CN117298130A - Chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress and application thereof - Google Patents

Chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress and application thereof Download PDF

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CN117298130A
CN117298130A CN202311534384.0A CN202311534384A CN117298130A CN 117298130 A CN117298130 A CN 117298130A CN 202311534384 A CN202311534384 A CN 202311534384A CN 117298130 A CN117298130 A CN 117298130A
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崔兰
娄卫爽
徐晴晴
孙梦瑶
杨硕晔
陈彦霖
屈凌波
张璐
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Henan University of Technology
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Abstract

The invention discloses a chitosan oligosaccharide-adriamycin biological material with enhanced cascade oxidative stress and application thereof, belonging to the technical field of pharmaceutical preparations, and comprising chitosan oligosaccharide, disulfide bond modified adriamycin, 4- (bromomethyl) -phenylboronic acid and gamma-polyglutamic acid, which are prepared into nano prodrugs in the process of preparing adriamycin prodrug monomer DOX-ss; copolymerizing DOX-ss with 4- (bromomethyl) -phenylboronic acid PBA and chitosan oligosaccharide COS to obtain a redox responsive polymer prodrug PBA-COS-ss-DOX; and (3) crosslinking and self-assembling the PBA-COS-ss-DOX and the gamma-polyglutamic acid gamma-PGA to obtain the PBA-COS-ss-DOX/gamma-PGA. The nano prodrug prepared by the invention has uniform particle size distribution, high DOX drug loading, stability under physiological conditions, and can specifically target liver cancer cells, realize the synergistic response enhancement of oxidation stress of oxidation reduction and esterase, promote drug release and achieve the high-efficiency and low-toxicity tumor inhibition effect.

Description

Chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress and application thereof
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a chitosan oligosaccharide-adriamycin biological material with enhanced cascade oxidative stress and application thereof.
Background
The hepatocellular carcinoma has hidden onset and high mortality, and chemotherapy is the most main treatment method aiming at the diseases in the liver which cannot be resected and advanced stage by operation, however, small molecule chemotherapy medicaments such as doxorubicin hydrochloride (DOX. HCl) and the like are easy to cause anemia, immune function reduction and multi-drug resistance of patients. The nano prodrug can reduce the toxic and side effects of chemotherapy to a certain extent, but still has the risks of early leakage of the drug, poor targeting of the drug release in liver cancer cells and the like.
The research shows that the transmembrane protein gamma-glutamine transpeptidase (GGT) is highly expressed on the surface of tumor cells with vigorous metabolism such as liver cancer and the like, and simultaneously participates in the decomposition and synthesis of glutathione GSH in cells, thereby being a main antioxidant on the cell membrane of mammals. In addition, tumor cell specific proliferation produces excessive Reactive Oxygen Species (ROS), which produce oxidative stress associated with imbalance in metabolic clearance, and are closely related to the pathogenesis of a variety of human diseases. However, the current redox responsive nano-prodrugs cannot well meet the requirements in terms of drug loading, oxidative stress regulation and control capability, active targeting and the like.
The anthracycline drug-Doxorubicin (DOX) clinically approved at present has remarkable toxic and side effects such as cardiotoxicity, myelosuppression, multi-drug resistance and the like. Drug molecules designed based on prodrug strategies have the equivalent effect of improving the physicochemical properties, biopharmaceutical or pharmacokinetic properties of the drug. How to amplify the stimulation signal in tumor tissue overcomes the limitations of low response efficiency, incomplete release and the like of the traditional prodrug, and is a great challenge for improving the activation efficiency of the prodrug.
Therefore, the ability to provide drugs with high drug loading rates, ROS surge, and active targeting of cascade oxidative stress enhancement is a problem that one skilled in the art would need to address.
Disclosure of Invention
In view of the above, the invention provides a chitosan oligosaccharide-adriamycin biological material with enhanced cascade oxidative stress, and nano prodrugs are prepared by the biological material, disulfide bonds are introduced during preparation, DOX-ss is prepared firstly, and then PBA and gamma-PGA are used as modifiers and COS is used as a carrier material; the preparation method is simple, the obtained PBA-COS-ss-DOX/gamma-PGA sample has uniform particle size distribution and stable physicochemical property, the drug loading rate of DOX is high, and oxidation stress in HepG2 cells is enhanced through the synergistic responsiveness of redox and esterase, DOX release is promoted, and the tumor inhibition effect with high efficiency and low toxicity is achieved.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress, which comprises chitosan oligosaccharide, doxorubicin prodrug monomers DOX-ss, 4- (bromomethyl) -phenylboronic acid and gamma-polyglutamic acid containing disulfide bonds, wherein the molar ratio of the chitosan oligosaccharide to DOX-ss and 4- (bromomethyl) -phenylboronic acid is 1:1:1 according to a certain saccharide unit, and the molar ratio of PBA-COS-ss-DOX to gamma-polyglutamic acid is 1:1 according to the ratio of amino, hydroxyl and carboxyl in a molecular chain; wherein the DOX-ss structure of the doxorubicin prodrug monomer is shown in a formula I;
as the invention concept same as the technical scheme, the invention also claims the application of the chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress in preparing nano prodrugs with enhanced cascade oxidative stress.
