CN109232875B - PH/reduction double-sensitive carrier material formed by Cys and derivatives thereof and polyester polymer, and preparation method and application thereof - Google Patents
PH/reduction double-sensitive carrier material formed by Cys and derivatives thereof and polyester polymer, and preparation method and application thereof Download PDFInfo
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract
The invention discloses a pH/reduction double-sensitive carrier material formed by Cys and derivatives thereof and a polyester polymer, and a preparation method and application thereof. The invention takes the aldehyde polyester polymer and Cys containing disulfide bond or derivatives thereof as raw materials, successfully prepares a new polymer with pH/reduction double sensitivity of imine bond and disulfide bond, and successfully constructs the nano-carrier for targeted delivery of the antitumor drug; drugs with different hydrophilicity and hydrophobicity can be physically wrapped through the interaction of the hydrophilicity and the hydrophobicity to form an environment dual-response type nano drug-carrying system with uniform and stable particle size. Under the tumor cell environment, the double-sensitive polymer nanoparticles can be rapidly disintegrated and the entrapped drug can be rapidly released, so that the whole system has good biosecurity, biodegradability and long-circulating stability, and has the characteristics of high-efficiency tumor targeting and high-efficiency tumor cell growth inhibition; in addition, the preparation method is simple, and has a good application prospect in the field of biological medicines.
Description
Technical Field
The invention belongs to the technical field of functional polymer materials and biological medicines. More particularly, relates to a pH/reduction double-sensitive carrier material formed by Cys and derivatives thereof and a polyester polymer, and a preparation method and application thereof.
Background
Cancer is one of the major diseases that endanger human health and cause human death. Clinically, advanced cancer patients are mainly treated by chemical drugs, but small-molecule anticancer drugs are easily destroyed in vivo due to low selectivity of the drugs, have short half-life and large toxic and side effects, and inevitably damage normal cells while killing cancer cells, so that the safe and effective transportation of the small-molecule anticancer drugs to the body is a problem to be solved urgently. With the cross development of nanotechnology and various disciplines, the traditional small-molecule antitumor drug is combined with the nanotechnology to form a nano controlled-release drug-carrying system, which is widely applied to the field of nano drugs. The system can improve the solubility and stability of the antitumor drug in water to a certain extent. The stimuli-responsive polymer system can respond to external environments such as temperature, illumination, enzyme, pH and the like, and in order to better release anticancer drugs, based on the nano drug delivery system, polymer nano carriers with multiple responsiveness, such as double pH responses, pH/reduction responses, pH/temperature/reduction responses and the like, are gradually paid attention by researchers.
Tumor tissue has a different microenvironment than normal tissue. In one aspect, a pH gradient exists between tumor tissue and normal tissue: the pH of normal tissue is about 7.4, while that of tumor tissue is around 6.8, and different stages of endosomes/lysosomes have different pH values, with the pH values of endosomes and lysosomes in early tumor cells being 6.0 and the late pH value being 5.0. The pH value of Lysosome (Lysosome) is lower than that of endosome, generally 4.0-5.0. After the polymer nanoparticles coated with the antitumor drug enter cells through endocytosis, most of the polymer nanoparticles enter endosomes/lysosomes. Therefore, in order to overcome multi-drug resistance, the carrier must escape as soon as possible when entering the endosome/lysosome, releasing the drug into the cytoplasm, and preventing the drug from being degraded by the acidic environment and enzymes in the endosome/lysosome, thereby improving the anti-tumor effect. On the other hand, studies have found that the specific microenvironment of tumor cells is not only manifested in changes in pH. Glutathione (GSH) is a thiol compound with reducibility commonly found in vivo, and compared with normal cells, the concentration of GSH in tumor cells is about 2-20 mM, which is 500-1000 times of that of normal cells. Therefore, in order to exert an antitumor effect, it is necessary to release the entrapped antitumor drug as soon as possible. Many reduction-sensitive drug delivery systems have been designed to promote the release of anticancer drugs in tumor cells. However, the existing pH or GSH sensitive nanocarriers have poor stability during long circulation of blood and cannot efficiently enter tumor cells during circulation. In addition, the existing nano delivery system for the antitumor drugs has the following defects: poor biocompatibility and biodegradability, toxic and side effects on normal tissues, low safety and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art, and provide a pH/reduction double-sensitive carrier material formed by Cys and derivatives thereof and a polyester polymer, so that the positioning controllable release of a drug in a tumor cell is realized, the whole system has good biosafety, biodegradability and long-circulating stability, has the characteristics of high-efficiency tumor targeting and high-efficiency inhibition of tumor cell growth, and can effectively solve the problems of poor systemic circulation stability, poor targeting property, poor biosafety and the like of a nano-drug carrier.
