CN110882396B - Preparation method and application of tumor microenvironment and redox stepwise responsive nano drug delivery system - Google Patents
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Abstract
The invention discloses a preparation method and application of a tumor microenvironment and redox stepwise responsive nano drug delivery system, relates to preparation of a high-load cis-platinum macromolecule prodrug and a double-end aldehydic polyethylene glycol cross-linked cis-platinum nano drug delivery system, and can be used for treating tumors. The invention takes polyethyleneimine as a framework, and adopts cystamine containing redox-responsive disulfide bond to react with succinic anhydride and then complex with cisplatin to obtain the cisplatin complex. The obtained cisplatin complex is covalently combined with polyethyleneimine to obtain a high-load cisplatin polymer prodrug, and the outer layer is crosslinked by adopting double-end aldehyde polyethylene glycol to obtain the cisplatin nano drug delivery system with tumor microenvironment and redox step-by-step responsiveness. Compared with the traditional chemotherapy medicament cisplatin, the invention can realize the removal of the microenvironment polyethylene glycol shell outside the tumor cell and the gradual responsive release of intracellular oxidation reduction, and ensures the effective uptake of the cisplatin delivery system and the responsive release of the medicament in the tumor, thereby better playing the anti-tumor role and having good clinical treatment application prospect.
Description
Technical Field
The invention relates to a preparation method of a cisplatin-loaded tumor microenvironment and redox step-by-step responsive nano drug delivery system, which can be used for treating tumors.
Background
Cisplatin (cis-diamminedicalloplatinum (II) and CDDP) is a platinum anti-tumor chemotherapeutic drug approved by the FDA in the United states and is a first-line therapeutic drug for treating various solid tumors at present, and shows a better anti-tumor effect in clinical treatment. After cisplatin enters cells, it cross-links DNA, inhibiting DNA replication and triggering tumor cell apoptosis and necrosis. Because the action of cisplatin in inhibiting DNA replication is nonspecific, cisplatin has strong systemic toxic and side effects, such as renal damage, neurotoxicity, bone marrow toxicity, anemia and the like, and has serious toxic and side effects and easy generation of tumor drug resistance, so that the cisplatin is limited in wide clinical application. Therefore, the cisplatin is subjected to pharmaceutical chemistry and pharmaceutical engineering modification, the treatment effect is improved, and the toxic and side effects are reduced, so that the cisplatin has important clinical application value.
The anti-tumor nano delivery system can avoid the peak-valley change of the blood concentration of intermittent administration, improve the selectivity and the anti-tumor effect by changing the distribution of the medicament in vivo and reduce the toxic and side effect of the medicament on tissues outside tumors, and has become one of the hotspots in the research and development field of cisplatin. However, most of the cisplatin nano-drug delivery systems reported at present have the problems of low platinum loading capacity, large carrier material dosage, easy removal of nanoparticles by reticuloendothelial system (RES) in blood circulation of an organism, difficulty in reaching tumor parts, poor drug release controllability and the like.
The polyethylene glycol (PEG) of the nano drug delivery system can cover up the nanoparticles from the influence of a host immune system, reduce immunogenicity and antigenicity, prolong the circulation time of the nanoparticles by reducing the recognition and removal of reticuloendothelial system macrophages in blood circulation, increase the continuous accumulation of the nano drug delivery system to tumor tissues by virtue of the EPR effect and enhance the targeting property of the drugs to tumor parts. However, when macrophage uptake in blood circulation is reduced due to the traditional PEG modified nanoparticles, uptake of the nanoparticles by tumor cells in tumor tissues is also reduced, so that drugs are prevented from entering the cells to play a pharmacological effect, and an anti-tumor effect is weakened to a certain extent. Moreover, the particle size of the nano drug delivery system cannot be effectively reduced by the common single-ended polyethylene glycol (PEG) modification, and the effective accumulation capacity of the tumor target site is poor [ Biomacromolecules,2017,18, 1342-. Therefore, the effective polyethylene glycol (PEG) modification method is adopted to timely remove the polyethylene glycol modification of the drug delivery system, and the method has important significance on the effect.
Polyethyleneimine (PEI) is a cationic polymer, the structure of the PEI contains a large number of amino groups, chemical modification is easy, high loading of a drug can be realized, and the specific proton sponge effect of PEI is favorable for the escape of a nano delivery carrier from a lysosome, the drug is released into cytoplasm, and the degradation of the lysosome to the drug is reduced, so that the treatment effect of PEI is improved. The high load of cisplatin is realized by using rich amino groups of polyethyleneimine as a framework in the earlier stage of the subject group, so that a cisplatin high-molecular prodrug containing a redox-responsive disulfide bond is obtained. The cisplatin polymer prodrug can keep stable in an extracellular environment by utilizing the great difference of the concentration of Glutathione (GSH) inside and outside cells, and can be broken in a high GSH environment in a tumor cell to release cisplatin, so that the rapid release of a medicament in the tumor cell is realized, and the stimulation response characteristic is generated.
