CN114288395A - Tumor microenvironment responsive in-situ nano vaccine and preparation method thereof - Google Patents
Tumor microenvironment responsive in-situ nano vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a tumor microenvironment responsive in-situ nano vaccine and a preparation method thereof, and the preparation method comprises the steps of preparing amphiphilic monomer molecules and self-assembling the amphiphilic monomer molecules into the tumor microenvironment responsive in-situ nano vaccine; the amphiphilic monomer molecule comprises a hydrophobic functional molecule, an environment-responsive connecting molecule and a hydrophilic functional molecule which are sequentially connected, and the step of preparing the amphiphilic monomer molecule comprises the following steps: connecting hydrophobic functional molecules and environment responsive connecting molecules to prepare an intermediate connecting product; connecting the intermediate connecting product with the hydrophilic functional molecule to prepare an amphiphilic monomer molecule; the hydrophobic functional molecule is a hydrophobic adjuvant molecule, and the hydrophilic functional molecule is a hydrophilic chemotherapeutic drug molecule, or the hydrophobic functional molecule is a hydrophobic chemotherapeutic drug molecule, and the hydrophilic functional molecule is a hydrophilic adjuvant molecule. The invention effectively improves the synthesis efficiency of the nano in-situ vaccine by integrally optimizing the preparation process.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a tumor microenvironment responsive in-situ nano vaccine and a preparation method thereof.
Background
The immune vaccine is one of the most potential treatment methods in the field of tumor treatment, and the ideal tumor vaccine can induce organisms to generate stronger specific anti-tumor immune response and has smaller toxic and side effects on the whole body. However, most tumor antigens have low specificity and weak antigenicity, so that the anti-tumor immune response induced by the antigens is weak, and the immune response can cause certain damage to normal healthy tissues and organs. In addition, in actual clinical treatment, even for the same type of tumor, there is a difference in antigen between different patients, and therefore, a specific certain tumor-specific antigen cannot be adapted to all patients.
Certain chemotherapeutic agents induce immunogenic death of tumor cells, producing tumor-specific antigens, and thus specific anti-tumor immune responses (also known as in situ vaccines). The simultaneous application of the nano material mediated immune adjuvant or other immune stimulators and chemotherapeutic drugs can improve the effect of in-situ vaccines and greatly enhance anti-tumor immune response, but the nano material has certain potential safety hazard.
Compared with the traditional in-situ vaccine taking the nano material as the carrier, the in-situ nano vaccine formed by the chemotherapeutic drug and the immunologic adjuvant does not need other nano material loads, and has high safety. The traditional preparation method of the in-situ nano vaccine without other nano material loading, for example:
CN113318224A discloses a method for preparing double-wheel nanoparticles, which comprises the following steps: mixing PSMA-MMP-9 polypeptide molecules (ESWTKKSPSPEFSGMGPQGIAGQR), 1, 2-dichloroethane and N-hydroxysuccinimide according to a certain mass ratio, stirring at room temperature for reaction for 2 hours, adding doxorubicin hydrochloride with the mass ratio of 0.1-1:1 to the polypeptide molecules, stirring at room temperature for reaction for 24 hours, dialyzing by using a dialysis bag, collecting substances with larger molecular weight, and freeze-drying to obtain PSMA-MMP-9-DOX; mixing monophosphosphatidylic acid A and N, N' -carbonyldiimidazole according to the mass ratio of 1:1, stirring at room temperature for reaction for 2 hours, adding PSMA-MMP-9-DOX with the mass 5 times that of monophosphoryl acid A, mixing, stirring at room temperature for reaction for 12 hours, dialyzing by using a dialysis bag, and collecting substances with larger molecular weight to obtain the MPLA-PSMA-MMP-9-DOX nanoparticles.
The synthesis efficiency of the preparation process of the in situ vaccine without additional nanomaterial loading as exemplified above needs to be further improved.
Disclosure of Invention
Based on the technical problems, one of the purposes of the invention is a preparation method of the tumor microenvironment responsive in-situ nano vaccine, and the synthesis efficiency is high when the method is adopted to prepare the tumor microenvironment responsive in-situ nano vaccine.
The purpose of the invention can be realized by the following technical scheme:
a preparation method of a tumor microenvironment responsive in-situ nano vaccine comprises the steps of preparing amphiphilic monomer molecules and self-assembling the amphiphilic monomer molecules into the tumor microenvironment responsive in-situ nano vaccine;
the amphiphilic monomer molecule comprises a hydrophobic functional molecule, an environment responsive connecting molecule and a hydrophilic functional molecule which are connected in sequence,
the step of preparing the amphiphilic monomer molecule comprises:
preparing an intermediate linking product by linking the hydrophobic functional molecule and the environment-responsive linking molecule;
connecting the intermediate connection product with a hydrophilic functional molecule to prepare the amphiphilic monomer molecule;
the hydrophobic functional molecules are hydrophobic adjuvant molecules, and the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, or the hydrophobic functional molecules are hydrophobic chemotherapeutic drug molecules, and the hydrophilic functional molecules are hydrophilic adjuvant molecules.
In some of these embodiments, the step of preparing the amphiphilic monomer molecule comprises:
mixing the hydrophobic functional molecule and a first activating agent to carry out a first activation reaction to prepare a first activation product;
mixing the first activation product and the environmentally responsive linker molecule for a first linking reaction to produce the intermediate linking product;
mixing the intermediate linkage product and a second activating agent to perform a second activation reaction to prepare a second activated product;
and mixing the second activation product and the hydrophilic functional molecule to perform a second connection reaction to prepare the amphiphilic monomer molecule.
