CN110604820A - Dual-sensitive polymer-drug conjugate and preparation method and application thereof - Google Patents
Dual-sensitive polymer-drug conjugate and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double-sensitive polymer-drug conjugate and a preparation method and application thereof, belonging to the fields of high-molecular chemistry and pharmaceutical preparations. Preparing an enzyme sensitive substrate into an enzyme sensitive substrate intermediate; then preparing the drug and cystamine dihydrochloride into a drug derivative containing disulfide bonds; then connecting the enzyme sensitive substrate intermediate with the drug derivative to prepare the dual sensitive drug derivative; finally, polyethylene glycol monomethyl ether and the drug derivative are condensed into the dual sensitive polymer-drug conjugate which is sensitive to glutathione reduction and sensitive to cathepsin B. The amphiphilic polymer micelle can be self-assembled into an amphiphilic polymer micelle in water, and a connecting bond is a disulfide bond and a dipeptide, so that the amphiphilic polymer micelle can be broken in response at a tumor part to release a medicament. 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 field of pharmaceutical preparations and the field of polymer chemistry, in particular to a dual-sensitive polymer-drug conjugate 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 bonding water-soluble polymers to chemotherapeutic drugs. With the development of synthesis and polymers, which are becoming a rapidly developing field, such conjugates began to enter the clinic in the 90 s of the 20 th century, as poly (L-glutamic acid) -paclitaxel copolymer. Most of the antitumor drugs are insoluble drugs such as paclitaxel, camptothecin, sorafenib and the like, the poor water solubility limits the bioavailability of the antitumor drugs, and the nano drug delivery system can improve the water solubility and increase the bioavailability. Other conjugates are also under development, such as peg-camptothecin in peg carriers. Polyethylene glycol is an FDA approved hydrophilic polymer with low toxicity and immunogenicity, but the fact is that at the tumor site, the polyethylene glycol linker may be difficult to break to release the drug, resulting in a significant decrease in anticancer effect. The tumor microenvironment sensitive drug delivery system which is rapidly developing at present can release drugs in response, and provides a new strategy for overcoming the obstacles of low solubility and site-specific delivery of chemotherapeutic drugs.
Gambogic Acid (GA, C)38H44O8) Is an extract in the Chinese medicinal gamboge, is one of main active compounds with anti-tumor effect, and has been applied for thousands of years. The research shows that GA has anticancer effect in many cancer types, such as prostate cancer and liver cancerBreast cancer, and the like, and the toxicity thereof is considered to be acceptable by research, and the toxicity is a hot spot of natural product anti-tumor research in recent years. But the current clinical research on the anti-tumor is limited due to poor water solubility, insignificant drug effect and low selectivity.
Glutathione (GSH) is a naturally occurring tripeptide in humans, and the concentration of glutathione in tumor tissues and lysosomes is much higher (about 2-10mM) than that in extracellular fluids (about 2-20 uM). Since tumor cells often develop resistance to chemotherapy due to high levels of GSH, some researchers have consumed GSH drugs, such as buthionine-sulfoximine (BSO), in anticipation of reducing GSH levels. However, BSO has limited and no specific effects, and can reduce the content of GSH in normal cells, thereby aggravating the side effects brought by radiotherapy and chemotherapy. The high-concentration glutathione in the tumor part can reduce disulfide bonds, and the low concentration of glutathione in normal tissues and blood vessels enables the disulfide bonds to exist stably. In addition, glutathione itself is oxidized at a high concentration after reduction of disulfide bonds, and thus is consumed.
Cathepsin B is a cysteine protease, an endopeptidase with a molecular weight of 30kDA, which is present intracellularly, particularly in lysosomes, in various animal tissues, but is not expressed in the normal extracellular environment. In particular pathological conditions, such as rheumatoid arthritis or tumor sites, a high expression of cathepsin B is observed, especially in various tumor sites. Tumor cells over secrete cathepsin B to aid in its metastasis, invasion, e.g. breakdown of high density collagen networks. 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 linking agents show effective drug release in tumor tissues.
Therefore, the research and development of various biological sensitive polymer-drug conjugates have bright prospect and practical significance in the targeted tumor treatment.
Disclosure of Invention
The invention aims to provide a glutathione reduction sensitive and cathepsin B sensitive double sensitive polymer-drug conjugate, wherein polyethylene glycol monomethyl ether and an insoluble drug are connected to a disulfide bond and a section of cathepsin B substrate through covalent bonds, so that the water solubility of the insoluble drug is improved, and meanwhile, due to the high content of the reduced glutathione and the cathepsin B in a tumor tissue and a tumor cell, the covalent bonds of the disulfide bond and the substrate can be broken, so that the effect of targeting the tumor tissue can be achieved, and the toxic and side effects on normal cells can be reduced.
The technical scheme adopted by the invention is as follows: a dual sensitive polymer-drug linker is a dual sensitive polymer-drug linker mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.
Wherein Y is valine-citrulline, forms a double sensitive polymer-drug linker mPEG-VC-SS-R, and has a structural formula shown as (I):
or Y is phenylalanine-arginine, forms a double sensitive polymer-drug connector mPEG-PA-SS-R, and has a structural formula shown as (II):
in the structural formula (I) and the structural formula (II), X is partially represented by a redox sensitive fragment disulfide bond; moiety Y refers to the cathepsin B substrate fragment valine-citrulline or phenylalanine-arginine; r is a pharmaceutical compound with carboxyl.
