Double-layer microneedle patch and preparation method thereof

Abstract

The invention discloses a double-layer microneedle patch and a preparation method thereof.The preparation method of the double-layer microneedle patch comprises the following steps that firstly, 3D printing is conducted through polydimethylsiloxane, and a PDMS mold similar to a machining microneedle is obtained; dispersing the medicine into the sericin solution to serve as an outer layer solution; dispersing germanium nanoparticles into the polycaprolactone solution to obtain an inner layer solution; and finally, adding the outer-layer solution into the needle-shaped groove of the PDMS mold, so that the needle-shaped groove is filled with the outer-layer solution, drying, adding the inner-layer solution, drying, and demolding to obtain the double-layer microneedle patch. When the double-layer microneedle patch is used for drug delivery, drugs can be dissolved and released in a short time, the inner layer is polycaprolactone (PCL) carrying photo-thermal agent germanium nanoparticles, degradation is slow, a photo-thermal effect is generated under irradiation of 808nm near-infrared light, residual tumor cells are induced to die immunogenicity, an immune microenvironment is programmed in a time sequence, and the double-layer microneedle patch has the advantages of being high in sensitivity, high in sensitivity and good in stability. Postoperative recurrence and metastasis of tumors are efficiently inhibited, and meanwhile operative wound repair is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of microneedle patches, and in particular to a double-layer microneedle patch and a preparation method thereof. Background Art

[0002] Cancer is one of the diseases that seriously threaten public health. For most solid tumors, surgery is still the preferred treatment option, but it is difficult to eradicate the tumor by surgery alone, and residual tumor cells lay hidden dangers for cancer recurrence and metastasis. It is reported that tumor recurrence and metastasis caused by incomplete surgical resection account for more than 90% of cancer deaths. A large number of studies have shown that inflammation caused by surgical trauma, infection, etc. can promote the occurrence, progression and metastasis of tumors. This type of inflammation is also called "pro-cancer inflammation". This type of inflammatory signal not only directly promotes the progression of tumor cells, but also participates in shaping the tumor suppressive microenvironment. Immune cells cannot recognize and kill residual tumor cells, and anti-tumor immunity is hindered, further promoting the occurrence and development of tumors. Therefore, the perioperative period is a critical time for postoperative tumor recurrence and progression. It is necessary to inhibit "pro-cancer inflammation" in the early perioperative period, minimize the impact of inflammation on the tumor microenvironment, and reverse the immunosuppressive microenvironment in the later stage to promote anti-tumor immunity, thereby inhibiting tumor growth and progression.

[0003] Traditional drug delivery methods include oral administration and intravenous injection, which have shortcomings such as first-pass effect in the liver and systemic cytotoxicity. Microneedles, as an effective local drug delivery system, have high drug bioavailability and are relatively safe, and have been widely studied. Compared with traditional transdermal microneedles, implantable microneedles have significant improvements, and their 100% degradable properties enable them to be safely used in surgical cavities. At present, most soluble drug-loaded microneedles are only single-layer structures with simple structures, relatively single modes of action, and often uncontrollable duration of action, which greatly limits the clinical application of microneedles. Summary of the invention

[0004] In view of this, the present invention provides a double-layer microneedle patch and a preparation method thereof. The double-layer microneedle patch of the present invention is to use sericin loaded with anti-inflammatory drugs as the outer layer, and then polycaprolactone loaded with germanium nanoparticles as the inner layer filling, which is directly implanted into the defect site after tumor surgery. In the early perioperative period, anti-inflammatory drugs are used to inhibit the "cancer-promoting inflammation" caused by the surgical operation. In the later period, germanium nanoparticles are used to exert photothermal effects under near-infrared light irradiation to remove the residual micro-tumor tissue after surgery, sequentially program the immune microenvironment, effectively inhibit tumor recurrence and metastasis, and promote surgical wound repair.

[0005] The preparation method of the double-layer microneedle patch of the present invention comprises the following steps:

[0006] S1. Use polydimethylsiloxane for 3D printing to obtain the PDMS mold used for processing microneedles;

[0007] S2, dispersing the drug in the sericin solution as the outer layer solution; dispersing germanium nanoparticles in the polycaprolactone solution to obtain the inner layer solution;

[0008] S3, adding the outer layer solution into the needle-shaped groove of the PDMS mold so that the outer layer solution fills the needle-shaped groove, adding the inner layer solution after drying, and demoulding after drying to obtain a double-layer microneedle patch.

[0009] Preferably, the height of the microneedles in the PDMS mold is 50 microns to 2 millimeters, the diameter of the bottom of the microneedles is 50 microns to 1 millimeter, and the number of the microneedle array is 1 to 500.

