A nano-drug of nerve growth factor antisense oligonucleotide and a preparation method and application thereof

Abstract

This invention belongs to the field of nanomedicine carriers, specifically relating to a nanomedicine carrying nerve growth factor antisense oligonucleotides, its preparation method, and its applications. This invention also relates to using cationic lipids and cofactor lipids as phospholipid materials to carry nerve growth factor antisense oligonucleotides to construct a cationic liposome delivery system. In the delivery system of this invention, the cationic liposomes bind to the highly negatively charged antisense oligonucleotides, which can better achieve the carrying capacity of the antisense oligonucleotides, helping to increase the uptake of antisense oligonucleotide drugs, and can be applied to the treatment of diseases such as inflammation and nerve damage.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine carriers, and more specifically, to the optimized construction and application of a stable, low-toxicity, and highly efficient cationic liposome delivery system carrying antisense oligonucleotides. This invention is particularly aimed at delivering antisense oligonucleotides of nerve growth factor to the corresponding sites via cationic liposomes, thereby downregulating the level of nerve growth factor and improving and treating related diseases. Background Technology

[0002] In recent years, nanomedicine has achieved significant breakthroughs, demonstrating great potential in the treatment of various diseases. Nucleic acid drugs, especially antisense oligonucleotides, have been widely studied and applied to intervene in the expression of specific genes. Antisense oligonucleotides, through complementary pairing with target mRNA sequences, can inhibit the translation of that mRNA, thereby reducing or preventing the synthesis of target proteins. Their design is relatively simple and they possess high specificity, making them an important method for disease treatment. Nerve growth factor (NGF) is a neurotrophic factor that plays a crucial role in the development and function of the nervous system by regulating the survival, proliferation, and differentiation of nerve cells, as well as the formation and maintenance of neural synapses. In the treatment of NGF-related diseases, there is still a need to develop novel drugs that deliver NGF antisense oligonucleotides to effectively reduce NGF expression and alleviate pain and other complications. However, antisense oligonucleotides themselves are difficult to permeate cell membranes, are easily degraded by nucleases in vivo, and have low stability. Therefore, the delivery technology of antisense oligonucleotides has always been a key issue in their application.

[0003] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, this invention utilizes cationic lipids and auxiliary lipids as phospholipid materials and employs a thin-film dispersion method to prepare cationic liposomes as carriers to transport antisense oligonucleotide drugs, constructing an antisense oligonucleotide-loaded liposome delivery system. This delivery system exhibits good uniformity and dispersibility, can bind to highly negatively charged antisense oligonucleotides, and achieves better antisense oligonucleotide loading. Its positively charged surface facilitates internalization into negatively charged cells, thereby exerting a therapeutic effect. The nerve growth factor-loaded antisense oligonucleotide liposomes prepared by this invention are highly efficient, stable, have low toxicity and side effects, and exhibit high delivery efficiency, significantly improving the effective drug uptake rate and greatly alleviating bladder dysfunction in rats. Specifically, this invention includes the following:

[0005] In a first aspect, the present invention provides a method for preparing a nanomedicine, the nanomedicine comprising a nerve growth factor antisense oligonucleotide and a liposome for delivering the antisense oligonucleotide, the preparation method comprising:

[0006] (1) Dissolve cationic lipids and auxiliary lipids in an organic solvent, mix the two solutions evenly, and then vacuum rotary evaporate until the lipid material forms a lipid film;

[0007] (2) Add the hydration medium to the lipid membrane in step (1) and perform hydration treatment at a hydration temperature of 40-70℃ to obtain a liposome suspension. Extrude the liposome suspension through a membrane method to obtain cationic liposomes.

[0008] (3) The cationic liposomes are added dropwise to the antisense oligonucleotide solution and incubated at room temperature to obtain the nanomedicine. The amount of cationic liposomes and antisense oligonucleotides added is controlled so that the nitrogen-to-phosphorus ratio of the nanomedicine is 1-10.

[0009] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the cationic lipid material is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), bis(octadecyl)dimethylammonium bromide (DDAB), trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTMA), and 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP).

[0010] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the auxiliary lipid material is selected from at least one of dioleoylphosphatidylethanolamine (DOPE), L-α-phosphatidylcholine (PC), sodium 1,2-dipalmitoyl-3-deoxycholate (DOPC), sodium 1,2-palmitoylphosphatidylglycerol (DPPG), and cholesterol.

[0011] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the liposomes have a particle size of 50-500 nm.

[0012] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the zeta potential of the liposomes is between +30 and +60 mV.

[0013] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the PDI value of the liposomes is 0.05-0.5.

[0014] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the hydration medium is selected from at least one of phosphate buffer, Tris-HCl, 5% glucose hydration solution, and deionized water.

[0015] In some embodiments, according to the method for preparing nanomedicines according to the present invention, the hydration time is 0.5-5 h.

[0016] A second aspect of the present invention provides a nanomedicine obtained by the preparation method described in the present invention.

[0017] A third aspect of the present invention provides the application of the nanomedicine described herein in the preparation of medicaments for nerve growth factor-related diseases.

[0018] This invention utilizes cationic lipids and auxiliary lipids to develop a novel nanomedicine that indirectly blocks the translation of nerve growth factor mRNA through antisense oligonucleotides, inhibiting the overexpression of nerve growth factor and thus improving and treating related diseases. The in vivo and in vitro effects are investigated using the treatment of interstitial cystitis as an example.

[0019] This invention offers the following advantages and effects: By employing the above-described scheme, a liposome delivery system carrying nerve growth factor antisense oligonucleotides can be obtained. This delivery system boasts advantages such as high efficiency, stability, low toxicity, and high delivery efficiency. The nerve growth factor antisense oligonucleotide liposome nanomedicine provided by this invention, when applied to the treatment of interstitial cystitis, improves drug adhesion and permeability at the mucosal site, enhances the effective uptake of antisense oligonucleotide drugs by cells, downregulates the overexpression of nerve growth factor, thereby inhibiting the expression of inflammation-related factors, significantly improving bladder function, and correspondingly improving urodynamic parameters, thus alleviating bladder damage and demonstrating a significant therapeutic effect. It also possesses advantages such as high stability, high biosafety, and efficient delivery. This invention provides more scientific basis for the future development of such nanomedicines and also provides basic data and references for the treatment of interstitial cystitis / bladder pain syndrome. Attached Figure Description

[0020] Figure 1 The graph shows the effect of hydration temperature on particle size and PDI of the cationic liposomes prepared in Examples 1-4 during the preparation process.

