Double-layer bionic microneedle drug release system and preparation method thereof

Abstract

The invention provides a double-layer bionic microneedle drug release system and a preparation method thereof, and belongs to the technical field of biological medicine. According to the invention, by designing different properties of the microneedle carrier materials on the inner layer and the outer layer, programmed sequential release of the drugs on the inner layer and the outer layer is realized. By designing the double-layer mushroom-shaped microneedle structure, the adhesive force and the hemostatic effect of the microneedle drug release device on the liver wound surface are enhanced, the water solubility of the hydrophobic therapeutic drug is improved by introducing the drug-loading particles, so that the hydrophobic therapeutic drug can be locally used, and the therapeutic drug-loading particles are conveyed into disease tissues by using the microneedle. Compared with the traditional adjuvant therapy for preventing liver primary tumor recurrence, the programmed sequential drug release device based on the microneedle constructed by the invention can realize direct delivery of drugs to potential metastatic lesions, and avoids whole-body adverse reactions caused by oral administration of drugs. Compared with local treatment methods such as hepatic artery interventional embolism and local radiotherapy, the double-layer bionic microneedle drug release system has the advantages of high targeting property, small toxic and side effects, convenience in operation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a double-layer bionic microneedle drug release system and a preparation method thereof. Background Art

[0002] Liver tumors are a major disease that seriously threatens human health, and liver resection is one of the important means of treating liver tumors. For tumors in certain special locations, such as tumors near the first, second, or third portal vein, liver resection with narrow resection margins is the only option. However, this surgical approach faces a serious problem - a high rate of local recurrence. This may be due to residual tumor cells at the surgical margins, causing local recurrence after surgery, which seriously affects the patient's prognosis. According to clinical research statistics, the local recurrence rate after narrow-margin liver resection for liver tumors can reach 30%-50%, making the prevention of postoperative local recurrence a key issue that needs to be urgently addressed in the field of liver tumor treatment.

[0003] Currently, there are limited options for preventing local recurrence after narrow-margin liver resection for liver tumors. Traditional adjuvant treatments, such as systemic targeted combined immunotherapy, involve oral or intravenous medications to inhibit tumor cell growth. However, these drugs, while circulating throughout the body, not only act on tumor cells but also have toxic side effects on normal tissues and organs, leading to adverse reactions such as nausea, vomiting, hair loss, and bone marrow suppression, severely impacting patients' quality of life and treatment compliance. Furthermore, oral medications undergo first-pass absorption through the gastrointestinal tract, resulting in low local concentrations in the liver and limited effectiveness at potential tumor metastases. Transarterial embolization involves injecting an embolic agent and chemotherapy drugs into the hepatic artery, causing ischemic necrosis of the tumor tissue and localized chemotherapy. However, this approach has not yet reached international consensus, and its effectiveness remains under debate. Localized radiotherapy utilizes high-energy radiation to irradiate the tumor site to inhibit tumor cell proliferation. However, while radiotherapy kills tumor cells, it also causes radiation damage to surrounding normal tissues, leading to radiation hepatitis and liver damage. In addition, radiotherapy is difficult to accurately cover all potential tumor metastases, and its effect is limited for tiny residual tumor cells.

[0004] Furthermore, existing drug delivery systems have numerous shortcomings. Conventional oral dosage forms, such as tablets and capsules, cannot achieve efficient localized drug delivery to the liver. While some novel drug delivery vehicles have improved drug delivery to some extent, they still struggle to meet the specialized needs of liver tumor treatment. For example, the in vivo stability, targeting, and controllable drug release of liposomes and nanoparticles still need to be further improved.

[0005] In this context, the development of an efficient, safe, targeted and programmed sequential drug delivery system has important clinical significance and application value for preventing local recurrence of liver tumors after narrow-margin liver resection. Summary of the Invention

