CN110604820B - Double-sensitive polymer-drug connector and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a double-sensitive polymer-drug connector and a preparation method and application thereof, belonging to the fields of polymer chemistry and pharmaceutical preparations. Preparing an enzyme sensitive substrate into an enzyme sensitive substrate intermediate; preparing the medicine and cystamine dihydrochloride into a medicine derivative containing disulfide bonds; then connecting the enzyme sensitive substrate intermediate with the drug derivative to prepare a dual sensitive drug derivative; finally, polyethylene glycol monomethyl ether and the drug derivative are condensed into a dual sensitive polymer-drug connector sensitive to glutathione reduction and sensitive to cathepsin B. The amphiphilic polymer micelle can be self-assembled into amphiphilic polymer micelle in water, the connection bond is disulfide bond and dipeptide, and the amphiphilic polymer micelle can be responsively broken at a tumor part to release a drug. The invention also discloses a preparation method of the mPEG-VC-SS-GA copolymer and application of the mPEG-VC-SS-GA copolymer as an anticancer drug carrier.
Description
Technical Field
The invention relates to the fields of pharmaceutical preparations and polymer chemistry, in particular to a double-sensitive polymer-drug connector with glutathione reduction sensitivity and cathepsin B sensitivity, and a preparation method and application thereof.
Background content
In the 70 s of the 20 th century, researchers have proposed the idea of covalently binding water-soluble polymers to chemotherapeutic agents. With the development of synthesis and polymers, it has become a rapidly evolving field, and this conjugate has entered the clinic, such as poly (L-glutamic acid) -taxol copolymer, beginning in the 90 s of the 20 th century. Most of the antitumor drugs are insoluble drugs, such as taxol, camptothecine, sorafenib and the like, the bioavailability of the antitumor drugs is limited by poor water solubility, and the nano drug-carrying system can improve the water solubility and the bioavailability. Other conjugates are also under development, such as polyethylene glycol-camptothecin in which polyethylene glycol is the carrier. Polyethylene glycol is an FDA approved hydrophilic polymer with low toxicity and immunogenicity, but the fact is that polyethylene glycol linkers may be difficult to cleave at tumor sites to release drugs, resulting in a significant decrease in anticancer efficacy. The drug delivery system sensitive to the tumor microenvironment, which is rapidly developing at present, can release drugs in a responsive way, and provides a new strategy for overcoming the barriers of low solubility and site-specific delivery of chemotherapeutic drugs.
Gambogic Acid (GA, C) 38 H 44 O 8 ) Is an extract of Chinese medicinal gamboge, is one of main active compounds with anti-tumor effect, and has been applied for thousands of years. Studies have shown that GA has an anticancer effect in many cancer types, such as prostate cancer, liver cancer, breast cancer, etc., and the toxicity thereof is considered to be acceptable, which has become a hot spot for the anti-tumor study of natural products in recent years. However, the clinical research of the traditional antitumor drugs is limited due to poor water solubility, insignificant drug effect and low selectivity.
Glutathione (GSH) is a naturally occurring tripeptide in humans, and has a concentration of glutathione in tumor tissue and cell lysosomes (about 2-10 mM) that is much higher than that in extracellular fluids (about 2-20 uM). Because of the high GSH content in tumor cells, which is often resistant to chemotherapy, some researchers use drugs such as thioflavin imide sulfate (BSO) to consume GSH, and it is desirable to reduce GSH content. However, the BSO has limited effect and no pertinence, and can reduce the GSH content of normal cells, thereby aggravating side effects caused by chemoradiotherapy. The high concentration of glutathione in the tumor part can reduce disulfide bonds, and the low concentration of glutathione in normal tissues and blood vessels can ensure that the disulfide bonds can exist stably. In addition, high concentrations of glutathione are themselves oxidized after disulfide bond reduction and are thus consumed.
Cathepsin B is a cysteine protease, a endopeptidase with a molecular weight of 30kDA, which is present in cells of various animal tissues, in particular in lysosomes, but is not expressed in normal extracellular environments. Under some specific pathological conditions, such as rheumatoid arthritis or tumor sites, high expression of cathepsin B can be seen, especially in various tumor sites. Tumor cells hypersecrete cathepsin B to aid in their metastasis and invasion, such as breakdown of a high density collagen network. Typical substrates for cathepsin B include valine-citrulline and phenylalanine-arginine. According to related research reports, some antibody drug conjugates using enzyme-sensitive small molecule peptide fragments as a linker show effective drug release in tumor tissues.
Accordingly, the development of various biosensitive polymer-drug conjugates has very bright prospect and practical significance in targeted tumor treatment.
Disclosure of Invention
The invention aims to provide a glutathione reduction-sensitive and cathepsin B-sensitive double-sensitive polymer-drug connector, polyethylene glycol monomethyl ether and a poorly soluble drug are connected to disulfide bonds and a section of cathepsin B substrate through covalent bonds, so that the water solubility of the poorly soluble drug is improved, and simultaneously, the disulfide bonds and the covalent bonds of the substrate can be broken due to high content of the reduction glutathione and cathepsin B in tumor tissues and tumor cells, the effect of targeting the tumor tissues can be achieved, and the toxic and side effects on normal cells can be reduced.
The invention adopts the technical scheme that: a dual-sensitive polymer-drug conjugate, which is a glutathione reduction-sensitive and cathepsin B-sensitive dual-sensitive polymer-drug conjugate mPEG-Y-SS-R.
Wherein Y is valine-citrulline, and forms a dual-sensitive polymer-drug connector mPEG-VC-SS-R, and the dual-sensitive polymer-drug connector has a structural formula shown as (I):
or Y is phenylalanine-arginine, and forms a dual-sensitive polymer-drug connector mPEG-PA-SS-R, and has a structural formula shown as (II):
in the structural formulas (I) and (II), the X part refers to a disulfide bond of a redox-sensitive fragment; the Y part refers to the cathepsin B substrate fragment valine-citrulline or phenylalanine-arginine; r is a drug compound with carboxyl.
