CN117024568A - ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof - Google Patents

ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof Download PDF

Info

Publication number
CN117024568A
CN117024568A CN202310811388.2A CN202310811388A CN117024568A CN 117024568 A CN117024568 A CN 117024568A CN 202310811388 A CN202310811388 A CN 202310811388A CN 117024568 A CN117024568 A CN 117024568A
Authority
CN
China
Prior art keywords
nucleic acid
atp
framework nucleic
tumor
stranded dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310811388.2A
Other languages
Chinese (zh)
Inventor
林云锋
张雨欣
战雨汐
毛陈晨
陈野
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan University
Original Assignee
Sichuan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sichuan University filed Critical Sichuan University
Priority to CN202310811388.2A priority Critical patent/CN117024568A/en
Publication of CN117024568A publication Critical patent/CN117024568A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention discloses an ATP response type vaccine functional framework nucleic acid, which is formed by four single-stranded DNA molecules through base complementary pairing; one of the four single-stranded DNA molecules is connected with CpG; the nucleotide sequence of the CpG is shown as SEQ ID NO. 5. The ATP responsive functionalized framework nucleic acid can respond to high-concentration ATP molecules released by tumor tissues after receiving radiotherapy to generate a passive targeting effect, and can be gathered around the tumor tissues after receiving low-dose radiotherapy, so that the ATP responsive functionalized framework nucleic acid plays a role in activating anti-tumor adaptive immunity in a directional manner, improves the treatment efficiency, reduces toxic and side effects brought by systemic administration, and has specific practical popularization and application values.

