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Preparation method and application of protein-polymer composite nanomaterial gene vector

A technology of composite nanomaterials and polymers, applied in the construction and application of gene delivery carriers, can solve the problems of poor biocompatibility and biodegradability, complex synthesis process, and low transfection efficiency

Active Publication Date: 2021-05-18
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although remarkable progress has been made in the design of polymeric carriers, research in this field has been plagued by complex synthesis processes, low transfection efficiency, poor biocompatibility and biodegradability, etc.

Method used

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  • Preparation method and application of protein-polymer composite nanomaterial gene vector
  • Preparation method and application of protein-polymer composite nanomaterial gene vector
  • Preparation method and application of protein-polymer composite nanomaterial gene vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation and characterization of protein-polymer composite nanomaterials

[0038] 1. Preparation method

[0039] 1) Dissolve bovine serum albumin (Sigma-Aldrich#SRE0096) in pH 8.5, 100mM borate buffer, and pass through a 30kD ultrafiltration tube (Amicon#UFC5030BK) to change the solution by centrifugation four times in this buffer to Remove a small amount of NH4 that may exist in the protein + , Diquinoline formic acid (BCA) method to measure the concentration and use this buffer to dilute the protein to 10mg / ml.

[0040] 2) Dissolve N-hydroxysuccinimide acrylate (Sigma-Aldrich#A8060) in dimethyl sulfoxide (Sigma-Aldrich#276855) to make a 20mg / ml solution, take 5.1μl and 100μl of 10mg / ml bovine serum The albumin solution was mixed evenly (N-hydroxysuccinimide acrylate: bovine serum albumin = 40:1 molar ratio), and reacted at room temperature 25°C for 6 hours to obtain protein solution A with surface double bond modification.

[0041]3) Dissolve acrylami...

Embodiment 2

[0046] Example 2: Application of protein-polymer composite nanomaterials to present genes

[0047] The composite nanomaterial prepared in Example 1 can be applied to gene transfection. The process is as follows:

[0048] 1) Mix the above-mentioned composite nanomaterial with nucleic acid, and place it at room temperature for 20 minutes to obtain a carrier / nucleic acid complex.

[0049] 2) The vector / nucleic acid complex was added to the serum-free medium of HEK293T cells in the exponential growth phase, and after 4 hours of transfection, the medium was replaced with a complete medium, and the culture was continued for 4 days.

[0050] 3) Observe the gene transfection effect of the vector in the cells by means of flow cytometer and fluorescence microscope.

[0051] The following are the specific experimental steps:

[0052] 1. Cellular Internalization of Composite Nanomaterial Carriers / Nucleic Acids

[0053] Before the internalization experiments of the composite nanomateri...

Embodiment 3

[0056] Embodiment 3: the optimization of preparation material of protein-polymer composite nanomaterial

[0057] 1. Optimal selection of crosslinking agent

[0058] Only replace the cross-linking agent, adopt the same preparation method as in Example 1 (the cross-linking agent of B4 is N,N'-methylenebisacrylamide) to prepare several other material systems, where B5 is prepared without adding a cross-linking agent , the crosslinking agent of B6 is poly(DL-lactide)-b-poly(ethylene glycol)-b-poly(DL-lactide)-diacrylate triblock copolymer (PolySciTech#AI102), The crosslinker for B7 was glyceryl dimethacrylate (Sigma-Aldrich #436895). The molecular structure of each crosslinking agent is as follows Figure 6 as shown in a.

[0059] Figure 6 b is the water contact angle of each material system measured by a surface interfacial tensiometer (DCAT21, Dataphysics). The larger the contact angle, the higher the hydrophobicity of the material. The results showed that N,N'-methylenebi...

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Abstract

The invention discloses a protein-polymer composite nanomaterial gene vector as well as a preparation method and application thereof. The vector is obtained by performing in-situ polymerization on a macromolecular shell on the surface of protein and synthesizing a nano material with a core-shell structure. The vector can effectively compound and compress nucleic acid, can protect the nucleic acid from being degraded by nuclease, can efficiently release the nucleic acid into cytoplasm through degradation of cationic groups in a polymer shell layer, achieves extremely high gene transfection efficiency, has high biocompatibility, low cytotoxicity and designable functionality, the preparation process and the purification process are simple, and the method has a wide application prospect in the related fields of gene therapy.

Description

technical field [0001] The invention belongs to the field of nanomaterials, and in particular relates to the construction and application of a gene delivery carrier combined with a protein and a polymer. Background technique [0002] Nucleic acid is considered a superior gene therapy agent, but it cannot directly enter cells by itself and is susceptible to nuclease degradation. Vectors commonly used for nucleic acid presentation are divided into two categories, viral vectors and non-viral vectors. Nonviral gene carriers, such as cationic liposomes and polymers, have been extensively studied due to their good safety, low immunogenicity and high gene load capacity. Although remarkable progress has been made in the design of polymeric vectors, research in this field has been plagued by complex synthesis processes, low transfection efficiency, and poor biocompatibility and biodegradability. [0003] Based on the technical problems existing in the existing carrier, the purpose ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C08F289/00C08F220/56C08F220/34C08F222/38A61K47/69A61K31/7088A61K48/00A61K47/64
CPCC12N15/87C08F289/00A61K47/6933A61K31/7088A61K48/005A61K47/643C08F220/56C08F220/34C08F222/385
Inventor 陈薇胡暄张晓鹏杨益隆
Owner ACADEMY OF MILITARY MEDICAL SCI
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