Injectable temperature-sensitive biological gel preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological gel preparations, in particular to an injectable temperature-sensitive biological gel preparation as well as a preparation method and application thereof, and the injectable temperature-sensitive biological gel preparation is prepared from the following components in parts by weight: 80 to 100 parts of exosome solution, 11 to 20 parts of poloxamer and 1 to 5 parts of long-chain polysaccharide hydrophilic polymer. The stem cell exosome hydrogel injection provided by the invention is prepared from temperature-sensitive hydrogel and stem cell exosome, the temperature-sensitive hydrogel is in a liquid state at low temperature and can be sucked into the needle cylinder and injected out, and when the body surface temperature exceeds 30 DEG C, the exosome of the temperature-sensitive hydrogel can form a gel state; the exosome injection gel can be conveniently fixed at an injection part, so that the exosome can be released for a longer time, the exosome injection gel is in a liquid state during filling and injection, the operation is convenient, and when the temperature is increased to 30 DEG C after injection, the hydrogel is in a gel state and is fixedly adhered to the injection part, so that the action time of the exosome is prolonged.

Description

Technical Field

[0001] The invention relates to the technical field of biogel preparations, and in particular to an injectable temperature-sensitive biogel preparation and a preparation method and application thereof. Background Art

[0002] The skin disease is an inflammatory skin disease. An inflammatory skin disease is a skin disease caused by an inflammatory response mediated by immune cells and manifested by symptoms such as itching and erythema. An inflammatory skin disease is a skin disease selected from the group consisting of atopic dermatitis, contact dermatitis and psoriasis vulgaris.

[0003] Modern people are prone to dry and thin facial skin due to long-term improper use of cosmetics and skin care products, premature exposure or improper modern medical cosmetic damage, use of steroid drugs, and long-term stay in air-conditioned rooms, which may cause the following skin diseases: eczematous dermatitis, seborrheic dermatitis, atopic dermatitis, contact dermatitis, acne skin, rosacea erythematosus dermatitis, etc. There are many reasons, and even similar or overlapping symptoms. Since the current medical treatment generally treats facial redness as a general skin allergy eczema, long-term use of steroid drugs causes skin atrophy, which greatly aggravates the damage to microvessels and the normal state of redness. Without proper treatment, the damaged sebum membrane barrier of the skin can be restored to health. In addition, the desire to quickly resolve skin problems accelerates the abuse of hormone drugs, which leads to dependence on skin problems and increasingly heavier medication. The result is a vicious cycle that leads to untreatable consequences. One day, the entire skin structure will collapse, and no matter how hard any drug is used, it will not be suppressed, and steroid rosacea will form, which becomes extremely difficult to treat. Dermatitis is difficult to cure because the inflammatory mechanism is complex and traditional treatments are limited. The latest research shows that exosomes released by mesenchymal stem cells are very helpful for treatment. In summary, this is a potential treatment method that is worth exploring, which is of great significance for inhibiting skin inflammation and promoting the healing of skin wounds (Suntanner, healthyself: Exosome therapy may enable better repair of sun, age-damaged skin); (Expert Opinion on Biological Therapy, 16:4, 489-506).

[0004] Studies have shown that in addition to being small in size and having a double-layer lipid structure, exosomes can easily penetrate the stratum corneum and penetrate into the dermis, which can effectively promote wound healing. Their effects include reducing inflammation, promoting angiogenesis, and promoting the proliferation and movement of skin cells. Exosomes are vesicles with a size of tens to hundreds of nanometers, which are composed of a phospholipid bilayer membrane with the same structure as the cell membrane. The exosomes contain proteins, nucleic acids (mRNA, miRNA, etc.), etc., which are called exosome cargo. It is known that the cargo of exosomes includes a wide range of signal transduction factors, and these signal transduction factors are specific to cell types and are regulated differently according to the environment of the secreting cells. It is known that exosomes are intercellular signal transduction mediators secreted by cells, and the various cell signals transmitted by them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis and necrosis of target cells. Depending on the nature and state of the cells from which the exosomes are derived, the exosomes contain specific genetic material and bioactive factors. Exosomes derived from proliferating stem cells regulate cell behaviors such as cell migration, proliferation, and differentiation, and recapitulate the characteristics of stem cells involved in tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