[0006] The purpose of the present invention is to provide a double-layer bionic microneedle drug delivery system and a preparation method thereof, which has the advantages of strong targeting, small toxic and side effects, and easy operation.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing a double-layer bionic microneedle drug release system, comprising the following steps: The anti-tumor drug is dissolved, mixed with the drug-loaded particles, subjected to ultrasonic emulsification, and evaporated and filtered to obtain a drug release device; The drug release device, polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator are mixed, filled into the microneedle template, and cured and cross-linked to obtain an outer layer; The hemostatic drug is dissolved, mixed with hyaluronic acid, and centrifuged to obtain the inner layer; The outer layer and the inner layer are overlapped, frozen at -100 to -60°C for 1 to 3 hours, and then subjected to vacuum freeze drying for 1 to 3 hours to obtain the double-layer bionic microneedle drug delivery system, comprising the outer layer and the inner layer, constituting multiple double-layer mushroom-shaped microneedle structures in the system; The outer layer contains a drug release device, and the drug release device contains anti-tumor drugs; The inner layer is filled with a hemostatic drug; The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong liver adhesion ability; In the microneedle structure, there is an expanded structure near the tip, and the maximum diameter of the expanded structure is 100-150 μm.

[0008] The present invention also provides a double-layer microneedle drug delivery system, comprising an outer layer and an inner layer, constituting a plurality of double-layer mushroom-shaped microneedle structures in the system; The outer layer contains a drug release device, and the drug release device contains anti-tumor drugs; The inner layer is filled with a hemostatic drug; The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong liver adhesion ability; In the microneedle structure, there is an expanded structure near the tip, and the maximum diameter of the expanded structure is 100-150 μm.

[0009] Preferably, the total height of the microneedles in the double-layer bionic microneedle system is 200-2000 μm, the inner layer height is 400-600 μm, and the base diameter of the needle body is 200-300 μm.

[0010] Preferably, the drug-releasing device uses drug-loaded particles as carriers of the anti-tumor drug. The drug-loaded particles are nano-scale particles, and the material thereof is a biocompatible and biodegradable polymer material.

[0011] Preferably, the material of the drug-loaded particles is one or more of distearoylphosphatidylethanolamine-polyethylene glycol, methoxypolyethylene glycol-poly (D, L-lactic acid), methoxypolyethylene glycol-polycaprolactone, methoxypolyethylene glycol-polylactic acid-glycolic acid copolymer, methoxypolyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, poly D-lactic acid, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, cholesterol, soybean lecithin, monooleylglycerol, dioleoyltrimethylammonium propane, sphingomyelin or distearoylphosphatidylglycerol.

[0012] Preferably, the height of the microneedles in the double-layer bionic microneedle system is 200-2000 μm.

[0013] Preferably, the raw materials of the inner layer include protein-based natural polymers, polysaccharide-based natural polymers or synthetic materials; The protein-based natural polymer includes collagen, gelatin, silk protein, albumin or spider silk protein The polysaccharide natural polymer or synthetic material includes chitosan, hyaluronic acid or sodium alginate; The synthetic material includes gelatin methacrylamide, polyvinyl alcohol, polycaprolactone or polylactic acid; The raw materials of the outer layer include polyethylene glycol diacrylate, protein-based natural polymers and / or polysaccharide-based natural polymers and a photoinitiator.

[0014] Preferably, the photoinitiator includes phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0015] Preferably, the hemostatic drug is selected from one or more of thrombin, tranexamic acid, and vitamin K.

[0016] Preferably, the anti-tumor drug is a drug for treating primary liver tumors.

[0017] Preferably, the anti-tumor drug is selected from one or more of lenvatinib, sorafenib, donafenib or bevacizumab.

[0018] Beneficial effects of the present invention: The present invention realizes the programmed sequential release of the inner and outer layers of drugs by designing the inner and outer layers of microneedle carrier materials to have different properties. By designing a double-layer mushroom-shaped microneedle structure, the adhesion of the microneedle drug release device to the liver wound is enhanced. By introducing drug-loaded particles, the water solubility of hydrophobic therapeutic drugs is improved so that they can be used locally. The therapeutic drug-loaded particles are delivered to the diseased tissue using microneedles. Compared with traditional adjuvant treatments for preventing primary liver tumors, the microneedle-based programmed sequential drug release device constructed by the present invention can achieve direct delivery of drugs to potential metastatic lesions, avoiding systemic adverse reactions caused by systemic drugs. Compared with methods such as transhepatic artery interventional embolization and local radiotherapy, the double-layer bionic microneedle drug release system has the advantages of strong targeting, low toxicity and side effects, and easy operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a transmission electron microscopy image of drug-loaded particles characterization; Figure 2 It is the release behavior curve in the characterization of drug-loaded particles; Figure 3 It is the cytotoxicity in the biological behavior of drug-loaded particles; Figure 4 It is the cellular uptake fluorescence microscopy and flow cytometry of the biological behavior of drug-loaded particles; Figure 5 It is the cellular uptake localization in the biological behavior of drug-loaded particles; Figure 6 This is a representation of a double-layered mushroom-shaped microneedle; Figure 7 It is the mechanical properties of the double-layered mushroom-shaped microneedles; Figure 8 It is a graph of the release behavior of targeted drugs in double-layer mushroom-shaped microneedles; Figure 9 This is a tumor in vivo imaging diagram of an embodiment; Figure 10 This is a graph showing changes in fluorescence values ​​during in vivo imaging of a tumor in an embodiment; Figure 11 This is a graph showing weight changes in experimental mice. DETAILED DESCRIPTION

