CN113332452A - Co-loaded immune adjuvant and green indocyanine polymer vesicle with triple pH/reduction/temperature stimulation responses, and preparation method and application thereof - Google Patents

Co-loaded immune adjuvant and green indocyanine polymer vesicle with triple pH/reduction/temperature stimulation responses, and preparation method and application thereof Download PDF

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CN113332452A
CN113332452A CN202110639519.4A CN202110639519A CN113332452A CN 113332452 A CN113332452 A CN 113332452A CN 202110639519 A CN202110639519 A CN 202110639519A CN 113332452 A CN113332452 A CN 113332452A
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张琳华
朱敦皖
郭勍
黄晨露
左月月
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Abstract

The invention discloses a pH/reduction/temperature triple stimulus response co-loaded immune adjuvant and indocyanine green polymer vesicle and a preparation method and application thereofA thin film with uniform layer; drying the film, hydrating, and adding NH4HCO3And mixing, carrying out ultrasonic treatment and dialysis to obtain a polymer vesicle solution; mixing the polymer vesicle solution with LHRH, reacting under the catalysis of triethylamine, and dialyzing to obtain the polymer vesicles; the invention adopts PCL-b-PEG-b-PCL as a carrier material of a polymer vesicle, carries photosensitizer ICG and TLR-7/8 agonist IMQ on a hydrophobic membrane layer, and carries bubble generating agent NH in a hydrophilic inner cavity4HCO3And LHRH targeting is connected to the surface of the polymersome to obtain the polymersome which can carry photosensitizer ICG and immunologic adjuvant IMQ together and has a targeting function and a pH/reduction/temperature triple stimulation response type for treating tumors through photo-thermal combined immunotherapy.

Description

Co-loaded immune adjuvant and green indocyanine polymer vesicle with triple pH/reduction/temperature stimulation responses, and preparation method and application thereof
Technical Field
The invention relates to the technical field of tumor treatment by photothermal-immune combination therapy, in particular to a pH/reduction/temperature triple-stimulus-response co-carried immune adjuvant and an indole green polymer vesicle, and a preparation method and application thereof.
Background
The polymer vesicle has high stability as a nano drug-carrying system. Compared with liposome nanoparticles, the polymer vesicle has higher stability, can form a thicker and more rigid double-layer structure when being self-assembled into the polymer vesicle, has more complete structure, and can avoid the leakage of medicaments. Compared to polymer micelles having a core-shell structure, polymer vesicles can be loaded with both hydrophilic and hydrophobic drugs more efficiently due to their bilayer structure similar to liposomes. Therefore, the polymersome can effectively load hydrophilic and hydrophobic drugs, avoid the drugs from being eliminated in vivo, and improve the drug loading rate and stability and the systemic delivery effect. The surface of the polymer vesicle can be modified by biological ligands so as to actively target tumor cells and effectively deliver drugs to tumor sites, thereby improving the treatment effect. The nano-carrier designed according to the tumor microenvironment can trigger drug release under specific stimulation, and realize the polymer vesicle targeted tumor local trigger drug release, thereby efficiently treating cancer.
Dendritic Cells (DCs) are the most potent Antigen Presenting Cells (APCs), innate immunity and adaptabilityPlays an important role in immunity. Immature DCs take up Tumor-associated antigens (TAAs), transfer them to Major Histocompatibility Complex I (MHCI) and load their migration to secondary lymphoid organs such as lymph nodes and spleen, where they differentiate from the immature state into the mature state. Mature DCs surface co-stimulatory molecules CD40, CD80, CD86, as well as MHCI and MHC ii expression are upregulated, producing various pro-inflammatory and regulatory cytokines. Mature DCs can present phagocytosed antigen to CD4+T cells and CD8+T cells, and activated CD4+T cells also further activate DCs through the interaction of CD40L with CD40, which in turn promotes CD8 through MHCI and the binding of CD80/86 to CD28 expressed by naive T cells+T cells react, producing cytotoxic effector T cells and memory T cells.
Toll-like receptors (TLRs) are predominantly expressed on macrophages and dendritic cells, TLR-7 is predominantly expressed on APCs, and TLR-8 is predominantly expressed on myeloid cells, such as macrophages, monocytes, and myeloid DCs. The TLR agonist can enhance the expression and cell migration capacity of DCs surface co-stimulatory molecules to promote the processing and presentation of TAA, and can promote DCs to produce cytokines to stimulate CD8+Proliferation and differentiation of T cells into Cytotoxic T Lymphocytes (CTLs) and CD4+T cells are proliferated and differentiated into Helper T cells (Th), so that Th type cell response is promoted, and anti-tumor response capability is stimulated. TLR-7/8 agonists can induce the maturation of DCs through Myeloid differentiation protein 88(My D88) dependent pathway and stimulate the production of cytokines including Interleukin 6 (IL-6), Interleukin-12 (Interleukin-12, IL-12), Tumor necrosis factor-alpha (TNF-alpha) and gamma Interferon (Interferon-gamma, IFN-gamma), and further activate CTLs and Th 1 cells. Activated CTLs can kill tumor cells by direct contact mediated cytotoxicity, and can also secrete cytokines such as IFN- γ that mediate local inflammation. CD8 with simultaneous small partial activation+Differentiation of T cells into memory CD8+T cells, which can be maintained for a long period without antigen stimulation, proliferate when antigen re-invades. CD4+T cells also play a crucial role in immune responses, CD4+Th 1 cells promote the generation and maintenance of memory CTLs phenotype by proliferation, while activation of Th 1 cells facilitates the activation of CTLs.
ICG is an amphiphilic tricarbocyanine dye with a large pi conjugated system, the structure determines that the dye has strong absorptivity in a near infrared spectrum region of 700-850nm and has the characteristic of low toxicity, and the dye is a near infrared imaging agent approved and applied to clinic by FDA and is also an effective NIR light absorber in laser-mediated photothermal therapy. Free ICG has the disadvantage of being prone to aggregation, degradation and poor photostability in aqueous solutions due to saturation of the double bonds in the conjugated chain, while it lacks target specificity, binds non-specifically to proteins and is rapidly cleared in the human body (half-life of 2-4 min). The nano drug-loaded system can effectively coat drugs to avoid the drugs from being removed in vivo, so that the drug-loaded rate and stability and the systemic delivery effect of the drugs are improved, and the polymer vesicle serving as a nano carrier has higher stability and can effectively deliver ICG to a tumor part, thereby improving the photo-thermal effect. As an FDA approved immunomodulator, IMQ has been used clinically in the treatment of anogenital warts, actinic keratosis and superficial basal cell carcinoma. Furthermore, IMQ is used as an TLR-7/8 agonist by multiple studies as an immune adjuvant to stimulate an immune response.
