Lipid Composition of the Cell Membrane Outer Leaflet Regulates Endocytosis of Nanomaterials through Alterations in Scavenger Receptor Activity

Abstract

Understanding the principles that guide the uptake of engineered nanomaterials (ENMs) by cells is of interest in biomedical and occupational health research. While evidence has started to accumulate on the role of membrane proteins in ENM uptake, the role of membrane lipid chemistry in regulating ENM endocytosis has remained largely unexplored. Here, we have addressed this issue by altering the plasma membrane lipid composition directly in live cells using a methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange method. Our observations, in an alveolar epithelial cell line and using silica nanoparticles, reveal that the lipid composition of the plasma membrane outer leaflet plays a significant role in ENM endocytosis and the intracellular fate of ENMs, by affecting nonspecific ENM diffusion into the cell, changing membrane fluidity, and altering the activity of scavenger receptors (SRs) involved in active endocytosis. These results have implications for understanding ENM uptake in different subsets of cells, depending on cell membrane lipid composition.

Graphical Abstract

Furthering the biomedical applications of nanotechnology requires a detailed understanding of the mechanisms through which engineered nanomaterials (ENMs) are internalized into mammalian cells and are translocated to subcellular locations. To date, a number of fundamental studies have focused on answering these important questionss.^1–3 Studies have also examined the role of membrane receptors in mediating ENM endocytosis.^4–6 However, the role of the plasma membrane lipid composition in regulating ENM uptake and intracellular fate has remained largely unknown.

Previous research on membrane receptors has revealed the important role of scavenger receptors (SRs), a family of transmembrane proteins known for their role in the uptake of modified low-density lipoproteins (LDLs),^7,8 in recognition and endocytosis of ENMs. The SRs of class A (SR-A) have - been shown to mediate the endocytosis of a number of pristine nanoparticles, such as silica, iron oxide, and polystyrene,^5,9–11 while the SRs of class B (SR-B) are primarily involved in the uptake of ENMs coated with proteins.^12,13 These receptors, however, are embedded in the cell plasma membrane, and it is thus possible that the membrane lipid composition could alter ENM endocytosis, either directly or by affecting the localization and function of membrane receptors.

The cell plasma membrane is an asymmetric lipid bilayer with different lipids in the outer leaflet (facing the outside environment) compared to those in the inner leaflet (facing the cytoplasm).^14–16 The lipid composition of the plasma membrane is different in different cell types and can change as a result of diet,¹⁷ circadian rhythm,^18,19 disease,^20,21 and cell cycle.²² In addition, various genetic diseases can perturb membrane lipid composition.^23,24 Therefore, understanding the role of membrane lipid chemistry and biophysical properties in the endocytosis of ENMs is essential for the design of ENMs for biomedical applications.

In the current study, we examined the role of the cell membrane lipid composition in the endocytosis of ENMs in live cells. A method based on methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange^25–27 was used to manipulate the lipid composition of the plasma membrane in live cells. Our approach allowed for the investigation of the role of lipid chemistry, such as backbone, headgroup, and acyl saturation in regulating ENM endocytosis. Plain silica nanoparticles (with a nominal diameter of 50 nm) in the absence and presence of serum were used as particle models. Silica particles have been explored in biomedical research,^28–30 and their cellular uptake via SRs has been extensively investigated.^1,2,31 Our results show that changes in the cell membrane lipid composition not only alter ENM endocytosis but also induce alterations in membrane biophysical properties and SR expression and activity.

RESULTS AND DISCUSSIONS

Confirmation of Outer Leaflet Lipid Exchange in Live Cells.

To examine the role of membrane lipids in the endocytosis of ENMs, we utilized the MαCD-catalyzed, lipid exchange method (Figure S1), introduced by Li and colleagues^25,27 and previously successfully used by us in red blood cells.²⁶ Using mass spectrometry (MS), an analysis of the total lipids clearly showed that the total lipid composition of A549 cells, a model airway epithelial cell line, was significantly altered by this method (Figure 1). For example, the total lipid composition of cells after exchange with 36:2 phosphatidylcholines (PC) showed a significant increase in the amount of total PCs while the amount of other lipid species decreased (Figure 1A). This reduction was particularly notable in sphingomyelins (SMs), one of the major lipids in the outer leaflet. Total cellular SM substantially increased after 24:0 SM exchange, indicating that significant levels of 24:0 SM were delivered to the outer leaflet of cell plasma membrane (Figure 1B). The total lipid compositions of cells after exchange with 18:0 SM and 18:1 SM are also provided (Figure S2A,B).

Figure 1.

Effect of lipid exchange on total phospholipid species and acyl chain distribution of phospholipids in A549 cells. (A) Abundance of phospholipids before and after exchange with 36:2 PC. (B) Abundance of phospholipids before and after exchange with 24:0 SM. (C) PC acyl chain distribution before and after exchange with 36:2 PC. (D) SM acyl chain distribution before and after exchange with 36:2 PC. (E) SM acyl chain distribution before and after exchange with 24:0 SM. (F) Efficiency of lipid exchange (amount of lipids added to cells relative to total cellular lipids after exchange). Two-way ANOVA with Sidak’s post hoc test was used for comparing the abundance of lipid species before and after exchange: (A) ****P(PC) < 0.0001 and ****P(SM and DSM) < 0.0001; (B) ****P(PC) < 0.0001 and ****P(SM and DSM) < 0.0001; (C) ****P(32:1 PC) < 0.0001, ****P(34:1 PC) < 0.0001, and ****P(36:2 PC) < 0.0001; (D) ****P(16:0 SM) < 0.0001, *P(22:0 SM) = 0.0118, not significant for 24:0 and 24:1 SMs (p > 0.05); (E) ****P(16:0 SM) < 0.0001, ****P(22:0 SM) < 0.0001, ****P(24:0 SM) < 0.0001, and ****P(24:1 SM) < 0.0001; (F) one-way ANOVA with Tukey’s post hoc test was used for exchange efficiency: P(18:0 vs 24:0 SMs) = 0.0213 and not significant for 18:0 SM vs 18:1 SM. Results are plotted on the basis of four independent replicates of the experiment (mean ± SD).

