CN110269931B - Preparation method of hydrogel tumor vaccine, hydrogel tumor vaccine prepared by preparation method and application of hydrogel tumor vaccine - Google Patents
Preparation method of hydrogel tumor vaccine, hydrogel tumor vaccine prepared by preparation method and application of hydrogel tumor vaccine Download PDFInfo
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Abstract
The invention discloses a preparation method of a hydrogel tumor vaccine, the hydrogel tumor vaccine prepared by the preparation method and application of the hydrogel tumor vaccine. The preparation method comprises the following steps: 1) Tumor cell pretreatment: adding a hydrophobic drug into tumor cells serving as a carrier to obtain pretreated tumor cells; 2) Preparation of hydrogel carrier: dissolving the polypeptide and inorganic base in buffer solution, and heating in water bath at 30-90deg.C for 5-50min to obtain hydrogel carrier; 3) Preparation of hydrogel tumor vaccine: and (3) slowly adding the pretreated tumor cells prepared in the step (1) into the hydrogel carrier prepared in the step (2) in a vortex state, uniformly mixing, and standing to obtain the hydrogel tumor vaccine. The hydrogel tumor vaccine combines tumor cell vaccine with hydrophobic drug, can enhance specific antigen presentation, inhibit immune evasion mechanism, and effectively inhibit tumor metastasis and recurrence.
Description
Technical Field
The invention relates to the field of biological medicine and tumor treatment, in particular to a preparation method of an individual tumor vaccine based on organism tumor cells, a tumor vaccine prepared by the preparation method and application thereof.
Background
Tumor recurrence and metastasis are the greatest challenges in tumor therapy. Traditional treatments such as surgery, chemotherapy and radiation therapy have limited efficacy against recurrent and metastatic tumors. Cancer immunotherapy has been recently receiving significant attention as a treatment for recurrent or metastatic tumors. Among them, tumor vaccines play an important role in the treatment for preventing cancer recurrence and metastasis, but the therapeutic effect of conventional tumor vaccines in different patients is greatly different. Most of the current tumor vaccines are directed to a single antigen molecule of a tumor cell or tissue, and more studies show that the development and progression of tumors are not caused by mutation of a single gene, but rather by mutation of genes or protein molecules of multiple targets. Along with the development of sequencing technology, the tumor neoantigen provides a new thought for the design of personalized tumor vaccines, however, the tumor neoantigen has long technical period and high cost, and is not beneficial to the long-term treatment of advanced and high-malignancy cancer patients. Therefore, there is a need in the art to construct a personalized tumor vaccine with a simple process for the efficient prevention of cancer recurrence and metastasis.
The tumor cells contain a large amount of tumor antigens, if the solid tumors of a tumor patient are subjected to surgical excision, the homologous tumor cells are collected, the antigen release behaviors are modified and efficiently regulated, and the tumor cells are hopeful to be used as personalized tumor vaccines to induce the same patient to generate high-strength specific immune responses and inhibit the recurrence and metastasis of the cancers. However, since a large number of immunonegative regulatory factors such as programmed death receptor 1/programmed death-ligand 1 (PD 1/PD-L1), interleukin 10 (IL-10), tryptophan degrading enzyme (IDO-1) and the like exist in the organism, the immunogenicity of tumor cells is greatly weakened, and the recognition of tumor antigens by an immune system is avoided. Therefore, the cancer cell vaccine and the immunomodulator are combined, the specific antigen presentation is enhanced, and meanwhile, the immune evasion mechanism is inhibited, so that a new thought is hopefully provided for the personalized treatment of recurrent and metastatic tumors.
Disclosure of Invention
The invention aims to provide a preparation method of a personalized hydrogel tumor vaccine based on body tumor cells, a tumor vaccine prepared from the hydrogel tumor vaccine and application of the tumor vaccine. The tumor vaccine prepared by the method can effectively inhibit tumor recurrence and metastasis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a preparation method of a hydrogel tumor vaccine is provided, comprising the following steps:
(1) Tumor cell pretreatment: adding a hydrophobic drug into tumor cells serving as a carrier to obtain pretreated tumor cells;
(2) Preparation of hydrogel carrier: dissolving polypeptide and inorganic base in buffer solution, and heating in water bath at 30-90deg.C for 5-50min to obtain hydrogel carrier, wherein the polypeptide is (FMOC) FMOC-KCRGDK, i.e. two amino groups of N-terminal lysine are protected by FMOC; and
(3) Preparation of hydrogel tumor vaccine: and (3) slowly adding the pretreated tumor cells prepared in the step (1) into the hydrogel carrier prepared in the step (2) in a vortex state until the pretreated tumor cells are uniformly mixed, and standing to obtain the hydrogel tumor vaccine.