Photothermal therapy and immunotherapy have been widely studied in recent years as a highly effective cancer treatment method. However, the single photothermal therapy is mainly used for ablation treatment of in-situ tumors, and cannot completely eradicate tumors with large volumes or effectively inhibit tumor metastasis and recurrence. The speed of killing tumor cells by a single immunotherapy is relatively slow, immune evasion phenomena of different degrees exist, the effect of immunotherapy is reduced, and meanwhile, the tumor immunotherapy can also generate side effects and adverse reactions by considering the difference of an immune system. Therefore, the combination of photothermal therapy and immunotherapy can improve the effect of cancer treatment. The photothermal-immune combination therapy of the tumor acts on the tumor through the combination of photothermal effect and immune response, avoids the side effect of cancer monotherapy, and simultaneously can enhance the specific ability of the immune system to kill the tumor, so that the tumor is a promising cancer treatment strategy with lower toxicity, more effectiveness and more accuracy. Therefore, there is a high need to design a triple stimulus responsive polymersome capable of co-delivering photosensitizer and immunomodulator according to the tumor microenvironment to realize targeted photothermal-immune combination therapy to effectively inhibit the growth, metastasis and recurrence of tumor.
Disclosure of Invention
The invention aims to provide a co-loading immune adjuvant and an indocyanine green polymer vesicle with triple stimulation responses of pH/reduction/temperature, and a preparation method and application thereof4HCO3And LHRH targeting is connected to the surface of the polymersome to obtain the polymersome which can carry photosensitizer ICG and immunologic adjuvant IMQ together and has a targeting function and a pH/reduction/temperature triple stimulation response type for treating tumors through photo-thermal combined immunotherapy.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a preparation method of a pH/reduction/temperature triple stimulus response co-loaded immune adjuvant and an indocyanine green polymer vesicle, which comprises the following steps:
(a) dissolving PCL-b-PEG-b-PCL, hydrophobic ICG, DSPE-PEG-Mal and immunologic adjuvant in an organic solvent, and then removing the organic solvent to form a uniform film;
(b) drying the film, hydrating, and adding NH4HCO3Mixing uniformly, then carrying out ultrasonic treatment and dialysis to obtain a polymer vesicle solution;
(c) and mixing the polymer vesicle solution and LHRH, reacting under the catalysis of triethylamine, and dialyzing to obtain the co-loaded immune adjuvant and green indocyanine polymer vesicles with the pH/reduction/temperature triple stimulus response.
Preferably, the immune adjuvant comprises a TLR-7/8 agonist, and the TLR-7/8 agonist is IMQ.
Preferably, the organic solvent is selected from one or a mixture of two of dichloromethane and methanol. More preferably, the mass ratio of the volume of the organic solvent to the PCL-ss-PEG-ss-PCL is (4-6) mL to (15-25) mg.
Preferably, the drying is vacuum drying, and the drying time is 10-24 h.
Preferably, the molecular weight of the PCL-b-PEG-b-PCL is 10000-24000, preferably 22500; wherein the mass percentage of the PCL hydrophobic chain segment is more than 33 percent; the PCL-b-PEG-b-PCL is preferably PCL7500-PEG7500-PCL7500。
Preferably, the solvent used for hydration is deionized water or a PBS solution; the hydration temperature is 60-70 ℃, and the time is 5-6 h;
the mass ratio of the addition volume of the solvent to the PCL-b-PEG-b-PCL is (3-10) mL: 20 mg.
Preferably, in the step (a),
the mass ratio of the PCL-b-PEG-b-PCL to the hydrophobic ICG is (15-25) to 1;
the mass ratio of the PCL-b-PEG-b-PCL to the immunologic adjuvant is (15-25) to 1;
the mass ratio of the PCL-b-PEG-b-PCL to the DSPE-PEG-Mal is (40-60) to 1.
Preferably, in the step (b),
NH4HCO3the final concentration of the additive (b) is 280-320 mM;
the ultrasonic treatment is ultrasonic treatment for 25-35 min under the ice bath condition;
the cut-off molecular weight of a dialysis bag used for dialysis is 8000-14000 Da; the dialysis time is 3-12 h.
Preferably, in the step (c),
the mass ratio of the PCL-b-PEG-b-PCL to the LHRH is (60-100) to 1;
the reaction is carried out for 10-12 h under the conditions of stirring and room temperature;
the cut-off molecular weight of a dialysis bag used for dialysis is 8000-14000 Da; the dialysis time is 4-8 h.
Preferably, the hydrophobic ICG is prepared by the following process:
dissolving hydrophilic ICG and tetrabutyl ammonium iodide in an organic solvent, and stirring for reaction at a dark room temperature to obtain a hydrophobic photosensitizer ICG, wherein the mass ratio of the tetrabutyl ammonium iodide to the hydrophilic ICG is (5-6): the organic solvent may be dichloromethane.
The invention provides a pH/reduction/temperature triple-stimulus-response co-loaded immune adjuvant and an indocyanine green polymer vesicle prepared by the preparation method.
The third aspect of the invention provides an application of the above-mentioned co-carried immune adjuvant with pH/reduction/temperature triple stimulation response and the indocyanine green polymer vesicle in preparation of an anti-tumor photothermal-immune combination therapy drug.
Compared with the prior art, the invention has the beneficial effects that at least:
(1) the invention adopts amphiphilic triblock copolymer PCL-b-PEG-b-PCL with good biocompatibility as a carrier material of polymer vesicles, prepares the polymer vesicles with uniform and stable particle sizes by using a thin film hydration ultrasonic dispersion method, can load photosensitizer ICG and TLR-7/8 agonist IMQ on a hydrophobic film layer and bubble generating agent NH into a hydrophilic inner cavity4HCO3And LHRH targeting is connected to the surface of the polymersome to obtain the polymersome which can carry photosensitizer ICG and immunologic adjuvant IMQ together and has a targeting function and a pH/reduction/temperature triple stimulation response type for treating tumors through photo-thermal combined immunotherapy.
(2) The invention designs a nano delivery system taking polymer vesicles as carriers, which carries photosensitizer ICG and TLR-7/8 agonist IMQ together, and can effectively increase ICG stability and IMQ solubility. The photo-thermal effect is exerted under the irradiation of near-infrared laser with the wavelength of 808nm to ablate primary tumor, NH is generated in the acidic environment of the tumor4HCO3Production of CO2Bubbles are formed to promote drug release, lysosome escape is realized, the activation and maturation of BMDCs are promoted, and CD8 is effectively activated+T cells and CD4+T cell immunity, generate strong T cell killing effect, promote the secretion of cell factors, simultaneously promote the generation of memory T cells, and achieve the effects of eliminating tumors and preventing tumor recurrence by photothermal combined immunity.
(3) The pH/reduction/temperature response type co-carried photosensitizer ICG and immune adjuvant IMQ-based polymer vesicle with targeting effect is designed by co-carrying the photosensitizer ICG and TLR-7/8 agonist IMQ on a hydrophobic membrane layer and wrapping a vesicle generating agent NH in a hydrophilic inner cavity4HCO3The compound preparation plays a role in killing tumors by light and heat under the irradiation of near-infrared laser, promotes lysosome escape and activates CD8+T cells and CD4+The immune reaction of T cells realizes photothermal synergistic immunity to play an anti-tumor effect, and effectively prevents tumor recurrence and lung metastasis.
(4) The pH/reduction/temperature response type polymer vesicle with targeting effect based on the co-carried photosensitizer ICG and the immunologic adjuvant IMQ, which is designed by the invention, realizes long blood circulation in the process of treating tumors through tail vein injection, enters tumor cells by utilizing the high permeability and retention effect of tumor tissues and LHRH targeting effect, realizes the purpose of photo-thermal combined immunotherapy of tumors in the acidic and reducing environment inside the tumors and under the irradiation of near-infrared laser, can effectively eliminate the tumors, and prevents the recurrence and the metastasis of the tumors. The polymersome has good biocompatibility and no toxicity to normal tissues and cells.