The invention uses disulfide bond to modify adriamycin, then uses 4- (bromomethyl) phenylboronic acid and gamma-polyglutamic acid as modifier to modify Chitosan Oligosaccharide (COS), wherein COS is natural cationic basic oligosaccharide, has strong affinity to cells, can interact with negatively charged cell membranes, and can carry out targeted drug delivery after COS functionalization modification; disulfide bonds are used as a stable reducing chemical bond, can be biodegraded by mercaptan, can promote the disintegration of nano particles and the effective release of medicines after being hydrolyzed by disulfide bond-containing carriers or prodrugs, and can regulate redox balance in the presence of high ROS in tumors, thereby improving the hydrophilic performance of a nano medicine system and effectively inhibiting the growth and metastasis of the tumors; 4- (bromomethyl) phenylboronic acid (PBA) has the characteristics of no toxicity and no immunogenicity, can specifically identify sugar antigen-sialic acid which is expressed on liver cancer cells, and can participate in intracellular redox regulation and control, and has strong sensitivity to active oxygen; the gamma-polyglutamic acid (gamma-PGA) is a biodegradable and nontoxic anionic polymer, the gamma-PGA is easy to form a complex network with a positive charge polymer through electrostatic action, so that nonspecific interaction with human serum albumin and the like is remarkably avoided, the in vivo stability of the nanoparticle is improved, in addition, the gamma-PGA can also interact with a transmembrane transport protein gamma-glutamine transpeptidase GGT on the surface of a liver cancer cell, and the transmembrane transport of the nanoparticle is promoted.
As the same invention conception as the technical scheme, the invention also claims a preparation method of the chitosan oligosaccharide-adriamycin nano prodrug with enhanced cascade oxidative stress, which is characterized in that adriamycin and a compound containing disulfide bonds are subjected to substitution reaction, then are subjected to copolymerization connection with the chitosan oligosaccharide and 4- (bromomethyl) -phenylboronic acid, and finally are subjected to cross-linking self-assembly with the gamma-polyglutamic acid.
Preferably, the substitution reaction is: firstly, carrying out substitution reaction on 2,2' -dithiodiethanol and methacryloyl chloride to obtain an intermediate 2- (2-hydroxyethyl) disulfonyl) ethyl methacrylate HSEMA; subsequently, doxorubicin hydrochloride and 4-dimethylaminopyridine were suspended in dry dichloromethane to give a doxorubicin mixed solution, which was then taken up in N 2 Adding triphosgene under protection, stirring, dispersing HSEMA in dichloromethane, dropwise adding into doxorubicin mixed solution, magnetically stirring, and substitution reacting to obtain a product containing two componentsSulfur-bonded doxorubicin prodrug monomer DOX-ss.
Preferably, the co-copolymeric linkages, cross-linked self-assembly are: adding an initiator into DOX-ss and chitosan oligosaccharide COS and 4- (bromomethyl) -phenylboronic acid PBA for copolymerization to obtain a polymer prodrug PBA-COS-ss-DOX, wherein the structure of the polymer prodrug PBA-COS-ss-DOX is shown as a formula II; then adding a cross-linking agent into PBA-COS-ss-DOX and gamma-polyglutamic acid gamma-PGA for self-assembly to obtain the chitosan oligosaccharide-doxorubicin nano prodrug PBA-COS-ss-DOX/gamma-PGA subjected to cascade oxidative stress;
preferably, the substitution reaction of 2,2' -dithiodiethanol and methacryloyl chloride is carried out in a molar ratio of 1:1, followed by the substitution reaction of free aliphatic hydroxyl groups, doxorubicin hydrochloride DOX HCl and triphosgene in a molar ratio of 1:1.
Preferably, the initiator is ceric ammonium nitrate, and the molar ratio of DOX-ss, chitosan oligosaccharide COS, 4- (bromomethyl) -phenylboronic acid PBA and the initiator is 1:1:1:1.
Preferably, the cross-linking agent is sodium tripolyphosphate, and the mass ratio of PBA-COS-ss-DOX, gamma-polyglutamic acid gamma-PGA and the cross-linking agent is 1:1.5:1.
Preferably, the particle size of the nano-prodrug PBA-COS-ss-DOX/gamma-PGA is 160-180nm.
The introduction of disulfide bonds endows doxorubicin with GSH response characteristics; secondly, DOX-ss is connected with COS and PBA atom transfer radical polymerization to generate PBA-COS-ss-DOX, and the introduction of PBA endows the polymer prodrug with active targeting and ROS response characteristics; finally, PBA-COS-ss-DOX and gamma-PGA are self-assembled through ionic crosslinking to generate PBA-COS-ss-DOX/gamma-PGA, the response characteristic of nano prodrug enzyme is endowed, the decomposition and synthesis paths of glutathione GSH in cells are regulated and controlled, and DOX is tightly connected with a crosslinking agent through disulfide bonds, so that the encapsulation rate and the drug loading rate of the drug are improved, and the encapsulation rate and the drug loading rate of the drug to the DOX are 96.33% and 27.52% respectively.
As the invention concept same as the technical scheme, the invention also claims the application of the nano prodrug prepared by the preparation method in preparing the anti-tumor drug.