The first purpose of the invention is to provide Cys-PCL which is a polymer sensitive to pH/reduction.
The second purpose of the invention is to provide a preparation method of the polymer with pH/reduction double sensitivity.
The third purpose of the invention is to provide the application of the Cys-PCL polymer or the polymer prepared by the method in the aspect of serving as or preparing a drug delivery carrier.
It is a fourth object of the present invention to provide a stimulus responsive drug delivery vehicle.
It is a fifth object of the present invention to provide a nano drug delivery system with a pH/reduction dual response.
The sixth purpose of the present invention is to provide the application of the Cys-PCL polymer or the stimulus-responsive drug delivery vehicle or the nano drug delivery system in preparing anticancer drugs.
The above purpose of the invention is realized by the following technical scheme:
a polymer Cys-PCL with pH/reduction double sensitivity is a polymer formed by Cys and PCL, and the structural formula of the polymer is shown as the following formula (I):
wherein n is 5-10000; cys is L-cystine dimethyl ester dihydrochloride, and PCL is polycaprolactone.
The polymer Cys-PCL has good biocompatibility, good biodegradability and low toxicity.
The invention also relates to a preparation method of the polymer with pH/reduction double sensitivity, which comprises the following steps:
s1, performing hydroformylation on a polyester polymer with hydroxyl groups at two ends through an esterification reaction to obtain a hydroformylation polyester polymer;
s2, grafting the aldehyde polyester polymer with disulfide bond-containing cystine ester or derivatives thereof through Schiff base reaction to obtain the polymer with pH/reduction dual response.
The invention develops a super-pH/reduction double-sensitive carrier material based on Cys containing corresponding reduction and derivatives thereof and polyester polymers, which can wrap different kinds of anti-tumor drugs, thereby obviously improving the bioavailability of the chemotherapeutic drugs, reducing adverse reactions, having excellent anti-tumor effect in vivo and having certain reference significance for the treatment of tumors.
Preferably, the polyester polymer is selected from any one of Polycaprolactone (PCL), polyethylene terephthalate (PET), polybutylene terephthalate (PBT) or Polyarylate (PAR).
More preferably, the polyester polymer is polycaprolactone.
Further, the weight average molecular weight of the polyester polymer is preferably 500-80000 Da, and more preferably 1000-10000 Da.
Preferably, the cystine ester is selected from any one of L-cystine dimethyl ester dihydrochloride (Cys), L-cystine ethyl ester dihydrochloride or L-cystine benzyl ester dihydrochloride.
More preferably, the cystine ester is selected from the group consisting of L-cystine dimethyl ester dihydrochloride.
Preferably, the step S1 is: and mixing the polyester polymer with hydroxyl at two ends with 4-chlorobenzoic acid (p-CBA), adding a reaction solvent, and carrying out esterification reaction under the protection of inert gas and the action of a catalyst to obtain the aldehyde-based polyester polymer.
Preferably, the molar ratio of the polyester polymer to the 4-chlorobenzoic acid is 1: 1-20.
More preferably, the molar ratio of the polyester polymer to the 4-chlorobenzoic acid is 1: 2.5-3.
Preferably, the catalyst is selected from one or two of 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU), N-hydroxysuccinimide (NHS), Dicyclohexylcarbodiimide (DCC) or 4-Dimethylaminopyridine (DMAP).
More preferably, the catalyst is DCC and DMAP.
Preferably, the molar ratio of the polyester polymer to the p-CBA to the DMAP to the DCC is 2: 5-6: 0.25-1.5: 3-7.
Preferably, the reaction solvent is dimethyl sulfoxide (DMSO), dichloromethane, chloroform, or tetrahydrofuran.
