Metal polyphenol-loaded microneedle array, and preparation method therefor and use thereof

Abstract

A metal polyphenol-loaded microneedle array, and a preparation method therefor and the use thereof. The microneedle array preparation method comprises the following steps: step 1, mixing a polyphenol solution and a metal salt solution to obtain a mixed solution, wherein the molar ratio of the polyphenol to the metal salt is 8:1 to 4:1; and step 2, dropwise adding the mixed solution obtained in step 1 to the surface of a microneedle array, allowing the mixed solution to deposit, and then cleaning same to obtain a metal polyphenol-loaded microneedle array. The microneedle array as a biomacromolecule intracellular delivery system can achieve delivery of ultra-large (15,000-20,000 bp) CRISPR / Cas9 plasmids, and achieves gene editing.

Description

A metal polyphenol-loaded microneedle array, preparation method, and application Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a metal polyphenol-loaded microneedle array, a preparation method and an application thereof. Background Art

[0002] Intracellular delivery of biomacromolecules refers to the delivery of biomacromolecules into target cells through specific methods, so that they can play relevant biological functions in the cells. However, biomacromolecules have high molecular weight, large size and mostly negative surface charge or a small amount of positive charge (some proteins and peptides), making it difficult for them to cross the cell membrane of the target cell and enter the cell interior. Currently reported macromolecular intracellular delivery carriers mainly include cationic liposomes, inorganic nanomaterials, polymer carriers, viral infection, cell-derived vesicles, etc. However, on the one hand, the biomacromolecules delivered by these methods have weak endosomal escape ability and are easily rapidly degraded by proteases; on the other hand, the method of releasing biomacromolecules from the carrier through stimulus response in the intracellular environment often requires chemical or biological modification of the biomacromolecules, which will reduce the biological activity of the macromolecules.

[0003] Microneedle arrays are a new and highly efficient drug delivery technology. Their structure is similar to that of traditional syringe needles, with the needle tips being symmetrically conical or asymmetrically beveled. Microneedle arrays feature multiple microscopic needle-like structures, with needle lengths typically ranging from a few hundred nanometers to a few millimeters. Because microneedles are of sufficient length and mechanical strength, they can penetrate cell membranes, reducing the retardation of the phospholipid bilayer on drugs. However, when using microneedles alone for drug delivery, it is difficult for drugs to accumulate on the microneedle surface, necessitating the addition of an excess of drug to dissolve in the solution. This not only places high demands on the drug's solubility, but also results in drug waste. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a metal polyphenol-loaded microneedle array, a preparation method and an application thereof.

[0005] The technical solution adopted in the present invention is:

[0006] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0007] Step 1: mixing a polyphenol solution and a metal salt solution to obtain a mixed solution; the molar ratio of the polyphenol to the metal salt is 4:1;

[0008] Step 2: Add the mixed solution obtained in step 1 dropwise onto the surface of the microneedle array. After the mixed solution is deposited, wash it; and a microneedle array loaded with metal polyphenols can be obtained.

[0009] Furthermore, the polyphenols in step 1 are one or two or more of anthocyanins, catechins, gallic acid, and tannic acid.

[0010] Furthermore, the metal ion in the metal salt is Fe 3+ 、Mn 2+ 、Fe 2+ One or two or more of the following.

[0011] Furthermore, the mixed solution in step 1 is vortexed for 10 to 20 seconds, and the sedimentation time in step 2 is 10 to 20 minutes.

[0012] A metal polyphenol-loaded microneedle array has a microneedle height of 1 μm, a microneedle bottom diameter of 0.8 μm, and a center distance between adjacent microneedles of 1.2 μm.

[0013] An application of a microneedle array loaded with metal polyphenols, wherein the microneedle array loaded with metal polyphenols is used as a delivery carrier.

[0014] Furthermore, the metal polyphenol-loaded microneedle array serves as a macromolecular intracellular delivery carrier.

[0015] Furthermore, the metal polyphenol-loaded microneedle array is used to deliver chicken ovalbumin, nucleic acid aptamers, small interfering RNA, polypeptides, and plasmids.

[0016] Furthermore, the metal polyphenol-loaded microneedle array is used in the preparation of medicines.

[0017] A recycling method for a metal polyphenol-loaded microneedle array comprises placing the metal polyphenol-loaded microneedle array in a solution with a pH of 3 to 5 and soaking the solution for 5 to 10 minutes to recycle the microneedle array.

