A double-layer biomimetic microneedle drug release system and a preparation method thereof

Abstract

The application provides a double-layer biomimetic microneedle drug release system and a preparation method thereof, and belongs to the technical field of biological medicines.The application realizes programmed sequential release of inner-layer and outer-layer drugs by designing different properties of the inner-layer and outer-layer microneedle carrier materials.The application enhances the adhesion and hemostatic effect of the microneedle drug release device on the liver wound surface by designing a double-layer mushroom-shaped microneedle structure, improves the water solubility of hydrophobic therapeutic drugs by introducing drug-loaded particles, so that the therapeutic drugs can be used locally, and uses the microneedle to deliver the therapeutic drug-loaded particles to the disease tissue.Compared with traditional adjuvant therapy for preventing recurrence of liver primary tumors, the programmed sequential drug release device based on the microneedle can realize direct delivery of drugs to potential metastatic lesions, and avoids systemic adverse reactions caused by oral administration.Compared with local treatment methods such as transhepatic arterial intervention embolization and local radiotherapy, the double-layer biomimetic microneedle drug release system has the advantages of high targeting, small toxic and side effects, and convenient operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a double-layer biomimetic microneedle drug release system and a preparation method thereof. BACKGROUND

[0002] Liver tumor is a major disease that seriously threatens human health, and liver resection is one of the important means for treating liver tumor. For some tumors in special positions, such as tumors close to the first, second or third hepatic portal, only narrow-margin liver resection can be performed. However, this surgical method faces a serious problem of high local recurrence rate. This may be due to the residual tumor cells at the surgical margin, causing postoperative local recurrence and seriously affecting the prognosis of patients. According to clinical research statistics, the local recurrence rate after narrow-margin liver resection for liver tumor can reach 30%-50%, which makes the prevention of postoperative local recurrence a key problem to be solved in the field of liver tumor treatment.

[0003] At present, the means for preventing local recurrence after narrow-margin liver resection for liver tumor are limited. Traditional adjuvant therapy methods, such as systemic use of targeted combined immunotherapy, inhibit tumor cell growth by oral or intravenous administration of drugs. However, these drugs not only act on tumor cells during systemic circulation, but also have toxic side effects on normal tissues and organs, leading to adverse reactions such as nausea, vomiting, hair loss, bone marrow suppression in patients, and seriously affecting the quality of life and treatment compliance of patients. In addition, due to the need for gastrointestinal absorption of oral drugs, the first-pass effect will be faced, and the local concentration of drugs in the liver is low, making it difficult to effectively act on potential tumor metastases. Transhepatic arterial interventional embolization therapy is to inject embolic agents and chemotherapeutic drugs into the hepatic artery to cause ischemic necrosis of tumor tissue and local chemotherapy. However, this method has not yet been internationally recognized, and its effectiveness is still under discussion. Local radiotherapy is to use high-energy rays to irradiate the tumor site to inhibit tumor cell proliferation. However, radiotherapy not only kills tumor cells, but also causes radiation damage to surrounding normal tissues, leading to problems such as radiation hepatitis and liver function damage. Moreover, radiotherapy cannot accurately cover all potential tumor metastases, and the effect of radiotherapy on residual microtumor cells is limited.

[0004] In addition, the existing drug delivery systems also have many shortcomings. Conventional drug dosage forms, such as tablets, capsules and other oral dosage forms, cannot achieve efficient drug delivery in the liver. Some new drug delivery carriers, although they have improved the delivery effect of drugs to some extent, still cannot meet the special needs of liver tumor treatment. For example, the stability, targeting and drug release controllability of liposomes, nanoparticles and other carriers in the body still need to be further improved.

[0005] In this context, developing an efficient, safe, targeted and programmable sequential drug delivery system has important clinical significance and application value for preventing local recurrence after narrow margin liver resection of liver tumor. SUMMARY

[0006] The application aims to provide a double-layer biomimetic microneedle drug delivery system and a preparation method thereof, which has the advantages of strong targeting, small toxic and side effects, and convenient operation.

