Hydrogel sustained-release microneedle loaded with PCLAF neutralizing antibody and preparation method of hydrogel sustained-release microneedle

Abstract

The invention relates to the technical field of biomedicine, and discloses a hydrogel sustained-release microneedle loaded with a PCLAF neutralizing antibody and a preparation method of the hydrogel sustained-release microneedle, the hydrogel sustained-release microneedle comprises a base substrate, the base substrate is provided with a microneedle side and an opposite back side; a solid microneedle extending from the microneedle side of the base substrate, the solid microneedle including: a gel polymer obtained by polymerizing methacryloyl gelatin and N-hydroxysuccinimide ester through a photoinitiator; the PCLAF neutralizing antibody is obtained by secreting a mouse hybridoma cell 2N52MM01-C of which the preservation number is CCTCC (China Center for Type Culture Collection) NO: 64139, and the PCLAF neutralizing antibody is obtained by secreting a mouse hybridoma cell 2N52MM01-C of which the preservation number is CCTCC NO: 64139; the PCLAF neutralizing antibody is dispersed in the gel polymer. The hydrogel sustained-release microneedle disclosed by the invention has the advantages of painless property, low trauma property, mild drug release, lower infection risk, higher stability, convenience in use and the like, and the hydrogel sustained-release microneedle (PCLAF-Nab-HMs) disclosed by the invention is beneficial to enriching the varieties of drugs for treating osteoporosis in the market.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and provides a hydrogel sustained-release microneedle loaded with a PCLAF neutralizing antibody and a preparation method thereof. Background Art

[0002] With the aging of the global population, osteoporosis has become a serious public health problem. Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and destruction of bone microstructure, which leads to increased bone fragility and susceptibility to fractures.

[0003] At present, the drugs for the treatment of osteoporosis mainly include bisphosphonates, calcitonin, selective estrogen receptor modulators (SERMs), parathyroid hormone (PTH) and, more recently, RANKL inhibitors. Although these drugs can increase bone density and reduce the risk of fractures to a certain extent, they still have some problems in terms of efficacy and safety. For example, long-term use of bisphosphonates may cause osteonecrosis of the jaw and atypical femoral fractures; calcitonin has low effectiveness and may cause side effects such as nausea and vomiting; SERMs have limited effect in preventing vertebral fractures and may increase the risk of thrombosis; although PTH is effective, its high price and daily injection method limit its use; RANKL inhibitors may increase the risk of infection and cancer. Therefore, improvements are urgently needed. Summary of the invention

[0004] The present invention provides a hydrogel sustained-release microneedle loaded with PCLAF neutralizing antibodies, which has the advantages of painlessness and low trauma, mild drug release, low infection risk, high stability, and convenient use. The hydrogel sustained-release microneedle (PCLAF-Nab-HMs) in the present invention helps to enrich the categories of osteoporosis treatment drugs on the market and provide a new research theoretical basis for drug development.

[0005] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0006] In a first aspect, the present application provides a hydrogel sustained-release microneedle loaded with a PCLAF neutralizing antibody, the hydrogel sustained-release microneedle comprising:

[0007] a base substrate having a microneedle side and an opposing back side;

[0008] Solid microneedles, the solid microneedles extending from the microneedle side of the base substrate, the solid microneedles comprising:

[0009] A gel polymer, wherein the gel polymer is obtained by polymerizing methacryloyl gelatin and N-hydroxysuccinimide ester by a photoinitiator;

[0010] PCLAF neutralizing antibody, the PCLAF neutralizing antibody is obtained by secretion of mouse hybridoma cells 2N52 MM01-C with a deposit number of CCTCC NO: 64139; the PCLAF neutralizing antibody is dispersed in a gel polymer;

[0011] Calcium ions, dispersed in gel polymer.

[0012] In the embodiments of the present application, the PCLAF neutralizing antibody can act on the PCLAF protein and have a certain effect thereon, thereby improving or curing osteoporosis to a certain extent.

[0013] The hydrogel sustained-release microneedles deliver PCLAF neutralizing antibodies directly through the skin to the target area, such as the bone site with osteoporosis, which can provide a continuous and stable drug release system to ensure that the drug is released into the blood at a stable rate over a long period of time. This sustained release method can avoid fluctuations in blood drug concentrations caused by oral medications and help maintain a more stable therapeutic effect. The compress is simple to use and does not require oral administration. This improves patient compliance and can reduce the impact of drugs on the systemic system after absorption through the gastrointestinal tract, thereby reducing the incidence of systemic side effects, such as reducing the burden on the liver and gastrointestinal tract.

