CN118873744A - PDRN-crosslinked collagen composition and preparation method thereof - Google Patents
PDRN-crosslinked collagen composition and preparation method thereof Download PDFInfo
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Abstract
The invention provides a PDRN-crosslinked collagen composition and application thereof, wherein the PDRN-crosslinked collagen composition comprises the following components in parts by mass: 2-5 parts of PDRN, 20-30 parts of collagen fiber, 25-40 parts of oxidized dextran and 300-400 parts of deionized water. The PDRN-crosslinked collagen composition is gelled in situ through the amine groups in the collagen fibers and the active carbonyl groups of oxidized glucan through Schiff base reaction, and is matched with the PDRN to form the PDRN-crosslinked collagen gel, the gel has excellent combination stability, mild reaction conditions, simple preparation operation, no need of adding chemical crosslinking agent impurities, environment friendliness, excellent combination stability, difficult degradation, convenient storage and simple preparation, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a PDRN-crosslinked collagen composition and a preparation method thereof.
Background
PDRN (Poly Deoxy Ribo Nucleotide), also known as "polydeoxyribonucleotide", is a DNA fragment of a specific size extracted from salmon germ cells. It is a deoxyribonucleotide polymer, is polymerized by 13 or more deoxyribonucleotide monomers, is one of the complexes of the raw materials of the generated DNA, and has the capability of stimulating the proliferation of collagen and elastic fiber. The treated PDRN basically has no immunogenicity, does not cause anaphylactic reaction when being used on a human body, and can be applied to the fields of anti-inflammation, tissue repair promotion, wound regeneration and the like. With the development of medical technology, PDRN is also gradually applied to the cosmetic field, improving skin quality, and reducing defects and wrinkles.
Collagen is a biopolymer, the main component of extracellular matrix, the main component of animal connective tissue, and the functional protein with the greatest content and the greatest distribution in mammals, accounting for 25% -30% of the total protein, and the triple helix collagen self-assembles in vivo to form a tightly arranged fiber structure, thus providing a good growth environment for cell adhesion, proliferation and differentiation. The collagen protein has a certain surface activity and good compatibility, and simultaneously has quite good moisturizing effect due to the fact that the collagen protein contains a large amount of hydroxyl groups, and has the effect of preventing tyrosine in skin from being converted into melanin, so that the collagen protein has the effects of purely natural moisturizing, whitening, wrinkle preventing, freckle removing and the like, and is widely applied to cosmetic products.
Chinese patent CN113384748B, a collagen dermis implant and a preparation method thereof, discloses a collagen dermis implant and a preparation method thereof, wherein the collagen is crosslinked in an alkaline aqueous solution through a chemical crosslinking agent, and chemical crosslinking agents such as glutaraldehyde, succinaldehyde and the like are required to be added, so that the product has the problems of poor biodegradability, residual crosslinking agent, inconvenience in storage and use and the like.
Therefore, development of the PDRN-crosslinked collagen composition which does not need to be added with a chemical crosslinking agent, has good biodegradability and stability and is convenient to store and use and the preparation method thereof have great significance.
Disclosure of Invention
In view of the problems of poor biodegradability, residual crosslinking agent and inconvenience in storage and use of the conventional PDRN-crosslinked collagen composition, the invention aims to provide the PDRN-crosslinked collagen composition which has the advantages of mild reaction conditions, simple preparation operation, no need of adding chemical crosslinking agent impurities, excellent combination stability, difficulty in degradation and convenience in storage, and is suitable for industrial production.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the PDRN-crosslinked collagen composition comprises the following components in parts by mass:
2-5 parts of PDRN, 20-30 parts of collagen fiber, 25-40 parts of oxidized dextran and 300-400 parts of deionized water.
Further, the collagen fiber is prepared by the following method:
Preparing collagen solution with concentration of 1-10mg/mL, adding 0.1-1M disodium hydrogen phosphate and/or sodium dihydrogen phosphate solution to adjust pH to 6-9, standing overnight, centrifuging, and collecting precipitate to obtain the collagen fiber.
Further, the oxidized dextran is prepared by the following method:
D1. adding beta-glucan into deionized water, and uniformly mixing;
D2. Dripping sodium periodate solution into the glucan solution obtained in the step D1, and stirring for reaction in a dark place;
D3. Stopping the reaction of the solution obtained in the step D2, dialyzing, purifying, and freeze-drying to obtain the oxidized dextran.
Still further, the molar ratio of beta-glucan to sodium periodate is (10-20): 1.
Further, the mass fraction of the sodium periodate solution of D2 is 3%; and D2, stirring and reacting for 20-24 hours in a dark place.
