EGFR-oriented monoclonal antibody modified PAMAM self-assembled transgenic composition and applications thereof

A composition and transgenic technology, applied in gene therapy, drug combination, medical preparations of non-active ingredients, etc., can solve the problems of increasing the steric hindrance of the combination of PAMAM molecules and DNA molecules, reducing transfection efficiency, and occurrence of hemolysis , to reduce cytotoxicity and hemolysis, promote tumor cell apoptosis, and inhibit tumor cell proliferation

Inactive Publication Date: 2014-12-10
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Many studies have shown that the PAMAM nano-gene delivery system has a good application prospect in in vitro transfection, but there are still several problems to be solved in the in vivo application of PAMAM as a nano-gene delivery carrier: 1) the transfection efficiency is relatively low; 2) In the in vivo environment, PAMAM can non-specifically bind a large number of negatively charged macromolecules and red blood cells, affecting transfection efficiency and causing hemolysis; 3) How to realize the targeting of PAMAM nano-gene delivery system
[0006] In order to solve the above problems, current studies mostly directly modify PAMAM molecules, such as modifying residues such as ornithine on the surface of...

Method used

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  • EGFR-oriented monoclonal antibody modified PAMAM self-assembled transgenic composition and applications thereof
  • EGFR-oriented monoclonal antibody modified PAMAM self-assembled transgenic composition and applications thereof
  • EGFR-oriented monoclonal antibody modified PAMAM self-assembled transgenic composition and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, preparation mAb / PAMAM G5 / DNA complex

[0053] 1. PAMAM G5 / DNA complex

[0054] First, the 10 mg / mL stock solution of PAMAM G5 (number average molecular weight: 28824.81) prepared above was dissolved in sterilized distilled water, oscillated and mixed to prepare a 1 mg / mL working solution, and stored at 4°C for later use. Before transfection, take 1.0 μg of pEGFP-N1 plasmid DNA (pEGFP-N1 plasmid was purchased from Clontech Company) and put it into EP tubes, add 0.7 μL, 1.4 μL, 3.5 μL, 7 μL, 14 μL, 21 μL, 28 μL and 42 μL respectively at a concentration of 1 mg / Add 500 μL of opti-MEM medium (purchased from invitrogen, catalog number 11058-021) to 5 mL of PAMAM G5 working solution, mix well and incubate at room temperature for 30 min to form a PAMAM G5 / DNA complex. In the formed PAMAM G5 / DNA complex, the nitrogen / phosphorus ratios of PAMAM G5 and DNA were 1:1, 2:1, 5:1, 10:1, 20:1, 30:1, 40:1 and 60, respectively. :1.

[0055] 2. mAb / PAMAM G5 / DNA complex a...

Embodiment 2

[0070] Example 2, Cytotoxicity Evaluation of mAb / PAMAM G5 / DNA Complex

[0071] The mAb / PAMAM G5 / DNA complex prepared in the above example 1 was subjected to cytotoxicity experiments. In the mAb / PAMAM G5 / DNA complex, the nitrogen / phosphorus ratios of PAMAM and DNA were 1:1, 5:1, and 10:1, respectively. , 20:1, 40:1 and 60:1; the mass ratio of mAb to DNA is 1:1. The cell proliferation was detected by MTT assay, the specific method is as follows:

[0072] HepG2, MCF-7, and 293T cells (purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated into 96-well plates at 10,000 cells per well, and transfected according to the optimal mass ratio after 24 hours, and the cells were randomly divided into Normal control group, PAMAM G5 / DNA group, and mAb / PAMAM G5 / DNA transfection group (the plasmid concentration is 0.5 μg / mL, each group has 6 replicate wells, repeated 3 times, and treated for 24 hours). The morphological...

Embodiment 3

[0077] Example 3. Application of mAb / PAMAM G5 / DNA complex in targeting DNA into cells

[0078] 1. mAb / PAMAM G5 / DNA complex improves DNA transfection efficiency and cell targeting

[0079] 1. The mAb / PAMAM G5 / DNA complex introduced DNA into HepG2 cells expressing EGFR

[0080] Prepare following compound according to the method for embodiment 1:

[0081] mAb / PAMAM G5 / DNA complex: the nitrogen / phosphorus ratio of PAMAM to DNA is 10:1, and the mass ratio of mAb to DNA is 0 (without monoclonal antibody), 0.1:1, 0.5:1, 1:1, 2:1, 5:1;

[0082] mAb / PAMAM G5 / DNA complex: the nitrogen / phosphorus ratio of PAMAM to DNA is 20:1, and the mass ratio of mAb to DNA is 0 (without monoclonal antibody), 0.1:1, 0.5:1, 1:1, 2:1, 5:1;

[0083] mAb / PAMAM G5 / DNA complex: the nitrogen / phosphorus ratio of PAMAM to DNA is 40:1, and the mass ratio of mAb to DNA is 0 (without monoclonal antibody), 0.1:1, 0.5:1, 1:1, 2:1, 5:1;

[0084] The mAb / PAMAM G5 / DNA complex prepared in Example 1 and the above-m...

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Abstract

The invention discloses an EGFR-oriented monoclonal antibody modified PAMAM self-assembled transgenic composition and applications thereof. The invention provides a composition for nucleic acid transgenosis, which is prepared by compounding a polyamidoamine-dendritic polymer of which the tail end is provided with amines, an EGFR-oriented monoclonal antibody, and a nucleic acid. The composition disclosed by the invention has the following advantages that 1) by taking an epidermal growth factor receptor (EGFR) as a therapeutic target, and using a monoclonal antibody and tumor cell EGFR induced endocytosis, the targeting of a PAMAM carrier to cells is increased; 2) through the modification of PAMAM by the monoclonal antibody (mAb), the ultra-strong positive charge intensity of the PAMAM carrier can be reduced, thereby facilitating the reduction of occurrence of cytotoxicity and hemolysis phenomena; and 3) an mAb/PAMAM G5/DNA composition is prepared by using a self-assembling method, and compared with chemical synthesis, the molecular self-assembling method can carry out modification on a system more conveniently and flexibly, and keep the bioactivity of ligands.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PAMAM self-assembled transgene composition modified by a monoclonal antibody against EGFR and its application. Background technique [0002] The emerging gene therapy method is to transfer exogenous gene fragments such as plasmid DNA, oligonucleotide, and small interfering RNA into the cell, regulate gene expression to correct the structural or functional disorder of the human gene, and prevent the progression of the disease. progress, kill diseased cells, so as to achieve the purpose of treatment. Because gene therapy can overcome the bottleneck of traditional treatment methods, gene therapy technology has been highly valued by various scientists and governments since its inception, and its development has been particularly rapid. , major malignant infectious diseases, genetic diseases) frontiers and hotspots. [0003] Vector technology has always been one of the core areas of g...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/48A61K47/42A61P35/00C12N15/87
Inventor 郝艳丽张小宁李军
Owner TSINGHUA UNIV
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