The invention bonds doxorubicin with redox cascade response groups, and the designed nano prodrug reaches liver cancer cell tissues through blood circulation. Gamma-polyglutamic acid gamma-PGA is rapidly hydrolyzed and shed by a transmembrane protein gamma-glutamyl transferase GGT which is highly expressed on the surface of liver cancer cells, and meanwhile, the decomposition and synthesis routes of glutathione GSH in the cells are regulated and controlled; in addition, 4- (bromomethyl) -phenylboronic acid PBA is covalently coupled with sialic acid on the surface of the cell, so that the nano-drug is promoted to enter the cell through endocytosis, and excessive Reactive Oxygen Species (ROS) generated by tumor cell specific proliferation is regulated and controlled; finally, disulfide bond-containing carriers or prodrugs react rapidly to glutathione GSH in cancer cells, so that the nano prodrugs are disintegrated and DOX is effectively released, apoptosis is promoted, meanwhile, the content of GSH in the cells after the reaction is reduced, reactive Oxygen Species (ROS) in tumors is locally increased, and oxidative stress in the cells is caused by unbalance of ROS production and metabolic clearance. The system can realize the effect of cascade amplification of the stimulation signal ROS through the oxidative stress regulation and control capability and the active targeting, overcomes the limitations of low activation efficiency, incomplete drug release and the like of the traditional prodrug, and finally realizes the purpose of 'self-amplifying' activation of the prodrug.
In summary, compared with the prior art, the invention has the following beneficial effects:
(1) Disulfide bonds in the prodrug monomers react with glutathione GSH in cancer cells rapidly, so that the nanometer prodrug is disintegrated and DOX is released effectively, and cells are promoted to undergo apoptosis;
(2) The chitosan oligosaccharide with low cytotoxicity and high biocompatibility is selected as the nano prodrug bracket, and the drug can be firmly linked to a polymer molecular chain through free radical polymerization and ionic crosslinking, so that the intermolecular acting force is strong;
(3) The PBA and the gamma-PGA selected by the invention can specifically recognize the glycoantigen-sialic acid and the transmembrane protein gamma-glutamyl transferase GGT on the surface of liver cancer cells; and PBA is sensitive to ROS, GGT participates in regulating and controlling the decomposition and synthesis of glutathione GSH, and cascade amplification of stimulation signal ROS is realized by utilizing the oxidative stress regulation and control capability and active targeting, so that the prodrug activation efficiency is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the feedstock of example 1 of the present invention with ethyl 2- ((2-hydroxyethyl) disulfonyl) methacrylate HSEMA, wherein A: methacryloyl chloride, B:2,2' -dithiodiethanol, C: HSEMA;
FIG. 2 is an infrared spectrum of the feedstock of example 1 of the present invention with ethyl 2- ((2-hydroxyethyl) disulfonyl) methacrylate HSEMA;
FIG. 3 is a mass spectrum of DOX-ss of example 1 of the present invention;
FIG. 4 is a graph showing the infrared comparison of DOX and DOX-ss for example 1 of the present invention;
FIG. 5 is an infrared spectrum of the present invention of examples 2 and 3 PBA-COS-ss-DOX/gamma-PGA;
FIG. 6 is a morphology characterization of PBA-COS-ss-DOX/gamma-PGA according to example 3 of the present invention, A: particle size potential, B: a TEM image;
FIG. 7 is a view showing the hemolysis of practical example 1 of the present invention, A: blank nanocarriers, B: free DOX and nano-drug, C: a rate of hemolysis;
FIG. 8 shows in vitro drug delivery under various conditions for PBA-COS-ss-DOX/gamma-PGA according to application example 2 of the present invention;
FIG. 9 shows the cell activities of the blank vector PBA-COS-ss/gamma-PGA of application example 3 of the present invention after 24 and 48 hours of interaction with HepG2 cells;
FIG. 10 shows the cell activity of the drug-loaded nanoparticle of application example 3 of the present invention after culturing with HepG2 for 24 hours and 48 hours;
FIG. 11 shows fluorescence imaging and fluorescence intensity analysis of PBA-COS-ss-DOX/gamma-PGA and HepG2 cells of application example 4 of the present invention after culturing for 4 and 24 hours;
FIG. 12 is a graph showing fluorescence images of ROS induced by co-incubation of free DOX and drug-loaded nanoparticles with HepG2 for 4 hours according to application example 5 of the present invention;
FIG. 13 is a graph showing fluorescence images of ROS induced by co-incubation of free DOX and drug-loaded nanoparticles with HepG2 for 24h according to application example 5 of the present invention;
FIG. 14 is a fluorescent intensity analysis of ROS after 4 and 24 hours incubation of free DOX and drug-loaded nanoparticles with HepG2, application example 5 of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The preparation of the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress mainly comprises the following steps:
preparation of GSH responsive prodrug (DOX-ss):
(1) Preparation of ethyl 2- ((2-hydroxyethyl) disulfonyl) methacrylate (HSEMA)
2,2' -dithiodiethanol (2.64 mL), triethylamine (4.20 mL), dry tetrahydrofuran (100 mL) were added to N-pass 2 To a 250mL dry three-necked flask of (2) in 50mL tetrahydrofuran was added dropwise to the three-necked flask with ice bath to 0℃followed by magnetic stirring (800 rpm) for 1h, followed by magnetic stirring at room temperature for 24h. The solvent was removed by filtration and rotary evaporation, the residue was diluted with ethyl acetate and washed twice with water and brine, respectively, the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give the product ethyl 2- ((2-hydroxyethyl) disulfonyl) methacrylate (HSEMA) as a pale yellow liquid.