Further, the termination reaction of D3 is to add diethylene glycol equivalent to beta-glucan for 0.5-4h.
Another object of the present invention is to provide a method for preparing a PDRN-crosslinked collagen composition.
A method of preparing a PDRN-crosslinked collagen composition according to any preceding claim, comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing the PDRN, the collagen fibers and deionized water, and continuously stirring;
s3, adding oxidized glucan into the solution obtained in the step S2, standing and crosslinking to obtain the PDRN-crosslinked collagen composition.
Further, the stirring speed of S2 is 200-400rpm, and the stirring time is 20-35min.
Further, the standing crosslinking temperature of S3 is 25-30 ℃ and the time is 40-60min.
It is another object of the present invention to provide the use of a PDRN-crosslinked collagen composition.
A PDRN-crosslinked collagen composition according to any preceding claim for use in the preparation of a tissue filler for medical use.
Compared with the prior art, the invention has the following advantages:
(1) The collagen fibers obtained by a specific method in the PDRN-crosslinked collagen composition component have a distinct fiber structure after self aggregation, and are mutually staggered to form a fiber net, so that a better fiber form can be formed, the fiber net has higher molecular regularity, and gel can be formed by matching with other components to have higher crosslinking degree, so that the PDRN-crosslinked collagen composition has lasting and excellent filling effect.
(2) The oxidized glucan obtained by a specific method in the PDRN-crosslinked collagen composition component has excellent biocompatibility, anti-inflammatory and antiallergic effects, and simultaneously has various effects of promoting fibroblast proliferation and collagen and elastin synthesis, free radical scavenging capability, resisting ultraviolet injury, promoting immune protection and the like, and the safety and the biocompatibility of the composition can be greatly improved by being matched with other components, and the effects of water locking, moisturizing, anti-aging and wrinkle removing of the composition are improved.
(3) According to the PDRN-crosslinked collagen composition, the amino groups in the collagen fibers and the active carbonyl groups of oxidized glucan in the components react to form Schiff base bonds through dehydration, spontaneous in-situ gelation is carried out, a gel network is formed based on the Schiff base bonds in a crosslinking mode, and the strength and the tissue adhesion of gel can be effectively improved; the PDRN is added to be matched with a gel network, so that the in-situ gel forming time can be shortened, the gel strength of the PDRN-crosslinked collagen composition can be regulated, the system stability can be improved, the metabolism can be promoted, the activity of epidermal cells can be enhanced, fibroblasts can be activated, the vascular synthesis capacity can be improved, the collagen cells can be repaired, the PDRN-crosslinked collagen composition gel which is difficult to degrade and good in stability can be formed, and the effects of quickly regenerating skin, removing wrinkles and resisting aging and promoting healthy and smooth skin can be achieved.
(4) The preparation method of the PDRN-crosslinked collagen composition has the advantages of mild reaction conditions, simple preparation operation, no need of adding chemical crosslinking agent impurities, excellent beautifying effect, environmental friendliness, excellent combination stability, difficult degradation, convenient storage and simple preparation, and is suitable for industrial production.
Detailed Description
For a better illustration of the objects, technical solutions and advantages of the present invention, the present invention is further illustrated by the following examples. It is apparent that the following embodiments are only some, but not all, embodiments of the invention; it should be understood that the embodiments of the present invention are only used for illustrating the technical effects of the present invention, and are not used for limiting the scope of the present invention.
The starting materials in the examples are all commercially available; unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The collagen of the invention is type I collagen, and is derived from rat tail type I collagen, and the collagen fibers in each of the examples and comparative examples are prepared by the following preparation methods.
A method for preparing collagen fibers, comprising the steps of:
preparing collagen solution with concentration of 5mg/mL, adding 0.5M disodium hydrogen phosphate solution to adjust pH to 6-9, standing overnight, centrifuging at 10000rpm, and collecting precipitate to obtain the collagen fiber.
The oxidized dextran described in each of the examples and comparative examples was prepared by the following preparation method.
A method for preparing oxidized dextran, comprising the following steps:
D1. Adding 10g of beta-glucan into 500mL of deionized water, and uniformly mixing;
D2. 100mL of 3wt.% sodium periodate solution is dripped into the glucan solution obtained in the step D1, and the mixture is stirred and reacted for 20 hours in a dark place;
D3. Adding diethylene glycol equivalent to beta-glucan into the solution obtained in the step D2, stirring for 2 hours, terminating the reaction, dialyzing, purifying and freeze-drying to obtain the oxidized glucan.