[0021] Figure 2 The graph shows the effect of the hydration medium on the particle size during the preparation of the cationic liposomes prepared in Examples 12-15.

[0022] Figure 3These are TEM images showing the effects of hydration media on the cationic liposomes prepared in Examples 12-15 during the preparation process: a, PBS; b, Tris-HCl; c, 5% Glucose; d, TEM image of liposomes in deionized water.

[0023] Figure 4 The graph shows the stability test results of the cationic liposomes prepared in Examples 12-15, with particle size and potential measured at 4°C over 25 days.

[0024] Figure 5 The graph shows the effect of different cationic lipid selections on particle size and PDI during the preparation of cationic liposomes prepared in Examples 16-19.

[0025] Figure 6 The graph shows the effect of different cationic lipid selections on the potential during the preparation of the cationic liposomes prepared in Examples 16-19.

[0026] Figure 7 The graph shows the effect of different nitrogen-to-phosphorus ratios on particle size and potential of the nerve growth factor-loaded antisense oligonucleotide liposomes prepared in Examples 23-27.

[0027] Figure 8 The graph shows the effect of different nitrogen-to-phosphorus ratios on the encapsulation efficiency of the nerve growth factor-loaded antisense oligonucleotide liposomes prepared in Examples 23-27.

[0028] Figure 9 The graph shows the effect of different nitrogen-to-phosphorus ratios on the drug loading rate of the nerve growth factor-loaded antisense oligonucleotide liposomes prepared in Examples 23-27.

[0029] Figure 10 The graph shows the effect of different nitrogen-to-phosphorus ratios on the uptake of nerve growth factor antisense oligonucleotides by the bladder urothelium of the liposomes prepared in Examples 23-27.

[0030] Figure 11 The graph shows the effect of different cationic lipid selections on the encapsulation efficiency during the preparation of the cationic liposomes prepared in Examples 23-27.

[0031] Figure 12 The graph shows the effect of different cationic lipid selections on drug loading during the preparation of the cationic liposomes prepared in Examples 23-27.

[0032] Figure 13 This is an agarose gel electrophoresis image showing the effect of different nitrogen-to-phosphorus ratios on the binding degree of cationic liposomes to NGF asODN of the nerve growth factor-carrying antisense oligonucleotide liposomes prepared in Examples 23-27.

[0033] Figure 14 This is a graph showing the toxicity analysis of SV-HUC-1 cells by the nerve growth factor-loaded antisense oligonucleotide liposomes prepared in Example 35.

[0034] Figure 15 This is a graph showing the toxicity analysis of different nerve growth factor-carrying antisense oligonucleotide liposomes prepared in Example 36 on SV-HUC-1 cells.

[0035] Figure 16 This is a qualitative analysis result of the uptake of nerve growth factor antisense oligonucleotides by the bladder urothelium using two-photon microscopy in Example 37. A is the control group; B is the nerve growth factor antisense oligonucleotide group; and C is the liposome group carrying nerve growth factor antisense oligonucleotides.

[0036] Figure 17 This is a graph showing the quantitative analysis results of nerve growth factor antisense oligonucleotide uptake by the bladder urothelium using two-photon microscopy in Example 37. A is the control group, B is the nerve growth factor antisense oligonucleotide group, and C is the liposome group carrying nerve growth factor antisense oligonucleotides.

[0037] Figure 18 The graph shows the effects of perfusion therapy on urodynamics in rats with cystitis in Example 38, including the control group, saline group, nerve growth factor antisense oligonucleotide group, cationic liposome group, and nerve growth factor antisense oligonucleotide liposome group. A is a comparison of MCC results among the groups, B is a comparison of ICI results among the groups, C is a comparison of Pdet results among the groups, and D is a comparison of BC results among the groups.

[0038] Figure 19 This is a graph showing the effect of nanomedicine on inflammatory factors in bladder epithelial tissue in Example 39. The left side is the saline group, and the right side is the liposome group carrying nerve growth factor antisense oligonucleotides.

[0039] Figure 20 This is a graph showing the effects of the control group, saline group, nerve growth factor antisense oligonucleotide group, cationic liposome group, and nerve growth factor antisense oligonucleotide liposome group on the expression levels of inflammatory factors in bladder epithelial tissue in Example 40. A, Nerve growth factor; B, PACAP; C, TGF-β; D, Piezo2; E, CCL-2; F, IL-6; G, IL-1β; H, IL-17; I, TNF-α; J, CCL3. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] The term "zeta potential" used in this invention refers to the sectional potential, an indicator used to measure the stability of colloidal dispersions. The zeta potential of the liposomes described in this invention is +30 to +60, indicating that the liposomes of this invention possess good stability.

[0044] The term "PDI value" used in this invention refers to the polymer dispersion index, an indicator used to describe the molecular weight distribution of polymers. The PDI value of the liposomes described in this invention is 0.05-0.5, indicating that the molecular weight distribution of the liposome polymers in this invention is concentrated and the liposomes are uniformly distributed throughout.

[0045] The term “MCC” used in this invention refers to maximum bladder capacity, which means the maximum amount of urine that the bladder can hold.

[0046] The term "ICI" as used in this invention refers to the intercontraction interval, which is the time interval between two bladder contractions.

[0047] The term "Pdet" used in this invention refers to the maximum detrusor pressure, which represents the maximum pressure value reached by the detrusor muscle (bladder excretory muscle) during urination.

[0048] The term "BD" used in this invention refers to bladder compliance, which represents the relationship between changes in bladder capacity and changes in detrusor pressure.

[0049] Nanomedicine

[0050] In one aspect, the present invention provides a nanomedicine comprising a nerve growth factor antisense oligonucleotide and a liposome for delivering the antisense oligonucleotide, wherein the positively charged liposome and the negatively charged antisense oligonucleotide are bound together by electrostatic adsorption. The liposome for delivering the antisense oligonucleotide is spherical, regularly shaped, and uniform in size, and the liposome comprises cationic lipids and auxiliary lipids.

[0051] In this invention, nanocarriers can effectively carry nucleic acid drugs and help them reach lesions, thus promoting their efficacy in vivo. Liposomes, by encapsulating hydrophilic or hydrophobic drugs, reduce systemic side effects and significantly improve therapeutic efficacy. Furthermore, liposomes can enhance drug permeability through biological membranes by altering membrane transport mechanisms, thereby improving drug efficacy and enhancing drug stability, protecting the drug from degradation.

