Nanometer delivery system for scar treatment and preparation method and application thereof

Abstract

The invention discloses a nano delivery system for scar treatment as well as a preparation method and application of the nano delivery system. The preparation method of the nano-delivery system comprises the following steps: firstly, forming a compound from a cationic substance and small interfering RNA (siRNA) through electrostatic interaction, and then forming nano-particles from the compound and a drug-loaded functional polymer through electrostatic interaction and hydrogen bond self-assembly. The nano delivery system provided by the invention is a novel nano system based on double-network physical crosslinking, so that the slow release of siRNA and drugs is realized, and the formation of keloids is reduced by improving the immune microenvironment at a focus, inhibiting the fibrosis degree of the keloids, so that the scars are effectively treated.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology. More specifically, it relates to a nano-delivery system for scar treatment, its preparation method and applications. Background Art

[0002] With the reduction in the mortality rate of severe burn-related diseases, the goal of burn wound care is shifting towards the effective management of burn scars. Keloid is a skin fibrosis disease caused by abnormal wound healing responses, generally manifested as large and hard raised scars. Different from hypertrophic scars, keloids grow beyond the boundaries of the injured skin and into the nearby normal skin, with the connective tissue's response to trauma exceeding the normal range, resulting in excessive deposition of the extracellular matrix and over-proliferation of fibroblasts. Among burn survivors, most patients are troubled by keloids, usually showing adverse functional and cosmetic effects such as pain, itching, and pigmentation. However, current treatment methods have not achieved completely satisfactory results and are usually accompanied by potential side effects and risks. The high recurrence rate and limited treatment success rate of keloids pose significant challenges and uncertainties to their management. These characteristics make the treatment of keloids a challenge, requiring comprehensive consideration of various treatment methods.

[0003] In addition, some drugs for scar treatment, such as chlorogenic acid, have high cytotoxicity and low bioavailability when used alone, posing challenges to practical applications. Because of the high cytotoxicity of the drug, the safe dosage is relatively low. If the dosage exceeds the safe dosage at one time, it will affect the survival of normal cells to a certain extent. And for the application scenarios of long-term use, there are currently no publicly available and relatively effective new dosage form products for scar treatment.

[0004] Delivery of exogenous nucleic acids to precisely regulate gene expression in specific cells is considered to have great potential in the treatment of various human diseases. Among them, the RNA interference technology of siRNA shows a promising gene regulation strategy, which can specifically target and silence disease-related genes, providing a better choice for traditional small molecule therapies. Although this technology has great potential, due to its hydrophilic and polyanionic properties, naked siRNA has low efficiency in crossing biological barriers and is easily degraded by ribonucleases, resulting in reduced therapeutic activity.

[0005] Therefore, the present invention aims to develop a nano-delivery system with dual sustained-release effects of drugs and siRNA to effectively treat scars, especially keloids. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, an object of the present invention is to provide a preparation method of a nano-delivery system for scar treatment and the nano-delivery system prepared by this method. Through the double sustained-release design of drugs and siRNA, the long-term release of drugs and siRNA can be achieved, solving the problems of poor stability and low bioavailability of scar treatment drugs, and easy degradation and poor stability of siRNA.

[0007] Another object of the present invention is to provide the application of the above nano-delivery system in the preparation of drugs for scar treatment.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a preparation method of a nano-delivery system for scar treatment, and the preparation method includes the following steps;

[0010] (1) Mix the cationic substance solution and the siRNA solution to obtain the complex solution A;

[0011] (2) Add the drug solution to the functional polymer I solution, and adjust the solution pH to 5-7 to obtain the mixed solution B;

[0012] (3) Mix the complex solution A and the functional polymer II solution to obtain the mixed solution C;

[0013] (4) Add the mixed solution B to the mixed solution C, self-assemble to obtain a nano-particle suspension, and centrifuge to obtain;

[0014] Wherein, the functional polymer I is selected from one of polylysine, cellulose, and polyvinyl alcohol;

[0015] The functional polymer II is selected from one of sodium alginate, carboxymethyl dextran, and guar gum.

