An RVG29-modified ziconopeptide-loaded liposome, its preparation method and application

Abstract

This invention discloses an RVG29-modified zicosin-loaded liposome, its preparation method, and its applications. The nanoliposomes are composed of phospholipid-polyethylene glycol-RVG29, lecithin, cholesterol, phosphatidic acid, and zicosin. Preparation involves obtaining RVG29 resin through multiple washing, resin detection, and condensation operations using 2-chlorotriphenylmethyl chloride resin, followed by cleavage and precipitation treatment with phospholipid-polyethylene glycol-active ester and cleavage reagent, and high-performance liquid chromatography purification. Lecithin, cholesterol, phosphatidic acid, and zicosin are then added to synthesize the product liposomes. The nanoliposomes of this invention are novel nasobrain nanoliposomes that improve gel adhesion and drug delivery. The liposome surface targets nicotinic acetylcholine receptors on the surface of neurons, achieving precise drug delivery without the involvement of small molecule cytotoxic drugs. This will improve treatment adherence for cancer pain patients and enhance treatment efficacy and quality of life.

Description

Technical Field

[0001] This invention relates to the preparation of liposome drug delivery systems, specifically to an RVG29-modified zicosinate-loaded liposome and its preparation method, as well as the application of RVG29-modified zicosinate-loaded liposome gel formulation in cancer pain and its pharmaceutical application. Background Technology

[0002] Cancer is one of the major diseases threatening human health. Although medical advancements have led to increasingly diverse treatment strategies for tumors and a declining cancer mortality rate, the management of pain during cancer progression and chronic pain caused by anti-tumor therapy remains a growing concern. This pain significantly impacts patients' daily lives and the quality of end-of-life care. Severe pain is also closely related to the incidence of depression and anxiety in cancer patients and can even affect the effectiveness of anti-cancer treatment. Currently, strong opioids are used in clinical practice for severe pain caused by cancer. However, due to the widespread distribution of opioid receptors, opioid use can cause severe constipation, delirium, cognitive impairment, respiratory depression, and other adverse reactions, and is prone to addiction and tolerance. Furthermore, some patients are insensitive to these drugs, and nearly half of cancer pain patients still do not experience effective pain control.

[0003] Furthermore, the use of opioids carries the risk of promoting disease progression in cancer patients. Ziconovide, a synthetic alternative to ω-conotoxin, specifically and efficiently blocks N-type calcium ion channels, thereby inhibiting nerve excitation transmission. It was approved by the US FDA in 2004 for the treatment of severe chronic pain. Compared to opioids, ziconovide offers more efficient and precise analgesia and exhibits no drug tolerance. However, due to its high systemic toxicity and short duration of action, continuous intrathecal infusion is the only clinical route for its application. Because ziconovide is a large molecule with a positive charge affecting its tissue penetration, traditional administration methods result in slow onset of action. Furthermore, invasive administration methods also present problems such as poor patient compliance and catheter-related complications. Summary of the Invention

[0004] To address the technical problems of this invention, this invention proposes an RVG29-modified zicosinate-loaded liposome in situ gel formulation, its preparation method, and its application.

[0005] The technical solution adopted in this invention is:

[0006] I. An RVG29-modified ziconopeptide-loaded liposome:

[0007] It is a nanoliposome, mainly composed of phospholipid-polyethylene glycol-RVG29, lecithin, cholesterol, phosphatidic acid, and zicosinate, with the following mass percentages: phospholipid-polyethylene glycol-RVG29 4%-4.85%, lecithin 45%-65.475%, cholesterol 15%-21.825%, phosphatidic acid 4.85%-16%, and zicosinate 3-20%.

[0008] The structural formula of the phospholipid-polyethylene glycol-RVG29 is as follows:

[0009] ;

[0010] The average molecular weight of polyethylene glycol is 1000 to 10000.

[0011] RVG29 is a rabies virus glycoprotein polypeptide that can specifically bind to acetylcholine receptors on the surface of neurons, with a molecular weight of 3266.62 Da.

[0012] Application of the RVG29-modified ziconopeptide-loaded liposomes in the preparation of cancer pain drugs and in the preparation of ziconopeptide-stable delivery and release gel formulations.

[0013] The gel formulation is a thermosensitive in-situ gel for nasal administration.

[0014] The thermosensitive in situ gel solidifies at a nasal cavity temperature (34 degrees Celsius), achieving stable release of zicosinopeptide. The liposome modification enables zicosinopeptide delivery to exhibit significant neuronal targeting.

[0015] II. A method for preparing RVG29-modified ziconopeptide-loaded liposomes, the method being implemented through the following scheme:

[0016] (1) Synthesis of RVG29:

[0017] RVG29 resin was prepared by repeatedly washing, testing, and condensing raw materials such as 2-chlorotriphenylmethyl chloride resin and FMOC-AA-OH.

[0018] (2) Phospholipid-polyethylene glycol modified RVG29:

[0019] The RVG29 resin obtained in step (1) was dissolved, cut, and precipitated using raw materials such as phospholipid-polyethylene glycol-active ester, cutting reagent, and anhydrous diethyl ether, and then purified by high performance liquid chromatography (HPLC) with a specific mobile phase.

[0020] (3) Synthesis of RVG29-modified liposomes:

[0021] RVG29-modified zicoseptide liposomes were prepared by synthesizing phospholipid-polyethylene glycol-RVG29 pure product, lecithin, cholesterol, phosphatidic acid and zicoseptide through water bath sonication and ultrasonic probe.

[0022] The specific steps (1) are as follows:

[0023] (11) Select a 30*250mm reactor, weigh 0.5g of 2-chlorotriphenylmethyl chloride resin and put it into the reactor, then add dichloromethane and soak for 30 minutes;

[0024] (12) The peptide synthesis starts from the C-terminus. Each amino acid in the peptide RVG29 is added to the centrifuge tube in sequence. Then, 0.15 mmol of organic solvent FMOC-AA-OH (N-[fluorenylmethoxycarbonyl]-L-alanyl-L-alanine) is weighed and added to the centrifuge tube. It is dissolved in 5 ml of dichloromethane and then 0.5 mmol of N,N-diisopropylethylamine is added and shaken well.

[0025] (13) Using a disposable pipette, add the solution obtained in step (12) into the reactor obtained in step (11). Bubble the reaction with nitrogen for 90 minutes. After the reaction is complete, add a mixture of 2 ml methanol and 4 ml dichloromethane and react for 20 minutes. Then wash the reactor.

[0026] (14) Washing: After the liquid in the reactor is drained by a circulating water vacuum pump, industrial grade DMF reagent is added to the reactor using a wash bottle. The volume of DMF reagent is about 3 times the volume of the resin, which is 3 times the volume of the solid 2-chlorotriphenylmethyl chloride resin in the reactor. Then, nitrogen is bubbled and washed for 30 seconds.

[0027] (15) Repeat the complete washing operation of step (14) 4 times, and finally end by draining the liquid in the reactor. That is, after repeating the operation 4 times, use a circulating water vacuum pump to drain the liquid in the reactor.

[0028] (16) After washing, remove Fmoc:

[0029] Add 20% piperidine / N,N-dimethylformamide solution to the reactor obtained in step (15) using a wash bottle. The solution volume is about 3 times the resin volume, which is 3 times the volume of the solid 2-chlorotriphenylmethyl chloride resin in the current reactor. Bubble the reaction with nitrogen for 20 minutes. Repeat the washing operation in step (14) once as the fifth washing. During the fifth washing, convert the industrial grade N,N-dimethylformamide into analytical grade N,N-dimethylformamide.

[0030] The 20% piperidine / N,N-dimethylformamide solution is a mixed solution of piperidine and N,N-dimethylformamide, containing a 20% mention fraction of piperidine, that is, 100 ml of the mixture contains 20 ml of piperidine and 80 ml of N,N-dimethylformamide.

