CN115477687A - Double-response block copolymer and preparation method and application thereof - Google Patents

Abstract

The invention relates to a double-response block copolymer, a preparation method and application thereof, wherein the block copolymer is a triblock copolymer FA-PEG-GPLGVRG-Plys with side chains connected with camptothecin CPT through disulfide bonds. The invention has good biocompatibility in physiological environment, shows selective rapid drug release and biotoxicity activation in tumor microenvironment (MMP 2 and reduction environment), can enhance the immune response level in vivo and improve the anti-tumor curative effect, and has good application prospect in the aspect of chemical drugs and RNAi combination of tumor to mobilize chemoimmunotherapy.

Description

Double-response block copolymer and preparation method and application thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a dual-response block copolymer, and a preparation method and application thereof.

Background

CD47 is a membrane receptor protein belonging to the immunoglobulin superfamily, and can interact with signal regulatory protein alpha (SIRPa) to inhibit phagocytosis of macrophages. CD47 is involved in a range of cellular activities including apoptosis, proliferation, adhesion and migration, and also plays an important role in immune and angiogenic responses. CD47 is widely expressed in human cells and is overexpressed in a variety of different cancer cells. CD47 thus has potential as a therapeutic target for certain cancers.

Research finds that by blocking a CD47/SIRP alpha immune checkpoint, macrophage migration to tumor tissues can be promoted, tumor-associated macrophages are caused to be functionally converted from a tumor-promoting M2-like phenotype to a tumor-killing M1-like phenotype, and an immune response to tumors is activated, so that tumor-associated macrophages can be promoted to kill tumors.

The method of using the monoclonal antibody against CD47 to bind with CD47, thereby blocking the interaction between CD47 and signal regulatory protein alpha (SIRP alpha) of cancer cells, so as to inhibit the growth and metastasis of tumors has achieved certain effects in the treatment of myeloma, leiomyosarcoma, lymphoma and breast cancer, but because of the wide expression of CD47 in human cells, the use of non-targeting antibodies causes a series of safety problems, thereby limiting the further clinical popularization of antibodies.

PD-1 (programmed death molecule 1) is a receptor protein on the T cell surface of immune cells, which interacts with a protein PD-L1 (apoptosis-ligand 1) expressed on the surface of tumor cells. The PD-L1 protein (apoptosis-ligand 1) is the ligand of PD-1, is related to the inhibition of the immune system, and can conduct inhibitory signals. Once PD-1 and PD-L1 bind, they transmit a negative regulatory signal to T cells, inducing T cells to go to a resting state, reducing T cell proliferation, making T cells unable to recognize cancer cells, and causing T cells to proliferate less or apoptosis, effectively relieving the immune response of the body, so that cancer cells can grow unscrupulously. Satisfactory results in cancer therapy were obtained using antibody blocking therapy (anti-PD-1 and PDL-1 antibodies, such as opdivo, keytruda, tecentriq and MPDL 320A). However, these antibodies are non-specific and can also bind to PD-1 or PDL-1 expressed on normal tissue cells, resulting in some degree of side effects, such as autoimmune diseases and the like.

Although some antibody drugs aiming at tumor immunotherapy are already promoted clinically, compared with small molecule drugs, the antibody drugs have more target protein types, and the affinity of the antibody drugs can be improved by protein engineering technology, so that the toxicity is reduced, and the like. However, antibody drugs have the disadvantages of more complicated molecular structure, higher production cost, and the like, and usually need to be administered by injection, and the antibody drugs usually only can act on proteins on the surface of cell membranes or outside cells, so that the application of the antibody drugs is limited. In contrast, nucleic acid drugs have great advantages.

The nucleic acid medicine can regulate the gene expressing related protein based on the base complementary principle, rather than combine with target protein, and through proper delivery system, the nucleic acid medicine may enter cell to act, so that the nucleic acid medicine may avoid the limitation of incapable medicine target in traditional small molecule medicine and antibody medicine and may regulate both intracellular and extracellular membrane protein. Meanwhile, the function basis of most nucleic acid medicaments is the base complementary pairing principle, the sequence design of the nucleic acid medicaments is very easy only by knowing the base sequence of a target gene, and the design of a chemical modification and delivery system is relatively independent from the design of the sequence; in contrast, in the process of discovering and optimizing small molecule and antibody drugs, the optimization of the properties such as activity, PK/PD and the like needs to change the structure, and a great deal of work is needed. Therefore, the nucleic acid medicine has incomparable advantages with other medicines, is safely and efficiently delivered into cells to play the function of the cells, realizes the long-term inhibition of pathogenic genes, and is an ideal treatment scheme. However, the half-life of nucleic acids in vivo is extremely short, and the degradation efficiency of nucleic acids by nucleases is also high, so that a new nucleic acid drug delivery vehicle needs to be sought.

Disclosure of Invention

The technical problems to be solved by the invention mainly comprise: 1. exogenous nucleic acid molecules need to be prevented from being rapidly cleared and have a prolonged half-life when introduced into the body; 2. nucleic acid drugs are sensitive to nucleases, so that the nucleic acid drugs are protected from degradation by nucleases in the process of in vivo circulation; 3. there is a need to increase the permeability of nucleic acid drugs in tumor tissues; 4. the binding efficiency of the nucleic acid drugs and target cells and the uptake efficiency of the cells are improved; 5. because the lysosome escape capacity of nucleic acid drugs is weak, the efficiency of lysosome escape needs to be improved; 6. the effect of killing tumor by only using nucleic acid medicine is weaker.

The invention provides a double-response type block copolymer, which is a triblock copolymer FA-PEG-GPLGVRG-Plys (ss-CPT) with side chains connected with camptothecin CPT through disulfide bonds.

The invention also provides a preparation method of the double-response block copolymer, which comprises the following steps:

(1) Synthesizing folic acid-polyethylene glycol-peptide segment-amino FA-PEG-GPLGVRG-NH by click reaction between azide and alkyne ₂ A molecule;

(2) With FA-PEG-GPLGVRG-NH ₂ As an initiator, lys (TFA) -NCA is polymerized to a PEG main chain by using NCA ring-opening polymerization reaction initiated by amino, and an amino protecting group TFA on a lysine side chain is removed through hydrolysis reaction to obtain hydrophilic folic acid-polyethylene glycol-peptide segment-polylysine FA-PEG-GPLGVRG-Plys;

(3) Covalently linking CPT-ss-OH prodrug molecule to FA-PEG-GPLGVRG-PLys side chain through carbamate bond to obtain dual-response block copolymer FA-PEG-GPLGVRG-Plys (ss-CPT).

FA-PEG-N in step (1) ₃ And alkyl-GPLGVRG-NH ₂ The mass ratio of (1) is 2.

FA-PEG-GPLGVRG-NH in step (2) ₂ And Lys (TFA) -NCA in a mass ratio of 1:1.

in the step (3), the mass ratio of FA-PEG-GPLGVRG-PLys to CPT-ss-OH is 3:1.

the invention also provides application of the double-response block copolymer in preparation of antitumor drugs.

The double-response block copolymer and siRNA are assembled into a nano prodrug compound FNP (ss-CPT & siRNA) carrying siRNA in aqueous solution through electrostatic interaction and hydrophobic interaction.

The principle of the invention is as follows:

(1) Aiming at the malignant negative regulation of tumor cells to T cells, small interfering RNA (siRNA) is used for silencing PD-L1 gene, the expression of PDL-1 of the tumor cells is reduced, the immune response of effector T cells to the tumor cells is activated, and the immunity of organisms to the tumor cells is increased.

(2) Aiming at the problems of short circulation time and easy degradation of naked siRNA in vivo, PEG-Plys (polyethylene glycol-polylysine) block copolymer is used for forming nanoparticles, so that the biocompatibility of siRNA in vivo is increased.

(3) Aiming at the problem that the formed nanoparticles are not targeted, FA-PEG-Plys block copolymers are formed by using folic acid modification on a carrier PEG-Plys (polyethylene glycol-polylysine), so that the formed nanoparticles have the function of tumor targeting and can increase the uptake of tumor cells to the nanoparticles.

(4) Aiming at the problem that the introduction of PEG (polyethylene glycol) shells increases the biocompatibility of nanoparticles, but also reduces the uptake efficiency of tumor cells to the nanoparticles, GPLGVRG peptide segments are introduced into FA-PEG-Plys block copolymers formed by folic acid modification to form FA-PEG-GPLGVRG-Plys triblock copolymers, wherein the introduced GPLGVRG peptide segments can respond to MMP2 efficiently expressed in the microenvironment of the tumor cells to remove the PEG shells from the nanoparticles, so that the problem of low cell uptake efficiency caused by the introduction of the PEG shells is solved, and the uptake efficiency and the transfection efficiency of the nanoparticles are increased.

(5) Aiming at the problems that the lethality of immunotherapy on tumor cells is weak, the lethality of chemotherapy on the tumor cells is strong, but the immune escape of the tumor cells is increased, the CPT (camptothecin) prodrug is introduced into the nanoparticles and is cooperated with small interfering RNA (si CD47 and si PDL-1) for anti-tumor treatment, so that the immune response of tumors is caused while the tumors are killed, and the anti-tumor effect is enhanced.

The invention verifies that:

1) By using ¹ The correctness of the structures of the F-P-G-P (ss-CPT) molecule and the intermediate thereof is verified by H NMR and FT-IR, and the pyrene fluorescence probe method is used for determiningFA-PEG-GPLGVRG-PLys (ss-CPT) [ abbreviation: F-P-G-P (ss-CPT)]Has a lower CAC (4.5 mg/L).

2) FNP (ss-CPT) [ nanoparticles formed by FA-PEG-GPLGVRG-PLys (ss-CPT) are examined by gel electrophoresis blocking experiments: the adsorption and protection capability of FNP (ss-CPT) ] to siRNA can completely adsorb siRNA when the N/P value is larger than or equal to 4, resist the competition of polyanion when the N/P value is larger than 8, can be stably stored in serum containing 10 percent, and finally, the nano prodrug compound with the N/P value of 16 is selected as a subsequent experimental material.

3) The particle size of the nano prodrug compound FNP (ss-CPT & siRNA) [ nano particles after the FNP (ss-CPT) adsorbs siRNA ] is measured to be about 95nm by using a nano particle sizer, and the surface potential is about-2.14 mV; the TEM picture shows an approximately spherical structure and uniform size distribution, and FNP (ss-CPT & siRNA) has good storage stability in PBS solution; the FNP (ss-CPT & siRNA) is proved to have good biocompatibility in a physiological environment through an erythrocyte hemolysis experiment, and can generate charge reversal and selectively destroy a biological membrane after MMP2 action.

