(611b) Ultra-Deformable Liposomes for Enhanced Drug Delivery

Introduction: Breast cancer is the second leading cause of cancer related deaths of women in the US [1]. Further, triple negative breast cancer (TNBC) has the highest likelihood of metastasis and poor prognosis when central fibrosis is present [2]. Our lab has demonstrated that nanoparticle elasticity plays a role in the regulation of cellular uptake [3]. Liposomal stiffness can be modulated by altering the composition of the lipid bilayer. This can be achieved by incorporating lipids of different acyl chain lengths or degrees of saturation, as well as the use of edge activators (i.e. surfactants). The incorporation of short acyl chain length phospholipids (less than 14) with unsaturated lipids, may disrupt packing of lipids within the bilayer, consequently increasing fluidity [4]. It is hypothesized that hypo-elastic, ultra-deformable liposomes (UDLs) are internalized preferentially due to two unique capabilities: (1) internalization via fusion with the cell membrane (a process of low energy dependence) [3] and (2) squeezing through small pores to permeate tissues [5]. Novel UDL formulations developed herein, have demonstrated significantly higher cell uptake in vitro and tumor accumulation in vivo relative to control liposomes.

Materials and Methods: UDLs were formulated with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) or 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1% lipophilic dye, either benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-perchlorate (DiO) or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) (DiR) for visualization in vitro or in vivo, respectively. Liposomes were synthesized using the thin film hydration method. Liposomal size and surface charge were determined via dynamic light scattering (DLS) and phase analysis light scatterin (PALs), respectively, using a Brookhaven Zeta-PALS analyzer (Brookhaven Instruments, Holtsville, NY, USA). To study the internalization of UDLs, MDA-MB-436, MDA-MB-231, 4T1, and MCF10A cells were seeded at 50,000 per well in a 24 well plate and incubated overnight at 37°C. Cells were treated with 200 μM lipid of UDLs for 4 hours at 37°C. Cells were rinsed twice with PBS prior to analysis with flow cytometry (Beckman Coulter, CytoFLEX). Quantification of liposome internalization by cells was determined by DiO fluorescence (ex/em: 484 nm), normalizing for background fluorescence. For in vivo studies, 6-8 week old female balb/c mice were implanted with subcutaneous orthotopic tumors in the mammary fat pad using 1 x 10⁶ 4T1 cells. Tumors were allowed to grow for 2-3 weeks until they reached 200 mm³ in volume. Mice were treated with 10 mg/kg UDL and imaged using an in vivo imaging system (IVIS) at 4, 8, 24, 48, 72, and 96 h post injection. Organs and tumors were excised and imaged ex vivo to determine liposomal biodistribution.

Results and Discussion: Novel UDL formulations were prepared using a combination of DOPC or DPMPC and short chain phospholipids (DHPC or DPMPC). Liposomes were characterized by size, polydispersity, and zeta potential (Fig 1a). UDLs demonstrated increased uptake in TNBC cells relative to DOPC or DPMPC controls, More specifically, DOPC-DHPC or DDPC liposomes demonstrated a 6-fold increase in MDA-MB-231 and MDA-MB-463 cell uptake compared to DOPC liposomes (Fig 1b). In vivo studies demonstrated that DPMPC-DHPC and DPMPC-DDPC UDLs increased tumor accumulation 1.5 and 2-fold relative to controls (Fig 1c).

Conclusions: The novel UDL formulations developed herein demonstrated enhanced cell uptake in breast cancer cells in vitro and increased tumor accumulation in vivo. In future studies, we will explore the effects of PEGylation on DPMPC UDLs in vivo.

References: [1] Barsky, S. H., Rao, C. N., Grotendorst, G. R., & Liotta, L. A. (1982). The American journal of pathology, 108(3), 276. [2] Takai, K., Le, A., Weaver, V. M., & Werb, Z. (2016). Oncotarget, 7(50), 82889–8290. [3] Guo, P., et al (2018). Nature communications, 9(1), 1-9. [4] Hussain, A., et al. (2017). International journal of nanomedicine, 12, 5087–5108. [5] Cevc, G., Schätzlein, A., & Richardsen, H. (2002). Biochimica et Biophysica Acta (BBA)-Biomembranes, 1564(1), 21-30.