[0031] (17) Complete resin testing:

[0032] Using a long-necked pipette, draw 10-20 grains of 2-chlorotriphenylmethyl chloro resin from the reactor and place them at the bottom of the detection tube. The detection tube is a separate glass tube used to hold the sampled resin for testing. Next, using a dropper, add two drops each of reagent A (5g ninhydrin - 100ml anhydrous ethanol) and reagent B (analytical grade pyridine) to the detection tube, ensuring full contact between the resin and reagents. Then, place the detection tube in a constant temperature of 100℃ for 2 minutes and observe the resin color.

[0033] If the resin shows a color, i.e., blue-purple, then the removal of Fmoc (fluorenemethyloxycarbonyl) is successful, and the next step can be performed.

[0034] If no color is developed, and no blue-purple color is developed, repeat steps (16)-(17) for Fmoc removal and subsequent operations until Fmoc removal is successful.

[0035] (18) Condensation: Weigh 0.5 mmol FMOC-AA-OH (the second amino acid at the C-terminus of the polypeptide) and 0.5 mmol 1-hydroxybenzotriazole and add them to a new centrifuge tube. Then dissolve them completely with 10 ml N,N-dimethylformamide, and then add 0.5 mmol dimethyl carbonate. After mixing, add the mixed solution from the new centrifuge tube to the resin that was dried in step (16), and bubble it with nitrogen for 1 h.

[0036] (19) Perform a second resin test: Observe the resin color as in step (16):

[0037] If there is color, repeat the shrinkage operation of step (17) above, and then proceed to the next step;

[0038] If there is no color, it indicates that the connection is complete. Then, repeat the washing-washing-detection-condensation-detection process for each amino acid.

[0039] The washing-washing-detection-condensation-detection process first involves washing step (15) once, and then repeating steps (16), (17), and (18) in sequence. Since the peptide has 29 amino acids, the process is repeated 28 times. Each repetition adds one amino acid until the peptide is attached. Finally, Fmoc removal is performed, and then washing step (14) is performed once to obtain RVG29 resin.

[0040] The aforementioned peptide linkage refers to the formation of a polypeptide by linking amino acids. The formation of FMOC-AA-OH is equivalent to activating amino acids, providing a carrier for the amino acids to link together. Finally, after the amino acid linkage is completed, Fmoc is removed to form the final polypeptide.

[0041] Step (2) specifically involves:

[0042] (21) Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester and add it to a centrifuge tube. Dissolve and mix it thoroughly with 100 ml of N,N-dimethylformamide.

[0043] The mixed solution was added to the RVG29 resin obtained in step (1) (i.e., the resin result after all amino acids were linked in step (19)), and 0.5 mmol N,N-diisopropylethylamine was added. The reaction was carried out under nitrogen bubbling for 3 h to obtain phospholipid-polyethylene glycol-RVG29-resin peptide.

[0044] (22) Cleavage of the peptide from the resin: Prepare a cleavage reagent consisting of trifluoroacetic acid, ethylenedithiol, triisopropylsilane, and water. For example, 100 ml of cleavage reagent consists of 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The cleavage reagent is added to the phospholipid-polyethylene glycol-RVG29-resin peptide obtained in step (21), followed by precipitation with diethyl ether and centrifugation and washing. The preparation amount is 10 mL of cleavage reagent per 1 g of resin, and each 1 mL of cleavage reagent is washed with 10 mL of diethyl ether by centrifugation.

[0045] In a cleavage solution containing peptides, the peptides will precipitate as white flocculent matter in diethyl ether. Then, by centrifugation, the precipitate will gather at the bottom of the centrifuge tube, thus separating the peptides and diethyl ether. After removing the diethyl ether, fresh diethyl ether is added and the solution is washed and centrifuged again to remove some of the cleavage reagents, peptide protecting groups, and other impurities.

[0046] The phospholipid-polyethylene glycol-RVG29-resin peptide was weighed, then cleaved for 2 hours with cleaving reagent, and then filtered to obtain filtrate. The filtrate was then added to anhydrous diethyl ether that had been pre-cooled at -20°C to precipitate and centrifuge to obtain crude product. The peptide was precipitated with 10 mL of diethyl ether for every 1 mL of cleaving reagent, and then the crude product was freeze-dried.

[0047] (23) The peptide was purified by high performance liquid chromatography (HPLC). The crude product was loaded onto the mobile phase A of the HPLC column. The HPLC used a 30 / 250 mm Daisogel 8 μm column. The mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, and the flow rate was 10 ml / min.

[0048] Then, after equilibration with 10% (v / v) acetonitrile-water (phase B) and 90% (v / v) phase A for 5 minutes, gradient elution was started. The initial conditions were 10% (v / v) phase B and 90% (v / v) phase A. Yes, this means the instrument started eluting, and the mixture in the instrument tubing contained 10% phase B liquid and 90% phase A liquid. Over 45 minutes, the volume fraction of phase B was gradually increased to 55%, while the volume fraction of phase A was gradually decreased to 45%, and this was maintained for 45 minutes to prepare, collect, and detect the sample peak.

[0049] (24) Finally, the purified crude product was transferred into a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain pure phospholipid-polyethylene glycol-RVG29.

[0050] The above 30 / 250mm indicates that the chromatographic column has a diameter of 30mm and a length of 250mm. 0.1% TFA / water indicates the volume ratio of the two components in the mixture, which contains 0.1% TFA and 99.9% water.

[0051] 0.1% TFA / acetonitrile indicates the volume ratio of the two components in the mixture, meaning the TFA / acetonitrile mixture contains 0.1% TFA and 99.9% acetonitrile. A liquid volume ratio of 10% B phase indicates that the liquid mixture in the chromatographic instrument tubing contains 10% B phase liquid and 90% A phase liquid.

[0052] The preparation of step (3) using the thin-film hydration method is specifically as follows:

[0053] (31) Accurately weigh 4-4.85 mg of phospholipid-polyethylene glycol-RVG29, 45-65.475 mg of lecithin, 15-21.825 mg of cholesterol, and 4.85-16 mg of phosphatidic acid and add them to a flask containing 20-30 ml of dichloromethane. Dissolve the flask thoroughly by ultrasonication in a water bath and then evaporate it by rotary evaporation at 40 degrees and 100 rpm until the organic solvent disappears to form a thin film.

[0054] The film is composed of lipid components that can only be suspended in water to form nano-sized liposomes after being dispersed, rather than becoming a precipitate or layered.

[0055] (32) Dissolve 3-20 mg of ziconopeptide in 40-50 ml of ultrapure water, add it to a flask with a membrane, mix thoroughly, resuspend, and extract into a centrifuge tube. Treat the centrifuge tube with an ultrasonic probe at 400-600 W for 4 minutes. The ultrasonic probe works for 2 seconds and then stops for 3 seconds. Repeat this process. After treatment, place the tube in a 4°C refrigerator for 8 hours to obtain the sample.

[0056] (33) After the step (32) is completed, the sample obtained is transferred into a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain RVG29 modified zicosine liposomes.

[0057] The 4 degrees Celsius mentioned in this invention refer to all temperatures.

[0058] III. A method for preparing an in-situ gel formulation of RVG29-modified zicosinate-loaded liposomes, the method comprising:

[0059] S1. Prepare the RVG29-modified ziconopeptide-loaded liposomes;

[0060] Preparation of S2, RVG29 modified ziconopeptide-loaded liposome in situ gel:

[0061] First, dissolve 0.5 mg of chitosan in 5 ml of 2% glacial acetic acid solution to form a chitosan solution. Mix 5 ml of chitosan solution, 2 mg of Bergsham 407 and 3 ml of deionized water, and slowly stir to dissolve at a low temperature of 4 degrees Celsius. Add deionized water to a total volume of 9 ml. Finally, add 1 ml of liposome suspension to the above solution system and slowly stir to mix at a low temperature of 4 degrees Celsius to obtain an in-situ gel formulation.