[0005] The most common application of poloxamer as a high molecular polymer is gel material. There are published patents: patents using poloxamer as a single gel matrix (e.g., Supository composition of the drug which undergoes the hepatic first-pass effect. WO9730693), patents for using different types of poloxamers in combination to have an appropriate phase transition temperature (patent publication number: CN1377706A), patents for using poloxamer in combination with other bioadhesive polymers to obtain bioadhesive in situ gels (e.g., Thermo reversible gel as a liquid pharmaceutical carrier for a galenic formulation. EP0551626), and patents for using poloxamer in combination with other polymers with specific properties to obtain in situ gels for special purposes, such as using poloxamer with highly water-absorbent cross-linked polymers to obtain gels for wound healing (Wound gels, US7,083,806), and using poloxamer with collagen to obtain gels for viral vectors (Hydrogel The administration routes involved include ocular administration (see above, CN1377706A; eye-droppable in vivo gel preparation and its preparation method, CN1397272A) and administration through body cavities, including oral, nasal, rectal and vaginal administration (RECTAL AND VAGINAL SUPPOSITORIES CONTAINING BIOADHESIIVE BROMOCRIPTINE AND POLOXAMER WO2006099877; a thermosensitive gel preparation and its preparation method CN1814253A; a novel vaginal pharmaceutical preparation, CN1872026A; a temperature-sensitive vaginal in situ gel preparation, CN1593386A). Different proportions of poloxamer P188 and P407 can be used to adjust the temperature-sensitive hydrogel to form a colloid at different temperatures, but the total concentration of poloxamer must be above 20%. The temperature-sensitive hydrogel of this total concentration of poloxamer at low temperature still has high viscosity and cannot be injected using a syringe. The relevant patents and applications of temperature-sensitive hydrogel are mainly based on colloid application, and there is no relevant disclosure on injections for the treatment and prevention of inflammatory skin and mucous membranes.

[0006] Currently, there is no clinically applicable exosome injectable thermosensitive gel for the prevention, inhibition, alleviation, improvement or treatment or repair of existing inflammatory symptoms of the skin and mucous membranes. Summary of the invention

[0007] The purpose of the present invention is to provide an injectable temperature-sensitive exosome gel injection, while solving the technical problems in the prior art that the exosome solution cannot stay in the inflammatory site and the temperature-sensitive gel cannot be treated using a syringe or microneedle.

[0008] The invention discloses an injectable thermosensitive biogel preparation, which comprises, by weight, 80-100 parts of an exosome solution, 10-20 parts of a poloxamer, and 1-5 parts of a long-chain polysaccharide hydrophilic polymer.

[0009] Furthermore, the concentration of the exosome solution is 10-1000×10 8 / ml, with a particle size of 50-200 nm.

[0010] Furthermore, the poloxamer is poloxamer 188 or poloxamer 188 combined with poloxamer 407.

[0011] Furthermore, the poloxamer 188 is 10-15 parts by weight, and the poloxamer 407 is 0-5 parts by weight.

[0012] Furthermore, other water-soluble drugs are included, and the weight of the other water-soluble drugs is less than or equal to 5 parts. The other water-soluble drugs are vitamins or their derivatives used for skin and mucous membrane care.

[0013] Furthermore, the other water-soluble drugs are placental polypeptides, vitamin C, vitamin B group, β-nicotinamide mononucleotide or pyrroloquinoline quinone, preferably vitamin C, β-nicotinamide mononucleotide or pyrroloquinoline quinone.

[0014] Furthermore, the long-chain polysaccharide hydrophilic polymer is selected from sodium hyaluronate, methylcellulose, chitosan or chondroitin sulfate.

[0015] A method for preparing an injectable thermosensitive biogel preparation comprises the following steps: Step 1: Poloxamer is added to the exosome solution and stirred to make it fully sol to serve as a thermosensitive gel matrix; Step 2, adding the long-chain polysaccharide hydrophilic polymer to the thermosensitive gel matrix of step 1 and stirring to make it fully sol; Step 3, adding other water-soluble drugs into the thermosensitive gel matrix prepared in step 2 and mixing them evenly, and then leaving them to stand to obtain the result.

