CN116003683B

CN116003683B - Nano gel blocking material, tumor multistep therapy series medicine based on RES-blockade strategy and application thereof

Info

Publication number: CN116003683B
Application number: CN202211633351.7A
Authority: CN
Inventors: 李子福; 杨祥良; 李峥
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2022-12-19
Filing date: 2022-12-19
Publication date: 2024-06-28
Anticipated expiration: 2042-12-19
Also published as: CN116003683A

Abstract

The invention belongs to the technical field of multidisciplinary intersection of chemistry, pharmacy, medicine and the like, and particularly relates to a nanogel blocking material, a tumor multistep therapy series medicine based on a RES-blockade strategy and application thereof. Aiming at synthesizing series of nanogels with different hardness, taking hard nanogels as RES-blockade blocking materials, using soft nanogels for delivering small-molecule chemotherapeutics, temporarily blocking reticuloendothelial systems by the hard nanogels, and solving the technical problems of low drug concentration enriched in tumor parts, poor tumor treatment effect and body injury caused by overlarge dosage of the existing RES-blockade blocking materials due to nonspecific uptake of drug-loaded nanoparticles by the reticuloendothelial systems.

Description

Nano gel blocking material, tumor multistep therapy series medicine based on RES-blockade strategy and application thereof

Technical Field

The invention belongs to the technical field of multidisciplinary intersection of chemistry, pharmacy, medicine and the like, and particularly relates to a nanogel blocking material, a tumor multistep therapy series medicine based on a RES-blockade strategy and application thereof.

Background

The use of nanomedicines to enhance the efficiency of delivery of small molecule chemotherapeutic drugs by enhancing permeation and retention effects has been a history of decades. Doxil, abraxane and other marketed nanomedicines have been widely used in clinical treatment of tumors. Although the pharmacokinetics, antitumor effect, and biosafety of nanomedicines have been significantly improved with the continued progress of research, only 0.7% of nanomedicines can be delivered to solid tumor sites. After intravenous injection, the surface of the nano-drug is rapidly coated by plasma protein and then recognized and cleared by macrophages in reticuloendothelial system (RES). Thus, there is a need for an adjuvant therapeutic strategy that can efficiently reduce plasma clearance rates and increase tumor enrichment.

Many methods have been applied to inhibit the clearance function of the reticuloendothelial system, including the use of toxic small molecule drugs to clear macrophages, CD47 to bind to the macrophage surface to block macrophage interactions with nanoparticles, or the use of large amounts of nanoparticles to saturate macrophages. However, toxic small molecule drugs such as methyl palmitate and the like can cause serious toxic side effects; CD47 may lead to unpredictable immunosuppression; macrophages can be temporarily and reversibly saturated with large amounts of nanoparticles, prolonging the nanomedicine circulation time, the RES-blockade strategy. Studies have shown that injection of large amounts of aggregated human serum albumin can temporarily block the reticuloendothelial system, prolonging the circulation time of nanoparticles in human blood. However, a large amount of nanoparticles may put additional burden on the reticuloendothelial system and liver, and thus it is important to improve the RES-blockade efficiency.

Many of the factors of RES-blockade have been optimized to improve their efficiency. For example, 1.5 hours is a suitable time interval for occlusion of the reticuloendothelial system and systemic administration; dosages are critical factors in increasing RES-blockade efficiency, and studies have found that 1 x10 ¹² nanoparticles significantly reduce liver clearance rates in mice. In addition, positively charged or larger size nanoparticles can block the reticuloendothelial system better, but the essential reason for this is still to increase macrophage uptake. The mechanical properties of nano-drugs have been shown to significantly affect drug delivery, tumor enrichment and deep penetration, however, the effect on macrophages and reticuloendothelial system is not clear, and therefore the RES-blockade strategy based on the mechanical properties of nanoparticles becomes a potential adjuvant therapy strategy.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a nanogel blocking material, a tumor multistep therapy series drug based on a RES-blockade strategy and application thereof, wherein series nanogels with different hardness are synthesized firstly, hard nanogels are used as RES-blockade blocking materials, soft nanogels are used for delivering small molecular chemotherapeutic drugs, and the non-specific uptake of drug-loaded nanoparticles by the reticuloendothelial system is solved by temporarily blocking the reticuloendothelial system by the hard nanogels, so that the technical problems of low concentration of the drug enriched in tumor parts, poor tumor treatment effect and body injury caused by overlarge dose of the existing RES-blockade blocking materials are solved.

In order to achieve the above object, the present invention provides a cellular endothelial system macrophage nanogel blocking material, which is a nanogel blocking material obtained by polymerizing monomers in an aqueous phase through an initiator in the presence of a crosslinking agent and a surfactant; the Young modulus of the nanogel blocking material can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer, and the average particle size of the nanogel blocking material can be regulated and controlled by using the surfactant; the Young's modulus of the nanogel blocking material is 300-600 kPa, preferably 400-500 kPa.

Preferably, the monomer includes one or more of a temperature responsive monomer, a pH responsive monomer, and a reduction responsive monomer; the temperature response type monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; the cross-linking agent is one or more of N, N-methylene bisacrylamide, divinylbenzene and N, N' -bis (acryloyl) cystamine.

Preferably, the crosslinking agent is a reduction-responsive crosslinking agent, further preferably N, N' -bis (acryloyl) cystamine; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (15-25) to 550.

Preferably, the monomers comprise a temperature-responsive monomer and a pH-responsive monomer, and the feeding molar ratio of the pH-responsive monomer to the temperature-responsive monomer is (3-8): 100; the feeding mole ratio of the cross-linking agent to the monomer is (10-15) 100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15): 550.

According to another aspect of the invention, there is provided a series of drugs for multi-step therapy of tumors based on the RES-blockade strategy, comprising said nanogel blocking material, and further comprising a nanogel anti-tumor drug; the nanogel anti-tumor drug is obtained by loading water-soluble micromolecular anti-tumor chemotherapeutic drug on nanogel through electrostatic adsorption; wherein the young's modulus of the nanogel blocking material is greater than the young's modulus of the nanogel anti-neoplastic drug; when in use, the nanogel blocking material is used for blocking reticuloendothelial system and reducing liver clearance rate, and the nanogel anti-tumor drug is used for delivering chemotherapeutic drugs to tumor sites to play an anti-tumor role.

Preferably, the Young's modulus of the nanogel anti-tumor drug is between 20 and 150kPa, and more preferably between 50 and 100kPa; the average particle diameter of the nano gel anti-tumor drug is 150-300 nm, and more preferably 200-240 nm; the average particle diameter of the nanogel blocking material is 150 to 300nm, and more preferably 200 to 240nm.

Preferably, the preparation method of the nanogel anti-tumor drug comprises the following steps:

S1: under the condition that a cross-linking agent and a surfactant exist, initiating the monomer to perform polymerization reaction in a water phase through an initiator to obtain nanogel, wherein the Young modulus of the nanogel can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer; the average particle size of the nanogel can be regulated and controlled by the use amount of the surfactant; the monomer includes one or more of a temperature responsive monomer, a pH responsive monomer, and a reduction responsive monomer;

S2: mixing and stirring the water solution of the nanogel in the step S1 and the water solution of the water-soluble small-molecule anti-tumor chemotherapeutic drug, so that the nanogel loads the water-soluble small-molecule anti-tumor chemotherapeutic drug through electrostatic adsorption to obtain the nanogel anti-tumor drug.

Preferably, the temperature responsive monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; the cross-linking agent is one or more of N, N-methylene bisacrylamide, divinylbenzene and N, N' -bis (acryloyl) cystamine.

Further preferably, the crosslinking agent in step S1 is a reduction-responsive crosslinking agent, and still further preferably N, N' -bis (acryloyl) cystamine; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (30-40) to 550.

Preferably, the monomers in the step S1 comprise pH-responsive monomers and temperature-responsive monomers, and the feeding molar ratio of the pH-responsive monomers to the temperature-responsive monomers is (3-8): 100; the feeding mole ratio of the cross-linking agent to the temperature responsive monomer is (1-5): 100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15): 550.

Preferably, the reaction temperature of the polymerization reaction in the step S1 is 70-85 ℃ and the reaction time is 4-8 h.

According to another aspect of the invention, there is provided the use of said series of medicaments for the manufacture of an anti-tumour medicament.

Preferably, the dosage of the nanogel blocking material in the series of drugs is 100-300 mg/kg; the dosage of the water-soluble small molecule anti-tumor chemotherapeutic in the nano gel anti-tumor drug is 4-6 mg/kg.

Further preferably, the series of medicaments is prepared for:

(1) Administering a dose of 100-300 mg/kg of the nanogel blocking material to a tumor patient;

(2) Waiting for a time interval of 0.5-4 h, and applying the nanogel anti-tumor drug in the series of drugs to the patient, wherein the dosage of the water-soluble small molecule anti-tumor chemotherapeutic drug in the nanogel anti-tumor drug is 4-6 mg/kg.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

(1) The invention provides a reticuloendothelial system macrophage nanogel blocking material, which is a nanogel blocking material obtained by initiating a polymerization reaction of a monomer in a water phase through an initiator in the presence of a cross-linking agent and a surfactant. Experiments have unexpectedly found that when the Young's modulus of the nanogel blocking material is between 400 and 500kPa, the nanogel blocking material has a good technical effect of blocking reticuloendothelial system macrophages.

