CN111569139A - Platelet lysate-loaded self-supporting self-assembled multilayer film and application thereof - Google Patents

Abstract

The first aspect of the invention provides a self-supporting self-assembled multilayer film loaded with platelet lysate, which comprises platelet lysate; and a self-supporting self-assembled multilayer film loaded with platelet lysate. The invention combines the platelet lysate and the self-supporting multilayer film for the first time and is used in the field of wound repair, the self-supporting multilayer film and the platelet lysate are perfectly matched, both the sources and the preparation are simple and convenient, the platelet lysate is directly adsorbed by the film, the change of protein conformation caused by chemical crosslinking can be avoided, and in addition, the platelet lysate can also avoid the defect of sudden release of growth factors caused by direct application along with the slow degradation of the film; in addition, the release (degradation) time can be accurately controlled by regulating the crosslinking degree of the membrane, so that the two membranes are combined to be used for the wound surface to be dressing and have feasibility.

Description

A self-supporting self-assembled multilayer film loaded with platelet lysate and its application

technical field

The invention relates to the field of wound repair, in particular to a self-supporting self-assembled multilayer film loaded with platelet lysate and its application.

Background technique

The repair of large-scale wounds caused by burns, trauma and chronic diseases remains a major clinical challenge. Due to the limited sources of autologous skin for transplantation, patients with large-area full-thickness skin wounds often require the intervention of artificial skin or some bioactive wound dressings. However, the current clinical application of skin materials is expensive, so skin materials that take into account both effectiveness and cost need to be developed urgently. Pathophysiologically, the wound healing process is the result of synergistic promotion of multiple aspects, including cell proliferation, angiogenesis, and extracellular matrix (ECM) deposition. Therefore, an ideal skin substitute not only needs to be easy to prepare and controllable in cost, but also can provide a good microenvironment for the wound to help restore normal blood supply.

In the present invention, we constructed an exponentially growing layer-by-layer (LBL) polyelectrolyte multilayer by alternating deposition of poly-L-lysine (PLL) and hyaluronic acid (HA) by means of pH regulation. Membranes (PEMs), whose degradation was regulated by a cross-linking agent, were bound to platelet lysate (PL) by means of osmosis and electrostatic adsorption, and the PEMs were then characterized and evaluated for their ability to adsorb and release growth factors. The bioactivity of composite PEMs was evaluated by assessing their proliferative, pro-migratory, and pro-angiogenic effects in vitro and examining their wound-healing potential in a rat full-thickness skin defect model.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a self-supporting self-assembled multilayer membrane loaded with platelet lysate and its application in view of the deficiencies in the prior art.

For realizing the above-mentioned purpose, the technical scheme that the present invention takes is:

A first aspect of the present invention is to provide a self-supporting self-assembled multilayer film loaded with platelet lysate, including platelet lysate; and a self-supporting self-assembled multilayer film loaded with platelet lysate.

Preferably, the self-supporting self-assembled multi-layer film is constructed by layer-by-layer self-assembly technology on the base material by alternating deposition of electrostatic attraction.

Preferably, in the self-supporting self-assembled multilayer film, polylysine and hyaluronic acid are respectively selected as polymer materials with positive and negative charges, and the selection of the last layer in the construction of the self-supporting self-assembled multilayer film It is realized by computer simulation method.

Preferably, the self-supporting self-assembled multilayer film is a polylysine-(hyaluronic acid-polylysine) _n -hyaluronic acid film.

Preferably, 5≤n≤10 in the self-supporting self-assembled multilayer film.

Further preferably, n=7 in the supported self-assembled multilayer film.

Preferably, the base material is polytetrafluoroethylene.