More preferably, the reaction solvent is anhydrous dichloromethane.
Preferably, the temperature of the esterification reaction is-50 to 25 ℃.
More preferably, the temperature of the esterification reaction is-10 to 10 ℃.
Preferably, the esterification reaction is carried out for 2-48 h under the stirring condition.
More preferably, the esterification reaction is carried out for 12-36 h under the stirring condition.
Preferably, the inert gas is nitrogen or argon.
More preferably, the inert gas is nitrogen.
Further, in order to improve the purity of the formylated polyester polymer, the product of step S1 was purified by removing DCU as a by-product and then further purified. The purification method is silica gel column chromatography or ether precipitation; preferably silica gel column chromatography.
Wherein the eluent for removing the byproduct DCU is selected from one or more of dichloromethane, ethyl acetate, acetone or methanol; preferred are dichloromethane and acetone.
The volume ratio of the dichloromethane to the acetone is preferably 2-20: 1; more preferably 5: 1.
The further purification eluent is one or more of dichloromethane, ethyl acetate, acetone or methanol; preferred are dichloromethane and methanol.
The volume ratio of the dichloromethane to the methanol is preferably 2-10: 1; more preferably 5: 1.
Preferably, in step S2, the molar ratio of the aldehyde-based polyester polymer to the cystine ester is 1-10: 1-10.
More preferably, in step S2, the molar ratio of the aldehydized polyester polymer to the cystine ester is 1: 1.
Further, in order to increase the purity of the polymer, the product of step S2 is purified. The product of step S2 is purified by gel chromatography or recrystallization using methanol, ethanol or acetone as eluent.
Preferably, the purification method of the product of step S2 is Sephadex LH-20 gel chromatography; the eluent is methanol.
Accordingly, the application of the Cys-PCL polymer or the polymer prepared by the method in the aspect of serving as or preparing a drug delivery carrier is also within the protection scope of the invention.
Preferably, the delivery carrier is a nano-drug carrier with pH/reduction dual responsiveness. The polymer Cys-PCL or the polymer prepared by the method is used as a nano carrier to load a drug.
The invention also relates to a stimulus-response type drug delivery carrier, which is prepared by preparing the polymer Cys-PCL or the polymer prepared by the method into nanoparticles.
The polymer Cys-PCL or the polymer prepared by the method can be prepared into nanoparticles which are uniform in particle size and stably loaded with different hydrophilic and hydrophobic drugs by a nano precipitation method or a single emulsion/double emulsion method.
The invention also relates to a nano drug delivery system with pH/reduction dual response, which comprises the polymer Cys-PCL or the polymer prepared by the method and a loaded drug.
The carried medicine includes hydrophilic medicine and hydrophobic medicine. Wherein, the hydrophilic drugs include, but are not limited to, doxorubicin hydrochloride, gemcitabine hydrochloride, irinotecan hydrochloride, fluorouracil or lentinan and other drugs. The hydrophobic drugs include, but are not limited to, Paclitaxel (PTX), methotrexate, camptothecin, adriamycin and the like.
Preferably, the mass ratio of the polymer to the carried medicine is 5-20: 1-2. If the mass ratio of the polymer to the loaded drug is too large, the drug loading rate is low, the number of the formed nanoparticles is small, and if the mass ratio of the polymer to the loaded drug is too small, the encapsulation rate is low, so that the drug waste is caused, and the obtained nanoparticles are unstable and easy to precipitate.
More preferably, the mass ratio of the polymer to the carried medicine is 5-10: 1.
The invention provides a method for preparing a nano drug delivery system with pH/reduction dual response by a nano precipitation method, which comprises the following steps:
s11, blending the polymer, the carried medicine and the stabilizer in an oil phase solvent to serve as an oil phase, and dripping the oil phase into a water phase at a certain speed under a stirring state to obtain the medicine-carrying nanoparticle solution with uniform and stable particle size.
S12, carrying out ultrafiltration, washing and freeze-drying on the obtained nanoparticle solution to obtain medicine-carrying nanoparticles, namely the nano medicine delivery system with pH/reduction dual response.
