Drug delivery system, preparation method, and use method

Abstract

The present invention relates to the field of biomedicine, and more specifically, to a drug delivery system for delivering drugs to cells. The drug delivery system disclosed herein comprises a drug delivery vehicle encapsulating a nucleic acid drug and photothermal nanoparticles. This drug delivery system pioneers an unprecedented approach to intracellular drug delivery, utilizing materials that are biocompatible, environmentally friendly, and pollution-free. Furthermore, compared to other methods, it is simpler and more convenient to operate, with lower production and application costs and a more economical and user-friendly approach.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a drug delivery system for delivering drugs to cells, and a preparation method and a use method thereof. Background Art

[0002] Transdermal drug delivery of small molecules involves delivering them through the skin barrier to produce their effects. However, many drugs, primarily but not exclusively, require entry into cells to express the relevant proteins to produce their desired effects. Current methods for delivering these drugs into cells include liposome transfection, electroporation, gene guns, and viral transfection. Liposomes have been extensively studied as delivery vehicles for both in vitro and in vivo. Synthetic liposomes encapsulating nucleic acid drugs can also be used to enter cells through fusion. Electroporation is a technique in which, under the influence of an electric field, small pores or openings are temporarily formed in the cell membrane, allowing nucleic acid drugs to enter the cell through these pores or openings. Gene guns use high-pressure gas as an accelerating force to temporarily create pores in the cell membrane, propelling the nucleic acid drug coated on the surface of gold particles into the host cell. Viral transfection involves constructing viral vectors, infecting cells with the virus, and integrating the gene into the host cell. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art in delivering nucleic acid drugs to cells, such as low transfection efficiency, relatively complex operation and preparation, and high application cost.

[0004] On the one hand, an embodiment of the present disclosure provides a drug delivery system, comprising a drug delivery carrier, wherein the drug delivery carrier encapsulates a nucleic acid drug and photothermal nanoparticles.

[0005] Optionally, the nucleic acid drug is a DNA drug or an RNA drug.

[0006] Optionally, the system further includes an infrared laser emitter, and the infrared laser emitter has an emission wavelength of 760nm-1mm.

[0007] Optionally, the infrared laser emitter is a near-infrared laser emitter; the infrared laser emitter has an emission wavelength of 760nm-2526nm and an illumination density of 3-4W / cm 2 .

[0008] Optionally, the infrared laser emitter emits laser with a wavelength of 808 nm and a light intensity of 4 W / cm 2 .

[0009] Optionally, the photothermal nanoparticles are compounds of metal and non-metal, metal nanoparticles, or non-metal nanoparticles.

[0010] Optionally, the compound of metal and non-metal is Mxene; the metal nanoparticles are gold nanoparticles or platinum nanoparticles; and the non-metal nanoparticles are polydopamine or graphene oxide.

[0011] Optionally, the drug delivery carrier is a microneedle patch.

[0012] Optionally, based on a 10*10 array of microneedle patches, the amount of the encapsulated nucleic acid drug is 100-600 ng.

[0013] Optionally, the raw materials for preparing the microneedle body include polyvinyl pyrrolidone and hyaluronic acid; wherein the molecular weight of polyvinyl pyrrolidone is 8-11 kDa, and the molecular weight of hyaluronic acid is 3.9 WDa; the mass ratio of polyvinyl pyrrolidone to hyaluronic acid is (10-30):3.

[0014] Optionally, the mass ratio of polyvinyl pyrrolidone to hyaluronic acid is 20:3.

[0015] Optionally, the backing material of the microneedle patch is polyvinyl pyrrolidone, which has a molecular weight of 32-38 WDa.

[0016] On the other hand, an embodiment of the present disclosure provides a method for preparing a drug delivery system, the drug delivery system comprising a microneedle patch encapsulated with a nucleic acid drug and photothermal nanoparticles;

[0017] The preparation method comprises the following steps:

[0018] S1: Prepare the needle tip working solution; including:

[0019] S11: dissolving raw materials for preparing the microneedle body to form a mixed solution 1;

[0020] S12: adding the photothermal nanoparticle solution and the nucleic acid drug solution to the mixed solution 1 obtained in S1, and mixing them evenly to obtain the needle tip working solution;

[0021] S2: prepare backing working solution;

[0022] S3: adding the needle tip working liquid and the backing working liquid into the mold in sequence, drying and solidifying the mold to obtain the microneedle patch.

