CN117004609A - siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof - Google Patents

siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof Download PDF

Info

Publication number
CN117004609A
CN117004609A CN202310979557.3A CN202310979557A CN117004609A CN 117004609 A CN117004609 A CN 117004609A CN 202310979557 A CN202310979557 A CN 202310979557A CN 117004609 A CN117004609 A CN 117004609A
Authority
CN
China
Prior art keywords
sirna
polypeptide
tumor
beta
nanoparticle
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
CN202310979557.3A
Other languages
Chinese (zh)
Inventor
杨莉
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 CN202310979557.3A priority Critical patent/CN117004609A/en
Publication of CN117004609A publication Critical patent/CN117004609A/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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Nanotechnology (AREA)
  • Endocrinology (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to the field of biological medicine, and mainly relates to siRNA for inhibiting TGF beta gene expression, nanoparticles loaded with siRNA for inhibiting TGF beta gene expression and application of the nanoparticles in anti-tumor treatment. The technical scheme for solving the technical problem is to provide an siRNA. The siRNA can inhibit TGF beta expression with high efficiency and further inhibit tumor proliferation. The TGF beta siRNA provided by the invention can efficiently inhibit the expression of TGF beta genes, the mRNA level can be regulated by more than 70%, the inhibition rate of protein expression reaches 90%, and simultaneously, the TGF beta siRNA can effectively inhibit tumor proliferation. The DP7-C/si TGF-beta complex can effectively treat tumors such as colon cancer and the like, and has good application prospect.