(2) The invention provides a series of tumor multi-step therapy medicines based on a RES-blockade strategy, which comprise the nanogel blocking material and the nanogel anti-tumor medicines. After the small molecule chemotherapeutic medicine such as doxorubicin hydrochloride is carried by the nanogel, the stability is obviously improved, and the pharmacokinetics behavior is obviously improved. Experiments show that the nanogel anti-tumor drug can be enriched in tumor parts by enhancing permeation and retention effects, wherein the soft (Young modulus is lower) nanogel anti-tumor drug can overcome a physical barrier formed by extracellular matrixes, is easier to penetrate blood vessels and permeate into the deep part of tumors, and has higher cell uptake efficiency, so that higher tumor enrichment amount and more excellent anti-tumor effect are realized.

(3) The invention provides a preparation method of a nanogel anti-tumor medicament with different hardness for carrying anti-tumor chemotherapeutic medicaments. The antitumor chemotherapeutic medicine, such as doxorubicin hydrochloride, can be carried on the nanogel through electrostatic adsorption by stirring in water solution, then free doxorubicin hydrochloride is removed by ultrafiltration, and the free doxorubicin hydrochloride is concentrated to proper concentration for storage and application, thus obtaining series of nano medicines with different hardness.

(4) The occlusion reticuloendothelial system needs a large amount of nanogels, and in order to avoid the damage to liver function caused by long-term occlusion of the liver, the nanogels need to have the capacity of being degraded by the liver while realizing the occlusion function. Meanwhile, the dense network structure of the hard nanogel can delay the degradation rate of glutathione to the nanogel, avoid the reduction of blocking efficiency caused by rapid degradation, and have good biocompatibility while ensuring the blocking efficiency

(5) The invention provides a series of tumor multistep therapy medicines based on a RES-blockade strategy, which utilizes a treatment strategy of reinforcing a reticuloendothelial system by blocking a hard nanogel blocking material and enriching tumors and resisting tumor curative effects of a soft nanogel anti-tumor medicine. The hard nanogel is injected preferentially, so that the uptake capacity of macrophages in a reticuloendothelial system can be reduced, and meanwhile, the hard nanogel is not easy to enrich in tumor parts and block the uptake of tumor cells due to weak deformability; then soft nano-drug is injected, because the reticuloendothelial system is blocked, the soft nano-drug can be more enriched at the tumor part, and can permeate to deeper tumor part due to the excellent deformability, so as to realize better anti-tumor effect. By fully utilizing the mechanical properties of the nanogel, the optimal anti-tumor treatment strategy is realized. Meanwhile, the hard nanogel can reduce the blocking dosage of RES-blockade and reduce the burden of the liver and the reticuloendothelial system by reducing the uptake capacity of macrophages to block the reticuloendothelial system. In addition, the RES-blockade strategy based on the hard nanogel can promote the anti-tumor effect of the medicines Doxil and Abraxane on the market, so that the preparation method has great potential for clinical transformation and application.

Drawings

FIG. 1 shows the particle size distribution and surface charge of the nanogels of different hardness series prepared in example 1.

FIG. 2 is a transmission electron microscope image of the nanogels of different hardness series prepared in example 1.

FIG. 3 is an atomic force microscope image and Young's modulus of the nanogels of different hardness series prepared in example 1.

FIG. 4 is a graph showing the triple responsiveness of the different hardness series nanogels prepared in example 1.

FIG. 5 shows the comparative properties and stability of doxorubicin hydrochloride loaded on nanogels of different hardness series prepared in example 1.

FIG. 6 is a liver enrichment of indocyanine green labeled nano-drugs of different hardness series prepared in example 4.

FIG. 7 shows the uptake of rhodamine B labeled hard nanogels prepared in example 2 in macrophages over time.

FIG. 8 shows the occlusion efficiency of the reticuloendothelial system by the different hardness series nanogels prepared in example 1.

FIG. 9 is the effect of the different hardness series nanogels of the blocked reticuloendothelial system prepared in example 1 on plasma half-life.

FIG. 10 shows the blocking efficiency of the different hardness series nanogels prepared in example 1 on macrophages.

Fig. 11 shows the blocking efficiency of the hard nanogel prepared in example 1 on reticuloendothelial system at various doses.

FIG. 12 shows the efficiency of the hard nanogel prepared in example 1 in blocking macrophages at various doses.

FIG. 13 shows the anti-tumor effect of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the different hardness series nanomedicines of example 3.

FIG. 14 shows the tumor rejection rate of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the different hardness series nanomedicines of example 3.

FIG. 15 shows tumor necrosis and proliferation after treatment with various hardness series nanomedicines of example 3 in combination with the RES-blockade strategy based on the hard nanogel of example 1.

FIG. 16 is a section of organ tissue after treatment based on the RES-blockade strategy of the hard nanogel of example 1 in combination with the different hardness series nanomedicine of example 3.

FIG. 17 shows blood biochemical and blood general index after treatment with various hardness series nanomedicine based on RES-blockade strategy for the hard nanogel of example 1 in combination with example 3.

FIG. 18 is the antitumor effect of RES-blockade strategy based on the hard nanogel of example 1 in combination with the marketed nanomedicine.

FIG. 19 is the tumor rejection rate of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the marketed nanomedicine.

FIG. 20 shows tumor necrosis and proliferation after combination of the RES-blockade strategy based on the hard nanogel of example 1 with the marketed nanomedicine treatment.

FIG. 21 is a section of organ tissue after treatment with a combination of a marketed nanomedicine based on the RES-blockade strategy for the rigid nanogel of example 1.

FIG. 22 is a graph showing blood biochemical and blood general index after combination of the RES-blockade strategy based on the hard nanogel of example 1 with the marketed nanomedicine treatment.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The invention provides a reticuloendothelial system macrophage nanogel blocking material, which is a nanogel blocking material obtained by initiating a monomer to generate a polymerization reaction in a water phase through an initiator under the condition that the monomer exists in a cross-linking agent and a surfactant. Experiments show that the Young modulus of the nanogel blocking material can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer, and the average particle size of the nanogel blocking material can be regulated and controlled by using the surfactant; moreover, it has been unexpectedly found that the nanogel blocking material exhibits a good technical effect of blocking macrophages of the reticuloendothelial system when the Young's modulus of the nanogel blocking material is 300 to 600kPa, more preferably 400 to 500 kPa.

The synthesis of the nanogel according to the invention can employ monomers commonly used for preparing biocompatible nanogels, including but not limited to one or more of temperature responsive monomers, pH responsive monomers, and reduction responsive monomers; in some embodiments, the temperature responsive monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide, and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; the cross-linking agent is one or more of N, N-methylene bisacrylamide, divinylbenzene and N, N' -bis (acryloyl) cystamine. The crosslinking agent is more preferably a reduction-responsive crosslinking agent, which in a preferred embodiment is N, N' -bis (acryloyl) cystamine.

In some embodiments, the initiator is one or more of potassium persulfate, sodium persulfate, and t-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (15-25) to 550.

In a preferred embodiment, the monomers comprise a temperature-responsive monomer and a pH-responsive monomer, wherein the temperature-responsive monomer is N-isopropyl methacrylamide, the pH-responsive monomer is methacrylic acid, and the feeding mole ratio of the pH-responsive monomer methacrylic acid to the temperature-responsive monomer N-isopropyl methacrylamide is (3-8): 100; the feeding mole ratio of the cross-linking agent to the monomer is (10-15) 100; within this range, a nanogel occlusion material having a superior occlusion effect of the reticuloendothelial system can be obtained. The mass ratio of the initiator to the temperature-responsive monomer is (5-15): 550.

According to the preferred embodiment of the invention, the crosslinking degree and further the hardness of the crosslinking agent N, N '-bis (acryloyl) cystamine and the temperature responsive monomer N-isopropyl methyl propionamide are regulated and controlled by regulating the feeding molar ratio of the crosslinking agent N, N' -bis (acryloyl) cystamine to the temperature responsive monomer N-isopropyl methyl propionamide, and experiments show that the higher the molar ratio is, the higher the hardness is and the weaker the deformability is. The surfactant sodium dodecyl sulfate can regulate and control the particle size of the nanogel, and the nanogel materials with different hardness are compared with the reticuloendothelial system blocking effect and the antitumor drug tumor enrichment and drug delivery effects by controlling the approaching of the particle sizes of the nanogels with different hardness in experiments.

