Multi-channel degradable hard microneedle shell and preparation method thereof

Abstract

The invention relates to a multi-channel degradable hard microneedle shell and a preparation method thereof, and belongs to the technical field of medical sanitary materials. The hard-dissolving microneedle shell has good physical properties, is hard in texture, biodegradable and good in molecular permeability, and the independent multi-channel microneedle can be used for loading living cells and supporting in-situ exchange of nutrient substances, so that subcutaneous delivery and in-situ regeneration of the living cells are realized. The preparation method comprises the steps of preparing a groove mold and a sleeve cover, filling PLGA solid blocks, heating the PLGA solid blocks to melt the PLGA solid blocks, performing vacuum-pumping treatment, performing pressurization treatment, performing freeze-thaw circulation, and performing demolding at room temperature. The invention has the beneficial effects of good physical properties, hard texture, biodegradability and good molecular permeability, and the independent multi-channel microneedle can be used for loading living cells, supports in-situ exchange of nutrient substances, and realizes subcutaneous delivery and in-situ regeneration of living cells.

Description

Technical Field

[0001] The invention belongs to the technical field of medical sanitary materials and relates to a multi-channel degradable hard microneedle shell and a preparation method thereof. Background Art

[0002] Microneedle (MN) technology is an important research direction in the field of drug delivery and medical treatment in recent years. It enhances the transport of skin molecules in a minimally invasive and painless way, and can quickly deliver drug molecules, genes, vaccines and other substances to the dermis and subcutaneous tissue layer, thereby improving their efficiency in the target tissue.

[0003] Although microneedle technology has been widely used in drug delivery, there are relatively few studies on the delivery of living cells. Currently, the only microneedle technology reported for the delivery of living cells is based on the cryo-microneedle method. This method has confirmed the viability and proliferation of delivered cells in mouse experiments. However, there are some limitations to the delivery of cells by cryo-microneedle, such as the need for a low temperature environment, the tight time for microneedle penetration, and the limited puncture depth.

[0004] In order to solve these problems, core-shell microneedle technology was proposed. This technology uses a hard microneedle shell to wrap a soft gel for drug delivery. It can not only effectively deliver drugs through the skin, but also has good in situ controlled release properties. However, the preparation of core-shell microneedles usually involves a layer-by-layer stacking method, which makes it difficult to make the wall thickness of the microneedle structure uniform. More importantly, the microneedle shell prepared in this way is a closed structure, which cannot achieve material exchange inside and outside the microneedle.

[0005] In addition, current microneedle therapy strategies mostly use microneedle delivery to achieve drug or cell release, but cannot make drugs or cells work in situ. Especially in the field of regenerative medicine, current microneedle therapy strategies are difficult to achieve in situ regeneration of living cells.

[0006] Therefore, a new microneedle technology is needed to prepare a microneedle shell that is connected inside and out, has uniform wall thickness, is hard in texture, and is degradable. This will not only help to achieve the loading and subcutaneous delivery of living cells at room temperature, but also have the potential to achieve in situ regeneration of living cells. It is a promising microneedle treatment strategy for the delivery of living cells and the development of regenerative medicine. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a multi-channel degradable hard microneedle shell and a preparation method thereof, which solves the problems of subcutaneous delivery of living cells at room temperature and in situ cell regeneration.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A multi-channel degradable hard microneedle shell comprises a base and microneedles arranged in a matrix on the base; the microneedles comprise a hollow cylindrical body, a conical needle tip is arranged on the side of the body away from the base; a plurality of channels are uniformly provided along the axial direction of the body, which are narrow inside and wide outside and have gradually changing sizes.

[0010] Optionally, it is prepared by pressurizing poly(lactic acid-glycolic acid) copolymer and can load living cells to achieve subcutaneous delivery and in situ regeneration of living cells at room temperature.

[0011] Optionally, the body diameter is 700 μm, the total length is 1300 μm, and the shell thickness is 100 μm.

[0012] Optionally, the channel has an inner width of 50 μm, an outer width of 70 μm, and a height of 900 μm.