[0018] The beneficial effects of the present invention are:

[0019] (1) The present invention performs functional modification on the surface of the microneedle array, and forms a nanocoating based on plant polyphenols on the modified microneedles. The nanocoating can effectively load different types of biomacromolecules and achieve cell penetration and delivery of biomacromolecules through the special structure of the microneedles;

[0020] (2) In the present invention, on the one hand, the microneedles can pierce the cell membrane and reduce the blocking effect of the phospholipid bilayer on drugs; on the other hand, the metal polyphenols have adhesive properties and can carry various biomacromolecules;

[0021] (3) The metal polyphenol coating of the present invention has strong adsorption properties and can also bind to biomacromolecules such as proteins and nucleic acids through hydrogen bonding, π-π stacking, and hydrophobic interactions;

[0022] (4) The phenolic hydroxyl groups in the metal polyphenol coating of the present invention are protonated, forming competitive chelation between proton hydrogen and metal ions, resulting in the disintegration of the coating; therefore, the carrier of the present invention can be recycled and reused. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a morphology of the metal polyphenol-loaded microneedle array obtained in Example 1 of the present invention, a is a scanning electron microscope image, the upper part is the unloaded coating, the lower part is the loaded coating, and b is an energy dispersive X-ray spectrum.

[0024] FIG2 is an X-ray photoelectron spectrum of the metal polyphenol-loaded microneedle array obtained in Example 1 of the present invention.

[0025] FIG3 is a schematic diagram of the cell puncture efficiency of the metal polyphenol-loaded microneedle array obtained in Example 1 of the present invention, a is the fluorescence staining result, and b is the statistical result.

[0026] FIG4 is a schematic diagram showing the intracellular delivery efficiency of biomacromolecules by the metal polyphenol-loaded microneedle array obtained in Example 1 of the present invention.

[0027] FIG5 is a schematic diagram showing the intracellular delivery efficiency of the biomacromolecule OVA of the metal polyphenol-loaded microneedle array and the microneedle array and metal polyphenol alone obtained in Example 1 of the present invention.

[0028] FIG6 is a schematic diagram showing the intranuclear delivery efficiency of the metal polyphenol-loaded microneedle array obtained in Example 1 of the present invention for oversized plasmids.

[0029] FIG7 shows the delivery efficiency of various biomacromolecules by the metal polyphenol-loaded microneedle arrays described in Examples 2 to 5 of the present invention.

[0030] FIG8 shows the nuclear delivery efficiency of the microneedle array loaded with metal polyphenols for plasmids described in Example 6 of the present invention, a is the result of GFP plasmid, and b is the result of RFP plasmid. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0033] Step 1: Mix the polyphenol solution and the metal salt solution to obtain a mixed solution; the molar ratio of polyphenol to metal salt is 4:1; the polyphenol is one or two or more of anthocyanin, catechin, gallic acid, and tannic acid. The metal ion in the metal salt is Fe 3+ 、Mn 2+ 、Fe 2+ One or two or more of the following.

[0034] Step 2: Add the mixed solution obtained in step 1 dropwise onto the surface of the microneedle array. After the mixed solution is deposited, rinse to obtain a microneedle array loaded with metal polyphenols. Vortex the mixed solution for 10-20 seconds. The deposition time in step 2 is 10-20 minutes.

[0035] A metal polyphenol-loaded microneedle array, wherein the microneedles have a height of 1 μm, a diameter at the bottom of the microneedles of 0.8 μm, and a center-to-center spacing of 1.2 μm between adjacent microneedles (the microneedle array used in the present invention is prepared using existing technologies, such as physical vapor deposition, and the improvement of the present invention does not lie in the preparation of the microneedles).

[0036] Example 1

[0037] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0038] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0039] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0040] Figure 1 is a morphological image of the metal polyphenol-loaded microneedle array obtained in Example 1. a is a scanning electron microscope image, with the top image showing the unloaded coating and the bottom image showing the loaded coating. b is an energy dispersive X-ray spectrometry image. As can be seen from the top image in Figure 1a, the microneedles are highly consistent in shape and size and are evenly arranged, with a height of approximately 887 nm. The microneedle array after loading with metal polyphenols does not show significant changes in size and shape, and the functional modification of the surface does not significantly affect the puncture performance of the microneedles. As can be seen from Figure 1b, the Fe element is evenly distributed on the microneedles.

