TWI564030B - Polymer microcell with high growth factor loading rate and manufacturing method thereof - Google Patents

Description

Polymer microcell with high growth factor loading rate and manufacturing method thereof

The present invention relates to a polymeric micelle, and more particularly to a polymeric microcell having a high growth factor loading rate and a method for producing the same.

Usually when people are traumatized by skin abrasions, cuts, etc., the most important thing is to clean the wounds, and wash off the dirt on the wound, such as sand, to avoid further infection. The most common use in hospitals to clean the wounds is saline. At home, the wounds can be cleaned with boiled cold water. When the wound is cleaned, it should be covered with a suitable dressing. The previous concept was to keep the wound dry, so it would be done by using a baking lamp or by blowing the wound with oxygen, or by not wrapping it to expose the wound. In a dry environment, the cells are allowed to die, so the wounds are more painful, and the wounds exposed to them are more susceptible to infection. New research has found that a moist wound bed is beneficial for angiogenesis and cell growth, so the new concept of wound care is to maintain a proper moistness to promote healing. There are many new dressings on the market to replace traditional gauze, which not only provides an environment suitable for wound healing, but also increases comfort, such as artificial skin, foam, and silicone dressing. A variety of dressings are available.

In several developmental processes of organisms, growth factors are key regulators of cell fate and differentiation; however, the bioavailability of growth factors is usually not high, because growth factors are easily inactive in general environments. And it is easy to decompose or aggregate the precipitate. Among all growth factors, Fibroblast growth factor 1 (FGF1) has been found to be a potential regulator in many types of cells and induces angiogenesis, a property of tissue regeneration. The essential. However, studies have found that FGF1 is not highly efficient to load via synthetic polymers, and has a short half-life characteristic due to enzymatic degradation and self-aggregation.

Hyaluronic acid (HA) is a kind of D-glucuronic acid and (1-β-3)-N-acetyl-D-glucosamine ((1-β-3) -N-acetyl-D-glucosamine) A polysaccharide composed of polysaccharide molecules. On the other hand, poly(ε-caprolactone, PCL) is stronger than the poly(lactide-co-glycolide) hydrophobic segment. Resistance to hydrolysis. When PCL is used as a constituent segment, such stability can lengthen the shelf life of the copolymer.

The faster the healing of the skin wound, that is, the shorter the time the skin lacking epidermal protection is exposed, the greater the chance of infection, and the faster return of normal life to the patient. However, in general, when a drug or a protein (for example, a growth factor) is directly coated with a carrier, a sudden release reaction is likely to occur, resulting in an excessive local concentration. It may be a method to combine dressings with growth factors, but how to make growth factors stably present in the dressing or make growth factors available in the case of poor growth factor loading rate. The state of slow release in the material is an urgent problem to be solved.

In order to solve the above problems, the present invention provides a polymeric micelle having a high growth factor loading ratio, comprising a hydrophilic backbone, a hydrophobic segment, and a crosslinking agent, wherein the growth factor is via A crosslinking agent is grafted to the polymer micelle, and the crosslinking agent is carbodiimide.

In a preferred embodiment of the present invention, the hydrophilic main chain is hyaluronic acid (HA), methoxy polyethylene glycol (mPEG), or poly-glutamic acid (poly- Γ-glutamic acid (PGA), preferably hyaluronic acid. The hydrophobic main chain is polycaprolactone (PCL), polyvalerolactone (PVL), poly(lactide-co-glycolide, PLGA), polylactic acid (polylactic acid). , PLA), polybutyro-lactone (PBL), polyglycolide or polypropiolactone (PPL), preferably polycaprolactone, wherein the molecular weight of the polycaprolactone is 2.7 kDa.

Generally, the cross-linking agent is mostly toxic or irritating, and must have a washable property for medical use, so the selection and treatment of the cross-linking agent become quite important. A carbonodiamine crosslinking agent according to a preferred embodiment of the present invention is 1-ethyl-3-(3-dimethylaminopropyl)carbodiamine (1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide (EDC), which is highly safe, and the small molecule urea product produced after cross-linking is water-soluble, washable or dialysis treated.

Growth factors tend to lose activity in the general environment, and easily decompose or aggregate and precipitate. The use of cross-linking agents to fix the residues and materials of growth factors can maintain the molecular weight of growth factors and avoid degradation, wherein the growth factor has a high loading rate. The cell line is 50% to 99%, and the growth factor of one of the polymer micelles is 90 μg/mg or more, and the polymer micet has only 5 to 40% of the growth factor release amount in the first 20 hours. . The invention adds EDC to fix the protein on the microcells, and still has a proliferative effect on the fibroblasts, and the animal experiment has an excellent effect on wound repair and angiogenesis in vivo experiments.