In step (1) of the method, preferably, the tumor cells may be solid tumor cell lines of various sources, preferably 4T1 breast cancer tumor cells, EMT6 breast cancer tumor cells, MCF-7 breast cancer tumor cells, CT26 colon cancer tumor cells, B16-F10 melanoma tumor cells, or may be surgical tumor sections obtained from tumor patients, from which primary tumor cells are isolated and cultured in vitro to obtain expanded tumor cells.
Preferably, the method step (1) comprises the following steps: the tumor cells serving as the carrier are placed in Foxp3 fixing solution for incubation for 10-60min, and the fixed and membrane-broken tumor cells can be obtained; adding a hydrophobic drug and an organic solvent into tumor cells, stirring at room temperature, centrifuging, washing with PBS, and removing free hydrophobic molecules to obtain pretreated tumor cells. Wherein, the PBS washing times are preferably 2-4 times, more preferably 3 times. Wherein the method comprises the steps ofPreferably, the ratio of the number of tumor cells in the pretreated tumor cells to the hydrophobic drug is 1X 10 7 And (5-200) mug, wherein the volume of the organic solvent is 1-10mL, and the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide and acetonitrile. Wherein, preferably, the stirring time is 1-10h, the centrifugal rotation speed is 2000rpm, and the centrifugal time is 3min. Wherein, preferably, the ratio of the number and volume of the tumor cells to the Foxp3 fixative is 1X 10 8 Individual cells/(1 to 10) mL.
In step (1) of the method, preferably, the hydrophobic drug may be a drug for the treatment of a tumor. For example, the hydrophobic drug may be a fluorescent molecule, such as coumarin 6, nile red; or a photoactive molecule such as chlorin e6, pyropheophorbide a, and the like; or chemotherapeutics, taxol, doxorubicin, camptothecins, platinum drugs, cabazitaxel, etc.; or small molecule immunomodulators such as JQ1, NLG919, etc.
In step (2) of the method, the polypeptides are regularly arranged in solution and interact with surrounding water molecules to form a hydrogel. The inorganic base is added to neutralize the carboxyl groups in the polypeptide so that it can be sufficiently dissolved in PBS, for example, at pH 7.4. Preferably, the buffer is selected from the group consisting of PBS buffer at pH 7.4, hepes buffer at pH 7.4, PB buffer at pH 7.4, and the like.
In step (2) of the method, preferably, the concentration of the polypeptide is 1-50mg/mL, and the molar ratio of carboxyl in the polypeptide to inorganic base is 2:1-1:5; preferably, the concentration of the polypeptide is 10mg/mL, and the molar ratio of carboxyl groups in the polypeptide to inorganic base is 1:1.
In step (2) of the present invention, preferably, the inorganic base is selected from anhydrous sodium carbonate or sodium bicarbonate.
In the step (2) of the method, the water bath heating temperature is preferably 40-60 ℃ and the water bath heating time is preferably 15-30min.
The polypeptide sequence (FMOC) FMOC-KCRGDK of the hydrogel carrier prepared by the invention can be combined with neuropilin-1 (Nrp-1) receptor on the surface of tumor cells due to CRGDK peptide segment, so that the penetration of vaccine into the tumor is promoted.
The invention also provides the hydrogel tumor vaccine prepared by the method.
The invention also provides application of the hydrogel tumor vaccine in preparing a medicament for preventing tumor recurrence and metastasis.
In the present invention, the type of the tumor is not particularly limited, and may be, for example, solid tumors such as melanoma, breast cancer, lung cancer, and colon cancer.
The hydrogel tumor vaccine has tumor-related antigens, can effectively promote tumor antigen to be presented in vivo, promote the maturation of dendritic cells, induce the organism to generate specific immune response, and promote the CD8 of tumor tissues + T cells infiltrate, so as to effectively prolong the immune memory effect and achieve the purpose of inhibiting tumor recurrence and metastasis.
