CN114432269A - Preparation method and application of stimuli-responsive nano drug delivery system - Google Patents

Preparation method and application of stimuli-responsive nano drug delivery system Download PDF

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CN114432269A
CN114432269A CN202210109147.9A CN202210109147A CN114432269A CN 114432269 A CN114432269 A CN 114432269A CN 202210109147 A CN202210109147 A CN 202210109147A CN 114432269 A CN114432269 A CN 114432269A
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cisplatin
delivery system
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张邦乐
贾舟延
王伟
何炜
侯丽爽
贠崇虓
张耀文
张堂睿
宦梦蕾
周四元
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Abstract

The invention discloses a preparation method and application of a stimuli-responsive nano drug delivery system. The invention takes polyethyleneimine as a framework, selects a disulfide bond with redox responsiveness to covalently combine a cisplatin complex and the polyethyleneimine to obtain a high-load cisplatin polymer conjugate, and then adopts hyaluronidase and calcium phosphate to assemble the conjugate to obtain a nano drug delivery system which can degrade and penetrate a tumor tissue microenvironment of a tumor matrix and has redox responsiveness. Compared with the traditional chemotherapy medicament cisplatin, the invention can realize the release of hyaluronidase in a tumor tissue microenvironment and the release of platinum medicaments in tumor cell redox response. And the cisplatin drug delivery system efficiently enters deep tumor tissues and quickly responds to release the drug in tumor cells by degrading the solid tumor matrix through hyaluronidase, so that the solid tumor resisting effect of the cisplatin drug delivery system is better exerted, and the cisplatin drug delivery system has a good clinical treatment application prospect.

Description

Preparation method and application of stimuli-responsive nano drug delivery system
Technical Field
The invention relates to a preparation method of a tumor tissue microenvironment capable of penetrating a tumor matrix and an oxidation-reduction responsive nano drug delivery system loaded with a cisplatin polymer conjugate and a hyaluronidase protein drug, and the nano drug delivery system can be used for treating tumors, especially solid tumors.
Background
Cisplatin (Cis-diamminedichloropsiclinum (II), cissplatin and CDDP) is a platinum-based anti-tumor chemotherapeutic drug, can be used for first-line treatment of various tumors, and shows a good anti-tumor effect in clinical treatment. Cisplatin initiates apoptosis of tumor cells by cross-linking and inhibiting DNA replication. Because the action of cisplatin in inhibiting DNA replication is nonspecific, cisplatin has strong kidney damage, neurotoxicity, bone marrow toxicity, anemia and other systemic toxic and side effects, and severe toxic and side effects and easy generation of tumor drug resistance limit the clinical wide application of cisplatin. Therefore, the cisplatin is subjected to pharmaceutical chemistry and pharmaceutical preparation transformation, 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, difficulty in reaching deep parts of solid tumor tissues, poor drug release controllability and the like.
Solid tumor tissues contain abundant tumor stroma, and pancreatic cancer tumor stroma accounts for up to 70-80% of the tumor volume. The tumor stroma, being a non-cellular component, may form a stromal barrier that prevents immune cells or therapeutic drugs (particularly nano-delivery systems) from reaching deep solid tumor tissue and tumor cells, thereby reducing the therapeutic effect. Hyaluronic acid is the major non-cellular component of the extracellular matrix of tumor tissue. The research has proved that the hyaluronidase can degrade the hyaluronic acid in the tumor stroma, thereby reducing the compactness of the tumor stroma, promoting the drug to be transported to the deep part of the tumor tissue and the tumor cells, and enhancing the curative effect of the drug. However, most normal tissues of human bodies contain hyaluronic acid, and serious toxic and side effects of the whole body can be caused by the degradation of non-specific non-tumor tissue parts. Therefore, the hyaluronidase forms a stimulation-responsive nano drug delivery system by adopting a nano assembly mode, so that the hyaluronidase is released in a tumor tissue microenvironment (pH6.5-6.8) and is less released in a normal tissue (pH7.0-7.4), the systemic toxic and side effects of the hyaluronidase can be effectively reduced, and the nano drug delivery system has important significance for clinical application and effect.
Polyethyleneimine (PEI) is a cationic polymer, and the structure of the PEI contains a large number of amino groups, so that chemical modification is easy, high loading of a medicament can be realized, and the treatment effect of the PEI is improved. The subject group uses polyethyleneimine as a framework, and realizes high load of cisplatin by utilizing abundant amino groups, so that the cisplatin polymer conjugate containing redox-responsive disulfide bonds is obtained. The cisplatin polymer conjugate can keep stable in extracellular environment by using the huge difference of the concentration of Glutathione (GSH) inside and outside cells, and can release cisplatin by breaking in the high GSH environment in tumor cells, thereby realizing the rapid release of drugs in the tumor cells and generating the stimulation response characteristic.