Preferably, the pharmaceutical compound having a carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azaserine, chlorophenylalanine, N-acetyl-L-phenylalanine and N-acetyl-L-valine.
More preferably, the Y is valine-citrulline, the pharmaceutical compound with carboxyl is gambogic acid, and the dual-sensitive polymer-drug conjugate mPEG-VC-SS-GA forming the glutathione reduction sensitivity and the cathepsin B sensitivity has a structural formula shown in (III):
wherein the X moiety is designated as a redox sensitive fragment disulfide bond; moiety Y refers to the cathepsin B substrate fragment valine-citrulline; the R part is gambogic acid.
Preferably, the mPEG specification is polyethylene glycol monomethyl ether mPEG 5000.
A preparation method of a dual-sensitive polymer-drug linker comprises the following steps: 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) connecting the enzyme sensitive substrate intermediate with a disulfide bond-containing drug derivative R-cystamine to prepare a double sensitive drug derivative; 4) polyethylene glycol monomethyl ether and a double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.
Preferably, in the above method for preparing the double sensitive polymer-drug conjugate, Y is valine-citrulline, and constitutes the double sensitive polymer-drug conjugate mPEG-VC-SS-R, the method comprises the following steps:
1) synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):
1.1) dissolving Fmoc-Val (N- (9-fluorenylmethoxycarbonyl) -L-valine), HOSu (N-hydroxysuccinimide), DCC (N, N' -dicyclohexylcarbodiimide) in tetrahydrofuran at 0 ℃, stirring for 24h, filtering, decompressing and rotary evaporating to obtain a white solid product Fmoc-Val-OSu;
1.2) combining Cit (citrulline) with NaHCO3Dissolving in distilled water, cooling to 0 deg.C, and adding solution of Fmoc-Val-OSu in DME (1, 2-dimethoxyethane) dropwise to Cit and NaHCO3Adding tetrahydrofuran to assist dissolving,stirring for 24h 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 value to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH value to 3-4, separating out a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, performing rotary evaporation and concentration, adding methyl tert-butyl ether, stirring overnight at 0 ℃, filtering, and performing 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 drug derivative R-cystamine (R-SS-NH)2) The synthesis of (2):
2.1) dissolving a pharmaceutical compound R with carboxyl in 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) reacting the reaction solution obtained in the step 2.1) with NaHCO3Washing the solution, collecting the 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 double sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):
3.1) dissolving Fmoc-Val-Cit obtained in the step 1) in a mixed solution of dichloromethane and methanol, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, and adding R-SS-NH obtained in the step 2)2Stirring the obtained mixture overnight, after the reaction is finished, concentrating under reduced pressure, adding ice water, storing overnight at 4 ℃, filtering, washing with water for three times, drying in vacuum, and purifying by column chromatography to obtain the 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 a pyridine solution, then dropwise adding the solution into a chloroform solution of mPEG, stirring the obtained mixture at 60 ℃ under the protection of nitrogen for 24 hours, washing the mixture with normal saline, drying the mixture with anhydrous magnesium sulfate, filtering the mixture to obtain an organic layer, concentrating the organic layer, washing the organic layer with 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 a type 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 reacting for 2 days at room temperature under the protection of nitrogen;
4.4) after the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, evaporating under reduced pressure, adding ether for washing, filtering, drying the obtained solid in vacuum, adding ultrapure water, stirring, filtering, dialyzing for 2 days, replacing release media for 5 times in 2h, 6h, 12h, 24h and 36h respectively, and freeze-drying to obtain the target product, namely the dual-sensitive polymer-drug conjugate mPEG-VC-SS-R.
Preferably, in the preparation method, the pharmaceutical compound R with carboxyl is Gambogic Acid (GA), the mPEG specification is mPEG5000, and the dual-sensitive polymer-gambogic acid conjugate 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 insoluble drug gambogic acid, is linked with valine-citrulline through disulfide bonds, has good release response performance, enhances the targeting property of the copolymerized drug, prolongs the retention time of the anticancer drug at a tumor part, and tests on the critical micelle concentration show that the polymer-drug copolymer is easy to form 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, the particle size is about 140nm, the accumulation of nano particles at a tumor part is facilitated, and the drug release is facilitated after GSH and enzyme responsive fracture. According to the invention, a polyethylene glycol monomethyl ether polymer targeted drug delivery technology is adopted, high-concentration glutathione and cathepsin B at a tumor part are taken as targets, and the developed polyethylene glycol monomethyl ether-valine-citrulline-S-S-gambogic acid copolymer drug delivery system is designed, so that the targeted therapeutic effect of gambogic acid is increased, the toxic and side effects are reduced, and the bioavailability is improved.
The polymer-drug copolymer has redox response and enzyme response performances, good water solubility and small toxic and side effects, and the hydrophilic section is polyethylene glycol monomethyl ether and the hydrophobic section is gambogic acid. In aqueous solution, amphiphilic polymer micelles are formed spontaneously due to the hydrophilic and hydrophobic effects, and the drug can be released at the tumor site in a response manner. The polymer-drug copolymer provided by the invention can be used as an anticancer drug carrier, and can effectively improve the water solubility of insoluble drugs.