Lipid exchange was further confirmed by an analysis of fatty acid distribution in cells before and after lipid exchange. Exchange with 36:2 PC altered the fatty acid distribution of PC species, leading to a significant increase in the amount of 36:2 PC in cells, accompanied by a reduction in 32:1 PC and 34:1 PC (Figure 1C). In addition, the amount of SM 16:0, the major SM species, was greatly reduced compared to untreated cells, while no appreciable change was observed for other SM species (Figure 1D). In contrast, the acyl chain distribution of SM species was completely changed after lipid exchange with 24:0 SM (Figure 1E). It should be noted that the fatty acid distribution of PCs did not change after exchange with SM species, confirming the specificity of the exchange (Figure S2C–E). A similar result was observed for 18:0 SM and 18:1 SM (Figure S3). The difference in the total percentage of lipids in cells before and after exchange relative to total lipids after exchange was used to measure the efficiency of each lipid. As expected, the exchange efficiency was highly dependent on the structure of lipids loaded in cyclodextrin. One unsaturation in each tail of 36:2 PC reduced the efficiency (~20%) significantly, while the presence of at least one saturated tail in the exchange of SMs (~above 60%) increased the exchange efficiency (Figure 1F). This structure dependency of lipid exchange is consistent with previous observations in the literature.^25,27

In addition to MS, thin layer chromatography (TLC) also confirmed the changes in the lipid composition of the cell plasma membrane (Figure S4). For instance, two bands were observed for SMs in control, due to cellular SMs having diverse fatty acid distributions, while only one band was observed after exchange with synthetic 24:0 SM (Figure S4). In 36:2 PC and inverse PC (iPC) exchange, bands corresponding to SMs were less intense, indicating a reduction in SM after exchange. Due to presence of phosphate at the surface of iPC resulting in negative charge of this lipid,³² the band corresponding to this lipid appeared below phosphatidylserine (PS) in the 36:2 iPC exchange lane (Figure S3). The amount of delivered lipids in cells did not appreciably change until at least 4 h after lipid exchange, as evidenced by both TLC and MS (Figure S5). This is congruent with previous studies showing that cells maintained their lipid environment after phospholipids exchange using MαCD and the related MβCD.^25,33

Confocal fluorescence microscopy was used as another evidence of lipid exchange. For this purpose, a mixture of 36:2 iPC:NBD-PE (9:1 mol ratio) was added to A549 cells in the presence and absence of MαCD. Confocal images, taken in the presence of MαCD, showed a strong fluorescence, indicating that 36:2 iPC was delivered to the outer leaflet of the cell plasma membrane (Figure S6A). In contrast, cells exposed to the lipid mixture in the absence of cyclodextrin showed no fluorescence (Figure S6B). Cells treated with sodium dithionite, a relatively membrane impermeable NBD quencher with access to the outer leaflet,²⁷ showed no NBD-PE fluorescence (Figure S6C). The presence of 36:2 iPC bands in cells after lipid exchange was also confirmed by TLC (Figure S6D).

Cells Were Metabolically Active After Lipid Exchange.

Cell metabolic activity after lipid exchange was examined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells in which the outer leaflet lipids were exchanged demonstrated comparable metabolic activity to control cells (Figure 2A). To evaluate whether lipid exchange has caused any damage to the cell membrane, annexin V, which has high affinity for PS exposed to the outer leaflet of cell plasma membrane, and propidium iodide (PI), a nucleic acid binding dye, were used.³⁴ Figure 2B shows a representative set of flow cytometry fluorescence dot plots of A549 cells following 24:0 SM exchange. Results indicate that lipid exchange, except for the cases of 36:2 PC and 18:1 SM, induced damage in the cell membrane (Figure 2C), as evidenced by increasing the number of cells in the positive quadrants for PI and Alexa Fluor 488 annexin V. However, cells recovered after 2 h of incubation in the complete growth medium (Figure 2C), while maintaining the latest lipid environment (Figure S5). Representative dot plots after exchange with each of the other lipids of choice, with and without a recovery step, is presented in Figure S7. The recovery step after lipid exchange was sufficient for cells to maintain metabolic activity and the lipid composition in all cases.

Figure 2.

Evaluation of cell proliferation and membrane integrity after lipid exchange. (A) Cells were grown and allowed to proliferate before and after exchange with PCs and SMs. (B) Example dot plots of flow cytometric analysis of PI and annexin V staining of A549 cells after exchange with 24:0 SM and incubation at 0 and 2 h of recovery time. Signals due to Alexa Fluor 488 annexin V and PI are on the x- and y-axes, respectively. Right quadrants are corresponded to positive annexin V staining, and upper right quadrant is corresponded to positive for PI staining. (C) Percentage of cells in PI and Alexa Fluor 488 annexin V positive quadrants before and after recovery in complete growth medium. Results are plotted on the basis of at least three independent replicates of the experiment (mean ± SD).

The MαCD-based lipid exchange method applied in the current study allowed for reconstitution of the lipids of the membrane outer leaflet and a systematic study of the role of lipid chemistry in nanomaterial endocytosis. Membrane integrity of cells after lipid exchange was dependent on the structure of lipids delivered to the cell; however, 2 h of recovery postexchange was enough to completely revive membrane integrity, regardless of the lipids delivered (Figure 2). Overall, results indicated that the outer leaflet of the plasma membrane was reconstituted with a different lipid composition and the cells remained metabolically active. While these results are congruent with the study from Li and London,^25,27 one has to be cautious in interpreting the composition of the lipids delivered, as metabolic processes such as lipid flip-flop might be altered by lipid exchange and could potentially affect lipid distribution in the outer leaflet. This was not examined in the current study but cannot be ruled out. In addition, while the overall metabolic activity of cells was studied after lipid exchange, the functionality of individual intracellular organelles might have changed after reconstruction of the outer leaflet lipid microenvironment due to the crosstalk between cellular organelles. Such changes are possible but were not examined herein.

ENM Characterization.

The size and ζ-potential of ENMs were measured in RPMI and RPMI supplemented in 10% FBS. The hydrodynamic diameter of ENMs in the absence of serum was 50.3 ± 4.1 nm, while significant aggregation, and a size of 1669.3 ± 65.4 nm (Figure 3), was observed in the presence of serum. The ζ-potential of ENMs was reduced from −20.4 ± 3.8 mV in RPMI to −5.5 ± 0.4 mV in the presence of serum. Changes in the size and charge of ENMs in the presence of serum are in agreement with the literature.^35,36 Approximately 500 proteins were detected in the nanoparticle “corona” using LC–MS/MS. Identified proteins, ranked on the basis of abundance, are given in Table 1. The abundant proteins (mass of protein i/mass of total proteins) in protein–silica complex did not follow the order in serum, as has been previously reported in the literature.³⁷ This suggests that specific proteins preferentially adsorbed on the surface of silica nanoparticles due to their physicochemical properties as their abundance in the biomolecular corona did not follow their abundance in FBS.

Figure 3.

Size distribution of nanoparticles after 1 h incubation at 25 °C dispersed in (A) RPMI and (B) RPMI supplemented with 10% FBS.

Table 1.

Top Abundant Proteins Content, in Units of Mass Protein i/Mass of Total Proteins (m/m), in RPMI Supplemented with 10% Fast Bovine Serum (FBS)

identified proteins	FBS (m/m)	protein–silica complex (m/m)
ALBU (albumin)	0.164	0.048 ± 0.001
TRFE (serotransferrin)	0.121	0.012 ± 0.002
FINC (fibronectin)	0.078	0.147 ± 0.004
CO3 (complement C3)	0.064	0.138 ± 0.002
A2MG (α-2-macroglobulin)	0.062	0.078 ± 0.005
CFAH (complement factor H)	0.055	0.077 ± 0.005
PLMN (plasminogen)	0.042	0.038 ± 0.003
FETA (α-fetoprotein)	0.029	0.012 ± 0.002
VTDB (vitamin D-binding protein)	0.027	0.005 ± 0
ITIH4 (interalpha-trypsin inhibitor)	0.024	0.027 ± 0.002
THYG (thyroglobulin)	0.023	0.008 ± 0.003
CFAB (complement factor B)	0.02	0.02 ± 0
FETUA (α-2-HS-glycoprotein)	0.018	0.005 ± 0
CO7 (complement component C7)	0.018	0.022 ± 0.002
MPRI (cation-independent mannose-6-phosphate receptor)	0.018	0.006 ± 0
CO4 (complement C4)	0.018	0.024 ± 0.002
FA5 (coagulation factor V)	0.013	0.073 ± 0.004
COMP (cartilage oligomeric matrix protein)	0.011	0.018 ± 0.004
THRB (prothrombin)	0.011	0.018 ± 0.002
TSP1 (thrombospondin-1)	0.008	0.051 ± 0.002

Changes in the Lipid Composition of the Plasma Membrane Affect ENM Internalization.

