ROS-responsive paclitaxel lipid prodrug, carboxyl liposome and carboxyl solid lipid preparation

Abstract

The invention provides an ROS (reactive oxygen species) response paclitaxel lipid prodrug, a carboxyl liposome and a carboxyl solid lipid preparation. The ROS-responsive paclitaxel lipid prodrug disclosed by the invention has good tumor inflammation environmental response capability, keeps a prodrug structure in a normal physiological environment, releases a parent drug paclitaxel at a high active oxygen site in tumors or inflammations, improves the biological activity of the drug, and reduces systemic side effects at the same time. The carboxyl lipidosome can be used for preparing a lipid nanoparticle preparation with carboxyl on the surface, the carboxyl has a certain bone targeting property due to the calcium ion chelating ability, and meanwhile, the pKa of the carboxyl is smaller than that of a physiological environment, so that the surface of the carboxyl has electronegativity and is not easily adsorbed with in-vivo protein; due to the characteristics, the nano-material has excellent targeting property and stability in vivo. The preparation method of the carboxyl solid lipid preparation is mild in condition, simple and easy to implement, the carboxyl solid lipid preparation can be efficiently enriched at the bone tumor part, paclitaxel is continuously released at the bone tumor part, and tumors are efficiently inhibited.

Description

Technical Field

[0001] The invention relates to the technical field of solid liposome drug delivery, in particular to a ROS-responsive paclitaxel lipid prodrug, a carboxyl liposome and a carboxyl solid lipid preparation. Background Art

[0002] Currently, treatment options for bone tumors primarily include surgery, radiotherapy, and medication. Surgery, while limited to localized lesions, can quickly alleviate pain and symptoms, but it carries the challenges of surgical difficulty and a high risk of recurrence. While radiotherapy can effectively control localized lesions, the radioactive elements present during treatment can damage hematopoietic stem cells in the bone marrow, leading to bone marrow suppression and other complications, hindering subsequent treatment. Currently, medication remains the primary treatment and adjunctive therapy for bone tumors, offering a wide range of applications. It can not only control localized tumors but also effectively kill metastases. Commonly used chemotherapy drugs in clinical practice include fluorouracil, doxorubicin, and paclitaxel. These drugs are poorly water-soluble and poorly absorbed, and the blood-bone barrier in bone tissue results in extremely low bioavailability at the site of bone tumors. Furthermore, these drugs are highly cytotoxic, not only targeting tumor cells but also damaging normal cells. Intravenous injection can produce significant side effects in the human body, including suppression of the immune system and bone marrow, digestive system impairment, and kidney damage. In order to improve the utilization rate of drugs in bone tumors and reduce the systemic toxic side effects of drugs, how to target the delivery of drugs to bone tumor sites and continuously release drugs has received increasing attention.

[0003] Common bone targeting strategies include the use of bisphosphonates, tetracycline and its analogs, nucleic acid aptamers or carboxyl-rich peptides as delivery materials, etc. Bisphosphonates are the most widely used bone-targeted drugs. The bisphosphonic acid group in their molecules can bind to Ca after deprotonation. 2+Chelation to form bidentate chelates gives bisphosphonates a high affinity for hydroxyapatite in bone tissue, allowing them to be retained in bone tissue. However, bisphosphonates require high concentrations for efficacy, and high-dose use carries the risk of osteonecrosis of the jaw. Furthermore, the vicious cycle between tumor cells, osteoblasts, and osteoclasts leads to bone damage that is difficult to recover from. Tetracycline and its analogs also have a high affinity for hydroxyapatite in bone tissue, but their use can have side effects such as inhibiting bone growth, significantly limiting their use in children and adolescents. In addition to bisphosphonates and tetracycline and their analogs, researchers are exploring the use of nucleic acid aptamers as delivery vehicles for chemotherapy drugs. Through specific recognition between aptamers and receptors, binding to target receptors allows drug delivery to bone. However, nucleic acid molecules have a certain degree of immunogenicity, and their use is often accompanied by uncertain immune responses and difficult-to-prevent side effects. In addition, carboxyl-rich peptide materials also have a high affinity for hydroxyapatite in bone tissue. Among them, the more common oligopeptides are aspartic acid and glutamic acid, both of which can also serve as ligands targeting bone tissue. However, aspartic acid and glutamic acid have weaker bone tissue affinity than bisphosphonates and also have corresponding immunogenicity, which carries certain risks in their use. Therefore, bone delivery drugs and their related drug delivery systems still face problems such as high risk of toxic side effects and insufficient targeting capabilities. There is an urgent need to develop a nano-delivery platform that adapts to the bone microenvironment to achieve precise treatment of chemotherapy drugs at the site of bone tumors. Summary of the Invention

[0004] The purpose of the present invention is to provide a ROS-responsive paclitaxel lipid prodrug, carboxyl liposomes and carboxyl solid lipid preparations to solve the problems of large toxic side effects or insufficient targeting ability of current bone-targeted drugs.

[0005] The present invention is achieved in that:

[0006] The present invention provides a ROS-responsive paclitaxel lipid prodrug, the structural formula of which is shown in formula (I):

[0007]

[0008] Wherein, R is unsubstituted or substituted by any of the following groups: C1-C 40 Alkyl, C2-C 40 Alkenyl, C2-C 40 Alkynyl, C3-C 20 Cycloalkyl, C3-C 30 Cycloalkoxy, 3-20 membered heterocyclic oxy, C 6-20 Aromatic group, C 6-20 aryloxy, 5-20 membered heteroaryl, 5-20 membered heteroaryloxy.

[0009] Preferably, the ROS-responsive paclitaxel lipid prodrug includes a racemate, stereoisomer, tautomer, or a pharmaceutically acceptable salt thereof as shown in structural formula (I).

[0010] Preferably, R is C 1-40 A substitution in an alkyl group.

[0011] Preferably, R is C 1-40 A substitution in a straight chain alkyl group.

[0012] Preferably, R is C 15 Straight chain alkyl substitution.

[0013] The present invention also provides a method for preparing the above-mentioned ROS-responsive paclitaxel lipid prodrug, comprising the following steps:

[0014] (1) 2,2'-[(1-methylethylidene)bis(thio)]bis-ethanol is reacted with palmitic acid in the presence of a condensing agent to obtain a dithioketal compound having a hydroxyl group at the end; the reaction formula is as follows:

[0015]

[0016] (2) reacting a dithioketal compound having a hydroxyl terminal with p-nitrophenyl chloroformate in the presence of a base catalyst to obtain a dithioketal compound modified with p-nitrobenzoate; the reaction formula is shown below:

[0017]

[0018] (3) The dithioketal compound modified with p-nitrobenzoate is reacted with paclitaxel under alkaline catalyst conditions to obtain a ROS-responsive paclitaxel lipid prodrug; the reaction formula is shown below:

[0019]

[0020] Furthermore, the condensing agent in step (1) is N,N-dimethyl-4-aminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the solvent for the reaction in step (1) is at least one of dichloromethane and tetrahydrofuran.

[0021] The step (1) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride for 3-10 times each, the organic phase is dried with anhydrous sodium sulfate, filtered, and then concentrated, purified, desolventized, and dried to obtain a dithioketal compound with a hydroxyl terminal.

[0022] Furthermore, the base catalyst in step (2) is at least one of triethylamine, N,N-dimethyl-4-aminopyridine and N,N-diisopropylethylamine; and the reaction solvent in step (2) is at least one of dichloromethane and tetrahydrofuran.

