CN115232306B

CN115232306B - Fluorinated modified linear poly beta-amino ester and preparation method and application thereof

Info

Publication number: CN115232306B
Application number: CN202210801394.5A
Authority: CN
Inventors: 殷黎晨; 吴凡
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2023-11-24
Anticipated expiration: 2042-07-08
Also published as: CN115232306A

Abstract

The invention discloses a fluorinated modified linear poly (beta-amino ester), a preparation method and application thereof, wherein fluorine-containing diacrylate and amino alcohol are used as monomers to synthesize the poly (beta-amino ester) through Michael addition reaction, and amine molecules are used for end capping to obtain the fluorinated modified linear poly (beta-amino ester). The prepared fluorinated modified linear poly (beta-amino ester) can be used as a carrier for simultaneously delivering oxygen and gene drugs into cells, wherein the oxygen can enhance DNA damage caused by ionizing radiation, the gene drugs can efficiently sensitize tumor radiotherapy by changing specific gene expression in tumor cells, and the combined action of the oxygen and the gene drugs can provide a new strategy for reducing radiotherapy resistance and enhancing tumor radiotherapy.

Description

Fluorinated modified linear poly beta-amino ester and preparation method and application thereof

Technical Field

The invention relates to the fields of chemical synthesis and biological medicine, in particular to a fluorinated modified linear poly beta-amino ester, a preparation method and application thereof.

Background

Radiotherapy is one of the most important modes of clinical treatment of malignant tumors, which can cause DNA damage by ionizing radiation (e.g., X-rays or gamma rays) and thus kill tumor cells. During radiation therapy, oxygen can stabilize ionizing radiation-induced DNA damage by providing lone pair electrons and promoting the production of Reactive Oxygen Species (ROS). Therefore, the oxygen content of the radiotherapy part has a critical influence on the radiotherapy effect. However, due to the abnormal vasculature of tumors, the Tumor Microenvironment (TME) of most solid tumors is highly hypoxic, resulting in resistance of the solid tumor to radiation therapy. At the same time, clinical radiotherapy faces another serious challenge, and radiotherapy is usually a local treatment, so that the far-end metastasis which is generated by tumor cannot be inhibited.

The strategy for improving the oxygen content of the tumor microenvironment can greatly improve the radiotherapy effect, and simultaneously does not cause additional toxic or side effect on the organism, so that the method is widely focused. Oxygen is a well-known sensitizer for radiotherapy, which is capable of fixing DNA molecular damage generated by radiation and promoting active oxygen production to promote death of tumor cells. The oxygen delivery carrier can directly release the oxygen at the tumor part by directly delivering the oxygen to the tumor part, and the existing oxygen delivery carrier can be used as an oxygen carrier material, such as Perfluorocarbon (PFC), and the like, has extremely high oxygen binding capacity, can adsorb oxygen in a high-oxygen environment and release the adsorbed oxygen in a hypoxia environment, but the existing oxygen delivery carrier is single in function, and the in-vivo clearance path is not clear.

Gene therapy is an emerging tumor treatment means, and the purpose of tumor prevention or treatment is achieved by delivering exogenous RNA or DNA into cells to influence the expression of related genes. The immune system is an important target for cancer gene therapy. The immune gene therapy of tumor can directly act on tumor cells to induce their immunogenic death or increase their sensitivity to the immune system of organism, and can also make target cells release immune stimulating cytokines such as GM-CSF, IL-12, CD40L, etc. by means of gene therapy. However, since gene drugs are easily degraded by in vivo nucleases and are themselves difficult to penetrate cell membranes, efficient delivery vehicles are required. Among them, cationic polymers are one of the most common gene vectors. However, most cationic polymers have low endocytosis levels and are difficult to escape from the inclusion bodies, and thus, their gene transfection efficiency is poor.

Therefore, an effective strategy is needed to be found, and the oxygen is delivered to the tumor part to relieve the hypoxia condition of the tumor microenvironment so as to enhance the damage of ionizing radiation to solid tumors, and meanwhile, the gene medicine is delivered efficiently to regulate the process of anti-tumor immunity induced by radiotherapy so as to realize comprehensive sensitization radiotherapy.

Disclosure of Invention

The invention aims to provide a fluorinated modified linear poly beta-amino ester, a preparation method and application thereof, and the fluorinated modified linear poly beta-amino ester can be used as a carrier for simultaneously delivering oxygen and gene drugs into cells, wherein the oxygen can enhance DNA damage caused by ionizing radiation, and the gene drugs can effectively sensitize tumor radiotherapy by changing specific gene expression in tumor cells and the combined action of the oxygen and the gene drugs.

In order to solve the technical problems, the invention provides the following technical scheme:

the first aspect of the invention provides a fluorinated modified linear poly-beta-amino ester having the following structural formula:

wherein n is an integer of 10 to 30; r is a C1-C20 fluorine-containing alkylene group; r is R ₁ Alkyl of C2-C10 substituted by hydroxy; r is R ₂ Is one of substituted or unsubstituted C3-C20 alkyl and substituted or unsubstituted C3-C20 heteroalkyl, wherein the C3-C20 heteroalkyl is at least one C-substituted alkyl with O or N.