Description

ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof
Technical Field
The invention particularly relates to an ATP response type vaccine functionalized framework nucleic acid and application thereof.
Background
Ionizing radiation generated by radiotherapy can cause DNA damage of tumor cells and induce the tumor cells to die by immunogenicity, namely, the tumor cells are subjected to external stimulus to die, and meanwhile, the non-immunogenicity is converted into immunogenicity, so that the process of generating anti-tumor immune response by a human body is mediated, a large number of damage related molecules can be generated in the process of generating ICD (tumor cell activation) by the tumor cells, and the innate and acquired immune responses can be finally activated to a certain extent. For example: intracellular ATP molecule release increases the aggregation of antigen presenting cells around tumor tissue, and heat shock proteins (HSP 70, HSP 90), calreticulin released by tumor cells promote phagocytosis of tumor antigens by dendritic cells and present these antigens to cytotoxic T cells. These effects of activating the immune system may enhance the adjuvanticity and antigenicity of the tumor, thereby eliciting an in situ vaccine effect. However, the immune effect is limited because the X-ray irradiation of tumor tissue can also cause the tumor immune microenvironment to be changed into an immunosuppressive microenvironment in the process of inducing adaptive immune response, and the change can cause the infiltration of regulatory T cells to be increased, so that various immunosuppressive phenomena such as infiltration of CTL (CD8+T cells) are reduced. However, the presence of these immunosuppressive factors makes the positive immune activation of radiotherapy insufficient to counteract the negative effects of immunosuppression, and thus the effectiveness of radiotherapy in treating tumors is very limited.
In recent years, more and more nanomaterials have been developed for delivering immunoadjuvants or tumor antigens to activate the anti-tumor immune response of the body. Framework Nucleic Acids (FNA) are considered to be one of the most potential drug delivery vehicles. It is a precise nanostructure that can be constructed to have various functions for a specific target by utilizing the self-assembly and recognition ability of DNA molecules. In particular, tetrahedral framework nucleic acid (tFNA) can efficiently break through the electrostatic barrier at the interface of cell membrane, and can freely enter and exit living cells. The structural characteristics of the nucleic acid molecule enable the tFNA to load various medicaments such as functional nucleic acid sequences (siRNA, antisense oligonucleotide, miRNA and the like) and small molecule chemotherapeutics. While tFNA itself has many unique advantages such as: has good chemical reactivity, multiple modification sites, mechanism stability in physiological environment, dynamic programmability of a framework structure, good biocompatibility and the like.
The professor Li Zigang to the transformation center for the development of biological medicine in the mountain of Shenzhen Bay laboratory/the team of Yin Feng researchers developed a nucleic acid-polypeptide drug based on tFNA for personalized immunotherapy of tumors. Because the drug is a nucleic acid-polypeptide reversible covalent conjugate containing active sulfonium salt center, has no targeting structure, does not have the function of precisely assisting tumor radiotherapy, has limited treatment efficiency, and needs to develop a drug with targeting effect, which can be gathered around tumor tissues after receiving low-dose radiotherapy, and can directionally play the role of activating antitumor adaptive immunity so as to improve the tumor treatment effect.
Disclosure of Invention
In order to solve the problems, the invention provides an ATP responsive vaccine functionalized framework nucleic acid, which is formed by four single-stranded DNA molecules through base complementary pairing;
one of the four single-stranded DNA molecules is connected with CpG;
the nucleotide sequence of the CpG is shown as SEQ ID NO. 5.
Further, one of the three other single stranded DNA molecules except CpG was linked to an OVAp, which is an ovalbumin polypeptide (257-264).
Still further, the OVAp is linked to a single stranded DNA molecule having a 5' -end modified aldehyde group by a Schiff base reaction.
Further, the nucleotide sequence of the single-stranded DNA molecule is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the CpG connected with the single-stranded DNA molecule is shown as SEQ ID NO. 4.
Further, the framework nucleic acid is formed by base complementary pairing of single-stranded DNA molecules with the sequences shown as SEQ ID NO. 1-4, wherein OVAp is connected to the single-stranded DNA molecules with the sequences shown as SEQ ID NO. 2.
Further, the vertices of the tetrahedral framework nucleic acids are blocked by Aapt; the nucleotide sequence of Aaptt is shown as SEQ ID NO. 6.
The invention also provides a preparation method of the ATP-responsive vaccine functionalized framework nucleic acid, which comprises the following steps:
1) Mixing a single-stranded DNA molecule with a nucleotide sequence of which the 5' -end is modified with an aldehyde group as shown in SEQ ID NO.2 with OVAp for Schiff base reaction, purifying by HPLC after the reaction is completed, and collecting a product of a maximum chromatographic peak of a response signal to obtain Op-S2;
2) Mixing single-stranded DNA molecules with sequences shown as SEQ ID NO.1 and SEQ ID NO. 3-4 with Op-S2, adding into a TM buffer solution, maintaining at 95 ℃ for 10min, and cooling at 4 ℃ for 20min to obtain tetrahedral framework nucleic acid;
or: mixing single-stranded DNA molecules with sequences shown as SEQ ID NO. 1-4, adding into TM buffer solution, maintaining at 95 ℃ for 10min, and cooling at 4 ℃ for 20min to obtain tetrahedral framework nucleic acid;
3) And (5) taking tetrahedral framework nucleic acid, and adding Aapts for mixing and incubating to obtain the product.
Further, the conditions for the HPLC purification in step 1) are: chromatographic column: XBridge Shield RP 18, 3.5um 4.6 mM, mobile phase A is 100mM aqueous triethylamine acetate, mobile phase B is acetonitrile, flow rate is 1.5mL/min, wavelength is 260nm, gradient elution procedure is: 0-9 min, mobile phase B is 5-15%, and mobile phase B is kept 95% for 9-11 min.
Further, the final concentration of 4 oligonucleotide chains in the buffer of step 2) is 1000nM; the incubation temperature in the step 3) is 20-30 ℃.
The invention finally provides the application of the ATP-responsive vaccine functionalized framework nucleic acid in preparing medicaments for treating tumors.
Further, the medicament is a medicament for treating melanoma.
According to the ATP responsive vaccine functional framework nucleic acid, the single-stranded DNA molecules connected with CpG and the other three single-stranded DNA molecules are self-assembled to form tFNA carrying an immune adjuvant, and the single-stranded DNA molecules modified by aldehyde groups at the 5' end are mixed with OVAp to perform Schiff base reaction, the OVAp is connected to the single-stranded DNA molecules, the single-stranded DNA molecules connected with CpG and the other two single-stranded DNA molecules are self-assembled to form tFNA carrying an antigen and the immune adjuvant, and meanwhile, an ATP aptamer is used for sealing the CpG structure, so that the ATP responsive vaccine functional framework nucleic acid can respond to high-concentration ATP molecules released after tumor tissues receive radiotherapy, and can generate a passive targeting effect, and can be gathered around tumor tissues after receiving low-dose radiotherapy, thereby directionally playing the role of activating anti-tumor adaptive immunity, improving the treatment efficiency, reducing toxic and side effects brought by systemic drug administration, and being particularly practical and application values.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: synthesis and characterization of ATP-responsive functionalized framework nucleic acids (Op-TGA); (a) OVAp-S2 generating a schematic; (b) HPLC results of OVA-S2; (c) mass spectrometry of OVAp-S2; (d) Op-TGA synthesis scheme; (e) PAGE analysis of Op-TGA; (f) mapping the Op-TGA morphology with TEM; AFM image of Op-TGA.
Fig. 2: controlled release studies of Op-TGA; the method comprises the steps of carrying out a first treatment on the surface of the (a) The fluorescence intensity of Cy5.5-Op-TG was varied with BHQ 3-Aaptt; (b) Schematic representation of ATP-induced dehybridization of double strand formed by BHQ 3-modified Aapt and cy 5.5-modified Op-TG; (c) In response to ATP, op-TG was released from Op-TGA (left to right: lane 1: op-TGA; lane 2: op-TGA+10mM ATP; lane 3: op-TGA+100mM ATP; lane 4: op-TGA+1M ATP; lane 5: op-TGA+0.5mM ATP; lane 6: op-TGA+50mM ATP; lane 7: op-TGA+5M ATP; lane 8: aapt; lane 9: make; d) after addition of 10mM ATP to Op-TGA, the fluorescence intensity of CY5.5 varies with time, (e) the relationship between the radiotherapy dose and the ATP concentration released by tumor cells, (f) the ATP concentration in tumor tissue was observed by in vivo imaging in mice, (h) in vivo distribution of Op-TG and Op-TGA at the organ level, (j) hemolysis of Op-TGA and its components.