4) The behavior of F-P-G-P (ss-CPT) molecules in response to reduction stimulation and release of CPT through disulfide bond breakage is verified by HPLC; the reverse behavior of FNP (ss-CPT & siRNA) in the presence of MMP2 and GSH (10 mM) in terms of particle size change and surface potential was verified by a nanometer particle sizer, respectively.

5) The release behavior of FNP (ss-CPT & siRNA) in MMP2 and reduction environment is investigated by adopting gel electrophoresis blocking experiment, the siRNA still has good siRNA adsorption capacity after the MMP2 acts, and siRNA is completely released after GSH acts; the dialysis method is used for detecting the release kinetics of FNP (ss-CPT & siRNA) in different environment stimulus responses, and the CPT accumulated release amount reaches 70 percent after incubation for 10 hours in a reducing environment.

6) The high uptake of FNP (ss-CPT & siRNA) by 4T1 cells and the diffusion behavior in cells are proved by LSCM and FCM, the FNP (ss-CPT & siRNA) pretreated by MMP2 shows enhanced intracellular diffusion characteristics, and CPT and sinRA can be rapidly released into cytoplasm to play a role.

7) The cytotoxicity of FNP (ss-CPT & siRNA) to 4T1 cells is detected by a CCK8 method, apoptosis of each group is visually observed by dead and live cell staining, and the result shows that the FNP (ss-CPT & siRNA) and FNP (ss-CPT & siRNA) + MMP2 have smaller IC50 values (1.47 and 1.18 mu M respectively).

8) The interference of siRNA to the mRNA and protein levels of a target gene is examined through qRT-PCR, western Blot and immunofluorescence staining, and the significant inhibition effect of both the mRNA and protein levels of siCD47 and siPDL1 transfected by FNP (ss-CPT) is verified.

9) Establishing 4T1 tumor-bearing mouse model for in vivo gene silencing and cancer inhibition experiment research, and showing FNP (ss-CPT) by result&siRNA) has significantly enhanced blood circulation time and highly efficient tumor enrichment ability; FNP (ss-CPT)&siCD47&siPDL 1) showed an absolutely superior tumor suppression effect in vivo antitumor therapy (IR: 67%); FNP (ss-CPT)&siCD 47) and FNP (ss-CPT)&siCD47&siPDL 1) treatment group, and FNP (ss-CPT)&siPDL 1) and FNP (ss-CPT)&siCD47&sipll 1) treatment group showed gene silencing effect at mRNA and protein levels of CD47 and PDL1, respectively; the expression of CD47 and PDL1 is down-regulated, so that the immunosuppression of macrophages can be relieved, and the CD68 in solid tumors can be increased ⁺ Macrophages and CD4 ⁺ 、CD8 ⁺ Infiltration of T cells; h by observing mouse body weight and vital organs&E, dyeing finds that the siRNA-loaded nano prodrug compound has good biological safety.

Advantageous effects

The invention has good biocompatibility in physiological environment, shows selective rapid drug release and biotoxicity activation in tumor microenvironment (MMP 2 and reducing environment), can enhance immune response level in vivo and improve anti-tumor curative effect, and has good application prospect in the aspect of chemical drugs and RNAi combination of tumor to mobilize chemoimmunotherapy.

Drawings

FIG. 1 shows FA-PEG-GPLGVRG-NH ₂ And (4) synthesizing.

FIG. 2 shows the synthesis of FA-PEG-GPLGVRG-PLys.

FIG. 3 is the synthesis of CPT-ss-OH prodrug molecule.

FIG. 4 shows the synthesis of FA-PEG-GPLGVRG-PLys (ss-CPT).

FIG. 5 shows the synthetic route of FA-PEG-GPLGVRG-PLys (ss-CPT).

Figure 6 is a fourier transform infrared spectrum of CPT prodrugs and intermediates thereof.

FIG. 7 shows FA-PEG-GPLGVRG-NH ₂ And FA-PEG-GPLGVRG-PLys at D ₂ Nuclear magnetic resonance hydrogen spectrum in O.

FIG. 8 shows FA-PEG-GPLGVRG-PLys (ss-CPT) in CDCl ₃ Medium nuclear magnetic resonance hydrogen spectrum.

Figure 9 is a high performance liquid chromatogram of CPT prodrug after GSH incubation.

FIG. 10 is a graph showing the relationship between the fluorescence intensity of pyrene and the concentration of amphipathic molecules.

FIG. 11 shows the result of gel electrophoresis of siRNA-loaded nanomicelle; (a) Gel electrophoresis results of nano prodrug complexes prepared from FNP (SS-CPT) and siRNA at different N/P values; (b) Gel electrophoresis results of FNP (ss-CPT & siRNA) with different N/P values under heparin sodium competition.

Figure 12 is the stability of free siRNA and siRNA loaded nanomicelles in serum.

FIG. 13 a) is the particle size distribution of the nanomicelle complex FNP (ss-CPT) and FNP (ss-CPT & siRNA); b) Is a transmission electron microscope picture; scale bar:100nm.

FIG. 14 shows the change in particle size in FNP (ss-CPT & siRNA) over 24 hours.

FIG. 15 shows the particle size change of FNP (ss-CPT & siRNA) after 2 hours incubation under different stimulation environments (MMP 2 and reducing environments).

FIG. 16 shows the results of gel electrophoresis of FNP (ss-CPT & siRNA) prepared at different N/P values after MMP2 incubation.

FIG. 17 is a transmission electron microscope image of FNP (ss-CPT & siRNA) incubated GSH; scale bar:100nm.

FIG. 18 shows the results of gel electrophoresis of FNP (ss-CPT & siRNA) prepared at different N/P values after GSH incubation.

FIG. 19 shows the cumulative release of CPT under different pH and GSH concentrations of FNP (ss-CPT & siRNA).

FIG. 20 a) is the hemolysis rate of siRNA-loaded nano-prodrug complexes at different concentrations; b) Photograph of 6 hours of incubation.

Fig. 21 a) is the flow cytometer analysis results after 4 h incubation of 4T1 cells with different agents; b) Quantitative for relative fluorescence.

FIG. 22 is a scanning microscope image of laser co-polymerization of free siRNA and FNP (ss-CPT & siRNA) incubated 4T1 cells for 4 hours.

FIG. 23 is a graph of confocal laser imaging after 2 or 8 hours of co-incubation of FNP (ss-CPT & siRNA) with/without MMP2 pretreatment with 4T1 cells.

FIG. 24 shows the effect of gene silencing by siCD47 on 4T1 cells after 48 hours; a) At the gene level; b) Is the expression at the protein level.

FIG. 25 is the gene silencing effect of siPDL1 on 4T1 cells after 48 hours; a) At the gene level; b) Is the expression situation of protein level.

FIG. 26 shows the expression of CD47 (a) and PDL1 (b) genes after different siRNAs were applied to 4T1 cells.

FIG. 27 shows the effect of CPT drugs on PDL1 gene level expression in 4T1 cells.

FIG. 28 is immunostaining for CD47 and PDL1 protein in 4T1 cells.

FIG. 29 shows the expression of CD47 and PDL1 protein levels in 4T1 cells.

Fig. 30 shows cell viability of 4T1 cells incubated with the nano-prodrug complexes of different sirnas after 48 hours.

FIG. 31 a) is the cell viability of 4T1 cells incubated with different concentrations of drug for 48 hours; b) Is the IC50 value.

FIG. 32 shows the staining results of live/dead cells after 4T1 cells were treated with different drug groups.

FIG. 33 is the pharmacokinetics and biodistribution of FNP (ss-CPT & siRNA); a) Pharmacokinetic profiles of free CPT and FNP (ss-CPT & siRNA); b) Drug content in major organs and tumors of mice 24 hours after drug injection.

FIG. 34 is the in vivo anti-tumor therapeutic effect; a) in vivo anti-tumor treatment protocol, b) tumor volume growth curve, c) post-treatment solid tumor photograph, d) post-treatment solid tumor weight and tumor inhibition rate.

Figure 35 is H & E and TUNEL staining of mouse solid tumor tissues after treatment.

FIG. 36 shows the expression of the CD47 (a) and PDL1 (b) genes in 4T 1-bearing tumors.

FIG. 37 shows CD47 and PDL1 protein level expression in 4T1 tumors.

FIG. 38 is immunohistochemical staining of mouse tumor tissue sections.

FIG. 39 shows immunofluorescent staining of tumor tissue sections.

Fig. 40 is a graph of the change in body weight of mice over the treatment period.

FIG. 41 is H & E staining of mouse major organ tissue sections.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

1. Synthesis of FA-PEG-GPLGVRG-Plys (ss-CPT):

1)FA-PEG-GPLGVRG-NH ₂ synthesis of (2)

Synthesis of folate-polyethylene glycol-peptide fragment-amino (FA-PEG-GPLGVRG-NH) with folate end cap by click reaction between azide and alkyne ₂ ) A molecule. The specific synthesis method comprises the following steps: in a 25mL Schlenk flask, dry anhydrous FA-PEG-N was dissolved with 2mL anhydrous DMF ₃ Yellow powder (0.02mmol, 100mg) was added to 2mL of a DMF solution in which alkyl-GPLGVRG (0.06mmol, 50mg) was dissolved. After mixing well, cuBr (35 mg) and TATB (15 mg) were added thereto under dark N ₂ The reaction is carried out for 24 hours under protection. Then, the precipitate was precipitated with glacial ethyl ether and dried to obtain FA-PEG-GPLGVRG-NH ₂ (105 mg) and stored at-20 ℃ until use.

2) Synthesis of FA-PEG-GPLGVRG-PLys

FA-PEG-GPLGVRG-NH ₂ For the initiator, the initiated NCA ring-opening polymerization will Lys (TFA) -NCA was polymerized to a PEG backbone, and an amino protecting group (TFA) in a lysine side chain was removed by hydrolysis to prepare a hydrophilic FA-PEG-GPLGVRG-PLys backbone molecule. First, to FA-PEG-GPLGVRG-NH ₂ Dehydrating, adding FA-PEG-GPLGVRG-NH into a 25mL Schlenk bottle ₂ (543mg, 0.05mmol), dissolved in a small amount of anhydrous DCM, followed by addition of 4mL of toluene and mixing, and draining off the water from the starting material with a cold trap. In an oxygen-free, anhydrous glove box, dry treated FA-PEG-GPLGVRG-NH was dissolved with 5mL of DMF/DCM (1 ₂ Then, 3mL of a DMF/DCM (1. The reaction solution was precipitated with precooled ether and washed to remove unreacted monomers and autopolymers, and finally dried by suction using a cold trap to obtain a white powder (839 mg, yield 89%) which was FA-PEG-GPLGVRG-PLys (TFA) and stored at-20 ℃ for later use.