[0007] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides a preparation method of a double-layer biomimetic microneedle drug delivery system, which comprises the following steps.

[0009] Dissolve the antitumor drug, mix it with the drug-loaded particles, perform ultrasonic emulsification treatment, and evaporate and filter to obtain a drug delivery device;

[0010] Mix the drug delivery device, polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator, fill a microneedle template, and solidify and crosslink to obtain an outer layer;

[0011] Dissolve the hemostatic drug, mix it with the hyaluronic acid, and centrifuge to obtain an inner layer;

[0012] Overlap the outer layer and the inner layer, freeze them at-100 to-60 DEG C for 1 to 3 hours, and then perform vacuum freeze-drying treatment for 1 to 3 hours to obtain the double-layer biomimetic microneedle drug delivery system, which comprises the outer layer and the inner layer and constitutes a plurality of double-layer mushroom-shaped microneedle structures in the system;

[0013] The outer layer contains the drug delivery device, and the drug delivery device contains the antitumor drug;

[0014] The inner layer is filled with the hemostatic drug;

[0015] The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong adhesion to the liver;

[0016] In the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is 100 to 150 microns.

[0017] The application further provides a double-layer microneedle drug delivery system, which comprises an outer layer and an inner layer and constitutes a plurality of double-layer mushroom-shaped microneedle structures in the system;

[0018] The outer layer contains the drug delivery device, and the drug delivery device contains the antitumor drug;

[0019] The inner layer is filled with the hemostatic drug;

[0020] The double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug delivery system to have strong adhesion to the liver.

[0021] In the microneedle structure, an enlarged structure exists adjacent to the tip, and the maximum diameter of the enlarged structure is 100-150 μm.

[0022] Preferably, the total height of the microneedle in the double-layer biomimetic microneedle system is 200-2000 μm, the height of the inner layer is 400-600 μm, and the base diameter of the needle body is 200-300 μm.

[0023] Preferably, the drug delivery device uses drug-loaded particles as the carrier of the antitumor drug, and the drug-loaded particles are nanoscale particles, and the material is a biocompatible and biodegradable polymer material.

[0024] Preferably, the material of the drug-loaded particles is one or more of distearoyl phosphatidyl ethanolamine-polyethylene glycol, methoxy polyethylene glycol-poly(D, L-lactic acid), methoxy polyethylene glycol-poly(caprolactone), methoxy polyethylene glycol-poly(lactic acid-glycolic acid), methoxy polyethylene glycol-poly(lactic acid), polyethylene glycol-poly(caprolactone), poly D-lactic acid, dioleoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, hydrogenated soybean phosphatidyl choline, cholesterol, soybean lecithin, glycerol monooleate, dioleoyl trimethyl ammonium propane, sphingomyelin, or distearoyl phosphatidyl glycerol.

[0025] Preferably, the height of the microneedle in the double-layer biomimetic microneedle system is 200-2000 μm.

[0026] Preferably, the raw material of the inner layer includes a protein natural polymer, a polysaccharide natural polymer, or a synthetic material.

[0027] The protein natural polymer includes collagen, gelatin, silk fibroin, albumin, or spider silk protein.

[0028] The polysaccharide natural polymer or synthetic material includes chitosan, hyaluronic acid, or sodium alginate.

[0029] The synthetic material includes gelatin methacrylamide, polyvinyl alcohol, polycaprolactone, or polylactic acid.

[0030] The raw material of the outer layer includes polyethylene glycol diacrylate, a protein natural polymer, and / or a polysaccharide natural polymer, and a photoinitiator.

[0031] Preferably, the photoinitiator includes phenyl-2,4,6-trimethylbenzoyl phosphonic acid lithium salt or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0032] Preferably, the hemostatic drug is selected from one or more of thrombin, tranexamic acid, and vitamin K.

[0033] Preferably, the anti-tumor drug is a drug for treating primary tumors of the liver.

[0034] Preferably, the anti-tumor drug is selected from one or more of regorafenib, sorafenib, donafenib, or bevacizumab.

