Ros / pH dual-responsive modified type xvii collagen microneedle patch loaded with bioactive substances and application thereof

Abstract

The present application relates to the field of biological materials, and particularly relates to a ROS / pH dual-response modified type XVII collagen microneedle patch loaded with bioactive substances and application, the microneedle patch is composed of boric acid modified type XVII collagen, bioactive substances and polymer substrate.Boric acid modified type XVII collagen and polyvinyl alcohol are double-crosslinked through physical and chemical levels to form collagen hydrogel, then load bioactive substance insulin-like growth factor, and form ROS / pH dual-response collagen microneedle patch loaded with bioactive substances with polymer substrate.When applied to the skin, the microneedle tip reaches the area where hair follicle stem cells exist in a painless and minimally invasive manner, and due to the change of active oxygen and pH environment in the skin tissue, the borate ester bond in the collagen hydrogel can be broken, thereby removing active oxygen and releasing the wrapped IGF-1 into the hair follicle stem cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological materials, in particular to a ROS / pH dual-responsive modified type XVII collagen microneedle patch loaded with bioactive substances and application thereof. BACKGROUND

[0002] Hair loss is becoming a global public concern, which is a hair disease characterized by gradual miniaturization of hair follicles and shortening of growth period, leading to reduced scalp hair. Although hair loss does not affect physical health, it has a negative impact on the psychological health of patients, leading to a decline in quality of life. Early diagnosis and treatment can significantly delay the progression of hair loss and improve prognosis. Treatments include drugs such as minoxidil and finasteride, which require long-term patient compliance, but the efficacy varies. Common side effects include sexual dysfunction, depressive mood changes, and skin irritation. Surgical effectiveness is influenced by the surgeon's proficiency, and is only suitable for patients with sufficient donor hair, in addition, unnatural hairline and donor area scarring are also frequently reported problems. In addition to genetic factors, the onset of androgenetic alopecia may also be influenced by other factors, such as environmental factors, changes in hormone levels, health status, etc.

[0003] Traditional treatments for androgenetic alopecia mainly include oral drugs, topical drugs, and laser therapy. However, these treatment methods have some limitations, such as oral drugs may cause side effects, topical drugs need to be used frequently and have limited effectiveness, and laser therapy is expensive and requires periodic treatment. The emergence of microneedle technology brings new ideas for hair loss treatment. Microneedle therapy uses tiny needle tips to pierce the skin surface, stimulating the skin's self-repair mechanism, promoting skin regeneration and hair follicle growth.

[0004] During the process of androgenetic alopecia, the level of oxidative stress around the hair follicle usually increases, leading to increased cellular oxidative damage and accelerated hair follicle regression. At the same time, the pH around the hair follicle may also change, and androgenetic alopecia may cause changes in the pH of the tissue around the hair follicle. At this time, the advantages of the environment-responsive microneedle over the ordinary microneedle are manifested. The environment-responsive microneedle is an innovative microneedle technology that adds an environmentally responsive material to the ordinary microneedle, which can automatically release active ingredients according to environmental conditions, and has more precise and efficient therapeutic effect. The environment-responsive microneedle may contain some ROS-sensitive drug carriers, such as compounds containing disulfide bonds. These drug carriers will release antioxidant or anti-inflammatory drugs when they detect an increase in ROS levels. At the same time, the environment-responsive microneedle may contain pH-sensitive drug carriers, such as compounds containing acidic or basic groups. These drug carriers will release drugs such as growth factors and anti-inflammatory drugs when they detect abnormal pH, which helps to regulate the acid-base balance of the environment around the hair follicle and promote hair follicle regeneration and growth. However, how to ensure that the drugs in the microneedle patch are released under the right conditions, and the release rate and amount meet the needs of treatment, is one of the technical problems to be solved. SUMMARY

[0005] The purpose of the present application is to provide a ROS / pH dual-responsive modified type XVII collagen microneedle patch loaded with bioactive substances and its application. The bioactive substances are combined with modified collagen hydrogel to develop a degradable microneedle patch made of physically inert polymer PVA, which can deliver bioactive substances to hair follicle stem cells in the androgenetic alopecia site.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The microneedle patch provided by the present application is composed of ROS / pH dual-responsive boron acid modified type XVII collagen protein gel

[0008] IGF-1 and a polymer base;

[0009] The polymer base is composed of a physically inert polymer PVA with good biocompatibility, and the preparation method is as follows: 4g PVA is dissolved in 20ml deionized water to prepare a 20% PVA solution, which is stored at room temperature for later use.