[0023] In another aspect, the present disclosure also provides a method for using a drug delivery system, wherein the drug delivery system includes a microneedle patch encapsulated with a nucleic acid drug and photothermal nanoparticles;

[0024] The method of use includes:

[0025] T1: placing the microneedle patch on the administration site of the drug administration recipient;

[0026] T2: irradiating the microneedle patch with an infrared laser light source to heat the photothermal nanoparticles until the temperature of the administration site reaches 50° C. to 80° C.;

[0027] T3: Maintain the temperature of the administration site at 50° C. to 80° C. and hold for 1 minute and cool for 1 minute for 5 to 10 cycles to facilitate the penetration of the nucleic acid drug into the administration site.

[0028] The technical solution of the present invention has the following advantages:

[0029] 1. The present invention provides a system for delivering drugs to cells. This intracellular drug delivery system based on the photothermal effect has created an unprecedented way of intracellular drug delivery. In addition, all materials used in the experimental process are biocompatible, environmentally friendly, pollution-free, and harmless materials.

[0030] 2. The present invention provides a system for delivering drugs to cells. Compared with other operation methods, this intracellular drug delivery system based on photothermal effect is simpler and more convenient to operate, has lower production and application costs, and is economical and friendly.

[0031] 3. The present invention provides a system for delivering drugs to cells, which has a wide range of applications. It can be applied not only to the skin and muscles, but also theoretically to other organs for local use. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 2 is a schematic structural diagram of a microneedle patch in an embodiment of the present disclosure; 2 represents a microneedle patch backing, and 1 represents a microneedle body disposed on the microneedle patch backing;

[0034] Figure 2 These are bright field photos and electron microscope photos of the microneedle patch in the embodiment of the present disclosure;

[0035] Figure 3 Schematic diagram of the mouse experiment process in the embodiment of the present disclosure;

[0036] Figure 4 This is the observation result of the small animal fluorescence imaging instrument in the embodiment of the present disclosure;

[0037] Figure 5 The results of observing mouse skin using a fluorescence microscope in the embodiments of the present disclosure are as follows;

[0038] Figure 6 The results of fluorescence microscopy observation of the skin after skin sectioning in the embodiment of the present disclosure; the arrow indicates the location of positive cells; in the figure, MN+GPC represents the results of the microneedle patch encapsulating nucleic acid drugs; MN+Mxene represents the results of the microneedle patch encapsulating photothermal nanoparticles; MN+Mxene+GPC represents the results of the microneedle patch encapsulating nucleic acid drugs and photothermal nanoparticles;

[0039] Figure 7 is a flow chart of a method for using the drug delivery system in an embodiment of the present disclosure;

[0040] Figure 8 Graphs showing experimental results of nucleic acid drugs in the drug delivery system of an embodiment of the present disclosure entering the cell membrane at different temperatures. DETAILED DESCRIPTION

[0041] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0042] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0043] The embodiments of the present disclosure provide a drug delivery system for delivering drugs to cells, comprising a drug delivery carrier that encapsulates nucleic acid drugs and photothermal nanoparticles.

[0044] The above-mentioned nucleic acid drug can be a DNA drug or an RNA drug.

[0045] The drug delivery system in the embodiment of the present disclosure also includes an infrared laser emitter, the power of the infrared laser emitter is adjustable, and the infrared laser emitter has an emission wavelength of 760nm-1mm. In one embodiment of the present disclosure, the infrared laser emitter can be a near-infrared laser emitter, which emits infrared light with a wavelength of 760nm-2526nm and an illumination density of 3-4W / cm 2 Further preferably, the emission laser wavelength is 808nm and the light intensity is 4W / cm 2 .

[0046] In the embodiment of the present disclosure, the drug delivery carrier may be a microneedle patch, such as Figure 1As shown, the drug delivery carrier includes a microneedle patch backing 2 and a microneedle body 1 disposed on the microneedle patch backing. Any material that is non-toxic, quickly soluble, biocompatible, and capable of encapsulating nucleic acid drugs can be used as a microneedle body material. In this embodiment, the microneedle body material includes polyvinyl pyrrolidone (PVP) and hyaluronic acid (HA); the molecular weight of PVP is 8-11 kDa, and the molecular weight of HA is 3.9 WDa. The mass ratio of polyvinyl pyrrolidone to hyaluronic acid is (10-30):3; in this embodiment, it can be preferably 20:3.

[0047] The backing material of the microneedle patch is polyvinyl pyrrolidone (PVP), which has a molecular weight of 32-38 WDa.

[0048] The amount of nucleic acid drug can be controlled by adding drugs of different concentrations; the nucleic acid drug is a DNA drug or an RNA drug. The nucleic acid drug used in this embodiment is a plasmid loaded with expression of green fluorescent protein (GFP) (plasmid GPC, nucleic acid sequence see SEQ ID NO.1).