[0035] Advantages of the present application:

[0036] The present application realizes the programmed sequential release of the inner layer and outer layer drugs by designing the properties of the inner layer and outer layer microneedle carrier materials to be different. The adhesion of the microneedle drug release device to the liver wound surface is enhanced by designing a double-layer mushroom-shaped microneedle structure. The water solubility of the hydrophobic therapeutic drug is improved by the introduction of drug-loaded particles, so that it can be used locally. The therapeutic drug-loaded particles are delivered to the diseased tissue by the microneedle. Compared with traditional adjuvant therapy for preventing primary tumors of the liver, the programmed sequential drug release device based on microneedles constructed in the present application can realize direct drug delivery to potential metastatic lesions, avoiding systemic adverse reactions caused by systemic drugs. Compared with methods such as transhepatic arterial intervention embolization and local radiotherapy, the double-layer biomimetic microneedle drug release system has the advantages of strong targeting, small toxicity and side effects, and easy operation. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a transmission electron microscope image in drug-loaded particle characterization;

[0038] Figure 2 is a release behavior curve in drug-loaded particle characterization;

[0039] Figure 3 is cytotoxicity in drug-loaded particle biological behavior;

[0040] Figure 4 is a fluorescence microscope and flow chart of cell uptake in drug-loaded particle biological behavior;

[0041] Figure 5 is cell uptake localization in drug-loaded particle biological behavior;

[0042] Figure 6 is a double-layer mushroom-shaped microneedle characterization graph;

[0043] Figure 7 is the mechanical property of the double-layer mushroom-shaped microneedle;

[0044] Figure 8 is a release behavior curve of the targeted drug in the double-layer mushroom-shaped microneedle;

[0045] Figure 9 is a tumor live imaging graph of the embodiment;

[0046] Figure 10 is a tumor live imaging fluorescence value change chart of the embodiment;

[0047] Figure 11 is an experimental mouse weight change chart. DETAILED DESCRIPTION

[0048] The application provides a preparation method of a double-layer biomimetic microneedle drug release system, comprising the following steps:

[0049] The antitumor drug is dissolved, mixed with the drug-loaded particles, subjected to ultrasonic emulsification treatment, evaporated and filtered to obtain a drug release device; the drug release device, polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator are mixed, the microneedle template is filled, and after solidification and crosslinking, an outer layer is obtained; 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 to-60 DEG C for 1 to 3 hours, and then subjected to vacuum freeze-drying treatment for 1 to 3 hours to obtain the double-layer biomimetic microneedle drug release system, which comprises the outer layer and the inner layer and constitutes a plurality of double-layer mushroom-shaped microneedle structures in the system; the outer layer contains the drug release device, and the drug release device contains the antitumor drug; the inner layer is filled with the hemostatic drug; the double-layer mushroom-shaped microneedle structure enables the double-layer microneedle drug release system to have strong adhesion liver penetration capacity; in the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is 100 to 150 microns.

[0050] In the present application, preferably, the anti-tumor drug is dissolved in a concentration of 200 μg anti-tumor drug (such as lenvatinib) in 0.5 mL dichloromethane solution; the drug-loaded particles are nanoscale DSPE-PEG particles, and the mixing is performed by adding the dissolved anti-tumor drug solution into 5 mL DSPE-PEG aqueous solution; the parameters of the ultrasonic emulsification treatment are ultrasonic emulsification at 100 W and 20 degrees Celsius for 6 min; the evaporation is performed by using a rotary evaporator to remove the organic solvent for 10 min, and the filtration is performed by using a 0.45 μM cartridge needle filter mixed cellulose (MCE) 13 mm; when the drug release device is mixed with polyethylene glycol diacrylate, hyaluronic acid and a photoinitiator, 1 mL of the prepared nanometer anti-tumor drug is 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 specifications of the microneedle template are 1*1 cm, and the needle height is 600 microns; the parameters of the centrifugation after filling the microneedle template are 4000 rpm for 10 min; the solidification and crosslinking are ultraviolet light solidification and crosslinking for 20 s; the hemostatic drug is dissolved in 1 mL ultrapure water, and the mixing with hyaluronic acid is performed by mixing with 200 mg of low molecular weight hyaluronic acid (20% w / v), and the parameters of the centrifugation after mixing are 4000 rpm for 10 min; the freezing temperature range of the overlapping of the outer layer and the inner layer is -100~-60℃, and the freezing time is 1~3 h; and the vacuum freeze-drying treatment time is 1~3 h.