Compared with the prior vaccine based on tumor specific polypeptide, the technical scheme of the invention has obvious advantages: the preparation and delivery technology of tumor mutant polypeptides is mature, but a tumor often has multiple multi-target mutations at the same time, so that the tumor polypeptide vaccine aiming at single or limited mutations has very limited treatment effect on the tumor and large individuation difference. The vaccine prepared by directly utilizing the tumor cells has all tumor antigens at the same time, and can be combined with chemotherapy, phototherapy or immunosuppression by loading different hydrophobic drugs into the tumor cells, so that the treatment effect on tumors is further enhanced.
The vaccine preparation process is suitable for preparing clinical tumor vaccine.
Taking tumor tissues of a patient after operation, separating primary tumor cells, amplifying in vitro, and collecting the tumor cells of the patient as raw materials to prepare the patient-specific hydrogel tumor vaccine.
Treatment of tumors includes four aspects of surgery, chemotherapy, radiation therapy, and immunotherapy. The vaccine of the invention can clinically combine these four treatments. The tumor tissue is removed by operation, the in vitro separation and enrichment tumor cells are used for preparing hydrogel tumor vaccines, optimized chemotherapy and radiotherapy schemes are selected, the toxic and side effects on organisms are reduced, and the hydrogel tumor vaccines are used for activating specific immune responses of the organisms and effectively inhibiting recurrence and metastasis of postoperative tumors.
Drawings
FIG. 1 is a transmission electron microscope image of the polypeptide prepared in the first embodiment of the present invention before, during and after the constant temperature water bath. With the extension of the constant temperature water bath time, the microscopic morphology of the polypeptide in the buffer solution gradually grows from nano particles with uniform morphology to form nano fibers, so that hydrogel is formed. The scale bar is 50 nanometers in the figure.
FIG. 2 is a scanning electron microscope image of a hydrogel prepared in accordance with an embodiment of the present invention, the hydrogel having a fibrous network microstructure.
FIG. 3 shows the mechanical properties of the hydrogels prepared in example one of the present invention. The frequency sweep of the rheometer was performed after the polypeptide had formed the hydrogel (4 h), with a strain of 1.0% and a hydrogel concentration of 1.0% by weight. The elastic modulus of the hydrogel was Pa, and the elastic modulus and the viscous modulus did not change much with the change of the scanning frequency, indicating that an elastic network was formed inside the gel.
FIG. 4 is a white light photograph and basic preparation steps at various stages of preparation of hydrogel tumor vaccine according to the third embodiment of the present invention. The concentration of the gel was 10mg/mL and the number of cells was 1X 10 8 And each.
FIG. 5 shows the effect of hydrogel tumor vaccine in accordance with the fourth embodiment of the present invention on inhibiting recurrence of 4T1 tumor. The hydrogel tumor vaccine was effective in inhibiting recurrence of in situ tumors when compared to normal saline and blank hydrogel groups after 27 days of administration. The figures represent that hydrogel tumor vaccines significantly inhibited the growth of 4T1 in situ tumors compared to saline and hydrogel.
FIG. 6 shows the inhibition of 4T1 tumor distal metastasis by the four hydrogel tumor vaccine of the present invention. a.4t1 distal metastasis growth curve over time; b. number of non-metastatic mice changes over time. The hydrogel tumor vaccine was effective in inhibiting distal metastasis of 4T1 tumors when compared to saline and blank hydrogel groups after 27 days of administration. The figures represent that hydrogel tumor vaccines are able to significantly inhibit the appearance and growth of distal metastases compared to normal saline and hydrogels.
Detailed Description
The present invention will be described with reference to the following embodiments, but the present invention is not limited to these embodiments.
The short peptide sequences used in the examples were synthesized by Shanghai Jier Biochemical Co. The photosensitive molecule indocyanine green ICG was purchased from the scientific company of belkyo carboline. The immunomodulator triphenylmethane triisocyanate (JQ 1) was purchased from seleck chemical technologies limited. Foxp3 fixative was purchased from sameiser technology (china) limited. 4T1 breast cancer cells were purchased from the American ATCC cell bank, and RPIM1640 medium and fetal bovine serum for cell culture were purchased from Gibco corporation. In this application, the remaining reagents and solvents were purchased from the national drug group (Shanghai) chemical reagent Co., ltd unless otherwise specified.