[0020] The present invention provides a method for preparing a double-layer bionic microneedle drug release system, comprising the following steps: The anti-tumor drug is dissolved, mixed with the drug-loaded particles, subjected to ultrasonic emulsification treatment, and evaporated and filtered to obtain a drug release device; the drug release device, polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator are mixed, filled into a microneedle template, and cured and cross-linked to obtain an outer layer; the hemostatic drug is dissolved, mixed with the hyaluronic acid and centrifuged to obtain an inner layer; the outer layer and the inner layer are overlapped, frozen at -100~-60℃ for 1~3 hours, and then vacuum freeze-dried for 1~3 hours to obtain the double-layer bionic microneedle drug release system, including an outer layer and an inner layer, constituting multiple double-layer mushroom-shaped microneedle structures in the system; the outer layer contains a drug release device, and the drug release device contains an anti-tumor drug; the inner layer is filled with a hemostatic drug; the double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug release system to have strong adhesion and liver penetration ability; in the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is 100~150μm.

[0021] In the present invention, preferably, the concentration of the anti-tumor drug dissolved is 200 μg of anti-tumor drug (such as lenvatinib) dissolved in 0.5 mL of dichloromethane solution; the drug-loaded particles are nano-sized DSPE-PEG particles, and the dissolved anti-tumor drug solution is added to 5 mL of DSPE-PEG aqueous solution during mixing; the parameters of the ultrasonic emulsification treatment are ultrasonic emulsification for 6 minutes under the conditions of 100W and 20 degrees Celsius; the evaporation is to remove the organic solvent using a rotary evaporator for 10 minutes, and the filtration is to filter using a 0.45 μM canned needle filter mixed with cellulose (MCE) 13 mm; when the drug release device is mixed with polyethylene glycol diacrylate, hyaluronic acid and photoinitiator, 1 mL of the prepared nano anti-tumor drug is taken and mixed with 0.25 mL of PEGDA (20% v / v) v), 125mg low molecular weight hyaluronic acid (10% w / v) and 3.2mg photoinitiator LAP (0.25% w / v) are mixed; the microneedle template specification is 1*1cm, and the needle height is 600 microns; the centrifugation parameter after filling the microneedle template is 4000rpm, and the centrifugation is 10 minutes; the curing and cross-linking is UV curing and cross-linking for 20s; the hemostatic drug dissolution is to dissolve 10mg of hemostatic drug (such as thrombin) in 1mL ultrapure water; when mixed with hyaluronic acid, it is mixed with 200mg low molecular weight hyaluronic acid (20% w / v), and the centrifugation parameter after mixing is 4000rpm, and the centrifugation is 10 minutes; the temperature range of freezing after the outer layer and the inner layer overlap is -100~-60℃, and the freezing time is 1~3h; the time of the vacuum freeze-drying treatment is 1~3h.

[0022] The present invention also provides a double-layer microneedle drug delivery system, comprising an outer layer and an inner layer, forming a plurality of double-layer mushroom-shaped microneedle structures within the system; the outer layer contains a drug delivery device containing an anti-tumor drug; the inner layer is filled with a hemostatic drug; the double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong adhesion and liver penetration capabilities; the microneedle structure has an enlarged structure near the tip, the maximum diameter of the enlarged structure being 100-150 μm. Preferably, the total height of the microneedles in the double-layer bionic microneedle system is 200-2000 μm, the outer layer height is 400-600 μm, and the base diameter of the needle body is 200-300 μm.