The uptake of fluorescent silica nanoparticles in A549 cells before and after lipid exchange was measured using flow cytometry (Figure 4A). Uptake studies were performed in RPMI, both with and without serum, to evaluate the role of the protein corona in nanoparticle uptake. The uptake of particles in lipid-exchanged cells in RPMI without serum was altered significantly after one hour incubation at 37 °C (Figure 4B). While the uptake of ENMs after 36:2 PC exchange did not change, uptake in 36:2 iPC, 18:0, and 24:0 SM was reduced significantly compared to the control. However, the uptake of ENMs after 18:1 SM exchange increased significantly with respect to untreated cells. This observation suggests that the native plasma membrane lipid microenvironment and the composition of the lipids delivered to the cell outer leaflet play an important role in nanoparticle internalization.

Figure 4.

Uptake of nanoparticles (0.01 mg/mL) before and after exchange with lipids of choice measured by tracing nanoparticle fluorescence in the absence and presence of serum at 37 °C using flow cytometry. (A) Representative histogram of flow cytometric analysis before and after nanoparticle exposure (black curve denotes the cells with no particles, and the red curve shows the cells after particle uptake). Median cell fluorescence intensity of nanoparticles following incubation with cells for (B) one hour in RPMI, (C) one hour in RPMI supplemented in 10% FBS, and (D) two hours in RPMI supplemented with 10% FBS. One-way ANOVA with Tukey’s post hoc test was used for comparing nanoparticle uptake before and after exchange: (B) no significant change was observed for 36:2 PC (P > 0.05), *P(36:2 iPC) = 0.0291, *P(18:0 SM) = 0.0247, **P(18:1 SM) = 0.0077, and no significant change observed for 24:0 SM (P < 0.05). (C) No significant change was observed (P < 0.05). (D) No significant change was observed for 36:2 PC (P > 0.05), ***P(36:2 iPC) = 0.0008, *P(18:0 SM) = 0.0124, no significant change was observed for 18:1 SM (P > 0.05), and *P(24:0 SM) = 0.0416. Results are plotted on the basis of four independent replicates of the experiment (mean ± SEM).

Differences in nanoparticle uptake on the basis of membrane lipid chemistry were abrogated in the presence of serum (Figure 4C). When the incubation time was increased to two hours, ENM uptake increased significantly, indicating that ENMs required more time to internalize in the presence of serum compared to the serum-free environment (Figure 4D). While the uptake of ENMs after 36:2 iPC, 18:0 SM, and 24:0 SM exchange was decreased significantly, uptake after 36:2 PC and 18:1 SM exchange was comparable with that of untreated cells. Combined, the results demonstrate that alterations in the lipid composition of the cell outer leaflet play a major role in the uptake of ENMs, particularly in the absence of serum.

Plasma Membrane Lipid Composition Alters the Intracellular Fate of ENMs.

To understand the intracellular fate of ENMs, we assessed their colocalization with early endosomal (EEA1) and lysosomal (LAMP1) markers using confocal microscopy. Control and lipid-exchanged cells were exposed to ENMs, dispersed in RPMI with no serum, at a concentration of 0.01 mg/mL, and allowed to incubate for one hour at 37 °C. ENMs were bound to and internalized by both treated and untreated cells (Figure S8A–F). To quantify the localization of NPs with the lysosome, Pearson’s correlation coefficient (PCC) and Manders’ overlap coefficient (MOC) were used. Approximately 100 cell regions where ENMs existed were selected and analyzed using PCC and MOC. ENMs were mostly found as free particles in the cytosol or associated with cell membrane but did not colocalize strongly with lysosomes, as the average PCC value was close to zero for both treated and untreated cells (Figure S8G). The MOC values further corroborated the PCC values and were also close to zero (Figure S8H). Some high MOC values for 36:2 PC and 36:2 iPC indicate an overlap of two intensities but not necessarily the colocalization.³⁸ Similar analysis was performed on cells stained for early endosomes after incubation with ENMs dispersed in RPMI for only 10 min to capture their colocalization with early endosomes. While the uptake of ENMs was significantly reduced at this shorter incubation time, it was possible to conclude that ENMs did not colocalize with early endosomes markers even after 10 min (Figure S9A–F). This lack of colocalization suggests that ENMs dispersed in the serum-free medium were internalized into cells mostly through passive diffusion, as receptor-mediated endocytosis is expected to result in colocalization with lysosomes and early endosomes.²

The uptake of ENMs in the presence of serum was further investigated using lysosome immunostaining. Treated and untreated cells were exposed to ENMs dispersed in RPMI in the presence of serum (0.01 mg/mL) for three hours of incubation at 37 °C. It was found that ENMs were internalized into untreated cells through endocytic pathways when serum was present. This was evidenced by the strong colocalization of green and red colors, which correspond to ENMs and lysosome, respectively (Figure 5A). The amount of internalized ENMs and their uptake pathway were changed in cells following lipid exchange (Figure 5B–F). The alteration in the uptake of ENMs was further corroborated by the fact that both PCC and MOC values were significantly higher in untreated cells compared to cells after lipid exchange (Figure 5G,H), revealing a strong colocalization and overlap of signals between ENMs and lysosomal markers in the control but not lipid-exchanged cells. In summary, the lipid microenvironment in the outer leaflet of cell plasma membrane triggered the displacement of the ENMs from the lysosomes into the cytosol but only in the presence of serum.

Figure 5.

Intracellular fate of nanoparticles (0.01 mg/mL) before and after exchange with lipids of choice was examined at 37 °C after three hours of incubation in complete medium (i.e., containing serum) using confocal microscopy: (A) No exchange, (B) 36:2 PC, (C) 36:2 iPC, (D) 18:0 SM, (E) 18:1 SM, and (F) 24:0 SM. To analyze colocalization of nanoparticles with lysosomes, (G) the Pearson correlation coefficient (varying between −1 and +1, with −1 for perfect negative correlation, 0 for perfect absence of correlation, and 1 for perfect correlation) and (H) Mander’s overlap coefficient of nanoparticles (varying between 0 and 1, with 0 for no overlap and +1 for perfect overlap) were calculated. One-way ANOVA with Tukey’s post hoc test was used for comparing nanoparticle uptake before and after exchange: (G) ****P (no exchange vs after exchange) < 0.0001, (H) ****P (no exchange vs after exchange) < 0.0001. Red, LAMP1 staining of lysosome (secondary antibody conjugated with Alexa-647); green, FITC fluorescent nanoparticles; and blue, nucleus stained with DAPI. Scale bar is 10 μm.

