Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Nov 6.
Published in final edited form as: Mol Pharm. 2023 Oct 2;20(11):5454–5462. doi: 10.1021/acs.molpharmaceut.3c00175

Enhancement of Anti-Inflammatory Effects of Synthetic High-Density Lipoproteins by Incorporation of Anionic Lipids

Minzhi Yu 1,2, Kristen Hong Dorsey 1,2, Troy Halseth 2,3, Anna Schwendeman 1,2,*
PMCID: PMC10916337  NIHMSID: NIHMS1969019  PMID: 37781907

Abstract

Phosphatidylserine (PS) is an anionic phospholipid component in endogenous high-density lipoprotein (HDL). With the intrinsic anti-inflammatory effects of PS and the correlation between PS content and HDL functions, it was hypothesized that incorporating PS would enhance the therapeutic effects of HDL mimetic particles. To test this hypothesis, a series of synthetic high-density lipoproteins (sHDLs) were prepared with an apolipoprotein A-I (ApoA-1) mimetic peptide, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS). Incorporating PS was found to improve the particle stability of sHDLs. Moreover, increasing PS content in sHDLs enhanced the anti-inflammatory effects on LPS-activated macrophages and endothelial cells. The incorporation of PS had no negative impact on cholesterol efflux capacity, in vivo cholesterol mobilization, and did not affect the pharmacokinetic profiles of sHDLs. Such results suggest the therapeutic potential of PS-containing sHDLs on inflammation resolution in atherosclerosis and other inflammatory diseases.

Keywords: Synthetic high-density lipoproteins, phosphatidylserine, anti-inflammation, reverse cholesterol transport

Graphical Abstract

graphic file with name nihms-1969019-f0001.jpg

1. Introduction

High-density lipoproteins (HDLs) are a group of endogenous nanoparticles consisting of apolipoproteins and diverse lipid components.1 As a major mediator in reverse cholesterol transport, HDL induces cholesterol efflux from peripheral cells and transfers cholesterol to the liver for elimination.2 Moreover, HDLs play a pivotal role in inflammation resolution by reducing pro-inflammatory cytokine release from immune effector cells,3 inhibiting endothelial activation,4 and scavenging oxidative lipid species.5 Low HDL or HDL-cholesterol (HDL-c) levels, altered HDL composition, and impaired HDL functions have been associated with varieties of diseases including cardiovascular diseases, autoimmune diseases, and infections.6–8

Biomimetic, synthetic HDLs (sHDLs) have attracted much interest in the past two decades. Mimicking components of endogenous HDLs, sHDLs are typically composed of ApoA-1 or its mimetics, as well as lipid components such as phospholipids and sphingolipids.9 Several sHDL candidates, such as CSL-111, CSL-112, CER-001 and ETC-642, have been developed and have entered clinical trials.10–12 Initially developed for atherosclerosis and optimized to promote cholesterol efflux, these sHDL candidates showed potent HDL-c elevating effects in Phase I/II clinical trials.13–14 However, phase II/III results only showed sub-optimal therapeutic benefits.15 Such clinical results called for a re-evaluation of the current HDL-c-focusing treatment strategies of sHDLs. Moreover, there has been an increasing recognition that HDL functionality, instead of HDL-c levels, is a better indicator of the protective effects of HDLs. Thus, there has been a paradigm shift for HDL replacement therapy from elevating HDL-c levels to enhancing sHDL functions.6 Developing sHDLs with more sophisticated functionalities is essential for the successful clinical translation of sHDLs.