Another object of the present invention is to provide a method for producing a polymer microcell, comprising the steps of: a. polymerizing a hydrophilic monomer as a main chain with a hydrophobic monomer to form a copolymer; b. Activating a carboxyl group of the copolymer in the presence of a crosslinking agent, wherein the crosslinking agent is a carbonic acid; and c. crosslinking a growth factor to the copolymer via the crosslinking agent to form a load growth a polymer microcapsule of a factor; wherein, in the case where the amount of the copolymer is fixed and the amount is assumed to be 1, the ratio of the crosslinking agent to the growth factor is between 0.5 and 5, and the concentration of the crosslinking agent is 0.05 to 0.5 mg/ In mL, the surface potential of the polymer micelle is -29 mV~-60 mV, the diameter of the polymer micelle is 30-220 nm, and the pH of the polymer micelle is about 7, and the stability is sustainable. Up to 1 month.

The present invention also provides a dressing for promoting wound healing comprising a polymeric microcell as described above and a matrix, wherein the matrix can be a cotton cloth or a gel.

On the other hand, when the drug or protein is coated directly with the carrier, it is easy to have a sudden release reaction, resulting in a situation where the local concentration is too high, such as Cross-linking mode can reduce the release situation and achieve the effect of controlled release.

The present invention prepares a novel copolymer constructed by grafting PCL (HA-g-PCL) onto hyaluronic acid. We used a specific cell concentration to show that HA-g-PCL is capable of self-aggregating in the micelles and recombining human FGF1 in water. The release profile of FGF1 in novel micelles by different methods was analyzed. The biological activity of the FGF1-loaded micelles was evaluated in vitro and in animal experiments. A FGF1 nanocarrier with high loading efficiency, high loading and sustained release has been developed. Therefore, the combination of hyaluronic acid and polycaprolactone of the present invention can produce a carrier having a high growth factor loading rate, which is a great advance in the development of wound dressings.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The first figure is (A) the molecular structure of hyaluronic acid (HA), poly(ε-caprolactone) (PCL), and HA-g-PCL; (B) the preparation process of HA-g-PCL.

The second figure is (1) ¹ H NMR spectrum of HA-g-PCL, showing a graft ratio of 6 to 8%; (B) HA-g-PCL infrared spectrum; (C) determining HA-g-PCL micro Critical Micelle Concentration (CMC); (D) TEM image of naked cells, indicated by arrows; (E) Evaluation of HA-g-PCL by hydrodynamic particle size and surface potential Stability of microvesicles during storage; (F) Stability of HA-g-PCL micelles at different pH values.

The third panel is the cytotoxicity analysis of different concentrations of HA-g-PCL micelles for fibroblast L929: (A) morphology and (B) survival rate; survival rate values were normalized relative to control group values. * represents p < 0.05.

The fourth graph is (A) the cumulative release rate of FGF1 encapsulated by three physical means; (B) the cumulative release rate of FGF1 micelles grafted with different concentrations of cross-linking agent EDC, and the "w/o EDC" group is ultrasonically oscillated. In the legal group, the cumulative release rate value is expressed as a percentage relative to the initial FGF1 loading; (C) the Western blotting method confirms that FGF1 is loaded on the micelle, and the amount of FGF1 loaded by the EDC-M group is higher than that of the physical package. Group (w/o EDC, for the ultrasonic oscillating method group). * indicates p < 0.05.

The fifth panel is (A) cumulative release rate of FGF1 by ELISA; (B) Western blotting method to calculate the amount of FGF1 on the cells after 21 days of release (200-fold dilution); based on semi-quantitative, EDC-M micelles More FGF1 was retained in the HA-g-PCL microsomes that encapsulated FGF1 than the physical (w/o EDC).

Figure 6 depicts the cell viability of L929 fibroblasts with native FGF1, loaded FGF1 micelles (day 0 of release assay), or loaded FGF1 micelles (day 21 of release assay), values processed relative to blank (0 ng The result of /mL FGF1) is normalized. * indicates p < 0.05.