[0016] Furthermore, in step 1, the stirring is performed at 6-10° C. for 50-60 min.

[0017] Furthermore, in step 2, the stirring is performed at 6-10° C. for 50-60 min.

[0018] Furthermore, in step 3, the stirring is performed at 6-10° C. for 20-30 min.

[0019] Furthermore, in step three, the step of standing at room temperature is 4-6 hours.

[0020] Furthermore, the injectable exosome thermosensitive hydrogel prepared in step 3 is stored at 0-10°C.

[0021] The invention discloses an injectable thermosensitive biogel preparation for preparing a medicine for improving acne, inflammatory and hypertrophic scar skin and mucous membranes.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. The stem cell exosome hydrogel injection provided by the present invention is made of a thermosensitive hydrogel and stem cell exosomes; the thermosensitive hydrogel is in liquid state at low temperature and can be sucked into a syringe and injected. When the temperature of the body surface exceeds 30°C, the exosomes will form a gel state; it is convenient to fix it at the injection site so that the exosomes can be released for a longer time. The exosome injection gel is in liquid state during filling and injection, which is convenient for operation. When the temperature rises to 30°C after injection, the hydrogel is in a gel state and fixedly adheres to the injection site to increase the action time of the exosomes; 2. When the exosome composition of the present invention is used as a thermosensitive biogel preparation or a topical preparation composition for energy damage to skin and mucous membranes, the exosomes, poloxamer 188 and long-chain polysaccharide hydrophilic polymers contained in the composition as active ingredients show significant effects on prevention, inhibition, alleviation, improvement or treatment. The use of poloxamer 188 and 407 and long-chain polysaccharide hydrophilic polymers can overcome the problem that poloxamer 188 and 407 alone gel too quickly and are difficult to inject and operate. The addition of long-chain polysaccharide hydrophilic polymers can extend its gelation time to a suitable time, and on the other hand increase its bioadhesiveness and repair properties. Therefore, the thermosensitive hydrogel used for exosome injection according to the present invention can be used as a mesodermal injection to prevent, inhibit, alleviate, improve or treat skin and mucous membrane aging and repair, and this effect cannot be predicted from conventional technology at all. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only represent some embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The particle size and concentration of Exos detected by NTA of stem cell exosomes in Example 1 of the present invention.

[0025] Figure 2 This is a surface ultrastructure image of the stem cell exosomes in Example 1 of the present invention taken using a transmission electron microscope TEM (scale bar = 100 nm).

[0026] Figure 3 The stem cell exosomes in Example 1 of the present invention were detected using flow cytometry to detect the protein expression levels of stem cell exosome surface markers CD9, CD63, and CD81.

[0027] Figure 4 This is a state diagram of the thermosensitive gel for stem cell exosome injection of formula 1 of the present invention. In the figure, the fluidity is good at 20°C and it is in a colloidal state at 35°C.

[0028] Figure 5 This is a state diagram of the thermosensitive gel for stem cell exosome injection of Formula 2 of the present invention. In the figure, at 20°C, the gel-like surface is severely attached to the wall, and at 30°C, it is in a colloidal state and cannot be injected using a syringe.

[0029] Figure 6 This is a state diagram of the stem cell exosome injectable thermosensitive gel of formula 3 of the present invention. In the figure, the fluidity is good at 20°C and it is still in a liquid state at 30°C.

[0030] Figure 7 This is a diagram showing the therapeutic effect of the control group of the present invention on acne vulgaris.

[0031] Figure 8 This is a diagram showing the therapeutic effect of Formula 1 of the present invention on rosacea.

[0032] Fig. 9 This is a diagram showing the therapeutic effect of Formula 2 of the present invention on acne vulgaris.

[0033] Fig.10 This is a diagram showing the therapeutic effect of Formula 3 of the present invention on acne vulgaris (since it is impossible to inject directly with a syringe, a roller needle is used for puncture and then colloid application).

