Compound of polynucleotide and hydroxyapatite as well as preparation method and application of compound

Abstract

The invention relates to the technical field of medical beauty, in particular to a compound of polynucleotide and hydroxyapatite as well as a preparation method and application of the compound. The compound provided by the invention comprises hydroxyapatite microspheres, polynucleotide and a carrier matrix, the mass ratio of the polynucleotide to the hydroxyapatite is (0.5-2): 1. The polynucleotide and the hydroxyapatite microspheres are compounded to improve the dispersity of the hydroxyapatite microspheres so as to reduce the side effects caused by the dispersity, the hydroxyapatite microspheres and the polynucleotide are synergistic, and when the hydroxyapatite microspheres and the polynucleotide are used for skin filling, a low-inflammation and nutrient-rich'regeneration microenvironment 'can be created, so that the skin filling effect is improved. The generation of healthier and more ordered collagenous fibers is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical aesthetics, in particular to a compound of polynucleotide and hydroxyapatite, a preparation method and application thereof. BACKGROUND

[0002] Hydroxyapatite is a kind of inorganic material with good biocompatibility, which has been widely used as a skin filler. Its working principle is mainly to provide immediate physical support through HAP microspheres, and then stimulate human fibroblasts to produce collagen, thereby achieving long-term and natural filling effect.

[0003] However, the existing HAP fillers have the following technical problems to be solved in practical application: Uneven distribution and easy aggregation: HAP microspheres are solid particles, which are prone to sedimentation or mutual aggregation in the injection carrier (such as gel or physiological saline) due to gravity, resulting in the need to shake repeatedly before injection. After injection, the microspheres may also locally aggregate in the tissue, forming hard knots, nodules or granular feeling, affecting the hand feeling and appearance.

[0004] Obvious side effects: As a foreign body, HAP can cause certain immune response after injection, leading to redness, pain, and inflammatory reaction. In some cases, uneven distribution can exacerbate these side effects, and even may cause problems such as Tyndall effect (skin light blue).

[0005] Biocompatibility needs to be improved: Although HAP itself has good biocompatibility, the inflammatory reaction accompanying the process of stimulating collagen regeneration affects the comfort and recovery period of treatment.

[0006] PN (Plydexyribnucletide, polynucleotide) is a DNA fragment extracted from the testes / sperm of salmon or other specific fish, which has excellent biological activity and safety. In the field of medical aesthetics, PN has been proven to have the following effects: Promote tissue repair and regeneration: by activating A2A adenosine receptors, promote fibroblast proliferation, angiogenesis and collagen synthesis.

[0007] Anti-inflammatory effect: can effectively reduce the expression of inflammatory factors (such as IL-6, TNF-α), and reduce inflammatory reaction.

[0008] Antioxidant and moisturizing: improve the overall condition of the skin.

[0009] At present, PN is mainly used in the form of water injection, essence, etc. for skin repair and anti-aging, but there is no report on its compounding with hydroxyapatite to solve the physical distribution and biocompatibility problems of HAP fillers. SUMMARY

[0010] The present application aims to provide a polynucleotide and hydroxyapatite complex, a preparation method thereof and an application thereof, so as to solve the technical problems of uneven distribution, easy agglomeration and side effects (such as redness, induration, pain) of existing hydroxyapatite fillers.

[0011] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions. One of the technical solutions of the present application provides a polynucleotide and hydroxyapatite complex, which comprises hydroxyapatite microspheres, polynucleotides and a carrier matrix. The mass ratio of the polynucleotides to the hydroxyapatite is 0.5-2:1.

[0012] The second technical solution of the present application provides a preparation method of the polynucleotide and hydroxyapatite complex, which comprises the following steps. The hydroxyapatite microspheres, the polynucleotides and the carrier matrix are mixed and dispersed to obtain the polynucleotide and hydroxyapatite complex.