Preferably, the pharmaceutical compound with a carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azoserine, phenylalanine, N-acetyl-L-phenylalanine and N-acetyl-L-valine.
More preferably, Y is valine-citrulline, and the pharmaceutical compound with carboxyl is gambogic acid, and the dual-sensitive polymer-pharmaceutical connector mPEG-VC-SS-GA for reducing sensitivity of glutathione and sensitivity of cathepsin B is formed, and has a structural formula shown in (iii):
wherein the X part refers to a redox-sensitive fragment disulfide bond; the Y part refers to the cathepsin B substrate fragment valine-citrulline; the R part is gambogic acid.
Preferably, the mPEG specification is polyethylene glycol monomethyl ether mPEG5000.
A method for preparing a dual-sensitive polymer-drug conjugate, comprising the steps of: 1) Connecting an enzyme sensitive substrate with Fmoc to prepare an enzyme sensitive substrate intermediate; 2) Preparing a drug compound R with carboxyl and cystamine dihydrochloride into a drug derivative R-cystamine containing disulfide bonds; 3) The enzyme sensitive substrate intermediate is connected with a disulfide bond-containing drug derivative R-cystamine to prepare a dual sensitive drug derivative; 4) The polyethylene glycol monomethyl ether and the double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and cathepsin B.
Preferably, the preparation method of the dual-sensitive polymer-drug conjugate, wherein Y is valine-citrulline, and the dual-sensitive polymer-drug conjugate mPEG-VC-SS-R is formed, and comprises the following steps:
1) Synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):
1.1 Fmoc-Val (N- (9-fluorenylmethoxycarbonyl) -L-valine), HOSu (N-hydroxysuccinimide), DCC (N, N' -dicyclohexylcarbodiimide) are co-dissolved in tetrahydrofuran at 0 ℃, filtered after stirring for 24 hours, and distilled under reduced pressure to obtain a white solid product Fmoc-Val-OSu;
1.2 Cit (citrulline) with NaHCO 3 Is co-dissolved in distilled water, cooled to 0 ℃, and the DME (1, 2-dimethoxyethane) solution of Fmoc-Val-OSu is added dropwise to Cit and NaHCO 3 Adding tetrahydrofuran to assist dissolution, and stirring for 24 hours at room temperature to obtain a reaction solution;
1.3 Dropwise adding saturated potassium carbonate into the reaction liquid obtained in the step 1.2) to adjust the pH to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH to 3-4, precipitating a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, rotationally concentrating, adding methyl tertiary butyl ether, stirring overnight at 0 ℃, filtering, and vacuum drying to obtain a white solid product which is an enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit);
2) Disulfide bond-containing pharmaceutical derivative R-cystamine (R-SS-NH) 2 ) Is synthesized by the following steps:
2.1 Dissolving a medicine compound R with carboxyl into a dichloromethane solution, cooling to 0 ℃, adding EDCI (N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride) and HOBT (1-hydroxybenzotriazole), activating the obtained mixture at 0 ℃ for 1h, sequentially adding a methanol solution of cystamine dihydrochloride and triethylamine, and stirring at normal temperature for 48h to obtain a reaction solution;
2.2 Reaction liquid obtained in the step 2.1) is treated with NaHCO 3 Washing the solution, collecting organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography, and vacuum drying to obtain disulfide bond-containing drug derivative R-cystamine (R-SS-NH) 2 )；
3) Synthesis of the dual sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):
3.1 Dissolving Fmoc-Val-Cit obtained in step 1) in a mixed solution of dichloromethane and methanol, cooling to 0deg.C, adding EDCI and HOBT, activating the obtained mixture at 0deg.C for 1 hr, and adding R-SS-NH obtained in step 2) 2 After the reaction, concentrating under reduced pressure, adding ice water, preserving at 4 ℃ overnight, filtering, washing with water for three times, drying in vacuum, purifying by column chromatography to obtain dual-sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R);
4) Synthesis of the double-sensitive Polymer-drug linker mPEG-VC-SS-R:
4.1 Dissolving succinic anhydride and DMAP (4- (dimethylamino) pyridine) in pyridine solution, then dropwise adding the mixture into a chloroform solution of mPEG, stirring the mixture at 60 ℃ for reaction for 24 hours, washing the mixture with physiological saline, drying the mixture with anhydrous magnesium sulfate, filtering to obtain an organic layer, concentrating the organic layer, washing the organic layer with diethyl ether, and drying the organic layer in vacuum to obtain a white solid product mPEG-COOH;
4.2 Dissolving mPEG-COOH in dichloromethane, adding EDCI, HOBT andactivating the molecular sieve in the dark at 0 ℃ to obtain mPEG-COOH solution;
4.3 Dissolving Fmoc-VC-SS-R obtained in the step 3) in THF, adding DBU (1, 8-diazabicyclo [5.4.0] undec-7-ene), stirring for 10 minutes, adding the obtained mixed solution into mPEG-COOH solution, and performing nitrogen protection reaction at room temperature for 2 days;
4.4 After the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, decompressing and evaporating, adding diethyl ether for washing, filtering, vacuum drying the obtained solid, adding ultrapure water, stirring, filtering, dialyzing for 2 days, respectively replacing release mediums for 5 times in 2h, 6h, 12h, 24h and 36h, and freeze-drying to obtain the double-sensitive polymer-drug connector mPEG-VC-SS-R of the target product.
Preferably, in the preparation method, the drug compound R with carboxyl is Gambogic Acid (GA), the specification of the mPEG is mPEG5000, and the dual-sensitive polymer-gambogic acid connector mPEG-VC-SS-GA is prepared.