Therefore, the invention adopts Polyethyleneimine (PEI) rich in amino-group-modifiable groups as a framework, and selects an oxidation-reduction responsive disulfide bond to highly load cisplatin on the PEI to obtain the cisplatin polymer conjugate. On the basis, a dual-responsiveness drug delivery system is designed, hyaluronidase, cisplatin polymer conjugate and calcium phosphate are selected to be assembled to form nanoparticles, and the cisplatin drug delivery system which is wrapped by shell calcium phosphate and has tumor tissue microenvironment and intracellular redox responsiveness is constructed. Due to the action of calcium phosphate, the drug delivery system is not easy to dissociate in normal tissues (pH 7.4, the calcium phosphate is not dissolved under neutral and alkaline conditions), after reaching tumor tissue parts, the calcium phosphate is firstly dissolved in a specific slightly acidic environment (pH6.5-6.8) of the tumor parts, hyaluronidase in the cisplatin drug delivery system is removed to play a role in degrading tumor matrixes, so that the density of the tumor matrixes is reduced, and after matrix barriers are damaged, PEI-cisplatin polymer conjugates can penetrate through the tumor tissues to easily reach deep parts of tumors and tumor cells, disulfide bonds of the cisplatin polymer conjugates are subjected to redox responsiveness triggering breakage and cisplatin release in an intracellular high GSH environment after entering the tumor cells, the effective transportation of the cisplatin drug delivery system and the response release of drugs in the tumors are ensured, and the antitumor therapeutic effect is better played.
Disclosure of Invention
The invention aims to provide a preparation method and application of a stimulus-responsive nano drug delivery system, which can degrade tumor matrixes to promote drugs to enter deep parts of tumor tissues and can highly load cisplatin drugs. The drug delivery system utilizes rich amino groups of polyethyleneimine and cystamine containing disulfide bonds to construct a cisplatin polymer conjugate, and can remarkably increase the loading capacity and redox-responsive drug release of cisplatin. On the basis, the hyaluronidase, the cisplatin polymer conjugate and the calcium phosphate are selected for assembly to construct a cisplatin nano drug delivery system which can degrade and penetrate through a tumor matrix and has tumor tissue microenvironment and redox responsiveness, and a new idea is provided for the research of antitumor drugs.
The technical scheme of the invention is that the preparation method of the stimulus-responsive nano drug delivery system specifically comprises the following steps:
(1) cisplatin is stirred at 37 ℃ in a dark place until the cisplatin is completely dissolved in III-grade ultrapure water, then the cisplatin is cooled to room temperature, silver nitrate with a corresponding molar ratio is added, the cisplatin is stirred at room temperature in a dark place for 48 hours, the cisplatin is centrifuged twice (5000rpm, 1 hour/time), and a supernatant is filtered by a 0.1 mu m water system filter to obtain a hydrated cisplatin solution (3mg/mL) for later use. 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 (7.73mg/mL) with a specific ratio are stirred and reacted at a certain temperature, and then the reaction is continuously stirred and reacted with polyethyleneimine with a certain molecular weight according to a corresponding grafting ratio. And (3) after the reaction is finished, dialyzing and purifying by using a dialysis bag with the molecular weight cutoff of 7000 (dialyzing in grade III ultrapure water for 4 times, and changing water every 2 hours), and freeze-drying to obtain the cisplatin polymer conjugate.
(3) Uniformly mixing a certain amount of hyaluronidase and cis-platinum polymer conjugate PEI-SS-Pt in an aqueous solution of calcium nitrate (50mmol/L) and disodium hydrogen phosphate (30mmol/L), and stirring and incubating for a certain time at a specific pH and temperature; and centrifuging the solution at a high speed, collecting the precipitate, and redissolving the precipitate in pure water to obtain a tumor tissue microenvironment and an oxidation-reduction 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 pH value of the reaction solution is 4.0-11.0, the total mass ratio of calcium phosphate generated by calcium nitrate and disodium hydrogen phosphate is 2-80%, the feeding mass ratio of hyaluronidase and cisplatin polymer conjugate is 1: 20-20: 1, the temperature is 5-60 ℃, and the stirring incubation time is 0.1-120 h.