Therefore, the invention adopts Polyethyleneimine (PEI) rich in amino-group-modifiable groups as a framework, and selects cystamine containing redox-responsive disulfide bonds as a raw material to highly load cisplatin on the PEI to obtain the cisplatin polymer prodrug. On the basis, a dual stepwise responsive drug delivery system is designed, double-end aldehyde-group PEG and a cisplatin polymer prodrug are selected for cross-linking modification, so that aldehyde groups of the aldehyde-group PEG react with amino groups of BPEI to form Schiff base (Schiff base) connecting bonds with acid sensitivity, particle size increase caused by conventional single-end pegylation modification is avoided, and the cisplatin drug delivery system with a tumor microenvironment and redox responsiveness, which is modified by shell PEG cross-linking, is constructed. Due to the shielding effect of PEG, the drug delivery system is not easy to dissociate and be easily absorbed by macrophages in normal tissues (pH 7.4), after reaching a tumor part, Schiff base acid-sensitive connecting bonds are firstly broken in a specific microenvironment of the tumor part (pH 5.5-6.8, the extracellular pH of the tumor part is slightly acidic due to a large amount of lactic acid generated by high-speed glycolysis under aerobic or anaerobic conditions), PEG of a shell in the cisplatin drug delivery system is removed and the endocytosis effect of the cis-platin is recovered, after the cis-platin is triggered and broken by redox responsiveness of disulfide bonds in a high GSH environment in a tumor cell and the cis-platin is released, the effective uptake of the cis-platin drug delivery system into the cell and the response release of drugs in the tumor are ensured, and the anti-tumor therapeutic effect is better exerted.
Disclosure of Invention
The invention aims to provide a preparation method and application of a cis-platinum nano drug delivery system with a tumor microenvironment and gradual redox responsiveness, which can highly load cis-platinum drugs. On the basis, double-end aldehyde-group PEG and a cisplatin polymer prodrug are selected for crosslinking, so that aldehyde groups of the aldehyde-group PEG react with amino groups of polyethyleneimine to form Schiff base (Schiff base) connecting bonds with acid sensitivity, a cisplatin nano drug delivery system with a tumor microenvironment and redox responsiveness is constructed, and a new idea is provided for research of antitumor drugs.
The invention takes polyethyleneimine as a framework, and adopts cystamine containing redox-responsive disulfide bond to react with succinic anhydride and then complex with cisplatin to obtain the cisplatin complex. The obtained cis-platinum complex is covalently combined with abundant amino groups of polyethyleneimine to obtain a high-load cis-platinum high-molecular prodrug, and the load of cis-platinum can reach 32.66%. Selecting two-end aldehyde PEG and cisplatin high-molecular prodrug for crosslinking, so that aldehyde group of the aldehyde PEG reacts with amino group of polyethyleneimine to form Schiff base (Schiff base) connecting bond with acid sensitivity characteristic, constructing and obtaining a cisplatin drug delivery system with a tumor microenvironment and redox responsiveness, and obtaining the high-efficiency and low-toxicity cisplatin anti-tumor drug through activity evaluation.
A preparation method of a tumor microenvironment and redox stepwise responsive cisplatin nano drug delivery system specifically comprises the following steps:
(1) stirring cisplatin at 37 deg.C in dark to completely dissolve in III grade ultrapure water, cooling to room temperature, adding silver nitrate at corresponding molar ratio, stirring at room temperature in dark for 48 hr, centrifuging twice (5000rpm, 1 hr/time), and filtering the supernatant with 0.1 μm water system filter to obtain hydrated cisplatin solution (3 mg. mL. solution)-1) And (5) standby. Cystamine dihydrochloride is dissolved in methanol at room temperature, stirred with a certain amount of triethylamine for 30min under the ice bath condition (0-4 ℃), added with 1, 4-dioxane solution of succinic anhydride with corresponding proportion and stirred for reaction for 1.5h at room temperature, the organic phase is distilled off under reduced pressure, and 0.3 percent of Na with corresponding amount is added2CO3Extracting the aqueous solution with diethyl ether for 3 times to obtain cystamine-sodium succinate aqueous solution. Slowly dripping the cystamine-sodium succinate aqueous solution into a hydrated cisplatin solution according to a certain proportion, stirring at room temperature in a dark place for 48 hours, concentrating the reaction solution, dialyzing and purifying by using a dialysis bag with the molecular weight cutoff of 100 (dialyzing in III-grade ultrapure water for 3 times, changing water every 2 hours), and freeze-drying to obtain the cisplatin complex.
(2) Carbonyl diimidazole and cisplatin complex aqueous solution (7.73 mg. mL) with specific ratio-1) Stirring the mixture for reaction at a certain temperature, and then continuously stirring the mixture for reaction with polyethyleneimine with a certain molecular weight according to a corresponding grafting proportion. After the reaction is finished, a dialysis bag with the molecular weight cutoff of 7000 is adopted for dialysis and purification (dialysis is carried out for 4 times in grade III ultrapure water, and water is changed every 2 hours), and then the cisplatin polymer prodrug is obtained by freeze drying.
(3) Adding a certain amount of 10 mg/mL-1Double end aldehyde group PEG aqueous solution and 1 mg/mL-1The cisplatin polymer prodrug aqueous solution is uniformly mixed and stirred at a specific temperature in the dark for reaction for a certain time. Upper part ofAnd dialyzing the reaction solution by using a dialysis bag with the molecular weight cutoff of 50000 to obtain a tumor microenvironment and redox stepwise responsive cisplatin nano drug delivery system.