In a 50mL round-bottomed flask, FA-PEG-GPLGVRG-PLys (TFA) (500 mg) was added and dissolved completely with a methanol solution of NaOH (20mL, 1M), and the reaction was stirred at room temperature for 24 hours. After the reaction, the reaction solution was transferred into a dialysis bag (MWCO: 10 kDa) and dialyzed (2L, 1M HCl aqueous solution and deionized water were each dialyzed for 48 hours, and the solution was replaced once for 8 hours). After dialysis, the mixture was lyophilized to give a white powder (FA-PEG-GPLGVRG-PLys, 410mg, 96% yield), which was stored at-20 ℃ until use.

3) Synthesis of CPT-ss-OH prodrug molecules

In a 250mL reaction flask, dissolve anhydrous CPT powder (500mg, 1.43mmol) with 80mL anhydrous DCM to a suspension in N ₂ 3mL of anhydrous DCM containing triphosgene (157mg, 0.53mmol) dissolved therein was added to the ambient and ice bath, and stirring was continued for 30 minutes while the ice bath was maintained, followed by the addition of anhydrous DCM (10 mL) containing DMAP (560mg, 4.6 mmol) dissolved therein until the CPT was completely dissolved. The mixture was stirred for 1 hour under ice bath and then transferred to room temperature for further reaction in the dark for 1 hour. In N ₂ Anhydrous THF (15 mL) containing 2-hydroxyethyl disulfide (1.75ml, 14.3 mmol) was added under protection and the reaction was carried out at room temperature under dark conditions for 24 hours. Subsequently, 50mL of DCM was added to the reaction solution, and washed successively with 0.1M aqueous HCl solution, saturated NaCl and water 3 times each, the organic phase was collected and dried, and the pressure was reducedThe organic solvent was then removed.

4) Synthesis of FA-PEG-GPLGVRG-PLys (ss-CPT)

CPT-ss-OH is covalently linked to FA-PEG-GPLGVRG-PLys side chains through carbamate bonds to synthesize amphiphilic CPT prodrug molecules, namely folic acid-polyethylene glycol-peptide fragment-polylysine (disulfide bond-CPT). In a 100mL round-bottomed flask, dissolve CPT-ss-OH yellow powder (70mg, 0.13mmol) in 15mL anhydrous DCM ₂ 1mL of anhydrous DCM containing triphosgene (15mg, 0.05mmol) dissolved therein was added at ambient and low temperature and stirring continued with ice bath until complete cooling. Then, DMAP (51mg, 0.42mmol) in dry DCM (1 mL) was added until complete dissolution of CPT-ss-OH, and the reaction was continued for 1 hour under ice-bath conditions, and then continued at room temperature with exclusion of light for 1 hour. Then at N ₂ While protecting, anhydrous DCM (20 mL) containing FA-PEG-GPLGVRG-PLys (210mg, 0.015mmol) was added dropwise, and the mixture was reacted at room temperature for 24 hours with exclusion of light. Subsequently, the reaction solution was precipitated with ether and dried on a cold trap. The resulting solid powder was dissolved in an appropriate amount of DMSO, and dialyzed against DMSO and pure water, respectively. The dialyzed sample was freeze-dried to give FA-PEG-GPLGVRG-PLys (ss-CPT) as a pale yellow powder (yield 71%).

2. The test method comprises the following steps:

(1) determination of critical micelle concentration of amphiphilic molecule FA-PEG-GPLGVRG-PLys (ss-CPT)

The assembling behavior of the amphiphilic molecules in the solvent is closely related to the concentration of the amphiphilic molecules, and when the concentration of the amphiphilic molecules in the solvent is lower than the Critical Aggregation Concentration (CAC), the nano-carrier cannot be formed. Fluorescence emission spectra of pyrene in different solvent environments (excitation wavelength λ) _ex =334 nm), the ratio of the fluorescence intensities (I) at 372nm and 383nm changes markedly ₃₇₂ /I ₃₈₃ ) Particularly obvious, therefore, pyrene is commonly used as a fluorescent probe to determine the critical aggregation concentration of amphiphilic molecules. The specific determination process comprises the following steps: preparation of 6.0X 10 by dissolving pyrene in methanol ^-5 M, spreading 30 mu L of mother liquor at the bottom of the test tube, and volatilizing methanol under the condition of keeping out of the light to form a pyrene film. 3mL of different concentrations (1.0X 10) were added to the tube ^-4 1.0 mg/mL) of amphiphilic molecules (FA-PEG-GPLGVRG-PLys (ss-CPT)) solution (final concentration of pyrene is 7.0X 10 ^-5 M)，And (3) carrying out water bath ultrasound for 30 minutes under the condition of keeping out of the sun, shaking in a shaking table at 37 ℃ for 1 hour, and standing at room temperature for 2 hours. The fluorescence intensity of pyrene in the prepared solution is measured by a fluorescence spectrophotometer, and the detection conditions are as follows: excitation wavelength lambda _ex =334nm, receiving and transmitting wavelength range lambda _em And (5) = 350-450 nm. The fluorescence emission intensities at 372nm and 383nm of different groups of solutions were collected and expressed as I ₃₇₂ /I ₃₈₃ The values are plotted on the ordinate and the log of the concentration of the amphipathic molecule (lg (C)) on the abscissa, to calculate the CAC value of the amphipathic molecule in water.

(2) Preparation and characterization of siRNA-loaded nano-micelle

1) The preparation of the CPT prodrug nano-complex utilizes the hydrophilic and hydrophobic action among CPT molecules to self-assemble into the CPT prodrug nano-complex in a buffer solution. Specifically, F-P-G-P (ss-CPT) was dissolved in PBS buffer (pH 7.4, 10 mM), sonicated in a probe-type sonicator for 10 minutes in an ice bath, and filtered through a 0.45 μm aqueous membrane to obtain a nanocomposite solution.

2) Preparation of siRNA-loaded nano-micelles: to prepare siRNA loaded nanoprecursor complexes, siRNA without enzymatic water dissolution was mixed with CPT prodrug nanoplex solution, the mixture was shaken for 30 seconds and then allowed to stand at room temperature for 30 minutes in the dark. The adsorption capacity of the carrier to siRNA was verified by blocking experiments with agarose gel electrophoresis. The nano prodrug assemblies formed by self-assembly of polymers PEG-PLys (ss-CPT), FA-PEG-GPLGVRG-PLys (ss-CPT)/siRNA, FA-PEG-GPLGVRG-PLys (ss-CPT)/siPDL 1, FA-PEG-GPLGVRG-PLys (ss-CPT)/siCD 47 and FA-PEG-GPLGVRG-PLys (ss-CPT)/siPDL 1/siCD47 are respectively named as NP (ss-CPT), FNP (ss-CPT & siRNA), FNP (ss-CPT & siCD 1), FNP (ss-CPT & siCD 47) and FNP (ss-CPT & siCD 47). Wherein the CPT drug loading in NP (ss-CPT) and FNP (ss-CPT) is 14.9wt% and 18.2wt%, respectively.

3) Characterization of the nanoprecursor complexes: determining the particle size, PDI and surface potential (zeta-potential) of the nano prodrug complex by using a nano particle sizer; the morphology was observed by TEM.

In order to explore the siRNA-carrying properties of the nano-prodrugs, agarose gel electrophoresis blocking experiments were performed. Complexes of FNP (ss-CPT & si RNA) at different nitrogen/phosphorus ratios (N/P, which refers to the ratio of moles of primary amine in the CPT prodrug to moles of phosphate groups in the siRNA) were added to the loading wells, containing 1. Mu.g siRNA per well. Electrophoresis was performed at 120V for 30 minutes, and the siRNA bands were observed using a gel imaging system.

In order to investigate the stability of the siRNA-loaded nano prodrug complex under the anion competition effect, heparin sodium (1 mg/mL) was added to the prepared nano complex, incubated at 37 ℃ for 30 minutes, and subjected to agarose gel electrophoresis.

To further verify the stability of the prodrug nanocomplex in serum, FBS (10%) was added to the prepared prodrug nanocomplex, incubated at 37 ℃, samples were taken at different time points, GSH (10 mM) and heparin sodium (1 mg/mL) were added for incubation, enzyme activity was stopped and siRNA was competitively released, -the samples were stored at 80 ℃, agarose gel electrophoresis was performed after all samples were collected, and the degradation of siRNA in each sample was observed.

In order to verify the storage stability of the siRNA-loaded nano prodrug complex, the prepared sample solution is stored in an environment at 4 ℃ in a dark place, taken out after 12 hours and 24 hours, and the particle size distribution and PDI of the sample solution are measured by a nano particle size analyzer.

The gel electrophoresis blocking experiment comprises the following specific methods: preparing 1.5% agarose gel with TAE buffer solution, heating to dissolve completely, cooling to 50-60 deg.C, adding SuperRed/GelRed (0.01%) and mixing, pouring into gel-making plate, preparing agarose gel, and performing 120V electrophoresis in TAE buffer solution for 30 min.

(3) Reduction/enzyme responsive behavior of siRNA-loaded nanomicelles

1) The FA-PEG-GPLGVRG-PLys (ss-CPT) prodrug molecule reduces and releases CPT, GSH is selected as a reducing agent, the intracytoplasmic environment is simulated, and the action of releasing CPT under the reducing environment by the FA-PEG-GPLGVRG-PLys (ss-CPT) prodrug molecule is detected by HPLC. CPT-ss-OH and F-P-G-P (ss-CPT) samples were tested for peak appearance in CPT and derivatives after incubation for 90 min with/without GSH (10 mM) respectively.

2) Reduction/enzyme response structural characterization of siRNA-loaded nanoprecursor complexes to study the depegylation of FNP (ss-CPT & siRNA) in response to MMP2, activated MMP2 (1 μ g/mL) was added to the prepared nanoprecursor solutions to mimic the tumor extracellular environment and the size of the nanoprecursor size and PDI value after 60 min incubation were determined using a nano-particle sizer.

In order to explore the influence of the FNP (ss-CPT & siRNA) nano-composite subjected to PEG removal on siRNA loading capacity, the nano-composite subjected to MMP2 incubation is subjected to agarose gel to observe the blocking condition of the FNP (ss-CPT & siRNA) nano-composite subjected to PEG removal on siRNA.

(4) Characterization of reduction response drug release behavior of siRNA-loaded nano-micelle

The behavior of CPT release under the environment simulating the reduction in tumor cells of the siRNA-carrying nano prodrug complex is determined by dialysis method, and the experiment shows that (1) the pH value is 7.4, (2) the pH value is 7.4+ GSH (10 mM), (3) the pH value is 5.0, and (4) the pH value is 5.0+ GSH (10 mM).