[0051] The present application also provides a double-layer microneedle drug release system, which comprises an outer layer and an inner layer, and constitutes 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 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 liver penetration ability; in the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is 100~150 μm. Preferably, the total height of the microneedle in the double-layer biomimetic microneedle system is 200~2000 μm, the height of the outer layer is 400~600 μm, and the base diameter of the needle body is 200~300 μm.

[0052] In the present application, preferably, the drug release device uses drug-loaded particles as the carrier of antitumor drugs, and the drug-loaded particles are nanoscale particles with biocompatible and biodegradable polymer materials. Preferably, the material of the drug-loaded particles is one or more of DSPE-PEG, MPEG-PDLLA, MPEG-PCL, MPEG-PLGA, MPEG-PLA, PEG-PCL, D-PLA, DOPC, DMPC, DPPC, HSPC, Cholesterol, Soy Lecithin, GMO, DOTAP, Sphingomyelin, or DSPG; further preferably, the material of the drug-loaded particles is DSPE-PEG. Preferably, the height of the microneedle in the double-layer biomimetic microneedle system is 200-2000 μm, and further preferably 600-1000 μm. Preferably, the raw material of the inner layer includes a protein natural polymer, a polysaccharide natural polymer, or a synthetic material; the protein natural polymer includes collagen, gelatin, silk fibroin, 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 material of the outer layer includes polyethylene glycol diacrylate and a photoinitiator. Preferably, the photoinitiator includes phenyl-2,4,6-trimethylbenzoyl phosphonic acid 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 antitumor drug is a drug for treating primary liver tumors. Preferably, the antitumor drug is selected from one or more of lenvatinib, sorafenib, donafenib, or bevacizumab, and in theory can also be applicable to other antitumor drugs targeting VEGFR.

[0053] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0054] The term abbreviations in the examples: lenvatinib is LEN, nanolenvatinib is LEN NP, and coumarin 6 is Coumarin6. Examples

[0055] First, prepare the therapeutic drug, drug-loaded material, and microneedle system carrier material, wherein the therapeutic drug and drug-loaded material are prepared into a drug release device by double emulsification method.

[0056] Prepare the therapeutic drug and drug-loaded particles into the aforementioned drug release device by emulsification evaporation method: dissolve 200 g of lenvatinib in 0.5 mL of dichloromethane solution, add to 5 mL of aqueous solution containing DSPE-PEG 1.13 mg, ultrasonic emulsification at 100 W and 20 degrees Celsius for 6 min, remove the organic solvent with a rotary evaporator for 10 min, and filter with a 0.45 m filter.

[0057] Then, prepare the double-layer mushroom-shaped biomimetic microneedle system loaded with lenvatinib nanoparticles by freeze-drying method and template method. The microneedle template is made of polydimethylsiloxane (PDMS) with a size of 1 cm x 1 cm, and each template is composed of 400 (20 x 20) needle tip grooves. The preparation process is as follows: mix 1 mL of prepared nanometer lenvatinib with 0.25 mL of PEGDA (20% v / v) and 125 mg of low molecular weight hyaluronic acid (10% w / v) and 3.2 mg of photoinitiator LAP (0.25% w / v), fill the microneedle template with a size of 1*1 cm and a needle height of 600 microns, centrifuge at 4000 rpm for 10 minutes. Then, after ultraviolet light curing and crosslinking for 20 s, the outer layer of the double-layer microneedle is obtained. Dissolve 10 mg of hemostatic drug thrombin in 1 mL of ultrapure water and mix with 200 mg of low molecular weight hyaluronic acid (20% w / v) as the inner layer microneedle system carrier material, then fill the same size microneedle template as above, centrifuge at 4000 rpm for 10 minutes. Overlap the outer layer and the inner layer after solidification and demolding, freeze in a-80 degrees Celsius refrigerator for 2 hours, demold, and then place in a vacuum freeze dryer for freeze-drying for 1.5 h, take out and place at room temperature for natural drying, and the aforementioned double-layer biomimetic microneedle drug release device is obtained.