The invention has the following outstanding advantages:
1. 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 cis-platinum complex and rich amino groups of polyethyleneimine are covalently combined to obtain a high-load cis-platinum polymer conjugate, wherein the load of cis-platinum can reach 32.66%. And assembling the cisplatin polymer conjugate with a hyaluronidase protein drug and calcium phosphate to obtain a cisplatin nano drug delivery system which is loaded by hyaluronidase and cisplatin and has a tumor tissue microenvironment and redox responsiveness. The cisplatin antineoplastic medicine with high efficiency and low toxicity and tumor matrix degradation effect is obtained through activity evaluation.
2. The activity evaluation experiment result of the tumor tissue microenvironment and the redox-responsive cisplatin nano drug delivery system shows that compared with the traditional chemotherapy drug cisplatin clinically used, the drug delivery system can degrade the matrix to promote the drug to enter the deep part of the tumor tissue, has the functions of tumor tissue microenvironment and redox-responsive drug release, can greatly reduce the blocking effect of the tumor matrix on the drug, reduce the systemic toxic and side effects of hyaluronidase and cisplatin while playing the effective anti-tumor effect, and has good clinical application prospect.
Drawings
FIG. 1 is a graph showing the particle size (A), PDI (B) and zeta potential (C) profiles of BPEI-SS-Pt/HAase @ CaP nano drug delivery system.
FIG. 2 is the stability of the BPEI-SS-Pt/HAase @ CaP nano drug delivery system. (A) Particle size of nanoparticles, (B) PDI.
FIG. 3 is an in vitro drug release experiment of the BPEI-SS-Pt/HAase @ CaP nano drug delivery system. (A) The shapes of the nanoparticles under different pH conditions, (B) the drug release curves of the nanoparticles under different pH conditions, and (C) the drug release curves of the nanoparticles under different GSH conditions.
FIG. 4 is a tumor stroma penetration experiment of the BPEI-SS-Pt/HAase @ CaP nano drug delivery system. (A) BPEI-SS-Pt group hyaluronic acid staining, (B) BPEI-SS-Pt/HAase @ Cap group hyaluronic acid staining, (C) BPEI-SS-Pt group green fluorescent labeling nanoparticle distribution, and (D) BPEI-SS-Pt/HAase @ Cap group green fluorescent labeling nanoparticle distribution.
FIG. 5 is the curve of the change of tumor volume in vivo anti-tumor activity experiment (A) of BPEI-SS-Pt/HAase @ CaP nano drug delivery system. (B) Mouse body weight change curve. n is 5, and n is 5,
Figure BDA0003494505650000061
。*P<0.05,**P<0.01, Cisplatin group compared with Control group,ξξP<0.01, BPEI-SS-Pt group is compared with Control group,#P<0.05,##P<0.01, comparing the BPEI-SS-Pt/HAase @ CaP group with the Control group,&P<0.05，&&P<0.01, BPEI-SS-Pt/HAase @ Cap group compared with Cisplatin group,ψP<0.05，ψψP<0.01 for the BPEI-SS-Pt/HAase @ CaP group compared to the BPEI-SS-Pt group.
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 polymer conjugate BPEI-SS-Pt
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. 202.4mg (1.98mmol) of succinic anhydride was weighed out and dissolved in 36.97mL of anhydrous 1, 4-dioxane at room temperature, and the solution was added to a solution of cystamine dihydrochloride in methanol while removing the ice bath, and the reaction was stirred at room temperature for 1.5 hours. 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.
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 load capacity of the cis-platinum in the BPEI-SS-Pt is 32.66-33.4% as determined by inductively coupled plasma mass spectrometry (ICP-MS).
Example 2
Preparation and characterization of tumor tissue microenvironment and redox-responsive cisplatin drug delivery system BPEI-SS-Pt/HAase @ CaP
16mg hyaluronidase was dissolved in 3.2mL, 4.8mL and 5.34mL of water, respectively. mu.L of calcium nitrate (50mmol/L) was added, and the pH of the solution was adjusted to 8.0 with aqueous NaOH (2 mol/L). According to the different weight ratios (1:5, 1:10 and 1:15) of the cisplatin polymer conjugate BPEI-SS-Pt and hyaluronidase (HAase), 3.2mL, 1.6mL and 1.06mL of BPEI-SS-Pt aqueous solutions (1mg/mL) were added and stirred at room temperature for 1 hour. mu.L of magnesium chloride (1mol/L) was added to the above mixture and stirred for 10 minutes. Finally, 330 mu L disodium hydrogen phosphate (30mmol/L) is added and stirred and incubated for 3h at room temperature, and after precipitation is obtained by high-speed centrifugation (25000rpm, 30min), the precipitate is redissolved by 6.4mL water to obtain BPEI-SS-Pt and hyaluronidase (HAase) nanoparticles with the series weight ratios of BPEI-SS-Pt/HAase @ CaP of 1:5, 1:10 and 1:15, and the nanoparticles are stored in a refrigerator at 4 ℃.