[0013] Optionally, the preparation material is monomethoxy polyethylene glycol polylactic acid glycolic acid copolymer, which can be completely liquefied at 60°C.

[0014] A method for preparing the above-mentioned multi-channel degradable hard microneedle shell comprises the following steps:

[0015] S1: preparing a groove mold and a cover according to the shape of the microneedle shell to form a matching microneedle mold, wherein the groove mold is made of an elastic material, and the cover is made of a metal material;

[0016] S2 fills the groove mold with a solid block of PLGA and heats it to melt it;

[0017] S3 vacuum treatment to fill PLGA into the microneedle groove of the groove mold;

[0018] S4 inserts the cover into the mold while it is still hot, and squeezes the PLGA liquid to make the cover and the mold completely fit together;

[0019] S5 uses a syringe to pressurize the mold;

[0020] S6 was subjected to freeze-thaw cycles;

[0021] S7 demoulding to obtain the microneedle shell.

[0022] Optionally, the elastic material is polydimethylsiloxane.

[0023] Optionally, the metal material is metallic copper.

[0024] Optionally, the vacuum treatment is performed at 60°C.

[0025] Optionally, the freeze-thaw cycle includes maintaining at 4°C for 2 hours, maintaining at -20°C for 2 hours, maintaining at 4°C for 2 hours, and maintaining at room temperature for 2 hours.

[0026] The beneficial effects of the present invention are:

[0027] 1. Good physical properties: The PLGA MN shell prepared by the present invention has a good molding state, uniform wall thickness, and hard texture. This property can effectively protect the internal substances of the microneedle during the microneedle puncture process, prevent it from compressing and deforming due to the puncture pressure, and thus maintain the good state of the internal substances.

[0028] 2. Good permeability: The obvious internal and external through-channels on the side wall of the microneedle provide good small molecule permeability. This property is conducive to the exchange of substances inside and outside the microneedle, providing a way for the living cells loaded inside the microneedle to absorb nutrients, and providing necessary nutritional support for the in-situ growth of living cells.

[0029] 3. Biosafety: The PLGA MN shell completely degrades in vivo in about 2 weeks, with no foreign matter remaining, and has good biosafety. This makes the present invention safer and more reliable in medical applications, and is particularly suitable for medical occasions that require biodegradation.

[0030] 4. Multifunctionality: The microneedle shell prepared by the present invention can not only be used to load and deliver living cells, but also support the in situ exchange of nutrients, achieving subcutaneous delivery and in situ regeneration of living cells. This multifunctionality provides new treatment strategies for fields such as regenerative medicine and cell therapy.

[0031] 5. Simplicity and controllability of the preparation method: The preparation method provided by the present invention is simple and controllable. Through computer-aided design and a specific mold preparation process, the size and shape of the microneedles can be accurately controlled to ensure the uniformity and consistency of the microneedle shell.

[0032] 6. Environmental friendliness: Due to the biodegradability of PLGA materials, the present invention not only provides an effective medical solution, but also meets environmental protection requirements and reduces the impact on the environment.

[0033] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0035] Figure 1 The overall morphology of the microneedle prepared by the present invention;

[0036] Figure 2 : are microscopic images of the present invention, wherein (A) is a microscopic image of a microneedle mold; (B) is an enlarged image of (A); (C) is a microscopic image of a microneedle cover; (D) is an enlarged image of (C); (E) is a microscopic image of a microneedle shell from a top view; (F) is a microscopic image of a microneedle shell from a side view;

[0037] Figure 3 This is a coronal scan image of the microneedle prepared in the present invention;

[0038] Figure 4 A sagittal scan image of the microneedle prepared in the present invention;

[0039] Figure 5 Characterization test results of the microneedle shell prepared by the present invention, wherein (A) is a stiffness test, and the inset shows the definition of the destructive force plotted on the y-axis; (B) is the mechanical behavior of anti-compression deformation; (C) is a growth factor permeability test;

[0040] Figure 6 The in vitro degradation of the microneedle shell prepared by the present invention, wherein (A) is the weight loss rate of PLGA MN after the shell is shaken and incubated at 37°C in a medium containing lipase for different time periods, and (B) is the lactic acid content in the solution;