[0058] The present application utilizes the double-layer mushroom-shaped biomimetic microneedle drug release device to prevent local recurrence after narrow margin liver resection of liver tumor by utilizing its programmed sequential drug delivery characteristics. When the double-layer microneedle is inserted into the liver incision, the inner layer of the microneedle dissolves first, releasing thrombin to promote hemostasis at the wound site, and then the outer layer loaded with lenvatinib nanoparticles dissolves, slowly releasing lenvatinib, which inhibits the proliferation of residual tumor through targeted action, preventing local recurrence of the tumor.

[0059] The therapeutic drug in this example is lenvatinib, and this example is a basic research on the application of the programmed drug release biomimetic double-layer microneedle device loaded with lenvatinib nanoparticles and thrombin in preventing recurrence after narrow margin resection of liver tumor.

[0060] Materials: Microneedle template, low molecular weight sodium hyaluronate, DSPE-PEG, lenvatinib, thrombin, PEGDA, LAP, trypan blue, etc.

[0061] Cells: Hep3B, Huh7, Hepa1-6. Among them, Huh7 and Hepa1-6 were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin; Hep3B was cultured in MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin.

[0062] Animals: SPF male c57BL / 6 mice, 6-8 weeks old, purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. Animal experiments were conducted under the approval and supervision of the Laboratory Animal Management Committee of West China Hospital of Sichuan University.

[0063] Experimental method:

[0064] Preparation of nano-drug-loaded particles: dichloromethane as emulsifying agent, DSPE-PEG as carrier material, LEN loaded in DSPE-PEG by the method described above, to obtain LEN nano-drug delivery device (LEN-DSPE-PEG), characterized by transmission electron microscopy (TEM), particle size, potential, etc. The characterization of the obtained drug-loaded particles is shown in Figure 1 .

[0065] Release function of nano-drug-loaded particles: the in vitro release capacity of LEN NP was evaluated by dialysis method. 1 mL of 1 mg LEN NP and 1 mL of 1 mg LEN free drug dissolved in DMSO were placed in dialysis bags with a molecular weight cutoff of 3500 Da, and were placed in 20 mL PBS solution containing 1% Tween 80 at pH=7.4 and pH=6.8, respectively, and were placed in a 37°C constant temperature water bath shaker (120 rpm) for release. According to the time interval of 3h, 6h, 9h, 12h, 24h, 36h, 48h, 72h, 1mL was taken each time, and the same volume of fresh PBS solution was added, and the concentration of released LEN was detected by HPLC. The release curves of nano-lenvatinib and free lenvatinib are shown in Figure 2 .

[0066] In vitro biological performance of nano-drug-loaded particles: the cytotoxicity of LEN NP and LEN on Hep3B, Huh7 and Hepa1-6 cells was investigated by CCK8 experiment. As shown in Figure 3As shown. The uptake of LEN NP by Hep3B, Huh7, and Hepa1-6 cells was investigated. In the cellular uptake experiment, a fluorescent hydrophobic coumarin was selected instead of LEN as the model drug. Qualitative and quantitative analyses of the cellular uptake capacity of the nanoparticles were performed using fluorescence microscopy and flow cytometry, respectively. Figure 4 As shown. Confocal microscopy was used to clarify the uptake and localization of nanoparticles within cells. (See figure.) Figure 5 As shown.