SRs are membrane proteins extensively reported as the major receptors in the uptake of ENMs, especially silica.^1,2,11,39 To understand how changes in the membrane lipid composition affect SR function, poly (I), a nonspecific SR ligand, was used to block the SRs.¹¹ Poly (C) was used as a negative control. Preincubation with poly (I) resulted in a significant reduction in ENM uptake in untreated cells, as well as cells in which lipid exchange had been performed using SMs (Figure 6A). This significant reduction in ENM uptake, however, was not observed in cells following lipid exchange with 36:2 PC and 36:2 iPC. Cell preincubation with poly (C) did not cause a significant decrease in ENM uptake in any of the cells. Further analysis of ENM uptake through SR activity was performed by quantifying the difference in uptake in the presence and absence of poly (I) subsequent to exchange with the SMs, which are poly (I)-alterable, as a percentage relative to the difference in the uptake of untreated cells (Figure 6B). It was observed that cells with 18:1 SMs exchange showed relatively high ENM uptake through SRs, ~50% relative to untreated cells. In addition, ENM uptake in cells after 18:0 and 24:0 SMs change was approximately ~20% and ~30%, respectively. These results suggest that changes in the lipid composition of the membrane outer leaflet can cause changes in the activity levels of SRs, depending on the lipid chemistry, thereby playing a role in ENM uptake.

Figure 6.

Assessing the role of SRs in the uptake of nanoparticles (0.01 mg/mL) in complete medium (i.e., containing serum) after two hours of incubation at 37 °C before and after exchange with lipids of choice using flow cytometry. (A) Cells before and after exchange were exposed to poly (I) at a concentration of 0.01 mg/mL to block scavenger receptors. Poly (C) was used as negative control for poly (I); (B) percentage of nanoparticle uptake through SRs after exchanges with SMs. One-way ANOVA with Tukey’s post hoc test was used for comparing nanoparticle uptake in the presence and absence of poly(I): **P(no exchange) = 0.0015, no significant change was observed for 36:2 PC and 36:2 iPC (P < 0.05), **P(18:0 SM) = 0.01, *P(18:1 SM) = 0.0232, and *P(24:0 SM) = 0.0238. Results are plotted on the basis of four independent replicates of the experiment (mean ± SEM).

There are several mechanisms that regulate cellular uptake of ENMs such as phagocytosis,^40–42 receptor-mediated endocytosis,^43–46 and passive diffusion.⁴² In the current study, we used a nonphagocytotic lung airway epithelial cell line, A549, in order to eliminate phagocytosis from these possibilities and focus on receptor-mediated endocytosis pathway and passive diffusion. Combined results from flow cytometry and confocal imaging indicated that passive diffusion was the common pathway for ENM internalization in the absence of serum (Figures 4–6). This was evidenced by the fact that ENMs and lysosomes did not colocalize at significant levels, suggesting that ENMs are mostly free in the cytosol or associated with the cell membrane.

Changes in Plasma Membrane Lipid Composition Affect the Expression and Activity of SRs.

Having demonstrated the role of native cell membrane SRs in NP uptake, we focused our studies on assessing the activity of SRs in a different lipid environment. For these studies, we narrowed our focus to two well-known SRs, MARCO and LDLR. Both receptors are expressed on the plasma membrane of A549 cells and have been reported to play a significant role in the uptake of silica particles.^10,11,47 Immunostaining and flow cytometry were used to detect the expression level of LDLR and MARCO (Figure 7A,B). It was found that lipid exchange affects the expression level of both receptors on the membrane. LDLR expression reduced significantly for cells following lipid exchange with 36:2 PC, 36:2 iPC, and 18:1 SM but not after exchange with 18:0 and 24:0 SMs (Figure 6C). The observed differences might be due to changes in LDLR recycling, leading to a lower density of receptors on the cell surface, as LDLRs traffic into and out of the cell interior every 10–20 min.^48,49 The expression level of MARCO receptors on the cell membrane showed a slight reduction after lipid exchange, although it was only significant in the case of 36:2 PC (Figure 6D).

Figure 7.

Expression level of LDLR and MARCO receptors was assessed using flow cytometry. (A) Representative histogram of the flow cytometric analysis of LDLR (the black curve is an isotypic control, the red curve shows cells before immunostaining, and the blue curve shows cells after immounostaining). (B) Representative histogram of flow cytometric analysis of MARCO (the black curve is an isotropic control, the red curve shows cells before immunostaining, and the blue curve shows cells after immounostaining). (C) LDLR expression before and after lipid exchange. (D) MARCO expression before and after lipid exchange. Black, isotype; red, cells only; and blue, cells stained with secondary antibody conjugated with Alexa Fluor 647. One-way ANOVA with Tukey’s post hoc test was used for comparing the level of expression after lipid exchange with no exchange: (C) **P(36:2 PC) = 0.0012, **P(36:2 iPC) = 0.0051, no significant change was observed for 18:0 and 24:0 SM (P > 0.05), ***P(18:1 SM) = 0.0003 and (D) *P(36:2 PC) = 0.311 and no significant change was observed for 36:2 iPC, 18:0, 18:1, and 24:0 SMs (P > 0.05). Results are plotted on the basis of four independent replicates of the experiment (mean ± SEM).

To measure the activity of LDLR and MARCO receptors, we developed an assay based on flow cytometry. Unlabeled human LDL and ox-LDL particles, known ligands of LDLR and MARCO, were employed for this assay.⁵⁰ Unlabeled ligands were preincubated with cells to occlude cell surface receptors. Cells with and without preincubation with unlabeled ligands were then exposed to fluorescent LDL and ox-LDL to assess receptor activity. Fluorescent LDL particles and LDLRs were colocalized as evidenced by confocal microscopy (Figure S10). Examples of the flow cytometric analysis of LDL and ox-LDL uptake is shown in Figures S11 and 12, respectively. Median cell fluorescence intensity (MFI) was used to evaluate ligand uptake and thus LDLR and MARCO receptor activity in cells.

Our results revealed that LDL uptake reduced significantly in untreated cells, due to its occlusion by unlabeled LDL particles, allowing for the measurement of the extent of LDLR activity. Interestingly, LDLR showed different activities in different lipid microenvironments. While cells subjected to 36:2 PC and 36:2 iPC exchange showed very low LDLR activity, higher LDLR activity was observed in cells subjected to exchange with SMs, especially 18:1 SM (Figure 8A,B). For example, LDLR activity for cells exchanged with 18:1 and 24:0 SMs was ~53% and 41% but was reduced to ~17% and 10% For cells subjected to 36:2 PC and 36:2 iPC exchange, respectively.

Figure 8.

