NK cell membrane coated prodrug self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses an NK cell membrane coated prodrug self-assembly nanoparticle as well as a preparation method and application thereof. The prodrug self-assembled nanoparticles are formed by self-assembling a bufalin carbonate prodrug and a paclitaxel carbonate prodrug independently or jointly through a nanoprecipitation method. The preparation method comprises the following steps: preparing bufalin carbonate prodrug, paclitaxel carbonate prodrug or double-prodrug self-assembled nanoparticles wrapped by an NK cell membrane, and further performing ultrasonic extrusion on the NK cell membrane, so that the nanoparticles are simple in preparation process and high in drug loading capacity. In-vitro cell experiments prove that the NK cell membrane can obviously improve the tumor cell uptake efficiency of the prodrug nanoparticles, enhance the anti-tumor effect of the prodrug nanoparticles, reduce acute toxicity and improve the administration safety, and can be further applied to clinical research in the future.

Description

Technical Field

[0001] The present invention relates to the field of anti-tumor drugs, and in particular to a NK cell membrane-wrapped prodrug self-assembly nanoparticle and a preparation method and application thereof. Background Art

[0002] Small molecule prodrug self-assembled nanoparticles refer to prodrugs that can self-assemble in water to form nanoparticles without the help of other excipients. The assembled nanoparticles can not only solve the problems of difficult encapsulation and low loading, but also can further encapsulate combined drugs to coordinate the ratio of combined drugs. Due to the special microenvironment of tumors, such as high reducibility and acidic environment, small molecule prodrugs containing chemical linkers (such as disulfide bonds) that are sensitive to the tumor microenvironment are designed to remain stable during blood circulation. In the tumor microenvironment, its chemical linker automatically breaks and quickly releases the raw material. Further self-assembly of different small molecule prodrugs to form mixed nanoprodrugs can achieve tumor-directed, fixed-ratio, and precise release at the same rate. However, the design of the linker needs to simultaneously examine its influence on the characteristics of small molecule prodrug self-assembled nanoparticles, such as particle size and stability. Therefore, small molecule prodrug self-assembled nanoparticles need to consider two key characteristics of prodrug self-assembly performance and inductive release.

[0003] NK cell membrane bionic nano-drug delivery is a nano-drug delivery system that uses NK cell membrane to wrap nano-carriers to form a "core-shell" structure. It not only retains the unique advantages of the nano-drug delivery system, but also solves the problem that nano-carriers are easily cleared from the body. At the same time, it adds the biological functions contained in the NK cell membrane [Rao L., Tian R., Chen X. Cell-membrane-mimicking nano-drugs against infectious diseases [J]. ACS nano, 2020, 14 (3): 2569-2574; Fang R. H., Kroll A. V., Gao W., et al. Cell membrane coating nanotechnology [J]. Advanced materials, 2018, 30 (23): 1706759.].

[0004] Paclitaxel and bufalin have good anti-tumor activity when used alone or in combination, but there are no reports on the study of NK cell membrane-encapsulated paclitaxel prodrug and bufalin prodrug self-assembled nanoparticles when used alone or in combination. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a NK cell membrane-wrapped prodrug self-assembled nanoparticle and a preparation method and application thereof, wherein the self-assembled nanoparticle has a simple preparation process and a high drug loading capacity, and significantly improves the selectivity of bufalin and / or paclitaxel to tumor cells, improves the cell uptake efficiency of bufalin prodrug and / or paclitaxel prodrug, and enhances the synergistic effect of bufalin and paclitaxel on tumor cells.

[0006] A NK cell membrane-wrapped prodrug self-assembling nanoparticle, firstly reacting a raw material with p-nitrophenyl chloroformic acid to generate p-nitrophenyl formate, then reacting with linoleic acid diol ester to generate a prodrug, and preparing the corresponding self-assembling nanoparticles with the prodrug through a nanoprecipitation method; finally, preparing the NK cell membrane-wrapped prodrug self-assembling nanoparticles through an ultrasonic extrusion method with the NK cell membrane; the raw material is one of bufalotoxin or paclitaxel or a mixture of the two; the linoleic acid diol ester is linoleic acid 2,2'-dithiodiethanol ester or linoleic acid 1,6-hexanediol ester.

[0007] As an improvement, the synthesis steps of the prodrug are as follows:

[0008] Step 1, weighing linoleic acid and 2,2'-dithiodiethanol or hexanediol according to a molar ratio of 1:2-10, adding anhydrous organic solvent A to dissolve, and then adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, wherein the molar ratio of dicyclohexylcarbodiimide, 4-dimethylaminopyridine to linoleic acid is 1-1.5:0.1-1:1, under sealed conditions, continuously stirring and reacting at a constant temperature of 30° C. for 2-14 hours, and after extraction and column chromatography purification, obtaining the target product linoleic acid diol ester, which is linoleic acid 2,2'-dithiodiethanol ester or linoleic acid 1,6-hexanediol ester;

[0009] Step 2, weighing the raw materials, weighing p-nitrophenyl chloroformic acid according to a molar ratio of 1:1 to 1:2 into a dry single-mouth reaction bottle pre-filled with anhydrous dichloromethane, and then adding an acid binding agent, and the molar ratio of the acid binding agent to the raw material is 1 to 1.5:1, under sealed conditions, continuously stirring and reacting at a constant temperature of 30° C. for 2 to 14 hours, and after extraction and column chromatography purification, the target product is obtained as the raw material-p-nitrophenyl formate;

[0010] Step 3, weigh the raw material obtained in step 2 - p-nitrophenyl formate, weigh 4-dimethylaminopyridine according to a molar ratio of 1:1 to 1:2, and then add it to anhydrous tetrahydrofuran to dissolve, weigh linoleic acid diol ester according to a molar ratio of 1:1 to 2, add it to the above reaction solution, stir and dissolve, seal and react for 4-12 hours under constant temperature and stirring at 45°C, and obtain the corresponding target product prodrug after extraction and column chromatography purification. Among them, the prodrug can be specifically linoleic acid 2,2'-dithiodiethanol-bufalin carbonate prodrug (LA-sso-BFL), linoleic acid 2,2'-dithiodiethanol-paclitaxel carbonate prodrug (LA-sso-PTX), linoleic acid 1,6-hexanediol-bufalin carbonate prodrug (LA-cco-BFL), or linoleic acid 1,6-hexanediol-paclitaxel carbonate prodrug (LA-cco-PTX).

