Glycerin entrapped with recombinant human-derived III-type collagen as well as preparation method and application of glycerol entrapped with recombinant human-derived III-type collagen

Abstract

The invention discloses glycerin entrapped with recombinant human collagen III and a preparation method and application thereof, and belongs to the technical field of medicines and cosmetics, the glycerin entrapped with recombinant human collagen III comprises phospholipid, glycerin, Tween 80, chitosan quaternary ammonium salt, sodium hyaluronate, recombinant human collagen III and a PBS buffer solution. Glycerin which has high deformation performance and can effectively penetrate through the stratum corneum is prepared, and double-layer modification of chitosan quaternary ammonium salt and sodium hyaluronate is carried out on the glycerol to deliver the recombinant human-derived III-type collagen, so that the problems that the recombinant human-derived III-type collagen is high in hydrophilicity, large in molecular weight and difficult to penetrate through the stratum corneum of the skin can be effectively solved; the bioavailability and the percutaneous penetration of the recombinant human III type collagen are obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and cosmetics, and specifically relates to a glycerol body encapsulating recombinant human type III collagen, a preparation method and an application thereof. Background Art

[0002] Recombinant human collagen type Ш (RHC) is obtained by optimizing and recombining the original gene sequence of human collagen type Ш using genetic engineering technology, and is highly consistent with the amino acid sequence of natural human collagen. Recombinant human type Ⅲ collagen can enhance skin elasticity, reduce wrinkles, deeply moisturize, and promote skin healing. However, due to the water solubility of recombinant human type Ⅲ collagen and its large molecular structure, it is difficult for it to penetrate the lipid structure between stratum corneum cells. Therefore, a special delivery system, such as liposomes, is needed to prolong the action time of recombinant human type Ⅲ collagen in the skin, so that it can penetrate the stratum corneum and eventually be effectively delivered to the deep layer of the skin, thereby achieving the therapeutic effect.

[0003] Glycerolsomes are a new type of liposomes prepared by adding a certain amount of glycerol to liposomes. As the concentration of glycerol increases, the hydrophobicity of the phospholipid bilayer decreases and the stability of the vesicles is significantly enhanced. This type of liposome can overcome the shortcomings of traditional liposomes such as poor stability, easy aggregation, and poor transdermal effect, and effectively improve the stability of liposomes and drug utilization. In addition, multilayer liposomes appropriately coated with chitosan and hyaluronic acid polyelectrolytes through electrostatic interactions can further improve the stability of liposomes and serve as a potential drug delivery system for transdermal delivery.

[0004] Currently, there is no report on the delivery of recombinant human type II collagen using chitosan-hyaluronic acid bilayer-modified glycerosomes. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a glycerosome encapsulating recombinant human type III collagen.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: a glycerol body encapsulating recombinant human type III collagen, comprising phospholipids, glycerol, a surfactant, chitosan quaternary ammonium salt, sodium hyaluronate, recombinant human type III collagen and a PBS buffer solution;

[0009] Wherein, based on the total mass of the raw materials, the phospholipid is 1-20%, the glycerol is 5-35%, the surfactant is 0.5-3.5%, the chitosan quaternary ammonium salt is 0.05-1%, the sodium hyaluronate is 0.1-0.4%, the recombinant human type III collagen is 0.1-0.5%, and the PBS buffer solution is supplemented to 100%.

[0010] As a preferred embodiment of the glycerol bodies of the present invention, the phospholipids include at least one of hydrogenated lecithin, soybean lecithin and egg yolk lecithin.

[0011] As a preferred embodiment of the glycerosomes of the present invention, the surfactant comprises at least one of Tween 80, sodium cholate, sodium deoxycholate and Spans.