The particle size, PDI and zeta potential of BPEI-SS-Pt/HAase @ Cap nanoparticles prepared from the hyaluronidase and cisplatin polymer conjugate in each mass ratio are respectively measured by a laser particle size analyzer, so that the influence of different mass ratios on the BPEI-SS-Pt/HAase @ Cap nanoparticles is researched. And the morphology of the prepared 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 nanoparticles prepared from hyaluronidase and BPEI-SS-Pt with different mass ratios are shown in FIG. 1. As a result, the particle diameters were 1345. + -.17 nm, 143. + -.14 nm and 232. + -.21 nm (FIG. 1A) at weight ratios of 1:5, 1:10 and 1:15, respectively, and the corresponding polydispersity indices (PDI) were 0.676. + -. 0.221, 0.287. + -. 0.024 and 0.401. + -. 0.013 (FIG. 1B), and the charges were-13. + -. 1.2mV, -17. + -. 1.7mV and-19. + -. 2.1mV, respectively (FIG. 1C). The particle size, PDI, the clinically effective amounts of cisplatin and HAase were taken together, preferably in a weight ratio of 1:10 for further study.
Example 3
Stability study of BPEI-SS-Pt/HAase @ CaP nano drug delivery system
The optimized BPEI-SS-Pt/HAase @ CaP nanoparticles (1:10) are stored at 4 ℃, the particle size of the nanoparticles is measured by a laser particle size analyzer, and the change condition of the particle size of the nanoparticles after being placed for different time 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 4
Research on tumor tissue microenvironment response and redox response characteristics of BPEI-SS-Pt/HAase @ CaP nanoparticles
The tumor tissue microenvironment and the redox response characteristics of the constructed nano drug delivery system are verified by observing the pH sensitivity and the redox stimulus responsiveness of the drug delivery system in vitro simulation tumor tissue microenvironment and the tumor cell internal and external reducibility levels (10mM GSH and 10 mu M GSH).
And (3) carrying out morphology investigation on BPEI-SS-Pt/HAase @ CaP nanoparticles under different pH conditions: and observing the change condition of the morphological characteristics of the nanoparticles after 4 hours under the conditions of pH 7.4 and pH6.5 by using a scanning electron microscope.
The drug release characteristics of the BPEI-SS-Pt/HAase @ CaP drug delivery system under different mediums are researched: (1) in order to simulate the release of the tumor tissue microenvironment, BPEI-SS-Pt/HAase @ CaP nanoparticles (1mL) were placed in 9mL of different media (Tris-HCl pH 7.4 and Tris-HCl pH 6.5). Setting different incubation time groups, centrifuging at 25000rpm for 30 minutes after the preset time is reached to remove nanoparticles, detecting the content of hyaluronidase in the supernatant by using a BCA kit, calculating the cumulative drug release amount of the hyaluronidase, and inspecting the pH response characteristic of the hyaluronidase; (2) when the release of platinum in cells is simulated, the cisplatin polymer conjugate aqueous solution is filled into a dialysis bag (molecular weight cut-off is 3500Da), and the external liquid is 10 mu M GSH physiological saline solution and 10mM GSH physiological saline solution respectively, and the cisplatin polymer conjugate aqueous solution is incubated in a water bath at 37 ℃. And taking 2mL of external liquid out of the dialysis bag at the set drug release time points of 0.5h, 1h, 2h, 4h, 6h, 12h and 24h, then adding 2mL of fresh dialysis medium, measuring the content of Pt by using ICP-MS, calculating the cumulative drug release amount of the drug, and inspecting the redox response characteristics of the drug.