The dual sensitive polymer-drug linker mPEG-VC-SS-GA (PVSG) sensitive to glutathione reduction and cathepsin B sensitivity designed by the invention takes the single sensitive polymer-drug linker mPE-SS-GA (PSG) reduced by glutathione as a reference, and the polymer-drug linker of the invention increases the solubility of drugs in water and evaluates the pharmacological action of the drugs in the aspect of cytotoxicity. In addition, compared with inclusion carriers such as micelle, liposome and the like, the polymer-drug conjugate has the advantages that the drug does not leak in the systemic circulation process, and the drug is a hydrophobic inner layer, so that the drug can be released more quickly when chemical bonds are broken.
Drawings
FIG. 1 shows the synthesis of 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 is a DSC measurement of PVSG.
FIG. 5 is the measurement of the particle size and potential of gambogic acid self-assembled nanoparticles;
wherein, A: particle size of PSG nanoparticles; b: PSG nanoparticle potential; c: particle size of PVSG nanoparticles: 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 a graph of the in vitro release of gambogic acid.
Fig. 8 is a sensitivity release diagram of gambogic acid nanoparticle glutathione.
Fig. 9 is a graph of sensitive release of gambogic acid nanoparticle cathepsin B.
FIG. 10 shows the inhibition of four cell proliferations by the double-sensitive polymer-gambogic acid conjugate (PVSG) of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
Glutathione reduction sensitive and cathepsin B sensitive double sensitive polymer-gambogic acid linker (mPEG-VC-SS-GA) (PVSG for short)
The preparation method comprises the following steps:
synthesis of Fmoc-valine-citrulline (Fmoc-Val-Cit)
1.1) dissolving Fmoc-Val (5g,14.73mmol), HOSu (1.70g,14.73mmol) and DCC (3.04g,14.73mmol) in tetrahydrofuran (50mL) at 0 ℃, stirring for 24h, filtering, decompressing and rotary evaporating to obtain a white solid product, namely Fmoc-Val-OSu, and carrying out the next reaction without purification.
1.2) Cit (0.4g,2.3mmol) with NaHCO3(0.19g,2.3mmol) in 50mL distilled water, cooled to 0 deg.C and gradually added dropwise to Cit and N a solution of Fmoc-Val-OSu (1g,2.29mmol) in 25mL DMEaHCO3The mixed solution of (1) was dissolved in 20mL of tetrahydrofuran, and the mixture was stirred at room temperature for 24 hours to obtain a reaction solution.
1.3) adding saturated potassium carbonate dropwise into the reaction liquid obtained in the step 1.2) to adjust the pH value to 8-9, extracting with 20mL ethyl acetate for three times, collecting a water layer, adding a citric acid solution to adjust the pH value to 3-4, so that a white gelatinous solid is separated out, filtering, dissolving the white gelatinous solid into a mixed solution of 25mL tetrahydrofuran and 10mL methanol, performing rotary evaporation concentration to 10mL in a 250mL round-bottom flask, adding 200mL methyl tert-butyl ether, stirring at 0 ℃ overnight, filtering, and performing vacuum drying to obtain a white solid product Fmoc-Val-Cit (0.5g, yield 44.3%).
Structural identification was performed with 1 HNMR. Fmoc-Val-OSU:1H NMR (600MHz, DMSO-d6) δ 8.15(d, J ═ 8.4Hz,1H),7.90(d, J ═ 7.5Hz,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 ═ 6.7Hz,6H), Fmoc-Cit: 1H NMR (600MHz, DMSO-d6) δ 8.13(d, J ═ 7.3Hz,1H),7.89(d, J ═ 7.5, 2H), 7.7.75 (ddc-d, 7.94H), 7.5H (ddc-d, 7.5H, 7.8.5H), 7.5H, 7.8.5H, 7.8, 7.5H, 7.8.8, 7H, 7.5H, 7H, 7.9H, 7.5H, 7.9H, 7.5H, 7.9, 1.98(H, J ═ 6.8Hz,1H),1.69(dq, J ═ 14.0,6.0,5.6Hz,1H),1.56(dtd, J ═ 13.9,9.2,5.5Hz,1H), 1.45-1.33 (m,2H),0.87(dd, J ═ 19.1,6.8Hz,6H).
2. Gambogic acid-cystamine (GA-SS-NH)2) Synthesis of (2)
2.1) Gambogic acid (1.57g,2.5mmol) was dissolved in dichloromethane solution (50mL), cooled to 0 deg.C, EDCI (623mg,3.25mmol) and HOBT (439mg,3.25mmol) were added and the mixture was activated at 0 deg.C for one hour. A solution of cystamine dihydrochloride (1.69g,7.5mmol) in methanol (30mL) was added to the mixed solution, triethylamine (500. mu.L, 3.75mmol) was added, and the mixture was stirred at room temperature for 48 hours to obtain a reaction solution.
