HPMC. The reaction took place in 0.05 M phosphate buffer at pH 7.2. Thus, a single viscosity measurement using the Size 300 viscometer was obtained in approximately one minute, and subsequent measurements could be obtained in rapid succession until the gel point was reached.

Hydrogel surface diffusion test

To simulate the behavior of polymer solutions on hydrophilic surfaces, the extent of droplet spreading and dripping on a high-water content biocompatible hydrogel polymer matrix at an inclination of approximately 30° was recorded. A biocompatible hydrogel polymer matrix was prepared by dissolving 0.10 g (0.04 mol arm equivalent) of 8-Arm-20k-NH2 in 7 mL of 0.05 M phosphate buffer at pH 7.4 in a petri dish, followed by the addition of 0.075 g (0.04 mol arm equivalent) of 8-Arm-15k-SG ester. The solution was stirred with a spatula for 10 to 20 seconds and allowed to gel, which typically took 5 to 10 minutes. The resulting polymer had a water content of 97.5%.

The test was conducted as follows: a polymer solution was first prepared in the usual manner. After thorough mixing, the polymer solution was dispensed dropwise through a 22-gauge needle onto the surface of a biocompatible hydrogel polymer matrix. The results are shown in Table 9B and are categorized into three main groups: 1) no spreading, with a compact droplet remaining in place; 2) mild spreading, with the droplet slowly dripping; and 3) severe spreading, with the droplet completely wetting the surface. Water falls into category 3.

Swelling and drying measurements

The degree of polymer swelling during degradation is quantified based on its liquid absorption. A known mass of polymer is placed in PBS at 37°C. At specified time intervals, the polymer is removed from the buffer solution, gently patted dry with a paper towel, and weighed. The percent mass increase is calculated based on the initial mass.

The fate of polymers in air under ambient conditions is quantified based on weight loss over time. A polymer film approximately 1 cm thick is placed on a surface at 20°C. Mass measurements are taken at set time intervals. The percent weight loss relative to the initial mass is calculated.

The percentage of water uptake by 8-Arm-20k-NH2/4-Arm-20k-SGA polymers containing 0%, 0.3%, and 1.0% HPMC was studied. The 1.0% HPMC polymer absorbed up to 30% of its weight in water until day 20. After day 20, the polymer had returned to approximately 10% of its weight in water. In comparison, the 0% HPMC polymer initially absorbed up to 10% of its weight in water but began to gradually lose water, hovering around 5% of its weight in water. The 0.3% HPMC polymer behaved in an intermediate manner. It initially absorbed up to 20% of its weight in water, but after one week returned to approximately 10% of its weight in water and continued to slowly lose water.

The percent weight loss of 8-arm-20k-AA/8-arm-20k-NH2 (75/25) and 4-arm-20k-SGA polymers containing 0.3% HPMC and 1.0% HPMC over 24 hours at ambient conditions is shown in FIG3 . Ambient conditions were approximately 20° C. and 30-50% relative humidity. The rate of water loss was fairly constant over 6 hours at approximately 10% per hour. After 6 hours, the rate slowed significantly as the polymer weight approached a constant value. The rate of water loss is expected to vary depending on polymer shape and thickness, as well as temperature and humidity.

Specific gravity measurement

The specific gravity of the polymer was obtained by preparing a polymer solution in the usual manner and pipetting 1.00 mL of the well-mixed solution onto an analytical balance. The measurements were performed in triplicate at 20° C. The specific gravity was calculated by using the density of water at 4° C. as a reference.

The specific gravity of the polymer is not significantly different from that of the buffer alone and is essentially the same as that of water. Exceptions may occur when the polymer solution is not filtered and air bubbles are embedded in the polymer matrix.

Barium sulfate suspension

For imaging purposes, barium sulfate was added as a contrast agent to several polymer formulations. Barium sulfate concentrations of 1.0%, 2.0%, 5.0%, and 10.0% (w/v) were investigated. The viscosity of the resulting polymer solutions was measured, and the effect of the addition of barium sulfate on the gelation time and injectability of the polymers was also investigated.

Barium sulfate concentrations of 1.0%, 2.0%, 5.0%, and 10.0% (w/v) were investigated. The opaque, milky white suspension formed a similarly opaque and white polymer. No change in gel time was observed. Qualitatively, the polymer appeared to have similar properties to the polymer without barium sulfate. All formulations were easily dispensed through a 22-gauge needle.

Viscosity measurements were performed at barium sulfate concentrations of 1.0%, 2.0%, 5.0%, and 10.0%. Viscosity remained relatively stable up to 2.0%, with a slight increase to approximately 2.5 cP at 5.0%. As the concentration approached 10.0%, the viscosity increased dramatically to nearly 10 cP. Therefore, a barium sulfate concentration of 5.0% was selected based on a balance between high contrast and similarity to the unmodified polymer formulation.

Hardness, elastic modulus and adhesion of biocompatible hydrogels

The hardness of the polymer was characterized using the TA.XT.plus Texture Analyzer using Exponent software version 6.0.6.0. This method follows the industry standard "Bloom test" for measuring the hardness of gelatin. In this test, a TA- ₈ ^1/4 " ball probe is used to penetrate the polymer sample to a specified depth and then return to its original position from the sample. The peak force measured is defined as the "hardness" of the sample. For the polymers studied, a test speed of 0.50 mm/sec, a penetration depth of 4 mm, and a trigger force of 5.0 g were used. The polymer was prepared directly in 5 mL vials at a 2.5 mL scale to ensure consistent sample size. The vials used were ThermoScientific/Nalgene LDPE vials, product number 6250-0005 (lot number 7163281060). Measurements were performed at 20°C. The polymer was allowed to stand at room temperature for approximately 1 hour before measurement. At least three samples were measured in triplicate. A sample plot generated by the Exponent software running the hardness test is shown in Figure 4. The peak in the plot represents the point at which the target penetration depth of 4 mm was achieved.

The elastic modulus of the polymers was characterized using the TA.XT.plus TextureAnalyzer model using Exponent software version 6.0.6.0. In this test, a TA-19 Kobe probe was used to compress a polymer cylinder of known dimensions until the polymer broke. The probe had a defined surface area of 1 ^cm2 . The modulus was calculated based on an initial slope of 10% of the maximum compressive stress. A test speed of 5.0 mm/min and a trigger force of 5.0 g were used for the polymers studied. The sample height was automatically detected by the probe. The polymers were prepared directly in 5 mL vial caps at a 2.5 mL scale to ensure consistent sample size. The vials used were ThermoScientific/Nalgene LDPE sample vials, product number 6250-0005 (lot number 7163281060). Measurements were performed at 20°C. The polymers were allowed to stand at room temperature for approximately 1 hour before measurement. At least three samples were measured. A sample curve generated by the Exponent software running the modulus test is shown in Figure 5. Polymers generally exhibit elastic behavior upon initial compression, as evidenced by the nearly linear curve.