Compared with the prior art, the invention has the following beneficial effects:
the polymer-drug copolymer improves the water solubility of the slightly soluble drug gambogic acid, has good release response performance through disulfide bond and valine-citrulline linkage, enhances the targeting property of the copolymer drug, prolongs the stay time of the anticancer drug at the tumor position, and the critical micelle concentration test shows that the polymer-drug copolymer of the invention easily forms micelles, and cell experiments show that the polymer-drug copolymer has good inhibition effect on liver cancer. The polymer-drug copolymer has the function of targeted intelligent drug release, has the particle size of about 140nm, is beneficial to the accumulation of nano particles at tumor positions, and is beneficial to the permeation of drugs after GSH and enzyme response rupture. According to the invention, by adopting a polyethylene glycol monomethyl ether polymer targeted drug delivery technology, high-concentration glutathione and cathepsin B at a tumor part are used as target points, and a developed polyethylene glycol monomethyl ether-valine-citrulline-S-S-gambogic acid copolymer drug delivery system is designed, so that the gambogic acid targeted therapeutic effect is increased, the toxic and side effects are reduced, and the bioavailability is improved.
The polymer-drug copolymer has oxidation-reduction response and enzyme response performances, good water solubility and small toxic and side effects, wherein the hydrophilic segment is polyethylene glycol monomethyl ether, and the hydrophobic segment is gambogic acid. Amphiphilic polymer micelle is spontaneously formed in aqueous solution due to hydrophilic and hydrophobic effects, and medicines can be responsively released at tumor sites. The application of the polymer-drug copolymer as an anticancer drug carrier can effectively improve the water solubility of insoluble drugs.
The polymer-drug connector mPEG-VC-SS-GA (PVSG) with glutathione reduction sensitivity and cathepsin B sensitivity is used as a control, and the polymer-drug connector increases the solubility of the drug in water and evaluates the pharmacological action of the drug in terms of cytotoxicity. In addition, compared with inclusion carriers such as micelles, liposome and the like, the polymer-drug connector has the advantages that the drug cannot leak in the systemic circulation process, and because the drug is a hydrophobic inner layer, when a chemical bond is broken, the drug can be released more rapidly, and researches prove that the polymer-drug connector can achieve better treatment effect by rapidly releasing the drug at a tumor part, and has very bright prospect and practical significance.
Drawings
FIG. 1 is the synthesis of the dual-sensitive polymer-gambogic acid linker mPEG-VC-SS-GA (PVSG).
FIG. 2 shows MALDI-TOF-MS detection of PVSG.
FIG. 3 shows MALDI-TOF-MS detection of mPEG-COOH (A) and PSG (B).
FIG. 4 shows DSC measurement of PVSG.
FIG. 5 is a measurement of particle size and potential of gambogic acid self-assembled nanoparticles;
wherein A: PSG nanoparticle particle size; b: PSG nanoparticle potential; c: PVSG nanoparticle particle size: d: PVSG nanoparticle potential.
FIG. 6 is a transmission electron microscope measurement of PVSG self-assembled nanoparticles;
wherein A: PVSG nanoparticles; b: PSG nanoparticles.
Fig. 7 is an in vitro release profile of gambogic acid.
FIG. 8 is a graph showing glutathione-sensitive release of gambogic acid nanoparticles.
FIG. 9 is a chart showing the sensitive release of gambogic acid nanoparticle cathepsin B.
FIG. 10 shows the inhibition of four cell proliferation by the dual sensitive polymer-gambogic acid linker (PVSG) of the present invention.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
Glutathione reduction-sensitive and cathepsin B-sensitive double-sensitive polymer-gambogic acid connector (mPEG-VC-SS-GA) (PVSG for short)
The preparation method comprises the following steps:
synthesis of Fmoc-valine-citrulline (Fmoc-Val-Cit)
1.1 Fmoc-Val (5 g,14.73 mmol), HOSu (1.70 g,14.73 mmol) and DCC (3.04 g,14.73 mmol) were co-dissolved in tetrahydrofuran (50 mL) at 0deg.C, stirred for 24h, filtered, and rotary distilled under reduced pressure to give Fmoc-Val-OSu as a white solid product which was used for the next reaction without purification.
1.2 Cit (0.4 g,2.3 mmol) with NaHCO 3 (0.19 g,2.3 mmol) was co-dissolved in 50mL distilled water, cooled to 0deg.C, and 25mL DME solution of Fmoc-Val-OSu (1 g,2.29 mmol) was gradually added dropwise to Cit and NaHCO 3 To the mixed solution of (2) was additionally added 20mL of tetrahydrofuran to aid dissolution, and the mixture was stirred at room temperature for 24 hours to obtain a reaction solution.
1.3 After the reaction solution obtained in the step 1.2) was added dropwise with saturated potassium carbonate to adjust the pH to 8-9, extracted three times with 20mL of ethyl acetate, the aqueous layer was collected, the pH was adjusted to 3-4 by adding citric acid solution, a white gelatinous solid was found to precipitate, filtration was performed, the white gelatinous solid was dissolved in a mixed solution of 25mL of tetrahydrofuran and 10mL of methanol, concentrated to 10mL by rotary evaporation in a 250mL round bottom flask, 200mL of methyl tert-butyl ether was added, stirring was carried out overnight at 0 ℃, filtration was carried out, and vacuum drying was carried out to obtain a white solid product Fmoc-Val-Cit (0.5 g, yield 44.3%).
