CN111569139A - Platelet lysate-loaded self-supporting self-assembled multilayer film and application thereof - Google Patents
Platelet lysate-loaded self-supporting self-assembled multilayer film and application thereof Download PDFInfo
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
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- A—HUMAN NECESSITIES
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/24—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/28—Polysaccharides or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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
Technical Field
The invention relates to the field of wound repair, in particular to a platelet lysate loaded self-supporting self-assembled multilayer film and application thereof.
Background
The repair of large areas of wounds caused by burns, wounds and chronic diseases remains a major clinical challenge. Patients who encounter extensive full-thickness skin wounds often require the intervention of artificial skin or some bioactive wound dressings due to the limited sources of autologous skin available for transplantation. However, the skin materials for clinical application are expensive, so that development of skin materials with both effectiveness and cost is urgently needed. Pathophysiologically, the wound healing process is the result of a synergistic promotion of multiple aspects, including cell proliferation, angiogenesis and deposition of extracellular matrix (ECM). Therefore, an ideal skin substitute not only needs to be easy to prepare, cost-controllable, but also be able to provide a good microenvironment for the wound to help reestablish a normal blood supply.
In the invention, index-growth type polyelectrolyte multilayer films (PEMs) with layer-by-layer self-assembly (LBL) are constructed by alternately depositing and accumulating levo-lysine (PLL) and Hyaluronic Acid (HA) through a pH regulation method, the degradation of the PEMs is regulated and controlled by a cross-linking agent, the PEMs are combined with Platelet Lysate (PL) in a permeation and electrostatic adsorption mode, and then the PEMs are characterized and the capacities of adsorbing and releasing growth factors are evaluated. The biological activity of composite PEMs was evaluated by assessing their proliferation, pro-migration and pro-angiogenesis effects in vitro and their wound healing potential in a rat full-thickness skin defect model.
Disclosure of Invention
The invention aims to provide a platelet lysate-loaded self-supporting self-assembled multilayer film and application thereof, aiming at the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
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.
Preferably, the self-supporting self-assembled multilayer film is built by electrostatic attraction alternating deposition on a base material by a layer-by-layer self-assembly technique.
Preferably, polylysine and hyaluronic acid are respectively selected from the self-supporting self-assembled multilayer film as polymer materials with positive and negative charges, and the selection of the final layer in the construction of the self-supporting self-assembled multilayer film is realized by a computer simulation method.
Preferably, the self-supporting self-assembled multilayer film is polylysine- (hyaluronic acid-polylysine)n-a hyaluronic acid membrane.
Preferably, n is 5 ≦ n ≦ 10 in the self-supporting self-assembling multilayer film.
Further preferably, n is 7 in the supported self-assembled multilayer film.
Preferably, the substrate material is polytetrafluoroethylene.
Preferably, the platelet lysate-loaded self-supporting self-assembled multilayer film is prepared by the following method:
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;
s4, immersing the self-supporting self-assembly multilayer film after S3 crosslinking in quintupling concentrated platelet lysate overnight to load the platelet lysate, then washing 3-5 times with water, and freeze-drying for 4-8 hours.
Further preferably, the platelet lysate-loaded self-supporting self-assembled multilayer film is prepared by the following method:
s1, selecting polytetrafluoroethylene as a substrate material, respectively performing 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 9.5 and a hyaluronic acid solution with the pH of 3mg/mL and the pH of 2.9, alternately rinsing the substrate material in water with the corresponding pH value, and drying the substrate material by blowing until 8 double layers are obtained, wherein the tail layer is the self-supporting self-assembled multilayer film of the hyaluronic acid layer;
s3, incubating the self-supporting self-assembly multilayer film obtained in the S2 in a combined solution containing 11mg/mL of N-hydroxysulfosuccinimide and 30mg/mL of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride overnight at 4 ℃ for crosslinking;
s4, immersing the self-supporting self-assembled multilayer film after S3 crosslinking in platelet lysate concentrated five times overnight to load the platelet lysate, then rinsing 3 times with water, and freeze-drying for 4 hours.
Preferably, the platelet lysate is prepared by the 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 Platelet Rich Plasma (PRP) 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.
