Preparation method of PLLA-nucleotide encapsulated nanoparticles, nanoparticles and application thereof

Abstract

The invention relates to a preparation method of PLLA-PN encapsulated nano-particles, nano-particles and application thereof, and the preparation method comprises the following steps: carrying out ultrasonic treatment on a PN solution, immediately carrying out vacancy for 5 seconds after carrying out ultrasonic treatment for 4 seconds, continuing for 5 minutes, taking out the PN solution, uniformly mixing for one time, and repeating for multiple times; the preparation method comprises the following steps: dissolving PLLA in dichloromethane to obtain a PLLA solution; mixing the PN solution subjected to ultrasonic treatment with a PLLA solution, then carrying out ultrasonic treatment, immediately carrying out vacancy for 5 seconds after carrying out ultrasonic treatment every 4 seconds, and continuing for 5 minutes to obtain a mixed solution; a PVA solution is added into the mixed solution, ultrasonic treatment is carried out, vacancy is carried out for 5 s after ultrasonic treatment is carried out every 4 s, and the operation lasts for 2 min; a raw material solution is obtained; and adding an isopropanol solution into the obtained raw material solution, and stirring to volatilize the organic solvent to obtain a PLLA-PN encapsulated nanoparticle solution. According to the invention, long-acting stable slow release of PN is realized through PLLA encapsulation.

Description

Technical Field

[0001] The present invention relates to the field of polymer materials and products, and particularly to a preparation method of PLLA-PN encapsulated nanoparticles, the nanoparticles and their applications. Background Art

[0002] With the intensification of aging, more and more people enter old age, and the aging of people will continue to intensify over time. Coupled with exposure to ultraviolet rays, environmental pollution, high work pressure, lack of sleep, etc., all these will exacerbate our aging.

[0003] Currently, in the field of medical aesthetics, PN (polynucleotide) is mainly used for repair and anti-aging. PN has the characteristics of macromolecules, long-chain double helix, and three-dimensional stereoscopic scaffold structure. Its unique three-dimensional scaffold structure can build extracellular matrix growth space in the dermis layer and play a tissue regeneration function. The three-dimensional structure can also help the skin to condense moisture, improve the water-locking and moisturizing ability, improve the skin texture, smooth fine lines, and achieve anti-aging effects. It is suitable for situations such as dull skin, aging, dry and water-deficient skin, rough and dull skin, skin relaxation, facial fine lines, lack of elasticity, and thinning of the skin.

[0004] However, currently, when applying PN in the industry, often only a single component is used, lacking a sustained-release effect. It will take effect quickly after entering the human body. However, since only a small amount of the product can be added, there is only a short-term and minor effect, without a long-term effect. For beauty seekers, they need to inject every month to achieve the anti-aging effect. However, the monthly skin breakage repair causes greater harm to human tissues. In the long run, it is actually not worth the loss. It can be seen that the current application of PN products faces problems such as a relatively short continuous action time, an unclear action effect, and an excessive effective dose. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a preparation method of PLLA-PN encapsulated nanoparticles, the nanoparticles and their applications. PN has the characteristics of macromolecules, long-chain double helix, and three-dimensional stereoscopic scaffold structure. The method provided by the present invention uses PLLA to encapsulate PN, which can make PN release slowly. Compared with the traditional single PN product, the encapsulated nanomaterial can play a long-term control role through the control of the sustained-release time, effectively improving the problem of the short action time of the traditional PN product; it can be applied to many medical aesthetic products, such as hydrodermabrasion needles, etc., improving the disadvantages of the current single PN hydrodermabrasion product with a small addition amount and a short maintenance time, and providing a new choice for the medical aesthetic field.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides a preparation method of PLLA-PN encapsulated nanoparticles, comprising the following steps:

[0008] S1. Dissolve PN in water to obtain a PN solution with a concentration of 10 mg / mL;

[0009] S2. Ultrasonically treat the PN solution described in S1, set the duty cycle to 45%, perform 4 s of ultrasonic treatment followed by 5 s of idle time immediately after each 4 s of ultrasonic treatment, take it out and mix well once after 5 min, and repeat this process multiple times until PN is completely dissolved in water;