[0049] The photothermal nanoparticles in the disclosed embodiments are metal-nonmetal compounds, metal nanoparticles, or nonmetal nanoparticles. Optionally, the metal-nonmetal compound is MXene, the metal nanoparticles are gold nanoparticles or platinum nanoparticles, and the nonmetal nanoparticles are polydopamine or graphene oxide. In this embodiment, the photothermal nanoparticles can be MXene (Shandong Enyan New Materials).

[0050] In this embodiment, a microneedle patch with a 10*10 array is prepared. In this embodiment, the needle body height is 670um, the needle tip size is 15um, the microneedle bottom size is 300*300um, the needle body shape is a quadrangular pyramid, the center distance between two adjacent microneedles is 500um, the backing size is 9.8*9.8mm, and the backing thickness is 1.5mm. The specific microneedle size and shape can be changed according to experimental needs. The amount of nucleic acid drug (a plasmid containing GFP encoding in the experiment, i.e., plasmid GPC) wrapped in each microneedle patch (the above-mentioned microneedle patch with a 10*10 array) is 500ng, and the amount of nanoparticles wrapped (Mxene in this embodiment) is 536ng. The amount of nucleic acid drugs and nanoparticles specifically contained can be changed according to experimental needs. The microneedle backing material is PVP with a molecular weight of 32-38WDa.

[0051] Each 10*10 array of microneedle patches contains 500 ng of plasmid GPC.

[0052] In the embodiments of the present disclosure, a method for preparing the drug delivery system is also provided, which is described by taking the preparation of a microneedle patch encapsulating a nucleic acid drug and photothermal nanoparticles as an example.

[0053] Material preparation

[0054] The preparation method of 1g / ml PVP solution is:

[0055] Take 5 g of powdered PVP, put it into a test tube, add deionized water to 5 ml, and shake for 20 minutes to fully dissolve it to obtain a 1 g / ml PVP solution.

[0056] The preparation method of 150mg / ml HA solution is as follows:

[0057] HA 750 mg was placed in a test tube, deionized water was added to 5 ml, and the mixture was shaken for 20 min to fully dissolve the mixture to obtain a 150 mg / ml HA solution.

[0058] Microneedle patch preparation

[0059] Step 1: Select the microneedle material.

[0060] The needle body material includes polyvinyl pyrrolidone (PVP) with a molecular weight of 8-11 kDa and a concentration of 1 g / ml, and hyaluronic acid (HA) with a molecular weight of 3.9 WDa and a concentration of 150 mg / ml. The microneedle backing working fluid material is PVP with a molecular weight of 32-38 WDa.

[0061] Step 2: Prepare the microneedle working fluid, including the needle tip (ie) working fluid and the backing working fluid.

[0062] The microneedle backing working fluid is a PVP solution with a concentration of 0.5 g / ml.

[0063] The needle tip working solution is prepared by taking the preparation of 400 μl needle tip working solution as an example. The volume ratio of PVP solution, HA solution and photothermal nanoparticle solution is (2-4): (2-4): 1. Specifically,

[0064] 1) Mix 133.92 μl of 1 g / ml PVP solution and 133.92 μl of 150 mg / ml HA solution, shake thoroughly to mix evenly, and then place in a vacuum pump and extract for 30 minutes to remove bubbles in the solution to obtain a mixed solution 1.

[0065] 2) Add MXene and GPC, a plasmid expressing green fluorescent protein, to Mixture 1 as needed. The tip working solution is designed to ensure that each microneedle patch contains 500 ng of GPC. Since the volume of the PDMS mold tip is 2.01 μl, to ensure that each microneedle patch contains 500 ng of GPC, the concentration of GPC in the tip working solution is:

[0066]

[0067] Therefore, 66.96 μl of 1.6 mg / ml Mxene solution and 65.2 μl of 1335.2 ng / μl GPC solution were added to the mixed solution 1, shaken thoroughly, and vacuumed to remove bubbles in the working solution to obtain a uniform needle tip working solution.

[0068] Step 3: After the two working solutions are prepared, take out the PDMS concave mold and start making microneedles.

[0069] First, use a pipette to draw the needle tip working fluid into the PDMS concave mold, ensuring that the needle tip working fluid covers the mold's pinhole array. Then, place the mold in a vacuum pump at -103 kPa and draw the vacuum for 3 hours. Humidify the vacuum pump during the extraction process to prevent water loss from the working fluid. After the extraction is complete, observe the mold under a stereoscope. If there are a few bubbles around the edge, manually remove them with the pipette. If more bubbles remain, continue to draw the vacuum until the working fluid fills the needle tip, recovering any excess working fluid.