Preferably, the mass ratio of the polymer to the carried drug to the stabilizer is 5-20: 1-2: 1-5.
More preferably, the mass ratio of the polymer to the carried drug to the stabilizer is 5:1: 1-1.25.
Preferably, in step S11, the stabilizer is distearoylphosphatidylethanolamine-polyethylene glycol stabilizer (DSPE-PEG), polyvinyl alcohol (PVA), phospholipid molecule, or the like.
More preferably, in step S11, the stabilizer is DSPE-PEG, and the weight average molecular weight of the polyethylene glycol segment is 500-5000.
Even more preferably, the weight average molecular weight of the polyethylene glycol segment in the DSPE-PEG is 2000, i.e. the stabilizerIs DSPE-PEG2K。
Preferably, the aqueous phase of step S11 is a PBS solution.
Preferably, the oil phase solvent in step S11 is an organic reagent miscible with water, such as dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, dioxane, pyridine, etc.
More preferably, the oil phase solvent is dimethyl sulfoxide or N, N-dimethylformamide.
Preferably, in step S11, the dropping rate is 0.01 to 1 mL/sec.
More preferably, in step S11, the dropping rate is 0.05 mL/sec.
Preferably, in step S12, the ultrafiltration speed of the nanoparticle solution is 1000-4000 rpm.
More preferably, in step S12, the ultrafiltration rate of the nanoparticle solution is 2500 rpm.
In addition, the invention also provides a method for preparing the nano drug delivery system with pH/reduction dual response by a single emulsion method, which comprises the following steps:
s21, dissolving the polymer and the carried medicine into an organic solvent which has a low boiling point and is not soluble in water together, and performing ultrasonic treatment to obtain an oil phase;
s22, mixing the obtained oil phase with a water phase containing a stabilizer, performing ultrasonic treatment, stirring at a constant speed to remove the organic solvent to obtain a drug-loaded nanoparticle solution with uniform and stable particle size, and freeze-drying to obtain drug-loaded nanoparticles, namely the nano drug delivery system with pH/reduction dual response.
In step S21, the organic reagent with a low boiling point and immiscible with water is chloroform, dichloromethane, acetone, ethyl acetate or acetonitrile.
Preferably, in step S21, the organic reagent with a low boiling point and immiscible with water is dichloromethane.
Preferably, in step S22, the concentration of the stabilizer is 0.01% to 0.5%.
More preferably, in step S22, the concentration of the stabilizer is 0.2%.
Preferably, in step S22, the stabilizer is distearoylphosphatidylethanolamine-polyethylene glycol stabilizer, polyvinyl alcohol (PVA), phospholipid molecule, or the like.
More preferably, in step S22, the stabilizer is PVA.
In addition, the invention also provides a method for preparing the nano drug delivery system with pH/reduction dual response by a dual emulsion method, which comprises the following steps:
s31, dissolving the polymer in an organic solvent which has a low boiling point and is insoluble in water, and performing ultrasonic treatment to obtain an oil phase;
s32, dissolving the carried medicine in water, mixing the medicine with the oil phase, and performing ultrasonic treatment to obtain a water-in-oil type emulsion, wherein the carried medicine is a hydrophilic medicine;
and S33, mixing the obtained water-in-oil emulsion oil phase with a water phase containing a stabilizer, performing ultrasonic treatment, stirring at a constant speed to remove an organic solvent to obtain a drug-loaded nanoparticle solution with uniform and stable particle size, and freeze-drying to obtain drug-loaded nanoparticles, namely the nano drug delivery system with pH/reduction dual response.
In step S31, the organic reagent with a low boiling point and immiscible with water is chloroform, dichloromethane, acetone, ethyl acetate or acetonitrile.
Preferably, in step S31, the organic reagent with a low boiling point and immiscible with water is dichloromethane.
Preferably, in step S33, the concentration of the stabilizer is 0.01% to 0.5%.
More preferably, in step S33, the concentration of the stabilizer is 0.25%.
Preferably, in step S33, the stabilizer is distearoylphosphatidylethanolamine-polyethylene glycol stabilizer, polyvinyl alcohol (PVA), phospholipid molecule, or the like.
More preferably, in step S33, the stabilizer is PVA.