[0010] Preferably, the mass concentration of the sericin solution is 8% to 15%. The preparation method of sericin of the present invention is to extract and prepare it by high temperature alkaline water method, specifically, the silk fibroin-deficient cocoon is cut into pieces, placed in 0.02 mol / L sodium carbonate solution, stirred and heated at 100°C for 1 hour, cooled and centrifuged to remove impurities, then dialyzed and freeze-dried to obtain pure sericin solid.

[0011] Preferably, the solvent in the polycaprolactone solution is dichloromethane, and the mass concentration of the polycaprolactone solution is 10% to 20%.

[0012] Preferably, the drug is a non-steroidal anti-inflammatory drug, including but not limited to meloxicam.

[0013] Preferably, the concentration of germanium nanoparticles in the inner layer solution is 500-1000 μg / mL. The germanium nanoparticles of the present invention are prepared by a microemulsion self-assembly method, specifically, deionized water (45 mL) and tetrahydrofuran (THF) (4 mL) are mixed, and then ammonia water (1.5 mL) and germanium dioxide (200 mg) are added. After reacting at room temperature for 1 hour, sodium borohydride (720 mg) is added and the stirring reaction is continued for 18 hours; the obtained product is collected by centrifugation, washed, and freeze-dried to obtain pure germanium nanoparticles.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The double-layer microneedle patch of the present invention is used for drug administration, which can dissolve and release drugs in a short period of time. The inner layer is polycaprolactone (PCL) loaded with photothermal agent germanium nanoparticles, which degrades slowly and produces a photothermal effect under 808nm near-infrared light irradiation, inducing immunogenic death of residual tumor cells, temporally programming the immune microenvironment, and effectively inhibiting postoperative tumor recurrence and metastasis, while promoting surgical wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is a flow chart of the preparation method of the double-layer microneedle patch of the present invention;

[0017] Figure 2 Schematic diagram of the structure of the double-layer microneedle patch of the present invention;

[0018] Figure 3 A physical picture of the double-layer microneedle patch of the present invention

[0019] Figure 4 This is a photothermal performance diagram of the double-layer microneedle patch of the present invention in vitro;

[0020] Figure 5 The double-layer microneedle patch of the present invention has biocompatibility. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1

[0023] A method for preparing a double-layer microneedle patch, the specific steps are as follows:

[0024] (1) Extracting sericin using high temperature alkaline water method

[0025] 5 g of fibroin-deficient cocoons were chopped up and placed in 200 mL of 0.02 mol / L sodium carbonate solution. The solution was stirred and heated at 100°C and 800 rpm for 1 hour to completely dissolve the sericin. The solution was cooled and centrifuged (3000 rpm, 5 min) to remove insoluble precipitates. The supernatant was collected and dialyzed with a dialysis bag for 2-3 days to obtain a clarified sericin solution, which was then freeze-dried to obtain a sericin protein solid.

[0026] (2) Preparation of germanium nanoparticles by microemulsion self-assembly method

[0027] Deionized water (45 mL) and tetrahydrofuran (THF) (4 mL) were mixed, and then ammonia (1.5 mL) and germanium dioxide (200 mg) were added. After reacting at room temperature for 1 hour, sodium borohydride (720 mg) was added and the reaction was continued for 18 hours. The obtained product was collected by centrifugation, washed, and freeze-dried to obtain pure germanium nanoparticles.

[0028] (3) Preparation of double-layer microneedle patches

[0029] S1. Use solidworks software to design the original microneedle master mold, and use Lite 600HD 3D printer and photo-crosslinking resin to print. Use polydimethyl siloxane (PDMS) to construct the PDMS mold for preparing microneedles through the master mold;

[0030] S2, dispersing meloxicam in a sericin solution with a mass concentration of 10% to obtain an outer layer solution with a meloxicam concentration of 5 mg / mL; dispersing germanium nanoparticles in a polycaprolactone solution to obtain an inner layer solution with a germanium nanoparticle concentration of 600 μg / mL; wherein the polycaprolactone solution is prepared by dissolving polycaprolactone in dichloromethane to obtain a polycaprolactone solution with a mass concentration of 10%;

[0031] S3. Add the outer layer solution into the needle-shaped groove of the PDMS mold, centrifuge at 2500 rpm for 3 minutes to allow the outer layer solution to fill the needle-shaped groove, centrifuge at 3000 rpm for 5 minutes, dry at 37°C for 4 hours, then add the inner layer solution, dry at 25°C for 24 hours, and then demold to obtain a double-layer microneedle patch.

[0032] Example 2

[0033] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 200 μg / mL.

