CN116396984A - Nucleic acid delivery vector and application of pharmaceutical composition with synergistic functions - Google Patents

Nucleic acid delivery vector and application of pharmaceutical composition with synergistic functions Download PDF

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CN116396984A
CN116396984A CN202210865687.XA CN202210865687A CN116396984A CN 116396984 A CN116396984 A CN 116396984A CN 202210865687 A CN202210865687 A CN 202210865687A CN 116396984 A CN116396984 A CN 116396984A
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nucleic acid
acid delivery
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sirna
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CN116396984B (en
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黄渊余
张天
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Suzhou Xuanjing Biotechnology Co ltd
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Abstract

The invention relates to the technical field of medicines, and discloses a nucleic acid delivery carrier and application of a functional synergistic pharmaceutical composition. The structure of the carrier is shown as a formula (I), and the preparation method comprises the following steps: in the presence of a reaction solvent I, mixing a compound shown in a formula (II), 2-bis (bromomethyl) -1, 3-propanediol, bis (2-hydroxyethyl) disulfide and L-lysine diisocyanate to react I, and then mixing the mixture with methoxy polyethylene glycol-hydroxyl to react II to obtain a reactant P1; the reaction III is carried out by mixing the reactant P1 with the compound represented by the formula (IV) in the presence of the reaction solvent II. The pharmaceutical composition comprises an siRNA and a nucleic acid delivery vehicle. The carrier can effectively induce immune cell death effect under the light stimulation, the pharmaceutical composition has endosome escape capability, and can carry out efficient delivery of nucleic acid, thereby realizing organic synergy of gene therapy, photodynamic therapy and immunotherapy and effectively inhibiting proliferation of various tumor cells.
Figure DDA0003758451500000011

Description

Nucleic acid delivery vector and application of pharmaceutical composition with synergistic functions
Technical Field
The invention relates to the technical field of medicines, in particular to a nucleic acid delivery carrier and application of a functional synergistic pharmaceutical composition.
Background
Immunotherapy, particularly Immune Checkpoint (ICB) therapy, aimed at activating T cells, increasing infiltration of T cells at the tumor site to enhance anti-tumor immune response, has the advantage of long duration of the activated immune response. Currently there are three immune checkpoints available in batches, CTLA-4, PD-1 and PDL1, respectively, where PD-L1 is highly expressed in tumor cells. PD-1 interferes with T cell antigen receptor mediated signaling pathways, protecting cancer cells from T cell attack; anti-PD-L1 is a disorder that eliminates T cell function. Clinical progress in cancer immunotherapy, represented by blocking PD-1/PD-L1 mab, has led to great stimulation. The interaction between PD-1 and PD-L1 is knocked out by using small interfering RNA such as siPD-L1 to down regulate the expression of cancer cell membrane PD-L1 protein, so that the inhibition of tumor cells on T cells is relieved. However, patients with cold tumors (non-immunogenic) have a low response rate to ICB therapy, and converting a "cold" tumor to a "hot" tumor (an immunogenic tumor with increased T cell infiltration) can be effective in increasing the response rate to ICB therapy.
Photodynamic therapy (PDT) can induce the production of Immunogenic Cell Death (ICD), release of tumor-associated antigens (TAAs) and lesion-associated molecular patterns (DAMPs), generate in situ vaccines to promote maturation of Dendritic Cells (DCs), significantly enhance antigen exposure, initiate and activate naive T cells, pass from lymph nodes into tumor sites through blood, and recognize killer cancer cells. But its therapeutic effect is severely impaired by dense tumor tissue, hypoxic tumor microenvironment. The excitation and emission wavelength of the photosensitizer small molecule in the near infrared first region is in the near infrared first region, and the photosensitizer small molecule has the characteristics and the faced problems: limited penetration depth (-1 mm) was not effective in penetrating deep tumor sites. More and more near infrared two-region photosensitizers are designed and synthesized to enhance the anti-tumor efficacy of PDT, on the one hand, the near infrared two-region photosensitizers generate reactive oxygen species ROS with cytotoxicity under NIR-II light stimulation; on the other hand, dying tumor cells release TAAs, including high mobility protein B1 (HMGB 1), calreticulin (CRT), adenosine Triphosphate (ATP) promotes DC maturation, which is then transported into lymph nodes, thereby activating T cells, exerting T cell mediated antitumor immunity, generating systemic immunity, inhibiting metastasis of tumors. In conclusion, the highly specific tumor cell death induced by near infrared two-zone photodynamic therapy does not impair the immunity of the body to the tumor, activates the polyclonal tumor-specific immune response, converts the "cold" tumor into the "hot" tumor, and stimulates the body's own immune response. PDT renders immune-resistant tumors more sensitive to the immune checkpoint therapy ICB, enhancing tumor immunotherapy effects.
Nano-drugs have unique roles in immunotherapy, and nano-particles accumulate in tumors through enhanced permeation and retention Effects (EPR), focusing the drug at the tumor site. Nanoparticles can be designed to interact with external energy sources, such as light, heat, magnetism, to enhance immunogenic cell death, and stability of the nanoparticles can be enhanced by adjusting the structure of the nanoparticles, such as surface modified polyethylene glycol (PEG), polyethylenimine (PEI). Nanoparticles can control the kinetics of drug release, and can be pre-programmed by particle chemistry, such as disulfide bonds; or may be controlled by response to an external stimulus, such as light or heat. The main delivery platforms at present comprise biomimetics, liposomes, no carriers, inorganic nanoparticles, polymer nanoparticles and the like, and polymer nanoparticles are widely focused on the aspects of the modifiable property and the biodegradability.
However, there is currently little research on tumor microenvironment-responsive and degradable photopolymer.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a nucleic acid delivery carrier and application of a functional synergistic pharmaceutical composition, wherein the nucleic acid delivery carrier has good cytotoxicity, and the pharmaceutical composition can be used for carrying out efficient delivery of nucleic acid, so that the organic synergy of gene therapy, photodynamic therapy and immunotherapy is realized, and proliferation of various tumor cells is effectively inhibited.
In order to achieve the above object, a first aspect of the present invention provides a nucleic acid delivery vector having a structure as shown in formula (I),
Figure BDA0003758451480000021
wherein m, n and p are each independently positive integers of 3-15, and q is a positive integer of 40-120.
In a second aspect, the present invention provides a method for preparing a nucleic acid delivery vector, comprising the steps of:
(1) In the presence of a reaction solvent I, mixing a compound shown in a formula (II), 2-bis (bromomethyl) -1, 3-propanediol and bis (2-hydroxyethyl) disulfide with L-lysine diisocyanate to perform a reaction I, and then mixing the mixture with methoxy polyethylene glycol-hydroxyl to perform a reaction II to obtain a reactant P1 shown in a formula (III);
(2) Mixing a reactant P1 with a compound shown in a formula (IV) in the presence of a reaction solvent II to perform a reaction III, so as to obtain a nucleic acid delivery vector shown in the formula (I);
Figure BDA0003758451480000031
Figure BDA0003758451480000041
Figure BDA0003758451480000042
wherein m, n and p are each independently positive integers of 3-15, and q is a positive integer of 40-120.
In a third aspect, the invention provides a functionally synergistic pharmaceutical composition comprising an siRNA and the nucleic acid delivery vector described above and/or the nucleic acid delivery vector prepared by the preparation method described above.
In a fourth aspect, the present invention provides a method of preparing a functionally synergistic pharmaceutical composition comprising the steps of: mixing the nucleic acid delivery carrier and/or the nucleic acid delivery carrier prepared by the preparation method with a micelle agent to obtain a nucleic acid delivery carrier micelle, and mixing the nucleic acid delivery carrier micelle with siRNA to obtain a reaction II.
In a fifth aspect, the present invention provides the use of at least one of the aforementioned nucleic acid delivery vector, the aforementioned nucleic acid delivery vector prepared by the aforementioned method, the aforementioned pharmaceutical composition and the pharmaceutical composition prepared by the aforementioned method for the preparation of an antitumor drug.
Through the technical scheme, the invention has the beneficial effects that:
the nucleic acid delivery carrier provided by the invention belongs to a photosensitizer with a main chain connected in series with NIR-II Aza-BODIPY, contains a tumor microenvironment response disulfide bond monomer, and has a side chain for modifying cholesterol cationic monomer to make the surface of cholesterol cationic monomer positive, and can generate toxic Reactive Oxygen Species (ROS) to kill tumor cells under 808nm laser irradiation; the nucleic acid delivery vector can carry out efficient delivery of nucleic acid (siRNA), realizes organic synergy of gene therapy, photodynamic therapy and immunotherapy, and more importantly, the photodynamic therapy can induce ICD effect, promote maturation of DC, recruit more Cytotoxic T Lymphocytes (CTL), convert cold tumors into hot tumors, generate systemic immunity at the same time, further inhibit metastasis and recurrence of tumors, and effectively improve sensitivity of tumors to immunotherapy. The nucleic acid delivery carrier provided by the invention can smoothly enter cells and realize escape of endosomes into cytoplasm to play a role through electrostatic effect adsorption of siPD-L1, and can lower the expression of PD-L1 protein by inhibiting the expression of PD-L1 gene, so as to relieve the 'cangue' of tumor cells to T cells and further amplify PDT-induced immunotherapy.
The functional synergistic pharmaceutical composition provided by the invention has better safety in vivo and in vitro, effectively realizes the escape and release of load nucleic acid, has higher gene silencing efficiency, can effectively inhibit the proliferation of tumor cells by RNAi and PDT combined treatment, and has good treatment effect in various tumor models.