Description

siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof
Technical Field
The invention belongs to the field of gene therapy, and mainly relates to an siRNA drug for inhibiting TGF beta gene expression and nanoparticles for delivering siRNA for inhibiting TGF beta gene expression.
Background
Cancer research has focused mainly on malignant cells and related gene regulation over the past few decades, and recently, the concept of Tumor Microenvironment (TME) has been widely accepted. The dynamic interactions between tumors and their microenvironment are thought to have a significant impact on carcinogenesis, as well as largely affecting the progress of tumorigenesis and development. Besides tumor cells, TMEs are composed of a variety of non-malignant cells such as fibroblasts, mesenchymal stem cells, blood lymphocytes, and a large number of infiltrating leukocytes.
In recent years, more and more researches show that tumor cells are in an immune tolerance state, and besides PD-L1 and CTLA-4 immune checkpoints, transforming growth factor-beta (Transforming growth factor beta and TGF beta) is also a gene for promoting tumor tolerance. Tgfβ is an important promoter of immune homeostasis and immune tolerance, inhibiting the expansion and function of various components of the immune system. Tgfβ also plays an important role in immunosuppression in the tumor microenvironment, and can regulate the production and function of many types of immune cells. It controls adaptive immunity by directly promoting expansion of Treg cells, inhibiting production and function of effector T cells and antigen presenting dendritic cells (DC cells). Particularly when the tumor has developed to a late stage, many tumor cells have developed a tolerance to the effects of tgfβ. While most tumor cells produce tgfβ by autocrine or paracrine, resulting in higher tgfβ concentrations in tumor tissue than physiological levels. At this time, tgfβ tends to promote EMT of tumor cells, promote infiltration and metastasis of tumors, participate in immune escape of tumors, and promote production of blood vessels to exert a cancer promoting effect. Tgfβ has therefore potential as a target for tumor therapy.
The small nucleic acid drug can silence specific disease genes through antisense principle targeting RNA, achieves the aim of treatment, has the advantages of high specificity, high efficiency, long acting and the like as a treatment strategy, greatly expands the number and types of targets available for treatment, and an efficient delivery system is key to the research and development and application of the small nucleic acid drug. At present, the effect of the small nucleic acid drug is often not as good as expected in the practical application process, and after the small nucleic acid drug is injected into a patient, how to remain in the body for a long time, how to allow therapeutic small nucleic acid to accurately enter targeted cells to perform therapeutic functions, and how to avoid injuring normal cells to the greatest extent are also facing great challenges. The poor efficacy of small nucleic acid drugs may also be due to differences in: (1) Challenges in siRNA target selection and sequence design, such as specificity, effective concentration, silencing efficiency, off-target effects, etc.; (2) Drug delivery modes (route, carrier, frequency, efficiency); (3) localized distribution of drug within tumor cells; (4) stage of cancer progression; (5) capability differentiation of the patient's immune system; (6) acquired drug resistance. Tumor heterogeneity, changes in tumor microenvironment, drug inactivation, decreased drug uptake or increased drug release by tumor cells, activation of tumor cell survival pathways, and epigenetic changes, etc. may also affect the use of small nucleic acid drugs. In addition, small nucleic acid drugs, especially RNAi drugs, need to act on mRNA in the cytoplasm or nucleus across the membrane to reach the site of action, and the delivery difficulty is great, and although chemical modification can solve the problems of stability and immunogenicity to some extent, small nucleic acid drugs still cannot exert the drug effect if they cannot enter cells to achieve endocytosis. DP7 polypeptide (VQWRIRVAVIRK) is a screening method for designing a novel antibacterial peptide with computer assistance, which is an antibacterial peptide having higher bacterial recognition specificity and stronger antibacterial activity by substituting 2 amino acids for template antibacterial peptide HH 2. The antibacterial peptide nanocrystallization can enable the antibacterial peptide to obtain better stability and reduce toxicity, and provides a new research direction for the application of the antibacterial peptide.
Disclosure of Invention
The invention aims to provide a new effective choice for small nucleic acid antitumor drugs.
In order to solve the technical problems described above, the present invention provides an siRNA that inhibits tgfβ gene expression. The sequence of the siRNA is as follows:
5'-CGGACUACUAUGCUAAAGATT-3' sense strand (SEQ ID No. 1)
The antisense strand (SEQ ID No. 2): 5'-UCUUUAGCAUAGUAGUCCGTT-3'.
The invention also provides the application of the siRNA for inhibiting TGF-beta gene expression in preparing antitumor drugs. Further, the tumor is melanoma or colon cancer.
The invention also provides a nanoparticle loaded with the siRNA. The nanoparticle is prepared by loading the siRNA for inhibiting TGF beta gene expression on the hydrophobically modified polypeptide; the sequence of the polypeptide is VQWRIRVAVIRK, and the hydrophobic modification is to couple a hydrophobic fragment at the nitrogen end of the polypeptide.
Wherein, polypeptide VQWRIRVAVIRK (SEQ ID No. 3) carbon end amidation modification is VQWRIRVAVIRK-NH2 in the siRNA loaded nanoparticle.
Wherein the hydrophobic segment is sterol compound or saturated straight chain fatty acid or PEG derivative; wherein the sterol compound is cholesterol compound or cholic acid compound; or the saturated straight chain fatty acid is at least one of C6-C20.
Wherein the sterol compound is at least one of butyrylated cholesterol, cholic acid or deoxycholic acid; or the PEG derivative is at least one of 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol or dipalmitoyl phosphatidylethanolamine-polyethylene glycol.
Wherein, the nitrogen end of the polypeptide is coupled with the hydrophobic segment in a way of being generated by amidation reaction of-CO-OH on the hydrophobic segment and-NH 2 on the polypeptide. Preferably, the hydrophobically modified polypeptide structure is:
the R in the polypeptide structure is sterol compound or saturated straight chain fatty acid or PEG derivative
Further, R in the above polypeptide structure is:
at least one of them.
Wherein the hydrophobic modified polypeptide and the small nucleic acid in the siRNA-loaded nanoparticle are prepared from raw materials in a mass ratio of 3-6:1. Preferably, the mass ratio of the hydrophobically modified polypeptide to the small nucleic acid is 5:1.
Furthermore, the nanoparticle loaded with siRNA is prepared by incubating hydrophobically modified polypeptide with siRNA.
Wherein, the nanoparticle loaded with siRNA is obtained by co-incubating hydrophobically modified polypeptide and siRNA in water or liquid culture medium for 5-15 min. The liquid culture medium is at least one of RPMI 1640, DMEM double-free culture medium or Optim culture medium.
Specifically, the method of the invention comprises the following steps:
a. weighing appropriate amount of DP7-C powder, adding sterilized water for dissolving, and spontaneously forming micelle
b. And incubating the siRNA of the TGF beta target gene for 15-20min at room temperature according to the mass ratio of (DP 7-C: siRNA or ASO=3:1-5:1) to obtain the DP7-C/siRNA complex.
The invention also provides application of the siRNA for inhibiting TGF beta gene expression or the nanoparticle loaded with the siRNA in preparing a medicine for treating tumors.
Further, the tumor is at least one of lung cancer, liver cancer, prostate cancer, bladder cancer, melanoma or colon cancer.
The invention also provides an anti-tumor drug which is prepared by adding pharmaceutically acceptable components into the siRNA for inhibiting TGF beta gene expression or the nanoparticle loaded with the siRNA. The medicine may further contain pharmaceutically acceptable auxiliary components.
Wherein, the medicine for treating tumor is in situ intratumoral injection.
The invention has the beneficial effects that: the invention provides an siRNA molecule with good effect aiming at TGF beta gene. The TGF beta siRNA can inhibit the expression of TGF beta gene effectively, the mRNA level is obviously reduced by more than 70%, the protein expression is obviously inhibited, the inhibition rate can reach 90%, and the TGF beta siRNA has obvious tumor inhibition function. The invention further uses the antimicrobial peptide derivative DP7-C as an siRNA delivery carrier. The invention also provides a DP 7-C-based small nucleic acid delivery system for TGF beta genes, and in-situ delivery of TGF beta siRNA can be used for treating tumors such as in-situ subcutaneous tumors. The DP7-C micelle disclosed by the invention has high-efficiency siRNA transmission capacity, has higher safety in vivo and in vitro, and is a good gene transmission carrier. The invention provides a new choice for the research and development of a new drug for treating tumors by using small nucleic acid drugs.
Drawings
Fig. 1: tgfβ is a candidate target for tumor immunotherapy: A. the expression result of immunohistochemical staining TGF beta in colon intestinal cancer and melanoma; B. the clinical database analyzes the correlation of tgfβ to the extent of immune cell infiltration in tumors.
Fig. 2: qPCR screening shows high knocking-down efficiency of TGF beta siRNA.