In some of these embodiments, the conditions of the second activation reaction comprise: the temperature is 32-42 ℃, and the time duration is 3.5-4.5 h.
In some embodiments, the hydrophobic functional molecule is a hydrophobic adjuvant molecule, and the hydrophilic functional molecule is a hydrophilic chemotherapeutic drug molecule, and the step of preparing the amphiphilic monomer molecule further comprises: and adding a catalyst to the second activation product and the hydrophilic functional molecule to perform the second linking reaction, wherein the catalyst comprises 4-dimethylaminopyridine.
In some of these embodiments, the conditions of the first activation reaction comprise: the temperature is 22-28 ℃, and the time duration is 2-4 h.
In some of these embodiments, the first activator comprises N, N' -carbonyldiimidazole; or/and the mass ratio of the hydrophobic functional molecule to the first activator is (0.75-1.5): 1-2.
In some of these embodiments, the conditions of the first ligation reaction comprise: the temperature is 22-28 ℃, and the time duration is 24-48 h.
In some of these embodiments, the mass ratio of the hydrophobic functional molecule and the environmentally responsive linker molecule is (0.75-1.5): (2-4).
In some of these embodiments, the second activator comprises 1, 2-dichloroethane and N-hydroxysuccinimide in a mass ratio of (3.3-6.6) to (1-2).
In some of these embodiments, the conditions of the second ligation reaction comprise: the temperature is 22-28 ℃, and the time duration is 24-48 h.
In some embodiments, the hydrophobic functional molecule is a hydrophobic chemotherapeutic drug molecule, the hydrophilic functional molecule is a hydrophilic adjuvant molecule, and the amount of the hydrophilic functional molecule is 5OD-10OD per 0.75mg-1.5mg of the hydrophobic functional molecule;
the hydrophobic functional molecules are hydrophobic adjuvant molecules, the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, and the dosage of the hydrophilic functional molecules corresponding to 0.75-1.5 mg of the hydrophobic functional molecules is 1.5-3 mg.
In some embodiments, the hydrophobic chemotherapeutic drug molecule comprises one or more of paclitaxel, oxaliplatin, indocyanine green, and hematoporphyrin and derivatives thereof.
In some of these embodiments, the hydrophilic adjuvant comprises one or more of QS21, CpG ODN, and analogs thereof.
In some of these embodiments, the hydrophilic chemotherapeutic drug molecule comprises one or more of doxorubicin hydrochloride, daunorubicin, and IR 820.
In some of these embodiments, the hydrophobic adjuvant molecule comprises monophosphoryl a and imiquimod and analogs thereof.
In some of these embodiments, the environmentally-responsive linker molecule comprises one or more of a matrix metalloproteinase-responsive polypeptide, a tumor-penetrating peptide, and an enzymatically-cleaved a-lactalbumin polypeptide.
In some embodiments, the environmentally-responsive linker molecule comprises one or more polypeptide molecules having an amino acid sequence as set forth in SEQ ID No.1 to SEQ ID No. 3.
The tumor microenvironment responsive in-situ nano vaccine prepared by the preparation method.
Compared with the traditional technical scheme, the invention has the following advantages:
according to the invention, through the integral optimization of the preparation process, in the process of preparing the amphiphilic monomer molecule, the hydrophobic functional molecule is connected to one end of the environment responsive connecting molecule, and then the hydrophilic functional molecule is arranged on the other end face of the environment responsive connecting molecule, so that the synthesis efficiency of the nano in-situ vaccine is effectively improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic structural diagram of a tumor microenvironment responsive in situ nano-vaccine prepared according to one embodiment of the present invention;
FIG. 2 is a graph showing the results of dynamic light scattering particle size detection of the tumor microenvironment responsive in situ nano vaccine prepared according to one embodiment of the present invention;
FIG. 3 is a graph comparing the results of various groups of treatments (including tumor microenvironment-responsive in situ nano-vaccines) on BMDCs in accordance with one embodiment of the present invention;
FIG. 4 is a graph comparing cytokine secretion after incubation of various groups of treatments (including tumor microenvironment-responsive in situ nano-vaccines) with BMDCs, in accordance with one embodiment of the present invention;
FIG. 5 is a graph comparing the effect of various groups of treatments (containing tumor microenvironment-responsive in situ nano-vaccines) on tumor mass in tumor-bearing mice according to one embodiment of the present invention;
FIG. 6 is a graph comparing the change in tumor volume of tumor-bearing mice for various groups of treatments (including tumor microenvironment-responsive in situ nano-vaccines) in accordance with an embodiment of the present invention;
FIG. 7 is a graph comparing survival time of groups of tumor-bearing mice treated with one embodiment of the present invention (containing tumor microenvironment-responsive in situ nano-vaccines);
FIG. 8 is a particle size distribution diagram of tumor microenvironment responsive nanoparticles prepared according to one embodiment of the present invention;
FIG. 9 is a transmission electron microscope used for observing the surface topography of the tumor microenvironment responsive nano vaccine prepared by one embodiment of the invention;
FIG. 10 is a graph comparing the effect of tumor cell treatment on nanoparticle uptake (A: Free DOX, B: NPs) in various groups according to an embodiment of the present invention;
FIG. 11 is a graph comparing the effects of BMDCs maturation promotion and activation following treatment with tumor fragment stimulation in various groups according to an embodiment of the present invention;
FIG. 12 is a graph comparing the antitumor ability (A: tumor volume in tumor-bearing mice, B: survival time in tumor-bearing mice) of each group of treatments according to one embodiment of the present invention;
FIG. 13 is a graph comparing the change in tumor volume of groups of tumor-bearing mice treated according to one embodiment of the present invention;
FIG. 14 is a graph comparing the survival time of tumor-bearing mice treated in various groups according to one embodiment of the present invention;
FIG. 15 is a graph comparing the synthesis efficiencies of example 1, example 4, example 5 and comparative example 1.