Drawings
FIG. 1 is a synthetic route diagram of the polymer PDNP of formula (I-1) in example 1;
FIG. 2 is a diagram of the reactant P1 of formula (III-1) in example 1 1 H NMR spectrum;
FIG. 3 is a polymer PDNP of the formula (I-1) in example 1 1 H NMR spectrum;
FIG. 4 is a schematic diagram of the structure of the pharmaceutical composition PDNP/siNC prepared in example 4;
FIG. 5 is a graph of particle size and potential of the pharmaceutical composition PDNP/sinC prepared in example 5;
FIG. 6 is an ultraviolet absorption spectrum and a fluorescence emission spectrum of the PDNP polymer obtained in example 1, wherein A is an ultraviolet absorption spectrum and B is a fluorescence emission spectrum;
FIG. 7 is an electrophoretogram of a gel blocking experiment of the pharmaceutical composition PDNP/siNC prepared in example 4;
FIG. 8 is a graph showing the generation of PDNP/siNC at various illumination times (0-330 s) using the DPBF probe in test example 3 1 O 2 Is not limited in terms of the ability to perform;
FIG. 9 is a graph of the CLSM images of test example 3 for evaluation of the power of the generation of active oxygen by siNC, lipo/siNC and PDNP/siRNA in cell line CT26, scale 50 μm, L representing the light under the conditions: wavelength 808nm, intensity 1W/cm 2 The time is 3min;
FIG. 10 is a CT26 cell viability graph after treatment of Naked SiNC, lipo/SiNC and PDNP/SiNC of different mass ratios prepared in example 6 of test example 4, A is no lightB is light (the condition of light irradiation is that the wavelength is 808nm and the intensity is 1W/cm) 2 Time 3 min);
FIG. 11 is a graph showing the evaluation of gene silencing effect of PDNP/siRNA in the pharmaceutical composition of test example 5, wherein A is a graph showing the silencing effect of PDNP/sIPD-L1 on CT26 cell line by qRT-PCR, B is the relative luciferase activity of 4T1-Luc cells after various treatments, and L represents light (light conditions: wavelength 808nm, intensity 1W/cm) 2 Time 3 min);
FIG. 12 is a graph showing the evaluation of the uptake capacity of PDNP/siRNA in the pharmaceutical composition of test example 6, wherein A is the cell uptake efficiency of the PDNP/Cy5-siRNA complex obtained by the flow cytometer, B is a graph of quantitative analysis of the average fluorescence intensity and the percentage of siRNA cellular uptake of FIG. 12A, C is the recorded cellular uptake of PDNP/Cy5-siRNA after treatment with different endocytosis inhibitors, and D is the quantitative analysis of cellular uptake by flow cytometry;
FIG. 13 is a graph of the endosome escape ability of the PDNP/siRNA complex of test example 7, wherein A is the confocal laser image and B is the pictorial quantification of laser confocal;
FIG. 14 is a graph of an immunogenic cell death study of pharmaceutical composition PDNP/siRNA of test example 8, wherein A is ATP secretion, B is exposed flow-through quantification of CRT, C is confocal assay of CRT, and D is release confocal assay of HMGB 1;
FIG. 15 is a graph of the pharmaceutical composition PDNP/siRNA of test example 9 in a model for treating colon cancer, wherein A is a tumor treatment schematic and grouping information, the tumor area is irradiated with 808nm laser after 6 hours, B is a tumor volume growth curve during treatment, C is the weight during treatment, D is the relative tumor weight of resected tumor after 11 days of treatment, E is the liver/body organ coefficient after 11 days of treatment, F is the spleen/body organ coefficient after 11 days of treatment, G is the survival curve of tumor-bearing mice;
FIG. 16 is a graph of the immune status change of tumor microenvironment and immune organs after evaluation of pharmaceutical composition PDNP/siRNA treatment in test example 10, wherein A is the flow cytometry analysis of CD80+CD86+T dendritic cells in tumor, B is the flow cytometry analysis of CD80+CD86+T dendritic cells in lymph nodes, C is the flow cytometry analysis of CD3+CD4+T dendritic cells in tumor, D is the flow cytometry analysis of CD3+CD8+T dendritic cells in tumor, E is the flow cytometry analysis of CD3+CD4+T dendritic cells in spleen, F is the flow cytometry analysis of CD3+CD8+T dendritic cells in spleen;
FIG. 17 is a graph showing in vivo safety assessment of the pharmaceutical composition PDNP/siRNA of test example 11;
FIG. 18 is a graph of in vivo safety assessment of drug composition PDNP/siRNA after treatment in test example 12, H & E staining of Balb/c mice heart, liver, spleen, lung, kidney, scale 100 μm;
FIG. 19 is a graph showing the results of a study of a pharmaceutical composition PDNP/siRNA in test example 13 in a model for treating breast cancer, wherein A is a tumor treatment schematic and grouping information, B is a tumor volume growth curve during treatment, C is a change in body weight of each group of mice during treatment, D is the weight of tumor excised after 25D of treatment, E is the tumor mass dissected after treatment, F is the expression of PD-L1 gene in tumor, G is the quantification of PD-L1 gene expression in tumor, and H is a biochemical index of each group of mice after treatment;
FIG. 20 is a study of a pharmaceutical composition PDNP/siRNA in test example 14 in a model of treating liver PDX tumors, wherein A is tumor treatment grouping information, B is tumor volume growth curve during treatment, C is tumor weight after 6D treatment, D is survival curve of tumor-bearing mice, E is PLK1 gene expression quantification after treatment, and F is liver/body organ factor and spleen/body organ factor after 6D treatment.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect, the present invention provides a nucleic acid delivery vector having a structure according to formula (I),
Figure BDA0003758451480000071
wherein m, n and p are each independently positive integers of 3-15, and q is a positive integer of 40-120.
The nucleic acid delivery carrier shown in the formula (I) belongs to a polymer photosensitizer with a main chain connected in series with NIR-II Aza-BODIPY (azaboron dipyrrole), contains a tumor microenvironment response disulfide bond monomer, and a side chain of the polymer photosensitizer modifies a cholesterol cation monomer, so that the surface of the polymer is positively charged, and siRNA can be effectively loaded; the strongest absorption wavelength of the nucleic acid delivery carrier is 830nm, the strongest emission wavelength is 960nm, the nucleic acid delivery carrier has two-region fluorescence and photodynamic capability, can generate toxic Reactive Oxygen Species (ROS) under 808nm laser irradiation to kill tumor cells, and has good cytotoxicity. When applied to drug-loaded nanoparticles, disulfide bonds on the backbone of the nucleic acid delivery vehicle may be responsively broken at high GSH levels in the tumor microenvironment, causing the nanoparticle to disintegrate, releasing the drug. Therefore, the nucleic acid delivery vector can carry out efficient delivery of nucleic acid (siRNA), realizes organic synergy of gene therapy, photodynamic therapy and immunotherapy, and more importantly, the photodynamic therapy can induce ICD effect, promote maturation of DC, recruit more CTLs, convert 'cold' tumor into 'hot' tumor, generate systemic immunity at the same time further inhibit metastasis and recurrence of tumor, and effectively improve sensitivity of tumor to immunotherapy.
According to the present invention, preferably, m, n, and p are each 3, and illustratively, m, n, and p are each 3, and q is 113 in the nucleic acid delivery vector, which has a structural formula shown in formula (I-1):
Figure BDA0003758451480000081
in a second aspect, the present invention provides a method for preparing a nucleic acid delivery vector, comprising the steps of:
(1) In the presence of a reaction solvent I, mixing a compound shown in a formula (II), 2-bis (bromomethyl) -1, 3-propanediol and bis (2-hydroxyethyl) disulfide with L-lysine diisocyanate to perform a reaction I, and then mixing the mixture with methoxy polyethylene glycol-hydroxyl to perform a reaction II to obtain a reactant P1 shown in a formula (III);
(2) Mixing a reactant P1 with a compound shown in a formula (IV) in the presence of a reaction solvent II to perform a reaction III, so as to obtain a nucleic acid delivery vector shown in the formula (I);
Figure BDA0003758451480000091
Figure BDA0003758451480000101
Figure BDA0003758451480000102
wherein m, n and p are each independently positive integers of 3-15, and q is a positive integer of 40-120.
The preparation method of the nucleic acid delivery vector provided by the invention has the advantages of simple steps, mild conditions and easiness in operation, and is beneficial to the production and development of the nucleic acid delivery vector.
In the present invention, the compound represented by formula (II), 2-bis (bromomethyl) -1, 3-propanediol, bis (2-hydroxyethyl) disulfide, L-lysine diisocyanate, methoxypolyethylene glycol-hydroxy and the compound represented by formula (IV) are commercially available, respectively, or may be prepared by themselves according to the methods disclosed in the prior art.
According to the invention, the amount of each raw material used in step (1) can be selected within a wide range. Preferably, the weight ratio of the compound shown in the formula (II) in the step (1), 2-bis (bromomethyl) -1, 3-propanediol, bis (2-hydroxyethyl) disulfide, L-lysine diisocyanate and methoxypolyethylene glycol-hydroxyl is 1:0.8-1.2:0.6-0.9:2-2.4:4.5-5.5. The inventors found that in this preferred embodiment, it is advantageous to increase the production efficiency of the reactant P1 represented by the formula (III), thereby increasing the yield of the nucleic acid delivery vector.
According to the invention, methoxypolyethylene glycol-hydroxy groups may be selected from those having conventional degrees of polymerization (mPEG-OH), preferably having a molecular weight of 2000 to 5000, e.g., mPEG 2000 -OH、mPEG 5000 -OH。
According to the present invention, in the above-described method for producing a nucleic acid delivery vector represented by formula (I), the conditions for reaction I can be selected within a wide range. Preferably, the conditions of reaction I include: the temperature is 45-55deg.C, specifically 45 deg.C, 47 deg.C, 49 deg.C, 51 deg.C, 53 deg.C, 55 deg.C, or any value between the two values; the time is 10-15h, and can be specifically 10h, 11h, 12h, 13h, 14h, 15h, or any value between the two values. The inventors found that in this preferred embodiment, it is advantageous to increase the yield of the nucleic acid delivery vector represented by formula (I).
According to the present invention, in the above-described method for producing a nucleic acid delivery vector represented by formula (I), the conditions for reaction II can be selected within a wide range. Preferably, the conditions of reaction II include: the temperature is 45-55deg.C, specifically 45 deg.C, 47 deg.C, 49 deg.C, 51 deg.C, 53 deg.C, 55 deg.C, or any value between the two values; the time is 10-15h, and can be specifically 10h, 11h, 12h, 13h, 14h, 15h, or any value between the two values. The inventors found that in this preferred embodiment, it is advantageous to increase the yield of the nucleic acid delivery vector represented by formula (I).