Preferably, the particle size of the drug-loaded nanoparticles is 50-200 nm.
More preferably, the particle size of the drug-loaded nanoparticles is 50-100 nm.
The invention firstly synthesizes aldehyde polyester by polyester polymer and 4-chlorobenzoic acid (p-CBA), and then uses L-type cystine dimethyl dihydrochloride bonded by disulfide bond or derivatives thereof to graft the product through Schiff base reaction, thereby obtaining the polymer with pH and reduction dual responsiveness.
The nano drug delivery system takes a specific stabilizer as a hydrophilic outer layer, simultaneously takes a super pH sensitive segment (PCL) as an intermediate layer in a polymer main chain and forms a hydrophobic core, and an anti-tumor drug is encapsulated in the hydrophobic core through hydrophobic interaction, so that the polymer nano particle is formed. Firstly, the special hydrophilic outer layer improves the blood circulation capability of the nano drug delivery system, then passively targets and accumulates in tumor cells through EPR effect, and effectively transports in cells and is captured by endosomes, and then escapes to cytoplasm under the acidic environment of endosomes/lysosomes. At the same time, high concentrations of GSH in the cytoplasm rapidly cleave the disulfide bonds, which can rapidly collapse the polymer nanoparticles, thereby initiating rapid release of the drug, and subsequently can efficiently induce apoptosis (fig. 1).
The rapid disintegration of the nano drug delivery system and the rapid release of the encapsulated drug have good application prospects in the aspect of inhibiting the proliferation of related tumor cells. Therefore, the application of the Cys-PCL polymer or the stimulus-responsive drug delivery carrier or the nano drug delivery system in preparing anticancer drugs is also within the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
(1) the super-pH/reduction double-sensitive carrier material with biodegradability, body circulation stability, targeting property and excellent biological safety is obtained by simply modifying the polyester polymer and organically combining the polyester polymer with Cys or derivatives thereof. In the presence of tumor cell acid environment and high concentration Glutathione (GSH), the breakage of any one of imine bond and disulfide bond can result in the disintegration of double-sensitive polymer nano-particle and the release of entrapped medicine.
(2) According to the invention, through different preparation technologies and methods of nanoparticles, the carrier material can successfully load drugs with different hydrophilicity and hydrophobicity, and nanoparticles with controllable particle size are further formed.
(3) The drug-loaded nanoparticles can be accumulated in tumor tissues through high permeability and high retention (EPR effect) of the tumor tissues, so that the passive targeting effect is realized; after the nanoparticles are taken up by tumor cells, under the action of high-concentration glutathione and low pH in the tumor cells, imine bonds and disulfide bonds are rapidly broken, a system is rapidly broken, and dual-responsiveness drug release is realized, so that the tumor cell proliferation is efficiently inhibited.
(4) The method has the advantages of simple reaction process, few reaction steps, short reaction period, good repeatability and the like, and has good application prospect and wide development space in the field of medicine.
Drawings
FIG. 1 is a schematic representation of the entire process of PTX-encapsulating Cys-PCL delivery from synthetic, autonomous loading to intracellular drug delivery according to the present invention.
FIG. 2 is a nuclear magnetic hydrogen spectrum of PCL-CHO in the present invention.
FIG. 3 is a nuclear magnetic hydrogen spectrum of Cys-PCL in the present invention.
FIG. 4 is a Transmission Electron Microscopy (TEM) result of Cys-PCL @ PTX nanoparticles formed by encapsulating PTX.
FIG. 5 is a graph of the results of Dynamic Light Scattering (DLS) of Cys-PCL @ PTX nanoparticles formed by grafting PTX.
FIG. 6 is a graph of drug release at different pH's at 37 ℃ of Cys-PCL @ PTX nanoparticles encapsulating PTX.
FIG. 7 is a graph of drug release at different concentrations of GSH at 37 ℃ encapsulating PTX-formed Cys-PCL @ PTX nanoparticles.
FIG. 8 is a graph of drug release of Cys-PCL @ PTX nanoparticles encapsulating PTX at different pH at 37 ℃ in combination with different concentrations of GSH.
FIG. 9 is a cytotoxicity diagram of PTX single drug, Cys-PCL vector, Cys-PCL @ PTX nanoparticles after acting on mammary tumor cells of 4T1 mice for 24 h.