Detailed Description
In order to facilitate an understanding of the present invention, the present invention will be described in more detail below. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments or examples set forth herein. Rather, these embodiments or examples are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments or examples only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of two or more of the associated listed items, including any and all combinations of two or more of the associated listed items, or all of the associated listed items.
The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way. Those skilled in the art will appreciate that variations and other uses are included within the spirit and scope of the invention as defined by the scope of the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the present invention, "first aspect", "second aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor are they to be construed as implicitly indicating the importance or quantity of the technical features indicated.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
In a first aspect, the invention provides a preparation method of a tumor microenvironment responsive in-situ nano vaccine, which comprises the steps of preparing amphiphilic monomer molecules and self-assembling the amphiphilic monomer molecules into the tumor microenvironment responsive in-situ nano vaccine;
the amphiphilic monomer molecule comprises a hydrophobic functional molecule, an environment responsive connecting molecule and a hydrophilic functional molecule which are connected in sequence,
the step of preparing the amphiphilic monomer molecule comprises:
preparing an intermediate linking product by linking the hydrophobic functional molecule and the environment-responsive linking molecule;
connecting the intermediate connection product with a hydrophilic functional molecule to prepare the amphiphilic monomer molecule;
the hydrophobic functional molecules are hydrophobic adjuvant molecules, and the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, or the hydrophobic functional molecules are hydrophobic chemotherapeutic drug molecules, and the hydrophilic functional molecules are hydrophilic adjuvant molecules.
The invention provides a tumor microenvironment responsive in-situ nano vaccine prepared by a preparation method, which is formed by connecting hydrophilic functional molecules (chemotherapeutic drugs or adjuvant molecules) and hydrophobic functional molecules (chemotherapeutic drugs or adjuvant molecules) through one or more connecting molecules, and self-assembling in water to form nano particles. When the amphiphilic nano-particles reach a tumor microenvironment, the functional connecting molecules are subjected to responsive fragmentation under the microenvironment condition, and chemotherapeutic drug molecules and adjuvant molecules are released. The chemotherapeutic drug molecule kills tumor cells, induces immunogenic death of the tumor cells to generate tumor specific antigens, and the adjuvant molecule acts on immune cells to enhance the specific immunoreaction of the tumor antigens so as to enhance the anti-tumor effect.
The tumor microenvironment responsive in-situ nano vaccine prepared by the preparation method provided by the invention has the particle size range of 110nm-250nm, such as 130nm, 150nm, 170nm, 190nm, 210nm, 240nm and 250nm, and the particle size range is beneficial to accumulation at a tumor part.
In one example, the step of preparing the amphiphilic monomer molecule comprises:
mixing the hydrophobic functional molecule and a first activating agent to carry out a first activation reaction to prepare a first activation product;
mixing the first activation product and the environmentally responsive linker molecule for a first linking reaction to produce the intermediate linking product;
mixing the intermediate linkage product and a second activating agent to perform a second activation reaction to prepare a second activated product;
and mixing the second activation product and the hydrophilic functional molecule to perform a second connection reaction to prepare the amphiphilic monomer molecule.
In one example, the conditions of the second activation reaction include: the temperature is 32-42 ℃, and the time duration is 3.5-4.5 h. The temperature of the second activation reaction of the present invention may be selected from, including but not limited to: 32 ℃, 32.5 ℃, 33 ℃, 33.5 ℃ and 34 ℃. The temperature of the second activation reaction of the present invention may be selected from, including but not limited to: 3.5h, 3.6h, 3.7h, 3.8h, 3.9h, 4.0h, 4.1h, 4.2h, 4.3h and 4.4 h. In one example, the hydrophobic functional molecule is a hydrophobic adjuvant molecule, and the hydrophilic functional molecule is a hydrophilic chemotherapeutic drug molecule, and the step of preparing the amphiphilic monomer molecule further comprises: and adding a catalyst to the second activation product and the hydrophilic functional molecule to perform the second linking reaction, wherein the catalyst comprises 4-dimethylaminopyridine. 0.8mg-1.6mg of 4-dimethylaminopyridine is correspondingly added into each 1.5mg-3mg of the hydrophilic functional molecules. The catalyst can improve the reaction efficiency by adjusting pH.
In one example, the conditions of the first activation reaction include: the temperature is 22-28 ℃, and the time duration is 2-4 h. In the conditions of the first activation reaction of the present invention, the temperature may be selected from, but is not limited to: 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C; the duration may be selected from, but is not limited to: 2h, 2.2h, 2.4h, 2.6h, 2.8h, 3.0h, 3.2h, 3.4h, 3.6h, 3.8h and 4 h.
In one example, the first activator comprises N, N' -carbonyldiimidazole.
In one example, the mass ratio of the hydrophobic functional molecule to the first activator is (0.75-1.5): (1-2). For example, 0.75:1, 0.75:1.5, 0.75:2, 1.5:1, 1.5:1.5, 1.5:2.
