CN115970059A - Compositions comprising elastin-like polypeptides and acellular dermal matrix and uses thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition containing elastin-like polypeptides and an acellular dermal matrix and application thereof. The compositions provided herein include elastin conserved sequences and multiple polypeptides that improve stability; the provided polypeptide is basically homologous or similar to a part of mammalian pig and human elastin and has good biological safety. Has remarkable capacity of promoting the production of skin elastin and angiogenesis; can improve the skin filling effect and has obvious synergistic effect; the provided polypeptide sequence is shorter and more stable; the whole polypeptide production process is short in time consumption, low in cost and easy to operate, is easy to realize large-scale production when being used as a three-dimensional soft tissue filler, has important commercial value in the aspects of medical and American product research and development, and has wide clinical application prospect.

Description

Compositions comprising elastin-like polypeptides and acellular dermal matrix and uses thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a composition containing elastin-like polypeptides and an acellular dermal matrix and application thereof.

Background

Porcine Acellular Dermal Matrix (PADM) is a dermal biomaterial that removes all cellular components. As a porcine-derived biological mesh, it is very common in clinical wound healing treatment. Since PADM plays a very similar role in promoting fibroblast, collagen and angiogenesis as human Acellular Dermal Matrix (ADM), it is often used to replace human ADM for clinical applications. PADM induces M2 macrophage polarization by stimulating collagen synthesis, stem cell proliferation and differentiation, and expression of associated growth factors, and releases a series of wound healing factors including Matrix Metalloproteinases (MMPs) and growth factors, promoting cell proliferation and angiogenesis, while remodeling the extracellular matrix (ECM).

Elastin is the main component of elastic fibres in animal tissues. Elastin is present in most connective tissues along with collagen and polysaccharides. In normal mammalian skin, especially human skin, the proportion of elastin proteins in the total skin protein is not very high, but plays a very important role in maintaining the tone, structure and elasticity of the skin. Elastin fibers are capable of stretching to several times their length and then rapidly return to their original dimensions after the tension is released. Thus, elastin contributes to the physiological elasticity of tissue. However, elastin does not penetrate skin sufficiently to provide substantial benefits, since its tightly cross-linked structure makes it difficult to dissolve. With the improvement of living standard, the most demand for beauty becomes a problem to be solved urgently. At present, no report about the application of elastin in acellular dermal matrix is found.

Disclosure of Invention

In view of the above, the present invention provides a composition comprising an elastin-like polypeptide and an acellular dermal matrix, and uses thereof. So that the composition has a synergistic effect, and the activity of promoting the generation of elastin and regenerating blood vessels is obviously increased; and has no acute or chronic toxic effect, and has wide clinical application value and prospect.

In order to achieve the above purpose, the invention provides the following technical scheme:

in one aspect, the invention provides a composition comprising an elastin-like polypeptide and an acellular dermal matrix, the composition comprising an elastin-like polypeptide and an acellular dermal matrix; the polypeptide comprises one or more of the amino acid sequences shown in Seq _ 1-12.

Preferably, the elastin-like polypeptide includes one or more of the amino acid sequences set forth in Seq _1, 3, 4, 5, 9, or/and 10.

The composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In another aspect, the present invention also provides a method for preparing an acellular dermal matrix, comprising the steps of:

(1) Cleaning, cutting off subdermal adipose tissue and removing hair;

(2) Removing epidermis of skin with 0.25% trypsin solution, chopping dermis, further placing into 0.25% trypsin solution for treatment reaction, and cleaning with 0.1% sodium dodecyl sulfate solution;

(3) Treating the cleaned skin with Dispase solution, washing with 0.1% sodium dodecyl sulfate solution, washing with phosphate buffer solution, pulverizing, and sterilizing with electron beam.

In another aspect, the present invention also provides the use of a composition as described above for the preparation of a micro-profiled material.

Preferably, the material is a soft tissue filler.

The invention also provides an elastin-like polypeptide comprising an amino acid sequence as set forth in Seq _1, seq _2, seq _3, seq _4, seq _5, seq _6, seq _7, seq _8, seq _9, seq _10, seq _11 or Seq _ 12.

Preferably, the polypeptide comprises an amino acid sequence as set forth in Seq _1, seq _3, seq _4, seq _5, seq _9 or Seq _ 10.

The invention also provides a pharmaceutical composition comprising an elastin-like polypeptide comprising one or more of the amino acid sequences set forth in Seq _1, seq _3, seq _4, seq _5, seq _9, or Seq _ 10.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

Compared with the prior art, the invention has the beneficial effects that:

(1) The compositions of the invention include elastin conserved sequences and multiple polypeptides that improve stability; the polypeptide of the invention is basically homologous or similar to a part of elastin of mammal pigs and human, and has good biological safety.

(2) The polypeptide provided by the invention has remarkable capacity of promoting the production of skin elastin and angiogenesis; the provided PADM and polypeptide composition has the capability of remarkably improving the skin filling effect and has an obvious synergistic effect;

(3) The polypeptide provided by the invention has shorter and more stable sequence; the whole polypeptide production process is short in time consumption, low in cost and easy to operate, is easy to realize large-scale production when being used as a three-dimensional soft tissue filler, has important commercial value in the aspects of medical and American product research and development, and has wide clinical application prospect.