Unless otherwise indicated, the equipment and testing methods used are those conventional in the art. Transmission electron micrographs were obtained by Tecnai transmission electron microscopy and scanning electron micrographs were obtained by Nova nano 450 scanning electron microscopy. The rheological properties of the vaccine were measured by a Kinexus rotarheometer.
Balb/c white mice, 4-5 weeks old, were purchased from Shanghai Laek laboratory animal Limited and tumor-bearing mouse models of 4T1 tumors were constructed by inoculating in vitro cultured 4T1 breast cancer cells with a mouse fat pad. The operation of the whole animal experiment strictly follows the relevant regulations of the animal care and use committee of Shanghai pharmaceutical research institute.
EXAMPLE one preparation of the hydrogel carrier of the present invention
Taking the (FMOC) FMOC-KCRGDK polypeptide sequence as an example: the permeable hydrogel carrier prepared by the constant temperature water bath method is used as a carrier of tumor cells, so that the vaccine is endowed with stability and injectability.
The specific operation process comprises the following steps:
5mg of (FMOC) FMOC-KCRGDK polypeptide and 0.92mg of anhydrous sodium carbonate are taken and dissolved together in 0.5mL of PBS buffer (pH 7.4), and the obtained solution is subjected to a constant temperature water bath at 70 ℃ until hydrogel is formed. Before (0 min), during (5 min after the start of the water bath), and after (50 min after the start of the water bath), a proper amount of hydrogel was taken and examined by transmission electron microscopy for the formation process and microscopic morphology of the hydrogel, respectively, as shown in FIG. 1. The specific microscopic space morphology was observed by scanning electron microscopy after hydrogel formation, and the results are shown in FIG. 2. The rheological property of the hydrogel was measured by a rheometer with a proper amount of the prepared hydrogel, and the result is shown in fig. 3.
EXAMPLE two preparation of pretreated tumor cells of the invention
Taking a mouse breast cancer 4T1 cell line as a model as an example: preparing a cell carrier by taking a mouse breast cancer 4T1 cell as a cell model; the photosensitive molecule indocyanine green (ICG) and the immunomodulator JQ1 are taken as model medicines and are loaded into 4T1 cells.
The specific operation process comprises the following steps:
collection of 4T1 tumor cells (1X 10) 7 After being incubated in 1mL of Foxp3 fixative for 30min, the solution is washed 3 times with PBS, and 5mL of methanol solution containing indocyanine green and JQ1 are added to be mixed uniformly, and the mixture is stirred for 4h, wherein the concentration of indocyanine green and JQ1 is 10 mug/mL. After 2h, centrifuging at 2000rpm for 3min to remove free drugs, and washing with PBS for 3 times to obtain 4T1 cells loaded with indocyanine green and JQ 1.
EXAMPLE three preparation of hydrogel tumor vaccine of the invention
The 4T1 cells loaded with indocyanine green and JQ1 obtained in the second embodiment are mixed with the hydrogel carrier obtained in the first embodiment to prepare a hydrogel tumor vaccine, the hydrogel tumor vaccine is inoculated to a Balb/c mouse loaded with 4T1 tumor, then the 4T1 tumor cells are inoculated to the mouse, and the protection effect of the vaccine on the mouse is observed.
The specific operation process comprises the following steps:
1mL of the hydrogel carrier obtained in example one was taken and slowly added to 1X 10 when vortexed 7 4T1 cells loaded with indocyanine green and JQ1 obtained in the second embodiment are fully vortex mixed and then are left to stand, and the hydrogel tumor vaccine can be obtained, and the specific process is shown in figure 4.
Example IV tumor recurrence and metastasis Using the hydrogel tumor vaccine of the invention
18 female Balb/c mice of 8 weeks of age were taken, respectivelyInoculating logarithmic growth 4T1 breast cancer cells on left armpit fat pad, and growing until tumor grows to 500mm 3 Then, most of the tumor tissues were surgically excised and randomly divided into three groups, the first group of saline, the second group of hydrogel carrier prepared in example one, and the third group of hydrogel tumor vaccine prepared in example three, were immunized. After immunization, 1X 10 was taken again 5 The right lower abdomen of the three groups of mice was inoculated with 4T1 tumor cells. The immunization was performed once every 6 days, and 3 times total. Mice were examined every two days for recurrence of left in situ tumor and growth of right distal metastatic tumor.