[0011] As an improvement, the organic solvent A in step 1 is one or more mixed solvents selected from anhydrous dichloroethane, anhydrous tetrahydrofuran, and anhydrous dimethyl sulfoxide.

[0012] As an improvement, the preparation method of the prodrug self-assembled nanoparticles is as follows: weigh the target product prodrug and dissolve it in organic solvent B to form a total concentration of 10 to 40 mg / mL, slowly add it dropwise into double distilled water under stirring, the volume ratio of organic solvent B to double distilled water is 1:4-10, spin dry or dialyze to remove the organic solvent, and self-assemble to obtain prodrug self-assembled nanoparticles, wherein the prodrug self-assembled nanoparticles are single prodrug or double prodrug self-assembled nanoparticles.

[0013] A further improvement is that a PEGylating agent is added in a molar ratio of 1 / 20-1 / 5 during stirring.

[0014] A further improvement is that the organic solvent B is one or a mixed solvent of ethanol, acetonitrile, methanol, tetrahydrofuran, and dimethyl sulfoxide.

[0015] A further improvement is that the PEGylation agent is dipalmitoylphosphatidylethanolamine-polyethylene glycol (DSPE 2k ), vitamin E polyethylene glycol succinate (TPGS) or linoleic acid-polyethylene glycol (LA-PEG 2k ).

[0016] As an improvement, the preparation steps of NK cell membrane are as follows: after centrifuging the NK-92MI cells, add a homogenate containing protease inhibitors, put them into ice cubes for homogenization, centrifuge the homogenate at 4°C for 10 minutes at a speed of 500g, then discard the precipitate, re-homogenize the supernatant, centrifuge it again at 4°C for 10 minutes at a speed of 500g, collect the supernatant, centrifuge it at 4°C for 50 minutes at a speed of 28000g, collect the final cell membrane precipitate and resuspend it in sterile phosphate buffer for later use.

[0017] As an improvement, the preparation steps of the ultrasonic extrusion method are as follows: first, the suspension containing NK cell membrane and the prodrug nanoparticle solution are evenly mixed, ultrasonicated for 1-3 minutes, and extruded 10 times under a 400nm filter membrane; then, the mixture is repeatedly extruded 10 times through an extruder under a 200nm filter membrane, and then concentrated by ultrafiltration using an ultrafiltration tube to obtain cell membrane-wrapped prodrug self-assembled nanoparticles.

[0018] Application of any of the above self-assembled nanoparticles of cell membrane-encapsulated prodrugs in the preparation of anti-tumor drugs.

[0019] A pharmaceutical composition comprises the self-assembled nanoparticles of the NK cell membrane-encapsulated prodrug.

[0020] As an improvement, the pharmaceutical combination further comprises at least one pharmaceutically acceptable carrier.

[0021] Beneficial effects:

[0022] Compared with the prior art, the NK cell membrane-encapsulated prodrug self-assembling nanoparticles and the preparation method and application thereof of the present invention have the following advantages:

[0023] 1. The present invention synthesizes paclitaxel or bufalin carbonate prodrug self-assembled nanoparticles, and single prodrug or double prodrug self-assembled nanoparticles can be prepared by nanoprecipitation method, and the nanoparticles have reducing environment-sensitive release characteristics;

[0024] 2. The present invention prepares NK cell membrane-wrapped prodrug self-assembled nanoparticles, further improving the selectivity and cellular uptake rate of single prodrug or double prodrug self-assembled nanoparticles for tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Different prodrug nanoparticles LA-cco-PTX / DSPE in the present invention 2k -NPs, LA-sso-PTX / DSPE 2k -NPs, LA-cco-BFL / DSPE 2k -Nps and LA-sso-BFLDSPE 2k - TEM images of NPs;

[0026] Figure 2 for LA-cco-BFL / DSPE 2k -NPs(A),LA-cco-PTX / DSPE 2k -NPs(B), LA-sso-BFL / DSPE 2k -NPs(C) and LA-sso-PTX / DSPE 2k-Release rate of NPs (D) in 0 mmol and 10 mmol DTT release medium;

[0027] Figure 3 For LssPB / DSPE 2k - Release rate graph of NPs in 0mmol and 10mmol DTT release medium;

[0028] Figure 4 SDS-PAGE (A) and Western immunoblotting (B) of NKCM / LssPB-NPs, where 1 represents MarKer and 2 represents LssPB / DSPE 2k -NPs, 3 represents NKCM / LssPB-NPs, and 4 represents NKCM;

[0029] Figure 5 For LssPB / DSPE 2k -NPs and NKCM / LssPB-NPs. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to specific embodiments.

[0031] The techniques used in the following examples are all conventional techniques in the art, and the materials used are all existing commercially available materials, without special explanation.

[0032] Example 1 Synthesis of Paclitaxel p-nitrophenyl formate (PTX-NPC)

[0033] Accurately weigh paclitaxel (PTX) (1.0 g, 1.17 mmol) and p-nitrophenyl chloroformic acid (NPC) (0.3 g, 1.5 mmol) in a dry single-mouth reaction bottle pre-loaded with 10 mL of dehydrated dichloromethane (DCM). At the same time, pipette N, N-diisopropylethylamine (DIPEA) (227 mg, 1.75 mmol) and add it to stir and dissolve. Then add a stirrer and seal the reaction at a constant temperature of 30 ° C overnight. The reaction solution was separated and purified by thin layer chromatography (TLC). Hexane and ethyl acetate (volume ratio 1:1) were prepared in advance as developing agents. The reaction solution was spot-controlled to find the position of PTX-NPC on the silica gel plate and mark it. Further separation was performed using a silica gel column, and the PTX-NPC spots were collected and concentrated to dryness to obtain the target compound.

