Liposome-polymer hybrid nanoparticles for nucleic acid vaccine delivery

A nanoparticle, nucleic acid vaccine technology, applied in liposome delivery, vaccines, DNA/RNA vaccination, etc., can solve the problems of strict protein cold chain requirements, limited application, complex production process of protein and RNA vaccines, etc.

Inactive Publication Date: 2021-05-14
AB&B BIO TECH CO LTD JS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional attenuated and inactivated vaccines fail to provide lifelong immunity and risk reversing toxicity; protein vaccines have the ability to trigger robust humoral immune responses against multiple microbial infections, but they are insufficient to induce T cell-mediated immune responses , thus limiting its application in cancer and chronic viral infections such as HIV
In addition, RNA and protein vaccines have at least the following defects: mRNA vaccines have been proven to be hepatotoxic, the production process of protein and RNA vaccines is complicated, and proteins have strict cold chain requirements, etc.
However, lipids are easily removed by RES

Method used

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  • Liposome-polymer hybrid nanoparticles for nucleic acid vaccine delivery
  • Liposome-polymer hybrid nanoparticles for nucleic acid vaccine delivery
  • Liposome-polymer hybrid nanoparticles for nucleic acid vaccine delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Design, Evaluation and Construction of DNA Vaccines

[0057] 1. Epitope screening

[0058] The protein sequences of LTB (1DJR), CFA / I (2HB0), CS6 (4B9J) and IpaB (3U0C) were obtained through the PDB database (https: / / www.rcsb.org / ), and the specific sequences are shown in SEQ ID NO: 3 ., SEQ ID NO: 4., SEQ ID NO: 5. and SEQ ID NO: 6. Shown. ABCpred and Bcepred were used to obtain linear B-cell epitopes, while DiscoTope2.0 and ElliPro were used to predict conformational B-cell epitopes. T cell epitopes are restricted by the major histocompatibility complex (MHC) and are divided into two classes, MHC-I and MHC-II. NetMHCpan4.0 and IEDB were used to obtain MHC-I and MHC-II, respectively, while RANKPEP was used for both MHC types.

[0059] Likewise, CTLPred and PAComplexc were used to obtain suitable CTL epitope candidates. The selected 16 B cell and T cell epitopes (shown in Table 1) have good hydrophilicity, good flexibility, high accessibility and strong a...

Embodiment 2

[0069] Example 2: Preparation and Characterization of Liposome-polymer Hybrid Nanoparticles Encapsulating MEG for Nucleic Acid Vaccine Delivery

[0070] 1. Preparation of liposome-polymer hybrid nanoparticles encapsulating MEG

[0071] Dissolve 240 μL of mPEG-PLGA (25 mg / mL) and 50 μg of plasmid DNA (pcDNA3.1, pSFV, pcDNA3.1-MEG, pSFV-MEG, pcDNA3.1-MEG-EGFP or pSFV-EMG-EGFP) in 3 mL of ethyl acetate ester, and the mixture was shaken to form an emulsion to obtain an organic phase. Meanwhile, 84 μL of lecithin (10 mg / mL) and 36 μL of DSPE-PEG2000-Mal (10 mg / mL) (9:1 molar ratio) were dissolved in 9 mL of 4% ethanol solution to obtain an aqueous phase.

[0072] The aqueous phase was preliminarily heated to 65°C, and then the organic phase was added dropwise to the preheated aqueous phase at a rate of 1 mL / min under slow stirring. Afterwards, the mixture was vortexed vigorously for 3 min, then stirred slowly at room temperature for 2 h to evaporate the organic phase. The obtain...

Embodiment 3

[0083] Example 3: Detection of the intracellular expression of MEG

[0084] 1. Detection of transfection efficiency

[0085] The L-02 cells were divided into 2×10 5 Cells / mL were inoculated into 24-well plates, cultured to 80%-90% cell confluence, and the transfection experiment was carried out. One hour before transfection, the medium in the culture plate was discarded, washed 3 times with PBS, and the anti-serum-free medium was replaced. Mix pcDNA3.1-MEG, pSFV-MEG, pcDNA3.1-MEG / LNPs, pSFV-MEG / LNPs, pcDNA3.1-MEG+Lipofectamine, pSFV-MEG+Lipofectamine with basic DMEM medium, add to cells middle. Incubate in an incubator for 4-6 hours, remove particles and liposome complexes, and add pre-warmed complete medium. The culture was continued for 48 h, and the transfection efficiency was detected by flow cytometry, and all experiments were repeated in triplicate.

[0086] like Figure 8 As shown in A, compared with pcDNA3.1-MEG and pSFV-MEG, the transfection efficiency of pcDNA3...

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Abstract

The invention relates to a liposome-polymer hybrid nanoparticle for nucleic acid vaccine delivery, which is characterized in that a biodegradable polymer mPEG-PLGA is used for packaging pcDNA3.1-MEG or pSFV-MEG to form a nanoparticle core, a hydrophilic surfactant PEG-DSPE2000-Mal is used as a nanoparticle shell, and a liposome monolayer (Lecithin) is arranged at the joint of the core and the shell. According to the invention, the transfection efficiency of the DNA vaccine is high, the encapsulation efficiency is high, the nucleic acid vaccine can be protected from being quickly degraded in a short time, and nucleic acid can be slowly released, so that the continuous expression of the antigen is realized, the circulation and action time of the antigen in vivo are improved, the continuous immunostimulation of the antigen on the body is enhanced, and the immune effect is enhanced.

Description

technical field [0001] The invention relates to the technical field of DNA vaccine delivery and protection, in particular to a liposome-polymer hybrid nanoparticle for nucleic acid vaccine delivery. Background technique [0002] In the 1990s, it was first demonstrated that DNA plasmids encoding antigens from pathogens can induce antigen-specific antibodies and T cell immune responses. Since then, many researchers have conducted extensive preclinical and clinical studies on DNA vaccines. Currently, DNA vaccines are licensed for the treatment of West Nile virus in horses (7 species), hematopoietic necrosis virus in salmon (8 species), melanoma in dogs (9 species), etc.; demonstrated that DNA vaccines can be used in larger animals elicit protective immunity. [0003] Traditional attenuated and inactivated vaccines fail to provide lifelong immunity and risk reversing toxicity; protein vaccines have the ability to trigger robust humoral immune responses against multiple microbi...

Claims

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Application Information

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IPC IPC(8): A61K9/52A61K9/127A61K39/00A61K47/36A61K47/28A61K47/34A61K47/24A61K47/10A61P37/04
CPCA61K9/1271A61K9/5146A61K9/5153A61K9/5161A61K39/00A61K2039/53A61P37/04
Inventor 安有才吴浩飞徐奇赵章婷张瑞环
Owner AB&B BIO TECH CO LTD JS
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