[0016] The present invention first forms a stable complex between the cationic substance and siRNA through electrostatic interaction, and then wraps the functional polymer nanoparticles loaded with drugs on the outer layer to achieve the double release of drugs and siRNA. That is, when acting on the affected area, the drug is first continuously released from the nanoparticles, and at the same time, the siRNA is sent to the cytoplasm to play a role due to the disintegration of the nanoparticles. siRNA is usually a double-stranded RNA fragment containing 19-23 base pairs. In the present invention, the sequence of siRNA is not particularly limited and can be designed according to actual treatment needs. The nano-delivery system of the present invention also solves the problems of poor stability and low bioavailability of natural drugs, as well as easy degradation and poor stability of siRNA.

[0017] Furthermore, the cationic substance is a cationic polymer or a cationic liposome.

[0018] Further, the cationic polymer is selected from one of polyethyleneimine, chitosan, and dextran sulfate.

[0019] Further, the cationic liposome is trimethyl-2,3-dioleyloxypropylammonium bromide or dimyristoyl trimethyl ammonium chloride.

[0020] Further, the drug is selected from one of chlorogenic acid, epigallocatechin gallate, or curcumin.

[0021] Chlorogenic acid, epigallocatechin gallate, or curcumin are natural drugs with various biological activities such as antioxidant and anti-inflammatory properties, and show great potential in inhibiting angiogenesis. However, they have high cytotoxicity when used alone, and their inherent low bioavailability poses challenges for practical applications. Because of the high cytotoxicity of these drugs, the safe dosage is relatively low. If the single dosage exceeds the safe dosage, it will affect the survival of normal cells to a certain extent. And for the application scenarios of long-term use, there are currently no publicly available new dosage form products of the above drugs that are more effective. The present invention realizes the dual sustained release of the above drugs and siRNA through the above nano-delivery system, providing new ideas for the treatment of scars with these drugs.

[0022] Further, the concentration of the cationic substance solution is 1 μg / ml - 6 μg / ml.

[0023] Further, the concentration of the siRNA solution is 0.1 - 2 μg / ml;

[0024] Further, in the complex solution A, the mass ratio of the cationic substance to siRNA is (1 - 6):(0.1 - 2).

[0025] Further, the concentration of the drug solution is 1 μg / ml - 100 μg / ml.

[0026] Further, the concentration of the functional polymer I solution is 0.1 mg / ml - 2 mg / ml.

[0027] Further, in the mixed solution B, the mass ratio of the drug to the functional polymer I is (1 - 100):(100 - 2000).

[0028] Further, the concentration of the functional polymer II solution is 0.1 mg / ml - 2 mg / ml.

[0029] Further, when mixing, the mass ratio of the complex solution A to the functional polymer II solution is (1 - 20):(1 - 20).

[0030] Further, during mixing, the volume ratio of the mixed solution B to the mixed solution C is (1 - 8):(8 - 1).

[0031] Further, the centrifugation conditions are centrifugation at 5000 rpm for 30 min.

[0032] The present invention also claims to protect the nano - delivery system for scar treatment prepared by the above - mentioned preparation method.

[0033] In the second aspect, the present invention provides the application of the above - mentioned nano - delivery system in the preparation of products for treating scars.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) Compared with single - drug treatment, the synergistic effect produced by multi - drug treatment will significantly enhance the curative effect. The nano - delivery system provided by the present invention is based on a multi - drug combination treatment strategy, which can synergistically play a therapeutic role through multiple pathways such as regulating angiogenesis, regulating the immune microenvironment, and inhibiting collagen deposition. At the same time, the combination of multiple drugs can reduce the single - drug dose, thereby reducing the side effects and drug resistance caused by single - drug treatment. Compared with traditional scar treatment materials, the nano - system provided by the present invention overcomes the disadvantage of the single function of traditional materials in terms of raw materials.

[0036] (2) In terms of structure, the nano - particles formed by self - assembly through electrostatic interaction in the nano - delivery system provided by the present invention have stable properties and can continuously release the content. The nano - particles not only have good biocompatibility, but also can effectively control the release of drugs, improve the drug utilization rate and continuously play a role, well meeting the needs of scar treatment.

[0037] (3) The preparation method of the nano - delivery system provided by the present invention, which can effectively inhibit angiogenesis and regulate the immune microenvironment, has a simple process, low production cost, and is easy to mass - produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings.

[0039] Figure 1 Shows the physicochemical property characterization of the nano - particles prepared in Example 1; A is the Fourier transform infrared spectroscopy diagram, B is the DLS measurement, C is the zeta potential experiment, and D is the SEM diagram.