[0041] Figure 7 : The effect of microneedle treatment on cell activity, wherein (A) is the activity of dermal cells after incubation with the microneedle shell for different time periods, and (B) is the activity of epidermal cells after incubation with the microneedle shell for different time periods;

[0042] Figure 8 Images quantifying the penetration depth of the microneedle shell in vitro. DETAILED DESCRIPTION

[0043] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0044] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0045] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0046] See also Figures 1 to 8 The present invention discloses a method for preparing a microneedle shell, comprising the following steps:

[0047] S1. Use Rhino software to draw the required microneedle structure: ① Design the microneedle shape based on the round-bottom low-adhesion 96-well plate, and design the microneedle length based on the full-thickness of the skin. The final designed microneedle is cylindrical + cone-shaped, with a cylinder height of 800μm and a cone height of 500μm; ② Based on the measured hair papilla diameter, design the microneedle internal width of 500μm; ③ The microneedle shell width is 100μm, and the overall width of the microneedle is 700μm; ④ There are 8 pores on the outer edge, which run through the cylinder + cone in height, are 100 long, 50μm wide, and have an overall porosity of 25%; ⑤ Design a 10×10 microneedle array with a spacing of 1.15mm; ⑥ There is a step with an area of ​​12mm×12mm and a height of 0.4mm above the array.

[0048] S2. Prepare the groove mold and cover to form a matching microneedle mold: ① The groove mold is made of polydimethylsiloxane (PDMS): a cylindrical groove mold with a diameter of 25 mm; the microneedles are 10×10 arrays with a spacing of 1.15 mm; the width of a single microneedle groove is 700 μm, the cylindrical part is 800 μm long, and the conical part is 500 μm long; the inner edge contains 8 symmetrically distributed small columns, which run through the cylinder + cone in height, are 100 long and 50 μm wide; there is a groove with an area of ​​12 mm×12 mm and a height of 400 μm above the array; there is another groove of 14 mm×14 mm×2.6 mm above. ② Cover, made of metallic copper: the microneedles are in a 10×10 array with a spacing of 1.15mm; a single microneedle is 500μm wide, the cylindrical part is 1200μm long, and the conical part is 357μm long; the top is a 14mm×14mm×2.6mm cylinder; there is also a handle with a diameter of 25mm and a height of 5mm.

[0049] S3. Preparation of microneedle shell: ① Fill the groove mold with PLGA solid blocks; ② Heat at 60℃ for 2h to completely melt the PLGA. During this process, constantly observe whether the melted PLGA in the mold is completely filled at the bottom of the mold, and add PLGA solid blocks to the unfilled parts; ③ Repeatedly vacuum at 60℃ in a vacuum drying oven to fill the PLGA into the microneedle groove of the groove mold; ④ Insert the cover into the mold while it is hot, squeeze the PLGA liquid, and make the cover and the mold completely fit; ⑤ Prepare a 50mL syringe with a closed needle end, pull out the syringe piston, put the entire microneedle mold into the 50mL syringe, replace the piston and pressurize the syringe for 12h (the pressure is about 2 standard atmospheres, i.e. 2.026*10 5 Pa, which can be replaced by other pressurization processes); ⑥ Take out the above mold and freeze-thaw cycle: 4℃2h→-20℃2h→4℃2h→room temperature2h; ⑦ Room temperature demolding: First, remove the cover and PLGA MN as a whole from the PDMS groove mold, and then use a surgical blade to gently separate the PLGA MN from the cover to obtain the PLGA MN shell. In addition, fill the groove mold with ethoxylated trimethylolpropane triacrylate (ETPTA) containing 0.25% photoinitiator (2959), vacuumize the mold to completely remove the bubbles in the mold, cover it with a cover, light cure it under ultraviolet light for 2min, and demold it to obtain the ETPTAMN shell.