The seventh picture shows the effect of loading FGF1 micelles on the wound healing of rat skin model; (A) the appearance of the wound area during the experiment and the quantification of the wound area; Blank: only the Guardian patch; FGF1: free FGF1 (1.5 ng) dispersed in 50 μL PBS; Micelles: nude cells (50 μg) dispersed in 50 μL PBS; FGF1-micelles (EDC-M): loaded FGF1 (1.5 Ng) of the micelles (50 μg) were dispersed in 50 μL of PBS; (B) wound morphology on day 8 was stained with hematoxylin-eosin (H&E); arrows indicated blood vessels; "E" represents wounds Epithelial formation; scale bar is 200 μm; (C) vascular area is quantified based on morphology. * indicates p < 0.05. (D) On the 8th day, the H&E staining was in a low magnification form; the arrow was referred to as the sebaceous gland; the scale bar was 200 μm; (E) the number of sebaceous glands per square centimeter based on morphology. * indicates p < 0.05.

Figure 8 is the result of a rat skin wound healing experiment (n=3); (A) a film used to carry free FGF1 or FGF1-loaded micelles; (B) contraction of wound area at different times; (C) Quantification of wound area data during the experiment; (D) H&E staining to observe the histology of wound repair on day 8; arrows refer to blood vessels; long arrows refer to residual colloids; (E) histologically quantified vascular area .

The term "microcell" as used in the specification of the present invention refers to a copolymer microparticle of hyaluronic acid grafted polycaprolactone, that is, HA-g-PCL microcell, unless otherwise specified.

The present invention is a polymer microcell having a high growth factor loading ratio, comprising a hydrophilic backbone, a hydrophobic segment, and a crosslinking agent. In a preferred embodiment of the invention, the hydrophilic backbone is hyaluronic The acid and hydrophobic backbone is polycaprolactone, which is loaded with the growth factor FGF1, and the crosslinker is carbamide. FGF1 was grafted onto the micelles of the hyaluronic acid grafted polycaprolactone copolymer via carboguanamine; FGF1 was protected from hydrolysis in an in vitro test and was still biologically active after 21 days of release. Tested in vivo to demonstrate the function of contracting wounds and promoting angiogenesis.

The materials used in the description of the present invention are as follows: hyaluronic acid (abbreviated as HA) (sodium salt, molecular weight 16 kDa), available from Czech Chem sro (Dolni Dobrouc, Czech). Tetrabutylammonium (TBA) hydroxide aqueous solution, stannous octoate (Sn(Oct) ₂ ), and 4,4′-diisocyanate dicyclohexylmethane (4,4′-diisocyanatodicyclohexylmethane, H ₁₂ MDI, 90%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Dodecanoyl (98%) and 1,4-diazabicyclo[2.2.2]octane (1,4-di-azabicyclo[2.2.2]octane, DABCO) were purchased from Acros Organics (Geel, Belgium) and Alfa Aesar (Heysham, Lancashire, UK). ε-Caprolactone was purchased from MP Biomedicals (Solon, CA, USA). Dimethyl sulfoxide (DMSO) and dichloromethane were purchased from JT Baker (Phillipsburg, NJ, USA). Sodium and hydrogen ion exchange resin were purchased from Rohm and Haas Shanghai Chemical Industry (China). Dialysis membranes (Spectra/Por4, MWCO: 14,000) and fluorescein isothiocyanate (FITC) were supplied by Spectrum Laboratories (Rancho Dominguez, CA, USA) and Sigma-Aldrich. 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) is commercially available from Tokyo Chemical Industry ( Tokyo, Japan). The pharmaceutical grade recombinant human FGF1 line was purchased from Renogen Biotech (Shanghai, China).

"About", "about" or "approximately" generally means within 20%, preferably 10%, and most preferably 5%. The numerical values in this paper are approximate to the measurement instruments, or the differences in the measurement. The numerical values in this paper are approximate values. If they are not clearly defined, the meaning of "about" or "approximate" may be implied. .

Multi-sample analysis was used for each experiment in the present specification. Values are expressed as mean ± standard deviation. In all experiments, each experiment was independently tested three times or more to ensure the reproducibility of the experiment. Statistical differences between the experimental groups were calculated using Student's t test. A p value of <0.05 is considered to be statistically significant.