Further, the substituent is a hydroxyl group or an amino group.

Further, R ism is an integer of 1 to 20; wherein is the site of attachment.

Further, the R ₁ Is thatWherein is the site of attachment.

Further, the R ₂ Is one of the following structures:

wherein is the site of attachment.

According to the second aspect of the invention, the fluorinated linear poly-beta-amino ester is prepared by taking fluorine-containing diacrylate and amino alcohol as monomers, synthesizing the poly-beta-amino ester through Michael addition reaction, and then blocking with amine molecules.

Further, the fluorine-containing diacrylate has the following structural general formula:

wherein m is an integer of 1 to 20.

Further, the amino alcohol is 4-amino-1-butanol or 5-amino-1-pentanol.

Further, the molar ratio of the fluorine-containing diacrylate to the amino alcohol is 1:0.8 to 1.5.

Further, the temperature of the Michael addition reaction is 30 to 80 ℃, for example 50 ℃.

Further, the preparation method further comprises the operation of carrying out multiple sedimentation on reactants of the Michael addition reaction and then vacuum-pumping the solvent.

Further, the specific operation of using amine molecules for the end capping treatment is: and dissolving the synthesized poly beta-amino ester and amine molecules in dichloromethane, and stirring for reaction to obtain the fluorinated modified linear poly beta-amino ester.

Further, the amine molecule is selected from one of spermine, 1, 3-diaminopropane, 1, 3-diamino-2, 2-dimethylpropane, 1, 3-pentanediamine, 2-methyl-1, 5-pentanediamine, 1, 11-diamino-3, 6, 9-trioxaundecane, 2- [ (3-aminopropyl) amine ] ethanol and 1- (3-aminopropyl) -4-methylpiperazine, and the corresponding structural formula is shown as follows:

further, the molar ratio of the fluorine-containing diacrylate to the amine molecule is 1:0.8 to 1.5.

Further, the molar ratio of fluorine-containing diacrylate, amino alcohol and amine molecule is 1.2:1:1.

the third aspect of the invention provides an application of the fluorinated modified linear poly beta-amino ester in preparing a sensitized anti-tumor radiotherapy medicament and/or a tumor gene therapy medicament.

Further, the fluorinated modified linear poly-beta-amino esters are useful as carriers for delivery of sensitizers and/or gene pharmaceuticals.

Further, the sensitizer is oxygen.

Further, the gene medicine is DNA, mRNA, siRNA or miRNA for promoting anti-tumor immune response.

Further, the fluorinated modified linear poly- β -amino ester is condensed with a genetic drug to form a nanocomposite.

Further, the nanocomposite is subjected to oxygen pre-saturation treatment, and the treated nanocomposite is used for simultaneously delivering oxygen and a genetic drug to a solid tumor.

The prepared fluorinated modified linear poly beta-amino ester is condensed with a gene drug to form a nano-composite, the nano-composite is injected to a tumor part after oxygen pre-saturation treatment, and the nano-composite can release oxygen at the tumor part to relieve hypoxia of a tumor microenvironment, so that the killing of ionizing radiation on tumor cells is enhanced; meanwhile, the formed nano-composite can be efficiently endocytosed by the tumor, and escapes from an endosome through a proton sponge effect, so that a gene medicine is released in tumor cytoplasm, and the expression of a target gene is changed, thereby realizing the purpose of gene therapy.

The invention has the beneficial effects that:

1. the invention designs a fluorinated modified linear poly beta-amino ester, which can simultaneously realize high-efficiency delivery of oxygen and gene drugs to tumor parts by introducing fluorine chains on a classical gene delivery carrier poly beta-amino ester and utilizing the unique fluorine effect of fluorine atoms; the fluorinated modified linear poly beta-amino ester can be condensed with a gene drug to form a nano-composite, and the nano-composite is injected to a tumor part after oxygen pre-saturation treatment, so that the radiotherapy can be effectively sensitized and the anti-tumor immunity caused by the radiotherapy can be enhanced, and the inhibition of the radiotherapy on the solid tumor can be enhanced; and simultaneously, the combination of the treatment of the apoptosis receptor-1 antibody (alpha PD-L1) can further strengthen the radiation therapy to trigger the systemic anti-tumor immune response and realize effective far-end tumor inhibition.

2. The fluorinated modified linear poly beta-amino ester is prepared by polymerizing two monomers, namely fluorine-containing diacrylate and amino alcohol, and using amine molecules to carry out end capping, and the preparation method is simple and the process is controllable; the fluorinated modified linear poly beta-amino ester has low cytotoxicity, can be used as a carrier for simultaneously delivering oxygen and gene medicaments, has remarkable treatment effect, and provides a new strategy for clinical application of radiotherapy sensitization.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of milky white liquid (a) and fluorinated modified linear poly-beta-amino ester (b) prepared in example 1;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the linear poly-beta-amino ester prepared in comparative example 1;

FIG. 3 is a graph showing particle size of nanocomposite formed by self-assembly of fluorinated modified linear poly- β -amino esters capped with different capping agents and siRNA;