[0010] The ROS / pH dual-responsive modified type XVII collagen protein is formed by reacting type XVII collagen with 3-amino-4-methoxybenzoic acid (AMB) using oxidative polymerization method, and then cross-linking with 3-aminobenzene boronic acid (ABA) under the action of cross-linking agent such as 2-morpholinoethanesulfonic acid (MES). The specific steps are as follows:

[0011] First, 1g of type XVII collagen was dissolved in 20ml of deionized water with 100mg of 3-amino-4-methoxybenzoic acid, and 136.5mg of ammonium persulfate (APS) was added as an oxidation polymerization catalyst. After 24h, the reaction solution was dialyzed in deionized water for at least 3 days (Mw=3500), and then freeze-dried to obtain Col17-AMB.

[0012] Secondly, 1g of modified type XVII collagen Col17-AMB was taken from the freeze-dried sample and dissolved in 2-morpholinoethanesulfonic acid (MES, 20ml, pH=5.0) buffer. At the same time, 0.26g of 3-aminobenzoic acid, 0.16g of 1-ethyl-3-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDC·HCl), and 0.06g of N-hydroxysuccinimide (NHS) were fully dissolved in the buffer and stirred in an ice bath for 2h. After 2h, the collagen solution was added and stirred for 48h. Dialysis and freeze-drying yielded boronate-modified type XVII collagen.

[0013] The collagen hydrogel was formed by physical and chemical cross-linking of boronate-modified type XVII collagen and polyvinyl alcohol (PVA). The 3-aminobenzoic acid in the boronate-modified type XVII collagen was chemically cross-linked with PVA to form a dynamic boronate ester bond, and the physical property of repeated freeze-thawing of PVA was used to synthesize boronate-modified type XVII collagen hydrogel. Taking 5% boronate-modified type XVII collagen hydrogel as an example: 50mg of boronate-modified type XVII collagen was dissolved in 1ml of deionized water, and 100mg of polyvinyl alcohol (PVA) was dissolved in 1ml of deionized water. The two solutions were mixed and ultrasonicated for 1min, then placed in a-20℃ refrigerator for 6h, then taken out and placed at room temperature for 3h, and repeated freeze-thawing several times to obtain 5% boronate-modified type XVII collagen hydrogel.

[0014] The bioactive substance IGF-1 was loaded in the following way: 200mg of freeze-dried boronate-modified type XVII collagen hydrogel was soaked in 10mL of PBS, then 65ul of 1mg / ml IGF-1 solution from GenScript was added, and the freeze-dried gel was soaked for 3 days to achieve drug loading equilibrium.

[0015] The application also provides a preparation method of the above-mentioned microneedle patch, specifically comprising:

[0016] First, 50mg of drug-loaded boronate-modified type XVII collagen hydrogel was placed on a microneedle PDMS mold, and a small medicine spoon was used to press the drug-loaded boronate-modified type XVII collagen hydrogel into the needle holes of the mold. The mold was centrifuged at 2200rpm for 15 minutes, and then the pressed mold was placed in a vacuum drying oven and repeatedly vacuumed three times.

[0017] Secondly, a 20% PVA solution is cast onto the mold loaded with the microneedle patch, and finally the covered mold is dried in a ventilated place for one day, after which the microneedle patch is carefully peeled off and stored in a sealed well plate for further study.

[0018] The ROS / pH dual-responsive collagen microneedle patch loaded with a bioactive substance of the present application is composed of borate-modified type XVII collagen hydrogel @ IGF-1 and a polymer matrix. When applied to the skin, the microneedle tips reach the area where hair follicle stem cells exist in a painless and minimally invasive manner. The borate ester bond in the collagen hydrogel is broken due to the presence of active oxygen in the environment and the influence of the skin pH environment, thereby removing active oxygen and releasing encapsulated IGF-1 into hair follicle stem cells. Compared with traditional androgenetic alopecia treatment drug minoxidil, the ROS / pH dual-responsive collagen microneedle patch provides an effective potential treatment method for androgenetic alopecia.

[0019] Compared with other treatment methods, the present application has the following beneficial effects:

[0020] (1) The ROS / pH dual-responsive collagen hydrogel @ IGF-1 contained in the present application is gelled by borate ester bond, which is broken due to the presence of active oxygen (ROS) in the environment and the influence of the skin pH environment, thereby removing active oxygen and releasing encapsulated IGF-1 into hair follicle stem cells.

[0021] (2) The microneedle patch of the present application is simple to prepare and low in cost, laying a foundation for large-scale preparation.