In one example, the conditions of the first ligation reaction include: the temperature is 22-28 ℃, and the time duration is 24-48 h. In the conditions of the first ligation reaction of the present invention, the temperature may be selected from, but is not limited to: 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C; the duration may be selected from, but is not limited to: 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h and 48 h.
In one example, the mass ratio of the hydrophobic functional molecule to the environment-responsive linker molecule is (0.75-1.5): (2-4). For example: 0.75:2, 0.75:2.5, 0.75:3, 0.75: 3.5, 0.75:4, 1.5:2, 1.5:2.5:, 1.5:3, 1.5: 3.5 and 1.5: 4.
In one example, the second activator comprises 1, 2-dichloroethane and N-hydroxysuccinimide in a mass ratio of (3.3-6.6) to (1-2). In the invention, the mass ratio of the 1, 2-dichloroethane to the N-hydroxysuccinimide is 3.3:1, 3.3: 1.5, 3.3: 2. 6.6:1, 6.6:1.5 and 6.6: 2.
In one example, the conditions of the second ligation reaction include: the temperature is 22-28 ℃, and the time duration is 24-48 h. In the conditions of the second ligation reaction of the present invention, the temperature may be selected from, but is not limited to: 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C; the duration may be selected from, but is not limited to: 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h and 48 h.
In one example, the hydrophobic functional molecule is a hydrophobic chemotherapeutic drug molecule, the hydrophilic functional molecule is a hydrophilic adjuvant molecule, and the dosage of the hydrophilic functional molecule is 5OD-10OD per 0.75mg-1.5mg of the hydrophobic functional molecule. For example, the amount of the hydrophilic functional molecule per 0.75mg of the hydrophobic functional molecule is 5OD, 6OD, 7OD, 8OD, 9OD, 10OD, the amount of the hydrophilic functional molecule per 1.5mg of the hydrophobic functional molecule is 5OD, 6OD, 7OD, 8OD, 9OD, 10OD, and the amount of the hydrophilic functional molecule per 1.5mg of the hydrophobic functional molecule is 5OD, 6OD, 7OD, 8OD, 9OD, 10 OD.
In one example, the hydrophobic functional molecule is a hydrophobic adjuvant molecule, the hydrophilic functional molecule is a hydrophilic chemotherapeutic drug molecule, and the amount of the hydrophilic functional molecule is 1.5mg to 3mg per 0.75mg to 1.5mg of the hydrophobic functional molecule. For example, the amount of the hydrophilic functional molecule per 0.75mg of the hydrophobic functional molecule is 1.5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2.0mg, 2.1mg, 2.2mg, 2.3mg, 2.4mg, 2.5mg, 2.6mg, 2.7mg, 2.8mg, 2.9mg, 3.0 mg.
In one example, the hydrophobic chemotherapeutic drug molecule comprises one or more of paclitaxel, oxaliplatin, indocyanine green, and hematoporphyrin and derivatives thereof.
In one example, the hydrophilic adjuvant comprises one or more of QS21, CpG ODN, and analogs thereof.
In one example, the hydrophilic chemotherapeutic drug molecules comprise one or more of doxorubicin hydrochloride, daunorubicin, and IR 820.
In one example, the hydrophobic adjuvant molecule comprises monophosphoryl a and imiquimod and one or more of its analogs.
In one example, the environmentally-responsive linker molecule comprises one or more of a matrix metalloproteinase-responsive polypeptide, a tumor penetrating peptide, and an enzymatically-cleaved a-lactalbumin polypeptide.
In one example, the environmentally-responsive linker molecule comprises one or more of the polypeptide molecules having the amino acid sequences shown in SEQ ID nos. 1 to 3.
In one example, the hydrophobic chemotherapeutic drug molecule is hematoporphyrin and derivatives thereof, the hydrophilic adjuvant is CpG ODN, and the environment-responsive linker molecule is a polypeptide molecule having an amino acid sequence shown in SEQ ID No. 3.
In one example, the hydrophilic chemotherapeutic drug molecule is doxorubicin hydrochloride, the hydrophobic adjuvant molecule is monophosphoryl a, and the environmentally-responsive linker molecule is a polypeptide molecule having an amino acid sequence as set forth in SEQ ID No.1 or SEQ ID No. 2.
In a second aspect, the invention provides a tumor microenvironment responsive in situ nano vaccine prepared by the preparation method. The tumor microenvironment responsive in-situ nano vaccine has a spherical structure formed by self-assembly of amphiphilic monomer molecules, wherein the amphiphilic monomer molecules comprise hydrophobic functional molecules, environment responsive connecting molecules and hydrophilic functional molecules which are sequentially connected, the hydrophobic functional molecules are arranged on the inner side of the spherical structure, the hydrophilic functional molecules are arranged on the outer side of the spherical structure, the hydrophobic functional molecules are hydrophobic adjuvant molecules, and the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, or the hydrophobic functional molecules are hydrophobic chemotherapeutic drug molecules, and the hydrophilic functional molecules are hydrophilic adjuvant molecules.
Example 1
A preparation method of a tumor microenvironment responsive in situ nano vaccine comprises the following steps:
s1: mixing hydrophobic molecule monophosphoryl lipid A (MPLA) and N, N '-carbonyldiimidazole according to the mass ratio of 0.75mg:2mg, stirring at room temperature for reaction for 2h, dialyzing with 1500Da dialysis bag to remove unreacted monophosphoryl lipid A and N, N' -carbonyldiimidazole, collecting large molecular weight substance, and lyophilizing.