Preferably, the rotation speed and time of the two-time centrifugation method are 160g, 10min and 250g, 15min in sequence.
The second aspect of the invention provides an application of the self-supporting self-assembled multilayer film loaded with the platelet lysate in the field of wound repair.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
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.
The construction of the self-supporting multilayer film is a continuous circulating process of positive and negative charge molecules A-B-A-B- … …, so it is unclear, and the last layer of A is made good or the last layer of B is made good.
Drawings
FIG. 1 is a graph of the percent wound healing results for a self-supporting self-assembled multilayer film of the present invention;
FIG. 2 is a graph showing the results of collagen fiber deposition rate of the self-supporting self-assembled multilayer film of the present invention;
FIG. 3 is a perfusion volume result for a self-supporting self-assembled multilayer film of the present invention;
FIG. 4 is a vascular volume result for a self-supporting self-assembled multilayer film of the present invention;
FIG. 5 shows the results of the number of blood vessels in the self-supporting self-assembled multilayer film of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
Example 1
This example provides a method for preparing a platelet lysate loaded self-supporting self-assembled multilayer film.
The final layer of the self-supporting self-assembled multilayer film was determined by molecular simulation methods. The structural formula of the HA and PLL repeat units is plotted by ChemBioDraw software and converted to 3D format by ChemBio 3D. The 3D structural formulae of PDGF-BB (PDB ID: 4QCI), TGF-. beta.1 (PDB ID: 1KLC) and bFGF (PDB ID: 1CVS) were downloaded from the PDB website (https:// www.rcsb.org /), and optimized with PyMOL software. Molecular docking simulations and subsequent binding affinity calculations were performed using AutoDock Vina software according to the disclosed method. The docking complex conformation at which the macromolecule repeat unit has the lowest binding energy for each protein was imaged by PyMOL, the polar bonds formed between the macromolecule and surrounding amino acid residues were generated, and such conformations were selected for further molecular dynamics analysis. Hydrogen and hydrophobic bonds between molecules and proteins were revealed using ligalot + software.
Further molecular dynamics simulations (MD) were performed using GROMOS 9643 a1 force field and SPC/E molecular water model to generate force field parameters and topology files for protein molecules. The force field parameters and topology files for the HA and PLL are generated using PRODRG software. A cubic box was used for the simulation and neutralization of the charge with chloride or sodium ions. The bond length of the hydrogen atoms was fixed by the shift algorithm and the electrostatic interaction was calculated using particle mesh method (PME). Energy minimization and pressure and temperature equilibration of the composite. Finally, a 20ns kinetic simulation was performed at 1 atmosphere and 80.3 ° f, during which coordinate recordings were taken every 100ps and the structural stability of the formed macromolecule and protein complexes was finally characterized by calculating the root mean square deviation of coordinates (RMSD). The results confirm that the stability of the complex structure formed between HA and the corresponding growth factor is superior to that formed by PLL and growth factor.
Selecting polytetrafluoroethylene as a substrate material, sequentially ultrasonically cleaning the polytetrafluoroethylene in ethanol, acetone and water for 15 minutes respectively, and then drying the polytetrafluoroethylene by using nitrogen; alternately immersing the dried substrate material into 1mg/mL polylysine solution with pH of 9.5 and hyaluronic acid solution with pH of 3mg/mL and pH of 2.9, alternately rinsing in double distilled water with corresponding pH values, and blow-drying until 8 self-supporting self-assembled multilayer films with double layers and the tail layer of the hyaluronic acid layer, namely EDC0 (uncrosslinked) are obtained; the self-supporting self-assembled multilayer film was incubated overnight at 4 ℃ in a combined solution containing 11mg/mL of N-hydroxysulfosuccinimide and 30mg/mL of 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride to obtain a crosslinked self-supporting self-assembled multilayer film, namely EDC30 (crosslinked).