[0010] S3. Dissolve PLLA to obtain a PLLA solution with a concentration of 10 mg / mL;

[0011] S4. Mix the PN solution after ultrasonic treatment in S2 with the PLLA solution obtained in S3, and perform ultrasonic treatment, set the duty cycle to 45%, perform 4 s of ultrasonic treatment followed by 5 s of idle time immediately after each 4 s of ultrasonic treatment, and continue for 5 min; to obtain a mixed solution;

[0012] S5. Add 25 mL of 2% PVA solution to the mixed solution obtained in S4, perform ultrasonic treatment, perform 4 s of ultrasonic treatment followed by 5 s of idle time immediately after each 4 s of ultrasonic treatment, and continue for 2 min; to obtain a raw material solution;

[0013] S6. Add 50 mL of 2% isopropanol solution to the raw material solution obtained in S5, stir at room temperature overnight to fully volatilize the organic solvent, and obtain a PLLA-PN encapsulated nanoparticle solution.

[0014] In a possible implementation, in S1, replace PN with PDRN.

[0015] In a possible implementation, for the ultrasonic treatment described in S2 and S4, ice-bath ultrasonic treatment is performed.

[0016] In a possible implementation, in S2, during the process of repeating the ultrasonic treatment multiple times, perform at least one nucleic acid electrophoresis on the PN solution to detect the molecular weight distribution of PN.

[0017] In a possible implementation, the organic solvent used for dissolution in S3 is dichloromethane.

[0018] In a possible implementation, the volume ratio of the PN solution in S2 to the PLLA solution in S3 is 5:1 to 1.5.

[0019] In a possible implementation, it further includes:

[0020] S7. Freeze-dry the PLLA-PN encapsulated nanoparticle solution obtained in S6 to obtain a PLLA-PN encapsulated nanoparticle lyophilized powder.

[0021] Second aspect, the present invention provides a nanoparticle with a core-shell structure, where the core is a nucleotide, specifically it can be PN (polynucleotide) or PDRN (polypolydeoxyribonucleotide), and at least a part of the outer region thereof is encapsulated with PLLA. This nanoparticle can be obtained according to the preparation method of the aforementioned PLLA-PN encapsulated nanoparticle.

[0022] Furthermore, the encapsulation rate of the PLLA is 90% - 95%.

[0023] Third aspect, the present invention provides the application of the above-mentioned nanoparticle (such as the application in preparing related medicaments), and the application includes:

[0024] Sustained-release administration of nucleotide molecules including PN, and the administration methods include subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, external application, and oral administration;

[0025] Applied to beauty and plastic surgery, including anti-skin aging, where the skin aging includes skin fine lines, relaxation, thinning, roughness and dullness, etc.; sensitive skin repair, skin anti-inflammatory and anti-allergic, skin moisturizing, skin cell regeneration and barrier repair, improving skin microcirculation;

[0026] Applied to the adjuvant treatment of inflammatory skin diseases, including acne, atopic dermatitis, psoriasis, and folliculitis.

[0027] The above technical solutions provided by this application have at least the following technical effects or advantages:

[0028] (1) In this application, PN (polynucleotide) or PDRN (polypolydeoxyribonucleotide) is encapsulated by poly-L-lactic acid (PLLA), achieving the long-term sustained release of PN or PDRN, and can effectively improve the action time and effect of PN or PDRN. When applied to medical aesthetic products, compared with traditional monomeric PN products, it can play a long-term control role by controlling the sustained release time, effectively improving the problem of short action time of traditional monomeric PN products.

[0029] (2) Since this application achieves the long-term sustained release of PN or PDRN by encapsulating PN or PDRN with PLLA, when applied to medical aesthetic products, users can tolerate a larger dose of PN or PDRN without causing a heavy burden on the human body. On the other hand, a larger dose of PN or PDRN also means more lasting and obvious effects. With one injection, the effective period is longer, avoiding the impact and harm on the user's body caused by long-term frequent use to maintain the effect.