FIG. 4 shows cytotoxicity of the fluorinated modified linear poly- β -amino esters prepared in example 1 and the linear poly- β -amino esters prepared in comparative example 1 to B16F10 cells at different concentrations;

FIG. 5 is f at different polymer/siRNA mass ratios ₈ Cytotoxicity of PsA, cPsA and PEI/siADAR1 nanocomplex on B16F10 cells; f (f) ₈ PsA is a nanocomposite obtained by condensing fluorinated modified linear poly beta-amino ester prepared in example 1 with siADAR1, cPsA is a nanocomposite obtained by condensing linear poly beta-amino ester prepared in comparative example 1 with siADAR1, and PEI/siADAR1 is a nanocomposite obtained by condensing Polyethyleneimine (PEI) with siADAR 1;

FIG. 6 is an oxygen carrying capacity of water, a fluorinated modified linear poly- β -amino ester nanocomposite with a backbone having fluorine numbers of 6, 8, 10, and a linear poly- β -amino ester nanocomposite with a backbone free of fluorine;

FIG. 7 is a photoacoustic imaging of B16F10 tumor oxygenation status before and 1, 2 and 4 hours after intratumoral injection of the fluorinated modified linear poly- β -amino ester nanocomposite and linear poly- β -amino ester nanocomposite, with tumor sites circled;

FIG. 8 shows endocytic levels of B16F10 cells after 4 hours of treatment with different polymer/Cy 3-siRNA nanocomposites;

FIG. 9 is a fluorescence confocal image of B16F10 cells after 4 hours of treatment with different polymer/Cy 3-siRNA nanocomposites, the figures representing co-localization rates of Cy3-siRNA with Lysotracker Deep Red stained inclusion bodies;

FIG. 10 shows the relative expression levels of ADAR1mRNA in B16F10 cells after various nanocomposite treatments;

FIG. 11 shows the relative expression levels of ADAR1mRNA in B16F10 tumors 24 hours after various intratumoral injections of the nanocomposites;

FIG. 12 is a graph showing the average tumor growth of mice in each group after various treatments;

FIG. 13 is a graph showing survival of mice in each group after various treatments;

FIG. 14 is an average tumor growth curve of distal tumors of each group of distal tumor model mice after various treatments;

FIG. 15 is a graph showing survival of mice from each group of distant tumor model mice after various treatments;

FIG. 16 shows maturation of mouse dendritic cells after various treatments;

FIG. 17 shows CD8 in B16F10 tumors of mice after various treatments ⁺ Quantitative statistics of effector T cells;

FIG. 18 shows TNF- α, IFN- γ and IFN- β concentrations in B16F10 tumors after various treatments;

FIG. 19 shows the concentration of TNF- α and IFN- γ in serum after various treatments;

in FIGS. 10 to 19, fPsA is denoted by f ₈ PsA, fPsN are all denoted f ₈ PsN。

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Example 1

This example relates to the preparation of fluorinated modified linear poly-beta-amino esters, the synthetic route being as follows:

the specific preparation process is as follows:

(1) Octafluoro-1, 6-hexanediol diacrylate (222 mg,0.6 mmol) and 4-amino-1-butanol (45 mg,0.5 mmol) were mixed, stirred at 50℃for 48 hours, then settled with glacial ethyl ether 3 times, and the solvent was removed in vacuo to give a milky white liquid.

(2) Dissolving the milky white liquid (200 mg) prepared in the step (1) and spermine (101 mg,0.5 mmol) in dichloromethane (1 mL), stirring for 4 hours at 50 ℃, then settling for 3 times with glacial ethyl ether, and vacuum pumping the solvent to obtain yellow liquid, namely the fluorinated modified linear poly beta-amino ester, abbreviated as f ₈ PBAE。

For the milky white liquid prepared in step (1) and f prepared in this example ₈ Nuclear magnetic hydrogen spectrum by PBAE ¹ H NMR) of the sample to be analyzed, ¹ the H NMR results are shown in FIG. 1, FIG. 1 (a) shows a milky white liquid ¹ H NMR, FIG. 1 (b) is f ₈ PBAE (PBAE) ¹ H NMR chart; further to f ₈ Characterization by Gel Permeation Chromatography (GPC) of PBAE, molecular weight M was determined _n 8000, M _w 11300 and a molecular weight distribution width of 1.41.

Example 2

This example relates to the preparation of a fluorinated linear poly-beta-amino ester by the same synthesis as in example 1, with the reactant octafluoro-1, 6-hexanediol diacrylate being replaced by hexafluoro-1, 5-pentanediol diacrylate to give a fluorinated linear poly-beta-amino ester, abbreviated as f ₆ PBAE。

Example 3

This example relates to the preparation of a fluorinated linear poly-beta-amino ester by the same synthesis as in example 1, with the reactant octafluoro-1, 6-hexanediol diacrylate being replaced by decafluoro-1, 7-heptanediol diacrylate to give a fluorinated linear poly-beta-amino ester, abbreviated as f ₁₀ PBAE。

Example 4

This example relates to fluorinated modified linear poly-beta-amino esters prepared from different capping agents by the same synthesis method as example 1, wherein the capping agents, spermine, are replaced by 1, 3-diaminopropane, 1, 3-diamino-2, 2-dimethylpropane, 1, 3-pentanediamine, 2-methyl-1, 5-pentanediamine, 1, 11-diamino-3, 6, 9-trioxaundecane, 2- [ (3-aminopropyl) amine ] ethanol, and 1- (3-aminopropyl) -4-methylpiperazine, respectively, to prepare the fluorinated modified linear poly-beta-amino esters with different capping agents.