[0040] Figure 2 Shows the chlorogenic acid drug release curve of the nano - particles prepared in Example 1.

[0041] Figure 3 Shows the biocompatibility determination of the nano - particles prepared in Example 1; A is the cell viability of HUVEC and HKF, and B is the blood compatibility determination.

[0042] Figure 4 Cell functional assay of the nanoparticles prepared in Example 1; A shows the proliferation of HUVEC after culturing with the nanoparticles. B shows the apoptosis of HUVEC after culturing with the nanoparticles. C shows the cell cycle of HUVEC and HKF after treatment with the nanoparticles.

[0043] Figure 5 Shows the situation of the nanoparticles prepared in Example 1 regulating the immune microenvironment; A shows the content of NO released by RAW264.7 after culturing with the nanoparticles, and B shows the expression of inflammatory factors of RAW264.7 after culturing with the nanoparticles. Detailed implementation manners

[0044] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0045] Example 1

[0046] A complex solution A was obtained by mixing and incubating a 4 μg / ml polyethyleneimine solution and a 1.3 μg / ml siRNA solution at a mass ratio of 4:1. A 50 μg / ml chlorogenic acid solution was added to a 0.25 mg / ml polylysine solution, and the pH of the solution was adjusted to 6 to obtain a mixed solution B, where the mass ratio of chlorogenic acid to polylysine was 1:5. The complex solution A was mixed with a 0.25 mg / ml sodium alginate solution at a mass ratio of 1:1 to obtain a mixed solution C; the mixed solution B was added to the mixed solution C at a volume ratio of 2:1, and self-assembled to obtain a nanoparticle suspension, and then centrifuged at 5000 rpm for 30 min to obtain the nanoparticles, namely the nano-delivery system.

[0047] Example 2

[0048] A complex solution A was obtained by mixing and incubating a 5 μg / ml chitosan solution and a 1.5 μg / ml siRNA solution at a mass ratio of 5:1.5. A 60 μg / ml epigallocatechin gallate solution was added to a 1 mg / ml polylysine solution, and the pH of the solution was adjusted to 6.5 to obtain a mixed solution B, where the mass ratio of epigallocatechin gallate to polylysine was 1:16. The complex solution A was mixed with a 0.5 mg / ml sodium alginate solution at a mass ratio of 1:2 to obtain a mixed solution C; the mixed solution B was added to the mixed solution C at a volume ratio of 4:1, and self-assembled to obtain a nanoparticle suspension, and then centrifuged at 5000 rpm for 30 min to obtain the nanoparticles, namely the nano-delivery system.

[0049] Example 3

[0050] A complex solution A was obtained by mixing a 3 μg / ml polyethyleneimine solution and a 1 μg / ml siRNA solution at a mass ratio of 3:1 and incubating. A 50 μg / ml curcumin solution was added to a 0.5 mg / ml cellulose solution, and the pH of the solution was adjusted to 6 to obtain a mixed solution B, where the mass ratio of curcumin to cellulose was 1:10. The complex solution A was mixed with a 1 mg / ml carboxymethyl dextran solution at a mass ratio of 2:1 to obtain a mixed solution C; the mixed solution B was added to the mixed solution C at a volume ratio of 2:1, and self-assembly was carried out to obtain a nanoparticle suspension, and then nanoparticles were obtained by centrifugation at 5000 rpm for 30 min, that is, the nano-delivery system.

[0051] Test Example 1

[0052] According to the method of Example 1, a series of nanoparticles (PCS) with concentration gradients were prepared. A Fourier transform infrared spectrometer was used to detect the FT-IR spectra of polylysine (PL), sodium alginate (SA), chlorogenic acid (CGA), and nanoparticles (PCS) in the range of 4000-400 cm -1 . The Fourier transform infrared spectra of PL, SA, CGA, and PCS are shown as A in Figure 1 . The characteristic peaks of CGA at 1686 cm -1 , 1639 cm -1 and 1518 cm -1 indicate the presence of benzene rings and -C=C-. The characteristic peaks at 1609 cm -1 and 3432 cm -1 show the presence of the carboxyl and hydroxyl groups of SA. The characteristic peaks at 3448 cm -1 and 1673 cm -1 are the presence of the amino and carboxyl groups of PL. By comparing the infrared curves of CGA, SA, and PL, the characteristic peaks of the above components are all reflected in the nanoparticle PCS curve, indicating the successful synthesis of PCS. Further, the zeta potential and DLS diameter of the nanoparticles were measured respectively. The results in B and C of Figure 1 show that the zeta potential of PCS is 26.45 mv and the diameter is 201.8 nm. The structure of the nanoparticles was observed by scanning electron microscopy. The prepared PCS suspension was dropped on a silicon wafer and air-dried naturally, and then imaged using a microscope. As can be seen from the SEM results in D of Figure 1 , the prepared nanoparticles are spherical particles. The above results indicate that the prepared nanoparticles with small particle size and positive charge have good external conditions for entering cells.