Embodiment 1 Synthesis of HA-g-PCL

A schematic diagram of the HA-g-PCL synthesis step is shown in the first figure (g is the meaning of grafting). Preparation of mono-functional polycaprolactone (PCL, molecular weight 2.7 kDa), first using Sn(Oct) ₂ as a catalyst to open ε-caprolactone, and then starting with 1-dodecanol Starter synthesis. Monofunctional PCL (1.08 x 10 ^-3 moles) was dissolved in 8 mL DMSO and H ₁₂ MDI (0.97 x 10 ^-3 moles) was added. Then, Sn(Oct) ₂ 3.3 mg and DABCO 6.5 mg dissolved in 1 mL of DMSO were added in sequence. The reaction was carried out at 60 ° C for 20 hours. The TBA-HA salt was prepared by mixing an equivalent amount of a TBA hydroxide aqueous solution with a hyaluronic acid (HA) solution for 3 hours, and further dried by a rotary evaporator and vacuum freeze-drying. TBA-HA salt (4.84 x 10 ^-4 mol) was dissolved in 100 mL of DMSO and added to the aforementioned PCL-containing reaction mixture, followed by the addition of DMSO containing 5.9 mg of Sn(Oct) ₂ in 1 mL and 11.8 mg of DABCO. The reaction was carried out at 60 ° C for 24 hours. The HA-TBA-g-PCL copolymer was transferred to the dialysis membrane and sealed at both ends and dialyzed against DMSO for 1 day, followed by dialysis against deionized water for 2 days. The water was completely evaporated to dryness and the crude material was further purified in dichloromethane to remove free PCL-OH fraction. The residue was dissolved in deionized water and the solution was eluted via an ion exchange resin to replace the quaternary ammonium ion with sodium ions. The solution was concentrated under vacuum on a rotary evaporator and lyophilized to give a dry powder of HA-g-PCL.

Characteristics of HA-g-PCL micelles

The ¹ H NMR spectrum of the HA-g-PCL micelles is shown in Figure 2A. The peak of amide-CH ₃ (amide-CH ₃ ) of HA was defined as δ = 1.9, and the terminal -CH _{3 of} PCL was defined as δ = 0.76. The PCL grafting ratio on the HA-g-PCL copolymer is based on the ratio of the HA terminal guanamine-CH ₃ peak to the PCL terminal-CH ₃ peak relative intensity in the ¹ H NMR spectrum, which is about 6 to 8%. The infrared spectrum of HA-g-PCL is shown in Figure B. It has an absorption peak at 1638 cm ^{-1 and} is a C=O carboxyl amide I group of HA. Another peak at 1727 cm ^-1 is the ester bond of PCL. The tin content of the copolymer was determined to be about 5 ppm via inductively coupled plasma mass spectrometry (ICP-MS). The Critical Micelle Concentration (CMC) of the aqueous dispersion was determined to be 4 μg/mL (as shown in Figure 2C). The TEM image shows that the micelles are spheres with a diameter of approximately 70 ± 30 nm (second D map). The microfluidic has a hydrodynamic diameter of approximately 186 nm and a surface potential of approximately -50 mV. The surface potential of the HA-g-PCL micelles showed that it was stably dispersed in an aqueous solution. The pH of the dispersion of HA-g-PCL micelles is about 7 (neutral). Under these conditions, the micelles can sustain their stability for up to 1 month (second E map). The stability of the microcells will decrease in an acidic environment, especially in an environment where the pH is less than 3 (second F map).

When measuring the particle size, different measurement methods may yield different results. For example, a microcell uses a multiangle static light scattering to measure an Rg of 22 nm, so the rotation diameter is 44 nm. The diameter of the same microcell measured by dynamic light scattering is 186 nm, and the difference between the two is due to the different definitions of the hydration radius.

Cytotoxicity of HA-g-PCL micelles

Murine L929 skin fibroblasts were used for cell culture studies. The cells were cultured in the presence of 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 1% penicillin-streptomycin-amphotericin B, and 1.5 g/L sodium bicarbonate. Dextrose Dulbecco's modified Eagle medium (DMEM) was incubated at 37 ° C in a humidified, 5% CO ₂ atmosphere. The survival rate of cells exposed to HA-g-PCL micelles was evaluated by the MTT method, wherein MTT was 3-(4,5-dimethylpyridin-2-yl)-2,5-diphenyl bromide (3). -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The cells were seeded in a 24-well plate (in 1 mL of medium) at a density of 5 x 10 ⁴ cells/well and cultured to a plate. After 24 hours, the cells were treated with 1 mL of medium containing different concentrations (0-2000 μg/mL) of HA-g-PCL. The control group was treated only with medium. After 24 hours of treatment, the MTT solution was added to each well and cultured for 4 hours. The purple crystals were dissolved in DMSO and the absorbance at a wavelength of 550 nm was examined.

The third panel shows the morphology and survival of L929 cells co-cultured with HA-g-PCL micelles for 24 hours. In the presence of micelles, the cells maintained a morphology similar to the control group and the cell concentration was up to 2000 μg/mL. When the concentration of micelles was below 1500 μg/mL, the effect of micelles on cell viability was not obvious. However, at a cell concentration of 2000 μg/mL, cell viability decreased to approximately 85% (as shown in Figure B). Based on these data, the micelles were not cytotoxic at concentrations below 1500 μg/mL. These concentrations are much higher than the cell concentration (i.e., 100 μg/mL) used to encapsulate FGF1 in the present invention.