[0034] Fig.11 The therapeutic effect of formula 6 and formula 5 of the present invention on inflammatory hyperplastic scar tissue is shown in FIG. DETAILED DESCRIPTION In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0035] Example 1 Characterization of stem cell exosomes Mesenchymal stem cells were cultured and exosomes were extracted. The quality of exosomes was tested. The population size distribution of exosomes was analyzed by nanoparticle size tracer analyzer, such as Figure 1 As shown: The particle size of exosomes is mainly distributed between 50-150nm, the median particle size is about 133nm, and the concentration is 1.3E+11 / ml (130 billion exosomes per ml). Transmission electron microscopy image of exosomes ( Figure 2 ) showed that the exosomes were complete and full in shape, presenting a typical cup-shaped state; the exosome identification analysis used nano-flow cytometry to detect CD9, CD63 and CD81 surface proteins, and the blank group was mesenchymal stem cells such as Figure 3 As shown: the exosome marker proteins CD9, CD63 and CD81 are positive, indicating that the extracted exosomes are from mesenchymal stem cells, and the extracted exosomes are frozen for subsequent use.

[0036] The preparation of different concentrations of poloxamer (P188 and P407) and long-chain polysaccharide hydrophilic polymer injectable thermosensitive gel or other configurations of thermosensitive gels for comparison specifically include the following steps: 1. Adjust the concentration of exosome suspension to 1×10 10 / mL, filtered through 0.22μm for later use.

[0037] 2. Weigh different amounts of poloxamer 188 and poloxamer 407 according to Table 1, put them into the exosome suspension and dissolve them at 6-10°C for 60 minutes. After dissolving evenly, set aside 3. According to Table 1, take different amounts of long-chain polysaccharide hydrophilic polymers (such as hyaluronic acid HA, methylcellulose CMC) or non-long-chain polysaccharide hydrophilic polymers (such as polyethylene glycol (PEG MW.150,000)) and put them into the exosome suspension and dissolve them at 6-10℃ for 60 minutes. After dissolving evenly, set aside. 4. Take different amounts of other water-soluble drugs (such as vitamin C, β-nicotinamide mononucleotide (NMN) or pyrroloquinoline quinone (PQQ)) and put them into the exosome suspension to dissolve at 6-10°C for 10 minutes. After the mixture is evenly dissolved, use sodium hydroxide to adjust the pH value to between 6.5 and 7.5. The detailed formula is shown in Table 1.

[0038] Table 1. Temperature-sensitive gels of different concentrations of poloxamer and other mixtures

[0039] 5. Test the temperature-sensitive gels of different formulas for gelling temperature injection test. Use 1 ml 26G (0.45х16mm, the most commonly used specification for subcutaneous injection and water light injection) needle to perform injection tests at different temperatures. See Table 2 and Figure 4-Figure 6 .

[0040] Table 2. Test on injectability of temperature-sensitive gels with different formula concentrations on gelling temperature

[0041] Inflammatory Skin Treatment Trial The treatment method is to inject 0.05 ml at one point on the entire face with a handheld needle, with an injection interval of 0.3-0.5 cm (40 billion exosomes for the entire face treatment). Each treatment uses 4 ml of injectable exosome thermal gel solution. The treatment is done every 2 weeks, for a total of 4 times, and the entire course of treatment lasts for 2 months.

[0042] Case 1 Control Group Treatment of acne vulgaris with only stem cell exosomes Figure 7 As shown in the results, direct injection of exosomes did not result in significant improvement before and after treatment, as the exosomes were unable to stay at the injection site for a long time.

[0043] Case 2 (Formula 1) The results of using formula 1 (needle size 30G) for the treatment of rosacea are as follows Figure 8 The rosacea around the nose was significantly improved before and after treatment with Formula 1 shown.

[0044] Case 3 (Formula 2) The results of using formula 2 (needle size 23G) for the treatment of acne vulgaris are as follows Fig. 9 There was no significant improvement before and after the treatment as shown, and Formula 2 was difficult to apply and mostly stayed on the skin surface.

[0045] Case 4 (Formulation 3, needle size 30G) Formula 3 was used to treat acne vulgaris. The results were as follows Fig.10 There was no significant improvement in acne vulgaris skin before and after the treatment as shown, because Formula 3 was easily lost and could not stay at the injection site.