[0014] In some embodiments, the solid microneedle comprises: 15% to 20% by mass of a gel polymer, 0.2% to 0.3% by mass of a PCLAF neutralizing antibody, 5 nM to 10 nM of calcium ions, and the remainder being a buffer.

[0015] In some embodiments, the PCLAF neutralizing antibody is bonded to methacryloyl gelatin.

[0016] In some embodiments, the buffer comprises one or more of a phosphate buffer having a mass concentration of 0.09 to 0.1 M and a sodium chloride buffer having a mass fraction of 0.89% to 0.91%.

[0017] In some embodiments, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

[0018] In some embodiments, the mass ratio of the PCLAF neutralizing antibody to calcium ions is 1:(20 to 25).

[0019] In some embodiments, the base substrate has a thickness of 800 to 900 microns and the solid microneedles have a length of 500 to 600 microns.

[0020] In some embodiments, the hydrogel sustained-release microneedles are attached to the skin surface of the target location. In the embodiments of the present application, the hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies can be arbitrarily combined together to form a patch or dressing to reduce pain and discomfort during use.

[0021] In a second aspect, the present application provides a method for preparing the hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies according to the first aspect, characterized in that the method comprises:

[0022] providing a buffer solution including calcium ions and a photoinitiator;

[0023] mixing a portion of the buffer solution with methacryloyl gelatin and heating to obtain a first mixed solution;

[0024] mixing the remaining portion of the buffer solution with the PCLAF neutralizing antibody and N-hydroxysuccinimide ester to obtain a second mixed solution;

[0025] The first mixed solution and the second mixed solution are mixed, and the mixture is shaped and concentrated and dried by a microneedle mold and cured by an ultraviolet lamp to obtain a hydrogel sustained-release microneedle.

[0026] In a second aspect, the present application provides a drug for preventing or treating osteoporosis, the drug comprising the hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies according to the first aspect.

[0027] This application has at least the following beneficial effects:

[0028] The hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies in the embodiments of the present application can effectively inhibit osteogenic differentiation, improve bone mass by promoting osteoblast differentiation, and delay the development of osteoporosis. Compared with intravenous injection of PCLAF-Nab, the hydrogel sustained-release microneedles (PCLAF-Nab-HMs) loaded with PCLAF neutralizing antibodies reduce the pain and discomfort of use, and the drug storage and carrying are more convenient. The drug can have a sustained-release effect, which can improve patient compliance. The hydrogel sustained-release microneedles are painless and low-traumatic, with mild drug release, lower risk of infection, and higher stability.

[0029] The hydrogel microneedles of the present application embodiment combine the unique properties of hydrogel materials and the advantages of microneedle technology, becoming a hot topic in microneedle drug delivery research. Hydrogels have high water content, softness, and good biocompatibility, making hydrogel microneedles almost painless and less traumatic when inserted into the skin, greatly improving patient acceptance. Hydrogel microneedles can achieve sustained release and precise delivery of drugs by adjusting the crosslinking density and composition of the hydrogel.

[0030] The present invention provides a novel osteoporosis treatment drug administered by microneedles, which has fewer side effects and a more convenient administration method. The present invention takes into account the bioavailability of the drug and the compliance of the patient, and adopts an improved drug delivery carrier and sustained-release technology to ensure that the drug can be released stably and continuously to effectively exert the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 paying creative work.

[0032] Figure 1 A flow chart of PCLAF-Nab-HMs of an embodiment of the present application and a finished product diagram of PCLAF-Nab-HMs are shown;

[0033] Figure 2 The structure of PCLAF-Nab-HMs of the embodiment of the present application under SEM electron microscope and its puncture force diagram are shown;

[0034] Figure 3 The graphs showing the changes in plasma drug concentration over time after PCLAF-Nab and PCLAF-Nab-HMs were respectively injected into the tail vein of 8-month-old rats in the example of the present application;

[0035] Figure 4 The bone scan three-dimensional reconstruction image and the bone parameter analysis statistical graph of the bone mass increase of 18-month-old mice after tail vein injection of PCLAF-Nab and use of PCLAF-Nab-HMs in the embodiment of the present application are shown;

[0036] Figure 5 Shown are the staining schematic diagrams of the calcein double labeling experiment after the tail vein injection of PCLAF-Nab and PCLAF-Nab-HMs in 18-month-old aged mice of the present application example and the statistical analysis diagram of the increase in bone formation rate;

[0037] Figure 6 A schematic diagram of cell staining and a statistical analysis diagram of the number of cells in an osteocalcin staining experiment after PCLAF-Nab was injected into the tail vein of 18-month-old mice and PCLAF-Nab-HMs were used in an embodiment of the present application;

[0038] Figure 7A schematic diagram of positive staining and a statistical analysis diagram of the number of cells in an osteoclast staining experiment after PCLAF-Nab was injected into the tail vein of 18-month-old mice and PCLAF-Nab-HMs were used in an embodiment of the present application;

[0039] Figure 8 This is a CT image of mouse bones taken using small animal Micro-CT.

