Microneedle delivery system for percutaneous delivery of NGF mRNA drugs

Abstract

The invention provides a microneedle delivery system for percutaneous delivery of an NGF (nerve growth factor) mRNA (messenger ribonucleic acid) drug, which comprises a frozen drug microneedle and a frozen microneedle base or a frozen microneedle handle, the frozen drug microneedle is composed of LNP-NGF mRNA and nuclease-free water in a mass ratio of 1: (80-100), and the frozen drug microneedle is attached to the frozen microneedle base or the frozen microneedle handle, and the sequence of the LNP-NGF mRNA is Sequence No.1. According to the technical scheme, the convenience of an emplastrum and the effectiveness of local precise administration are integrated, the defects of other administration modes are avoided, the quick humoral immune response is achieved after administration, meanwhile, the application is convenient in a diabetic foot animal model, and the wide application prospect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery systems, and particularly relates to a microneedle delivery system for transdermal delivery of NGF mRNA drugs. Background Art

[0002] Diabetes-induced sweating disorder is a disorder of sweat excretion caused by autonomic nerve dysfunction due to diabetes. It is mostly manifested as dry and cool skin on the lower extremities, reduced sweating, or even no sweating, while excessive sweating occurs on the upper body, especially the face and chest. This condition can be manifested as excessive thermoregulatory sweating and excessive mental sweating, etc. The danger of diabetes-induced sweating disorder is that the effect of adrenaline against insulin is weakened, and insulin overdose and hypoglycemia are likely to occur. Modern medicine believes that diabetes-induced sweating disorder belongs to secondary hyperhidrosis or diabetic autonomic neuropathy. Since the secretory function of sweat glands is innervated by small, unmyelinated sympathetic C nerve fibers, when diabetic patients develop diabetic autonomic neuropathy, the function of small fiber nerves is damaged, which in turn affects the sweating function. Therefore, strictly controlling blood sugar levels and treating the damaged nerves are effective methods for treating sweating disorders caused by diabetic autonomic neuropathy.

[0003] Multiple lines of evidence indicate that relevant animal models respond well to exogenous NGF administration. Two Phase II clinical trials by Genentech have shown that administration of recombinant human NGF (rhNGF) can effectively improve symptoms related to diabetic polyneuropathy and HIV-related neuropathy.

[0004] One NGF administration route in the prior art, as disclosed in the invention patent of Patent CN 114933657B published on August 23, 2022, uses lipid microparticles as a delivery carrier to encapsulate mRNA encoding a recombinant protein or an NGF protein variant in the delivery carrier, and the prepared composition is used by injection; this mRNA can be transcribed in vivo into a recombinant protein containing an NGF protein variant and a heterologous signal peptide linked to the NGF protein variant, and can be used to relieve, prevent, and / or treat central and / or peripheral nervous system diseases. However, the administration means of the prior art have not effectively promoted the recovery of sweating dysfunction in diabetic feet. Summary of the Invention

[0005] In view of the above problems, the technical solution of the present invention, in a first aspect, provides a microneedle delivery system for mRNA drugs, including frozen drug microneedles composed of LNP-NGF mRNA and nuclease-free water with a mass ratio of 1:80 - 100, and a frozen microneedle base or a frozen microneedle handle to which the frozen drug microneedles are attached, and the sequence of the LNP-NGF mRNA is Sequence No.1.

[0006] In a second aspect, a method for preparing a microneedle delivery system for an mRNA drug is proposed, which includes synthesizing NGF mRNA through in vitro transcription mediated by T7 RNA polymerase, and encapsulating the synthesized NGF mRNA with LNP to obtain the LNP-NGF mRNA.

[0007] In a third aspect, the application of the above microneedle delivery system for an mRNA drug in the preparation of a drug for diabetic sudomotor dysfunction.

[0008] In a fourth aspect, the application of the above microneedle delivery system for an mRNA drug in a diabetic foot animal model.

[0009] In a fifth aspect, the application of the above microneedle delivery system for an mRNA drug in a mouse paw diabetic foot model.