[0013] The third technical solution of the present application provides an application of the polynucleotide and hydroxyapatite complex in preparing a medical and cosmetic filling drug.

[0014] The fourth technical solution of the present application provides an application of the polynucleotide and hydroxyapatite complex in preparing a skin and soft tissue filling drug.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) Achieving uniform suspension and solving the problem of uneven distribution: Based on the steric hindrance effect and three-dimensional network structure of polynucleotides (PN), the composition of the present application has excellent physical stability, and does not need to be shaken excessively before injection, and can be more uniformly distributed in the tissue after injection, thereby fundamentally avoiding the hardening and particle feeling caused by the aggregation of microspheres.

[0016] (2) Significantly reducing side effects: The strong anti-inflammatory activity of PN can effectively inhibit the local inflammatory response caused by hydroxyapatite (HAP) microspheres as a foreign body, thereby reducing redness, pain and shortening the recovery period after injection. At the same time, the uniform distribution also reduces the mechanical stimulation to the local tissue, further reducing the risk of side effects.

[0017] (3) Synergistic effect, improving the overall treatment effect: HAP provides a "skeleton": providing immediate physical support and long-term collagen stimulation; PN provides an "environment": on the one hand, it creates a uniform physical environment, and on the other hand, it creates a low-inflammatory, nutrient-rich "regenerative microenvironment" to promote the generation of healthier and more ordered collagen fibers. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Macrodispersion results of samples prepared for Examples and Comparative Examples; Figure 2 Microtubule morphology of samples prepared for Examples and Comparative Examples; Figure 3 Histological section comparison. DETAILED DESCRIPTION

[0019] Various illustrative embodiments of the present application are now described in detail below. The embodiments discussed herein should be understood to be non-limiting examples of the application. The description of the embodiments is intended to be illustrative, and not to limit the scope of the application, as defined by the claims.

[0020] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and each range of values should be considered as having been specifically stated.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law. Nothing herein is to be construed as an admission that the application is not entitled to antedate such disclosure by virtue of prior application.

[0022] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples provided herein. The description and examples are illustrative only and are not intended to limit the scope of the application.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0024] The raw materials used in the present application can be obtained commercially or prepared by prior art.

[0025] The room temperature described in the present application is 25±2℃.

[0026] The present application provides a complex of polynucleotide and hydroxyapatite, comprising hydroxyapatite microspheres, polynucleotide and carrier matrix. The mass ratio of the polynucleotide and hydroxyapatite is 0.5-2:1, for example, 0.5:1, 1:1, 1.5:1 or 2:1, etc.; the concentration of the hydroxyapatite microspheres in the polynucleotide and hydroxyapatite complex is 5-30 mg / mL, for example, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL, etc. In the present application, the hydroxyapatite (HAP) microspheres: as the main filling and collagen regeneration stimulating component; In some embodiments of the present application, the HAP is purchased from Shanghai Bierong Biotechnology Co., Ltd.

[0027] In the present application, the polynucleotide (PN): as a key suspension stabilizer and biological activity regulator, the long-chain polymer structure of PN forms a three-dimensional network in solution, which can effectively wrap and suspend HAP microspheres through steric hindrance effect, preventing their sedimentation and aggregation, and its biological activity can neutralize the inflammation caused by HAP.

[0028] In the present application, the carrier matrix can be any carrier acceptable in medicine, and in some embodiments of the present application, the carrier matrix includes physiological saline, phosphate buffer or hyaluronic acid gel, which provides a suspension medium for PN and HAP; The concentration of the hyaluronic acid gel is 5 mg / ml.

[0029] In the present application, the particle size of the hydroxyapatite microspheres is 15-50 μm, to ensure that they can be effectively phagocytosed and stimulate collagen, while avoiding excessive immune response.

[0030] In the present application, the polynucleotide is of DNA type, with a molecular weight range of 1000 kDa-10000 kDa; the polynucleotide is derived from DNA fragments extracted from the testes / sperm of salmon or other specific fish.