[0023] The step (2) further includes a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride 1-3 times each, the organic phase is dried with anhydrous sodium sulfate, filtered, and then concentrated, purified, desolventized, and dried to obtain a dithioketal compound modified with p-nitrobenzoate.

[0024] Furthermore, the base catalyst in step (3) is at least one of triethylamine, N,N-dimethyl-4-aminopyridine and N,N-diisopropylethylamine; and the reaction solvent in step (3) is at least one of dichloromethane and tetrahydrofuran.

[0025] The step (3) also includes a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride 3-5 times each, the organic phase is dried with anhydrous sodium sulfate, filtered, and then concentrated, purified, desolvated, and dried to obtain the ROS-responsive paclitaxel lipid prodrug.

[0026] Furthermore, in steps (1), (2) and (3), the purification step uses a silica gel column chromatography separation method to purify the product; the developing solvent of the silica gel column chromatography separation method is at least one of petroleum ether and ethyl acetate.

[0027] The present invention provides two carboxyl liposomes, one of which is a single-tail carboxyl liposome, the structural formula of which is shown in the following formula (V).

[0028]

[0029]

[0030] The other is a double-tail carboxyl-terminated liposome, the structural formula of which is shown in the following formula (VI).

[0031]

[0032] Wherein, R in formula (V) and formula (VI) is unsubstituted or substituted by any of the following groups: C1-C 40 Alkyl, C2-C 40 Alkenyl, C2-C 40 Alkynyl, C3-C 20 Cycloalkyl, C3-C 30 Cycloalkoxy, 3-20 membered heterocyclic oxy, C 6-20 Aromatic group, C 6-20Aryloxy, 5-20 membered heteroaryl, 5-20 membered heteroaryloxy, methyl-hexanoic acid, ethyl-hexanoic acid.

[0033] Preferably, the carboxyl liposome comprises a racemate, stereoisomer, tautomer or a pharmaceutically acceptable salt thereof as shown in structural formula (V) or formula (VI).

[0034] Preferably, R is substituted by one of methyl-hexanoic acid and ethyl-hexanoic acid.

[0035] Preferably, R is substituted with one of propionic acid, succinic acid and glutaric acid.

[0036] Preferably, R is substituted with one of linear propionic acid, succinic acid and glutaric acid.

[0037] The preparation method of the above-mentioned single-end carboxyl liposome comprises the following steps:

[0038] (1) β-alanine tert-butyl ester, aspartic acid di-tert-butyl ester and glutamic acid di-tert-butyl ester are subjected to amidation reaction with acryloyl chloride under alkaline conditions to obtain a double-bond modified Boc-protected amino acid compound; the reaction formula is shown below:

[0039]

[0040] Wherein, X is propionate, succinate or glutarate.

[0041] (2) The double-bond modified Boc-protected amino acid compound is subjected to an addition reaction with hexadecyl mercaptan under alkaline conditions to obtain a hexadecyl-modified Boc-protected amino acid derivative; the reaction formula is shown below:

[0042]

[0043] (3) The hexadecyl-modified Boc-protected amino acid derivative is deprotected under acid-catalyzed conditions to obtain a lipid monomer with a single hydrophobic tail modified with a carboxyl group, namely, a single tail carboxyl liposome; the reaction formula is shown below:

[0044]

[0045] Furthermore, the alkaline conditions in step (1) and step (2) are provided by at least one of triethylamine, pyridine and N,N-diisopropylethylamine, and the solvent for the reaction in step (1) and step (2) is at least one of dichloromethane and tetrahydrofuran.

[0046] The step (1) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, removing the precipitate, removing the solvent by vacuum rotary evaporation, redissolving with ethyl acetate, and then washing with saturated ammonium chloride and saturated sodium chloride for 3-5 times; removing the reaction solvent from the organic phase by vacuum rotary evaporation, and then purifying and desolvating the product to obtain a double-bond-modified Boc-protected amino acid compound.

[0047] The step (2) further includes a purification step, which specifically comprises the following steps: after the reaction is completed, concentrating, dissolving, washing, drying, purifying, and desolvating to obtain a hexadecyl-modified Boc-protected amino acid derivative.

[0048] Furthermore, the purification steps in step (1) and step (2) adopt a silica gel column chromatography method to analyze and purify the product; the developing solvent of the silica gel column chromatography method is at least one of petroleum ether and ethyl acetate.

[0049] Furthermore, the acid catalysis condition in step (3) is provided by at least one of trifluoroacetic acid and acetic acid, and the reaction solvent in step (3) is at least one of dichloromethane and tetrahydrofuran.

[0050] The step (3) further includes a purification step, which specifically comprises the following steps: after the reaction is completed, the carboxyl-modified single-tail lipid monomer is obtained by concentration and drying.

[0051] The method for preparing double-end carboxyl liposomes provided by the present invention comprises the following steps:

[0052] (1) (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol, benzyl bromide and a reaction solvent are subjected to a substitution reaction at 0°C under base catalysis. After the reaction is completed, water is used to terminate the reaction to obtain a benzylbenzene-protected cyclic acetone molecule; the reaction formula is as follows:

[0053]

[0054] (2) Decomposing the product obtained in step (1) at a certain temperature under acidic conditions; the reaction formula is as follows:

[0055]

[0056] (3) The product obtained in step (2) is subjected to an esterification reaction with palmitic acid under alkaline conditions and a condensing agent, and the double lipid tail is connected; the reaction formula is shown below:

[0057]

[0058] (4) The product obtained in step (3) is reacted with hydrogen in the presence of a catalyst under a certain pressure (0.2 to 0.6 MPa) to remove the benzyl protection to obtain a hydroxyl-modified double-tailed lipid molecule; the reaction formula is shown below:

[0059]

[0060] (5) The product obtained in step (4) is subjected to an esterification reaction with 3-(triphenylmethylthio)propionic acid under conditions of a base catalyst and a condensing agent; the reaction formula is shown below:

[0061]

[0062] (6) The product obtained in step (5) is subjected to a decomposition reaction under acidic conditions to obtain a thiol-modified double-tailed lipid molecule; the reaction formula is as follows:

[0063]

[0064] (7) The product obtained in step (6) is subjected to an addition reaction with the product obtained in step (1) during the preparation of single-tail carboxyl liposomes under alkaline conditions to obtain a Boc-protected double-tail lipid molecule; the reaction formula is shown below:

[0065]

[0066] (8) The product obtained in step (7) is deprotected from Boc under acidic conditions to obtain a lipid monomer with a carboxyl-modified dipalmitate tail, i.e., a double-tail carboxyl liposome; the reaction formula is shown below:

[0067]

[0068] Furthermore, in step (1), the reaction solvent is at least one of dichloromethane, tetrahydrofuran, toluene, benzene, N,N-dimethylformamide and acetone; and the base catalysis condition is provided by at least one of NaH, K2CO3 or 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0069] The step (1) further comprises a purification step, which specifically comprises the following steps: after the substitution reaction is completed, the reaction solvent is removed by rotary evaporation under reduced pressure, and then the product is purified, concentrated and dried.

[0070] Furthermore, in step (2), the reaction solvent is at least one of water, tetrahydrofuran and acetone; and the acidic condition is provided by at least one of acetic acid, trifluoroacetic acid and hydrochloric acid.

