Composite drug-loaded extracellular vesicle inhalation preparation as well as preparation method and application thereof

Pending Publication Date: 2021-06-01
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production cost of liposomes is high, the stability is poor, and the leakage of loaded drugs is prone to occur during storage.

Method used

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  • Composite drug-loaded extracellular vesicle inhalation preparation as well as preparation method and application thereof
  • Composite drug-loaded extracellular vesicle inhalation preparation as well as preparation method and application thereof
  • Composite drug-loaded extracellular vesicle inhalation preparation as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation and Characterization of TRAIL Genetically Modified MSC Stem Cells

[0033] 1. TRAIL genetic engineering modification of MSC cells: Subculture P3-P5 umbilical cord-derived mesenchymal stem cells (UC-MSCs), and the MSCs in good condition were divided into 1×10 6 Cell culture six-well plate at the density of cells / well, 37°C, 5% CO 2 overnight in the cell culture incubator; according to the method described in the inventor's published literature (Yuan et al., Cytotherapy.2015Jul; 17(7):885-96.doi:10.1016 / j.jcyt.2015.03.603.), the expression human The full-length TRAIL lentivirus was used at a virus concentration of MOI=3, and 8 μg / mL polybrene was used to enhance transfection, and TRAIL genetic engineering was performed on MSCs. After transfection and incubation for 6-24 hours (optimally 10 hours), high Cells expressing full-length TRAIL, that is, MSCflT; replace with fresh DMEM / F12 medium containing 10% FBS (fetal bovine serum), and continue to cult...

Embodiment 2

[0037] Example 2 Preparation and identification of MSC extracellular vesicles (EV-T) expressing TRAIL

[0038] 1. EV-T preparation: thaw the frozen MSCflT cell culture supernatant at 4°C, first centrifuge at low speed (4°C, 10min, 1000g) to remove dead cells and cell debris, and filter through a 0.22μm filter membrane to remove cells larger than 220nm Then use 100kD ultrafiltration centrifugation (4°C, 3000g, 10min) to concentrate the EV-T solution 3 times, and finally precipitate EV by ultracentrifugation (4°C, 120000g, 2h) -T, resuspend the EV-T pellet in PBS solution filtered through a 0.22 μm filter membrane, aliquot and freeze at -80°C for future use.

[0039] 2. Observation of EV-T morphology: Observe the morphology and size of the prepared EV-T through a transmission electron microscope. The results are as follows: figure 2 As shown, it was observed that EV-T presents a typical membrane vesicle structure of extracellular vesicles, with a diameter of about 60-100nm.

Embodiment 3

[0040] Embodiment 3 establishes the standard curve of HPLC quantitative determination Dina

[0041] First prepare 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL, 6.25μg / mL and 3.125μg / mL Dina standard samples for establishing the standard curve. The model of the chromatographic column is selected as C18 (4.6*150mm) and the particle size of the filler is 5 μm according to the references, and the mobile phase composition of the chromatographic analysis is determined as: A phase H 2 O (add 0.05% H 3 PO 4 ), phase B HPLC grade acetonitrile (adding 0.05% H 3 PO 4 ), the column temperature was 40°C, and the flow rate was 0.8mL / min. Mobile phase gradient time: 0~6min grade (5%B), 6.1~15min grade (5%~95%B), 15.1~20min grade (95%B), 20.1~25min (5%B). The detection wavelength is 220 / 254nm, and the injection volume is 20μl. HPLC detects that the main peak of its Dina appears at about 11.5min, and the standard curve result of detecting Dina is as follows image 3 As shown, the fitted standa...

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Abstract

According to the composite drug-loaded extracellular vesicle inhalation preparation and the preparation method and application thereof, the extracellular vesicle inhalation preparation is used for treating lung tumors in an oral inhalation administration mode, the residence time of treatment drugs in a body can be effectively prolonged, distribution of the drugs in other non-target tissues and organs in the body is reduced. The toxic and side effects of the medicine are reduced while the treatment effect is enhanced, and the medicine shows a good treatment effect of remarkably inhibiting the growth of drug-resistant lung tumors in an animal model.

Description

technical field [0001] The invention relates to a composite drug-loaded extracellular vesicle inhalation preparation, a preparation method and application thereof. Background technique [0002] Pulmonary inhalation drug preparation is a dosage form in which the raw material drug dissolved or dispersed in a suitable medium is delivered to the corresponding part of the lung in the form of aerosol to exert local or systemic effects. Pulmonary inhalation drug delivery is a non-invasive drug delivery method that can locally deliver antineoplastic drugs to lung tumor tissue and enter the blood through the alveolar surface. Compared with other administration methods such as oral and intravenous injection, pulmonary inhalation preparations can reduce the degradation of protein or nucleic acid drugs by various enzymes abundant in the digestive system or blood, improve pharmacokinetics, and better exert the drug's effect. Therapeutic effect. The in vivo efficacy of inhaled aerosol p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/72A61K9/12A61K47/46A61K38/17A61K31/519A61P35/00
CPCA61K9/0078A61K9/12A61K47/46A61K38/1709A61K31/519A61P35/00
Inventor 袁正强苏奎袁倩黎淑怡候欢孙健武黄超鸿
Owner GUANGDONG UNIV OF TECH
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