[0040] Cell line collection

[0041] The mouse hybridoma cells 2N52 MM01-C provided in this application have a deposit number of CCTCC NO: 64139 and are deposited in Guangdong Microbiological Culture Collection Center (GDMCC); address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The strain was received and registered by the collection center on March 4, 2024, and was detected as a viable biological material by the collection center on March 19, 2024. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0043] PCLAF (PCNA clamp associated factor), whose Chinese name is proliferating cell nuclear antigen clamp associated factor, is a protein that plays a role in cell proliferation, DNA repair and cell cycle regulation. It is related to proliferating cell nuclear antigen (PCNA), which is a key factor that stabilizes the template during DNA replication. Studies have found that when PCLAF neutralizing antibodies are applied to cells to allow them to interact with PCLAF, the gene sequence encoding PCLAF is shown in SEQ ID No. 2, which can effectively inhibit osteogenic differentiation, while improving bone mass by promoting osteoblast differentiation and delaying the development of osteoporosis. Therefore, the present application uses hydrogel sustained-release microneedles containing PCLAF neutralizing antibodies to delay the development of osteoporosis.

[0044] The PCLAF-Nab-HMs of the examples of the present application have a prolonged half-life and reduced dosing frequency compared to the single PCLAF neutralizing antibody.

[0045] The PCLAF-Nab-HMs of the present application example can reverse the inhibitory effect of PCLAF on the osteogenic differentiation of BMSCs. The mRNA levels of Runx2, Sp7, and Alp in the BMSC cells treated with PCLAF-Nab-HMs are significantly increased compared with those in the PCLAF treatment group, and the effect of GCA-Nab-MSNs is equivalent to that of the GCA-Nab treatment group alone;

[0046] The PCLAF-Nab-HMs of the present application example can improve bone mass, increase bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and bone formation rate, and reduce trabecular separation (Tb.Sp), and its therapeutic effect is equivalent to that of mice treated with GCA-Nab alone;

[0047] The PCLAF-Nab-HMs of the present application example can increase the number of OCN+ osteoblasts and reduce the number and area of ​​adipocytes, and the degree of improvement is comparable to that of mice treated with PCLAF-Nab alone.

[0048] Example 1 Preparation of PCLAF neutralizing antibodies

[0049] The mouse hybridoma cells 2N52 MM01-C with the deposit number of CCTCC NO: 64139 stored in the laboratory are induced by the PCLAF antigen, i.e., SEQ ID No. 1, so that the mouse hybridoma cells can stably secrete PCLAF neutralizing antibodies. The mouse hybridoma cells are amplified, 1 mL of the hybridoma cells are transferred into a 100 mL culture bottle, and DMEM culture medium containing 10% FBS is regularly added to the 37°C constant temperature cell culture incubator for cell amplification and culture for 10-12 days. Among them, the amino acid sequence 54-77 of the PCLAF antigen is as shown in SEQ ID No. 1: 54-77: AC-CVRPTPKWQKGIGEFFRLSPKDSE-NH2.

[0050] The Protein A affinity chromatography column was rinsed with ultrapure water and then equilibrated with an equilibrium buffer; the treated hybridoma cell supernatant was loaded onto the affinity chromatography column, and after loading, it was rinsed with an equilibrium buffer. The elution was performed with an elution buffer, and the elution peak was collected, neutralized with a Tris buffer, and desalted to PBS7.4 to obtain PCLAF-Nab.

[0051] Example 2 Construction of PCLAF-Nab-HMs

[0052] The preparation steps include: adding 0.05g of photoinitiator LAP to 20ml of 10mM Ca 2+The solution was heated in a water bath at 40-50°C for 15 minutes to dissolve, and shaken once every 3 minutes to obtain solution A.

[0053] The above solution A was equally divided into two parts, one part was added with 3 g of methacryloyl gelatin (GelMA), heated in a water bath at 40-50°C for 50 minutes, and shaken once every 10 minutes to obtain solution B. The other part was added with 4 mg of PCLAF-Nab and 30 μg of acryloyl polyethylene glycol NHS ester (NHS), dissolved at 37°C, and incubated at 4°C for 18-24 hours to obtain solution C.