Comparative example 1

This comparative example relates to the preparation of a linear poly-beta-amino ester, which is prepared by replacing only the reactant octafluoro-1, 6-hexanediol diacrylate with 1, 6-hexanediol diacrylate, as in example 1, to give a linear poly-beta-amino ester, abbreviated cPBAE.

The prepared cPBAE is subjected to ¹ The characterization by H NMR was carried out, ¹ h NMR results are shown in fig. 2; further characterization of cPBAE by Gel Permeation Chromatography (GPC), molecular weight M was determined _n 8500, M _w 13000 the molecular weight distribution width was 1.53.

Performance study

1. Effect of different capping agents on nanocomposite particle size

Preparation of nanocomposites: f prepared in example 1 and example 4 using different end-capping agents ₈ PBAE were dissolved in diethyl pyrocarbonate (DEPC) water, each configured as 5mg/mL stock solution, and then mixed with DEPC aqueous solutions (0.1 mg/mL) of negatively charged siRNA, respectively, at the same mass ratio (polymer/sirna=100:1). Vortex for 15 seconds, incubate for 30 minutes at 37 ℃, different nanocomposites can be obtained.

The particle size of the nanocomposite formed by the fluorinated modified linear poly-beta-amino ester capped with different capping agents and the siRNA is characterized by using a nano-particle size meter, the characterization result is shown in figure 3, the particle size of the nanocomposite is related to the type of the fluorinated modified linear poly-beta-amino ester capping agent, and when the capping agent adopts spermine, the particle size of the obtained nanocomposite is minimum and is about 200nm.

2. Cytotoxicity of cells

(1) Investigation of Polymer f ₈ Cytotoxicity of PBAE and cPBAE

B16F10 cells were seeded into 96-well plates (1.0X10) ⁴ Cells/well), for 24 hours. Replace medium with 90. Mu.L/well serum free DMEM (Dulbecco's modified Eagle medium), mix f ₈ PBAE, cPBAE or PBS were added to the wells and the following three groups of experiments were performed depending on the type of substance added: f (f) ₈ PBAE group, cPBAE group and blank group with PBS added, each set with the following concentration gradients: 10. 20, 40, 60, 80, 100. Mu.g/mL, incubation at 37℃for 4 hours, and further incubation was continued for 20 hours with serum-free DMEM replaced with DMEM containing 10% FBS, and cell viability was determined by MTT (3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyltetrazolium bromide).

The test results are shown in FIG. 4, where fPBAE refers to f ₈ The cell viability of the PBAE, on the ordinate, is the percentage of the absorbance of cells after sample treatment compared to the absorbance of the blank cells. cPBAE showed concentration-dependent cytotoxicity with a cell viability of about 40% at a concentration of cPBAE of 100 μg/mL. And f ₈ The cell survival rate of the PBAE group is more than 80% in the concentration range of 10-100 mug/mL, indicating f ₈ PBAEs have lower cytotoxicity, mainly because fluorinated modifications can reduce the interaction of cationic polymers with cell membranes, thus reducing their damage to cell membranes.

(2) Investigation from different polymers f ₈ Cytotoxicity of PBAE, cPBAE, PEI and siADAR 1-prepared nanocomposites

According to the method for preparing the nanocomposite, different polymers f are prepared ₈ PBAE, cPBAE, PEI preparation of f with different mass ratios with siADAR1 respectively ₈ PBAE/siADAR1 (abbreviated as f ₈ PsA)、cPBAE/siADAR1(Abbreviated cPsA), PEI/siADAR1 nanocomplex.

To examine cytotoxicity of different polymers/siADAR 1 nanocomposites in different ratios, B16F10 cells were seeded into 96-well plates (1.0X10) ⁴ Cells/well), for 24 hours. The medium was replaced with serum-free DMEM (90. Mu.L/well) and f was then applied ₈ PsA, cPsA or PEI/siADAR1 nanocomplex (polymer/siADAR 1 mass ratio 60, 80, 100 or 120, 1. Mu.g siADAR1/mL, 10. Mu.L) was added to the wells, incubated at 37℃for 4 hours, and serum-free DMEM was replaced with DMEM containing 10% FBS for an additional 20 hours, and the viability of the cells was determined by MTT method.

The test results are shown in FIG. 5, where fPBAE refers to f ₈ PsA, cPBAE refers to cPsA and PEI refers to PEI/siADAR1. From the figure, f ₈ The cytotoxicity of the PsA nanocomposite is much less than that of the cPsA and PEI/siADAR1 nanocomposite. And at f ₈ The cell viability of the PsA nanocomposite was still greater than 80% at a mass ratio of polymer to siADAR1 of up to 120.