According to the invention, the amount of each raw material used in step (2) can be selected within a wide range. Preferably, the weight ratio of the reactant P1 of formula (III) to the compound of formula (IV) in step (2) is 2.5-3.5:1. The inventors found that in this preferred embodiment, it is advantageous to further increase the fluorescence effect and cytotoxicity of the nucleic acid delivery vector represented by formula (I).
According to the present invention, in the method for preparing a nucleic acid delivery vector represented by the above formula (I), the conditions for reaction III can be selected within a wide range. Preferably, the conditions of reaction III include: the temperature is 45-55deg.C, specifically 45 deg.C, 47 deg.C, 49 deg.C, 51 deg.C, 53 deg.C, 55 deg.C, or any value between the two values; the time is 10-15h, and can be specifically 10h, 11h, 12h, 13h, 14h, 15h, or any value between the two values. The inventors found that in this preferred embodiment, it is advantageous to increase the yield of the nucleic acid delivery vector represented by formula (I).
According to the present invention, preferably, the reaction solvent I and the reaction solvent II are each independently selected from at least one of anhydrous N, N-dimethylformamide, anhydrous dichloromethane, anhydrous tetrahydrofuran, and anhydrous dimethyl sulfoxide. More preferably, reaction solvent I and reaction solvent II are anhydrous N, N-dimethylformamide. The inventors found that in this preferred embodiment, it is advantageous to improve the reaction efficiency in the preparation of the nucleic acid delivery vector represented by formula (I).
According to the invention, the reactant P1 can be separated from the reaction solution obtained in reaction II by a separation method which is conventional in the art. Preferably, step (1) further comprises: dialyzing the reaction solution obtained in the reaction II to obtain a dialysate I, and drying the dialysate I to obtain a reactant P1 shown in the formula (III). For example, the reaction solution obtained in reaction II may be mixed with a small amount of water, and then the target reactant P1 may be trapped using a 7000MW dialysis bag.
According to the invention, the nucleic acid delivery vehicle can be separated from the reaction solution obtained in reaction III by separation methods conventional in the art. Preferably, step (2) further comprises: dialyzing the reaction solution obtained in the reaction III to obtain a dialysate II, and drying the dialysate II to obtain the nucleic acid delivery vector shown in the formula (I).
In the present invention, drying may be performed by a drying method conventional in the art, for example, vacuum freeze drying, hot air drying, freeze drying, etc., preferably, freeze drying is used for the drying of dialysate I and dialysate II, and the specific freeze drying process includes: precooling dialysate I or dialysate II at-90deg.C to-70deg.C for 25-35min, and freeze-drying for 40-60 hr.
In a third aspect, the invention provides a functionally synergistic pharmaceutical composition comprising an siRNA and the nucleic acid delivery vector described above and/or the nucleic acid delivery vector prepared by the method described above.
The pharmaceutical composition formed by the nucleic acid delivery carrier and the siRNA has ideal particle size and potential, can smoothly enter cells and realize the escape of endosomes into cytoplasm to play a role, and can inhibit the expression of PD-L1 genes, down regulate the expression of PD-L1 proteins, relieve the 'cangue' of tumor cells to T cells and further amplify PDT-induced immunotherapy; the pharmaceutical composition has better safety in vivo and in vitro, still keeps higher gene silencing efficiency for a long time, can effectively inhibit proliferation of tumor cells, and has good treatment effect in various tumor models (CT 26 colorectal cancer, 4T1-Luc breast cancer and cancer tissue xenograft of liver cancer patients).
According to the invention, the amounts of nucleic acid delivery vehicle and siRNA in the pharmaceutical composition can be selected within a wide range. Preferably, the weight ratio of nucleic acid delivery vehicle to siRNA is 10-100:1. The inventors have found that in this preferred embodiment, it is advantageous to improve the inhibition of tumor cell growth by the pharmaceutical composition.
According to the invention, the siRNA can be selected from RNA drugs (siRNA pairs) which can be loaded on cholesterol cations in the field and have specific targeting therapeutic targets, for example, the siRNA can be an siRNA pair which selectively silence the sIPD-L1 of PD-L1 protein highly expressed by tumor cells, the sIPLK1 of specific targeting PLK1 protein or the sIPL of specific targeting firefly luciferase. Preferably, the siRNA comprises a sense strand and an antisense strand which are fully reverse complementary, the nucleotide sequences of the sense strand and the antisense strand are shown in any one pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6, respectively;
siPD-L1 sense strand (SEQ ID No. 1): 5'-AGACGUAAGCAGUGUUGAA-3';
siPD-L1 antisense strand (SEQ ID No. 2): 5'-UUCAACACUGCUUACGUCUCC-3';
siPLK1 sense strand (SEQ ID No. 3): 5 '-UGAAAGAAUCACCUCCUUAdT-3';
siPLK1 antisense strand (SEQ ID No. 4): 5 '-UAAGGAGGGUGAUCUUCUUCUCUCAdTdT-3';
the sense strand of siFL (SEQ ID NO. 5): 5 '-CCCUUAUUCCUUCCUUCGCdTDT-3';
the siFL antisense strand (SEQ ID NO. 6): 5'-GCGAAGAAGGAGAAUAGGGdTdT-3';
the 19 nucleotides of the siPD-L1 antisense strand starting from the 5' end are fully reverse-complementary to the 19 nucleotides of the corresponding siPD-L1 sense strand; the 3 'end of the siPD-L1 antisense strand is ligated with 2 additional nucleotides, thereby forming a 3' overhang consisting of 2 nucleotides upon complementary pairing of the siPD-L1 antisense strand.
In a fourth aspect, the present invention provides a method for preparing a functionally synergistic pharmaceutical composition, comprising the steps of: mixing the nucleic acid delivery carrier and/or the nucleic acid delivery carrier prepared by the preparation method with a micelle agent to obtain a nucleic acid delivery carrier micelle, and mixing the nucleic acid delivery carrier micelle with siRNA to obtain a reaction II.
In the invention, after the nucleic acid delivery carrier forms a nucleic acid delivery carrier micelle, the nucleic acid delivery carrier micelle is combined with siRNA through electrostatic adsorption, so that the pharmaceutical composition is obtained, and the preparation process is simple and convenient to operate.
According to the present invention, preferably, the weight ratio of nucleic acid delivery vehicle to siRNA is 10-100:1.
According to the present invention, preferably, the siRNA contains a sense strand and an antisense strand which are completely reverse complementary, and the nucleotide sequences of the sense strand and the antisense strand are shown in any one pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6, respectively.
According to the present invention, a micelle agent capable of forming a micelle with the nucleic acid delivery vehicle represented by formula (I) in the art may be selected. Preferably, the micelle agent is ultrapure water; the micelle agent is used in an amount of 0.1 to 0.3mL relative to 1mg of the nucleic acid delivery vehicle. The inventors found that in this preferred embodiment, it is advantageous to improve the micelle efficiency to the nucleic acid delivery vector represented by formula (I), and the micelle effect is more excellent.
According to the invention, preferably, the process of mixing reaction I comprises: dissolving the nucleic acid delivery carrier in a solvent, dripping the solvent into a micelle agent, stirring the mixture for 25 to 35 minutes at the temperature of 5 to 40 ℃ and the rotating speed of 80 to 150rpm to obtain a mixed reaction solution, and dialyzing III the mixed reaction solution to obtain the nucleic acid delivery carrier micelle. The inventors have found that under this preferred embodiment, it is advantageous to optimize the micelle forming effect on the hydrophilic nucleic acid delivery vehicle, thereby increasing the yield of the pharmaceutical composition.
In the present invention, the solvent of the nucleic acid delivery vehicle may be a solvent conventionally used in the art capable of dissolving the nucleic acid delivery vehicle, and preferably the solvent is at least one selected from the group consisting of anhydrous N, N-dimethylformamide, anhydrous methylene chloride, anhydrous tetrahydrofuran and anhydrous dimethyl sulfoxide, more preferably N, N-dimethylformamide.
In accordance with the present invention, the conditions for mixing reaction II can be selected within a wide range. Preferably, the conditions of mixing reaction II include: the temperature is 5-40deg.C, specifically 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, or any value between the two values; the time is 25-35min, and can be 25min, 27min, 29min, 31min, 33min, 35min, or any value between the above two values.
According to a particularly preferred embodiment of the present invention, the method for preparing a pharmaceutical composition comprises the steps of:
(1) In the presence of a reaction solvent I, mixing a compound shown in a formula (II), 2-bis (bromomethyl) -1, 3-propanediol, bis (2-hydroxyethyl) disulfide and L-lysine diisocyanate in a weight ratio of 1:0.8-1.2:0.6-0.9:2-2.4, reacting for 10-15h at a temperature of 45-55 ℃, mixing the mixture with methoxy polyethylene glycol-hydroxyl (the weight ratio of the compound shown in the formula (II) to methoxy polyethylene glycol-hydroxyl is 1:4.5-5.5), reacting for 10-15h at a temperature of 45-55 ℃ to obtain a reaction solution I, dialyzing the reaction solution I to obtain a dialysate I, and freeze-drying the dialysate I to obtain a reactant P1 shown in a formula (III);
(2) Mixing a reactant P1 with a compound shown in a formula (IV) according to a weight ratio of 2.5-3.5:1 in the presence of a reaction solvent II, reacting for 10-15h at a temperature of 45-55 ℃ to obtain a reaction solution II, dialyzing the reaction solution II to obtain a dialysate II, and freeze-drying the dialysate II to obtain a nucleic acid delivery carrier shown in the formula (I);
(3) Dissolving a nucleic acid delivery carrier shown in a formula (I) in a solvent, dripping the nucleic acid delivery carrier into a micelle agent, stirring the mixture for 25-35min at a temperature of 5-40 ℃ and a rotating speed of 80-150rpm to obtain a mixed reaction solution, dialyzing the mixed reaction solution III to obtain a nucleic acid delivery carrier micelle, mixing the nucleic acid delivery carrier micelle with siRNA with a nucleotide sequence shown as SEQ ID No.1, and standing the mixture at a temperature of 5-40 ℃ for 25-35min to obtain a nano mixture, wherein the weight ratio of the nucleic acid delivery carrier to the siRNA is 10-100:1.