In the specific preparation process, in the step (1), the molar ratio of silver nitrate to cisplatin is 1: 2-2: 1, the molar ratio of cystamine dihydrochloride, triethylamine and succinic anhydride is 1:2: 0.5-1: 20:2, the molar ratio of sodium carbonate to cystamine dihydrochloride is 1: 1-5: 1, and the molar ratio of cystamine-sodium succinate to hydrated cisplatin is 1: 2-2: 1.
In the step (2), the molar ratio of carbonyldiimidazole to cisplatin complex is 1: 0.5-5: 1, the reaction temperature is 20-50 ℃, and the reaction time is 0.5-5 h. The polyethyleneimine is Linear Polyethyleneimine (LPEI) or Branched Polyethyleneimine (BPEI), the molecular weight range is 600-50K Da, the molar ratio of the cisplatin complex to amino groups on the polyethyleneimine is 1: 50-1: 1, the reaction temperature is 0-100 ℃, and the reaction time is 5-48 h.
In the step (3), the molecular weight of PEG in the double-end aldehyde-group PEG is 200-50K Da, the feeding mass ratio of the double-end aldehyde-group PEG to the cis-platinum polymer prodrug is 1: 100-5: 1, the temperature is 4-100 ℃, and the stirring reaction time is 0.1-120 h.
The activity evaluation experiment result of the tumor microenvironment and redox stepwise responsive cisplatin nano drug delivery system shows that compared with the traditional chemotherapy drug cisplatin clinically used, the drug delivery system can realize the gradual drug release of the tumor microenvironment and the redox responsive cisplatin, can greatly reduce the toxic and side effects of the cisplatin while playing the role of effectively resisting tumors, and has good clinical application prospect.
Drawings
FIG. 1 is a particle size distribution diagram of PEG- (BPEI-SS-Pt) nanoparticles obtained by crosslinking reaction for 24h (A), 48h (B), 72h (C).
FIG. 2 is the stability of PEG- (BPEI-SS-Pt) nanoparticles.
FIG. 3 is the in vitro drug release profile of PEG- (BPEI-SS-Pt) nanoparticles under different medium conditions.
FIG. 4 is an in vitro cell-ball penetration experiment of PEG- (BPEI-SS-Pt) nano-drug delivery system under different pH conditions.
FIG. 5(A) graph of tumor volume changes in nude mice. n is equal to 4, and n is equal to 4,
*P<0.05，**P<0.01 is that Cisplatin group is compared with Control group,&&P<0.01 is PEG- (BPEI-SS-Pt) group compared with Control group. (B) Body weight change of nude mice. n is equal to 4, and n is equal to 4,
*P<0.05，**P<0.01 is that Cisplatin group is compared with Control group,##P<0.01 is Cisplatin group compared to PEG- (BPEI-SS-Pt) group. (C) TUNEL immunofluorescent staining of tumor tissue and caspase 3 and ki67 staining of immunohistochemistry (50 μm).
Detailed Description
The following examples further describe embodiments of the present invention. The following embodiments further illustrate the technical problems and technical solutions of the present invention in detail. It should be understood that the following description is only exemplary of the present invention and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
EXAMPLE 1 preparation of cisplatin Complex
675.0mg (2.23mmol) of cisplatin is weighed, added into 225mL grade III ultrapure water, stirred at 37 ℃ in the dark until the cisplatin is completely dissolved, cooled to room temperature, added with 758.2mg (4.45mmol) of silver nitrate, and stirred at room temperature in the dark for reaction for 48 hours. After completion of the reaction, the reaction mixture was centrifuged 2 times (5000rpm, 1 hour each), and the supernatant was collected and filtered through a 0.1 μm water filter to obtain a hydrated cisplatin solution.
499.4mg (2.18mmol) of cystamine dihydrochloride is dissolved in 24.6mL of methanol at room temperature. To a solution of cystamine dihydrochloride in methanol was added 445.7mg (4.36mmol) of triethylamine under ice bath conditions and stirred for 30 min. Succinic anhydride 202.4mg (1.98mmol) was dissolved in 36.97mL of anhydrous 1, 4-bisIn an oxygen hexacyclic ring, the solution is added into a methanol solution of cystamine dihydrochloride, the ice bath is removed, and the reaction is stirred at room temperature for 1.5 h. After the reaction, the organic solvent was removed by rotary evaporation of the reaction mixture, and 0.3% Na was added2CO377mL of aqueous solution is extracted by diethyl ether for 3 times, the volume of the diethyl ether used in each extraction is 150mL, the aqueous phase is collected, and the residual diethyl ether is removed by rotary evaporation to obtain the cystamine-sodium succinate solution.
77mL of cystamine-sodium succinate solution is slowly added into 200mL of hydrated cisplatin solution in a dropwise manner, and the mixture is stirred at room temperature in the dark for reaction for 48 hours. And after the reaction is finished, concentrating the reaction solution under reduced pressure to about 10mL, transferring the reaction solution into a dialysis bag with the molecular weight cutoff of 100, dialyzing the reaction solution in 3000mL III-grade ultrapure water for 3 times, changing water every 2h, and freeze-drying the reaction solution after the dialysis is finished to obtain the yellow powdery cis-platinum complex.