[0034] Mass spectrum ESI (-) is m / z = 1017.3306 (MH) - .

[0035] Example 2 Synthesis of Bufalin-p-Nitrophenyl Formate (BFL-NPC)

[0036] Accurately weigh bufalin (BFL) (0.3 g, 0.78 mmol) into a dry single-mouth reaction bottle pre-loaded with 10 mL of dehydrated tetrahydrofuran (THF), and at the same time, pipette pyridine (0.072 g, 0.78 mmol) and add it to stir and dissolve, then add p-nitrophenyl chloroformate (0.156 g, 0.78 mmol), 45 ° C, seal and react for 6 hours. The reaction is complete. Separate and purify by silica gel column chromatography (with petroleum ether / ethyl acetate volume ratio of 2:1 as the mobile phase) to obtain 180 mg of a white solid. The corresponding target compound was synthesized by nuclear magnetic resonance and mass spectrometry identification.

[0037] Mass spectrum ESI (+) is m / z = 552.2581 (M+H) + ; 1 H-NMR 1 H NMR (400MHz, CDCl3) δ8.41-8.24(tt,4H),7.84(dd,J=9.7,2.6Hz,1H,H-22),7.52-7.35(tt,4H),7.24(d,J=1.7Hz, 1H, H-21), 6.27 (d, J = 9.7Hz, 1H, H-23), 5.12 (s, 1H, H-3), 2.57-2.42 (m, 1H), 1.00 (s, 3H, H-19), 0.72 (s, 3H, H-18).

[0038] Example 3 Synthesis of linoleic acid 2,2'-dithiodiethanol ester (LA-ss-OH)

[0039] Weigh 0.6g of linoleic acid, 0.3g of 2-hydroxyethyl disulfide, 0.504g of N,N'-dicyclohexylcarbodiimide (DCC), and 0.065g of 4-dimethylaminopyridine (DMAP) in a dry single-mouth reaction bottle equipped with a stirrer, add 15mL of anhydrous DCM and stir to dissolve, seal and stir to react overnight at a constant temperature of 30°C. Use column chromatography to separate and purify the reaction solution, collect the target points, and finally use a rotary evaporator to remove petroleum ether and ethyl acetate from the target solution to obtain the target LA-ss-OH. The corresponding target compound was synthesized by nuclear magnetic resonance identification.

[0040] Mass spectrum ESI (+) is m / z = 417.2471 (M+H) + ; 1 H NMR (400MHz, CDCl3) δ5.44-5.27(m,4H,-CH=CH-), 4.35(t,2H,-CH2-), 3.89(t,2H,-CH2-), 2.91(dt,J=16,13.6Hz,4H,-ss-CH2-), 2.77(t,2H,-CH2-).

[0041] Example 4 Synthesis of linoleic acid 2,2'-dithiodiethanol-paclitaxel carbonate prodrug (LA-sso-PTX)

[0042] Accurately weigh PTX-NPC (110 mg, 0.11 mmol) and DMAP (17 mg 0.14 mmol) in a dry single-mouth reaction bottle pre-installed with 5 mL of dehydrated DCM and a stirrer. At the same time, take LA-ss-OH (90 mg 0.22 mmol) and add it to the above reaction solution, stir and dissolve, and then seal and react overnight at 45 ° C under constant temperature stirring. Use silica gel column for separation and purification, use n-hexane and ethyl acetate (volume ratio 1:1) as eluents, collect the target points, and finally use a rotary evaporator to remove n-hexane and ethyl acetate from the target solution to obtain the target LA-sso-PTX. The corresponding target compound was synthesized by nuclear magnetic resonance and mass spectrometry identification.

[0043] Mass spectrum ESI (+) is m / z = 1318.5320 (M + Na) + ; 1 H NMR (400MHz, CDCl3)8.14(2H,d,J=7.2Hz),7.77(2H,d,8.8Hz),7.61(1H,m),7.52(3H,m),6.30(1H,s,10-H),6.25(1H,m,13- H),6.01(1H,dd,J=9.2,3.4Hz,3'-H),5.69(1H,d,J=8.8Hz,2-H),5.43(1H,d,J=2.4Hz,2'-H),5.43-5.28(4H,m,-CH=CH-),4. 98(1H,d,J=7.6Hz,5-H),4.5-4.16(4H,dt,-COO-CH2-),2.91(dt,J=16,13.6Hz,4H,-ss-CH2-),2.77(t,2H,-CH2-),2.78(2H ,t,-CH=CH-CH2-CH=CH-),2.56(1H,m,6α-H)2.45(3H,s,4-COCH3),2.30(2H,t,-CH2CO-),2.22(3H,s,10-COCH3),2.05(4H,m,

[0044] -CH2CH=CH-CH2-CH=CH-CH2-),1.94(3H,m),1.68(3H,s,19-H),1.14(3H,s,16-H),0.89(3H,t,-CH3).

[0045] Example 5 Synthesis of linoleic acid 2,2'-dithiodiethanol-bufotoxin carbonate prodrug (LA-sso-BFL)

[0046] Weigh BFL-NPC (220 mg) and DMAP (60 mg) and add them to a dry single-mouth reaction bottle pre-installed with 5 mL of dehydrated DCM and a stirrer. At the same time, take LA-ss-OH (200 mg) and add it to the above reaction solution, stir and dissolve it, and then seal and react overnight at 45 ° C under constant temperature stirring. Use silica gel column for separation and purification, use n-hexane and ethyl acetate (volume ratio 3:1) as eluents, collect the target points, and finally use a rotary evaporator to remove n-hexane and ethyl acetate from the target solution to obtain the target LA-sso-BFL. The corresponding target compound was synthesized by nuclear magnetic resonance and mass spectrometry identification.