[0071] The step (2) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, the solvent is removed by extraction and vacuum rotary evaporation, and then the product is purified, concentrated and dried.

[0072] Furthermore, in the step (3), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the alkaline condition is 4-dimethylaminopyridine; and the reaction solvent in the step (3) is at least one of dichloromethane and tetrahydrofuran.

[0073] The step (3) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride for 3-5 times, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the product is purified, concentrated, and dried.

[0074] Furthermore, in the step (4), the reaction solvent is at least one of methanol or ethanol; and the catalyst is at least one of platinum or platinum-carbon.

[0075] The step (4) also includes a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is filtered, the solvent is removed by rotary evaporation under reduced pressure, and the product is purified, concentrated, and dried.

[0076] Furthermore, in the step (5), the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and the base catalyst is 4-dimethylaminopyridine; and the reaction solvent in the step (5) is at least one of dichloromethane and tetrahydrofuran.

[0077] The step (5) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride for 3-5 times, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the product is purified, concentrated, and dried.

[0078] Furthermore, the acidic conditions in step (6) are provided by at least one of trifluoroacetic acid or acetic acid, and the reaction solvent in step (6) is at least one of dichloromethane or tetrahydrofuran.

[0079] The step (6) also includes a purification step, which specifically comprises the following steps: concentration, purification and drying after the reaction is completed.

[0080] Furthermore, the alkaline condition in step (7) is provided by triethylamine; and the reaction solvent in step (7) is at least one of dichloromethane and tetrahydrofuran.

[0081] The step (7) further comprises a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction system is washed with saturated ammonium chloride and saturated sodium chloride for 3-5 times, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then the product is purified, concentrated, and dried.

[0082] Furthermore, in steps (1), (2), (3), (4), (5), (6), and (7), the purification step adopts a silica gel column chromatography separation method to purify the product; the developing solvent of the silica gel column chromatography separation method is at least one of petroleum ether or ethyl acetate.

[0083] Furthermore, the acidic condition of step (8) is provided by at least one of trifluoroacetic acid or acetic acid, and the reaction solvent in step (8) is at least one of dichloromethane or tetrahydrofuran.

[0084] The step (8) also includes a purification step, which specifically comprises the following steps: after the reaction is completed, the reaction is concentrated and dried.

[0085] The present invention also provides a bone-targeted carboxyl solid lipid preparation loaded with ROS-responsive paclitaxel lipid prodrug, and the preparation method of the solid lipid preparation is as follows:

[0086] (1) dissolving the ROS-responsive paclitaxel lipid prodrug, at least one carboxyl liposome, other lipid materials, and cholesterol (Chol) in chloroform to obtain a mixed organic phase solution;

[0087] (2) The mixed organic phase solution in step (1) is added dropwise to the aqueous solution. After the addition is completed, an emulsion is formed by fine ultrasonication. The organic solvent chloroform is evaporated by heating in a water bath. After freeze-drying, the solid lipid preparation is sterilized by ultraviolet irradiation.

[0088] The other lipid materials in step (1) are hydrogenated soybean lecithin (HSPC) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0089] In the step (1), the molar ratio of carboxyl liposomes, other lipid materials and cholesterol is 40:21.5:38.5.

[0090] The molar ratio of the other lipid material components hydrogenated soybean phospholipids (HSPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in step (1) is 20:1.5.

[0091] In the step (1), the mass ratio of the ROS-responsive paclitaxel lipid prodrug to hydrogenated soybean phospholipid (HSPC) is 1:4.

[0092] The aqueous solution in step (2) is at least one of PBS or ultrapure water.

[0093] The ultrasonic frequency in step (2) is 35 Hz.

[0094] The volume ratio of chloroform to the aqueous solution is 1:3.

[0095] The particle size of the obtained solid lipid preparation is less than or equal to 600 nm. Preferably, the particle size of the obtained solid lipid preparation is 100-300 nm.

[0096] The carboxyl solid lipid preparation loaded with ROS-responsive paclitaxel lipid prodrug is used as an anti-bone tumor drug.

[0097] Compared with the prior art, the present invention has the following advantages:

[0098] The ROS-responsive paclitaxel lipid prodrug of the present invention has good responsiveness to the tumor inflammation environment, maintains the prodrug structure under normal physiological conditions, releases the parent drug paclitaxel at sites with high reactive oxygen species in tumors or inflammation, thereby enhancing the biological activity of the drug and reducing systemic side effects.

[0099] The carboxyl liposomes of the present invention can be used to prepare lipid nanoparticle preparations with carboxyl groups on the surface. Since the carboxyl group has the ability to chelate calcium ions, it has a certain bone targeting property. At the same time, since the pKa of the carboxyl group is lower than that in the physiological environment, its surface has a negative charge and is not easily adsorbed by proteins in the body. These characteristics give it excellent targeting and stability in the body.

[0100] The carboxyl solid lipid preparation of the present invention has good bone targeting and target in situ drug release capabilities, remains stable in the blood circulation in the body, targets bone tumor sites, and increases in situ retention time. At the same time, the prodrug responds to the highly reactive oxygen environment of the tumor and releases the parent drug paclitaxel. The preparation greatly improves drug utilization, reduces its toxic side effects, and enhances the drug's bone targeting ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figures 1 to 3 They are respectively the hydrogen nuclear magnetic resonance spectra of the compounds represented by formula (II), formula (III) and formula (IV) in Example 1.

[0102] Figures 4 to 10 They are the hydrogen nuclear magnetic resonance spectra of βAlatBuMA, Asp(tBu)2MA, Glu(tBu)2MA, βAlatBu-S-16, βAla-S-16, Asp-S-16, and Glu-S-16 compounds in Example 2, respectively.

[0103] Figures 11 to 16 They are respectively the hydrogen nuclear magnetic resonance spectra of the compounds represented by formula (V), formula (VI), formula (VII), formula (VIII), formula (IX) and formula (X) in Example 3.

[0104] Figures 17 to 20They are the hydrogen nuclear magnetic resonance spectra of Asp(tBu)2-dS-16, Asp-dS-16, βAla-dS-16, and Glu-dS-16 compounds in Example 4, respectively.

[0105] Figure 21 The particle size and potential of Pre-Glu-S16-S, Pre-Glu-dS16-S, Pre-Asp-S16-S, Pre-Asp-dS16-S, Pre-Ala-S16-S and Pre-Ala-dS16-S solid lipid nanoparticles in Example 5.

[0106] Figure 22 This is the in vivo fluorescence distribution diagram of Pre-Glu-S16-S, Pre-Glu-dS16-S, Pre-Asp-S16-S, Pre-Asp-dS16-S, Pre-Ala-S16-S and Pre-Ala-dS16-S solid lipid nanoparticles in Example 6 after 12 hours.

[0107] Figure 23 This is the in vivo fluorescence image of Pre-Glu-S16-S, Pre-Glu-dS16-S, Pre-Asp-dS16-S and Pre-Ala-S16-S solid lipid nanoparticles in Example 6.

[0108] Figure 24 This is the in vitro drug release of the drug-loaded solid lipid nanoparticles Asp-dS16-S and Ala-S16-S in Example 7.

[0109] Figure 25 The cytotoxicity of the drug-loaded solid lipid nanoparticles Asp-dS16-S and Ala-S16-S in Example 8.