FIGS. 1 and 2 show the nuclear magnetic hydrogen spectrum analysis and the infrared spectrum analysis characterization of HSEMA, and as shown in the figure, the characteristic peak at 7.26ppm is the chemical shift characteristic peak of deuterated chloroform. As shown in fig. 1, the characteristic peaks of methacryloyl chloride are: CH at 6.51 and 6.05ppm 2 Methylene proton peak of =c, -CH at 2.02ppm 3 Methyl proton peak of (2)The method comprises the steps of carrying out a first treatment on the surface of the Characteristic peaks of dithiodiethanol are: 3.93ppm of-CH close to-OH 2 2.87ppm of-CH near-S-S 2 Methylene proton peaks of (2); compared with methacryloyl chloride and dithiodiethanol, HSEMA has the chemical shift of the methylene proton peak at one side of dithiodiethanol shifted to the left due to the formation of esters, i.e. the methylene proton peak near the ester group at 4.43ppm and the methylene proton peak near-S-S-at 2.98 ppm. The above results indicate that the synthesis of ethyl 2- (2-hydroxyethyl) disulfonyl) methacrylate (HSEMA) was successful with the formation of esters from the reaction of methacryloyl chloride and dithiodiethanol. HSEMA:1HNMR (500 MHz, CDCl) 3 )δ7.26(s,113H),6.22(s,8H),5.68(s,8H),4.49-4.35(m,4H),3.90(dt,J＝46.0,19.3Hz,27H),3.07-2.95(m,7H),2.89(t,J＝5.6Hz,25H),3.56-2.08(m,90H),2.37-1.73(m,96H),2.29-1.73(m,88H),2.01(d,J＝5.8Hz,6H),1.96(s,39H),1.67(d,J＝26.4Hz,8H),1.73-1.58(m,15H),1.60(dd,J＝64.3,33.2Hz,25H),1.59(dd,J＝69.4,38.3Hz,31H)。
FIG. 2 is an infrared spectrum of HSEMA, dithiodiethanol and methacryloyl chloride. As can be seen from fig. 2, the characteristic peaks of methacryloyl chloride are: 2928cm -1 at-CH 3 Is 1451cm -1 at-CH of 3 Peak of deformation vibration, 1782cm -1 Acid chloride stretching vibration peak at 1717cm -1 The C=O stretching vibration peak is 1629cm -1 The C=C stretching vibration peak, 884cm -1 The out-of-plane bending vibration peak at c=c; characteristic peaks of dithiodiethanol are: 3300cm -1 O-H stretching vibration peak at 2928cm -1 、2873cm -1 Where is-CH 2 、CH 3 Is 539cm -1 The stretching vibration peak of-S-S-; HSEMA was found to be 3300cm compared to methacryloyl chloride -1 The stretching vibration peak of O-H appears at 2928cm -1 、2873cm -1 where-CH appears 2 、CH 3 And at 539cm -1 A stretching vibration peak of-S-S-appears at the position; HSEMA was found to be 1782cm compared to dithiodiethanol -1 The vibration peak of the acid chloride at the position disappears and 1717cm -1 A stretching vibration peak of c=o appears at; 1629cm -1 Is discharged fromNow c=c stretching vibration peak and 884cm -1 Out-of-plane bending vibration peak at c=c, and at 1167 and 1045cm -1 Characteristic peaks of the ester appear there. The above results indicate that the reaction of methacryloyl chloride with dithiodiethanol succeeded in producing HSEMA.
(2) Preparation of DOX-ss prodrugs
Doxorubicin hydrochloride (DOX HCl,58.00 mg) and 4-dimethylaminopyridine (48.87 mg) were suspended in 20mL of dry dichloromethane under N 2 Triphosgene (274.16 mg) was added under protection and magnetically stirred at room temperature for 30min. HSEMA (20.18. Mu.L) prepared above was dispersed in 6mL of methylene chloride, added dropwise to the above doxorubicin mixed solution, and magnetically stirred at room temperature for 24 hours. The solvent was removed by filtration and rotary evaporation, the residue was diluted with ethyl acetate and washed once with water, twice with 1.0M hydrochloric acid and brine, respectively. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated by rotary evaporation to give DOX-ss as a red solid powder. The solid was subjected to mass and infrared spectroscopic analysis, see fig. 3 and 4. FIG. 3 is a mass spectrum of DOX-ss, m/z 791 being the molecular ion peak, i.e., the relative molecular mass of DOX-ss; FIG. 4 is an infrared spectrum of DOX-ss at 805 and 1220cm -1 Characteristic absorption peak of (C) and DOX =c-O-CH 3 In relation, at 1285 and 1412cm -1 Representative of C-OH and C-C characteristic peaks of DOX; at 1614cm -1 The double carbonyl stretching vibration peak of DOX appears at 539cm -1 There appears a stretching vibration peak of-S-S-. The connection between DOX and HSEMA is successful, and the synthesis of DOX-ss is successful.