Experimental results:
anti-tumor recurrence and metastasis experiments find that the hydrogel tumor vaccine obtained in the third embodiment can effectively inhibit recurrence of 4T1 in-situ tumor after operation. 40 days after surgery, in-situ tumors of both groups of mice immunized with normal saline and hydrogel carrier all recurred and grew to 3000cm 3 Left and right. In contrast, mice immunized with the hydrogel tumor vaccine had substantially no recurrence of in-situ tumors, as shown in fig. 5. Meanwhile, the detection result of the right distal metastasis also shows that the occurrence and development of the distal metastasis cannot be inhibited by the immunization of physiological saline and a hydrogel carrier. Whereas hydrogel tumor vaccine immunization was effective in inhibiting the production of distant metastases, the results are shown in figure 6.
SEQUENCE LISTING
<110> Shanghai pharmaceutical institute of China academy of sciences
<120> a method for preparing hydrogel tumor vaccine, hydrogel tumor vaccine prepared therefrom and use thereof
<130> DI18-8049-XC47
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
<213> Artificial
<220>
<221> CHAIN
<222> (1)..(6)
<223> Artificial sequence
<400> 1
Lys Cys Arg Gly Asp Lys
1 5
Claims (8)
1. The preparation method of the hydrogel tumor vaccine is characterized by comprising the following steps:
(1) Tumor cell pretreatment: the tumor cells serving as the carrier are placed in Foxp3 fixing solution for incubation for 10-60min, and the fixed and membrane-broken tumor cells can be obtained; adding a hydrophobic drug and an organic solvent into tumor cells, stirring at room temperature, centrifuging, washing with PBS, and removing free hydrophobic molecules to obtain pretreated tumor cells;
(2) Preparation of hydrogel carrier: dissolving polypeptide and anhydrous sodium carbonate in PBS buffer solution with pH of 7.4, and heating in water bath at 70 ℃ for 50min after full dissolution to obtain a hydrogel carrier, wherein the polypeptide is (FMOC) FMOC-KCRGDK, two amino groups of lysine at the N end of the polypeptide are protected by FMOC, the concentration of the polypeptide is 1-50mg/mL, the mass of the polypeptide is 5mg, and the mass of the anhydrous sodium carbonate is 0.92mg; and
(3) Preparation of hydrogel tumor vaccine: and (3) slowly adding the pretreated tumor cells prepared in the step (1) into the hydrogel carrier prepared in the step (2) in a vortex state until the pretreated tumor cells are uniformly mixed, and standing to obtain the hydrogel tumor vaccine.
2. The method of manufacturing according to claim 1, characterized in that: the ratio of the number of tumor cells in the pretreated tumor cells to the hydrophobic drug is 1×10 7 And (5-200) mug, wherein the volume of the organic solvent is 1-10mL, and the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide and acetonitrile.
3. The method of manufacturing according to claim 1, characterized in that: the number-volume ratio of the tumor cells to the Foxp3 fixative is 1×10 8 Individual cells/(1 to 10) mL.
4. A production method according to any one of claims 1 to 3, characterized in that: the tumor cells are selected from 4T1 breast cancer tumor cells, EMT6 breast cancer tumor cells, MCF-7 breast cancer tumor cells, CT26 colon cancer tumor cells and B16-F10 melanoma tumor cells.
5. A production method according to any one of claims 1 to 3, characterized in that: the tumor cells are operation tumor cut pieces obtained from a tumor patient, primary tumor cells are separated from the operation tumor cut pieces, and the operation tumor cut pieces are cultured in vitro to obtain amplified tumor cells.
6. A production method according to any one of claims 1 to 3, characterized in that: the hydrophobic drug is fluorescent molecule, photosensitive molecule, chemotherapy drug or small molecule immunomodulator.
7. A hydrogel tumor vaccine obtainable by the method of any one of claims 1-3.
8. Use of the hydrogel tumor vaccine of claim 7 in the preparation of a medicament for preventing tumor recurrence and metastasis.
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