[0012] As a preferred embodiment of the glycerol body of the present invention, the average particle size of the glycerol body is 60-400 nm, and the particle size distribution index PDI of the glycerol body vesicle is 0.1-0.5.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing glycerosomes encapsulating recombinant human type III collagen, comprising:

[0014] Dissolve recombinant human type III collagen, glycerol, and surfactant in PBS buffer to obtain an aqueous phase;

[0015] Slowly and evenly add lecithin to the water phase and continue stirring at room temperature until hydration is complete;

[0016] The obtained liposome suspension is subjected to ultrasonic treatment to obtain glycerol bodies encapsulating recombinant human type III collagen;

[0017] Dissolve chitosan quaternary ammonium salt in PBS buffer, stir thoroughly to dissolve, and add dropwise the above-obtained glycerol bodies encapsulating recombinant human type III collagen under continuous stirring to obtain chitosan quaternary ammonium salt-modified chitosan quaternary ammonium salt-modified glycerol bodies encapsulating recombinant human type III collagen;

[0018] Sodium hyaluronate was dissolved in PBS buffer, stirred thoroughly to dissolve, and the chitosan quaternary ammonium salt-modified glycerol body obtained above was added dropwise under continuous stirring to obtain chitosan quaternary ammonium salt-sodium hyaluronate double-layer-modified glycerol body encapsulating recombinant human type III collagen.

[0019] As a preferred embodiment of the preparation method of the present invention, the temperature of the hydration treatment is 15 to 35° C., and the time of the hydration treatment is 3 to 24 hours.

[0020] As a preferred embodiment of the preparation method of the present invention, the amplitude of the ultrasonic treatment is 30 to 80 μm, the power of the ultrasonic treatment is 180 to 530 W, and the time of the ultrasonic treatment is 60 to 300 s.

[0021] Another object of the present invention is to overcome the deficiencies in the prior art and provide a chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerol body encapsulating recombinant human type III collagen for use in the preparation of cosmetics.

[0022] Another object of the present invention is to overcome the deficiencies in the prior art and provide a chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerosome encapsulating recombinant human type III collagen for use in the preparation of medicines.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention can prepare vesicles with high deformability and stability by compounding ingredients such as phospholipids, glycerol, Tween 80, chitosan quaternary ammonium salt, sodium hyaluronate, etc. in specific proportions, thereby effectively promoting the effective utilization of recombinant human type III collagen.

[0025] (2) The present invention deposits charged polymers, nanoparticles or active molecules onto the surface of glycerol bodies through electrostatic interactions, thereby modifying them. This can effectively enhance the stability of glycerol bodies, increase the encapsulation efficiency of collagen, and reduce its permeation behavior in vesicles, thereby achieving slow and sustained release and improving the bioavailability of recombinant human type III collagen.

[0026] (3) The chitosan-hyaluronic acid bilayer modified glycerol bodies loaded with recombinant human type III collagen prepared by the present invention can significantly enhance the stability of liposomes and their affinity to the skin, while improving the permeability of drugs; the modified liposomes effectively improve the transdermal effect of drugs by regulating the interaction between surface charge, particle size and skin barrier, and show significant advantages in the delivery of recombinant human type III collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0028] Figure 1 The graphs showing the appearance and particle size changes of the glycerol bodies of Example 3 of the present invention stored at 4° C., 25° C. and 45° C. on days 1, 30 and 60.

[0029] Figure 2 This is a graph showing the total reflection-Fourier infrared spectroscopy scanning results of the present invention on RHC powder, Comparative Example 1, Example 4, and Example 16.

[0030] Figure 3 This is a graph showing the in vitro release test results of Example 4, Example 16, Comparative Example 1, and Comparative Example 2 of the present invention.

[0031] Figure 4 This is a test result diagram of transdermal absorption of Example 4, Example 16, Comparative Example 1, and Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] The present invention relates to recombinant human type III collagen and other raw materials used in experiments, all of which are common commercially available products.

[0036] Examples 1 to 20

[0037] An embodiment of the chitosan-hyaluronic acid double-layer modified glycerol body encapsulating recombinant human type III collagen of the present invention;

[0038] The glycerol body comprises phospholipids, glycerol, a surfactant, chitosan quaternary ammonium salt, sodium hyaluronate, recombinant human type III collagen (RHC) and PBS buffer solution, wherein the surfactant is Tween 80; the mass percentage of each component in the glycerol body is shown in Table 1.