[0054] Solutions B and C were sterile filtered and then returned to 37°C, and solutions B and C were mixed to obtain a mixed solution D.

[0055] The mixed solution D was added to the microneedle mold and centrifuged at 3000 rpm for 5 min 5-9 times to remove bubbles until the bubbles disappeared.

[0056] The mixture was concentrated in an oven at 37°C for 5-6 hours, cured with an ultraviolet lamp (405 nm) for 30-40 seconds, dried in an oven at 37°C for 12-18 hours, and demolded to obtain microneedles.

[0057] The process of the preparation method of PCLAF-Nab-HMs in this embodiment and the finished product of PCLAF-Nab-HMs are shown in FIG. Figure 1 Figure A is a flow chart of the preparation method, and Figure B is a finished product of PCLAF-Nab-HMs.

[0058] PCLAF-Nab-HMs were observed by SEM electron microscope. Figure 2 Among them, Figure A shows the structure of PCLAF-Nab-HMs under SEM electron microscope; Figure B shows the puncture force diagram of PCLAF-Nab-HMs microneedle.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 2 is that no Ca is added 2+ .

[0061] Example 3 Drug Half-Life Detection

[0062] The 18-month-old mice were divided into a control group and a treatment group. The control group was injected with a single antibody, namely PCLAF-Nab, through the tail vein of the mice. The treatment group was injected with CLAF-Nab-HMs prepared in Example 2 above through the skin. The antibody concentration was measured at regular intervals starting from the day of injection for 6 consecutive days. Blood was collected from the inner canthus, and the concentration of mouse plasma antibodies was detected using an ELISA kit. The concentration changes of the two groups of drugs were compared. Compared with PCLAF-Nab, PCLAF-Nab-HMs prolonged the half-life of the drug in mice.

[0063] Example 4

[0064] 18-month-old mice were randomly divided into a control group, a PCLAF-Nab treatment group, and a PCLAF-Nab-HMs treatment group. The control group was injected with PBS through the tail vein, and treatment group 1 was injected with PCLAF neutralizing antibodies (dosage was 1 mg / kg) through the tail vein once every 3 days for 2 months. Treatment group 2 used PCLAF-Nab-HMs on the skin once every 6 days for 2 months. Then the mice in each group were killed and the bilateral femurs were collected. The femur specimens on one side were fixed with formaldehyde and placed in a sample tube for CT scanning, and then micro-CT analysis and three-dimensional reconstruction were performed to observe the bone mass. The results are shown in Figure 2. Figure 3 and Figure 4 .

[0065] from Figure 3 It can be seen from the bone scan reconstruction images that compared with the PBS control group, the bone mass of the PCLAF-Nab treatment group and the PCLAF-Nab-HMs treatment group increased significantly, and the effect of the PCLAF-Nab-HMs treatment group was better.

[0066] Figure 4 In the figure, A is a three-dimensional reconstruction of bone scans showing the increase in bone mass in 18-month-old mice after tail vein injection of PCLAF-Nab and use of PCLAF-Nab-HMs; B is a statistical analysis of bone parameters showing the increase in bone mass, including bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N).

[0067] from Figure 4 The Micro-CT analysis parameters of AC showed that the bone volume fraction (BV / TV), number of trabeculae (Tb.N) and trabecular thickness (Tb.Th) of mice in the PCLAF-Nab-treated group and the PCLAF-Nab-HMs-treated group were significantly increased, and the effect was better in the PCLAF-Nab-HMs-treated group.

[0068] In conclusion, PCLAF-Nab-HMs can reverse the effects of PCLAF on bone mass and bone turnover to improve bones.

[0069] OCN, Trap staining and calcein double labeling experiments

[0070] The femur on the other side was decalcified in 10% EDTA decalcification solution for 2-3 weeks, embedded in paraffin, and sectioned, and then stained with OCN and Trap, respectively. The calcein double labeling experiment was also carried out at the same time. The control group mice and the treatment group mice were intraperitoneally injected with calcein 10 days and 3 days before sacrifice. After the mice were sacrificed, the femur was kept for hard tissue sectioning and calcein fluorescence was detected. The results are shown in Figures 5 to 7 .

[0071] Figure 5 Calcein double labeling experiment was performed on 18-month-old mice after tail vein injection of PCLAF-Nab and PCLAF-Nab-HMs. Figure A is a staining diagram, and Figure B is a statistical analysis of the increase in bone formation rate. Figure 5 It can be seen that compared with the PBS control group, the bone formation of 18-month-old mice treated with PCLAF-Nab and PCLAF-Nab-HMs was significantly increased, and the effect of the PCLAF-Nab-HMs treatment group was better.