[0010] The technical solution of the present invention combines the convenience of a plaster and the effectiveness of local precise drug delivery, avoids the deficiencies of other drug delivery methods, has a rapid humoral immune response after drug administration, and is also convenient to use in a diabetic foot animal model, showing broad application prospects. Description of the Drawings

[0011] Figure 1 It is a plasmid map of NGF-EGFP and a schematic diagram of NGF mRNA;

[0012] Figure 2 It is the HE staining result of a mouse paw, where the sweat gland depth of C57BL / 6 mice is 250 - 820 μm;

[0013] Figure 3 It is a schematic diagram of the structure of a frozen microneedle for a mouse footpad and the mold size;

[0014] Figure 4 It is a schematic diagram of the effect of a frozen microneedle transdermal experiment. Detailed Embodiments

[0015] First, a brief description is given of the relevant technical fields involved in the specification. The terms appearing in this specification should be interpreted according to the common knowledge of those skilled in the relevant fields and the general data in the relevant fields.

[0016] NGF (Nerve Growth Factor) is a polypeptide substance mainly present in the cell tissues within the target areas where sympathetic neurons and some sensory neurons are distributed. Its biological activity mainly maintains the functions of sympathetic nerves and sensory nerves. It can induce the synthesis of neurotransmitters, protein phosphorylation, methylation, and the synthesis of enzymes required for gene expression similar to Ras proteins. It can selectively nourish sympathetic ganglion neurons and small fiber sensory neurons in the peripheral nervous system, has nerve growth stimulating activity, and participates in the growth regulation and differentiation of sympathetic neurons and certain sensory neurons. NGF first binds to the specific receptor NGFR on the cell surface. NGFR is a transmembrane protein, and its tyrosine kinase activity is activated after binding to NGF, initiating the downstream signal transduction pathway.

[0017] Microneedle drug delivery is a new type of local transdermal drug delivery technology that combines the convenience of patch and the effectiveness of intradermal injection drug delivery, avoiding the deficiencies of other drug delivery methods and having multiple advantages such as not touching the nerves, being safe, painless, and highly permeable.

[0018] LNP (Lipid Nanoparticle): The oligonucleotides encapsulated in lipid nanoparticles are protected from enzymatic degradation during delivery and are effectively delivered into cells. In the cells, the contents in the carrier particles are released and translated into therapeutic proteins. The lipid molar ratio determines the lipid composition of the particles and affects their size, polydispersity, and efficacy.

[0019] The following examples further illustrate the content of the present invention, but should not be construed as a limitation to the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention belongs to the scope of the present invention.

[0020] Sources of reagents, culture media, kits, instruments, or consumables, etc.:

[0021] Nuclease-free water (RNA free H2O) comes from: Beijing Coolaber Technology Co., Ltd., PBS buffer comes from: Beijing Coolaber Technology Co., Ltd.,

[0022] The source of the frozen microneedle handle is: self-made,

[0023] T7 RNA polymerase comes from: Beijing Tsingke Biotechnology Co., Ltd.,

[0024] The microfluidic chip comes from: Beijing Tsingke Biotechnology Co., Ltd.,

[0025] Preparation steps of LNPs (Prepare LNP encapsulation):

[0026] Under RNase-free working conditions, dissolve each lipid in absolute ethanol. After mixing in proportion, form a uniform lipid film by rotary evaporation at 60 °C and vacuum dry for 24 hours to remove trace solvents. Dissolve mRNA in citrate buffer (pH 4.0, 50 mM) and adjust the final concentration to 1 mg / mL. Pump the lipid ethanol solution and the mRNA buffer into a microfluidic chip respectively, and control the flow rate ratio (lipid phase: aqueous phase) to be 3:1 to mix the aqueous and organic phases. Dialyze the LNPs in a storage buffer (PBS, pH 7.4) using an appropriate molecular weight cut-off (MWCO) tube to remove unencapsulated cargo, excess lipid components, and the organic solvent - ethanol. After sterile filtration through a 0.22 μm filter, store in PBS buffer to ensure the stability and activity of LNP-mRNA.

[0027] The base sequences in the following examples are shown in the table below:

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[0039] Example 1

[0040] The microneedle delivery system of the mRNA drug of this embodiment includes a frozen drug microneedle composed of LNP-NGF mRNA (taken from Example 3) and nuclease-free water in a mass ratio of 1:80-100, and a frozen microneedle base or a frozen microneedle handle to which the frozen drug microneedle is attached, and the sequence of the LNP-NGF mRNA is Sequence No.1. The polydimethylsiloxane (PDMS) mold is preferably circular with a diameter of 24 mm, in order to match the 50 ml centrifuge tube for convenient centrifugation so that the liquid can fill the needle tip after centrifugation. After centrifugation, the handle is dipped in enzyme-free water and inserted into the groove, and is frozen at -80°C for 15 minutes and then taken out for use. The needle length of the frozen drug microneedle is 550-650μm, the tip is located within a length range of 10-15μm from its top to its base, the base diameter is 280-320μm, and the frozen drug microneedle is on the attachment surface of the frozen microneedle base or the frozen microneedle handle at a density of 2-3 particles / mm 2 Array arrangement.