Drawings

FIG. 1 is a graph of the ability of a polypeptide to produce elastin;

FIG. 2 is a graph of the ability of different concentrations of polypeptide compositions to produce elastin;

FIG. 3 is a graph of filling results 10 days after implantation of different materials;

FIG. 4 is a graph of filling results 30 days after implantation of different materials;

fig. 5 is a graph of HE staining of implants of the PADM + polypeptidyl.

Detailed Description

The invention discloses a composition containing elastin-like polypeptide and acellular dermal matrix and application thereof. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.

Example 1: preparation of porcine acellular dermal matrix

Obtaining fresh pig skin from local slaughterhouse, thoroughly cleaning, cutting subcutaneous adipose tissue and removing hair, and cutting the obtained skin into 5 × 5 × 0.3 cm ³ The small blocks are stored in a refrigerator at the temperature of 20 ℃ below zero for standby. The treated pigskin was treated with a 0.25% trypsin (Biosharp) solution at 25 ℃ for 18 hours to remove the skin epidermis, and then the dermal section was cut into sections of 0.5X 0.3 cm ³ The fragments of (a); shaking with 0.25% trypsin solution at 25 ℃ for 12 hours on a shaker; the pigskin was washed with 0.1% sodium dodecyl sulfate (SDS, chinese medicine) solution for 12 hours at room temperature, followed by 560U/L Dispase (Roche) solution for 12 hours at 25 ℃. Next, the pigskin was washed with 0.1% SDS at room temperature for 12 hours and then twice with Phosphate Buffered Saline (PBS) (15 minutes each). And further crushing the treated pigskin by using a crusher, and sending the pigskin to a tin-free Futeng irradiation company for electron beam sterilization to obtain the pig acellular dermal matrix. It was freeze-dried and stored in a-80 ℃ refrigerator.

Example 2: screening for active Polypeptides

BLAST homology analysis is carried out on human and pig elastin sequences, sequences with higher conservation degree among species are taken, and elastin polypeptides are designed according to the principle of higher polypeptide stability. The peptide sequence was synthesized by Kinseri. The synthetic polypeptides are recorded as polypeptides 1 to 12; the amino acid sequence is shown in Seq _ 1-12. Namely respectively corresponding to:

polypeptide 1: D-Gly D-Ala D-Val D-Val D-Pro D-Gln;

polypeptide 2D-Phe D-Pro D-Gly Ala Leu Val Pro D-Gly D-Gly D-Val

Polypeptide 3D-Pro D-Gly D-Val D-Gly D-Pro

Polypeptide 4: D-Thr D-Gly D-Lys Leu D-Pro D-Tyr D-Gly

Polypeptide 5, D-Ala D-Arg D-Phe Pro Gly D-Val D-Gly D-Val

Polypeptide 6, D-Lys D-Pro D-Gly Lys Val Pro Gly D-Val D-Gly D-Leu

Polypeptide 7, D-Pro D-Gly D-Val Tyr Pro Gly Gly Val D-Leu D-Pro D-Gly

Polypeptide 8, D-Gln D-Pro D-Gly Val Pro Leu Gly D-Tyr D-Pro D-Ile

Polypeptide 9, D-Leu D-Pro D-Gly Val D-Pro D-Thr D-Gly

Polypeptide 10, D-Gly D-Gly D-Ala Phe D-Ala D-Gly D-Ile

Polypeptide 11, D-Lys D-Ala D-Pro Lys Leu Pro Gly Gly Tyr Gly D-Leu D-Pro D-Tyr

The polypeptide 12 is D-Aal D-Gly D-Tyr Pro Thr Gly Thr D-Gly D-Val D-Gly.

The elastin polypeptide solution was prepared with physiological saline, and 1.5% elastin polypeptide solution was injected subcutaneously (wt/wt) into SD rats (3 per group), respectively, and the amino acid sequence of the negative control polypeptide was shown as Seq _ 13 (GRADSP). Three skin samples were taken from the treatment sites of each animal and the results averaged for each animal. FIG. 1 is a graph of the ability of polypeptides to produce elastin; as can be seen in fig. 1, there was a significant increase in elastin levels (measured as the ratio of elastin micrograms (μ g) to dry weight per milligram (mg) of fat-free skin) in rats using polypeptides 1, 3, 4, 5, 9 and 10 after 30 days compared to the negative control polypeptide. Therefore, the screened polypeptides 1, 3, 4, 5, 9 and 10 can promote the production of skin elastin. Elastin quantification was accurately quantified using the method of Sandberg, et al, (connecting Tissue research. 25: 139-48, 1990).