2.2) reaction solution obtained in step 2.1) was treated with 1mM NaHCO3Washing the solution (20mL) for 3 times, collecting 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-NH2(1.45g,76％)。
The product was characterized by ESI-MS, 1HNPR and IR. ESI M/z 763.3[ M + H ] +,1H NMR (600MHz, DMSO-d6)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 ═ 7.0Hz,1H),5.60(dt, J ═ 10.2,2.8Hz,1H),5.06(q, J ═ 7.8Hz,2H),3.50(t, J ═ 5.8Hz,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, 2.0, 1.8H), 1.13.8H, 1H, 2.47 (dd, 8H), 1H, 13.8.8H, 1H, 13.8H, 1H.
Synthesis of Fmoc-valine-citrulline-gambogic acid (Fmoc-VC-SS-GA)
Fmoc-Val-Cit (0.5g,1mmol) was dissolved in 50mL dichloromethane and 10mL methanol, cooled to 0 deg.C, EDCI (0.25g,1.3mmol) and HOBT (0.18g,1.3mmol) were added, the resulting mixture was activated at 0 deg.C for 1h, and GA-SS-NH was added2(1.69g,7.5mmol) in dichloromethane (30mL) and triethylamine (267. mu.L, 2mmol), 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 the mixture was stored at 4 ℃ overnight, filtered, washed with water three times, and dried under vacuum. Purification was performed by column chromatography (methanol: dichloromethane 1: 11) to obtain 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 (600MHz, DMSO-d6) 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 ═ 6.0Hz,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 mainly comprises the following two steps: (1) treating mPEG5000 with succinic anhydride to convert all hydroxyl groups into acid groups, and (2) connecting mPEG5000-COOH with Fmoc-VC-SS-GA to obtain mPEG-VC-SS-GA.
4.1) adding toluene and mPEG5000 into a container, refluxing with toluene for 2h to remove water in the mPEG5000(10g,2mmol), cooling, performing rotary evaporation under reduced pressure to remove the toluene, and dissolving the residue in 50mL of chloroform to obtain a chloroform solution of mPEG 5000; succinic anhydride (1g,10mmol) and DMAP (733mg,6mmol) are dissolved in 10mL pyridine solution, then the solution is dropwise added into chloroform solution of mPEG5000, the obtained mixture is stirred at 60 ℃ under the protection of nitrogen for 24 hours, then the mixture is washed with 100mL physiological saline for three times, after being dried with anhydrous magnesium sulfate, an organic layer is filtered and taken, the organic layer is concentrated, and after being washed with 50mL diethyl ether for three times, the organic layer is dried in vacuum, and a white solid product, namely mPEG5000-COOH (6.63g,65 percent) is obtained.
4.2) mPEG5000-COOH (0.5g,0.1mmol) was dissolved in 20mL dichloromethane, EDCI (0.025g,0.13mmol) and HOBT (0.018g,0.13mmol) and 1g were addedActivating the molecular sieve type molecular sieve in the dark at 0 ℃ for 30min to obtain mPEG-COOH solution.
4.3) Fmoc-VC-SS-GA (0.5g,0.4mmol) was dissolved in 15mL THF and DBU (0.8mmol,120ul) was added and stirred for ten minutes to remove the Fmoc group. And adding the mixed solution with the Fmoc group removed into the mPEG5000-COOH solution, and reacting for 2 days at room temperature under the protection of nitrogen.
4.4) after the reaction was completed, washing with 50mL of distilled water three times, followed by extraction with chloroform three times, combining the organic layers, evaporating under reduced pressure, adding ether for washing, followed by filtration, vacuum-drying the resulting solid, adding 20mL of ultrapure water, stirring, filtering, and dialyzing for 2 days (dialysis medium was 1L of 5% DMF ultrapure water, dialysis bag MWCO ═ 5kDa), while changing the release medium for 5 times at 2h, 6h, 12h, 24h, and 36h, respectively. After freeze drying, collecting yellow fluffy solid, namely the target product dual-sensitive polymer-gambogic acid connector mPEG-VC-SS-GA (PVSG), wherein the yield is as follows: 45 percent. The product was structurally characterized by MALDI-TOF-MS as shown in FIG. 2.
Comparative example (II): glutathione reducing Single-sensitization Polymer-drug linker mPEG-SS-GA (PSG)
The preparation method comprises the following steps:
1. gambogic acid-cystamine (GA-SS-NH)2) Synthesis of (2)
1.1) Gambogic acid (1.57g,2.5mmol) was dissolved in dichloromethane solution (50mL), cooled to 0 deg.C, EDCI (623mg,3.25mmol) and HOBT (439mg,3.25mmol) were added and the mixture was activated at 0 deg.C for one hour. A solution of cystamine dihydrochloride (1.69g,7.5mmol) in methanol (30mL) was added to the mixed solution, triethylamine (500. mu.L, 3.75mmol) was added, and the mixture was stirred at room temperature for 48 hours to obtain a reaction solution.
1.2) reaction solution obtained in step 2.1) was treated with 1mM NaHCO3Washing the solution (20mL) for 3 times, collecting 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-NH2。
Synthesis of mPEG-SS-GA (PSG)
2.1) adding toluene and mPEG5000 into a container, refluxing with toluene for 2h to remove water in the mPEG5000(10g,2mmol), cooling, performing rotary evaporation under reduced pressure to remove the toluene, and dissolving the residue in 50mL of chloroform to obtain a chloroform solution of mPEG 5000; succinic anhydride (1g,10mmol) and DMAP (733mg,6mmol) are dissolved in 10mL pyridine solution, then the solution is dropwise added into chloroform solution of mPEG5000, the obtained mixture is stirred at 60 ℃ under the protection of nitrogen for 24 hours, then the mixture is washed with 100mL physiological saline for three times, after being dried with anhydrous magnesium sulfate, an organic layer is filtered and taken, the organic layer is concentrated, and after being washed with 50mL diethyl ether for three times, the organic layer is dried in vacuum, and a white solid product, namely mPEG5000-COOH (6.63g,65 percent) is obtained.