The DOX-ss structure is shown as a formula I;
example 2 preparation of PBA-COS-ss-DOX Polymer prodrug:
the chitosan oligosaccharide is modified by adopting atom transfer radical polymerization. COS (102.00 mg) was dissolved in 10mL of 2% (V/V) acetic acid at 30℃and N 2 Magnetically stirring for 30min under protection; the initiator ceric ammonium nitrate (27.41 mg) was added and stirred for 5min. Subsequently, at 60 DEG CAt this time DOX-ss (39.56 mg) was added dropwise to the solution; after 1h, N-diethylaminoethyl acrylate (8.56. Mu.L) and PBA (10.74 mg) were added, respectively, and magnetic stirring was continued for 24h at a given saccharide unit, ceric ammonium nitrate, DOX-ss, N-diethylaminoethyl acrylate and PBA molar ratio (1:1:1:1:1). The pH-GSH-ROS responsive PBA-COS-ss-DOX samples were prepared by dialysis purification for 1 day and freeze-drying. The structure of the PBA-COS-ss-DOX is shown as a formula II;
example 3 preparation of PBA-COS-ss-DOX/gamma-PGA:
sodium tripolyphosphate is taken as an ion crosslinking agent, sodium tripolyphosphate (2 mg/mL,20 mL) and anionic polymer gamma-polyglutamic acid (gamma-PGA, 1.5mg/mL,40 mL), PBA-COS-ss-DOX (1 mg/mL,40 mL) are self-assembled, magnetically stirred for 3H at room temperature, and then the sample PBA-COS-ss-DOX/gamma-PGA is prepared after dialysis purification and freeze drying, GSH and H are studied 2 O 2 And effect on PBA-COS-ss-DOX/gamma-PGA drug release in the presence of GGT. Carrying out infrared spectrum analysis on the PBA-COS-ss-DOX and the PBA-COS-ss-DOX/gamma-PGA, and observing the morphology of the drug-loaded nano particles; see fig. 5 and 6; FIG. 5 shows the IR spectrum analysis of PBA-COS-ss-DOX and PBA-COS-ss-DOX/gamma-PGA, as shown in the figure, PBA-COS-ss-DOX is 3300cm compared to DOX-ss -1 The treatment is-NH in COS 2 Is formed by overlapping telescopic vibration of (C) and telescopic vibration of-OH, and is 1380cm in length -1 The C=C skeleton vibration peak of benzene ring shows that DOX-ss is successfully connected with COS and PBA; PBA-COS-ss-DOX/gamma-PGA is 1640 and 1548cm compared to PBA-COS-ss-DOX -1 The stretching vibration of-c=o in the amide group indicates that there is an amide group in γ -PGA. The results show that PBA-COS-ss-DOX/gamma-PGA is successfully coupled; FIG. 6 is a graph of a morphology characterization of drug-loaded nanoparticles. The particle size of PBA-COS-ss-DOX/gamma-PGA is 160-180nm, and the potential is-17.20 mV. FIG. 6 (B) is a transmission electron microscope image of PBA-COS-ss-DOX/gamma-PGA, showing that the drug-loaded nanoparticles are regular spheres, uniform in size and flat in surface. And the encapsulation rate and drug loading rate of DOX in PBA-COS-ss-DOX/gamma-PGA are respectively96.33% and 27.52%.
According to the results of the characterization, the invention successfully prepares the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress, and the prodrug has good physical stability and good dispersibility, and can be used as an excellent delivery carrier of antitumor drugs.
Application example 1 experiment for verifying biocompatibility of nanocarriers and drug-loaded nanoparticles
5mL of mouse blood was placed in a glass tube, EDTA-2Na was immediately added to prevent coagulation, and the mixture was stirred for 10 minutes to remove fibrin, thereby obtaining defibrinated blood. Then, 10mL of physiological saline was added and centrifuged at 1000rpm for 15min to pellet blood cells (RBC). Pouring out the supernatant, adding normal saline, centrifuging, and repeating the operation until the supernatant is red-free, wherein the precipitate is red blood cells. Subsequently, RBCs were prepared as a 2% cell suspension in physiological saline, and hemolysis was analyzed and stored at 4 ℃.
DOX, DOX@PBA-COS/gamma-PGA and PBA-COS-ss-DOX/gamma-PGA were prepared at a range of concentrations (calculated as DOX content) of 0.1, 0.2, 0.5, 1.0, 2.0. Mu.g/mL, respectively, and further PBA-COS/gamma-PGA and PBA-COS-ss/gamma-PGA solutions were prepared at concentrations of 10, 20, 50, 100, 200. Mu.g/mL, respectively.
Physiological saline (negative control, 0% hemolysis) and 1% TritonX-100 (positive control, 100% hemolysis) were added to RBC cell suspensions respectively (0.5 mL of 2% erythrocyte suspension was transferred to a 1.5mL centrifuge tube, then 0.5mL of nanoparticles of different concentrations were added), mixed uniformly, incubated in a incubator at a constant temperature of 37℃for 2 hours, after incubation was completed, placed in a high-speed centrifuge for centrifugation at 3000rpm for 5 minutes, 100. Mu.L of supernatant was taken into a 96-well plate, absorbance was detected at a wavelength of 540nm, and the hemolysis rate was calculated according to formula 1.
Hemolytic Rate(％)＝(A sample -A negative control )/(A positive control -A negative control )×100％(1)
Wherein A is sample Absorbance of the experimental group at 540nm, A negative Absorbance of negative control group at 540nm, A positive The absorbance of the positive control group at 540nm is plotted with the nanoparticle concentration as abscissa and the hemolysis rate as ordinate, see fig. 7; FIG. 7 is a graph of a hemolysis analysis of nanoparticles. When the hemolysis rate is less than 5%, the biocompatibility of the test sample is good. The experimental data show that the hemolysis rate is lower than 5% in the nano particle test concentration range, which indicates that the biocompatibility of PBA-COS/gamma-PGA, PBA-COS-ss/gamma-PGA, free DOX, nano medicine DOX@PBA-COS/gamma-PGA and PBA-COS-ss-DOX/gamma-PGA used as drug carriers is good.