(5) Evaluation of biocompatibility of siRNA-loaded nanomicelle

The blood compatibility of the FA-PEG-GPLGVRG-PLys (ss-CPT) molecules with the nano-micelles formed by siRNA and the nano-complexes after the de-PEGylation strategy was evaluated by the hemolysis experiment of erythrocytes.

(6) Cellular uptake and intracellular distribution of siRNA-loaded nanomicelles

1) Qualitative analysis of cellular uptake: in order to visually observe the siRNA uptake capacity of the cells, the siRNA content in the 4T1 cells was qualitatively analyzed by a flow cytometer. Cy 3-labeled siRNA (labeled as siRNA) was added to each dish inoculated with 4T1 cells ^Cy3 ) And FNP (ss-CPT)&siRNA ^Cy3 ) (siRNA concentration is 5 nM), further culturing for 4 hours, adding 1. Mu.L Hoechst33342 dye to stain cell nucleus for 15 minutes, washing with PBS for 3 times, and observing intracellular siRNA by flow cytometry ^Cy3 And (3) fluorescence distribution. SiRNA ^Cy3 The detection excitation wavelength was 488nm, and hoechst33342 the detection excitation wavelength was 405nm.

2) Quantitative analysis of cellular uptake: to further examine siRNA uptake by 4T1 cells, FCM was chosen to quantify siRNA uptake by cells. Cy 3-labeled siRNA (Note) in 6-well plates seeded with 4T1 cellsAs siRNA ^Cy3 )、NP(ss-CPT&siRNA ^Cy3 )、FNP(ss-CPT&siRNA ^Cy3 )、FNP(ss-CPT&siRNA ^Cy3 ) + MMP2 (MMP 2 pretreatment 30 min) and Lipo2000&siRNA ^Cy3 (siRNA concentration of 5 nM), continued culture for 4 hours, trypsin digestion and cell collection, cell washing with PBS 2 times, adding 400 u L PBS solution preparation cell suspension, for flow cytometry detection, siRNA ^Cy3 The detection excitation wavelength is 488nm.

3) Visual observation of intracellular distribution of siRNA-loaded nano prodrug complexes: in the experiment, alexa Flour 647-NHS is covalently linked with primary ammonia on FA-PEG-GPLGVRG-PLys (ss-CPT) to form an Alexa Flour 647 labeled FA-PEG-GPLGVRG-PLys (ss-CPT) molecule (denoted as FNP) ^AF674 ) And used for subsequent experiments. The intracellular fluorescence distribution conditions of different incubation times are visually observed through a laser confocal microscope, and FNP is respectively added into a laser confocal dish inoculated with 4T1 cells ^AF647 (ss-CPT&siRNA ^Cy3 ) And FNP pretreated with MMP2 ^AF647 (ss-CPT&siRNA ^Cy3 ) The solution (siRNA concentration of 5 nM), continued to culture for 2 and 8 hours. Washed 2 times with PBS, followed by intracellular fluorescence co-localization distribution by confocal laser microscopy. FNP ^AF674 Detection of siRNA at 635nm excitation wavelength ^Cy3 The excitation wavelength was detected at 488nm.

(7) In vitro gene silencing experiment of siRNA-loaded nano micelle

1) mRNA level Gene silencing efficiency study: to 6-well plates seeded with 4T1 cells were added siNC, siCD47, siPDL1 and siCD47& siPDL1 (siRNA modified by 5'Chol-2' OMe; siRNA final concentration 50 nM) transfected with Lipo2000 and FNP (ss-CPT), respectively, and the culture was continued for 6 hours, after which the complete culture was replaced followed by culture for 42 hours. Total RNA was extracted by TRIzol method, and the transcription level of the target protein (PDL 1 and CD 47) was measured.

2) Protein level gene silencing efficiency investigation: to 6-well plates seeded with 4T1 cells, lipo2000& siRNA and FNP (ss-CPT & siRNA), (siRNA being 5'Chol-2' OMe-modified siRNA including siNC, siPDL1, siCD47 and siPDL1& siCD 47) were added, respectively, and the culture was continued for 6 hours, followed by replacement of the complete culture solution and culture for 42 hours. The cells were collected to extract whole proteins, and the expression levels of the proteins of interest (CD 47 and PDL 1) were measured by the Western Blot method.

3) Cellular level Immunofluorescence (IF) staining: different siRNAs (siNC, siPDL1 and siCD47, all siRNA concentrations were 50 nM) were added to 35-mm dishes seeded with 4T1 cells, and after 6 hours of continuous culture, the complete culture broth was replaced and the culture was continued for 42 hours. Performing immunofluorescence staining:

i. the medium was discarded, rinsed 3 times with pre-cooled PBS for 5 minutes each, and 1mL of paraformaldehyde (4%) solution was added to fix the cells for 1 hour at room temperature.

Carefully remove the liquid, rinse 3 times with pre-cooled PBS (5 minutes each), add 500 μ L of 3% BSA solution to the bottom of the dish and block for 1 hour.

Carefully remove the liquid, add 100 μ L primary antibody (anti-CD 47 or anti-PDL 1) diluted with 3% BSA solution to the bottom of the dish, and incubate in a refrigerator at 4 ℃ overnight.

Carefully remove the liquid, rinse 3 times with pre-cooled PBS (5 minutes each), add 100 μ L of secondary antibody [ CoraLite488-conjugated giated anti-antigen-rabbitIgG (H + L) ] diluted with 3% BSA solution to the bottom of the dish, incubate for 1 hour at room temperature in the dark.

Carefully remove the liquid, rinse 3 times with pre-cooled PBS (5 min each), add 200 μ L PBS solution containing Hoechst33342 dye (5 μ g/mL) to the bottom of the dish, stain the nuclei, incubate at room temperature in the dark for 30 min, rinse 3 times with pre-cooled PBS (5 min each), add 500 μ L PBS, and store in the dark at 4 ℃ in a refrigerator.

And vi, using a laser confocal microscope to collect image analysis, wherein the detection excitation wavelength of the secondary antibody is 488nm, and the detection excitation wavelength of the hoechst33342 is 405nm.

(8) In vitro cancer inhibition experiment of siRNA-loaded nano micelle

1) Cytotoxicity experiments: the toxicity of different samples on 4T1 cells was tested by CCK 8. mu.L of different concentrations of siRNA, CPT, NP (ss-CPT), FNP (ss-CPT & siRNA) and FNP (ss-CPT & siRNA) + MMP2 (MMP 2 pre-treatment for 30 minutes) solutions (N/P: 16, 1, CPT concentrations of 0, 0.01, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 and 100. Mu.M, respectively) were added to the 96-well plates inoculated with 4T1 cells, and the culture was continued in the incubator for 48 hours. Discarding the culture solution, washing with PBS for 2 times, adding 100 μ L of CCK 8-containing culture solution (CCK 8 diluted 10 times) into each well, culturing for 3-4 hr, shaking and mixing uniformly in dark, measuring absorbance at 450nm, and calculating average survival rate of cells in each group. The pure culture solution group is a blank control group.

2) Staining live and dead cells: the cell death status after 24 hours incubation of 4T1 cells with PBS, siRNA, CPT, NP (ss-CPT), FNP (ss-CPT) and FNP (ss-CPT & siRNA) (N/P is 16, CPT concentration is 2. Mu.M) was examined using Calcein-AM/PI live/dead cell double staining kit, respectively.

(9) Pharmacokinetics and tissue distribution in siRNA-loaded nanomicelles

1) Establishing a mouse tumor-bearing model, and when the tumor volume is about 100mm ³ At this time, the subsequent experiments were performed in random groups.

2) Establishing a mouse tumor-bearing model through blood pharmacokinetics research, and when the tumor volume is about 100mm ³ At this time, tumor-bearing mice were randomly grouped (3 mice per group), and free CPT solution or FNP (ss-CPT) was injected via tail vein&siRNA) nano prodrug complex solution, the injection dose is 3mg/kg CPT. Blood was taken at predetermined time points (0, 1,2,4,8, 10, 12, 24 hours) after the injection of the drug, respectively, and the CPT content in plasma [ μ g (drug amount)/mL (plasma volume) was measured].

3) Study of drug distribution in tissues tumor-bearing mice (3 mice per group) were sacrificed 24 hours after drug administration by tail vein injection of the drug and the CPT content in the major organs and tumors was determined [ μ g (drug amount)/g (tissue or organ weight) ].

In vivo cancer inhibition experiment of siRNA carrying in Ribogum

1) In vivo antitumor therapy: tumor-bearing mice were randomly divided into 6 groups (5 mice per group), and PBS, CPT, NP (ss-CPT), FNP (ss-CPT) and the like were administered through tail vein&siCD47)、FNP(ss-CPT&siPDL 1) and FNP (ss-CPT)&siCD47&sipll 1) solution (dose: 3mg/kg CPT and 60nmol/kg siRNA). The administration was performed once every 3 days for 6 times, and the body weight and tumor-bearing volume (mm) of the mice were recorded once every 2 days ³ ). After 19 days of tumor-bearing mice treatment, the mice were sacrificed and solid tumors were removed and weighed for tumor Inhibition (IR).

2) Evaluation of tumor tissue gene silencing efficiency: randomly cutting a solid tumor into small pieces, adding 1mL of TRIzol grinding cells, extracting total RNA in tissues according to a total RNA extraction method, and performing qRT-PCR to determine the mRNA transcription level.

Another tumor tissue is cut into small pieces, precooled protein lysate is added, the tissue is ground in ice bath, holoprotein is extracted according to a holoprotein extraction method and quantified, and a Western Blot method is carried out after high-temperature boiling denaturation to determine the protein translation level

The collected tumor tissues were immersed in 4% paraformaldehyde solution for 24 hours for fixation, paraffin-embedded and sectioned for immunofluorescence staining analysis:

i. tissue sections were deparaffinized.

Antigen microwave repair, adding 10mM citrate buffer, microwave setting temperature 92-96 deg.C, 10 minutes.

iii.3% BSA for 1 h, add primary antibody dropwise and incubate overnight at 4 deg.C, wash with PBS for 5 min (3 times); dripping Cy3 labeled secondary antibody, keeping the room temperature away from light for 1 hour, and washing with PBS for 5 minutes (3 times);

v. Add Hoechst33342 dye dropwise, incubate 30 min at room temperature in the dark, wash 5 min with PBS (3 times).

Sealing the anti-quenching sealing tablet, and storing at 4 ℃ in a dark place.