In some embodiments, the preparation method of the nanogel blocking material of the invention specifically comprises the following steps:

(1) Preparation of the nanogel: the method comprises the steps of dissolving the weighed N-isopropyl methyl propionamide and sodium dodecyl sulfate in water, dissolving the weighed N, N '-bis (acryloyl) cystamine in ultrapure water, and if the cross-linking agent N, N' -bis (acryloyl) cystamine is used in a large amount and cannot be dissolved in ultrapure water, dissolving the cross-linking agent N, N '-bis (acryloyl) cystamine in a small amount of ethanol, then adding the cross-linking agent N, N' -bis (acryloyl) cystamine into the aqueous solution, and then adding methacrylic acid into the aqueous solution. The solution was subjected to 3 cycles of pumping-argon to remove oxygen (or oxygen and ethanol) from the system. The deoxygenated solution was heated to 80 ℃, then the weighed potassium persulfate was dissolved in a small amount of water, and added to the above system via syringe to initiate polymerization.

(2) Purification of the nanogel: and cooling the prepared nano gel water solution to room temperature, and adding the cooled nano gel water solution into an ultrafiltration tube. Centrifuging to remove unreacted monomer and other impurities, concentrating the nanogel, adding ultrapure water after concentration, centrifuging and washing for 3 times, detecting the solid content of the washed and concentrated nanogel, and storing at 4 ℃.

Based on the blocking material, the invention uses the hard nanogel material to temporarily block macrophages in the reticuloendothelial system, and then uses the soft nanogel to deliver small molecule chemotherapeutic drugs such as doxorubicin hydrochloride for anti-tumor treatment research. Further provides a series of medicines for tumor multi-step therapy based on the RES-blockade strategy, which comprises the nanogel blocking material and the nanogel anti-tumor medicines; the nanogel anti-tumor drug is obtained by loading water-soluble micromolecular anti-tumor chemotherapeutic drug on nanogel through electrostatic adsorption; wherein the young's modulus of the nanogel blocking material is greater than the young's modulus of the nanogel anti-neoplastic drug; when in use, the nanogel blocking material is used for blocking reticuloendothelial system and reducing liver clearance rate, and the nanogel anti-tumor drug is used for delivering chemotherapeutic drugs to tumor sites and treating tumors.

In some embodiments of the invention, a nanogel blocking material is prepared, wherein the temperature responsive monomer N-isopropyl methacrylamide imparts excellent hydrophilicity, stability and biocompatibility to the nanogel, and the nanogel has sufficient deformation space under physiological conditions compared with the N-isopropyl acrylamide at a higher volume phase transition temperature; wherein pH response monomer methacrylic acid endows the nanogel with negative charge, enhances stability, endows the nanogel with the capability of carrying a positively-charged water-soluble micromolecule drug, and the carboxyl can enhance the hydrophilicity of the nanogel after phase transition; the crosslinking agent N, N '-bis (acryloyl) cystamine endows the nanogel network structure, the deformation capacity, namely the hardness of the nanogel is regulated and controlled by changing the molar ratio of the N, N' -bis (acryloyl) cystamine to N-isopropyl methacrylamide, and the hardness is higher as the molar ratio is higher; the particle size of the nano gel is regulated and controlled by the surfactant sodium dodecyl sulfate, so that the surfactant sodium dodecyl sulfate meets the use requirement, and the more the material is added, the smaller the particle size of the nano gel is.

In the tumor multi-step therapy series medicine based on the RES-blockade strategy, both the nanogel blocking material and the nanogel anti-tumor medicine relate to the preparation of nanogel. The temperature response monomer N-isopropyl methacrylamide and the pH response monomer methacrylic acid are crosslinked by N, N' -bis (acryloyl) cystamine in ultrapure water, and series of nanogels with different hardness are obtained by an emulsion polymerization mode. Wherein potassium persulfate initiates polymerization reaction, and sodium dodecyl sulfate regulates and controls the hydration particle size of the nano gel with different hardness. The nano gel obtained by the reaction is ultrafiltered to remove unreacted monomers and other impurities, washed by ultrapure water, concentrated and stored at the temperature of 4 ℃.

In some embodiments, the Young's modulus of the nanogel anti-neoplastic agent is between 20 and 150kPa, more preferably between 50 and 100kPa; experiments show that the nanogel anti-tumor drug with smaller Young's modulus (hardness) range has higher tumor deep penetration efficiency compared with the nanogel anti-tumor drug with larger hardness.

The particle size of the nanogel blocking material and the nanogel anti-tumor drug in the series of drugs can be regulated and controlled by regulating and controlling the dosage of the surfactant according to the requirement. In some embodiments, the average particle size of the nanogel anti-tumor drug is 150-300 nm, more preferably 200-240 nm; the average particle diameter of the nanogel blocking material is 150 to 300nm, more preferably 200 to 240nm.

In some embodiments, the preparation method of the nanogel anti-tumor drug comprises the following steps:

S1: under the condition that a cross-linking agent and a surfactant exist, initiating the monomer to perform polymerization reaction in a water phase through an initiator to obtain nanogel, wherein the Young modulus of the nanogel can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer; the average particle size of the nanogel can be regulated and controlled by the use amount of the surfactant; the monomer comprises one or more of a temperature responsive monomer, a pH responsive monomer and a reduction responsive monomer; the temperature response type monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; the cross-linking agent is one or more of N, N-methylene bisacrylamide, divinylbenzene and N, N' -bis (acryloyl) cystamine.

The soft nanogel for loading the water-soluble micromolecular anti-tumor chemotherapeutic drug and the hard nanogel for preparing the blocking material can be prepared by the same preparation method, wherein the types of raw materials such as monomers, cross-linking agents, initiators, surfactants and the like can be the same. In contrast, the hardness, i.e., young's modulus, of the nanogel is controlled by controlling the different molar ratios of the crosslinking agent to the monomer to accommodate different requirements for use as an occlusive material or for delivery of anti-tumor drugs; correspondingly, the average granularity of the nanogel with different hardness can be regulated by regulating the dosage of the surfactant so as to regulate the granularity of the nanogel blocking material and the nanogel anti-tumor drug.

In a preferred embodiment, when the soft nanogel for loading the anti-tumor chemotherapeutic is prepared, the monomers comprise a temperature-responsive monomer and a pH-responsive monomer, wherein the temperature-responsive monomer is N-isopropyl methacrylamide, the pH-responsive monomer is methacrylic acid, and the feeding mole ratio of the pH-responsive monomer methacrylic acid to the temperature-responsive monomer N-isopropyl methacrylamide is (3-8): 100; the feeding mole ratio of the cross-linking agent to the temperature responsive monomer is (1-5): 100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15): 550. The crosslinking agent is a reduction-responsive crosslinking agent, such as N, N' -bis (acryloyl) cystamine; the initiator is one or more of potassium persulfate, sodium persulfate and tert-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (30-40) to 550. The nanogel anti-tumor drug obtained in the range can permeate into deeper tumor parts due to the excellent deformability of the nanogel anti-tumor drug, so that a better anti-tumor effect is realized.

In some embodiments, the polymerization reaction during the preparation of the hard nanogel used as an occlusion material or the soft nanogel used for delivering a chemotherapeutic agent has a reaction temperature of 70-85 ℃ and a reaction time of 4-8 hours.

The crosslinking agent of the invention preferably adopts a reduction-responsive crosslinking agent, and the crosslinking agent with reduction responsiveness can endow the prepared nanogel with reduction responsiveness; or a monomer with reduction response can also be adopted, so that the prepared nanogel has reduction response. The occlusion reticuloendothelial system needs a large amount of nanogels, and in order to avoid the damage to liver function caused by long-term occlusion of the liver, the nanogels need to have the capacity of being degraded by the liver while realizing the occlusion function. Meanwhile, the dense network structure of the hard nanogel can delay the degradation rate of glutathione to the nanogel, avoid the reduction of blocking efficiency caused by rapid degradation, and have good biocompatibility while ensuring the blocking efficiency.

The water-soluble small molecule antitumor drug carried in the nanogel antitumor drug can be various small molecules common in the field, including but not limited to one or more of doxorubicin hydrochloride, metformin and indocyanine green.

The series of medicaments provided by the invention can be used for preparing antitumor medicaments. In some embodiments, the dose of the nanogel blocking material in the series of drugs is 100-300 mg/kg; preferably 200mg/kg; the dosage of the water-soluble small molecule anti-tumor chemotherapeutic in the nano gel anti-tumor drug is 4-6 mg/kg. When the medicine is specifically applied, the medicine can be used in the following mode:

(2) Waiting for a time interval of 0.5-4 hours, preferably 1.5 hours, and administering the nanogel anti-tumor drug in the series of drugs to the patient, wherein the dosage of the water-soluble small molecule anti-tumor chemotherapeutic drug in the nanogel anti-tumor drug is 4-6 mg/kg, preferably 4mg/kg.