[0062] The liposome suspension is prepared by suspending the previously freeze-dried liposomes in ultrapure water.

[0063] The chitosan solution contains 0.1 mg / ml of chitosan, and the glacial acetic acid solution in the chitosan solution contains 2% glacial acetic acid. The ratio of chitosan solution, Bergsham 407, deionized water, and liposome suspension is 5 ml: 2 mg: 9 ml: 1 ml. The low temperature is 4 degrees Celsius to maintain the gel liquid state and stability.

[0064] The in-situ gel formulation of the liposome can be used in the treatment of severe chronic pain (cancer pain treatment) and in pharmaceutical applications. It does not involve small molecule cytotoxic drugs. It exerts its analgesic effect on the site-neurons by specifically targeting ziconopeptide through the ligand RVG29, reducing systemic side effects. The application of the thermosensitive in-situ gel allows the liposome to deliver ziconopeptide to the central nervous system non-invasively via the nose, changing the traditional intrathecal injection method of ziconopeptide and improving patient compliance.

[0065] This invention successfully encapsulates zicosinate in liposomes, establishes a nasal delivery pathway using resin RVG29, and improves the sustained release and safety of zicosinate.

[0066] This invention, based on the preparation of phospholipid-polyethylene glycol-RVG29, synthesizes liposomes containing negatively charged phosphatidic acid via thin-film hydration technology to efficiently encapsulate zicosinate. Furthermore, a thermosensitive gel containing chitosan is synthesized, which is then combined with the liposomes to form novel nasobrain nanoliposomes.

[0067] The beneficial effects of this invention are:

[0068] The liposomes prepared by this invention can efficiently encapsulate ziconopeptide, and a novel nasobrain nanoliposome was prepared.

[0069] The nanoliposomes of this invention can be conveniently sprayed into the nasal cavity in a liquid state and solidify and adhere at nasal cavity temperature. The positively charged chitosan acts as a "double-sided adhesive" between the negatively charged liposomes and nasal mucin, further improving gel adhesion and drug delivery. The liposome surface targets nicotinic acetylcholine receptors on the surface of neurons, achieving precise drug delivery.

[0070] The nanoliposomes of this invention do not involve small molecule cytotoxic drugs, which will improve treatment adherence, treatment efficacy and quality of life for cancer pain patients. Attached Figure Description

[0071] Figure 1 This is a mass spectrum of the RVG29 peptide from an embodiment of the present invention.

[0072] Figure 2 This is a high-resolution liquid chromatography-mass spectrometry diagram of phospholipid-polyethylene glycol-RVG29 according to an embodiment of the present invention.

[0073] Figure 3 This is a transmission electron microscope image of RVG29-modified liposomes according to an embodiment of the present invention.

[0074] Figure 4 This is a particle size diagram of RVG29-modified liposomes according to an embodiment of the present invention.

[0075] Figure 5 This is a graph showing the toxicity results of RVG29 modified liposomes on HT-22 cells according to an embodiment of the present invention.

[0076] Figure 6 This is a graph showing the mechanical pain measurement results of mice treated with bone cancer pain using the RVG29-modified zicosinate-loaded liposome in situ gel formulation. Group A: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of the RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 80 ng of zicosinate. Group B: Blank control group (mice injected with physiological saline into the femur). Group C: Lewis lung cancer cells were injected into the femur of mice. Group D: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of the RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 40 ng of zicosinate.

[0077] Figure 7This is an example of the treatment of bone cancer pain in mice with thermal pain using an RVG29-modified zicosinate-loaded liposome in situ gel formulation. In this example, A: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of an RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 80 ng of zicosinate; B: Blank control group (mice injected with saline in the femur); C: Lewis lung cancer cells were injected into the femur of mice; D: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of an RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 40 ng of zicosinate.

[0078] Figure 8 This is a graph showing the in vivo side effects assay of the RVG29-modified zicosinate-loaded liposome in situ gel formulation. (a) represents the rotator bar test, and (b) represents the tremor score. Among them, A is the blank control; B is the RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 80 ng of zicosinate; C is the RVG29-modified zicosinate-loaded liposome in situ gel formulation containing 40 ng of zicosinate administered via nasal administration; D is the intrathecal injection of 80 ng of zicosinate solution; and E is the intrathecal injection of 40 ng of zicosinate solution.

[0079] Figure 9 The images show cellular uptake of RVG29-modified liposomes. (a) shows a mouse hippocampal neuronal cell line, and (b) shows a mouse microglia cell line. Group A consists of blank liposomes labeled with a green fluorescent probe on the cell membrane, while Group B consists of RVG29-modified liposomes labeled with a green fluorescent probe on the cell membrane.

[0080] Figure 10 These are organ fluorescence distribution images of RVG29 modified liposome in situ gel formulations. (a) shows the fluorescence distribution of important organs throughout the body, and (b) shows the fluorescence distribution of the brain. Group A is RVG29 modified liposome in situ gel formulation labeled with indocyanine green 5.5, and Group B is the blank control. Detailed Implementation

[0081] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0082] The embodiments of the present invention are as follows: Example 1:

[0083] Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel formulation

[0084] (1) Synthesis of phospholipid-polyethylene glycol-RVG29:

[0085] A 30*250mm reactor was selected. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis began at the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was then added to the reactor from the previous step using a disposable pipette. The reaction was bubbled under nitrogen for 90 minutes. After the reaction, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for another 20 minutes. Washing was then performed: the liquid in the reactor was evacuated using a circulating water vacuum pump. Industrial-grade DMF was added to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This process was repeated four times. After washing, perform Fmoc removal: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Perform resin testing: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Then, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. Color development indicates successful Fmoc removal; if no color development occurs, repeat the Fmoc removal and subsequent steps. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml of N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix. Add this mixture to the dried resin and react under nitrogen bubbling for 1 h. Perform a second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove the Fmoc and perform the first wash operation again.

[0086] Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml of N,N-dimethylformamide, mix it and add it to the dried resin, then add 0.5 mmol of N,N-diisopropylethylamine, and bubble the reaction under nitrogen for 3 h.

[0087] The peptides were cleaved from the resin. A cleavage reagent was prepared (using 100 ml of reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 10 mL diethyl ether. For sedimentation, anhydrous diethyl ether was pre-cooled to -20°C. 1 mL of cleavage reagent was used to precipitate the peptides with 10 mL diethyl ether. After drying the resin, the peptides were weighed, and the cleavage buffer was added for 2 hours. The residue was then filtered to obtain the filtrate. The filtrate was then added to diethyl ether to precipitate the peptides, centrifuged, and the crude product was lyophilized.

[0088] Peptide purification was performed using HPLC with a 30 / 250 mm Daisogel 8 μm column. Mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, flow rate: 10 mL / min. Samples were loaded via pump A, followed by equilibration with 10% acetonitrile for 5 min. Gradient elution was then initiated with 10% phase B, increasing the phase B concentration to 55% within 5 minutes and maintaining this concentration for 45 minutes to prepare and collect the target peak.

[0089] Finally, the sample was transferred to a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain a pure product.

[0090] Relevant characteristics such as Figure 1 and Figure 2 As shown, Figure 1 The relative atomic mass of the RVG29 polypeptide measured by the mass spectrum was 3266 Daltons, which is consistent with the relative atomic mass of RVG29, indicating that the preparation was successful; Figure 2 The image shows the time-of-flight mass spectrum of the phospholipid-polyethylene glycol-RVG29 polymer. The polymer's relative atomic mass distribution is between 5000 and 6000, indicating that the phospholipid-polyethylene glycol-RVG29 was successfully prepared.