Structural identification was performed with 1 HNMR. Fmoc-Val-OSU:1H NMR (600 MHz, DMSO-d 6) delta 8.15 (d, J=8.4 Hz, 1H), 7.90 (d, J=7.5 Hz, 2H), 7.75 (dd, J=14.2, 7.5Hz, 2H), 7.46-7.28 (m, 4H), 4.41-4.19 (m, 4H), 2.82 (s, 4H), 2.21 (dq, J=13.5, 6.7Hz, 1H), 1.03 (d, J=6.7 Hz, 6H) Fmoc-Val-Cit 1H NMR (600 MHz, DMSO-d 6) delta 8.13 (d, J=7.3 Hz, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.75 (dd, J=10.8, 7.5Hz, 2H), 7.46-7.28 (m, 5H), 5.94 (dd, 9 Hz), 1.9 Hz, 6.7.7 Hz, 1H), 1.03 (d, J=6.7.7 Hz, 1H), 1.9 (d, 6H), 1.7.9 Hz,1H (d, 6H), 7.9 Hz,1H (d, 7.7.5 Hz, 1H), 7.9 Hz,1H (d=7.9, 7.5Hz, 2H), 7.9 Hz, 2H), 7.46-7.28 (d, 2H), 7.9.7.7.9 Hz, 7.9 Hz, 7.7.7.9 Hz, 2H), 7.7.7.7.7H (2H, 4H).
2. Gambogic acid-cystamine (GA-SS-NH) 2 ) Is synthesized by (a)
2.1 Gambogic acid (1.57 g,2.5 mmol) was dissolved in dichloromethane solution (50 mL), after cooling to 0 ℃, EDCI (627 mg,3.25 mmol) and HOBT (439 mg,3.25 mmol) were added and the mixture was activated at 0 ℃ for one hour. A solution of cystamine dihydrochloride (1.69 g,7.5 mmol) in methanol (30 mL) was added to the mixed solution, triethylamine (500. Mu.L, 3.75 mmol) was added, and the mixture was transferred to room temperature and stirred for 48 hours to obtain a reaction solution.
2.2 (1 mM NaHCO) to the reaction solution obtained in step 2.1) 3 Washing the solution (20 mL) for 3 times, collecting the organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography (petroleum ether/ethyl acetate 1:2), and vacuum drying to obtain yellow solid product GA-SS-NH 2 (1.45g,76％)。
The product was characterized by ESI-MS, 1HNPR and IR. ESI m/z 763.3[ m+h ] +,1H NMR (600 mhz, dmso-d 6) 7.70-7.9 (m, 1H), 7.61 (dd, j=7.1, 1.6hz, 1H), 6.59-6.50 (d, 1H), 5.94 (t, j=7.0 hz, 1H), 5.60 (dt, j=10.2, 2.8hz, 1H), 5.06 (q, j=7.8 hz, 2H), 3.50 (t, j=5.8 hz, 1H), 3.28 (dd, j=14.5, 8.5hz, 1H), 3.10 (dd, j=14.3, 5.1hz, 1H), 2.94-2.51 (m, 11H), 2.51 (q, j=2.0 hz, 1H), 2.25 (dd, j=13.8, 4.7hz, 1.98 hz), 3.28 (dd, j=14.5.5 hz, 1H), 3.10 (dd, 1.5 hz, 1H), 3.20.20 (d, 1H).
Synthesis of Fmoc-valine-citrulline-gambogic acid (Fmoc-VC-SS-GA)
Fmoc-Val-Cit (0.5 g,1 mmol) was dissolved in 50mL of dichloromethane and 10mL of methanol, cooled to 0deg.C, EDCI (0.25 g,1.3 mmol) and HOBT (0.18 g,1.3 mmol) were added, and after activation of the resulting mixture at 0deg.C for 1h, GA-SS-NH was added 2 (1.69 g,7.5 mmol) in dichloromethane (30 mL) and triethylamine (267. Mu.L, 2 mmol) were added and the resulting mixture was stirred overnight. After the reaction was completed, the solution was concentrated under reduced pressure, 100mL of ice water was added, and stored at 4 ℃ overnight, filtered, washed three times with water, and dried under vacuum.Purification by column chromatography (methanol: dichloromethane 1:11) gave Fmoc-VC-SS-GA in 67.8% yield.
The product was structurally characterized by ESI-MS and 1H NPR. ESI m/z 1241.5[ M+H ] +,1H NMR (600 MHz, DMSO-d 6) delta 8.35 (s, 1H), 7.89 (s, 2H), 7.78 (s, 2H), 7.43 (s, 6H), 6.56 (ddd, J=9.8, 6.3,3.2Hz, 1H), 5.94 (t, J=6.0 Hz, 1H), 5.66 (d, 1H), 5.38 (s, 2H), 5.28-4.98 (m, 2H), 4.23 (s, 5H), 3.93 (s, 1H), 3.61 (s, 3H), 2.95-2.52 (m, 11H), 2.17-0.93 (m, 35H), 0.65-0.91 (s, 6H)
Synthesis of mPEG-VC-SS-GA (PVSG)
The synthesis from Fmoc-VC-SS-GA to mPEG-VC-SS-GA is mainly divided into the following two steps: (1) Treatment of mPEG5000 with succinic anhydride converts all hydroxyl groups to acidic groups, (2) mPEG5000-COOH is linked to Fmoc-VC-SS-GA to give mPEG-VC-SS-GA.
4.1 Toluene and mPEG5000 are added into a container, water of mPEG5000 (10 g,2 mmol) is removed by refluxing with toluene for 2h, after cooling, toluene is removed by rotary evaporation under reduced pressure, and the residue is dissolved in 50mL chloroform to obtain chloroform solution of mPEG 5000; succinic anhydride (1 g,10 mmol) and DMAP (733 mg,6 mmol) were dissolved in 10mL of pyridine solution, then added dropwise to chloroform solution of mPEG5000, the resulting mixture was stirred under nitrogen protection at 60 ℃ for reaction for 24h, then washed three times with 100mL of physiological saline, dried over anhydrous magnesium sulfate, filtered to obtain an organic layer, concentrated, washed three times with 50mL of diethyl ether, and dried under vacuum to obtain a white solid product, namely mPEG5000-COOH (6.63 g, 65%).
4.2 mPEG5000-COOH (0.5 g,0.1 mmol) in 20mL dichloromethane, EDCI (0.025 g,0.13 mmol) and HOBT (0.018 g,0.13 mmol) were added 1gThe molecular sieve is activated for 30min in the dark at 0 ℃ to obtain mPEG-COOH solution.