[0039] The preparation method of the glycerol body containing recombinant human type III collagen is as follows:

[0040] (1) Prepare the raw materials of each component according to Table 1;

[0041] (2) dissolving glycerol, Tween 80, and recombinant human type III collagen in PBS buffer to obtain an aqueous phase;

[0042] (3) adding phospholipids to the aqueous phase and stirring at room temperature until the phospholipids are completely dissolved;

[0043] (4) using an ultrasonic cell disruptor to ultrasonically treat the hydration product obtained in step (3), wherein the amplitude of the ultrasonic treatment is 70 μm, the time of the ultrasonic treatment is 180 s, the working cycle of the ultrasonic probe of the ultrasonic cell disruptor is 3 s, and the interval time between two adjacent working cycles is 3 s, to obtain glycerol bodies encapsulating recombinant human type III collagen;

[0044] (5) adding the glycerol bodies obtained above dropwise into chitosan quaternary ammonium salt aqueous solutions of different concentrations to obtain chitosan quaternary ammonium salt-modified glycerol bodies;

[0045] (6) The liposomes obtained above are added dropwise to sodium hyaluronate aqueous solutions of different concentrations to obtain chitosan quaternary ammonium salt-sodium hyaluronate bilayer-modified glycerol bodies.

[0046] Table 1

[0047]

[0048] Comparative Example 1

[0049] This comparative example provides a method for preparing common liposomes (without chitosan or hyaluronic acid) encapsulating recombinant human type III collagen:

[0050] Weigh 1.5 g of phospholipids, 2.5 g of glycerol, and 20 mg of recombinant human type III collagen, add PBS buffer to 10 g, hydrate at 25°C overnight to obtain a liposome suspension, and use an ultrasonic cell disruptor to ultrasonically disrupt the liposome suspension for 180 s (70 μm, 3 s on, 3 s off).

[0051] Comparative Example 2

[0052] This comparative example provides a recombinant human type III collagen directly mixed with PBS buffer at a concentration of 2 mg / mL.

[0053] Effect Example 1

[0054] The products obtained in the above embodiments and comparative examples were used as samples and the following tests were performed:

[0055] (1) Each sample was sealed in a transparent glass sample bottle and its appearance was observed;

[0056] (2) The average particle size of the vesicles in the sample was determined by Zeta potential and nanoparticle size analyzer. Each sample was placed in a transparent sample bottle, sealed and stored at 4°C for 30 days. The sample was diluted to an appropriate concentration with deionized water and vortexed on a vortex oscillator to mix evenly. The particle size, PDI and Zeta potential of the sample were analyzed by Zeta potential and nanoparticle size analyzer.

[0057] (3) The encapsulation efficiency of recombinant human type III collagen in each sample was tested. The specific test steps were as follows: a certain amount of sample was dialyzed in a dialysis bag with a molecular weight of 300,000 Da for 12 h, the liquid in the bag was taken out and fixed to volume, and then methanol was used to break the emulsion and fix the volume, and then the sample was filtered with a water filter membrane (pore size 220 nm) and the content of recombinant human type III collagen therein was detected by high performance liquid chromatography, and recorded as W 1。 A certain amount of sample was taken and demulsified with methanol and fixed to volume. The content of recombinant human type III collagen was obtained by the same filtration and detection method, recorded as W2, and the final encapsulation rate (%) was obtained as W1 / W2×100%;

[0058] The test results are shown in Table 2.

[0059] Table 2

[0060]

[0061]

[0062]

[0063] Examples 1 to 5 and Example 10 respectively explored the effects of different concentrations of glycerol on the preparation of glycerol bodies;

[0064] Examples 4 and 6 to 10 respectively explored the effects of different Tween 80 concentrations on the preparation of glycerosomes;

[0065] Examples 11 to 15 respectively explored the effects of different chitosan quaternary ammonium salt concentrations on the preparation of glycerol bodies;

[0066] Examples 13 and 16 to 20 respectively explored the effects of different sodium hyaluronate concentrations on the preparation of glycerol bodies;

[0067] As shown in Table 2, the stability of glycerol bodies is affected by the concentrations of glycerol and Tween 80. A certain concentration of glycerol can reduce the hydrophobicity of liposomes, increase the stability and encapsulation rate of liposomes. At the same time, Tween 80 at an appropriate concentration can be inserted into the phospholipid bilayer, reduce the fluidity of liposomes, and work together with glycerol to stabilize the vesicles. In addition, chitosan and hyaluronic acid can rely on electrostatic effects and adsorb on liposomes layer by layer. Due to the influence of the distribution state of chitosan and hyaluronic acid in water at different concentrations, the particle size of liposomes shows a trend of first increasing, then decreasing, and then increasing. At an appropriate concentration, the double-layer modification of chitosan and hyaluronic acid can fix the vesicles, increase the stability of liposomes and delay the release of recombinant human type III collagen.