In a fifth aspect, the present invention provides the use of at least one of the aforementioned nucleic acid delivery vector, the aforementioned nucleic acid delivery vector prepared by the aforementioned method, the aforementioned pharmaceutical composition and the pharmaceutical composition prepared by the aforementioned method for the preparation of an antitumor drug.
In the invention, the antitumor drug can contain at least one of the nucleic acid delivery vector, the nucleic acid delivery vector prepared by the preparation method, the pharmaceutical composition and the pharmaceutical composition prepared by the method, and can also contain pharmaceutically acceptable auxiliary materials and/or other compounds capable of inhibiting tumor growth.
Wherein the auxiliary materials are pharmaceutically acceptable auxiliary materials such as stabilizer, excipient, solvent and the like. Antitumor drugs can be formulated by methods known in the art into the following forms: tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols or sprays for inhalation, sterile aqueous or oily solutions or suspensions for parenteral (including intravenous, intramuscular or infusion) or sterile emulsions.
According to the present invention, preferably, the type of tumor is selected from at least one of colorectal cancer, breast cancer and liver cancer.
The present invention will be described in detail by examples.
In the following examples, the compounds represented by the formula (II) and the formula (IV) are provided by the national institute of sciences chemistry, the Showa subject group; bis (2-hydroxyethyl) disulfide was purchased from sigma alderThe product number of the Liqi (Shanghai) trade company is 380474; l-lysine diisocyanate was purchased from Shanghai Ala Ding Shiji Co., ltd and product number L193461;2, 2-bis (bromomethyl) -1, 3-propanediol was purchased from Shanghai Ala Ding Shiji Co., ltd and product number B152042; mPEG (polyethylene glycol) 2000 -OH is available from hadamard reagent limited under product number 89171Y, molecular weight 2000; mPEG (polyethylene glycol) 5000 -OH is available from hadamard reagent limited under product number 89171U and has a molecular weight of 5000; the mouse colon cancer cells CT26 and the mouse breast cancer cells 4T1 are all purchased from the cell resource center of Shanghai life science research institute of Chinese sciences; other materials and reagents are conventional commercial products.
In the following examples, ultraviolet absorption spectra were measured using an ultraviolet spectrophotometer (UVPC 1601) manufactured by shimadzu instruments, inc; fluorescence emission spectra were measured using a fluorescence spectrometer (Cary Eclipse) manufactured by valian company, usa; particle size and potential were measured using a dynamic light scattering instrument (Malvern Nano-s) manufactured by Malvern instruments, inc., UK; 1 h NMR spectrum 13 C NMR was measured using a liquid Nuclear magnetic resonance Spectroscopy instrument (Bruker Avance 400) manufactured by Bruker Corp; other parameters are detected using instruments and methods conventional in the art.
In the following examples, siPD-L1 is an siRNA pair specifically targeting PD-L1 protein, the nucleotide sequences of the sense strand and the antisense strand of which are shown as SEQ ID No.1 and SEQ ID No.2, designed and synthesized by Soviet Rabo Biotechnology Co., ltd.,
Sense strand (SEQ ID No. 1): 5'-AGACGUAAGCAGUGUUGAA-3';
antisense strand (SEQ ID No. 2): 5'-UUCAACACUGCUUACGUCUCC-3';
SiPLK1 is a siRNA pair specifically targeting PLK1 protein, the nucleotide sequences of the sense strand and the antisense strand of the siRNA pair are shown as SEQ ID No.3 and SEQ ID No.4, and the siRNA pair is designed and synthesized by Suzhou Rabo biotechnology Co., ltd,
sense strand (SEQ ID No. 3): 5 '-UGAAAGAAUCACCUCCUUAdT-3';
antisense strand (SEQ ID No. 4): 5 '-UAAGGAGGGUGAUCUUCUUCUCUCAdTdT-3';
siFL is an siRNA pair specifically targeting FL protein, the nucleotide sequences of the sense strand and the antisense strand of which are shown as SEQ ID No.5 and SEQ ID No.6, designed and synthesized by Soviet Rabo biotechnology Co., ltd,
sense strand (SEQ ID No. 5): 5 '-CCCUUAUUCCUUCCUUCGCdTDT-3';
antisense strand (SEQ ID No. 6): 5'-GCGAAGAAGGAGAAUAGGGdTdT-3';
siNC (negative controlled siRNA) is a non-specific RNA pair with similar performance to that of SiPD-L1, is mainly used for early physical characterization, has the nucleotide sequences of a sense strand and an antisense strand shown as SEQ ID No.7 and SEQ ID No.8, is designed and synthesized by Suzhou Rabo biotechnology Co., ltd,
sense strand (SEQ ID No. 7): 5 '-CCUUUGAGGCAUACUUCAAAdTdT-3';
Antisense strand (SEQ ID No. 8): 5 '-UUUGAAGUGCCUCAAGGdTdT-3';
cy5-siRNA (Cy 5-labeled siRNA) is Cy5-labeled siRNA, the nucleotide sequences of the sense strand and the antisense strand of which are shown as SEQ ID No.9 and SEQ ID No.10, designed and synthesized by Soviet Rabo Biotechnology Co., ltd,
sense strand (SEQ ID No. 9): 5'-Cy5 UUCUCUCCCGAACGUGUCACGUTTDT-3';
antisense strand (SEQ ID No. 10): 5 '-ACGUGACACGUCGGAGAAdTDT-3'.
In the examples below, the room temperature was 25.+ -. 5 ℃ unless otherwise specified.
Example 1
(1) Accurately weighing 20.75mg of compound shown in formula (II), 20.95mg of 2, 2-bis (bromomethyl) -1, 3-propanediol, 15.40mg of bis (2-hydroxyethyl) disulfide and 45.22mg of L-lysine diisocyanate, adding into 5mL of anhydrous N, N-dimethylformamide, reacting for 12h at 50 ℃, and adding 102.31mg of m-PEG 5000 -OH, continuously reacting at 50deg.C for 12 hr to obtain reaction solution I, adding small amount of water into the reaction solution I, intercepting target polymer with 7000MW dialysis bag, and lyophilizing the dialysate I for 48 hr after dialysisTo a reactant P1 represented by the formula (III-1);
(2) Weighing 60mg of the reactant P1 obtained in the step (1), 20mg of the compound shown in the formula (IV) and adding into 5mL of anhydrous N, N-dimethylformamide, reacting for 12 hours at 50 ℃ to obtain a reaction liquid II, adding a small amount of water into the reaction liquid II, intercepting a target polymer by using a 7000MW dialysis bag, placing the dialysis liquid II into a refrigerator at the temperature of minus 80 ℃ for precooling for 30 minutes after dialysis is finished, and then freeze-drying for 48 hours to obtain the polymer PDNP shown in the formula (I-1) as a nucleic acid delivery carrier.
The synthetic route of the polymer PDNP of formula (I-1) in example 1 is shown in FIG. 1, and the structural formula and polymerization degree of the reactant P1 of formula (III-1) and the polymer PDNP of formula (I-1) are determined by nuclear magnetic resonance spectroscopy. The reactant P1 represented by the formula (III-1) 1 The H NMR spectrum is shown in FIG. 2, and the polymer PDNP shown by the formula (I-1) 1 The H NMR spectrum is shown in FIG. 3. As can be seen from FIG. 2, reactant P1 1 The H NMR spectrum showed the compounds of formula (II) (peak a), m-PEG 5000 Characteristic peaks of (peaks b and c), bis (2-hydroxyethyl) disulfide (peak d), confirm successful attachment of these monomers to the polymer chain; as can be seen from FIG. 3, the polymer PDNP 1 In the H NMR spectrum, in addition to the compounds of the formula (II) (peak a), m-PEG 5000 (peaks b and c), characteristic peaks of bis (2-hydroxyethyl) disulfide (peak d), characteristic peaks of the compound (peaks e, f and g) represented by formula (IV) also appear, confirming successful synthesis of the polymer PDNP.
Figure BDA0003758451480000151
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Figure BDA0003758451480000161
Example 2
(1) 20.75mg of the compound represented by the formula (II), 16.6mg of 2, 2-bis (bromomethyl) -1, 3-propanediol, 12.45mg of bis (2-hydroxyethyl) disulfide, 41.5mg of L-lysine diisocyanate were accurately weighed and added to 5mL of anhydrous N, N-dimethylformamideIn amide, the mixture was reacted at 45℃for 15 hours, and 93.4mg of m-PEG was added thereto 5000 -OH, continuing to react for 15 hours at the temperature of 45 ℃ to obtain a reaction solution I, adding a small amount of water into the reaction solution I, intercepting a target polymer by using a 7000MW dialysis bag, and freeze-drying the dialysis solution I for 60 hours after dialysis is finished to obtain a reactant P1 shown in a formula (III-1);
(2) 50mg of the reactant P1 obtained in the step (1) and 20mg of the compound shown in the formula (IV) are weighed and added into 5mL of anhydrous N, N-dimethylformamide, the mixture is reacted for 15 hours at the temperature of 45 ℃ to obtain a reaction liquid II, a small amount of water is added into the reaction liquid II, a 7000MW dialysis bag is used for intercepting a target polymer, after dialysis is finished, the dialyzate II is placed into a refrigerator at the temperature of minus 90 ℃ for precooling for 25 minutes, and then freeze drying is carried out for 60 hours to obtain the polymer PDNP shown in the formula (I-1).