IR and of cisplatin Complex1The H NMR analysis results were as follows: IR (KBr) 3387,3194,3082,2943,1636,1543,1396,1150,1049,636cm-1。1H NMR(400MHz,D2δ 2.46-2.48(6H, m),2.85(t,2H, J ═ 6.4Hz),3.17-3.23(2H, m),3.52(t,2H, J ═ 6.4Hz) ppm. The obtained product has the same structure with the target product through the identification of infrared and nuclear magnetic resonance hydrogen spectrums.
EXAMPLE 2 preparation of cisplatin Polymer prodrug BPEI-SS-Pt
Weighing 48.8mg (2 mu mol) of Branched Polyethyleneimine (BPEI) to dissolve in 5mL of grade III ultrapure water, and performing ultrasonic treatment at 60 ℃ for 15min until complete dissolution to obtain a colorless and clear BPEI aqueous solution; cisplatin complex 77.3mg (143. mu. mol) was dissolved in 10mL of grade III ultrapure water, and stirred at room temperature until completely dissolved, to give a yellow clear and transparent cisplatin complex aqueous solution. Adding 25.6mg (158 mu mol) of carbonyldiimidazole into the cisplatin complex aqueous solution, stirring for 1h under the ice bath condition, removing the ice bath, adding the BPEI aqueous solution into the reaction solution after the reaction solution returns to the room temperature, and stirring for reaction for 24h at the room temperature in a dark place. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed in 2000mL of grade III ultrapure water for 4 times with water change every 2 hours. After dialysis, yellow solid BPEI-SS-Pt is obtained by freeze drying.
The infrared spectrum analysis result of the product is as follows: IR (KBr) 3564,2962,1717,1636,1558,1508,1458,1339,1026,806,706cm-1. The obtained product has the same structure with the target product through infrared spectrum identification. The load of the cis-platinum in the BPEI-SS-Pt is 32.66 percent as determined by inductively coupled plasma mass spectrometry (ICP-MS).
Example 3 two-terminal hydroformylation of PEG2000Preparation of the Material PEG-DiAlde
387.2mg (2.5mmol) of p-aldobenzoic acid are placed in 50mL of dichloromethane, 721.8mg (3.75mmol) of EDCI are added with stirring at room temperature, and 9.4mg (75. mu. mol) of DMAP are added with stirring for 0.5h while cooling on ice after the reaction solution is clear and transparent. After returning to room temperature in an ice-free bath, 1g (2.5mmol) of PEG was added2000The reaction was stirred for 1 h. The reaction mixture was concentrated to 5mL under reduced pressure, washed with a saturated aqueous sodium chloride solution 5 times, and then added with anhydrous sodium sulfate and left overnight. Filtering, adding 10 times volume of ethyl acetate into the filtrate, standing at 4 deg.C for 6 hr, and vacuum filtering. After the crude product was purified 2 more times by dissolution in dichloromethane-precipitation with glacial ethyl ether, the solid was dried under vacuum at 20 ℃ for 12 h. Dissolving the product in grade III ultrapure water, dialyzing, and freeze-drying (molecular weight cutoff is 1000) to obtain the product. Calculating the yield, measuring the melting point, and characterizing the structure by adopting a nuclear magnetic resonance hydrogen spectrum and an infrared spectrum.
The experimental results are as follows: para-aldehyde benzoic acid and PEG are covalently connected in dichloromethane to obtain PEG with two aldehyde groups at two ends2000283.1mg of product, 26.8% yield, 51.0 ℃ -51.4 ℃ melting point measured by a precision melting point tester. IR (KBr): 3433, 2885, 1716, 1701, 1465, 1342, 1280, 1149, 964, 840cm-1。1H NMR(400MHz,D2O): δ 10.00(s,2H),8.19(d, J ═ 7.6Hz,4H),8.02(d, J ═ 7.6Hz,4H),4.56-4.52(m,4H),3.91-3.84(m,4H),3.84-3.28(m, 182H). The obtained product is identified by infrared and nuclear magnetic resonance hydrogen spectrum and has the same structure with the literature reports [ Biomaterials,2017,147,53-67]。
Example 4 preparation and characterization of tumor microenvironment and Redox progressive responsiveness cisplatin drug delivery System PEG- (BPEI-SS-Pt)
The cross-linking is carried out according to the mass ratio of 1:9 between the two ends of aldehyde-modified PEG-DiAlde and BPEI-SS-Pt. The amount of each component was 1 mg/mL-1BPEI-SS-Pt macromolecule prodrug water solution50mL of the mixture is subjected to ultrasonic treatment for 1h at 50 ℃ in a dark place. Weighing aldehydized PEG2000Dissolving 10.0mg and 1mL of III-grade ultrapure water into clear and transparent solution by oscillation to obtain solution with concentration of 10 mg/mL-1Aldehyde-based PEG of2000An aqueous solution. Absorbing the aldehyde-group PEG aqueous solution with the corresponding volume according to the mass ratio, adding the aldehyde-group PEG aqueous solution into the aqueous solution of BPEI-SS-Pt, stirring for 24 hours, 48 hours and 72 hours at normal temperature in the dark, using a dialysis bag with the molecular weight cutoff of 50000Da, quickly dialyzing for 2 hours in third-grade ultrapure water at normal temperature in the dark by stirring, and storing in a refrigerator at 4 ℃.