From the above test results, it is apparent that the fluorinated modified linear poly-beta-amino ester prepared by the present invention and the nanocomposite prepared from the condensed gene drug thereof all show low cytotoxicity.

3. Oxygen carrying capacity

(1) Investigation from different polymers f ₈ PBAE、f ₆ PBAE、f ₁₀ Oxygen carrying capacity of PBAE, cPBAE and siADAR1 prepared nanocomposites

According to the above-mentioned method for preparing nanocomposite, the monomer prepared in examples 1 to 3 contains fluorinated modified linear poly-beta-amino ester f having 8,6, 10 fluorine atoms ₈ PBAE、f ₆ PBAE、f ₁₀ PBAE and the monomer prepared in comparative example 1 straight-chain, fluorine-free, poly-beta-amino ester cPBAE were prepared with the same mass ratio f (polymer/siADAR 1 mass ratio 100) as siADAR1, respectively ₈ PsA、f ₆ PsA、f ₁₀ PsA, cPsA nanocomplexes.

The different nanocomposites prepared above were placed in a sterile oxygen chamber (oxygen flow = 5L/min) and oxygen was bubbled for 5 min to obtain an oxygen pre-saturated nanocomposite. The oxygen pre-saturated nanocomposite (2 mL) was diluted with deionized water (4 mL) and the oxygen concentration of the solution was measured by a portable oxygen meter.

As shown in FIG. 6, the addition of oxygen to water presaturated the monomer backbone containing eight fluorine atoms was measured for f ₈ After PsA, the oxygen concentration in the system increased from 8.32mg/L to 11.92mg/L in 100 seconds, and the oxygen concentration could be maintained for more than 15 minutes, indicating f ₈ The PsA nanocomposite has good oxygen carrying capacity. While adding oxygen pre-saturated cPsA, f to water ₆ PsA or f ₁₀ The oxygen concentration in the system after PsA is significantly lower than f ₈ PsA, which indicates that the polymerization oxygen carrying capacity is strongest when the number of fluorine atoms in the monomer backbone is eight.

(2) Nanocomposite to alleviate tumor microenvironment hypoxia

The hypoxia level of the tumor tissue after different nano-composites is further measured by photo-acoustic imaging in the tumor-bearing mice of B16F 10. Tumor volume was selected to be about 100mm ³ B16F10 tumor-bearing mice of (2), respectively intratumorally injecting freshly prepared oxygen pre-saturated F ₈ PsA or cPsA nanocomposite (0.5 mg siADAR1/kg, 50. Mu.L). The tumor sites were photo-acoustic imaged in Oxy-hem dual spectral mode (750 and 850 nm) using a Visualsonic Vevo LAZER photo-acoustic (PA) imaging system 0,1, 2 and 4 hours after intratumoral injection. The total tumor oxygen saturation was analyzed by the data analysis software of the Vevo LAZER imaging system and the results are shown in fig. 7. The results show that the injection of oxygen presaturated f ₈ After PsA nanocomposite, intratumoral oxyhemoglobin signal was significantly enhanced and remained almost unchanged for 4 hours. In contrast, little oxyhemoglobin signal was detected within 4 hours after injection of the oxygen pre-saturated cPsA nanocomposite. The above results demonstrate that f, pre-saturated by injection of oxygen ₈ The PsA nano-composite can effectively relieve the microenvironment of hypoxia in tumors.

4. Effects of fluorinated modified Linear Polybeta-amino esters on intracellular kinetics of tumor cells

(1) Fluorinated modified linear poly beta-amino ester for improving endocytosis level of tumor cells to gene medicine

According to the preparation method of the nano-composite, the nano-composite is put into practiceFluorinated modified straight-chain poly-beta-amino ester f having 8 fluorine atoms in monomer prepared in example 1 ₈ PBAE and the monomer prepared in comparative example 1 straight-chain, fluorine-free, poly-beta-amino ester cPBAE were prepared with the same mass ratio (Polymer/Cy 3-sinC mass ratio 100) f, respectively, as Cy3-sinC ₈ PsN, cPsN nanocomplex; furthermore, PEI and Cy3-siNC were prepared in a mass ratio of 5:1 to give PEI/Cy3-siNC nanocomposite.

B16F10 cells were seeded into 96-well plates (1.0X10) ⁴ Cells/well), for 24 hours. The medium was then replaced with serum-free DMEM and f ₈ PsN or cPsN nanocomplex and PEI/Cy3-siNC nanocomplex were added to the wells in an amount of 0.3 μg Cy 3-siNC/well and incubated for 4 hours. The medium was discarded, washed 3 times with 20U/mL heparin sodium in PBS, and lysed by addition of RIPA lysate (100. Mu.L/well) for 20 min. The content of Cy3-siNC in the resulting lysates was determined by means of a microplate reader (λex=550 nm, λem=580 nm). The BCA kit measures intracellular protein content and cellular uptake is expressed as "μg Cy3-siRNA/mg protein", where Cy3-siRNA refers to Cy3-siNC. Referring specifically to FIG. 8, f ₈ PsN nanocomplex (corresponding to fPBAE in the figure) showed the highest level of cellular uptake, significantly higher than the commercial agent PEI. This suggests that fluorinated modified linear poly- β -amino esters can promote uptake of the nanocomposite by tumor cells.