[0091] 2) Synthesis of RVG29-modified ziconopeptide-loaded liposomes

[0092] A thin-film hydration method was employed. 4.8 mg of phospholipid-polyethylene glycol-RVG29, 60.7 mg of lecithin, 20.2 mg of cholesterol, and 9.5 mg of phosphatidic acid were accurately weighed and added to a round-bottom flask. 30 ml of dichloromethane was added, and the mixture was thoroughly dissolved by sonication in a water bath. The solution was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 4.78 mg of zicosinate was dissolved in 50 ml of ultrapure water and added to the round-bottom flask. After thorough mixing and resuspending, the solution was extracted into centrifuge tubes and treated with a 450 W ultrasonic probe in an ice bath for 4 minutes (2 seconds on, 3 seconds off). After treatment, the sample was stabilized at 4°C for 8 hours. Finally, the sample was transferred to lyophilization dishes and freeze-dried for 24 hours to obtain the pure product.

[0093] The average particle size was determined to be 151.8 ± 1.1 nm and the zeta potential was 52.6 ± 0.3 nm using a nanoparticle size and zeta potential analyzer. The drug loading of ziconopeptide was determined to be 85% by high-performance liquid chromatography.

[0094] The obtained liposomes were analyzed by transmission electron microscopy and their particle size distribution as follows: Figure 3 and Figure 4 As shown, Figure 3 Transmission electron microscopy can be used to observe hollow liposomes under negative pressure.

[0095] Figure 4 The visible particle size distribution shows that the diameter of the liposomes is concentrated at 151 nm.

[0096] 3) Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel

[0097] First, dissolve 0.5 mg of chitosan in 5 ml of 2% glacial acetic acid solution. Then, mix 5 ml of chitosan solution, 2 mg of Bergsham 407, and 3 ml of deionized water. Stir slowly at 4 degrees Celsius to dissolve the mixture, and add deionized water to bring the total volume to 9 ml. Finally, add 1 ml of liposome suspension to the above system and stir slowly at 4 degrees Celsius to mix.

[0098] Test verification of Example 1:

[0099] 1) Cytotoxic effects of RVG29-modified ziconopeptide liposomes:

[0100] The RVG29-modified zicosinate-loaded liposomes obtained in Example 1 were used. HT22 cells (mouse hippocampal neuronal cell line) were seeded in 96-well plates at a density of 5 x 10^3 / well and cultured overnight in an incubator (37°C, 5% CO2). Then, aqueous solutions and liposome solutions containing zicosinate at final concentrations of 1 μg / mL, 5 μg / mL, and 50 μg / mL were added, along with a liposome-only group (without zicosinate) at a final concentration of 2.5 mg / mL. Each concentration was used in 6 replicates. After incubation for 24 h, cell viability was assessed using the CCK-8 assay: 10 μL of CCK-8 solution was added to each well, and the plate was incubated for another 2 h. The absorbance was then measured at 450 nm using a multi-mode microplate reader, and the cell growth inhibition rate was calculated using the following formula:

[0101] Cell growth inhibition rate (%) = (1 - AT / Ac) x 100%

[0102] In the formula, Ar is the absorbance value at 450 nm of the experimental group, and Ac is the absorbance value at 450 nm of the blank control group.

[0103] The results are as follows Figure 5 As shown, the cell survival rate of the experimental group was not significantly affected compared with the control group, indicating that the RVG29-modified zicosinate-loaded liposomes did not exhibit cytotoxicity in vitro.

[0104] 2) The efficacy of RVG29-modified ziconopeptide liposome in situ gel formulation in the treatment of cancer pain

[0105] The analgesic effect of the RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1 was observed by intranasal administration. A c57 mouse bone cancer pain model was constructed using Lewis lung cancer cells. On the seventh day after modeling, mice were randomly divided into four groups of six each. The following treatments were performed: Group A: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of 4 μL of the RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1 (containing 80 ng zicosinate); Group B: Blank control group (mice injected with physiological saline into the femur); Group C: Lewis lung cancer cells were injected into the femur of mice; Group D: Lewis lung cancer cells were injected into the femur of mice, followed by intranasal administration of 4 μL of the RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1 (containing 40 ng zicosinate). Mechanical pain and thermal pain behavior were measured before and after administration, and behavioral curves were generated.

[0106] The results are as follows Figure 6 and Figure 7 As shown, compared with the blank control group, mice with bone cancer showed significant pain on day 7 of the model, but this pain could be improved by the RVG29-modified zicosinate-loaded liposome in situ gel formulation. Giving mice containing higher concentrations of zicosinate nanoliposomes significantly prolonged the duration of treatment.

[0107] 3) Side effects of RVG29-modified ziconopeptide liposome in-situ gel formulation

[0108] The RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1 was administered intranasally to observe its in vivo side effects. Healthy mice were used for the rotating bar test and tremor assessment. Mice were placed on a rotating bar and rotated at 10 rpm for 3 minutes for three consecutive days. Afterwards, they were randomly divided into 5 groups of 6 mice each, and treated as follows: Group A: blank control; Group B: 4 μL of the RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1, containing 80 ng of zicosinate, administered intranasally; Group C: 4 μL of the RVG29-modified zicosinate-loaded liposome in situ gel formulation obtained in Example 1, containing 80 ng of zicosinate, administered intranasally; Group D: 80 ng / 8 μL zicosinate solution administered intrathecally; Group E: 40 ng / 8 μL zicosinate solution administered intrathecally. After administration, mice were placed on the rotating bar at 5 rpm for 60 s at various time points to adapt.

[0109] Then, the rotating bar mode was set to uniform acceleration mode. Over 3 minutes, the rotating bar was rotated at a uniform acceleration from 5 revolutions per minute to 25 revolutions per minute. Timing was started, and the time the mouse remained on the rotating bar was recorded (maximum dwell time was 3 minutes). A behavior-performance curve was created, and the results are as follows: Figure 8 As shown in (a). Simultaneously, tremor intensity was assessed in mice via nasal administration of RVG29-modified ziconopeptide-loaded liposome in situ gel formulation and intrathecal injection of 80 ng ziconopeptide solution. A 4-point scale was used to score tremor intensity: 0: normal; 1: mild (limited to certain areas of the body, such as the head, neck, forelimbs, and tail); 2: moderate (tremor in multiple body parts, including the head, upper body, abdomen, and trunk); 3: severe (severe tremor throughout the body). Behavioral-performance curves were constructed, and the results are shown in Figure 1. Figure 8 As shown in (b).

[0110] The results showed that, at the dose that produced an analgesic effect, the nasal delivery of the RVG29-modified zicosinate-loaded liposome in situ gel formulation did not produce significant side effects. In contrast, the current clinical application of intrathecal injection of zicosinate affected the coordinated movement of mice and produced tremors after administration. The RVG29-modified zicosinate-loaded liposome in situ gel formulation can significantly improve the safety of drug application.

[0111] Example 2:

[0112] 1) Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel formulation

[0113] Phospholipid-polyethylene glycol-RVG29 synthesis: A 30*250mm reactor was used. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis started from the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was added to the reactor from the previous step using a disposable pipette, and the reaction was carried out under nitrogen bubbling for 90 minutes. After the reaction was completed, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for 20 minutes. Washing was then performed: After the liquid in the reactor was evacuated using a circulating water vacuum pump, industrial-grade DMF was added to the reactor through a wash bottle, with the reagent volume approximately 3 times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This operation was repeated 4 times. After washing, Fmoc removal is performed: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Resin testing is then performed: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Next, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Then, heat the test tube at 100℃ for 2 minutes and observe the resin color. If the resin shows color, Fmoc removal is successful; if no color develops, repeat the Fmoc removal and subsequent operations. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix into the dried resin. React under nitrogen bubbling for 1 h. Second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove Fmoc and perform the first wash operation again. Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml N,N-dimethylformamide, mix and add into the dried resin. Add 0.5 mmol N,N-diisopropylethylamine and react under nitrogen bubbling for 3 h.The peptide was cleaved from the resin. A cleavage reagent was prepared (using 100 mL of cleavage reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 1 mL of cleavage reagent using 10 mL of diethyl ether. For precipitation preparation: anhydrous diethyl ether was pre-cooled at -20°C. 1 mL of cleavage reagent was used to precipitate the peptide with 10 mL of diethyl ether. After drying the resin, the peptide was weighed, and the cleavage reagent was added for 2 hours. The filtrate was then filtered. The filtrate was then added to diethyl ether for precipitation, centrifuged, and the crude product was lyophilized. The peptide was purified by HPLC using a 30 / 250 mm Daisogel 8 μm column. The mobile phase was: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, and the flow rate was 10 mL / min. The sample was loaded onto pump A, and then equilibrated with 10% acetonitrile for 5 minutes before gradient elution was initiated. The initial concentration of phase B was 10%, and the concentration was increased to 55% within 5 minutes and maintained for 45 minutes to prepare and collect the sample peak for detection. Finally, the sample was transferred to a lyophilization dish and freeze-dried for 24 hours to obtain the pure product.