4.3 Fmoc-VC-SS-GA (0.5 g,0.4 mmol) was dissolved in 15mL THF, DBU (0.8 mmol,120 ul) was added and stirred for ten minutes to remove Fmoc groups. The Fmoc group removed mixed solution was added to the mPEG5000-COOH solution and the reaction was allowed to proceed for 2 days at room temperature under nitrogen.
4.4 After the reaction was completed, the reaction mixture was washed three times with 50mL of distilled water, then extracted three times with chloroform, the organic layers were combined, evaporated under reduced pressure, washed with diethyl ether, then filtered, and the obtained solid was dried in vacuo, then 20mL of ultrapure water was added, stirred, filtered, and dialyzed for 2 days (dialysis medium was 1l of ultrapure water of 5% dmf, dialysis bag mwco=5 kDa), and the release medium was exchanged 5 times during 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, respectively. After freeze drying, collecting yellow fluffy solid, namely the target product double-sensitive polymer-gambogic acid connector mPEG-VC-SS-GA (PVSG), and obtaining the yield: 45%. The product was structurally characterized by MALDI-TOF-MS as shown in FIG. 2.
(II) comparative examples: glutathione reduced singleplex sensitive Polymer-drug linker mPEG-SS-GA (PSG)
The preparation method comprises the following steps:
1. gambogic acid-cystamine (GA-SS-NH) 2 ) Is synthesized by (a)
1.1 Gambogic acid (1.57 g,2.5 mmol) was dissolved in dichloromethane solution (50 mL), after cooling to 0 ℃, EDCI (627 mg,3.25 mmol) and HOBT (439 mg,3.25 mmol) were added and the mixture was activated at 0 ℃ for one hour. A solution of cystamine dihydrochloride (1.69 g,7.5 mmol) in methanol (30 mL) was added to the mixed solution, triethylamine (500. Mu.L, 3.75 mmol) was added, and the mixture was transferred to room temperature and stirred for 48 hours to obtain a reaction solution.
1.2 (1 mM NaHCO) to the reaction solution obtained in step 2.1) 3 Washing the solution (20 mL) for 3 times, collecting the organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography (petroleum ether/ethyl acetate 1:2), and vacuum drying to obtain yellow solid product GA-SS-NH 2 。
Synthesis of mPEG-SS-GA (PSG)
2.1 Toluene and mPEG5000 are added into a container, water of mPEG5000 (10 g,2 mmol) is removed by refluxing with toluene for 2h, after cooling, toluene is removed by rotary evaporation under reduced pressure, and the residue is dissolved in 50mL chloroform to obtain chloroform solution of mPEG 5000; succinic anhydride (1 g,10 mmol) and DMAP (733 mg,6 mmol) were dissolved in 10mL of pyridine solution, then added dropwise to chloroform solution of mPEG5000, the resulting mixture was stirred under nitrogen protection at 60 ℃ for reaction for 24h, then washed three times with 100mL of physiological saline, dried over anhydrous magnesium sulfate, filtered to obtain an organic layer, concentrated, washed three times with 50mL of diethyl ether, and dried under vacuum to obtain a white solid product, namely mPEG5000-COOH (6.63 g, 65%).
2.2 mPEG5000-COOH (0.5 g,0.1 mmol) in 20mL dichloromethane, EDCI (0.025 g,0.13 mmol) and HOBT (0.018 g,0.13 mmol) were added 1gThe molecular sieve is activated for 30min in the dark at 0 ℃ to obtain mPEG-COOH solution.
2.3 GA-SS-NH is taken 2 (0.153 g,0.20 mmol)) and triethylamine (26. Mu.L, 0.20 mmol) were added to the mPEG5000-COOH solution and the reaction was nitrogen-blanketed at room temperature for 2 days.
2.4 After the reaction was completed, the reaction mixture was washed three times with 50mL of distilled water, then extracted three times with chloroform, the organic layers were combined, evaporated under reduced pressure, washed with diethyl ether, then filtered, and the obtained solid was dried in vacuo, then 20mL of ultrapure water was added, stirred, filtered, and dialyzed for 2 days (dialysis medium was 1l of ultrapure water of 5% dmf, dialysis bag mwco=5 kDa), and the release medium was exchanged 5 times during 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, respectively. After freeze drying, a yellow fluffy solid was collected, which was the target product mPEG-SS-GA (PSG). The product was identified by MALDI-TOF-MS and the results are shown in FIG. 3.
FIG. 2 shows MALDI-TOF-MS mass spectra of PVSG, and FIG. 3 shows MALDI-TOF-MS mass spectra of mPEG-COOH (A) and PSG (B). The molecular ion peak in each mass spectrogram is identical to the synthesized molecule, and the main fragment ion peak energy corresponds to the related structure, thus proving that the structure is correct.
DSC measurement of PVSG
1mg of GA (A), 10mg of mPEG-COOH (B), 10mg of PSG (D), 10mg of PVSG (E) and a physical mixture (C) of 10mg of mPEG-COOH and 1mg of GA are respectively taken, and the temperature rise rate is 20 ℃/min and the temperature rise interval is 25 ℃ to 260 ℃ in DSC measurement.
As shown in FIG. 4, the gambogic acid absorbs heat at about 80 ℃, and as can be seen from the curve A and the curve C, the absorption heat peak of the gambogic acid can still be seen in the physical mixture, and the absorption heat peaks of the PSG and the PVSG disappear, which means that free gambogic acid does not exist, and the absorption heat of the PVSG is deviated compared with that of the PSG, and is possibly influenced by instrument fluctuation or VC fragments.