S2: dissolving the high molecular weight substance obtained in S1 in 150 μ L DMSO, adding functional linker molecule consisting of polypeptide 2mg, stirring at room temperature for reaction for 24 hr, dialyzing with 2000Da dialysis bag to collect the high molecular weight substance, and lyophilizing.
S3: 1, 2-dichloroethane and N-hydroxysuccinimide were mixed with the reaction product of S2 in a mass ratio of 3.3mg:2mg, dissolved in 200. mu.L of DMSO, and reacted at 40 ℃ for 4 hours with stirring.
S4: adding hydrophilic chemotherapeutic drugs of adriamycin hydrochloride 1.5mg and 4-dimethylaminopyridine 1.6mg into the S3 reaction product, stirring and reacting for 48 hours at room temperature, dialyzing by using a 3000Da dialysis bag to retain a high molecular weight substance, and freeze-drying to prepare the tumor microenvironment responsive in-situ nano vaccine.
The structure of the tumor microenvironment responsive in situ nano vaccine prepared by the embodiment is shown in fig. 1, and the dynamic light scattering particle size detection result is shown in fig. 2.
The tumor microenvironment responsive in situ nano vaccine (abbreviated as "nanoparticle") prepared in this example was tested as follows:
1. effect of tumor microenvironment-responsive in situ Nanoprotein on BMDCs (bone marrow dendritic cells) maturation
Flow cytometry to detect the maturation promoting condition of the nanoparticles to BMDCs cells comprises the following steps: tumor cells (B-16 cells) and tumor microenvironment responsive in-situ nano vaccines are incubated for 12h (dosage is calculated according to doxorubicin hydrochloride 2 mu M), BMDCs and tumor cells containing nanoparticles are incubated for 24h, cells are collected, flow antibodies such as CD11C, CD40 and CD80 are marked, and detection is carried out by a flow cytometer. PBS buffer, free doxorubicin hydrochloride of the same concentration, and a mixture of free doxorubicin hydrochloride + MPLA of the same concentration were used as controls. Wherein Free doxorubicin hydrochloride is represented by Free DOX, Free doxorubicin hydrochloride + MPLA is represented by Free DM, and nanoparticles are represented by NPs.
The results are shown in FIG. 3. The graphical results show that the tumor microenvironment responsive in situ nano-vaccine can effectively promote the maturation of BMDCs.
2. ELISA method for determining influence of tumor microenvironment responsiveness in situ nano vaccine on secretion of BMDCs (BMDCs) cytokines
BMDCs were collected and plated in 96-well plates. The tumor cells (B-16 cells) and the tumor microenvironment responsive in-situ nano vaccine are incubated for 12h (dosage is calculated according to adriamycin hydrochloride 2 mu M), then the tumor cells containing the nanoparticles and BMDCs in a 96-well plate are incubated for 24h, and then the supernatant is obtained by centrifugation. The BMDCs supernatant was assayed for the amount of the cytokine TNF-. alpha.according to the ELISA kit protocol. And (3) measuring the absorbance OD value at 450nm by using a microplate reader, drawing a standard curve according to the absorbance and the concentration of the standard substance, and calculating the concentration of the sample. PBS buffer, free doxorubicin hydrochloride of the same concentration, and a mixture of free doxorubicin hydrochloride + MPLA of the same concentration were used as controls. Wherein Free doxorubicin hydrochloride is represented by Free DOX, Free doxorubicin hydrochloride + MPLA is represented by Free DM, and nanoparticles are represented by NPs.
The results are shown in fig. 4, which illustrates that the tumor microenvironment-responsive in situ nano-vaccine can effectively promote the secretion of TNF-alpha.
3. Anti-tumor curative effect of tumor microenvironment responsive in-situ nano vaccine in tumor-bearing mice
A melanoma mouse ectopic tumor transplantation model is established by selecting female C57BL/6 mice which are bred in a SPF-level breeding room and are healthy for 6 weeks. The method comprises the following steps:
after the experimental animals were adapted to the breeding environment, the hair on the back and groin of the mice was removed, and B-16 cells (about 5X 10) in logarithmic growth phase and in good culture state were subcutaneously inoculated into the right groin5One/only). The tumor volume of the mouse to be inoculated is about 50mm3Tumor-bearing mice with similar growth conditions were randomly divided into four groups of PBS buffer solution, free doxorubicin hydrochloride + MPLA, and nanoparticles, and equal volumes of fresh sterile PBS buffer solution, free doxorubicin hydrochloride + MPLA, and nanoparticle solution were administered at day 6, day 12, and day 18 of tumor cell inoculation (dose was calculated as doxorubicin hydrochloride 0.1 mg/ml, MPLA 20 μ g/ml). Carefully observing the survival condition, the diet condition and the mental state of the mice, measuring and recording the length and the width of the tumor-bearing mice and the tumor volume (mm) of the mice by using a digital vernier caliper every two days3) 1/2 × length × width2. When the tumor volume reaches 2000mm3The mice were judged to be dead. The mice were sacrificed in unison and the tumors were removed and the tumor mass was recorded. (wherein Free doxorubicin hydrochloride is represented by Free DOX, Free doxorubicin hydrochloride + MPLA is represented by Free DM, and nanoparticles are represented by NPs).