Fig. 3: detection of the interfering effect of the DP 7-C/simfβ complex in CT26 cells and promotion of tumor apoptosis: RNA expression detection results of TGF beta; protein expression detection results of TGF beta; DP7-C/siTGF beta stimulates flow detection of DC cell maturation; ct26 cell migration light mapping; E. white light photographing and statistical result of plate cloning experiment
Fig. 4: DP 7-C/simfβ complex for the treatment of CT26 mouse subcutaneous tumors: A. after the siRNA drug is delivered to the tumor in situ, the tumor of each group is photographed; B. tumor volume statistics for each group; C. tumor weight statistics for each group; D. effector T cell flow statistics.
Fig. 5: DP7-C/siTGF beta composite nanoparticle anti-tumor mechanism research: A. fluorescent photographing of apoptotic cells in tumor tissues; B. KI67 histochemical results in tumor tissue; C. CD31 organization results in tumor tissue.
Fig. 6: important organ HE staining results.
Detailed Description
Transforming growth factor-beta (TGF-beta) is a gene that promotes tumor tolerance. Tgfβ is an important promoter of immune homeostasis and immune tolerance, inhibiting the expansion and function of various components of the immune system. Tgfβ also plays an important role in immunosuppression in the tumor microenvironment, and can regulate the production and function of many types of immune cells. It is believed that the TGF-beta gene of tumor cells may serve as a potential target.
The invention designs and screens out an siRNA molecule aiming at TGF beta gene (TGF beta 1) with good effect through a large amount of work in the early stage, and obtains a TGF beta siRNA sequence with high-efficiency silencing through a large amount of optimization in the early stage:
sense strand 5'-GCAACAUGUUGGUCAUUAUTT-3'
Antisense strand 5'-AUAAUGACCAACAUGUUGCTT-3'.
The TGF beta siRNA can inhibit the expression of TGF beta gene with high efficiency, so that the mRNA level of the TGF beta siRNA is obviously reduced, the protein expression is obviously inhibited, and the TGF beta siRNA has a function of obviously inhibiting tumors.
Because how to remain in the body for a long time after the small nucleic acid medicine, how to allow the therapeutic small nucleic acid to accurately enter the targeted cells to play a therapeutic role, and how to avoid injuring normal cells to the greatest extent, the method also faces great challenges. Therefore, it is necessary to match the siRNA with an appropriate delivery system to ensure its own function.
For this reason, the present invention further uses the antibacterial peptide derivative DP7-C as a delivery system for siRNA molecules against tgfβ gene of the present invention, and a novel nano-drug is prepared. DP7-C is simple to prepare, can self-assemble into nano-micelle in aqueous solution, presents positive electricity in aqueous solution, has good monodispersity and Zeta potential, and is stable in freeze-drying and redissolution.
The polypeptide structure of the DP7-C is as follows:
wherein R is:
based on this DP7-C delivery system, delivery of tgfβ sirnas targeting macrophages can be used for treatment of solid tumors. The solid tumors comprise lung cancer, liver cancer, prostate cancer, bladder cancer, melanoma or colon cancer.
The DP7-C micelle disclosed by the invention has high-efficiency siRNA transmission capacity and also has higher safety in vitro and in vivo. In the embodiment of the invention, the DP7-C micelle loaded with the TGF-beta siRNA successfully realizes the efficient systemic introduction of the siRNA to tumor cells and effectively inhibits the growth of various tumor models, thereby obtaining a novel TGF-beta siRNA drug.
The present invention will be described in detail below with reference to the drawings and examples, and it should be noted that the examples are further illustrative of the technical aspects of the present invention and do not indicate that the present invention is limited to only these examples.
The main reagents used in the examples of the present invention:
(1) Small interfering RNA siRNA (synthesized by Shanghai Ji Ma company).
(2) The butyrylcholesterol modified antibacterial peptide DP7 (DP 7-C) is self-synthesized.
(3) TGF-beta antibodies (ab 179695, abcam)
(4) TUNEL detection kit (A113-01, novain)
Cell lines and experimental animals according to embodiments of the present invention:
(1) The murine melanoma cell line B16F10 was purchased from ATCC.
(2) The mouse colon cancer cell line CT26 was purchased from ATCC.
(3) C57BL/6J female mice purchased from Experimental animal technologies, inc. of Beijing vitamin Toril.
EXAMPLE 1 high correlation of TGF-beta expression levels with various degrees of infiltration of immune cells