[0053] Test Example 2

[0054] The sustained release of drugs from nanoparticles is essential for the treatment of scars. The PCS was resuspended in PBS and incubated in a constant temperature shaker at 37 °C at a speed of 100 rpm. The PCS suspension was taken out at certain time intervals and centrifuged, and the cumulative release rate of CGA was calculated. As can be seen from Figure 2 it, for the nanoparticles prepared in Example 1, the release rate of CGA was about 17.6% at 1 h and about 23.5% at 48 h. The experimental results showed that the nanoparticles prepared in Example 1 could continuously release drugs.

[0055] Test Example 3

[0056] Good biocompatibility is the first condition that a nanosystem must meet as a scar treatment material. Human umbilical vein endothelial cells (HUVEC) were selected as a cell model to study the abnormal angiogenesis at the keloid site, and keloid fibroblasts (HKF) were selected as a cell model to study the extracellular matrix deposition at the keloid site. The cytotoxicity of PCS was detected by Cell Counting Kit-8 (CCK-8). Based on the nanoparticles PCS prepared in Example 1, the effects on the viability of the two types of cells at 48 h were investigated. The control group was the sample group without nanoparticles. As shown in Figure 3 A, under the treatment of the nanoparticles, there was no obvious cytotoxicity in HUVEC and HKF within 48 h, and the cell survival rate was above 70%. The PCS was immersed in 1 mL of PBS, and 100 μl of citrated anticoagulant rabbit whole blood diluted 10 times with PBS was added. It was incubated in a constant temperature shaker at 37 °C at a speed of 100 rpm for 1 h. Then it was centrifuged at 4 °C and 1500 rpm for 5 min. The absorbance of the supernatant was measured at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Deionized water was used as the positive control and PBS as the negative control. As shown in Figure 3 B, compared with the control group, the blood compatibility of the nanoparticles prepared in Example 1 was less than 5%, demonstrating its good blood compatibility. In summary, the nanoparticles prepared in Example 1 had good biocompatibility.

[0057] Test Example 4

[0058] Inhibiting excessive angiogenesis and excessive fibroblast proliferation can effectively inhibit scar formation. HUVEC and HKF were respectively seeded in 6-well plates and cultured for 48 h. Then, the control group was replaced with the basal medium, and the experimental group was replaced with the PCS solution and cultured for another 48 h. Subsequently, the corresponding experiments were carried out using a cell proliferation kit, a cell apoptosis kit, and a cell cycle kit, and quantitative detection was performed using a flow cytometer. Figure 4 The analysis results of flow cytometry in A showed that the cell proliferation rate of the nanoparticle PCS treatment group was about 84% of that of the control group, indicating that the nanoparticles had an obvious inhibitory effect on the proliferation of HUVEC and HKF. As shown in Figure 4As shown in B, compared with the control group, PCS nanoparticles can effectively induce apoptosis and necrosis of HUVEC cells. Among them, in the early apoptosis stage, the number of cells in this stage after PCS treatment is 0.599% more than that in the control group. In the late apoptosis stage, the number of cells in this stage after PCS treatment is 2.4875% more than that in the control group. From Figure 4 As seen from the flow cytometry detection results in C, compared with the control group, after HUVEC and HKF were treated with PCS, there were no significant differences in the G1 and G2 phases, while there were significant differences in the S phase, indicating that PCS mainly exerts its function by affecting the S phase in the cell cycle. Compared with the control group, the proportion of cells in the S phase of the PCS group in HUVEC decreased by 1.7%, and the proportion of cells in the S phase of the PCS group in HKF decreased by 9.26%, indicating that PCS can inhibit cell DNA synthesis.