[0046] Hypertrophic scar treatment Case 5 For hypertrophic scar treatment, a handheld needle is used to inject into the scar: Distal end: Use Formula 6, hand-held needle (needle size 26G) 0.03 ml 1 point, inject directly into the scar area at intervals of 0.3-0.5cm, and inject a total of 1 ml (a total of 10 billion stem cell exosomes). After injection, use Formula 6 to apply on the surface of the injection site, using 2 ml (a total of 20 billion stem cell exosomes).

[0047] Proximal end: Use Formula 5 (needle size 23G), hold the needle and inject 0.03 ml at 1 point, 0.3-0.5 cm apart, inject 1 ml in total (a total of 10 billion stem cell exosomes are used). After injection, apply Formula 5 on the surface of the injection site, using 2 ml (a total of 20 billion stem cell exosomes are used).

[0048] Treatment was given once every 4 weeks, for a total of 3 times, and the entire course of treatment lasted for 3 months.

[0049] The results are as follows Fig.11 As shown, the hypertrophic scar was injected with formula 5 at the proximal end and formula 6 at the distal end. After treatment, it can be found that the scar at the proximal end is still very obvious but the inflammatory response is slightly reduced, the scar at the distal end is softened, and the boundary of the angiogenesis scar becomes less obvious.

[0050] The above are the implementation methods listed in this embodiment, but this embodiment is not limited to the above optional implementation methods. Those skilled in the art can arbitrarily combine the above methods to obtain other various implementation methods. Anyone can derive other various forms of implementation methods under the inspiration of this embodiment. The above specific implementation methods should not be understood as limiting the scope of protection of this embodiment. The scope of protection of this embodiment shall be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. An injectable thermosensitive biogel preparation, characterized in that: The composition comprises 80-100 parts of exosome solution, 11-20 parts of poloxamer, and 1-5 parts of long-chain polysaccharide hydrophilic polymer in parts by weight.

2. The injectable thermosensitive biogel preparation according to claim 1, characterized in that: The concentration of the exosome solution is 10-1000×10 8 / ml, with a particle size of 50-200 nm.

3. The injectable thermosensitive biogel preparation according to claim 1, characterized in that: The poloxamer is poloxamer 188 or poloxamer 188 combined with poloxamer 407.

4. The injectable thermosensitive biogel preparation according to claim 3, characterized in that: In parts by weight, the poloxamer 188 is 10-15 parts, and the poloxamer 407 is 0-5 parts.

5. The injectable thermosensitive biogel preparation according to claim 1, characterized in that: It also includes other water-soluble drugs, the weight of which is less than or equal to 5 parts, and the other water-soluble drugs are vitamins or their derivatives used for skin and mucous membrane care.

6. The injectable thermosensitive biogel preparation according to claim 1, characterized in that: The long-chain polysaccharide hydrophilic polymer is selected from sodium hyaluronate, methylcellulose, chitosan or chondroitin sulfate.

7. A method for preparing an injectable thermosensitive biogel preparation according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1: Poloxamer is added to the exosome solution and stirred to make it fully sol to serve as a thermosensitive gel matrix; Step 2, adding the long-chain polysaccharide hydrophilic polymer to the thermosensitive gel matrix of step 1 and stirring to make it fully sol; Step 3, adding other water-soluble drugs into the thermosensitive gel matrix prepared in step 2 and mixing them evenly, and then leaving them to stand to obtain the result.

8. The method for preparing an injectable thermosensitive biogel preparation according to claim 7, characterized in that: The stirring in step 1 is performed at 6-10° C. for 50-60 min.

9. The method for preparing an injectable thermosensitive biogel preparation according to claim 7, characterized in that: In step 3, the mixture is allowed to stand at room temperature for 4-6 hours.

10. An application of an injectable thermosensitive biogel preparation, characterized in that: A product for improving acne, inflammatory and hypertrophic scar skin and mucous membranes is prepared using a gel preparation prepared by an injectable thermosensitive biogel preparation as described in any one of claims 1 to 6 or a gel preparation prepared by a method for preparing an injectable thermosensitive biogel preparation as described in any one of claims 7 to 9.

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