2.2) mPEG5000-COOH (0.5g,0.1mmol) was dissolved in 20mL dichloromethane, EDCI (0.025g,0.13mmol) and HOBT (0.018g,0.13mmol) and 1g were addedActivating the molecular sieve type molecular sieve in the dark at 0 ℃ for 30min to obtain mPEG-COOH solution.
2.3) taking GA-SS-NH2(0.153g,0.20mmol)) and triethylamine (26. mu.L, 0.20mmol) were added to the mPEG5000-COOH solution and reacted for 2 days at room temperature under nitrogen.
2.4) after the reaction was completed, washing with 50mL of distilled water three times, followed by extraction with chloroform three times, combining the organic layers, evaporating under reduced pressure, adding diethyl ether for washing, followed by filtration, vacuum-drying the resulting solid, adding 20mL of ultrapure water, stirring, filtering, and dialyzing for 2 days (dialysis medium was 1L of 5% DMF ultrapure water, dialysis bag MWCO ═ 5kDa), while changing the release medium for 5 times at 2h, 6h, 12h, 24h, and 36h, respectively. After freeze drying, collecting yellow fluffy solid, namely 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 is a MALDI-TOF-MS mass spectrum of PVSG, and FIG. 3 is a MALDI-TOF-MS mass spectrum of mPEG-COOH (A) and PSG (B). The molecular ion peak in each mass spectrogram is identical with the synthesized molecule, and the main fragment ion peak energy corresponds to the related structure, so that the structure is proved to be correct.
DSC measurement of (III) 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 25-260 ℃ in DSC measurement.
As shown in fig. 4, the gambogic acid has absorbed heat at about 80 ℃, and it can be seen from curve a and curve C that the absorbed heat peak of gambogic acid can still be seen in the physical mixture, and the absorbed heat peaks of PSG and PVSG disappear, indicating that there is no free gambogic acid and the absorbed heat of PVSG is shifted from PSG, possibly due to the influence of the instrument fluctuation or the influence of VC segment.
(IV) examination of hydration volume of PVSG
Precisely weighing 5mg PVSG, placing the PVSG into a50 mL centrifuge tube, adding 2mL, 5mL, 10mL and 20mL of distilled water into three parallel samples respectively, shaking up and down to ensure that the PVSG is rapidly dissolved, carrying out 60W ultrasonic treatment for one minute, passing through a 0.45um filter membrane, carrying out detection by dynamic light scattering, and taking the particle size and PDI as investigation parameters, wherein the results are shown in Table 1.
Table 1 effect of hydration volume on nanoparticles
The results in Table 1 show that the particle size and PDI of PVSG-NPs are small at 5mL and 10mL when the hydration volume is increased from 2mL to 20mL, and the hydration volume is 10mL, i.e. the concentration is 0.5mg/mL, the particle size is 142 + -4.71 and the PDI is 0.259 + -0.048 in the reference and considering the dosage requirements of the subsequent experiments.
(V) PVSG ultrasound time review
Precisely weighing 5mg PVSG, placing in a50 mL centrifuge tube, adding 10mL distilled water into three parts of parallel samples, shaking up and down to dissolve rapidly, performing ultrasonic treatment at 60W for 1min, 3min and 5min, filtering with 0.45um filter membrane, detecting by dynamic light scattering, taking particle size and PDI as investigation parameters, and obtaining the results shown in Table 2
TABLE 2 Effect 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 conversely, the particle size and PDI are increased at 5min, so that the polymer is easy to break when the ultrasonic time is too long, and the ultrasonic time is selected for one minute when the ultrasonic time is 1min and 3min which have no obvious difference.
The final prescription process is as follows: precisely weighing 5mg of PVSG, placing the PVSG in a50 mL centrifuge tube, adding 10mL of distilled water, shaking up and down, carrying out ultrasonic treatment for 1min at 60W, filtering through a 0.45um filter membrane, treating PSG by the same method, and taking an equivalent amount of GA to obtain PVSG-NPs and PSG-NPs.
(VI) measurement of particle size and potential of PVSG self-assembled nanoparticles
PSG-NPs and PVSG-NPs were prepared according to the optimal recipe, and the particle size, potential and PDI (25 ℃, n ═ 3) of the conjugate were measured by Dynamic Light Scattering (DLS) method, and the results are shown in fig. 5. The particle size of PSG-NPs is determined to be 117 +/-4.71 nm, PDI is 0.315 +/-0.016, the particle size of PVSG-NPs is 142 +/-4.71 nm, the particle size of PDI is 0.190 +/-0.036, the particle size of the PVSG-NPs is larger than that of the PVSG-NPs, but the particle sizes of the PSG-NPs and the PDI are both smaller than 200nm, and the PSG-NPs and the PDI can enter tumor tissues through an EPR effect. Both potentials were around 0, which is due to the stealth effect of mPEG, indicating that mPEG successfully encapsulated GA.