[0068] The glycerosomes prepared in the embodiment of the present invention are yellow, transparent and uniform liquids, wherein the liposome structure is stable, the vesicle particle size does not change much after being stored at 4° C. for 60 days, and the encapsulation rate of recombinant human type III collagen is as high as 75.98%.

[0069] Effect Example 2

[0070] The present invention performs total reflection-Fourier infrared spectroscopy scanning on RHC powder, Example 4, Example 16, and Comparative Example 1.

[0071] Use a medicine spoon to scoop a small amount of RHC powder, or use a Pasteur pipette to absorb a trace amount of liposome liquid sample and drop it on the laser probe of the total reflection Fourier transform infrared spectrometer to collect FTIR infrared spectra of different samples.

[0072] like Figure 2 As shown in the figure, after modification with chitosan quaternary ammonium salt, the stretching vibration of liposome hydroxyl group changes from 3334.96cm -1 Low wave number 3309.18cm -1 As the phospholipid head group C=O moves, the stretching vibration absorption peak area of ​​phospholipid head group C=O also decreases. -1 The characteristic peak of the acetyl group of chitosan quaternary ammonium salt appeared at 3309.18 cm-1, indicating that chitosan quaternary ammonium salt was successfully adsorbed onto the surface of RHC-LIP through electrostatic interaction. In addition, after further addition of sodium hyaluronate, the stretching vibration of the liposome hydroxyl group increased from 3309.18 cm-1 to 3309.18 cm-1. -1 High wave number 3315.79cm -1 Mobile, 1736.87cm -1 The characteristic peak of the acetyl group of the quaternary ammonium salt of chitosan that appeared at 4° disappeared, indicating that sodium hyaluronate was modified onto the surface of RHC-LIP-QCS through electrostatic interaction.

[0073] Effect Example 3

[0074] The present invention conducts in vitro release tests on Example 4, Example 16, Comparative Example 1 and Comparative Example 2.

[0075] Accurately measure 1 mL of sample and add it to a 300,000 Da dialysis bag, clamp it with a clamp and put it into 25 mL of PBS (pH = 7.4) buffer solution, and react in a shaker at 37°C and 100 r / min. Take out 1 mL of release medium from the external solution at 6h, 8h, 12h, 24h, and 48h, and add 1 mL of fresh medium at the same time. Then, the taken liquid is measured by HPLC to calculate the release amount of nanoparticle RHC. The calculation formula for the total release is as follows:

[0076] Cumulative drug release (%) = ((C i *25+C (i-1) *1+...+C1*1) / total amount of drug in sample)*100%

[0077] i is the number of times taken out.

[0078] Results Figure 3 It can be seen that RHC is released rapidly within 0-12h and then tends to be slow. At 12h, the release rates of RHC-PBS, RHC-LIP, RHC-LIP-QCS, and RHC-LIP-QCS-HA are 83.72%, 60.2%, 40.34%, and 33.12%, respectively, indicating that the layer-by-layer modification of QCS and HA on the liposome surface can significantly affect the release rate of RHC and make it slowly released.

[0079] Effect Example 4

[0080] The present invention performs transdermal absorption test on Example 4, Example 16, Comparative Example 1 and Comparative Example 2.

[0081] First, prepare FITC-labeled RHC. Add fluorescein isothiocyanate (FITC) to 2 mL of 5 mg / mL RHC solution (prepared in 0.1 M, pH 9.0 carbonate buffer) and stir at room temperature in the dark for 5 hours to complete the cross-linking reaction. Subsequently, the labeled collagen solution is dialyzed multiple times using carbonate buffer to remove unbound FITC. After dialysis, the solution is freeze-dried and stored in the dark at -20°C.

[0082] FITC-labeled RHC was used to prepare different samples for transdermal detection.

[0083] The pig skin was placed between the supply pool and the receiving pool of the Franzs diffusion cell, 2 ml of sample was added to the supply pool and placed in the transdermal diffusion tester for 24 hours. After the transdermal test was completed, the pig was taken out and the skin surface was gently rinsed with water for 15 seconds, and the skin moisture was absorbed with filter paper. The skin was photographed with a fluorescence microscope and its fluorescence intensity was calculated using Image software.