[0031] The present application also provides a preparation method of the polynucleotide and hydroxyapatite complex as described above, comprising the following steps: Mixing the hydroxyapatite microspheres, polynucleotide and carrier matrix, and dispersing to obtain the polynucleotide and hydroxyapatite complex.

[0032] In the present application, the dispersion temperature is room temperature, and the time is 4 h.

[0033] In the present application, PN not only can exert its known biological activity, but also can serve as an excellent physical suspension agent, fundamentally solving the distribution problem of HAP microspheres. Its mechanism is not a single factor, but is based on the unique physicochemical properties of PN, and is realized through multiple synergistic effects: (1) Core mechanism: steric hindrance effect PN is a long-chain polymer composed of a large number of deoxy nucleotide units. In aqueous solution, it is stretched due to the negative charge of the phosphate group, forming a huge "random coil" structure. When mixed with HAP microspheres, the long chains of PN will adsorb on the surface of the microspheres, and the unabsorbed part will stretch into the solution, forming a layer of flexible "molecular brush". When two microspheres approach each other, the respective "molecular brush" layers will penetrate and compress each other, causing the local osmotic pressure to rise sharply and the entropy (freedom) of the polymer chain to decrease, thereby generating a strong repulsive force, effectively preventing the aggregation of microspheres.

[0034] (2) Auxiliary mechanism one: physical interception of three-dimensional network structure When PN reaches a certain concentration, its long-chain molecules will form a dynamic three-dimensional polymer network through chain entanglement and hydrogen bonding in the entire solution system, similar to a micro "hydrogel". The grid size of this network is smaller than the diameter of the HAP microspheres, which can physically "imprison" the microspheres in a small "cage", effectively intercepting and supporting the microspheres, preventing them from settling under the action of gravity. (3) Auxiliary mechanism two: increase the viscosity of the system The three-dimensional network formed by PN can significantly increase the macroscopic viscosity of the entire injection system. According to Stokes' law (the settling velocity of particles is inversely proportional to the viscosity of the fluid), the increase in viscosity greatly slows down the settling velocity of HAP microspheres, achieving apparent stable suspension within the product shelf life. (4) Auxiliary mechanism three: electrostatic repulsion effect The PN molecular chain carries a large number of negative charges, which significantly increases the Zeta potential (absolute value) of the microsphere surface after adsorption on the surface of the HAP microspheres, enhancing the electrostatic repulsion between the microspheres, providing additional protection for the stability of the system.

[0035] In summary, PN builds an extremely stable suspension system through the synergistic effect of four mechanisms: steric hindrance, network interception, viscosity increase, and electrostatic repulsion, completely solving the problem of physical distribution of HAP microspheres.

[0036] The application also provides the use of the above-mentioned polynucleotide and hydroxyapatite complex in the preparation of a medical and cosmetic filling drug.

[0037] The application also provides the use of the above-mentioned polynucleotide and hydroxyapatite complex in the preparation of a skin soft tissue filling drug.

[0038] When the complex filler of the application is used for skin soft tissue filling, the final effect is not only filling, but also a comprehensive improvement of filling + repair + anti-aging, making the skin texture more delicate and natural.

[0039] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0040] Example 1 The polynucleotide (PN), hydroxyapatite microspheres (HAP) and pure phosphate buffer were mixed, uniformly dispersed, and then placed at room temperature for 4 hours on a shaker to obtain a polynucleotide and hydroxyapatite compound, the mass ratio of PN and HAP was 0.5:1, and the concentration of HAP in the compound was 10 mg / mL.

[0041] Example 2 The difference from Example 1 is only that the mass ratio of polynucleotide and hydroxyapatite is 1:1.

[0042] Example 3 The difference from Example 1 is only that the mass ratio of the polynucleotide and hydroxyapatite compound is 1.5:1.

[0043] Example 4 The difference from Example 1 is only that the mass ratio of polynucleotide and hydroxyapatite is 2:1.

