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Application of PEG (polyethylene glycol)-PLA (Poly Lactic Acid) nano-material-coated HBV (Hepatitis B Virus)-CpG (Cytosine Phosphate Guanosine) in prevention and/or treatment of hepatitis B

A technology of PEG-PLA and nanomaterials, which is applied in the application field of HBV-CpG coated with PEG-PLA nanomaterials in the prevention and/or treatment of hepatitis B, and can solve application limitations, toxic and side effects limiting applications, and unfavorable hydrophobicity Release of insoluble drugs and other issues to achieve the effect of improving immunity and clearing hepatitis B virus

Active Publication Date: 2013-08-07
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies have shown that CpG ODN can be used as an adjuvant in the prevention and treatment of hepatitis B, but the existing research on the treatment of hepatitis B with CpG ODN has not achieved the effect of completely eradicating hepatitis B, and the excessive toxic and side effects limit its application.
[0005] Polylactic acid (PLA) is a class of synthetic biodegradable polymers that have been approved by the US FDA for tissue engineering, medical materials, drug carriers, etc., but PLA is not conducive to the release of insoluble drugs due to its strong hydrophobicity. , the drug loading rate of polar drugs is low, so its application is limited

Method used

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  • Application of PEG (polyethylene glycol)-PLA (Poly Lactic Acid) nano-material-coated HBV (Hepatitis B Virus)-CpG (Cytosine Phosphate Guanosine) in prevention and/or treatment of hepatitis B
  • Application of PEG (polyethylene glycol)-PLA (Poly Lactic Acid) nano-material-coated HBV (Hepatitis B Virus)-CpG (Cytosine Phosphate Guanosine) in prevention and/or treatment of hepatitis B
  • Application of PEG (polyethylene glycol)-PLA (Poly Lactic Acid) nano-material-coated HBV (Hepatitis B Virus)-CpG (Cytosine Phosphate Guanosine) in prevention and/or treatment of hepatitis B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Design and screening of HBV-CpG

[0051] 1. Design of HBV-CpG: According to the characteristics of GenBank HBV gene sequence, a series of CpG-containing oligonucleotide sequences derived from HBV gene were designed and synthesized: A1, A2, A3, A5, A6, A7, A8, B1, B2 and B3, as shown in Table 1, where the capital letters of nucleotides are modified by phosphorothioate bonds. The above sequences were all synthesized by Shanghai Sangon Biosynthesis Co., Ltd.

[0052] Table 1. Design of HBV-CpG

[0053]

[0054] 2. Screening of HBV-CpG:

[0055] (1) Preparation of human peripheral blood mononuclear cells (PBMC):

[0056] Peripheral blood buffy coat was taken from healthy persons (with informed consent) in Hefei Blood Center, Anhui Province, diluted with an equal amount of 1×PBS, and subjected to density gradient centrifugation by Ficoll-Hypaque method, and the mononuclear cells at the interface of the upper and middle layers were absorbed. The white clo...

Embodiment 2

[0062] Embodiment 2: Nanomaterial PEG-PLA coats HBV-CpG

[0063] Weigh 10.0mg of PEG 5000 -PLA 10000 (This laboratory designed and synthesized, and the synthesis method is detailed in references Yang XZ, Dou S, Sun TM, Mao CQ, Wang HX, Wang J. Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy. Journal of Controlled Release .2011; 156: 203-11.), supplemented with 1.0 mg cationic lipid BHEM-Chol (designed and synthesized in this laboratory, the synthesis method is detailed in references Yang XZ, Dou S, Sun TM, Mao CQ, Wang HX, Wang J.Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy.Journal of Controlled Release.2011; 156:203-11.) was dissolved in 0.5mL chloroform, and 0.2mg of HBV-CpG was added to the PBS solution ( The volume is 25 μL), ultrasonicated in a probe-type ultrasonic breaker (VC130 type, Sonics, the United States), the output power is 80 watts, and the frequency of ultras...

Embodiment 3

[0064] Embodiment 3: NP (HBV-CpG) synergistically enhances HBsAg vaccine antibody response

[0065] 1. Immunization of experimental animals

[0066]Male BALB / C mice aged 6 to 8 weeks, 20 to 22 g (purchased from Shanghai Slack Experimental Animal Co., Ltd.), were randomly divided into four groups, 6 mice in each group. Group I: PBS control; Group II: HBsAg vaccine alone group: each mouse was immunized with recombinant HBsAg vaccine (purchased from Shenzhen Kangtai Biological Products Co., Ltd.) (2 μg per mouse per injection); Group III: empty nano Carrier combined with HBsAg vaccine group: 500ug PEG-PLA nanomaterial empty carrier (NPs) was mixed with 2 μg of HBsAg to make a homogeneous suspension, and each mouse was immunized; Group IV: NP (HBV-CpG) combined with HBsAg vaccine group of the present invention: 500ugPEG-PLA was coated with 10μg HBV-CpG to form nanoparticle NP (HBV-CpG), and then mixed with 2μg HBsAg to make a homogeneous suspension to immunize each mouse. The mi...

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Abstract

The invention relates to an HBV (Hepatitis B Virus)-CpG (Cytosine Phosphate Guanosine) oligonucleotide sequence capable of inducing alpha-interferon expression, and further relates to a nano-particle prepared by coating the HBV-CpG oligonucleotide sequence by using a nano material PEG-PLA, and relates to an application of the nano-particle in prevention and treatment of hepatitis B, wherein the oligonucleotide sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2; the nano material PEG-PLA is a segmented copolymer prepared from polyethylene glycol and poly lactic acid; and the nano material PEG-PLA has the general formula of PEGm-PLAn, wherein m represents the number-average molecular weight of a PEG chain segment and can be 2000, 5000 or 10000, and n represents the number-average molecular weight of a PLA chain segment and can be 5000 or 10000. The CpG(HBV-CpG) from HBV gene sources coated by the nano material PEG-PLA not only can be used for preparing a prophylactic vaccine for hepatitis B but also can be used for preparing a therapeutic vaccine for hepatitis B.

Description

technical field [0001] The present invention relates to two CpG oligonucleotide sequences (HBV-CpG) that are derived from HBV genome, the present invention also relates to the nanoparticle that is made by coating HBV-CpG by nano material PEG-PLA, the present invention also relates to nano material PEG -PLA-coated HBV-CpG is used for the prevention and / or treatment of hepatitis B. Background technique [0002] Chronic hepatitis B virus infection is a problem that endangers global human health. At present, there are at least 360 million hepatitis B virus (HBV) carriers in the world, and more than one-third of them are in China. The existing hepatitis B surface antigen vaccine (HBsAg) is used as a preventive vaccine by inducing humoral immune response, but about 10% of vaccinators still cannot produce protective antibodies, and the vaccine has no therapeutic effect on chronic hepatitis B patients. Existing anti-hepatitis B drugs such as interferon and lamivudine can inhibit vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/117A61K39/39A61K47/34A61P1/16A61P31/20
Inventor 魏海明吕树娟田志刚王均孙汭
Owner UNIV OF SCI & TECH OF CHINA
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