[0047] Mass spectrum ESI (+) is m / z = 829.4733 (M+H) + ; 1 H-NMR (400MHz, CDCl3) δ7.84 (dd, J=9.7, 2.5Hz, 1H, H-22), 7.24 (d, J=1.7Hz, 1H, H-21),6.27(d,J=9.8Hz,1H,H-23),5.54-5.26(m,4H,-CH=CH-),4.98(s,1H,H-3 ),4.36(dt,J=17.2,6.6Hz,4H,-COO-CH2-),3.02-2.85(m,4H,-ss-CH2-),2.77( t,J=6.4Hz,2H,-CH2-),0.96(s,3H,H-19),0.89(3H,t,-CH3),0.70(s,3H,H-18).

[0048] Example 6 Preparation of LA-sso-BFL Nanoparticles (LA-sso-BFL-NPs)

[0049] First, a LA-sso-BFL anhydrous ethanol solution with a mixing concentration of 20 mg / mL was prepared.

[0050] Secondly, under stirring at 800 rpm, 200 μL of the anhydrous ethanol solution was sucked up by a pipette and slowly and steadily added dropwise into approximately 2 mL of ultra-high purity distilled water (ultrapure water).

[0051] Finally, the anhydrous ethanol was removed by rotary evaporation using a rotary vacuum evaporator to obtain LA-sso-BFL nanoparticles (LA-sso-BFL-NPs).

[0052] In addition, LA-sso-BFL / DSPE 2k -NPs were prepared in the same manner as above, except that DSPE was added to the LA-sso-BFL anhydrous ethanol. 2k (Final concentration is 5 mg / mL).

[0053] Example 7 Preparation of LA-sso-PTX Nanoparticles (LA-sso-PTX-NPs)

[0054] First, a LA-sso-PTX anhydrous ethanol solution with a mixed concentration of 20 mg / mL was prepared. Secondly, under stirring at 800 rpm, 200 μL of the anhydrous ethanol solution was slowly and steadily added to approximately 2 mL of ultra-high purity distilled water (ultrapure water). Finally, anhydrous ethanol was removed by rotary evaporation using a rotary vacuum evaporator to obtain LA-sso-PTX nanoparticles (LA-sso-PTX-NPs).

[0055] LA-sso-PTX / DSPE 2k The preparation method of -NPs was the same as above, except that DSPE was added to LA-sso-PTX anhydrous ethanol. 2k (Final concentration is 5 mg / mL).

[0056] Example 8 Preparation of LA-sso-PTX / LA-sso-BFL Nanoparticles (LssPB-NPs)

[0057] First, a mixed solution of LA-sso-PTX and LA-sso-BFL in anhydrous ethanol with a total concentration of 20 mmol was prepared, and the molar ratio of the two was 1:1. Secondly, under stirring at 800 rpm, 200 μL of the above anhydrous ethanol solution was slowly and steadily added to ultra-high purity distilled water (ultrapure water) with a volume of about 2 mL. Finally, anhydrous ethanol was removed by rotary evaporation using a rotary vacuum evaporator to obtain LssPB-NPs.

[0058] LssPB / DSPE 2k The preparation method of -NPs was the same as above, except that DSPE was added to the mixed solution of LA-sso-PTX and LA-sso-BFL in anhydrous ethanol. 2k (Final concentration is 5 mg / ml).

[0059] Example 9 Synthesis of 1,6-hexanediol linoleate (LA-cc-OH)

[0060] Weigh 0.6g of linoleic acid, 0.2g of 1,6-hexanediol, 0.5g of N,N'-dicyclohexylcarbodiimide (DCC), and 0.065g of 4-dimethylaminopyridine (DMAP) in a dry single-mouth reaction bottle equipped with a stirrer, add 8mL of anhydrous DCM and stir to dissolve, seal and stir to react overnight at a constant temperature of 30°C. Use column chromatography to separate and purify the reaction solution, collect the target points, and finally use a rotary evaporator to remove petroleum ether and ethyl acetate from the target solution to obtain the target LA-cc-OH. The corresponding target compound was synthesized by nuclear magnetic resonance identification.

[0061] Mass spectrum ESI (+) is m / z = 381.3375 (M+H) + ; 1 H NMR (400MHz, CDCl3) δ5.38-5.33(m,4H,-CH=CH-), 4.07(t,2H,-CH2-), 3.64(t,2H,-CH2-), 2.77(t,2H,-CH2-).

[0062] Example 10 Synthesis of linoleic acid 1,6-hexanediol-paclitaxel carbonate prodrug (LA-cco-PTX)

[0063] Accurately weigh PTX-NPC (110 mg, 0.11 mmol) and DMAP (17 mg 0.14 mmol) in a dry single-mouth reaction bottle pre-installed with 5 mL of dehydrated DCM and a stirrer, and at the same time, take LA-cc-OH (90 mg 0.21 mmol) and add it to the above reaction solution, stir and dissolve, and then seal and react overnight at 45 ° C. Use a silica gel column for separation and purification, use n-hexane and ethyl acetate (volume ratio 1:1) as eluents, collect the target points, and finally use a rotary evaporator to remove n-hexane and ethyl acetate from the target solution to obtain the target LA-cco-PTX.

[0064] Mass spectrum ESI (+) is m / z = 1282.6288 (M + Na) + ; 1H-NMR(400MHz, CDCl3)8.15(2H,d,J=7.2Hz),7.75(2H,d,8.8Hz),7.61(t,J=7 .4Hz,2H),7.52(3H,m),6.96(d,J=9.3Hz,1H),6.30(1H,s,10-H),6.25(1H,m,1 3-H),5.99(1H,dd,J=9.3,2.5Hz,3'-H),5.69(1H,d,J=8.8Hz,2-H),5.42(1H, d,J=2.4Hz,2'-H),5.41-5.29(4H,m,-CH=CH-),4.98(1H,d,J=7.6Hz,5-H),4.1 5(2H,m,-COO-CH2-),4.04(t,J=6.7Hz,2H,-COO-CH2-),3.82(d,J=7.0Hz,1H) ,2.77(t,2H,-CH=CH-CH2-CH=CH-),2.56(1H,m,6α-H)2.47(3H,s,4-COCH3),2. 29(2H,t,-CH2CO-),2.23(3H,s,10-COCH3),2.05(4H,m,-CH2CH=CH-CH2-CH=CH -CH2-),1.94(3H,m),1.68(3H,s,19-H),1.14(3H,s,16-H),0.89(3H,t,-CH3).