(5) The preparation method of the pH/reduction/temperature response type co-carried photosensitizer ICG and immune adjuvant IMQ-based polymer vesicle with the targeting effect, which is designed by the invention, has the advantages of simple process, low cost and short preparation period.
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In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a graph showing the stability of the particle size of the polymersome prepared in example 1 of the present invention, an ultraviolet absorption spectrum, a graph showing the change in particle size under different conditions, the in vitro release of IMQ under different conditions, and a laser beam at 808nm (1.5W/cm)2) Semi-quantitative PBS (phosphate buffer solution) and F (fluorescent phosphate buffer solution) of DPBF (double-stranded fluorogenic probe) under irradiationreactive oxygen species generation profiles of ree ICG, ARL, and AIRL;
FIG. 2 is a graph of in vitro photothermal temperature rise, cooling-temperature rise cycle and near infrared thermal imaging of the polymersome AIRL, AIR, IRL and AIL prepared in example 1 of the present invention and free drug;
FIG. 3 is a laser confocal graph and a flow chart of CT26 cellular uptake of polymersome and free drug prepared in example 1 of the present invention;
FIG. 4 is a graph showing the toxicity test results of CT26 cells by the polymersome and free drug prepared in example 1 of the present invention;
FIG. 5 is a laser confocal image and a flow chart of the cellular active oxygen detection of the polymersome and the free drug prepared in example 1 of the present invention;
FIG. 6 is an ICG fluorescence image and a tumor local ICG mean fluorescence intensity histogram of the polymersome and free drug prepared in example 1 of the present invention in BALB/C mice;
FIG. 7 is a near infrared thermal imaging graph and a photothermal temperature curve graph of the polymer vesicle and the free drug prepared in example 1 of the invention in a BALB/C mouse;
FIG. 8 is a flow chart, an uptake flow chart and a laser confocal chart of toxicity of the polymersome and the free drug on DCs prepared in example 1 of the present invention;
FIG. 9 is a graph showing the flow results of the Transwell system evaluating the effect of polymersomes prepared in example 1 of the present invention on the maturation and activation of BMDCs under laser irradiation;
FIG. 10 is a graph showing the inhibition of tumor growth in the proximal and distal ends of BALB/C mice by the polymersome and free drugs prepared in example 1 of the present invention, a graph showing survival curves and a graph showing weight changes;
FIG. 11 is a graph of H & E staining in the major organs of BALB/C mice;
FIG. 12 is a graph showing flow-through results of maturation and activation of DCs in lymph nodes in BALB/C mice treated with polymersomes prepared in example 1 of the present invention;
FIG. 13 is a graph showing flow results of mouse splenocyte proliferation in BALB/C mice treated with polymersomes prepared in example 1 of the present invention;
FIG. 14 is a graph showing CD3 of splenocytes from BALB/C mice treated with polymersomes prepared in example 1 of the present invention+CD4+、CD3+CD8+A T cell population distribution flow chart;
FIG. 15 is a graph showing the flow results of mouse spleen memory T cell assay after BALB/C mice are treated with the polymersome prepared in example 1 of the present invention;
FIG. 16 is a graph showing the anti-lung metastasis effect of BALB/C mice treated with the polymersome prepared in example 1 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
The invention provides a preparation method of a pH/reduction/temperature triple-stimulus-response co-loaded immune adjuvant and an indocyanine green polymer vesicle, which comprises the following steps:
(a) dissolving PCL-b-PEG-b-PCL, hydrophobic ICG, DSPE-PEG-Mal and immunologic adjuvant in an organic solvent, and then removing the organic solvent to form a uniform film;
(b) drying the film, hydrating, and adding NH4HCO3Mixing uniformly, then carrying out ultrasonic treatment and dialysis to obtain a polymer vesicle solution;
(c) and mixing the polymer vesicle solution and LHRH, reacting under the catalysis of triethylamine, and dialyzing to obtain the co-loaded immune adjuvant and green indocyanine polymer vesicles with the pH/reduction/temperature triple stimulus response.
The technical solution provided by the present invention is further illustrated below with reference to specific examples.
The hydrophobic ICG was prepared by the following method:
dissolving hydrophilic ICG and tetrabutyl ammonium iodide in dichloromethane, and stirring and reacting under the condition of keeping out of the sun and room temperature to obtain a hydrophobic photosensitizer ICG, wherein the ratio of the volume of the dichloromethane to the mass of the hydrophilic ICG is 1 mL: 1mg, tetrabutylammonium iodide and hydrophilic ICG in a mass ratio of 5.72: 1.
example 1
This example is a preparation method of a pH/reduction/temperature triple stimulus responsive co-loaded immune adjuvant and a green indocyanine polymer vesicle, comprising the following steps:
(a) adding PCL7500-PEG7500-PCL750020mg, hydrophobic ICG 1mg, DSPE-PEG-Mal0.4mg and TLR-7/8 agonist IMQ 1mg are dissolved in 5mL of mixed solvent of dichloromethane and methanol, then dichloromethane and methanol are removed by rotary evaporation and a layer of uniform thin film is formed on the flask;
(b) vacuum drying the film for 12h, adding 5mL deionized water, hydrating in a constant temperature drying oven at 65 ℃ for 5h, cooling to room temperature, and adding NH4HCO3Uniformly mixing to make the final concentration of the mixture be 300mM, then carrying out ultrasonic treatment for 30min under the ice bath condition, and dialyzing for 3h by adopting a dialysis bag with the molecular weight cutoff of 8000-14000Da to obtain a polymer vesicle solution;
(c) mixing the polymer vesicle solution with LHRH 0.25mg, reacting for 10h under the catalysis of 1 μ L triethylamine under stirring at room temperature, and dialyzing for 6h by using a dialysis bag with the molecular weight cutoff of 8000-14000Da to obtain the co-loaded immune adjuvant and the green indole phthalocyanine polymer vesicle with pH/reduction/temperature triple stimulation response.
In order to better characterize the effect of the co-loaded immune adjuvant and the green indocyanine polymersome with pH/reduction/temperature triple stimulation response, the pH/reduction/temperature response type polymer vesica with targeting effect ABC @ ICG-LHRH (AIL) based on the encapsulated photosensitizer ICG without encapsulating IMQ is prepared by adopting the same method as the embodiment 1, and NH is not encapsulated4HCO3The photosensitizer ICG and the immunologic adjuvant IMQ are carried togetherThe polymer vesicle ICG-IMQ-LHRH (IRL) with targeting effect and the pH/reduction/temperature response type polymer vesicle ABC @ ICG-IMQ (AIR) without targeting modification based on the co-loaded photosensitizer ICG and the immune adjuvant IMQ; the preparation method comprises the following steps:
a method of making a green indocyanine-encapsulated polymersome that does not encapsulate IMQ with a pH/reduction/temperature triple stimulus response, the method of making being essentially the same as in example 1 except that no TLR-7/8 agonist IMQ is added in step (a).