(IV) investigation of PVSG hydration volume
Precisely weighing 5mg PVSG, placing into a50 mL centrifuge tube, adding 2mL, 5mL, 10mL and 20mL distilled water into the parallel samples in three parts, shaking up and down to quickly dissolve the PVSG, performing ultrasonic treatment at 60W for one minute, filtering with a 0.45um filter membrane, detecting by dynamic light scattering, and taking particle size and PDI as investigation parameters, wherein the results are shown in Table 1.
TABLE 1 influence of hydration volume on nanoparticles
The results in Table 1 show that the particle size and PDI of PVSG-NPs are smaller for both 5mL and 10mL when the hydration volume is increased from 2mL to 20mL, and that the hydration volume is selected to be 10mL, i.e., 0.5mg/mL concentration, 142+ -4.71 particle size, and 0.259+ -0.048 PDI for the reference and in consideration of the subsequent experimental dosage requirements.
(V) PVSG ultrasonic time investigation
Precisely weighing 5mg PVSG, placing into 50mL centrifuge tube, adding 10mL distilled water into parallel sample three times, shaking up and down to dissolve rapidly, respectively ultrasonic treating at 60W for 1min, 3min, and 5min, filtering with 0.45um filter membrane, detecting by dynamic light scattering, and measuring particle size and PDI as investigation parameters, and the result is shown in Table 2
TABLE 2 influence of ultrasound time on gambogic acid nanoparticles
The results in Table 2 show that when the ultrasonic power is increased from 1min to 5min, the particle size and PDI of PVSG-NPs are minimum at 3min, and the particle size and PDI are increased instead at 5min, so that the polymer is easy to break due to overlong ultrasonic time, and the ultrasonic time is selected to be one minute due to no obvious difference between 1min and 3min, and the time is saved.
The final prescription process is as follows: precisely weighing 5mg PVSG, placing into a50 mL centrifuge tube, adding 10mL distilled water, shaking up and down, performing ultrasonic treatment at 60W for 1min, passing through a 0.45um filter membrane, treating PSG by the same method, and taking equivalent GA to obtain PVSG-NPs and PSG-NPs.
Measurement of particle size and potential of PVSG self-assembled nanoparticles
PSG-NPs and PVSG-NPs were prepared with the optimal prescription, and the particle size, potential and PDI (25 ℃, n=3) of the conjugate were tested by Dynamic Light Scattering (DLS) method, and the results are shown in FIG. 5. As a result of measurement, the particle size of PSG-NPs was 117.+ -. 4.71nm, the particle size of PDI was 0.315.+ -. 0.016, the particle size of PVSG-NPs was 142.+ -. 4.71nm and the particle size of PDI was 0.190.+ -. 0.036, and it was found that the latter was larger than the former, but both were smaller than 200nm, and that it could be entered into tumor tissue by EPR effect. Both potentials were around 0, due to the stealth effect of mPEG, indicating that mPEG successfully encapsulated GA.
Determination of PVSG self-assembled nanoparticles by transmission electron microscopy
And (3) observing the PSG-NPs (B) and the PVSG-NPs (A) by a transmission electron microscope (Transmission electron microscope, TEM), sucking micelle solution by a pipette gun, dripping the micelle solution onto a 200-mesh copper net, naturally airing, dripping 0.2% phosphotungstic acid for dyeing, sucking the materials after 3-5min, naturally airing, and observing the morphology of the two.
As can be seen from fig. 6, TEM analysis confirmed that the nanoparticles were spherical, uniform in size, and slightly smaller in particle size than DLS detection, which is probably caused by evaporation of water, and in summary, characterization results demonstrated successful preparation of micelles.
In vitro release experiments of PVSG self-assembled nanoparticles
The in vitro release is carried out by adopting a dialysis method, the accumulated release amount is taken as an index, a culture medium containing 1% Tween 80 is taken as a release medium, and the L-arginine is used for preparing the gambogic acid preparation, so that whether PSG and PVSG exist stably at normal temperature is compared and examined. The specific implementation method is as follows:
1) 22mg of L-arginine and 10mg of gambogic acid are dissolved in a culture medium of 1% Tween 80, and vortexed. Namely the control preparation L-GA.
2) Taking L-GA, PSG and PVSG prepared in the same batch, preparing a solution with 2mg/mL of gambogic acid equivalent content by taking a culture medium of 1% Tween 80 as a medium, and respectively adding the solution into dialysis bags with the same length (MWCO: 1kDa; pretreatment: the dialysis bag was boiled with 2% (W/V) sodium bicarbonate and 1mmol/L EDTA for 10min, cooled to room temperature and stored at 4℃), carefully air bubbles removed, tightly sealed, placed in an eggplant-type bottle to which 40mL of dissolution medium had been added, incubated at 37℃ with a shaking incubator at 100rpm, 3mL were sampled at time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h, and isothermal equal volume of release medium was immediately replenished after sampling. The sample is filtered by a microporous membrane with the diameter of 0.45 μm, the subsequent filtrate is taken, the ultraviolet condition sample injection analysis is carried out, the peak area is recorded, the standard curve is carried in, the medicine content of the preparation leaked into the medium at different time points is calculated, the release amount and the accumulated release amount of the medicine are calculated, a release curve (namely, the accumulated release amount-time curve of the medicine) is drawn, and the test result is shown in figure 7.
As shown in FIG. 7, L-GA release was fast, nearly 80% had been released at 12h, whereas PSG-NPs released slower than PVSG-NPs than L-GA, and it was seen that PVSG-NPs released more cumulatively than PSG-NPs at 72h, and PSG-NPs released more in normal conditions, but there was no significant difference (P > 0.05) between the two groups.
Glutathione sensitivity Release assay of PVSG self-assembled nanoparticles
The tumor microenvironment was prepared by taking acetic acid-sodium acetate buffer solution with pH5.0 and GSH concentration of 10mM as release medium, respectively taking two micelles of PSG-NPs and PVSG-NPs to dissolve in the acetic acid-sodium acetate buffer solution (equivalent GA 1 mg/mL), performing in vitro release by dialysis (40 mL release medium), and taking the group without GSH as a control, sampling 3mL at the time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h, and supplementing isothermal and equal volume release medium, and the result is shown in FIG. 8.