Nano-drugs have been used for decades in anti-tumor research. Physicochemical properties of the nano-drug, such as particle size, charge, morphology, composition, surface modification, etc., are continuously optimized to enhance the anti-tumor efficacy of the nano-drug and reduce toxic and side effects. However, most nano-drugs are rapidly combined with plasma proteins after being injected into the circulatory system, and then are recognized and cleared by macrophages in the reticuloendothelial system, so that the plasma half-life of the nano-drugs is greatly shortened, and only 0.7% of the nano-drugs can reach the tumor sites, which severely limits the anti-tumor efficacy of the nano-drugs. The macrophage in reticuloendothelial system is cleared or the uptake capacity of the macrophage is inhibited, so that the plasma circulation time of the nano-drug can be obviously prolonged, more nano-drugs can reach the tumor part, the tumor enrichment is improved, and the anti-tumor curative effect is enhanced. Among the various RES-blockade strategies, temporary occlusion of the reticuloendothelial system with large numbers of biocompatible nanoparticles is one of the most promising occlusion strategies. The traditional Chinese medicine composition has the advantages that on one hand, toxic and side effects are negligible; on the other hand, no unexpected immunosuppressive effects are caused; at the same time, the transience and reversibility are also important guarantees of the clinical development of the RES-blockade strategy. However, based on this strategy, how to reduce the blocking dose as much as possible to alleviate the metabolic burden of the human body is a matter of urgent consideration.

The nanogel is a widely used nano drug carrier, and similar to other nano carriers, can carry small molecular drugs and is enriched at tumor sites by enhancing permeation and retention effects, thereby remarkably solving the problems of poor targeting and strong toxic and side effects of the small molecular drugs. The nanogel has the following advantages over other nanocarriers. Firstly, the interior of the water-soluble nano gel can form a large number of hydrogen bonds with water molecules, so that the water-soluble nano gel has excellent hydrophilicity, stability and biocompatibility, and the biodegradability of the nano gel can be realized by using a crosslinking agent containing response bonds, such as disulfide bonds with reduction response, so that the nano gel has higher safety. Secondly, different monomers can be utilized to endow the nanogel with different functions, such as hydrophilicity, pH responsiveness, reduction responsiveness and the like, so that the nanogel meets different application conditions. In addition, with the progress of research, researchers find that mechanical properties, particularly hardness, as an entirely new parameter significantly affect the pharmacokinetic behavior of nano-drugs, while the network structure of nanogels imparts excellent mechanical properties thereto, and that the hardness and deformability thereof are directly affected by the network density thereof, generally the greater the network density, the higher the hardness, and the weaker the deformability. Meanwhile, experimental researches show that the hard nanogel can remarkably reduce the uptake capacity of macrophages, and has more excellent reticuloendothelial system blocking efficiency than the soft nanogel under the same administration dosage, so that the invention provides the hard nanogel as the first-choice blocking material of the RES-blockade strategy.

Experiments of the invention also find that the water-soluble micromolecular chemotherapeutic drugs such as doxorubicin hydrochloride are carried on the soft nanogel in an electrostatic adsorption mode, so that the physical and chemical properties, especially the hardness, of the nanogel are not obviously affected, and the stability of the doxorubicin hydrochloride in a salt solution and under physiological conditions is obviously improved. Meanwhile, under the load of the soft nanogel, more doxorubicin hydrochloride is delivered to the tumor area compared with the hard nanogel, and the soft nanogel can deliver the doxorubicin hydrochloride to the deep part of the tumor so as to further enhance the anti-tumor curative effect.

The invention utilizes the hard nanogel to block the reticuloendothelial system, prolongs the circulation time of the drug-loaded soft nanogel, enables more soft nanogel to be enriched at the tumor part, and the soft nanogel can deliver small molecular chemotherapeutic drug doxorubicin hydrochloride to the deep part of the tumor, so that the optimal anti-tumor effect can be realized based on the treatment strategy of enhancing the tumor enrichment and anti-tumor curative effect of the soft nanogel based on the hard nanogel-blocked reticuloendothelial system.

In some embodiments of the invention, the temperature responsive monomer N-isopropyl methacrylamide and the pH responsive monomer methacrylic acid are prepared into the nanogel through an emulsion polymerization mode, and the hardness of the nanogel is regulated and controlled through the molar ratio of the cross-linking agent N, N' -bis (acryloyl) cystamine to the monomer N-isopropyl methacrylamide; the invention provides a tumor multistep therapy series medicine based on a RES-blockade strategy, which specifically comprises a medicine A serving as a macrophage blocking material and a medicine B serving as an anti-tumor therapy, wherein the medicine A is hard nanogel prepared by the method, and the medicine B is a nano medicine prepared by carrying a small-molecule anti-tumor chemotherapy medicine such as doxorubicin hydrochloride on soft nanogel. The RES-blockade strategy can be used for obviously reducing the intake of the nano-drug by the liver, improving the enrichment quantity of the drug B at the tumor part and enhancing the anti-tumor curative effect of the nano-drug; meanwhile, the RES-blockade strategy based on the hard nanogel can also enhance the anti-tumor curative effect of the marketed nano medicines Doxil and Abraxane, and has clinical conversion value.

The raw material components used in the invention are all available commercially, and the reagents used in the examples of the invention are all chemically pure.

When the nanogel is prepared in the following embodiment of the invention, the feeding mole ratio of the cross-linking agent and the temperature responsive monomer N-isopropyl methacrylamide is defined as X:100, the corresponding cross-linking degree of the nanogel is X%, and the prepared nanogel is expressed as X% NGs.

The following are examples:

Example 1

A series of nanogels with different hardness comprises 2% ngs, 5% ngs, 10% ngs and 15% ngs, and the preparation method comprises the following steps:

(1) Preparation: 550mg of N-isopropyl-methylpropionamide was weighed, 35/30/25/20mg (corresponding to a degree of crosslinking of 2%/5%/10%/15%) of sodium dodecyl sulfate was weighed, and 80mL of ultrapure water was added thereto for ultrasonic dissolution. 22.5/56.3/112.6/168.9mg (corresponding to 2%/5%/10%/15%) of N, N' -bis (acryl) cysteamine were weighed respectively, 0.5/1/2/3mL (corresponding to 2%/5%/10%/15%) of ethanol was added respectively, sonicated and dissolved, and then added to the above solution. 3.65mL of methacrylic acid was dissolved in 6.35mL of ultrapure water, and 50. Mu.L was added to the above solution. Pumping the obtained solution for 10min, then introducing argon, circulating for three times, and fully removing oxygen in the solution. The resulting solution was heated to 80℃for 10min. 10mg of potassium persulfate was weighed, added with 0.5mL of ultrapure water, dissolved by ultrasonic, and then added to the above solution by means of a syringe to initiate polymerization for a period of 6 hours.

(2) Purifying: after the reaction is finished, after the solution obtained in the step (1) is restored to room temperature, the solution is transferred into a ultrafiltration tube, the interception value is 10kDa, unreacted monomers and other impurities are removed by centrifugation at 2000rpm, meanwhile, excessive water is removed, the solution is repeatedly added with ultrapure water for cleaning for 3 times after concentration, and finally the solution is concentrated to 8mL.

(3) Quantification: and (3) taking 300 mu L of the concentrated solution obtained in the step (2), drying and weighing, and calculating the solid content in the concentrated solution.

(4) And (3) storing: according to the calculation result obtained in the step (3), the concentrated solution is diluted to 20mg/mL to respectively obtain 2 percent of NGs, 5 percent of NGs, 10 percent of NGs and 15 percent of NGs of nanogels, and the nanogels are stored at the temperature of 4 ℃.

Example 2

A rhodamine B marked serial nanogel RhB@NGs with different hardness comprises RhB@2% NGs, rhB@5% NGs, rhB@10% NGs and RhB@15% NGs, and the preparation method comprises the following steps:

(1) Preparation of RhB-HEMA: 0.5g of rhodamine B,75mg of 4-dimethylaminopyridine and 2.6g of N, N' -dicyclohexylcarbodiimide were weighed, 52.5mL of anhydrous dichloromethane was added, ultrasonic dissolution was performed, the obtained solution was vacuumed to remove dissolved oxygen, stirred for 30 minutes under the protection of argon, and then 1.55mL of hydroxyethyl methacrylate was added. The mixed solution was stirred under argon at 20℃for 25h. The reaction product is purified by a silica gel column chromatography, the eluent is a mixed solution of dichloromethane and methanol in a ratio of 90:10, and finally, a dry solid powder product RhB-HEMA is obtained by a rotary evaporation method.

(2) Preparation of rhodamine B-labeled series of nanogels rhb@ngs of different hardness: 550mg of N-isopropyl-methylpropionamide was weighed, 35/30/25/20mg (corresponding to a degree of crosslinking of 2%/5%/10%/15%) of sodium dodecyl sulfate was weighed, and 80mL of ultrapure water was added thereto for ultrasonic dissolution. 22.5/56.3/112.6/168.9mg (corresponding to 2%/5%/10%/15%) of N, N' -bis (acryl) cysteamine was weighed out respectively, 0.5/1/2/3mL (corresponding to 2%/5%/10%/15%) of ethanol was added respectively, dissolved by ultrasound, and then added to the above solution. 255 μg RhB-HEMA was added to the above solution and dissolved with stirring. 3.65mL of methacrylic acid was dissolved in 6.35mL of ultrapure water, and 50. Mu.L was added to the above solution. Pumping the obtained solution for 10min, then introducing argon, circulating for three times, and fully removing oxygen in the solution. The resulting solution was heated to 80℃for 10min. 10mg of potassium persulfate was weighed, added with 0.5mL of ultrapure water, dissolved by ultrasonic, and then added to the above solution by means of a syringe to initiate polymerization for a period of 6 hours.