Not entrapping NH4HCO3The preparation method of the co-loaded immune adjuvant and the indocyanine green polymersome with triple stimulation response of pH/reduction/temperature is basically the same as that of example 1, except that NH is not added in the step (b)4HCO3。
A method of preparing a pH/reduction/temperature triple stimulus responsive co-loaded immune adjuvant and an indocyanine green polymersome without targeted modification, which is substantially the same as example 1 except that DSPE-PEG-Mal is replaced with an equal amount of DSPE-PEG-Me in step (a); and step (c) is absent.
Example 2
Characterization of the polymersome prepared in example 1 of the present invention includes particle size, potential, PDI, and particle size stability.
The above prepared polymer vesicle suspensions of AIR rl, AIR, IRL, AIL and blank polymer vesicle were diluted to 200 μ g/mL with deionized water at room temperature and then measured for particle size, PDI and potential, and the results are shown in table 1.
TABLE 1 particle size, PDI, Zeta potential of different polymersomes
Figure BDA0003106703980000101
As shown in Table 1, the polymer vesicles with a particle size of 100-200nm, which is much smaller than 700nm, can be gathered at the tumor site by EPR effect. The polymer vesicles have good dispersity and can be uniformly distributed, and the circulation half-life period of the polymer vesicles in vivo can be prolonged by negative charge
After the suspension of the polymer vesicle AIRL prepared in example 1 was diluted with deionized water at room temperature to an appropriate concentration, the particle sizes within 6 days were measured respectively with a Nano-ZS particle size analyzer, as shown in a in fig. 1;
FIG. 1A is a graph showing the particle size stability of the polymersome AIRL prepared in example 1 of the present invention. The result shows that the particle size of the polymersome ABC @ ICG-IMQ-LHRH is not obviously changed, which indicates that the polymersome is not aggregated and is relatively stable;
example 3
Ultraviolet absorption spectrum of the polymersome prepared in example 1 of the present invention.
The polymeric vesicle AIRL suspension prepared in example 1, Free IMQ solution and Free ICG solution were diluted with deionized water at room temperature to a suitable concentration, and then the absorption spectra thereof were measured by a Lambda35 UV-visible spectrophotometer.
Fig. 1B is a uv absorption spectrum of the polymersome AIRL, free ICG and free IMQ prepared in example 1 of the present invention. The results demonstrate that both ICG and IMQ are successfully encapsulated in the hydrophobic layer of polymersomes.
Example 4
The drug loading and encapsulation efficiency of the polymersome prepared in example 1 of the present invention.
The nanoparticles of example 1 (c) were lyophilized by high speed centrifugation (30 min/time, 23000rpm/min, resuspended in deionized water), and 1mg of the nanoparticles were dissolved in 1mL of dimethylsulfoxide and diluted by a factor of two. The absorbance was measured using a Lambda35 UV-Vis spectrophotometer. The ICG and IMQ measurement wavelengths were 790nm and 330nm, respectively. And calculating the drug loading and encapsulation efficiency of the ICG and the IMQ in the sample by adopting a standard curve method. The drug loading and encapsulation efficiency formulas are as follows:
Figure BDA0003106703980000111
Figure BDA0003106703980000112
the results are shown in table 2:
TABLE 2 drug loading, encapsulation efficiency of ICG and IMQ of different polymersomes
Figure BDA0003106703980000113
From table 2, it can be seen that the drug loading rate and the encapsulation efficiency of the polymer vesicle carrier single-loading and double-loading drugs are both high, indicating that the polymer vesicle carrier has good drug loading performance as a drug carrier.
Example 5
The polymer vesicles prepared in example 1 of the present invention have particle size distributions under different conditions.
The polymersome AIRL suspension prepared in example 1 was diluted with pH 7.4PBS, pH5.5PBS, 10mM GSH + pH 7.4PBS, 10mM GSH + pH5.5PBS, respectively. And meanwhile, setting a laser group, diluting the sample with the four groups of PBS under different conditions, and irradiating for 5min by 808nm near-infrared laser. The Nano-ZS particle size analyzer is used for measuring the particle size distribution of the polymer vesicles under different conditions.
FIG. 1C is a graph showing the variation of the particle size of the polymersome AIRL prepared in example 1 of the invention under different conditions. The results show that under reduction, acidity or laser irradiation, the polymersome structure is destroyed, the particle size is increased, two broad peaks can be generated, the dispersibility is increased, and the stability is reduced. These results indicate that the polymersome AIRL has a pH/reduction/temperature triple responsiveness.
Example 6
In vitro release of IMQ in polymersomes prepared in example 1 of the invention.
The polymersome AIRL suspension (332. mu.g/mL) prepared in example 1 was placed in an MWCO 8000-14000Da dialysis bag, and the release solutions were 0.1% Tween 80-PBS (pH 7.4), 0.1% Tween 80-PBS (pH 5.5), and 0.1% Tween 80-10mM GSH-PBS (pH 5.5), respectively. Meanwhile, in order to examine the influence of near-infrared laser irradiation on drug release, a sample is irradiated by 808nm laser (1.5W/cm)25min) was added to the dialysis bag and placed in 0.1% Tween 80-10mM GSH-PBS (pH 5.5). Three in each groupIn parallel, 20mL of release solution was shaken at 37 ℃ on a shaker at 120rpm/min in the dark, and after a certain period of time (1h, 2h, 4h, 6h, 12h, 1d, 2d, 3d, 4d, 5d, 7d, 9d, 11d, 14d) all release solutions were removed and 20mL of the corresponding release solution was replenished. Adding a mixed solution of dichloromethane and methanol (V/V ═ 1:1) into the collected release solution for extraction, standing for layering, carefully absorbing an organic reagent layer, repeating the steps three times, drying the organic reagent solution containing the IMQ in a fume hood, dissolving the organic reagent solution with a mixed solution of dichloromethane and methanol (V/V ═ 1:1), detecting the absorbance of the IMQ at a wavelength of 330nm by using a Lambda35 ultraviolet-visible spectrophotometer, and calculating the release amount of the IMQ in the polymer vesicle by combining a standard curve of free IMQ in the release solution.
Fig. 1D shows the in vitro release of IMQ of polymersome AIRL prepared in example 1 of the present invention under different conditions. The results show that the acidic environment, the reducing environment and laser irradiation can accelerate the release of IMQ in the polymer vesicle AIRL.
Example 7
The polymer vesicle prepared in example 1 of the present invention has active oxygen generation.
To the polymersome AIRL suspension, ARL, Free ICG, and PBS (0.5mL, ICG concentration 10 μ g/mL, each sample diluted to 200 μ g/mL with a PBS-ethanol (V/V ═ 4:6) mixed solution prepared in example 1, an ethanol solution (2mg/mL, 40 μ L) of 1,3-Diphenylisobenzofuran (1,3-Diphenylisobenzofuran, DPBF) was added, and a near infrared laser (808 nm) (1.5W/cm) was used in the dark2) The light was irradiated for 0, 1, 2, 3, 4, 5min, and the absorbance at 410nm was measured using a Lambda35 UV-Vis spectrophotometer.
FIG. 1E shows the in vitro photodynamic effects of the polymersomes AIRL, ARL, Free ICG prepared according to the invention under different conditions. The result shows that the ICG still has good photodynamic effect after being wrapped and loaded by the AIRL.
Example 8
In vitro photothermal studies of polymersomes prepared in example 1 of the present invention.