[0110] Figure 26 This is a graph showing the tumor inhibition effect of drug-loaded solid lipid nanoparticles Asp-dS16-S and Ala-S16-S in Example 9.

[0111] Figure 27 The changes in body weight, tumor volume, and tumor weight of the model mice after injection of drug-loaded solid lipid nanoparticles Asp-dS16-S and Ala-S16-S in Example 9. DETAILED DESCRIPTION

[0112] Example 1 Preparation of ROS-responsive paclitaxel lipid prodrug.

[0113] 196 mg (1 mmol, 1 eq) of 2,2'-[(1-methylethylidene)bis(thio)]bis-ethanol and 512 mg (2 mmol, 2 eq) of palmitic acid were dissolved in 20 mL of dichloromethane. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (477.5 mg, 2.5 eq) and 4-dimethylaminopyridine (61 mg, 0.5 eq) were added. Reaction progress was monitored by TLC. After 12 h of reaction completion, the mixture was washed with saturated ammonium chloride and brine, dried, and desolventized. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 400 mg of the pure product in an approximately 92% yield.

[0114] The obtained product was identified and characterized by nuclear magnetic resonance technology (see Figure 1 ), the specific results are as follows:

[0115] 1 H NMR(400MHz,Chloroform-d)δ4.24(t,J=7.0Hz,2H),3.79(t,J=6.1Hz,2H),2.87(dt,J=1 0.7, 6.5Hz, 4H), 2.31 (t, J = 7.6Hz, 2H), 1.63 (s, 8H), 1.25 (s, 24H), 0.89 (d, J = 6.6Hz, 3H).

[0116] The structural formula of the above product is shown in the following formula (II).

[0117]

[0118] 218 mg (0.5 mmol, 1 eq) of the product represented by formula (II) and 202 mg (1 mmol, 2 eq) of p-nitrophenyl chloroformate were dissolved in 10 mL of dichloromethane. 51 mg (0.5 mmol, 1 eq) of triethylamine was added, and the reaction progress was monitored by TLC. After 18 h of reaction completion, the mixture was washed with saturated ammonium chloride and brine, dried, and desolventized. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 270 mg of the pure product in an approximately 90% yield.

[0119] The obtained product was identified and characterized by nuclear magnetic resonance technology (see Figure 2 ), the specific results are as follows:

[0120] 1H NMR (400MHz, Chloroform-d) δ8.32–8.25(m,2H),7.43–7.37(m,2H),4.44(t,J=7.0Hz,2H),4.24(t,J=7.0Hz,2H),3. 00(t,J=7.0Hz,2H),2.88(t,J=7.0Hz,2H),2.30(t,J=7.5Hz,2H),1.64(s,8H),1.25(s,24H),0.87(d,J=7.1Hz,3H).

[0121] The structural formula of the above product is shown in the following formula (III).

[0122]

[0123] 120 mg (0.2 mmol, 1 eq) of the product represented by formula (III) above and 171 mg (0.2 mmol, 1 eq) of paclitaxel were dissolved in 10 mL of dichloromethane. 51 mg (0.5 mmol, 1 eq) of triethylamine was added, and the reaction progress was monitored by TLC. After 18 h of complete reaction, the product was washed with saturated ammonium chloride and saturated brine, dried, and desolventized. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 255 mg of the pure product in an approximately 97% yield.

[0124] The obtained product was identified and characterized by nuclear magnetic resonance technology (see Figure 3 ), the specific results are as follows:

[0125] 1H NMR(400MHz,Chloroform-d)δ8.18–8.13(m,2H),7.78–7.73(m,2H),7.63–7.34(m,11H),7.09(d,J=9.4Hz,1H),6.29(d,J=3.2Hz,2H),6 .01(dd,J=9.4,2.7Hz,1H),5.69(d,J=7.1Hz,1H),5.44(d,J=2.7Hz,1H),4.98(dd,J=9.8,2.3Hz,1H),4.45(dt,J=10.9,5.9Hz,1H),4.3 1(q,J=7.4,6.9Hz,3H),4.24–4.14(m,3H),3.82(d,J=6.9Hz,1H),2.89(t,J=6.9Hz,2H),2.82(t,J=6.9Hz,2H),2.61–2.38(m,6H),2.25 (d,J=13.6Hz,6H),1.94(d,J=1.4Hz,3H),1.69(s,3H),1.58(d,J=4.9Hz,11H),1.32–1.21(m,27H),1.14(s,3H),0.88(t,J=6.7Hz,3H).

[0126] The structural formula of the above product is shown in the following formula (IV).

[0127]

[0128]

[0129] The compound represented by formula (IV) is the ROS-responsive paclitaxel lipid prodrug prepared in this example.

[0130] Example 2 Synthesis of single-tail carboxyl liposomes.

[0131] 2.73 g (15 mmol, 1 eq) of β-alanine tert-butyl ester was dissolved in 20 mL of dichloromethane, and 6.3 mL (45 mmol, 3 eq) of triethylamine was slowly added in an ice bath. After the addition was complete, 1.47 mL (18 mmol, 1.2 eq) of acryloyl chloride was added dropwise to the mixture. After reacting for 2 h, the mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was filtered, the filtrate was dried, and ethyl acetate was added to dissolve the mixture. The mixture was washed with saturated ammonium chloride 3-5 times, washed with saturated brine 3-5 times, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 2.4 g of the target product βAlatBuMA with a yield of about 80%.

[0132] The obtained product βAlatBuMA was identified and characterized by nuclear magnetic resonance technology (see Figure 4 ), the specific results are as follows:

[0133] 1 H NMR(400MHz,Chloroform-d)δ6.25(dd,J=17.0,1.5Hz,2H),6.07(dd,J=17.0,10.3Hz,1H) ,5.61(dd,J=10.3,1.5Hz,1H),3.54(q,J=6.1Hz,2H),2.47(t,J=6.0Hz,2H),1.43(s,9H).

[0134] By changing the reaction raw material β-alanine tert-butyl ester to aspartic acid di-tert-butyl ester or glutamate di-tert-butyl ester in equal proportions, two other amino acid tert-butyl ester products containing double bond modifications with different carbon numbers, Asp(tBu)2MA (1.3 g, yield of about 85%) and Glu(tBu)2MA (1.128 g, yield of about 89%), can be prepared.

[0135] The products Asp(tBu)2MA and Glu(tBu)2MA were identified and characterized by nuclear magnetic resonance (NMR) technology (see Figure 5 and Figure 6 ), the specific results are as follows:

[0136] Asp(tBu)2MA: 1 H NMR (400MHz, Chloroform-d) δ6.60 (d, J=8.0Hz, 1H), 6.30 (dd, J=

[0137] 17.1,1.5Hz,1H),6.14(dd,J=17.0,10.2Hz,1H),5.67(dd,J=10.2,1.5Hz,1H),4.75(dt,J=8.2, 4.3Hz, 1H), 2.91 (dd, J=17.1, 4.3Hz, 1H), 2.78 (dd, J=17.1, 4.3Hz, 1H), 1.45 (d, J=10.0Hz, 18H).

[0138] Glu(tBu)2MA: 1 H NMR (400MHz, DMSO-d6) δ8.35 (d, J=7.7Hz, 1H), 6.30 (dd, J=17.1,

[0139] 10.2Hz,1H),6.11(dd,J=17.1,2.1Hz,1H),5.64(dd,J=10.2,2.2Hz,1H),4.23 (m,1H),2.33–2.20(m,2H),1.94(m,1H),1.78(m,1H),1.41(d,J=3.8Hz,18H).