Fig. 3: in vitro study of the immune response enhancing effect of Op-TG; schematic of Op-TG enhanced immune response; (b) Aggregation of OVA-TG in DCs by TEM, scale: 1 μm (left), 500nm (right); (c) Representative flow cytometer results show the maturity of DCs at different treatments, as well as the flow cytometer analysis of cd45+cd11c+cd80+cd86+ DCs; (d) Measuring IL-12p70, IL-1 beta and TNF-alpha levels secreted by DCs after different treatments; (e) Representative flow cytometer results showed the efficiency of DC antigen presentation and flow cytometry analysis of the antigen status of DCs presentation (cd45+cd11c+h-2 kb+); immunofluorescence analysis of antigen presented by DCs, scale: 20 μm; (g) DCs stimulate lymphocyte proliferation; (h) DCs from different treatment groups stimulated IFN- γ levels secreted by spleen lymphocytes.
Fig. 4: research on synergistic antitumor effect of ATP-responsive adjuvant-functionalized framework nucleic acid (TGA) and radiotherapy; schematic of experimental design; (b) Individual tumor growth curves for different groups of mice B16-F10 tumors; (c) a statistical tumor growth curve; error bars represent mean ± SEM (n=6); (d) tumor photographs of different groups; (e) tumor weights of different treatment groups; (f) H & E staining (scale: 50 μm); (g) TUNEL staining (scale: 50 μm) of the different treatment groups; (h) IHC staining of CD31 (red) in tumor tissue of different treatment groups, scale: 50 μm.
Fig. 5: research of the immune activation effect of TGA generated in coordination with radiotherapy-immunotherapy; (a) Representative flow cytometer results show the maturity of DCs in lymph nodes under different treatments; (b) Flow cytometry analysis of cd45+cd1c+cd80+cd86+ mature DCs in lymph nodes; (c) Immunofluorescence analysis of DC maturation in lymph nodes (scale: 50 μm); (d) Immunofluorescent staining of Treg cells in tumor tissue (mere), scale: 20 μm; (e) Representative flow cytometer results show that cd3+cd8+ T cells in tumor tissue and flow cytometry analyze cd3+cd8+ T cells in tumor tissue; (f) Measuring IFN-gamma and TNF-alpha levels in the tumor 7 days after the different treatments; (g) Representative flow cytometer results show Tem in peripheral blood and cd3+cd8+cd44+cd62l-Tem analyzed by flow cytometer; (h) Representative flow cytometer results show flow cytometer analysis of Tcm and cd3+cd8+cd44+cd62l+tcm in spleen under different treatments.
Fig. 6: ATP-responsive functionalized framework nucleic acid (Op-TGA) assisted radiation therapy for treatment of melanoma expressing OVA antigen and immune activation assays; schematic of experimental design; (b) statistically tumor growth curves; mean standard deviation ± SEM (n=6); (c) tumor weights of different treatment groups; survival of OVA-B16 tumor mice after different treatments; TUNEL staining of different treatment groups, scale: 50 μm; (f) IHC staining of CD31 (red) in tumor tissue of different treatment groups, scale: 50 μm; (g) Representative flow cytometry results and analysis of cd45+cd11c+cd80+cd86+ mature DCs in lymph nodes; (h) Observing the T cell typing in spleen by immunofluorescence method; (i) Fluorescence images of cd8+ T cells and cd4+ T cells in tumor and tumor margin tissue; (j) Representative flow cytometry results and analysis of cd3+ T cells in tumor tissue, scale: 100 μm; (k) Representative flow cytometry results and analysis of cd3+cd8+ T cells in tumor tissue; (l) Representative flow cytometer results and flow cytometer analyses of cd3+cd8+cd44+cd62l-Tem in peripheral blood; (m) representative flow cytometer results and flow cytometer analyses of cd3+cd8+cd44+cd62l+tcm in spleen; re-challenge tumor growth curves for untreated and rt+ova-TGA groups. Error bars reflect the mean standard deviation (n=6).
Detailed Description
The principles, reagents and equipment used in the specific embodiments of the invention are all known products and are obtained by purchasing commercial products, wherein the nucleotide sequence information involved in the preparation process is as follows:
example 1 preparation and characterization of ATP-responsive functionalized framework nucleic acids (Op-TGA) of the invention
1. Ligation of the backbone chain of tetrahedral framework nucleic acids to antigenic peptides
The framework chain of a tetrahedral framework nucleic acid with the 5 '-end modified aldehyde group (5' -end modified aldehyde group-S2, CHO-S2) and OVAp are covalently connected into Op-S2 through Schiff base reaction, and the specific connection principle is shown in figure 1a;
chemical name of OVAp: ovalbumin polypeptide (257-264), CAS number: 1262751-08-5; canonic SMILES, ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu; structural formula:
the amino group of the OVAp and the modified aldehyde group on the framework chain of the tetrahedral framework nucleic acid (tFNA) are subjected to Schiff base reaction (reaction conditions are that the aldehyde group modified S2 and the OVAp are respectively dissolved by absolute ethyl alcohol, the two are mixed, stirring and heating are carried out at about 50 ℃, a few drops of acetic acid solution are dropwise added as a catalyst, stirring and reacting for 2 hours) are carried out, covalent connection synthesis is carried out, and then the synthesis product Op-S2 is purified by High Performance Liquid Chromatography (HPLC).
The conditions for HPLC purification were: chromatographic column XBridge Shield RP 18 3.5um 4.6*50mm, mobile phase A is 100mM triethylamine acetate aqueous solution, mobile phase B is acetonitrile, the flow rate is 1.5mL/min, the elution method is gradient elution, and the elution conditions are as follows: mobile phase B was maintained from 5% to 15% (9 min), followed by mobile phase B95% for 2min, monitoring the product elution at a wavelength of 260nm, and collecting the effluent of the peak of maximum response signal, as shown in fig. 1B, and determining from fig. 1B that the purified Op-S2 had a purity of 95% or higher.
The relative molecular mass of the purified product is analyzed by a liquid chromatography-mass spectrometer (LC-MS), the result is shown in figure 1c, the molecular mass of the target product is 20586.7Da, the purity reaches 98.07+/-0.3 percent, the molecular weight is consistent with the molecular weight of the designed Op-S2 (theoretical molecular weight is 20586 g/mol), the synthesized product is the expected Op-S2, and the successful connection of the OVAp and the skeleton chain is determined.
2. Synthesis of ATP-responsive functionalized framework nucleic acids (Op-TGA)
Four single strands of S1, op-S2, S3 and CpG-cAapt-S4 are dissolved in a TM buffer (pH 8), the final concentration of the 4 oligonucleotide chains is 1000nM, the system is placed in a PCR instrument and maintained at 95 ℃ for 10min after full mixing, and then cooled at 4 ℃ for 20min, so that tetrahedral framework nucleic acid is self-assembled to form Op-TG carrying CpG and OVAp, and then the Op-TGA (vaccine functionalized framework nucleic acid carrying antigen and immunoadjuvant) is formed by incubating with Aapt (ATP aptamer) at a molar ratio of 2:1 for 2h at room temperature to close the CpG-cAapt arm chain extending from the vertex of the Op-TG. In particular as shown in figure 1d.
The molecular weight sizes of Op-TG, op-TGA, and Op-tFNA were analyzed using 8% polyacrylamide gel electrophoresis (PAGE). Wherein Op-tFNA is a tetrahedral framework nucleic acid obtained by dissolving four single strands of S1, op-S2, S3 and S4 in TM buffer (ph=8) at a final concentration of 1000nM, mixing thoroughly, maintaining the system in PCR instrument at 95 ℃ for 10min, and cooling at 4 ℃ for 20min.
The specific method for analyzing the molecular weight is as follows: electrophoresis was performed at constant pressure of 90V for 100min at room temperature, and the running buffer was TAE. After 15min staining by Gel red, differences in migration rates of the synthesized product and modified single strand in the Gel were observed using a chemiluminescent imager. The result is shown in FIG. 1e. As can be seen from fig. 1 e: the mobility of Op-TGA is slower than Op-TG, which is slower than Op-tFNA. This is consistent with the trend of slower mobility with higher molecular weight, demonstrating successful synthesis of Op-TGA.
3. Morphological characterization of Op-TGA
Characterization of the synthesized Op-TGA morphology using transmission electron microscopy, results in fig. 1f, the tetrahedral structure of the vertex-extended arm chain is observed from the figure and its size is about 15 nm.
Characterization of the synthesized Op-TGA morphology using an atomic force microscope resulted in fig. 1g, from which the tetrahedral structure of the extended arm chain at the vertices was observed and whose size was about 15nm, consistent with transmission electron microscopy results.
4. ATP response verification of Op-TGA
4.1 closing of Aaptt switch
Aaptt (BHQ-3-Aaptt) carrying fluorescent phagocytic group BHQ-3 and Op-TG (CY 5.5-Op-TG) carrying fluorescent group CY5.5 were incubated at a molar ratio of 2:1 at room temperature. Fluorescence intensity of CY5.5 at different time points was recorded using a multifunctional microplate reader after adding the key chain Aapt. The results are shown in FIG. 2a.
Results: the addition of BHQ-3-Aaptt to CY5.5-Op-TG showed a gradual decrease in fluorescence intensity of the reaction system over time, which demonstrated successful closing of the Aaptt switch.
4.2 Aaptt switch on
Op-TGA carrying fluorescent phagocytic group BHQ-3 and fluorescent group CY5.5 and 10mM ATP are mixed in TM buffer, and put into a multifunctional microplate reader for incubation at constant temperature of 37 ℃, and change of fluorescence intensity of system CY5.5 at different time points is observed. The results are shown in FIGS. 2b and d.
Results: when 10mM ATP was added to the Op-TGA, the fluorescence intensity of CY5.5 increased rapidly and became equilibrated after 10min, demonstrating that the Op-TGA was able to successfully release Op-TG by response to ATP.