The adhesion properties of the polymer were characterized by using the TA.XT.plus TextureAnalyzer using Exponent software version 6.0.6.0. In the adhesion test, a TA-57R impact probe with a diameter of 7 mm was used to contact the polymer sample for a specific length of time with a limited force and then returned to its original position from the sample. An exemplary graph generated by the Exponent software running the adhesion test is given in Figure 6. The drawing begins when the probe hits the surface of the polymer. A target force is applied to the sample within a specified time unit, which is represented by the constant force area in the figure. The probe then returns to its original position from the sample, and the adhesion between the probe and the sample is measured as "adhesion", which is the peak force required to remove the probe from the sample. Other properties measured include adhesion energy or adhesion work, and the "ductility" of the material. Adhesion energy is simply the area under the curve representing adhesion. Thus, a sample with high adhesion and low adhesion energy will feel qualitatively very sticky but can be removed cleanly with a quick pull. A sample with high adhesion and high adhesion energy will also feel very sticky, but removal of the material will be more difficult and may be accompanied by polymer stretching, fiber formation, and adhered residue. The elasticity of a polymer is proportional to the measured "ductility," which is the distance the polymer stretches when adhered to the probe before the bond fails. For the polymers studied, a test speed of 0.50 mm/sec, a trigger force of 2.0 g, a contact force of 100.0 g, and a contact time of 10.0 seconds were used. Polymers were prepared directly in 5 mL vials at a scale of 1.0 to 2.5 mL to ensure consistent sample size. The vials used were Thermo Scientific/Nalgene LDPE vials. Measurements were performed at 20°C. The polymers were allowed to stand at room temperature for approximately 1 hour before measurement. As reference materials, the adhesion properties of standards and were measured. All measurements were performed in triplicate. The average and standard deviation were calculated.

The effects of HPMC addition on the mechanical properties of the polymers were investigated, along with the effect of adding the degradable 8-Arm-20k-AA amine. Under the described hardness testing conditions, the addition of 0.3% HPMC reduced the hardness of the polymer by approximately half. This corresponds to a slight decrease in the elastic modulus. The polymer containing 1.0% HPMC exhibited approximately the same hardness as the 0.3% HPMC polymer, but exhibited a slight decrease in the elastic modulus. The discrepancies between the hardness and elastic modulus measurements are likely due to experimental error. The polymer solutions were not filtered, so the presence of air bubbles may have increased the error. Exposure of the polymer to air can also alter its water content, substantially changing the material's physical properties.

The addition of the degradable 8-Arm-20k-AA amine was found to have little effect on the measured values of hardness or elastic modulus. Measurements of a standard commercial Post-It ^™ Note were also included for reference. The adhesion force of the polymer was found to be approximately 40 mN, approximately three times lower than the Post-It ^™ Note value. The adhesion properties of the polymer were not found to change with the addition of the degradable amine.

Figure 7 shows the hardness versus degradation time of 4.8% solutions of 8-Arm-20k-AA/8-Arm-20k-NH2 (70/30) and 4-Arm-20k-SGA containing 0.3% HPMC. Error bars represent the standard deviation of three samples. The polymers degraded over 18 days. The hardness of the polymers strongly correlated with the extent of degradation. Swelling may also play a role in the early stages.

The effects of various formulation additives on polymer properties were investigated. The gelation time, degradation time, hardness, adhesion, and elastic modulus of polymers prepared by varying the combination of 1% HPM, 2% chlorhexidine, and 1% denatonium benzoate were measured. The polymer properties remained largely unchanged, with the exception of the formulation containing 2% chlorhexidine, which exhibited a decrease in hardness and elastic modulus. Visual inspection of the polymers revealed that these changes were due to the detergent present in the Nolvasan solution used, rather than the chlorhexidine; the detergent caused heavy foaming during mixing, resulting in gelation of the aerated polymer.

Optical clarity

The optical clarity of viscous solutions was measured using a Thermo Scientific GENESYS 10S UV-Vis spectrophotometer. 1.5 mL of the sample solution was transferred to a quartz cuvette. A buffer solution without additives was used as a reference. The stabilized % transmittance of the sample at 650 nm was recorded.

To measure the light transmission of the polymer, 1 mL of the polymer solution was filtered through a 5 μm filter into a cuvette before gelation. The cuvette was then placed horizontally so that the polymer gelled as a film on one side of the cuvette. The film thickness was found to be 3 mm. The polymer was allowed to cure at room temperature for 15 minutes, after which the % light transmission was measured at 400, 525, and 650 nm relative to air.

All viscous solutions considered were found to have acceptable to excellent optical clarity (greater than 97% transmittance) over the concentration range used. For highly viscous solutions, bubble formation was observed during mixing, which was resolved by adding a defoamer or by using a syringe filter (see Table 9C).

The polymer exhibits excellent optical clarity across the visible spectrum. The lowest % transmission relative to buffer is only 97.2% while the highest is 99.7%. The decrease in % transmission at lower wavelengths is likely due to some energy absorption near the ultraviolet region.

Drug Elution: General Procedure

Thermo Scientific GENESYS 10S UV-Vis spectrophotometer was used to quantify the release of different drugs from several polymers. First, a reference drug or drug solution was dissolved in a suitable solution. Typically, phosphate buffered saline (PBS), ethanol, or dimethyl sulfoxide (DMSO) was used as a solvent. Next, the optimal absorption peak for identifying and quantifying the drug was determined by scanning the drug solution between 200 and 1000 nm. Using the selected absorption peak, a reference curve was established by measuring the peak absorbance at different concentrations of the drug. Different drug concentration solutions were prepared using an analytical pipette using standard dilution techniques. A linear fit of absorbance to drug concentration produced a general equation for converting the measured absorbance of the eluted sample into drug concentration.

The polymer was prepared with a known drug dose in the same manner as a physician would administer it in a clinical setting. However, in this case, the polymer was molded into a cylinder approximately 18 mm in diameter. The polymer cylinder was then placed in a 50 mL Falcon tube containing a set amount of PBS and placed at 37°C. The temperature was maintained in a digitally controlled water bath.