The polymersome AIRL, AIR, IRL, AIL suspension and Free IC prepared by the method of example 1G. PBS (500. mu.L, 10. mu.g/mL ICG) was added to a 1.5mL centrifuge tube at 1.5W/cm2Irradiating for 5min under 808nm near-infrared laser, detecting the temperature change condition with time by a near-infrared thermal imager, and recording the near-infrared thermal imaging graph of each group at the highest temperature under the laser irradiation.
To the polymersome AIRL suspension prepared in example 1, Free ICG (500. mu.L, 10. mu.g/mL ICG) was added into a 1.5mL centrifuge tube at 1.5W/cm2Irradiating for 5min under 808nm laser, closing the laser for 5min, repeating the above steps for three times to obtain a temperature rise-cooling circulation curve, and detecting the temperature change with time by using a near-infrared thermal imager.
Fig. 2A, C is a graph of the temperature profile with time and the thermal imaging at the highest temperature for different sets of samples under laser irradiation. The results show that the highest temperature of the drug treatment groups exceeds 43 ℃, and the drug treatment groups can kill tumor cells. The highest temperature of the polymersome group is higher than that of the Free ICG group, and the polymer vesicle group has good photothermal conversion efficiency. NH due to laser irradiation4HCO3The ICG is easier to release in the vesicle due to the gas generating vesicle function, so the in vitro photothermal effect of the polymer vesicle AIRL is better than that of IRL.
Fig. 2B is a temperature rise-cooling cycle curve of the sample with or without laser irradiation. The results show that after three temperature increases-cooling, the generated heat of the AIRL is still higher than that of the free ICG, and the good photo-thermal performance is shown, which indicates that the light stability of the polymer vesicle AIRL is stronger than that of the free ICG.
Example 9
Examination of CT26 cellular uptake of polymersome prepared in example 1 of the present invention.
CT26 cells were diluted to 6X 10 with complete medium4Each/mL single cell suspension was cultured in a confocal laser culture dish at a volume of 1mL per dish, after overnight incubation for cell attachment, the medium was aspirated, and 1mL of Free ICG + IMQ, AIR, and AIRL drug medium mixtures (final concentration of ICG and IMQ was 10. mu.g/mL) were added to each dish. And simultaneously, setting a laser group, and adding the medicines. The laser group was applied to 808nm near infrared laser (1.5W/cm) after 2h of cell culture2) Irradiating for 5min, and culturing in culture box at 37 deg.C,5％CO2Saturated humidity) for 2h, and the non-laser group was incubated directly for 4 h. Washing with PBS once, fixing cells with 4% tissue fixative for 10min, removing 4% tissue fixative by aspiration, washing with PBS twice, adding Hoechst, and culturing for 5 min. The staining solution was aspirated, washed twice with PBS, and finally 200. mu.L of PBS was added to observe the uptake of AIRL by the cells with CLSM.
CT26 cells were diluted to 5X 10 with complete medium5Inoculating single cell suspension per mL in 12-well plate at volume of 1mL per well, incubating overnight for cell adherence, removing culture medium by aspiration, adding 1mL PBS, Free ICG + IMQ, AIR, and AIR respectively (final concentration of ICG and IMQ is 10 μ g/mL), incubating for 2 hr, and irradiating with 808nm near infrared laser (1.5W/cm)2) Irradiating for 5min, and culturing in incubator (37 deg.C, 5% CO)2Saturated humidity) for 2h, and the non-laser group for 4 h. The cells were harvested and centrifuged (1500rpm/s, 5min), 300. mu.L PBS was added to resuspend the cells and sieve in a flow tube, and intracellular ICG uptake was quantified using a flow cytometer.
Fig. 3A is a laser confocal image of the uptake of polymersomes and free drug by CT26 cells. The results indicate that polymersomes can facilitate ICG entry into cells. The laser irradiation generates heat, so that the permeability and the fluidity of cell membranes can be enhanced, the accumulation of drugs in tumor cells can be increased, meanwhile, the laser can break the polymer vesicle to form small fragments, and the release of ICG is promoted, so that the laser irradiation can remarkably increase the ICG uptake of CT26 cells. LHRH modification can promote accumulation of polymersomes in CT26 cells. Fig. 3B is a flow chart of results of cell uptake by flow cytometry, consistent with confocal laser detection results.
Example 10
Toxicity of the polymersome prepared in example 1 of the present invention on CT26 cells was examined.
CT26 cells were diluted to 5X 10 with complete medium4Single cell suspensions per mL were seeded in 96-well plates at a volume of 100 μ L per well. After overnight incubation for cell attachment, the medium was aspirated, 100. mu.L of PBS, Free ICG + IMQ, IRL, AIR, and AIRL drug medium mixtures (IMQ and ICG concentrations 5, 10, 15, 20. mu.g/mL) were added, and a laser set was set simultaneouslyThe medicines are added in the same way, and a negative control group and a blank control group are arranged, wherein each group of the non-laser group is provided with 6 multiple holes, and each group of the laser group is provided with 3 multiple holes. The laser irradiation group used 808nm near infrared laser (1.5W/cm) after adding medicine for 2h2) Irradiating for 5min, and placing 96-well plate into incubator (37 deg.C, 5% CO)2Saturated humidity) for 24h, and the non-laser group directly puts the 96-well plate into an incubator (37 ℃, 5% CO)2Saturated humidity) for 24 h. mu.L of RPMI-1640 culture solution mixed with MTS was added to each well, incubated in an incubator for a certain period of time, and then the absorbance value was measured at 490nm on a microplate reader.
Figure BDA0003106703980000151
FIG. 4A, B is a graph showing the toxicity test results of polymersome on CT26 cells. The result shows that the AIRL has no obvious toxicity to CT26 cells no matter whether laser exists or not, which indicates that the nano preparation carrier has no cytotoxicity. Under the condition of no laser irradiation, the polymersome and free medicine are nontoxic to CT26 cells within a certain concentration range (ICG concentration is less than 20 mu g/mL). Under the laser irradiation condition, the ICG-encapsulated polymersome showed concentration dependence on the toxicity of CT26 cells. The laser irradiation can destroy the structure of the polymer vesicle, and the AIRL can release CO2The air bubbles, to some extent, damaged the tumor cells, so the toxic effect of the AIRL on CT26 cells was the strongest.
Example 11
Investigation of the generation of active oxygen in CT26 cells in the polymersome prepared in example 1 of the present invention.
CT26 cells were diluted to 4X 10 with complete medium5The single cell suspension per mL is inoculated in a laser confocal dish in a volume of 1mL per dish, after overnight incubation and cell adherence, the culture medium is aspirated, 1mL of PBS, Free ICG + IMQ, AIR and AIRL drug culture medium mixed liquor (the final concentration of ICG and IMQ is 10 mug/mL) is respectively added in each dish, and the incubation is carried out for 4 h. The cells were washed gently with PBS once and 600. mu.l Lcaboxy-H was added2DCFDA (6. mu.g/mL), cells were incubated at 37 ℃ for 15 min. The cells were washed gently once with PBS, where the laser group was near 808nmInfrared laser (1.5W/cm)2) Irradiating for 5 min. Cells were fixed in 4% tissue fixative for 10min at room temperature, washed twice with PBS and stained with 600 μ L Hoechst (8 μ g/mL) for 5 min. The cells were washed once with PBS and finally observed under CLSM with 200. mu.L PBS.