[0041] Example 2

[0042] The difference from Example 1 is that this example includes frozen drug microneedles composed of LNP-NGF mRNA (taken from Example 3) and nuclease-free water in a mass ratio of 1:100.

[0043] As shown in Table 1, the needle length of the frozen drug microneedle of Example 2 is 600 μm, the tip is located within a length range of 10-15 μm from the top to the base, the base diameter is 300 μm, and the frozen drug microneedle is arranged in a 10×7 array on the attachment surface of the frozen microneedle handle with a size of 6 mm×4 mm;

[0044] Table 1. Size and specification data of frozen drug microneedles

[0045] Unit / μm Unit / μm Unit / μm Unit / piece <![CDATA[Unit / mm 2 > Unit / mm Unit / μm Needle length Bottom diameter Tip distance Quantity array Microneedle area Groove depth Tip 600 300 600 10*7 6*4 1.5 10-15

[0046] Example 3 Preparation of LNP-NGF mRNA

[0047] In this embodiment, NGF mRNA is synthesized by in vitro transcription mediated by T7 RNA polymerase, and the synthesized NGF mRNA is encapsulated in LNP prepared by the above-mentioned LNP preparation work to obtain the LNP-NGF mRNA. Wherein, the LNP formula is D-Lin-MC3-DMA, DSPC, cholesterol and PEG2000-C-DMG, and the lipid molar ratio is 50:10:38.5:1.5. The specific steps of synthesizing NGF mRNA by in vitro transcription mediated by T7 RNA polymerase include:

[0048] Pseudo-UTP modification,

[0049] EGFP codon optimization,

[0050] 3' end poly (A) tailing, co-transcriptional 5' end capping and EGFP tagging.

[0051] The mRNA obtained in this example is encapsulated by LNP and delivered into cells, and can stably express the NGF encoding protein.

[0052] The NGF-EGFP plasmid map and NGF mRNA schematic diagram in the above table are as follows Figure 1 .

[0053] Example 4

[0054] Take the LNP-NGF mRNA obtained in Example 3, and then perform the following steps under sterile conditions to obtain the finished product of the microneedle delivery system of mRNA drugs:

[0055] S1: thoroughly mix the LNP-NGF mRNA and nuclease-free water at a mass ratio of 1:100;

[0056] S2: Take 100 μl of the mixture and place it in the cylindrical PDMS microneedle negative mold until it covers the groove at the bottom of the mold. Centrifuge at 4000 rpm for 3 minutes at 4°C to ensure that all the pinhole cavities are completely filled.

[0057] S3 Then, the frozen microneedle handle is wetted with nuclease-free water and inserted into the groove of the microneedle mold. After freezing at -80°C for 15 minutes, the mold is demolded to obtain the finished product of the microneedle delivery system for mRNA drugs, which is kept at -80°C for a long time.

[0058] Example 5

[0059] The diabetic foot model of mouse paw pad was constructed. The experimental animals were 14-week-old BKS.Cg-Leprdb, db / db) diabetic mice and control mice (BKS-Lepr, db / m) from Beijing Weishanglide Biotechnology Co., Ltd. They were fed with radiation-sterilized feed and purified water freely in an SPF-grade experimental environment and fed with high-fat and high-protein feed for 4 weeks to 18 weeks of age. The results of HE staining of mouse paw pads are shown in the figure. Figure 2 , the sweat gland depth of C57BL / 6 mice is 250-820μm.

[0060] Example 6

[0061] A microneedle delivery system for mRNA drugs was designed based on the diabetic foot model of mouse paw pads. Schematic diagram of the microneedle design of the mouse paw pad and the PDMS frozen microneedle handle are shown in Figure 1. Figure 3 .