As shown in FIG. 8, when glutathione was added, the release amounts of PSG-NPs and PVSG-NPs were both increased, but compared with each other, the release amount of PSG-NPs was increased by about 15% after 72 hours, and the release amount of PVSG-NPs was increased by about 25%, indicating that the redox performance of PVSG-NPs was superior to that of PSG-NPs, but the two groups of data did not have significant difference (P > 0.05).
Cathepsin B-sensitive assay of (ten) PVSG self-assembled nanoparticles
10mM GSH and CB in 10mL sodium acetate buffer is used as a release medium. PSG and PVSG (eq GA 500. Mu.g) were dissolved in 2mL of acetic acid-sodium acetate buffer, respectively. CB (lyophilized solid, 1.2 mg) was dissolved in 1mL of 25mM sodium acetate/1 mM EDTA buffer (pH 5.0) and stored frozen at-80℃after stabilization at 4℃for 8 h. CB was incubated at 37℃in 100. Mu.L of 30mM DTT/15mM EDTA buffer (pH 5.0) before the test, 50. Mu.L of buffer (50 UI/mL) was added to the micelle solution, and the reaction system was shaken at 37℃with 3mL samples at time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h while supplementing an isothermal equal volume of release medium, and the results were shown in FIG. 9.
The results in FIG. 9 show that after cathepsin B is added, the release curves of the PSG-NPs are very similar to those of the PSG-NPs without CB, the release of the PVSG-NPs is improved, the release amount of the PSG-NPs is remarkably improved (P < 0.05) compared with that of the PSG group, and the PVSG-NPs have excellent performance.
(eleven) pharmacodynamic test of PSVG
Precisely weighing GA powder, PSG-NPs and PVSG-NPs 1mg each in an EP tube, sterilizing by ultraviolet, dissolving the medicine (100 mu L/900 mu L) in a blank culture medium containing Tween 80, diluting the blank culture medium to 100 mu M equivalent GA concentration, and storing as mother liquor. When HepG2 cells were grown to 80%, cells were collected, diluted with medium, counted, transferred to 96-well cell culture plates at 1X 104 cells/well (100. Mu.L), cultured in a 5% CO2 incubator at 37℃for 24 hours, three drugs were diluted in proportion to 6 concentration gradients of 8. Mu.M, 4.0. Mu.M, 2.0. Mu.M, 1.0. Mu.M, 0.5. Mu.M, 0.25. Mu.M (eq. GA), and three parallel groups were set for each concentration of 100. Mu.L, drug was added, and finally the drug was administered in 96-well plates at concentrations of 4.0. Mu.M, 2.0. Mu.M, 1.0. Mu.M, 0.5. Mu.M, 0.25. Mu.M, 0.125. Mu.M (eq. GA), taking sterile PBS as a blank control group, additionally arranging a solvent control group, respectively culturing for 24 hours, 48 hours and 72 hours in a 5% CO2 incubator at 37 ℃, adding MTT solution (20 mu L, 0.5%) in a dark place, incubating for 4 hours in the incubator at 37 ℃, taking out the 96-well plate, pouring out the liquid in the plate, air-drying in an ultra-clean bench, adding DMSO (150 mu L/hole) for dissolution, shaking for 5 minutes on a shaking table, fully dissolving purple crystals, detecting OD value with an enzyme-labeled instrument at 570nm wavelength, preserving data results, calculating cell inhibition rates of each group, and drawing a histogram according to the inhibition rate results.
The same procedure was used for BV2 cells, RAW264.7 cells and HEK293 cells, and the results are shown in Table 3 and FIG. 10.
TABLE 3 IC50 values of drugs against four cells
From table 3 and fig. 10, cytotoxicity of the three drugs to normal cells and toxicity to tumor cells were detected by MTT method, respectively, and it was found that in the three normal cell lines, PSG-NPs and PVSG-NPs were much less toxic than GA, especially at high concentration, whereas PVSG-NPs were much more sensitive and release than PSG-NPs, thus exhibiting higher normal cytotoxicity, but still having significant difference compared to GA drug, in the three cell lines, PSG-NPs and PVSG-NPs had minimal cytotoxicity to BV2 cells, and BV2 cells still survived more than 80% at high concentration, whereas RAW264.7 and HEK293 survived less than BV2, probably because RAW264.7 can ingest the drugs, GA drug kills macrophages to 5% or less at high concentration, and both had some effect on survival rate of macrophages, but IC50 was significantly higher than GA drug. In cytotoxicity test on HepG2 cell line, the 24h IC50 of GA bulk drug is higher than IC50 value of normal cells, it can be seen that both micelles have concentration dependence, but PSG-NPs have weak killing ability on tumor cells, and can not be released completely under the wrapping action of PEG, while PVSG-NPs have killing effect on tumor cells less than GA at 24h, and PVSG-NPs have higher killing effect than GA at 48h and 72h, and three drugs have concentration dependence and time dependence, but compared with the PVSG-NPs, the release speed is faster, and the response ability is higher than that of PSG-NPs.
Claims (7)
1. A dual-sensitive polymer-drug conjugate is characterized in that the dual-sensitive polymer-drug conjugate is glutathione reduction sensitive and cathepsin B sensitive, the dual-sensitive polymer-drug conjugate is mPEG-Y-SS-R,
wherein, the specification of mPEG is polyethylene glycol monomethyl ether mPEG5000, Y is valine-citrulline, R is a drug compound with carboxyl, and the double-sensitive polymer-drug connector mPEG-VC-SS-R is formed, and the structural formula is shown as (I):
（Ⅰ）
the medicine compound with carboxyl is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azoserine, phenylalanine, N-acetyl-L-phenylalanine and N-acetyl-L-valine.