[0052] In this invention, cationic liposomes possess the following advantages: good biocompatibility, good stability, and extended cycle time. Furthermore, their positive surface charge allows for the adsorption of negatively charged antisense oligonucleotide drugs onto the liposomes and also enables them to bind to the cell membrane through electrostatic interactions, facilitating internalization into cells. These advantages make cationic liposomes highly promising for the delivery of antisense oligonucleotide drugs.

[0053] As a preferred embodiment of the present invention, the cationic lipid is selected from trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), bis(octadecyl)dimethylammonium bromide (DDAB), trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTMA), and 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), with DOTAP being the most preferred.

[0054] As a preferred embodiment of the present invention, the auxiliary lipid material is selected from dioleoylphosphatidylethanolamine (DOPE), L-α-phosphatidylcholine (PC), sodium 1,2-dipalmitoyl-3-deoxycholate (DOPC), sodium 1,2-palmitoylphosphatidylglycerol (DPPG), and cholesterol, preferably DOPE.

[0055] Preferably, the particle size of the nerve growth factor-carrying antisense oligonucleotide liposome is 50-500 nm, for example, it can be 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0056] When the particle size of the antisense oligonucleotide liposomes carrying nerve growth factor (NGF) is within the aforementioned range, the liposomes readily adhere to the bladder mucosa, enhancing the effective uptake of the antisense oligonucleotide drug by the bladder urothelial cells and thus fully leveraging the NGF-inhibiting effect of the antisense oligonucleotide. When the particle size of the antisense oligonucleotide drug is too small (e.g., less than 50 nm), these drug particles may be too small to be effectively recognized by bladder urothelial cells, failing to trigger these cellular uptake mechanisms and resulting in ineffective drug delivery into the cells, thus failing to inhibit NGF expression. When the particle size of the antisense oligonucleotide drug is too large (e.g., greater than 500 nm), these larger particles may be too large to penetrate the bladder urothelial barrier or be taken up by cells through endocytosis.

[0057] Preferably, the dispersion coefficient of the nerve growth factor-carrying antisense oligonucleotide liposomes is in the range of 0.05-0.5, for example, it can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0058] When the dispersion coefficient of the nerve growth factor-loaded antisense oligonucleotide liposomes is within the aforementioned range, the delivery system exhibits better morphology and greater stability, which is beneficial for promoting bladder uptake of antisense oligonucleotides, thereby resulting in better therapeutic effects. However, when the dispersion coefficient is too large (e.g., greater than 0.5), the size distribution of the drug particles is highly uneven. This unevenness leads to uneven distribution of the drug in the bladder urothelium, affecting bioavailability and efficacy. When the dispersion coefficient is too small (e.g., less than 0.05), the preparation difficulty and cost increase. Furthermore, an overly uniform particle size distribution causes liposome aggregation or precipitation, which is detrimental to the loading of antisense oligonucleotides, further affecting the therapeutic effect.

[0059] Preferably, the potential of the nerve growth factor-carrying antisense oligonucleotide liposome is +30 to +60 mV, for example, +30 mV, +35 mV, +40 mV, +45 mV, +50 mV, +55 mV, +60 mV, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0060] When the potential of NGF-loaded antisense oligonucleotide liposomes is at the above-mentioned values, they can better bind to negatively charged cell membranes through electrostatic interactions, thereby being internalized. When the potential of NGF-loaded antisense oligonucleotide liposomes is too low (e.g., less than 30 mV), the electrostatic interaction between the liposomes and the negatively charged bladder urothelial cell membrane is weakened, and the liposomes may not be able to bind to the cell membrane effectively, thus reducing the chance of internalization and affecting the uptake efficiency of antisense oligonucleotide drugs, which may lead to ineffective inhibition of NGF expression. When the potential of NGF-loaded antisense oligonucleotide liposomes is too high (e.g., greater than 60 mV), the excessively high potential may reduce the stability of the liposomes or cause excessively strong interactions with the cell membrane, resulting in cell membrane damage and affecting drug delivery efficiency.

[0061] Preparation method

[0062] Secondly, the present invention provides a method for preparing nanomedicines, the method comprising the following steps:

[0063] (1) Dissolve cationic lipids and auxiliary lipids in an organic solvent, mix the two solutions evenly, and then vacuum rotary evaporate until the lipid material forms a lipid film;

[0064] (2) Add the hydration medium to the lipid membrane in step (1) and perform hydration treatment at a hydration temperature of 40-70℃ to obtain a liposome suspension. Extrude the liposome suspension through a membrane method to obtain cationic liposomes.

[0065] (3) The cationic liposomes are added dropwise to the antisense oligonucleotide solution and incubated at room temperature to obtain the nanomedicine. The amount of cationic liposomes and antisense oligonucleotides added is controlled so that the nitrogen-to-phosphorus ratio of the nanomedicine is 1-10.

[0066] In a preferred embodiment, the preparation method of the present invention includes:

[0067] (1) Dissolve cationic lipids and auxiliary lipids in an organic solvent, mix the two solutions evenly, and then vacuum rotary evaporate until the lipid material forms a lipid film;

[0068] (2) Add hydration medium to (1) and hydrate at a hydration temperature of 40-70℃ using a rotary evaporator for 0.5-5 hours. Sonicate until the liposome suspension is clear and transparent.

[0069] (3) The liposome suspension in (2) is squeezed through a carbonate membrane with a pore size of 15 nm-8 μm to prepare cationic liposomes.

[0070] (4) The cationic liposomes obtained in (3) are added dropwise to the antisense oligonucleotide solution and incubated at room temperature to obtain the nanomedicine.

[0071] As a preferred technical solution of the present invention, the molar ratio of cationic lipid to auxiliary lipid in step (1) is 10:1 to 1:10, for example, it can be 10:1, 5:1, 1:1, 1:5, 1:10, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0072] Preferably, the concentration of the cationic lipid is 10-200 mg / mL, for example, it can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, etc., but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0073] Preferably, the concentration of the auxiliary lipid is 10-200 mg / mL, for example, it can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, etc., but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0074] Preferably, the organic solvent is selected from chloroform, dichloromethane, and acetone, and the volume of chloroform is 1-5 mL, for example, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0075] As a preferred technical solution of the present invention, the hydration medium in step (2) is preferably DEPC water with a volume of 1 mL.