Application example 2 verification experiment of pH response, ROS response, GSH and GGT responsive drug release of drug-loaded nanoparticle sample
The PBA-COS-ss-DOX, the gamma-PGA and the ionic crosslinking agent sodium tripolyphosphate are crosslinked by the crosslinking agent, so that the PBA-COS-ss-DOX/gamma-PGA forms a compact network structure. 5mg of PBA-COS-ss-DO X/gamma-PGA drug-loaded nanoparticles were dispersed in 5mL of pH7.4, 7.4 (1 mM GSH), 7.4 (10 mM GSH), 5.5 (1 mM GSH), 5.5 (10 mM GSH) and 6.5 (10 mM GSH, 100. Mu. M H), respectively 2 O 2 10U/mL GGT) in PBS. The above liquids were placed in dialysis bags with a molecular weight cut-off of 500Da, respectively, and then placed in 50mL PBS buffer solution under the corresponding conditions, and continuously shaken at 100rpm in a constant temperature shaking box at 37 ℃. 3mL of release solution was aspirated at regular intervals and 3mL of fresh PBS buffer under different conditions was supplemented. Measuring absorbance of the sample at 480nm absorption wavelength by using an ultraviolet spectrophotometer, calculating the accumulated drug release rate Qt according to a standard curve of DOX & HCl under different pH conditions and a formula 2, and drawing a relation chart of the accumulated drug release rate and the release time, wherein the relation chart is shown in fig. 8;
wherein C is n To supplement the concentration of the release solution collected prior to fresh PBS solution, V 0 To oscillate the volume of PBS buffer in the tank for drug release, V i For each sample volume, C i The drug concentration in the release liquid in the ith replacement is given, and m is the total drug amount of the drug-loaded nano particles.
Fig. 8 is an in vitro release profile of drug-loaded nanoparticles. After 48h, the release rate of PBA-COS-ss-DOX/gamma-PG A at pH7.4 was 13.63%, which was a 3.65-fold improvement in release rate at pH 5.5 (49.79%). In addition, the introduction of GSH further increases the drug release rate of PBA-COS-ss-DOX/gamma-PGA to 25.76% at pH7.4 and 10mM GSH, and particularly to 85.37% at pH 5.5 and 10mM GSH. Finally, at pH 6.5,10mM GSH,100 μ M H 2 O 2 And the cumulative drug release rate under the condition of 10U/mL GGT is as high as 93.63%, which is about 6.87 times that under the condition of pH 7.4. The result shows that the active and passive dual targeting enhanced drug-loaded nanoparticle PBA-COS-ss-DOX/gamma-PGA has better drug release characteristics. As disulfide bonds are introduced, the PBA-COS-ss-DOX/gamma-PGA has obvious GSH response performance, so that the PBA-COS-ss-DOX/gamma-PGA has better drug release performance on the basis of the original pH/ROS/GGT response performance, which is beneficial to prolonging the circulation time of the drug in blood and improving the uptake of the drug by cells, thereby avoiding the premature release of the drug in microenvironment and improving the uptake efficiency of the drug.
Application example 3 cytotoxicity test verification
Taking 2mg of each nano-carrier to be dissolved in serum-free DMEM culture medium to prepare 200 mug/mL solution, and preparing the solution into the concentrations of 200 mug/mL, 100 mug/mL, 50 mug/mL, 20 mug/mL and 10 mug/mL by using cell culture solution; the free DOX and the drug-loaded nano particles take DOX concentration as quantitative basis, and concentration gradients are set to be 2, 1, 0.5, 0.2 and 0.1 mug/mL; they were added separately to 96-well plates, 5 parallel wells were set for each concentration, and the blank was completely incubated with fresh DMEM. After further incubation at 37℃in an incubator for 24 and 48 hours, respectively, the old medium was removed and washed twice with pre-warmed PBS to wash away residual nanoparticles. Subsequently 100. Mu.L of 20% MTT was added to the 96-well plate. After incubation for 4h at 37 ℃, MTT was blotted off, 100 μl DMSO was added to each well, absorbance was measured at 490nm wavelength after shaking for 10min, and cell viability was calculated according to equation 3 compared to the blank control group:
wherein Abs sample Absorbance of cells at 490nm after incubation of nano drug carrier, abs control Absorbance of the medium at 490nm, abs cell Absorbance at 490nm of untreated cells. Similarly, pH7.4 (10 mM GSH), 6.5 (10 mM GSH) and 6.5 (10 mM GSH,10 μ M H) were set 2 O 2 10U/mL GGT) were cultured in fresh DMEM complete medium, and after 12h, old medium was removed, DOX and PBA-COS-ss-DOX/gamma-PGA were added to 96-well plates, followed by the same procedure. The results are shown in fig. 9 and 10; FIG. 9 is a graph of cytotoxicity of nanocarriers against HepG 2. After PBA-COS-ss/gamma-PGA and HepG2 cells act for 24 and 48 hours, the sensitivity of the HepG2 cells to foreign substances slightly reduces along with the increase of the concentration of the nano-carrier, but the cell activity is more than 80%, so that the PBA-COS-ss/gamma-PGA has smaller toxicity to cells and better biocompatibility in a certain concentration range.