Activity of LDLR and MARCO receptors was assessed before and after exchange with lipids of choice using flow cytometry. Unlabeled LDL and ox-LDL particles were used to occlude the LDLR and MARCO receptors, respectively. Labeled LDL and ox-LDL were used to assess their uptake in the presence or absence of unlabeled particles. (A) Uptake of fluorescent LDL particles with or without LDLR obstruction. (B) LDLR activity after exchange. (C) Uptake of fluorescent ox-LDL particles with or without MARCO obstruction. (D) MARCO activity after lipid exchange. Two-tailed unpaired t test was used to compare LDL uptake in the presence and absence of unlabeled LDL in each group: (A) ***P(no exchange) = 0.0001, **P(36:2 PC) = 0.0022, **P(18:0 SM) = 0.0020, **P(18:1 SM) = 0.0098, **P(24:0 SM) = 0.0063, no significant change was observed for 36: iPC (P > 0.05) and (C) ***P(no exchange) = 0.0039, **P(36:2 PC) = 0.0014, **P(18:0 SM) = 0.034, *P(18:1 SM) = 0.0141, *P(24:0 SM) = 0.0353, no significant change was observed for 36: iPC (P > 0.05). Results are plotted on the basis of at least three independent replicates of the experiment (mean ± SEM).

A similar assay was used to assess the activity of MARCO receptors. Cells showed appreciable MARCO activity, after exchange with SMs, especially for 18:1 SM (~77% activity compared to that of the control). The activity of MARCO receptors in cells subjected to 36:2 PC was still considerable (close to 50%). However, negligible MARCO activity was observed after 36:2 iPC exchange (Figure 8C,D). In summary, our findings reveal that the LDLR and MARCO activities are affected by the lipid microenvironment, signaling that the lipid composition in which these receptors are embedded play an important role in their activities.

Plasma Membrane Lipid Composition Affects Membrane Biophysical Properties but not the Localization of LDLR.

We next examined the effect of lipid chemistry on membrane lipid packing and LDLR localization. For these studies, we overexpressed human LDLR in A549 cells, performed lipid exchange, and prepared giant plasma membrane vesicles (GPMVs) using established protocols.^51,52 GPMVs isolated directly from living cells have been utilized as a model for protein localization in ordered lipid domains (also known as rafts).^53–55 To visually examine the localization of LDLR in ordered vs disordered phases in the membrane, cells were transfected with human LDLR tagged with GFP (Figure S13). Additionally, Rhod-DOPE (18:1 PE), which preferentially partitions into the disordered phase, was used to examine membrane phase separation.⁵³ Membrane lipid exchange affected membrane phase separation. As shown in Figure 9, ordered membrane domains (dark, nonfluorescent regions) were observed in untreated cells and in cells subjected to lipid exchange with SMs. This was particularly notable after lipid exchange with the long-chain 24:0 SM (Figure 9F). However, no phase separation was observed after lipid exchange with unsaturated 36:2 PC and iPC (Figure 9B,C). These findings clearly show that membrane lipid chemistry affects membrane phase separation.

Figure 9.

Localization of LDLR in GPMVs isolated from transfected cells before and after lipid exchange, observed using confocal microscopy: (A) No exchange, (B), 36:2 PC, (C) 36:2 iPC, (D) 18:0 SM, (E) 18:1 SM, and (F) 24:0 SM. (G) TMADPH anisotropy on cells before and after exchange to evaluate the lipid packing of the cell plasma membrane. One-way ANOVA with Tukey’s post hoc test was used for comparing anisotropy after lipid exchange with no exchange: (G) ****P(36:2 iPC) < 0.0001, *P(24:0 SM) = 0.0476, and no significant change was observed for 36:2 PC, 18:0, and 24:0 SM (P < 0.05). Rhod-PE (18:1 PE) was used to identify the disordered membrane domain (red), and LDLR-tagged GFP was identified after cell transfection (green). Results are plotted on the basis of three independent replicates of the experiment (mean ± SEM). Scale bar is 5 μm.

To further examine the effect of lipid chemistry on lipid packing of the membrane, we employed (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate) (TMA-DPH) anisotropy on cells before and after lipid exchange (Figure 9G). This assay showed a significant increase in anisotropy (indicative of increased membrane order) of cells after 24:0 SM exchange and a significant reduction in anisotropy (decreased membrane order) following 36:2 iPC exchange, which is in agreement with the findings of GMPV imaging. Changes in membrane biophysical properties after exchange with other lipids were not significant enough to be captured in anisotropy, although they could be qualitatively observed with confocal microscopy (Figure 9A–F). LDLR localized preferentially with 18:1 PE in cells with and without lipid exchange, indicating that it localizes in disordered membrane domain. In summary, whenever phase separation was observed, LDLR was found to preferentially localize in the disordered phase. This was not affected by the increased domain formation in the membrane caused by lipid exchange. It should be noted that it is possible that changes in lipid packing might have occurred in the disordered phases, affecting LDLR function. Such changes, however, could not be captured by anisotropy and microscopy studies and require more sensitive techniques.

ENM uptake in cells was significantly altered by lipid exchange in the absence of serum (Figure 4). The role of lipids in ENM uptake is likely understood by considering membrane biophysical properties after lipid exchange. After membrane reconstitution with 36:2 PC, there is no significant difference in nanoparticle uptake compared to the control (Figure 4B) and changes in membrane order compared to the control are negligible (Figure 9G). In contrast, a tight lipid packing is observed after exchange with 18:0 and 24:0 SMs, due to their long and saturated tails, suggesting that ENMs diffuse into cells in lower amounts compared to untreated cells. Note that, while significant change in anisotropy of cells following 18:0 exchange was not observed, ordered lipid phases appeared in GMPVs after the exchange (Figure 9D). This does not explain the increase in ENM uptake after 18:1 SM exchange, although changes in membrane packing could be too small to discern with anisotropy and GPMV imaging. Of course, changes in the activity of other membrane receptors cannot be ruled out. Together, results suggest that ENM uptake in the absence of serum is at least in part controlled by passive diffusion, which can be affected by membrane packing. This complements our previous study, showing a difference in nanoparticle-induced membrane disruption depending on membrane lipid chemistry and order.^32,56,57 Electrostatic interactions are also likely to play a role. The significant reduction in ENM uptake after lipid exchange with 36:2 iPC is likely due to the increased phosphate (PO₃⁻) concentration on the membrane and the electrostatic repulsion between the membrane and SiO⁻ on the particle surfaces.

ENM uptake in the presence of serum was significantly reduced compared to serum free medium (Figure 4C). This is likely due to changes in particle size and charge in the presence of serum. The reduced endocytosis was observed regardless of the membrane lipid composition. However, increasing the incubation time between the ENMs and the cells in the presence of serum increased particle uptake, approximately to the same level observed in the absence of serum. Significant differences in the uptake of ENMs depending on membrane lipid composition were again observed (Figure 4D). In this case, significant colocalization with lysosomal markers indicated a role for receptor-mediated endocytosis.^1,2 Note that, in this case, passive diffusion still cannot be ruled out, as individual particles still exist after incubation with serum. However, changes in membrane lipid chemistry resulted in displacement of ENMs from the lysosomes (Figure 4), hinting at changes in the mechanisms of receptor-mediated uptake following lipid exchange.