Embodiment 2 Grafting or physical loading of FGF1 onto HA-g-PCL micelles by EDC (1) EDC chemical grafting

Three different concentrations of aqueous solutions (0.05 mg/mL, 0.1 mg/mL, and 0.5 mg/mL) were prepared by cross-linking agent EDC, and 1 mL of EDC aqueous solution was mixed with 1 mg of HA-g-PCL powder and ultrasonically oscillated. Half an hour to activate the carboxyl group of HA-g-PCL. After shaking, the mixture was centrifuged at 14,000 rpm for 15 min at 4 ° C, and the supernatant was removed and 1 mL of FGF1 solution (100 μg/mL) was added to disperse the micelles and complete the grafting reaction. FGF1 microtubule generation and groups with different EDC concentrations were named EDC-L (EDC concentration 0.05 mg/mL), EDC-M (EDC concentration 0.1 mg/mL), and EDC-H (EDC concentration 0.5 mg) /mL).

(2) Conventional physical methods

In addition, FGF1 was loaded onto the micelles by three conventional physical methods: sonication, lyophilization or electrospraying as a control group:

1. Ultrasonic shock method: 1 mg of HA-g-PCL and FGF1 (100 μg/1 mL) were vortexed for 30 seconds with ultrasound, and then placed overnight at 4 °C to form FGF1-loaded micelles.

2. Freeze vacuum drying method: FGF1 solution (100 μg/deionized water 0.1 mL) was dropped into 1 mg HA-g-PCL powder, the mixture was vortexed with ultrasonic waves and placed in a freeze dryer (FDU-1200, Eyela, Tokyo, Japan). The lyophilized powder was further dissolved in 1 mL of deionized water to form micelles.

3. Electrostatic spray method: The solution was prepared by the steps of ultrasonic vibration method, and then electrostatically sprayed from the tip of the needle at a rate of 200 μL/min with a voltage of 20 kV, and the nozzle was 10 cm away from the collection point.

The microcapsules were centrifuged at 14,000 rpm for 15 minutes at 4 ° C in an aqueous solution containing non-associated FGF1, and the loading ratio was calculated. The amount of FGF1 in the supernatant was assessed by protein quantitative analysis (Bradford assay). In protein quantification, Coomassie Brilliant Blue G-250 (Bio-Rad, Richmond, VA, USA) was used to bind the protein in the supernatant and further via a microplate spectrometer The absorbance at a wavelength of 595 nm was measured, and the results are shown in Table 1 and Table 2. The loading rate was calculated by the following formula: load ratio = [(total FGF1-supernatant FGF1) / total FGF1] x 100%.

Table 1 Loaded FGF1 micelles prepared by EDC chemical grafting method

The particle size, surface potential, and protein quantitative analysis (Bradford assay) of the loaded FGF1 micelles produced by the various methods described above were evaluated as shown in Table 1 and Table 2. The values of particle size, surface potential, and loading rate of loaded FGF1 micelles prepared by EDC grafting varied with EDC concentration. The surface electric power is between -39.6~-43.1mV, but the measured surface potential values will be slightly different due to different measuring instruments, and may differ by about 10mV. Therefore, the surface of the loaded FGF1 micelle prepared by EDC grafting in this case Electricity is available from -29 to -54 mV. When a higher concentration of EDC is utilized (eg, 0.5 mg/mL, EDC-H group), the surface potential of the FGF1 micelles is slightly increased. At the same time, the micelles are more compact (ie smaller particle size). The higher the concentration of EDC, the higher the loading rate of FGF1. The FGF1 loading rates of the EDC-L, EDC-M, and EDC-H groups were approximately 61%, 87%, and 92%.

In addition, a 1.5 μg repeated loading rate experiment of total FGF1/mg HA-g-PCL was carried out, in which the supernatant FGF1 was analyzed by an enzyme-linked immunosorbent assay (ELISA). The human FGF1 immunoassay kit (Antigenix, Huntington, NY, USA) was used to detect the absorbance of proteins in the supernatant at a wavelength of 450 nm. FGF1 micro-prepared by ultrasonic oscillating method, freeze vacuum drying method and electrostatic spray method The loading rate of the cells was about 32%, 48%, and 30% as measured by ELISA, respectively, while the EDC-M of the present invention was 85%, which was much higher than the coverage of physical packaging of FGF1, and EDC. The loading rate of the loaded FGF1 micelles was 90 μg/mg or more, while the maximum loading of FGF1 of EDC-M cells was above 175 μg/mg.