The results are shown in FIGS. 5, 6 and 7. The graphical result shows that the tumor microenvironment responsive in situ nano vaccine can effectively inhibit the growth of the tumor.
Example 2
A preparation method of a tumor microenvironment responsive in situ nano vaccine comprises the following steps:
s1: mixing hydrophobic molecule monophosphoryl lipid A (MPLA) and N, N '-carbonyldiimidazole according to the mass ratio of 1.5mg:1mg, stirring at room temperature for reaction for 4h, dialyzing by a 1500Da dialysis bag to remove unreacted monophosphoryl lipid A and N, N' -carbonyldiimidazole, and reserving a high molecular weight substance for freeze-drying.
S2: dissolving the substance obtained in S1 in 150 μ L DMSO, adding functional linker molecule consisting of polypeptide 4mg, stirring at room temperature for reaction for 48h, dialyzing with 2000Da dialysis bag to collect large molecular weight substance, and lyophilizing.
S3: 1, 2-dichloroethane and N-hydroxysuccinimide were mixed with the reaction product of S2 in a mass ratio of 6.6mg:1mg, dissolved in 200. mu.L of DMSO, and reacted at 40 ℃ for 4 hours with stirring.
S4: adding hydrophilic chemotherapeutic medicine adriamycin hydrochloride 3mg and 4-dimethylamino pyridine 0.8mg into the S3 reaction product, stirring and reacting for 24h at room temperature, dialyzing with a 3000Da dialysis bag to obtain a high molecular weight substance, and freeze-drying.
The structure of the tumor microenvironment responsive in situ nano vaccine prepared in this embodiment is shown in fig. 1, the dynamic light scattering particle size detection result is shown in fig. 8, the surface morphology of the tumor microenvironment responsive in situ nano vaccine observed by a transmission electron microscope is shown in fig. 9, and it can be seen from fig. 9 that the tumor microenvironment responsive in situ nano vaccine has uniform particle size and uniform distribution in an aqueous solution as seen by a transmission electron microscope.
The tumor microenvironment responsive in situ nano vaccine (abbreviated as "nanoparticle") prepared in this example was tested as follows:
1. study on uptake condition of tumor cell E.G7-OVA to tumor microenvironment responsive in situ nano vaccine
E.G7-OVA cells in logarithmic growth phase at 1.5X 105The cell density is inoculated on a 24-pore plate, after the cells are stabilized, Free DOX and a nanoparticle solution (the final DOX content is 2 mu M) are added into the 24-pore plate, the cells are respectively incubated for 2, 6 and 12 hours, the cells are collected into an EP tube after the incubation is carried out for a specific time, the cells are washed by PBS, after 4 percent of tissue cell fixing solution is fixed, the responsiveness of the tumor cells to small molecule drugs DOX and the tumor microenvironment is observed by a laser scanning confocal microscopeUptake of in situ nano-vaccine.
FIG. 10 is a graph comparing the uptake of nanoparticles (A: Free DOX, B: NPs) by tumor cells treated in each group, and the results are shown as follows: the tumor microenvironment responsive in-situ nano vaccine can promote the medicine intake of the tumor.
2. Effect of tumor microenvironment responsive in situ nano vaccine on maturation and activation of BMDCs
Tumor cells E.G7-OVA at 2X 105The cells were inoculated in 24-well plates, incubated overnight, and Free DOX and Free M were added&D (Free MPLA and DOX) and a nanoparticle solution are added into a 24-well plate to enable the final concentration of DOX to be 2 mu M and the concentration of MPLA to be 1 mu g/mL, incubation is continued for 24h, then a culture medium containing cell debris is added into the 24-well plate in which BMDCs are paved in advance, incubation is continued for 24h, and the expression condition of a BMDCs surface marker is detected by using a fluorescent dye-labeled antibody, so that the influence of the nano-tumor microenvironment responsive in-situ nano vaccine on BMDCs maturation promotion is evaluated.
And (3) stimulating BMDCs cells by using tumor cell fragments according to the method, incubating for 24h, centrifuging to collect BMDCs supernatant, and detecting the condition of cytokines secreted by the BMDCs by using a corresponding ELISA kit.
FIG. 11 is a graph showing the effect of BMDCs maturation and activation after stimulation with tumor fragments in each group. According to the results shown in the figure, the tumor microenvironment responsive in-situ nano vaccine can promote the maturation of BMDC and the secretion of interleukin 1 beta and interleukin 6.
3. Evaluation of tumor-inhibiting effect of tumor microenvironment responsive in-situ nano vaccine
Female mice C57BL/6 injection 5X 10 at 6-8 weeks5G7-OVA tumor cells to construct subcutaneous lymphoma model, and after the tumor volume reaches a certain degree, the tumor cells are administered to mice with PBS, Free DOX and Free M&D and nanoparticle solution (DOX: 0.1 mg/mouse, MPLA: 20. mu.g/mouse) treatment, mice body weight and tumor volume were recorded every other day, drug treatment was 1 time every 5 days via tail vein for 3 times.
FIG. 12 is a graph showing the results of the antitumor abilities of each group (A: tumor volume of tumor-bearing mice, B: survival time of tumor-bearing mice), and it can be seen from the results shown in the figure that the tumor microenvironment-responsive in situ nano-vaccine can effectively inhibit the growth of lymphoma and prolong the survival time of mice.
