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Lung gene delivery system and preparation method and application thereof

A gene delivery and lung technology, applied in gene therapy, pharmaceutical formula, drug delivery, etc., can solve the problems of weakened anti-inflammatory effect, inability to achieve mucus layer penetration, inability to achieve gene transfection, etc., and achieve high-efficiency gene transfection Effect

Active Publication Date: 2020-10-09
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Thus, positively charged nanocomplexes capable of efficiently mediating cell membrane penetration are trapped by mucin in the mucus layer through electrostatic adsorption, leading to weakened anti-inflammatory effects in vivo
The commonly used method in the prior art to overcome the mucus layer barrier is mainly to modify the surface of the delivery carrier by PEGylation to increase the level of penetration into the mucus layer by reducing the interaction between the gene delivery system and mucin; however, the PEGylated The delivery system also reduces its uptake by cells, making it impossible to achieve efficient gene transfection
The commonly used gene delivery methods in the prior art are mainly viral vectors and non-viral vectors. Among them, viral vectors have potential safety hazards such as immunogenicity, and non-viral vectors such as liposomes and cationic polymers have low efficiency; High transfection efficiency often fails to achieve efficient mucus layer penetration

Method used

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  • Lung gene delivery system and preparation method and application thereof
  • Lung gene delivery system and preparation method and application thereof
  • Lung gene delivery system and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Dissolve siTNF-α and DPLL in DEPC water (pH 7.4) at concentrations of 0.1 mg / mL and 0.5 mg / mL, respectively. Then the DPLL solution was added to the siTNF-α solution at different ratios (DPLL / siTNF-α = 0.5 / 1, 1 / 1, 2 / 1, 5 / 1, 10 / 1 and 20 / 1, w / w), Vortex for 5 s and incubate at 37 °C for 30 min to obtain DsT solution. For RCDsT ternary nanocomposites, RC was dissolved in DEPC water (pH 7.4) at a concentration of 10 mg / mL at different ratios (RC / siTNF-α = 1 / 1, 2 / 1, 5 / 1, 10 / 1, 20 / 1, 30 / 1 and 40 / 1, w / w) were added to the prepared DsT solution (DPLL / siTNF-α = 5 / 1, w / w), vortexed for 5 s, 37 °C The RCDsT ternary nanocomposite was obtained by incubating for 30 min.

Embodiment 2

[0059] RAW 264.7 cells were seeded into 96-well plates at a density of 2 × 10 4 / Wells were incubated with DMEM containing 10% FBS at 37°C, 5% CO 2 Incubate under the condition for 24 h. Add FAM-siRNA-containing RGDsT (w / w / w = 40 / 5 / 1) nanocomplexes and RCDsT (w / w / w = 40 / 5 / 1) nanocomposite. Continue to incubate at 37°C for 4 h, rinse with PBS containing sodium heparin (20 U / mL) three times, and lyse with RIPA lysate (100 µL / well). respectively with a microplate reader ( lambda ex / lambda em = 492 / 518 nm) and BCA kit to determine FAM-siRNA and protein content. Cell uptake efficiency is expressed in μg FAM-siRNA / mg protein.

[0060] RAW 264.7 cells were seeded into 6-well plates at a density of 4 × 10 5 / well, and cultured in DMEM containing 10% FBS at 37°C, 5% CO 2 Incubate under the condition for 24 h. Add FAM-siRNA-containing RGDsT (w / w / w = 40 / 5 / 1) nanocomplexes and RCDsT (w / w / w = 40 / 5 / 1) nanocomposite. Continue to incubate at 37 °C for 4 h, rinse with PBS c...

Embodiment 3

[0067]DsT (w / w = 5 / 1, 2 μg Cy3-siRNA, 20 μL) nanocomplex containing Cy3-siRNA and RCDsT (w / w / w = 40 / 5 / 1, 2 μg Cy3-siRNA, 20 μL) nanocomposites were fully mixed with cystic fibrosis patient sputum (3%, w / w, 200 μL), and incubated at 37 °C for 30 min. Then the sample was transferred to a small dish dedicated to CLSM, observed under a microscope, and the Brownian motion of each group of nanoparticles was recorded. The recording time was 20 s, and the data analysis software was Imaris. The Brownian motion of DsT nanocomposites and RCDsT nanocomplexes in sputum was observed by multi-particle tracking technique (MPT). Such as Figure 9 As shown in a, the Brownian motion of the DsT nanocomposite was severely hindered in sputum, while the Brownian motion of the RCDsT nanocomposite was more severe. mean square displacement ( ) shows quantitatively that when the particle motion time is 10 s, the RCDsT particles About 1000 times that of DsT particles ( Figure 9 b). Furthermore, ...

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Abstract

The invention provides a lung gene delivery system as well as a preparation method and application thereof. The lung gene delivery system has the capability of efficiently overcoming mucus layers andcell membrane barriers. RBP and cis-aconitic anhydride are used for preparing RC, a polymer solution and a nucleic acid solution are mixed and incubated, then the RC solution is added, and incubationis conducted to obtain the lung gene delivery system. An RCDsT nano-composite is used for treating ALI diseases, after the RCDsT nano-composite is injected into the lung airway, the RCDsT nano-composite can effectively penetrate through the mucus layer and is effectively taken in by cells, and therefore, siTNF-alpha and RBP combined anti-inflammatory treatment for ALI is achieved. Briefly speaking, the work provides another method for solving the contradiction between mucus permeation and cellular uptake, and a new method capable of realizing oligopeptide / siRNA co-delivery is provided for treating ALI in a mode of direct administration in the lung airway.

Description

technical field [0001] The invention relates to the fields of gene loading and short peptide delivery, in particular to a preparation method and application of a lung gene delivery system RCDsT that efficiently overcomes mucus layer and cell membrane barriers, and is used for the treatment of acute lung injury. Background technique [0002] In the strategy of using pulmonary airway drug delivery to treat ALI, due to the existence of the mucus layer barrier, the traditional gene delivery system often cannot achieve efficient mucus layer penetration while meeting high transfection efficiency. Prior art CN102178957A discloses a respiratory tract homing siRNA atomized nano drug delivery system. The drug delivery system uses albuterol guanidinated chitosan after ultrasonic atomization treatment as a carrier material to wrap siRNA that interferes with the target disease-causing gene It is made, and also discloses the preparation method of the above-mentioned airway homing siRNA at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K31/713A61K9/00A61K47/34A61K47/42A61P11/00C08G73/02
CPCA61K31/713A61K9/007A61K47/34A61K47/42A61P11/00C08G73/028
Inventor 殷黎晨杨剑东
Owner SUZHOU UNIV
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