The experimental results are as follows: the scanning electron microscope detection results of the BPEI-SS-Pt/HAase @ CaP nanoparticles under different pH conditions show that the BPEI-SS-Pt/HAase @ CaP nanoparticles with the pH of 7.4 are complete and spherical in structure, and the structure of the BPEI-SS-Pt/HAase @ CaP nanoparticles is destroyed at the pH of 6.5. The nanoparticles released more hyaluronidase in the pH6.5 solution was also found by detecting the released hyaluronidase. Namely, the BPEI-SS-Pt/HAase @ CaP nanoparticles can be disintegrated to release hyaluronidase under the slightly acidic condition of tumor parts, and play the roles of degrading matrix and enhancing the effect of medicament penetrating through a matrix barrier to reach deep tumors.
In vitro drug release results under different medium conditions show that under the conditions of blood circulation and normal extracellular environment (10 mu M GSH), the disulfide bond of the BPEI-SS-Pt/HAase @ CaP nanoparticle has no obvious response to the low-concentration GSH, and the drug release speed and the drug release amount are lower (figure 3). Under the condition of simulating tumor cells (10mM GSH), the drug release amount is rapidly increased, and the disulfide bond rupture response release of Pt is realized. The results show that the BPEI-SS-Pt/HAase @ CaP nano delivery system has better tumor tissue microenvironment and redox response characteristics, can release hyaluronidase and cisplatin polymer conjugates in the tumor tissue microenvironment, degrade hyaluronic acid in a tumor matrix, promote the cisplatin polymer conjugates to enter deep parts of tumor tissues, and release drugs in response to high GSH concentration in cells after nanoparticles are taken into the cells so as to achieve efficient antitumor effect.
Example 5
In-vivo tumor matrix degradation and penetration behavior research of tumor tissue microenvironment and redox-responsive drug delivery system BPEI-SS-Pt/HAase @ CaP
The effective delivery of the antitumor drug into the deep tumor tissue is the key to improving and improving the drug action. The experiment adopts 5-FAM fluorescence labeled nanometerDrug delivery system BPEI-SS-Pt/HAase @ CaP. Selecting 6-8 weeks old C57 mice as experimental models, 100 mu L of pancreatic cancer cells Panc02 (density of 2 x 10)7one/mL) and 100 mu L of matrigel, and inoculating the mixture to the subcutaneous tissue of the right back of the mouse until the subcutaneous tumor volume of the mouse is 80mm3When the tumor is administrated, 5-FAM fluorescence labeled cis-platinum polymer conjugate BPEI-SS-Pt is adopted in a control group, and the Pt content is kept the same during administration. And taking out a tissue section after administration for 1d, staining hyaluronic acid by an immunohistochemistry method, and observing the distribution condition in tumor tissues of the fluorescence labeling nanoparticles under a fluorescence microscope.
The experimental results are as follows: the in vivo tumor stroma degradation and drug penetration research results of the BPEI-SS-Pt/HAase @ CaP nanoparticles show (figure 4), the control group BPEI-SS-Pt has no degradation effect on the tumor stroma (figure 4A), and the cisplatin polymer conjugate is mainly distributed at the edge of the tumor tissue and has less drug penetration in the deep tissue (figure 4B) in the tumor tissue; however, the BPEI-SS-Pt/HAase @ CaP group showed significant degradation of tumor stroma, significant vacuoles were formed (FIG. 4A), and the amount of drug infiltrated into deep tissue portions was significantly increased compared to the control group BPEI-SS-Pt (FIG. 4B).
Example 6
Evaluation of in vivo antitumor Effect of BPEI-SS-Pt/HAase @ CaP drug delivery System
Selecting a C57 mouse with the age of 6-8 weeks as an experimental model, and carrying out the experiment on 100 mu L of mouse pancreatic cancer cells Panc02 (the density is 2 multiplied by 10)7one/mL) and 100 mu L of matrigel are mixed and inoculated under the skin of the right back of the mouse until the subcutaneous tumor volume of the mouse is 80mm3The intratumoral administration is carried out, a normal saline group, a cis-platinum polymer conjugate BPEI-SS-Pt group and a BPEI-SS-Pt/HAase @ CaP group are arranged, the Pt content is kept the same during administration, and the administration is carried out 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.
The experimental results are as follows: the in vivo anti-tumor activity experiment of the BPEI-SS-Pt/HAase @ CaP nano drug delivery system shows that (figure 5), at 20 days after the drug administration, the tumor volumes of mice in the cis-platinum group (Cisplatin group) and the cis-platinum polymer conjugate BPEI-SS-Pt group are inhibited, and the tumor volume of the mice in the BPEI-SS-Pt/HAase @ CaP group is the smallest and the anti-tumor effect is the best (as figure 5A) compared with the mice in the normal saline Control group (Control group) which is not treated with the drug. The results in FIG. 5B show that the body weight of the mice in the cisplatin group was significantly reduced after administration, the body weight of the mice in the normal saline, BPEI-SS-Pt group and BPEI-SS-Pt/HAase @ CaP group was less affected, and the toxic and side effects were significantly reduced.