(2) Fluorinated modified linear poly beta-amino ester for promoting gene medicine inclusion body escape

B16F10 cells were seeded onto confocal dedicated petri dishes (20 mm diameter, 3X 10) ⁴ Cells/dish), for 24 hours. Replacement of the Medium with serum-free DMEM, f ₈ PsN or cPsN nanocomposites (polymer/Cy 3-sinC mass ratio 100) were added to the wells in an amount of 1. Mu.g Cy 3-sinC/dish and incubated for 4 hours at 37 ℃. The medium was discarded, washed 3 times with PBS solution containing 20U/mL heparin sodium, stained with Hoechst33258 (5. Mu.g/mL) and Lysotracker Deep Red (200 nM) for 30 minutes and 1 hour, respectively, observed by confocal laser scanning microscopy and photographed. The co-localization rates of Cy3-siNC and Lysotracker Deep Red were calculated using Image J, and the test results are shown in FIG. 9. Experimental results show that B16F10 cells and F ₈ After a PsN nanocomposite co-incubation of 4 hours,significant amounts of green fluorescent siRNA appeared in the cells, with a co-localization rate between green and red fluorescence (inclusion bodies) of only 27.2%. The above results indicate that the fluorinated modified linear poly-beta-amino ester can promote the escape of the inclusion bodies of the gene medicine.

(3) Fluorinated modified linear poly beta-amino ester for promoting transfection ability of gene medicine outside body

In vitro test: B16F10 cells were seeded into 6-well plates (2.0X10) ⁴ Cells/well), for 24 hours. The medium was then replaced with serum-free DMEM (2 mL), f ₈ PsN、f ₈ PsA or cPsA nanocomposites (polymer/siRNA mass ratio 100) were added to the wells in an amount of 2. Mu.g siRNA/well and incubated for 4 hours at 37 ℃. Serum-free DMEM was exchanged for DMEM containing 10% fbs for further incubation for 20 hours. The Trizol reagent is used for extracting the total RNA, primeScript RT kit of the cells and carrying out reverse transcription to obtain cDNA. The cDNA was analyzed by a SYBR Premix Ex Taq kit in a real-time PCR system, and the ratio of the expression level of ADAR1mRNA in the cells after the addition of the sample to the cells after the addition of the equal volume of PBS was determined. Referring specifically to FIG. 10 (where fPsA denotes f ₈ PsA, fPsN denotes f ₈ PsN), the experimental result shows that f ₈ ADAR1 gene silencing efficiency of PsA (-70%). This suggests that the fluorinated modified linear poly- β -amino esters can increase gene silencing efficiency by promoting cellular uptake and endosomal escape of the nanocomposite.

Animal in vivo test: further verifying the ability of the fluorinated modified linear poly beta-amino ester to promote in vivo transfection of gene drugs when the tumor volume of the B16F10 tumor-bearing mice reaches 200mm ³ When randomly divided into 4 groups of 4. Intratumoral injection of PBS, f ₈ psN, cPsA or f ₈ PsA nanocomposite (0.5 mg siRNA/kg, 50. Mu.L). The tumor tissue of the mice was collected 24 hours after administration. 30mg of the obtained tumor tissue was weighed, homogenized and total RNA was extracted with Trizol reagent, and RT-PCR was performed by the same procedure as in vitro gene silencing to calculate the ratio of the expression level of ADAR1mRNA in the tumor tissue after the addition of the sample to the tumor tissue after the addition of an equal volume of PBS. The results are shown in FIG. 11 (fPsA denotes f in the figure ₈ PsA, fPsN denotes f ₈ PsN), the experimental result is consistent with the in vitro gene silencing result, f ₈ PsA nano complexThe ADAR1 gene silencing efficiency of the compound (66.7%) was significantly higher than that of the cPsA nanocomposite (5.1%).

5. In vivo anti-tumor efficacy

Tumor volume of 70mm ³ The B16F10 tumor-bearing mice of (C) were randomly divided into 8 groups. 50 μl PBS, oxygen pre-saturated cPsA nanocomposite, oxygen pre-saturated f were injected intratumorally on days 0, 2 and 4, respectively ₈ PsN nanocomposite or oxygen presaturated f ₈ PsA nanocomposites. After 2 hours of intratumoral injection, X-ray radiation radiotherapy is carried out on the tumor parts of the mice in groups 3-8, and the rest parts of the mice are shielded and protected by a lead plate during radiotherapy. The dose of the injected nano-composite is 0.5mg siRNA/kg, and the dose of radiotherapy is 6Gy. Finally, on days 5, 7 and 9, mice from groups 5 and 8 were injected intravenously with αPD-L1 (1 mg/kg). Tumor volume and body weight of mice were measured every two days. Tumor volume = length x width ² /2. Tumor volume of mice reaches 1000mm ³ When it is regarded as dead. The number of remaining mice survived was continuously recorded for 60 days after the first treatment.