[0041] Figure 2 shows the X-ray photoelectron spectrum of the metal polyphenol-loaded microneedle array obtained in Example 1. The figure shows a significant increase in the CO single bond ratio on the functionalized microneedles compared to those without metal polyphenol loading. This is due to the successful loading of tannic acid onto the microneedle surface. The successful loading of metal polyphenols onto the microneedle array enables the microneedles to bind and load biomacromolecules.

[0042] The functionalized modified microneedle array obtained in Example 1 was used to test the cell puncture efficiency as follows:

[0043] Place the microneedle in a cell culture dish (needle facing up) and add a 1x10 5cells / mL of cell solution. Place the culture dish in a centrifuge at room temperature, 50g, and centrifuge for 60-100 seconds. Gently dislodge the cells from the microneedles with PBS (pH 7.4). Stain the cells with Calcein AM / EthD-1 to verify microneedle penetration efficiency. Cells stained with both Calcein AM and EthD-1 indicate cells punctured by the microneedles. The results are shown in Figure 3.

[0044] The image shows that Calcein-AM easily penetrates the membranes of living cells, becoming trapped inside and emitting strong green fluorescence. EthD-1, however, cannot penetrate the membranes of living cells, but can penetrate disordered regions of dead cell membranes to reach the nucleus, where it intercalates into the cell's DNA double helix, producing red fluorescence. Live cells successfully punctured by the microneedle are stained with both Calcein AM and EthD-1. Statistically, the modified microneedle array SNNs achieve a cell puncture efficiency of approximately 98%.

[0045] The functionalized modified microneedle array obtained in Example 1 was used to test the intracellular delivery of biomacromolecules in the following manner:

[0046] When the cells grew to a density of about 80-90%, the culture medium was aspirated and 2 mL of 0.25% trypsin was added. After the cells shrank and rounded under a microscope, 3 mL of complete culture medium was added to terminate the trypsin action. The cells were dispersed and detached by pipetting. The cells were centrifuged at 200 g for 3 min, the supernatant was discarded, and the cell concentration was adjusted to 1 × 10 with culture medium containing 10% FBS. 5 cells / mL.

[0047] The functionalized microneedles prepared above were placed in a cell culture dish. 20 μL of a biomacromolecule at a concentration of 1 μg / μL was dripped onto the microneedle surface and allowed to stand on ice until the liquid settled. Unbound biomacromolecules were then rinsed with phosphate-buffered saline (PBS, pH 7.4). The cell suspension was added, and the cell culture dish was centrifuged at 50 g for 60–100 s. The microneedles were removed and the cells were gently dislodged with phosphate-buffered saline (PBS, pH 7.4). Different biomacromolecules, including hen ovalbumin (OVA), a nucleic acid aptamer (AS1411), a small interfering RNA (siRNA), a peptide, and a plasmid, were prepared according to the above method. The results are shown in Figure 4. Fluorescence physically adsorbed on the cell membrane was quenched by the addition of 0.04% trypan blue. Fluorescence in the cells was observed using laser confocal microscopy, and the biomacromolecule delivery efficiency was quantified by flow cytometry.

[0048] It can be seen from the figure that the modified microneedle SNNs have an intracellular presentation efficiency of about 90% for biological macromolecules in 293T, Hela and 4T1 cells.

[0049] The microneedle array obtained in Example 1 was loaded with OVA (SNNs) according to the above method, and its intracellular delivery efficiency was tested in accordance with the above method compared with the microneedle array (NNs) and metal polyphenol (MPN) without metal polyphenol loading. The results are shown in Figure 5. As can be seen from the figure, the modified microneedle SNNs can significantly improve the intracellular delivery efficiency of the biomacromolecule OVA. From the test results, it can be seen that the microneedle NNs and metal polyphenol MPN have a synergistic effect, which can greatly improve their intracellular delivery efficiency, making it have practical application value. It is not just a simple superposition of effects.

[0050] The modified microneedle array from Example 1 was tested for its nuclear delivery efficiency of oversized plasmids using the same method described above. The results are shown in Figure 6. As can be seen from the figure, compared to the traditional carrier Lipofectamine (liposomes), the transfection efficiency of Lipofectamine is ~75% after 24 hours, while the transfection efficiency of the functionalized microneedles is ~95% after 6 hours. These results demonstrate that the modified microneedle SNNs can significantly shorten the transfection time of biomacromolecules and improve transfection efficiency.