And vii, observing the prepared section through a laser confocal microscope, collecting images, analyzing, and detecting the excitation wavelength of the secondary antibody 488nm and the excitation wavelength of the hoechst33342 for 405nm.

3) Tissue immunofluorescence staining to detect immune cell infiltration: detection of CD4 in different tumor tissues by immunofluorescence staining ⁺ T、CD8 ⁺ The infiltration of T and macrophages were labeled with anti-CD4, anti-CD8 and anti-CD68, respectively, stained with Cy3 fluorescently labeled secondary antibody, stained with Hoechst33342, observed with confocal laser microscopy, and image analysis was performed.

4) Tissue H & E staining analysis: after the tumor-bearing mice are treated for 3 weeks, the main organs (heart, liver, spleen, lung and kidney) and tumor tissues of the mice are collected and subjected to H & E staining, the prepared sections are observed under an inverted fluorescence microscope, and images are collected for analysis. 5) Apoptosis staining analysis: different tumor tissues are subjected to apoptosis staining analysis by a TdT-mediated deoxyuridine triphosphate Nick-End Labeling (TUNEL), and are subjected to chromogenic Labeling by a DAB chromogenic method (diaminobenzidine method), observed under a fluorescent microscope, and images are collected and analyzed.

3. And (3) test results:

1) Structural characterization and performance analysis of the Block copolymer molecule FA-PEG-GPLGVRG-PLys (ss-CPT):

from the FT-IR spectrum (FIG. 6), it can be seen that the interaction with FA-PEG-N ₃ In contrast, in FA-PEG-GPLGVRG-NH ₂ In the infrared spectrum at 2100cm ^-1 The characteristic absorption peak of the azide disappears, which is attributed to the fact that the azide group is consumed by click reaction, and the original characteristic absorption peak of the azide disappears; furthermore, at 1650cm ^-1 A new absorption peak appeared due to stretching vibration of newly formed carbon-nitrogen bond (C-N = N) and stretching vibration of peptide bond (-CO-NH-) in peptide fragment on the new coupling, indicating successful coupling of peptide fragment at the end of FA-PEG. And 1535cm in the IR spectrum of F-P-G-PLys ^-1 The absorption peak of (2) is a deformation vibration absorption peak in an amido bond (-CO-NH-) on the peptide segment of the polylysine, which indicates that the lysine is successfully polymerized on the PEG main chain. Furthermore, 1750cm appeared in the IR spectrum of F-P-G-P (ss-CPT) compared to F-P-G-PLys ^-1 Absorption peaks attributed to the presence of C = O (carbonate) in the carbonate linkage (-O-CO-O-) on CPT derivatives (CPT-ss-) and in the carbamate linkage (-NH-CO-O-) grafted to lysine side chains, indicate the successful synthesis of FA-PEG-GPLGVRG-PLys (ss-CPT) molecules.

By passing ¹ H NMR further confirmed the correctness of the structures of the FA-PEG-GPLGVRG-PLys (ss-CPT) molecules and their intermediates. As shown in FIG. 7, in D ₂ When O is a solvent, in FA-PEG-GPLGVRG-NH ₂ Of molecules ¹ Chemical positions in H NMR spectrum of delta 7.57 and 6.73ppmThe shift belongs to a proton peak on a benzene ring of folic acid at the end of a PEG chain, the delta 1.66-1.40ppm of chemical shift belongs to a proton peak on a methylene of arginine on the peptide chain, the 0.80ppm of chemical shift belongs to a proton peak on a methyl on the peptide chain, and FA-PEG-GPLGVRG-NH is proved according to the peak position and the integral condition of each proton peak in a spectrogram ₂ Successful formation of the molecule.

In the F-P-G-PLys molecule ¹ In the H NMR spectrum (FIG. 7), 7.75ppm of chemical shift was assigned to the proton peak on the quaternary carbon of folic acid at the end of PEG chain, δ 7.62 and 6.76ppm of chemical shift were assigned to the proton peak on the benzene ring of folic acid, δ 4.24ppm was assigned to the proton peak on the quaternary carbon at α position on lysine, β 3.95-3.40ppm was assigned to the proton peak on methylene (-CH 2-CH 2-) at mPEG main chain, δ 2.94ppm of chemical shift was assigned to the proton peak on methylene at ω position of lysine side chain, δ 1.85-1.25ppm of chemical shift was assigned to the proton peak on methylene at b-d position of lysine side chain, δ 0.85ppm of chemical shift was assigned to the proton peak on methyl group of peptide chain, successful synthesis of F-P-G-PLys molecule was confirmed based on the coupling coefficient of each proton peak and the split of peak in the spectrum, and the degree of polymerization of lysine on F-P-G-PLys was calculated as 25.0.0.0.0.

With CDCl ₃ Determination of FA-PEG-GPLGVRG-PLys (ss-CPT) molecules as solvents ¹ Multiple proton peaks appear at chemical shifts of delta 8.50-7.50ppm in the H NMR spectrum, and aromatic proton peaks attributed to CPT prove the successful synthesis of FA-PEG-GPLGVRG-PLys (ss-CPT) molecules according to the peak positions of the protons in the molecules in FIG. 8. The integral area ratio of the characteristic proton peak on the CPT to the main chain proton peak on the PEG is calculated to obtain that each PEG molecule is averagely grafted with 6.9 CPT molecules.

The peak appearance of FA-PEG-GPLGVRG-PLys (ss-CPT) molecules in the presence/absence of GSH (10 mM) is verified by HPLC, and the result is shown in figure 9, in an HLPC chromatogram of FA-PEG-GPLGVRG-PLys (ss-CPT) after GSH incubation, a new elution peak appears at the position with retention time of 11.69 minutes, and the peak appearance position is the same as that of CPT, which indicates that disulfide bonds in FA-PEG-GPLGVRG-PLys (ss-CPT) molecules can be reduced by GSH, and CPT is released, so that the method lays a foundation for later use in-vitro in-vivo environmental response anticancer technical material.

The Critical Aggregation Concentration (CAC) of the amphipathic molecules FA-PEG-GPLGVRG-PLys (ss-CPT) is determined by pyrene fluorescence probe method, and the result is shown in FIG. 10, and the CAC of the FA-PEG-GPLGVRG-PLys (ss-CPT) molecules is calculated to be as low as 4.5mg/L (3.4X 10) ^-7 mol/L) far lower than other amphiphilic self-assembly nano-carriers in the existing research, which shows that FA-PEG-GPLGVRG-PLys (ss-CPT) molecules can self-assemble to form a nano-composite at a lower concentration, have higher nano-carrier stability when the concentration is higher than the CAC value, can greatly reduce the risk of disassembly in the dilution environment such as blood circulation and the like, and are beneficial to later application in organisms.

2) Structural characterization and stability analysis of the siRNA-loaded nanomicelle:

siRNA adsorption encapsulation capacity of the nano prodrug: the siRNA loading capacity of siRNA-loaded nano prodrug complexes with different N/P values and the stability of siRNA-loaded nano prodrug complexes in anion (heparin sodium) competition are respectively verified through a gel electrophoresis blocking experiment. As shown in FIG. 11 (a), when the N/P value is 4 or more, siRNA can be completely blocked in the loading well, which indicates that FNP (ss-CPT) and siRNA can be completely complexed at this N/P value, which is very advantageous compared to other reported materials, indicating that FNP (ss-CPT) has good adsorption ability to siRNA and can be used as a delivery vehicle for siRNA for subsequent experiments.

The siRNA loading capacity of the siRNA-loaded nano prodrug compound under the action of anion competition is further explored by adding heparin sodium into the prepared nano prodrug compound. As a result, as shown in FIG. 11 (b), after 1mg/mL heparin sodium incubation treatment, siRNA could be completely blocked in the loading well in each group of samples with N/P value >8, indicating that FNP (ss-CPT) has strong siRNA-loading ability, and the CPT prodrug carrier is presumed to be capable of successfully delivering siRNA to tumor sites in response to complex biological environment. In order to improve the bioavailability of siRNA and reduce unnecessary waste, siRNA-loaded nanocomposites used in subsequent experiments were prepared with an N/P value of 16 if not specifically stated.

Serum stability of siRNA loaded nanoprecursor complexes: the stability of FNP (ss-CPT & siRNA) in serum and the ability to protect against ribonuclease were investigated by adding serum to the nano-prodrug complex, and the results are shown in FIG. 12. Within 30 minutes after the initiation of incubation, the band of free siRNA decreased significantly and disappeared completely after 1 hour, indicating that free siRNA was degraded by rnase soon after being exposed to FBS. In contrast, in FNP (ss-CPT & siRNA), although some reduction in siRNA bands was observed at different incubation times, a clear siRNA band was observed after 24 hours of incubation. It is presumed that a small amount of edge siRNA is degraded by nuclease during initial incubation, but due to the protection of the PEG layer of the nanoparticle, macromolecules such as nuclease are prevented from entering the core of the nanoparticle and contacting the siRNA, thereby protecting the siRNA from degradation by ribonuclease and facilitating the siRNA to remain stable during blood circulation.

The particle size distribution (FIG. 13 a), PDI and surface potential of different samples were measured by a nanometer particle sizer, and the specific data are shown in Table 1. The results showed that the average particle sizes of the prepared FNP (ss-CPT) and FNP (ss-CPT & siRNA) were 102.43 nm and 95.09nm, respectively, and the particle sizes showed lower PDI values, indicating that the prepared nanocomposites had better homogeneity. The average particle size of the carrier loaded with siNRA is slightly reduced, which is due to the fact that electrostatic repulsion of cations is reduced after siRNA is coated, the electrostatic action inside the carrier is increased, so that the inside of the carrier is tighter, the slight change of the external dimension is caused, and the improvement of the stability of the carrier and the capability of protecting siRNA can be facilitated. Meanwhile, before and after coating siRNA, the z potential of the nano-composite is changed from +1.85mV to-2.14 mV, which is probably because the addition of the negative charge siRNA neutralizes the positive charge of a part of lysine residues in FNP (ss-CPT), and the outer layer of PEG is added, the final carrier shows a certain negative potential, the negative nano-carrier can reduce the risk of aggregation of the nano-composite to a certain extent, and evades the adsorption of negative charge macromolecules (such as albumin and the like) in the blood circulation process, thereby reducing the clearance of a clearance mechanism by organisms, prolonging the blood circulation time and ensuring that more drugs can be delivered to a target site.

Meanwhile, images of the nano prodrug complex were captured by TEM, and as shown in fig. 13 (b), FNP (ss-CPT) and FNP (ss-CPT & siRNA) nanoparticles appeared spherical in morphology and uniform in particle size distribution.