[0044] Comparative Example 1 The hydroxyapatite microspheres and pure phosphate buffer were mixed to obtain a hydroxyapatite microsphere solution with a concentration of 10 mg / mL.

[0045] Comparative Example 2 The polynucleotide and pure phosphate buffer were mixed to obtain a polynucleotide solution with a concentration of 20 mg / mL.

[0046] Test Example 1 Influence of PN Concentration on Hydroxyapatite Dispersion (Macro and Micro) The samples of Examples 1-4 and Comparative Example 1 were placed, and the initial state and the precipitation after 8 days of standing were observed and recorded.

[0047] Figure 1 is a macroscopic dispersion result chart, and Figure 1 It can be seen that all the samples are initially white and turbid, with no obvious difference; after standing for 8 days, a large amount of white precipitate appears at the bottom of the sample bottle of Comparative Example 1, and the upper liquid is basically clear, indicating that the dispersion is very poor; the sample of Example 1 has obvious precipitation, but less than the control group; the sample of Example 2 has reduced precipitation, and the dispersion effect is improved; the sample of Example 3 has significantly reduced precipitation, and the system still maintains a good turbid state; the sample of Example 4 has the least precipitation and the best suspension effect.

[0048] After standing, the samples were properly treated, and the micro-morphology, particle size and dispersion uniformity of the hydroxyapatite microspheres were observed using a scanning electron microscope (SEM).

[0049] Figure 2 is a micro-morphology result chart, wherein Figure 2 It can be seen that the hydroxyapatite microsphere particles in Comparative Example 1 are relatively large (about 10-50 μm) and irregular in shape, and have a serious agglomeration phenomenon; the hydroxyapatite microsphere particles in Example 1 and Example 2 have a reduced size and a reduced degree of agglomeration; the hydroxyapatite microsphere particles in Example 3 and Example 4 are rounder and have the best uniformity of dispersion and almost no agglomeration.

[0050] In summary, the polynucleotide can significantly improve the dispersibility of hydroxyapatite. In combination with the macroscopic and microscopic results, the dispersing and anti-agglomeration effects of the HAP microspheres reach the optimum when the polynucleotide concentration is 15 mg / mL.

[0051] Test Example 2: Suspension stability and rheological analysis 5 mL of the samples of Example 4 and Comparative Example 1 were injected into 10 mL transparent scale centrifuge tubes, and the tubes were vertically placed in a 25°C thermostat for standing. The supernatant height was recorded at 0, 1, 6, 24 and 48 hours, and the sedimentation rate was calculated.

[0052] Table 1: Sedimentation rate test results (%)

[0053] As shown in Table 2, when the mass ratio of the polynucleotide and hydroxyapatite is 2:1, the suspension stability of the HAP microspheres is improved by an order of magnitude.

[0054] Test Example 3: In vitro anti-inflammatory and regenerative effect evaluation Cell culture: Human dermal fibroblasts were cultured.

[0055] Anti-inflammatory test: Cells were induced to be inflamed by LPS, wherein the control group (without LPS): only 10% FBS-containing medium was added, without adding LPS and any sample. LPS model group: The original culture medium was discarded, and 100 μL of serum-free medium containing 1 μg / mL LPS (final concentration) was added to induce inflammation. LPS+Comparative Example 1 group: The original culture medium was discarded, 50 μL of serum-free medium containing 1 μg / mL LPS was added first, and then 50 μL of serum-free medium containing 1 μg / mL HAP was added to make HAP reach the set concentration. LPS+Comparative Example 2 group: The original culture medium was discarded, 50 μL of serum-free medium containing 1 μg / mL LPS was added first, and then 50 μL of serum-free medium containing 2 μg / mL PN was added. LPS+the application group: Discard the original culture medium, first add 50 μL of serum-free culture medium containing 1 μg / mL LPS, and then add 50 μL of serum-free culture medium containing the composite sample (the mass ratio of PN to HAP is 2:1, and the concentration of HAP is 1 μg / mL).