Example 3: effect of polypeptide compositions on elastin expression

Polypeptides 1, 3, 4, 5, 9 and 10 (corresponding to Seq _1, 3, 4, 5, 9 and 10) were mixed in equal amounts to form a polypeptide composition, which was injected subcutaneously into SD rats (3 per group) in groups of low dose (concentration 0.15%, wt/wt), medium dose (1.5%, wt/wt) and high dose (10.5%, wt/wt), three skin samples were removed from the treatment site of each animal and the results of each animal were averaged, and the amino acid sequence of the negative control is shown as Seq _ 13. FIG. 2 is a graph of the ability of a polypeptide composition to produce elastin. As can be seen in fig. 2, the polypeptide compositions at these concentrations can significantly increase elastin production, with comparable results at 1.5% and 10.5% concentrations. The elastin of the rats in the medium-dose group is more than 4 times of that of the negative control polypeptide, and has significant difference. The provided polypeptide also has excellent capability of promoting the production of skin elastin after being combined. It should be noted that the mixing of the same amount of each polypeptide in this embodiment is only for the convenience of experimental operation and should not be taken as a limitation on the content of each component of the polypeptide.

Example 4: polypeptide composition and filling effect after pig acellular dermal matrix implantation

Depilating the back of a male Balb/c mouse aged 6 weeks, confirming that the depilating is clean and has no inflammation, and randomly dividing the depilating into a PADM group, a polypeptide group and a PADM + polypeptide group; 6 mice per group; the PADM group is only injected with PADM, the polypeptide group is only injected with the polypeptide composition with a medium dosage, and the PADM + polypeptide group is simultaneously injected with the polypeptide composition with a medium dosage; the dosage of the polypeptide is 200 mug/Kg, and the dosage of the PADM is 10 g/Kg (calculated by wet weight). Injecting 0.5ml of corresponding product into each mouse subcutaneously, observing whether outflow exists after injection, and enabling the mice successfully injected to enter cages for observation. The status of the mice was observed daily. Table 1 is a comparative table of toxicity observations in vivo for each group of mice.

TABLE 1 Observation of the toxic Effect of the Polypeptides and PADM in mice

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As shown in Table 1, after the polypeptide and PADM provided by the invention are injected into mice, no obvious influence is caused on factors such as normal diet, excretion and exercise of the mice, all recorded ethological indications are normal, and no death phenomenon of the mice is found one day, one week and 30 days after the injection. Proved that after the polypeptide and the PADM are implanted into mice, the polypeptide and the PADM have no obvious acute or chronic toxic effect on the mice.

The size of the implants in each group was measured at 10 and 30 days each, and sacrificed and dissected after 30 days of measurement. It was observed that 10 days after implantation, the development size of the PADM + polypeptide group and PADM group did not differ much, and there was no inflammation in all three groups. After 30 days of implantation, the PADM + polypeptide group and PADM group implants were hemispherical, and the implant volume of PADM + polypeptide group was significantly different from that of other two groups, and the volume was significantly increased. FIG. 3 is a graph of fill results 10 days after implantation of different materials; FIG. 4 is a graph of the filling results 30 days after implantation of different materials; as shown in fig. 3 and 4, compared with the PADM combination polypeptide group, the PADM and polypeptide group have significantly larger volume than the PADM group, so that the skin filling effect is significantly improved, and the PADM and polypeptide combination has a synergistic effect.

The implants of the PADM + polypeptidyl were HE stained and photographs taken. FIG. 5 is a graph of HE staining of implants from the PADM + polypeptide set (scale bar 100 μm); as can be seen from fig. 5, the implant can be roughly divided into 3 layers from the surface to the inside, and is loose, compact and uniform; the surface of the implant has a multilayer tissue structure and is provided with abundant blood vessels; the inside has dense cells and capillary vessels. It can be seen that PADM and polypeptides are suitable for cellular invasion and growth.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (10)

1. A composition comprising an elastin-like polypeptide and an acellular dermal matrix; the elastin-like polypeptide includes one or more of the amino acid sequences shown as Seq _ 1-12.

2. The composition of claim 1, wherein the elastin-like polypeptide comprises one or more of the amino acid sequences set forth in Seq _1, 3, 4, 5, 9, or/and 10.

3. The composition of claim 1, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

4. A method for preparing an acellular dermal matrix, comprising the steps of:

(1) Cleaning, cutting off subdermal adipose tissue and removing hair;

(2) Removing epidermis of skin with 0.25% trypsin solution, chopping dermis, further placing into 0.25% trypsin solution for treatment reaction, and cleaning with 0.1% sodium dodecyl sulfate solution;

(3) Treating the cleaned skin with Dispase solution, washing with 0.1% sodium dodecyl sulfate solution, washing with phosphate buffer solution, pulverizing, and sterilizing with electron beam.

5. Use of a composition according to claim 1 for the preparation of a micro-sculpturing material.

6. Use according to claim 5, wherein the material is a soft tissue filler.

7. An elastin-like polypeptide, comprising an amino acid sequence as set forth in Seq _1, seq _2, seq _3, seq _4, seq _5, seq _6, seq _7, seq _8, seq _9, seq _10, seq _11, or Seq _ 12.

8. An elastin-like polypeptide comprising an amino acid sequence as set forth in Seq _1, seq _3, seq _4, seq _5, seq _9 or Seq _ 10.

9. A pharmaceutical composition comprising the polypeptide of claim 8.

10. The pharmaceutical composition of claim 9, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

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