Example 3
A preparation method of a tumor microenvironment responsive in situ nano vaccine comprises the following steps:
s1: mixing hydrophobic molecule hematoporphyrin and N, N '-carbonyl diimidazole according to the mass ratio of 1mg:1.5mg, stirring and reacting for 3h at room temperature, dialyzing by using a 800Da dialysis bag to remove unreacted hematoporphyrin and N, N' -carbonyl diimidazole, and freeze-drying.
S2, dissolving the substance obtained in S1 in 150 μ L DMSO, adding 3mg of functional linker molecule composed of polypeptide, stirring at room temperature for 36h, dialyzing with 2000Da dialysis bag to collect the high molecular weight substance, and lyophilizing.
S3: 1, 2-dichloroethane and N-hydroxysuccinimide in a mass ratio of 5mg:1.5mg were mixed with the reaction product of S2 and dissolved in 200. mu.L of DMSO, and the reaction was stirred at 40 ℃ for 4 hours.
S4: and adding a hydrophilic immune adjuvant CpG ODN 7OD into the S3 reaction product, stirring and reacting for 36h at room temperature, and dialyzing by using a 6000Da dialysis bag to retain the high molecular weight substance to obtain the tumor microenvironment responsive in-situ nano vaccine.
The structure of the tumor microenvironment responsive in situ nano vaccine prepared by the embodiment is shown in figure 1.
The tumor microenvironment responsive in situ nano vaccine (abbreviated as "nanoparticle") prepared in this example was tested as follows:
a melanoma mouse ectopic tumor transplantation model is established by selecting a female C57BL/6 mouse which is bred in an SPF-level breeding room and is healthy for 6 weeks old. After the experimental animals were adapted to the breeding environment, the hair on the back and groin of the mice was removed, and B-16 cells (about 5X 10) in logarithmic growth phase and in good culture state were subcutaneously inoculated into the right groin5One/only). The tumor volume of the mouse to be inoculated is about 50mm3Then, tumor-bearing mice with similar growth conditions are randomly divided into four groups of PBS buffer solution, free hematoporphyrin + CpG ODN and nanoparticles, and are respectively administered to an equal body on the 6 th day, the 12 th day and the 18 th day after tumor cell inoculationFresh sterile PBS solution, free hematoporphyrin + CpG ODN and four groups of nano-particles (dosage is calculated according to 0.2mg of hematoporphyrin and 10 mug of CpG ODN per group), and the tumor part is irradiated by laser for 5min 24h after the administration. Carefully observing the survival condition, the diet condition and the mental state of the mice, measuring and recording the length and the width of the tumor-bearing mice and the tumor volume (mm) of the mice by using a digital vernier caliper every two days3) 1/2 × length × width2. When the tumor volume reaches 2000mm3The mice were judged to be dead. Wherein Free hematoporphyrin is represented by Free HEM, Free hematoporphyrin + CpG ODN is represented by Free HC, and nanoparticles are represented by NPs).
The results are shown in fig. 13 and 14, which illustrate that the tumor microenvironment-responsive in situ nano-vaccine can effectively inhibit the growth of tumors.
Example 4
This example is a variation of example 1, and the major variation from example 1 involves the addition of 2mg of 4-dimethylaminopyridine in step S4. The rest are referred to example 1.
Example 5
This example is a variation of example 1, and the reaction conditions including step S3, which are the main variations from example 1, include: the reaction was stirred at room temperature for 24 h.
Comparative example 1
The comparative example is a variation of example 1, and the preparation method of the tumor microenvironment responsive in situ nano vaccine of the comparative example comprises the following steps:
s1: functional connecting molecules consisting of polypeptide are mixed with activators 1, 2-dichloroethane and N-hydroxysuccinimide to activate carboxyl terminals of the polypeptide molecules, the mass ratio of the functional connecting molecules to the activators 1, 2-dichloroethane and N-hydroxysuccinimide is 2mg to 3.3mg to 2mg, and the mixture is stirred and reacted for 4 hours at the temperature of 40 ℃.
S2: adding hydrophilic chemotherapeutic drug doxorubicin hydrochloride 1.5mg and 4-dimethylaminopyridine 1.6mg into the substance obtained in S1, stirring at room temperature for reaction for 48h, dialyzing with a 1000Da dialysis bag to obtain a high molecular weight substance, and freeze-drying.
S3: mixing hydrophobic molecule monophosphoryl lipid A (MPLA) and N, N '-carbonyldiimidazole according to the mass ratio of 0.75mg:2mg, stirring for reaction for 2h at room temperature, and dialyzing by a 1500Da dialysis bag to remove unreacted monophosphoryl lipid A and N, N' -carbonyldiimidazole;
s4: and mixing the S2 and the obtained substance of S3, stirring and reacting for 24 hours at room temperature, and dialyzing by using a 3000Da dialysis bag to obtain the high molecular weight substance, thereby obtaining the tumor microenvironment responsive in-situ nano vaccine.
For example 1, example 4, example 5 and comparative example 1, ultraviolet absorption of DOX and the solution of the synthesized product was measured by an ultraviolet spectrophotometer, and as a result, the ultraviolet maximum absorption wavelength was found to be around 480 nm. And drawing a DOX concentration standard curve by using the DOX standard substance through a fluorescence spectrophotometer, and measuring the absorbance of each synthetic product at 480nm so as to determine the DOX concentration in each synthetic product and obtain the DOX content in the synthetic product. Finally, the synthesis efficiency is calculated by the following formula:
the synthesis efficiency (amount of DOX in the synthesis product ÷ amount of DOX charged into the reaction) × 100%.