[0076] Preferably, the hydration temperature is 40-70℃, for example, it can be 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0077] Preferably, the hydration time is 0.5-5h, for example, it can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0078] Preferably, the rotary evaporation rate is 100-200 rpm, for example, it can be 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0079] Preferably, the ultrasonic power is 200-600W, for example, it can be 200W, 300W, 400W, 500W, 600W, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0080] Preferably, the ultrasound time is 10-50 min, for example, it can be 10 min, 20 min, 30 min, 40 min, 50 min, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0081] As a preferred technical solution of the present invention, the pore size of the polycarbonate membrane in step (3) is selected from 15nm, 30nm, 50nm, 100nm, 200nm, 400nm, 800nm, 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 8μm, etc., and is preferably a 200nm polycarbonate membrane.

[0082] As a preferred embodiment of the present invention, the amount of antisense oligonucleotide in step (4) is determined according to the different nitrogen-to-phosphorus ratios of the nerve growth factor antisense oligonucleotide liposomes. The nitrogen-to-phosphorus ratio is 1-10, for example, it can be 1, 2, 4, 8, 10, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable. In a preferred embodiment, the amount of cationic liposomes and antisense oligonucleotides added is controlled so that the nitrogen-to-phosphorus ratio of the nanomedicine is 4-8. The nitrogen-to-phosphorus ratio within the above range has a significant impact on cell viability and cell membrane fusion or transmembrane ability, while significantly reducing toxicity and improving the therapeutic effect of the nerve growth factor antisense oligonucleotide drug.

[0083] As a preferred embodiment of the present invention, the ratio (mass ratio) of cationic liposomes to antisense oligonucleotides can be varied within the range of 1:1 to 10:1. Preferably, to significantly enhance the stability of the drug delivery system, while improving the encapsulation efficiency, drug loading rate, and biocompatibility of the drug delivery system, and strengthening the effective uptake of antisense oligonucleotide drugs by the bladder urothelium, the ratio of cationic liposomes to antisense oligonucleotides is preferably 4:1 to 9:1, more preferably 4:1 to 8.5:1, and even more preferably 4.2:1 to 8.5:1 (so that the nitrogen-to-phosphorus ratio of the nanomedicine is 4-8), for example 4.2:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, and 8.5:1. Preferably, the room temperature incubation time is 0.5-2 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0084] The antisense oligonucleotide liposomes carrying nerve growth factor described in this invention are prepared using a thin-film dispersion method. This method is highly adaptable, simple to operate, and produces cationic liposomes with high stability. Furthermore, by optimizing the process conditions and the nitrogen-to-phosphorus ratio, the stability of the drug delivery system can be further enhanced, while also improving the encapsulation and drug loading rates, improving biocompatibility, and strengthening the effective uptake of antisense oligonucleotide drugs by the bladder urothelium.

[0085] Thirdly, the present invention provides a nanomedicine, wherein the nanomedicine is a liposome containing the aforementioned nerve growth factor antisense oligonucleotide, wherein the cationic liposome carries the nerve growth factor antisense oligonucleotide, for treating related diseases, such as interstitial cystitis / bladder pain syndrome, neurological diseases, rheumatoid arthritis, chronic pain syndrome, etc.

[0086] Preferably, the particle size of the nanomedicine is 80-250 nm, for example, it can be 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0087] Preferably, the dispersion coefficient of the nanomedicine is in the range of 0.1-0.5, for example, it can be 0.1, 0.2, 0.3, 0.4, 0.5, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0088] Preferably, the potential of the nanomedicine is +30mV to +60mV, for example, it can be +30mV, +40mV, +50mV, +60mV, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0089] All reagent kit materials used in this invention are commercially available; for devices, conditions (temperature, time, etc.), substances, dosages, methods, etc. not specifically described in this invention, those known in the art or those who are skilled in the art can determine them using conventional techniques.

[0090] Each liposome was prepared according to the following method:

[0091] Cationic liposomes were prepared using a thin-film dispersion method. Each selected cationic lipid and each auxiliary lipid were mixed in a 1:1 molar ratio. 69.8 μL of the cationic lipid solution and 74.4 μL of the auxiliary lipid solution were taken from a 50 mg / mL lipid solution and placed in a 50 mL round-bottom flask. 1-2 mL of solvent was added to completely dissolve the lipids, and the mixture was vacuum-evaporated in a 40 °C water bath for 30 min until a lipid film formed on the inner wall of the flask. The flask was then placed in a 40 °C vacuum drying oven and vacuum-dried for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flask, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. The state of the liposomes was observed using a TEM transmission electron microscope, and their size and distribution were measured using a particle size analyzer. To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

[0092] Example 1: Preparation of cationic liposomes

[0093] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature is 50℃, the prepared liposomes are spherical, with a smooth and uniform surface distribution, a particle size of 188.5±1.4nm, and a PDI of 0.42±0.12. Figure 1 ).

[0094] Example 2 Preparation of cationic liposomes

[0095] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature was 55℃, the prepared liposomes were spherical, with a smooth and uniform surface distribution, a particle size of 172.7±3.6nm, and a PDI of 0.29±0.04. Figure 1 ).

[0096] Example 3: Preparation of cationic liposomes

[0097] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature was 60℃, the prepared liposomes were spherical, with a smooth and uniform surface distribution, a particle size of 159.1±2.6nm, and a PDI of 0.27±0.09. Figure 1 ).

[0098] Example 4: Preparation of cationic liposomes

[0099] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature was 65℃, the prepared liposomes were spherical, with smooth and uniform surface distribution, a particle size of 146.2±1.2nm, and a PDI of 0.25±0.03. Figure 1 ).

[0100] Example 5: Preparation of cationic liposomes

[0101] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DDAB and PC, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of DDAB and PC solutions (50 mg / mL) were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of acetone was added to completely dissolve the lipids, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nanoextruder to prepare single-chamber cationic liposomes. When the hydration temperature is 55℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 166.5±1.8nm, and a PDI of 0.52±0.07.

[0102] Example 6 Preparation of cationic liposomes

[0103] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DOTMA and DOPC, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DOTMA and DOPC solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of chloroform was added to completely dissolve the lipid materials, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 1 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals with 1 s intervals, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nanoextruder to prepare single-chamber cationic liposomes. When the hydration temperature is 40℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 177.6±1.9nm, and a PDI of 0.46±0.06.