In contrast to the simple composition of sHDLs, endogenous HDLs present very complex and highly dynamic proteomic and lipidomic profiles, which are closely associated with the functions of HDLs.1, 16 Among various bioactive components of endogenous HDLs, anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), and phosphatidylserine (PS), have been shown to have a significant association with the protective functions of HDLs.17 Notably, a strong positive correlation was found between PS abundance and HDL functions such as cholesterol efflux, anti-inflammation, and anti-oxidation capacities.17–18

PS is an anionic lipid mainly located in the inner leaflet of cell membranes. On apoptotic cells, PS is translocated to the outer leaflet of membranes, serving as an ‘eat me’ signal to phagocytes such as macrophages.19 Activation of PS receptors induces anti-inflammatory and immunosuppressive responses in phagocytes, which enables the silent clearance of apoptotic cells.19–20 Based on the correlation between PS content and HDL functions, as well as the intrinsic anti-inflammatory effects of PS, it was hypothesized that introducing PS could increase the therapeutic effects of sHDLs.18, 21 This hypothesis has been recently examined by Darabi et al., PS-containing sHDLs showed greater anti-inflammatory effects by modulating Akt1/2/3- and p38 MAPK- mediated signaling pathways.22 As a continuous effort to investigate the impacts of PS on the functionality of HDL mimetics, in the present study, a series of PS-containing sHDLs was prepared using POPC, POPS, and an ApoA-1 mimetic peptide 22A. The impacts of PS incorporation on particle characteristics, anti-inflammatory effects, in vitro and in vivo cholesterol efflux capacities, and pharmacokinetic profiles were investigated.

2. Materials and Methods

2.1. Materials

22A peptide (PVLDLFRELLNELLEALKQKLK) was synthesized by Genscript Inc. (Piscataway, NJ). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-3-phospho-L-serine sodium salt were (POPS-Na) were purchased from NOF America Corporation (White Plains, NY). LPS from Escherichia coli O111 (L2630):B4 was purchased from Sigma. Mouse IL-6, TNF-α, MCP-1 ELISA kits were purchased from Invitrogen.

2.2. Cell culture

RAW264.7 and J774.A1 cells were obtained from ATCC. Both cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (10,000 U/mL). HUVEC cells were obtained from Lonza (Morristown, NJ) and cultured in endothelial cell growth media EGM-2 purchased from Lonza. Cells were used before passage 7. All cells were cultured in a 37 °C incubator with 5% CO2.

2.3. Preparation and characterization of sHDLs

sHDLs composed of 22A, POPC and POPS were prepared by the lyophilization-rehydration method as described previously.23–24 22A and POPC were dissolved and mixed in acetic acid in lipid to peptide weight ratio of 2:1. For POPS-containing sHDLs, 5%, 10%, 25% or 50% of POPC (mass ratio) was replaced by POPS-Na during preparation. The mixture was then freeze-dried. The lyophilized powder was rehydrated by PBS (pH 7.4), followed by 3 thermocycles. For POPC-sHDLs and 5% POPS/POPC-sHDLs, the thermocycle conditions are room temperature (5 min) and ice bath (5 min) trice. For 10%, 25% and 50% POPS/POPC-sHDLs, the thermocycle conditions are 37 °C (5 min) and ice bath (5 min) trice. The particle size and zeta potential of different sHDLs were examined by dynamic light scattering (DLS) using Malvern Zetasizer Nano ZSP (Westborough, MA). The purity of sHDLs was analyzed by gel permeation chromatography (GPC) on a Tosoh TSK gel G3000SWxl column with a PBS flow rate of 1 mL/min and UV detection at 220 nm. For transmission electron microscopy (TEM) observation, different sHDLs were loaded on a carbon film-coated 400 mesh copper grid from Electron Microscopy Sciences (Hatfield, PA), negatively stained with 1% (w/v) uranyl formate and dried. The samples were imaged with 100kV Morgagni TEM. For particle stability evaluation, different sHDLs were incubated in PBS (pH 7.4) at a 22A concentration of 1 mg/mL at 37 °C. At different time points, the particle size of sHDLs was analyzed by DLS.

2.4. Cytotoxicity assay

RAW 264.7 cells were seeded to 96-well plates at a density of 5 × 104 cells/well. After overnight incubation, cells were incubated with different sHDLs with different 22A concentrations for 24 h. At the end of the incubation, the cell viability was determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay following the protocol provided by the manufacturer.