[0030] (3) The PN nanoparticles provided by this application achieve the purpose of long-term sustained release of polynucleotide PN through the encapsulation technology, with a high encapsulation rate, significant long-term sustained release effect and very stable performance.

[0031] (4) After the PLLA-PN encapsulated nanoparticle solution provided in this application is prepared, it can be put into use immediately. If long-term storage or long-distance transportation is required, it can be further prepared into freeze-dried powder for long-term storage, and the effect and activity of the PLLA-PN encapsulated nanoparticles are not affected. Description of the Drawings

[0032] Figure 1 It is the nucleic acid electrophoresis result diagram after the S2 repeated ultrasonic treatment 10 times in Example 1 of this application;

[0033] Figure 2 It is the particle size distribution diagram of the obtained encapsulated nanoparticles in Example 1 of this application. Detailed Embodiments

[0034] The following combines the drawings to exemplarily illustrate the specific implementation solutions of the present invention.

[0035] Example 1

[0036] This example provides a preparation method of PLLA-PN encapsulated nanoparticles. In this example, polynucleotide (PN) is used as the material to be encapsulated, and the method includes the following steps:

[0037] S1. Drug dissolution: Dissolve PN in ultrapure water to obtain a PN solution with a concentration of 10 mg / mL, and the total volume of the PN solution is 25 mL;

[0038] S2. Ultrasonic treatment of the PN solution on ice: Use a 6 mm horn, set the duty cycle to 45%, after each 4 s of ultrasonic treatment, immediately leave it idle for 5 s, and continue for a total of 5 min, then take it out and mix it once; repeat several times; after repeating the ultrasonic operation 10 times, perform nucleic acid electrophoresis to observe the molecular weight distribution of PN, and judge whether to continue ultrasonic treatment according to the judged molecular weight distribution of PN. The observation results are as Figure 1 shown;

[0039] S3. PLLA dissolution: Dissolve PLLA in dichloromethane to obtain a PLLA solution with a concentration of 10 mg / mL, and the total volume of the PLLA solution is 5 mL;

[0040] S4. Mix the PN solution and the PLLA solution, and perform ultrasonic treatment on ice: Set the duty cycle to 45%, after each 4 s of ultrasonic treatment, immediately leave it idle for 5 s, and continue for a total of 5 min; obtain a mixed solution;

[0041] S5. Add 25 mL of 2% PVA solution to the mixed solution obtained in S4, and perform ultrasonic treatment: After each 5 s of ultrasonic treatment, immediately leave it idle for 5 s, and continue for a total of 2 min; obtain a raw material solution;

[0042] S6. Add 50 mL of 2% isopropanol solution to the raw material solution obtained in S5, and stir overnight at room temperature to fully volatilize the organic solvent, thus obtaining the PLLA-PN encapsulated nanoparticle solution;

[0043] S7. Freeze-dry the PLLA-PN encapsulated nanoparticle solution obtained in S6 to obtain the freeze-dried powder of PLLA-PN encapsulated nanoparticles.

[0044] It should be noted that generally, after obtaining the PLLA-PN encapsulated nanoparticle solution in S6, the preparation is completed because the PLLA-PN encapsulated nanoparticles obtained in this example are usually used in the form of a solution; however, considering the situations of long-term storage and long-distance transportation, S7 is added in this example to prepare the PLLA-PN nanoparticle solution into a freeze-dried powder, which can be dissolved in a solvent to obtain a solution for use before use, and freeze-drying preservation will not affect the structure and activity of the PLLA-PN nanoparticles.

[0045] In this example, dichloromethane is preferentially used as the solvent in S3, and the main reasons are as follows:

[0046] 1. Good solubility

[0047] Dichloromethane has good solubility in many polymers, organic compounds, and inorganic materials, and is suitable as a solvent in nanoparticle preparation.

[0048] When preparing PLLA-PN encapsulated nanoparticles, dichloromethane can dissolve the polymer, facilitating subsequent emulsification or solvent volatilization.