[0104] Example 7 Preparation of cationic liposomes

[0105] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DODAP and DPPG, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DODAP and DPPG solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of dichloromethane was added to completely dissolve the lipid materials, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 2 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals with 1 s intervals, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature is 70℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 163.6±1.4nm, and a PDI of 0.52±0.05.

[0106] Example 8: Preparation of cationic liposomes

[0107] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP to cholesterol was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and cholesterol solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of dichloromethane was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 2 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature is 45℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 189.6±2.4nm, and a PDI of 0.50±0.05.

[0108] Example 9: Preparation of cationic liposomes

[0109] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DPPG and DDAB, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DPPG and DDAB solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of dichloromethane was added to completely dissolve the lipid materials, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 2 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nanoextruder to prepare single-chamber cationic liposomes. When the hydration temperature is 65℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 208.3±2.2nm, and a PDI of 0.43±0.06.

[0110] Example 10 Preparation of cationic liposomes

[0111] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP to cholesterol was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and cholesterol solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of dichloromethane was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 2 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature is 70℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 196.6±2.6nm, and a PDI of 0.46±0.07.

[0112] Example 11 Preparation of cationic liposomes

[0113] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DOTMA and DPPG, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DOTMA and DPPG solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of dichloromethane was added to completely dissolve the lipid materials, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried under vacuum for 1 h to remove organic solvents. 1 mL of hydration medium was added to the flasks, and hydration was carried out by rotary hydration for 2.5 h, depending on the hydration temperature. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic disruptor at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solutions were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration temperature is 65℃, the prepared liposomes are spherical, with smooth and uniform surface distribution, a particle size of 187.6±2.1nm, and a PDI of 0.41±0.11.

[0114] Example 12 Preparation of cationic liposomes

[0115] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 60 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was PBS, the prepared liposomes had a particle size of 405.6 ± 1.5 nm. Figure 2 The particles are spherical and evenly distributed. Figure 3 a).

[0116] Example 13 Preparation of cationic liposomes

[0117] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 60 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was Tris-HCl, the prepared liposomes had a particle size of 143.8 ± 2.1 nm. Figure 2 The particles are spherical and evenly distributed. Figure 3 b).

[0118] Example 14 Preparation of cationic liposomes

[0119] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 60 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was 5% glucose hydration solution, the prepared liposomes had a particle size of 125.9 ± 0.9 nm. Figure 2 The particles are spherical and evenly distributed. Figure 3 c).

[0120] Example 15 Preparation of cationic liposomes

[0121] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DOTAP and DOPE was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 60 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the liposomes were repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was deionized water, the prepared liposomes had a particle size of 130.2 ± 1.3 nm. Figure 2 The particles are spherical and evenly distributed. Figure 3 d).

[0122] Example 16 Preparation of cationic liposomes

[0123] Cationic liposomes were prepared using a thin-film dispersion method with a 1:1 molar ratio of DOTAP and DOPE. 69.8 μL and 74.4 μL of 50 mg / mL DOTMA and DOPE solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of acetone was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the solution was rotary hydrated at 60 °C for 1.5 h. After hydration, the solution was allowed to cool to room temperature. The solutions were then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals with 1 s intervals, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Then, using a nano-extruder, the liposomes were repeatedly extruded through a polycarbonate membrane with a pore size of 200 nm to prepare single-chamber cationic liposomes. When the hydration medium was 5% glucose hydration solution, the prepared liposomes had a particle size of 197.8 ± 2.1 nm and a PDI of 0.13 ± 0.02. Figure 5 The potential is 35.6 ± 1.9 mV. Figure 6 ).

[0124] Example 17 Preparation of cationic liposomes

[0125] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DDAB to PC was 1:1. 69.8 μL and 74.4 μL of DDAB and PC solutions (50 mg / mL) were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a thin lipid film formed on the inner wall of the flask. The flasks were then placed in a vacuum drying oven at 40 °C for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the solution was rotary hydrated at 55 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was Tris-HCl aqueous solution, the prepared liposomes had a particle size of 233.8 ± 2.6 nm and a PDI of 0.23 ± 0.02. Figure 5 The potential is 22.6 ± 2.9 mV. Figure 6 ).

[0126] Example 18 Preparation of cationic liposomes

[0127] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DOTMA and DOPC, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DOTMA and DOPC solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of chloroform was added to completely dissolve the lipid materials, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 65 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was 5% glucose hydration solution, the prepared liposomes had a particle size of 129.8 ± 4.3 nm and a PDI of 0.22 ± 0.02. Figure 5 The potential is 9.1 ± 1.2 mV. Figure 6 ).

[0128] Example 19 Preparation of cationic liposomes

[0129] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DODAP and DPPG, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DODAP and DPPG solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of chloroform was added to completely dissolve the lipids, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 40 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium was PBS buffer, the prepared liposomes had a particle size of 253.8 ± 5.8 nm and a PDI of 0.28 ± 0.02. Figure 5 The potential is 16.8 ± 2.5 mV. Figure 6 ).

[0130] Example 20 Preparation of cationic liposomes

[0131] Cationic liposomes were prepared using a thin-film dispersion method. The molar ratio of DODAP to cholesterol was 1:1. 69.8 μL and 74.4 μL of 50 mg / mL DODAP and cholesterol solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of chloroform was added to completely dissolve the liposomes, and the solutions were then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the solution was rotary hydrated at 50 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium is Tris-HCl aqueous solution, the prepared liposomes have a particle size of 173.8±2.1 nm and the particles are spherical.

[0132] Example 21 Preparation of cationic liposomes

[0133] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DOTAP and DOPC, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DOTAP and DOPC solutions were accurately pipetted into 50 mL round-bottom flasks, respectively. 1-2 mL of acetone was added to completely dissolve the lipid materials, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 60 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium is 5% glucose hydration solution, the prepared liposomes have a particle size of 146.5±1.9nm and the particles are spherical.