We collected tumor specimens from colorectal and melanoma patients, fixed with 4% formaldehyde for 48 hours, embedded sections, and stained for tgfβ targets by histochemical staining. The results are shown in FIG. 1A, and TGF beta is highly expressed in colorectal cancer and melanoma patients, so that the TGF beta can be used as a knockdown target point of tumor immunotherapy. By subsequent bioinformatics ((http:// timer. Cistrom. Org /)) analysis, it was also found that the expression level of TGF-beta was highly correlated with the degree of infiltration of various immune cells (FIG. 1B). It is shown that it can be used as knockdown target for various tumor immunotherapy.
EXAMPLE 2 siRNA screening against TGF beta
The chemokine receptor TGF beta exists on the surface of immune cells, can guide the immune cells to reach inflammation and tumor sites, and cancer cells expressing the TGF beta can generate an immunosuppressive microenvironment. The invention deduces that therapeutic blocking of TGF beta signals can inhibit inflammatory cell aggregation and infiltration, thereby reversing the immunosuppressive state of tumor microenvironment and activating anti-tumor CD8 + T cell response.
Aiming at TGF beta genes, the invention designs and screens the siRNA (siTGF beta) for targeted inhibition of the TGF beta genes so as to obtain the siTGF beta with good inhibition and anti-tumor effects.
In the early stage of the experiment, three siRNAs to TGF beta genes are selected and synthesized by software design and optimization in the early stage. First, three siRNAs synthesized to target TGF-beta gene were formed into DP 7-C/siTGF-beta complex with DP7-C, respectively.
According to the preferred ratio of siTGF beta to DP7-C C obtained by the previous experiment, siTGF beta is dissolved to 1 mug/mu l by DEPC water, 2 mug of siTGF beta is absorbed and mixed uniformly in 200 mu l of culture medium, 10 mug of DP7-C is added, and after incubation for 10 minutes at room temperature, the mixture is gently added into cells. By comparing cell transfection and qPCR experiments, we selected siRNA2 with highest knockdown efficiency for subsequent in vivo and in vitro anti-tumor experiments (FIG. 2). The sequence of siRNA2 is as follows:
sense strand 5'-CGGACUACUAUGCUAAAGATT-3'
Antisense strand 5'-UCUUUAGCAUAGUAGUCCGTT-3'.
We further validated the knockdown efficiency of the preferred siRNA2 (labeled as sitgfβ in subsequent experiments) on tgfβ mRNA and protein, and the results are shown in fig. 3A and 3B, with the DP7-C/sitgfβ treatment group significantly down-regulating mRNA expression level (fig. 3A) and protein expression level (fig. 3B) of tgfβ gene in CT26 cells.
Example 3 functional assay of DP7-C/siTGF beta Complex
Tgfβ is an important promoter of immune homeostasis and immune tolerance, inhibiting the function of various components of the immune system. Tgfβ also plays an important role in immunosuppression in the tumor microenvironment, and can regulate the production and function of many types of immune cells. TGF beta can regulate proliferation, differentiation, apoptosis, epithelial-mesenchymal transition and metastasis of cancer cells, inhibit production and function of effector T cells and antigen presenting dendritic cells (DC cells), and control adaptive immunity. Intervention with tgfβ thus provides a viable therapeutic approach for cancer patients. Therefore, in this project we target TGF beta gene, further verify the effectiveness of DP7-C/siTGF beta composite nanoparticles.
1. Experiments to promote maturation of DC cells
Dendritic cells derived from mouse bone marrow were extracted, and differentiation of bone marrow cells in the DC direction was induced by the addition of 20ng/mL of cytokine rmGM-CSF. On day 6 of incubation, siTGF beta was dissolved with DEPC water to 1. Mu.g/. Mu.l, 2. Mu.g of siTGF beta was pipetted into 200. Mu.l medium and mixed well, 10. Mu.g DP7-C was added, incubated at room temperature for 10 min and then gently added to DC cells. While the untreated group, the DP7-C/siScramble group, was established. After 48 hours, DCs were collected and the proportion of mature DCs in each group was detected using CD11c, CD80, CD86 flow antibody markers. The results showed that DP7-C/siTGF beta was effective in promoting DC cell maturation (FIG. 3C)
2. Experiments to inhibit tumor cell proliferation and migration
DP7-C (10 ug)/siTGF beta (2 ug) was gently added to CT26 cells after 10 min incubation at room temperature. After 48 hours, the cells were digested and 2X 10 5 Each CT26 cell was added to a Transwell chamber, and 600. Mu.l of medium containing 15% FBS was added to the well plate lower chamber. After conventional incubation for 24h, the Transwell cells were removed, the medium in the wells was discarded, washed 2 times with calcium-free PBS, the upper non-migrating cells were gently rubbed off with a cotton swab, 4% formaldehyde was fixed for 30 min, and the cells were air-dried appropriately. Dyeing with 0.1% crystal violet for 30-60min, and washing with PBS for 3 times. The upper chamber was gently rubbed off with a cotton swab. Five fields were counted randomly under a 40-fold microscope. The results showed that DP7-C/siTGF beta was effective in inhibiting tumor cell migration (FIG. 3D).