Example 3
(1) Accurately weighing 20.75mg of compound shown in formula (II), 24.9mg of 2, 2-bis (bromomethyl) -1, 3-propanediol, 18.6mg of bis (2-hydroxyethyl) disulfide and 49.8mg of L-lysine diisocyanate, adding into 5mL of anhydrous N, N-dimethylformamide, reacting for 10 hours at 55 ℃, and adding 114mg of m-PEG 5000 -OH, continuing to react for 10 hours at 55 ℃ to obtain a reaction solution I, adding a small amount of water into the reaction solution I, intercepting a target polymer by using a 7000MW dialysis bag, and freeze-drying the dialysis solution I for 40 hours after dialysis is finished to obtain a reactant P1 shown in a formula (III-1);
(2) 70mg of the reactant P1 obtained in the step (1) and 20mg of the compound shown in the formula (IV) are weighed and added into 5mL of anhydrous N, N-dimethylformamide, the mixture is reacted for 10 hours at the temperature of 55 ℃ to obtain a reaction liquid II, a small amount of water is added into the reaction liquid II, a 7000MW dialysis bag is used for intercepting a target polymer, after dialysis is finished, the dialyzate II is placed into a refrigerator at the temperature of-70 ℃ for precooling for 35 minutes, and then freeze drying is carried out for 40 hours to obtain the polymer PDNP shown in the formula (I-1).
Example 4
Weighing 10mg of the polymer PDNP prepared in example 1, adding 500 mu L of anhydrous N, N-dimethylformamide solution, slowly dripping the solution into 2mL of ultrapure water after dissolving, rotating the solution at a rotating speed of 100rpm for 30min by using a magnetic stirrer to obtain a mixed reaction solution, dialyzing the mixed reaction solution for 12h by using a dialysis bag with a molecular weight of 7000MW, and collecting a dialysis solution to obtain PDNP micelles; mixing the PDNP micelle and siRNA (adopting siNC) at room temperature (the weight ratio of the PDNP polymer to the siRNA is respectively 0.5:1, 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 and 100:1), and standing for 30min to obtain the PDNP/siRNA pharmaceutical composition, namely PDNP/siNC.
The structure of the pharmaceutical composition PDNP/siRNA prepared in example 4 is schematically shown in FIG. 4.
Implementation of the embodiments example 5
Weighing 10mg of the PDNP polymer prepared in example 1, adding 500 mu L of anhydrous N, N-dimethylformamide solution, slowly dripping the solution into 1mL of ultrapure water after dissolving, rotating the solution at room temperature for 35min at a rotating speed of 80rpm by using a magnetic stirrer to obtain a mixed reaction solution, dialyzing the mixed reaction solution for 12h by using a dialysis bag with a molecular weight of 7000MW, and collecting a dialysis solution to obtain PDNP micelles; mixing the PDNP micelle and siRNA (adopting siNC) at room temperature (the weight ratio of the PDNP polymer to the siRNA is respectively 0.5:1, 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 and 100:1), and standing for 25min to obtain the PDNP/siRNA pharmaceutical composition, which is called PDNP/siNC for short.
The nano-composite PDNP/siRNA prepared in example 5 has uniform particle size, proper size and proper potential, and is beneficial to drug delivery, and the particle size and the potential of the nano-composite PDNP/siRNA are detected by a dynamic light scattering instrument, and the result is shown in figure 5.
Example 6
Weighing 10mg of the PDNP polymer prepared in example 1, adding 500 mu L of anhydrous N, N-dimethylformamide solution, slowly dripping the solution into 3mL of ultrapure water after dissolving, rotating the solution at a rotating speed of 150rpm for 25min by using a magnetic stirrer to obtain a mixed reaction solution, dialyzing the mixed reaction solution for 12h by using a dialysis bag with a molecular weight of 7000MW, and collecting a dialysis solution to obtain PDNP micelles; mixing the PDNP micelle and siRNA (adopting siNC) at room temperature (the weight ratio of the PDNP polymer to the siRNA is respectively 0.5:1, 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 and 100:1), and standing for 35min to obtain the PDNP/siRNA pharmaceutical composition, which is called PDNP/siNC for short.
Test example 1
The PDNP polymer prepared in example 1 was subjected to uv absorption spectrum and fluorescence emission spectrum analysis, and the results are shown in fig. 6. As can be seen from fig. 6, the PDNP polymer prepared in example 1 has a strong ultraviolet absorption peak at 830nm, and a strong broad fluorescence emission at 900-1100nm, and a strongest fluorescence emission at 960nm, which indicates that the deep penetration capability of the PDNP material can play a stronger role in photodynamic therapy.
Test example 2 gel blocking assay and physical Properties of PDNP/siRNA
0.8g of agarose is weighed, 40mL of TBE buffer is added, and the mixture is heated and dissolved into 2 weight percent agarose gel in a microwave oven with high fire; then 4. Mu.L of 1/10000 of Super Red Gel Red are added TM (Beijing, china) and gently stirring with a glass rod; pouring into a mold, and standing at room temperature for 30min; after cooling and solidifying, 10. Mu.L of PDNP/siNC solution prepared in example 4 (final concentration of siRNA particles: 25 pmol) was taken, 2. Mu.L of loading buffer was added, and after blowing and mixing, it was added to agarose gel loading wells; electrophoresis was performed for 20min at 100V using 1 XTBE buffer (Tris/Acetate/EDTA buffer) as running buffer, and then imaging was performed by a gel imager with free siRNA (Naked siNC) as a control, and the results are shown in FIG. 7.
The ability of the PDNP polymer to bind siRNA loaded drugs at different weight ratios of PDNP polymer to siRNA in example 4 was analyzed by gel blocking experiments. As shown in fig. 7, the free siRNA bands were not blocked on agarose gel, apparent migration occurred and band brightness was high, when the weight ratio of PDNP polymer to siRNA was 10:1, the siRNA bands in lanes were completely disappeared, the siRNA was retained in the gel-loaded wells, the PDNP polymer completely assembled the siRNA in nanoparticles, and the siRNA was completely bound to the PDNP polymer to form a stable complex. Based on the gel retardation results, it is known that the weight ratio of the PDNP polymer to the siRNA is 10-100:1, and the PDNP polymer material is completely loaded with the siRNA.
In the following test examples, without specific explanation, all of the PDNP/siRNA was carried out using the PDNP/siRNA complexes prepared in example 4 at a weight ratio of PDNP polymer to siRNA of 50:1.
Test example 3 evaluation of the active oxygen generating ability of pharmaceutical composition
1, 3-Diphenylisobenzofuran (DPBF) is a common use 1 O 2 The detection indicator has an ultraviolet absorption peak at 420nm and can be fast combined with 1 O 2 The reaction is carried out, and the ultraviolet absorption value is reduced after the reaction. Reactive Oxygen Species (ROS) detection using DPBF is therefore performed as follows: taking 10 mu L of mother solution DPBF solution, adding 0.99mL of DMF, uniformly mixing, and adding the mixture into 19mL of water under an ultrasonic state to obtain working solution; dissolving 3mg of PDNP/siRNA prepared in example 4 (weight ratio of PDNP to siRNA is 50:1) in 1mL of DMF to obtain a PDNP/siRNA solution with concentration of 3mg/mL, taking a 96-well plate, adding 190 mu L of working solution, adding 10 mu L of PDNP/siRNA solution, detecting an ultraviolet absorption value of 300-600nm by a standard Ma Yongmei as a 0s absorption control curve, adding 190 mu L of working solution and 10 mu L of PDNP/siRNA solution into a new well, and irradiating with a 808nm laser with intensity of 1W/cm 2 The detection was performed after 10s irradiation, the previous operation was repeated, the irradiation time (0-330 s) was changed, and the detection data was recorded, and as a result, the curves from top to bottom in fig. 8 correspond to the times 0s, 30s, 60s, 90s, 120s, 150s, 180s, 210s, 240s, 270s, 300s, 330s in this order, as shown in fig. 8.
The free siRNA (siNC group), lipo/siNC group, PDNP/siNC prepared in example 4 (weight ratio of PDNP to siRNA 50:1), and PDNP/siNC+L group were evaluated for their ability to generate active oxygen in cell line CT26 using DCFH-DA probe, which was oxidized to green fluorescent DCF by active oxygen after hydrolysis by cellular esterase, and thus the level of active oxygen was expressed by the intensity of green fluorescence. The method comprises the following specific steps: CT26 cells were plated in confocal microscope dishes with a cell density of 1X 10 per dish 5 After 24h, the original medium was aspirated, and Opti-MEM medium containing the PDNP/siNC complex (50:1 weight ratio of PDNP to siRNA, prepared in example 4), free siRNA (Naked siNC) or Lipo/siNC (complex obtained by transfecting siNC with transfection reagent Lipofectamine 2000 as positive control) was added, and culture was continued in a 37℃cell incubator for 3h; DCFH-DA (10 mM) probe was diluted to 10. Mu.M using Opti-MEM medium as working solution,absorbing and removing all liquid in the culture dish, adding the diluted DCFH-DA working solution, incubating in a cell culture box at 37 ℃ in the dark for 30min, washing cells for 2 times by using Opti-MEM culture medium after 30min, irradiating PDNP/siNC+L group by using 808nm laser, and ensuring the light intensity to be 1W/cm 2 Illumination time is 4min, hoechst 33342 dye 1: after 1000 dilution, the mixture was added to a petri dish for 10min and washed 3 times with 1 XPBS buffer; the results are shown in FIG. 9, where nucleic is a nuclear cell nucleus image and Merge is a combined image, using Confocal Laser Scanning Microscopy (CLSM) observation and recording.
The results of FIG. 8 show that over time, DPBF is oxidized by active oxygen generated by the PDNP/siNC complex and the characteristic peak at 430nm is decreased; the results in fig. 9 show that the green fluorescence of the PDNP/siNC complex is significantly stronger after treatment in the different groups than in the remaining groups. The above results demonstrate that the PDNP/siNC complex is effective in generating active oxygen with cytotoxicity under light conditions.