[0059] Experimental Example 5

[0060] Currently, it is considered that the inflammatory response plays an important promoting role in the pathological formation and development of scars. Different from the acute inflammatory response, one of the causes of scar formation is due to the continuous action of inflammatory factors and inflammatory responses for several weeks, resulting in progressive damage and repair of local tissues. A pro-inflammatory model was constructed by inducing macrophages (RAW264.7) to polarize into the M1 type using lipopolysaccharide (LPS). RAW 264.7 was inoculated in a 6-well plate and cultured for 24 h. Then, the blank control group was replaced with the basal medium, the positive control group was replaced with the complete medium containing 25 μg / ml LPS, and the experimental group was replaced with the PCS solution containing 25 μg / ml LPS to continue culturing the cells. After 48 h, the supernatant was taken and the content of NO in the supernatant was detected according to the instructions. From Figure 5 As seen from the detection results in A, there was a significant difference in the NO content in the supernatant between the LPS treatment group and the control group, indicating that the inflammatory model constructed by inducing RAW264.7 to polarize into the M1 type with LPS was successfully established. The results showed that after co-culturing RAW264.7 with PCS and LPS, compared with the LPS group, the content of NO decreased significantly and there was no significant difference from the control group. After culturing with PCS, the release of NO by macrophages could be significantly reduced. Further, RT-qPCR was used to detect and analyze the expression of inflammatory factors in RAW264.7 after PCS treatment. Figure 5 The results in B showed that compared with the LPS group, the gene expressions of TGF-β1, IL-13, and TNF-α in the co-culture group of PCS and LPS all decreased significantly.

[0061] The nanoparticles prepared in Examples 2 and 3 are also small-sized spherical particles, which can continuously release drugs, have good biocompatibility, can inhibit excessive angiogenesis and excessive proliferation of fibroblasts, effectively inhibit scar formation, and reduce the inflammatory response.

[0062] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A preparation method of a nano-delivery system for scar treatment, characterized in that, The preparation method comprises the following steps: (1) Mixing the cationic substance solution and the siRNA solution to obtain a complex solution A; (2) adding the drug solution to the functional polymer I solution and adjusting the pH of the solution to 5-7 to obtain a mixed solution B; (3) mixing the complex solution A and the functional polymer II solution to obtain a mixed solution C; (4) adding the mixed solution B to the mixed solution C to self-assemble to obtain a nanoparticle suspension, which is then centrifuged to obtain; Wherein, the functional polymer I is selected from one of polylysine, cellulose, and polyvinyl alcohol; The functional polymer II is selected from one of sodium alginate, carboxymethyl dextran and guar gum.

2. The preparation method according to claim 1, characterized in that, The cationic substance is a cationic polymer or a cationic liposome; Preferably, the cationic polymer is selected from one of polyethyleneimine, chitosan, and dextran sulfate; Preferably, the cationic liposome is trimethyl-2,3-dioleoyloxypropylammonium bromide or dioleoylpropyltrimethylammonium chloride.

3. The preparation method according to claim 1, characterized in that, The drug is selected from one of chlorogenic acid, epigallocatechin gallate, and curcumin.

4. The preparation method according to claim 1, characterized in that, The concentration of the cationic substance solution is 1 μg / ml-6 μg / ml; Preferably, the concentration of the siRNA solution is 0.1-2 μg / ml; Preferably, in the complex solution A, the mass ratio of the cationic substance to the siRNA is (1-6):(0.1-2).

5. The preparation method according to claim 1, characterized in that, The concentration of the drug solution is 1 μg / ml-100 μg / ml; Preferably, the concentration of the functional polymer I solution is 0.1 mg / ml-2 mg / ml; Preferably, in the mixed solution B, the mass ratio of the drug to the functional polymer I is (1-100):(100-2000).

6. The preparation method according to claim 1, characterized in that, The concentration of the functional polymer II solution is 0.1 mg / ml-2 mg / ml; Preferably, the mass ratio of the complex solution A to the functional polymer II solution is (1-20):(1-20).

7. The preparation method according to claim 1, characterized in that The volume ratio of the mixed solution B to the mixed solution C is (1-8):(8-1).

8. The preparation method according to claim 1, wherein The centrifugation condition is 5000 rpm for 30 min.

9. A nano-delivery system for scar treatment, characterized in that, The method is described in any one of claims 1 to 8.

10. Use of the nano delivery system according to claim 9 in preparing a product for treating scars.

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