(256d) Functionalized Ferri-Liposomes for Hyperthermia Induced Glioma Targeting and Brain Drug Delivery

1. Introduction

Consisting of tight junctions between endothelial cells of the microvasculature of the central nervous system (CNS), the blood-brain barrier (BBB) keeps large microorganisms or hydrophilic molecules in the circulating blood from penetrating into the cerebrospinal fluid [1-2]. However, limited by current technologies, the delivery of therapeutic agents or imaging molecules in the brain is also blocked by these highly selective tight junctions. Currently, targeting of malignant brain tumors (such as glioblastoma multiforme (GBM)) and the delivery of therapeutic agents into the brain remains a big obstacle because of the existence of the blood-brain barrier and the difficulties in finding suitable candidates for specific tumor locating. In the current study, an optimized in vitro BBB model was established using mouse brain endothelial cells and astrocytes. This in vitro model was confirmed by an immunofluorescent (IF) stain and evaluating the trans-endothelial electrical resistance (TEER) before testing. In the meantime, superparamagnetic iron oxide nanoparticle (SPIONs) loaded thermosensitive liposomes were developed for better BBB penetration and selective targeting of the brain glioma under mild hyperthermia conditions introduced by an alternating magnetic field (AMF). To specifically target the glioma cells, a new cell-penetrating peptide (CPP) and an anti-glioma antibody (Ab) were also conjugated onto the liposome surface. With the encapsulation of SPIONs and doxorubicin (DOX) inside the functionalized liposome, a DOX drug release assay and cell uptake studies were carried out and the in vitro BBB model was used to indicate the permeability of these functionalized ferri-liposomes.

2. Materials and Methods

A. Material Synthesis and Characterization

CPP and Ab were first conjugated onto mal-DSPE-mPEG2000 by a well-established chemical reaction and the PEGylated ferri-liposomes were then prepared and encapsuled with iron oxide and DOX using the rehydration method [3]. Briefly, lipids were dissolved in chloroform and vacuum-dried overnight. Then, thin lipid films were rehydrated with an ammonium sulfate solution (PH=5.5) and extruded through 100nm filters to determine their diameters. The liposome solution was then gel-filtered with a HEPES buffer (PH=7.4) and DOX was loaded into the liposomes during an overnight incubation due to the pH gradient effect. The nanoparticles were then characterized by zeta potential to determine their charge and dynamic light scattering for hydrodynamic diameter. TEM was used to assess the iron oxide inner core diameter as well as the liposome diameter.

B. Drug Release Assay and Experimental Samples Tested Through the Blood-Brain Barrier Model

For the in vitro blood- brain barrier model, both mouse brain endothelial cells (b.End3, ATCC CRL-2299) and astrocytes (C8-D1A, ATCC CRL-2541) were first cultured in complete media (DMEM with 10% FBS and 1% P/S) in a flask at 37^oC in a humidified incubator with 5% CO₂ to reach confluency before being moved to inserts. Then, astrocytes at a 10⁵cells/cm²density were seeded onto the bottom of the 24 well plates in complete DMEM media. After 24 hours of adhesion, endothelial cells were seeded onto the upper side of the inserts at the same density and the inserts were then placed in the 24-well plates containing astrocytes. The model was evaluated and confirmed using TEER and FITC-dextran transport. After such confirmation, the permeability and DOX drug release efficiency from several functionalized, traditional thermosensitive (TTSL), lyso-thermosensitive (LTSL) and non-thermosensitive (NTSL) liposomes were tested against the BBB model in an effort to increase BBB passage and deliver anti-tumor drugs for glioblastoma multiforme effectively while diminishing cytotoxicity. Each experiment was conducted in triplicate and differences between means were determined using ANOVA followed by student t-tests.

3. Results and Discussion

A. Material Characterization

Dynamic light scattering revealed that the hydrodynamic diameters of the samples ranged from 100nm to 150nm. TEM images show that the iron core was about 5-10nm for all of the samples.

B. Experimental Samples Tested Through the Blood-Brain Barrier Model

The permeability of FITC-Dextran across the model confirmed that the model was successfully established and the TEER value of this co-cultured model was around 150 Ohms/cm². Results showed that the functionalized ferri-liposomes had great permeability using the in vitro BBB model. In addition, compared to NTSL and TTSL, functionalized LTSL had significantly higher drug release efficiency and cell uptake ability towards tumor cells at its transition temperature (42 ^oC) induced by the AMF.

4. Conclusions

An in vitromodel of blood-brain barrier was established using a co-culture method. The model was confirmed by comparing the permeability trend of FITC-dextran in serum-free medium and the TEER value with previous research. The results suggest a possibility to manipulate magnetic nanocarriers penetration across the blood-brain barrier by modifying the surface chemistry. Moreover, among all the thermosensitive nanocarriers, LSTL is suggested to be a good candidate for brain tumor drug delivery based on its good drug release results and BBB penetration results. Such data lay the foundation for the modification of liposomes to increase glioma targeting efficiency.

5. Acknowledgment

The authors would like to thank Northeastern University for funding this research.

6. References

[1] Christian P, et al. Magnetically enhanced nucleic acid delivery. Ten years of magnetofectionâ??Progress and prospects, Advanced Drug Delivery Reviews. 2011; 63: 1300-1331.

[2] Richard G, et al. BOLD functional MRI in disease and pharmacological studies: room for improvement?, Magnetic Resonance Imaging. 2007; 25: 978-988.

[3] Li L, et al. Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy. J of Controlled Release. 2013;168(2):142-150.