[0070] The fourth step is to add backing working liquid to the PDMS concave mold so that the backing liquid covers the convex mold surface in an arc shape to avoid backing defects caused by water loss during the curing process. Then place it at a temperature of 20°C and an air humidity of 15% for 24 hours to dry and cure. Use tweezers or tape to peel off the microneedle patch wrapped with nucleic acid drugs and photothermal nanoparticles, such as Figure 2 shown.

[0071] How to use the microneedle patch

[0072] With respect to the drug delivery system provided in the above embodiment, the present disclosure also provides a method for using the drug delivery system, such as Figure 7 As shown, this may include:

[0073] T1: placing the microneedle patch on the administration site of the drug administration recipient;

[0074] T2: irradiating the microneedle patch with an infrared laser light source to heat the photothermal nanoparticles until the temperature of the administration site reaches 50° C. to 80° C.;

[0075] T3: Maintain the temperature of the administration site at 50° C. to 80° C. and hold for 1 minute and cool for 1 minute, for 5 to 10 cycles, so that the nucleic acid drug can penetrate into the administration site.

[0076] Examples & Comparative Examples

[0077] Using infrared laser light source to irradiate, the photothermal nanoparticles in the microneedle patch generate heat. The photothermal nanoparticles conduct heat to the cell membrane, causing local disorder of the cell membrane, thereby allowing the drug to enter the cell.

[0078] like Figure 8The molecular dynamics simulation experimental results shown in the figure show that the average resistance of nucleic acid drugs (such as DNA) to cross the cell membrane at a temperature of 323K (50°C) is less than the average resistance to cross the cell membrane at a temperature of 303K (30°C), which means that DNA can more easily penetrate the cell membrane at 50°C.

[0079] The inventors of the present disclosure have found through research that when the temperature at the administration site is maintained at 50°C to 80°C, nucleic acid drugs can quickly enter the cell membrane without causing damage to the cells.

[0080] Animal experiments

[0081] 1. Combine Figure 3 The mouse experiment process shown in the figure is as follows: the experimental C57BL / 6N mice were anesthetized and the backs were locally depilated. The microneedle patch containing nucleic acid drugs and photothermal nanoparticles was inserted into the depilated area on the backs of the mice. After waiting for 20 minutes, the microneedle backing was irradiated with a near-infrared laser with an emission wavelength of 808nm and an illumination density of 4w / cm 2 The irradiation time was 1 min, followed by 1 min interval and then irradiation, and the cycle was repeated 5 times. The nucleic acid drug in the microneedle patch encapsulating the nucleic acid drug and photothermal nanoparticles was replaced with deionized water as a control.

[0082] 2. Data collection is required at the end of this experiment. First, use a small animal fluorescence imager for observation. Observe once every 24 hours and 48 hours after the experiment. Anesthetize the experimental mice with isoflurane and set aside. After opening the software, adjust the relevant settings. Set the excitation wavelength to 480nm, the emission wavelength to 520nm, and the exposure time to 200s, Binning to 2, and Fstop to 2 as needed. Then take pictures to collect data. The results are shown in the figure. Figure 4 In the figure, blank MN represents the control result, and MN+Mxene+GFP represents the result of the microneedle patch encapsulating nucleic acid drugs and photothermal nanoparticles. After the data acquisition is completed, the experimental image can be adjusted through the software, such as the luminescence type of the fluorescent part, the fluorescence threshold setting in the image, etc., to make the experimental comparison more beautiful. The results show that the MN+Mxene+GFP group has stronger fluorescence than the blank MN group, indicating that GFP is expressed in the microneedle-treated part, indicating that the GPC plasmid has been delivered into the cell and can express green fluorescent protein. The results of fluorescence microscopy observation of the skin are shown in Figure 5 Finally, the mouse skin was sliced ​​and observed at the cellular level. Figure 6 It can be seen that the expression of green fluorescent protein is consistent with the spacing of microneedles.