[0134] Example 22 Preparation of cationic liposomes

[0135] Cationic liposomes were prepared using a thin-film dispersion method. The lipid materials, DOTMA and PC, were in a 1:1 molar ratio. 69.8 μL and 74.4 μL of 50 mg / mL DOTMA and PC solutions, respectively, were accurately pipetted into 50 mL round-bottom flasks. 1-2 mL of acetone was added to completely dissolve the lipid materials, and the mixture was then rotary evaporated under vacuum at 40 °C for 30 min until a lipid film formed on the inner wall of the flask. The flasks were then placed in a 40 °C vacuum drying oven and dried for 1 h to remove organic solvents. Depending on the hydration medium, 1 mL of hydration medium was added to the flask, and the mixture was rotary hydrated at 70 °C for 1 h. After hydration, the solution was allowed to cool to room temperature. The solution was then sonicated using an ultrasonic homogenizer at 50% power for 3 s intervals of 1 s, for a total sonication time of 60 s, until the lipid suspension became clear and transparent. Finally, the solution was repeatedly extruded through a 200 nm pore size polycarbonate membrane using a nano-extruder to prepare single-chamber cationic liposomes. When the hydration medium is 5% glucose hydration solution, the prepared liposomes have a particle size of 166.8±2.5nm and the particles are spherical.

[0136] Example 23: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0137] Accurately weigh 37.9 mg of DOTAP and 37.2 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 60℃ and 130 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 200 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, liposomes carrying nerve growth factor antisense oligonucleotides with a nitrogen-to-phosphorus ratio of 1 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 30 minutes to obtain the nerve growth factor-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 184.2 ± 2.6 nm, and the delivery potential was 55.1 ± 1.6 mV. Figure 7 The encapsulation rate was 65.65%. Figure 8 The drug loading rate was 5.73%. Figure 9 ).

[0138] Example 24: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0139] Accurately weigh 37.9 mg of DOTAP and 37.2 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 60℃ and 130 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 200 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, liposomes carrying nerve growth factor antisense oligonucleotides with a nitrogen-to-phosphorus ratio of 2 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 30 minutes to obtain the nerve growth factor-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 220.6 ± 2.5 nm, and the delivery potential was 47.3 ± 1.5 mV. Figure 7 The encapsulation rate was 71.34%. Figure 8 The drug loading rate was 5.73%. Figure 9 ).

[0140] Example 25: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0141] Accurately weigh 37.9 mg of DOTAP and 37.2 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 60℃ and 130 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 200 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, liposomes carrying nerve growth factor antisense oligonucleotides with a nitrogen-to-phosphorus ratio of 4 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 30 minutes to obtain the nerve growth factor-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 211.3 ± 2.9 nm, and the delivery potential was 50.4 ± 1.1 mV. Figure 7 The encapsulation rate was 84.72%. Figure 8 The drug loading rate was 4.58%. Figure 9 ).

[0142] Example 26: Preparation of Nerve Growth Factor-Loaded Antisense Oligonucleotide Liposomes

[0143] Accurately weigh 37.9 mg of DOTAP and 37.2 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 60℃ and 130 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 200 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, liposomes carrying nerve growth factor antisense oligonucleotides with a nitrogen-to-phosphorus ratio of 8 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 30 minutes to obtain the nerve growth factor-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 199.8 ± 1.2 nm, and the delivery potential was 53.3 ± 2.6 mV. Figure 7 The encapsulation rate was 90.65%. Figure 8 The drug loading rate was 5.01%. Figure 9 ).

[0144] Example 27 Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0145] Accurately weigh 37.9 mg of DOTAP and 37.2 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 60℃ and 130 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 200 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, nerve growth factor-loaded antisense oligonucleotide liposomes with a nitrogen-to-phosphorus ratio of 10 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 30 minutes to obtain the nerve growth factor-loaded antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 200.5 ± 1.9 nm, and the delivery potential was 49.8 ± 1.8 mV. Figure 7 The encapsulation rate was 91.53%. Figure 8 The drug loading rate was 4.62%. Figure 9 ).

[0146] Example 28: Preparation of Nerve Growth Factor-Loaded Antisense Oligonucleotide Liposomes

[0147] Accurately weigh 23.1 mg of DOTMA and 14.5 mg of PC into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC-containing water and hydrate by rotary evaporation at 60℃ and 140 rpm for 1.5 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 400 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 2 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 40 minutes to obtain NGF-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 359.2 ± 3.5 nm, the potential was 40.6 ± 1.8 mV, the encapsulation efficiency was 76.53%, and the drug loading rate was 3.22%.

[0148] Example 29: Preparation of Nerve Growth Factor-Loaded Antisense Oligonucleotide Liposomes

[0149] Accurately weigh 32.4 mg of DODAP and 39.4 mg of DOPC into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC-containing water and hydrate by rotary evaporation at 50℃ and 150 rpm for 1.5 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 400 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 4 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 50 min to obtain NGF-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 368.2 ± 2.8 nm, the potential was 43.2 ± 2.1 mV, the encapsulation efficiency was 78.66%, and the drug loading rate was 3.28%.

[0150] Example 30: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0151] Accurately weigh 23.1 mg of DDAB and 37.3 mg of DPPG into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of acetone, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 70℃ and 160 rpm for 2 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 800 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 10 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 60 minutes to obtain NGF-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 730.2 ± 2.5 nm, the potential was 53.2 ± 2.1 mV, the encapsulation efficiency was 74.56%, and the drug loading rate was 3.42%.

[0152] Example 31: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0153] Accurately weigh 37.9 mg of DOTAP and 14.5 mg of PC into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of chloroform, vortex for 2 min to fully dissolve, then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. Add a fixed volume of DEPC-containing water, and hydrate by rotary evaporation at 60℃ and 170 rpm for 2.5 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a 200 nm pore size polycarbonate membrane to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding antisense oligonucleotides and varying amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 5 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 40 minutes to obtain NGF-carrying antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 222.5 ± 1.3 nm, the potential was 52.6 ± 2.3 mV, the encapsulation efficiency was 82.92%, and the drug loading rate was 3.26%.

[0154] Example 32: Preparation of Nerve Growth Factor-Loaded Antisense Oligonucleotide Liposomes

[0155] Accurately weigh 23.1 mg of DDAB and 19.3 mg of cholesterol into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of acetone, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. Add a fixed volume of 5% glucose hydration solution and hydrate at 65℃ and 180 rpm for 3 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a 200 nm pore size polycarbonate membrane to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM label). By adding a fixed amount of antisense oligonucleotides and varying amounts of cationic liposomes, nerve growth factor-loaded antisense oligonucleotide liposomes with a nitrogen-to-phosphorus ratio of 10 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 60 min to obtain nerve growth factor-loaded antisense oligonucleotide liposomes. The particle size of the delivery system was measured to be 165.2 ± 1.9 nm, the potential was 49.2 ± 2.3 mV, the encapsulation efficiency was 75.43%, and the drug loading rate was 3.31%.

