Novel acid-sensitive nano-carrier simultaneously loaded with siRNA and cis-platinum prodrug as well as preparation method and application thereof
A technology of prodrug and endosome, applied in the field of new acid-sensitive nanocarriers and its preparation, can solve the problems of small capacity, difficult preparation and strong immunogenicity of siRNA, and achieve the effect of increasing chemotherapy sensitivity and accelerating intracellular release
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Embodiment 1
[0045] A novel acid-sensitive nanocarrier loaded with siRNA and cisplatin prodrug at the same time, its preparation process is as follows:
[0046] (1) Synthesis of brominated polyethylene glycol (Meo-PEG-Br):
[0047] Dissolve polyethylene glycol (Meo-PEG-OH) and triethylamine in dichloromethane, add α-bromoisobutyryl bromide dropwise in an ice-salt bath, stir and react at room temperature for 20 to 30 hours, Wash the reaction solution with 50ml of sodium hydroxide and hydrochloric acid aqueous solution, and finally wash with deionization, collect the organic phase, dry with anhydrous magnesium sulfate, concentrate the solution, add cold ether to precipitate the product, and collect the white powder product after vacuum drying; Wherein, the consumption of polyethylene glycol, triethylamine, α-bromoisobutyryl bromide is respectively 1.6mmol, 9mmol, 9mmol, and the concentration of sodium hydroxide and hydrochloric acid aqueous solution is 1.0mol / L; figure 1 is the H NMR spectr...
Embodiment 2
[0056] According to the above method, the fluorescently labeled siRNA (such as cy5-siRAN / FAM-siRAN, which can be ordered directly from Gemma Gene) was loaded into the nanoparticles, and after ultrafiltration and washing 3 times, the obtained upper layer nano solution was dispersed in 1mL PBS. This nanoparticle solution was transferred into dialysis tubing, which was then placed in 37°C aqueous PBS (pH=7.4 or pH=6.0). At the indicated time points, 5 μL of the nanoparticle solution was withdrawn and added to 100 μL of the DMSO solution. Measure the fluorescence intensity of the fluorochrome using a spectrofluorometer. The cumulative release rate of siRNA is calculated by the following formula: cumulative release (%)=(M t / M ∞ )×100; where M t is the siRNA released from the nanoparticles at the indicated time points, M ∞ is siRNA loaded in nanoparticles. Figure 6 is the controlled release profile of siRNA from nanoparticles, given by Figure 6 It can be seen that the nano...
Embodiment 3
[0058] The cells were seeded in a 6-well plate, and 1 mL of medium containing 10% fetal calf serum was added to incubate for 24 hours to allow them to fully adhere to the wall. Nanoparticles loaded with fluorescent dye-labeled siRNA and cisplatin prodrug were then added. After 4 hours of incubation, the medium was removed and the cells were washed three times with PBS. Lysotracker green and Hoechst 33342 were added to mark endosomes and nuclei, respectively, and the endosome escape of nanoparticles was observed by confocal laser microscopy. Figure 7 Fluorescent photos of nanoparticles loaded with fluorescent dye-labeled siRNA and cisplatin prodrug and cells incubated for 1 hour, 3 hours and 6 hours. Depend on Figure 7 It can be seen that siRNA can escape from endosomes and enter the cytoplasm as time goes by. The experimental results prove that the above-mentioned synthesized nanocarriers can respond to the weak acid environment, which is beneficial for siRNA to enter the ...
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