Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method

A sensitive, anti-tumor technology, applied in anti-tumor drugs, medical formulations with non-active ingredients, and medical formulations containing active ingredients, etc., can solve the problem of unstable oral administration route, low expression efficiency, nanoparticle carrier Toxic and side effects are great, achieving the effect of favorable phagocytosis, strong antigen presentation, and small toxic and side effects

Inactive Publication Date: 2013-08-14
NANKAI UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problems that the nanoparticle carriers currently used in experimental tumor immunotherapy have high toxicity and side effects, unstable oral administration route, low expression efficiency and difficulty in carrying DNA through the gastric acid environment and entering the intestinal tract to efficiently express tumor antigens. A novel acid-resistant nanoparticle oral DNA antitumor vaccine with pH-sensitive properties

Method used

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  • Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method
  • Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method
  • Antiacid nano oral deoxyribonucleic acid (DNA) anti-tumor vaccine with potential of hydrogen (pH) sensitive characteristic and preparation method

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preparation example Construction

[0034] 1. Preparation of Chitosan Nanoparticles

[0035] 1.1. Dissolve long-chain chitosan with a molecular weight of 28,000 to 32,000 in double-distilled water, adjust the pH to 5.2, and stir at 55°C for 30 minutes to form liquid A;

[0036] 1.2, while adding 25mM Na 2 SO 4 Mix the solution with the required DNA plasmid and heat to 55°C, stir at a constant speed for 30 minutes to form solution B;

[0037]1.3, then mix the two solutions with liquid A and liquid B at a volume ratio of 1:1, and mix immediately, and keep the prepared chitosan nanoparticles at 4°C after 5 minutes;

[0038] 2. Activation of the carboxyl group on the side chain of alginic acid

[0039] Dissolve alginic acid powder into double distilled water, add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the alginic acid solution by molar Mix at a ratio of 1:5:5, adjust the pH value to 9.0, stir overnight at room temperature, and store the activated alginic acid at 4...

Embodiment 1

[0062] 1. Preparation of acid-resistant nanoparticle oral DNA anti-tumor vaccine with pH-sensitive properties:

[0063] (1) Preparation of chitosan nanoparticles: Dissolve long-chain chitosan with a molecular weight of 28,000 to 32,000 in double distilled water, adjust the pH value to 5.2, and stir at 55° C. for 30 minutes.

[0064] (2) At the same time, 25mM Na 2 SO 4 The solution was mixed with the Legumain DNA plasmid, heated to 55°C, and stirred at a constant speed for 30 minutes. Mix two kinds of solutions with volume ratio 1: 1 subsequently, and carry out mixing at once, after 5 minutes, the prepared chitosan nanoparticle is kept at 4 ℃;

[0065] (3) Activation of the carboxyl group on the side chain of alginic acid: dissolve the alginic acid powder in double distilled water, mix the alginic acid solution with the added N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride was mixed at a molar ratio of 1:5:5, and the pH value was adjust...

Embodiment 2

[0071] An acid-resistant nanoparticle oral DNA anti-tumor vaccine with pH-sensitive properties expresses the encoded protein in normal BALB / c mice.

[0072] (1) intragastric administration and dosage: each BALB / c mouse was given an anti-acid nanoparticle oral DNA anti-tumor vaccine containing 150 μg of DNA plasmid or 10 8 CFU of Salmonella, once daily;

[0073] (2) Repeat the above steps 5 times;

[0074] (3) In vivo fluorescence expression detection of BALB / c tumor-bearing mice after oral immunization: the tumor-bearing mice were sacrificed, and Peyer's lymph nodes in the small intestine were collected to observe the acid-resistant nanoparticles encoding the pH-sensitive properties of green fluorescent protein orally administered DNA The expression of anti-tumor vaccines was compared with other control groups.

[0075] The result is as Figure 7 As shown, GFP was highly expressed in macrophages in mouse Peyer's lymph nodes. Figure 8 The results of flow cytometry detectio...

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Abstract

Provided are an antiacid nano particle oral deoxyribonucleic acid (DNA) anti-tumor vaccine with the potential of hydrogen (pH) sensitive characteristic and a preparation method. The DNA oral vaccine is nano particles formed by combining chitosan with alginic acid for surface finish and encoded tumor specific antigen Legumain protein DNA plasmids. The DNA oral vaccine is capable of being efficiently phagocytized by dendritic cells and macrophage in peyer's patches and expressing the encoded tumor antigen to activate immune damage of a host for tumor cells. The nano particles have small toxic and side effects and a strong antigen presentation role. Specifically, the DNA plasmids and chitosan are led to be in static negative crosslinking, and then, alginic acid is used for surface finish of chitosan nano particles. By means of immunofluorescent staining and flow cytometry, growth and transfer conditions of breast cancer of a mouse are estimated, immune response of the mouse is analyzed, and antigen presentation capability and anti-tumor effect of the antiacid nano particle oral DNA anti-tumor vaccine with the potential of pH sensitive characteristic are determined.

Description

technical field [0001] The present invention relates to a novel anti-acid nanoparticle oral DNA anti-tumor vaccine with pH-sensitive properties and a treatment method using it. It belongs to the field of genetic engineering pharmaceutical technology. Background technique [0002] Breast cancer has become a common tumor in women. According to the statistics of the National Cancer Institute (NCI), between 2004 and 2008, the average age of patients diagnosed with breast cancer was 61 years old, and the annual incidence rate among women was about 0.0122%. The incidence rate of Asians is about 0.0937%; between 2003 and 2007, the average age of death of breast cancer patients was 68 years old, and the annual mortality rate among women was 0.024%, among which the annual mortality rate of Asians / Pacific Islanders was 0.0122%; The rate and death rate are the first and second among women, respectively. The current treatment of breast cancer is mainly traditional therapy, namely sur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/00A61K9/10A61K47/36A61P35/00
Inventor 刘赜谭小月刘淑熊敏吕丹向荣
Owner NANKAI UNIV
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