[0156] Example 33: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0157] Accurately weigh 46.26 mg of DDAB and 54.5 mg of DOPG into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of dichloromethane, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 45℃ and 200 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 400 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding a fixed amount of antisense oligonucleotides and different amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 7 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 50 min to obtain antisense oligonucleotide liposomes carrying NGF. The particle size of the delivery system was 334.2 ± 2.3 nm, the potential was 51.2 ± 2.3 mV, the encapsulation efficiency was 73.46%, and the drug loading rate was 3.22%.

[0158] Example 34: Preparation of antisense oligonucleotide liposomes carrying nerve growth factor

[0159] Accurately weigh 46.26 mg of DDAB and 54.4 mg of DOPE into a round-bottom flask, with a molar ratio of 1:1. Add 1-2 mL of acetone, vortex for 2 min to fully dissolve, and then perform rotary evaporation at 40℃, 60 rpm, and 0.07-0.09 MPa for 30 min. After evaporation, add a fixed volume of DEPC water and hydrate by rotary evaporation at 45℃ and 200 rpm for 1 h. After hydration, allow the solution to cool to room temperature. Use an ultrasonic cleaner with parameters set to 300 W to sonicate until the liposome suspension is clear and transparent. Then, use a nanoextruder to repeatedly extrude the liposomes through a polycarbonate membrane with a pore size of 400 nm to prepare single-compartment cationic liposomes. The synthetic sequence of the nerve growth factor antisense oligonucleotide is: 5′GCCCGAGACGCCTCCCGA3′ (5′ end FAM labeled). By adding a fixed amount of antisense oligonucleotides and different amounts of cationic liposomes, antisense oligonucleotide liposomes carrying nerve growth factor (NGF) with a nitrogen-to-phosphorus ratio of 9 were prepared. The cationic liposomes were slowly added dropwise to the antisense oligonucleotide solution, gently shaken, and incubated at room temperature for 50 min to obtain antisense oligonucleotide liposomes carrying NGF. The particle size of the delivery system was measured to be 365.3 ± 2.1 nm, the potential was 52.2 ± 2.5 mV, the encapsulation efficiency was 74.55%, and the drug loading rate was 3.16%.

[0160] Example 35: Cytotoxicity of Nerve Growth Factor-Loaded Antisense Oligonucleotide Liposomes

[0161] The cytotoxicity of nerve growth factor-loaded antisense oligonucleotide liposomes was evaluated using the CCK-8 assay. SV-HUC-1 cells were cultured at 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 50 μL per well in 96-well plates, followed by 100 μL of complete culture medium. The plates were incubated at 37°C with 5% CO2 for 24 h. Cells were then incubated with 50 μL of antisense oligonucleotides or nerve growth factor-loaded antisense oligonucleotide liposomes at a final antisense oligonucleotide concentration of 100 nM for 18 h at prepared N:P ratios of 1:1, 2:1, 4:1, 8:1, and 10:1. Negative control and blank control wells were included, with three replicates per well. After treatment, 20 μL of CCK-8 reagent was added to each well, and the mixture was shaken and incubated for another 4 h. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader. When the N:P ratio was 8, cell viability remained above 80%, indicating that the delivery system had weak inhibitory effects on bladder epithelial cells and demonstrated biocompatibility. Figure 14 ).

[0162] Example 36: Cytotoxicity of Different Liposomes Carrying Nerve Growth Factor Antisense Oligonucleotides

[0163] The cytotoxicity of nerve growth factor-loaded antisense oligonucleotide liposomes was evaluated using the CCK-8 assay. SV-HUC-1 cells were seeded at a density of 5 × 10⁴ cells / well in 96-well plates, with 50 μL seeded per well and supplemented with 100 μL of complete culture medium. The cells were incubated at 37°C and 5% CO₂ for 24 h. Cells were then incubated with 50 μL of antisense oligonucleotides or different nerve growth factor-loaded antisense oligonucleotide liposomes at a final antisense oligonucleotide concentration of 100 nM and a nitrogen-to-phosphorus ratio of 8:1 for 18 h. Negative control and blank control wells were included, with three replicates per well. After treatment, 20 μL of CCK-8 reagent was added to each well, and the mixture was shaken and cultured for another 4 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Figure 15 ).

[0164] Example 37: Effect of Nanomedicines on Uptake by Bladder Urothelial Cells

[0165] Twenty-four hours after perfusion, three rats in each group were euthanized. Frozen sections were prepared, and the bladders were immediately removed and placed in centrifuge tubes pre-filled with 4% paraformaldehyde fixative. The sections were fixed at 4°C in the dark for 24 hours. After fixation, the rats were washed three times with PBS buffer for 5 minutes each time. The samples were dehydrated with 20% sucrose at 4°C for 48 hours. A special embedding box was prepared, and a layer of embedding medium was added. When a layer of solidified embedding medium formed at the bottom, the tissue was placed in the center of the embedding medium, air bubbles were removed, and another layer of embedding medium was added. Sectioning could begin after the embedding medium had completely solidified. After OCT embedding, the embedding box was placed in a liquid nitrogen tank to immerse the tissue and create a frozen block. The frozen sections were then cut into 8μm sections using a -25°C cryostat, collected on glass slides, and air-dried. 5-10μL of anti-fluorescence attenuation mounting medium was added, and the slides were mounted with clean coverslips. The slides were then photographed using a two-photon microscope for qualitative analysis. Figure 16 ) and quantitative analysis ( Figure 17 In the presence of liposomes, a large amount of green fluorescence was detected in urothelial cells, indicating that liposomes mediated the delivery of antisense oligonucleotide drugs and significantly improved drug uptake by the bladder epithelium.

[0166] Example 38: Effects of nanomedicine infusion therapy on urodynamics in a rat model of cystitis.