And (3) measuring the content of platinum in the nanoparticles by using inductively coupled plasma mass spectrometry (ICP-MS). The particle size, PDI and zeta potential of the PEG- (BPEI-SS-Pt) nanoparticles prepared by crosslinking for 24h, 48h and 72h are respectively measured by a laser particle size analyzer so as to explore the influence of different crosslinking time on the PEG- (BPEI-SS-Pt) nanoparticles. And the morphology of the PEG cross-linked nanoparticles is examined by adopting a transmission electron microscope and a scanning electron microscope.
The experimental results are as follows: the particle size, PDI and zeta potential measurement experiment results of the cross-linked nanoparticles obtained by performing cross-linking reaction on PEG-DiAlde and BPEI-SS-Pt for 24h, 48h and 72h are shown in Table 1, and the results show that the particle size obtained by cross-linking PEG-DiAlde and BPEI-SS-Pt high-molecular prodrug for 72h is 135.8 +/-7.1 nm, the dispersion coefficient PDI is 0.292 +/-0.007, the particle size distribution range is narrow (<0.3), and the requirements of a nano delivery system for tumor treatment are met. The zeta potential of the nano delivery system is 13.1 +/-1.9 mV, and the nano particles are positively charged. Comparing the peak shapes of the nanoparticles formed at different time points, as shown in fig. 1, compared with 24h and 48h, the particle size of the nanoparticles formed in 72h is a single peak type with normal distribution, the particle size distribution is narrow, and finally 72h is selected as the cross-linking reaction time for preparing a tumor microenvironment and a redox step-by-step responsive cisplatin delivery system. The loading of cisplatin in the cross-linked nano drug delivery system PEG- (BPEI-SS-Pt) was calculated to be 21.74%.
TABLE 1 particle size, PDI and zeta potential of PEG- (BPEI-SS-Pt) nanoparticles
Example 5 stability Studies of PEG- (BPEI-SS-Pt) nanoparticles
The optimized PEG- (BPEI-SS-Pt) nanoparticles are stored at 4 ℃, the particle size of the nanoparticles is measured by a laser particle size tester, and the change condition of the particle size of the nanoparticles after being placed for different times is observed to study the stability of the nanoparticles.
The experimental results are as follows: the stability study results (as shown in fig. 2) show that the particle size of the nanoparticles after 28 days is relatively small, and the stability is good under the storage condition of 4 ℃.
Example 6 study on tumor microenvironment sensitivity and redox characteristics of PEG- (BPEI-SS-Pt) nanoparticles
The tumor microenvironment and redox stepwise response characteristics of the constructed nano drug delivery system are verified by observing the pH sensitivity and redox sensitivity of the drug delivery system in vitro simulation tumor microenvironment and the in-vitro and in-vitro reducing levels (10mM GSH and 10 mu M GSH) of tumor cells.
1. Nuclear magnetic hydrogen spectrum determination of PEG- (BPEI-SS-Pt) nanoparticles under different pH conditions
Dissolving three parts of PEG cross-linked nanoparticle freeze-dried powder with the same quantity in a 4mL centrifuge tube by 550 mu L of heavy water, adjusting the pH values of two parts of PEG cross-linked nanoparticle solutions to be 6.5 and 7.4 by using a small quantity of deuterium chloride and deuterium sodium oxide respectively after the PEG cross-linked nanoparticle freeze-dried powder is completely dissolved, directly dissolving the other part of PEG cross-linked nanoparticle solution by using the heavy water only, and transferring a sample into a nuclear magnetic tube to perform nuclear magnetic resonance hydrogen spectrometry.
2. Study on drug release characteristics of PEG- (BPEI-SS-Pt) drug delivery system under different media
Simulating in vivo and tumor tissue microenvironment, adding 50 μ L of prepared PEG- (BPEI-SS-Pt) nanoparticle aqueous solution into III-grade ultrapure water to constant volume of 2mL, filling into a dialysis bag (molecular weight cut-off 3500Da, external solution pH 7.4, 10 μ M GSH PBS solution), and incubating in water bath at 37 deg.C. After 4h incubation the external solution was adjusted to pH 6.5, 10. mu.M GSH in PBS. After further incubation for 4h at this condition, the external solution was adjusted to pH 7.4 and incubated in a 37 ℃ water bath under 10mM GSH conditions. Taking out 2mL of drug-releasing external liquid from the dialysis bag at the set drug-releasing time points of 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h and 48h, then adding 2mL of fresh dialysis medium, measuring the content of Pt by ICP-MS, calculating the accumulated drug-releasing amount of the drug, and inspecting the pH value of the tumor microenvironment and the redox response characteristic.
The experimental results are as follows: the nuclear magnetic hydrogen spectrum detection results of the PEG- (BPEI-SS-Pt) nanoparticles under different pH conditions show that the characteristic peak of aldehyde group is not observed in the PEG cross-linked nanoparticles only dissolved by heavy water and adjusted to pH 7.4, and the peak of aldehyde group of 9.95ppm appears in a dissolution medium with pH 6.5. Namely, under the slightly acidic condition of the tumor part, the Schiff base connecting bond in the PEG- (BPEI-SS-Pt) can be broken, and the PEG shell is removed, thereby being beneficial to further uptake of nanoparticles into cells and playing an effective anti-tumor role.