(3) Purifying: after the reaction is finished, after the solution obtained in the step (2) is restored to room temperature, the solution is transferred into a ultrafiltration tube, the interception value is 10kDa, unreacted monomers and other impurities are removed by centrifugation at 2000rpm, meanwhile, excessive water is removed, the solution is repeatedly added with ultrapure water for cleaning for 3 times after concentration, and finally the solution is concentrated to 8mL.

(4) Quantification: taking 300 mu L of the concentrated solution obtained in the step (3), drying and weighing, and calculating the solid content in the concentrated solution.

(5) And (3) storing: and (3) diluting the concentrated solution to 20mg/mL according to the calculated result obtained in the step (4) to obtain RhB@2% NGs, rhB@5% NGs, rhB@10% NGs and RhB@15% NGs respectively, and storing the concentrated solution at the temperature of 4 ℃.

Example 3

A series of nano-drugs with different hardness comprises DOX@2% NGs, DOX@5% NGs, DOX@10% NGs and DOX@15% NGs, and the preparation method comprises the following steps:

(1) Drug loading: 10mg of doxorubicin hydrochloride was weighed, 5mL of ultrapure water was added, and the mixture was sonicated, and then added to 5mL of the nanogel solution (2% ngs, 5% ngs, 10% ngs, 15% ngs) obtained in example 1, and mixed and stirred for 48 hours.

(2) Purifying: transferring the solution obtained in the step (1) into a ultrafiltration tube, centrifuging at 2000rpm to remove the non-carried doxorubicin hydrochloride, and concentrating the nano-drug.

(3) Quantification: and (3) adding 50 mu L of the nano-drug solution obtained in the step (2) into 2.95mL of dimethyl sulfoxide, uniformly mixing, detecting by an ultraviolet spectrophotometer, detecting the wavelength to be 483nm, and calculating the concentration of the doxorubicin hydrochloride in the nano-drug solution obtained in the step (2) according to a standard curve of the doxorubicin hydrochloride in the dimethyl sulfoxide at the wavelength of 483 nm.

(4) And (3) storing: according to the calculated result obtained in the step (3), diluting the nano-drug concentrated solution until the concentration of doxorubicin hydrochloride is 1mg/mL, respectively obtaining DOX@2% NGs, DOX@5% NGs, DOX@10% NGs and DOX@15% NGs, and storing at the temperature of 4 ℃.

Example 4

The indocyanine green marked serial nano medicine with different hardness comprises ICG@2% NGs, ICG@5% NGs, ICG@10% NGs and ICG@15% NGs, and the preparation method comprises the following steps:

(1) Drug loading: 5mg of indocyanine green is weighed, 5mL of ultrapure water is added for ultrasonic dissolution, and then the obtained mixture is respectively added into 5mL of nano-drug solution (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs and DOX@15% NGs) obtained in the example 3, and the mixture is mixed and stirred for 48 hours.

(2) Purifying: transferring the solution obtained in the step (1) into a ultrafiltration tube, centrifuging at 2000rpm to remove the non-carried indocyanine green with a cut-off value of 10kDa, and concentrating the indocyanine green labeled nano-drug.

(3) Quantification: adding 50 mu L of the nano-drug solution obtained in the step (2) into 2.95mL of dimethyl sulfoxide, uniformly mixing, detecting by an ultraviolet spectrophotometer, detecting the detection wavelength to be 783nm, and calculating the concentration of indocyanine green in the indocyanine green marked nano-drug solution obtained in the step (2) according to a standard curve of indocyanine green in the dimethyl sulfoxide at the wavelength of 783 nm.

(4) And (3) storing: according to the calculated result obtained in the step (3), the nano-drug concentrated solution is diluted to the concentration of indocyanine green of 1.5mg/mL, ICG@2% NGs, ICG@5% NGs, ICG@10% NGs and ICG@15% NGs are respectively obtained, and the obtained mixture is stored at the temperature of 4 ℃.

Example 5

Detection of hydrated particle size of serial nanogels with different hardness

10. Mu.L of the nanogel concentrate obtained in example 1 (2%, 5%, 10%, 15%) were each dispersed in 1mL of PBS buffer, and the detection temperature was 37℃and the equilibration time was 15min.

Detection of zeta potential of nanogel with different hardness series

10. Mu.L of the nanogel concentrate obtained in example 1 (2%, 5%, 10%, 15%) was dispersed in 1mL of ultrapure water, and the mixture was examined by dynamic light scattering at 37℃for 15min.

Fig. 1, content a and content B, show the particle size distribution and surface charge, respectively, of the nanogels of different hardness series prepared in example 1. It can be seen that the nanogels of 2%, 5%, 10% and 15% ngs prepared in this example were uniformly distributed in PBS buffer at 37deg.C, and the average hydrated particle size was about 220nm. The surface charges are all negative charges and increase slightly with increasing degree of crosslinking.

Example 6

Detection of appearance of series of nanogels with different hardness

Dispersing the nanogel concentrated solution (2%NGs, 5%NGs, 10%NGs and 15%NGs) obtained in the example 1 with ultrapure water until the concentration is 0.01mg/mL, taking 10 mu L of the dispersed solution, dripping the dispersed solution onto a carbon support membrane, naturally drying, dripping 10 mu L of 1% aqueous solution of phosphotungstic acid onto the carbon support membrane after drying, dyeing for 2min, sucking the excessive phosphotungstic acid solution along the edge of the carbon support membrane by using filter paper after dyeing, dripping 10 mu L of ultrapure water for 1min, sucking the excessive ultrapure water along the edge of the carbon support membrane by using filter paper after washing, and observing by using a transmission electron microscope after natural drying.

FIG. 2 is a transmission electron microscope image of the nanogels of different hardness series prepared in example 1. It can be seen that the nano-gels of different hardness series are spherical with uniform particle size distribution.

Example 7

Detection of the height and Young's modulus of series of nanogels of different hardness

The cover glass is soaked in 1% polyethyleneimine aqueous solution for 24 hours, positive charges are modified on the surface of the cover glass, the nanogel concentrated solution (2% ngs, 5% ngs, 10% ngs and 15% ngs) obtained in the embodiment 1 is respectively dispersed to the concentration of 0.01mg/mL by using ultrapure water, 100 mu L of the dispersed solution is dripped on the positively modified cover glass, electrostatic absorption is carried out for 10 minutes, redundant dispersed solution is absorbed, 300 mu L of ultrapure water is dripped to wash out unadsorbed nanogel, then an atomic force microscope is used for detection, the detection environment is a liquid phase, the image is obtained as a contact mode, and the Young modulus is detected as a tapping mode.

Fig. 3, panel a and panel B, are atomic force microscope images and young's modulus, respectively, of the different hardness series nanogels prepared in example 1. It can be seen that the nano-gels of different hardness series are spherical with uniform particle size distribution, the nano-gel with low crosslinking degree is easier to collapse, the height is lower, the nano-gel with high crosslinking degree is not easy to collapse, and the height is higher. The hardness, i.e., young's modulus, of the nanogel increases gradually with increasing degree of crosslinking.

Example 8

Triple responsiveness of series of nanogels of different hardness

(1) Temperature responsiveness: 10. Mu.L of the nanogel concentrate obtained in example 1 (2%, 5%, 10%, 15%) was dispersed in 1mL of ultrapure water, and the temperature was measured by dynamic light scattering at 25-55deg.C with a temperature interval of 1℃and an equilibration time of 1min.

(2) PH responsiveness: 10. Mu.L of the nanogel concentrate obtained in example 1 (2%, 5%, 10%, 15%) was dispersed in 1mL of ultrapure water, the pH was adjusted to 3 to 9, and the measurement was performed by dynamic light scattering at 25℃for 15min.

(3) Reduction responsiveness: 10 mu L of the nanogel concentrated solution obtained in example 1 (2%, 5%, 10%, 15% NGs) was respectively dispersed in 1mL of ultrapure water containing or not containing 10mM glutathione, incubated at room temperature for 24 hours, 10 mu L of the dispersed solution was dropped onto a carbon support membrane, naturally dried, 10 mu L of a 1% aqueous solution of phosphotungstic acid was dropped onto the carbon support membrane after drying, dyeing was carried out for 2 minutes, after dyeing was completed, 10 mu L of ultrapure water was dropped and washed for 1 minute by sucking away excess phosphotungstic acid along the edge of the carbon support membrane with filter paper, after washing was completed, and observation was carried out by a transmission electron microscope after naturally drying.

FIG. 4 is a graph showing the triple responsiveness of the different hardness series nanogels prepared in example 1. Wherein, the content A is temperature response, and the hydrogel particle size of the nanogel is gradually reduced along with the temperature rise, and the shrinkage degree is reduced along with the rise of the crosslinking degree; content B is pH responsive, and it can be seen that the hydrogel particle size gradually increases with increasing pH, and the degree of swelling decreases with increasing degree of crosslinking; content C is the restore responsiveness. It can be seen that after 24h incubation with glutathione, the structure of the nanogel was significantly destroyed.