[0023] In the present invention, preferably, the drug release device uses drug-loaded particles as carriers of anti-tumor drugs, and the drug-loaded particles are nano-sized particles, and the material thereof is a polymer material with good biocompatibility and biodegradability. Preferably, the material of the drug-loaded particles is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), methoxy polyethylene glycol-poly (D, L-lactic acid) (MPEG-PDLLA), methoxy polyethylene glycol-polycaprolactone (MPEG-PCL), methoxy polyethylene glycol-polylactic acid-glycolic acid copolymer (MPEG-PLGA), methoxy polyethylene glycol-polylactic acid (MPEG-PLA), polyethylene glycol-polycaprolactone (PEG-PCL), poly D-lactic acid (D-PLA), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Cholesterol), soy lecithin (Soy The drug-loaded particles may be made of one or more of the following: lecithin, monoolein (GMO), dioleoyltrimethylammonium propane (DOTAP), sphingomyelin, or distearoylphosphatidylglycerol (DSPG); further preferably, the drug-loaded particles are made of DSPE-PEG. Preferably, the microneedles in the double-layer biomimetic microneedle system have a height of 200-2000 μm, more preferably 600-1000 μm. Preferably, the inner layer comprises a natural protein polymer, a natural polysaccharide polymer, or a synthetic material; the natural protein polymer includes collagen, gelatin, silk protein, albumin, or spider silk protein; the natural polysaccharide polymer or synthetic material includes chitosan, hyaluronic acid, or sodium alginate; the synthetic material includes gelatin methacrylamide, polyvinyl alcohol, polycaprolactone, or polylactic acid; and the outer layer comprises polyethylene glycol diacrylate and a photoinitiator. Preferably, the photoinitiator includes phenyl-2,4,6-trimethylbenzoylphosphonate lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. Preferably, the hemostatic drug is selected from one or more of thrombin, tranexamic acid, and vitamin K. Preferably, the anti-tumor drug is a drug for treating primary liver tumors. Preferably, the anti-tumor drug is selected from one or more of lenvatinib, sorafenib, donafenib or bevacizumab, and in theory, it can also be applied to other anti-tumor drugs targeting VEGFR.

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The abbreviations of terms in the examples are: lenvatinib is LEN, nano-lenvatinib is LEN NP, and coumarin 6 is Coumarin 6. Example

[0026] First, the therapeutic drug, drug-carrying substance and microneedle system carrier material are prepared, wherein the therapeutic drug and drug-carrying substance are made into a drug release device through a double emulsification method.

[0027] The therapeutic drug and drug-loaded particles were prepared into the aforementioned drug delivery device by emulsification and evaporation: 200 μg of lenvatinib was dissolved in 0.5 mL of dichloromethane solution and added to 5 mL of an aqueous solution containing 1.13 mg of DSPE-PEG. Ultrasonic emulsification was performed at 100 W and 20 degrees Celsius for 6 minutes. The organic solvent was removed by rotary evaporation for 10 minutes, and the solution was filtered through a 0.45 μM canned syringe filter with mixed cellulose (MCE) 13 mm.

[0028] A double-layer mushroom-shaped biomimetic microneedle system loaded with lenvatinib nanoparticles was then prepared via freeze-drying and template methods. The microneedle templates were made of polydimethylsiloxane (PDMS), measuring 1 cm × 1 cm, and each template consisted of 400 (20 × 20) needle-tip grooves. The preparation process was as follows: 1 mL of prepared nanolenvatinib was mixed with 0.25 mL of PEGDA (20% v / v), 125 mg of low-molecular-weight hyaluronic acid (10% w / v), and 3.2 mg of photoinitiator LAP (0.25% w / v). The mixture was then filled into a 1 x 1 cm microneedle template with a needle height of 600 μm. The mixture was centrifuged at 4000 rpm for 10 minutes. After cross-linking by UV light for 20 seconds, the template was removed to obtain the outer layer of the double-layer microneedles. 10 mg of the hemostatic drug thrombin was dissolved in 1 mL of ultrapure water and mixed with 200 mg of low-molecular-weight hyaluronic acid (20% w / v), the carrier material for the inner microneedle system. The mixture was then filled into a microneedle template of the same specifications as described above and centrifuged at 4000 rpm for 10 minutes. The cured and de-molded outer layer was overlapped with the inner layer and frozen in a -80°C freezer for 2 hours. The membrane was then removed and freeze-dried in a vacuum freeze dryer for 1.5 hours. The device was then dried naturally at room temperature to obtain the aforementioned double-layer biomimetic microneedle drug delivery device.