The test results are shown in FIG. 12 (in the figure, fPsA denotes f ₈ PsA, fPsN denotes f ₈ PsN), tumors of mice injected with PBS only intratumorally grew rapidly. While the oxygen is singly injected to presaturate f ₈ The anti-tumor efficacy of the PsA nanocomposite or Radiation Therapy (RT) alone was not evident, with tumor inhibition rates of-13% and-54% at 14 days, respectively. RT+f ₈ PsN has the tumor inhibiting effect of obviously enhancing the tumor inhibiting rate (about 70 percent), and shows that the nano-composite can effectively sensitize radiotherapy by relieving hypoxia at tumor parts. Combination treatment group rt+f ₈ The PsA has obviously enhanced tumor inhibition effect, and the tumor inhibition rate of the PsA is far higher than that of an RT+cPsA group with an inhibition rate of 59 percent. The anti-tumor curative effect is further enhanced (tumor inhibition rate is 95 percent) after the alpha PD-L1 is combined. In contrast, the tumor inhibition rate of tumor inhibition of RT+αPD-L1 was only 59%. This may be due to immune checkpoint inhibitors, which facilitate promotion of T cell killing of the tumor by overcoming immune escape of the tumor cells. Survival of the mice in each group after treatment is shown in FIG. 13 (fPsA indicates f in the figure ₈ PsA, fPsN denotes f ₈ PsN), consistent with the tumor inhibiting effect, RT+f during the 22 day observation period ₈ PsA and rt+f ₈ The survival of mice in group psa+αpd-L1 mice was 100% and significantly better than the other treatment groups. The above results indicate that f ₈ PsA can jointly promote the anti-tumor curative effect of radiotherapy by relieving tumor hypoxia and enhancing organism anti-tumor immunity, and the anti-tumor curative effect of radiotherapy can be further enhanced after being treated by combining alpha PD-L1.

6. In vivo distal tumor suppression

B16F10 cells (1.0X10) were inoculated subcutaneously on both left and right sides of C57BL/6 mice ⁶ /v), a B16F10 melanoma distal tumor model was constructed. When the tumor volume is as long as 70mm ³ At this time, the B16F10 tumor-bearing mice were randomly divided into 8 groups (8 per group). 50 μLPBS, oxygen presaturated cPsA nanocomposite, oxygen presaturated f were injected intratumorally on days 0, 2 and 4, respectively ₈ PsN nanocomposite or oxygen presaturated f ₈ PsA nanocomposites. After 2 hours of intratumoral injection, X-ray irradiation radiotherapy is performed on tumor sites of mice in groups 3-8. During X-ray irradiation, the mice are shielded and protected by a lead plate except for the right tumor (including the left far tumor). Volume and body weight of in situ tumor and distal tumor of mice were measured every two days. When the tumor volume at any side reaches 1000mm ³ Mice were identified as dead. The number of remaining mice survived was continuously recorded during 38 days after the first treatment.

The inhibition of distant tumors in each group is shown in FIG. 14 (fPsA denotes f in the figure ₈ PsA, fPsN denotes f ₈ PsN), RT+f on carcinoma in situ ₈ After psa+αpd-L1 treatment, the growth of distant tumors in mice that were not directly treated was significantly inhibited. The inhibition rate of the distal tumor is 68 percent at 14 days. In contrast, mice receiving only unilateral radiation therapy had a distal tumor inhibition of only 13%.

The survival of mice is shown in FIG. 15 (fPsA indicates f in the figure ₈ PsA, fPsN denotes f ₈ PsN)，RT+f ₈ psa+αpd-L1 treatment also significantly prolonged survival in tumor-bearing mice. Rt+f on day 20 after first treatment ₈ Mice in the PsA+αPD-L1 group had 100% survival. Whereas mice receiving only one-sided radiation therapy all died. The above results indicate f ₈ PsA nanocompositesCan enhance the far-end effect of radiotherapy.

7. Effects of fluorinated modified Linear Polyβ -amino ester treatment on individual immune cells

Freshly collected tumor tissue was sheared to homogenate and filtered through a nylon mesh (200 mesh), and cells were collected by centrifugation (4 ℃,1500rpm,5 min). Adding erythrocyte lysate, standing for 3 minutes at room temperature, and washing for 3 times by PBS to obtain the B16F10 tumor single cell suspension. Lymph nodes at the groin of mice were collected, ground with a sterile syringe core, and filtered through a nylon mesh (200 mesh). Cells were collected by centrifugation (4 ℃,1500rpm,5 minutes) and washed 3 times with PBS to obtain a lymph node single cell suspension. Taking prepared tumor and lymph node single cell suspension (1×10) ⁶ Cell/tube), anti-C16/32 was blocked for 10 min, stained with fluorescent antibodies for 30 min, centrifuged (4 ℃,1500rpm,5 min) and washed 3 times with PBS, and flow cytometry detected.

The antibodies and dilution factors used for T cell staining were: anti-CD3-FITC (1/50), anti-CD8a-APC (1/50) and anti-CD4-PE (1/50). The antibodies and dilution factors used for staining dendritic cells were: anti-CD11c-FITC (1/50), anti-CD86-PE (1/50) and anti-CD80-APC (1/50).