2. The dual-sensitive polymer-drug conjugate of claim 1, wherein the drug compound with a carboxyl group is gambogic acid, comprising glutathione reduction-sensitive and cathepsin B-sensitive dual-sensitive polymer-drug conjugate mPEG-VC-SS-GA having a structural formula as shown in (iii):
(Ⅲ)。
3. a method of preparing a dual sensitive polymer-drug conjugate according to claim 1 or 2, comprising the steps of: 1) Connecting an enzyme sensitive substrate with Fmoc to prepare an enzyme sensitive substrate intermediate; 2) Preparing a drug compound R with carboxyl and cystamine dihydrochloride into a drug derivative R-cystamine containing disulfide bonds; 3) The enzyme sensitive substrate intermediate is connected with a disulfide bond-containing drug derivative R-cystamine to prepare a dual sensitive drug derivative; 4) The polyethylene glycol monomethyl ether and the double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and cathepsin B.
4. A method of preparation according to claim 3, characterized in that the method of preparation comprises the steps of:
1) Synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):
1.1 The Fmoc-Val, HOSu and DCC are dissolved in tetrahydrofuran at 0 ℃, are stirred for 24 hours, are filtered and are subjected to reduced pressure rotary evaporation, and a white solid product Fmoc-Val-OSu is obtained;
1.2 Cit and NaHCO) 3 Is co-dissolved in distilled water, cooled to 0 ℃, and the DME solution of Fmoc-Val-OSu is added dropwise to Cit and NaHCO 3 Adding tetrahydrofuran to assist dissolution, and stirring for 24 hours at room temperature to obtain a reaction solution;
1.3 Dropwise adding saturated potassium carbonate into the reaction liquid obtained in the step 1.2) to adjust the pH to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH to 3-4, precipitating a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, rotationally concentrating, adding methyl tertiary butyl ether, stirring overnight at 0 ℃, filtering, and vacuum drying to obtain a white solid product which is an enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit);
2) Disulfide bond-containing pharmaceutical derivative R-cystamine (R-SS-NH) 2 ) Is synthesized by the following steps:
2.1 Dissolving a medicine compound R with carboxyl into a dichloromethane solution, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, sequentially adding a methanol solution of cystamine dihydrochloride and triethylamine, and stirring at normal temperature for 48h to obtain a reaction solution;
2.2 Reaction liquid obtained in the step 2.1) is treated with NaHCO 3 Washing the solution, collecting organic layer, drying with anhydrous magnesium sulfate, filtering, separating by column chromatography, and vacuum drying to obtain disulfide bond-containing drug derivative R-cystamine (R-SS-NH) 2 )；
3) Synthesis of the dual sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):
3.1 Fmoc obtained in step 1)Dissolving Val-Cit in mixed solution of dichloromethane and methanol, cooling to 0deg.C, adding EDCI and HOBT, activating the obtained mixture at 0deg.C for 1 hr, and adding R-SS-NH obtained in step 2) 2 After the reaction, concentrating under reduced pressure, adding ice water, preserving at 4 ℃ overnight, filtering, washing with water for three times, drying in vacuum, purifying by column chromatography to obtain dual-sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R);
4) Synthesis of the double-sensitive Polymer-drug linker mPEG-VC-SS-R:
4.1 Dissolving succinic anhydride and DMAP in pyridine solution, then dropwise adding the solution into chloroform solution of mPEG, stirring the obtained mixture under the protection of nitrogen at 60 ℃ for reaction for 24 hours, washing the mixture with physiological saline, drying the mixture with anhydrous magnesium sulfate, filtering to obtain an organic layer, concentrating the organic layer, washing the organic layer with diethyl ether, and drying the organic layer in vacuum to obtain a white solid product mPEG-COOH;
4.2 Dissolving mPEG-COOH in dichloromethane, adding EDCI, HOBT and 4A type molecular sieve, and activating in the dark at 0 ℃ to obtain mPEG-COOH solution;
4.3 Dissolving Fmoc-VC-SS-R obtained in the step 3) in THF, adding DBU, stirring for 10 minutes, adding the obtained mixed solution into mPEG-COOH solution, and performing nitrogen protection reaction at room temperature for 2 days;
4.4 After the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, decompressing and evaporating, adding diethyl ether for washing, filtering, vacuum drying the obtained solid, adding ultrapure water, stirring, filtering, dialyzing for 2 days, respectively replacing release mediums for 5 times in 2h, 6h, 12h, 24h and 36h, and freeze-drying to obtain the double-sensitive polymer-drug connector mPEG-VC-SS-R of the target product.
5. The method according to claim 4, wherein in step 4.4), the dialysis, dialysis bag has a molecular weight of 5kDa and the dialysis medium is distilled water.
6. The method according to claim 3 or 4, wherein the pharmaceutical compound R having a carboxyl group is gambogic acid.
7. Use of a dual-sensitive polymer-drug conjugate according to claim 1 or 2 for the preparation of an antitumor drug.
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| CN108752507A (en) * | 2018-05-12 | 2018-11-06 | 辽宁大学 | A kind of enzyme sensitivity and isotope of redox-sensitive double-response type copolymer and its preparation method and application |
| CN108794654A (en) * | 2018-05-14 | 2018-11-13 | 辽宁大学 | A kind of biodegradable isotope of redox-sensitive type polymer and its preparation method and application |
| CN114377151B (en) * | 2022-01-24 | 2024-02-20 | 辽宁大学 | Dual-response polymer prodrug micelle and preparation method thereof |
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| CN107335060A (en) * | 2016-04-28 | 2017-11-10 | 北京大学 | A kind of small molecule conjugate and its nano prodrug system based on rgd peptide-chemotherapeutics |
| CN108101825A (en) * | 2016-11-25 | 2018-06-01 | 上海青润医药科技有限公司 | Disubstituted maleic amide class connexon for antibody-drug conjugate and its preparation method and application |
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