[0022] (3) The microneedle material contains IGF-1 and collagen, which stimulates cell proliferation and differentiation, promotes the activation and growth of hair follicles; the microneedle patch made of collagen hydrogel has a certain sustained release effect and is slowly released in the body for several days after use. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the bright field image of borate-modified type XVII collagen hydrogel before and after gelling.

[0024] Figure 2 is the injectability verification of the hydrogel by a syringe.

[0025] Figure 3 is the rheological property of borate-modified type XVII collagen hydrogel with different ratios.

[0026] Figure 4 is the nuclear magnetic detection spectrum of type XVII collagen and borate-modified type XVII collagen.

[0027] Figure 5are the FTIR detection patterns of collagen XVII, AMB modified collagen XVII and borate modified collagen XVII.

[0028] Figure 6 are the antioxidant abilities of borate modified collagen XVII hydrogels with different ratios.

[0029] Figure 7 are the in vitro release situations of borate modified collagen XVII in four different environments.

[0030] Figure 8 are the in vivo release situations of borate modified collagen XVII microneedles after being applied on the back of mice.

[0031] Figure 9 are the super-depth-of-field microscope pictures of borate modified collagen XVII microneedles.

[0032] Figure 10 are the SEM pictures of borate modified collagen XVII microneedles.

[0033] Figure 11 are the in vitro degradation situations of borate modified collagen XVII microneedles after being pressed on 6% agarose.

[0034] Figure 12 are the in vivo degradation situations of borate modified collagen XVII microneedles after being pressed on the back of mice.

[0035] Figure 13 Animal experiment.

[0036] Figure 14 H&E staining results of the back skin of mice.

[0037] Figure 15 Immunofluorescence results of the back skin of mice.

[0038] Figure 16 Biocompatibility experiment results.

[0039] Figure 17 Cell migration experiment results.

[0040] Figure 18 are the preparation and use process diagrams of the microneedle patch of the present application. DETAILED DESCRIPTION

[0041] The technical solutions of the present application are further explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0042] Preparation of boronic acid-modified Col17

[0043] First, 1 g of Col17 was dissolved in 20 ml of deionized water with 100 mg of 3-amino-4-methoxybenzoic acid and 136.5 mg of ammonium persulfate (APS) as an oxidation polymerization catalyst. After 24 h, the reaction solution was dialyzed in deionized water for at least 3 days (Mw = 3500), and then lyophilized to obtain the modified Col17-AMB.

[0044] Second, 1 g of the modified Col17 was taken from the lyophilized sample and dissolved in a 2-morpholinoethanesulfonic acid (MES, 20 ml, pH = 5.0) buffer. At the same time, 3-aminobenzoic acid (ABA, 0.26 g), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 0.16 g), and N-hydroxysuccinimide (NHS, 0.06 g) were fully dissolved in 20 ml of MES buffer at pH = 5.0 and stirred in an ice bath for 2 h. After 2 h, the collagen solution was added and stirred for 48 h. Dialysis and lyophilization yielded the boronic acid-modified Col17, i.e., Col17-BA.

[0045] Preparation of boronic acid-modified Col17 hydrogel

[0046] 50 mg of boronic acid-modified Col17 was weighed and dissolved in 1 ml of deionized water, and 100 mg of polyvinyl alcohol (PVA) was weighed and dissolved in 1 ml of water. The two solutions were mixed and placed in a -20°C refrigerator for 6 h, then taken out and placed at room temperature for 3 h. The freeze-thaw process was repeated several times to obtain the boronic acid-modified Col17 hydrogel.

[0047] Encapsulation of bioactive substance IGF-1

[0048] 200 mg of lyophilized boronic acid-modified Col17 hydrogel was soaked in 10 mL of PBS, and then 65 μl of 1 mg / ml IGF-1 solution from GenScript was added. After 3 days of soaking, the lyophilized gel reached drug loading equilibrium, and the drug-loaded boronic acid-modified Col17 hydrogel was obtained.

[0049] Preparation of ROS / pH dual-responsive collagen microneedle patch

[0050] Take 50 mg of drug-loaded boron acid-modified type XVII collagen hydrogel on the microneedle PDMS mold, use a small medicine spoon to press the gel into the needle hole of the mold as the microneedle tip, and centrifuge the mold at 2200 rpm for 15 minutes; then put the pressed mold into a vacuum drying oven and repeatedly vacuum three times; secondly, pour 20% PVA solution into the PDMS mold loaded with the needle head to completely cover the needle head and just fill the mold, the thickness is about 0.5 ml, finally, place the covered mold in a well-ventilated place to dry for one day, carefully peel off the microneedle patch, and store the obtained microneedle patch in a sealed well plate for further study.