[0050] The microneedle molds and PLGA MN shells obtained in Examples 1 to 3 above were subjected to macroscopic and microscopic photography, characterization testing, biosafety assessment, live cell loading, and skin puncture experiments:

[0051] The appearance image of the microneedle mold: the microneedle mold was photographed under a stereo microscope and an upright fluorescence microscope; the PLGAMN shell was photographed under a digital camera and an inverted fluorescence microscope; the PLGA MN shell was observed by a scanning electron microscope after gold spraying; 0.1% rhodamine B dye solution was added to the PLGA MN shell during the preparation process and photographed under a laser confocal microscope. The microneedle groove mold is composed of PDMS, a 10×10 array, and the diameter of each microneedle groove from the inside to the outside is 500-700μm, the spacing is 1150μm, and the column width is 50-70μm. The microneedle cover is composed of PDMS, a 10×10 array, and the diameter of each microneedle groove is 500μm, and the spacing is 1150μm.

[0052] The appearance of PLGA MN shell: PLGA MN shell was photographed under a digital camera and an inverted fluorescence microscope; PLGAMN shell was observed under a scanning electron microscope after being treated with gold spraying; 0.1% rhodamine B dye was added to the PLGA MN shell during the preparation process and photographed under a laser confocal microscope. Figures 2 to 4 As shown, the microneedle shell has a thin-walled milky white structure in the shape of a cylinder + cone. The overall size of the microneedle is about 14 mm, the diameter of a single microneedle is about 700 μm, the thickness of the microneedle shell is about 100 μm, and 8 symmetrically distributed pore structures that run through the side wall are clearly visible. The pores are 50-70 μm wide and 900 μm long.

[0053] Physical characterization test of PLGA MN shell: Use a universal material testing machine to perform compression tests. Place the PLGA MN shell, the PLGA MN shell loaded with 200μL room temperature cell suspension for 5 / 10 / 15min, and the PLGA MN shell loaded with frozen cells on the universal material testing machine, respectively. Measure the compression deformation of PLGA MN under different loads, and calculate its maximum destructive force. Cut the sealing film into sheets of about 2cm×2cm, and stack 10 sheets of film into an artificial membrane with a thickness of 1270μm. Artificial pressure is applied to make the microneedle penetrate the artificial membrane, and a vernier caliper is used to measure the final penetration depth of the microneedle. Soak the PLGA MN shell in human epidermal growth factor, and regularly test the peptide chain concentration of the solution to evaluate the small molecule permeability of the microneedle. Lipase was dissolved in DMEM / F12 medium at a concentration of 1 mg / mL. The PLGA MN shell was immersed in the above solution and shaken at 37°C and 200 rpm / min. The contents of lactic acid and glycolic acid in the solution after different degradation times were detected. The weight loss of the microneedles was measured after freeze-drying to evaluate the in vitro degradation performance of the microneedles.

[0054] Biosafety assessment: Soak the PLGA MN shell in DMEM or F12 medium for 24 hours, filter and sterilize it, and use it as the experimental group culture medium for CCK8 cell activity detection. Ordinary DMEM or F12 medium is used as the control group culture medium. Dermal cells and epidermal cells are cultured separately in 96-well plates. After the cells adhere to the wall for 3 days, the experimental group and control group culture medium are used to replace the culture medium of the cell wells of different groups (DMEM medium is used for dermal mesenchymal cells, and F12 medium is used for epidermal cells); 10% CCK8 reagent is added after continuing to culture for 24h, 48h, and 72h, and the absorbance at 450nm is detected by an enzyme reader to evaluate the CCK8 cell activity of the two cells.

[0055] Live cell loading experiment: First, PLGA MN shell and ETPTAMN shell were placed in sterile cell culture separation dishes, sterilized with UV light for 15 minutes, 200 μL of dermal cell suspension at room temperature was added to each microneedle shell, the dish cover was closed, and the vacuum pump was used for debubbling for 2 minutes, and the cell sedimentation was observed by taking pictures. Then, the dermal cell suspension containing 5% cell freezing solution was loaded in the microneedle mold, and the frozen cell microneedles were obtained by gradient freezing (-20℃2h→demembrane→-80℃ storage), and a new PLGA MN shell was placed in another cell culture dish, the frozen cell microneedles were taken out, frozen in a -196℃ liquid nitrogen box for 1 hour, and the frozen cell microneedles were inserted into the PLGA MN shell on ice, and the cell loading was observed by taking pictures.