CT26 cells were diluted to 5X 10 with complete medium5The single cell suspension per mL was inoculated into 12-well plates at a volume of 1mL per well, after overnight incubation for cell attachment, the medium was aspirated, and 1mL of PBS, Free ICG + IMQ, AIR, AIRL drug medium mixture (final concentration of ICG and IMQ is 10 μ g/mL) was added and incubated for 4 h. The cells were gently washed once with PBS and 600. mu.L carboxy-H was added2DCFDA (6. mu.g/mL), cells were incubated at 37 ℃ for 15min and the cells were washed once gently with PBS. Laser group was added with 1mL of PBS and then treated with 808nm near-infrared laser (1.5W/cm)2) After 5min of irradiation, the cells were washed once with PBS. The cells were collected and centrifuged (1500rpm/s, 5min), 300. mu.L of PBS was added to resuspend the cells, the cells were sieved in a flow tube, and the intracellular reactive oxygen species production was quantitatively determined by a flow cytometer.
Fig. 5A is a confocal laser image of reactive oxygen species detection of CT26 cells. The results show that ICG is unable to produce the photodynamic effect without laser light. Fluorescence signals appear in cells of the drug-treated group CT26 under the irradiation of near-infrared laser, which indicates that the photosensitizer ICG can induce the generation of ROS in the cells under the irradiation of the laser. And the AIRL has better PDT effect because the AIRL is more taken up by cells. FIG. 5B is a flow chart showing the generation of reactive oxygen species in cells, which is consistent with the confocal laser detection results.
Example 12
Characterization of polymersomes prepared in example 1 of the present invention by in vivo imaging.
Female BALB/C mice were inoculated subcutaneously with CT26 cells (1X 10)6One/only) until the tumor volume reaches 200mm3In this case, the mice were randomly divided into 4 groups, and each group was injected with PBS, Free ICG + IMQ, AIR, and AIRL, respectively, via tail vein. Near-infrared in vivo imaging of mice was performed at 6h, 24h, 48h, and 72h after dosing to obtain tissue distribution maps (λ ex 704nm, λ em 735nm) of ICG.
Fig. 6A shows the accumulation of polymersome at different times at the tumor site by in vivo imaging of mice. After administration, the fluorescence intensity of the administered group reached a maximum at 24 h. The Free ICG + IMQ group fluorescence signal is always lower than that of the polymersome group, which shows that the polymersome drug-carrying system can effectively reduce the liver clearance rate of ICG and has good long-circulation characteristic. Within 72h, the fluorescence signal of the polymer vesicle AIRL group is always stronger than that of the AIR group, which shows that the LHRH targeting effect can enhance the accumulation of the polymer vesicle at the tumor part and prolong the retention time, thereby realizing the tumor targeted therapy. The results show that the polymer vesicle AIRL has excellent drug-loading performance, realizes high accumulation of the drug at the tumor part by utilizing the tumor high permeability and retention effect, and simultaneously realizes the targeting effect of the drug to the tumor by the modification of LHRH, thereby effectively playing the anti-tumor effect. FIG. 6B is a bar graph of ICG mean fluorescence intensity at the mouse tumor site, again demonstrating the above conclusions.
Example 13
In vivo photothermal studies of polymersomes prepared in example 1 of the present invention.
Female BALB/C mice were inoculated subcutaneously with CT26 cells (1X 10)6One/one) mouse to be tumor-bearing, the proximal tumor volume of the mouse to be tumor-bearing is 50-70mm3In the case of the treatment, PBS, Free ICG + IMQ, AIL, IRL, AIR, and AIRL (6mg/kg ICG) were injected into the tail vein, and 1.5W/cm was applied to the tumor site 24 hours and 48 hours after the administration2808nm for 5 min. The change of the highest temperature of the tumor part of the mouse along with the time is observed by using a near infrared thermal imaging instrument.
FIG. 7A shows the local maximum temperature near-IR thermography of tumors after 24h and 48h of dosing for each group. FIG. 7B, C is a photothermal warming profile 24h and 48h after dosing. The results show that the temperature in the free group, although slightly above 43 ℃ at 24h post-dose, is lower and not sufficient to effectively inhibit tumor growth. The temperature of the polymer vesicle group exceeds 48 ℃, which can cause irreversible damage to tumor cells, and ICG can be enriched in the local tumor to play a photothermal role through in vivo long circulation and EPR effect to effectively inhibit tumor growth. The LHRH modified AIRL can target tumor tissues and enable local drugs of tumors to be accumulated more, so that the photothermal effect of the polymer vesicle AIRL is better than that of IRL and AIR. At 48h after administration, the polymersome group exceeds 43 ℃, and can still cause tumor cell damage.
Example 14
Evaluation of toxicity of polymersomes prepared in example 1 of the present invention to DCs.
BMDCs (1X 10) were collected and cultured up to day 66piece/mL) centrifugation (450g, 5min), cell resuspension in 12-well plate culture (each hole 1mL), 2h after 12h to 12-well plate adding PBS, Free ICG + IMQ, IRL, AIRL polymer vesicle (ICG concentration 10 u g/mL) and cell co-incubation 24h, then collecting cells and centrifugation (450g, 5min), PBS washing once, using antibody FITC-anti-mouse-CD11c at 4 ℃ in dark condition staining for 30 min. The viability of BMDCs was determined according to the instructions of Annexin V-APC/7-AAD double-staining apoptosis detection kit and detected by flow cytometry.
Fig. 8A is a flow chart of the results of toxicity tests on DCs with polymersome and free drug, which shows that neither polymersome nor free drug has toxic effects on BMDCs.
Example 15
The uptake of DCs by the polymersome prepared in example 1 of the present invention was examined.
DC 2.4 cells were diluted to 6X 10 with complete medium4The single cell suspension per mL is cultured in a laser confocal dish in a volume of 1mL per dish, after overnight incubation and cell adherence, the culture medium is aspirated, and 1mL of Free ICG + IMQ, IRL and AIRL drug culture medium mixed liquor (the final concentration of ICG and IMQ is 10 mug/mL) is respectively added into each dish for incubation for 4 hours. Wash once with PBS, add 250. mu.L of Lyso-Tracker Red staining solution prepared and preincubated at 37 ℃ and co-incubate with cells at 37 ℃ for 60 min. The staining solution was aspirated and the cells were washed once with 1mL PBS. Cells were fixed with 4% tissue fixative for 10min at room temperature and the fixative was washed away with PBS. Add 200. mu.L Hoechst (8. mu.g/mL) to incubate for 5min, aspirate the staining solution, wash twice with PBS, and finally add 200. mu.L PBS to observe under laser confocal.
DC 2.4 cells were diluted to 5X 10 with complete medium5The single cell suspension/mL was inoculated into 12-well plates at a volume of 1mL per well, after overnight incubation and cell attachment, the medium was aspirated, and 1mL of Free ICG + IMQ, IRL, and AIRL drugs were addedThe culture medium mixture (final concentration of ICG and IMQ is 10 μ g/mL), after incubation for 4h, cells were harvested and centrifuged (450g, 5min), washed once with 1mL PBS, and 300 μ LPBS resuspended cells were added and sieved in a flow tube, and the nanoparticle phagocytosis efficiency of DCs was determined using a flow cytometer.