[0114] 2) Synthesis of RVG29-modified ziconopeptide-loaded liposomes

[0115] A thin-film hydration method was employed. 4 mg of phospholipid-polyethylene glycol-RVG29, 45 mg of lecithin, 15 mg of cholesterol, and 16 mg of phosphatidic acid were accurately weighed and added to a round-bottom flask. 30 ml of dichloromethane was added, and the mixture was dissolved thoroughly by sonication in a water bath. The solution was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 20 mg of zicosinate was dissolved in 50 ml of ultrapure water and added to the round-bottom flask. The mixture was thoroughly mixed, resuspended, and extracted into centrifuge tubes. The samples were then treated with a 450 W ultrasonic probe in an ice bath for 4 minutes (2 seconds on, 3 seconds off). After treatment, the samples were stabilized at 4°C for 8 hours. Finally, the samples were transferred to lyophilization dishes and freeze-dried for 24 hours to obtain the pure product.

[0116] 3) Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel

[0117] First, dissolve 0.5 mg of chitosan in 5 ml of 2% glacial acetic acid solution. Then, mix 5 ml of chitosan solution, 2 mg of Bergsham 407, and 2 ml of deionized water. Stir slowly at 4 degrees Celsius to dissolve the mixture, and add deionized water to bring the total volume to 8 ml. Finally, add 2 ml of liposome suspension to the above system and stir slowly at 4 degrees Celsius to mix. Transfer the sample to a lyophilization dish and freeze-dry for 24 hours to obtain the pure product.

[0118] Example 3:

[0119] 1) Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel formulation

[0120] Phospholipid-polyethylene glycol-RVG29 synthesis: A 30*250mm reactor was used. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis started from the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was added to the reactor from the previous step using a disposable pipette, and the reaction was carried out under nitrogen bubbling for 90 minutes. After the reaction was completed, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for 20 minutes. Washing was then performed: After the liquid in the reactor was evacuated using a circulating water vacuum pump, industrial-grade DMF was added to the reactor through a wash bottle, with the reagent volume approximately 3 times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This operation was repeated 4 times. After washing, Fmoc removal is performed: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Resin testing is then performed: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Next, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Then, heat the test tube at 100℃ for 2 minutes and observe the resin color. If the resin shows color, Fmoc removal is successful; if no color develops, repeat the Fmoc removal and subsequent operations. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix into the dried resin. React under nitrogen bubbling for 1 h. Second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove Fmoc and perform the first wash operation again. Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml N,N-dimethylformamide, mix and add into the dried resin. Add 0.5 mmol N,N-diisopropylethylamine and react under nitrogen bubbling for 3 h.The peptide was cleaved from the resin. A cleavage reagent was prepared (using 100 mL of cleavage reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 1 mL of cleavage reagent using 10 mL of diethyl ether. For precipitation preparation: anhydrous diethyl ether was pre-cooled at -20°C. 1 mL of cleavage reagent was used to precipitate the peptide with 10 mL of diethyl ether. After drying the resin, the peptide was weighed, and the cleavage reagent was added for 2 hours. The filtrate was then filtered. The filtrate was then added to diethyl ether for precipitation, centrifuged, and the crude product was lyophilized. The peptide was purified by HPLC using a 30 / 250 mm Daisogel 8 μm column. The mobile phase was: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, and the flow rate was 10 mL / min. The sample was loaded onto pump A, and then equilibrated with 10% acetonitrile for 5 minutes before gradient elution was initiated. The initial concentration of phase B was 10%, and the concentration was increased to 55% within 5 minutes and maintained for 45 minutes to prepare and collect the sample peak for detection. Finally, the sample was transferred to a lyophilization dish and freeze-dried for 24 hours to obtain the pure product.

[0121] 2) Synthesis of RVG29-modified ziconopeptide-loaded liposomes

[0122] A thin-film hydration method was employed. 4.85 mg of phospholipid-polyethylene glycol-RVG29, 65.475 mg of lecithin, 21.825 mg of cholesterol, and 4.85 mg of phosphatidic acid were accurately weighed and added to a round-bottom flask. 40 ml of dichloromethane was added, and the mixture was thoroughly dissolved by sonication in a water bath. The solution was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 3 mg of zicosinate was dissolved in 40 ml of ultrapure water and added to the round-bottom flask. The mixture was thoroughly mixed, resuspended, and extracted into a centrifuge tube. The film was then treated with a 450 W ultrasonic probe at an ice bath for 4 minutes (2 seconds on, 3 seconds off). After treatment, the film was stabilized at 4°C for 8 hours. The average particle size was determined to be 132.3 ± 1.6 mm, and the zeta potential was 30.6 ± 1.0 mm using a nanoparticle size and zeta potential analyzer. High-performance liquid chromatography (HPLC) determined the zicosinate drug loading to be 80%.

[0123] 3) Preparation of RVG29-modified ziconopeptide-loaded liposome in situ gel

[0124] First, dissolve 0.5 mg of chitosan in 5 ml of 2% glacial acetic acid solution. Then, mix 5 ml of chitosan solution, 2.85 mg of Bergsham 407, and 3 ml of deionized water. Stir slowly at 4 degrees Celsius to dissolve the mixture, and add deionized water to bring the total volume to 9 ml. Finally, add 1 ml of liposome suspension to the above system and stir slowly at 4 degrees Celsius to mix.

[0125] Comparative Example 1:

[0126] (1) Synthesis of phospholipid-polyethylene glycol-RVG29:

[0127] A 30*250mm reactor was selected. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis began at the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was then added to the reactor from the previous step using a disposable pipette. The reaction was bubbled under nitrogen for 90 minutes. After the reaction, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for another 20 minutes. Washing was then performed: the liquid in the reactor was evacuated using a circulating water vacuum pump. Industrial-grade DMF was added to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This process was repeated four times. After washing, perform Fmoc removal: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Perform resin testing: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Then, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. Color development indicates successful Fmoc removal; if no color development occurs, repeat the Fmoc removal and subsequent steps. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix. Add this mixture to the dried resin and bubble under nitrogen for 1 hour. Perform a second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove the Fmoc and perform the first wash operation again.

[0128] Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml of N,N-dimethylformamide, mix it and add it to the dried resin, then add 0.5 mmol of N,N-diisopropylethylamine, and bubble the reaction under nitrogen for 3 h.

[0129] The peptides were cleaved from the resin. A cleavage reagent was prepared (using 100 ml of reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 10 mL diethyl ether. For sedimentation, anhydrous diethyl ether was pre-cooled to -20°C. 1 mL of cleavage reagent was used to precipitate the peptides with 10 mL diethyl ether. After drying the resin, the peptides were weighed, and the cleavage buffer was added for 2 hours. The residue was then filtered to obtain the filtrate. The filtrate was then added to diethyl ether to precipitate the peptides, centrifuged, and the crude product was lyophilized.

[0130] Peptide purification was performed using HPLC with a 30 / 250 mm Daisogel 8 μm column. Mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, flow rate: 10 mL / min. Samples were loaded via pump A, followed by equilibration with 10% acetonitrile for 5 min. Gradient elution was then initiated with 10% phase B, increasing the phase B concentration to 55% within 5 minutes and maintaining this concentration for 45 minutes to prepare and collect the target peak.