The constructed cisplatin nano-delivery system BPEI-SS-Pt/HAase @ CaP can realize the oxidation reduction response drug release in a tumor site microenvironment and cells, hyaluronidase released through the tumor site microenvironment stimulation response can effectively degrade a matrix rich in solid tumors, so that the tumor matrix density is reduced, the matrix barrier is damaged, the drugs are promoted to penetrate through tumor tissues and easily reach deep tumor cells and tumor cells, the oxidation reduction response triggering breakage and the cisplatin release are realized in the intracellular high GSH environment after entering the tumor cells, the response release of the cisplatin delivery system in the tumors is realized, the anti-tumor effect of the cisplatin nano-delivery system is better exerted, the cisplatin nano-delivery system has important guiding significance on the research of new dosage forms of chemotherapeutic drugs cisplatin, and the cisplatin nano-delivery system has good clinical treatment application prospects.

Claims (7)

1. A preparation method of a stimulus-responsive nano drug delivery system is characterized in that polyethyleneimine which is rich in amino-group-modifiable groups is used as a framework, and cystamine containing redox-responsive disulfide bonds is selected to react with succinic anhydride and then is complexed with cisplatin to obtain a cisplatin complex; the obtained cisplatin complex is covalently combined with polyethyleneimine to obtain a high-load cisplatin polymer conjugate, and then the high-load cisplatin polymer conjugate is assembled with hyaluronidase protein drugs and calcium phosphate to obtain a cisplatin nano drug delivery system which is loaded by hyaluronidase and cisplatin together, can be degraded and penetrates through a tumor matrix and has a tumor tissue microenvironment and redox responsiveness.
2. The method of preparing a stimuli-responsive nano-drug delivery system according to claim 1, comprising in particular the steps of:
stirring the cisplatin 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 system filter to obtain a 3mg/mL hydrated cisplatin solution for later use; cystamine dihydrochloride is dissolved in methanol at room temperature, stirred with a certain amount of triethylamine for 30min at the ice bath condition of 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, and freeze-drying to obtain a cisplatin complex;
stirring carbonyl diimidazole with a specific ratio and 7.73mg/mL cisplatin complex aqueous solution at a certain temperature for reaction, then continuously stirring the mixture and polyethyleneimine with a certain molecular weight according to a corresponding grafting ratio for reaction, dialyzing and purifying the reaction product by using a dialysis bag with the intercepted molecular weight of 7000 after the reaction is finished, and freeze-drying the product to obtain the cisplatin polymer conjugate PEI-SS-Pt;
step (3) uniformly mixing a certain amount of hyaluronidase and cis-platinum polymer conjugate PEI-SS-Pt in 50mmol/L calcium nitrate and 30mmol/L disodium hydrogen phosphate aqueous solution, and stirring and incubating for a certain time at a specific pH and temperature; and (3) centrifuging the solution at a high speed, collecting the precipitate, and redissolving the precipitate in water to obtain a tumor tissue microenvironment and an oxidation-reduction responsive cisplatin nano drug delivery system.
3. The preparation method of the stimuli-responsive nano drug delivery system according to claim 2, wherein 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.
4. The preparation method of the stimuli-responsive nano drug delivery system according to claim 2, wherein 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 hours.
5. The method of claim 2, wherein the polyethyleneimine is Linear Polyethyleneimine (LPEI) or Branched Polyethyleneimine (BPEI) with a molecular weight range of 600-50K Da. 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.
6. The method for preparing the stimuli-responsive nano drug delivery system according to claim 2, wherein in the step (3), the pH of the reaction solution is 4.0 to 11.0, the total mass ratio of calcium phosphate generated by calcium nitrate and disodium hydrogen phosphate is 2 to 80%, the mass ratio of hyaluronidase and cisplatin polymer conjugate is 1:20 to 20:1, the temperature is 5 to 60 ℃, and the stirring incubation time is 0.1 to 120 hours.
7. Use of a stimuli-responsive nano-delivery system prepared according to claims 1-6 in the treatment of anti-tumor drugs.
CN202210109147.9A 2022-01-28 2022-01-28 Preparation method and application of stimuli-responsive nano drug delivery system Pending CN114432269A (en)

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