[0167] Urodynamic bladder manometry was performed using a transurethral catheterization method. The computer collecting data was connected to the BL-420N urodynamic pressure sensor, and a three-way connector was used. One end of the connector connected to the 19G BD bladder manometry catheter, the other end to the infusion pump tubing, and the third end to the pressure sensor. The infusion pump and tubing were connected, and air was purged from the three-way connector, sensor, and pump tubing. The infusion pump rate was set to 0.1 mL / min. After inducing SD rats with 2-3% isoflurane inhalation anesthesia, they were fixed in a supine position on a rat board. The rat urethral orifice was disinfected three times with povidone-iodine. The urethra was lifted with micro-forceps, and the 19G BD manometry catheter was inserted into the bladder to a depth of approximately 3 cm. Normal saline infusion was initiated, and the urodynamic curve image acquired in the computer software was observed. Simultaneously, the manometry instrument was zeroed against atmospheric pressure, and pressure measurements were taken and relevant data recorded. MCC, ICI, Pdet, and BC were observed in each rat for at least 20 minutes until the urodynamic curve stabilized, at which point monitoring was discontinued. Urodynamic testing was performed on rats in each group before and after modeling and after drug administration to compare differences in urodynamic parameters. Based on the test results ( Figure 18 Compared with the blank control group, the CYP-saline group showed significantly reduced maximum bladder capacity and bladder compliance, as well as decreased maximum intravesical pressure, indicating bladder dysfunction after modeling. Compared with the blank control group, the CYP-CLs / asODN group showed increased maximum bladder capacity, while the CYP-CLs and CYP-asODN groups showed significantly reduced maximum bladder capacity. This demonstrates that perfusion of nerve growth factor-loaded antisense oligonucleotide liposomes improved corresponding bladder urodynamic parameters, significantly enhanced bladder function, and mitigated bladder injury.

[0168] Example 39: Study on the effect of nanomedicines on the inflammatory state of bladder epithelial tissue

[0169] Immunohistochemical staining was used to further quantify the expression of inflammation-related factors in the bladder urothelium. Paraffin sections were dewaxed to water, antigen retrieval was performed, and after natural cooling, slides were placed in PBS (pH 7.4) and washed three times (5 min each time) on a destaining shaker. After blocking endogenous peroxidase, serum blocking was performed. The blocking solution was gently shaken off, and primary antibody was added to the slides. The slides were then incubated overnight at 4°C in a humidified chamber. Slides were placed in PBS (pH 7.4) and washed three times (5 min each time) on a destaining shaker. After slightly drying the slides, secondary antibody (HRP-labeled) of the corresponding species to the primary antibody was added to the circle to cover the tissue, and the slides were incubated at room temperature for 50 min. Slides were placed in PBS (pH 7.4) and washed three times (5 min each time) on a destaining shaker. After slightly drying the slides, freshly prepared diaminobenzidine chromogenic solution was added to the circle. The chromogenic time was controlled under a microscope. A positive result was brownish-yellow. The slides were rinsed with tap water to stop the chromogenic process. Hematoxylin was used to counterstain cell nuclei, and the sections were dehydrated and mounted. The results were interpreted under a white light microscope. Hematoxylin stained cell nuclei blue, while diaminobenzidine showed a brownish-yellow positive expression. Based on the staining results ( Figure 19 As can be seen from the data, after staining the same bladder urothelial region, the staining intensity of each factor, including nerve growth factor, PACAP, Piezo2, CCL2, IL-6, and TGF-β, was significantly reduced in the group of liposomes carrying nerve growth factor antisense oligonucleotides. This indicates that the instillation treatment inhibited the expression of the above factors in the urothelial tract, and that liposomes carrying nerve growth factor antisense oligonucleotides have a positive effect in the treatment of interstitial cystitis / bladder pain syndrome.

[0170] Example 40: Study on the effect of nano-drugs on the expression levels of inflammatory factors in bladder epithelial tissue

[0171] Immunohistochemical quantitative analysis results were performed using the H-Score evaluation method. The expression levels of each factor were assessed by multiplying the staining intensity by the percentage of positively stained cells and then using the H-Score scoring method. The results showed that ( Figure 20 Compared with the saline-treated group, the expression levels of Piezo2 and IL-6 were significantly reduced in the nerve growth factor-loaded antisense oligonucleotide liposome group. Compared with the cationic liposome group, the expression levels of PACAP, CCL2, and TGF-β were significantly downregulated. Compared with other experimental groups, the expression levels of IL-1β, IL-17, TNF-α, and CCL3 in the rat bladder decreased significantly after nerve growth factor-loaded antisense oligonucleotide liposome perfusion treatment. These results indicate that nerve growth factor-loaded antisense oligonucleotide liposome treatment can effectively inhibit the expression of inflammatory factors.

[0172] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for preparing a nanomedicine, characterized in that, The nanomedicine comprises a nerve growth factor antisense oligonucleotide and a liposome for delivering the antisense oligonucleotide, and the preparation method includes: (1) Dissolve cationic lipids and auxiliary lipids in an organic solvent, mix the two solutions evenly, and then vacuum rotary evaporate until the lipid material forms a lipid film; (2) Add the hydration medium to the lipid membrane in step (1) and perform hydration treatment at a hydration temperature of 40-70℃ to obtain a liposome suspension. Extrude the liposome suspension through a membrane method to obtain cationic liposomes. (3) The cationic liposomes are added dropwise to the antisense oligonucleotide solution and incubated at room temperature to obtain the nanomedicine. The amount of cationic liposomes and antisense oligonucleotides added is controlled so that the nitrogen-to-phosphorus ratio of the nanomedicine is 1-10.

2. The method for preparing nanomedicine according to claim 1, characterized in that, The cationic lipid material is selected from at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide, dioctadecyl dimethylammonium bromide, trimethyl-2,3-dioleenoyloxypropylammonium chloride, and 1,2-dioleoyloxy-3-(dimethylamino)propane.

3. The method for preparing nanomedicine according to claim 1, characterized in that, The auxiliary lipid material is selected from at least one of dioleoylphosphatidylethanolamine, L-α-phosphatidylcholine, sodium 1,2-dipalmitoyl-3-deoxycholate, sodium 1,2-palmitoylphosphatidylglycerol, and cholesterol.

4. The method for preparing nanomedicine according to claim 1, characterized in that, The liposomes have a particle size of 50-500 nm.

5. The method for preparing nanomedicine according to claim 1, characterized in that, The zeta potential of the liposomes is between +30 and +60 mV.

6. The method for preparing nanomedicine according to claim 1, characterized in that, The liposome PDI value is 0.05-0.

5.

7. The method for preparing nanomedicine according to claim 1, characterized in that, The hydration medium is selected from at least one of phosphate buffer, Tris-HCl, 5% glucose hydration solution, and deionized water.

8. The method for preparing nanomedicine according to claim 1, characterized in that, The hydration time is 0.5-5 hours.

9. A nanomedicine, characterized in that, The preparation method according to any one of claims 1-8 is obtained.

10. The application of the nanomedicine according to claim 9 in the preparation of drugs for nerve growth factor-related diseases.

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