[0084] The experimental results are from Figure 4 It can be seen that compared with the RHC-PBS sample, after RHC was encapsulated by glycerol bodies, the fluorescence intensity of the retained amount in the skin increased by 1.26 times; after the glycerol bodies were further modified with chitosan, the fluorescence intensity increased by 1.89 times; after the glycerol bodies were double-layer modified with sodium hyaluronate, the fluorescence intensity increased by 2.23 times, which shows that glycerol bodies can significantly improve the transdermal performance of collagen due to their excellent deformability, and the positive charge of chitosan and the adhesion of hyaluronic acid to the skin can further enable the double-layer modified glycerol bodies to be more tightly adsorbed on the negatively charged skin surface, significantly improving the penetration of glycerol bodies loaded with collagen.

[0085] The present invention provides a chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerol body encapsulating recombinant human type III collagen, and a preparation method and application thereof. In order to solve the problems of strong water solubility, large molecular weight and poor skin permeability, a chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerol body is selected to encapsulate the recombinant human type III collagen, which can effectively improve its permeability in the skin.

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A glycerol body encapsulating recombinant human type III collagen, characterized in that: include, Phospholipids, glycerol, surfactant, chitosan quaternary ammonium salt, sodium hyaluronate, recombinant human type III collagen and PBS buffer solution; Wherein, based on the total mass of the raw materials, the phospholipid is 1-20%, the glycerol is 5-35%, the surfactant is 0.5-3.5%, the chitosan quaternary ammonium salt is 0.05-1%, the sodium hyaluronate is 0.1-0.4%, the recombinant human type III collagen is 0.1-0.5%, and the PBS buffer solution is supplemented to 100%.

2. The glycerol body according to claim 1, characterized in that: The phospholipids include at least one of hydrogenated lecithin, soybean lecithin, and egg yolk lecithin.

3. The glycerol body according to claim 1 or 2, characterized in that: The surfactant includes at least one of Tween 80, sodium cholate, sodium deoxycholate and Spans.

4. The glycerol body according to claim 3, characterized in that: The average particle size of the glycerol body is 60-400 nm, and the particle size distribution index PDI of the glycerol body vesicle is 0.1-0.

5.

5. The method for preparing the glycerol body according to any one of claims 1 to 4, characterized in that: include, Dissolve recombinant human type III collagen, glycerol, and surfactant in PBS buffer to obtain an aqueous phase; Slowly and evenly add lecithin to the water phase and continue stirring at room temperature until hydration is complete; The obtained liposome suspension is subjected to ultrasonic treatment to obtain glycerol bodies encapsulating recombinant human type III collagen; Dissolve chitosan quaternary ammonium salt in PBS buffer, stir thoroughly to dissolve, and add dropwise the above-obtained glycerol bodies encapsulating recombinant human type III collagen under continuous stirring to obtain chitosan quaternary ammonium salt-modified chitosan quaternary ammonium salt-modified glycerol bodies encapsulating recombinant human type III collagen; Sodium hyaluronate was dissolved in PBS buffer, stirred thoroughly to dissolve, and the chitosan quaternary ammonium salt-modified glycerol body obtained above was added dropwise under continuous stirring to obtain chitosan quaternary ammonium salt-sodium hyaluronate double-layer-modified glycerol body encapsulating recombinant human type III collagen.

6. The preparation method according to claim 5, characterized in that: The temperature of the hydration treatment is 15 to 35° C., and the time of the hydration treatment is 3 to 24 hours.

7. The preparation method according to claim 6, characterized in that: The amplitude of the ultrasonic treatment is 30 to 80 μm, the power of the ultrasonic treatment is 180 to 530 W, and the time of the ultrasonic treatment is 60 to 300 s.

8. Use of the chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerol body encapsulating recombinant human type III collagen as claimed in any one of claims 1 to 4 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that: The cosmetic comprises a facial cleanser.

10. Use of the chitosan quaternary ammonium salt-sodium hyaluronate double-layer modified glycerosomes encapsulating recombinant human type III collagen as claimed in any one of claims 1 to 4 in the preparation of medicines.

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