CT26 cells with good growth state were cultured according to 1X 10 3 The cells/well were inoculated into 6-well plates and incubated overnight. The next day, the medium was replaced with fresh double medium, and vehicle delivery was performed in the experimental setting blank (Untreated group)The independent siRNA group (DP 7-C/siScamble group, 10ug/2 ug) and the vector delivery siTGF beta group (DP 7-C/siTGF beta group, 10ug/2 ug) were 3 groups in total. The above-mentioned grouping reagent was added to CT26 cells, and the culture was continued for 2 weeks. The supernatant was then discarded and carefully rinsed with PBS. Cells were fixed by adding an appropriate amount of 4% paraformaldehyde fixative solution to each well for about 10 minutes. The fixing solution is discarded, the crystal violet dye solution is added for dyeing for 10-15 minutes, the dye solution is slowly and carefully washed by flowing water, and the dye solution is dried in the air and photographed. The results showed that DP 7-C/siTGF-beta was effective in inhibiting tumor cell proliferation (FIG. 3E).
EXAMPLE 4 in situ injection of DP7-C/siTGF beta Complex for treatment of CT26 mouse colon cancer subcutaneous tumor model
C57BL/6J female mice with the age of about 6-8 weeks are kept in SPF experimental animal houses after quarantine for one week, the state of the mice is observed one day before tumor cell inoculation, and the hair at the root of the right thigh is removed. CT26 tumor cells were collected and counted in conventional culture, and serum-free DMEM basal medium was used to resuspend the cells to a cell concentration of 1X 10 6 And each mL. Subcutaneous inoculation of right thigh roots of mice was performed in an ultra clean bench in SPF animal houses with 100. Mu.l each of tumor cell suspensions (1X 10 5 Individual cells/individual) mice were randomly divided into 2 groups after inoculation, and group-marked.
The mice were observed daily for their status and tumor growth the following day of inoculation. When the subcutaneous tumor of the mice grows to 40mm 3 When the preferred siTGF-beta is dissolved to 1ug/ul with DEPC water, 12ug of siTGF-beta is pipetted and mixed with 60ug DP7-C, after 10 minutes incubation at room temperature, DEPC water is added to a volume of 100ul. DP 7-C/siTGF-beta (60 ug/12 ug) complexes were injected in situ within the tumor. Tumor volumes were injected once every three days and recorded prior to injection. Six times after administration, a significant inhibition of CT26 subcutaneous tumor growth was observed. The results are shown in figure 4, which shows that mice treated with DP 7-C/siffβ significantly inhibited colorectal cancer growth compared to the other two groups (n=5 per group, figure 4A). 2011.802 + -780.364 mm to NS group 3 ,DP7-C/siScramble1745.234±530.769mm 3 In comparison, the tumor volume of the DP7-C/siTGF beta treatment group is obviously reduced to 690.451 +/-326.260 mm 3 (FIG. 4B). Furthermore, at the same time, the NS group 7.370 + -3.32 g DP7-C/siScramble group 4.178 + -0Compared to 73, the tumor weight of the DP7-C/siTGFβ treated group was significantly reduced to 2.776 + -0.344 g (FIG. 4C).
After the treatment, CD8 in the tumors of each group of mice was detected + T ratio. The results are shown in FIG. 4D, CD8 after DP 7-C/siTGF-beta treatment + The T cell proportion of (c) is significantly increased, indicating that silencing tgfβ expression enhances the anti-tumor immune response.
Furthermore, the TUNEL assay results on tumor tissue sections showed that DP 7-C/simfβ treated tumor tissues showed strong apoptosis signals compared to the other two groups (fig. 5A). The KI67 and CD31 immunohistochemical results showed reduced tumor cell proliferation and reduced angiogenesis in the tumor tissue of the DP 7-C/siTGF-beta treated group (FIGS. 5B-C).
Overall, these results indicate that DP7-C polypeptide nanocarriers are capable of significantly inhibiting proliferation of subcutaneous tumors, with good therapeutic ability against tumors when administered in situ.
Example 5 safety evaluation of intratumoral injection delivery of DP7-C/siTGF beta complexes
At present, safety is an important investigation index for developing siRNA drugs. After the end of DP 7-C/simfβ treatment, mice were sacrificed, dissected and the major organs were removed and fixed by soaking in 4% paraformaldehyde to preserve tissue morphology. After paraffin embedding and slicing, HE staining was performed and whether the major organ tissues and cell morphology of the mice were changed was observed under a microscope to evaluate whether DP 7-C/simfβ had toxic or side effects on the major organs of the mice.
The results showed that the heart, liver, spleen, lung and kidney tissue structures of each group of mice were clear, the histiocyte morphology was normal, and no obvious pathological changes were seen (fig. 6). HE staining results show that DP 7-C/simfβ in situ injection does not cause significant toxic side effects and abnormalities.