Collect elution samples daily by pouring PBS solution over the polymer. Record the volume of the collected sample. Place the polymer in a volume of fresh PBS equal to the volume of the collected sample and return to 37°C. Analyze the elution sample by first diluting the sample in a suitable solvent using an analytical pipette so that the measured absorbance is within the range determined by the reference curve. Record the dilution factor. Calculate the drug concentration from the measured absorbance using the reference curve and the dilution factor. Calculate the drug amount by multiplying the drug concentration by the sample volume. Calculate the elution percentage for that day by dividing the drug amount by the total amount of drug administered.

Drug eluting: chlorhexidine

A peak found between 255 and 260 nm was selected and a reference curve was constructed by measuring the peak absorbance of chlorhexidine at 0, 0.5, 1, 2.5, 5, 10, 20, 40 and 50 ppm. Concentrations exceeding 50 ppm did not show linear behavior in peak absorbance.

The polymer was prepared using a commercial Nolvasan solution corresponding to a 2% chlorhexidine dose (50 mg). The elution volume was 2 mL PBS per 1 g of polymer. The eluted samples were stored at 20° C. The eluted samples were analyzed by diluting them 1000-fold with dimethyl sulfoxide (DMSO) in a quartz cell.

The elution behavior of chlorhexidine was similar to previous experiments with other small molecules. Almost half of the chlorhexidine was released within the first three days. Then, the elution rate slowed significantly over the next three to four days, followed by another large release of chlorhexidine due to polymer degradation (Figure 8).

The elution behavior of steroidal drugs, triamcinolone acetonide and methylprednisolone is similar. The first few days usually show an elution rate that increases, presumably due to the release of weakly bound surface drugs. Then, elution is relatively constant at a rate related to drug solubility. Eventually, the remaining drug in the polymer is released with the start of degradation. Several examples of controlling the elution behavior formed are provided in Figures 9, 10 and 11. The drug can be released over a short period of time (several weeks) or over a long period of time (several years, planned).

Example 12: General Procedure for Preparing Polymerizable Biocompatible Formulations

Several representative formulations of adhesive and non-adhesive films, along with details of the specific reactions, are listed in Table 10. These films had thicknesses ranging from 100 to 500 μm and could be layered with different formulations in composite films.

Table 10. (A) Summary of reaction details for several representative film formulations; (B) More detailed list of selected reaction details including molar numbers (film thicknesses from 100 to 500 μm).

(A)

(B)

Example 13: Preparation and application of kit

Several test kits were prepared using previously tested polymer formulations. The materials used to assemble the test kits are listed in Table 11 and the formulations used are listed in Table 12. The test kits typically consist of two syringes: one containing the solid components and another containing a liquid buffer. The syringes are connected via a mixing tube and a one-way valve. The contents of the syringes are mixed by opening the valve and repeatedly transferring the contents of one syringe to the other for 10 to 20 seconds. The used syringe and mixing tube are then removed and discarded, and a dispensing unit, such as a needle or cannula, is assembled to the syringe in use, and the polymer solution is discharged until gelation begins. In other embodiments, a viscous solution hinders the dissolution of the solid components, so a third syringe is used. The third syringe contains a concentrated viscous buffer that enhances the viscosity of the solution once all components have dissolved. In some embodiments, the optical clarity of the polymer obtained is improved by adding a syringe filter.

All formulations tested were easily dispensed through a 22-gauge needle. The mixing action between the two syringes was turbulent and clearly introduced a significant amount of air bubbles. Gentle mixing produced a bubble-free, transparent material. Additionally, it was found that the use of a syringe filter was able to remove air bubbles without any change in polymer properties.

Table 11. Materials used to manufacture the kits, including suppliers, part numbers, and lot numbers.

Table 12. Detailed contents of four different kits; solid components are in one syringe, while liquid components are in another syringe; a mixing tube connects the two syringes.

Several additional test kits were prepared using the polymer formulations that performed best in the initial trials. The materials used to assemble the test kits are listed in Table 13. The test kits typically consist of two syringes, one containing the solid components and the other containing the liquid buffer. The syringes were loaded by removing the plunger, adding the components, purging the syringes with a gentle stream of nitrogen for 20 seconds, and then replacing the plunger. Finally, the plunger was depressed as much as possible to reduce the internal volume of the syringe. The specifications for the amounts of the chemical components in the test kits are listed in Table 14A. A summary of the prepared batch of test kits is listed in Table 14B.

The syringes are directly connected after removing the caps, with the male part locking into the female part. The contents of the syringes are mixed by repeatedly transferring the contents of one syringe to the other for 10 to 20 seconds. The used syringe is then removed and discarded, and the syringe in use is fitted with a dispensing element, such as a needle or cannula, and the polymer solution is discharged until gelation begins. In other embodiments, a viscous solution hinders the dissolution of the solid components, so a third syringe is used. The third syringe contains a concentrated, viscous buffer that increases the viscosity of the solution once all components have dissolved.

All formulations tested were easily dispensed through a 22 gauge needle. The mixing action between the two syringes was turbulent and a significant amount of air bubbles were apparently introduced. The use of a syringe filter was found to be able to remove the air bubbles without any change in polymer properties.

The prepared test kits, along with one oxygen absorber per bag, were placed in foil pouches. The pouches were heat-sealed using a CHTC-280PROMAX benchtop chamber sealing unit. Two different sealing modes were studied: under nitrogen and under vacuum. The sealing settings under nitrogen were: 30 seconds vacuum, 20 seconds nitrogen, 1.5 seconds heat seal, and 3.0 seconds cool down. The sealing settings under vacuum were: 60 seconds vacuum, 0 seconds nitrogen, 1.5 seconds heat seal, and 3.0 seconds cool down.

Table 13. Materials used to manufacture the kits, including suppliers, part numbers, and lot numbers.

describe supplier 12mL male Luer lock syringe 5mL female Luer lock syringe, purple QOSINA Male Luer lock cap, no opening QOSINA Female Luer Dispenser Cap Without Opening, White QOSINA 100cc oxygen absorption bag IMPAK 6.25" x 9" OD PAKVF4 Mylar Foil Bag IMPAK

Table 14. Kit component specifications for 8Arm-20k-AA/8Arm-20-NH2 and 4Arm-20k-SGA formulations containing 60%, 65%, 70%, and 75% degradable amines (A). Batch formulation summary (B).