2.5. Cholesterol efflux assay

J774.A1 cells were seeded in 24-well plates at a density of 2 × 105 cells/well. Cells were incubated overnight with 1 μCi/ml [3H] cholesterol in DMEM containing 0.3% fatty acid-free bovine serum albumin (BSA) and 5 μg/mL ACAT inhibitor Sandoz 58–035. At the end of the incubation, the media was discarded and cells were then washed with PBS twice. Cells were then equilibrated in DMEM containing 0.3% BSA and 5 μg/mL ACAT inhibitor Sandoz 58–035 for 24 h. The media was discarded, and cells were washed with PBS again. The [3H] cholesterol labeled cells were incubated with different sHDLs for 4 h at 22A concentrations of 5, 10, 25 or 50 μg/mL in 0.5 mL DMEM containing 0.3% BSA. Cells treated with PBS in the same media were used to measure background cholesterol efflux. At the end of incubation, media were collected, and cells were lysed with 0.5 mL 0.1% SDS and 0.1 N NaOH. The cell culture media and cell lysate were centrifuged at 400 g for 10 min. 200 μL supernatant was collected. The radioactive counts were measured using liquid scintillation counting by Perkin Elmer Tri-Carb 2910TR (Waltham, MA). The cholesterol efflux percentage was calculated by dividing the media count by the sum of the media and cell counts. Background cholesterol efflux was subtracted from cholesterol efflux percentages of treatment groups.

2.6. Anti-inflammatory study

The anti-inflammatory effects of sHDLs were evaluated using method described previously with modifications.25 For LPS-induced inflammation, RAW 264.7 cells were seeded to 24-well plates at a density of 2 × 105/well and incubated overnight to allow attachment. Cells were co-incubated with 100 ng/ml LPS and sHDLs at a 22A concentration of 10, 20, or 50 μg/ml for 12 h. The cell culture media were collected. The cytokine concentrations were quantified by ELISA. For TNF-α-induced inflammation, HUVEC cells were seeded to 12-well plates and cultured to reach confluence before experiments. HUVEC cells were then incubated with sHDLs and 2 ng/ml TNF-α for 12 h. The cytokine concentrations in cell culture media were quantified by ELISA.

2.7. Efferocytosis assay

Efferocytosis assay was conducted using Efferocytosis kit (Cayman Chemicals, Ann Arbor MI) according to instructions from the manufacturer with modification. Briefly, for effector cell labeling, RAW264.7 cells were labeled with CytoTell Blue in PBS at 37 °C for 30 min. The cells were then washed twice with complete media, seeded into 12-well plates at a density of 3×105/well, and incubated overnight. Fluorescent labeled apoptotic bait cells were prepared by incubating non-labeled RAW 264.7 cells with CFSE at 37 °C for 30 min. Apoptosis of the CFSE labeled cells was induced by incubation with staurosporine for 6 h. For the co-incubation study, effector cells, bait cells (3×105/well), and different sHDLs (22A concentration 100 μg/mL) were incubated for 18 h. For the pre-treatment study, effector cells were pre-treated with different sHDLs (22A concentration 100 μg/mL), followed by the addition of bait cells. After incubation, the effector cells were collected by gentle scraping. The cell suspension was analyzed by flow cytometry. The percentage of CFSE/CytoTell Blue double-positive cells in effector cells was calculated.

2.8. In vivo cholesterol mobilization study

Sprague-Dawley rats (8–9 weeks old) were purchased from Charles River Breeding Laboratories (Portage, MI). Animals were randomly assigned to 3 groups with 4 rats per group and fasted 8 h prior to the administration of sHDLs. POPC-sHDL, 25% POPS/POPC-sHDL and 50% POPS/POPC-sHDL were injected to rats through tail vein at a 22A dose of 50 mg/kg. At different time points, approximately 300 μL blood samples were drawn from the jugular vein. Serum was isolated by centrifuging blood samples at 8,000 rpm for 10 minutes at 4°C. The serum samples were stored at −80 °C until analysis. The serum concentration of phospholipids (PL), total cholesterol (TC), and free cholesterol (FC) were quantified enzymatically using Wako detection kits (Wako Chemicals, Richmond, VA) following protocols provided by the manufacturer. Serum aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were quantified using AST and ALT colorimetric activity assay kit (Cayman Chemical, Ann Arbor, MI).