[0140] 589 mg (3 mmol, 1 eq) of βAlatBuMA was dissolved in 2 mL of dichloromethane, and 1.56 g of hexadecylmercaptan (6 mmol, 2 eq) was added, followed by 42 μL of triethylamine (0.3 mmol, 0.1 eq). The reaction progress was monitored by TLC. After 72 h of reaction completion, the solvent was removed and concentrated, and ethyl acetate was added to dissolve the product. The product was washed with saturated ammonium chloride and saturated brine, dried, and concentrated. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain the pure product βAlatBu-S-16 (744 mg, yield of about 54%).

[0141] The product βAlatBu-S-16 was identified and characterized by nuclear magnetic resonance technology (see Figure 7 ), the specific results are as follows:

[0142] 1 H NMR(400MHz,Chloroform-d)δ6.22(s,1H),3.49(q,J=6.0Hz,2H),2.79(t,J=7.3Hz,2H),2.55–2.49(m,2H), 2.48–2.40(m,4H),1.62–1.52(m,3H),1.46(s,9H),1.36(t,J=7.4Hz,2H),1.25(s,24H),0.91–0.85(m,3H).

[0143] 744 mg of βAlatBu-S-16 was dissolved in 2 mL of DCM, and 4 mL of a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio 1:1) was added. The reaction was monitored by TLC. After reacting at room temperature for 2 h, the solvent was removed and concentrated by rotary evaporation to obtain 570 mg of the pure product βAla-S-16 with a yield of approximately 88%.

[0144] The product βAla-S-16 was identified and characterized by nuclear magnetic resonance technology (see Figure 8 ), the specific results are as follows:

[0145] 1 H NMR (400MHz, DMSO-d6) δ7.95(t,J=5.9Hz,1H),3.21(q,J=6.8Hz,2H),2.62(t,J=7.5Hz,2H) ,2.32(dt,J=21.8,7.3Hz,4H),1.46(q,J=7.9Hz,2H),1.22(s,26H),0.84(t,J=6.5Hz,3H).

[0146] By changing the reaction raw material βAlatBuMA to Asp(tBu)2MA and Glu(tBu)2MA in equal proportions, two other carboxyl-modified single-tail lipid monomers Asp-S-16 (709 mg, yield 89%) and Glu-S-16 (367 mg, yield 96%) can be prepared.

[0147] The products Asp-S-16 and Glu-S-16 were identified and characterized by nuclear magnetic resonance technology (see Figure 9 and Figure 10 ), the specific results are as follows:

[0148] Asp-S-16: 1 H NMR(400MHz,DMSO-d6)δ8.18(d,J=7.9Hz,1H),4.46(d,J=8.0Hz,1H),

[0149] 2.62(t,J=7.8Hz,3H),2.37(t,J=7.6Hz,2H),1.22(s,28H),0.84(t,J=6.7Hz,3H).

[0150] Glu-S-16: 1 H NMR (400MHz, DMSO-d6) δ8.15 (d, J=7.9Hz, 1H), 4.21 (dd, J=9.5, 5.4Hz,

[0151] 1H),2.63(t,J=7.6Hz,2H),2.38(q,J=7.3Hz,2H),2.26(t,J=7.8Hz,2H),1.94(dq,J=14.7,7. 7,7.2Hz,1H),1.75(p,J=7.6Hz,1H),1.53–1.44(m,2H),1.22(s,26H),0.84(t,J=6.6Hz,3H).

[0152] Example 3 Synthesis of thiol double-tailed liposomes.

[0153] 725 mg (30.2 mmol, 2 eq) of NaH (60%) was added to a 250 mL flask, along with 50 mL of N,N-dimethylformamide. The mixture was mixed thoroughly in an ice-water bath. 2 g (15.1 mmol, 1 eq) of (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol was gradually added dropwise. After cooling to 0°C, 3.8 g (22.7 mmol, 1.5 eq) of benzyl bromide was added dropwise. After two hours of reaction, the mixture was returned to room temperature and allowed to react overnight. After completion of the reaction, ice water was added dropwise to quench the reaction. The solvent was concentrated by rotary evaporation under reduced pressure, followed by extraction with ethyl acetate, drying over anhydrous sodium sulfate, and filtering. After solvent removal, the crude product was separated by silica gel column chromatography using petroleum ether and ethyl acetate as eluents to obtain 2.4 g of the pure product in an approximately 64% yield.

[0154] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 11 ), the specific results are as follows:

[0155] 1 H NMR(400MHz,Chloroform-d)δ7.35–7.12(m,5H),4.59–4.40(m,2H),4.23(p,J=6.0Hz,1H),3.98(dd,J=8 .3, 6.4Hz, 1H), 3.67 (dd, J=8.3, 6.3Hz, 1H), 3.44 (ddd, J=33.9, 9.8, 5.6Hz, 2H), 1.32 (d, J=23.0Hz, 6H).

[0156] The structural formula of the above product is shown in the following formula (V).

[0157]

[0158] 2.4 g (10.8 mmol, 1 eq) of the product represented by formula (V) was dissolved in 24 mL of 70% acetic acid solution, heated to 65°C, and allowed to react for 2 h. After the reaction was complete, the temperature was cooled to room temperature. Saturated sodium bicarbonate was added to adjust the pH to neutral, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and desolventized. The product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 1.62 g of the pure product in an approximately 82% yield.

[0159] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 12 ), the specific results are as follows:

[0160] 1 H NMR (400MHz, Chloroform-d) δ7.49–7.16 (m, 5H), 4.55 (s, 2H), 3.89 (tt, J = 5.9, 4.0Hz, 1H), 3.76–3.48 (m, 4H).

[0161] The structural formula of the above product is shown in the following formula (VI).

[0162]

[0163] 1.62 g (8.8 mmol, 1 eq) of the product represented by formula (VI) was dissolved in 25 mL of dichloromethane. 4.7 g (22.1 mmol, 2.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.54 g (4.4 mmol, 0.5 eq) of 4-dimethylaminopyridine were added to the solution and mixed for 30 minutes. Then, 5.68 g (22.1 mmol, 2.5 eq) of palmitic acid was added to the solution. After reacting for 24 hours, the mixture was filtered, washed with saturated ammonium chloride, dried over anhydrous sodium sulfate, and desolventized. The mixture was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 3.6 g of the pure product in an approximately 62% yield.

[0164] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 13 ), the specific results are as follows:

[0165] 1 H NMR(400MHz,Chloroform-d)δ7.32(tt,J=11.5,6.0Hz,5H),5.28–5.20(m,1H),4.61–4.49(m,2H),4.38–4.16(m,2H) ,3.63–3.52(m,2H),2.30(dt,J=16.7,7.5Hz,4H),1.60(dt,J=10.7,7.3Hz,4H),1.25(s,48H),0.88(t,J=6.7Hz,6H).

[0166] The structural formula of the above product is shown in the following formula (VII).

[0167]

[0168] 3.6 g (5.5 mmol, 1 eq) of the product represented by formula (VII) was dissolved in 20 mL of ethanol. 360 mg (10% w / w) of Pd / C was weighed and added to a reactor, and the ethanol solution was then poured into the reactor. Hydrogenation was carried out at 0.4 MPa for 6 h, and the reaction was filtered through diatomaceous earth. After concentrating the solvent, the product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 2.3 g of the pure product in a yield of approximately 73%.