The results are shown in fig. 15, in which the synthesis efficiency of example 1 is 26.2%, the synthesis efficiency of example 4 is 20.7%, the synthesis efficiency of example 5 is 18.7%, and the synthesis efficiency of comparative example 1 is 16.7%.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features. The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Claims (10)
1. The preparation method of the tumor microenvironment responsive in-situ nano vaccine is characterized by comprising the steps of preparing amphiphilic monomer molecules and self-assembling the amphiphilic monomer molecules into the tumor microenvironment responsive in-situ nano vaccine;
the amphiphilic monomer molecule comprises a hydrophobic functional molecule, an environment responsive connecting molecule and a hydrophilic functional molecule which are connected in sequence,
the step of preparing the amphiphilic monomer molecule comprises:
linking the hydrophobic functional molecule and the environment-responsive linker molecule to produce an intermediate linking product;
connecting the intermediate connecting product and the hydrophilic functional molecule to prepare the amphiphilic monomer molecule;
the hydrophobic functional molecules are hydrophobic adjuvant molecules, and the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, or the hydrophobic functional molecules are hydrophobic chemotherapeutic drug molecules, and the hydrophilic functional molecules are hydrophilic adjuvant molecules.
2. The method for preparing the tumor microenvironment-responsive in situ nano vaccine of claim 1, wherein the step of preparing the amphiphilic monomer molecule comprises:
mixing the hydrophobic functional molecule and a first activating agent to carry out a first activation reaction to prepare a first activation product;
mixing the first activation product and the environmentally responsive linker molecule for a first linking reaction to produce the intermediate linking product;
mixing the intermediate linkage product and a second activating agent to perform a second activation reaction to prepare a second activated product;
and mixing the second activation product and the hydrophilic functional molecule to perform a second connection reaction to prepare the amphiphilic monomer molecule.
3. The method for preparing the tumor microenvironment-responsive in situ nano vaccine of claim 2, wherein the conditions of the second activation reaction include: the temperature is 32-42 ℃, and the time duration is 3.5-4.5 h.
4. The method for preparing the tumor microenvironment responsive in-situ nano vaccine of claim 2, wherein the hydrophobic functional molecule is a hydrophobic adjuvant molecule, and the hydrophilic functional molecule is a hydrophilic chemotherapeutic drug molecule, and the step of preparing the amphiphilic monomer molecule further comprises: and adding a catalyst to the second activation product and the hydrophilic functional molecule to perform the second linking reaction, wherein the catalyst comprises 4-dimethylaminopyridine.
5. The method for preparing the tumor microenvironment-responsive in situ nano vaccine of any one of claims 2 to 4, wherein the conditions of the first activation reaction include: the temperature is 22-28 ℃, and the time duration is 2-4 h; or/and, the first activator comprises N, N' -carbonyldiimidazole; or/and the mass ratio of the hydrophobic functional molecule to the first activator is (0.75-1.5): 1-2; and/or the first and/or second light sources,
the conditions of the first ligation reaction include: the temperature is 22-28 ℃, and the time duration is 24-48 h; or/and the mass ratio of the hydrophobic functional molecule to the environment-responsive connecting molecule is (0.75-1.5) to (2-4); and/or the first and/or second light sources,
the second activator comprises 1, 2-dichloroethane and N-hydroxysuccinimide in a mass ratio of (3.3-6.6) to (1-2).
6. The method for preparing the tumor microenvironment-responsive in situ nano vaccine of any one of claims 2 to 4, wherein the conditions of the second ligation reaction include: the temperature is 22-28 ℃, and the time duration is 24-48 h.
7. The method for preparing the tumor microenvironment responsive in situ nano vaccine of any one of claims 2 to 4, wherein the hydrophobic functional molecule is a hydrophobic chemotherapeutic drug molecule, the hydrophilic functional molecule is a hydrophilic adjuvant molecule, and the amount of the hydrophilic functional molecule is 5OD to 10OD per 0.75mg to 1.5mg of the hydrophobic functional molecule;
the hydrophobic functional molecules are hydrophobic adjuvant molecules, the hydrophilic functional molecules are hydrophilic chemotherapeutic drug molecules, and the dosage of the hydrophilic functional molecules corresponding to 0.75-1.5 mg of the hydrophobic functional molecules is 1.5-3 mg.
8. The method of any one of claims 1 to 4, wherein the hydrophobic chemotherapeutic drug molecule comprises one or more of paclitaxel, oxaliplatin, indocyanine green, and hematoporphyrin and derivatives thereof, and/or wherein the hydrophilic adjuvant comprises one or more of QS21, CpG ODN, and analogs thereof, and/or wherein the hydrophilic chemotherapeutic drug molecule comprises one or more of doxorubicin hydrochloride, daunorubicin, and IR820, and/or wherein the hydrophobic adjuvant molecule comprises one or more of monophosphoryl A and imiquimod and analogs thereof; or/and the environment-responsive linker molecule comprises one or more of a matrix metalloproteinase-responsive polypeptide, a tumor penetrating peptide, and an enzymatically hydrolyzed alpha-lactalbumin polypeptide.
9. The method for preparing the tumor microenvironment-responsive in situ nano vaccine of claim 8, wherein the environment-responsive linker molecule comprises one or more polypeptide molecules with amino acid sequences shown as SEQ ID No.1 to SEQ ID No. 3.
10. The tumor microenvironment responsive in situ nano vaccine prepared by the preparation method of any one of claims 1 to 9.
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