Example 1
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing 3 parts of PDRN, 25 parts of collagen fibers and 300 parts of deionized water, and continuously stirring at 300rpm for 30min;
S3, adding 30 parts of oxidized dextran into the solution obtained in the S2, standing and crosslinking at 25 ℃ for 60 minutes to obtain the PDRN-crosslinked collagen composition.
Example 2
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing 2 parts of PDRN, 20 parts of collagen fibers and 320 parts of deionized water, and continuously stirring at 300rpm for 30min;
S3, adding 26 parts of oxidized dextran into the solution obtained in the S2, standing and crosslinking at 25 ℃ for 60 minutes to obtain the PDRN-crosslinked collagen composition.
Example 3
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing 5 parts of PDRN, 30 parts of collagen fibers and 390 parts of deionized water, and continuously stirring at 300rpm for 30 minutes;
S3, adding 37 parts of oxidized dextran into the solution obtained in the S2, standing and crosslinking at 26 ℃ for 40min to obtain the PDRN-crosslinked collagen composition.
Comparative example 1
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
S2, mixing 5 parts of PDRN, 10 parts of collagen and 100 parts of deionized water, magnetically stirring for 2 hours at 4 ℃, adding 2mL of glutaraldehyde (the concentration is 50%), stirring for 2 hours at 4 ℃, and refrigerating for 4 hours;
S3, heating the mixture obtained in the step S2 in a water bath at 45 ℃ for 8 hours, adding 1500mL of ammonia physiological saline (ammonia concentration is 2%) for reaction for 2 hours, purifying by using the physiological saline, and purifying by using 1000mL of the physiological saline each time for 1 hour for 4 times to obtain the PDRN-crosslinked collagen composition.
In comparison to example 1, this comparative example prepared a PDRN-crosslinked collagen composition by adding a chemical crosslinking agent.
Comparative example 2
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
s2, fully mixing 3 parts of PDRN, 25 parts of collagen and 300 parts of deionized water, and continuously stirring at 300rpm for 30min;
S3, adding 30 parts of oxidized dextran into the solution obtained in the S2, standing and crosslinking at 25 ℃ for 60 minutes to obtain the PDRN-crosslinked collagen composition.
In comparison with example 1, this comparative example uses a collagen component instead of a collagen fiber component.
Comparative example 3
A method of preparing a PDRN-crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing 3 parts of PDRN, 25 parts of collagen fibers and 300 parts of deionized water, and continuously stirring at 300rpm for 30min;
s3, adding 30 parts of glucan into the solution obtained in the S2, standing and crosslinking at 25 ℃ for 60 minutes to obtain the PDRN-crosslinked collagen composition.
In comparison to example 1, the present comparative example uses a dextran component instead of the oxidized dextran component.
Comparative example 4
A method of preparing a crosslinked collagen composition comprising the steps of:
s1, weighing all the components according to mass parts;
s2, fully mixing 25 parts of collagen fibers with 300 parts of deionized water, and continuously stirring at 300rpm for 30min;
S3, adding 30 parts of oxidized dextran into the solution obtained in the S2, standing and crosslinking at 25 ℃ for 60 minutes to obtain the PDRN-crosslinked collagen composition.
In comparison to example 1, this comparative example lacks a PDRN component.
The performance test is carried out on the samples, and the specific test process is as follows:
(1) Mechanical property test
Examples 1-3 and comparative examples 1-4 were tested for push force using a 26G disposable sterile syringe needle, flow scanned at 25 ℃ using a laminar clamp at a shear rate of 0.001s -1-1000s-1, a sample shear viscosity value corresponding to a shear rate of 2.5s -1 was taken, an oscillation test was performed according to a strain of 5%, and a G' value corresponding to a 1Hz position was taken with the scan frequency as the abscissa.
The actual results of the obtained examples and comparative examples are shown in the following Table 1:
TABLE 1 mechanical Properties of examples 1-3 and comparative examples 1-4
As can be judged from Table 1, examples 1 to 3 of the present invention have more stable mechanical properties, less pushing force, good fluidity and more excellent shear viscosity and elastic modulus than comparative examples 1 to 4.
The PDRN-crosslinked collagen composition prepared by adding the chemical crosslinking agent of comparative example 1 exhibited high push force and high shear viscosity, but the elastic modulus was low, and the hardness was too high, resulting in poor use experience; while comparative example 2 uses collagen instead of the collagen fiber component so that the degree of crosslinking is not high, the pushing force is small, but the shear viscosity and elastic modulus are low compared with examples; comparative example 3 failed to form a gel; comparative example 4 shows poor stability in both shear viscosity and elastic modulus, indicating that the addition of PDRN helps to improve gel stability.
(2) Gel time test
In particular, the rheological analyses of examples 1 to 3 and comparative examples 1 and 3 were performed to compare the gel times, and a dynamic rheological test was performed at 37℃using a rheometer, and a time sweep oscillation test was performed at a frequency of 1Hz for 300 seconds at a strain of 5%. The pre-gel solution was strain scanned to verify the linear response. The gel point is determined when the torsional modulus (G ') exceeds the loss modulus (G').
Wherein comparative example 3 failed to form a gel because comparative example 1 had been crosslinked to form a gel, the specific results of the gel time could not be tested, and therefore no record was made.
The actual results of the obtained examples and comparative examples are shown in the following Table 2:
TABLE 2 gel times for examples 1-3 and comparative examples 2, 4
As can be seen from Table 2, examples 1-3 of the present invention have shorter and more stable gel times than comparative examples 2 and 4, demonstrating that the addition of the PDRN-crosslinked collagen composition of the present invention to the PDRN in combination with other components can reduce the in situ gel formation time for ease of use.
(3) Animal experiment
200. Mu.L of PDRN-crosslinked collagen compositions of examples 1-3 and comparative examples 1-4 prepared by the present invention were subcutaneously injected into female nude mice with five weeks of age, and the skin injected from the back was subjected to pathological analysis at 7d, 14d and 28d, respectively, to a depth of subcutaneous (including epidermis, dermis and a part of subcutaneous tissue), and then suturing was performed, 1g of epidermal tissue was weighed, 9g of PBS (phosphate buffer solution) having pH of about 7.2-7.4 was added, the samples were homogenized sufficiently with a homogenizer, centrifuged for about 20 minutes (2000-3000 rpm), the supernatant was collected carefully, absorbance at 450nm was measured using an Elisa kit assay, and the concentration of inflammatory factor IL-1 alpha was recorded and measured.
The actual results of the obtained examples and comparative examples are shown in the following Table 3:
TABLE 3 inflammatory response test results for examples 1-3 and comparative examples 1-4
The female nude mice of each of examples 1-3 have no abnormal appearance after 28 days of injection, have lower IL-1 alpha factor content, have no abnormal change of surrounding tissues, and do not cause intradermal reaction of the female nude mice, which indicates that the PDRN-crosslinked collagen composition is safe and qualified in intradermal reaction test.
The IL-1 alpha factor content of the female nude mice of the comparative example 3 is obviously higher than that of the nude mice of the examples 1-3 after 7 days of injection; the IL-1 alpha factor secretion level of the female nude mice of the comparative example 3 is obviously increased after 14 days of injection; the IL-1 alpha factor content of the female nude mice of the comparative example 3 is higher after 28 days of injection, and a more obvious inflammatory reaction occurs.
At the same time, at 0h, 1d, 7d, and 14d after injection, whether filling effect is present or not is observed, and whether adverse reaction occurs or not is observed.
TABLE 4 detection results for examples 1-3 and comparative examples 1-4
With the increase of injection time, the samples of examples 1-3 showed no filling effect after 0h injection, and showed an obvious filling effect after 1 day, which indicates that the PDRN-crosslinked collagen composition of the present invention has schiff base reaction to form in-situ gel, and still had a certain filling effect after 14 days of injection, which indicates that the gel of the PDRN-crosslinked collagen composition is not easy to diffuse and absorb, and can meet the requirement of long-term filling effect, and the subcutaneous tumor of female nude mice has a vanishing trend after 14 days of injection, which indicates that the synthesized PDRN-crosslinked collagen composition is nontoxic and has good biodegradability.
The comparative example 1 shows an obvious filling effect after 0h injection, and an unnatural hard lump appears under the skin of a female nude mouse can be observed by touching, and obvious lumps can be still touched after 15 days of injection, which indicates that the composition combining the PDRN and the crosslinked collagen through chemical crosslinking has higher hardness, poorer biodegradability and poor use feeling; comparative example 3 failed to form a gel, and a short subcutaneous bulge appeared upon injection in the form of a solution, but did not form a tumor upon touch, and rapidly degraded, failing to maintain a long-term filled state.
(4) Cosmetic effect test
The PDRN-crosslinked collagen compositions of examples 1-3 and comparative examples 1, 2, 4 were injected on the face of 6 subjects, respectively, and the 6 subjects selected were 30-60 years old, healthy, with obvious facial lines, fish tail lines, intereyebrow lines, eye grooves, forehead lines, lower eyelid pits, other facial pits, etc., with unlimited initial skin scores, and skin scores were summarized by the VAS scoring method before injection, 1 month after injection, 2 months after injection. Wherein the VAS total score is 10 minutes, the range value is 0-10, the 10 score is very good in skin state, and 0 is poor in skin state.