4.3 Effect of different concentrations of ATP on Op-TGA dissociation
1. Mu.M Op-TGA was incubated with ATP at 0.5mM, 10mM, 50mM, 100mM, 1M, 5M concentrations in TM buffer for 2h (37 ℃). The dissociation of Op-TG from Aaptt in the Op-TGA structure was observed by polyacrylamide gel electrophoresis (PAGE). The result is shown in FIG. 2c.
Results: dissociation of Aapt and Op-TG was observed in the PAGE gel when ATP was added to Op-TGA at different concentrations. The results demonstrate that Op-TG was successfully released in the presence of ATP.
4.4 ATP response of Op-TGA to tumor-bearing model
4.4.1 ATP concentration of tumor cells in vitro
B16-F10 and DC 2.4 cells were cultured on 6-well plates and after cell attachment received X-ray irradiation at different doses (1 Gy, 2Gy, 3Gy, 4 Gy). After 16h the ATP concentration in the supernatant was detected using an enhanced ATP detection kit. The result is shown in FIG. 2e.
Results: the B16-F10 cells release ATP molecules outside the cells in a dose-dependent manner, the higher the dose of irradiation, the more ATP molecules in the cell supernatant.
4.4.2 ATP concentration in tumor tissue in vivo
A3 Gy dose of X-ray radiation was administered locally to tumors of B16-F10 tumor-bearing mice. After 16h, the luciferase and luciferin in the ATP detection kit are mixed to generate a detection working solution, and the detection working solution is injected into tumor tissues. After 2min, tumor-bearing mice were subjected to in vivo imaging using a small animal in vivo imager, and ATP production at tumor lesions was observed. The results are shown in FIG. 2f.
Results: the tumor lesion chemiluminescent signal of the X-ray irradiated mice is significantly enhanced, which indicates that radiotherapy can lead to a significant increase in ATP concentration in tumor tissue.
4.4.3 distribution of op-TGA in tumor-bearing models
The living body imaging observation material is distributed in a tumor-bearing model after radiotherapy; CY5-Op-TG and CY5-Op-TGA (CY 5 fluorophore modified on backbone chain) were injected into tumor-bearing mice at the end of radiotherapy for 16h via tail vein, respectively, and their distribution in mice was observed by in vivo imaging. The results are shown in FIG. 2h and FIG. 2i.
Results: it was observed that Op-TGA was able to aggregate in large amounts around tumor tissue and in small amounts in the kidneys and liver, confirming that Op-TGA had ATP response to tumors, whereas Op-TG without ATP response was mainly aggregated in the kidneys and liver of mice.
4.7 hemolysis
After Op-TGA and its components were contacted with C57BL/J mouse blood using quantitative colorimetry, the total hemoglobin in mouse whole blood and the free hemoglobin released into plasma were measured for their haemolysis coefficients to assess the in vitro acute haemolytic properties of the nanoparticles. The experimental method refers to medical nanomaterial detection and evaluation standards issued by national center for nanometer, and comprises the following specific steps:
(1) The hemoglobin standard is dissolved in DPBS buffer solution to prepare 1.6mg/mL hemoglobin standard solution, and the solution is kept at 4 ℃ for standby.
(2) A solution of cyanmethemoglobin was prepared using 1L of Drabkin's reagent with 500. Mu.L of 30% Brij 35 reagent.
(3) The hemoglobin standard solution and the cyanmethemoglobin solution were mixed in different proportions to prepare a series of standard solutions with concentration gradients, the standard solution concentrations are shown in table 1, and absorbance of each standard solution at 540nm was measured using an enzyme-labeled instrument to calculate hemoglobin concentration and draw a standard curve.
Table 1 standard liquid preparation
(4) The formulation of the quality control solution is shown in table 2.
TABLE 2 preparation of quality control solutions
(5) The peripheral blood of the C57 mouse was collected with an anticoagulation blood collection tube, and a part of the obtained blood was centrifuged (800 g, centrifugation 15 min) to collect the supernatant, and reacted with a cyanidation methemoglobin solution at 1:1, and incubating for 20min with shaking for measuring plasma free hemoglobin.
(6) The whole blood obtained was mixed with a solution of methemoglobin cyanide at 250:1, and incubating for 20min with shaking for measuring whole blood hemoglobin.
(7) Preparation of samples: the synthesized Op-TGA (1. Mu.M) and the components with equal concentration were mixed with whole blood of mice, incubated in a water bath at 37℃for 3h and shaken once for 30min. Followed by centrifugation (800 g, centrifugation 15 min). And observing the hemolysis, and then incubating the centrifugal supernatant and the cyanide methemoglobin solution for 20min by mixing and shaking in an equal volume ratio. And the samples (4) and (5) were placed in an enzyme-labeled instrument to measure absorbance at 540nm, while positive (10 mg/mL poly L-lysine solution) and negative control (40% polyethylene glycol aqueous solution) were set.
(8) The hemoglobin content of each sample was calculated from the standard curve and the haemolysis coefficient of Op-TGA and its components was calculated.
Results: as can be seen, little hemolysis was observed after the Op-TGA and its components, respectively, were contacted with the blood of the mice.
Example 2 preparation and characterization of ATP-responsive functionalized framework nucleic acids (TGAs) of the invention
1. Preparation of TGA
Four single strands of S1, S2, S3 and CpG-cAapt-S4 were dissolved in a TM buffer (pH 8) and the final concentration of the 4 oligonucleotide strands was 1000nM, after thorough mixing, the system was placed in a PCR apparatus at 95℃for 10min, then cooled at 4℃for 20min to obtain a tetrahedral framework nucleic acid carrying CpG, and then incubated at room temperature for 2h using Aapt with it in a molar ratio of 2:1 to block the CpG-cAapt arm strand extending from the apex of the tetrahedral framework nucleic acid, thus forming TGA (vaccine functionalized framework nucleic acid carrying only immune adjuvants).
2. Morphological characterization of TGA
The resultant TGA morphology was characterized using transmission electron microscopy and atomic force microscopy, and the tetrahedral structure of the vertex-extended arm chain was observed from the morphology and AFM images and was comparable in size to Op-TGA in example 1.
The beneficial effects of Op-TG, op-TGA, TGA prepared in examples 1 and 2 are further illustrated by the following test examples.
Experimental example 1 study of the mechanism of action of Op-TG of the present invention
The function of functionalized framework nucleic acid (Op-TG) on Dendritic Cells (DCs) derived from mouse bone marrow was investigated at the cellular level by immunocolloidal gold techniques, flow cytometry, immunofluorescence assays, see in particular fig. 3a.
1. OVAp tracer
The immune colloidal gold technique traced the OVAp in ultra-thin sections of BMDCs. DCs were treated with OVAp, op-SG (double strand formed by Op-S2 and CpG-cAapt-S4 without tFNA structure), op-TG, respectively, and incubated for 12h. The supernatant was removed, washed 2 times with PBS, scraped cells were collected in a 5mL centrifuge tube and centrifuged at 1200rpm for 10min. After rewarming the electron microscope fixing solution, the cells were resuspended and fixed for 2min. Transfer to a 1.5ml centrifuge tube, centrifuge at 2000rpm for 10min, wash with PBS 2 times (1000 rpm,5 min). The obtained cells were further fixed, dehydrated, impregnated and embedded to prepare ultra-thin sections of cells. And then, the prepared cell ultrathin section is marked by sequentially using an Anti-Ova primary antibody and a 10nm immune colloidal gold coupling secondary antibody, and the internalization condition and the positioning of the material are analyzed by using a transmission electron microscope. The results are shown in FIG. 3b.
Results: it was observed that Op-TG was largely phagocytized by BMDCs, and that the phagocytized Op-TG was mainly accumulated in vesicles and lysosomes (unilamellar vesicles) inside DCs, which are necessary processes for the DCs to present exogenous antigens, and that the acidic environment in lysosomes could promote dissociation of OVAp from tFNAs, compared to free OVAp and Op-SG without tFNAs.
3. Surface costimulatory molecule expression of DCs
Flow cytometry was used to analyze the expression of costimulatory molecules on the surface of DCs. BMDCs were treated with 250nM OVAP, op-SG (double strand formed by Op-S2 and CpG-cAapt-S4 without tFNA structure), op-TG, respectively, while negative control groups were set and incubated for 24h.
Preparation of mouse bone marrow derived dendritic cells (BMDCs)
(1) The culture medium is prepared for standby, and the culture medium is RPMI 1640 complete culture medium containing 20ng/mL Granulocyte-macrophage colony stimulating factor (Granulocyte-macrophage colony stimulating factor, GM-CSF) and 10ng/mL Interleukin-4 (Interrukin-4, IL-4).
(2) The cervical dislocation of the male C57BL/J mice with age of 6-8 weeks after anesthesia is killed, the mice are soaked in 70% ethanol for 3min, the tibia-fibula and the femur are taken out by using a surgical instrument after high-temperature high-pressure sterilization, and the mice are washed by 70% ethanol and then placed in a culture dish with PBS.
(3) Tissue was removed from the dish as much as possible and the tibia and femur were separated, and an incision was made (excess resections were not made) with surgical scissors at each end of the tibia and femur. Bone marrow was rapidly flushed from an incision in one end of the leg bone into a 15mL centrifuge tube containing PBS using a 1mL syringe containing PBS.