[0029] This invention utilizes a double-layered, mushroom-shaped biomimetic microneedle drug delivery device, leveraging its programmed sequential drug delivery properties to prevent local recurrence of liver tumors after narrow-margin liver resection. When the double-layered microneedles penetrate the liver resection wound, the inner layer of the microneedles dissolves first, releasing thrombin and promoting hemostasis. Subsequently, the outer layer, loaded with the lenvatinib nanoparticle, dissolves, slowly releasing lenvatinib. This targeted effect inhibits the proliferation of residual tumors and prevents local tumor recurrence.

[0030] The therapeutic drug in this case is lenvatinib. This case is a basic research on the application of a programmed drug-release bionic double-layer microneedle device loaded with lenvatinib nanoparticles and thrombin in preventing recurrence after narrow-margin resection of liver tumors.

[0031] Materials: microneedle template, low molecular weight sodium hyaluronate, DSPE-PEG, lenvatinib, thrombin, PEGDA, LAP, trypan blue, etc.

[0032] Cells: Hep3B, Huh7, and Hepa1-6. Huh7 and Hepa1-6 were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin; Hep3B was cultured in MEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin.

[0033] Animals: SPF male c57BL / 6 mice, 6–8 weeks old, were purchased from Sichuan Weitonglihua Laboratory Animal Technology Co., Ltd. All animal experiments were performed under the approval and supervision of the Laboratory Animal Care Committee of West China Hospital, Sichuan University.

[0034] Experimental methods: Preparation of nanoparticles loaded with drug, using dichloromethane as emulsifier and DSPE-PEG as carrier material, LEN was loaded into DSPE-PEG by the method described above, and LEN loaded nanoparticles (LEN-DSPE-PEG) were obtained. The nanoparticles were characterized by transmission electron microscopy (TEM), particle size, potential and other methods. Figure 1 shown.

[0035] Release function of nano drug-loaded particles: The dialysis method was used to evaluate the in vitro release ability of LEN NP. 1 mL of LEN NP containing 1 mg and 1 mL of free LEN drug dissolved in DMSO were placed in dialysis bags with a molecular weight cutoff of 3500 Da, respectively, and placed in 20 mL of PBS solution containing 1% Tween 80 at pH = 7.4 and pH = 6.8, respectively, and placed in a 37°C constant temperature water bath shaker (120 rpm) for release. Samples were taken at time intervals of 3h, 6h, 9h, 12h, 24h, 36h, 48h, and 72h, with 1 mL taken each time. At the same time, the same volume of fresh PBS solution was added, and the released LEN concentration was detected by HPLC. The release curves of nano-lenvatinib and free lenvatinib were obtained as shown below. Figure 2 shown.

[0036] In vitro biological performance of drug-loaded nanoparticles: CCK8 assay was used to investigate the cytotoxic effects of LEN NP and LEN on Hep3B, Huh7 and Hepa1-6 cells. Figure 3As shown. The uptake of LEN NP by Hep3B, Huh7 and Hepa1-6 cells was investigated. In the cell uptake experiment, hydrophobic coumarin with fluorescence was selected instead of LEN as the model drug, and the cell uptake ability of the nanoparticles was qualitatively and quantitatively analyzed by fluorescence microscopy and flow cytometry, respectively. Figure 4 As shown. The uptake and localization of nanoparticles in cells are clearly defined using confocal microscopy. Figure 5 shown.

[0037] Preparation of microneedles: A mushroom-shaped biomimetic double-layer microneedle device loaded with lenvatinib nanoparticles and thrombin for programmed drug release was prepared by freeze-drying and template methods. The prepared microneedles were characterized by scanning electron microscopy. Figure 6 shown.