[0169] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 14 ), the specific results are as follows:

[0170] 1 H NMR(400MHz,Chloroform-d)δ5.08(p,J=5.0Hz,1H),4.35–4.20(m,2H),3.81–3.68 (m,2H),2.33(dt,J=9.2,7.5Hz,4H),1.63(s,4H),1.26(s,48H),0.92–0.85(m,6H).

[0171] The structural formula of the above product is shown in the following formula (VIII).

[0172]

[0173] 1.2 g (2 mmol, 1 eq) of the product represented by formula (VIII) was dissolved in 10 mL of DCM, and 1.1 g (5 mmol, 2 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 130 mg (1 mmol, 0.5 eq) of 4-dimethylaminopyridine were added. After complete dissolution, 1.4 g (4 mmol, 2 eq) of 3-(tritylthio)propionic acid was added. The reaction progress was monitored by TLC. After 24 h of complete reaction, the mixture was washed with saturated ammonium chloride and brine, dried, and desolventized. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 1.8 g of the pure product in an approximately 99% yield.

[0174] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 15 ), the specific results are as follows:

[0175] 1 H NMR(400MHz,Chloroform-d)δ7.44–7.35(m,6H),7.29–7.22(m,6H),7.22–7.16(m,3H),5.19(ttd,J=6.2,4.3,1.9Hz,1H),4.28–4.18( m,2H),4.13–4.04(m,2H),2.43(t,J=7.3Hz,2H),2.32–2.15(m,6H),1.55(d,J=12.0Hz,4H),1.23(d,J=2.0Hz,48H),0.89–0.82(m,6H).

[0176] The structural formula of the above product is shown in the following formula (IX).

[0177]

[0178] 900 mg (1 mmol, 1 eq) of the product represented by Formula (IX) was dissolved in 2 mL of dichloromethane, and 0.34 mL of trifluoroacetic acid (3 mmol, 3 eq) was added. After stirring for 30 minutes, 0.48 mL of triethylsilane (3 mmol, 3 eq) was added dropwise. The reaction progress was monitored by TLC. After 1 hour of complete reaction, the solvent was removed by rotary evaporation, and the crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain the pure dithiol-terminated lipid (SH-dS-16, 604 mg, approximately 92% yield).

[0179] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 16 ), the specific results are as follows:

[0180] 1 H NMR(400MHz,Chloroform-d)δ5.29(dddd,J=10.2,8.5,6.0,4.2Hz,1H),4.40–4.26(m,2H),4.22–4.09(m,2H),2.77(dtd,J=8.1,6.1 ,2.1Hz,2H),2.71–2.62(m,2H),2.36–2.25(m,4H),1.62(dtd,J=10.3,7.8,7.4,2.5Hz,4H),1.34–1.20(m,48H),0.90–0.86(m,6H).

[0181] The structural formula of the above product is shown in the following formula (X).

[0182]

[0183] Example 4 Synthesis of double-tail carboxyl-terminated liposomes.

[0184] 360 mg (1.2 mmol, 1 eq) of Asp(tBu)2MA was dissolved in 2 mL of dichloromethane, followed by the addition of 1.56 g (2.4 mmol, 2 eq) of SH-dS-16 and 17 μL of triethylamine (0.12 mmol, 0.1 eq). The reaction progress was monitored by TLC. After 96 h of complete reaction, the solvent was removed and the product was dissolved in ethyl acetate. The product was washed with saturated ammonium chloride and saturated brine, dried, and concentrated. The crude product was separated by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain 956 mg of pure Asp(tBu)2-dS-16 in an approximately 82% yield.

[0185] The product was identified and characterized by nuclear magnetic resonance technology (see Figure 17 ), the specific results are as follows:

[0186] 1H NMR(400MHz,Chloroform-d)δ6.60(d,J=7.9Hz,1H),5.30–5.22(m,1H),4.67(dt,J= 8.3,4.3Hz,1H),4.31(ddd,J=12.5,8.6,4.3Hz,2H),4.22–4.11(m,2H),2.95–2.68(m ,6H),2.62(t,J=7.0Hz,2H),2.52(hept,J=7.2Hz,2H),2.31(ddd,J=7.6,6.0,3.5Hz, 4H),1.64–1.57(m,4H),1.45(d,J=5.4Hz,18H),1.25(s,48H),0.87(t,J=6.7Hz,6H).

[0187] 956 mg of Asp(tBu)2-dS-16 was dissolved in 2 mL of DCM, and 4 mL of a mixture of trifluoroacetic acid and dichloromethane (1:1 by volume) was added. The reaction was monitored by TLC. After reacting at room temperature for 2 h, the solvent was removed by rotary evaporation to obtain 715 mg of the pure product Asp-dS-16 in an approximately 87% yield.

[0188] The identification and characterization were performed using nuclear magnetic resonance technology (see Figure 18 ), the specific results are as follows:

[0189] 1 H NMR(400MHz,Chloroform-d)δ5.30(d,J=6.6Hz,1H),4.17(dt,J=11.8,5.2Hz,2H),3.55(q,J=6.1Hz,2H),2.80(dt,J=14.3,7 .1Hz,4H),2.62(q,J=5.8,4.7Hz,4H),2.51(t,J=7.1Hz,2H),2.33(q,J=7.4,6.7Hz,4H),1.25(s,48H),0.87(t,J=6.7Hz,6H).

[0190] By changing the reaction raw material Asp(tBu)2MA to βAlatBuMA or Glu(tBu)2MA in equal proportions, two other carboxyl-modified double-tail lipid monomers βAla-dS-16 (390 mg, yield 90%) and Glu-dS-16 (470 mg, yield 73%) were prepared.

[0191] The identification and characterization were performed using nuclear magnetic resonance technology (see Figure 19 and Figure 20 ), the specific results are as follows:

[0192] βAla-dS-16:1 H NMR (400MHz, Chloroform-d) δ5.28 (s, 1H), 4.31 (d, J = 12.3Hz, 2H), 4.17

[0193] (dt,J=13.4,6.8Hz,2H),3.58–3.51(m,2H),2.78(q,J=8.2,7.7Hz,4H),2.64–2.60(m, 4H), 2.46 (d, J = 7.5Hz, 2H), 2.32 (q, J = 6.9Hz, 4H), 1.25 (s, 48H), 0.87 (t, J = 6.8Hz, 6H).

[0194] Glu-dS-16: 1 H NMR (400MHz, Chloroform-d) δ5.27 (dt, J=10.0, 4.6Hz, 1H), 4.66 (d, J=

[0195] 6.7Hz,1H),4.31(dt,J=10.6,5.3Hz,2H),4.18(dd,J=11.9,5.7Hz,2H),2.89–2.77(m,4H),2.64(t,J=7.1Hz,2H),2.56(dt,J=14 .0,7.0Hz,4H),2.32(t,J=7.6Hz,4H),2.21(tt,J=14.6,7.1Hz,2H),1.60(t,J=7.2Hz,4H),1.25(s,48H),0.88(t,J=6.6Hz,6H).

[0196] Example 5 Solid lipid nanoformulation and preparation and characterization.