In vitro FGF1 release curve

In a short-term release experiment, 100 μg of FGF1 was loaded onto 1 mg of HA-g-PCL in 1 mL of deionized water. The loaded FGF1 micelles were divided into EDC-L, EDC-M, EDC-H, and ultrasonic shock method, freeze vacuum drying method, and electrostatic spray method through the aforementioned different EDC concentrations as a control group. FGF1 freely suspended in the solution was separated from the aqueous medium by centrifugation. The micelles loaded with FGF1 were redispersed in 1 mL of deionized water and maintained at 37 °C. Remove 1 mL of supernatant at the appropriate time and add 1 mL of fresh deionized water. The amount of FGF1 suspended in the supernatant at each time point was determined by the protein quantitative method (Bradford assay).

The concentration curve of the cumulative release of FGF1 micelles in a physical manner (ultrasonic shock method, freeze vacuum drying method, and electrostatic spray method) is shown in FIG. 4A. The micelles that physically encapsulate FGF1 have a 40-60% burst release in the first 20 hours. After 48 hours, the amount of FGF1 released by the micelles was very small and almost undetectable. The micelles loaded with EGF grafted with FGF1 had only 5-40% burst release in the first 20 hours, while the release profile of this example was as shown in Figure 4B, and the burst was about 20-35%. Amount, compared with the cells that physically wrap FGF1, the release profile For a lot of relaxation, the phenomenon of sudden release is greatly reduced.

In addition, as the EDC dosage was from low (0.05 mg/mL) to moderate (0.1 mg/mL), the release rate of FGF1 decreased. Further increase in EDC dosage from medium (0.1 mg/mL) to high (0.5 mg/mL) did not affect the release rate of FGF1. As a result of ELISA, the initial amount of FGF1 in the EDC-M group was about 1270 ng per milligram of HA-g-PCL. The loading of FGF1 on the micelles was additionally confirmed by Western blotting (as shown in Figure 4C). Based on the band strength, the loading of micelles loaded with FGF1 (EDC-M group) grafted with 0.1 mg/mL of EDC was about 1174 ng, which was higher than the group of micelles loaded with FGF1 without EDC grafting (w /o EDC, the table does not add EDC group, this is the ultrasonic oscillating method group).

Considering the load rate and reducing the occurrence of bursts, the EDC-M group was selected for long-term release testing. In a long-term release experiment, 1.5 μg of FGF1 was loaded onto 1 mg of HA-g-PCL in 1 mL of deionized water. The FGF1 micelles prepared by the EDC-M group and the ultrasonic vibration method were used as a control group. The loading and release were determined by ELISA. The initial load on the micelles and the amount of FGF1 remaining after 21 days were also verified by Western blotting. The amount of FGF1 on the micelles was determined based on the band intensities and the FGF1 standard.

Figure 5A shows the ELISA results from the EDC-M long-term release experiment. FGF1 was released from EDC-M at the same rate (slope was 0.0159% per hour). The micelles that physically encapsulated FGF1 released very little after 5 days (w/o EDC, ultrasonic shock group). Western blot results The fifth B plot shows that the molecular weight of FGF1 in the EDC-M group was not changed after 21 days compared with the FGF1 control group. After 21 days, physically packaged In the coated micelles, the actual amount of FGF1 remaining in the micelles was approximately 450 ng (HA-g-PCL per milligram), while the EDC-M micelles were approximately 762 ng (per milligram of HA-g-PCL).

Microcell-loaded FGF1 biological activity

To verify the biological activity of FGF1, the activity of loaded FGF1 micelles on fibroblasts was determined by MTT assay. L929 fibroblasts were implanted into a 24-well plate of general medium at a density of 2 x 10 ⁴ cells/well for 24 hours. The medium was then changed to contain different concentrations of FGF1 (0-10 ng/mL) or the first release test day 0 (control group), or about 20 μg of loaded FGF1 micelles after 21 days. All groups were tested in four replicates. Considering the load rate and reducing the occurrence of burst, the EDC-M group was selected for bioactivity testing.

To verify the activity of FGF1 on the micelles after 21 days, the effect of FGF1-loaded micets on fibroblast proliferation was compared to native FGF1 at a standard concentration of 0-10 ng, as shown in Figure 6. EDC-M micelles continued to stimulate fibroblast proliferation after 21 days of release. The cell viability after 21 days of treatment with EDC-M micelles relative to the control group was 120%. The cell viability of the micelles encapsulating FGF1 via the physical side was only slightly increased (about 106%) at 21 days of culture.