[0038] Microneedle Performance Evaluation: The mechanical strength of DMN@LEN NP / Th was tested using an electronic universal testing machine. A double-layer microneedle patch containing a 20×20 array was fixed on a stainless steel plate with the needle tips facing upward. The distance between the microneedle tips and the sensor was set to 0.5 cm. The sensor was then moved perpendicular to the stainless steel plate at a constant speed. After the sensor contacted the microneedle tip, the sensor displacement and corresponding resistance were recorded. Three parallel samples were tested in each group. For details, see Figure 7 shown.

[0039] In vitro drug release from microneedles. Figure 8 The release behavior of DMN@LEN NP / Th and DMN@LEN / Th in PBS solution at pH 6.8 (simulating the weakly acidic environment of the tumor microenvironment) was investigated. The two DMNs were placed in 20 mL of PBS solution containing 1% Tween 80 at pH 6.8 and pH 7.4, respectively. Release was performed in a 37°C constant temperature shaker (120 rpm). Samples of 1 mL were taken at intervals of 3, 6, 9, 12, 24, 48, and 72 hours, and the same volume of fresh PBS solution was added. The released LEN concentration was measured by HPLC.

[0040] In vivo animal experiment. C57BL / 6 mice were used as model animals to study the tumor treatment effect of the microneedle system in vivo. First, 100.0 μL of Luc-Hepa1-6 cells (cell concentration of 5×10 7 The tumor xenografted mice were randomly divided into 4 groups as follows: (1) Control group, no treatment; (2) double-layer microneedle group loaded with lenvatinib at a dose of 10 mg / kg; (3) double-layer microneedle group loaded with lenvatinib at a dose of 20 mg / kg; (4) Double-layer microneedle group loaded with lenvatinib at a dose of 40 mg / kg.

[0041] As the volume of the inoculated tumor increased to 200.0 mm 3 , and treatment began. 90% of the tumor volume was removed, and 10% remained. Mice were treated according to different grouping methods. The microneedles were attached subcutaneously to the tumor site for 24 hours and then removed. Changes in mouse tumor size were recorded using in vivo imaging. Body weight was recorded every 3 days during the experiment, and in vivo imaging was taken every 7 days to record changes in fluorescence values. The experiment ended after 28 days of treatment. The results of the in vivo imaging experiment are as follows Figure 9 As shown, the fluorescence value changes as Figure 10 As shown, weight changes Figure 11 shown.

[0042] Figure 1 The particle size of LEN NPs is shown. The nanoparticles are well dispersed, with a uniform size distribution and no agglomeration. The Malvern particle size analyzer measured the particle size to be 157.2 ± 4.98 nm. Due to dehydration and shrinkage of the nanoparticles during TEM sample preparation, the particle size was slightly reduced. Figure 2 The release curves of LEN NPs in release media with different pH values ​​are shown. The release rate of LEN NPs in weakly acidic media is faster than that in neutral media. Compared with free drug, the drug release from nanoparticles is slow.

[0043] Figure 3 It was shown that in three liver cancer cell lines, Huh 7, Hep3B, and Hepa1-6, nano-lenvatinib can achieve a cell killing effect similar to that of lenvatinib.

[0044] Figure 4 Fluorescence microscopy showed that nano-lenvatinib could be taken up by three cell lines, and quantitative analysis by flow cytometry showed that the amount of nano-lenvatinib taken up by cells increased with time.

[0045] Figure 5 In the study, confocal microscopy showed that after cells took up nano-lenvatinib, lysosomal escape could occur, allowing the drug to take effect.

[0046] Figure 6 The AC super-depth-of-field microscope shows a double-layer mushroom-shaped microneedle structure; the DF scanning electron microscope shows a double-layer mushroom-shaped microneedle structure. As can be seen from the figure, in the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is about 150μm. The total height of the microneedle in the double-layer bionic microneedle system is about 600μm, the inner layer height is about 500μm, and the base diameter of the needle body is about 250μm.

[0047] Figure 7 The mechanical strength of the double-layer mushroom-shaped microneedles is shown.

[0048] Figure 8 The drug release curves of the double-layer mushroom-shaped microneedles in different pH media are shown. In slightly acidic release media, the drug release is greater and faster.

[0049] Figure 9 In vivo imaging shows the anti-recurrence effect of double-layer microneedles with different drug loading levels on subcutaneous tumors in mice. A dose of 10mg / kg can achieve significant anti-recurrence effects.