(VII) determination of PVSG self-assembled nanoparticles by transmission electron microscope
And (3) carrying out Transmission Electron Microscope (TEM) observation on PSG-NPs (PSG-NPs) (PSB) and PVSG-NPs (PSG-NPs) (A), sucking the micellar solution by using a pipette, dripping the micellar solution onto a 200-mesh copper net, naturally drying the solution, dripping 0.2% phosphotungstic acid for dyeing, sucking the solution after 3-5min, naturally drying the solution, and observing the forms of the PSG-NPs and the PVSG-NPs (PVSG-NPs) (A).
As can be seen from fig. 6, TEM analysis confirmed that the nanoparticles were spherical, uniform in size, and slightly smaller than DLS detection, which is probably due to water evaporation, and the characterization results, taken together, demonstrated successful preparation of micelles.
(VIII) in vitro Release assay of PVSG self-assembled nanoparticles
The in vitro release is carried out by dialysis, the accumulated release amount is used as an index to simulate the in vivo environment, a culture medium containing 1% of Tween 80 is used as a release medium, and L-arginine is used for preparing a garcinolic acid preparation, and the stable existence of PSG and PVSG at normal temperature is contrastingly examined. The specific implementation method comprises the following steps:
1) dissolving 22mg L-arginine and 10mg gambogic acid in a culture medium containing 1% Tween 80, and vortexing. Namely the control preparation L-GA.
2) The L-GA, PSG and PVSG prepared in the same batch were prepared into a solution with a gambogic acid equivalent content of 2mg/mL using a medium of 1% tween 80 as a medium, and added into dialysis bags of the same length (MWCO: 1 kDa; pretreatment: boiling the dialysis bag in 2% (W/V) sodium bicarbonate and 1mmol/L EDTA for 10min, cooling to room temperature, storing at 4 deg.C, carefully removing bubbles, sealing tightly, placing into an eggplant-shaped bottle containing 40mL dissolution medium, incubating at constant temperature of 37 deg.C and constant temperature shaking box of 100rpm, sampling for 3mL at time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h, and immediately supplementing isothermal release medium with equal volume. Filtering the sample with 0.45 μm microporous membrane, collecting filtrate, analyzing by ultraviolet condition sample injection, recording peak area, introducing standard curve, calculating drug content leaked into medium from preparation at different time points, calculating drug release amount and cumulative release amount, and drawing release curve (i.e. drug cumulative release amount-time curve), with the test result as shown in FIG. 7.
As shown in FIG. 7, L-GA release was fast, nearly 80% released already at 12h, while PSG-NPs released slower than PVSG-NPs than L-GA, and it can be seen that PVSG-NPs released more than PSG-NPs at 72h, and under normal conditions PSG-NPs released more than PSG-NPs, but there was no significant difference between the two groups (P > 0.05).
(nine) glutathione sensitivity release experiment of PVSG self-assembled nanoparticles
Selecting an acetic acid-sodium acetate buffer solution with pH of 5.0 and GSH concentration of 10mM as a release medium, respectively dissolving two micelles of PSG-NPs and PVSG-NPs in an acetic acid-sodium acetate buffer solution (equivalent GA 1mg/mL), performing in-vitro release (40mL release medium) by a dialysis method, simultaneously taking a group without GSH as a control, sampling 3mL at time points of 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h, and simultaneously supplementing isothermal release medium with the same volume, wherein the two micelles are respectively dissolved in the acetic acid-sodium acetate buffer solution, and the results are shown in figure 8.
As shown in FIG. 8, the release amounts of PSG-NPs and PVSG-NPs were increased after glutathione was added, but compared with the release amounts of PSG-NPs increased by about 15% and PVSG-NPs increased by about 25% after 72h, which indicates that the redox performance of PVSG-NPs is better than that of PSG-NPs, but the two sets of data have no significant difference (P > 0.05).
(ten) cathepsin B sensitivity experiment of PVSG self-assembly nanoparticles
10mL of acetic acid-sodium acetate buffer containing 10mM GSH and CB was used as the release medium. PSG and PVSG (eq GA 500. mu.g) were dissolved in 2mL of acetic acid-sodium acetate buffer, respectively. CB (lyophilized solid, 1.2mg) was dissolved in 1mL of 25mM sodium acetate/1 mM EDTA buffer (pH5.0), stabilized at 4 ℃ for 8 hours, and then stored at-80 ℃ by freezing. CB is placed in 100 mu L of 30mM DTT/15mM EDTA buffer solution (pH5.0) for incubation at 37 ℃ before the test, 50 mu L of LCB buffer solution (50UI/mL) is added into the micellar solution, and the reaction system is shaken at constant temperature at 37 ℃ for sampling 3mL every 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h and 72h time points, and simultaneously is supplemented with isothermal equal volume of release medium, and the result is shown in FIG. 9.
As shown in the results of FIG. 9, the PSG-NPs release curve was very close to that of the release curve without the addition of CB, and it was found that PVSG-NPs release was significantly increased compared to the PSG group (P <0.05), indicating excellent performance of PVSG-NPs.