2.9. Statistical analysis

Data analysis was conducted using GraphPad Prism 8. Statistical significance was determined using a two-tailed unpaired Student’s t-test for 2 groups of data or one-way analysis of variance (ANOVA) for data of more than 2 groups except for those stated otherwise. A p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Preparation and characterization of sHDLs

The phase transition temperature as well as the charge of phospholipids have been known to affect the assembly and particle characteristics of sHDLs.26–27 To investigate the effects of POPS on particle characteristics of sHDLs, a series of POPC/POPS-sHDLs with different POPS contents were prepared and characterized in terms of particle size, surface charge, morphology, as well as particle stability. Following rehydration of the lyophilized 22A and phospholipid mixture, three heat-cooling cycles were conducted to allow the assembly of sHDLs. Due to the low phase transition temperature and aggregation tendency of POPC, a lower heating temperature was used for the preparation of 0%- and 5%-POPS/POPC-sHDLs. As shown in Figure 1A, replacing POPC with POPS up to 50% total lipid did not lead to significant changes in particle size of sHDLs. The zeta potential of sHDLs decreases with increasing POPS percentage, which is expected due to the anionic nature of POPS (Figure 1B). GPC analysis showed a more uniform particle size distribution for sHDLs with higher POPS content (Figure 1C). Consistent with the GPC results,TEM images (Figure 1D) showed that sHDLs with no or low PS contents presented more heterogeneous shapes with signs of particle aggregation. In contrast, 25%- and 50%-POPS/POPC-sHDLs presented more uniformly distributed, tightly bound particles. When incubated at 37 °C, a fast increase in particle size of POPC-sHDLs was observed, indicating aggregation of POPC-sHDLs. Meanwhile, sHDLs containing higher POPS percentages showed improved particle size stability manifested with slower aggregation and smaller size of aggregates (Figure 1E).

Figure 1.

Figure 1.

Open in a new tab

Particle size (A) and zeta potential (B) of different sHDLs measured by DLS (n = 3, mean ± SD). (C) GPC chromatogram of different sHDLs. (D) TEM images of POPC-sHDL (D1), 5% POPS/POPC-sHDL (D2), 10% POPS/POPC-sHDL (D3), 25% POPS/POPC-sHDL (D4), and 50% POPS/POPC-sHDL (D5). (E) Particle sizes of sHDLs at different time points when incubated at 37°C (n = 3, mean ± SD).

3.2. Cellular toxicity

To investigate whether the introduction of PS affects the safety profile of sHDLs, the cell viability after 24 h incubation with different sHDLs was evaluated on RAW264.7 macrophages. As shown in Figure 2, no significant cellular toxicity was found on for all formulations with 22A concentrations up to 500 μg/ml, suggesting good compatibility of POPS/POPC-sHDLs.

Figure 2.

Figure 2.

Open in a new tab

Relative cell viability of RAW264.7 cells incubated with different sHDLs. Cells without treatment were used as 100% (n = 6, mean ± SD).

3.3. Cholesterol efflux assay

Since the PS content was reported to be positively associated with the cholesterol efflux capacity of endogenous HDLs, it was hypothesized that supplementing PS would enhance the cholesterol efflux capacity of sHDLs.17, 21 To test this hypothesis, the cholesterol efflux capacity of sHDLs with different POPS contents was compared on J774 A.1 macrophage loaded with 3[H]-cholesterol. As shown in Figure 3, all sHDLs could induce significant cholesterol efflux in a concentration-dependent manner. However, no statistical difference was observed among sHDLs with different POPS contents, suggesting that POPS did not significantly affect the cholesterol efflux capacity of sHDLs.