Preparation of platelet lysate: PRP was extracted and then concentrated PL was obtained by liquid nitrogen freeze-thaw. The study was conducted according to the principles of the declaration of helsinki and was approved by the independent ethics committee of the sixth national hospital affiliated with the university of shanghai transportation (shanghai, china) for collecting samples and using them in scientific experiments. Each participant signed an informed consent. Platelet rich plasma (160 g, 10min and 250g, 15min in sequence) was collected from 50ml venous blood of 40 healthy adults by two centrifugation; carrying out freeze thawing on the platelet-rich plasma in a liquid nitrogen-37 ℃ water bath for three times of circulation to destroy a platelet membrane, so as to release the intracellular growth factors of the platelet-rich plasma, thereby obtaining a platelet lysate; the obtained platelet lysate was freeze-dried and reconstituted with physiological saline, one fifth of the amount of the original solution, to obtain a platelet lysate concentrated five times.
Immersing the self-supporting self-assembled multilayer film into platelet lysate concentrated by five times overnight to enrich the platelet lysate in the film, then washing the film for 3 times by deionized water, and freeze-drying the film for 4 hours to obtain the self-supporting self-assembled multilayer film loaded with the platelet lysate, namely EDC0@ PL and EDC30@ PL.
Detection examples
A total of 25 adult SD rats (age 2 months, weight 250 ± 15g, each group n 5) were used in this example. The experiment was approved by the animal experiments ethics committee of the sixth national hospital, Shanghai, affiliated Shanghai university of transportation. The relevant guidelines were strictly followed at each stage of the experiment. Prior to the experiment, each group of dressings was sterilized with plasma beam radiation. Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (2.5%, 30 mg/kg). After subsequent sterilization of the surgical site with iodophor, a standard circular defect (20mm in diameter) was dissected in full thickness by a skilled surgeon 1cm lateral to the spinal column on the dorsal side of the rat. Four sets of dressings, EDC0, EDC30, EDC0@ PL and EDC30@ PL, prepared from example 1 were then placed at the skin defect, respectively. The wound was covered with gauze and held by needle thread, and the control group was covered with gauze only without the dressing filled in.
1.1 wound closure and histological analysis of defective skin treated with different dressings:
experimental results as shown in fig. 1, in order to study the effect of platelet lysate loaded self-supporting self-assembled multilayer films in vivo, a standard rat full-thickness skin defect model (20mm) was constructed in this example, and no adverse reaction was observed in each group throughout the experiment. The general image shows that all the film dressings have good hydrophilicity, can well cover the wound surface and keep the wound surface dry. In addition, the course of wound healing was recorded after different treatments on days 0, 3, 7 and 14. While the wound size decreased over time for all five groups, the EDC0@ PL group had a smaller wound size than the other groups within 3 days. While the EDC30@ PL group also showed a relatively high healing rate in 7 days and 14 days. Meanwhile, unlike in vitro studies, at day 14, the EDC0 and EDC30 groups alone also exhibited some restorative effects compared to the untreated group, indicating that the multilayer film dressing not loaded with PL also helped the wound healing. The multilayer film of uncrosslinked EDC0@ PL degraded faster (more growth factors released early) in the early stages of healing (3 and 7 days) and repaired the wound surface better than EDC30@ PL.
And collecting various groups of wound surface tissues for H & E staining, so that the healing quality of the wound surface can be further evaluated. Based on the H & E staining results, at 7 days, EDC0@ PL and EDC30@ PL treated wounds formed more neoepithelial tissue than the other three groups. By day 14, the largest neoepithelium was in the EDC30@ PL group, followed by EDC0@ PL, EDC0 and EDC30 in that order and the control group.
1.2 Masson trichrome staining of wound tissue to evaluate collagen deposition:
as shown in fig. 2, the wounds treated with EDC0 and EDC30 membranes, and in particular PL-loaded EDC0 and EDC30 membranes, showed wavy collagen fibrils deposited in the defect area at 7 days, compared to the untreated control. At 14 days, the wound surface treated with PL loaded membrane exhibited a large number of collagen fibers and was ordered, similar to the collagen arrangement of normal skin. This suggests that PL promotes 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 indicates that the cross-linked membrane (EDC30) carrying PL can achieve better therapeutic effects through long-lasting controlled release of PL.