(A)

(B)

preparation Buffer pH Sealing method Remark 60/40 7.46 Nitrogen

preparation Buffer pH Sealing method Remark 60/40 7.58 Nitrogen 60/40 7.72 Nitrogen 70/30 7.58 vacuum 70/30 7.58 vacuum No nitrogen purge for syringes 65/35 7.58 vacuum 75/25 7.58 vacuum 75/25 7.58 vacuum 75/25 7.58 Nitrogen 65/35 7.58 vacuum 65/35 7.58 Nitrogen

Several test kits were prepared for beta testing. The materials used to assemble the test kits are listed in Table 15. The test kits typically consist of two syringes, one containing the solid components and the other containing the liquid buffer. The syringes were loaded by removing the plungers, adding the components, purging the syringes with a gentle stream of inert gas for 10 seconds, and then replacing the plungers. Finally, the plungers were depressed as far as possible to reduce the internal volume of the syringes.

Alternatively, a single syringe test kit can be prepared by loading the solid component into a female syringe together with a phosphate buffered saline buffer in solid form. The test kit is then used in a manner similar to the dual syringe test kit, except that the user can use a variety of liquids of the specified amount in the male syringe. Typically, any substance provided in a liquid solution for injection can be used. Some examples of suitable liquids are water, saline, Kenalog-10, Depo-Medrol, and Nolvasan.

The syringes are directly connected after removing the caps, with the male part locking into the female part. The contents of the syringes are mixed by repeatedly transferring the contents of one syringe to the other for 10 to 20 seconds. The used syringe is then removed and discarded, and a dispensing unit, such as a needle, nozzle, or brush, is fitted to the syringe in use, and the polymer solution is discharged until gelation begins.

The prepared test kits, along with one oxygen absorber packet and one indicator silica gel packet per bag, were placed in foil pouches. A label displaying the product and company name, contact information, batch and lot number, expiration date, and recommended storage conditions was affixed to the pouch. A radiation sterilization indicator, which changes color from yellow to red upon exposure to sterilizing radiation, was also affixed to the upper left corner of the pouch. The pouches were heat-sealed using a CHTC-280PROMAX benchtop chamber sealing unit. The vacuum sealing settings were: 50 seconds of vacuum, 1.5 seconds of heat sealing, and 5 seconds of cooling.

The example of the sterile test kit batch of detailed description preparation is listed in Table 15.Previous research discovery, if the syringe of loading is not purged with nitrogen before changing piston during test kit preparation, the test kit that has been flushed with nitrogen relative to syringe, this sterile test kit shows gel time and increases about 30 seconds.No significant difference is found between the test kit sealed under vacuum and the test kit sealed under nitrogen.When the vacuum-sealed test kit loses its seal, it is easy to observe, so decide to carry out vacuum sealing as standard procedure to all test kits.In the test kit, comprise oxygen absorption bag and silica gel bag on the influence of long-term storage stability at present.

Table 15. Materials used to manufacture the kits, including suppliers and part numbers.

Table 16: Example specifications for kit components for 8-ARM-AA-20K/8-ARM-NH2-20K and 4-ARM-SGA-20K formulations containing 75% degradable amines (A). Batch formulation summary (B).

(A)

(B)

Components Batch number and specifications 8-arm-20k-AA 0.029-0.031g 8 arms-20k-NH2 0.009-0.011g 4-arm-20k-SGA 0.079-0.081g Phosphate buffer powder 0.03-0.06g Nolvasan (2% chlorhexidine) 2.50 mL, 1% denatonium benzoate batch 64 Gel time (s) 150 Degradation time (days) 11

The test kit preparation time was recorded. It took an average of 1.5 minutes to load a buffer syringe, and an average of 4 minutes to load a solid syringe. It took about 1.5 minutes to vacuum seal a test kit. Therefore, the estimated time for preparing a test kit was 7 minutes or about 8 test kits per hour. The test kit preparation time can be improved by premixing all solids in the correct proportions so that only one solid mass needs to be measured, and by optimizing the vacuum sealing procedure by reducing the vacuum cycle time.

All formulations tested were easily dispensed through 23 to 34 gauge needles. As expected, higher gauge numbers exhibited lower flow rates. The mixing action between the two syringes was turbulent and a significant amount of air bubbles was evident. The use of a syringe filter was found to be able to remove the air bubbles without any change in polymer properties.

For the single syringe system, the effect of using phosphate powder was studied. Figure 12 shows the effect of varying the amount or concentration of solid phosphate on polymer gelation time and solution pH. The system was found to be relatively insensitive to the amount of phosphate, tolerating up to a two-fold difference without significant change.

Kit sterilization and testing

The sealed kits are packaged in large FedEx boxes. Each box is sterilized by electron beam radiation according to a standard procedure developed at NUTEK Corporation. A copy of the standard sterilization procedure document is included with this report.

For each batch of sterilized kits, gel time and degradation time tests were performed on randomly selected kits to verify the viability of the material. Previous studies included runs of kits without sterilization or control kits and concluded that the environmental conditions during kit shipment did not play a significant role in gel time variations.

The sterilized kits were sent to NAMSA for sterility validation according to USP <71>. The kits were confirmed to be sterile.

After sterilization, no physical changes of monomers and phosphate buffer solutions were observed. Previous experiments have shown that the gel time of polymers increases consistently by about 30 seconds after sterilization. For example, a polymer with a gel time of 90 seconds will exhibit a gel time of 120 seconds after sterilization. The pH of the sterile buffer does not change, so it is suspected that some monomers degrade during the sterilization process. This is determined by preparing non-sterile polymers of varying concentrations and comparing gel time, degradation time, and mechanical properties with the sterilized polymer (Figure 13). Existing data show that monomers experience about 15 to 20% degradation during sterilization. Therefore, 5% polymer will behave similarly to 4% polymer after sterilization. Additional experiments have been planned to establish detailed quality control calibration curves.

Storage stability

Sterilized kits were stored at 5°C. Some kits were stored at 20°C or 37°C to investigate the effect of temperature on storage stability. Kit stability was primarily quantified by recording changes in gel time, which is proportional to the extent of monomer degradation. The temperature of 37°C was maintained by completely immersing the kits in a water bath, thus representing a worst-case scenario with respect to humidity.

The storage stability of the test kits was investigated by placing some test kits at 5°C, 20°C or 37°C and measuring the change in gel time at defined intervals. The test kits were prepared and sealed according to the procedures detailed in the previous section. The results are shown in Figure 14. Over 16 weeks, no significant change in gel time was observed for the test kits stored at 5°C and 20°C. At 37°C, the gel time began to increase at a constant rate after about 1 week. The foil pouches proved to be an effective moisture barrier. The indicator silica gel packs showed only slight signs of moisture absorption, as evidenced by the color. Long-term data is still in the process of being collected.