[0067] Microneedle fabrication: A programmed drug-release mushroom-shaped biomimetic bilayer microneedle device loaded with lenvatinib nanoparticles and thrombin was prepared using a freeze-drying method and a template method. The prepared microneedles were characterized by scanning electron microscopy, see details below. Figure 6 As shown.

[0068] 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 upwards. The distance between the microneedle tips and the sensor was set to 0.5 cm. Subsequently, the sensor was moved perpendicular to the stainless steel plate at a certain speed. After the sensor contacted the microneedle tip, the sensor displacement and corresponding resistance were recorded. Three parallel samples were tested in each group. See details below. Figure 7 As shown.

[0069] In vitro drug release via microneedles. Specifically, as follows: Figure 8 As shown. 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, and released in a constant-temperature shaker at 37 °C (120 rpm). Samples were taken at time intervals of 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h, with 1 mL taken each time and the same volume of fresh PBS solution added simultaneously. The concentration of released LEN was detected by HPLC.

[0070] In vivo animal experiments. The efficacy of the microneedle system in tumor treatment was studied in vivo using c57BL / 6 mice as a model animal. First, 100.0 μL of Luc-Hepa1-6 cells (cell concentration 5 × 10⁻⁶) were subcutaneously injected into the right hind limb of the mice. 7 Mice with tumor xenografts were randomly divided into 4 groups, as follows:

[0071] (1) Control group, no treatment was given;

[0072] (2) The double-layer microneedle group with a lenvatinib dosage of 10 mg / kg;

[0073] (3) double-layer microneedle group with 20 mg / kg of lenvatinib;

[0074] (4) double-layer microneedle group with 40 mg / kg of lenvatinib.

[0075] As the volume of the inoculated tumor increased to 200.0 mm 3 , the treatment was started. 90% of the tumor volume was removed, and the remaining 10% was treated according to the different grouping methods. The microneedles were removed after being attached to the tumor site for 24 h. The change in the size of the tumor in the mice was recorded using live imaging. The body weight was recorded every 3 days during the experiment, and live imaging was taken every 7 days to record the change in fluorescence value. The experiment ended after 28 days of treatment. The results of the live imaging experiment are shown in Figure 9 , the change in fluorescence value is shown in Figure 10 , and the change in body weight is shown in Figure 11 .

[0076] Figure 1 The particle size of the LEN NP is shown, and the nanoparticles have good dispersity and uniform particle size distribution without aggregation. The Malvern particle size analyzer measurement result is 157.2 ± 4.98 nm. Due to the dehydration and shrinkage of the nanoparticles during the TEM sample preparation process, the particle size is slightly reduced;

[0077] Figure 2 The release curve of the LEN NP in different pH release media is shown. The release rate of the LEN NP in weakly acidic media is faster than that in slightly neutral release media; compared with the free drug, the drug release of the nanoparticles exhibits slow release.

[0078] Figure 3 In three hepatoma cell lines, Huh 7, Hep3B, and Hepa1-6, it is seen that the nanolenvatinib can achieve a similar cell killing effect as lenvatinib.

[0079] Figure 4 In the fluorescence microscope, it is shown that the nanolenvatinib can be taken up by the three cell lines, and the flow cytometry quantitative analysis shows that the amount of nanolenvatinib taken up by the cells increases with time.

[0080] Figure 5 In the confocal microscope, it is shown that after the cells take up the nanolenvatinib, lysosomal escape can occur, thus enabling the drug to act.

[0081] Figure 6A-C super-depth-of-field microscope shows the double-layer mushroom-shaped microneedle structure; D-F scanning electron microscope shows the 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, the maximum diameter of the enlarged structure is about 150 μm, the total height of the microneedle in the double-layer biomimetic microneedle system is about 600 μm, the height of the inner layer is about 500 μm, and the base diameter of the needle body is about 250 μm.

[0082] Figure 7 The mechanical strength of the double-layer mushroom-shaped microneedle is shown.

[0083] Figure 8 The drug release curves of the double-layer mushroom-shaped microneedle in different pH media are shown. In the slightly acidic release medium, the drug is released more quickly.