(eleven) pharmacodynamic assay of PSVG
Accurately weighing 1mg of GA powder, PSG-NPs and PVSG-NPs in an EP tube, performing ultraviolet sterilization, dissolving the medicine (100 mu L/900 mu L) in a blank culture medium containing Tween 80, diluting the medicine to 100 mu M equivalent GA concentration in the blank culture medium, and storing the diluted medicine as a mother solution. When HepG2 cells grew to 80%, the cells were collected, diluted with medium, counted, transferred to 96-well cell culture plates at 1 × 104/well (100 μ L), cultured in 5% CO2 incubator at 37 ℃ for 24h, diluted in proportion to 6 concentration gradients of 8 μ M, 4.0 μ M, 2.0 μ M, 1.0 μ M, 0.5 μ M, 0.25 μ M (eq.GA), diluted in 100 μ L per well, dosed to 96-well plates, dosed to 4.0 μ M, 2.0 μ M, 1.0 μ M, 0.5 μ M, 0.25 μ M, 0.125 μ M (eq.GA), three parallel groups per concentration, sterile PBS as a blank control in one round of the periphery of 96-well plates, a solvent control group was set, incubated in 37 ℃ and 5% CO2 incubator for 24h, 48h, 72h, and then incubated in constant temperature incubator (37.5%) for 4h, and a constant temperature solution (37 ℃ 4.0.0.GA), and (3) taking out a 96-well plate, pouring out liquid in the plate, air-drying in an ultra-clean bench, adding DMSO (150 mu L/hole) for dissolving, shaking on a shaking table for 5min to fully dissolve purple crystals, detecting an OD value by using an enzyme labeling instrument at a wavelength of 570nm, storing data results, calculating the inhibition rate of each group of cells, and drawing a histogram according to the inhibition rate results.
The same procedure was performed 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 on four cells
As shown in table 3 and fig. 10, the toxicity of the three drugs to normal cells and the toxicity of the three drugs to tumor cells were respectively detected by MTT method, and it was found that PSG-NPs and PVSG-NPs are much less toxic than GA in the three normal cell lines, especially at high concentration, and the sensitivity and release of PVSG-NPs are higher than PSG-NPs, thus presenting higher normal cytotoxicity, but still having significant difference compared to GA bulk drug, in the three cell lines, two micelles of PSG-NPs and PVSG-NPs have the smallest cytotoxicity to BV2 cell, and BV2 cell still survives more than 80% at high concentration, while the survival rate of RAW264.7 and HEK293 is less than 2, probably because RAW264.7 can take in drugs, GA bulk drug kills macrophages to less than 5% at high concentration, and the survival rate of two micelles also has a certain effect on macrophages, however, IC50 was significantly higher than the GA drug substance. In a cytotoxicity test on a HepG2 cell line, the 24h IC50 of GA bulk drug is higher than the IC50 value of normal cells, and it can be seen that both micelles have concentration dependence, but PSG-NPs have weaker killing capacity on tumor cells and cannot be completely released under the encapsulation effect of PEG, PVSG-NPs have less killing effect on the tumor cells at 24h than GA, at 48h and 72h, the high-concentration PVSG-NPs have stronger killing effect than GA, and all three drugs have concentration dependence and time dependence, but compared with the above, the release speed of the PVSG-NPs is higher, and the response capacity is higher than that of the PSG-NPs.
Claims (9)
1. A dual sensitive polymer-drug conjugate is characterized in that the dual sensitive polymer-drug conjugate is a dual sensitive polymer-drug conjugate mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B,
wherein Y is valine-citrulline, forms a double sensitive polymer-drug linker mPEG-VC-SS-R, and has a structural formula shown as (I):
or Y is phenylalanine-arginine, forms a double sensitive polymer-drug connector mPEG-PA-SS-R, and has a structural formula shown as (II):
wherein R is a pharmaceutical compound with carboxyl.
2. The dual sensitive polymer-drug conjugate of claim 1, wherein the drug compound having a carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azaserine, chlorophenylalanine, N-acetyl-L-phenylalanine, and N-acetyl-L-valine.
3. The dual sensitive polymer-drug conjugate of claim 2, wherein Y is valine-citrulline and the drug compound having a carboxyl group is gambogic acid, and the dual sensitive polymer-drug conjugate mPEG-VC-SS-GA that is sensitive to glutathione reduction and sensitive to cathepsin B has the structural formula shown in (iii):
4. the dual sensitive polymer-drug conjugate of claim 1,2 or 3, wherein mPEG specification is polyethylene glycol monomethyl ether mPEG 5000.
5. The method for preparing a dual sensitive polymer-drug conjugate of claim 1,2 or 3, 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) connecting the enzyme sensitive substrate intermediate with a disulfide bond-containing drug derivative R-cystamine to prepare a double sensitive drug derivative; 4) polyethylene glycol monomethyl ether and a double-sensitive drug derivative are condensed into a double-sensitive polymer-drug connector mPEG-Y-SS-R sensitive to glutathione reduction and sensitive to cathepsin B.