[0065] Example 11 Synthesis of linoleic acid 1,6-hexanediol-bufalin carbonate prodrug (LA-cco-BFL)

[0066] Weigh BFL-NPC (220 mg) and DMAP (60 mg) and add them to a dry single-mouth reaction bottle pre-installed with 5 mL of dehydrated DCM and a stirrer. At the same time, take LA-cc-OH (200 mg) and add it to the above reaction solution, stir and dissolve it, and then seal and react overnight at 45°C under constant temperature stirring. Use a silica gel column for separation and purification, use n-hexane and ethyl acetate (volume ratio 3:1) as eluents, collect the target points, and finally use a rotary evaporator to remove n-hexane and ethyl acetate from the target solution to obtain the target LA-cco-BFL. The corresponding target compound was synthesized by nuclear magnetic resonance identification.

[0067] Mass spectrum ESI (+) is m / z = 793.5623 (M+H) + ; 1H-NMR (400MHz, CDCl3) δ7.85 (dd, J=9.7, 2.5Hz, 1H, H-22), 7.24 (d, J=1.7Hz, 1H, H-21), 6.27 (d, J=9.6Hz, 1H, H-23), 5.57-5.27 (m, 4H, -CH=CH-), 4.96 (s,1H,H-3),4.14(ddt,J=23.5,13.4,5.6Hz,4H,-COO-CH2-),2.77(t,J=6 .5Hz,2H,-CH2-),0.96(s,3H,H-19),0.89(3H,t,-CH3),0.70(s,3H,H-18).

[0068] Example 12 Preparation of LA-cco-BFL Nanoparticles (LA-cco-BFL-NPs)

[0069] First, prepare a LA-cco-BFL anhydrous ethanol solution with a mixed concentration of 20 mg / mL. Secondly, under stirring at 800 rpm, use a pipette to draw 200 μL of the anhydrous ethanol solution and slowly and steadily add it dropwise into 2 mL of ultra-high purity distilled water (ultrapure water). Finally, use a rotary vacuum evaporator to perform rotary evaporation to remove the anhydrous ethanol, thereby obtaining LA-cco-BFL nanoparticles (LA-cco-BFL-NPs).

[0070] In addition, LA-cco-BFL / DSPE 2k The preparation method of -NPs was the same as above, except that DSPE was added to the LA-cco-BFL anhydrous ethanol. 2k (Final concentration is 5 mg / mL).

[0071] Example 13 Preparation of LA-cco-PTX Nanoparticles (LA-cco-PTX-NPs)

[0072] First, a LA-cco-PTX anhydrous ethanol solution with a mixed concentration of 20 mg / mL was prepared. Secondly, under stirring at 800 rpm, 200 μL of the anhydrous ethanol solution was slowly and steadily added to 2 mL of ultra-high purity distilled water (ultrapure water). Finally, anhydrous ethanol was removed by rotary evaporation using a rotary vacuum evaporator to obtain LA-cco-PTX nanoparticles (LA-cco-PTX-NPs).

[0073] The preparation method of LA-cco-PTX / DSPE2k-NPs was the same as above, except that DSPE2k (final concentration was 5 mg / mL) was added to the anhydrous ethanol of LA-cco-PTX.

[0074] Example 14 Preparation of LA-cco-PTX / LA-cco-BFL Nanoparticles (LccPB-NPs)

[0075] First, a mixed solution of LA-cco-PTX and LA-cco-BFL in anhydrous ethanol with a total concentration of 20 mmol was prepared, and the molar ratio of the two was 1:1. Secondly, under stirring at 800 rpm, 200 μL of the above anhydrous ethanol solution was slowly and steadily added to 2 mL of ultra-high purity distilled water (ultrapure water). Finally, anhydrous ethanol was removed by rotary evaporation using a rotary vacuum evaporator to obtain LccPB-NPs.

[0076] LccPB / DSPE 2k- The preparation method of NPs was the same as above, except that DSPE was added to the mixed solution of LA-cco-PTX and LA-cco-BFL in anhydrous ethanol. 2k (Final concentration is 5 mg / ml).

[0077] Example 15 Characterization of Prodrug Nanoparticles

[0078] The particle size and Zeta potential of the nanoparticles were measured using a nanoparticle size analyzer (Bruke particle size and Zeta potential analyzer) using the principle of dynamic light scattering. Steps: First, dilute the prepared prodrug nanoparticles with ultrapure water to an appropriate concentration (0.05 mg / mL), place them in a nanoparticle size analyzer to measure the light intensity distribution diagram, and use a transmission electron microscope to observe the size and surface of the nanoparticles. The test steps are as follows: On a copper mesh, drop an appropriate amount of diluted prodrug nanoparticles (0.5 mg / mL) and dry with infrared. Place the copper mesh under a transmission electron microscope to carefully observe its appearance and take pictures. Figure 1 It shows that the four nanomorphologies are regular, round and evenly dispersed.

[0079] The results are as follows: LA-cco-PTX / DSPE 2k -NPs have a particle size of 173.9±1.323nm, a PDI of 0.09±0.019, and a Zeta of -18.9±2.7mV. 2k -NPs have a particle size of 126.5±2.72nm, a PDI of 0.206±0.009, and a Zeta of -11.7±1.33mV. 2k -NPs have a particle size of 274.4±2.75nm, a PDI of 0.134±0.025, and a Zeta of -7.16±0.497mV. 2kThe particle size of -NPs is 150.2±1.664nm, PDI is 0.095±0.012, and Zeta is -21.6±0.153mV. The particle size of LSSPB-NPs is 108.2±0.513nm, PDI is 0.1±0.016, and Zeta is -3.9±0.43mV.