[0072] Figure 6 Osteocalcin staining (OCN staining) was performed after PCLAF-Nab and PCLAF-Nab-HMs were injected into the tail vein of 18-month-old mice. Figure 5 A is a schematic diagram of OCN+ positive cell staining. Figure 5 B is a statistical analysis of the number of OCN-positive (OCN+) cells; Figure 6 It can be seen that compared with the PBS control group, the OCN of 18-month-old mice treated with PCLAF-Nab and PCLAF-Nab-HMs + The osteoblasts increased significantly, and the effect was better in the PCLAF-Nab-HMs treatment group;

[0073] Figure 7 The osteoclast staining experiment (Trap staining) was performed after the tail vein injection of PCLAF-Nab and PCLAF-Nab-HMs in 18-month-old mice. Figure 6 A is a schematic diagram of Trap+ positive staining. Figure 6 B is the statistical analysis of the number of Trap positive (Trap+) cells; Figure 7 It can be seen that after PCLAF-Nab and PCLAF-Nab-HMs were used in 18-month-old mice, the Trap + The number of osteoclasts increased significantly, and the effect was better in the PCLAF-Nab-HMs treatment group.

[0074] Bone scan test

[0075] The PCLAF-Nab-HMs microneedles of Example 2 and the microneedles prepared in Comparative Example 1 were applied to multiple elderly mice under the same conditions. The two groups were applied at the same time. The concentration of effective active substances in the microneedles, such as PCLAF-Nab-HMs, was consistent with that in the comparative example. The application site was the skin surface of the target bone area. After 2 months of use, CT imaging was used for detection. The results are shown in Figure 8 .

[0076] Figure 8 The left picture shows the bone scan result of mice injected with microneedles of comparative example 1, and the right picture shows the bone scan result of mice injected with microneedles of example 2. It can be seen from the figure that with the addition of Ca 2+ The group showed more bone mass, indicating that the PCLAF-Nab-HMs of Example 2 has a significant effect on the prevention and treatment of osteoporosis. After testing, the effect of the comparative example is inferior to that of the example in terms of osteoporosis treatment effect.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A hydrogel sustained-release microneedle loaded with PCLAF neutralizing antibodies, characterized in that: The hydrogel sustained-release microneedle comprises: a base substrate having a microneedle side and an opposing back side; A solid microneedle extending from the microneedle side of the base substrate, the solid microneedle comprising: A gel polymer obtained by polymerizing methacryloyl gelatin and N-hydroxysuccinimide ester via a photoinitiator; PCLAF neutralizing antibody, the PCLAF neutralizing antibody is obtained by secretion of mouse hybridoma cells 2N52 MM01-C with a deposit number of CCTCCNO: 64139; the PCLAF neutralizing antibody is dispersed in the gel polymer; Calcium ions are dispersed in the gel polymer.

2. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The solid microneedle comprises: 15% to 20% by mass of gel polymer, 0.2% to 0.3% by mass of PCLAF neutralizing antibody, 5nM to 10nM of calcium ion, and the remainder is buffer.

3. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The PCLAF neutralizing antibody is bonded to the methacryloyl gelatin.

4. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The buffer comprises one or more of a phosphate buffer having a mass concentration of 0.09 to 0.1 M and a sodium chloride buffer having a mass fraction of 0.89% to 0.91%.

5. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoyl phosphate.

6. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The mass ratio of the PCLAF neutralizing antibody to the calcium ions is 1:(20 to 25).

7. The hydrogel sustained-release microneedle according to claim 1, characterized in that: The base substrate has a thickness of 800 to 900 micrometers, and the solid microneedles have a length of 500 to 600 micrometers.

8. The hydrogel sustained-release microneedle according to claim 7, characterized in that: The hydrogel sustained-release microneedles adhere to the skin surface at the target location.

9. A method for preparing the hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies according to any one of claims 1 to 8, characterized in that: The method comprises: providing a buffer solution including calcium ions and a photoinitiator; mixing a portion of the buffer solution with methacryloyl gelatin and heating to obtain a first mixed solution; mixing the remaining portion of the buffer solution with the PCLAF neutralizing antibody and N-hydroxysuccinimide ester to obtain a second mixed solution; The first mixed solution and the second mixed solution are mixed, and the mixture is shaped and concentrated and dried by a microneedle mold and cured by an ultraviolet lamp to obtain the hydrogel sustained-release microneedle.

10. A drug for preventing or treating osteoporosis, characterized in that: The drug comprises the hydrogel sustained-release microneedles loaded with PCLAF neutralizing antibodies as described in any one of claims 1-8.

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