[0049] 2. Low boiling point (40 °C)

[0050] Dichloromethane has a low boiling point and is easy to remove by evaporation or vacuum distillation, thus quickly forming nanoparticles.

[0051] This property is particularly important in the emulsification-solvent evaporation method because the rapid solvent volatilization helps the solidification of nanoparticles.

[0052] 3. Immiscible with water

[0053] Dichloromethane is immiscible with water and is suitable for use in water-in-oil (W / O) or oil-in-water (O / W) emulsification systems.

[0054] During the emulsification process, dichloromethane, as the oil phase, can encapsulate the water-soluble drug PN to form nanoparticles.

[0055] 4. Forming nanoparticles with small particle size

[0056] The low viscosity and high volatility of dichloromethane contribute to the formation of nanoparticles with smaller particle sizes.

[0057] During the emulsification process, dichloromethane diffused rapidly into the aqueous phase, promoting the rapid solidification of the nanoparticles and thus reducing particle aggregation.

[0058] It should be noted that for environmental protection and safety considerations, other low-toxic solvents (such as ethyl acetate) can be tried to replace dichloromethane, but the above indicators are not as good as dichloromethane.

[0059] Duty cycle = ultrasonic emission time / total cycle time. The duty cycles set in S2 and S4 of this embodiment are finally determined by the applicant through multiple experimental attempts, but it should be noted that the setting of different experimental duty cycles for different samples requires preliminary exploration. In the actual implementation process of this application, there will be different adaptation duty cycles for raw materials with different concentrations and addition amounts, which can be flexibly adjusted according to actual conditions.

[0060] Next, the performance of the nanoparticles obtained in this example is characterized:

[0061] 1. Nucleic Acid Electrophoresis

[0062] As mentioned above, in S2, after repeating the ultrasonic operation 10 times, nucleic acid electrophoresis was performed to observe the molecular weight distribution of PN and compare it with the same PN solution without ultrasonic treatment. The results are shown in Figure 1 shown.

[0063] 2. Particle size detection

[0064] The particle size data of the nanoparticles detected are shown in Table 1 below:

[0065] Table 1-Particle size data of nanoparticles obtained in Example 1

[0066] MI (nm) 454 MN (nm) 302 MA (nm) 401 CS 14.96 SD 143.3 PDI 0.0998 Mz 455.5 si 0.151 Ski 0.1881 Kg 1.143

[0067] The particle size distribution of the nanoparticles obtained by the test is as follows Figure 2 shown.

[0068] 3. Encapsulation rate detection

[0069] The raw material solution after adding 2% isopropanol solution in part S6 was taken and divided into two equal volumes and placed in centrifuge tubes, one of which was marked as A and the other as B. After standing at room temperature for 2 hours, tube B was slowly centrifuged at 500g for 3 minutes, and the precipitate was discarded (the precipitate obtained by detection was PLLA molecules that did not form nanoparticles).

[0070] Place tube A and tube B in a freeze dryer and freeze-dry overnight; then add equal volumes of ultrapure water to dissolve them respectively;

[0071] The absorbance was detected at 247 nm; (the absorbance of tube B / the absorbance of tube A)% was the encapsulation efficiency.

[0072] After detection, the encapsulation rate of PN was 91.98%.

[0073] Example 2

[0074] This example provides a method for preparing PLLA-PDRN encapsulated nanoparticles. In this example, polydeoxyribonucleotide (PDRN) was used as the material to be encapsulated, and the operation steps were basically the same as those in Example 1, except that the raw material PN in Example 1 was replaced with PDRN.

[0075] In step S2 of this example, nucleic acid electrophoresis was also carried out and compared with the same PDRN solution without ultrasonic treatment. The results are as Figure 1 shown.