Example of syringe kit preparation

A syringe kit has been developed in which the components are stored in two syringes: a male and a female syringe. The female syringe contains a mixture of white powders. The male syringe contains a buffer solution. The two syringes are connected and the contents are mixed to produce a liquid polymer. The liquid polymer is then sprayed or applied to the sutured wound, where it covers the entire suture line. During this process, the polymer enters the gaps left by the stitches and prevents infection. At the wound site, the liquid polymer transforms into a solid gel and remains there for over two weeks. During this time, the wound heals and remains infection-free.

The components required for preparing this test kit are disclosed in Table 17 and Table 18. For the preparation of the powder components of this test kit to be filled into a female syringe, the piston of a 5mL female Luer lock syringe is removed and the syringe is covered with a suitable cap. 8-arm-20k-AA (0.028g, an acceptable weight range is 0.0270g to 0.0300g), 8-arm-20k-NH2 (0.012g, an acceptable weight range is 0.0100g to 0.0130g), 4-arm-20k-SGA (0.080g, an acceptable weight range is 0.0790g to 0.0820g) and 0.043g of freeze-dried phosphate buffer powder (0.043g, an acceptable weight range is 0.035g to 0.052g) are carefully weighed and poured into the syringe. The syringe is then flushed with nitrogen/argon at a rate of 5 to 10L/min for approximately 10 seconds and the piston is replaced to seal contents. The syringe is then turned over so that the cap is facing upward. The syringe cap is then loosened and air gaps in the syringe are minimized by expelling as much air as possible from the syringe. A typical compressed powder volume is 0.2 mL. The syringe cap is then tightened until it is finger-tight.

The liquid component was prepared in a 500 mL batch size by pouring 50 mL of a commercial 2% chlorhexidine solution, 450 mL of distilled water, and 1.5 g of HPMC into a sterile container. The sterile container was then covered and shaken vigorously for 10 seconds. The solution was allowed to sit at ambient conditions for 16 hours, allowing the foam to dissipate and any remaining HPMC to dissolve.

Prepare a liquid/buffer syringe by removing the piston of the male/female Luer lock syringe and then capping the syringe with a suitable cap. Transfer 2.5 mL of the buffer/liquid solution into the syringe via a pipette. Then, flush the syringe with nitrogen/argon at a rate of 5 to 10 L/min for about 5 seconds. Then replace the syringe's piston to seal the contents. The syringe is then flipped so that the cap is facing the top, and the syringe cap is loosened, minimizing air gaps by expelling as much gas as possible from the syringe. Then, tighten the syringe cap until it is finger-tight.

Table 17. Components used to make solid components for female syringes

Components Technical Name 8-arm-20k-AA 8-arm PEG amine acetate, HCl salt, MW 20k 8 arms-20k-NH2 8-arm PEG amine (hexaglycerol), HCl salt, MW 20k 4-arm-20k-SGA 4-arm PEG succinimidyl glutaramide (pentaerythritol), MW 20k Commercial 2% chlorhexidine solution Freeze-dried phosphate buffered saline powder

Table 18. Materials used to manufacture the kits, including suppliers, part numbers, and lot numbers.

illustrate supplier Part Number Supplier Catalog Number 10mL Luer lock syringe BD CM-0003 309604 No-Spout Female Luer Dispenser Tip Cap, White QOSINA CM-0004 65119 5mL female Luer lock syringe, purple PP QOSINA CM-0005 C3610 Male Luer lock cap, no opening, PP QOSINA CM-0006 11166

Example of syringe kit preparation

Another syringe kit has been developed in which the solid component, a white powder mixture, is stored in a female syringe. A standard male syringe is used to draw up a drug solution, such as that containing Kenalog. The two syringes are connected and the contents are mixed to produce a liquid polymer. This liquid polymer is then delivered to the target site.

The components required for preparing this test kit are disclosed in Table 17 and Table 18. For the preparation of the powder components of this test kit to be filled into a female syringe, the piston of a 5mL female Luer lock syringe is removed and the syringe is covered with a suitable cap. 8-arm-20k-AA (0.0125g, an acceptable weight range is 0.012g to 0.013g), 8-arm-20k-NH2 (0.075g, an acceptable weight range is 0.007g to 0.008g), 4-arm-20k-SGA (0.040g, an acceptable weight range is 0.040g to 0.042g) and 0.018g of freeze-dried phosphate buffer powder (0.043g, an acceptable weight range is 0.017g to 0.022g) are carefully weighed and poured into the syringe. The syringe is then flushed with nitrogen/argon at a rate of 5 to 10L/min for approximately 10 seconds and the piston is replaced to seal contents. The syringe is then turned over so that the cap is toward the top. The cap is then loosened and the air gap in the syringe is minimized by expelling as much air as possible from the syringe. The syringe cap is then tightened until the cap is finger tight.

Example 14: General Procedure for Preparing Polyglycol-Based Biocompatible Hydrogel Polymer Matrices

A biocompatible preformulation based on polyglycol was prepared by mixing 0.028 g of 8-arm-AA-20k, 0.012 g of 8-arm-NH2-20K, and 0.080 g of 4-arm-SGA-20K. 2.50 mL of culture medium was added to the preparation. The preparation was mixed for approximately 10 seconds, and 1 mL of the mixture solution was aspirated using a mechanical high-precision pipette. The biocompatible preformulation components based on polyglycol were polymerized to form a biocompatible hydrogel polymer matrix based on polyglycol. The polymerization time of 1 mL of liquid was collected and then verified by the lack of flow of the remaining liquid.

Example 15: General Procedure for Preparing Polyglycol-Based Biocompatible Hydrogel Polymer Matrix and Stem Cells

A polyglycol-based biocompatible preformulation was prepared by mixing 0.0125 g of 8-arm-AA-20k, 0.0075 g of 8-arm-NH2-20K, and 0.040 g of 4-arm-SGA-20K. 1.0 mL of culture medium was added to the preparation. The preparation was mixed for approximately 10 seconds, and 1 mL of the mixture solution was aspirated using a mechanical high-precision pipette. The polyglycol-based biocompatible preformulation components were polymerized to form a polyglycol-based biocompatible hydrogel polymer matrix. The polymerization time of 1 mL of liquid was collected and then verified by the lack of flow of the remaining liquid. Polymerized polyglycol-based biocompatible hydrogel polymer matrix sheets of different sizes were placed in different wells of a 24-well plate. 0.5 mL of adult mesenchymal stem cells were seeded onto the polymer matrix at different densities. The stem cells diffused and incorporated into the polyglycol-based biocompatible hydrogel polymer matrix. The incorporation of the stem cells into a biocompatible polyglycol-based hydrogel polymer matrix was demonstrated by removing the polymer matrix sheet 10 days after stem cell addition and using the sheet to expand the cells in culture. The incorporated stem cells remained viable, as shown by their ability to proliferate in culture.