[0080] Example 16 LA-cco-PTX / DSPE 2k- NPs or LA-sso-PTX / DSPE 2k In vitro release of NPs

[0081] Take 0.1 mL of LA-cco-PTX / DSPE obtained in Example 2 2k -NPs or LA-sso-PTX / DSPE 2k -NPs sample (concentration of 1 mg / mL), then placed in 2 mL of 30% ethanol-0.01M PBS (pH=7.4), wherein the PBS contains dithiothreitol (DTT) at concentrations of (0 mM and 10 mM). After completing the above operations, place in a water bath (37°C), shake at a speed of 100 rpm, and finally select the following: 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h, these 9 release time points, take 100 μL of release solution and add 300 μL of acetonitrile, first mix evenly with a vortex mixer, then centrifuge with a centrifuge, and use a high performance liquid chromatograph to determine the content of paclitaxel, and calculate the release rate through the experimental data.

[0082] LA-cco-PTX / DSPE 2k -NPs hardly released in the release medium with or without DTT, while LA-sso-PTX / DSPE 2k -NPs have a release effect in different concentrations of DDT solution, especially in 10mmol, the release is most significant, such as Figure 2 (A) The release rate reached more than 80% in the first 4 h, indicating that the paclitaxel prodrug containing a disulfide bond has sensitive reduction responsiveness.

[0083] Example 17 LA-cco-BFL / DSPE 2k -NPs or LA-sso-BFL / DSPE 2k In vitro release of NPs

[0084] Take 0.1 mL of LA-cco-BFL / DSPE obtained in Example 2 2k -NPs or LA-sso-BFL / DSPE 2k-NPs sample (concentration of 1 mg / mL), then placed in a volume of 2 mL of 30% ethanol-0.01M PBS (pH = 7.4), where the PBS contains DTT at concentrations of (0 mM and 10 mM). After completing the above operations, place it in a water bath (37°C) and shake it at a speed of 100 rpm. Finally, select the following 9 release time points: 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h. Take 100 μL of release solution and add 300 μL of acetonitrile. First mix it evenly with a vortex mixer, then centrifuge it with a centrifuge, and use a high performance liquid chromatograph to determine the content of toad venom. The release rate is calculated based on the experimentally measured data. LA-cco-PTX / DSPE 2k -NPs were hardly released in the release medium with different concentrations of DTT, while LA-sso-PTX / DSPE 2k -NPs have a release effect in different concentrations of DDT solution, especially in 10mmol, the release is most significant, such as Figure 2 (B) The release rate reached more than 80% in the first 4 h, indicating that the paclitaxel prodrug containing a disulfide bond has sensitive reduction responsiveness.

[0085] Example 18 In vitro release of LssPB-NPs

[0086] Take 0.1mL of the LssPB-NPs sample obtained in Example 2 (concentration of 1mg / mL), and then put it into 2mL of 30% ethanol-0.01M PBS (pH=7.4), wherein the concentration of DTT contained in PBS is (0mM and 10mM). After completing the above operation, place it in a water bath (37°C) and shake it at a speed of 100rpm. Finally, select the following: 0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h, these 9 release time points, take 100μL of release solution and add 300μL of acetonitrile, first mix it evenly with a vortex mixer, then centrifuge it with a centrifuge, and use a high performance liquid chromatograph to determine the content of bufalin and paclitaxel, and calculate the release rate through the experimental data.

[0087] The results are as follows Figure 3 As shown in the figure, it can be seen that LssPB-NPs hardly release paclitaxel and bufalotoxin in a solution without DDT, but quickly release paclitaxel and bufalotoxin in a 10 mmol release medium, and the release rate reaches more than 80% in the first 4 hours, indicating that LssPB-NPs containing disulfide bonds have sensitive reduction responsiveness and can release paclitaxel and bufalotoxin synchronously.

[0088] Example 19 Preparation of NK cell membrane

[0089] NK-92MI cells purchased from Shanghai Gaining Biotechnology Co., Ltd. were revived and cultured to a number of 1×10 8 After centrifugation, add homogenate (containing protease inhibitor mixture, 20μL / 4mL), put it in ice and homogenize it. The homogenate is centrifuged at 4°C for 10 minutes at a speed of 500g. Then, the precipitate is discarded, the supernatant is re-homogenized, centrifuged again at 4°C for 10 minutes at a speed of 500g, the supernatant is collected, and centrifuged at 4°C for 50 minutes at a speed of 28000g. The final cell membrane precipitate is collected and resuspended in sterile phosphate buffered saline (PBS) for subsequent experiments. The content of cell membrane protein and supernatant protein components is significantly different by SDS-PAGE electrophoresis analysis.

[0090] Example 20 Preparation and Characterization of NK Cell Membrane-Encapsulated LssPB-NPs

[0091] First, the NK cell membrane-containing suspension (protein content of 1 mg / mL) and the prodrug nanoparticle solution (1 mM) were mixed evenly, and then ultrasonic power was 200 W, ultrasound was performed for 3 min, and extrusion was performed 10 times under a 400 nm filter membrane; then, the mixture was repeatedly extruded 10 times under a 200 nm filter membrane, and then concentrated by ultrafiltration using an ultrafiltration tube to obtain cell membrane-wrapped prodrug self-assembled nanoparticles, which were labeled as NKCM / LssPB-NPs.

[0092] The particle size, dispersion coefficient, and Zeta potential of NKCM / LssPB-NPs were measured using a nanoparticle size potential analyzer. The particle size of NKCM / LSSPB-NPs was 103.2±0.703nm, and the PDI was 0.119±0.015. Zeta was -9±2.64mV. SDS-PAGE and Western Blot were used to simultaneously detect the protein types of NKCM, LssPB-NPs, and NKCM / LssPB-NPs. The surface of NKCM / LssPB-NPs was covered with NK cell membrane-related protein components ( Figure 4 ). Transmission electron microscopy confirmed that the surface of the nanoparticles was covered with NK cell membranes ( Figure 5 ).

