第一届亚洲药物制剂科学和技术研讨会

发布于:2021-07-26 14:14:24

附件 3. 论文模版 Comparison of Methods for Quantifying siRNA Encapsulated into Poly (lactide-co-glycolide) Nanoparticles Dongmei Cun , Camilla Foged , Mingshi Yang , Linda Boye Jensen , Sven Fr?kj? r , Hanne 1: Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen. Universitetsparken 2, DK-2100 Copenhagen, dcun@farma.ku.dk 2: Novo Nordisk A/S, Novo Nordisk Park B6.2.086, DK-2760 M?l?v, Denmark ABSTRACT SUMMARY: Three different strategies to determine siRNA encapsulation efficiency in PLGA nanoparticles were compared. Results show that siRNA can be quantitatively removed from PLGA by extraction. However, the indirect method tends to give false positive results because a considerable part of siRNA was lost during preparation as shown by the direct method and tracking of radio-labeled siRNA. INTRODUCTION: The encapsulation efficiency of antisense oligonucleotides (ODNs) has been measured after hydrolysis of NPs with 0.5 N NaOH [3]. Shikha et al. [4] used an extraction method applying a buffer/CHCl3 biphasic solvent system followed by UV spectrophotometry to determine the encapsulation efficiency of plasmid DNA. It was proved that the technique could quantitatively and completely remove DNA and preserve the degree of super-coiling of the encapsulated plasmid. However, an indirect method has commonly been employed to evaluate the encapsulation efficiency of DNA since the direct method is often associated with incompletely extraction [5, 6]. In the indirect method, the DNA loading of nanoparticles is determined by subtracting the amount of non-encapsulated DNA from the total amount of DNA. It is difficult to compare literature results obtained with the methods mentioned above due to different preconditions. In this present study, three different procedures for determination of encapsulation efficiency were compared to assess their applicability for siRNA-loaded PLGA NPs. EXPERIMENTAL METHODS: 1. Preparation of siRNA-loaded PLGA NPs SiRNA-loaded NPs were prepared using a double emulsion solvent evaporation technique. 2. Determination of siRNA loading percent and encapsulation efficiency 2.1 NaOH hydrolysis procedure About 2 mg of NPs were dissolved in 300 ? l 0.5 N NaOH under magnetic stirring at RT. The concentration of siRNA in the solution was determined by UV absorbance at 260 nm. 2.2 Extraction procedure About 2 mg NPs were dissolved in CHCl3 and the CHCl3 layer was extracted four times with TE buffer (p

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