Test example 4 evaluation of cell viability and toxicity of pharmaceutical compositions
Toxicity and evaluation of the PDNP/siRNA complex on CT26 cells were analyzed using the MTT method, and the ability of the PDNP/siRNA complex to kill CT26 cells under light conditions. MTT is a water-soluble compound that can be reduced to insoluble formazan by succinate dehydrogenase on the mitochondrial membrane, specifically by the following steps: CT26 cells were plated on 96-well plates, 1X 10 per well 4 Individual cells, 96 well plates containing cells were incubated at 37℃with 5% by volume CO 2 The resulting PDNP/siNC complex, free siRNA (Naked siNC) and Lipo/siNC (complex obtained by transfecting siNC with transfection reagent Lipofectamine 2000 as positive control) were diluted to the corresponding volumes (100. Mu.L total volume per well) with transfection medium (Opti-MEM), the 1640 medium in 96 well plates was then aspirated, and the culture medium was changed to the Opti-MEM medium containing PDNP/siNC, naken or Lipo/siNC as described above, after 4 hours of transfection, 100. Mu.L of 1640 complete medium was supplemented per well and the culture was continued for 20 hours, the prepared MTT mother liquor (5 mg/mL) was diluted to 100. Mu.g/mL of working solution using 1640 medium, the medium in 96 well plates was aspirated, and the culture medium was addedThe prepared working solution was placed in an incubator, after 4 hours, all the solution in each well was pipetted off, 100 μl DMSO was added, incubated at 37 ℃ for 10min to allow the formazan produced to be fully dissolved, and absorbance was detected at wavelengths 540 and 650nm using an enzyme-labeled instrument, as shown in fig. 10A.
In evaluating the killing ability of the PDNP/siRNA complex on CT26 cells, the cells are subjected to light treatment after 3 hours of transfection, and the light intensity is 1W/cm 2 The illumination time was 4min, and the other steps were consistent with the MTT method described above, and the results are shown in FIG. 10B.
FIG. 10A shows that at a weight ratio of PDNP to siNC of 50:1, PDNP/siNC still had no significant toxicity to tumor cells; fig. 10B shows that the PDNP/siNC complex shows strong cytotoxicity to CT26 cells under light conditions and is dose dependent. The result shows that the PDNP/siNC compound does not have the effect of killing tumor cells, has good biological safety, and can generate active oxygen to kill the tumor cells after photodynamic activation.
Test example 5 evaluation of Gene silencing Effect of pharmaceutical compositions
Evaluation of PDNP/siRNA Complex mediated Gene silencing efficacy of cell PD-L1 referring first to the method of example 4, siRNA was subjected to CT26 cell line culture in accordance with the method of test example 4 to form a Nakend siPD-L1 group, a Lipo/siPD-L1 group, a PDNP/siPD-L1 group and a PDNP/siPD-L1+L group, respectively, with the weight ratio of PDNP to siRNA being 50:1, and total RNA of each group was extracted and assayed using Nanodrop; synthesizing cDNA by using a rapid reverse transcription kit, preparing a gDNA removal reaction system, preparing a template mRNA 1 mug/tube, and heating at 42 ℃ for 3min; then preparing a reverse transcription reaction system, adding the reverse transcription reaction system into the gDNA removal reaction system, heating at 42 ℃ for 15min, and continuously heating at 95 ℃ for 3min to carry out reverse transcription; qPCR: preparing a reaction system by using a Superreal fluorescent quantitative premixing kit, and setting a reaction program to be 95 ℃ for 15min;95 ℃ for 3s;60 ℃ for 20s; and (5) circulating for 40 times, performing qPCR reaction, and detecting the relative content of the target gene PD-L1 mRNA by taking GAPDH as an internal reference.
The silencing effect of PDNP/siPD-L1 on CT26 cell line gene was examined by qRT-PCR and the results are shown in FIG. 11A. FIG. 11A shows that PDNP/siPD-L1 was effective in inhibiting/modulating the expression level of mRNA of PD-L1 compared to the Mock group, PD-L1 mRNA expression was reduced by 60% in the PDNP/siPD-L1 group compared to the Mock control group.
To evaluate the ability of the PDNP/siRNA complex to mediate inhibition of expression of target genes at the cellular level, the relative luciferase activity of 4T1-Luc cells after different treatments was examined. Compounding the PDNP/siFL prepared in example 1 with siRNA (siFL) against Luciferase (weight ratio of PDNP to siRNA: 50:1) according to the method described in example 4, and transfecting the siFL with a transfection reagent Lipo 2000 to obtain a complex Lipo/siFL as a positive control; cell culture was performed according to the method of example 4, after plating for 24 hours, 1. Mu.g of siFL-containing PDNP/siFL or Lipo/siFL (system 25. Mu.LOpti-MEM) was transfected per well, three replicate wells were set per study sample, 2mL of fresh complete medium DMEM was added per well for 4 hours after transfection, culture was continued, all medium was aspirated, cells were gently washed with pre-chilled 1 xBS, PBS was then aspirated, 100. Mu.L of 1 Xcell lysate was added per well, and shaking was performed at room temperature for 20min to allow sufficient lysis of cells, cell lysate (containing lysed cells) was transferred to lmL centrifuge tubes, centrifugation was performed at 12000rpm for 30s at room temperature, 5. Mu.L of supernatant was aspirated into assay plates, 50. Mu.L of substrate was added per well, and the results were shown in FIG. 11B using a microplate reader. Compared with Mock, PDNP/siPD-L1 was able to significantly inhibit/modulate the mRNA expression level of luciferases. The expression level of mRNA was similar compared to the commercial lipid positive Lipo vector, indicating that PDNP/siPD-L1 can act as a suitable vector for reducing the expression level of mRNA.
Test example 6 study of endocytosis and endocytosis mechanism of pharmaceutical composition
CT26 cell culture was performed by the method of test example 4, and the polymer of PDNP prepared in example 1 was compounded with Cy 5-labeled siRNA (Cy 5-siRNA) according to the method described in example 4 (weight ratio of PDNP to siRNA: 50:1), and the compound Lipo/siRNA obtained by transfection of Cy5-siRNA with Lipofectamine 2000 was used as a positive control, and after 3 hours of transfection, 808nm light/no light treatment was performed on the group of PDNPs/siRNAs, and after 4 hours of transfection, cell uptake was examined by flow cytometry, and the results are shown in FIGS. 12A-12B. As shown in fig. 12A and 12B, the average fluorescence intensity and phagocytosis rate of the PDNP/siRNA complex gradually increased with time, proving that it has time dependence; the average fluorescence intensity and phagocytosis rate of the PDNP/siRNA complex at 4h transfection was slightly weaker than the commercial reagent Lipo2000, probably due to the easier entry of the lipid compounds into the cell membrane.
CT26 cell cultures were performed with various inhibitors to block the cellular internalization pathway, including Chlorpromazine (blocking clathrin-mediated endocytosis), amiloride (blocking macrophyte) and Genistein (blocking vesicle-mediated endocytosis) with the aim of finding the cell-dependent manner of PDNP/siRNA complex entry, while the cells were placed at 4 ℃ to block cell-dependent energy internalization, CT26 cells pre-incubated with different endocytosis inhibitors were subjected to PDNP/siRNA complex treatment for 4h, and Cy 5-labeled siRNA fluorescence was detected using confocal microscopy and flow cytometry, as shown in fig. 12C-12D. The results of FIGS. 12C-12D are similar to those obtained in FIGS. 12A-12B, with the average fluorescence intensity and phagocytosis rate of the PDNP/siRNA complex being weaker at 37℃than those of the commercial reagent Lipo2000, with 4℃and Genistein exhibiting the most significant inhibition of phagocytosis, indicating that endocytosis of the PDNP/siRNA complex is primarily energy dependent, via clathrin-mediated pathways, and that part of the pharmaceutical composition can also act by both small and giant potion into tumor cells using the small cell mediated endocytosis inhibitor Chlorpromazine (CPZ) and the giant potion inhibitor Amiloride (AMR).
Test example 7 test of the ability of pharmaceutical compositions to escape endosomes
CT26 cell culture was performed by referring to the method in test example 4, the specific position of the PDNP/siNC complex was localized using Cy 5-labeled siRNA, the nuclei were stained with the nuclear dye Hoechst 33342, lysosomes were labeled using Lysotracker, and the endosome escape ability of the PDNP/siNC complex obtained using a laser confocal microscope was tested, and the results are shown in FIG. 13. Free sipc is difficult to break through the physiological barrier into tumor cells, and sipc can be delivered into tumor cells using the commercial reagent Lipo2000, which Lipo2000 is unevenly dispersed in tumor cells due to heterogeneity of tumor cells and instability of Lipo2000, possibly resulting in uneven enrichment in lysosomes. After the PDNP/siNC complex is used for delivering the siNC, due to the stability of a PDNP carrier, the nano particles can obviously observe the accumulation of fluorescence of Cy5 in lysosomes at 0.5h, and the fluorescence curves of the two are distributed similarly, so that the siNC and the lysosomes have stronger co-localization. At 4h, the fluorescence intensity of cy5 gradually increased, and the fluorescence curves of both still showed a similar distribution. At 8h, the fluorescence curve of Cy5 deviated from that of the green lysosome, indicating that the PDNP/siNC complex escaped from the lysosome. When the laser is externally added for 8 hours, the deviation of red fluorescence from green fluorescence can be obviously observed from the confocal image, and the fluorescence curves of the red fluorescence and the green fluorescence show different distributions, which indicates that the light can promote the RNA to escape from the lysosome, which is favorable for the RNA to play a role in cytoplasm, and further highlights the advantages of the material.
Test example 8 study of immunogenic cell death of pharmaceutical compositions
CT26 cell culture was performed according to the method of test example 4, and then the ATP was indirectly detected according to the luciferin-producing fluorescence, as follows: diluting ATP standard solution with detection solution to proper concentration gradient, preparing ATP detection working solution, placing each sample and standard substance in 100 μl on ice, adding 100 μl ATP detection working solution into detection plate, and standing at room temperature for 5min; the suspension medium and the standard substance in the cell culture dish were each 20. Mu.L and added to the detection wells for detection, and the results are shown in FIG. 14A.