Embodiment 3 Animal wound healing effect of micelles loaded with FGF1

All animal experiments were ethical and approved by the National Taiwan University Laboratory Animal Care and Use Committee. 200-250 g Sprague Dawley (SD) rats (purchased from Lesco Biotech Co., Ltd.) were anesthetized with a mixture of oxygen and isoflurane, and 1.5 x 1.5 cm ^{2 was} cut from the back of the rat. The skin forms a wound. The effect of micelles loaded with FGF1 on the wound was evaluated. The wound area is covered with a band-aids (band group) or a vasing patch. Add one of the following ingredients: 1.5 ng FGF1 is dispersed in 50 μL PBS (natural FGF1), 50 μg HA-g-PCL is dispersed in 50 μL of PBS (naked cells) and loaded FGF1 micelles (50 μg of micelles and 1.5 ng of FGF1, EDC-M) were dispersed in 50 μL of PBS and changed every two days.

The contraction of the wound was recorded every 2 days. On day 8, the rats were sacrificed with CO ₂ and the skin tissue of the back was removed. The removed tissue was then fixed in 4% formaldehyde for one day and then rinsed with PBS and embedded in paraffin after dehydration. The embedded samples were sliced and stained with hematoxylin-eosin (H&E) and placed under an upright microscope. The ratio of the area of blood vessels and sebaceous glands per square millimeter of the fresh part of the wound was calculated by ImageJ software.

As shown in Figure AA, wounds treated with FGF1-loaded micelles (EDC-M) remained moist and the wound area decreased over time. The group treated with FGF1-loaded micelles (EDC-M) had an optimal recovery rate of 82% after 8 days. The recovery rate of the group treated with native FGF1 and naked micelles was similar, about 76%. The group treated with the band-aid had a 68% wound recovery rate. Eight days of wound histology showed that FGF1, nude cells, and FGF1-loaded micelles caused more microangiogenesis than the untreated control group, as shown in Figure B. The average number of microvessels produced by the FGF1-loaded mice was three times higher than that of the untreated control group, as shown in Figure 7C. The micelles loaded with FGF1 had more sebaceous gland tissue around the regenerated skin than the other three groups. Average sebum per square millimeter of area of the microcapsules loaded with FGF1 The number of glands is greater than 50% compared to the designer or nude cell group, as shown in Figure 7E.

Animal experiment with FGF1 wound treatment, using Tegaderm as a comparison

200-250 g Sprague Dawley (SD) rats were anesthetized with a mixture of oxygen and isoflurane, and 1.5 x 1.5 cm ² of skin was cut to form a wound. A gel film (Gel for short) is 4 mL of gelatin-poly(γ-glutamic acid (γ-PGA) solution (10% type A bloom 300 gelatin and 1% γ) -PGA) and 400 μL of 1.7% EDC (prepared according to Biorheology 2007; 44:17-28) applied to the wound area. To prepare a gel containing FGF1, free FGF1 (400 μg) or FGF1-loaded micelles (EDC-M group, 2 mg HA-g-PCL nominally 400 μg FGF1, or actual load about 350 μg FGF1), A gelatin-y-PGA solution (4 mL) was added before adding 400 μl of EDC. The colloidal film was cut to a size of 1.5 x 1.5 cm ² to cover the wound, as shown in Figure 8A. Each gel film has a dry weight of 6.5 mg, containing 15 μg of free FGF1 ("Gel+FGF1") or 88.1 μg of FGF1 (ie 75 μg HA-g-PCL micelles and 13.1 μg FGF1; Referred to as "Gel+FGF1-micelles"). In addition, Tegaderm was used as a control group, which is a waterproof and breathable dressing produced by 3M Company.

The effect of adding a wound dressing loaded with FGF1 micelles is shown in Figure 8. All wounds contracted at a similar rate, as shown in Figures 8 and C. Wound histology on day 8 showed that both FGF1 and FGF1-loaded micelles (in the gelatin membrane) caused more microvascular formation, as shown in Figures 8 and E. On the other hand, Tegaderm or film without FGF1 shows Less angiogenesis. Loaded FGF1 micelles are similar to native FGF1 (buried in glue) in wound healing. This result shows that loading of FGF1 micelles can be combined with wound dressings to enhance wound healing.

Generally, in a neutral environment, the electrical properties of the microcells and growth factors are negative and weakly attractive to each other; although the formation of microvesicles may cause a small number of growth factors to adsorb or produce entanglement, there is no specific bond. Under the structure, the efficiency of coating can be low, and the control group of the present case uses the direct mixing coating, and the efficiency is also very poor. In addition, generally, the method of treating growth factors will use no cross-linking agent as much as possible, except that in the process of production, the step of washing away the cross-linking agent is increased, and more importantly, the cross-linking agent causes degeneration of growth factors and loss of activity. No growth factors were detected.