After 48 hours of injection, 6 injection sites have no symptoms such as red swelling, ecchymosis and the like, and have no allergic reaction.
The actual results of the obtained examples and comparative examples are shown in the following Table 3:
TABLE 3 cosmetic effects of examples 1-3 and comparative examples 1,2, 4
As can be judged from table 3, the PDRN-crosslinked collagen composition of examples 1 to 3 of the present invention effectively improved the skin state of the subject before and after injection, reduced wrinkles, increased skin elasticity, and an obvious skin lightening and whitening effect; the PDRN-crosslinked collagen composition of comparative example 1, after injection, improved the wrinkle state of the subject by filling means, and had a more remarkable improvement effect, but had poor biodegradability, and had a slight local skin stiffness; comparative example 2 and comparative example 4 have some improvement effect, but the improvement amplitude is not slightly different from comparative examples 1 to 3.
It is known from the comprehensive tables 1,2 and 3 that the invention synergistically plays the roles of PDRN, collagen fiber and oxidized dextran through specific component proportions, utilizes the amino groups in the specific collagen fiber and the active carbonyl groups of the oxidized dextran in the components to form Schiff base bonds through dehydration reaction, spontaneously and in-situ gels, and forms a gel network based on the Schiff base bonds in a crosslinking way, so that the strength and tissue adhesion of the gel can be effectively improved; the PDRN is added to be matched with a gel network, so that the in-situ gel forming time can be shortened, the gel strength of the PDRN-crosslinked collagen composition can be regulated, the system stability can be improved, the metabolism can be promoted, the activity of epidermal cells can be enhanced, fibroblasts can be activated, the vascular synthesis capacity can be improved, the collagen cells can be repaired, the PDRN-crosslinked collagen composition gel which is difficult to degrade and good in stability can be formed, and the effects of quickly regenerating skin, removing wrinkles and resisting aging and promoting healthy and smooth skin can be achieved.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. The PDRN-crosslinked collagen composition is characterized by comprising the following components in parts by mass:
2-5 parts of PDRN, 20-30 parts of collagen fiber, 25-40 parts of oxidized dextran and 300-400 parts of deionized water.
2. The PDRN-crosslinked collagen composition according to claim 1, wherein: the collagen fiber is prepared by the following method:
Preparing collagen solution with concentration of 1-10mg/mL, adding 0.1-1M disodium hydrogen phosphate and/or sodium dihydrogen phosphate solution to adjust pH to 6-9, standing overnight, centrifuging, and collecting precipitate to obtain the collagen fiber.
3. The PDRN-crosslinked collagen composition according to claim 1, wherein: the oxidized dextran is prepared by the following method:
D1. Adding beta-glucan into deionized water, and uniformly mixing;
D2. Dripping sodium periodate solution into the glucan solution obtained in the step D1, and stirring for reaction in a dark place;
D3. Stopping the reaction of the solution obtained in the step D2, dialyzing, purifying, and freeze-drying to obtain the oxidized dextran.
4. The PDRN-crosslinked collagen composition according to claim 3, wherein: the mol ratio of the beta-glucan to the sodium periodate is (10-20): 1.
5. The PDRN-crosslinked collagen composition according to claim 3, wherein: the mass fraction of the sodium periodate solution is 3%; and D2, stirring and reacting for 20-24 hours in a dark place.
6. The PDRN-crosslinked collagen composition according to claim 3, wherein: the termination reaction is to add diethylene glycol equivalent to beta-glucan for 0.5-4h.
7. A method of preparing a PDRN-crosslinked collagen composition according to any one of claims 1 to 6, comprising the steps of:
s1, weighing all the components according to mass parts;
S2, fully mixing the PDRN, the collagen fibers and deionized water, and continuously stirring;
s3, adding oxidized glucan into the solution obtained in the step S2, standing and crosslinking to obtain the PDRN-crosslinked collagen composition.
8. The method of preparing a PDRN-crosslinked collagen composition according to claim 7, wherein: and S2, stirring at 200-400rpm for 20-35min.
9. The method of preparing a PDRN-crosslinked collagen composition according to claim 7, wherein: s3, standing and crosslinking at 25-30 ℃ for 40-60min.
10. A PDRN-crosslinked collagen composition according to any preceding claim for use in the preparation of a tissue filler for medical use.
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