FIG. 10 is a cytotoxicity graph of PTX single drug, Cys-PCL vector, Cys-PCL @ PTX nanoparticles after acting on breast tumor cells of 4T1 mice for 48h, respectively.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 Synthesis of an aldehyde-based polyester Polymer PCL-CHO
1. The synthesis of the aldehyde polyester polymer PCL-CHO comprises the following steps:
(1) mixing Polycaprolactone (PCL)2K) (4.03g, 2.01mmol), 4-chlorobenzoic acid (p-CBA) (0.82g, 5.46mmol) and 4-Dimethylaminopyridine (DMAP) (0.33g, 0.27mmol) are blended and placed in a 50mL round-bottom flask, 30mL of anhydrous dichloromethane is added, then the mixture is placed in a cold trap, the temperature is controlled at 0 ℃, Dicyclohexylcarbodiimide (DCC) (1.34g, 6.51mmol) is added under the protection of nitrogen, and the mixture is stirred at 0 ℃ for 24 hours to obtain an aldehyde-based polyester product;
(2) performing rotary evaporation on the obtained product, performing silica gel column chromatography, eluting by using dichloromethane/acetone with the volume ratio of 5:1 as an eluent, and removing a byproduct DCU; and eluting by using dichloromethane/methanol with the volume ratio of 5:1 as a purifying eluent, and further purifying the product to finally obtain a white waxy solid, namely the hydroformylation polyester polymer PCL-CHO with the yield of 90.3%. The reaction process is as follows:
the structural formula of the obtained PCL-CHO is shown as a formula (II).
2. Results
The nuclear magnetic hydrogen spectrum of the aldehyde-based polyester polymer PCL-CHO is shown in FIG. 2. As can be seen from FIG. 2, the proton absorption peak at-10.12 ppm corresponds to the proton absorption peak at-8.21 ppm and-7.98 ppm of the newly generated aldehyde group, and the proton absorption peak at-4.37 ppm, 4.07ppm, 2.32ppm, 1.66ppm and 1.39ppm of the newly generated aldehyde group represent the proton absorption peaks of the polycaprolactone methylene group. The above results indicate that successful hydroformylation of PCL gives PCL-CHO.
In addition, when the molar ratio of polycaprolactone to 4-chlorobenzoic acid is 1: 1-20, the esterification reaction temperature is-50-25 ℃, and the esterification reaction time is 2-48 hours, the yield of the final product is high, and the performance of the obtained PCL-CHO product is good; wherein, when the molar ratio of the polycaprolactone to the 4-chlorobenzoic acid is 1: 2.5-3, the esterification reaction temperature is-10 ℃, and the esterification reaction time is 12-36 hours, the yield of the product is higher, and the performance of the obtained PCL-CHO product is better.
Example 2 Synthesis of Cys-PCL Polymer having a double sensitivity to pH/reduction
1. The synthesis of polymer Cys-PCL with pH/reduction double sensitivity includes the following steps:
(1) putting L-cystine dimethyl ester dihydrochloride (Cys) (0.34g,1.01mmol) and an aldehyde polyester polymer PCL-CHO (2.05g,1.02mmol) into a 50mL round-bottom flask together, adding 30mL ethanol, refluxing overnight, performing rotary evaporation, purifying the obtained product by Sephadex LH-20 gel chromatography with methanol as an eluent to finally obtain a yellow waxy solid, namely the polymer Cys-PCL with pH/reduction double sensitivity, wherein the reaction yield is 86.7%.
The structural formula of the obtained polymer Cys-PCL is shown as the formula (I):
2. results
The nuclear magnetic hydrogen spectrum of the polymer Cys-PCL is shown in FIG. 3. As can be seen from FIG. 3, the signal at 10.12ppm was found to have disappeared in response to the absorption of the proton on the newly formed aldehyde group, and 8.12ppm and 7.95ppm represent the absorption of the proton on the benzene ring, 7.51ppm represents the absorption of the proton on the imine bond, 4.35ppm and 4.06ppm represent the absorption of the methyl proton, and 2.29ppm and 1.80ppm represent the absorption of the proton on the methylene group. The above results indicate the successful synthesis of Cys-PCL.