Fig. 8B is a confocal laser map of uptake of polymersomes and free drug by DCs. The result shows that the free drug and the lysosome are co-localized, and the lysosome escape phenomenon does not exist. Compared with IRL, the DCs in the AIRL group have stronger green fluorescence signal in cytoplasm and less green fluorescence signal overlapping with red lysosome, which indicates that NH is entrapped and loaded4HCO3The polymersome AIRL has lysosome escape in DCs, promotes drug release and enhances the uptake of the polymersome by the DCs. Fig. 8C is a graph of flow results of uptake of polymersomes and free drug by DCs, consistent with laser confocal results.
Example 16
The invention provides an in vitro Transwell experiment of polymersome prepared in example 1.
CT26 cells (1X 10)5pieces/mL) were co-incubated with PBS, AIL, IRL, AIRL polymersomes (ICG and IMQ final concentration 10. mu.g/mL) in the upper compartment for 2h, 1.5W/cm2The 808nm near infrared laser of each group of CT26 cells was irradiated for 5min, and at the same time, a laser-free group containing AIRL polymer vesicles was set. BMDCs (1X 10) were collected and cultured up to day 66pieces/mL) and centrifuged (450g, 5min), resuspended and cultured in the lower layer of Transwell plates (1 mL per well), the upper compartment CT26 cell remnants and drug cocktail were mixed with the lower BMDCs (1X 10)6one/mL) for 24 h. Then, semi-adherent BMDCs in the upper layer of the lower chamber were collected and centrifuged (450g, 5min), the supernatant was stored, and the cell pellet was washed once with PBS, and stained with antibodies Cy5.5-anti-mouse-CD11c, PE-anti-mouse-CD40, APC-anti-mouse-CD80, FITC-anti-mouse-CD86, APC-anti-mouse-MHC I, and FITC-anti-mouse-MHC II, respectively, at 4 ℃ for 30min in the dark, washed once with PBS, and then 300. mu.L of PBS was added to resuspend the cells and sieved in a flow tube. The expression of the BMDCs surface co-stimulatory molecules CD40, CD80 and CD86 and MHC I and MHC II was examined by flow cytometry. Mouse ELISA assay according to instructionsThe assay kit detects cytokines including TNF-alpha and INF-gamma in the supernatant culture solution.
FIGS. 9A-E are flow charts of the expression of surface molecules of DCs, showing that tumor cell remnants from CT26 cells co-cultured with polymersome AIL under laser irradiation induced slight maturation of BMDCs. In contrast, the cell remnants of CT26 cells co-cultured with polymersome IRL and AIRL after PTT treatment as TAA significantly promoted the maturation of BMDCs under the action of TLR-7/8 agonist IMQ, and the effect of AIRL was higher than IRL. The AIRL has weak effect of promoting the maturation of DCs only under the action of an immunological adjuvant IMQ. FIGS. 9F-G are cytokine assays showing that the trend in the secretion levels of IFN-. gamma.and TNF-. alpha.for each group is substantially consistent with the results of flow assays for cell surface molecules.
Example 17
The invention provides an antitumor study of polymersome prepared in example 1.
CT26 cells (1X 10) were first subcutaneously inoculated in the right back of BALB/C mice6One/only) to establish a proximal tumor model, the left back of which was subcutaneously inoculated with CT26 cells (5X 10)5One/only) establish a distal tumor model. When the proximal tumor volume reaches 50-70mm3Mice were randomized into 9 groups (8 per group): (1) PBS; (2) PBS + laser; (3) free ICG + IMQ; (4) free ICG + IMQ + laser; (5) AIL + laser; (6) IRL + laser; (7) AIR + laser; (8) an AIRL; (9) AIRL + laser, 150 μ L of each group of drugs (6mg/kg ICG) were given tail vein injections to each group on day 0 and day 5, respectively. At 24h and 48h post-dose, the laser group used 1.5W/cm2Irradiating the near-end tumor part of the mouse with 808nm near-infrared laser for 5 min. The body weight and tumor volume at both ends of the mice were measured and recorded every other day after self-administration when the tumor volume exceeded 3000mm3Mice were sacrificed and mouse survival curves were plotted. The tumor volume calculation formula is as follows:
tumor size (mm)3) Long diameter of tumor (tumor) × (short diameter of tumor)2/2
FIG. 10A, B is a graph showing inhibition of tumor growth in proximal and distal mice, respectively. Under the action of laser, the proximal tumor of each polymersome group in the mouse is completely eliminated within 24 days. Under laser irradiation, the AIL group has no obvious inhibition effect on the growth of the distal tumor, and the AIRL group can better delay the growth of the distal tumor through photothermal-immune combined treatment under laser irradiation.
Fig. 10C is a graph representing survival curves of mice, and shows that all of the AIRL group mice survived under laser irradiation within 40 days.
Figure 10D is a graph representing the change in body weight of mice during treatment. The results show that the body weight of each group of mice is not obviously reduced, and the polymersome has good biocompatibility.
Example 18
Histopathological study of polymersomes prepared in example 1 of the present invention.
At 24 days of treatment, mice were sacrificed and dissected, their hearts, livers, spleens, lungs, kidneys were taken and washed with normal saline, placed in 4% tissue fixative overnight, tissues were cut into appropriate sizes with a razor blade in a petri dish containing absolute ethanol, dehydrated, paraffin-embedded, and sectioned. Staining was performed according to Hematoxylin-eosin staining kit instructions (H & E) and finally photographing was performed using a microscope.
FIG. 11 is H & E staining diagram of mouse major organs, and the results show that each polymer vesicle has no obvious toxic and side effect on normal tissues and organs and has excellent biocompatibility.
Example 19
The invention provides a research on the in vivo anti-tumor immune mechanism of the polymersome prepared in the example 1.
CT26 cells (1X 10) were first subcutaneously inoculated in the right back of BALB/C mice6one/mL), the left back of the mouse was inoculated subcutaneously with CT26 cells (5 × 10) the day before administration5individual/mL) was modeled for distal tumors. When the primary tumor volume reaches 50-70mm3Mice were randomized into 9 groups (16 per group): (1) PBS; (2) PBS + laser; (3) free ICG + IMQ; (4) free ICG + IMQ + laser; (5) AIL + laser; (6) IRL + laser; (7) AIR + laser; (8) an AIRL; (9) AIRL + laser, 150 μ L of each group of drugs (6mg/kg ICG) were injected tail vein on day 0 and day 5, respectively. Laser groups utilized 1 at 24h and 48h after two doses, respectively.5W/cm2Irradiating the near-end tumor part of the mouse with 808nm near-infrared laser for 5 min. And the in vivo immune mechanism study was performed according to the following protocol. Lymph nodes were collected and stained after decapitation of mice on day 15 post-dose, and the maturation levels of BMDCs were analyzed using flow cytometry. Meanwhile, spleens were excised, ground, filtered, lysed with erythrocyte lysis buffer for 2min, and cells were washed twice with 10 volumes of PBS. The resulting cells were further stained with different antibodies, respectively: 1) anti-CD 4, anti-CD 8, anti-Ki 67; 2) anti-CD 3, anti-CD 4, anti-CD 8.