Preferably, the self-supporting self-assembled multilayer film loaded with platelet lysate is prepared by the following method:

S1. Select polytetrafluoroethylene as the base material, and ultrasonically treat them in ethanol, acetone, and water for 15 minutes, respectively, and then dry them with a nitrogen stream;

S2. Alternately immerse the dried base material in polylysine solution of 1-5mg/mL, pH 9-10 and hyaluronic acid solution of 1-5mg/mL, pH 2.5-3.5, and alternately in Rinse in water with corresponding pH value, blow dry until 5-10 double layers are obtained, and the last layer is a self-supporting self-assembled multilayer film of hyaluronic acid layer;

S3. The self-supporting self-assembled multilayer film obtained in S2 was prepared in a solution containing 9-13 mg/mL of N-hydroxysulfosuccinimide and 25-35 mg/mL of 1-ethyl-3-[3-dimethyl The combined solution of aminopropyl]carbodiimide hydrochloride was incubated overnight at 0-4°C for cross-linking;

S4. Immerse the S3 cross-linked self-supporting self-assembled multilayer film in five-fold concentrated platelet lysate overnight to load the platelet lysate, then rinse with water for 3-5 times, and freeze-dry for 4-8 hours.

Further preferably, the self-supporting self-assembled multilayer film loaded with platelet lysate is prepared by the following method:

S1. Select polytetrafluoroethylene as the base material, and ultrasonically treat it in ethanol, acetone and water for 15 minutes, and then dry it with a nitrogen stream;

S2. Alternately immerse the dried substrate material in 1 mg/mL polylysine solution with pH 9.5 and 3 mg/mL hyaluronic acid solution with pH 2.9, and alternately rinse in water with corresponding pH value, blow Dry until 8 double layers are obtained, and the last layer is a self-supporting self-assembled multilayer film of a hyaluronic acid layer;

S3. The self-supporting self-assembled multilayer film obtained in S2 was prepared in a solution containing 11 mg/mL of N-hydroxysulfosuccinimide and 30 mg/mL of 1-ethyl-3-[3-dimethylaminopropyl] Incubate overnight at 4°C in a combined solution of carbodiimide hydrochloride for cross-linking;

S4. The S3 cross-linked self-supporting self-assembled multilayer film was immersed in five-fold concentrated platelet lysate overnight to load the platelet lysate, then washed with water 3 times, and freeze-dried for 4 hours.

Preferably, the preparation step of the platelet lysate comprises:

S4-1. Collect platelet-rich plasma from 50 ml of venous blood from each of 40 healthy donors by two centrifugation methods;

S4-2, freeze and thaw platelet-rich plasma (PRP) for three cycles to destroy the platelet membrane, thereby releasing growth factors;

S4-3. After the liquid obtained in S4-2 is centrifuged to remove the precipitate, the supernatant of the platelet lysate is obtained, freeze-dried, and reconstituted with physiological saline that is one-fifth of the original solution to obtain five times the amount of the original solution. Concentrated platelet lysate.

Preferably, the rotation speed and time of the two centrifugal methods are 160g, 10min and 250g, 15min in sequence.

The second aspect of the present invention is to provide the application of the self-supporting self-assembled multilayer membrane loaded with platelet lysate in the field of wound repair as described above.

The present invention adopts the above technical scheme, compared with the prior art, has the following technical effects:

The invention combines platelet lysate and self-supporting multi-layer membrane for the first time and is used in the field of wound repair. The self-supporting multi-layer membrane and platelet lysate are a perfect match, both of which are easy to source and prepare, and platelet lysis is directly adsorbed through the membrane. In addition, with the slow degradation of the membrane, the platelet lysate can also avoid the drawbacks of direct application of growth factor burst release; in addition, by regulating the degree of membrane crosslinking, the The time of release (degradation) is controlled, so it is very feasible to combine the two for wound dressings.

The construction of the self-supporting multilayer film of the present invention is a process of continuous circulation of positively and negatively charged molecules A-B-A-B-..., so it is not clear whether it is better to let A be the last layer or B be the last layer. Molecular simulations A and B are the affinity of polylysine and hyaluronic acid with the main growth factors (PDGF, TGF, bFGF) in platelet lysate, and it is calculated that hyaluronic acid as the last layer can have a positive effect on the three growth factors. Better Affinity, a methodological innovation that uses simulation for the first time instead of screening of materials.

Description of drawings

Fig. 1 is the wound healing percentage result of the self-supporting self-assembled multilayer film of the present invention;

Fig. 2 is the collagen fiber deposition rate result of the self-supporting self-assembled multilayer film of the present invention;

Fig. 3 is the blood perfusion volume result of the self-supporting self-assembled multilayer film of the present invention;

Fig. 4 is the blood vessel volume result of the self-supporting self-assembled multilayer film of the present invention;

Figure 5 is the result of the number of blood vessels of the self-supporting self-assembled multilayer film of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

It should be noted that the embodiments of the present invention and the features of the embodiments may be combined with each other under the condition of no conflict.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it is not intended to limit the present invention.