[0056] Skin puncture and degradation experiment: 6-week-old female nude mice were anesthetized with 1% sodium pentobarbital. The skin tissue of the nude mice was stretched and completely punctured into the PLGA MN shell stained with rhodamine B. The skin was fixed with a transparent breathable dressing. The dressing was changed regularly and photos were taken to record the degradation of the microneedles.

[0057] During the experiment, the experimental mice showed normal eating and activity behaviors, and did not show any abnormal behaviors or symptoms. Physical examination showed that there were no abnormal changes in the weight, hair condition, and activity level of the mice. During the experiment, all mice did not show any obvious discomfort or adverse reactions. These observations indicate that the microneedle shell used during the experiment had no negative impact on the health of the mice, and the experimental conditions were safe for the mice.

[0058] On the first day after the experiment, the microneedles were still intact, with a smooth surface and no obvious signs of degradation. As time went on, especially on the 7th day in the mice, we could observe that the microneedles began to show obvious signs of degradation. The appearance of the microneedles gradually became blurred and depressions appeared on the surface, indicating that the PLGA material had begun to degrade. On the 14th day after the experiment, most of the microneedles had been completely degraded, leaving only a small amount of residue on the skin surface. These residues were also completely absorbed in the next few days, leaving no foreign matter residue.

[0059] In addition, through skin puncture experiments, we found that the skin of experimental mice did not show adverse reactions such as redness, swelling, bleeding or infection during the microneedle insertion and degradation process. This further proves the safety of the microneedle shell of the present invention in animals.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A multi-channel degradable hard microneedle shell, characterized in that: It comprises a base and microneedles arranged in a matrix on the base; the microneedles comprise a hollow cylindrical body, a conical needle tip is arranged on the side of the body away from the base; a plurality of channels are evenly arranged on the body along its axial direction, which are narrow inside and wide outside and have gradually changing sizes.

2. The multi-channel degradable hard microneedle housing according to claim 1, characterized in that: It is made of pressurized poly(lactic acid-glycolic acid) copolymer and can load living cells to achieve subcutaneous delivery and in situ regeneration of living cells at room temperature.

3. The multi-channel degradable hard microneedle housing according to claim 1, characterized in that: The body diameter is 700 μm, the total length is 1300 μm, and the shell thickness is 100 μm.

4. The multi-channel degradable hard microneedle housing according to claim 1, characterized in that: The channel has an inner width of 50 μm, an outer width of 70 μm, and a height of 900 μm.

5. The multi-channel degradable hard microneedle housing according to claim 2, characterized in that: The preparation material is monomethoxy polyethylene glycol polylactic acid glycolic acid copolymer, which can be completely liquefied at 60°C.

6. A method for preparing the multi-channel degradable hard microneedle shell according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: preparing a groove mold and a cover according to the shape of the microneedle shell to form a matching microneedle mold, wherein the groove mold is made of an elastic material, and the cover is made of a metal material; S2 fills the groove mold with a solid block of PLGA and heats it to melt it; S3 vacuum treatment to fill PLGA into the microneedle groove of the groove mold; S4 inserts the cover into the mold while it is still hot, and squeezes the PLGA liquid to make the cover and the mold completely fit together; S5 uses a syringe to pressurize the mold; S6 was subjected to freeze-thaw cycles; S7 demoulding to obtain the microneedle shell.

7. The method according to claim 6, characterized in that: The elastic material is polydimethylsiloxane.

8. The method according to claim 6, characterized in that: The metal material is copper.

9. The method according to claim 6, characterized in that: The vacuum treatment was performed at 60°C.

10. The method according to claim 6, characterized in that: The freeze-thaw cycle includes maintaining at 4°C for 2 hours, maintaining at -20°C for 2 hours, maintaining at 4°C for 2 hours, and maintaining at room temperature for 2 hours.

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