Cationic elaioplast and its adenovirus composition, its preparing method and use

A technology of cationic liposomes and complexes, which is applied in the directions of liposome delivery, drug combination, pharmaceutical formulations, etc., can solve problems such as difficulty in obtaining in vivo curative effect, difficulty in determining liposomes, etc., and achieves improvement of raw materials and dosage, transfection The effect of improved efficiency and high encapsulation efficiency

Active Publication Date: 2010-07-14
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there have been reports in the field that try to use liposome-encapsulated adenovirus to overcome the above problems, and some positive effects have been observed in in vitro experiments, but it is still difficult to obtain curative effect in vivo
And to make liposome-encapsulated adenovirus achieve good results, it is necessary to carry out a large number of explorations in the formulation of liposomes, the ratio of adenovirus and liposomes, etc., and there are many kinds of raw materials that can be prepared liposomes , very difficult to determine good liposomes

Method used

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  • Cationic elaioplast and its adenovirus composition, its preparing method and use
  • Cationic elaioplast and its adenovirus composition, its preparing method and use
  • Cationic elaioplast and its adenovirus composition, its preparing method and use

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Embodiment 1

[0044] Embodiment 1 Cationic liposome formulation and preparation process of the present invention are preferred

[0045] Cationic liposomes were selected as the target liposome type, and after preliminary screening tests, dioleoylphosphatidylethanolamine (DOPE), N-[1-(2,3-dioleoyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA), cholesterol (CHOL) as the basic components, and then optimize the formula.

[0046] The used raw material of the preparation method of liposome of the present invention and consumption are carried out optimal (seeing table 1) by the scope that initial screening obtains, process parameter and main technique are determined as follows:

[0047] According to Table 1, take 20-50 grams of DOPE, 20-50 grams of DOTMA, and 5-15 grams of cholesterol. The above raw materials are all solid; dissolve them with an organic solution of chloroform and methanol, and rotate them at a temperature not exceeding 40°C. Vacuum dry for 2 hours, add sterilized water, an...

Embodiment 2

[0052] The preparation of embodiment two cationic liposomes of the present invention

[0053] Dissolve 60mg of DOPE, 60mg of DOTMA and 6mg of cholesterol in 100ml of organic solvent of chloroform and methanol (volume ratio is 3:1, V / V), the temperature does not exceed 40 ℃, rotary evaporation, vacuum drying for 2 hours, add Sterilized water, under the protection of nitrogen, ultrasonically disrupted to obtain cationic liposomes. After aliquoting, store at 4°C-8°C for future use.

[0054] The transmission electron micrograph of the prepared cationic liposome is shown in figure 1 , it can be seen that the cationic liposome prepared by this method presents a multilayer vesicular structure with a particle size within a certain range. Prepared cationic liposomes are detected with a Malvern particle size analyzer, and the distribution diagram of the particle size curve is shown in figure 2 , it can be seen that the particle size of this cationic liposome is about 100nm, the dist...

Embodiment 3

[0055] Example 3 Construction and preparation of recombinant human endostatin adenovirus

[0056] The flow chart for the construction of recombinant human endostatin adenovirus is shown in Figure 4 (Refer to the published Chinese patent application for the construction process and the involved gene sequences, vectors, instruments and reagents: 200510021720 A recombinant human endostatin adenovirus and its preparation method and use), the process is as follows:

[0057] a. Constructing the coding sequence of human endostatin and the cloning vector pUC18 into pUC18-endo;

[0058] b. Using pUC18-endo as a template, use the PCR primers synthesized in step a to amplify the recombinant human endostatin gene containing IL-2 gene signal peptide.

[0059] c. Constructing the recombinant human endostatin gene obtained in step b into the pAdenoVator-CMV5 shuttle vector;

[0060] d. Co-transform Escherichia coli with the vector obtained in step c and the plasmid pAdenoVatorΔE1E3 contai...

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Abstract

A cationic liposome is proportionally prepared from DOPE, DOTMA and cholesterol and can be used to encapsulate the object (adenovirus carrier) to obtain a recombinant human endostatin adenovirus-cationic liposome composition carrying the active antineoplastic genes for suppressing the growth of tumor. Its preparing process is also disclosed.

Description

technical field [0001] The invention belongs to the field of gene therapy pharmaceutical preparations, and specifically relates to a cationic liposome, its adenovirus complex, their preparation method and application. Background technique [0002] Adenoviral vectors are currently one of the most potential viral vectors in gene therapy. They have a wide range of target cell types, the ability to efficiently infect dividing cells and non-dividing cells, and high import efficiency, which is not available in retroviral vectors. ; ② easy preparation and purification, high titer, large capacity, ultracentrifugation can produce high titer (10 13 PFU / ml) virus vector; ③The foreign gene carried is not integrated into the host chromosome, avoiding the risk of insertion mutation; ④It can carry a large-capacity foreign fragment (up to 36kb), which is much higher than that of retrovirus ; ⑤ At the same time, adenovirus can efficiently carry out gene transfer in exvivo and invivo pathway...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K47/28A61K47/18A61K9/127A61P35/00
Inventor 杨莉魏于全陈俐娟赵霞
Owner SICHUAN UNIV
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