Example 16: General Procedure for Preparing Polyglycol-Based Biocompatible Hydrogel Polymer Matrix and Stem Cells

A biocompatible preformulation based on polyglycol was prepared by mixing 0.0125 g of 8-arm-AA-20k, 0.0075 g of 8-arm-NH2-20K, and 0.040 g of 4-arm-SGA-20K. 1.0 mL of culture medium containing adult mesenchymal stem cells was added to the preparation. The preparation was mixed for approximately 10 seconds, and 1 mL of the mixture solution was aspirated using a mechanical high-precision pipette. The biocompatible preformulation components based on polyglycol were polymerized to form a biocompatible hydrogel polymer matrix based on polyglycol. The polymerization time of 1 mL of liquid was collected and then verified by the lack of flow of the remaining liquid.

Additional components can be added to the formulation at any point during the combination of the biocompatible preformulation compound based on polyglycol. When additional components are added, the formulation can be solid, liquid, polymeric, gelled, or any combination thereof. Additional components can be combined with the formulation or diffused through the formulation and retained with the formulation for a determined period of time. In one example, a biocompatible hydrogel polymer matrix based on polyglycol is formed and then a growth factor is added. The growth factor is incorporated into the biocompatible hydrogel polymer matrix based on polyglycol. Additional components include, but are not limited to, biomolecules, antibiotics, anticancer agents, anesthetics, antivirals, or immunosuppressants.

Example 17: Cell Viability in a Polyglycol-Based Biocompatible Hydrogel Polymer Matrix

A single cell suspension of mesenchymal stem cells in D15 (DMEM, high glucose, 15% fetal bovine serum) was prepared and the cells were counted. 1 mL of cells with a density of 2 x 10 ⁴ / mL was added to a 50 mL tube. The cells were maintained at room temperature and prepared just before being added to the preformulation. A female syringe containing a biocompatible preformulation based on polyglycol was prepared by mixing 0.0125 g of 8-arm-AA-20k, 0.0075 g of 8-arm-NH2-20K, and 0.040 g of 4-arm-SGA-20K in a female syringe. An 18G needle was attached to the male syringe and filled with 1 mL of PBS. The next step was performed within 90-120 seconds. The needle was removed from the male syringe and the male syringe was connected to the female syringe containing the preformulation. PBS was pushed from the male syringe into the female syringe, and the mixing process was started by repeatedly pushing PBS from one syringe into another, 20 times being sufficient for mixing. After the final injection, push the entire contents into the male syringe. Attach an 18G needle to the male syringe and eject the liquid formulation into a 50mL tube containing 1mL of mesenchymal stem cells. Carefully mix the cells as the preformulation is ejected into the tube. Be careful not to mix the cells by drawing the needle, as this can cause cell stress.

Aliquots of the preformulation containing mesenchymal stem cells were placed in the chambers of a 4-chamber tissue culture slide at 50, 100, 200, and 400 μL. The preformulation was allowed to gel for 2 minutes. 200 μL of D15 was added to each chamber. Three of these slides were prepared for three time points: 0, 2, and 24 hours. Cells were stained with membrane-permeable 3', 6'-di(O-acetyl)-2', 7'-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, tetraacetoxymethyl ester, and membrane-impermeable ethidium homodimer-1, 1 μL/ml propidium iodide. Cells were imaged using bright field and fluorescence microscopy. Live cells fluoresce green, while dead cells fluoresce red. At the 2-hour time point, only one dead cell was observed in multiple field observations. One live cell had a punctate cytoplasm. The remaining cells were alive and had a typical ellipsoidal morphology in the hydrogel polymer matrix. At the 24 hour time point, greater than 95% of cells were viable.

Example 18: Methods for Determining the Properties of Cells in a Polyglycol-Based Biocompatible Hydrogel Polymer Matrix Use the program

The proliferation rate, viability, and structural characteristics of mesenchymal stem cells were evaluated after incorporation into a biocompatible hydrogel polymer matrix.

To measure the proliferation rate of mesenchymal stem cells, a cell proliferation assay was performed. A biocompatible preformulation comprising a polyglycol-based compound and a suitable buffer as described in Example 14 was prepared. 100 μl of the preformulation was coated on a 24-well plate to produce a coating <5 mm thick. Stem cells were seeded on the coated plate at different cell densities (1x10 ³ , 5x10 ³ , 10x10 ³ and 20x10 ³ cells). Cells were incubated at 37°C, 5% CO ₂ in growth medium. For each sample, an AQueous Non-Radioactive (MTS) assay was performed on the 2nd, 7th and 10th day after inoculation to confirm that the cells were proliferating. Growth medium was removed from each well and replaced with 500 μl of fresh culture medium, and incubated at 37°C, 5% CO ₂ for at least 1 hour. 100 μl of MTS reagent was added to each well and incubated at 37°C, 5% CO ₂ for 3 hours. Use a microplate reader to measure and record the absorbance at 490nm. Use wells containing the preparation but without any cells as blanks. Similarly, wells with only culture medium without any cells are used as blanks. Obtain the reading of each sample by subtracting the blank. Draw an absorbance-time graph. Absorbance is proportional to cell number, where a significant increase in absorbance indicates cell viability and proliferation. Calculate the fold change of proliferation.

To demonstrate the viability of adult mesenchymal stem cells, staining assays were performed on cells seeded on coated 24-well plates as previously described in this example on days 2, 7, and 10. The culture medium was removed and the cells were washed twice with phosphate saline buffer. 0.5 ml of a staining solution containing calcein-am (10 μg/ml) and propidium iodide (100 μg/ml) was added to each well and the plate was incubated at 37°C for 5-10 minutes. The cells were washed with phosphate saline buffer and imaged immediately. Live cells fluoresce green and dead cells fluoresce red.

To prove that adult mesenchymal stem cells maintain their structure, a staining test was performed on cells seeded on a coated 24-well plate as previously described in this embodiment. The culture medium was removed and the cells were washed twice with phosphate buffer. The cells were fixed at room temperature for 10 minutes with 4% paraformaldehyde and subsequently washed twice with phosphate buffer. Cytoplasmic WGA stain (wheat germ agglutinin; 488 green fluorescence) was added to the washed cells and the cells were incubated at room temperature for 10 minutes. The stain was removed and the cells were washed twice with phosphate buffer. Nuclear TO-PRO-3 iodide stain (red fluorescence) was added to the cells, and the cells were incubated at room temperature for 10 minutes. The stain was removed and the cells were washed twice with HBSS buffer. Anti-fading reagent Pro-long gold was added to the cells, and the cells were covered with coverslips. 3D confocal microscopy was performed to visualize the structure and adhesion of the cells. Overall, stem cells maintained their physicochemical properties.