[0056] After incubation of each group for 24 hours, the concentrations of inflammatory factors IL-6 and TNF-α in the supernatant were detected by ELISA, and the test results are shown in Table 2.

[0057] Collagen promotion test: HDFs were treated with different samples. After 48 hours, the secretion amount of type I procollagen in the supernatant was detected by ELISA, and the test results are shown in Table 3.

[0058] Table 2: Inflammatory factor levels in cell supernatant (pg / mL)

[0059] Table 3: Type I procollagen secretion amount in cell supernatant (ng / mL)

[0060] As can be seen from Tables 1 and 2, in terms of anti-inflammation, the anti-inflammatory effect of the application group is significantly better than that of the HAP group alone, which proves that PN can effectively inhibit the inflammation that may be caused by HAP; in terms of collagen promotion, the PICP secretion amount of the application group is significantly higher than the simple addition of the HAP group and the PN group, which proves that there is a synergistic effect (1+1>2) between PN and HAP in promoting collagen regeneration.

[0061] Test Example 4: Evaluation of filling effect and biocompatibility in animals 8-week-old mice were selected, and 0.2 mL of the composite sample of Example 4, Comparative Example 1 (10 mg / mL HAP), and the normal group (PBS solution) were subcutaneously injected on both sides of the back of the mice, respectively.

[0062] Macroscopic evaluation (redness, induration palpation score) and histological analysis (H&E staining) were performed at 1 day, 1 week, 4 weeks, and 8 weeks after injection.

[0063] Table 4: Score and symptom performance correspondence table

[0064] Table 5: Animal palpation score (n=5, average)

[0065] The results of H&E staining (4 weeks) are shown in Table 5, and the results are shown in Table 6. Figure 3 Figure 3 ​It can be seen from the comparative example 1 that the HAP microspheres are aggregated and surrounded by a large number of inflammatory cell infiltrations; the inventive group PN+HAP microspheres are uniformly distributed and have very few inflammatory cell infiltrations.

[0066] Conclusion: The animal experiment proves that the PN / HAP compound of the application has better biocompatibility in vivo, significantly reduces the risk of redness and induration after injection, and promotes more uniform and higher quality collagen regeneration.

[0067] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. A compound of polynucleotide and hydroxyapatite, characterized in that, Includes hydroxyapatite microspheres, polynucleotides, and a carrier matrix; The mass ratio of the polynucleotide to hydroxyapatite is 0.5 to 2:

1.

2. The polynucleotide and hydroxyapatite complex according to claim 1, characterized in that, The carrier matrix includes physiological saline, phosphate buffer, or hyaluronic acid gel.

3. The polynucleotide and hydroxyapatite complex according to claim 1, characterized in that, The concentration of hydroxyapatite microspheres in the polynucleotide and hydroxyapatite complex is 5~30 mg / mL.

4. The polynucleotide and hydroxyapatite complex according to claim 1, characterized in that, The hydroxyapatite microspheres have a particle size of 15~50 μm.

5. The polynucleotide and hydroxyapatite complex according to claim 1, characterized in that, The polynucleotide is of DNA type and has a molecular weight range of 1000KD to 10000KD.

6. A method for preparing the polynucleotide and hydroxyapatite complex according to any one of claims 1 to 5, characterized in that, Includes the following steps: Hydroxyapatite microspheres, polynucleotides, and a carrier matrix were mixed and dispersed to obtain a complex of polynucleotides and hydroxyapatite.

7. The preparation method according to claim 6, characterized in that, The dispersion was carried out at room temperature for 4 hours.

8. The use of the polynucleotide and hydroxyapatite compound according to any one of claims 1 to 5 in the preparation of medical aesthetic filler drugs.

9. The use of the polynucleotide and hydroxyapatite compound according to any one of claims 1 to 5 in the preparation of a skin soft tissue filling drug.

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