[0197] The components cholesterol (Chol), hydrogenated soybean lecithin (HSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and lipid materials (Glu-S-16, Asp-S-16, Ala-S-16 obtained in Example 2 and Ala-dS-16, Glu-dS-16, Asp-dS-16 obtained in Example 4) were dissolved in 2 mL of chloroform at a molar ratio of 38.5%:20%:1.5%:40%, respectively, and then glyceryl monostearate (GMS) was dissolved therein at 1 / 4 of the mass of HSPC, and the total mass of the components was 10 mg. After complete dissolution, 2 mL of chloroform solution containing different lipid materials was dropped into 6 mL of ultrapure water respectively, and emulsified and ultrasonicated for 3 min at an ultrasonic frequency of 35 Hz using a cell crusher. The obtained emulsion was heated in a water bath at 57 ° C with stirring, and chloroform was evaporated and then freeze-dried. After ultraviolet irradiation for 30 min, solid lipid nanoformulations (NPs) Pre-Glu-S16-S, Pre-Asp-S16-S, Pre-Ala-S16-S, Pre-Glu-dS16-S, Pre-Asp-dS16-S and Pre-Ala-dS16-S were obtained, respectively, and stored at 4 ° C. The hydration kinetic particle size, dispersion index and potential of NPs were determined by dynamic light scattering. Figure 21 The preparation method of the drug-loaded solid lipid nanoformulation is the same as above, except that GMS is replaced by the ROS-responsive paclitaxel lipid prodrug obtained in Example 1.

[0198] Dynamic light scattering analysis showed that the particle sizes of Pre-Glu-S16-S, Pre-Glu-dS16-S, Pre-Asp-S16-S, Pre-Asp-dS16-S, and Pre-Ala-S16-S ranged from 200 to 300 nm, with polydispersity coefficients ranging from 0.1 to 0.3. The particle size of Pre-Ala-dS16-S was approximately 600 nm. Zeta potential measurements showed that all NPs were negatively charged, ranging from -20 to -40 mV. In summary, with the exception of Pre-Asp-dS16-S NPs, the remaining NPs maintained a size of approximately 200 nm and were negatively charged, facilitating precise enrichment of bone tumors through the EPR effect and ion chelation effects. Furthermore, all NPs exhibited excellent stability over a two-week period, with their sizes remaining largely unchanged.

[0199] Example 6 NPs bone targeting experiment.

[0200] First, all NPs were preliminarily screened. In order to identify the distribution location of the preparation in the body, 1 mol% of DiD dye was added to the chloroform solution when preparing NPs according to Example 5. The subsequent steps remained the same. Normal female ICR mice were divided into 7 groups and injected intravenously with normal saline and NPs with DiD dye. Fluorescence imaging was performed as shown in Figure 5. Figure 22 As shown. In vivo imaging of mice showed that at 12 hours, the four preparations, Pre-Glu-S16-S, Pre-Glu-dS16-S, Pre-Asp-S16-S, and Pre-Ala-dS16-S, had a large amount of residual fluorescence in the liver, spleen, and lungs, while only a weak fluorescence signal was found in the leg bones. This indicates that these NPs have weak bone targeting abilities and are concentrated in the liver, spleen, and lungs. However, 12 hours after injection, strong fluorescence signals were observed in the leg bones for Pre-Asp-dS16-S and Pre-Ala-S16-S, while the fluorescence signals in the liver, spleen, and lungs were not significantly different from those in the other preparation experimental groups. This indicates that Pre-Asp-dS16-S and Pre-Ala-S16-S have superior bone targeting abilities compared to other preparations.

[0201] Considering that the bone targeting ability of the two preparations Pre-Asp-S16-S and Pre-Ala-dS16-S is weak and the particle size is larger than the other four preparations, these preparations were excluded from the subsequent animal experimental model studies. Mice were imaged at 6, 12, and 24 hours to observe the in vivo distribution and retention time of NPs. Figure 23 As shown. In vivo imaging shows that Pre-Glu-S16-S and Pre-Glu-dS16-S preparations have a large amount of residual fluorescence in the liver, spleen and lung organs at 24 hours, and only weak fluorescence signals in the leg bones, indicating that the bone targeting retention ability of these two NPs is weak. However, Pre-Asp-dS16-S and Pre-Ala-S16-S can still be observed in the leg bones 24 hours after injection. At the same time, the fluorescence signals in the liver, lungs and spleen are not significantly different from those of the other preparation experimental groups. This shows that Pre-Asp-dS16-S and Pre-Ala-S16-S have better bone targeting ability than other preparations.

[0202] Example 7 In vitro hydrolysis experiment of bone-targeted paclitaxel lipid prodrug carboxyl solid lipid formulations (Asp-dS16-S and Ala-S16-S).

[0203] The Asp-dS16-S and Ala-S16-S formulations screened for strong bone targeting ability in Example 6 were used. Samples of Asp-dS16-S (100 μg) and Ala-S16-S (75 μg) were added to 500 μL of release medium (acetonitrile / PBS buffer, pH 7.4, v / v = 1 / 1) and 10 mM H₂O₂ (acetonitrile / PBS buffer, pH 7.4, v / v = 1 / 1). All mixtures were incubated at 37°C on a shaker (150 rpm). At 0, 12, 24, 48, 72, 96, 120, and 144 h, 500 μL of acetonitrile was added to dissolve the released paclitaxel (PTX). The resulting mixed solutions were centrifuged, and the collected supernatant was filtered through an organic filter. Three samples were tested at each time point. The PTX content in the sample was determined by high performance liquid chromatography. The in vitro hydrolysis experiment at different pH values ​​was performed in the same manner as above. The experimental results are shown in Figure 2. Figure 24 As shown ( Figure 24 Corresponding to the release medium containing 10 mM H2O2).

[0204] Experiments demonstrated that Asp-dS16-S and Ala-S16-S exhibited similar hydrolysis behaviors, which depended on the presence of H2O2 in the solution. Within 24 hours of adding reactive oxygen species (ROS), both Asp-dS16-S and Ala-S16-S hydrolyzed and released over 40% of PTX, while in the absence of ROS, less than 3% of PTX was released, confirming the ROS (ROS) responsiveness of the formulation. The hydrolysis data demonstrated that Asp-dS16-S and Ala-S16-S remain stable under normal physiological conditions, preventing premature PTX release. However, when exposed to the high ROS environment at the tumor site, the formulation rapidly releases PTX.

[0205] Example 8 In vitro cytotoxicity.

[0206] The cytotoxicity of Asp-dS16-S and Ala-S16-S against MDA-MB-231 cells and MCF-7 cells was detected by CCK-8. The commercially available preparation Taxol was used as a control. The cells were cultured in a culture medium containing 1% penicillin / streptomycin and 10% fetal bovine serum in an environment of 5% CO2 and 37°C. MDA-MB-231 cells and MCF-7 cells (2000 / well) were inoculated on a 96-well plate and cultured for 24 hours. The culture medium was then removed and replaced with 100 μL of fresh culture medium containing different concentrations of Taxol, Asp-dS16-S or Ala-S16-S. After incubation for 72 hours, the culture medium was discarded and 100 μL of CCK-8 culture medium was added to each well. After incubation in the dark for 1.5 hours, the absorbance at 450 nm was measured using a microplate reader. Untreated cells were used as the blank control group. Cell viability was calculated as (OD sample -OD blank ) / (OD control –OD blank )×100%, where OD is the absorbance of each well at 450 nm.