Example 9

Influence of carried doxorubicin hydrochloride on series nanogel properties with different hardness

(1) Hydrated particle size: 10. Mu.L of the nano-drug concentrate obtained in example 3 (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs, DOX@15% NGs) was dispersed into 1mL of PBS buffer, and the detection was carried out by dynamic light scattering method at 37℃for 15min.

(2) Temperature responsiveness: 10 mu L of the nano-drug concentrate (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs, DOX@15% NGs) obtained in example 3 was respectively dispersed into 1mL of ultrapure water, and the detection was carried out by a dynamic light scattering method at a temperature ranging from 25 to 55 ℃ at a temperature interval of 1 ℃ for 1min.

(3) Morphology: dispersing the nano-coagulation drug condensed liquid (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs and DOX@15% NGs) obtained in the example 3 with ultrapure water to a concentration of 0.01mg/mL, taking 10 mu L of the dispersed liquid, dripping the dispersed liquid onto a carbon support film, naturally drying, dripping 10 mu L of 1% phosphotungstic acid aqueous solution onto the carbon support film after drying, dyeing for 2min, sucking the excessive phosphotungstic acid solution along the edge of the carbon support film by using filter paper after dyeing, dripping 10 mu L of ultrapure water for cleaning for 1min, sucking the excessive ultrapure water along the edge of the carbon support film by using filter paper after cleaning, naturally drying, and observing by using a transmission electron microscope.

(4) Height and young's modulus: immersing the cover glass in a 1% polyethyleneimine aqueous solution for 24 hours, modifying positive charges on the surface of the cover glass, dispersing the nano-drug concentrated solution (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs and DOX@15% NGs) obtained in the example 3 with ultrapure water to a concentration of 0.01mg/mL, taking 100 mu L of the dispersed liquid, adding the dispersed liquid to the positively modified cover glass, carrying out electrostatic adsorption for 10 minutes, absorbing excessive dispersion liquid, then dripping 300 mu L of ultrapure water to wash off the unadsorbed nano-drug, and then detecting with an atomic force microscope, wherein the detection environment is a liquid phase, the image is obtained as a contact mode, and the Young modulus is detected as a tapping mode.

(5) Stability: 10 mu L of the nano-drug concentrate (DOX@2% NGs, DOX@5% NGs, DOX@10% NGs, DOX@15% NGs) obtained in example 3 was dispersed in 1mL of PBS buffer solution or 10% FBS solution, respectively, and the detection was carried out by a dynamic light scattering method at 37℃for 15min at 24h intervals.

FIG. 5 shows the comparative properties and stability of doxorubicin hydrochloride loaded on nanogels of different hardness series prepared in example 1. Wherein the content A is the particle size distribution of the nanogel before and after drug loading and PBS buffer solution under the condition that the hydration particle size is 37 ℃, and the particle size distribution of the nanogel before and after drug loading is basically unchanged; the content B is the temperature response of the nanogel before and after drug loading, and the temperature response of the nanogel before and after drug loading can be seen to be basically unchanged; the content C is a transmission electron microscope image of the nanogel before and after the medicine is loaded, so that the particle size and the morphology of the nanogel before and after the medicine is loaded are basically unchanged; the content D is an atomic force microscope image of the nanogel before and after the medicine is loaded, so that the particle size, the morphology and the height of the nanogel before and after the medicine is loaded are basically unchanged; content E is Young's modulus of the nanogel before and after drug loading, and the hardness of the nanogel before and after drug loading can be seen to be basically unchanged; content F is the stability of the drug-loaded nanogel in PBS buffer solution and 10% FBS solution, and it can be seen that the hydrated particle size of the drug-loaded nanogel is basically unchanged with time, and has excellent stability.

Example 10

Enrichment of nanogels of different hardness in the liver

32 Mice were randomly divided into two groups, including 2% NGs and 15% NGs, and indocyanine green labeled nano-drug (icg@2% NGs and icg@15% NGs) obtained in example 4 was injected by tail vein at a dose of 4mg/kg indocyanine green. Then 4 mice were sacrificed at 1h, 4h, 8h and 24h, respectively, livers were stripped for in vitro imaging and fluorescence intensity was quantified.

Fig. 6, content a and content B, show that the liver enrichment of indocyanine green labeled nano-drugs of different hardness series prepared in example 4 shows that the enrichment of hard nanogel at the liver is significantly higher than that of soft nanogel, and the retention capacity is stronger.

Example 11

Effect of incubation time on macrophage uptake of hard nanogels

RAW264.7 cells were added to 6-well plates at a concentration of 5X 10 ⁵ cells/well, and cultured in an incubator for 12 hours to adhere the RAW264.7 cells. After adherence, the upper medium was aspirated, 3mL of serum-free medium containing rhodamine B-labeled hard nanogel (RhB@2% NGs, rhB@5% NGs, rhB@10% NGs, rhB@15% NGs) obtained in example 2 was added, the nanogel concentration was 50 μg/mL, the upper medium was aspirated in an incubator for 0h, 0.5h, 1h, 2h, 4h, 6h, washed 3 times with PBS buffer, cells were digested with pancreatin, collected centrifugally and fluorescent intensity was detected with a flow cytometer, the detection channel was PE, and the influence of incubation time on uptake of hard nanogel by macrophages was counted.

FIG. 7 shows the uptake of rhodamine B labeled hard nanogel prepared in example 2 in macrophages over time, and it can be seen that RAW 264.7 cells uptake of hard nanogel was highest at 1.5 h.

Example 12

Effect of hardness on efficiency of nanogel-blocked reticuloendothelial System

(1) Effect on tumor and liver enrichment: a model of 4T1 breast cancer subcutaneous tumor was constructed by subcutaneously seeding 1X 10 ⁶ 4T1 cells at the back of female BALB/C mice near the right hind limb in a volume of 100. Mu.L. After the tumor volume reached 200mm ³, the mice were randomly divided into three groups, including Control, 2% -blockade and 15% -blockade. The dose of the nanogel (2% ngs and 15% ngs) with different hardness obtained in example 1 was 200mg/kg by tail vein injection of physiological saline. After 1.5h, the indocyanine green obtained in example 4 was injected into the tail vein to label the soft nanogel, the injection dose was 4mg/kg of indocyanine green, then the mice were anesthetized at 1h, 2h, 4h, 8h, 12h, 24h, tumors and livers were imaged in vivo, and fluorescence intensity was quantified.

(2) Effect on plasma half-life of subsequent nanomedicine: female BALB/C mice were randomly divided into three groups, including Control, 2% -blockade and 15% -blockade. The dose of the nanogel (2% ngs and 15% ngs) with different hardness obtained in example 1 was 200mg/kg by tail vein injection of physiological saline. After 1.5h, the indocyanine green-labeled soft nanogel obtained in example 4 was injected into the tail vein at an injection dose of 6mg/kg of indocyanine green, and then 80. Mu.L of blood was taken through the tail vein at 5min, 15min, 0.5h, 1h, 2h, 4h, 8h, and 12h, respectively, and added to a 1.5mL EP tube containing 10. Mu.L of dipotassium ethylenediamine tetraacetate, followed by mixing. Then, the mixture was centrifuged at 2000rpm for 10 minutes, and 10. Mu.L of the plasma was dispersed in 50. Mu.L of dimethyl sulfoxide, followed by detection with an ELISA reader at an excitation wavelength of 783 nm. The concentration of indocyanine green in blood and the injection dose percentage were calculated according to standard curve, and the relevant pharmacokinetic parameters were calculated using software DAS 2.0.

FIG. 8 shows the occlusion efficiency of the reticuloendothelial system by the different hardness series nanogels prepared in example 1. Wherein, the content A is enrichment of indocyanine green marked soft nanogel in tumor and liver after the nanogel of different hardness series blocks reticuloendothelial system, the content B is semi-quantitative for tumor enrichment, and the content C is semi-quantitative for liver enrichment. The method can be used for finding that the blocking efficiency of the hard nanogel to the reticuloendothelial system is higher, and can obviously reduce the enrichment of indocyanine green marked soft nanogel in the liver and enhance the enrichment of tumors.

Fig. 9 shows the effect of the different hardness series nanogel blocking reticuloendothelial system prepared in example 1 on the plasma half-life of indocyanine green-labeled soft nanogel, and it can be seen that the plasma half-life of indocyanine green-labeled soft nanogel can be significantly prolonged after the hard nanogel blocking the reticuloendothelial system.