To distinguish the pathways of ENM uptake, an approach based on blocking of SRs was implemented. Using gene silencing techniques, Prapainop and colleagues revealed the important role of LDLR and MARCO in the uptake of silica nanoparticles with core diameters of 50 and 200 nm.¹¹ The same group also found that MARCO receptors recognize silica particles in the presence of serum,¹⁰ in a manner that is similar to specific binding of MARCO to its ligands. In agreement with these studies, ENM uptake decreased significantly in untreated cells in which SRs were blocked (Figure 6). It should also be noted that ENMs were internalized into the cells in significant amounts even after blocking SRs. This shows that passive diffusion and also other receptors, such as mannose/lectin receptors, might be involved in the uptake of ENMs.^58–60 ENM uptake was also reduced after lipid exchange with SMs but not with 36:2 PC and 36:2 iPC, further suggesting that other pathways could be involved in ENM internalization.

Importantly, changes in membrane lipids induced changes in the expression or activity of LDLR and MARCO. There is no direct correlation between the expression level of receptors and their activity. For example, the expression level of LDLR decreased after 18:1 SM exchange, but LDLR showed very high activity. A similar trend was observed for MARCO receptor after exchange with 36:2 PC. Evidence from the literature corroborates the role of lipids in receptor function, primarily through protein sorting.⁶¹ For example, protein 24, which modulates the budding of vesicles from the Golgi apparatus, is regulated by sphingomyelin through interactions of its transmembrane domain with this lipid species but not with other lipids.⁶²

Lipid-induced changes in the activity of LDLR and MARCO can be through direct lipid–protein interactions or occur indirectly through changes in membrane properties. For example, an exchange with 24:0 SM is expected to increase the thickness of membrane, resulting in a hydrophobic mismatch between the receptor and the bilayer,^63–65 which can result in various perturbations in receptor function. In addition to the hydrophobic mismatch, lipid composition can alter lateral pressure in the membrane, leading to changes in protein conformation.^64,66 Changes in membrane fluidity, as observed in Figure 9, can also affect endocytosis, as well as passive diffusion, through changes in spontaneous curvature and bending stiffness.^67,68 For example, it has been reported that polyunsaturated fatty acids reduce membrane bending rigidity, facilitating endocytosis.⁶⁷ Importantly, our results on the uptake of ENMs through SRs (Figure 6B) match with observations on the activity of LDLR after exchange with SMs (Figure 8B). In particular, SR activity after exchange with 18:1 might further corroborate the interaction of SM with transmembrane domain of the protein.⁶² This high activity after 18:1 SM exchange was also observed for MARCO, except that, in that case, lipid exchange with 36:2 PC led to a similar increase in MARCO activity. Therefore, lipid composition can affect transmembrane protein recruitment, conformation, activity, and localization through specific lipid–protein interactions or through its effect on membrane properties, as is the case here with the SRs.

To understand whether changes in LDLR partitioning in lipids affected its activity, GPMVs were utilized. GPMVs are biological membrane models that contain complex compositional lipids and proteins of cell plasma membrane. GPMVs are disconnected from the cytoskeleton and thus have larger ordered domains compared to the domains found in the cell.^55,69 GPMVs have been used by several studies to evaluate the partitioning of membrane proteins in ordered lipid domains.^51,53,55 In the current study, changes in the lipid composition of the membrane were found to affect large-scale phase separation in GPMVs (Figure 9). While this observation was further confirmed by anisotropy, both anisotropy and confocal imaging are sensitive to larger scale changes in membrane properties and unable to detect smaller, nano- and micron-scale changes. Regardless, results do indicate alterations in membrane phase segregation, which could have contributed to differences in the uptake and intracellular fate of ENMs.

CONCLUSION

In summary, the results of this study reveal that the lipid composition of the cell plasma membrane plays an important role in the uptake of ENMs either through changes in membrane fluidity, thus affecting passive diffusion, or by changing receptor expression and affecting receptor-mediated endocytosis. With the membrane lipid composition changing in a wide variety of conditions, including various diseases,^21,22 and various reports on differential nanoparticle uptake between healthy and diseased cells,^70,71 these results have important implications in the judicious design of ENMs, and other exogenous compounds, for selective uptake in certain subsets of cells. How exactly lipids alter the function of receptors and how such alterations can be used for manipulating the cell endocytic machinery remain important questions for future research. Methods outlined in the current study allow for further investigation into these questions, the answers to which are important for both biomedical and environmental nanotechnology.

MATERIAL AND METHODS

Materials. The lipids of choice, including 1,2-dioleoyl-sn-glycero-3-phosphocholine, 36:2 (Δ9-Cis) (DOPC), 2-((2,3 bis(oleoyloxy)-propyl)dimethylammonio)ethyl hydrogen phosphate, 36:2 inverse PC (iPC), 18:0 sphingomyelin (SM) (d18:1/18:0), 18:1 SM (d18:1/18:1(9Z)), 24:0 SM, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (16:0 NBD-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). Methyl-α-cyclodextrin (MαCD) was purchased from AraChem (Tilburg, Netherlands). Roswell Park Memorial Institute (RPMI) medium 1640, Opti-MEM, fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS) (200 mg/L KCl, 200 mg/L KH₂PO₄, 8 g/L NaCl, and 2.16 g/L Na₂HPO₄), penicillin and streptomycin, 0.25% trypsin with EDTA (1X), apoptosis assay kit (Alexa Fluor 488 labeled-Annexin-V/propidium iodide (PI) staining kit) (V13241), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), unlabeled low density lipoprotein (LDL), unlabeled oxidized LDL (ox-LDL), Dil LDL, and BODIPY FL LDL were purchased from Thermo Fisher Scientific (Waltham, MA). Bovine serum albumin (BSA), formaldehyde, anf dl-1,4-dithiothreitol (DTT) were obtained from Fisher Scientific (Pittsburgh, PA). Goat anti-rabbit IgG H&L (Alexa Fluor 647) (ab150079), rabbit IgG, polyclonal-isotype control (ab37415), anti-LDL receptor antibody (ab30532), anti-MARCO antibody (ab231046), anti-EEA1 antibody-early endosome marker (ab2900), anti-LAMP1 antibody-lysosome marker (ab24170), and MTT Assay Kit (Cell Proliferation) (ab211091) were procured from Abcam (Cambridge, MA). Saponin from quillaja bark (S7900), polyinosinic acid potassium salt, poly (I), (P4154), and polycytidylic acid potassium salt, poly (C), (P4903), were purchased from MilliporeSigma (St. Louis, MO). FuGENE HD transfection reagent (E2311) was obtained from Promega (Madison, WI). Human low density lipoprotein receptor LDLR (GFP-tagged) transcript variant 1 and precision shuttle mammalian vector with C-terminal tGFP tag were obtained from ORIGENE (Rockville, MD). Fluorescent silica NPs (50 nm) were purchased from Micromod Partkeltechnologie GMBH (Rostock, Germany).

Cell Culture.

A549 alveolar epithelial cells were purchased from the American Type Culture Collection (ATCC CCL-185, Manassas, VA). RPMI 1640 medium supplemented with 10% FBS and 100U/mL penicillin and streptomycin were used for A549 cells culture, and cells were incubated at 37 °C with 5% CO₂ in a humidified environment.

Lipid Exchange in Live Cell. Lipid exchange in the outer leaflet of live cells was performed using the protocol from Li and colleagues.^25,27 Briefly, the desired amount of lipids of choice dissolved in organic solvents was dried using a SpeedVac vacuum concentrator (Thermofisher, Watham, MA) for 20 min, and multilamellar vesicles were then prepared in RPMI prewarmed at 70 °C. MαCD dissolved in DPBS at 200 mM stock solution was then added to the vesicle suspension at a final concentration 40 mM and incubated at 37 °C for 30 min, resulting in the loading of lipids from MLVs to MαCD. The final concentration of PCs in lipid-loaded MαCD was 3 mM while that of SMs was 1.5 mM. Cells were cultured in plates and washed three times with DPBS to remove residual cell culture media prior to performing the lipid exchange. The lipid-loaded MαCD was then added to the plates at the desired concentration (1.5 mL for 10 cm dish, 0.5 mL per well for a 6-well plate, 0.3 mL per well for a 12-well plate, and 0.2 mL per well for a 24-well plate) and incubated for approximately 45 min at room temperature. Afterward, the supernatant was aspirated and cells were washed three times with DPBS. Cells were then incubated at 37 °C for 2 h in complete growth medium unless otherwise noted, allowing cells to recover from lipid exchange. All assays performed on cells in this study were given two hours of recovery time in complete growth medium unless otherwise noted.

Thin Layer Chromatography (TLC).

After lipid exchange, the supernatant was decanted. Lipids were then extracted from the cells with three mL of 3:2 (v/v) hexane/isopropanol, incubated at room temperature for 30 min, with vortex-mixing every five min, and dried using a SpeedVac. Dried lipids redissolved in chloroform or pure lipid standards were applied to Silica Gel 60 TLC plates (MilliporeSigma) and chromatographed in 65:25:5 (v/v) chloroform/methanol/28.0–30.0 (v/v) % ammonium hydroxide or 4:8:38:50 (v/v) acetic acid/water/methanol/chloroform. The solvents were allowed to migrate the entire height of the plate as the plate was dried at room temperature. Afterward, the plate was sprayed with 3% cupric acetate (w/v) and 8% phosphoric acid (v/v). Sprayed plates were then placed in an oven at 180 °C for 5–10 min for charring. Subsequently, to determine the lipid bands, the plates were imaged.

Flow Cytometric Analysis of Cells After Lipid Exchange.

The Alexa Fluor 488 annexin V and propidium iodide (PI) kit (Invitrogen Carlsbad, CA, V13241) were used to detect live cells, exposure of phosphotidylserine (PS), and cell membrane integrity.³⁴ To check lipid membrane scramblase, annexin V, which binds to PS exposed to the outer membrane leaflet of cell plasma membrane, was used. In addition, PI, a nucleic acid binding dye, was used to check the membrane integrity. Briefly, after lipid exchange, the cells were harvested and washed with cold DPBS (without Ca²⁺ and Mg²⁺). Cells were then resuspended in annexin V binding buffer 1× (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl₂, pH 7.4). Afterward, Alexa Fluor 488 annexin and PI (100 μg/mL) were added to the cells and incubated at room temperature for 15 min in the dark. After incubation, the samples were analyzed on a FACSAria Special Order Research Product cytometer/sorter (BD Bioscience, San Jose, CA). Both annexin V (Ex/Em: 488/499 nm) PI fluorescence (Ex/Em: 535/617 nm) were excited with a 488 nm laser. Emission was detected with a 530 ± 15 nm bandpass filter for annexin V, while a 610 ± 10 nm bandpass filter was used to detect PI emission. FlowJo version 10 software (FlowJo, Ashland, OR) was used to analyze the results.

Cell Proliferation Assay.

To determine the growth rate of cells before and after exchange, the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit, measuring of the conversion of water-soluble MTT compound to an insoluble formazan product, was used (Abcam). The cells were seeded in a 96-well plate at a density of 8000 cells per well with and without lipid exchange. The supernatant was aspirated, and cells were trypsinzed after 24, 48, and 72 h according to the manufacturer’s protocol. Briefly, 50 μL of serum free medium and MTT reagent were added to each well, and incubated for three hours at 37 °C. Afterward, 150 μL of MTT solvent was added to each well and mixed for 15 min. Finally, the absorbance was measured at 590 nm using a Synergy H1 microplate reader spectrophotometer (Biotek, Winooski, VT).

Confirming the Localization of Lipids in the Outer leaflet.

To confirm the lipid exchange, a mixture of 40 mM MαCD with 3 mM 1:9 (mol/mol) NBD-DPPE/iPC was prepared as described earlier. The lipid-loaded MαCD was added to the cells plated on 35 mm plates with 15 mm diameter glass coverslips. After lipid exchange was carried out, the supernatant was discarded and the cells were washed three times with DPBS, fixed with paraformaldehyde (PFA) or 10 min, and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min to stain nuclei. Freshly prepared 1 M dithionite in 1 M Tris buffer (PH 10) was added to the plates at a final concentration of 50 mM. NBD and DAPI fluorescence were then imaged using a Nikon Eclipse Ti inverted microscope using lasers at 364 and 488 nm (g), respectively.

Nanoparticle Characterization.

To measure the size distribution and ζ-potential of nanoparticles, particles (0.01 mg/mL) were suspended in RPMI or RPMI with the presence of 10% serum. A Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) was used for this measurement.

Nanoparticle Uptake Using Flow Cytometry.

The uptake of fluorescent NPs before and after lipid exchange was examined using flow cytometry.^2,72 After lipid exchange was performed on A549 cells at ~90% confluency in 12-well plates and two hours of recovery time were allowed in complete growth medium, the media was aspirated and washed three times with DPBS. Fresh nanoparticle dispersions at a concentration of 0.01 mg/mL in serum free medium (RPMI) were added to each well. The media was discarded after one hour of incubation with NPs at 37 °C, and cells were washed three times with DPBS. Afterward, the cells were trypsinized and resuspended in DPBS prior to flow cytometry analysis.

To examine the effect of the biomolecular corona on the uptake of NPs, particles (0.01 mg/mL) dispersed in complete growth medium (RPMI plus 10% FBS (Lot# 26219001)) were added to the plated cells. The cells were place in an incubator at 37 °C for the required amount of time prior to uptake assessment as described above. For this assay, the excess and loosely bound proteins were not removed by centrifugation to further simulate physiologically relevant of NPs uptake after interaction with biomolecules.

Cell Immunostaining.

To examine the intracellular fate of NPs, lysosomal and endosomal imaging was performed. Cells were seeded on a glass coverslips (35 mm plates with 15 mm diameter), and a similar procedure to flow cytometry was used for this assay as described above. Briefly, first, cells were washed with DPBS for two or three times, and formalin at 4% (v/v) was used to fix the cells. Subsequently, cells were permeabilized with saponin (0.1% wt/v, from quillaja bark) for 5 min. To block the nonspecific binding, bovine serum albumin (1 wt %/v) was used and cells were incubated in the blocking solution for 30 min at RT. Afterward, primary antibody 1:200 rabbit polyclonal to LAMP1 (lysosomal) or EEA1 (endosomal) was added to samples for one hour incubation at RT, following three washes with DPBS. Secondary antibody to rabbit IgG-H&L, AlexaFluor 647 goat polyclonal (1:400), was then added to samples and incubated in the dark at RT for one hour. For nucleus staining, DAPI was added to samples for 5 min incubation at RT, which were washed with DPBS three times prior to confocal imaging. The cells were then imaged using a Nikon confocal microscope (Nikon Eclipse Ti inverted microscope) with lasers at 364 nm (DAPI), 488 nm (green-labeled NPs), and 640 nm (LAMP1, EEA1 antibody). The expression levels of LDL and MARCO receptors on cell plasma membrane with and without lipid exchange were assessed with the similar protocol using flow cytometry. A primary antibody with a dilution ratio of 1:100 rabbit polyclonal to LDL receptor and 1:200 to MARCO receptor was used, followed by a secondary antibody with a dilution ratio of 1:200 Alexa Fluor 647 goat polyclonal secondary antibody to rabbit IgG-H&L. Cells were then resuspended in DPBS and analyzed by flow cytometry.

Receptor Activity Assays.

The activity of LDL and MARCO receptors before and after lipid exchange in cells were examined using flow cytometry. The seeded cells (0.18 × 10⁶/well plates) were incubated overnight at 37 °C in complete growth medium.

Subsequently, supernatant was removed, rinsed once with starvation medium, RPMI supplemented with 0.3% BSA, and then incubated in serum-starved medium for 24 h. At the end of the incubation, cells were rinsed with DPBS containing calcium and magnesium supplemented with 0.3% BSA. The lipid exchange was performed two or three hours prior to the end of the serum starvation period. To specifically block the LDL receptor uptake and MARCO receptor uptake, cells were pretreated with unlabeled LDL or unlabeled oxLDL to occlude the LDL and MARCO receptors, respectively. To pretreat the cells, unlabeled LDL and ox-LDL, diluted in DPBS (containing Ca²⁺ and Mg²⁺) with 0.3% BSA at a concentration of 250 μg/mL, were added to cells and incubated for 40 and 60 min, respectively. To assess the uptake, 10 μg/mL labeled LDL and oxLDL, diluted in DPBS (with Ca²⁺ and Mg²⁺) with 0.3% BSA, were added to the cells and incubated for 2 h at 37 °C. Afterward, cells were rinsed two times with DPBS at RT. Cells were then trypsinized and resuspended in 200 μL in DPBS (with Ca²⁺ and Mg²⁺) with 0.3% BSA containing 7-AAD, a nucleic acid binding dye. Finally, uptake was analyzed using flow cytometry. In this analysis, after digital compensation to exclude the overlap intensity of dyes, 7-AAD negative live cells were gated first, followed by scatter. Single cells were gated using an area versus height dot plot, and the geometric mean fluorescence intensity (MFI) of the resulting cells was determined. To evaluate the LDLR activity in different lipid microenvironments, the following equation was used:

$$\text{receptor}\text{activity}(\mathrm{\%})=\left[\frac{{\left({\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{U}}-{\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{L}}\right)}_{\text{after}\text{exchange}}}{{\left({\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{U}}-{\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{L}}\right)}_{\text{no}\text{exchange}}}\right]\times 100$$

where ${\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{U}}$ is $\mathrm{M}\mathrm{F}\mathrm{I}$ in the absence of unlabeled LDL or ox-LDL and ${\mathrm{M}\mathrm{F}\mathrm{I}}_{\mathrm{L}}$ is $\mathrm{M}\mathrm{F}\mathrm{I}$ in the presence of unlabeled LDL or ox-LDL particles.

Plasmid Purification.

Calcium chloride treated chemically competent Eschericia coli (E. coli) cells were prepared as described in detail by Sambrook et al.⁷³ E. coli cells, harboring individual LDLR plasmid constructs, were grown in 50 mL of Luria–Bertani (LB) broth media containing ampicillin overnight at 37 °C while shaking. The following day, bacterial culture was harvested by centrifugation at 4 °C and plasmid isolation was performed according to provided protocols by the manufacturer’s protocol (PureLink HiPure Plasmid Filter Midiprep Kit, ThermoFisher, Waltham, MA). The plasmid DNA was then quantified and quality confirmed before performing the transfection experiments using a NanoDrop One C apparatus (ThermoFisher, Madison, WI).

Cell Transfection.

A549 cells were plated (3 × 10⁵ cells per well) in a 6-well plate in three ml of complete growth medium the day before transfection. of FuGENE HD reagent (FuGENE HD/DNA: 3.5/1) was used to transfect cells. Plasmid DNA diluted in Opti-MEM medium (0.02 μg/μL) was added to. FuGENE HD reagent. The solution was then pipetted up and down for 15 times.. After 10 min incubation at RT, 150 μL of the complex was added the cells. Finally, the cells along with transfection complex were placed in an incubator for 24 h at 37 °C and 5% CO₂ for further experiments.

Isolation of GPMV from Cells.

GPMV isolation from A549 cells were performed according to established protocols.^51,52 Briefly, the medium of cells grown to confluence was removed from the cells. Then, the cells were washed with GPMV buffer for three times (10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂ pH 7.4). GPMV buffer was supplemented with 2 mM DTT and 25 mM formaldehyde and was then added to cells. Subsequently, cells were incubated at 37 °C for 90 min while shaking at speed of 70 rpm. To stain lipid membrane, Rhod-PE (18:1 PE) diluted to 0.5 μg/mL in EtOH was added to the cells. Cells were gently rocked at room temperature for 15 min. Supernatants containing GPMVs were decanted and placed to a 1.5 microcentrifuge tube. Tubes were then placed on ice for 40 min to precipitate GPMVs at the bottom of the tube. To image GPMVs, 300 μL of GPMV solution was drawn by a syringe from the bottom of the tube and injected into a custom-made chamber, in which two coverslips cover two sides of a channel with a thickness of ~2 mm. GPMVs were imaged using a Nikon confocal microscope (Nikon Eclipse Ti inverted microscope, oil-immersed 100× objective, pinhole set to 1.5 μm). The fluorophores were excited using the 488 nm line of a 40 mW argon laser (GFP) and the 543 nm line of a HeNe laser (Rhod-PE).

Fluorescence Anisotropy.

TMA-DPH anisotropy was used to assess cell membrane properties with and without lipid exchange. TMA-DPH contains a cationic trimethylammonium substitute that allows it to locate in the outer leaflet of the plasma membrane, providing information on lipid packing in that leaflet. The lipophilic fluorescent probe TMA-DPH (Ex/Em: 355/433 nm) was dissolved in ethanol and added to A549 cells detached and resuspended in 2 mL of DPBS at a concentration of 40 nM. Anisotropy was measured at 25 °C using a Fluorolog-3 spectrofluorometer (Horiba Scientific, Edison, NJ).

Statistical Analysis.

A minimum of three independent replicates was performed. Graphpad Prism software package (La Jolla, CA) was used to analyze data in this study. A P-value of <0.05 was considered statistically significant. The symbols denoting data significance can be found in caption of each figure in the main text. Several methods depending on statistical comparison were used in this study. Details also can be found in the caption of each figure.