[0131] Finally, the sample was transferred to a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain a pure product.

[0132] (2) Synthesis of RVG29-modified liposomes: The thin-film hydration method was used. 4.8 mg of phospholipid-polyethylene glycol-RVG29, 60.7 mg of lecithin, and 20.2 mg of cholesterol were accurately weighed and added to a round-bottom flask. 30 ml of dichloromethane was added and the mixture was dissolved thoroughly by sonication in a water bath. The solution was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 4.78 mg of ziconopeptide was dissolved in 50 ml of ultrapure water and added to the round-bottom flask. After thorough mixing and resuspending, the solution was extracted into a centrifuge tube and treated with a 450 W ultrasonic probe in an ice bath for 4 minutes (2 seconds on, 3 seconds off). After treatment, the sample was stabilized at 4°C for 8 hours. Afterward, the sample was transferred to a lyophilization dish and freeze-dried for 24 hours to obtain the pure product.

[0133] The average particle size was determined to be 101.2 ± 0.6 nm and the zeta potential was 1.05 ± 0.05 using a nanoparticle size and zeta potential analyzer. The drug loading of ziconopeptide was determined to be 19.4% using high-performance liquid chromatography.

[0134] Comparative Example 2:

[0135] (1) Synthesis of phospholipid-polyethylene glycol-RVG29:

[0136] A 30*250mm reactor was selected. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis began at the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was then added to the reactor from the previous step using a disposable pipette. The reaction was bubbled under nitrogen for 90 minutes. After the reaction, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for another 20 minutes. Washing was then performed: the liquid in the reactor was evacuated using a circulating water vacuum pump. Industrial-grade DMF was added to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This process was repeated four times. After washing, perform Fmoc removal: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Perform resin testing: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Then, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. Color development indicates successful Fmoc removal; if no color development occurs, repeat the Fmoc removal and subsequent steps. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix. Add this mixture to the dried resin and bubble under nitrogen for 1 hour. Perform a second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove the Fmoc and perform the first wash operation again.

[0137] Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml of N,N-dimethylformamide, mix it and add it to the dried resin, then add 0.5 mmol of N,N-diisopropylethylamine, and bubble the reaction under nitrogen for 3 h.

[0138] The peptides were cleaved from the resin. A cleavage reagent was prepared (using 100 ml of reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 10 mL diethyl ether. For sedimentation, anhydrous diethyl ether was pre-cooled to -20°C. 1 mL of cleavage reagent was used to precipitate the peptides with 10 mL diethyl ether. After drying the resin, the peptides were weighed, and the cleavage buffer was added for 2 hours. The residue was then filtered to obtain the filtrate. The filtrate was then added to diethyl ether to precipitate the peptides, centrifuged, and the crude product was lyophilized.

[0139] Peptide purification was performed using HPLC with a 30 / 250 mm Daisogel 8 μm column. Mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, flow rate: 10 mL / min. Samples were loaded via pump A, followed by equilibration with 10% acetonitrile for 5 min. Gradient elution was then initiated with 10% phase B, increasing the phase B concentration to 55% within 5 minutes and maintaining this concentration for 45 minutes to prepare and collect the target peak.

[0140] Finally, the sample was transferred to a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain a pure product.

[0141] Synthesis of RVG29-modified liposomes: The RVG29-modified liposomes were synthesized using a thin-film hydration method. 4.8 mg of phospholipid-polyethylene glycol-RVG29, 60.7 mg of lecithin, 20.2 mg of cholesterol, and 9.5 mg of phospholipid-polyethylene glycol-carboxyl group were accurately weighed and added to a round-bottom flask. 30 ml of dichloromethane was added and the mixture was sonicated in a water bath until fully dissolved. The solution was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 4.78 mg of ziconopeptide was dissolved in 50 ml of ultrapure water and added to the round-bottom flask. After thorough mixing and resuspending, the mixture was extracted into centrifuge tubes and treated with a 450 W ultrasonic probe for 4 minutes (2 seconds on, 3 seconds off) in an ice bath. After treatment, the samples were stabilized at 4°C for 8 hours. Finally, the samples were transferred to lyophilized dishes and freeze-dried for 24 hours to obtain the pure product.

[0142] The average particle size was determined to be 104.2 ± 0.2 nm and the zeta potential was 4.74 ± 0.12 using a nanoparticle size and zeta potential analyzer. The drug loading of ziconopeptide was determined to be 12.8% using high-performance liquid chromatography.

[0143] Comparative Example 3: rvg29 neuronal targeting (cellular uptake)

[0144] (1) Synthesis of phospholipid-polyethylene glycol-RVG29:

[0145] A 30*250mm reactor was selected. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis began at the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was then added to the reactor from the previous step using a disposable pipette. The reaction was bubbled under nitrogen for 90 minutes. After the reaction, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for another 20 minutes. Washing was then performed: the liquid in the reactor was evacuated using a circulating water vacuum pump. Industrial-grade DMF was added to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This process was repeated four times. After washing, perform Fmoc removal: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Perform resin testing: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Then, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. Color development indicates successful Fmoc removal; if no color development occurs, repeat the Fmoc removal and subsequent steps. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix. Add this mixture to the dried resin and bubble under nitrogen for 1 hour. Perform a second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove the Fmoc and perform the first wash operation again.

[0146] Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml of N,N-dimethylformamide, mix it and add it to the dried resin, then add 0.5 mmol of N,N-diisopropylethylamine, and bubble the reaction under nitrogen for 3 h.

[0147] The peptides were cleaved from the resin. A cleavage reagent was prepared (using 100 ml of reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 10 mL diethyl ether. For sedimentation, anhydrous diethyl ether was pre-cooled to -20°C. 1 mL of cleavage reagent was used to precipitate the peptides with 10 mL diethyl ether. After drying the resin, the peptides were weighed, and the cleavage buffer was added for 2 hours. The residue was then filtered to obtain the filtrate. The filtrate was then added to diethyl ether to precipitate the peptides, centrifuged, and the crude product was lyophilized.

[0148] Peptide purification was performed using HPLC with a 30 / 250 mm Daisogel 8 μm column. Mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, flow rate: 10 mL / min. Samples were loaded via pump A, followed by equilibration with 10% acetonitrile for 5 min. Gradient elution was then initiated with 10% phase B, increasing the phase B concentration to 55% within 5 minutes and maintaining this concentration for 45 minutes to prepare and collect the target peak.

[0149] Finally, the sample was transferred to a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain a pure product.

[0150] (2) Liposome synthesis: The thin-film hydration method was used. 60.7 mg of lecithin, 20.2 mg of cholesterol, and 0.5 mg of cell membrane green fluorescent probe were accurately weighed and added to a flask. Group A had no other components added, while Group B had an additional 4.8 mg of phospholipid-polyethylene glycol-RVG29 added. 30 ml of dichloromethane was added to each group, and the mixture was thoroughly dissolved by sonication in a water bath. The mixture was then rotary evaporated at 40°C and 100 rpm until the organic solvent disappeared, forming a thin film. 50 ml of ultrapure water was added to each of the two flasks, thoroughly mixed, resuspended, and extracted into centrifuge tubes. The flasks were then treated with a 450 W sonication probe for 4 minutes on ice (2 seconds on, 3 seconds off). After treatment, the flasks were stabilized at 4°C for 8 hours. The final results were: Group A - blank liposomes labeled with cell membrane green fluorescent probe; Group B - RVG29 modified liposomes labeled with cell membrane green fluorescent probe.