[0207] like Figure 25 As shown in the data, the toxicity of Taxol is higher than that of Asp-dS16-S and Ala-S16-S; the toxicity of Asp-dS16-S and Ala-S16-S preparations is related to the PTX concentration, but their toxicity is much lower than that of the commercially available preparation Taxol.

[0208] Example 9 In vivo anti-bone tumor activity determination.

[0209] The present invention established an MDA-MB-231 cell bone metastasis model in NU / NU nude mice to evaluate the therapeutic effects of Asp-dS16-S and Ala-S16-S preparations. The specific method is as follows: MDA-MB-231 cells cultured to the logarithmic growth phase were collected, washed three times with PBS, and diluted to 2×10 7 Cell suspension of 100 μg / mL. NU / NU nude mice were anesthetized with 60 mg / kg sodium pentobarbital injected intraperitoneally, and 20 μL of cell suspension was injected into the tibial bone marrow cavity. After the tumor had grown for 6 days, treatment was started, and Asp-dS16-S, Ala-S16-S, commercially available Taxol and normal saline were injected intravenously. The PTX dosage was 10 mg / kg, and the drug was administered once every 3 days for a total of 8 times. The mice were weighed every 3 days to monitor their overall health and growth and development. The tumor volume was also measured to evaluate the anti-tumor effect. The tumor volume was calculated as follows: (length × width 2) / 2. Observation was continued for 30 days. When the tumor grew to a diameter of 1.5 cm, the mice were anesthetized with ethylfluorouracil to reduce pain. The mice were killed using appropriate methods and the tumors of each group were weighed. Figure 26 、 Figure 27 On day 30, the tumor volumes of the untreated group and the Taxol group were approximately 2470 cm 3 and 1753cm 3 The tumor volume of the Asp-dS16 group was approximately 625 cm 3 , slightly smaller than the marketed group and the untreated group; in contrast, the average tumor volume of the Ala-S16-S group was 343 cm 3 No significant weight loss was observed. These results demonstrate that the Ala-S16 formulation exhibits the best anti-tumor efficacy and a favorable safety profile. Tumors excised and weighed after the animals were sacrificed at the end of the experiment demonstrate that the Ala-S16-S formulation effectively inhibits bone tumor growth.

[0210] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A ROS-responsive paclitaxel lipid prodrug, characterized in that Its structural formula is shown in the following formula (I): Wherein, R is unsubstituted or substituted by any of the following groups: C1-C 40 Alkyl, C2-C 40 Alkenyl, C2-C 40 Alkynyl, C3-C 20 Cycloalkyl, C3-C 30 Cycloalkoxy, 3-20 membered heterocyclic oxy, C 6-20 Aromatic group, C 6-20 aryloxy, 5-20 membered heteroaryl, 5-20 membered heteroaryloxy.

2. The ROS-responsive paclitaxel lipid prodrug according to claim 1, characterized in that R is C 15 Straight chain alkyl substitution.

3. The method for preparing the ROS-responsive paclitaxel lipid prodrug according to claim 1, characterized in that: The steps include: (1) reacting 2,2'-[(1-methylethylidene)bis(thio)]bis-ethanol with palmitic acid in the presence of a condensing agent to obtain a dithioketal compound having a terminal hydroxyl group; (2) reacting a dithioketal compound having a hydroxyl terminal with p-nitrophenyl chloroformate under alkaline catalyst conditions to obtain a dithioketal compound modified with p-nitrobenzoate; (3) The dithioketal compound modified with p-nitrobenzoate is reacted with paclitaxel under alkaline catalyst conditions to obtain a ROS-responsive paclitaxel lipid prodrug.

4. A carboxyl liposome, characterized in that: Its structural formula is shown in the following formula (V) or formula (VI): Wherein, R is unsubstituted or substituted by any of the following groups: C1-C 40 Alkyl, C2-C 40 Alkenyl, C2-C 40 Alkynyl, C3-C 20 Cycloalkyl, C3-C 30 Cycloalkoxy, 3-20 membered heterocyclic oxy, C 6-20 Aromatic group, C 6-20 Aryloxy, 5-20 membered heteroaryl, 5-20 membered heteroaryloxy, methyl-hexanoic acid, ethyl-hexanoic acid.

5. Carboxyl liposome according to claim 4, characterized in that, R is substituted by one of linear propionic acid, succinic acid and glutaric acid.

6. A method for preparing the carboxyl liposome represented by formula (V) according to claim 4, characterized in that: The steps include: (1) subjecting β-alanine tert-butyl ester, aspartic acid di-tert-butyl ester or glutamic acid di-tert-butyl ester to an amidation reaction with acryloyl chloride under alkaline conditions to obtain a double-bond modified Boc-protected amino acid compound; (2) reacting a double-bond-modified Boc-protected amino acid compound with hexadecyl mercaptan under alkaline conditions to obtain a hexadecyl-modified Boc-protected amino acid derivative; (3) removing the Boc-protected amino acid derivative modified with hexadecyl under acid catalysis to obtain a lipid monomer with a single hydrophobic tail modified with a carboxyl group, which is the carboxyl liposome represented by formula (V) in claim 4.

7. A method for preparing the carboxyl liposome represented by formula (VI) according to claim 4, characterized in that: The steps include: (1) (S)-(2,2-dimethyl-1,3-dioxolane-4-yl)methanol, benzyl bromide and a reaction solvent undergo a substitution reaction under base catalysis to produce a benzylbenzene-protected cyclic acetone molecule; (2) decomposing the product obtained in step (1) under acidic conditions; (3) esterifying the product obtained in step (2) with palmitic acid under alkaline conditions and a condensing agent to insert a double lipid tail; (4) reacting the product obtained in step (3) with hydrogen under catalyst conditions to remove the benzyl protection to obtain a hydroxyl-modified double-tail lipid molecule; (5) subjecting the product obtained in step (4) to an esterification reaction with 3-(triphenylmethylthio)propionic acid in the presence of a base catalyst and a condensing agent; (6) subjecting the product obtained in step (5) to a decomposition reaction under acidic conditions to obtain a thiol-modified double-tailed lipid molecule; (7) subjecting the product obtained in step (6) to an addition reaction with the product obtained in step (1) of claim 6 in the presence of a base to obtain a Boc-protected double-tail lipid molecule; (8) The product obtained in step (7) is deprotected from Boc under acidic conditions to obtain a lipid monomer having a carboxyl-modified dipalmitate tail, which is the carboxyl liposome represented by formula (VI) in claim 4.

8. A method for preparing a carboxyl solid lipid preparation, characterized in that: The steps include: (1) dissolving the ROS-responsive paclitaxel lipid prodrug of claim 1, at least one carboxyl liposome of claim 4, other lipid materials, and cholesterol in chloroform to obtain a mixed organic phase solution; (2) The mixed organic phase solution in step (1) is added dropwise to the aqueous solution. After the addition is completed, an emulsion is formed by fine ultrasonication, and chloroform is evaporated by heating in a water bath. After freeze-drying, the emulsion is sterilized by ultraviolet irradiation to obtain a carboxyl solid lipid preparation.

9. The method for preparing the carboxyl solid lipid formulation according to claim 8, wherein Other lipid materials in step (1) include hydrogenated soybean lecithin and distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

10. The carboxyl solid lipid preparation prepared according to the method of claim 8 or 9.

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