The in vitro drug release results under different medium conditions show that the PEG shell of the PEG- (BPEI-SS-Pt) nanoparticle can not be removed under the conditions of blood circulation and normal extracellular environment (pH 7.4, 10 mu M GSH), the disulfide bond has no obvious response to low-concentration GSH, and the drug release speed and the drug release amount are low (figure 3). And under the condition of a tumor microenvironment (pH 6.5 and 10 mu M GSH), the Schiff base connecting bond in the PEG- (BPEI-SS-Pt) nanoparticle is sensitively broken, the PEG shell is removed, the drug release amount is still lower, but after the nanoparticle without the PEG shell is inserted into a cell, namely under the condition of simulating tumor cells (pH 7.4 and 10mM GSH), the drug release amount is rapidly increased, and the disulfide bond breakage response release of Pt is realized. The results show that the PEG- (BPEI-SS-Pt) nano delivery system has better tumor microenvironment and gradual response characteristics of oxidation reduction, can remove the PEG water-soluble shell in the tumor microenvironment, increase the uptake of nanoparticles into cells, and simultaneously respond to and release drugs under the condition of high GSH concentration in the cells so as to achieve efficient anti-tumor effect.
Example 7 study of tumor microenvironment and in vitro tumor cell sphere penetration behavior of PEG- (BPEI-SS-Pt) in a Redox stepwise responsive cisplatin NanoDriver System
The effective delivery of the antitumor drug into the deep tumor tissue is the key to improving and improving the drug action. Studies have shown that monolayer cell cultures do not represent completely in vivo tumors, because they differ in cell heterogeneity, nutrients, oxygen gradients, cell-cell interactions, matrix deposition, gene expression profiles, etc., and thus produce different drug responses in vitro and in vivo, making the in vitro-in vivo drug evaluation poorly correlated. Compared to monolayer cell cultures, tumor cell spheres represent a more realistic in vitro model of tumor conditions. The experiment adopts a droplet overlapping method to construct A549 cell spheres for the research of the penetrating behavior of the tumor cell spheres, and the specific method is as follows:
0.3g of agarose was weighed into a 50mL beaker, and 15mL of RPMI medium (2.0%) was added. Sealing the beaker with aluminum foil or cover, sterilizing in autoclave at 121 deg.C for 20 min, cooling to about 90 deg.C, taking out sterilized agarose solution, and transferring to sterile ultra-clean bench. To avoid cooling solidification of the agarose, the beaker containing the agarose was placed in a water bath preheated to 70 ℃ throughout the plating. mu.L of agarose (less than 100. mu.L will not completely cover the bottom of the cell plate) is added to each well of a 48-well cell culture plate (flat bottom) and the agarose plated in the plate solidifies within a few seconds. The 100 mu L agarose layer is easy to generate a proper concave surface, is beneficial to the aggregation of cell suspension and is easy to form cell balls. The agarose cell plate is cooled to room temperature, sealed and placed in a lunch box, and can be placed at 4 ℃ in the dark for about 10 days after preparation.
To prepare 400 μm diameter cell spheres at the appropriate time, a549 single cell suspension was diluted to a density of 250, 500, 1000, 1500, 2000, 3000 cells per well and a cell suspension volume of 400 μ L seeded on agarose gel plates. After inoculation, the plates were placed in a 5% cell incubator at 37 ℃ and observed, the solution was changed every two days, the liquid was slowly removed along the edge of the well wall using a 200 μ L pipette, avoiding aspiration of the cell pellet formed, and fresh culture medium was slowly and gently added along the well wall. The growth of the cell balls in each well was observed every day, and the appropriate cell density was selected for the subsequent experiments.
The preparation of the PEG- (BPEI-SS-Pt) -CY5 fluorescent nanoparticles carries out fluorescent labeling on the PEG- (BPEI-SS-Pt) cross-linked nanoparticles according to the fluorescent material grafting mass ratio of 5%. Inoculating 400 mu L of A549 single-cell suspension to an agarose gel plate according to the density of 3000 cells per hole, changing the solution once every two days, generating macroscopic cell spheres when the cell spheres are cultured to the 10 th day, respectively adjusting the pH of the environment outside the cell spheres to 6.5 and 7.4, respectively, administering the solution according to the Pt concentration of 2 mu g/mL, incubating the cell spheres for 4h, slowly removing the culture solution along the hole wall, slowly washing the cell spheres along the hole wall by PBS for 2 times, adding 400 mu L of 4% paraformaldehyde fixing solution into each hole, fixing for 30min at room temperature, removing the fixing solution, washing for 2 times by PBS, adding 0.5 mu g/mL DAPI cell staining solution (the concentration of DAPI stock solution is 5 mu g/mL, diluting by 10 times by methanol) for dyeing for 40min at room temperature, removing the staining solution, transferring the cell spheres to a 4mL centrifuge tube, washing the surfaces of the cell spheres by PBS at 37 ℃, after the cell pellet settled naturally, the supernatant was aspirated and repeated 3 times. Dispersing the cell ball suspension at the bottom in the middle of a confocal culture dish, sealing and airing, and sealing with glycerol. And (3) carrying out tomography observation on the penetration capacity of the nanoparticles under different pH conditions by using a laser confocal microscope.