Example 1

This embodiment provides a method for preparing a self-supporting self-assembled multilayer film loaded with platelet lysate.

The final layers of the self-supporting self-assembled multilayer films were determined by molecular modeling methods. The structural formulas of the HA and PLL repeating units were drawn by ChemBioDraw software and converted to 3D format by ChemBio3D. The 3D structural formulas of PDGF-BB (PDB ID: 4QCI), TGF-β1 (PDB ID: 1KLC) and bFGF (PDB ID: 1CVS) were downloaded from the PDB website (https://www.rcsb.org/), and used PyMOL software optimized. Molecular docking simulations and subsequent binding affinity calculations were performed using AutoDock Vina software according to published methods. PyMOL generated images of the docking complex conformations at which the polymer repeat unit had the lowest binding energy for each protein and polar bonds formed between the polymer and surrounding amino acid residues, and picked out such conformations for further use molecular dynamics analysis. Hydrogen and hydrophobic bonds between molecules and proteins were displayed with Ligplot+ software.

The force field parameters and topology files of the protein molecules were generated using the GROMOS96 43A1 force field and the SPC/E molecular water model by performing further molecular dynamics simulations (MD) using GROMACS software. The force field parameters and topology files for HA and PLL were generated using PRODRG software. Use a box of cubes for the simulation and neutralize the charge with chloride or sodium ions. The bond lengths of the hydrogen atoms were fixed by the SHAKE algorithm, and the electrostatic interactions were calculated using the particle mesh method (PME). Energy minimization of the complex, and equilibration of pressure and temperature. Finally, kinetic simulations for 20 ns were performed at 1 atmosphere pressure and 80.3 °F, with coordinate recordings taken every 100 ps, and the resulting macromolecular and protein complexes were finally represented by calculating the coordinate root mean square deviation (RMSD). structural stability. The results confirmed that the structure stability of the complex formed between HA and corresponding growth factors was better than that of PLL and growth factors.

Teflon was selected as the base material, and ultrasonically cleaned in ethanol, acetone, and water for 15 minutes, respectively, and then blown dry with nitrogen; the dried base material was alternately immersed in 1 mg/mL polylysine, pH 9.5. solution and 3 mg/mL, pH 2.9 hyaluronic acid solution, and alternately rinsed in double-distilled water at the corresponding pH value, and dried until 8 bilayers were obtained and the last layer was a self-supporting self-assembled polyamide layer of hyaluronic acid. layered film, namely EDCO (uncrosslinked); the self-supporting self-assembled multilayer film was prepared in a solution containing 11 mg/mL of N-hydroxysulfosuccinimide and 30 mg/mL of 1-ethyl-3-[3 - Dimethylaminopropyl]carbodiimide hydrochloride combined solution was incubated overnight at 4°C to obtain cross-linked self-supporting self-assembled multilayer films, ie EDC30 (cross-linked).

Preparation of platelet lysate: PRP was extracted, and then concentrated PL was obtained by liquid nitrogen freeze-thaw method. This study was conducted in accordance with the principles of the Declaration of Helsinki, and the research protocol was approved by the Independent Ethics Committee of the Sixth People's Hospital affiliated to Shanghai Jiaotong University (Shanghai, China) for collecting samples and using them in scientific experiments. Each participant signed an informed consent form. Platelet-rich plasma was collected from 50 ml venous blood of 40 healthy adults by double centrifugation (160 g, 10 min and 250 g, 15 min in sequence); the platelet-rich plasma was subjected to three cycles of freezing and thawing in a liquid nitrogen-37°C water bath to destroy the platelet membrane , so as to release its intracellular growth factors to obtain platelet lysate; freeze-dry the obtained platelet lysate, and reconstitute it with physiological saline that is one-fifth of the original solution to obtain a five-fold concentrated platelet lysate. .

