I'm starting my PhD project, and need suggestions.
1. Here I'm working on liposome drugs modifications, which I use Lipodox (HSPC: Chol: PEG2000-DSPE= 3: 2: 0.3), when I tried for the first time, there was a white transparent membrane in the flask bottom after evaporation using HEPES 10 mm 80 microml, so far I read many references it should be clear membrane. The condition is 50 degrees C; 1 hour before adding HEPES).
2. Here also using DSPE-PEG-Maleimide (3.4 DA), which will be attached to the liposomal surface, which percentage is proper for this modification, since (point 1, has a white membrane on the flask), is this effect to the DSPE-PEG-Maleimide attachment?
3. Is there any suggestions, which better way to combine DOTAP into a liposome drug to enhance the encapsulation itself? Since using Lipodox and which best ratio condition for this?
Dear Wenny Ambarsari
You can try "Mozafari method" (see below for References***), or "Heating method" or their modifications. Note that these techniques do not require use of toxic solvents or harsh procedures.
For instance you can try this modified protocol:
The heating method ***** was combined with a gradient of pH. The lipids were hydrated in a citrate solution (pH 4), and mixed in a bath-ultrasound for 1 min. The mixture was extruded as it has been described in the previous methods, and the excess of citrate was removed by ultrafiltration (Amicon with a cut-off membrane of 10,000 MWCO membrane, Millipore, Billerica, USA). The incorporation of drug (2 mg/mL) dissolved in glucose 5% was achieved by adding the drug solution together with Hepes solution (pH 7.8). This mixture was heated at the corresponding lipids transition temperature 60º C for 30 min. Afterwards, it was cooled at 4º C.
Modified Heating Method: This method has been modified by adding citrate and Hepes buffers to reach a gradient of pH. This change was because the amount of drug incorporated into liposomes without difference of pH between inside and outside of the vesicles, was very low, (around 6%). However, when the drug was in a solution of pH 7.8, the encapsulation rate was higher than 20%.
Ref: Zalba, S., Navarro, I., Trocóniz, I. F., de Ilarduya, C. T., & Garrido, M. J. (2012). Application of different methods to formulate PEG-liposomes of oxaliplatin: evaluation in vitro and in vivo. European journal of pharmaceutics and biopharmaceutics, 81(2), 273-280.
*** Some References for "Mozafari method":
1. Colas, J.C.; Shi, W.; Rao, V.M.; Omri, A.; Mozafari, M.R.; Singh, H. Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting. Micron 2007, 38(8), 841-847, https://doi.org/10.1016/j.micron.2007.06.013
2. Putri, D.C.A.; Dwiastuti, R.; Marchaban, M.; Nugroho, A.K. Optimization of mixing temperature and sonication duration in liposome preparation. J Pharm Sci Comm 2017, 14(2), 79-85, https://doi.org/10.24071/jpsc.00728
3. Poudel, A.; Gachumi, G.; Wasan, K.M.; Dallal Bashi, Z.; El-Aneed, A.; Badea, I. Development and characterization of liposomal formulations containing phytosterols extracted from canola oil deodorizer distillate along with tocopherols as food additives. Pharmaceutics 2019, 11(4), 185, https://doi.org/10.3390/pharmaceutics11040185
4. Zarrabi, A.; Zarepour, A.; Khosravi, A.; Alimohammadi, Z.; Thakur, V.K. Synthesis of Curcumin Loaded Smart pH-Responsive Stealth Liposome as a Novel Nanocarrier for Cancer Treatment. Fibers 2021, 9(3), 19, https://doi.org/10.3390/fib9030019
***** Reference for "Heating method":
M.R. Mozafari, Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett. 10 (2005) 711-719.
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