[0093] Example 21 Cytotoxicity Evaluation

[0094] HepG2 human liver cancer cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) in the vigorous logarithmic growth phase were obtained and digested with trypsin. The cells were centrifuged at 1500 rpm for 5 min and resuspended in 10% FBSDMEM (high glucose) medium. The cell concentration was adjusted to 5 × 10 4 / mL, and then added to a 96-well cell culture plate, 100μL per well, and continued to culture in a cell culture incubator (37°C, 5% CO2) for 24h. Aspirate the culture medium, add 100μL of 10% FBSDMEM (high glucose) culture medium containing the same concentration of the test drug (bufalin, paclitaxel, bufalin + paclitaxel group, different prodrug nanoparticle solutions, among which the bufalin + paclitaxel group is mixed in equal molar ratios, and the total concentration after mixing is the same as that of a single prodrug nanoparticle solution), and add a culture medium with a concentration of 0.1% DMSO to the negative control group. Each group has 3 replicates. After continuing to culture for 48h after adding the drug, add 20μL of 5mg / mL MTT reagent to each well and place it at 37°C for 4h. Add 100μL of DMSO to each well, shake and mix, and measure the absorbance value of each well at a wavelength of 570nm.

[0095] Calculate the cell growth inhibition rate:

[0096] Inhibition rate (%) = (1-absorbance of the test drug group / absorbance of the negative group) × 100%.

[0097] The results showed that when the concentration of the tested drug was 0.1 μM, the average inhibition rate of cell growth of bufalin was 40.1%, and LA-cco-BFL / DSPE 2k- NPs and LA-sso-BFL / DSPE 2k The average cell growth inhibition rates of LA-sso-BFL / DSPE2k-NPs were 8.7% and 33.2%, respectively. This indicates that LA-sso-BFL / DSPE2k-NPs maintained the anti-tumor activity of bufalin. The average cell growth inhibition rate of paclitaxel was 27.0%, and that of LA-cco-PTX / DSPE 2k -NPs and LA-sso-PTX / DSPE 2k -NPs had an average cell growth inhibition rate of 5.4% and 24.6%, respectively. 2k- NPs maintained the antitumor activity of paclitaxel. The average cell growth inhibition rate of the combination group of bufalin and paclitaxel was 30.2%, the average cell growth inhibition rate of the LssPB-NPs group was 28.3%, and the average cell growth inhibition rate of the NKCM / LssPB-NPs group was 37.2%, indicating that NK cell membrane-modified prodrug nanoparticles can significantly improve the antitumor effect of the combination of the two drugs.

[0098] Example 22 Cellular uptake experiment

[0099] LssPB-NPs@C-6 loaded with coumarin-6 (C-6) was prepared by nano-coprecipitation method. The specific preparation method was similar to that in Example 8. A certain proportion of C-6 and prodrug were dissolved in anhydrous ethanol, and the concentration of C-6 was 2 mM. NKCM / LssPB-NPs@C-6 wrapped with NK cell membrane was further prepared, and the corresponding C-6 concentration was determined by liquid phase. According to the HepG2 cell suspension (5×103 / well), it was added to a 96-well plate and cultured for 24 hours. The culture medium was aspirated, and LssPB-NPs@C-6 and NKCM / LssPB-NPs@C-6 were added. After administration, the fluorescence intensity of tumor cells was observed by fluorescence microscopy at different time points (0.5h, 1h, 3h, 6h), and statistical data were obtained by Image software.

[0100] The results showed that the fluorescence intensity of the NKCM / LssPB-NPs@C-6 group was significantly stronger than that of the LssPB-NPs@C-6 group at different time points, indicating that NKCM / LssPB-NPs significantly improved the uptake efficiency of double prodrug nanoparticles in HepG2 cells.

[0101] Example 23 Feedback on drug administration safety from the median lethal dose

[0102] 120 healthy KM mice, half male and half female, were taken and raised separately. After the mice adapted to the laboratory environment for one week, they were randomly divided into 6 groups, 10 mice in each group, half male and half female. One group was the control group of bufotolin-0.05% TPGS solution, and the mice were intravenously injected with a dose of 0.88-5 mg / kg; the other group was the NKCM / LssPB-NPs administration group, and the total dose of intravenous administration of mice was 3.2-20 mg / kg. The administration volume was 0.2 mL / 20 g body weight, and the animals were fasted and deprived of water for 3 hours after administration. The possible toxic reactions and deaths of the animals immediately after administration and within 14 days were carefully observed and recorded in detail.

[0103] The results showed that when the total dosage of NKCM / LssPB-NPs was 20 mg / kg, the mortality rate was 0, indicating that the median lethal dose was greater than 20 mg / kg. The median lethal dose of bufalotoxin was 1.85 mg / kg. It can be seen that the NKCM / LssPB-NPs of the present invention effectively improve the safety of drug administration.

[0104] In summary, the present invention further provides the characterization of bufalin carbonate prodrug self-assembled nanoparticles, paclitaxel carbonate prodrug self-assembled nanoparticles and double prodrug self-assembled nanoparticles. The average particle size of bufalin carbonate prodrug self-assembled nanoparticles and paclitaxel carbonate prodrug self-assembled nanoparticles determined by dynamic light scattering is 120-270nm, and the zeta potential is -7 to -21mV. Transmission electron microscopy observation shows that the morphology of the nanoparticles is regular spherical and has good dispersibility. The average particle size of the double prodrug self-assembled nanoparticles is 108nm, which is significantly lower than that of the single prodrug self-assembled nanoparticles.