[0062] Experimental Example 1: Frozen microneedle transdermal experiment

[0063] Using the mouse paw pad diabetic foot model of Example 5 and the microneedle delivery system of the mRNA drug based on this model, a cryogenic microneedle transdermal experiment was carried out. The specific experimental steps are as follows:

[0064] Since db / db mice can mimic the characteristics of human diabetic neuropathy (DN) (including progressive loss of sensory function and electrophysiological disorders), and the density of nerve fibers in the skin of 18-week-old db / db mice is reduced, which is closely related to sensory loss and is considered to be the early stage of DN. Through the acetylcholine-induced sweating experiment on 18-week-old db / db mice, diabetic mice with sweating dysfunction were screened as the diabetic foot model. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital at an anesthetic dose of 50 mg / kg. The cryogenic microneedles stored at -80 °C were soaked in liquid nitrogen for 10 seconds before application to ensure the high strength of the cryogenic microneedles, and then quickly taken out and inserted into the mouse paw pads.

[0065] To evaluate the transdermal property of the microneedles, first shave the hair on the back skin of the mice, and use depilatory cream to remove the exposed mouse hair. The cryogenic microneedles were inserted into the back skin of the mice, and then marked and photographed. After recording, the mice were euthanized, and the back skin at the microneedle application site was separated for H&E staining.

[0066] See Figure 4 , the test results confirmed that the microneedles can penetrate the back skin of the mice, and the microneedle channels can quickly close.

[0067] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A microneedle delivery system for percutaneous delivery of NGF mRNA drugs, characterized in that, It includes frozen drug microneedles composed of LNP-NGF mRNA and nuclease-free water with a mass ratio of 1:80 - 100, and a frozen microneedle base or a frozen microneedle handle to which the frozen drug microneedles are attached. The sequence of the LNP-NGF mRNA is Sequence No.

1.

2. The microneedle delivery system for transdermal delivery of NGF mRNA drugs according to claim 1, wherein It includes frozen drug microneedles composed of LNP-NGF mRNA and nuclease-free water with a mass ratio of 1:

100.

3. The microneedle delivery system for transdermal delivery of NGF mRNA drug according to claim 1, wherein The needle length of the frozen drug microneedle is 550-650 μm, the tip is located within the length range of 10-15 μm from its top to its base, the base diameter is 280-320 μm, and the frozen drug microneedles are arranged in an array on the attachment surface of the frozen microneedle base or the frozen microneedle handle at a density of 2-3 needles / mm 2 2.

4. The microneedle delivery system for percutaneous delivery of NGF mRNA drug according to claim 1, wherein The needle length of the frozen drug microneedles is 600 μm, the tip is within the length range of 10 - 15 μm from its top to its base, the base diameter is 300 μm, and the frozen drug microneedles are arranged in a 10×7 array on the attachment surface of a frozen microneedle handle with a size of 6 mm×4 mm.

5. The preparation method of the microneedle delivery system for the percutaneous delivery of NGF mRNA drug according to any one of claims 1-4, characterized in that, The preparation method includes synthesizing NGF mRNA by in vitro transcription mediated by T7 RNA polymerase, and encapsulating the synthesized NGF mRNA with LNP to obtain the LNP-NGF mRNA.

6. The preparation method of the microneedle delivery system for transdermal delivery of NGF mRNA drug according to claim 4, wherein, The specific steps of synthesizing NGF mRNA by in vitro transcription mediated by T7 RNA polymerase specifically include the following steps: Pseudo-UTP modification, EGFP codon optimization, 3'-end Poly(A) tailing, co-transcriptional 5'-end capping, and EGFP tagging.

7. The preparation method of the microneedle delivery system for the mRNA drug according to claim 5, wherein, It also includes the following steps completed under sterile conditions: fully mixing the LNP-NGF mRNA and nuclease-free water with a mass ratio of 1:100, taking 100 μl of the mixture into a cylindrical PDMS microneedle female mold until the bottom groove of the mold is covered, and centrifuging at 4000 rpm for 3 minutes at 4°C to ensure that all the needle hole cavities are completely filled; subsequently, wetting the frozen microneedle handle with nuclease-free water and inserting it into the groove of the microneedle mold, freezing for 15 minutes at -80°C and then demolding, and storing it at -80°C for a long time.

8. Application of the microneedle delivery system of any one of the mRNA drugs described in claims 1 - 7 in the preparation of drugs for diabetic sudomotor dysfunction.

9. Application of the microneedle delivery system of any one of the mRNA drugs described in claims 1 - 7 in a diabetic foot animal model.

10. Application of the microneedle delivery system of any one of the mRNA drugs described in claims 1 - 7 in a mouse paw pad diabetic foot model.

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