(4) The collected bone marrow cells were filtered through a 40 μm cell sieve and centrifuged at 1500rpm for 5min. The supernatant was discarded, 1mL of red blood cell lysate was added for resuspension, 9mL of PBS was added after 3min of lysis, and centrifugation was performed at 1500rpm for 5min. The supernatant was discarded and washed 1 time with PBS.
(5) The supernatant was discarded and the pre-formulated medium was added for resuspension. 10. Mu.L of the cell suspension was mixed with 40. Mu.L of trypan blue and the viable cell count was counted using a cytometer.
(6) Cells were placed at 2106 cells/well in 6-well plates and placed in a cell incubator (37 ℃,5% co 2). After 24 hours, the liquid is completely changed, and then the liquid is changed every two days. On day seven of culture, loosely adhered cells were collected, most of which were immature DCs. )
The test cells were collected in flow tubes, resuspended in 100 μ L Staining buffer after one wash with PBS and blocked for 30min using blocking antibodies. After washing 2 times with a starting buffer, 100. Mu. L Staining buffer was added for resuspension, 1. Mu.L of the corresponding detection antibody was added sequentially, staining was performed by standard cell Staining protocols, and the corresponding FMO groups were set. The immune marker of bone marrow-derived dendritic cell is CD45 + CD11c + CD80 + CD86 + . The fluorescent labels used were as follows: CD45-APC/CY7, CD11c-APC, CD80-PE, CD86-BV421. The result is shown in FIG. 3c.
Results: the effect of Op-TG on promoting CD80 and D86 expression is most remarkable, the effect of Op-SG is not obvious, in addition, the Op-SG without tFNas structure has some effect on the maturation of DCs, but is obviously lower than that of Op-TG, and free OVAp has no obvious stimulation effect on the maturation of DCs, which further explains that the effect of Op-TG on promoting the maturation of DCs is enhanced by improving the phagocytosis of DCs on CpG.
4. Secretion of cytokines
ELISA detects cytokine secretion. BMDCs were treated with OVAp, op-SG, op-TG, respectively, while negative controls were set up and incubated for 24h. Cell supernatants were collected. And enzyme-linked immunosorbent assayDetection of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-12 using an accessory assay (ELISA) kit p70 (IL-12p 70 ) Is a secretion situation of (a). The result is shown in FIG. 3d.
Results: op-TG is able to significantly promote the secretion of large amounts of IL-12P70, IL-1 β and TNF- α by BMDCs.
5. Presentation efficiency of tFNAs-loaded OVAp
Flow cytometry was used to examine the efficiency of presentation of tFNAs loaded with OVAp, BMDCs were treated with Op-SG, op-TG, respectively, and incubated for 36h. Collecting cells to be tested, wherein the staining method comprises the following steps: the test cells were collected in flow tubes, resuspended in 100 μ L Staining buffer after one wash with PBS and blocked for 30min using blocking antibodies. After washing 2 times with a starting buffer, 100. Mu. L Staining buffer was added for resuspension, 1. Mu.L of the corresponding detection antibody was added sequentially, staining was performed by standard cell Staining protocols, and the corresponding FMO groups were set. The cell marker was CD45 + CD11c + H-2Kb + . The fluorescent labels used were as follows: anti-mouse-CD45-APC/CY7, anti-mouse-CD11c-PE/CY7, anti-mouse-H-2Kb-APC. The result is shown in FIG. 3e.
Results: the highest antigen presenting efficiency of the Op-TG treatment group is obviously higher than that of the Op-SG.
6. Effect of OVAp on DCs
Immunofluorescence was used to examine whether the delivery platform was capable of presenting the carried OVAps to the surface of DCs, BMDCs were treated with Op-TG or Op-SG (control) for 36H, stained with Anti-mouse-H-2kb-FITC, DAPI, and the expression of OVAps on the surface of BMDCs was observed using a Laser Scanning Confocal Microscope (LSCM). The results are shown in FIG. 3f.
Results: the expression of H-2 kb-SINFETL (green fluorescence) was highest in Op-TG-treated DCs. The H-2 kb-SINFETL expression was significantly lower in the Op-SG treated group than in the Op-TG.
7. Influence of Op-TG on lymphocyte proliferation
The CCK-8 method was used to determine the proliferation rate of lymphocytes and to investigate the ability of Op-TG-treated BMDCs to promote lymphocyte proliferation. Mouse spleen lymphocytes were used as effector cells, and BMDCs treated with OVAp, op-SG and Op-TG for 24 hours were co-cultured with spleen lymphocytes at different ratios for 48 hours, respectively, to determine whether the BMDCs were able to promote proliferation of spleen lymphocytes. The results are shown in FIG. 3g.
Results: DCs from Op-TG treated groups were able to significantly promote spleen lymphocyte proliferation and were superior to DCs from OVAp and Op-SG treated groups. When the ratio of BMDCs to spleen lymphocytes was 1: at 100, the proliferation rate of spleen lymphocytes in the Op-TG treatment group reaches 62.1+ -3.3%.
8. Influence of Op-TG on spleen lymphocytes
BMDCs treated for 24h in the different treatment groups were incubated with mouse spleen lymphocytes while a blank control group was set, incubated for 24h, cell supernatants were collected and assayed for secretion of interferon gamma (IFN- γ) using an ELISA kit. The results are shown in FIG. 3h.
Results: DCs from Op-TG treated groups were able to stimulate spleen lymphocytes to secrete higher levels of IFN- γ, whereas OVAp was far less potent than Op-TG groups.
Test example 2 efficacy study of TGA of the invention
And (3) establishing a B16-F10 mouse melanoma animal model, and exploring the effect of the TGA amplification radiotherapy induced anti-tumor immune response of the ATP response.
1. Inhibition of melanoma by TGA
B16-F10 mouse melanoma animal model is established, male C57BL/6 mice with the age of 6-8 weeks are selected, and the back hair of the mice is shaved. B16-F10 melanoma cells (1X 10) 6 And C57BL/6 mice were inoculated subcutaneously on the backs. Tumor growth was observed every 2 days after inoculation. After the tumor grows to about 1000mm3, the mice with the tumor volume close to that of the mice are screened for subsequent experiments. The tumor-bearing mice obtained above were randomly divided into 6 groups of 12 animals each, and the grouping conditions are shown in Table 3.
Table 3: grouping of anti-tumor treatment experiments
Tumor-bearing mice of groups 4, 5, and 6 were treated with Radiation (RT) at a dose of 3Gy on days 1, 4, 7, and 10, respectively. All treatment groups were dosed equally (1 μm) by tail vein injection on day 2, day 5, day 8, and day 11, while tumor volumes and body weights of mice were measured to draw tumor growth curves. Finally, mice were sacrificed on day 15, tumor tissues and organs were obtained for subsequent study.
The results are shown in FIGS. 4a-e, from which: compared with the Saline group, the SGA group which is not subjected to radiotherapy hardly shows the effect of inhibiting the growth of tumors, while the treatment group which is subjected to pure radiotherapy and pure TGA only shows partial tumor inhibition effect, and the growth of melanoma is obviously inhibited when the radiotherapy is combined with the TGA. And the quality measurement of the isolated melanoma tissue on the 15 th day further proves that the combined use of radiotherapy and TGA has the most obvious effect of inhibiting the melanoma of mice.
2. Effect of TGA on tumor tissue
2.1H & E staining of tumor tissue
After the sample is obtained and fixed, paraffin embedding is carried out, and paraffin sections are manufactured. Dewaxing paraffin sections, soaking the paraffin sections in hematoxylin dye liquor for 10min, and flushing the paraffin sections with running water to remove the dye liquor. It was then immersed in a saturated aqueous solution of lithium carbonate for 30s and rinsed off with running water. Then the mixture is dyed for 2min by using eosin solution and washed by running water. Finally, gradient ethanol from low to high is used for dehydration. Finally, xylene is used for transparency, and resin sealing sheets are used. And (5) observing and drawing by a machine.
The results are shown in FIG. 4f, from which it can be seen that: h & E staining results of tumor tissue sections showed that tumor necrosis area was further enlarged when radiotherapy was used in combination with TGA compared to radiotherapy or TGA treatment alone.
2.2TUNEL staining analysis of apoptosis in tumor tissues
The tumor tissues of each experimental group are stained by using a terminal deoxynucleotidyl transferase labeling analysis kit, the apoptosis condition of tumor cells is observed, and a fluorescence image is shot by adopting a forward fluorescence microscope.
The results are shown in FIG. 4g, from which: the proportion of apoptotic cells (green fluorescence) in tumor tissue in the rt+tga group was significantly higher than in the other treatment groups.
2.3 CD31 immunohistochemical analysis of tumor tissue was used to assess tumor angiogenesis and thus determine malignancy of tumors.
Tissue sections were stained with anti-mouse CD31 primary antibody followed by incubation with a secondary antibody coupled to 3-amino-9-ethylcarbazole (AEC).
The results are shown in FIG. 4h, which shows the results: the significant reduction of CD31 positive areas in tumor tissue in the rt+tga group, i.e. a significant reduction of angiogenesis in the tumor, suggests the ability of radiation therapy in combination with TGA to inhibit melanoma invasion and metastasis.
Test example 3 efficacy study of the Op-TGA of the invention (1)