Claims (13)

1. An siRNA that inhibits tgfβ gene expression, characterized in that the siRNA sequence is as follows:
sense strand 5'-GCAACAUGUUGGUCAUUAUTT-3'
Antisense strand 5'-AUAAUGACCAACAUGUUGCTT-3'.
2. Nanoparticles loaded with siRNA, characterized in that the nanoparticles are prepared by loading the siRNA inhibiting TGF-beta gene expression of claim 1 with hydrophobically modified polypeptides; the sequence of the polypeptide is VQWRIRVAVIRK, and the hydrophobization modification is to couple a hydrophobic fragment at the nitrogen end of the polypeptide; preferably, the polypeptide VQWRIRVAVIRK is modified into VQWRIRVAVIRK-NH2 through carbon end amidation.
3. Nanoparticle loaded with siRNA according to claim 2, characterized in that the hydrophobic fragment is a sterol compound or a saturated linear fatty acid or PEG derivative; further, the sterol compound is cholesterol compound or cholic acid compound; or the saturated straight chain fatty acid is at least one of C6-C20.
4. The siRNA loaded nanoparticle of claim 3, wherein the sterol compound is at least one of but diacylated cholesterol, cholic acid or deoxycholic acid; or the PEG derivative is at least one of 1, 2-dioleoyl-SN-glycerol-3-phosphorylethanolamine-polyethylene glycol, distearoyl phosphatidylethanolamine-polyethylene glycol or dipalmitoyl phosphatidylethanolamine-polyethylene glycol.
5. The siRNA loaded nanoparticle of claim 4, wherein the nitrogen terminus of the polypeptide is coupled to the hydrophobic moiety by amidation of-CO-OH on the hydrophobic moiety with-NH 2 on the polypeptide.
6. The siRNA loaded nanoparticle of claim 4, wherein the hydrophobically modified polypeptide has the structure:
and R is a sterol compound or saturated straight-chain fatty acid or PEG derivative.
7. The siRNA loaded nanoparticle of claim 6, wherein R in the polypeptide structure is:
at least one of them.
8. The siRNA-loaded nanoparticle according to claim 2, wherein the nanoparticle is prepared from the hydrophobically modified polypeptide and the small nucleic acid in a mass ratio of 3-6:1; preferably, the mass ratio of the hydrophobically modified polypeptide to the small nucleic acid is 5:1.
9. The siRNA loaded nanoparticle of claim 3, wherein the siRNA loaded nanoparticle is prepared by co-incubating a hydrophobically modified polypeptide with an siRNA.
10. The siRNA loaded nanoparticle according to claim 9, characterized in that it is obtained by co-incubating the hydrophobically modified polypeptide with siRNA in water or liquid medium for 5-15 min; further, the liquid culture medium is at least one of RPMI 1640, DMEM double-free culture medium or Optim culture medium.
11. Use of the siRNA that inhibits expression of tgfβ gene of claim 1 or the siRNA loaded nanoparticle of any one of claims 2 to 10 in the preparation of a medicament for treating a tumor.
12. An antitumor drug characterized by comprising the siRNA for inhibiting TGF-beta gene expression according to claim 1 or the siRNA-loaded nanoparticle according to any one of claims 2 to 10, and a pharmaceutically acceptable component.
13. The use according to claim 11 or the antitumor drug according to claim 12, characterized in that the tumor is at least one of lung cancer, liver cancer, prostate cancer, bladder cancer, melanoma or colon cancer.
CN202310979557.3A 2023-08-06 2023-08-06 siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof Pending CN117004609A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310979557.3A CN117004609A (en) 2023-08-06 2023-08-06 siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310979557.3A CN117004609A (en) 2023-08-06 2023-08-06 siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof

Publications (1)

Publication Number Publication Date
CN117004609A true CN117004609A (en) 2023-11-07

Family

ID=88561447

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310979557.3A Pending CN117004609A (en) 2023-08-06 2023-08-06 siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof

Country Status (1)

Country Link
CN (1) CN117004609A (en)

Similar Documents

Publication Publication Date Title
CN108220293B (en) Small interfering nucleic acid, pharmaceutical composition and application thereof
WO2017190695A1 (en) Egfr gene expression-suppressing sirna, precursor of same, and applications thereof
Tang et al. A simple self-assembly nanomicelle based on brain tumor-targeting peptide-mediated siRNA delivery for glioma immunotherapy via intranasal administration
CN113846047A (en) Kidney targeting drug-loaded exosome and application and drug for treating kidney diseases
US11028391B2 (en) Methods and compositions to inhibit metastasis and to treat fibrosis and to enhance wound healing
CN113388571A (en) Liver targeted drug-loaded exosome and application and drug for treating liver diseases
CN115772232A (en) GPC 3-targeted chimeric antigen receptor mononuclear/macrophage and construction method and application thereof
Yang et al. Efficient inhibition of ovarian cancer by recombinant CXC chemokine ligand 10 delivered by novel biodegradable cationic heparin-polyethyleneimine nanogels
CN113528427A (en) Colorectal targeted drug-loaded exosome, application thereof and drug for treating colorectal diseases
Yan et al. Nano-adjuvants and immune agonists promote antitumor immunity of peptide amphiphiles
CN117004609A (en) siRNA for inhibiting TGF beta gene expression, nanoparticle loaded with siRNA and application thereof
CN114191539B (en) Exosome nano particle for compositely co-carrying small molecule nucleic acid and active protein, and preparation method and application thereof
CN108403665B (en) Prostate cancer targeted drug delivery carrier modified by EpDT3 aptamer, delivery system, preparation method and application thereof
CN117821457A (en) SiRNA for inhibiting CCR2 gene expression, nano particle loaded with siRNA and application thereof
CN117025603A (en) SiRNA (small interfering RNA) and nanoparticle for inhibiting signal transduction and transcriptional activator protein 3 gene expression and application thereof
JP2022512894A (en) Methods and compositions for the treatment of breast cancer using antisense
WO2023138451A1 (en) Sirna pharmaceutical composition for inhibiting her2 and her3
CN101289653A (en) Nicotine-processed dendritic cells for preventing and treating tumors
KR102574252B1 (en) Composition for Preventing or Treating Pancreatic Cancer Comprising Peptide Nucleic Acid Complex
CN114377140B (en) Application of hydrophobically modified polypeptide in preparation of microRNA related nucleic acid delivery system
CN113817677A (en) Use of pantothenic acid or derivatives thereof and alpha-D-glucose-1, 6-bisphosphate or derivatives thereof for promoting DC migration
CN117925617A (en) SiRNA and nanoparticle for inhibiting expression of CD47-SIRP alpha signal axis gene and application thereof
CN116059235A (en) siRNA pharmaceutical composition for inhibiting various solid tumors
CN116999565A (en) Multiple small nucleic acid delivery system and multiple target vaccine
CN117598980A (en) Injectable nucleic acid hydrogel in-situ tumor vaccine 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