[0105] The present invention provides a study on the in vitro release characteristics of bufalin carbonate prodrug self-assembled nanoparticles, paclitaxel carbonate prodrug self-assembled nanoparticles or double prodrug self-assembled nanoparticles. 2k -NPs or LA-sso-BFL / DSPE 2k -NPs) in PBS (pH = 7.4) medium, the drug release and degradation rate increased with the increase of reducing agent concentration at 10 nM, showing a certain reduction sensitivity. 2k -NPs) can release paclitaxel and bufalin synchronously.

[0106] The present invention provides a preparation method and characterization of NK cell membrane-encapsulated double prodrug self-assembled nanoparticles. Through tumor cell proliferation experiments and acute toxicity evaluations, it is shown that the NK cell membrane-encapsulated double prodrug self-assembled nanoparticles provided by the present invention can enhance the killing effect of the double prodrug self-assembled nanoparticles on tumor cells, and the median lethal dose of the nanoparticles to mice is significantly higher than that of the original drug. It can be used to prepare products related to the prevention or treatment of liver cancer.

[0107] The above is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A NK cell membrane-encapsulated prodrug self-assembled nanoparticle, characterized in that: Firstly, the raw material is reacted with p-nitrophenyl chloroformic acid to generate p-nitrophenyl formate, and then reacted with linoleic acid diol ester to generate a prodrug, and the prodrug is prepared by a nanoprecipitation method to obtain corresponding self-assembled nanoparticles; finally, the NK cell membrane is reacted with an ultrasonic extrusion method to obtain self-assembled nanoparticles of the prodrug wrapped in the NK cell membrane; the raw material is one of bufalotoxin or paclitaxel or a mixture of the two; the linoleic acid diol ester is linoleic acid 2,2'-dithiodiethanol ester or linoleic acid 1,6-hexanediol ester.

2. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 1, characterized in that: The synthesis steps of the prodrug are as follows: Step 1, weighing linoleic acid and 2,2'-dithiodiethanol or hexanediol according to a molar ratio of 1:2-10, adding anhydrous organic solvent A to dissolve, and then adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, wherein the molar ratio of dicyclohexylcarbodiimide, 4-dimethylaminopyridine to linoleic acid is 1-1.5:0.1-1:1, under sealed conditions, continuously stirring and reacting at a constant temperature of 30° C. for 2-14 hours, and after extraction and column chromatography purification, obtaining the target product linoleic acid diol ester, which is linoleic acid 2,2'-dithiodiethanol ester or linoleic acid 1,6-hexanediol ester; Step 2, weighing the raw materials, weighing p-nitrophenyl chloroformic acid according to a molar ratio of 1:1 to 1:2 into a dry single-mouth reaction bottle pre-filled with anhydrous dichloromethane, and then adding an acid binding agent, and the molar ratio of the acid binding agent to the raw material is 1 to 1.5:1, under sealed conditions, continuously stirring and reacting at a constant temperature of 30° C. for 2 to 14 hours, and after extraction and column chromatography purification, the target product is obtained as the raw material-p-nitrophenyl formate; Step 3, weigh the raw material obtained in step 2 - p-nitrophenyl formate, weigh 4-dimethylaminopyridine according to a molar ratio of 1:1 to 1:2, and then add it to anhydrous tetrahydrofuran to dissolve, weigh linoleic acid glycol ester according to a molar ratio of 1:1 to 2, add it to the above reaction solution, stir and dissolve, and seal the reaction under constant temperature stirring at 45° C. for 4-12 hours. After extraction and column chromatography purification, the corresponding target product prodrugs are obtained.

3. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 2, characterized in that: In step 1, the organic solvent A is one or more mixed solvents selected from anhydrous dichloroethane, anhydrous tetrahydrofuran, and anhydrous dimethyl sulfoxide.

4. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 2, characterized in that: The preparation method of the prodrug self-assembled nanoparticles is as follows: weigh the target product prodrug and dissolve it in organic solvent B to form a total concentration of 10-40 mg / mL, slowly add it dropwise into double distilled water under stirring conditions, the volume ratio of organic solvent B to double distilled water is 1:4-10, spin dry or dialyze to remove the organic solvent, and self-assemble to obtain prodrug self-assembled nanoparticles.

5. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 4, characterized in that: While stirring, add PEGylating agent at a molar ratio of 1 / 20-1 / 5.

6. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 4, characterized in that: The organic solvent B is one or more mixed solvents selected from ethanol, acetonitrile, methanol, tetrahydrofuran and dimethyl sulfoxide; the PEGylation agent is dipalmitoylphosphatidylethanolamine-polyethylene glycol, vitamin E polyethylene glycol succinate or linoleic acid-polyethylene glycol.

7. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 1, characterized in that: The steps for preparing NK cell membrane are as follows: after centrifuging NK-92MI cells, add a homogenate containing protease inhibitors, put them into ice cubes for homogenization, centrifuge the homogenate at 4°C for 10 minutes at a speed of 500g, then discard the precipitate, re-homogenize the supernatant, centrifuge it again at 4°C for 10 minutes at a speed of 500g, collect the supernatant, centrifuge it at 4°C for 50 minutes at a speed of 28000g, collect the final cell membrane precipitate and resuspend it in sterile phosphate buffer for later use.

8. The NK cell membrane-encapsulated prodrug self-assembled nanoparticles according to claim 7, characterized in that: The preparation steps of the ultrasonic extrusion method are as follows: first, the suspension containing NK cell membrane and the prodrug nanoparticle solution are evenly mixed, ultrasonicated for 1-3 minutes, and extruded 10 times under a 400nm filter membrane; then, the mixture is repeatedly extruded 10 times through an extruder under a 200nm filter membrane, and then concentrated by ultrafiltration using an ultrafiltration tube to obtain cell membrane-wrapped prodrug self-assembled nanoparticles.

9. Use of self-assembled nanoparticles based on any one of the cell membrane-encapsulated prodrugs of claims 1 to 8 in the preparation of anti-tumor drugs.

10. A pharmaceutical composition, characterized in that Self-assembled nanoparticles comprising the NK cell membrane-encapsulated prodrug as described in any one of claims 1-9.

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