Example 19: Elution of cells from a polyglycol-based biocompatible hydrogel polymer matrix

The polyglycol-based biocompatible hydrogel polymer matrices of the examples were prepared. Additional polyglycol-based biocompatible hydrogel polymer matrices were prepared using the preformulated compounds and cells of Table 13. The polymer matrices were weighed and placed in separate Falcon tubes. 2 mL of buffer per gm of polymer matrix was added to the Falcon tubes. The Falcon tubes were placed in a water bath maintained at 37°C. After 24 hours, the buffer was carefully removed and replaced with fresh buffer to maintain a constant volume. This extraction process was repeated until each polymer matrix was completely dissolved. The polymer matrix dissolved within two weeks.

The elution behavior of cells under different biocompatible preformulation components was tested. The cell elution profile varies with different biocompatible preformulation components. Cells can spread while being retained in the polymer matrix, released by degradation of the polymer matrix, or any combination thereof. The composition of the biocompatible preformulation components can be selected to control the release of cells at a predetermined time.

In some cases, the polymer matrices containing cells described herein also contain additional components, such as buffers, growth factors, antibiotics, or anticancer agents. The composition of the biocompatible preformulation components and the additional components can be varied to control the release of cells and/or the additional components.

In some cases, cells from any of the cell-containing polymer matrices described in this example can be released from the polymer matrix in a manner based on the pore size of the polymer matrix. In some cases, the cells remain viable after being released from the polymer matrix.

Example 20: Polyglycol-based biocompatible preformulations for disease treatment

A biocompatible preformulation based on polyglycol containing 0.0125 g 8-arm-AA-20K, 0.0075 g 8-arm-NH2-20k, 0.040 g 4-arm-SGA-20K, mesenchymal stem cells, and a suitable culture medium was combined in the presence of 1.0 mL of water. The liquid formulation was delivered by direct injection to the site of tissue injury in the liver. The biocompatible preformulation mixture based on polyglycol polymerized in vivo at the delivery site to form a biocompatible hydrogel polymer matrix based on polyglycol at the target site within 4 minutes. The biocompatible hydrogel polymer matrix culture medium component based on polyglycol was configured to influence the physical, chemical, and biological environment surrounding the stem cells during and after administration to the target site.

A biocompatible polyglycol-based hydrogel polymer matrix is retained at the target site, where stem cells are released over a two-week period. The released stem cells need to interact and integrate with the target tissue through the incorporation of appropriate physical and cellular signals. Therefore, the polyglycol-based biocompatible hydrogel polymer matrix culture medium contains modifying factors, such as bioactive proteins, that are critical for successful tissue generation. Mesenchymal stem cells begin to differentiate at the target site between 7 and 14 days, resulting in improved liver function.

Example 20: Polyglycol-based biocompatible hydrogel polymer matrix for disease treatment

A biocompatible polyglycol-based hydrogel polymer matrix was prepared by adding 1 mL of water to a preformulation containing 0.0125 g of 8-Arm-AA-20K, 0.0075 g of 8-Arm-NH2-20K, 0.040 g of 4-Arm-SGA-20K, mesenchymal stem cells, and a suitable culture medium. After gelation, the hydrogel polymer matrix was delivered directly to the site of tissue injury in the liver. The polyglycol-based biocompatible hydrogel polymer matrix culture medium component was configured to influence the physical, chemical, and biological environment surrounding the stem cells during and after administration to the target site in the liver.

A biocompatible hydrogel polymer matrix based on polyglycol is retained at the target site, where the stem cells are released over a two-week period. The released stem cells need to interact and integrate with the target tissue through the incorporation of appropriate physical and cellular signals. Therefore, the biocompatible hydrogel polymer matrix culture medium based on polyglycol contains modifying factors, such as bioactive proteins, that are critical for successful tissue generation. Mesenchymal stem cells begin to differentiate at the target site between 7 and 14 days, resulting in improved liver function.

Example 21: Polyglycol-based biocompatible hydrogel polymer matrix for growth factor delivery

A biocompatible preformulation based on polyglycol comprising 0.028g 8-arm-AA-20K, 0.012g 8-arm-NH2-20k, 0.08g 4-arm-SGA-20K, growth factors and a buffer solution was combined in the presence of 2.5mL of water. The liquid formulation was delivered by direct injection into the tissue injury site. The biocompatible preformulation mixture based on polyglycol was polymerized in vivo at the delivery site to form a biocompatible hydrogel polymer matrix based on polyglycol at the target site. The biocompatible hydrogel polymer matrix based on polyglycol was configured to release growth factors at the target site. The growth factors were configured to recruit cells from the body to the polymer matrix site, where the recruited cells could form tissues on and throughout the polymer matrix.

An alternative way to incorporate growth factors into a polyglycol-based biocompatible hydrogel polymer matrix is to integrate a DNA plasmid encoding the gene and a mammalian promoter into the polymer matrix. Delivery of a polyglycol-based biocompatible hydrogel polymer matrix containing DNA programs local cells to produce their own growth factors.

Example 22: Pore Size Determination

The pore diameter was estimated from the molecular weight of each arm of the combined components. The pore diameter was calculated based on the number of PEG units per arm and a carbon-carbon-carbon bond length of 0.252 nm and a bond angle of 110°. Fully extended chains were assumed to account for the bond angles and complete reactivity of all functional end groups to form the pore network. The pore diameter was further refined by its correlation with the inverse of the swelling ratio of the biocompatible hydrogel:

ξ≈L*(V _p /V _s ) ^-1/3 (Equation 1)

where _Vp is the polymer volume, _Vs is the volume of the swollen gel, L is the calculated pore diameter, and ξ is the swollen pore diameter. Based on equilibrium swelling experiments, the ratio of _Vp to _Vs is estimated to be around 0.5.

For multi-component mixtures containing reactive esters, a weighted average of the pore sizes of each component and the ester was used. For example, for a polymer composed of 4-arm-20k-AA, 8-arm-20k-NH2, and 4-arm-20k-SGA, the pore sizes obtained from 4-arm-20k-AA and 4-arm-20k-SGA were averaged with the pore sizes obtained from 8-arm-20k-NH2 and 4-arm-20k-SGA.