The self-supporting self-assembled multilayer membrane was immersed in five-fold concentrated platelet lysate overnight to enrich the platelet lysate in the membrane, rinsed with deionized water 3 times, and freeze-dried for 4 hours to obtain the load. Self-supporting self-assembled multilayer films of platelet lysates, namely EDC0@PL and EDC30@PL.

Detection Example

A total of 25 adult SD rats (age=2 months, body weight=250±15g, n=5 in each group) were used in this example. The experiment was approved by the Animal Experiment Ethics Committee of Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University. The relevant guidelines were strictly followed at each stage of the experiment. The dressings of each group were sterilized with plasma beam radiation before the experiment. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital (2.5%, 30 mg/kg). Following subsequent disinfection of the surgical site with iodophor, a standard circular defect (diameter = 20 mm) was excised in full thickness at 1 cm on the dorsal side of the rat along the paraspinal region by a skilled surgeon. Then, four groups of dressings, EDC0, EDC30, EDC0@PL and EDC30@PL prepared in Example 1, were placed on the skin defect, respectively. The wound was covered with gauze and fixed by needle and thread, and the control group was covered with gauze only without the filling of the dressing.

1.1 Wound closure and histological analysis of defect skin treated with different dressings:

The experimental results are shown in Figure 1. In order to study the effect of the platelet lysate-loaded self-supporting self-assembled multilayer film in vivo, a standard rat full-thickness skin defect model (20mm) was constructed in this example, and the During the experiment, no adverse reactions were observed in each group. The macroscopic images showed that all membrane dressings had good hydrophilicity and could cover the wound well and keep the wound dry. In addition, the process of wound healing after different treatments at 0, 3, 7 and 14 days was recorded. Although the wound size of all five groups decreased over time, the wound size of the EDC0@PL group was smaller than that of the other groups within 3 days. And at 7 days and 14 days, the EDC30@PL group also showed a rather high healing rate. Meanwhile, different from the in vitro study, at day 14, the EDC0 and EDC30 groups alone also showed a certain pro-reparative effect compared with the untreated group, indicating that the multi-layer film dressing without PL loading also contributed to the wound healing. In the early stage of healing (3 days and 7 days), the multi-layer film of uncrosslinked EDC0@PL could repair the wound better than EDC30@PL due to its faster degradation (more growth factors could be released in the early stage).

The wound tissues of each group were collected for H&E staining to further evaluate the quality of wound healing. According to the results of H&E staining, at 7 days, the wounds treated with EDC0@PL and EDC30@PL had more neo-epithelial tissue formation than the other three groups. By day 14, the EDC30@PL group had the most neoplastic epithelium, followed by EDC0@PL, EDC0 and EDC30, and the control group.

1.2 Masson's trichrome staining was performed on the wound tissue to evaluate its collagen deposition:

The results are shown in Figure 2. Compared with the untreated control group, wounds treated with EDC0 and EDC30 membranes, especially PL-loaded EDC0 and EDC30 membranes, showed wavy collagen fibers deposited in the defect area at 7 days. At 14 days, wounds treated with PL-loaded membranes exhibited abundant collagen fibers in an orderly arrangement, which was similar to that of normal skin. This suggests that PL can promote epithelial regeneration and collagen remodeling. In addition, at 14 days, some hair follicles and sebaceous glands were also found in the wound tissue treated with EDC30@PL, which further indicated that the cross-linked membrane (EDC30) was loaded with PL, and through the long-term controlled release of PL, better performance was obtained. treatment effect.

1.3 Assess blood flow and functional vascular formation in wounds treated with different dressings:

As shown in Figure 3-5, the laser Doppler imaging system was used to evaluate the wound blood flow, and the MoorLDIReview software was used to quantify the blood flow results. Compared with the other three groups, the membrane-treated group loaded with PL had better blood supply at the wound surface. The EDC0@PL group had the most abundant blood supply in the early stage (3 and 7 days). However, except for the EDC30@PL group, all other groups showed decreased blood perfusion on postoperative day 14, indicating that the cross-linked membrane could continuously promote blood supply to the wound area through long-term controlled release of PL. In addition, at 14 days, micro-CT scan analysis of vascular perfusion tissue showed that PL-loaded membranes, especially the EDC30@PL group, had more neovascularization.