[0084] Figure 9 The in vivo imaging of the anti-recurrence effect of the double-layer microneedle with different drug loads on the subcutaneous tumor of mice is shown. The drug dosage of 10 mg / kg can achieve obvious anti-recurrence effect.

[0085] Figure 10 The mice in the group not receiving drug-loaded microneedle treatment have heavier body weight and heavier tumor load than the mice in the group receiving drug-loaded microneedle treatment.

[0086] Figure 11 The in vivo imaging of the mice in the group not receiving treatment shows that the fluorescence intensity is stronger than that of the group receiving drug-loaded microneedle treatment.

[0087] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A method for preparing a bilayer biomimetic microneedle drug delivery system, characterized in that, Includes the following steps: The antitumor drug is dissolved, mixed with drug-loaded particles, subjected to ultrasonic emulsification, and filtered to obtain a drug delivery device. The drug delivery device, polyethylene glycol diacrylate, hyaluronic acid and photoinitiator are mixed, filled into a microneedle template, and cured and crosslinked to obtain the outer layer; Dissolve the hemostatic drug, mix it with hyaluronic acid, and centrifuge to obtain the inner layer; The outer and inner layers are overlapped and frozen at -100~-60℃ for 1~3 hours, and then vacuum freeze-dried for 1~3 hours to obtain the double-layer biomimetic microneedle drug delivery system, which 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, which contains an antitumor drug; The inner layer is filled with hemostatic drugs; The double-layered mushroom-shaped microneedle structure gives the double-layered microneedle drug delivery system a strong ability to adhere to the liver; In the microneedle structure, there is an enlarged structure near the tip, and the maximum diameter of the enlarged structure is 100~150μm.

2. A dual-layer microneedle drug delivery system, characterized in that, It consists of an outer layer and an inner layer, forming multiple double-layered mushroom-shaped microneedle structures in the system; The outer layer contains a drug release device, which contains an antitumor drug; The inner layer is filled with hemostatic drugs; The double-layered mushroom-shaped microneedle structure gives the double-layered microneedle drug delivery system a strong ability to adhere to the liver; In the microneedle structure, there is a swollen structure near the tip, and the maximum diameter of the swollen structure is 100~150μm; The drug delivery device uses drug-loaded particles as carriers for anti-tumor drugs. The drug-loaded particles are nano-sized particles made of biocompatible and biodegradable polymer materials.

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

4. The dual-layer biomimetic microneedle drug delivery system according to claim 2, characterized in that, The drug-loaded particles are made of one or more of the following: distearylphosphatidylethanolamine-polyethylene glycol, methoxy polyethylene glycol-poly(D,L-lactic acid), methoxy polyethylene glycol-polycaprolactone, methoxy polyethylene glycol-polylactic acid-glycolic acid copolymer, methoxy polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, poly-D-lactic acid, dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, cholesterol, soybean lecithin, monooleate glycerol, dioleoyltrimethylammonium propane, sphingomyelin, or distearylphosphatidylglycerol.

5. The dual-layer biomimetic 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 polymers include collagen, gelatin, silk protein, albumin, or spider silk protein; The polysaccharide-based natural polymers or synthetic materials include chitosan, hyaluronic acid, or sodium alginate; The synthetic materials include gelatin methacrylamide, polyvinyl alcohol, polycaprolactone, or polylactic acid; The outer layer is made of polyethylene glycol diacrylate, protein-based natural polymers and / or polysaccharide-based natural polymers and photoinitiators.

6. The dual-layer biomimetic microneedle drug delivery system according to claim 5, characterized in that, The photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphonate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

7. The dual-layer biomimetic 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.

8. The dual-layer biomimetic microneedle drug delivery system according to claim 2, characterized in that, The anti-tumor drug is a drug for treating primary liver tumors.

9. The dual-layer biomimetic microneedle drug delivery system according to claim 8, characterized in that, The antitumor drug is selected from one or more of lenvatinib, sorafenib, donafenib, or bevacizumab.

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