CT26 cell culture was performed according to the method described in test example 4, in which cells were digested and fixed with 4% by volume paraformaldehyde for 10min for CRT exposure, 0.5% Trito-100 was used for HMGB1, cells were permeabilized for 20min, 1 XPBS containing 1% BSA was added to a petri dish, the dish was blocked at room temperature for 30min, 1 XPBS diluted antibody containing 1% BSA was added, cell membrane surface Calreticulin (CRT) exposure was detected by flow cytometry, the above antibody-added cells were left at room temperature for 4h, and the petri dish was placed in a refrigerator at 4℃overnight; washing cells with 1 XPBS for 2-3 times, adding a secondary antibody solution related to CRT and high mobility group protein B1 (HMGB 1), incubating for 1h at room temperature in a dark place, and for flow cytometry detection, directly incubating the secondary antibody after directly incubating for 4h on the same day, and performing flow detection; in the confocal detection, cell nuclei are required to be dyed, DAPI dye is added for 5min, cells are washed for 2-3 times by using 1 XPBS, and light shielding is required after secondary antibodies are added in the operation, and the result is shown in figures 14B-14D, and G1 is Mock; g2 is Nakes SiPD-L1; g3: lipo/siPD-L1; g4, PDNP/siNC; g5, PDNP/siNC+L; g6 PDNP/siPD-L1; g7 PDNP/siPD-L1+L. .
ATP, CRT, HMGB1 serve as important biomarkers for immunogenic death. As shown in fig. 14A, the extracellular secretion of ATP from the PDNP/siNC complex group (PDNP/sinc+l group) was 8.35 times that from the untreated Mock control group, indicating that the laser irradiation was effective for promoting ATP secretion by physiological activities of tumor cells. As shown in the flow and confocal results of fig. 14B and 14C, the extracellular exposure of the PDNP/siNC complex to green calreticulin after additional laser irradiation can be clearly observed, and calreticulin acts as a signal for the tumor cell "eat me" and can promote phagocytosis of tumor cells by T cells. Meanwhile, HMGB1 release from the nucleus to the outside of the cell occurs during immunogenic death. As shown in fig. 14D, after light irradiation, the red fluorescence intensity in the cell nucleus was significantly decreased, thereby accelerating extracellular release of HMGB1, whereas in the remaining groups not irradiated with light, the fluorescence intensity in the cell nucleus was not significantly changed, indicating that only the tumor cells generated active oxygen, but the immunogenic death of the tumor cells could be promoted. The results show that the PDNP/siNC compound can generate active oxygen under the irradiation of near infrared 808nm light, the active oxygen can kill tumor cells, the immunogenic death of the tumor cells is never promoted, the surface exposure of CRT (cathode ray tube) is promoted, the release of HMGB1 and the secretion of ATP are promoted.
Test example 9 study in a pharmaceutical composition for treatment of colorectal cancer model
The armpit of 6-8 weeks female/male BALB/c mice was inoculated with about 100. Mu.L of mouse colon cancer cells CT26, or transplanted 3-5mm 3 When the tumor grows to 100mm 3 When left and right, mice were randomly divided into 5 groups, G1: PBS group, G2: PDNP/siNC group, G3: PDNP/sinc+l group, G4: PDNP/siPD-L1 group, G5: PDNP/siPD-l1+l group. Intratumorally injecting the corresponding medicaments of each group into mice at the 1 st and 4d, wherein the dosage of siRNA is 0.5mg/kg; after 6 hours, the tumor site was irradiated with 808nm laser light for 3min at 1W/cm 2 During this period, the body weight and tumor volume of the mice were recorded daily, and the volume calculation formula=1/2×length×width 2 The method comprises the steps of carrying out a first treatment on the surface of the When the tumor volume of the PBS group mice reached 2000mm 3 Euthanizing the mice, collecting tumors, hearts, livers, lungs, kidneys, spleens and blood of the mice, recording the weights of the tumors, spleens and livers of the mice, and keeping the residual mice as a life time observation; taking a part of the whole blood for blood cell analysis, centrifuging the rest whole blood, taking a supernatant for serum biochemical analysis, embedding and freezing the organ paraformaldehyde and OCT gel, and then carrying out hematoxylin/eosin staining analysis.
Fig. 15A is a schematic of tumor treatment and grouping information. After 6h of administration, the tumor area of the illuminated group was irradiated with 808nm laser light. Fig. 15B is a tumor volume growth curve during treatment. The G1 group grew faster as no inhibition was added, and at 14 days the tumor volume increased 40-fold compared to the initial. Similar to the results of the cell experiments of fig. 10A, the G2 group showed similar growth behavior to the G1 group in the tumor growth without laser irradiation. Because the siPD-L1 released in the G4 group can inhibit PL-L1 in tumor cells and enhance the phagocytosis of the tumor cells by T cells, the tumor growth of the G4 group can be inhibited to a certain extent. Compared with the G3 group, the G5 group effectively inhibits the tumor growth due to the active oxygen induced tumorigenic death generated in the photodynamic process and the enhanced phagocytosis of the tumor cells by the siPD-L1T cells, which synergistically improve the tumor microenvironment. The above results are also further confirmed from the quality of the dissected tumor in fig. 15D. The body weight of mice did not change significantly throughout the treatment period, demonstrating the safety of PDNP as a vehicle. FIGS. 15E and 15F are liver/body weight ratios and spleen/body weight ratios obtained by weighing the liver and spleen, and no significant changes in liver/body weight ratios and spleen/body weight ratios were observed, further demonstrating the biosafety of PDNP and the lack of stress type immune response in the liver and spleen under the irradiation of externally applied laser, which resulted in damage to the normal body. As shown in the survival curve of fig. 15G, it was found that only one of the five mice died within the observation period of 35 days in the G5-treated group, while all the other mice in each group died within 35 days.
Test example 10
In the treatment of mice, tumor photodynamic method and immune checkpoint inhibition method were used, and the mice tumor, spleen and draining lymph node of 6d after treatment in test example 9 were subjected to immunoassay. The method comprises the following specific steps: taking tumor, spleen and drainage lymph node of a 6d mouse, placing the tumor, spleen and drainage lymph node in 1 XPBS buffer solution, shearing the tissues by using a shearing knife, placing the crushed tissues in a 200-mesh molecular sieve for grinding, preparing single-cell suspension, collecting grinding liquid of each tissue, 300g, centrifuging for 5min, pouring out supernatant, reserving bottom cell sediment, adding 1-3mL of erythrocyte lysate, and standing for 20min; adding 1 XPBS (phosphate buffer solution) with the volume twice that of the lysate, uniformly mixing by using a suction pipe, centrifuging for 5min at 300g, pouring out supernatant, reserving cell sediment, ensuring that red blood cells are removed completely, and repeating the operation if the red blood cells are not removed completely; adding 3-5mL 1 XPBS buffer, transferring to a new molecular sieve, collecting and filtering single cell suspension, 300g, centrifuging for 5min; 1 XPBS 1-3mL containing 1% BSA was added and placed on a shaker at room temperature for 1h of blocking, after which the cells were stained with various antibodies, the antibodies used to differentiate immune cell populations were as follows: cd3+, cd4+, cd8-, cd3+, cd4-, cd8+, cd11c+, cd80+, cd86+, light-shielding for 1 hour at room temperature, washing the cells 2-3 times with 1×pbs buffer, adding 300 μl of 1×pbs buffer for flow cytometry analysis, and evaluating the immune status change results of tumor microenvironment and immune organs after PDNP/siRNA complex treatment as shown in fig. 16.
FIGS. 16A and 16B are statistics of CD80+CD86+DC cells in tumor and lymph nodes, respectively, and active oxygen generated after 808 laser irradiation promotes tumor immune death, and released ATP, HMGB1 and CRT promote maturation of DC cells, so that in G5 tumor and lymph nodes we detected that mature DC cells are significantly higher than in the other groups, quantitative results of lymph node mature DC also indicate that siPD-L1 can also enhance maturation of DC cells, which may be related to improvement of immunogenic death promoting tumor microenvironment further enhancing DC phagocytosis tumor-related antigens in lymph nodes. Fig. 16C and 16E are changes in cd3+cd4+ T cells in tumor and lymph node, respectively, and fig. 16D and 16F are changes in cd3+cd8+ T cells in tumor and lymph node, respectively. Because of the photodynamic induced dual synergistic effect of tumor immunogenic death and siPD-L1, the number of cytotoxic T lymphocytes in the G5 group was significantly higher than in the remaining groups, thus killing the tumor most strongly, which was also demonstrated from the tumor growth curve and dissected tumor mass. The result shows that the PDNP/siNC compound can induce CT26 tumor-bearing mice to generate systemic immune response under the irradiation of light, and can further develop the immune regulation effect of organisms and play an anti-tumor immunity role by combining immune checkpoint siPD-L1 therapy.
Test example 11
To evaluate the recovery of normal levels in mice treated with the PDNP/siRNA complex, whole blood from tumor-bearing mice was collected for blood analysis after 11d of treatment with reference to the method in test example 9, and the in vivo safety factor of the PDNP/siRNA complex was evaluated, and the results are shown in fig. 17. As shown in fig. 17, parameters including WBC (white blood cell count), RBC (red blood cell count), lym (lymphocyte ratio), HCT (hematocrit), MCV (mean red blood cell volume), MCH (mean red blood cell hemoglobin content), MCHC (mean red blood cell hemoglobin concentration), RDW-CV (red blood cell distribution width variation coefficient) and the like are included. The detection of treated whole blood of mice shows that under the irradiation of the PDNP/siNC compound and the laser, various physiological indexes of the mice have no obvious change compared with PBS group, further shows that the biological safety of the mice can be used as an excellent RNA delivery carrier without causing systemic immune toxicity, probably due to the inertia of the PDNP in normal physiological environment and the specific release of the PDNP in tumor microenvironment.
Test example 12
To evaluate the safety level of organs in the tissues of mice after treatment with the PDNP/siRNA complex, heart, liver, spleen, lung, kidney tissues of tumor-bearing mice were collected for H & E staining analysis after 11d of treatment with the method of reference test example 9, and the evaluation results of the anti-tumor safety factor in the PDNP/siRNA complex are shown in fig. 18. From fig. 18, it can be seen that the various tissue organs of the mice were not abnormal, further demonstrating the excellent biosafety of the PDNP/siNC complex and the potential as an RNA delivery vehicle.