[0051] Example 2

[0052] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0053] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0054] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0055] The microneedle array was placed in a cell culture dish, and 10 μL (1.0 μg / μL) of nucleic acid aptamer (AS1411) was dripped onto the microneedle surface. The solution was allowed to settle on ice, and then unbound biomacromolecules were rinsed with PBS (pH 7.4). The prepared microneedles were placed in a cell culture dish, the cell suspension was added, and the microneedles were centrifuged at 50 g for 90 seconds to complete the intracellular delivery of the macromolecules. The results are shown in Figure 7, which shows that the modified microneedle array SNNs have an intracellular delivery efficiency of approximately 90% for AS1411.

[0056] Example 3

[0057] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0058] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0059] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0060] The microneedle array was placed in a cell culture dish, and 10 μL (1.0 μg / μL) of chicken ovalbumin (OVA) was added dropwise to the microneedle surface. The mixture was allowed to stand on ice until the liquid settled, and then unbound biomacromolecules were rinsed with PBS (pH 7.4). The prepared microneedles were placed in a cell culture dish, the cell suspension was added, and the mixture was centrifuged at 50 g for 90 seconds to complete the intracellular delivery of the macromolecules. The results are shown in Figure 7, which shows that the modified microneedle array SNNs have an intracellular presentation efficiency of about 90% for OVA.

[0061] Example 4

[0062] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0063] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0064] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0065] The microneedle array was placed in a cell culture dish, and 10 μL (1.0 μg / μL) of small interfering RNA (siRNA) was dripped onto the microneedle surface. The solution was allowed to settle on ice, and then unbound biomacromolecules were rinsed with PBS (pH 7.4). The prepared microneedles were placed in a cell culture dish, the cell suspension was added, and the microneedles were centrifuged at 50 g for 90 seconds to complete the intracellular delivery of the macromolecules. The results are shown in Figure 7, which shows that the modified microneedle array SNNs have an intracellular delivery efficiency of approximately 90% for siRNA.

[0066] Example 5

[0067] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0068] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0069] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0070] The microneedle array was placed in a cell culture dish, and 10 μL (1.0 μg / μL) of peptide was added dropwise to the microneedle surface. The mixture was allowed to stand on ice until the liquid settled, and then unbound biomacromolecules were rinsed with PBS (pH 7.4). The prepared microneedles were placed in a cell culture dish, the cell suspension was added, and the mixture was centrifuged at 50 g for 90 seconds to complete the intracellular delivery of the macromolecules. The results are shown in Figure 7, which shows that the modified microneedle array SNNs have an intracellular presentation efficiency of about 90% for the peptide.

[0071] Example 6

[0072] A method for preparing a microneedle array loaded with metal polyphenols comprises the following steps:

[0073] Step 1: Add 5 μL of 20 mg / mL tannic acid solution and 5 μL of 5 mg / mL Fe-containing solution to 500 μL of acetone. 3+ The metal salt solution was vortexed for 20 seconds.

[0074] Step 2: 20 μL of the mixed solution was added dropwise to the surface of the microneedle array and allowed to stand for 20 minutes to allow the liquid to settle. The excess unbound tannic acid and metal salt solution was then rinsed with phosphate buffered saline (PBS, pH 7.4).

[0075] The microneedle array was placed in a cell culture dish, and 10 μL (0.2 μg / μL) of plasmid was added dropwise to the microneedle surface. The solution was allowed to settle on ice, and then unbound biomacromolecules were rinsed with PBS (pH 7.4). The prepared microneedles were placed in a cell culture dish, the cell suspension was added, and the microneedles were centrifuged at 50 g for 90 seconds to complete the intracellular delivery of the macromolecules. The results are shown in Figure 7, which shows that the modified microneedle array SNNs have an intracellular presentation efficiency of about 90% for the plasmid.

[0076] GFP plasmid and RFP plasmid were selected and processed according to the above method, and the results are shown in Figure 8. It can be seen from the figure that the modified microneedle SNNs have an intracellular presentation efficiency of about 95% for the plasmid.