[0050] Figure 10 It was shown that the mice that did not receive drug-loaded microneedle treatment weighed more and had heavier tumor burden than the mice that received drug-loaded microneedle treatment.

[0051] Figure 11 The fluorescence intensity of in vivo imaging of mice in the untreated group was stronger than that in the group treated with drug-loaded microneedles.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a double-layer bionic microneedle drug delivery system, characterized in that: The following steps are involved: The anti-tumor drug is dissolved, mixed with the drug-loaded particles, subjected to ultrasonic emulsification, and filtered to obtain a drug release device; The drug release device, polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator are mixed, filled into the microneedle template, and cured and cross-linked to obtain an outer layer; The hemostatic drug is dissolved, mixed with hyaluronic acid, and centrifuged to obtain the inner layer; The outer layer and the inner layer are overlapped, frozen at -100 to -60°C for 1 to 3 hours, and then subjected to vacuum freeze drying for 1 to 3 hours to obtain the double-layer bionic microneedle drug delivery system, comprising the outer layer and the inner layer, constituting multiple double-layer mushroom-shaped microneedle structures in the system; The outer layer contains a drug release device, and the drug release device contains anti-tumor drugs; The inner layer is filled with a hemostatic drug; The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong liver adhesion ability; In the microneedle structure, there is an expanded structure near the tip, and the maximum diameter of the expanded structure is 100-150 μm.

2. A double-layer microneedle drug delivery system, characterized in that: It includes an outer layer and an inner layer, forming multiple double-layer mushroom-shaped microneedle structures in the system; The outer layer contains a drug release device, and the drug release device contains anti-tumor drugs; The inner layer is filled with a hemostatic drug; The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong liver adhesion ability; In the microneedle structure, there is an expanded structure near the tip, and the maximum diameter of the expanded structure is 100-150 μm.

3. The double-layer microneedle drug delivery system according to claim 2, characterized in that: The total height of the microneedles in the double-layer bionic microneedle system is 200-2000 μm, the inner layer height is 400-600 μm, and the base diameter of the needle body is 200-300 μm.

4. The double-layer microneedle drug delivery system according to claim 1, characterized in that: The drug-releasing device uses drug-loaded particles as carriers of anti-tumor drugs. The drug-loaded particles are nano-scale particles, and the material of the drug-loaded particles is a biocompatible and biodegradable polymer material.

5. The double-layer bionic microneedle drug delivery system according to claim 4, characterized in that: The material of the drug-loaded particles is one or more of distearoylphosphatidylethanolamine-polyethylene glycol, methoxypolyethylene glycol-poly (D, L-lactic acid), methoxypolyethylene glycol-polycaprolactone, methoxypolyethylene glycol-polylactic acid-glycolic acid copolymer, methoxypolyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, poly D-lactic acid, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, cholesterol, soybean lecithin, monooleylglycerol, dioleoyltrimethylammonium propane, sphingomyelin or distearoylphosphatidylglycerol.

6. The double-layer bionic microneedle drug delivery system according to claim 2, characterized in that: The raw materials of the inner layer include protein-based natural polymers, polysaccharide-based natural polymers or synthetic materials; The protein-based natural polymer includes collagen, gelatin, silk protein, albumin or spider silk protein; The polysaccharide natural polymer or synthetic material includes chitosan, hyaluronic acid or sodium alginate; The synthetic material includes gelatin methacrylamide, polyvinyl alcohol, polycaprolactone or polylactic acid; The raw materials of the outer layer include polyethylene glycol diacrylate, protein-based natural polymers and / or polysaccharide-based natural polymers and a photoinitiator.

7. The double-layer bionic microneedle drug delivery system according to claim 6, characterized in that: The photoinitiator includes phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

8. The double-layer bionic microneedle drug delivery system according to claim 2, characterized in that: The hemostatic drug is selected from one or more of thrombin, tranexamic acid, and vitamin K.

9. The double-layer bionic microneedle drug delivery system according to claim 2, characterized in that: The anti-tumor drug is a drug for treating primary liver tumors.

10. The double-layer bionic microneedle drug delivery system according to claim 9, characterized in that: The anti-tumor drug is selected from one or more of lenvatinib, sorafenib, donafenib or bevacizumab.

Images