In addition, when the molar ratio of the PCL-CHO to the Cys is 1-10: 1-10, the obtained polymer Cys-PCL has stable structure and better comprehensive performance; when the molar ratio of the PCL-CHO to the Cys is 1:1, the prepared polymer Cys-PCL has stable structure and better biocompatibility, can entrap drugs with different hydrophilicity and hydrophobicity, has low pH and GSH rapid reduction dual responsiveness, and has good application prospect in the aspect of being used as a drug delivery carrier.
Example 3 preparation and characterization of drug Cys-PCL @ PTX drug-loaded nanoparticles, in vitro drug release and cell-to-cell experiments
1. The Cys-PCL @ PTX drug-loaded nanoparticle is prepared by the following nano precipitation method, and comprises the following steps:
(1) 10mg of Cys-PCL and 2.5mg of DSPE-PEG2K2mg of PTX are dissolved in 1.0mL of DMSO together to be used as an oil phase, and the oil phase is dripped into 20mL of PBS solution at the speed of 0.05 mL/sec under the stirring state to obtain a drug-loaded nanoparticle solution with uniform and stable particle size;
(2) and (3) carrying out ultrafiltration concentration on the obtained nanoparticle solution at 2500rpm, fixing the volume to 2mL, and freeze-drying to obtain the Cys-PCL @ PTX drug-loaded nanoparticle.
2. The obtained Cys-PCL @ PTX drug-loaded nanoparticles are characterized, and the results of in vitro drug release investigation and cytotoxicity investigation are respectively as follows:
(1) DLS detection shows that the medicine carrying nanometer particle of the present invention has particle size of 80nm and no precipitate produced in refrigerator at 4 deg.c. The Transmission Electron Microscope (TEM) result graph and the Dynamic Light Scattering (DLS) result graph are shown in fig. 4 and 5, respectively. The result shows that the Cys-PCL @ PTX nano-particle is spherical, has uniform size and narrow particle size distribution.
(2) The drug-loaded nanoparticles prepared by the invention are respectively used for carrying out drug release experiments in PBS solutions with different pH values (5.5, 6.5 and 7.4), different concentrations of glutathione (0mM, 2mM, 10mM and 20mM) and different pH values combined with different concentrations of glutathione (pH 5.5+2mM GSH, pH 6.5+2mM GSH, pH 5.5+10 mu M GSH and pH 6.5+10 mu M GSH) at 37 ℃, and the concentration of PTX cumulatively released by a nano system within 140h in the PBS solution is detected.
The experimental results are respectively shown in fig. 6, fig. 7 and fig. 8, which prove that the nano drug-loaded system of the invention has dual responsiveness of pH/GSH reduction, and the drug release becomes faster and the cumulative release amount of the drug increases with the decrease of pH or the increase of GSH concentration.
(3) The PTX single drug, the Cys-PCL vector and the Cys-PCL @ PTX drug-loaded nano-particles respectively act on the mammary tumor cells of the 4T1 mouse for 24 hours or 48 hours.
The experimental results are respectively shown in fig. 9 and fig. 10, and the simple carrier material Cys-PCL has no obvious cytotoxicity; the single-drug PTX and Cys-PCL @ PTX drug-loaded nanoparticle groups have obvious time-dependent and concentration-dependent cytotoxicity; compared with single-drug PTX, the drug-loaded nanoparticle group has no obvious difference in the capacity of inhibiting cell proliferation. The results show that the carrier material has better biological safety, and the nanoparticles after carrying the medicine have better cell proliferation inhibition capability; can realize the positioning controllable release of the medicine in the tumor cells, has stable systemic circulation, has good biosecurity and biodegradability in the whole system, and has the characteristics of high-efficiency tumor targeting and high-efficiency tumor cell growth inhibition.
In addition, the mass ratio of the polymer to the loaded drug is found to be too large, so that the drug loading rate is lower, and the number of the formed nanoparticles is less; if the mass ratio of the polymer to the carried drug is too small, the encapsulation efficiency is low, the drug is wasted, and the formed nanoparticles are unstable and easy to precipitate. When the mass ratio of the polymer Cys-PCL to the loaded drug is 5-20: 1-2, the obtained nanoparticles are uniform and stable in particle size, proper in drug loading and good in drug action effect; wherein when the mass ratio of the polymer Cys-PCL to the carried medicine is 5-10: 1, the medicine carrying effect and the medicine action effect are optimal.