[0077] The present invention modifies metal polyphenols onto the surface of a microneedle array to obtain an efficient, safe, and platform-applicable intracellular delivery carrier for biomacromolecules. The application of microneedle carriers can effectively deliver a variety of biomacromolecules with different molecular weights and isoelectric points into cells, and does not require modification of the biomacromolecules, and can maintain the activity of the biomacromolecules. The microneedles have sufficient length and mechanical strength to pierce the cell membrane, destroying the blocking effect of the phospholipid bilayer on drugs, and can allow biomacromolecules to escape from lysosomes, greatly improving the delivery rate of drugs. The modified microneedles overcome the problem of small nucleic acid loading capacity (no more than 5,000bp) existing in many viral vectors, and can efficiently achieve the delivery of ultra-large-sized (15,000-20,000bp) CRISPR / Cas9 plasmids, and achieve effective gene editing of multiple gene sites in different cells, which has broad application prospects in the biomedical field.

[0078] The self-assembly of metals and polyphenols is affected by the pH of the solution, and the stability of the self-assembled coating is pH-dependent. At low pH, most of the phenolic hydroxyl groups on the polyphenol molecules are protonated, resulting in competitive chelation between the protonated hydrogen and the metal ions, leading to MPN instability and even disintegration. Therefore, washing the microneedles with a solution of appropriate pH can disintegrate the MPN coating, making it recyclable.

[0079] From the test results of the examples, it can be seen that when the microneedle carrier obtained by the present invention transfects super-sized plasmids, compared with the traditional infectious agent Lipofectamine (liposomes), Lipofectamine has a transfection efficiency of ~75% in 24 hours, and the microneedles after MPN functionalization modification have a transfection efficiency of ~95% in 6 hours. The microneedle SNNs modified with MPN significantly shorten the transfection time of super-sized plasmids and improve the transfection efficiency. The microneedles modified with MPN functionalization can deliver six biomacromolecules: chicken ovalbumin (OVA), nucleic acid aptamer (AS1411), small interfering RNA (siRNA), peptide, GFP plasmid and RFP plasmid into a variety of cells with a delivery efficiency of more than 90%, achieving high throughput, high efficiency and universal applicability of the vector. The microneedles modified with MPN functionalization can be washed with a suitable solution to disintegrate the MPN covering the microneedles, making them recyclable. After 2-3 rounds of recycling, the microneedle delivery efficiency of biomacromolecules can still be higher than 90%.

Claims

1. A preparation method of a metal-loaded polyphenol microneedle array, characterized in that, It includes the following steps: Step 1: Mix a polyphenol solution and a metal salt solution to obtain a mixed solution; the molar ratio of polyphenol to metal salt is 8:1 to 1:4; Step 2: Drop the mixed solution obtained in Step 1 onto the surface of the microneedle array. After the mixed solution is deposited, wash it; thus, a microneedle array loaded with metal polyphenol can be obtained.

2. The preparation method of a metal-loaded polyphenol microneedle array according to claim 1, characterized in that, In Step 1, the polyphenol is one or two or more of anthocyanin, catechin, gallic acid, and tannic acid.

3. The preparation method of a metal-loaded polyphenol microneedle array according to claim 1, wherein The metal ion in the metal salt is Fe 3+ , Mn 2+ , Fe 2+ or one or more of them.

4. The preparation method of a metal-loaded polyphenol microneedle array according to claim 1, characterized in that The mixed solution in Step 1 is vortexed for 10 - 20 s, and the deposition time in Step 2 is 10 - 20 min.

5. The micro-needle array loaded with metal polyphenols obtained by the preparation method according to any one of claims 1 to 4, characterized in that, The height of the microneedle is 1 μm, the bottom diameter of the microneedle is 0.8 μm, and the center distance between adjacent microneedles is 1.2 μm.

6. The application of a metal-loaded polyphenol microneedle array as described in claim 5, characterized in that, The microneedle array loaded with metal polyphenol is used as a delivery carrier.

7. Use of a microneedle array loaded with metal polyphenols according to claim 6, characterized in that, The microneedle array loaded with metal polyphenol is used as a macromolecule intracellular delivery carrier.

8. Use of a microneedle array loaded with metal polyphenols according to claim 6, characterized in that, The microneedle array loaded with metal polyphenol is used to deliver ovalbumin, aptamer, small interfering ribonucleic acid, polypeptide, and plasmid.

9. Use of a microneedle array loaded with metal polyphenols according to claim 6, characterized in that, The application of the microneedle array loaded with metal polyphenol in the preparation of drugs.

10. The recycling method of a metal polyphenol-loaded microneedle array according to claim 5, characterized in that, Place the microneedle array loaded with metal polyphenol in a solution with pH = 3 - 5 and soak it for 5 - 10 min; thus, the microneedle array can be recovered.

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