[0051] Example 5 Antioxidant capacity determination of modified type XVII collagen hydrogel

[0052] Prepare boron acid-modified type XVII collagen hydrogel with Wt% of 2.5%, 5%, and 7.5% respectively (i.e. for example, 2.5% boron acid-modified type XVII collagen hydrogel is 25 mg of boron acid-modified type XVII collagen added to 1 ml of deionized water and mixed with 1 ml of 10% PVA solution; 5% boron acid-modified type XVII collagen hydrogel is: take 50 mg of boron acid-modified type XVII collagen, add 1 ml of deionized water to dissolve, and then mix with 1 ml of 10% PVA solution). Take 3 50 mg hydrogels each and place them in 5 ml centrifuge tubes, a total of 9 tubes, then use the DPPH kit from Shanghai Yuan Ye Biological Technology Co., Ltd. to test the antioxidant capacity of the hydrogel.

[0053] Example 6 In vitro degradation capacity of microneedle patch

[0054] Take 3 g of agarose in a 50 ml beaker and add 50 ml of deionized water to make a 6% agarose solution, then heat the agarose solution to boiling in a microwave oven, pour it into a cell culture dish repeatedly three times, and then put it in a 4°C refrigerator for one hour to freeze and solidify. Use an inverted fluorescence camera to take a picture of the microneedle patch at 0 min to observe the shape of the needle tip in bright field, place the microneedle on the 6% agarose, press it with a 1 kg weight and time for 1 min, then take a picture of the microneedle degradation at 1 min, and finally take a picture of the microneedle patch completely degraded at the fourth minute.

[0055] Example 7 In vitro degradation capacity of microneedle patch

[0056] Take the boron acid-modified type XVII collagen microneedle and press it on the back of a mouse for 5, 10, 15, and 20 minutes, then use an inverted fluorescence camera to take a picture of the needle tip in bright field.

[0057] Example 8 In vitro release capacity

[0058] Bovine serum albumin (BSA) loaded with rhodamine B dyeing boric acid modified type XVII collagen hydrogel was placed in a 15 ml centrifuge tube to simulate the release of IGF-1 and the centrifuge tube was divided into four groups, 10 ml 0.2 mM H2O2 + pH 7.4 PBS solution, 0.2 mM H2O2 + pH 5 PBS solution, pH 7.4 PBS solution, pH 5 PBS solution were added respectively. The centrifuge tube was placed in a shaking bed at 37°C and shaken every hour, 1 ml sample was taken and the same 1 ml solution was added. After 24 h, it was added to a 96-well plate and photographed in a live imaging instrument.

[0059] Example 9 in vivo release ability

[0060] After the rhodamine B dyed boric acid modified type XVII collagen microneedle was pressed on the back of the mouse for 20 min, the release was photographed in the live imaging instrument every day.

[0061] Example 10 hair follicle regeneration experiment

[0062] The specific experimental steps are as follows:

[0063] (1) Androgenetic alopecia model of mice

[0064] Twenty-four 6-week-old male C57BL / 6 mice were subcutaneously injected with testosterone solution (0.2%, w / v, 0.1 mL / cm 2 ) on the back of the C57BL / 6 mouse for 35 consecutive days to induce the AGA model; During the entire experiment (35 days), each animal was injected with 0.2 mL of 0.2% testosterone solution every day. On the 14th day after injection, the hair on the back skin was removed by shaving a 2x3 cm area. The mice were randomly divided into 4 groups: ① PBS group ② minoxidil group ③ COL17-BA MNs group ④ IGF-1@COL17-BA MNs group. The mouse skin became powdery and the skin became thick after modeling, indicating that the modeling was successful.

[0065] (2) Animal grouping

[0066] On the 14th day after injection, the hair on the back skin was removed by shaving a 2x3 cm area. The mice were randomly divided into 4 groups:

[0067] ① PBS group, the dosage of the drug was 200 μl PBS, and the back was smeared

[0068] ② Minoxidil group, the dosage of the drug was 200 μl minoxidil, and the back was smeared.

[0069] ③ COL17-BA MNs group, the back was pressed for 15 min.

[0070] ④ IGF-1@COL17-BA MNs group, the back was pressed for 15 min.

[0071] (3) On the 14th day after androgen modeling, shave a 2x3 cm area to remove the hair on the back of the mouse, and start dosing on the 14th day, once every two days. Start taking photos on the 15th day to record the hair growth on the back of the mouse, once every five days.

[0072] It was observed that after treatment, the IGF group showed the first blue skin and hair growth trend on the 11th day after starting dosing, followed by the minoxidil group and the blank microneedle group, which showed a hair growth trend on the 16th day, while the PBS group also had a little hair growth. The results are shown in the accompanying figures, which show that the use of the drug-loaded microneedle patch of the application significantly promotes the speed of hair follicle regeneration, which is greater than that of the control group.