Example 4 preparation of drug-loaded nanoparticles
1. The preparation method of the drug-loaded nanoparticles with pH/reduction dual response by a single emulsion method comprises the following steps:
(1) dissolving the polymer Cys-PCL and the carried medicine methotrexate into dichloromethane together, and performing ultrasonic treatment to obtain an oil phase;
(2) mixing the obtained oil phase with a water phase PBS buffer solution (the concentration of polyvinyl alcohol is 0.2%) containing polyvinyl alcohol serving as a stabilizer, performing ultrasonic treatment, stirring at a constant speed to remove a solvent dichloromethane to obtain a drug-loaded nanoparticle solution with uniform and stable particle size, and freeze-drying to obtain drug-loaded nanoparticles, namely the drug-loaded nanoparticles with pH/reduction dual response.
Example 5 preparation of drug-loaded nanoparticles
1. The preparation method of the drug-loaded nanoparticles with pH/reduction dual response by a dual emulsion method comprises the following steps:
(1) dissolving the polymer Cys-PCL in dichloromethane, and performing ultrasonic treatment to obtain an oil phase;
(2) dissolving a water-soluble drug gemcitabine hydrochloride in water, mixing the gemcitabine with the oil phase, and performing ultrasonic treatment to obtain a water-in-oil type emulsion;
(3) mixing the obtained water-in-oil emulsion oil phase with a water phase containing a stabilizer PVA (the concentration of the PVA is 0.25%), performing ultrasonic treatment, stirring at a constant speed to remove an organic solvent dichloromethane to obtain a drug-loaded nanoparticle solution with uniform and stable particle size, and freeze-drying to obtain drug-loaded nanoparticles, namely the drug-loaded nanoparticles with pH/reduction dual response.
The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it is not intended that the present invention necessarily depends on the above embodiment for implementation. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (9)
1. A polymer Cys-PCL with pH/reduction double sensitivity is a polymer formed by Cys and PCL, and the structural formula of the polymer Cys-PCL is shown as the following formula (I):
wherein n = 5-10000; cys is L-cystine dimethyl ester dihydrochloride, and PCL is polycaprolactone.
2. A preparation method of a polymer with pH/reduction double sensitivity is characterized by comprising the following steps:
s1, subjecting the polyester polymer with hydroxyl at two ends to hydroformylation through esterification reaction to obtain a hydroformylation polyester polymer;
s2, grafting the aldehyde polyester polymer with disulfide bond-containing cystine ester or derivatives thereof through Schiff base reaction to obtain the polymer with pH/reduction dual response;
the polyester polymer is polycaprolactone.
3. The preparation method according to claim 2, wherein the cystine ester is selected from any one of dimethyl L-cystine dihydrochloride, ethyl L-cystine dihydrochloride or benzyl L-cystine dihydrochloride.
4. Use of the polymer Cys-PCL according to claim 1 or the polymer prepared by the method according to any one of claims 2 to 3 for the preparation of a drug delivery vehicle.
5. A stimulus-response type drug delivery carrier is characterized in that the polymer Cys-PCL of claim 1 or the polymer prepared by the method of any one of claims 2 to 3 is prepared into nanoparticles to obtain the stimulus-response type drug delivery carrier.
6. A nano drug delivery system with pH/reduction dual response, comprising the polymer of claim 1 or the polymer prepared by the method of any one of claims 2 to 3, and a drug carried therein.
7. The nano-delivery system of claim 6, wherein the drug loaded comprises a hydrophilic drug and a hydrophobic drug.
8. The nano drug delivery system of claim 6, wherein the mass ratio of the polymer to the drug loaded is 5-20: 1-2.
9. Use of the polymer Cys-PCL of claim 1, or the stimuli-responsive drug delivery vehicle of claim 5, or the nano-delivery system of any one of claims 6 to 8 for the preparation of an anti-cancer drug.
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