Example 13

Effect of hardness on nanogel-blocked macrophage efficiency

RAW264.7 cells were added to 6-well plates at a concentration of 5X 10 ⁵ cells/well, and cultured in an incubator for 12 hours to adhere the RAW264.7 cells. After adherence, the upper medium was aspirated, and 2mL of serum-free medium containing the nanogels of different hardness (2% ngs versus 15% ngs) obtained in example 1 was added at a nanogel concentration of 400 μg/mL. After 1.5h incubation, 1mL of serum-free medium containing rhodamine B-labeled soft nanogel obtained in example 2 was added, the nanogel concentration was 50 μg/mL, incubated in an incubator for 0.5h, the upper medium was aspirated, washed 3 times with PBS buffer, cells were digested with pancreatin, collected by centrifugation and fluorescence intensity was detected with a flow cytometer, the detection channel was PE, and relative cell uptake was calculated from the fluorescence intensity.

Fig. 10 shows the blocking efficiency of the different hardness series nanogels prepared in example 1 on macrophages, and it can be seen that the hard nanogels can significantly reduce the uptake of rhodamine B labeled soft nanogels by RAW 264.7 cells.

Example 14

Effect of dose on efficiency of hard nanogel blocking reticuloendothelial System

A model of 4T1 breast cancer subcutaneous tumor was constructed by subcutaneously seeding 1X 10 ⁶ 4T1 cells at the back of female BALB/C mice near the right hind limb in a volume of 100. Mu.L. When the tumor volume reached 200mm ³, the mice were randomly divided into four groups, including Control, 100mg/kg, 200mg/kg and 300mg/kg. The hard nanogel obtained in example 1 was injected with physiological saline and different doses through the tail vein. After 1.5h, the indocyanine green labeled soft nanogel obtained in example 4 was injected into the tail vein at a dose of 4mg/kg indocyanine green, and then mice were anesthetized at 1h, 2h, 4h, and 10h, tumors and livers were imaged in vivo, and fluorescence intensity was quantified.

Fig. 11 shows the blocking efficiency of the hard nanogel prepared in example 1 on reticuloendothelial system at various doses. Wherein, the content A is enrichment of indocyanine green marked soft nanogel in tumor and liver after different doses of hard nanogel block reticuloendothelial system, the content B is enrichment semi-quantitative of tumor, and the content C is enrichment semi-quantitative of liver. It can be seen that the blocking efficiency of the hard nanogel to the reticuloendothelial system increases with increasing dose at doses not higher than 200 mg/kg; when the dose exceeds 200mg/kg, the blocking efficiency does not further increase with the increase of the dose.

Example 15

Effect of dose on hard nanogel-blocked macrophage efficiency

RAW264.7 cells were added to 6-well plates at a concentration of 5X 10 ⁵ cells/well, and cultured in an incubator for 12 hours to adhere the RAW264.7 cells. After adherence, the upper medium was aspirated, and 2mL of serum-free medium containing the hard nanogel obtained in example 1 was added at nanogel concentrations of 100. Mu.g/mL, 200. Mu.g/mL, 300. Mu.g/mL, and 400. Mu.g/mL. After 1.5h incubation, 1mL of serum-free medium containing rhodamine B-labeled soft nanogel obtained in example 2 was added, the nanogel concentration was 50 μg/mL, incubated in an incubator for 0.5h, the upper medium was aspirated, washed 3 times with PBS buffer, cells were digested with pancreatin, collected by centrifugation and fluorescence intensity was detected with a flow cytometer, the detection channel was PE, and relative cell uptake was calculated from the fluorescence intensity.

Fig. 12 shows the macrophage blocking efficiency of the hard nanogel prepared in example 1 at different doses, and it can be seen that the intake of rhodamine B-labeled soft nanogel by RAW 264.7 cells gradually decreases with increasing dose of the hard nanogel.

Example 16

Influence of RES-blocakde strategy based on hard nanogel on anti-tumor effect of soft nano medicine

(1) Antitumor effect: a model of 4T1 breast cancer subcutaneous tumor was constructed by subcutaneously seeding 1X 10 ⁶ 4T1 cells at the back of female BALB/C mice near the right hind limb in a volume of 100. Mu.L. When tumor volume reached 100mm ³, mice were randomly divided into seven groups, including Control(G1)、Free DOX(G2)、DOX@2％NGs(G3)、DOX@15％NGs(G4)、2％-blockade+DOX@2％NGs(G5)、15％-blockade+DOX@2％NG(G6) and 15% -blockade +DOX@15% NGs (G7), recorded as day 0. On the 1 st and 4 th days after grouping, the Control, free DOX, DOX@2% NGs and DOX@15% NGs are respectively injected with physiological saline, free doxorubicin hydrochloride and the nano-drugs (DOX@2% NGs and DOX@15% NGs) obtained in the example 3 through tail veins, wherein the injection dosage is 4mg/kg of doxorubicin hydrochloride; the group of 2% -blockade +DOX@2% NGs was injected with 200mg/kg of the 2% NGs obtained in example 1 by tail vein, and after 1.5 hours, with 4mg/kg of doxorubicin hydrochloride, and with 4mg/kg of doxorubicin hydrochloride, with only the DOX@2% NGs obtained in example 3 by tail vein; 15% -blockade +DOX@2% NG group was injected with 200mg/kg of 15% NGs obtained in example 1 by tail vein on day 1, and with 4mg/kg of doxorubicin hydrochloride after 1.5 hours, and with 4mg/kg of doxorubicin hydrochloride by tail vein alone, and with 4mg/kg of doxorubicin hydrochloride; the 15% -blockade +DOX@15% NG group was injected with 200mg/kg of the 15% NGs obtained in example 1 by tail vein on day 1, and with 4mg/kg of doxorubicin hydrochloride after 1.5 hours, and with 4mg/kg of doxorubicin hydrochloride by tail vein alone, and with 4mg/kg of doxorubicin hydrochloride. From day 0, the long side (a) and short side (b) of the mouse subcutaneous tumor were measured daily with vernier calipers according to the calculation formula: tumor volume v=a×b ²/2, tumor volume was calculated. After the end of the 15 th day measurement, mice were sacrificed, the tumor was peeled off, weighed and photographed, and the tumor suppression rate was calculated from the tumor volume and mass.

(2) Apoptosis and proliferation: fixing and slicing the isolated tumor obtained in the step (1) by using 4% paraformaldehyde, staining by H & E, TUNEL and Ki67, and then quantifying the ratio of apoptosis to proliferation area by using software to evaluate the necrosis, apoptosis and proliferation degree of tumor cells.

(3) Security assessment: in the antitumor effect evaluation process of step (1), the body weight of the mice was weighed every day from day 0, and after the end of the measurement on day 15, the main organs (heart, liver, spleen, lung, kidney) were peeled off, fixed with 4% paraformaldehyde and sectioned, and H & E staining was performed to evaluate the tissue toxicity. Meanwhile, blood is taken from mice for blood biochemical and blood routine detection, and nano drug toxicity is estimated.

FIG. 13 shows the anti-tumor effect of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the different hardness series nanomedicines of example 3. Wherein, the content A is the change relation of tumor volume with time after administration, the content B is the weight of the tumor after treatment, and the content C is the tumor photo after treatment, and compared with the soft nanogel, the anti-tumor effect of the soft nano drug and the hard nano drug can be obviously enhanced after the reticuloendothelial system is blocked by the hard nanogel; the content D is the change relation of the weight of the mice with time after administration, and the fact that the weight of the mice is reduced slightly and restored to the normal level after administration can be seen, and the RES-blockade strategy of the hard nanogel has good safety in combination with nano medicines of different hardness series.

FIG. 14 shows the tumor rejection rate of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the different hardness series nanomedicines of example 3. Wherein, the content A is the tumor inhibition rate based on the tumor volume, the content B is the tumor inhibition rate based on the tumor weight, and the tumor inhibition rate of the soft nano-drug and the hard nano-drug can be obviously improved after the reticuloendothelial system is blocked by the hard nano-gel.

FIG. 15 shows tumor necrosis and proliferation after treatment with various hardness series nanomedicines of example 3 in combination with the RES-blockade strategy based on the hard nanogel of example 1. Wherein content A is H & E, tunel and Ki67 staining images of tumor sections, content B is quantification of the necrotic area ratio of the tumor sections, and content C is quantification of the proliferation area ratio of the tumor sections. It can be seen that the hard nanogel blocking reticuloendothelial system can obviously increase the ratio of the sparseness of tumor cells to the necrotic area of the tumor section and reduce the ratio of the proliferation area of the tumor section.

FIG. 16 is an H & E stained image of an organ tissue section after treatment with a different hardness series of nanomedicine based on the RES-blockade strategy for the hard nanogel of example 1 in combination with the different hardness series of nanomedicine of example 3, which shows that the RES-blockade strategy for the hard nanogel has substantially no effect on the organ and good safety.

FIG. 17 shows blood biochemical and blood general index after treatment with various hardness series nanomedicine based on RES-blockade strategy for the hard nanogel of example 1 in combination with example 3. Wherein content A is glutamic pyruvic transaminase, content B is glutamic oxaloacetic transaminase, content C is glutamic oxaloacetic transaminase/glutamic pyruvic transaminase, content D is creatine kinase, content E is urea nitrogen, content F is creatinine, content G is white blood cell, content H is red blood cell, and content I is platelet. The RES-blockade strategy of the hard nanogel and various indexes of the nanometer medicaments of different hardness series are all in the normal value range after the treatment, and the safety is good.