The experimental results are as follows: the research result of the in-vitro tumor cell sphere penetration behavior of the PEG- (BPEI-SS-Pt) nanoparticle shows that (figure 4), under the condition of physiological condition pH 7.4, the shell of the PEG- (BPEI-SS-Pt) nanoparticle cannot be removed, the water-soluble shell blocks the uptake, cell entry and transportation of the nanoparticle, the fluorescence intensity is gradually weakened along with the increase of the depth of laser confocal tomography, and the nanoparticle cannot effectively penetrate into the cell sphere. Under the condition that the pH of a tumor microenvironment is 6.5, the pH-sensitive Schiff base connecting bond is broken, the PEG shell is removed, the cell-entering capability of the nanoparticles is recovered, strong fluorescence can still be observed along with the increase of the tomography depth, and the drugs can penetrate into the cell spheres. The results of in vitro tumor cell sphere penetration experiments show that the nanoparticles have tumor microenvironment response characteristics, the PEG water-soluble shell can be removed under the subacid condition, and the cell sphere penetration and drug transport capacity are increased, so that the effective anti-tumor effect is achieved, and a basis is provided for in vivo drug evaluation.
Example 8 evaluation of in vivo antitumor Effect of PEG- (BPEI-SS-Pt) NanoTavery System
Selecting 6-8 weeks old BALB/c nude mice as experimental models, and 100 mu L human non-small cell lung cancer cell A549 (density is 2 multiplied by 10)7one/mL) is inoculated under the skin of the right back of the mouse until the volume of the subcutaneous tumor of the mouse is 100mm3The administration is carried out at the same time. Subcutaneous tumor-inoculated BALB/c nude mice were divided into 3 groups (4 mice per group)) The solvent, the cisplatin positive control drug and the PEG- (BPEI-SS-Pt) nanoparticle are respectively administered in a tail vein injection mode. The dosage of the positive control drug cis-platinum is 3mg/kg, and the dosage of the PEG- (BPEI-SS-Pt) is the same as the Pt amount of the free drug cis-platinum, and the dose is administered once every 2 days for 3 times. Tumor volume and body weight of mice were measured every 2 days. The length and width of the mouse tumor are measured by a digital vernier caliper, and the tumor volume is calculated by the formula of V ═ a × b2) And/2, a is the longest diameter of the tumor and b is the shortest diameter of the tumor. At day 20 of observation after dosing, the experiment was terminated, the mice sacrificed, tumor tissue collected and fixed in 4% paraformaldehyde, paraffin embedded, and processed into 5mm thick sections. Paraffin section is dewaxed and rehydrated, HE staining is carried out, immunofluorescence and immunohistochemical staining are carried out on the tumor tissue section at the same time, a TUNEL kit is adopted to detect the number of apoptotic cells in the tumor tissue, and related antigens are adopted to measure the expression quantity of apoptosis pathway related protein caspase 3 and cell nucleus proliferation related antigen Ki 67.
The experimental results are as follows: the experiment of the in vivo antitumor activity of the PEG- (BPEI-SS-Pt) nano drug delivery system shows that the average tumor volume of untreated blank Control (Control) mice is close to 2000mm at 20 days after the drug administration (figure 5)3And the positive drugs are effectively inhibited in tumor volume of mice in cis-platinums (Cisplatin group) and PEG- (BPEI-SS-Pt) nanoparticle group, and the nano drug delivery system can achieve the in vivo anti-tumor activity equivalent to or better than that of the clinical drug Cisplatin (as shown in figure 5A). The results of fig. 5B show that the positive drug significantly decreased the body weight of the cisplatin group mice after administration, whereas compared to the cisplatin group, the constructed tumor microenvironment and redox-progressive responsive cisplatin nano drug delivery system PEG- (BPEI-SS-Pt) had less effect on the body weight of the mice and significantly reduced toxic and side effects. Results of immunofluorescent staining of TUNEL (fig. 5C) showed that the PEG- (BPEI-SS-Pt) nano-delivery system was able to cause more apoptosis than the control and free drug groups, and immunohistochemical staining of caspase 3 results showed that caspase 3 activity was higher in the PEG- (BPEI-SS-Pt) nano-delivery system group than the control and free drug groups. Cell proliferation associated antigen marker ki67 stainingColor results indicate that cell proliferation ability in both the cis-platinum group and the nanoparticle group was effectively inhibited compared to the control group.
The cisplatin nano-drug delivery system PEG- (BPEI-SS-Pt) constructed by the invention can realize gradual response drug release of tumor site microenvironment and intracellular redox, and can ensure effective uptake of the cisplatin drug delivery system and response release of drugs in tumors, thereby better playing the anti-tumor role of the cisplatin drug delivery system, having important guiding significance for the research of new dosage forms of chemotherapeutic drugs cisplatin, and having good clinical treatment application prospect.
Claims (9)
1. A preparation method of a tumor microenvironment and redox stepwise responsive nano drug delivery system is characterized in that: reacting cystamine containing redox-responsive disulfide bond with succinic anhydride and complexing with cisplatin to obtain cisplatin complex by using polyethyleneimine rich in amino-modifiable groups as a framework; the obtained cis-platinum complex is covalently combined with polyethyleneimine to obtain a high-load cis-platinum polymer prodrug, wherein the load of cis-platinum can reach 32.66%; and the outer layer is crosslinked by adopting double-end aldehyde polyethylene glycol, so that aldehyde groups of the aldehyde PEG react with amino groups of PEI to form Schiff base connecting bonds with acid sensitivity, and the cis-platinum nano drug delivery system with a tumor microenvironment and gradual redox responsiveness modified by PEG crosslinking of the shell is obtained.