[0083] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention. Sequence Listing <110> Beijing University of Aeronautics and Astronautics <120> Intracellular drug delivery method based on photothermal nanoparticles <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2217 <212> DNA <213> Artificial sequence <400> 1 ggatccatgg gacaaatagt gacattcttc caggaagtgc ctcatgtaat agaagaggtg 60 atgaacattg ttctcattgc actgtctgta ctagcagtgc tgaaaggtct gtacaatttt 120 gcaacgtgtg gccttgttgg tttggtcact ttcctcctgt tgtgtggtag gtcttgcaca 180 accagtcttt ataaaggggt ttatgagctt cagactctgg aactaaacat ggagacactc 240 aatatgacca tgcctctctc ctgcacaaag aacaacagtc atcattatat aatggtgggc 300 aatgagacag gactagaact gaccttgacc aacacgagca ttattaatca caaattttgc 360 aatctgtctg atgcccacaa aaagaacctc tatgaccacg ctctttatgag cataatctca 420 actttccact tgtccatccc caacttcaat fotatgagg caatgagctg cgattttaat 480 gggggaaaga ttagtgtgca gtacaacctg agtcacagct atgctggggga tgcagccaac 540 cattgtggta ctgttgcaaa tggtgtgtta cagactttta tgaggatggc ttggggtggg 600 agctacattg ctcttgactc aggccgtggc aactgggact gtattatgac tagttatcaa 660 tatctgataa tccaaaatac aacctgggaa gatcactgcc aattctcgag accatctccc 720 atcggttatc tcgggctcct ctcacaaagg actagagata tttatattag tagagaattg 780 ctaggcacat tcacatggac actgtcagat tctgaaggta aagacacacc agggggatat 840 tgtctgacca ggtggatgct aattgaggct gaactaaaaat gcttcgggaa cacagctgtg 900 gcaaaatgta atgagaagca tgatgaggaa ttttgtgaca tgctgaggct gtttgacttc 960 aacaaacaag ccattcaaag gttgaaagct gaagcacaaa tgagcattca gttgatcaac 1020 aaagcagtaa atgctttgat aaatgaccaa cttataatga agaaccatct acgggacatc 1080 atgggaattc catactgtaa ttacagcaag tattggtacc tcaaccacac aactactggg 1140 agaacatcac tgcccaaatg ttggcttgta tcaaatggtt catacttgaa cgagacccac 1200 ttttctgatg atattgaaca acaagctgac aatatgatca ctgagatgtt acagaaggag 1260 tatatggaga ggcaggggaa gacaccattg ggtctagttg acctctttgt gttcagtaca 1320 agtttctatc ttattagcat cttccttcac ctagtcaaaa taccaactca taggcatatt 1380 gtaggcaagt cgtgtcccaa acctcacaga ttgaatcata tgggcatttg ttcctgtgga 1440 ctctacaaac agcctggtgt gcctgtgaaa tggaagagag gcggtggcgg tatggtgagc 1500 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 1560 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 1620 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 1680 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 1740 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 1800 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1860 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 1920 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 1980 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 2040 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 2100 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 2160 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agatatc 2217

Claims

1. A drug delivery system, characterized in that: The system comprises a drug delivery carrier, wherein the drug delivery carrier encapsulates nucleic acid drugs and photothermal nanoparticles; the photothermal nanoparticles are Mxene; the system further comprises an infrared laser emitter, wherein the infrared laser emitter is a near-infrared laser emitter; the infrared laser emitter is an infrared laser emitter with an emission wavelength of 808 nm. , light density is 4 W / cm 2 ; The nucleic acid drug is a DNA drug or an RNA drug; The drug delivery carrier is a microneedle patch; the raw materials for preparing the microneedle body include polyvinyl pyrrolidone and hyaluronic acid; wherein the molecular weight of polyvinyl pyrrolidone is 8-11 The molecular weight of hyaluronic acid is 3.9 ; The mass ratio of polyvinyl pyrrolidone to hyaluronic acid is (10-30):

3.

2. The system according to claim 1, wherein: Based on a 10*10 array of microneedle patches, the amount of nucleic acid drug encapsulated is 100-600ng.

3. The system according to claim 1 or 2, characterized in that The mass ratio of the polyvinyl pyrrolidone to the hyaluronic acid is 20:

3.

4. The system according to claim 1 or 2, characterized in that The backing material of the microneedle patch is polyvinyl pyrrolidone, which has a molecular weight of 32-38 WDa .

5. A method for preparing the drug delivery system according to any one of claims 1 to 4, characterized in that: The drug delivery system includes a microneedle patch coated with nucleic acid drugs and photothermal nanoparticles; The preparation method comprises the following steps: S1: Prepare the needle tip working solution; including: S11: dissolving raw materials for preparing the microneedle body to form a mixed solution 1; S12: adding the photothermal nanoparticle solution and the nucleic acid drug solution to the mixed solution 1 obtained in S1, and mixing them evenly to obtain the needle tip working solution; S2: prepare backing working solution; S3: adding the needle tip working liquid and the backing working liquid into the mold in sequence, drying and solidifying the mold to obtain the microneedle patch.

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