TABLE 1 Property characteristics of siRNA-loaded nanomicelle complexes

Analyzing the structural stability of the siRNA-loaded nano micelle compound: the storage stability of the nanocomposite in solution was examined by measuring the change in particle size of the siRNA loaded nanoprecursor complex at 4 ℃. As shown in FIG. 14, FNP (ss-CPT & siRNA) showed almost no change in particle size around 24 hours, the average particle size was maintained around 95nm, and no significant change in surface potential was observed, indicating that the nano prodrug complex had good storage stability under physiological conditions.

3) And (3) analyzing the stimulation response capability of the siRNA-loaded nano-micelle compound:

the nano prodrug complex MMP2 stimulates response behavior: the de-PEGylation behaviour of FNP (ss-CPT & siRNA) in response to MMP2 was examined by determining the particle size distribution of FNP (ss-CPT & siRNA) before and after MMP2 incubation. As a result, as shown in fig. 15, particles having a smaller particle size (several tens of nm) appeared after 1 hour of incubation of MMP2 after activation compared to the nanocomposite prepared freshly, which is probably due to the fact that after adding MMP2, part of the nanocomposite had removed the PEG shell and the particle size of the nanocomposite became smaller. In addition, the surface potential of the enzyme-responsive nanocomplex became +7.24mV, and this charge reversal was probably due to the exposure of excess lysine residues by depegylation (positive charge remaining after electrostatic interaction with siRNA). The results show that the nano-composite after MMP2 response has smaller particle size due to the removal of the PEG protective layer, and is beneficial to improving the uptake of tumor cells and the penetration of deep tissues of the tumor; on the other hand, the negative charge PEG layer is removed to expose redundant cations, so that the nano-composite is positively charged and is more easily absorbed by a negatively charged cell membrane, and the endocytosed positively charged nano-composite is easy to break through an endosome membrane and escape into cytoplasm to help the efficient internalization of the drug.

The adsorption capacity of the nano-composite to siRNA after MMP2 response is observed through an agarose gel electrophoresis experiment, and the result is shown in figure 16, when the N/P value is more than or equal to 4, siRNA can still be adsorbed and blocked in the loading hole by the carrier, which shows that the PEG removal effect of MMP2 can not damage the adsorption capacity of the carrier to siRNA. The depegylated nanocomplexes had better blocking ability for siRNA compared to the nanocomplexes before MMP2 incubation (fig. 11 a), probably because the depegylated nanocomplexes exposed more cations and were able to bind more fully to siRNA.

Analysis of ability of the nano prodrug complex to reduce stimulus response: once the PEG-removed siRNA-carrying nano prodrug compound is endocytosed into a cell, the exposed positive charges can help the PEG-removed siRNA-carrying nano prodrug compound escape from an endosome to cytoplasm, and the CPT prodrug is reduced into a CPT technical drug under the action of high GSH reduction level in tumor cytoplasm, so that the hydrophilic-hydrophobic water balance of the nano compound is destroyed, and the released siRNA plays a role in silencing a target protein. GSH was added to the prepared nano prodrug complex to simulate the cytoplasmic reducing environment, and the change in particle size of the incubated nano complex was measured by a nano-particle sizer, and as a result, as shown in fig. 15, after 1 hour of incubation in a 10mM GSH environment, the nano complex showed smaller (tens of nanometers) and larger (hundreds of nanometers and several micrometers) particle size distribution, which is probably because GSH destroyed the disulfide bond connecting CPT, breaking the self-assembly equilibrium of nano-micelles, and the resulting CPT aggregated due to hydrophobicity to show macroscopic precipitation and flocculation. Meanwhile, in the TEM image (fig. 17) of the nanocomposite after GSH incubation, no obvious nanoparticle boundary can be found, indicating that the original morphology of the nanocomposite after GSH action is obviously damaged.

The release behavior of the nano-composite to the siRNA after GSH response was observed through agarose gel electrophoresis experiments, and the results are shown in fig. 18, compared with the group without GSH, after GSH incubation, the siRNA is almost released by the nano-prodrug composites with different N/P values under the competition of heparin sodium, which indicates that GSH destroys the basic structure of the nano-carrier, and significantly reduces the adsorption capacity of the nano-carrier to the siRNA. In conclusion, in a tumor microenvironment, the siRNA carrying nano prodrug compound can respond to MMP2 to generate a de-PEG effect so as to realize endosome escape, respond to GSH in cytoplasm to release CPT and siRNA, and respectively play the interference effects of chemotherapy and RNA.

4) Stimulation response in vitro drug release of siRNA-loaded nano micelle compound

The release behavior of CPT in different tumor microenvironments was studied by dialysis, and FNP (ss-CPT & siRNA) was incubated in PBS buffer with/without GSH (10 mM) to simulate the reducing environment inside/outside tumor cells, and as a result, as shown in fig. 19, the cumulative release amount of CPT after 48 hours was less than 3.5% (almost negligible) in the normal human physiological environment (pH 7.4), even though the cumulative release amount in the acidic endosomal environment (pH 5.0) was only 4.5%. In contrast, the cumulative release of CPT in the neutral reducing environment (pH 7.4, 10mM GSH) was up to 70% in 10 hours, and the cumulative release of CPT in the acidic reducing environment (pH 5.0, 10mM GSH) was up to 91% in 48 hours, all of which showed a total CPT release of 95% or more. Indicating that the prodrug of the nano prodrug complex can completely release the CPT drug after responding to a reducing environment.

5) Evaluation of biocompatibility of siRNA-loaded nano-micelle complex:

the biocompatibility of FNP (ss-CPT & siRNA) was analyzed by erythrocyte hemolysis experiment, and as a result, as shown in FIG. 20, almost no hemolysis of erythrocytes was observed in the FNP (ss-CPT & siRNA) group after 6 hours of incubation with erythrocytes, the hemolysis rate was only 1.5% at a carrier concentration of 0.25mg/mL relative to PBS, and the hemolysis rate was only 4.4% at a concentration of 1 mg/mL. However, the hemolysis rates of Tween 20 in concentrations of 0.25 and 1mg/mL were as high as 83.5% and 92.5%, respectively, and the above results indicate that FNP (ss-CPT & siRNA) has lower hemolysis of erythrocytes.

After the FNP (ss-CPT & siRNA) is incubated by MMP2, positive charges exposed by PEG removal can be adsorbed by cell membranes, and the hemolysis condition of the FNP (ss-CPT & siRNA) pretreated by the MMP2 shows that the hemolysis rate of red blood cells is increased from 1.5% to 11.3% at the concentration of 0.25mg/mL, and is increased from 4.4% to 51.3% at the concentration of 1 mg/mL.

6) The siRNA-loaded nano micelle compound has the following cell uptake capacity and intracellular diffusion:

cellular uptake: quantitative analysis of the condition of promoting 4T1 cells to take up siRNA by different materials through flow cytometry is carried out, and free siRNA is respectively detected ^Cy3 、NP(ss-CPT&siRNA ^Cy3 )、FNP(ss-CPT&siRNA ^Cy3 ) And FNP (ss-CPT)&siRNA ^Cy3 ) + uptake efficiency of MMP2 (MMP 2 pretreatment 30 min) after 4 h incubation of 4T1 cells, lipo2000&siRNA ^Cy3 Is a positive control. As a result, as shown in fig. 21, 4T1 cells hardly absorbed free siRNA, and the efficiency of siRNA absorption was significantly improved in all the other vector groups. Is particularly expressed as FNP (ss-CPT)&siRNA) group is significantly higher than NP (ss-CPT)&siRNA), which may help the endocytosis of folate-modified nanocomposites due to the folate receptor highly expressed by tumor cells, thereby improving FNP (ss-CPT)&siRNA) uptake efficiency; FNP (ss-CPT) after MMP2 pretreatment&siRNA) compared to untreated FNP (ss-CPT)&siRNA) without significant difference, taking into account the previous results that FNP (ss-CPT) after treatment with MMP2&siRNA) has higher hemolytic rate and certain toxicity, so the cellular uptake efficiency of the nano-micelle compound treated by MMP2 is improved to a certain extent.

The uptake of free siRNA and FNP (ss-CPT & siRNA) was visually observed by confocal laser microscopy, and the results are shown in fig. 22, and in the free siRNA group, only a portion of 4T1 cells in the visual field were observed to have weak green fluorescence (Cy 3 labeled siRNA) after 4 hours of co-incubation; however, in the FNP (ss-CPT & siRNA) group, all 4T1 cells in the field showed bright and clear green fluorescence, indicating that the nano prodrug complex can assist cellular internalization of siRNA.

Intracellular diffusion: further, FNP (ss-CPT) was observed by a confocal laser microscope&siRNA) Diffusion behavior after uptake by cells. For ease of observation, F-P-G-P (ss-CPT) and siRNA were labeled with Alexa Fluor 647 and Cy3 dyes, respectively, and counted as FNP ^AF647 And siRNA ^Cy3 The distribution of intracellular fluorescence after 2 or 8 hours of co-culture with 4T1 cells is shown in FIG. 23. It can be observed that FNP (ss-CPT)&siRNA) after 2 hours of incubation, bright red (fluorescence of Alexa Fluor 647) and green fluorescence (fluorescence of Cy 3) appeared in 4T1 cells and had a very high co-localization coefficient (0.92), which decreased to 0.83 after 8 hours of incubation, indicating that the ingested F-P-G-P (ss-CPT) and siRNA separated and diffused to some extent. In contrast, FNP (ss-CPT) pretreated by MMP2&siRNA) group decreased from 0.95 at 2 hours to 0.68 at 8 hours, indicating that the nanocomplexes after MMP2 treatment were more easily separated in the cell. This is probably due to charge reversal after depegylation, which promotes endosome escape of the nanoprobe complex, consistent with the previous dramatic increase in red blood cell hemolysis rate. The nano prodrug complex entering the cytoplasm can respond to GSH and activate the CPT prodrug to release CPT and siRNA.

7) siRNA sequence screening:

silencing of siRNA with different sequences on mRNA and protein levels was detected by qRT-PCR and Western Blot methods.

1) CD47 gene silencing siRNA screening: the patent designs and synthesizes 4 siRNA sequences for silencing mouse-derived CD47, which are respectively counted as CD47-1, CD47-2, CD47-3 and CD47-4, lipo2000 is used as a transfection reagent, siNC is negative control interfering RNA, the expression of each group relative to CD47 mRNA level and protein level is shown in figure 24, the siCD47-3 is observed to have the highest gene silencing effect, and the siCD47-3 is selected as siRNA for silencing CD47 genes in later experiments.