To sum up, although platelet-rich plasma (PRP) and its derivatives have been widely used in clinical practice, there are still limitations such as burst release of growth factors, rapid in situ degradation, and poor tissue in situ. The invention can effectively solve the limitation of its clinical application by carrying PL through the self-supporting self-assembled multilayer film constructed by the LBL method. The membrane showed significant advantages for GFs enrichment. PL-loaded dressings showed great potential in promoting skin wound healing. Meanwhile, by controlling the degree of cross-linking, the controlled release behavior of PL can be precisely tuned.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the embodiments and protection scope of the present invention. For those skilled in the art, they should be able to realize that all equivalents made by using the description and illustrations of the present invention The solutions obtained by substitutions and obvious changes shall all be included in the protection scope of the present invention.

Claims (10)

1. A self-supporting self-assembled multilayer film loaded with platelet lysate is characterized by comprising platelet lysate; and a self-supporting self-assembled multilayer film loaded with platelet lysate.

2. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 1, wherein the self-supporting self-assembled multilayer film is constructed by alternate electrostatic attraction deposition on a base material by layer-by-layer self-assembly techniques.

3. The platelet lysate-loaded self-supporting self-assembled multilayer film according to claim 2, wherein polylysine and hyaluronic acid are respectively selected as the polymer materials with positive and negative charges in the self-supporting self-assembled multilayer film, and the selection of the final layer in the self-supporting self-assembled multilayer film construction is realized by a computer simulation method.

4. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 3, wherein the self-supporting self-assembled multilayer film is polylysine- (hyaluronic acid-polylysine)_n-a hyaluronic acid membrane.

5. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 4, wherein n is 5. ltoreq. n.ltoreq.10 in the self-supporting self-assembled multilayer film.

6. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 2, wherein the base material is polytetrafluoroethylene.

7. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 1, wherein the platelet lysate loaded self-supporting self-assembled multilayer film is prepared by a method comprising:

s1, selecting polytetrafluoroethylene as a substrate material, respectively carrying out ultrasonic treatment on the polytetrafluoroethylene in ethanol, acetone and water for 15 minutes, and then drying the polytetrafluoroethylene by using nitrogen flow;

s2, alternately immersing the dried substrate material into a polylysine solution with the pH of 9-10 and a hyaluronic acid solution with the pH of 1-5mg/mL and the pH of 2.5-3.5, alternately rinsing in water with corresponding pH values, and drying until 5-10 double layers are obtained, wherein the tail layer is a self-supporting self-assembled multilayer film of a hyaluronic acid layer;

s3, incubating the self-supporting self-assembly multilayer film obtained in the S2 in a combined solution containing 9-13mg/mL of N-hydroxysulfosuccinimide and 25-35mg/mL of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride at 0-4 ℃ overnight for crosslinking;

and S4, immersing the self-supporting self-assembled multilayer film after the S3 crosslinking in quintupling concentrated platelet lysate overnight to load the platelet lysate, then washing the platelet lysate for 3 to 5 times by using water, and freeze-drying the platelet lysate for 4 to 8 hours to obtain the self-supporting self-assembled multilayer film loaded with the platelet lysate.

8. The platelet lysate loaded self-supporting self-assembled multilayer film of claim 7, wherein the platelet lysate is prepared by steps comprising:

s4-1, collecting platelet-rich plasma from 50ml venous blood of each of 40 healthy donors by two-time centrifugation;

s4-2, carrying out three times of circulating freeze thawing on the platelet-rich plasma to destroy platelet membranes so as to release growth factors;

s4-3, centrifuging the liquid obtained in S4-2 to remove precipitates, obtaining a supernatant of the platelet lysate, freeze-drying the supernatant, and redissolving the supernatant by using physiological saline one fifth of the amount of the original solution to obtain the platelet lysate concentrated by five times.

9. The platelet lysate-loaded self-supporting self-assembled multilayer film according to claim 7, wherein the rotation speed and time of the two centrifugation methods are 160g, 10min and 250g, 15min in sequence.

10. Use of a platelet lysate loaded self-supporting self-assembled multilayer film according to any one of claims 1 to 9 in the field of wound repair.

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