A B16 tumor-bearing murine model was established and the treatment was administered in groups according to the same method as test example 2, and the effect of ATP-responsive Op-TGA amplified radiation therapy-induced antitumor immune responses was investigated.
(1) Assessing maturation of DCs in vivo using flow cytometry
B16 tumor-bearing mice 7 days after treatment were sacrificed and Tumor Draining Lymph Nodes (TDLNs) were collected for analysis of DCs function. The isotype control was set with corresponding fluorescence using staining with anti-mouse APC/CY7-CD45 antibody, anti-mouse PE/CY7-CD11c antibody, anti-mouse BV421-CD86 antibody, anti-mouse PE-CD80 antibody, and DCs cells were CD45+CD11c+CD80+CD86+. See in particular fig. 5a and b:
results: tumor-bearing mice treated with TGA in combination with RT had significantly higher (32.6%) rates of mature DCs in TDLNs (CD45+CD11c+CD80+CD86+) than either RT or TGA alone. The maturation rate of DCs in TDLNs was also significantly increased in tumor-bearing mice in TGA group compared to Saline or SGA group, demonstrating the effectiveness of the TGA delivery platform, consistent with the results of in vitro experiments.
(2) Multiplex immunofluorescent staining assay to assess maturation of DCs in vivo
B16 tumor-bearing mice 7 days after treatment were sacrificed and tumor draining lymph nodes were collected. Labeling was performed using CD11c, CD80c, CD86 antibodies. See in particular FIG. 5c
Results: the proportion of the three-color fluorescent overlap in the TDLNs of the RT+TGA group was highest and significantly higher than in the RT group. TGA alone, the proportion of mature DCs in TDLNs was also increased.
(3) Multiple immunofluorescence analysis regulatory T cells (Tregs), the presence of which can suppress the anti-tumor immune response of the body and promote the occurrence and development of tumors. We therefore analyzed the infiltration of Tregs in tumor tissue. The results are shown in FIG. 5d, from which it can be seen that: when TGA is used in combination with radiotherapy, the infiltrated Tregs in tumor tissue is significantly reduced.
(4) Flow cytometry was used to analyze the proportion of cd8+ T cells in Tumor Infiltrating Lymphocytes (TILs), TILs in each group of tumor tissues were extracted by Percoll gradient separation, and anti-mouse CD3 antibody, anti-mouse CD4 antibody, anti-mouse CD8 antibody were added sequentially, followed by staining. Corresponding isotype controls and FMO controls were set simultaneously. Fig. 5e:
results: the proportion of cd8+ T cells in TILs increased only slightly after RT alone, whereas the proportion of cd8+ T cells increased significantly when TGA alone or TGA in combination with radiotherapy.
(5) Tumor-associated cytokine secretion was detected by ELISA by preparing homogenates of tumor tissues from different treatment groups. The result is shown in FIG. 5f.
Results: the levels of TNF- α in tumors were significantly higher when TGA was used in combination with radiation therapy than when either radiation therapy alone or TGA therapy was used. Meanwhile, the combined treatment strategy of TGA and radiotherapy also obviously improves the IFN-gamma level in tumor tissue homogenate.
(6) Tumor-bearing mice were analyzed for memory T cells using flow cytometry, and 7 days after treatment, tumor-bearing mice were analyzed for effector memory T cells (Tem) in peripheral blood and central memory T cells in spleen after treatment was completed. The results are shown in FIG. 5g and FIG. 5h.
Results: after combination therapy with TGA and radiotherapy, the proportion of Tem in the peripheral blood of mice was significantly higher than that of Tcm in the spleen for the experimental group treated with TGA alone.
Test example 4 efficacy study of the Op-TGA of the invention (2)
1. In vivo therapeutic measurement of ATP-responsive vaccine functionalized framework nucleic acid in conjunction with radiation therapy
OVA-B16-F10 cell lines specifically expressing OVA antigen were selected as cell models. Male C57BL/6J mice aged 6-8 weeks were inoculated subcutaneously (1X 10) 6 And/or just). Tumor growth was observed every 2 days after inoculation. To the extent that the tumor grows to about 1000mm 3 About, mice with closely tumor volumes were screened for subsequent experiments. The randomization was divided into 4 groups: (1) saline; (2) RT; (3) rt+ovap; (4) rt+op+tga; (5) rt+op-TGA. And carrying out local X-ray irradiation radiotherapy on the tumors of the other 4 groups except the (1) group on the 1 st day, the 4 th day, the 7 th day and the 10 th day respectively, wherein the radiotherapy dosage is 3Gy, and the total irradiation dosage is 12Gy after four times of radiotherapy. The corresponding drug was then injected into the bodies of all the group tumor-bearing mice by tail vein the following day after the end of each radiotherapy. See fig. 6a-d for specific results:
results: during the treatment period, tumor volumes were measured and growth curves for each group of tumors were plotted. The results are shown in figure 6b, where there was no statistical difference in tumor growth from the radiation treatment alone when the free OVAp was used in combination with radiation. The OVAp is covalently connected with TGA and combined with radiotherapy, so that the growth of melanoma is obviously inhibited, and the inhibition rate reaches 92.8%. The tumor tissue of the mice after the treatment is obtained and the quality is measured, and the result is consistent with the tumor volume. At the same time, we also recorded the survival of tumor-bearing mice during treatment, and Op-TGA could significantly improve survival of mice receiving repeated radiation therapy during treatment.
2. Tumor tissue observation
Tumor tissues of each experimental group were stained with TUNEL staining kit and Anti-CD31 antibody, and apoptosis of tumor cells and generation of tumor blood vessels were observed, respectively.
The specific results are shown in FIG. 6e. The results show that: the cells in the tumor tissue of the RT+Op-TGA group were extensively apoptotic (green fluorescence), while the proportion of apoptotic cells was not significantly changed in the RT+Op+TGA group compared to the RT group. The results of CD31 staining showed (FIG. 30 c) that the platelet-endothelial cell adhesion molecule CD31 was highly expressed in Saline, RT and RT+OVAp groups, suggesting that there was more angiogenesis in the tumor tissue and thus a higher malignancy of the tumor, while CD31 expression in RT+OVAp+TGA and RT+Op-TGA groups was significantly reduced, indicating a lower malignancy of the tumor.
3. Treatment of maturation of DCs in early TDLNs
Maturation of DCs in early TDLNs was analyzed using flow cytometry. The results are shown in FIG. 6g. The results show that: tumor-bearing mice treated with op+tga in combination with radiotherapy had significantly higher proportion of mature DCs in TDLNs (cd45+cd11c+cd80+cd86+) (44.3%, statistical result). And is significantly higher than the radiation therapy group. There was no significant increase in the maturation rate of DCs in tumor-bearing murine TDLNs in the rt+ovap group compared to the radiation-only group. Whereas the proportion of mature DCs was significantly increased in the rt+op-TGA group compared to the rt+ovap+tga group, demonstrating that carrying OVAp in a covalently linked manner could achieve a stronger immune response, demonstrating the effectiveness of vaccine functionalization Op-TGA.
4. Post-treatment tumor tissue condition
4.1 lymphocytes
Multiplex immunofluorescence analysis the spleen and tumor tissues of the mice after the end of treatment were observed for lymphocytes infiltrating around the tumor tissues, and for T lymphocytes infiltrating in the tissues, respectively. The specific results are shown in FIG. 6h and FIG. 6i.
Results: FIG. 6h shows that the RT+Op-TGA group induced a large number of CTLs in the spleen (green fluorescence: CD8+ T cells).
Fig. 6i shows: the RT+Op-TGA group induced more T lymphocyte infiltration around tumor tissue and was dominated by cytotoxic T cells (green fluorescence: CD8+ T cells).
4.2TILs
Flow cytometry analyzed the proportion of TILs in tumor tissue, the results are shown in fig. 6j and k; the results show that: after treatment with rt+op-TGA, more TILs were present in tumor tissue. And the proportion of CD3+CD8+T cells in TILs is significantly increased.
4.3 peripheral blood and spleen
Peripheral blood and spleen were collected from mice of different treatment groups after the end of treatment. The ratio of Tem (cd3+cd8+cd62l-cd44+) in the peripheral blood of mice to Tc (cd3+cd8+cd62l+cd44+) in the spleen tissue of mice was identified using flow cytometry. The results are shown in FIGS. 6l and m.
The results show that: the proportion of Tem in the peripheral blood and the proportion of Tcm in the spleen of mice after RT+Op-TGA treatment are both significantly higher than those of RT and RT+OVAp groups and also superior to those of RT+OVAp+TGA groups, which indicates that Op-TGA combined with radiotherapy produces a strong immune memory effect.
5. Tumor volume after treatment
Four times of Op-TGA and radiotherapy combined treatment of OVA-B16-F10 tumor-bearing mice, and after 20 days of treatment, eight mice were selected to be inoculated with 1×10 tumor on opposite sides of in situ tumor 6 And OVA-B16-F10 cells. Tumor volumes were measured every two days and tumor growth curves were drawn. Healthy mice of the same week-old and sex were set up and vaccinated with an equal amount of OVA-B16-F10 cells as controls. The result is shown in FIG. 6n.
The results show that: no tumor regrowth was observed in the Op-TGA and RT combination mice over the 20 day observation period, whereas tumor growth was rapid following inoculation of untreated healthy mice.