[0076] The PLLA-PN encapsulated nanoparticles and PLLA-PDRN encapsulated nanoparticles provided in Example 1 and Example 2 of this example can be used to prepare related medicaments and applied to:

[0077] Sustained-release administration of nucleotide molecules including PN, and the administration methods include subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, external application, and oral administration;

[0078] Applied to beauty and plastic surgery, including anti-skin aging, and the skin aging includes skin fine lines, relaxation, thinning, roughness and dullness, etc.; sensitive skin repair, skin anti-inflammatory and soothing (skin inflammation, acne, water-oil imbalance, skin pigmentation and dullness, etc.), skin moisturization, skin cell regeneration and barrier repair, improving skin microcirculation;

[0079] Applied to the adjuvant treatment of inflammatory skin diseases, including acne, atopic dermatitis, psoriasis, and folliculitis.

[0080] The various embodiments in this specification are described in a progressive manner. For the same or similar parts between the various embodiments, reference can be made to each other. The key points of each embodiment are the differences from other embodiments. The above embodiments are only used to illustrate the technical solutions of the present application, rather than limiting the present application; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application.

Claims

1. A preparation method of PLLA-PN encapsulated nanoparticles, characterized in that, It includes the following steps: S1. Dissolve PN in water to obtain a PN solution with a concentration of 10 mg / mL; S2. Ultrasonically treat the PN solution described in S1, set the duty cycle to 45%, after every 4 s of ultrasonic treatment, immediately leave it idle for 5 s, take it out and mix it evenly after 5 min, and repeat this process multiple times until PN is completely dissolved in water; S3. Dissolve PLLA to obtain a PLLA solution with a concentration of 10 mg / mL; S4. Mix the PN solution after ultrasonic treatment in S2 with the PLLA solution obtained in S3, and perform ultrasonic treatment, set the duty cycle to 45%, after every 4 s of ultrasonic treatment, immediately leave it idle for 5 s, and continue for 5 min; A mixed solution is obtained; S5. Add 25 mL of 2% PVA solution to the mixed solution obtained in S4, and perform ultrasonic treatment, after every 4 s of ultrasonic treatment, immediately leave it idle for 5 s, and continue for 2 min; A raw material solution is obtained; S6. Add 50 mL of 2% isopropyl alcohol solution to the raw material solution obtained in S5, stir at room temperature overnight to fully volatilize the organic solvent, and obtain a PLLA-PN encapsulated nanoparticle solution.

2. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, characterized in that, In S1, replace PN with PDRN.

3. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, characterized in that, The ultrasonic treatment described in S2 and S4 is ultrasonic treatment on ice.

4. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, wherein, In S2, during the process of repeatedly performing ultrasonic treatment multiple times, at least one nucleic acid electrophoresis is performed on the nucleotide solution to detect the molecular weight distribution of the nucleotide.

5. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, characterized in that, The solvent used for dissolution in S3 is dichloromethane.

6. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, wherein, The volume ratio of the nucleotide solution in S2 to the poly-L-lactic acid solution in S3 is 5:1 to 1.

5.

7. The preparation method of PLLA-PN encapsulated nanoparticles according to claim 1, characterized in that, It also includes: S7. Freeze-dry the PLLA-PN encapsulated nanoparticle solution obtained in S6 to obtain PLLA-PN encapsulated nanoparticles in the form of a freeze-dried powder.

8. A nanoparticle, characterized in that, The nanoparticle is a core-shell structure, the core is a nucleotide, and at least part of its exterior is encapsulated with poly-L-lactic acid.

9. A nanoparticle according to claim 8, wherein, The encapsulation rate of the poly-L-lactic acid is 90% - 95%.

10. Application of the nanoparticle according to claim 8, the application includes: Sustained-release administration of nucleotide molecules including PN, and the administration methods include subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, intravenous injection, topical application, and oral administration; Applied to beauty and plastic surgery, including anti-skin aging, and the skin aging includes skin fine lines, relaxation, thinning, roughness and dullness; Sensitive skin repair, skin anti-inflammatory and soothing, skin moisturizing, skin cell regeneration and barrier repair, improving skin microcirculation; Applied to the adjuvant treatment of inflammatory skin diseases, including acne, atopic dermatitis, psoriasis, and folliculitis.

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