Purpose: Glioblastoma multiforme (GBM) is a grade IV brain tumor that exhibits low patient survival of less than 15 months and poor chemotherapeutic response due to the presence the blood brain barrier (BBB). Current efforts in GBM therapy are focused on designing drug delivery systems that can overcome the BBB and actively target the tumor with multiple therapeutics and functionality. The current study aims to prepare and test two distinct liposomal delivery systems that can be able to deliver temozolomide, elacridar and RG7388 for GBM therapy in vitro.
Methods: The two different liposomal systems were prepared by two distinct techniques. The first system consisted of multilamellar liposomes (MLL) which were prepared by hydration of a thin film, followed by membrane extrusion to form unilamellar liposomes (ULL) and subsequent freeze thawing to produce the nanosized MLL. The second system consisted of a vesicle in vesicle system in the form of PLGA nanoparticles in liposomes (PLL); which were prepared by nanoprecipitation and freeze drying prior to reconstitution in the liposomal hydration buffer. The PLL were optimized for particle size by varying concentrations and types of PLGA, PVA, surfactant and organic phase as well as sonication time.
GBM targeted therapy was investigated by the surface modification of all formulations with folic acid conjugation. The folic conjugate was synthesized by reacting activated folic acid to PEG-bisamine followed by a reaction between the intermediate with cholesteryl chloroformate to generate Fol-PEG-Chol conjugate. The completion of reaction was confirmed by thin layer chromatography. The conjugate was characterized by DSC, FTIR, mass spectroscopy and 1H NMR. Folic acid concentration was measured by UV spectroscopic analysis. The addition of the conjugate to the liposomes was carried out by preparation of micelles of the Fol-PEG-Chol and tween 80.
Encapsulation efficiency for MLL was maximized by testing different initial drug concentrations. All formulations were characterized for particle size, zeta potential and liposomal lamellarity was evaluated by 31P NMR and cryo-TEM.
Results: The formation of the conjugate was confirmed by the DSC, FTIR, mass spectroscopy and 1H NMR. The conjugate yield was 94% and the folate content was determined to be 86 ± 2.6%. The particle size for the ULL was 99 ± 0.9 nm, while that of the MLL freeze thawed for 5 and 11 cycles was 135.2 ± 5.9 nm and 168.6 ± 1.5 nm, respectively. The phenomenon of change in lamellarity was confirmed with 31P NMR and cryo-TEM. However, a small oligolamellar vesicle population was also observed with increase in freeze thaw cycles.
An increase in the particle size to 204.2 ± 1.6nm and 198.4 ± 1.7nm was observed for the drug loaded conjugated MLL and PLL. A low polydispersity index was found for all formulations, while all liposomes were negatively charged with an average zeta potential of -20 ± 3.1 mV. Fig 1. shows most optimal encapsulation for all three drugs was achieved when the drug initial load was adjusted to 5mg. Further, Fig 2. shows that when the conjugate is added after the formation of the liposomes, a higher encapsulation is achieved for both formulations.
The most optimal particle size for the PLL was observed with 0.1g/dL PLGA, P-127 and 2.5% PVA for sample preparation and the sonication time was adjusted to 4 min. P-127 was selected as the surfactant, which gave lower particle size over tween 80 and F-68. Higher particle size of liposomes was observed when methylene chloride and acetone were used as the organic phase. The encapsulation efficiency of RG7388 alone in the PLL was found to be 78 ± 3.55%, while that of PLGA folate conjugated liposomes it was 25.1 ± 2.48%.
Conclusion: The present study demonstrates that the PLL system offers an enhanced method to deliver multiple drugs as compared to the MLL. While both systems show good particle size and ability to deliver temozolomide, elacridar and RG7388 simultaneously, the PLL system also offers the opportunity to allow for delayed release of the encapsulated drug due to the presence of an additional barrier for drug diffusion.
Tasneem Arsiwala– Creighton University, Nebraska