FIG. 10 is a toxicity analysis of drug-loaded nanoparticles in HepG2 cells, with free DOX and drug-loaded nanoparticles exhibiting time and dose dependence on the toxic effects of HepG2 cells. Compared with free DOX, PBA-COS-ss-DOX/gamma-PGA has lower toxicity after 24 and 48 hours, because free DOX is a small molecular drug which can easily enter the cell nucleus to prevent the replication and transcription of DNA, thereby killing tumor cells; the particle size of the drug-loaded nano particles is larger, and the drug-loaded nano particles enter cells mainly through endocytosis of the cells, so that DOX is released slowly, and the effective concentration of the drug in the cell nucleus is lower. Then, under TME conditions (pH 6.5,10mM GSH), cytotoxicity was increased due to the drug-loaded nanoparticles exhibiting good pH and GSH response characteristics, consistent with drug release results in vitro. After 24h of interaction of PBA-COS-ss-DOX/gamma-PGA with HepG2 cells, half-lethal concentration (IC) at pH 6.5 and 10mM GSH 50 ) About 0.81 mug/mL, the early design study shows that the nano-carrier also has better ROS and GGT sensitive characteristics, and has pH of 6.5,10mM GSH,10 mu M H 2 O 2 And IC under conditions of 10U/mL GGT 50 Down to 0.66 μg/mL, probably due to the tight disulfide bond to DOX, H 2 O 2 And the addition of GGT accelerates chain cleavage, exposing disulfide bonds, and allowing more complete reaction of disulfide bonds with GSH. After the culture time was prolonged to 48 hours, cytotoxicity of each group was further improved. PBA-COS-ss-DOX/gamma-PGA still exhibited good redox, acid and enzyme sensitive properties at pH 6.5,10mM GSH, 10. Mu. M H 2 O 2 And IC under conditions of 10U/mL GGT 50 Reduce to 0.19 mug/mL.
Application example 4 cell uptake assay validation
HepG2 cells according to 10 5 Cell Density of/well after plating in 12 well plates, old media was removed and 1mL of media of different pH and GSH (pH 7.4, 7.4 (10 mM GSH), 6.5 (10 mM GSH) and 6.5 (10 mM GSH, 10. Mu. M H) was added to each well 2 O 2 10U/mL GGT)), and placing the cells in a 37 ℃ cell incubator for culturing for 12 hours, replacing fresh culture medium, adding DOX and PBA-COS-ss-DOX/gamma-PGA (0.5 mug/mL) respectively, culturing for 24 hours, washing with PBS for 2 times, adding 0.5mL Hoechst33342 and culturing for 15 minutes, washing with PBS again for 2 times, adding 0.5mL of 4% tissue cell fixative solution into each hole for fixing for 20 minutes, washing with PBS, and observing with a fluorescence microscope, wherein the result is shown in FIG. 11;
FIG. 11 is a fluorescence imaging analysis of drug-loaded nanoparticles after 4 and 24 hours of interaction with HepG2 cells. As can be seen, at 6.5 (10 mM GSH,10 μ M H) 2 O 2 And 10U/mL GGT), the fluorescence intensity of DOX of PBA-COS-ss-DOX/gamma-PGA after 24h treatment of HepG2 cells is 2.05 times that of the corresponding free DOX, 1.29 times that of TME, weak acidity of TME and excessive GSH induce absorption of drug-loaded nanoparticles by cells compared with normal tissue environment, so that HepG2 cells are in a range of 6.5 (10 mM GSH,10 mu M H) 2 O 2 And 10U/mL GGT, the uptake of PBA-COS-ss-DOX/gamma-PGA is strongest, and DOX enters the nucleus more.
Application example 5 intracellular oxidative stress enhancement experiment verification
After HepG2 cells were seeded in 6-well plates to adhere, the old medium was removed,2mL of medium (pH 7.4, 7.4 (10 mM GSH), 6.5 (10 mM GSH) and 6.5 (10 mM GSH, 10. Mu. M H) of different pH and GSH were added per well 2 O 2 10U/mL GGT)), then the culture medium in each well is replaced by an equal volume of fresh culture medium containing 10% serum, DOX and PBA-COS-ss-DOX/gamma-PGA (0.5 mug/mL) are added respectively, the old culture medium is removed after 4 hours of administration, PBS is used for washing twice, the intracellular active oxygen indicator DCFH-DA (which shows bright green fluorescence after the cells are oxidized) is added for incubation, the cells are incubated in a cell incubator at 37 ℃ for 20min, and the mixture is inverted and evenly mixed every 3min to ensure that the probes and the cells are fully contacted. The cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. The fluorescence intensity of ROS was observed with a fluorescence microscope, and the results are shown in fig. 12, 13 and 14;
FIGS. 12 and 13 are fluorescent plots of ROS production after 4 and 24 hours of the effect of the PBA-COS-ss-DOX/gamma-PGA prodrug on HepG2 cells, with the free DOX group having weak fluorescence after 4 and 24 hours and no significant change from the control group under simulated tumor microenvironment conditions, demonstrating that treatment of HepG2 cells with free DOX did not cause ROS changes in HepG2 cells. While after treatment of HepG2 cells with PBA-COS-ss-DOX/gamma-PGA for 4 and 24h, ROS green fluorescence was at pH 6.5,10mM GSH, 10. Mu. MH 2 O 2 And 10U/mL GGT, indicating an increase in ROS content in the HepG2 cells.
FIG. 14 shows fluorescence intensity analysis of ROS produced by PBA-COS-ss-DOX/gamma-PGA pro-drugs after acting on HepG2 cells, without significant change in fluorescence intensity of ROS in HepG2 of the free DOX treated group compared to the control group, and without enhancement after 24 h; and after 4h the ROS fluorescence intensity in HepG2 of the PBA-COS-ss-DOX/gamma-PGA treated group was at pH 6.5,10mM GSH, 10. Mu. M H 2 O 2 And the maximum under the condition of 10U/mL GGT is 6.94 times of the pH of 7.4, 7.48 times of free DOX under the same condition, and P is less than 0.001, and the statistical difference is provided; after 24h, ROS fluorescence intensity in HepG2 of PBA-COS-ss-DOX/gamma-PGA treated group was at pH 6.5,10mM GSH, 10. Mu. M H 2 O 2 And 10.49 times the ROS fluorescence intensity at pH7.4 at 10U/mL GGT, 15.36 times that of free DOX at the same conditions, P < 0.001, with statistical differences. PBA-COS-ss-DOX/gamma-PGA enters cells by endocytosis,then, the tumor cells escape into cells through lysosomes, and oxidation-reduction reactions occur in the cells to trigger GSH down-regulation-ROS up-regulation mechanisms, so that the ROS content of the tumor cells is increased, and DOX is released into cell nuclei. The addition of disulfide bonds causes PBA-COS-ss-DOX/gamma-PGA to have obvious GSH responsiveness, and can induce HepG2 cells to generate excessive ROS, thereby promoting liver cancer cells to generate immunogenic death, and the synergistic DOX plays an anti-tumor effect, thus providing potential choices for inhibiting tumor recurrence and metastasis for a long time.