Thus, the present invention, via the above examples, proposes novel HA-g-PCL micelles that can be used to efficiently load high amounts of growth factors. FGF1 was loaded onto the EDC grafting reaction to have a high loading rate (>85%) on the micelles and a less pronounced burst release than the loaded FGF1 micelles made by other methods. In vitro, FGF1 loaded by EDC grafting continued to be released from the micelles and continued to be released even after 21 days. The FGF1-loaded HA-g-PCL micelles after 21 days of release were collected and in vitro experiments showed that FGF1 on HA-g-PCL micelles still has the ability to stimulate fibroblast proliferation. The molecular weight of FGF1 in the micelles did not change after more than 21 days of release, indicating that HA-g-PCL micelles should protect FGF1 from hydrolysis. Both naked microvessels and loaded FGF1 micelles have a role in promoting wound contraction in a rat skin wound model.

Claims (20)

A polymer microcell having a high growth factor loading ratio, comprising a hydrophilic main chain, a hydrophobic segment, and a crosslinking agent, wherein the growth factor is grafted to the polymer microcell via a crosslinking agent, And the crosslinking agent is carbodiimide; wherein any of the hydrophilic main chain and the hydrophobic segment has a group of -COOH. The polymer microcapsule according to claim 1, wherein the hydrophilic main chain is hyaluronic acid (HA), methoxy polyethylene glycol (mPEG), or polyglutamic acid. (poly-γ-glutamic acid, PGA). The polymer microcapsule according to claim 1, wherein the hydrophobic main chain is polycaprolactone (PCL), polyvalerolactone (PVL), polylactic acid-glycolic acid ( Poly(lactide-co-glycolide), PLGA), polylactic acid (PLA), polybutyro-lactone (PBL), polyglycolide or polypropiolactone (PPL). The polymer microcapsule according to claim 1, wherein the carbon diamine is 1-ethyl-3-(3-dimethylaminopropyl)carbodiamine (1-ethyl-3- (3-dimethylaminopropyl)carbodiimide, EDC). The polymer microcell as described in claim 1, wherein the growth The load factor of the factor is 50~99%. The polymer microcapsule according to claim 1, wherein the growth factor loading of the polymer microcapsule is 90 μg/mg or more. The polymer microcapsule according to claim 1, wherein the polymer microcell has only 5 to 40% of the growth factor release amount in the first 20 hours. The polymer microcapsule according to claim 1, wherein the growth factor is Fibroblast growth factor 1 (FGF1). The polymer microcapsule according to claim 8, wherein the high loading ratio of the FGF1 is 61% to 92%. The polymer microcapsule according to claim 8, wherein the maximum loading amount of the FGF1 of one of the polymer micelles is 175 μg/mg or more. The polymer microcapsule according to claim 8, wherein the polymer microcell has only 20 to 35% of the growth factor burst release amount in the first 20 hours. A method for producing a polymer microcapsule according to claim 1, comprising the steps of: a. polymerizing a hydrophilic monomer as a main chain with a hydrophobic monomer to form a hydrophilic backbone; a copolymer of a hydrophobic segment; wherein the hydrophilic backbone, any one of the hydrophobic segments has a group of -COOH ^- ; b. activating the copolymer in the presence of a crosslinking agent a carboxyl group, wherein the crosslinking agent is carbamine; and c. crosslinking a growth factor to the copolymer via the crosslinking agent to form a growth factor-loaded polymer micelle. The manufacturing method according to claim 12, wherein the ratio of the crosslinking agent to the growth factor is between 0.5 and 5. The manufacturing method according to claim 12, wherein the surface potential of the polymer micelle is -29 mV to -60 mV. The manufacturing method according to claim 12, wherein the polymer micelle has a diameter of 30 to 220 nm. The manufacturing method according to claim 12, wherein the polymer micelle has a pH of about 7 and can maintain its stability for one month. The production method according to claim 12, wherein the carbamine is 1-ethyl-3-(3-dimethylaminopropyl)carbodiamine. A polymer microcell for use in a growth factor, wherein the polymer microcell is a polymer microcell as described in any one of claims 1 to 4. A dressing for promoting wound healing, comprising the polymer micelle and a substrate according to any one of claims 1 to 4. A dressing for promoting wound healing as described in claim 19, wherein the substrate is a cotton cloth or a gel.