2. The method of claim 1, wherein the nano drug delivery system comprises: due to the shielding effect of PEG, the drug delivery system is not easy to dissociate and be absorbed by macrophage in normal tissue, after reaching the tumor part, the Schiff base acid-sensitive connecting bond is firstly broken in the specific microenvironment of the tumor part, the PEG of the shell in the cisplatin drug delivery system is removed and the endocytosis effect is recovered, after endocytosis, the disulfide bond is subjected to redox responsiveness triggering breakage in the high GSH environment in the tumor cell and cisplatin is released, the effective uptake endocytosis of the cisplatin drug delivery system and the response release of the drug in the tumor are ensured, and the antitumor therapeutic effect is better exerted.
3. The method for preparing a tumor microenvironment and redox cascade responsive nano drug delivery system of claim 1, comprising the steps of:
stirring the cisplatin in the step (1) at 37 ℃ in a dark place until the cisplatin is completely dissolved in III-grade ultrapure water, cooling to room temperature, adding silver nitrate in a corresponding molar ratio, stirring at room temperature in a dark place for 48 hours, centrifuging twice, and filtering the supernatant by using a 0.1-micron water filter to obtain 3 mg/mL-1The hydrated cisplatin solution is ready for use;
dissolving cystamine dihydrochloride in methanol at room temperature, stirring with a certain amount of triethylamine at the ice bath condition of 0-4 ℃ for 30min, adding a 1, 4-dioxane solution of succinic anhydride in a corresponding proportion, stirring and reacting at room temperature for 1.5h, distilling under reduced pressure to remove an organic phase, adding a corresponding amount of 0.3% Na2CO3Extracting the aqueous solution with diethyl ether for 3 times to obtain cystamine-sodium succinate aqueous solution;
slowly dripping the cystamine-sodium succinate aqueous solution into a hydrated cisplatin solution according to a certain proportion, stirring at room temperature in a dark place for 48 hours, concentrating the reaction solution, dialyzing and purifying by using a dialysis bag with the molecular weight cutoff of 100, and freeze-drying to obtain a cisplatin complex;
step (2) carbonyl diimidazole and 7.73 mg/mL in specific ratio-1Stirring cisplatin complex aqueous solution for reaction at a certain temperature, then continuously stirring and reacting with polyethyleneimine with a certain molecular weight according to a corresponding grafting proportion, dialyzing and purifying by using a dialysis bag with the molecular weight cutoff of 7000 after the reaction is finished, and freeze-drying to obtain a cisplatin polymer prodrug;
step (3) adding a predetermined amount of 10 mg/mL-1Double end aldehyde group PEG aqueous solution and 1 mg/mL-1The cisplatin polymer prodrug aqueous solution is uniformly mixed and stirred in the dark at a specific temperature for reaction for a certain time; and dialyzing the reaction solution by using a dialysis bag with the molecular weight cutoff of 50000 to obtain a tumor microenvironment and redox stepwise responsive cisplatin nano drug delivery system.
4. The method of claim 3, wherein the nano drug delivery system comprises: in the step (1), the molar ratio of silver nitrate to cisplatin is 1: 2-2: 1, the molar ratio of cystamine dihydrochloride to triethylamine to succinic anhydride is 1:2: 0.5-1: 20:2, the molar ratio of sodium carbonate to cystamine dihydrochloride is 1: 1-5: 1, and the molar ratio of cystamine-sodium succinate to hydrated cisplatin is 1: 2-2: 1.
5. The method for preparing a tumor microenvironment and redox cascade responsive nano drug delivery system according to claim 3, wherein the nano drug delivery system comprises: in the step (2), the molar ratio of the carbonyldiimidazole to the cis-platinum complex is 1: 0.5-5: 1, the reaction temperature is 20-50 ℃, and the reaction time is 0.5-5 h.
6. The method for preparing a tumor microenvironment and redox cascade responsive nano drug delivery system according to claim 3, wherein the nano drug delivery system comprises: in the step (2), the polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine, and the molecular weight range is 600-50K Da.
7. The method for preparing a tumor microenvironment and redox cascade responsive nano drug delivery system according to claim 3, wherein the nano drug delivery system comprises: in the step (2), the molar ratio of the cis-platinum complex to the amino group on the polyethyleneimine is 1: 50-1: 1, the reaction temperature is 0-100 ℃, and the reaction time is 5-48 h.
8. The method for preparing a tumor microenvironment and redox cascade responsive nano drug delivery system according to claim 3, wherein the nano drug delivery system comprises: in the step (3), the molecular weight of PEG in the double-end aldehyde-group PEG is 200-50K Da, the feeding mass ratio of the double-end aldehyde-group PEG to the cis-platinum polymer prodrug is 1: 100-5: 1, the temperature is 4-100 ℃, and the stirring reaction time is 0.1-120 h.
9. The application of the tumor microenvironment and the redox progressive responsive nano drug delivery system prepared by the preparation method according to any one of claims 1 to 8 in preparing anti-lung cancer drugs.
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