Figure 3.

Figure 3.

Open in a new tab

Cholesterol efflux effects of different sHDLs (n = 3, mean ± SD).

3.4. Anti-inflammatory effects

The anti-inflammatory effects of different sHDLs were investigated on LPS-treated RAW 264.7 macrophages and HUVEC cells. As shown in Figure 4, while LPS greatly increased the production of pro-inflammatory cytokines, sHDL treatment reduced the cytokine secretion on both cell lines. A greater reduction in proinflammatory cytokines was observed on cells treated with sHDLs composed of a higher POPS percentage. To investigate whether the increased anti-inflammatory effects of POPS-containing sHDLs were due to increased LPS neutralization, an LPS binding assay was conducted. No increase in LPS binding capacity was found in POPS-containing sHDLs in LPS binding assays (Figure S1). Moreover, the anti-inflammatory effects of 25%- and 50%-POPS/POPC-sHDLs retained when cells were treated with sHDLs and LPS separately (Figure S2). Such results suggested that the enhanced anti-inflammatory effects of POPS-containing sHDLs which is not completely dependent on LPS neutralization effects of sHDLs.

Figure 4.

Figure 4.

Open in a new tab

Pro-inflammatory cytokine levels from LPS-treated RAW264.7 (A, B, and C) or HUVEC (D and E) cells. (n = 3, mean ± SD). *p<0.05; **p<0.01; ***p<0.005, ****p<0.0001 compared to POPC-sHDLs (0% POPS group). #p<0.05; ##p<0.01; ###p<0.005, ####p<0.001 compared to LPS-only group.

3.5. Efferocytosis assay

Efferocytosis is an essential physiological process to clear apoptotic cells by phagocytes. Serving as an ‘eat me’ signal on apoptotic cells, PS plays important roles in facilitating efferocytosis process.28 The effects of PS-containing sHDLs on efferocytosis efficiency of macrophages were evaluated on RAW 264.7 macrophages. As shown in Figure 5A, when effector cells are co-incubated with sHDLs and bait cells, efferocytosis efficiency decreased as POPS content in sHDLs increased. When macrophages were pretreated with sHDLs prior to efferocytosis assay (Figure 5B), POPC-sHDL, 5%- or 10%- POPS/POPC-sHDLs did not alter the efferocytosis efficiency of macrophages compared to control cells. However, pre-incubating cells with 25%- and 50%-POPS/POPC-sHDL led to a slight increase in the percentage of double-positive in effector cells, suggesting an enhanced efferocytosis activity of macrophages.

Figure 5.

Figure 5.

Open in a new tab

Percentage of CytoTell Blue/CFSE double-positive effector cells after (A) co-incubated or (B) pretreated with different sHDLs (n = 3, mean ± SD). *p<0.05; **p<0.01; ***p<0.005, ****p<0.0001 compared to POPC-sHDLs (0% POPS) group. #p<0.05; ##p<0.01; ###p<0.005 compared to mock group.

3.6. In vivo PK/PD studies

The phospholipid composition has greatly impact on PK/PD properties of sHDLs.29–30 To evaluate how POPS affects the in vivo behavior of sHDLs, the PK/PD profiles of sHDLs composed with different POPS contents were evaluated were investigated in Sprague Dawley rats. As shown in Figure 6, i.v. infusion of sHDLs induced a significant increase in serum cholesterol levels in rats. Compared to the POPC-sHDLs-treated group, rats treated with 25%- and 50%-POPS/POPC-sHDLs showed slightly higher total cholesterol and free cholesterol levels in 2 h post-injection. No significant difference was found in phospholipid levels between different groups at all timepoints. The serum ALT and AST levels were masured at different time points following i.v. infusion of sHDLs to evaluate the safety profiles of different sHDLs. As seen in Figure 7, no significant elevation (> 3-folds baseline) in ALT or AST was observed. Any changes in ALT and AST levels were transient, as ALT and AST levels returned back to baseline at 48 h.