Claims (10)

1. An ATP-responsive vaccine functionalized framework nucleic acid, characterized in that: the framework nucleic acid is formed by four single-stranded DNA molecules through base complementation pairing;
one of the four single-stranded DNA molecules is connected with CpG;
the nucleotide sequence of the CpG is shown as SEQ ID NO. 5.
2. The ATP-responsive vaccine functionalized framework nucleic acid of claim 1, wherein: one of the three other single stranded DNA molecules except CpG was linked to an OVAp, an ovalbumin polypeptide (257-264).
3. The ATP-responsive vaccine functionalized framework nucleic acid of claim 2, characterized in that: the OVAp is linked to a single-stranded DNA molecule having a 5' -end modified aldehyde group by a Schiff base reaction.
4. The ATP-responsive vaccine functionalized framework nucleic acid of claim 3, wherein: the nucleotide sequence of the single-stranded DNA molecule is shown as SEQ ID NO. 2.
5. The ATP-responsive vaccine functionalized framework nucleic acid of claim 1, wherein: the nucleotide sequence of the CpG connected with the single-stranded DNA molecule is shown as SEQ ID NO. 4.
6. The ATP-responsive vaccine functionalized framework nucleic acid of any one of claims 1-5, wherein: the framework nucleic acid is tetrahedral framework nucleic acid formed by base complementary pairing of single-stranded DNA molecules with the sequences shown as SEQ ID NO. 1-4, wherein OVAp is connected to the single-stranded DNA molecules with the sequences shown as SEQ ID NO. 2.
7. The ATP-responsive vaccine functionalized framework nucleic acid of claim 6, wherein: the vertices of the tetrahedral framework nucleic acids are blocked by Aapt; the nucleotide sequence of Aaptt is shown as SEQ ID NO. 6.
8. A method for preparing the ATP-responsive vaccine functionalized framework nucleic acid of any one of claims 1 to 7, characterized in that: it comprises the following steps:
1) Mixing a single-stranded DNA molecule with a nucleotide sequence of which the 5' -end is modified with an aldehyde group as shown in SEQ ID NO.2 with OVAp for Schiff base reaction, purifying by HPLC after the reaction is completed, and collecting a product of a maximum chromatographic peak of a response signal to obtain Op-S2;
2) Mixing single-stranded DNA molecules with sequences shown as SEQ ID NO.1 and SEQ ID NO. 3-4 with Op-S2, adding into a TM buffer solution, maintaining at 95 ℃ for 10min, and cooling at 4 ℃ for 20min to obtain tetrahedral framework nucleic acid;
or: mixing single-stranded DNA molecules with sequences shown as SEQ ID NO. 1-4, adding into TM buffer solution, maintaining at 95 ℃ for 10min, and cooling at 4 ℃ for 20min to obtain tetrahedral framework nucleic acid;
3) And (5) taking tetrahedral framework nucleic acid, and adding Aapts for mixing and incubating to obtain the product.
9. The method of manufacturing according to claim 8, wherein: the conditions for the HPLC purification in step 1) are: chromatographic column: XBridge Shield RP 18, 3.5um 4.6 mM, mobile phase A is 100mM aqueous triethylamine acetate, mobile phase B is acetonitrile, flow rate is 1.5mL/min, wavelength is 260nm, gradient elution procedure is: 0-9 min, mobile phase B is 5-15%, and mobile phase B is kept 95% for 9-11 min.
And/or: the final concentrations of the 4 oligonucleotide chains in the solution of step 2) are all 1000nM;
and/or: the incubation temperature in the step 3) is 20-30 ℃.
10. Use of the ATP-responsive vaccine functionalized framework nucleic acid of any one of claims 1 to 6 for the manufacture of a medicament for the treatment of a tumor, characterized in that: the medicine is used for treating melanoma.
CN202310811388.2A 2023-07-03 2023-07-03 ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof Pending CN117024568A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310811388.2A CN117024568A (en) 2023-07-03 2023-07-03 ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310811388.2A CN117024568A (en) 2023-07-03 2023-07-03 ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof

Publications (1)

Publication Number Publication Date
CN117024568A true CN117024568A (en) 2023-11-10

Family

ID=88625197

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310811388.2A Pending CN117024568A (en) 2023-07-03 2023-07-03 ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof

Country Status (1)

Country Link
CN (1) CN117024568A (en)

Similar Documents

Publication Publication Date Title
CN108992666B (en) Cationic phospholipid-polymer hybrid nanoparticle vaccine adjuvant of targeting co-carried antigen and TLR agonist, preparation method and application
Shi et al. Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine
Li et al. Dendritic cell-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites elicits enhanced anti-cancer immune response in glioblastoma
Zhang et al. Tumor microenvironment responsive FePt/MoS 2 nanocomposites with chemotherapy and photothermal therapy for enhancing cancer immunotherapy
CN110124018B (en) Calcium phosphate-lipid nano vaccine simulating necrotic tumor cells and application thereof
CN110846281B (en) Anti-tumor vaccine based on exosomes
CN108938568B (en) Phospholipid hybrid polymer vesicle (DCs) vaccine based on co-entrapped antigen and double immune agonists as well as preparation method and application of DCs vaccine
CN108969771B (en) Mannose-modified co-carried antigen and double immune agonist phospholipid hybrid polymer vesicle and preparation method and application thereof
Yang et al. Preparation and antitumor effects of nanovaccines with MAGE-3 peptides in transplanted gastric cancer in mice
CN109998998A (en) A kind of nano vaccine and preparation method thereof
Teng et al. Bi-functional gold nanocages enhance specific immunological responses of foot-and-mouth disease virus-like particles vaccine as a carrier and adjuvant
Bhargava et al. Comparative assessment of lipid based nano-carrier systems for dendritic cell based targeting of tumor re-initiating cells in gynecological cancers
KR20160132044A (en) Vaccine delivery systems using yeast cell wall particles
CN112089834B (en) Preparation and application of pachyman nano adjuvant based on graphene oxide and adjuvant/antigen co-delivery vaccine
Zuo et al. Macro-microporous ZIF-8 MOF complexed with lysosomal pH-adjusting hexadecylsulfonylfluoride as tumor vaccine delivery systems for improving anti-tumor cellular immunity
CN113332452A (en) Co-loaded immune adjuvant and green indocyanine polymer vesicle with triple pH/reduction/temperature stimulation responses, and preparation method and application thereof
CN111973741B (en) preparation method of pH response type therapeutic vaccine based on mesoporous silicon nanoparticles co-entrapped with antigen and photosensitizer
CN117024568A (en) ATP (adenosine triphosphate) response type vaccine functional framework nucleic acid and application thereof
CN114225029B (en) Nanoparticle with sound-sensitive response and application thereof
CN116162138A (en) Dendritic polypeptides and uses thereof
CN110101853B (en) Dandelion type heterogeneous nano vesicle and application thereof
CN113384689B (en) Boron alkene nanometer vaccine and preparation method and application thereof
CN114177281B (en) Nano vaccine and preparation method and application thereof
CN111407897B (en) Avidin nanoparticle based on co-combination of fluorescent dye and Coomassie brilliant blue, preparation method thereof and application thereof in construction of tumor vaccine
CN111763246B (en) Pharmaceutical composition for preventing and/or treating cervical cancer and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination