Supporting Information. Maximizing the Supported Bilayer Phenomenon: LCP Liposomes Comprised Exclusively
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1 S-1 Supporting Information Maximizing the Supported Bilayer Phenomenon: LCP Liposomes Comprised Exclusively of PEGylated Phospholipids for Enhanced Systemic and Lymphatic Delivery Matthew T. Haynes and Leaf Huang* The Center for Nanotechnology in Drug Delivery, Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC *Correspondence: Leaf Huang
2 S-2 Materials and Methods 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}12-sn-glycero-3-phosphate (NBD-PA) was purchased from Avanti Polar Lipids (Alabaster, AL). S1.1 Preparation of Fully-PEGylated LCP Nanoparticles of Variable Precipitate Core Size and PEG Chain Length. Preparation of larger (30-40 nm) CaP cores was performed following a similar method to that of smaller cores, employing instead a mixed surfactant system in the emulsion. 1 Encapsulation efficiency was characterized radiometrically using a gamma counter (the Nucleus, Oak Ridge, TN) or through a fluorescence method. For final LCP preparation, the protocol was modified in terms of cholesterol content as needed for preparation of a transparent thin film in variation of PEG chain length. For fully-pegylated DSPE-PEG 1000 and DSPE- PEG 350, dissolution in a small amount of tetrahydrofuran prior to aqueous dispersion facilitated optimal particle quality as well. S1.2 Determination of Pharmacokinetic Parameters for 100 mol% DSPE-PEG 2K LCP. For kinetics calculations, mice were assumed to possess a total blood volume of 7.5% total body weight. Mean residence time (MRT) was determined via noncompartmental analysis using Phoenix WinNonlin (Version 6.3, Pharsight Corporation; Mountain View, CA). The data for PCP incorporating PEG2000 was shown to fit well under a two-compartment model through the same software, where after the distribution and elimination half-lives were calculated from a linearization of the early and late phases of the kinetic profile. S1.3 Ex Vivo Protein Adsorption onto LCP Nanoparticles of Varying Outer Leaflet PEG Chain Lengths. LCP nanoparticles of varying PEG chain lengths were incubated with serum and saline for 2 hours at 37 C, and purified via aqueous and organic washes for BCA analysis.
3 S-3 Results were background-subtracted from solvent, and normalized to absorbance generated from the DSPE-PEG coated LCP particles (PEG2000). S1.4 Western Blot Determination of Sigma Receptor Expression Levels. A variety of cell lines were cultured to explore their overall expression levels of sigma receptor, including bladder cancer (UMUC3), unmodified breast cancer (4T1), and engineered breast cancer (4T1 GFP/Luc), using the traditionally-targeted H460 lung cancer line as a positive control. Relative receptor expression levels were characterized through a standard Western Blot protocol. S1.5 In Vitro Cell Uptake of LCP Nanoparticles. PCP and LCP nanoparticles labeled with trace amounts (1 mol%) of NBD-PA (labeling the inner leaflet of the lipid bilayer alone) and targeted with either 20% anisamide (AA) or 20% mannose (MAN) ligand, were incubated with (respectively) sigma receptor-expressing H460 lung cancer cells and mannose receptorexpressing JAWSII dendritic cells for 4 hours in DMEM containing 10% FBS at 37 C and 5% CO 2, after which cells were washed with PBS, trypsinized, washed again with PBS, and characterized for fluorescent signal with a BD FACSCanto II Flow Cytometer.
4 S-4 Figures and Tables Fig. S1A LCP Coated Exclusively by Alternative Phospholipopolymers. LCP cores of varying CaP precipitate diameters were resuspended effectively with a variety of lipid-polymer conjugates serving as the sole phospholipid in the formulation. (A) DSPE-PEG conjugates of varying PEG chain length can be employed to produce uniform dispersions of CaP precipitates. (B) CaP precipitates of larger core size are amenable to modification with DSPE-PEG 2000, although requiring moderate cholesterol (2:1 PEG:Chol) for optimal resuspension. Scale bar = 50 nm. Fig. S1B LCP Isolated via Sucrose Density Gradient Centrifugation. LCP cores were coated with DSPE-PEG 2000 containing trace amounts of both 177 Lu and one of the following components: NBD-OPA, NBD-OPC, or DSPE-PEG 2000-FITC. (A) Schematic of the centrifuge tube, with solutions of sucrose at various weight percentages established as discrete density layers. (B) Sample results with fully-pegylated LCP preparations containing either 0.3% NBD-OPA or 1% DSPE-PEG-FITC. The red arrow represents the interface between the 20% and 40% sucrose bands.
5 BCA Absorbance (norm.) S-5 PCP Particle Size (nm) PDI Zeta Potential (mv) PEG ± 8.2 PEG ± 6.4 PEG ± 6.8 PEG ± 4.3 Table S1: Characterization of PCP Liposomes. Particle size was determined via dynamic light scattering (DLS), and surface potential by laser Doppler electrophoresis, using a Malvern Zeta-Sizer (Nano-ZS) PEG2000 PEG1000 PEG350 Fig. S2A Ex Vivo Protein Adsorption onto LCP Nanoparticles of Varying Outer Leaflet PEG Chain Lengths. LCP nanoparticles of varying PEG chain lengths were incubated with serum and saline for 2 hours at 37 C, and purified via aqueous and organic washes for BCA analysis. Results were background-subtracted from solvent, and normalized to absorbance generated from the DSPE-PEG 2000-coated LCP particles (PEG2000).
6 %ID/g %ID % Injected Dose S % PEG 100% PEG Time (hours) Fig. S2B Circulation Kinetics of LCP after i.v. Injection. LCP nanoparticles encapsulating both 177 Lu and oligonucleotides were dispersed with outer-leaflet lipids from a thin film through simple addition of absolute ethanol and rapid dilution in water at 65 C. Particles were injected via tail vein into BALB/c mice and monitored radiometrically through serial sampling (n = 3) Blue = Large CaP Cores (30-40 nm) Red = Small CaP Cores (10-15 nm) Blood Liver Spleen Kidneys Lung Heart Fig. S2C Biodistribution of 177 Lu-encapsulated LCP Nanoparticles. CaP precipitates encapsulating trace amounts of 177 Lu of varying core size were modified exclusively with PEGylated phospholipids (DSPE-PEG 2000), and administered via tail vein to BALB/c mice (n = 3), with biodistribution determined radiometrically after organ harvest 24 hours post-injection.
7 Fig. S3 Systemic Nanoparticle Absorption after Subcutaneous Administration. LCP particles encapsulating trace 177 Lu were coated with either 100 mol% (PCP) or 20 mol% (LCP) DSPE-PEG 2k, modified in each case with 20% mannose ligand, and administered subcutaneously to BALB/c mice in 10% sucrose (n = 3). After 72 hours, major organs were harvested, and accumulation was determined radiometrically. *p<0.05; **p<0.01 S-7
8 S-8 Fig. S5 In Vitro Cell Uptake of LCP Nanoparticles. PCP and LCP nanoparticles containing trace amounts of NBD-OPA and targeted with either anisamide (AA) or mannose (MAN) ligand, were incubated with (respectively) sigma receptor-expressing H460 lung cancer cells and mannose receptorexpressing JAWSII dendritic cells and analyzed via flow cytometry. References 1. Tseng, Y.-C.; Xu, Z.; Guley, K.; Yuan, H.; Huang, L., Lipid-Calcium Phosphate Nanoparticles for Delivery to the Lymphatic System and SPECT/CT Imaging of Lymph Node Metastases. Biomaterials 2014, 35, 16,
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