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Novel nano gene vector and preparation method thereof

A gene carrier and nanotechnology, which can be used in preparations for in vivo experiments, pharmaceutical formulations, and medical preparations with inactive ingredients, etc. problem, to achieve the effect of good product stability, high safety and good dispersibility

Active Publication Date: 2018-03-27
山西省肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In short, both inorganic nanomaterials and organic nanomaterials have problems such as toxicity, degradability, stability, biological safety, biocompatibility and preparation process.

Method used

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  • Novel nano gene vector and preparation method thereof
  • Novel nano gene vector and preparation method thereof
  • Novel nano gene vector and preparation method thereof

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preparation example Construction

[0035] A preparation method of a novel nano-gene carrier, the steps are as follows:

[0036] (1) Synthesis of melanin nanoparticles (MNP)

[0037] Take 20mg of melanin, add 4ml of 0.1mol / L NaOH solution to ultrasonic vibration, mix and dissolve; then slowly add 2.5ml of 0.1mol / L HCl solution, and ultrasonic vibration, adjust the pH value to 7-8, centrifuge 15 min; wash with double distilled water and centrifuge for 3-10 times; freeze-dry to obtain the water-soluble melanin nanoparticles.

[0038] (2) Modification of melanin nanoparticles

[0039] Weigh 1 mg of melanin nanoparticles, dissolve them in 1 ml of ultrapure water, and oscillate until they are completely dissolved; take 2 mg of polylysine and dissolve them in 1 ml of ultrapure water, and oscillate until they are completely dissolved. Add 1 ml of MNP aqueous solution dropwise into 1 ml of polylysine aqueous solution, and stir overnight at 25°C with magnetic force. The next day, the solution was transferred to an ult...

Embodiment 1

[0044] Synthesis and Characterization of Melanin Nanoparticles (MNPs)

[0045](1) Synthesis of melanin nanoparticles (MNP)

[0046] Dissolve 20 mg of melanin in 4 ml of sodium hydroxide solution, ultrasonically shake until completely dissolved, add 2.5 ml of hydrochloric acid to neutralize, parallel cell crushing, centrifugation, washing with water, and freeze-drying. Weigh the obtained melanin nanoparticles and calculate the yield.

[0047] (2) Characterization of melanin nanoparticles (MNP)

[0048] A. Electron microscope morphology detection: Take a small amount of MNP aqueous solution, ultrasonically disperse it and drop it on a filmed copper grid to prepare an electron microscope sample, use TEM to observe its morphology and structure, choose 20 fields of view to take pictures under the condition of 50,000 times, and then take pictures Scan to the computer, use the image analysis system to calculate the average particle size, such as figure 1 Shown is the TEM image of ...

Embodiment 2

[0053] Construction of Multifunctional Nanogene Carrier (MNP-PLL-SIRNA)

[0054] ①Modification of melanin nanoparticles

[0055] Weigh 1 mg of melanin nanoparticles, dissolve them in 1 ml of ultrapure water, and oscillate until they are completely dissolved; take 2 mg of polylysine and dissolve them in 1 ml of ultrapure water, and oscillate until they are completely dissolved. Add 1ml of MNP aqueous solution dropwise into 1ml of polylysine aqueous solution, and magnetically stir overnight at 25°C. Transfer the solution into an ultrafiltration centrifuge tube (3000KD), add ultrapure water for centrifugal washing, and centrifuge 5 times at 3500rpm / min.

[0056] ② Construction of multifunctional nanogene carrier (MNP-PLL-SIRNA)

[0057] SIRNA: First put the RNA dry powder tube in a hand-held centrifuge for centrifugation, then add 125ul DEPC water to dissolve 0.5ODRNA dry powder, make it 10pmol / ul, and distribute it for use. According to different mass ratios, 10 pmol / ul RNA a...

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Abstract

The invention relates to the field of gene treatment, and in particular relates to an MNP / PLL / SIRNA novel nano gene vector as well as preparation and dual curative effect application thereof. The novel nano gene vector is composed of melanin nano particles synthesized by a human body endogenous substance, polylysine PLL and a gene SIRNA, wherein the melanin nano particles are modified with polylysine, so that MNP-PLL is synthesized, and then MNP-PLL is combined with a negative charge SIRNA by virtue of electrostatic effect, so that the MNP / PLL / SIRNA nano gene vector is formed. The method provided by the invention has the advantages that a synthetic process is simple, and the cost is relatively low; the prepared nano composite carries a positive charge self, is small in particle size, can easily enter a human body and enter cell nucleuses to play effect, and the product is good in stability, good in dispersibility, good in biocompatibility and high in safety; and processes of entering into the human body via a caudal vein carrying out gene therapy and photothermal therapy are respectively easy to operate.

Description

technical field [0001] The invention relates to the field of gene therapy, in particular to a novel MNP / PLL / SIRNA nanometer gene carrier and its construction and dual curative effect application. Background technique [0002] Cancer, namely malignant tumor, is the primary disease that seriously endangers human health and causes people's death. Currently, cancer treatment strategies include chemotherapy, radiotherapy, surgical resection, and biological therapy. However, these measures will inevitably produce toxic side effects on normal tissues, making it difficult to continue treatment or relapse. In 2003, China's State Food and Drug Administration (SFDA) defined gene therapy as a medical treatment based on changing the genetic material of cells. [0003] In essence, gene therapy refers to the delivery of exogenous genetic material to target cells to correct or compensate for gene defects, thereby achieving therapeutic purposes. It starts from the root of the disease, dir...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K31/713A61K41/00A61K49/00A61P35/00
CPCA61K41/0052A61K49/00A61K31/713Y02A50/30
Inventor 张瑞平杨曦王斌全范博范曲立程霞高伟纪钰徐雯李小燕
Owner 山西省肿瘤医院