Test example 13
Referring to the method in test example 9, a study of PDNP/siRNA complexes in the treatment of recurrent breast cancer models was performed with breast cancer tumor cells 4T1 as mouse breast cancer cells, the experiment divided into 5 groups, G1: PBS group, G2: PDNP/siNC group, G3: PDNP/sinc+l group, G4: PDNP/siPD-L1 group, G5: PDNP/siPD-l1+l groups, the results are shown in fig. 19.
Fig. 19A is a schematic of tumor treatment and grouping information. Mice were vaccinated with 4T1-Luc tumor cells at day-7, and tumors were excised and the excised tumor sites were sutured until tumor volume reached 500mm 3. After 15 and 18 days of injection of the different drugs and light exposure, focal fluorescence was tracked by in vivo imaging and tumor volumes were recorded. Fig. 19B shows the change in volume growth of tumors in the recurrence model, since the 4T1 tumor model was prone to recurrence, tumor growth was not inhibited in G1 and G2 groups, whereas since G4 group siPD-L1 could shield PD-L1 expressed on the tumor cell surface, phagocytosis of tumors by immune system was enhanced, and recurrence of tumors was alleviated to some extent. The active oxygen generated in the photodynamic process of the G3 group induces the tumor cells to generate immunogenic death, and the growth of recurrent tumor is obviously inhibited. The G5 group has almost no recurrent tumorigenesis under the double functions of photodynamic and immune checkpoint blocking SiPD-L1, and proves the superiority of photodynamic combined immune checkpoint blocking therapy in tumor treatment. Fig. 19C shows the change in weight of mice during different treatments, with no significant change in weight of mice after different treatments, and with a certain increase in weight compared to the pre-treatment, similar to the results obtained in the CT26 anti-tumor model before. Fig. 19D is the mass of the dissected tumor after treatment, and the results obtained are similar to the tumor growth curve of fig. 19B, G5 being barely detectable under both photodynamic and RNA therapy. Fig. 19E is a measurement of survival of mice following various treatments, during which the non-lased group died almost entirely within 40 days, whereas significantly prolonged survival was observed in lased G3 and G5 groups, possibly associated with a stronger penetration depth of the two-zone photodynamic therapy. FIGS. 19F and 19G are, respectively, expression profile and gray scale quantification of tumor PD-L1 after treatment, and similar to the expected results, the use of PDNP/siNC complex to carry siPD-L1 can reduce the expression of PD-L1 in tumors, which helps to enhance the killing power and sensitivity of photodynamic to tumor cells, and to extend the inventory time of mice. FIG. 19H shows the change of biochemical indicators of various groups after treatment, including ALT (glutamic pyruvic transaminase), AST (glutamic pyruvic transaminase), CK-MB (creatine kinase), UREA (UREA ammonia) and other parameters. The absence of significant changes in each parameter compared to the PBS group, closer to the normal range, suggests an important potential in the prevention of tumor recurrence using the PDNP/siNC complex after surgical resection.
Test example 14
Referring to the method of example 4, the siRNA was used with siPLK1 to prepare a PDNP/siPLK1 complex at a weight ratio of PDNP to siRNA of 50:1. Referring to the method in test example 9, a study of PDNP/siRNA complexes in treating a PDX patient-derived liver cancer model was performed, with liver cancer tumor cells being patient-derived xenograft liver cancer cells, and the experiments were divided into 5 groups: g1: PBS group, G2: PDNP/siNC group, G3: PDNP/sinc+l group, G4: PDNP/siPLK1 group, G5: PDNP/siplk1+l groups, the results are shown in fig. 20.
Fig. 20A is a schematic of tumor treatment. Fig. 20B is a graph of the growth of each group of tumors after treatment with different drugs during the treatment period, since PDX grew faster in nude mice, and the treatment was terminated on the sixth day, and the tumor volumes of mice were recorded daily during the treatment period. Due to the synergistic effect of photodynamic and RNA interference therapy, the growth of the G5 group tumor is obviously inhibited, and the tumor volume of the G1 group tumor is up to 2000mm in the treatment period of 6 days 3 The growth of the other groups of tumors is also inhibited to a certain extent. FIG. 20C shows a therapeutic knotThe tumor mass of post-bundle resected mice was found from the quantitative results to be significantly weaker in the G5 group than the other group due to the synergistic effect of photodynamic and RNA interference. Fig. 20D shows the change in survival rate of nude mice during treatment, in which group G1, G2, G4 tumors had completely died within about 15 days of observation period of 40 days, whereas group G3 tumors survived only 1/5 of nude mice in 40 days, and group G5 showed significant survival rate, and only two mice died within 40 days of observation period. FIG. 20E shows the reduction of PLK1 mRNA expression in treated nude mice tumors by the synergistic effect of photodynamic and RNA interference in the G5 group. Fig. 20F shows that there was no significant difference in liver/body mass index for each group of mice after the course of treatment, and that the spleen was significantly reduced in the G5 group of mice compared to the other groups, which may be associated with inhibition of tumor metastasis by photodynamic therapy and RNA interference therapy.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims (10)

1. A nucleic acid delivery vector is characterized in that the structure of the nucleic acid delivery vector is shown as a formula (I),
Figure FDA0003758451470000011
formula (I), wherein m, n, p are each independently a positive integer of 3-15, and q is a positive integer of 40-120.
2. A method of preparing a nucleic acid delivery vector comprising the steps of:
(1) In the presence of a reaction solvent I, mixing a compound shown in a formula (II), 2-bis (bromomethyl) -1, 3-propanediol and bis (2-hydroxyethyl) disulfide with L-lysine diisocyanate to perform a reaction I, and then mixing the mixture with methoxy polyethylene glycol-hydroxyl to perform a reaction II to obtain a reactant P1 shown in a formula (III);
(2) Mixing a reactant P1 with a compound shown in a formula (IV) in the presence of a reaction solvent II to perform a reaction III, so as to obtain a nucleic acid delivery vector shown in the formula (I);
Figure FDA0003758451470000021
Figure FDA0003758451470000031
Figure FDA0003758451470000032
Wherein m, n and p are each independently positive integers of 3-15, and q is a positive integer of 40-120.
3. The preparation method according to claim 2, wherein the weight ratio of the compound represented by the formula (II), 2-bis (bromomethyl) -1, 3-propanediol, bis (2-hydroxyethyl) disulfide, L-lysine diisocyanate and methoxypolyethylene glycol-hydroxyl group in the step (1) is 1:0.8-1.2:0.6-0.9:2-2.4:4.5-5.5;
preferably, the molecular weight of the methoxy polyethylene glycol-hydroxyl group is 2000-5000;
preferably, the conditions of reaction I include: the temperature is 45-55 ℃ and the time is 10-15h;
preferably, the conditions of reaction II include: the temperature is 45-55deg.C, and the time is 10-15h.
4. A process according to claim 2 or 3, wherein the weight ratio of the reactant P1 of formula (III) to the compound of formula (IV) in step (2) is 2.5-3.5:1;
preferably, the conditions of reaction III include: the temperature is 45-55deg.C, and the time is 10-15h.
5. The production method according to claim 2 or 3, wherein the reaction solvent I and the reaction solvent II are each independently selected from at least one of anhydrous N, N-dimethylformamide, anhydrous dichloromethane, anhydrous tetrahydrofuran, and anhydrous dimethyl sulfoxide;
Preferably, the step (1) further comprises: dialyzing the reaction solution obtained in the reaction II to obtain a dialyzate I, and drying the dialyzate I to obtain a reactant P1 shown in the formula (III);
preferably, the step (2) further includes: and (3) dialyzing the reaction solution obtained in the reaction III to obtain a dialysate II, and drying the dialysate II to obtain the nucleic acid delivery vector shown in the formula (I).
6. A functionally synergistic pharmaceutical composition, characterized in that it contains siRNA and a nucleic acid delivery vector according to claim 1 and/or a nucleic acid delivery vector prepared according to the method of any one of claims 2 to 5.
7. The pharmaceutical composition of claim 6, wherein the weight ratio of the nucleic acid delivery vehicle to the siRNA is 10-100:1;
preferably, the siRNA comprises a sense strand and an antisense strand that are fully reverse complementary, the nucleotide sequences of the sense strand and the antisense strand being shown as any one pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6, respectively.
8. A method of preparing a functionally synergistic pharmaceutical composition comprising the steps of: mixing the nucleic acid delivery vector according to claim 1 and/or the nucleic acid delivery vector prepared by the preparation method according to any one of claims 2 to 5 with a micelle agent to obtain a nucleic acid delivery vector micelle, and mixing the nucleic acid delivery vector micelle with siRNA to obtain a reaction II.
9. The method of claim 8, wherein the weight ratio of the nucleic acid delivery vector to the siRNA is 10-100:1;
preferably, the siRNA comprises a sense strand and an antisense strand which are fully reverse complementary, the nucleotide sequences of the sense strand and the antisense strand are shown in any one pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6, respectively;
preferably, the micelle agent is ultrapure water; the micelle agent is used in an amount of 0.1 to 0.3mL relative to 1mg of the nucleic acid delivery vehicle;
preferably, the process of mixing reaction I comprises: after dissolving the nucleic acid delivery carrier in a solvent, dripping the nucleic acid delivery carrier into the micelle agent, stirring the mixture for 25 to 35 minutes at the temperature of 5 to 40 ℃ and the rotating speed of 80 to 150rpm to obtain a mixed reaction solution, and dialyzing III the mixed reaction solution to obtain a nucleic acid delivery carrier micelle;
preferably, the conditions of the mixing reaction II include: the temperature is 5-40deg.C, and the time is 25-35min.
10. Use of at least one of the nucleic acid delivery vector according to claim 1, the nucleic acid delivery vector produced according to the method of any one of claims 2 to 5, the pharmaceutical composition according to claim 6 or 7 and the pharmaceutical composition produced according to the method of claim 8 or 9 in the manufacture of an antitumor drug;
Preferably, the tumor is of a type selected from at least one of colorectal cancer, breast cancer and liver cancer.
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