[0073] Example 11 Biocompatibility experiment

[0074] MTT method was used to explore the toxicity of boron-modified type XVII collagen on L929 cells, so as to evaluate the biocompatibility of boron-modified type XVII collagen. First, the cells were inoculated on a 96-well microplate (10 4 cells per well) overnight, and the boron-modified type XVII collagen hydrogel (2 g) and the IGF-loaded boron-modified type XVII collagen (2 g) were added to 2 ml of DMEM culture medium for extraction overnight, then the two extraction solutions were co-incubated with the cells cultured overnight, and after 24 h of incubation, the cell viability was evaluated by measuring the absorbance at 490 nm with a microplate reader to evaluate the biocompatibility of boron-modified type XVII collagen. The results are shown in the accompanying figures Figure 16 , which show that boron-modified type XVII collagen does not produce toxicity to cells.

[0075] Example 12 Cell migration experiment

[0076] First, draw three horizontal lines as marker lines on the bottom surface of a six-well plate with a straight ruler and a marker pen. Then divide into three groups: Control, COL17-BA, IGF@COL17-BA, add 2 ml of the prepared cell suspension to each well, with a cell density of about 6x10 6 per well, and evenly spread. After the cells grow, use a ruler to draw two vertical lines with a 200 ul gun head perpendicular to the plate and the marker lines, so that the scratch intersects with the marker lines, forming several intersection points as fixed detection points. Then discard the old culture medium and rinse it gently with PBS for two to three times until the cells scraped off are washed clean. According to the grouping, add the drug-containing medium or serum-free medium. After the scratch, cleaning and liquid addition are completed, take photos of different magnifications under a microscope as a 0h control. Place in a 37℃, 5% CO2 incubator. At the required time point, take out the cells and observe the scratch width at the same position under a microscope and take photos. Finally, use ImageJ software to analyze the migration area.

[0077] With the above ideal embodiments according to the present application as the inspiration, through the above description, relevant staff can make various changes and modifications without deviating from the scope of the technical idea of the present application. The technical scope of the present application is not limited to the content in the specification, and the technical scope must be determined according to the scope of claims.

Claims

1. A ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances, characterized in that: The microneedle patch is composed of bioactive substances, boric acid-modified type 17 collagen and a polymer matrix polyvinyl alcohol; The boric acid-modified type 17 collagen is formed by reacting type 17 collagen with 3-amino-4-methoxybenzoic acid (AMB) by oxidative polymerization, and then cross-linking with 3-aminophenylboronic acid (ABA) under the action of 2-morpholineethanesulfonic acid (MES) cross-linking agent.

2. The ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances according to claim 1, characterized in that: The preparation method of the polymer matrix is ​​as follows: polyvinyl alcohol is dissolved in deionized water to prepare a 20% PVA solution, and the solution is stored at room temperature for later use.

3. The ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances according to claim 1, characterized in that: The biologically active substance is insulin-like growth factor IGF-1.

4. A method for preparing a ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances according to claim 1, characterized in that: The specific steps of the preparation method of the microneedle patch are as follows: (1) Weigh boric acid-modified type 17 collagen and dissolve it in deionized water. Then weigh polyvinyl alcohol and dissolve it in deionized water. Mix the two solutions by ultrasonication for 1 minute, freeze them in a -20°C refrigerator for 6 hours, take them out, and thaw them at room temperature for 3 hours. Repeat the freeze-thaw cycle to obtain boric acid-modified type 17 collagen hydrogel. (2) Soak the boric acid-modified type XVII collagen hydrogel in PBS, then add IGF-1 solution and soak for 3 days to allow the freeze-dried gel to reach drug loading equilibrium; (3) Place the drug-loaded boric acid-modified type 17 collagen hydrogel on the microneedle PDMS mold, use a small medicine spoon to press the drug-loaded boric acid-modified type 17 collagen hydrogel into the needle hole of the mold as the microneedle tip, and centrifuge the mold at 2200 rpm for 15 minutes; then place the pressed mold in a vacuum drying oven and repeatedly evacuate it three times; (4) Pour 20% PVA solution onto the mold containing the microneedle patch. Finally, place the covered mold in a ventilated place to dry for one day, and then peel off the microneedle patch.

5. An application of the ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances according to claim 1, characterized in that: The ROS / pH dual-responsive modified type 17 collagen microneedle patch loaded with bioactive substances is used to prepare a patch for treating androgen-induced alopecia.

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