PDL1 gene silencing siRNA screening: the invention designs and synthesizes 4 siRNA sequences for silencing murine PDL1, which are respectively counted as siPDL1-1, siPDL1-2, siPDL1-3 and siPDL1-4, lipo2000 is used as a transfection reagent, siNC is negative control interfering RNA, the expression of each group relative to CD47 mRNA level and protein level is shown in figure 25, the siPDL1-4 is observed to have the highest gene silencing effect, and the siPDL1-4 is selected as siRNA for PDL1 gene silencing in later experiments.

siRNA sequences used

siNC：negative control small interfering RNA

8) In vitro gene silencing evaluation of siRNA-loaded nano-micelle complex

mRNA level expression assay: silencing of CD47 and PDL1 genes in 4T1 cells by modified siRNA (5 'Chol-2' OMe-siRNA) was investigated by qRT-PCR. As shown in FIG. 26 (a), compared with the siNC group, the CD47 gene expression levels in the transfection group containing siCD47 were all significantly reduced, and no significant difference was observed in the expression levels in the siCD47 group, indicating that the modified siRNA did not affect the silencing effect of the siCD47 gene. In addition, the expression level (32.1%) of CD47 mRNA in the FNP (ss-CPT & siCD 47) group was comparable to the level (23.4%) of the Lipo2000& siCD47 group, and the group containing siPDL1 had no significant effect on the expression level of CD47, indicating that FNP (ss-CPT) was able to successfully deliver siCD47 into the cytoplasm to exert a specific inhibitory effect.

Meanwhile, the effect of siPDL1 on the expression level of PDL1 gene was also observed, and the relative expression results of each group are shown in FIG. 26 (b). Compared with the siNC group, the PDL1 gene expression level in the transfection group containing the siPDL1 is obviously reduced, which indicates that the siPDL1 has obvious interference effect. The expression levels of DPL1 of FNP (ss-CPT & siPDL 1) and FNP (ss-CPT & siCD47& siPDL 1) are 40.7% and 36.8%, respectively, and the expression levels are not obviously different from those of the group consisting of Lipo2000& siPDL1 and Lipo2000& siCD47& siPDL1, which indicates that FNP (ss-CPT) has high transfection effect and is consistent with the result of cell uptake.

Compared with siNC, PDL1 expression (158.5%) in the FNP (ss-CPT) treated group was significantly increased, while CD47 expression level was not affected; in contrast, there was also a significant increase in the expression level (130.0%) of PDL1 in the FNP (ss-CPT & siCD 47) group, indicating that CPT could up-regulate the expression of PDL1 to some extent. In addition, the expression level of PDL1 after CPT and FNP (ss-CPT) treatment alone was examined, and as a result, as shown in fig. 27, the PDL1 gene expression level was significantly increased at 1 and 2 μ M CPT concentrations, indicating that CPT at low concentrations was able to up-regulate the expression of PDL 1. Compared with the sNC and the FNP (ss-CPT), the expression level of PDL1 of 4T1 cells in the FNP (ss-CPT & siPDL 1) and FNP (ss-CPT & siCD47& siPDL 1) groups is obviously reduced, the CPT increases the expression level of PDL1 of the tumor cells and partially offsets the silencing level of the siPDL1, and finally the expression of PDL1 of the tumor cells is reduced. In the treatment process of CPT, the introduction of siPDL1 can effectively down-regulate the expression of immune checkpoint PDL1, thereby inducing more efficient tumor killing effect.

Protein level expression assay: the effect of modified siCD47 and siPDL1 (5 'Chol-2' OMe-siRNA) on the expression of CD47 and PDL1 proteins in 4T1 cells was first visualized by immunofluorescence, with sNC as control, CD47 and PDL1 proteins labeled with anti-CD47 and anti-PDL1, respectively, and primary antibodies to CD47 and PDL1 labeled with fluorescent antibodies. The result is shown in fig. 28, it can be seen that the significant green fluorescence in the siNC group, and under the same test conditions, only weak green fluorescence can be seen in the siCD47 group and the siPDL1 group, and the distribution is sparse, which indicates that the modified siCD47 and siPDL1 still have significant silencing effect of protein expression level.

Subsequently, the expression of CD47 and PDL1 protein levels in 4T1 cells was further examined by the Western Blot method, and the results are shown in fig. 29. In the transfection group containing siCD47, the CD47 protein expression level in 4T1 cells is obviously reduced, and compared with Lipo2000& siCD47, the silencing effect of FNP (ss-CPT & siCD 47) is not obviously different. PDL1 has similar protein silencing effect to CD47 and is basically consistent with qRT-PCR results, which shows that FNP (ss-CPT) can successfully deliver siRNA into cytoplasm and play a specific silencing effect on a target gene.

9) In vitro cancer inhibition capability analysis of siRNA-carrying nano micelle compound

Analysis of cancer cytotoxicity: the siRNA used in the experiment is designed and synthesized aiming at 4T1 cells of mouse origin, and 4T1 tumor cells homologous with Balb/c mice are used to cause cellular immunity more easily, so the 4T1 cells are selected as model cancer cells of in vitro and in vivo cancer inhibition experiments in the experiment. To examine the effect of transfection process on 4T1 cell activity, siRNA-loaded nanoprug complexes with N/P value of 16 were selected as the subjects, free siRNA and CPT as controls. The effect of different siRNA sequences on cytotoxicity was first explored and as a result, as shown in fig. 30, the cytotoxicity of siNC, siCD47 and sipl 1 on 4T1 cells at the same FNP (ss-CPT) concentration was not different, so the siNC sequences were used to examine the effect of different nano prodrug complexes on 4T1 cell survival. As a result, as shown in FIG. 31, free siRNA hardly produced cytotoxicity, and the cell survival rate was 95% or more even at 800nM concentration, probably because free siRNA was easily degraded by RNase and hardly taken up by cells, and thus had little effect on the cell survival rate. Based on the data for cell viability in fig. 31 for different groups, IC50 values were calculated for each group (fig. 30 b), and it was found that the toxicity of different nanopharmaceutical complexes was greater than that of free CPT. Interestingly NP (ss-CPT) was similar cytotoxic to free CPT at low concentrations (< 3. Mu.M), but at high concentrations it was significantly more cytotoxic than free CPT, probably related to depletion of intracellular GSH by the CPT prodrug upon release, suggesting that the toxic effect of the responsive prodrug was somewhat superior to that of the free drug. In addition, the cytotoxicity of the surface folate-modified nano prodrug complex (FNP (ss-CPT) and FNP (ss-CPT & siRNA)) is significantly higher than that of the NP (ss-CPT) group, which is probably because the internalization capability of the folate-modified nano material can be promoted by the highly-expressed folate receptor on the surface of the tumor cells. Although the IC50 value (1.47) of FNP (ss-CPT & siRNA) group was slightly smaller than that of FNP (ss-CPT) group (1.56), there was no significant difference, so it could not be stated that the addition of siRNA could increase the toxicity of the nano prodrug complex to 4T1 cells. Earlier uptake experiments showed that depegylation of MMP2 could improve the transfection efficiency of siRNA, and also showed that the uptake of vector was synchronously improved, thus explaining that the cytotoxicity of MMP2 pretreated FNP (ss-CPT & siRNA) group was greater than that of untreated FNP (ss-CPT & siRNA) group.

And (3) staining and detecting dead and live cells: the cell viability of the 4T1 cells incubated with different prodrug complexes for 24 hours was visualized by confocal laser microscopy, and the live and dead cells were selectively stained with green fluorescent calcein AM and red fluorescent PI, respectively, as shown in fig. 32. The red color of dead cells was hardly observed in the field of view of the free siRNA group, indicating that the free siRNA did not kill the cells. The number of cells was significantly reduced in the free CPT, NP (ss-CPT), FNP (ss-CPT) and FNP (ss-CPT & siRNA) groups compared to the PBS group. Compared with the free CPT and NP (ss-CPT) groups, the red fluorescent cells in the FNP (ss-CPT) and FNP (ss-CPT & siRNA) groups are remarkably increased, which indicates that the red fluorescent cells have higher cytotoxicity, and the result is consistent with the cell survival rate determined by a CCK8 method.

10 Analysis of in vivo cancer suppressing ability of siRNA-loaded nanomicelle complexes

A subcutaneous 4T1 cell tumor-bearing mouse model is constructed for evaluating the gene silencing condition and the anti-tumor effect of the siRNA-carrying nano prodrug compound on the living body of animals.

Pharmacokinetic analysis and drug tissue distribution: the pharmacokinetics and biodistribution of FNP (ss-CPT & siRNA) after tail vein injection in mice were first examined and the results are shown in FIG. 33 (a). The concentration of free CPT in plasma is very low (2.33. Mu.g/mL) and as the blood circulates, it drops very quickly to a concentration of only 0.29. Mu.g/mL after 24 hours, indicating that hydrophobic drugs tend to aggregate in the blood and are rapidly cleared. In contrast, the FNP (ss-CPT & siRNA) content (6.22 μ g/mL) in blood was significantly higher than that of the free CPT group, although there was also a time-dependent concentration decrease trend, but the rate of removal was significantly slowed, and the CPT content remained at 1.84 μ g/mL after 24 hours, which is a significant difference probably because the PEG protective layer, proper size and negative charge on the surface of the nano prodrug complex can effectively escape the capture and removal of the reticuloendothelial system (RES), and to some extent, can increase the blood circulation time of the nano drug.

In addition, after 24 hours of tail vein injection of the nano-drug, the distribution of the CPT drug in the major organs and tumors was observed, and the results are shown in fig. 33 (b). Compared with free CPT, CPT in the FNP (ss-CPT & siRNA) group is mainly concentrated at tumor tissues, and the obvious distribution difference is that on one hand, the blood circulation time is increased due to the PEG (polyethylene glycol) concealment capability of the nano prodrug compound, and the enrichment of the drug in the tumor is realized through the EPR (ethylene propylene rubber) effect of the solid tumor; on the other hand, the addition of folic acid in the nano-medicine can actively stay in tumor cells with high expression of folic acid receptors, thereby realizing high-efficiency tumor enrichment capacity. In addition, free CPT and the nano prodrug complex were also distributed in the liver and kidney, indicating that the drug was metabolically cleared in the body via the liver and kidney. In contrast, the concentration of the nano prodrug compound in the liver is lower than that of free CPT, which shows that the PEG nano prodrug compound has certain hiding capacity and can escape from the interception and phagocytosis of macrophages such as Kupffer cells (Kupffer cells) and the like, thereby improving the blood circulation time of the nano prodrug compound and the enrichment at tumor tissues and providing feasibility for the efficient treatment of tumor resistance.