1.3 assessment of blood flow and formation of functional vessels in wounds treated by different dressings:
as shown in fig. 3-5, the blood flow of the wound was evaluated using a laser doppler imaging system and the blood flow results were quantified using moorlreeview software. The membrane treatment group loaded with PL has better blood supply at the wound surface than the other three groups. The EDC0@ PL group was most abundant in early (3 and 7 days) blood supply. However, all other groups showed reduced blood perfusion at day 14 post-surgery except the EDC30@ PL group, indicating that the cross-linked membrane continuously promoted blood flow to the wound area through the long-acting controlled-release PL. In addition, at 14 days, microct scan analysis of vascular perfused tissues showed that PL loaded membranes, especially the EDC30@ PL group, had more neovascularization.
In summary, although Platelet Rich Plasma (PRP) and its derivatives have been widely used in clinical practice, there are still limitations of burst release of growth factors, rapid in situ degradation, and poor tissue in situ. The invention can effectively solve the limitation of clinical application by carrying PL through the self-supporting self-assembly multilayer film constructed by the LBL method. This membrane showed significant advantages for GFs enrichment. Dressings loaded with PL have shown great potential in promoting healing of skin wounds. At the same time, by controlling the degree of crosslinking, the controlled release behavior of PL can be precisely tuned.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the 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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| CN112107733A (en) * | 2020-09-14 | 2020-12-22 | 上海市第六人民医院 | Temperature-sensitive hydrogel-high-dispersion nanoparticle system for injection type loaded platelet lysate and application of temperature-sensitive hydrogel-high-dispersion nanoparticle system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007140573A1 (en) * | 2006-04-24 | 2007-12-13 | Axcelon Biopolymers Corporation | Nanosilver coated bacterial cellulose |
| CN103702674A (en) * | 2011-06-27 | 2014-04-02 | 爱默蕾大学 | Compositions, uses, and preparation of platelet lysates |
| CN107929810A (en) * | 2017-12-01 | 2018-04-20 | 浙江大学 | A kind of LBL self-assembly film and its preparation method and application |
| CN109153734A (en) * | 2016-03-24 | 2019-01-04 | 斯泰玛特斯，生物技术药物改造有限公司 | Gellan gum hydrogel, preparation, method and application thereof |
| CN110144124A (en) * | 2019-05-07 | 2019-08-20 | 华中科技大学 | A kind of composite material and its preparation and application of quaternized chitin and fibroin albumen |
-
2020
- 2020-05-26 CN CN202010456167.4A patent/CN111569139A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007140573A1 (en) * | 2006-04-24 | 2007-12-13 | Axcelon Biopolymers Corporation | Nanosilver coated bacterial cellulose |
| CN103702674A (en) * | 2011-06-27 | 2014-04-02 | 爱默蕾大学 | Compositions, uses, and preparation of platelet lysates |
| CN109153734A (en) * | 2016-03-24 | 2019-01-04 | 斯泰玛特斯，生物技术药物改造有限公司 | Gellan gum hydrogel, preparation, method and application thereof |
| CN107929810A (en) * | 2017-12-01 | 2018-04-20 | 浙江大学 | A kind of LBL self-assembly film and its preparation method and application |
| CN110144124A (en) * | 2019-05-07 | 2019-08-20 | 华中科技大学 | A kind of composite material and its preparation and application of quaternized chitin and fibroin albumen |
Non-Patent Citations (2)
| Title |
|---|
| QIAN TANG ET AL: "A free-standing multilayer film as a novel delivery carrier of platelet lysates A free-standing multilayer film as a novel delivery carrier of platelet lysates", 《BIOMATERIALS》 * |
| 曾戎，屠美编著: "《生物医用仿生高分子材料》", 31 October 2010, 华南理工大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112107733A (en) * | 2020-09-14 | 2020-12-22 | 上海市第六人民医院 | Temperature-sensitive hydrogel-high-dispersion nanoparticle system for injection type loaded platelet lysate and application of temperature-sensitive hydrogel-high-dispersion nanoparticle system |
| CN112107733B (en) * | 2020-09-14 | 2024-02-23 | 上海市第六人民医院 | Temperature-sensitive hydrogel-high-dispersion nanoparticle system of injection type platelet lysate loaded and application thereof |
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