FIG. 12 is a graph of flow results of surface molecule expression of mouse lymph node DCs after different treatments. Free and AIRL groups stimulated little maturation of DCs in the absence of laser irradiation, but only with the help of immunoadjuvants. The AIL + laser group only weakly stimulated expression of surface molecules of lymph node DCs, consistent with in vitro Transwell results. Due to the targeting of LHRH to tumors and NH4HCO3The AIRL + laser group significantly promoted lymph node DCs maturation compared to other laser groups for the release-promoting effect of the drug, thereby highly expressing co-stimulatory molecules (CD80, CD86) and adhesion factors (CD40) and presenting antigen to T lymphocytes to activate subsequent immune responses.
Fig. 13 is a representative flow chart and histogram of T cell proliferation in the splenocytes from each group of mice at day 15 post-treatment. IRL + laser group and AIR + laser group CD4+And CD8+Higher levels of T cell proliferation and with targeting and blebbing polymersomes AIRL + laser group CD4+And CD8+Higher levels of T cell proliferation. Other laser groups also have different degrees of CD4+And CD8+T cells proliferate, but the proliferation level is low, Free group and AIRL group without laser irradiation are CD4 only under the action of immune adjuvant+And CD8+T cell proliferation was not evident.
FIG. 14 is a graph of CD4 in splenocytes from groups of mice at day 15 post treatment+T(CD3+CD4+) And CD8+T(CD3+CD8+) Representative flow charts and bar graphs of cell activation levels. IRL + laser group, AIR + laser group and AIR + lasAll of the er groups can stimulate CD4+And CD8+Stimulation of the AIRL + laser group of polymersomes with targeting and bubble generation CD4 with T cell proliferation and compared to the other laser groups+And CD8+T cells have better proliferation capacity. These results indicate that the polymersome AIRL with targeting effect and bubble generation effect under laser irradiation can significantly induce CD4+And CD8+T cells proliferate, effectively promoting cellular immune responses.
Example 20
The detection and anti-lung metastasis evaluation of memory T cells of the polymersome, the free drug and the PBS prepared in the embodiment 1 of the invention.
CT26 cells (1X 10) were injected via tail vein on day 21 post-dose5one/mL), mice were killed by decapitation on day 22 after administration, soaked in 75% alcohol for 2min, and splenocytes were collected to analyze memory T cells. The spleen cell suspension is centrifuged (450g, 5min), the supernatant is discarded, then flow antibodies FITC-anti-mouse-CD8, PE-anti-mouse-CD4, Cy5.5-anti-mouse-CD62L and APC-anti-mouse-CD44 are used for labeling, the spleen cell suspension is incubated at 4 ℃ in the dark for 30min, 1mL of PBS is added for washing once, and finally 300 muL of PBS is added for resuspension of cells, the cells are sieved in a flow tube, and the determination is carried out by a flow cytometer.
CT26 cells (1X 10) were injected via tail vein on day 21 post-dose5one/mL), lungs were dissected after decapitation of mice on day 35 after administration, lung tissues were washed with physiological saline and fixed in Bouin's solution, taken out after overnight and photographed to record tumor nodules, and then tissues were cut into appropriate sizes with a razor blade in a petri dish containing absolute ethanol, dehydrated, paraffin-embedded, and sliced. According to H&E staining kit instructions for staining. Finally, photographing is carried out by using a microscope.
FIG. 15 shows the immunological memory CD4 in the splenocytes of mice in each group+And CD8+Representative flow and histogram of T cell levels. Flow results show CD4 in spleen of AIRL + laser group+And CD8+T cells all express a higher proportion of TCMAnd TEMIt is demonstrated that the AIRL + laser group induced the most effective memory CD4+T and CD8+T cell immune response, the strongest antitumor memory immune effect.
FIG. 16 is a photograph and H & E staining of lung tissue of each group of mice. The whole lung and H & E staining plots show that metastatic nodules of the lung were found in all but the IRL + laser, AIR + laser and other groups that produced potent immunological memory effects. Therefore, the above results suggest that the photothermal combined immune therapy based on the polymersome AIRL can effectively inhibit the lung metastasis of the tumor and prevent the metastasis and recurrence of the tumor.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (10)

1. A preparation method of a pH/reduction/temperature triple-stimulus-responsive co-loaded immune adjuvant and a green indocyanine polymer vesicle is characterized by comprising the following steps:
(a) dissolving PCL-b-PEG-b-PCL, hydrophobic ICG, DSPE-PEG-Mal and immunologic adjuvant in an organic solvent, and then removing the organic solvent to form a uniform film;
(b) drying the film, hydrating, and adding NH4HCO3Mixing uniformly, then carrying out ultrasonic treatment and dialysis to obtain a polymer vesicle solution;
(c) and mixing the polymer vesicle solution and LHRH, reacting under the catalysis of triethylamine, and dialyzing to obtain the co-loaded immune adjuvant and green indocyanine polymer vesicles with the pH/reduction/temperature triple stimulus response.
2. The method of claim 1, wherein the immunoadjuvant comprises a TLR-7/8 agonist and the TLR-7/8 agonist is IMQ.
3. The method as claimed in claim 1, wherein the molecular weight of PCL-b-PEG-b-PCL is 10000-24000, and the mass percentage of the PCL hydrophobic segment is more than 33%.
4. The preparation method according to claim 1, wherein the solvent used for the hydration is deionized water or a PBS solution; the hydration temperature is 60-70 ℃, and the time is 5-6 h;
the mass ratio of the addition volume of the solvent to the PCL-b-PEG-b-PCL is (3-10) mL: 20 mg.
5. The production method according to any one of claims 1 to 4, wherein in the step (a),
the mass ratio of the PCL-b-PEG-b-PCL to the hydrophobic ICG is (15-25) to 1;
the mass ratio of the PCL-b-PEG-b-PCL to the immunologic adjuvant is (15-25) to 1;
the mass ratio of the PCL-b-PEG-b-PCL to the DSPE-PEG-Mal is (40-60) to 1.
6. The production method according to any one of claims 1 to 4, wherein in the step (b),
NH4HCO3the final concentration of the additive (b) is 280-320 mM;
the ultrasonic treatment is ultrasonic treatment for 25-35 min under the ice bath condition;
the cut-off molecular weight of a dialysis bag used for dialysis is 8000-14000 Da; the dialysis time is 3-12 h.
7. The production method according to any one of claims 1 to 4, wherein in the step (c),
the mass ratio of the PCL-b-PEG-b-PCL to the LHRH is (60-100) to 1;
the reaction is carried out for 10-12 h under the conditions of stirring and room temperature;
the cut-off molecular weight of a dialysis bag used for dialysis is 8000-14000 Da; the dialysis time is 4-8 h.
8. The method of claim 1, wherein the hydrophobic ICG is prepared by:
dissolving hydrophilic ICG and tetrabutyl ammonium iodide in an organic solvent, and stirring for reaction at a dark room temperature to obtain a hydrophobic photosensitizer ICG, wherein the mass ratio of the tetrabutyl ammonium iodide to the hydrophilic ICG is (5-6): 1.
9. the co-carried immunoadjuvant and the green indocyanine polymer vesicle with triple stimulation response of pH/reduction/temperature, which are prepared by the preparation method of any one of claims 1 to 8.
10. Use of the pH/reduction/temperature triple stimulus responsive co-loaded immunoadjuvant and the green indocyanine polymer vesicles of claim 9 in the preparation of anti-tumor photothermal-immune combination therapy drugs.
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