In vivo antitumor effect: the in vivo cancer inhibition capability of the nano prodrug compound is investigated through 4T1 tumor-bearing mice, the tumor volume change curve of each group of mice in the treatment process is determined, after 19 days of treatment, the tumors of the mice are collected, weighed and photographed, and the result is shown in figure 34, compared with the PBS group, the tumor inhibition effect of the free CPT group is not greatly different, on one hand, the reason is probably that the blood circulation time of the hydrophobic CPT drug is short, the hydrophobic CPT drug is easily eliminated by RES, and the drug concentration at the solid tumor is too low, so that the effective treatment effect cannot be obtained; on the other hand, the dosage of the free CPT and the nano prodrug compound are lower, so that the inhibition effect on the tumor growth is weaker. The nano prodrug compound FNP (ss-CPT), FNP (ss-CPT & siCD 47), FNP (ss-CPT & siPDL 1) and FNP (ss-CPT & siCD47& siPDL 1) groups have the advantages that although the tumor volume is continuously increased, the growth is relatively slow, in addition, the tumor inhibition effects are relatively obvious, the tumor inhibition rates are respectively about 33 percent, 45 percent, 51 percent and 67 percent, which are superior to that of free CPT, and the positive correlation exists between the nano prodrug compound FNP and the free CPT, and the long blood circulation time and the high tumor medicine enrichment amount are realized. Among the treatment groups, FNP (ss-CPT & siCD47& siPDL 1) group showed the most significant antitumor effect, showing the least tumor volume.

After 19 days of anti-tumor treatment, tumor tissues of the mice were collected, and H & E staining and TUNEL staining were performed to examine pathological changes and apoptosis of tumor cells, and the results are shown in fig. 35. As can be seen from the results of H & E stained sections, the tumor cells in the PBS group are arranged in a close cell morphology rule, while the FNP (ss-CPT & siCD 47), FNP (ss-CPT & siPDL 1) and FNP (ss-CPT & siCD47& siPDL 1) groups have a reduced number of tumor cells, show a distinct necrotic region by nuclear fragmentation, and are accompanied by an inflammatory cell infiltration phenomenon. In addition, the TUNEL method can reveal apoptotic cells by labeling the dUTP nick ends of the DNA fragments. The results of TUNEL stained sections showed an increase in brown (TUNEL positive) cells in the free CPT and FNP (ss-CPT) groups compared to the PBS control group, indicating that the anticancer drug CPT can increase apoptosis of tumor cells. The FNP (ss-CPT & siCD 47), FNP (ss-CPT & siPDL 1) and FNP (ss-CPT & siCD47& siPDL 1) groups show a large number of TUNEL positive signals, wherein the positive rate in the FNP (ss-CPT & siCD47& siPDL 1) group is more obvious, and the nano prodrug compound is proved to improve the apoptosis level of tumor cells.

In vivo gene silencing: CD47 and PDL1 mRNA transcript levels in tumors of Balb/c tumor-bearing mice after the end of the administration were detected by qRT-PCR experiments, respectively. As shown in FIG. 36 (a), there was no significant difference in the CD47 transcript levels in the free CPT and FNP (ss-CPT) groups compared to the PBS group, and the CD47 expression was significantly reduced in the groups containing siCD47 with a silencing efficiency of about 50%. The expression level of PDL1 was significantly increased in each of the free CPT, FNP (ss-CPT) and FNP (ss-CPT & siCD 47) groups compared to the PBS group (fig. 36 b), because the CPT drug was able to up-regulate the expression of PDL1 after the action, which is consistent with the in vitro cell experiment results. And the mRNA expression level of PDL1 in each group containing siPDL1 is obviously reduced, which shows that siPDL1 can down-regulate the up-regulation expression of PDL1 caused by CPT, thereby achieving the aim of combined treatment. In addition, western Blot analysis was performed on protein levels in tumor tissues, and as shown in fig. 37, in the group containing siCD47 and sipl 1, the levels of protein expression of corresponding CD47 and PDL1 were significantly reduced, which is consistent with the quantitative result in qRT-PCR, indicating that siRNA can significantly inhibit the expression of the target protein.

The results of measuring the expression levels of CD47 and PDL1 in the tumor tissues after the treatment by immunohistochemical method are shown in FIG. 8, and for CD47, the expression level of CD47 was higher in the PBS, CPT, FNP (ss-CPT) and FNP (ss-CPT & siPDL 1) groups than in the FNP (ss-CPT & siCD 47) and FNP (ss-CPT & siCD47& siPDL 1) groups. Similarly, the level of PDL1 protein in the group of sipll 1-loaded nanoprodrug complexes was also significantly reduced, and the expression level of PDL1 in the CPT-containing sipll 1-free group was increased. The above results are consistent with qualitative and quantitative results, so that the siRNA-loaded nano prodrug complex effectively reduces the expression level of the target proteins (CD 47 and PDL 1) in tumor tissues.

Immunological evaluation: the high expression CD47 of the tumor can be combined with a membrane protein SIRP-alpha on macrophages to generate a 'do not eat me' signal, can escape from the monitoring and phagocytosis of the macrophages, and simultaneously prevents the 'isohexia' signal from being presented to T lymphocytes so as to block immune response. Blocking the binding of the antibody to SIRP-alpha through a CD47 antibody or RNAi technology is used for relieving the inhibition effect on macrophages, and then T cells are activated. As shown in FIG. 38, the CD68 protein in the tumor tissue was labeled with Cy3 fluorescent antibody (red fluorescence), and the nucleus was stained with Hoechst33342 (blue fluorescence), and FNP (ss-CPT) was observed&siCD 47) and FNP (ss-CPT)&siCD47&siPDL 1) group exhibited a significant increase in red fluorescence in the field of section, in contrast to the expression level of CD47, CD68 ⁺ Macrophage numbers were inversely correlated with CD47 expression levels, indicating that down-regulation of CD47 can increase CD68 ⁺ Macrophage infiltration.

Research shows that the surface of tumor cells can up-regulate PDL1 expression, can be combined with PD1, and can inhibit proliferation and activation of T lymphocytes, thereby escaping from the elimination of immune cells. Researchers typically restore the body's cellular immune mechanisms by specifically blocking the binding of PD1 and PDL1 through antibody or RNAi technology to eliminate tumor cells. Thus, CD4 in treated tumor tissue was determined by immunofluorescence ⁺ And CD8 ⁺ Infiltration of T cells to further investigate the therapeutic effect of different nanopragent complexes. As shown in FIG. 39, CD4 and CD8 proteins in tumor tissues were labeled with Cy3 fluorescent antibody (red fluorescence), and nuclei were stained with Hoechst33342 (blue fluorescence), and FNP (ss-CPT) was observed&siPDL 1) and FNP (ss-CPT)&siCD47&siPDL 1) group, and the results in FIG. 38 show that PDL1 expression is negatively correlated with the number of tumor-infiltrating T cells, indicating that the down-regulation of PDL1 gene enhances CD4 ⁺ And CD8 ⁺ Infiltration of T cells. Notably, in FNP (ss-CPT)&siCD 47), showing a clear trend of increased red fluorescence, indicating that the down-regulation of CD47 enhances the tumor tissue CD68 ⁺ The number of macrophages, in turn, enhances the infiltration of T cells in tumor tissue.

The results show that the inhibition of the expression of CD47 and PDL1 can relieve the immune suppression of macrophages and promote the proliferation of T lymphocytes on the one hand, and can increase the CD68 in tumors to a certain extent on the other hand ⁺ Macrophages and CD4 ⁺ 、CD8 ⁺ T lymphocyte infiltration. The combination of siCD47 and siPDL1 can improve the immune response level of the organism so as to improve the anti-tumor effect.

Evaluation of biological safety: biosafety evaluations were performed by monitoring the body weight changes of the mice during treatment and pathological analysis of major organs. The body weight change curves of the mice in each group are shown in fig. 40, and it can be clearly observed that the body weight of the mice in the PBS group rises faster, probably because the tumor cells proliferate faster; the body weight of the free CPT group mice is slowly increased even without increasing, but the tumor growth speed of the free CPT group mice is similar to that of PBS (phosphate buffer solution), which indicates that the body weight of the mice except the tumors has a descending trend, and also indicates that the free CPT has certain systemic toxicity in vivo; however, mice with the other 4 nano prodrug complex groups showed a small upward trend in body weight, indicating that the nano prodrug complexes did not cause significant toxic side effects.

After the treatment is finished, the H & E staining results of the major organs of the mice are shown in fig. 41, compared with the PBS group, the tissue sections of the tissues and organs in the 4 kinds of nano prodrug compound groups have substantially no morphological difference, which indicates that the nano prodrug compound has good biological safety and is relatively safe in vivo, and can be tried for preclinical testing.

Claims (7)

1. A block copolymer of the dual response type, characterized in that: the segmented copolymer is a triblock copolymer FA-PEG-GPLGVRG-Plys (FA-PEG-GPLGVRG-Plys) with side chains connected with camptothecin CPT through disulfide bonds, and is marked as FA-PEG-GPLGVRG-Plys (ss-CPT).

2. A method for preparing a dual-response block copolymer, comprising the steps of:

(1) Synthesizing folic acid-polyethylene glycol-peptide segment-amino FA-PEG-GPLGVRG-NH by click reaction between azide and alkyne ₂ A molecule;

(2) With FA-PEG-GPLGVRG-NH ₂ As an initiator, lys (TFA) -NCA is polymerized to a PEG main chain by using NCA ring-opening polymerization reaction initiated by amino, and an amino protecting group TFA on a lysine side chain is removed through hydrolysis reaction to obtain hydrophilic folic acid-polyethylene glycol-peptide segment-polylysine FA-PEG-GPLGVRG-Plys;

(3) Covalently linking CPT-ss-OH prodrug molecule to FA-PEG-GPLGVRG-PLys side chain through carbamate bond to obtain dual-response block copolymer FA-PEG-GPLGVRG-Plys (ss-CPT).

3. The production method according to claim 2, characterized in that: FA-PEG-N in step (1) ₃ And alkyl-GPLGVRG-NH ₂ The mass ratio of (1) is 2.

4. The method of claim 2, wherein: FA-PEG-GPLGVRG-NH in step (2) ₂ And Lys (TFA) -NCA at a mass ratio of 1.

5. The method of claim 2, wherein: in the step (3), the mass ratio of FA-PEG-GPLGVRG-PLys to CPT-ss-OH is 3.

6. Use of the block copolymer of claim 1 for the preparation of an antitumor drug.

7. Use according to claim 6, characterized in that: the double-response block copolymer and siRNA are assembled into a nano prodrug compound FNP (ss-CPT & siRNA) carrying siRNA in aqueous solution through electrostatic interaction and hydrophobic interaction.

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