Example 17

Influence of RES-blocakde strategy based on hard nanogel on antitumor effect of marketed nano-drug

(1) Antitumor effect: a model of 4T1 breast cancer subcutaneous tumor was constructed by subcutaneously seeding 1X 10 ⁶ 4T1 cells at the back of female BALB/C mice near the right hind limb in a volume of 100. Mu.L. When the tumor volume reached 100mm ³, the mice were randomly divided into seven groups, including Control(G1)、Doxil(G2)、2％-blockade+Doxil(G3)、15％-blockade+Doxil(G4)、Abraxane(G5)、2％-blockade+Abraxane(G6)、15％-blockade+Abraxane(G7),, which was recorded as day 0. On the 1 st and 4 th days after grouping, control, doxil and Abraxane groups were respectively injected with physiological saline, doxil and Abraxane through tail veins at doses of 3mg/kg doxorubicin hydrochloride and 10mg/kg paclitaxel; 2% -blockade +Doxil and 2% -blockade +Abraxane groups are injected into 2% NGs obtained in example 1 through tail vein, the injection dose is 200mg/kg, and after 1.5h, doxil or Abraxane is injected through tail vein, the injection dose is 4mg/kg of doxorubicin hydrochloride and 10mg/kg of paclitaxel; 15% -blockade +Doxil and 15% -blockade +Abraxane groups were injected with 200mg/kg of 15% ngs obtained in example 1 by tail vein, and after 1.5h Doxil or Abraxane was injected by tail vein at a dose of 4mg/kg of doxorubicin hydrochloride and 10mg/kg of paclitaxel. From day 0, the long side (a) and short side (b) of the mouse subcutaneous tumor were measured daily with vernier calipers according to the calculation formula: tumor volume v=a×b ²/2, tumor volume was calculated. After the end of the 15 th day measurement, mice were sacrificed, the tumor was peeled off, weighed and photographed, and the tumor suppression rate was calculated from the tumor volume and mass.

FIG. 18 is the antitumor effect of RES-blockade strategy based on the hard nanogel of example 1 in combination with the marketed nanomedicine. Wherein, the content A is the change relation of tumor volume with time after administration, the content B is the weight of the tumor after treatment, and the content C is the tumor photo after treatment, and compared with the soft nanogel, the anti-tumor effect of the nano-drug on the market can be obviously enhanced after the reticuloendothelial system is blocked by the hard nanogel; content D is the change relation of the weight of the mice with time after administration, and the fact that the weight of the mice is reduced slightly and restored to normal level after administration can be seen, and the RES-blockade strategy of the hard nanogel has good safety in combination with the marketed nano-drug.

FIG. 19 is the tumor rejection rate of the RES-blockade strategy based on the hard nanogel of example 1 in combination with the marketed nanomedicine. Wherein, the content A is the tumor inhibition rate based on the tumor volume, the content B is the tumor inhibition rate based on the tumor weight, and the tumor inhibition rate of the nano-drug on the market can be obviously improved after the reticuloendothelial system is blocked by the hard nano-gel.

FIG. 20 shows tumor necrosis and proliferation after combination of the RES-blockade strategy based on the hard nanogel of example 1 with the marketed nanomedicine treatment. Wherein content A is H & E, tunel and Ki67 staining images of tumor sections, content B is quantification of the necrotic area ratio of the tumor sections, and content C is quantification of the proliferation area ratio of the tumor sections. It can be seen that the hard nanogel blocking reticuloendothelial system can obviously increase the ratio of the sparseness of tumor cells to the necrotic area of the tumor section and reduce the ratio of the proliferation area of the tumor section.

FIG. 21 is an H & E stained image of organ tissue sections after combination of the RES-blockade strategy based on the hard nanogel of example 1 with the marketed nanomedicine treatment, which can be seen to have substantially no effect on the organ and good safety with the combination of the RES-blockade strategy of the hard nanogel with the marketed nanomedicine treatment.

FIG. 22 is a graph showing blood biochemical and blood general index after combination of the RES-blockade strategy based on the hard nanogel of example 1 with the marketed nanomedicine treatment. Wherein content A is glutamic pyruvic transaminase, content B is glutamic oxaloacetic transaminase, content C is glutamic oxaloacetic transaminase/glutamic pyruvic transaminase, content D is creatine kinase, content E is urea nitrogen, content F is creatinine, content G is white blood cell, content H is red blood cell, and content I is platelet. The RES-blockade strategy of the hard nanogel combined with the nanometer medicament on the market can be seen to ensure that all indexes are in the normal value range after the treatment, and the safety is good.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. The macrophage nanogel blocking material of reticuloendothelial system is characterized in that the material is a nanogel blocking material obtained by initiating polymerization reaction of monomers in water phase through an initiator in the presence of a cross-linking agent and a surfactant;

the Young modulus of the nanogel blocking material can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer, and the average particle size of the nanogel blocking material can be regulated and controlled by using the surfactant; the Young's modulus of the nano gel blocking material is 300-600 kPa;

The monomer comprises a temperature-responsive monomer and a pH-responsive monomer, wherein the temperature-responsive monomer is one or more of N-isopropyl methacrylamide, N-isopropyl acrylamide and N-ethyl acrylamide; the pH response type monomer is one or more of methacrylic acid, acrylic acid and 2-acrylamido-2-methyl-1-propane sulfonic acid; the cross-linking agent is one or more of N, N-methylene bisacrylamide, divinylbenzene and N, N' -bis (acryloyl) cystamine; the feeding mole ratio of the cross-linking agent to the monomer is (10-15) 100;

The feeding mole ratio of the pH response type monomer to the temperature response type monomer is (3-8): 100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15) to 550.

2. The nanogel blocking material of claim 1 wherein the initiator is one or more of potassium persulfate, sodium persulfate, and t-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (15-25) to 550.

3. A multistep tumor therapy drug based on RES-blockade strategy, comprising a nanogel blocking material according to claim 1 or 2, further comprising a nanogel anti-tumor drug; the nanogel anti-tumor drug is obtained by loading water-soluble micromolecular anti-tumor chemotherapeutic drug on nanogel through electrostatic adsorption; wherein the young's modulus of the nanogel blocking material is greater than the young's modulus of the nanogel anti-neoplastic drug; the Young's modulus of the nano gel antitumor drug is 20-150 kPa;

when in use, the nanogel blocking material is used for blocking reticuloendothelial system and reducing liver clearance rate, and the nanogel anti-tumor drug is used for delivering chemotherapeutic drugs to tumor sites to play an anti-tumor role.

4. The RES-blockade policy-based tumor multistep therapy drug according to claim 3, wherein the average particle size of the nanogel anti-tumor drug is 150-300 nm, and the average particle size of the nanogel blocking material is 150-300 nm.

5. The RES-blockade policy-based tumor multistep therapy drug according to claim 3, wherein the preparation method of the nanogel anti-tumor drug comprises the steps of:

s1: under the condition that a cross-linking agent and a surfactant exist, initiating the monomer to perform polymerization reaction in a water phase through an initiator to obtain nanogel, wherein the Young modulus of the nanogel can be regulated and controlled by regulating and controlling the molar ratio of the cross-linking agent to the monomer; the average particle size of the nanogel can be regulated and controlled by the use amount of the surfactant; the monomers include temperature-responsive monomers and pH-responsive monomers;

6. The RES-blockade policy-based tumor multistep therapy drug of claim 5, wherein the initiator is one or more of potassium persulfate, sodium persulfate, and t-butyl hydroperoxide; the surfactant is one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate and lecithin; the mass ratio of the surfactant to the temperature-responsive monomer is (30-40) 550;

The monomer comprises a pH responsive monomer and a temperature responsive monomer, and the feeding molar ratio of the pH responsive monomer to the temperature responsive monomer is (3-8) 100; the feeding mole ratio of the cross-linking agent to the temperature responsive monomer is (1-5): 100; the mass ratio of the initiator to the temperature-responsive monomer is (5-15) to 550.

7. Use of a RES-blockade strategy based tumor multistep therapy drug according to any one of claims 3 to 6 in the manufacture of an anti-tumor drug.

8. The use of claim 7, wherein the dose of the nanogel blocking material in the RES-blockade policy-based tumor multistep therapy drug is 100-300 mg/kg; the dosage of the water-soluble small molecule anti-tumor chemotherapeutic in the nano gel anti-tumor drug is 4-6 mg/kg.

9. The use of claim 7, wherein the RES-blockade strategy-based tumor multistep therapy drug is prepared for:

(1) Applying a 100-300 mg/kg dose of the nanogel blocking material to a tumor patient;

(2) And waiting for a time interval of 0.5-4 h, and applying a nanogel anti-tumor drug in the RES-blockade strategy-based tumor multi-step therapy drug to the patient, wherein the dosage of the water-soluble small molecule anti-tumor chemotherapeutic drug in the nanogel anti-tumor drug is 4-6 mg/kg.