Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells

Abstract

Lipid nanoparticles are used widely as anticancer drug and gene delivery systems. Internalizing into the target cell is a prerequisite for the proper activity of many nanoparticulate drugs. We show here, that the lipid composition of a nanoparticle affects its ability to internalize into triple-negative breast cancer cells. The lipid headgroup had the greatest effect on enhancing cellular uptake compared to other segments of the molecule. Having a receptor-targeted headgroup induced the greatest increase in cellular uptake, followed by cationic amine headgroups, both being superior to neutral (zwitterion) phosphatidylcholine or to negatively-charged headgroups. The lipid tails also affected the magnitude of cellular uptake. Longer acyl chains facilitated greater liposomal cellular uptake compared to shorter tails, 18:0 > 16:0 > 14:0. When having the same lipid tail length, unsaturated lipids were superior to saturated ones, 18:1 > 18:0. Interestingly, liposomes composed of phospholipids having 14:0 or 12:0-carbon-long-tails, such as DMPC and DLPC, decreased cell viability in a concertation dependent manner, due to a destabilizing effect these lipids had on the cancer cell membrane. Contrarily, liposomes composed of phospholipids having longer carbon tails (16:0 and 18:0), such as DPPC and HSPC, enhanced cancer cell proliferation. This effect is attributed to the integration of the exogenous liposomal lipids into the cancer-cell membrane, supporting the proliferation process. Cholesterol is a common lipid additive in nanoscale formulations, rigidifying the membrane and stabilizing its structure. Liposomes composed of DMPC (14:0) showed increased cellular uptake when enriched with cholesterol, both by endocytosis and by fusion. Contrarily, the effect of cholesterol on HSPC (18:0) liposomal uptake was minimal. Furthermore, the concentration of nanoparticles in solution affected their cellular uptake. The higher the concentration of nanoparticles the greater the absolute number of nanoparticles taken up per cell. However, the efficiency of nanoparticle uptake, i.e. the percent of nanoparticles taken up by cells, decreased as the concentration of nanoparticles increased. This study demonstrates that tuning the lipid composition and concentration of nanoscale drug delivery systems can be leveraged to modulate their cellular uptake.

Keywords: Cancer; Cell signaling; Lipid; Liposome; Metabolism; Targeting.

Figure 1. The uptake of liposomes (100μM) composed of various lipid compositions by triple negative 4T1 breast cancer cells.

(A) Schematic representation of the systematic screening approach of the study. The effect of lipid head moieties, fatty acid chains length and saturation, and cholesterol on the cellular uptake were studied. (B) The effect of different lipid head groups (PA, PE, PC, PS and PG) on cellular uptake were quantified over time. (C) The effect of the lipid tail fatty acid saturation was compared. (D) The effect of the acyl chain length on cellular uptake was studied. Phospholipids with different fatty acyl chain length (18, 16 and 14-carbon-long tails) were compared (I) over 24 hours, using flow cytometry (II). Error bars represent standard deviation from 3 independent repeats. *Significant difference between the reference formulation and the other formulations, where *p<0.05, **p<0.01, ***p<0.001 according to a Student’s t-test with a two-tailed distribution with equal variance.

Figure 2. Effect of cholesterol and the ratio of liposomes-per-cell on the cellular uptake.

(A, B) The uptake of liposomes composed of HSPC (18:0) or DMPC (14:0), with or without cholesterol, was studied at 4°C and 37°C. (C) CryoTem images of the effect of cholesterol on a DPPC liposome structure, transforming from a faceted to rounded structure upon adding 40mole% cholesterol into the membrane. (II) DMPC liposomes without/with cholesterol (40mole%). Scale bars represent 10nm. (D) (I) 4T1 cells were incubated with HSPC liposomes at increasing concentrations and the uptake was recorded. (II) The efficiency of liposomal uptake (the percent of liposomes taken up from the solution relative to their concentration in the media) was measured. Error bars represent standard deviation from 3 independent repeats. *Significant difference, where *p<0.05, **p<0.01, ***p<0.001 according to a Student’s t-test with a two-tailed distribution with equal variance.

Figure 3. The effect of the liposome lipid composition on the viability of cancer cells.

(A) Cancer cell viability 48-hours after incubating with different liposomes(5mM), with and without cholesterol, normalized to the viability of untreated breast cancer cells. (B) Cancer cell proliferation rate as a function of treatment with different formulations. (C) The effect of DMPC liposomes(5mM), enriched with cholesterol at different concentrations, on the viability of triple-negative 4T1 cancer cells. (D) The effect of DMPC liposomes at different concentrations(0.1mM-5mM) on the viability of 4T1 cells. (E) The effect of DLPC (12:0) liposomes on cancer cell viability. (F) Cell membrane permeability, measured using propidium iodide fluorescence intensity over time after incubating 4T1 cells with DMPC liposomes. (G) Cell cycle analysis by flow cytometry after incubating the cells with HSPC, DPPC and DMPC-liposomes compared to untreated cell. The percentage of cells in the G1(protein synthesis phase), S (DNA synthesis phase) and G2(pre mitosis) phases are presented. Error bars represent standard deviation from 3 independent repeats. *Significant difference between the untreated group and the other formulations treated groups, where *p<0.05, **p<0.01, ***p<0.001 according to a Student’s t-test with a two-tailed distribution with equal variance.

Figure 4. The lipid formulation can destabilize the membrane of the cancer cell.

(A) Representative images of DMPC and HSPC-liposomes (5mM) uptake by 4T1 cells after a 48 hr incubation. Liposome’s lipid layer was labeled red (Rhodamine), cell nucleus was labeled blue (Hoechst) and cell membrane was labeled green (Alexa Fluor 488), overlay images, scale bars represent 20μm. (B) Representative images demonstrate membrane destabilization after incubation with DMPC-liposomes after 48 hours, scale bars represent 2μm.