In conclusion, the invention constructs a cascade oxidative stress enhanced chitosan-doxorubicin nano prodrug, firstly introduces disulfide bonds, firstly prepares DOX-ss, and then uses PBA and gamma-PGA as modifier and COS as carrier materials; the preparation method is simple, the obtained PBA-COS-ss-DOX/gamma-PGA nano prodrug has uniform particle size distribution, high DOX drug loading quantity, stability under physiological conditions, and capability of specifically targeting liver cancer HepG2 cells, realizing the synergistic response enhancement of oxidation stress of oxidation reduction and esterase, promoting drug release and achieving the high-efficiency and low-toxicity tumor inhibition effect. Thus, the PBA-COS-ss-DOX/gamma-PGA prodrug is an excellent and highly efficient nano-drug delivery system.
The various embodiments are described in a progressive manner, each embodiment focusing on differences from the other embodiments, and identical and similar parts between the various embodiments are sufficient to be seen with each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A chitosan oligosaccharide-doxorubicin biological material with enhanced cascade oxidative stress, which is characterized by comprising chitosan oligosaccharide, doxorubicin prodrug monomer DOX-ss containing disulfide bonds, 4- (bromomethyl) -phenylboronic acid and gamma-polyglutamic acid; wherein the DOX-ss structure of the doxorubicin prodrug monomer is shown in a formula I;
2. use of the chitosan oligosaccharide-doxorubicin biomaterial of claim 1, which has been subjected to cascade oxidative stress enhancement, for the preparation of a nano-prodrug having been subjected to cascade oxidative stress enhancement.
3. A preparation method of a chitosan oligosaccharide-adriamycin nano prodrug with enhanced cascade oxidative stress is characterized in that adriamycin and a compound containing disulfide bonds are subjected to substitution reaction, then are subjected to copolymerization connection with chitosan oligosaccharide and 4- (bromomethyl) -phenylboronic acid, and finally are subjected to cross-linking self-assembly with gamma-polyglutamic acid.
4. The method for preparing the chitosan oligosaccharide-doxorubicin nano-prodrug with enhanced cascade oxidative stress according to claim 3, wherein the substitution reaction is as follows: firstly, carrying out substitution reaction on 2,2' -dithiodiethanol and methacryloyl chloride to obtain an intermediate 2- (2-hydroxyethyl) disulfonyl) ethyl methacrylate HSEMA; subsequently, doxorubicin hydrochloride and 4-dimethylaminopyridine were suspended in dry dichloromethane to give a doxorubicin mixed solution, which was then taken up in N 2 Adding triphosgene under protection, stirring, dispersing HSEMA in dichloromethane, dropwise adding into doxorubicin mixed solution, magnetically stirring, and substitution reaction to obtain doxorubicin prodrug monomer DOX-ss containing disulfide bond.
5. The method for preparing the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress according to claim 3, wherein the copolymerization connection and the cross-linking self-assembly are as follows: adding an initiator into DOX-ss and chitosan oligosaccharide COS and 4- (bromomethyl) -phenylboronic acid PBA for copolymerization to obtain a polymer prodrug PBA-COS-ss-DOX, wherein the structure of the polymer prodrug PBA-COS-ss-DOX is shown as a formula II; then adding a cross-linking agent into PBA-COS-ss-DOX and gamma-polyglutamic acid gamma-PGA for self-assembly to obtain the chitosan oligosaccharide-doxorubicin nano prodrug PB A-COS-ss-DOX/gamma-PGA subjected to cascade oxidative stress;
6. the preparation method of the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress according to claim 4, wherein the substitution reaction is carried out on 2,2' -dithiodiethanol and methacryloyl chloride according to a molar ratio of 1:1, and the substitution reaction is carried out on doxorubicin hydrochloride DOX. HCl and triphosgene according to a molar ratio of 1:1.
7. The method for preparing the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress according to claim 5, wherein the initiator is ceric ammonium nitrate, and the molar ratio of DOX-ss, chitosan oligosaccharide COS, 4- (bromomethyl) -phenylboronic acid PBA and the initiator is 1:1:1:1:1.
8. The method for preparing the chitosan oligosaccharide-doxorubicin nanometer prodrug with enhanced cascade oxidative stress according to claim 5, wherein the cross-linking agent is sodium tripolyphosphate, and the mass ratio of PBA-COS-ss-DOX, gamma-polyglutamic acid gamma-PGA and the cross-linking agent is 1:1.5:1.
9. The method for preparing a nano-prodrug of chitosan oligosaccharide and doxorubicin with enhanced oxidative stress according to claim 6, wherein the particle size of the nano-prodrug PBA-COS-ss-DOX/gamma-PGA is 160-180nm.
10. The use of the nano-prodrug prepared by the preparation method of claim 9 in the preparation of antitumor drugs.
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