[0034] Example 3

[0035] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 400 μg / mL.

[0036] Example 4

[0037] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 800 μg / mL.

[0038] Example 5

[0039] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 1000 μg / mL.

[0040] Example 6

[0041] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 250 μg / mL.

[0042] Example 7

[0043] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 500 μg / mL.

[0044] Example 8

[0045] A double-layer microneedle patch was prepared by referring to the method of Example 1, except that the concentration of germanium nanoparticles in the inner layer solution was 750 μg / mL.

[0046] Comparative Example 1

[0047] A double-layer microneedle patch was prepared by referring to the method of Example 1, the only difference being that the inner layer solution did not contain germanium nanoparticles.

[0048] The in vitro photothermal performance test of the double-layer microneedle patches prepared in Examples 1 to 5 and Comparative Example 1 is as follows:

[0049] The double-layer microneedle patches prepared in Examples 1 to 5 and Comparative Example 1 were immersed in 350 μL of double distilled water, respectively, and exposed to a near-infrared laser of 808 nm (diameter 13 mm, power 0.5 W / cm2) for 5 minutes. The temperature of the water was measured every one minute using a digital thermometer to test the photothermal performance of the double-layer microneedle patches. The experimental results are shown in FIG. Figure 4 , indicating that the double-layer microneedle patch prepared in the embodiment of the present invention has a good in vitro photothermal effect.

[0050] The double-layer microneedle patches prepared in Examples 6-8 and Comparative Example 1 were used to treat tumors in mice after surgery, as follows:

[0051] The in vivo anti-tumor effect of the double-layer microneedle patch was observed in female Balb / c mice (5-6 weeks old) bearing tumors on their backs. First, 1×106 CT26 cells (100 μL) were subcutaneously injected into the right back of each mouse. When the tumor volume reached about 100 mm3, the mice were divided into 4 groups, 95% of the tumor was surgically removed, and the following treatments were performed: the control group, only the tumor was surgically removed and sutured; the pure drug group, the microneedles loaded with meloxicam were placed in the tumor resection site and sutured; the pure light group, the microneedles loaded with germanium nanoparticles were placed in the tumor resection site and sutured, and near-infrared irradiation (808nm, 0.5W / cm2, 5min) was given after surgery, and light treatment was given every other day, for a total of 3 times; the experimental group, the microneedles loaded with meloxicam and germanium nanoparticles were placed in the tumor resection site and sutured, and the postoperative light treatment was the same as the light group. The mice were killed 12 days after surgery, and the tumor tissues were dissected and photographed.

[0052] Mouse embryonic fibroblasts (NIH3T3) and human umbilical vein endothelial cells (HUVEC) were cultured overnight in a 48-well plate at a density of 2×104 cells / well. Subsequently, the double-layer microneedle patches prepared in Examples 6-8 and Comparative Example 1 were gently introduced into the plate, and after culturing for 24 hours, the cell proliferation was detected by CCK-8 method, and the cell viability was detected by calcein-AM and propidium iodide double staining method. The blank control group was not treated with microneedles.

[0053] From the experimental results Figure 5 It can be seen that the double-layer microneedle patches prepared in Examples 6-8 of the present invention have good biocompatibility.

[0054] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing a double-layer microneedle patch, characterized in that: The following steps are involved: S1. Use polydimethylsiloxane for 3D printing to obtain the PDMS mold used for processing microneedles; S2, dispersing the drug in the sericin solution as the outer layer solution; dispersing germanium nanoparticles in the polycaprolactone solution to obtain the inner layer solution; S3, adding the outer layer solution into the needle-shaped groove of the PDMS mold so that the outer layer solution fills the needle-shaped groove, adding the inner layer solution after drying, and demoulding after drying to obtain a double-layer microneedle patch.

2. The preparation method according to claim 1, characterized in that: The height of the microneedles in the PDMS mold is 50 microns to 2 millimeters, the diameter of the bottom of the microneedles is 50 microns to 1 millimeter, and the number of the microneedle array is 1 to 500.

3. The preparation method according to claim 1, characterized in that: The mass concentration of the sericin solution is 8% to 15%.

4. The preparation method according to claim 1, characterized in that: The solvent in the polycaprolactone solution is dichloromethane, and the mass concentration of the polycaprolactone solution is 10% to 20%.

5. The preparation method according to claim 1, characterized in that: The drug is a non-steroidal anti-inflammatory drug, including but not limited to meloxicam.

6. The preparation method according to claim 1, characterized in that: The concentration of germanium nanoparticles in the inner layer solution is 500-1000 μg / mL.

7. A double-layer microneedle patch, characterized in that: Prepared by any one of the methods of claims 1 to 6.

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