The test results are shown in FIGS. 16 and 17 (fPsA denotes f in the figure ₈ PsA, fPsN denotes f ₈ PsN)。RT+f ₈ Dendritic cell maturation levels in PsA mice were increased to-3 fold in RT. RT+f ₈ The PsA+αPD-L1 group increased to 5-fold that of the RT group. The differentiation of T cells in tumors after different treatments is shown in FIG. 17, and the results show that RT+f is used ₈ PsA and rt+f ₈ Intratumoral CD8 treated with PsA+αPD-L1 ⁺ The T cell ratio is significantly increased to-1.5 and-2 times that of RT group.

8. Effects of fluorinated modified Linear Polyβ -amino ester treatment on expression levels of individual inflammation-associated cytokines

The TNF-alpha, IFN-gamma and IFN-beta levels in tumor tissues and serum of each group of mice after treatment were determined by ELISA experiments, respectively, following the procedure provided by ELISA kit.

Cytokine changes in tumor tissue are shown in FIG. 18 (denoted by fPsA in the figuref ₈ PsA, fPsN denotes f ₈ PsN)，RT+f ₈ TNF- α and IFN- γ concentrations in the tumors of the PsA group mice were 54.2 pg/100. Mu.g tumor tissue and 259.7 pg/100. Mu.g tumor tissue, respectively. Significantly higher than the RT+cPsA group 27.9 pg/100. Mu.g tumor tissue and 88.2 pg/100. Mu.g tumor tissue. TNF- α and IFN- γ concentrations were further increased to about 2-fold in the RT+αPD-L1 group when further combined with αPD-L1 treatment, at 74.7pg/100 μg tumor tissue and 305.5pg/100 μg tumor tissue, respectively. The same trend is also shown for the intratumoral IFN- β concentration, RT+f ₈ PsA group concentration was 16.4 pg/100. Mu.g tumor tissue, RT+f ₈ The concentration of PsA+αPD-L1 group was 26.8pg/100 μg tumor tissue, well above the RT and RT+cPsA groups. Cytokine changes in mouse serum are shown in FIG. 19 (fPsA indicates f in the figure ₈ PsA, fPsN denotes f ₈ PsN) via RT+f ₈ The IFN-gamma and TNF-alpha concentration in serum of mice treated by PsA+alpha PD-L1 is obviously higher than that of RT and RT+alpha PD-L1 groups, and the anti-tumor curative effect is further proved.

The invention discloses a fluorinated modified linear poly beta-amino ester, which can simultaneously realize high-efficiency delivery of oxygen and gene medicine to tumor parts. The delivery strategy is applicable to various solid tumors, and can enhance DNA damage generated by radiotherapy by relieving the hypoxia microenvironment of the solid tumors, thereby overcoming the radiotherapy resistance of the solid tumors. In addition, the body anti-tumor immune response is activated by means of gene therapy, and the remote effect of radiotherapy is amplified, so that the systemic effect of local radiotherapy is realized, and the defect that the remote metastasis of tumors cannot be treated by solid tumor radiotherapy is overcome.

Two siRNAs as described above: the sense and antisense sequences of siADAR1 and siNC are shown in Table 1:

TABLE 1 sense and antisense sequences of siADAR1, siNC

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. The application of the fluorinated modified linear poly beta-amino ester in preparing a sensitization anti-tumor radiotherapy medicament and/or a tumor gene therapy medicament is characterized in that the fluorinated modified linear poly beta-amino ester is used as a carrier for delivering a sensitization agent and/or a gene medicament, wherein the sensitization agent is oxygen, and the gene medicament is siRNA for promoting an anti-tumor immune response;

the fluorinated modified linear poly-beta-amino ester has the following structural general formula:

2. The use according to claim 1, wherein R ism is an integer of 1 to 20; the R is ₁ Is->The R is ₂ Is one of the following structures:

3. the use according to claim 1, wherein the fluorinated linear poly- β -amino ester is obtained by synthesizing poly- β -amino ester by michael addition reaction using fluorine-containing diacrylate and amino alcohol as monomers, and then capping with amine molecules.

4. The use according to claim 3, wherein the fluorine-containing diacrylate has the following general structural formula:

wherein m is an integer of 1 to 20.

5. Use according to claim 3, characterized in that the amino alcohol is 4-amino-1-butanol or 5-amino-1-pentanol.

6. Use according to claim 3, characterized in that the amine molecule is selected from one of spermine, 1, 3-diaminopropane, 1, 3-diamino-2, 2-dimethylpropane, 1, 3-pentanediamine, 2-methyl-1, 5-pentanediamine, 1, 11-diamino-3, 6, 9-trioxaundecane, 2- [ (3-aminopropyl) amine ] ethanol, 1- (3-aminopropyl) -4-methylpiperazine.

7. Use according to claim 3, characterized in that the molar ratio of fluorine-containing diacrylate to amino alcohol is 1:0.8 to 1.5; the temperature of the Michael addition reaction is 30-80 ℃.