Claims (39)

1. A fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix comprising a fully synthetic, polydiol-based, biocompatible hydrogel polymer, the polymer comprising at least one first monomer unit bonded to at least one second monomer unit via at least one amide bond, wherein the polymer forms a matrix that encapsulates: (a) at least one cell or virus; and (b) A culture medium that supports the growth of at least one of the cells or viruses. The hydrogel polymer is derived from the first monomer unit of 8-arm PEG-acetic acid and 8-arm PEG-amine and the second monomer unit of 4-arm PEG-succinimide-glutaramide; and (c) Phosphate buffer and viscosity enhancer When the fully synthetic, polydiol-based biocompatible biodegradable hydrogel polymer matrix is implanted into a target site in an animal, the biodegradation of the biocompatible biodegradable hydrogel polymer matrix provides controlled release of at least one cell or virus to the target site in the animal. 2. The fully synthesized, biocompatible, biodegradable hydrogel polymer matrix based on polydiol according to claim 1, wherein the 8-arm PEG-acetamide is either the 8-arm PEG-acetamide (hexaglycerol) HCl salt of MW 20000 or the 8-arm PEG-acetamide (hexaglycerol) TFA salt of MW 20000. 3. A fully synthesized, biocompatible, biodegradable hydrogel polymer matrix based on polydiol according to any one of claims 1-2, wherein the cells are selected from mammalian cells, insect cells, protozoan cells, bacterial cells, or fungal cells. 4. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the mammalian cells are stem cells. 5. A fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to any one of claims 1-2 and 4, wherein the culture medium contains growth factors. 6. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the culture medium contains growth factors. 7. The fully synthesized, biocompatible, biodegradable hydrogel polymer matrix based on polydiol according to any one of claims 1-2, 4 and 6, wherein the ratio of 8-arm PEG-acetic acid to 8-arm PEG-amine is 1:1, 70:30 or 75:25. 8. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the ratio of 8-arm PEG-acetic acid to 8-arm PEG-amine is 1:1, 70:30, or 75:25. 9. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 5, wherein the ratio of 8-arm PEG-acetic acid to 8-arm PEG-amine is 1:1, 70:30, or 75:25. 10. A fully synthesized, biocompatible, biodegradable hydrogel polymer matrix based on polydiol according to any one of claims 1-2, 4, 6 and 8-9, wherein the animal is human. 11. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the animal is human. 12. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 5, wherein the animal is human. 13. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 7, wherein the animal is human. 14. A fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to any one of claims 1-2, 4, 6, 8-9 and 11-13, wherein the polydiol-based biocompatible hydrogel polymer matrix is bioabsorbed within 14 to 180 days. 15. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the polydiol-based biocompatible hydrogel polymer matrix is bioabsorbed within 14 to 180 days. 16. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 5, wherein the polydiol-based biocompatible hydrogel polymer matrix is bioabsorbed within 14 to 180 days. 17. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 7, wherein the polydiol-based biocompatible hydrogel polymer matrix is bioabsorbed within 14 to 180 days. 18. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 10, wherein the polydiol-based biocompatible hydrogel polymer matrix is bioabsorbed within 14 to 180 days. 19. A fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to any one of claims 1-2, 4, 6, 8-9, 11-13, and 15-18, wherein the controlled release of the at least one cell or virus to a target site in an animal body comprises diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 20. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix of claim 3, wherein the controlled release of the at least one cell or virus to a target site in an animal body includes diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 21. The fully synthetic, polydiol-based biocompatible biodegradable hydrogel polymer matrix of claim 5, wherein the controlled release of the at least one cell or virus to a target site in an animal body includes diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 22. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix of claim 7, wherein the controlled release of the at least one cell or virus to a target site in an animal body includes diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 23. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix of claim 10, wherein the controlled release of the at least one cell or virus to a target site in an animal body includes diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 24. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix of claim 14, wherein the controlled release of the at least one cell or virus to a target site in an animal body includes diffusion of the at least one cell or virus from the polydiol-based biocompatible hydrogel polymer matrix. 25. A fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to any one of claims 1-2, 4, 6, 8-9, 11-13, and 15-18, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished by the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 26. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 3, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished through the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 27. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 5, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished through the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 28. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 7, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished through the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 29. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix of claim 10, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished through the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 30. The fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 14, wherein the controlled release of at least one cell or virus to a target site in an animal body is at least partially accomplished through the degradation and bioabsorption of the polydiol-based biocompatible hydrogel polymer matrix. 31. The fully synthesized, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to claim 1, wherein the viscosity enhancer is HPMC. 32. A fully synthetic, polydiol-based, biocompatible, biodegradable preformulation comprising: (a) At least one fully synthetic, polydiol-based first composition comprising 8-arm PEG-acetic acid and 8-arm PEG-amine; (b) At least one fully synthetic, polydiol-based second composition comprising 4-arm PEG succinimide glutaramide; (c) Phosphate buffer; (d) Viscosity enhancers; (e) at least one cell or virus; and (f) A culture medium that supports the growth of at least one of the cells or viruses; The polydiol-based biocompatible biodegradable preformulation is at least partially polymerized and/or gelled in the presence of water to form a polydiol-based biocompatible biodegradable hydrogel polymer matrix encapsulating the cells or viruses, and the polydiol-based biocompatible biodegradable hydrogel polymer matrix is capable of biodegrading in a subject and releasing the at least one cell or virus. 33. The fully synthetic, polydiol-based, biocompatible, biodegradable preformulation according to claim 32, wherein the cells are stem cells. 34. The fully synthesized, polydiol-based, biodegradable preformulation according to any one of claims 32-33, wherein the culture medium comprises a buffer solution. 35. The fully synthesized, polydiol-based biocompatible biodegradable preform for any one of claims 32-33, wherein the polydiol-based biocompatible biodegradable preform gels to form a polydiol-based biocompatible hydrogel polymer matrix between 20 seconds and 10 minutes. 36. The fully synthesized, polydiol-based biocompatible biodegradable preform according to claim 34, wherein the polydiol-based biocompatible biodegradable preform gels to form a polydiol-based biocompatible hydrogel polymer matrix between 20 seconds and 10 minutes. 37. The fully synthesized, polydiol-based, biocompatible, biodegradable preformulation according to claim 32, wherein the viscosity enhancer is HPMC. 38. A fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix, said fully synthetic, polydiol-based, biodegradable hydrogel polymer matrix being formed from a fully synthetic, polydiol-based, biocompatible, biodegradable preformulation according to any one of claims 32-37. 39. Use of a fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix in the preparation of a medicament for treating a disease, wherein the polymer matrix is a fully synthetic, polydiol-based, biocompatible, biodegradable hydrogel polymer matrix according to any one of claims 1-31.