6. The method of claim 5, wherein Y is valine-citrulline and constitutes dual susceptible polymer-drug conjugate mPEG-VC-SS-R, comprising the steps of:
1) synthesis of enzyme sensitive substrate intermediate Fmoc-valine-citrulline (Fmoc-Val-Cit):
1.1) dissolving Fmoc-Val, HOSu and DCC in tetrahydrofuran at 0 ℃, stirring for 24h, filtering, decompressing and rotary evaporating to obtain a white solid product Fmoc-Val-OSu;
1.2) combining Cit with NaHCO3Dissolving in distilled water, cooling to 0 ℃, and dropwise adding DME solution of Fmoc-Val-OSu to Cit and NaHCO3Adding tetrahydrofuran for assisting dissolution, and stirring at room temperature for 24h 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 value to 8-9, extracting with ethyl acetate, collecting a water layer, adding a citric acid solution to adjust the pH value to 3-4, separating out a white gelatinous solid, filtering, dissolving the obtained white gelatinous solid in a mixed solution of tetrahydrofuran and methanol, performing rotary evaporation and concentration, adding methyl tert-butyl ether, stirring overnight at 0 ℃, filtering, and performing 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 drug derivative R-cystamine (R-SS-NH)2) The synthesis of (2):
2.1) dissolving a pharmaceutical compound R with carboxyl in 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) reacting the reaction solution obtained in the step 2.1) with NaHCO3Washing the solution, collecting the 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 double sensitive drug derivative Fmoc-valine-citrulline-R (Fmoc-VC-SS-R):
3.1) dissolving Fmoc-Val-Cit obtained in the step 1) in a mixed solution of dichloromethane and methanol, cooling to 0 ℃, adding EDCI and HOBT, activating the obtained mixture at 0 ℃ for 1h, and adding R-SS-NH obtained in the step 2)2Stirring the obtained mixture overnight, concentrating under reduced pressure after the reaction is finished, adding ice water, storing at 4 deg.C overnight, filtering, washing with water for three times, vacuum drying, and performing column chromatographyPurifying to obtain the double 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 a pyridine solution, then dropwise adding the pyridine solution into a chloroform solution of mPEG, stirring the obtained mixture at 60 ℃ under the protection of nitrogen for 24 hours, washing the obtained mixture with physiological saline, drying the obtained product with anhydrous magnesium sulfate, filtering the obtained product to obtain an organic layer, concentrating the organic layer, washing the organic layer with 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 a type 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, stirring for 10 minutes, adding the obtained mixed solution into mPEG-COOH solution, and reacting for 2 days at room temperature under the protection of nitrogen;
4.4) after the reaction is finished, washing with distilled water, extracting with chloroform, combining organic layers, evaporating under reduced pressure, adding ether for washing, filtering, drying the obtained solid in vacuum, adding ultrapure water, stirring, filtering, dialyzing for 2 days, replacing release media for 5 times in 2h, 6h, 12h, 24h and 36h respectively, and freeze-drying to obtain the target product, namely the dual-sensitive polymer-drug conjugate mPEG-VC-SS-R.
7. The method according to claim 6, wherein in step 4.4), the dialysis bag has a molecular weight of 5kDa, and the dialysis medium is distilled water.
8. The preparation method according to claim 6 or 7, wherein the pharmaceutical compound R with carboxyl is gambogic acid, and the mPEG has the specification of mPEG 5000.
9. Use of the dual sensitive polymer-drug conjugate of claim 1,2 or 3 for the preparation of an anti-tumor drug.
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Cited By (3)
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|---|---|---|---|---|
| CN111494318A (en) * | 2020-04-28 | 2020-08-07 | 华中科技大学同济医学院附属协和医院 | Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof |
| CN111870579A (en) * | 2020-07-17 | 2020-11-03 | 山东大学 | Tumor-targeted nano micelle, preparation method and application of nano micelle as drug carrier |
| CN115093434A (en) * | 2022-06-21 | 2022-09-23 | 中国中医科学院中药研究所 | Gambogic acid nanometer preparation and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108478804A (en) * | 2018-05-08 | 2018-09-04 | 辽宁大学 | A kind of polyacrylic acid-S-S- block copolymer drugs and preparation method thereof |
| 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 |
| CN114377151A (en) * | 2022-01-24 | 2022-04-22 | 辽宁大学 | Dual-response type polymer prodrug micelle and preparation method thereof |
-
2019
- 2019-10-17 CN CN201910988030.0A patent/CN110604820B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108478804A (en) * | 2018-05-08 | 2018-09-04 | 辽宁大学 | A kind of polyacrylic acid-S-S- block copolymer drugs and preparation method thereof |
| 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 |
| CN114377151A (en) * | 2022-01-24 | 2022-04-22 | 辽宁大学 | Dual-response type polymer prodrug micelle and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 薛大权主编: "《聚乙二醇在医药学领域的应用及技术》", 30 April 2011, 华中科技大学出版社, pages: 99 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111494318A (en) * | 2020-04-28 | 2020-08-07 | 华中科技大学同济医学院附属协和医院 | Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof |
| CN111870579A (en) * | 2020-07-17 | 2020-11-03 | 山东大学 | Tumor-targeted nano micelle, preparation method and application of nano micelle as drug carrier |
| CN115093434A (en) * | 2022-06-21 | 2022-09-23 | 中国中医科学院中药研究所 | Gambogic acid nanometer preparation and preparation method thereof |
| CN115093434B (en) * | 2022-06-21 | 2023-06-23 | 中国中医科学院中药研究所 | Gambogic acid nano preparation and preparation method thereof |
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