[0151] Cell uptake validation: Mouse hippocampal neuronal lines and mouse microglia were seeded at a density of 3 × 10⁵ cells per well in 35 mm confocal culture dishes and cultured at 37°C for 24 hours. Once cell confluence reached 60%-70%, groups A and B were co-incubated with the cells at a working concentration of 5 μg / mL for 4 hours. After incubation, the cells were washed three times with pre-cooled PBS buffer to thoroughly remove unintegrated liposomes. Cells were then fixed with 4% paraformaldehyde and treated with 4',6-diamidindo-2-phenylindole.

[0152] Nuclear counterstaining was performed. Finally, fluorescence images were acquired using an inverted laser confocal microscope (Olympus IX81-FV1000) to observe and analyze the intracellular localization and uptake of liposomes. Results are as follows: Figure 9 As shown in (a) and (b), the blank liposomes could not form good colocalization with the two cell lines, while the RVG29 modified liposomes were well localized to the mouse hippocampal neuron line rather than the mouse microglia line, indicating that the RVG29 modified liposomes have good neuronal targeting.

[0153] Comparative Example 4:

[0154] (1) Synthesis of phospholipid-polyethylene glycol-RVG29:

[0155] A 30*250mm reactor was selected. 0.5g of 2-chlorotriphenylmethyl chloride resin was weighed and placed into the reactor, followed by soaking in dichloromethane for 30 minutes. Peptide synthesis began at the C-terminus. 0.15mmol of FMOC-AA-OH was weighed and added to a centrifuge tube, dissolved in 5ml of dichloromethane, and then 0.5mmol of N,N-diisopropylethylamine was added and shaken well. The solution was then added to the reactor from the previous step using a disposable pipette. The reaction was bubbled under nitrogen for 90 minutes. After the reaction, a mixture of 2ml methanol and 4ml dichloromethane was added and reacted for another 20 minutes. Washing was then performed: the liquid in the reactor was evacuated using a circulating water vacuum pump. Industrial-grade DMF was added to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. The reactor was washed under nitrogen bubbling for 30 seconds, and then the liquid in the reactor was evacuated again using a circulating water vacuum pump. This process was repeated four times. After washing, perform Fmoc removal: Add 20% piperidine / N,N-dimethylformamide solution to the reactor using a wash bottle, with the reagent volume approximately three times the resin volume. Bubble the reaction under nitrogen for 20 minutes. Wash again, and during the fifth wash, replace the industrial-grade N,N-dimethylformamide with analytical-grade N,N-dimethylformamide. Perform resin testing: Use a long-necked pipette to place 10-20 resin particles from the reactor at the bottom of a test tube. Then, use a dropper to add two drops each of test reagents A and B to the test tube, ensuring sufficient contact between the resin and the reagents. Heat the test tube at 100℃ for 2 minutes and observe the resin color. Color development indicates successful Fmoc removal; if no color development occurs, repeat the Fmoc removal and subsequent steps. Condensation: Weigh 0.5 mmol FMOC-AA-OH (C-terminal second position) and 0.5 mmol 1-hydroxybenzotriazole into a centrifuge tube, dissolve them thoroughly in 10 ml N,N-dimethylformamide, then add 0.5 mmol dimethyl carbonate and mix. Add this mixture to the dried resin and bubble under nitrogen for 1 hour. Perform a second resin test: Observe the resin color. If there is color, repeat the condensation operation. If there is no color, the ligation is complete. Repeat the first wash, Fmoc removal, second wash, first resin test, condensation, and second resin test until peptide incorporation is complete. Finally, remove the Fmoc and perform the first wash operation again.

[0156] Phospholipid-polyethylene glycol modification: Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester into a centrifuge tube, dissolve it thoroughly in 100 ml of N,N-dimethylformamide, mix it and add it to the dried resin, then add 0.5 mmol of N,N-diisopropylethylamine, and bubble the reaction under nitrogen for 3 h.

[0157] The peptides were cleaved from the resin. A cleavage reagent was prepared (using 100 ml of reagent as an example): 95 mL trifluoroacetic acid + 1 mL water + 2 mL ethylenedithiol + 2 mL triisopropylsilane. The reagent volume was 1 g resin + 10 mL cleavage reagent, followed by centrifugation and washing with 10 mL diethyl ether. For sedimentation, anhydrous diethyl ether was pre-cooled to -20°C. 1 mL of cleavage reagent was used to precipitate the peptides with 10 mL diethyl ether. After drying the resin, the peptides were weighed, and the cleavage buffer was added for 2 hours. The residue was then filtered to obtain the filtrate. The filtrate was then added to diethyl ether to precipitate the peptides, centrifuged, and the crude product was lyophilized.

[0158] Peptide purification was performed using HPLC with a 30 / 250 mm Daisogel 8 μm column. Mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, flow rate: 10 mL / min. Samples were loaded via pump A, followed by equilibration with 10% acetonitrile for 5 min. Gradient elution was then initiated with 10% phase B, increasing the phase B concentration to 55% within 5 minutes and maintaining this concentration for 45 minutes to prepare and collect the target peak.

[0159] Finally, the sample was transferred to a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain a pure product.

[0160] (2) Liposome synthesis: The thin-film hydration method was used. 60.7 mg of lecithin, 20.2 mg of cholesterol, and 1 mg of indocyanine green 5.5 were accurately weighed and added to a flask. Group A was supplemented with 4.8 mg of phospholipid-polyethylene glycol-RVG29, while Group B was not supplemented. 30 ml of dichloromethane was added to each group and the mixture was fully dissolved by sonication in a water bath. The mixture was then rotary evaporated at 40 degrees Celsius and 100 rpm until the organic solvent disappeared, forming a thin film. 50 ml of ultrapure water was added to each of the two flasks, mixed thoroughly, resuspended, and extracted into centrifuge tubes. The mixture was then treated with a 450 W ultrasonic probe in an ice bath for 4 minutes, with a 2-second working and 3-second pause. After treatment, the mixture was placed in a 4-degree Celsius refrigerator for 8 hours to stabilize.

[0161] (3) Preparation of liposome in situ gel

[0162] First, dissolve 0.5 mg of chitosan in 5 ml of 2% glacial acetic acid solution. Then, mix 5 ml of the chitosan solution, 2 mg of Bergsham 407, and 3 ml of deionized water, and stir slowly at 4°C until dissolved. Add deionized water to a total volume of 9 ml. Finally, add 1 ml of liposome suspensions from group A and group B to the above system and stir slowly at 4°C to mix. The final results are: Group A: Indocyanine Green 5.5-labeled RVG29-modified liposome in situ gel formulation; Group B: Blank control.

[0163] Organ distribution: After administering reagents from groups A and B intranasally to mice, and waiting for 4 hours, the mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, and brain tissues were removed. Indocyanine green 5.5 fluorescence was immediately imaged using a small animal in vivo optical imaging system (IVIS Lumina SeiesIII). Fluorescence intensity statistics are shown below. Figure 10 As shown in (a) and (b), the results indicate that the RVG29 modified liposome in situ gel formulation mainly targets the central brain, without significant distribution to important organs throughout the body.

[0164] As can be seen from this implementation, the RVG29-modified zicosinate-loaded liposome of the present invention achieves efficient encapsulation of the positively charged drug zicosinate. Nasal administration of RVG29-modified liposomes is a novel delivery method from the periphery to the brain. Furthermore, the thermosensitive in situ gel system constructed in this invention is particularly suitable for nasal administration of RVG29-modified zicosinate-loaded liposomes, exhibiting good central nervous system targeting without significant systemic exposure.

[0165] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0166] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. An RVG29-modified ziconopeptide-loaded liposome, characterized in that, It is mainly composed of phospholipid-polyethylene glycol-RVG29, lecithin, cholesterol, phosphatidic acid, and ziconopeptide, with the following mass percentages: phospholipid-polyethylene glycol-RVG29 4%-4.85%, lecithin 45%-65.475%, cholesterol 15%-21.825%, phosphatidic acid 4.85%-16%, and ziconopeptide 3-20%.

2. The RVG29-modified ziconopeptide-loaded liposome according to claim 1, characterized in that, The structural formula of the phospholipid-polyethylene glycol-RVG29 is as follows: ； The average molecular weight of polyethylene glycol is 1000 to 10000. RVG29 is a rabies virus glycoprotein polypeptide.

3. The application of the RVG29-modified zicosinate-loaded liposomes according to any one of claims 1-2, characterized in that, Application in the preparation of drugs for treating cancer pain.

4. The application of the RVG29-modified ziconopeptide-loaded liposomes according to any one of claims 1-2, characterized in that, Application in the preparation of ziconopeptide delivery gel formulations.

5. The application according to claim 4, characterized in that, The gel formulation is a thermosensitive in-situ gel for nasal administration.

6. The method for preparing RVG29-modified zicosinate-loaded liposomes according to any one of claims 1-2, characterized in that: The method is implemented through the following scheme: (1) Synthesis of RVG29: The specific steps (1) are as follows: (11) Weigh 0.5g of 2-chlorotriphenylmethyl chloro resin and put it into the reactor, then add dichloromethane and soak for a period of time; (12) Starting from the C-terminus, add each amino acid in the polypeptide RVG29 into the centrifuge tube in sequence. Then weigh 0.15 mmol of organic solvent FMOC-AA-OH into the centrifuge tube, dissolve it with dichloromethane, and then add 0.5 mmol of N,N-diisopropylethylamine and shake well. (13) Using a disposable pipette, add the solution obtained in step (12) into the reactor obtained in step (11). Bubble the reaction with nitrogen for 90 minutes. After the reaction is complete, add a mixture of methanol and dichloromethane and react for 20 minutes. Then wash the reactor. (14) After the liquid in the reactor is drained, industrial grade DMF reagent is added to the reactor using a washing bottle, and then nitrogen is bubbled and washed. (15) Repeat the complete washing operation of step (14) 4 times, and finally end by draining the liquid in the reactor. (16) After washing, remove Fmoc: Add 20% piperidine / N,N-dimethylformamide solution to the reactor obtained in step (15) using a wash bottle. The solution volume should be 3 times the resin volume. Bubble the reaction with nitrogen for 20 minutes. Repeat the washing operation in step (14) once as the fifth wash. In the fifth wash, replace the industrial grade N,N-dimethylformamide with analytical grade N,N-dimethylformamide. (17) Complete resin testing: Place the 2-chlorotriphenylmethyl chloride resin from the reactor at the bottom of the detection tube. Then, use a dropper to add two drops each of test reagent A and B to the detection tube, ensuring full contact between the resin and the reagents. Next, heat the test tube at 100℃ for 2 minutes and observe the resin color. If the resin shows color, the removal of Fmoc is successful, and the next step can be performed. If no color develops, repeat steps (16)-(17) for Fmoc removal and subsequent operations until Fmoc removal is successful; (18) Condensation: Weigh FMOC-AA-OH and 1-hydroxybenzotriazole and add them to a new centrifuge tube. Then dissolve them completely with N,N-dimethylformamide and add dimethyl carbonate. After mixing, add the mixed solution from the new centrifuge tube to the resin dried in step (16) and bubble it with nitrogen for 1 hour. (19) Conduct a second resin test: Observe the resin color: If there is color, repeat the shrinkage operation of step (18) above, and then proceed to the next step; If there is no color, it indicates that the connection is complete. Then, repeat the washing-washing-detection-condensation-detection process for each amino acid. The washing-washing-detection-condensation-detection process first involves washing step (15) once, then repeating steps (16), (17), and (18) until peptide inoculation is completed. Finally, Fmoc removal is performed, and then washing step (15) is performed once to obtain RVG29 resin. (2) Phospholipid-polyethylene glycol modified RVG29: Step (2) specifically involves: (21) Weigh 0.5 mmol of phospholipid-polyethylene glycol-active ester and add it to a centrifuge tube. Dissolve and mix it thoroughly with N,N-dimethylformamide. The mixed solution was added to the RVG29 resin obtained in step (1), and then N,N-diisopropylethylamine was added. The reaction was carried out under nitrogen bubbling for 3 h to obtain phospholipid-polyethylene glycol-RVG29-resin peptide. (22) Cutting the peptide from the resin: Prepare a cutting reagent consisting of trifluoroacetic acid, ethylenedithiol, triisopropylsilane and water. Add the phospholipid-polyethylene glycol-RVG29-resin peptide to the cutting reagent and cut for 2 hours. Then filter to obtain the filtrate. Add the filtrate to anhydrous diethyl ether that has been pre-cooled at -20°C to precipitate and centrifuge to obtain the crude product. Then freeze-dry the crude product. (23) The peptide was purified by high performance liquid chromatography (HPLC). The crude product was loaded onto the mobile phase A of the HPLC column. The HPLC used a 30 / 250 mm Daisogel 8 μm column. The mobile phases were: A: 0.1% TFA / water, B: 0.1% TFA / acetonitrile, and the flow rate was 10 ml / min. Then, after equilibration with 10% acetonitrile water (phase B) and 90% phase A for 5 minutes, gradient elution was started. The initial conditions were 10% phase B and 90% phase A. The volume fraction of phase B was gradually increased to 55% over 45 minutes, while the volume fraction of phase A was gradually decreased to 45% and maintained for 45 minutes to prepare and collect the sample peaks for detection. (24) Finally, the purified crude product was transferred into a freeze-drying dish and freeze-dried in a freeze dryer to obtain pure phospholipid-polyethylene glycol-RVG29. (3) Synthesis of RVG29-modified liposomes: RVG29-modified zicoseptide liposomes were prepared by synthesizing phospholipid-polyethylene glycol-RVG29 pure product, lecithin, cholesterol, phosphatidic acid and zicoseptide through water bath sonication and ultrasonic probe.

7. The preparation method according to claim 6, characterized in that: The specific steps (3) are as follows: (31) Weigh 4-4.85 mg of phospholipid-polyethylene glycol-RVG29, 45-65.475 mg of lecithin, 15-21.825 mg of cholesterol, and 4.85-16 mg of phosphatidic acid and add them to a flask containing dichloromethane. Dissolve the phospholipids by sonication in a water bath and then evaporate them by rotary evaporation at 40 degrees Celsius and 100 rpm until the organic solvent disappears to form a thin film. (32) Dissolve 3-20 mg of ziconopeptide in 40-50 ml of ultrapure water, add it to a flask with a membrane, mix thoroughly, resuspend, and extract into a centrifuge tube. Treat the centrifuge tube with an ultrasonic probe at 400-600 W for 4 minutes. After treatment, place it in a refrigerator at 4 degrees Celsius for 8 hours to stabilize and obtain the sample. (33) After the step (32) is completed, the sample obtained is transferred into a freeze-drying dish and freeze-dried in a freeze dryer for 24 hours to obtain RVG29 modified ziconopeptide liposomes.

8. A method for preparing an in-situ gel formulation of RVG29-modified zicosinate-loaded liposomes based on any one of claims 1-2 or the preparation method of any one of claims 6-7, characterized in that: The method includes: S1. Prepare the RVG29-modified ziconopeptide-loaded liposomes; Preparation of S2, RVG29 modified ziconopeptide-loaded liposome in situ gel: First, chitosan is dissolved in glacial acetic acid solution to form a chitosan solution. The chitosan solution, poloxamer 407 and deionized water are mixed and stirred at 4 degrees Celsius to dissolve. Finally, liposome suspension is added to the above solution and stirred at 4 degrees Celsius to obtain an in-situ gel formulation.

9. The method for preparing the in-situ gel formulation according to claim 8, characterized in that: The chitosan solution contains 0.1 mg / ml of chitosan, and the glacial acetic acid solution in the chitosan solution contains 2 wt% glacial acetic acid. The ratio of chitosan solution, poloxamer 407, deionized water, and liposome suspension is 5 ml: 2 mg: 9 ml: 1 ml.

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