Figure 6.

Figure 6.

Open in a new tab

Total cholesterol (A), free cholesterol (B), cholesterol ester (C) and phospholipids (D) levels in rat serum after i.v. injection of different sHDLs at 22A 50 mg/kg (n = 4, mean ± SD). *p<0.05, **p<0.01 for 25% POPS/POPC-sHDL compared to POPC-sHDL. ##p<0.005, ###p<0.001 for 50% POPS/POPC-sHDL compared to POPC-sHDL.

Figure 7.

Figure 7.

Open in a new tab

Serum ALT and AST levels after i.v. injection of different sHDLs. (N = 4, mean ± SD).

4. Discussion

Despite accounting for less than 1% of phospholipid content, anionic phospholipids such as PG, PS, and PI are associated with protective functions of HDLs, such as cholesterol efflux, anti-inflammation, and anti-oxidation effects31. Among various anionic lipids, PS has generated great research interest with its intrinsic anti-inflammatory capacities.21 However, the impact of PS on the functionality of sHDL has not been fully examined. In this study, a series of sHDLs with different POPS contents was prepared. The effects of PS on particle characteristics, cholesterol efflux, anti-inflammatory effects, efferocytosis, as well as pharmacokinetic profiles were evaluated.

Substituting POPC to POPS significantly altered the physiochemical properties of sHDLs, which may greatly impact on their biological functions. Increasing POPS content in sHDLs led to a reduction of zeta potential of sHDLs without impacting the main particle size. POPS-containing sHDLs presented a more uniform particle size distribution and greater size stability when incubated at 37 °C. The improved particle stability could be attributed to two reasons. First, POPS has a higher phase transition temperature of 14 °C compared to −2 °C of POPC. Thus, with the increasing POPS content, the fluidity of the lipid membrane of sHDLs decreases, which prevents the aggregation of particles.29 Second, negatively charged POPS creates electrostatic repulsion between particles, which is expected to increase the colloidal stability of sHDLs. The impacts of altered surface charge and increased particle stability of POPS-containing sHDLs on sHDL biological functions including anti-inflammatory effects, cholesterol efflux and PK/PD profiles were further examined.

sHDLs exert anti-inflammatory effects through multiple mechanisms including direct neutralization of endotoxins and regulatory effects on inflammatory signaling pathways.25 Interestingly, POPS/POPC-sHDLs showed increased anti-inflammatory effects in a POPS-content-dependent manner when evaluated on LPS-activated cells. However, no increase in LPS binding capacity was found in POPS-containing sHDLs in LPS binding assays, suggesting the enhanced anti-inflammatory effects were not due to increased LPS neutralization. Additionally, 25%- and 50%-POPS/POPC-sHDLs showed anti-inflammatory effects when pretreated cells were activated with LPS in absence of sHDLs. Thus, it suggested that the enhanced anti-inflammatory effects of 25%- and 50%-POPS/POPC-sHDLs were related to regulating inflammatory response of cells. Several mechanisms related to anti-inflammatory effects of PS nanoparticles have been proposed. For example, in a recent study by Darabi et al., it was reported that PS incorporation into recombinant HDL particles led to more potent anti-inflammatory effects by modulating Akt1/2/3- and p38 MAPK signaling pathways.22 In other studies, PS-containing liposomes were found to drive the repolarization of macrophages from pro-inflammatory M1 to anti-inflammatory M2 phenotype.32 The results suggest that POPS incorporation would be an effective strategy to enhance the anti-inflammatory effects of sHDLs.

Efferocytosis, a process where apoptotic cells are cleared by macrophages and other phagocytes in a non-inflammatory manner, is essential for the maintenance of homeostasis and inflammation resolution.33 Impaired efferocytosis causes the secondary necrosis of apoptotic cells, which contributes to unresolved inflammation in various chronic inflammatory diseases.33–35 Serving as an ‘eat me’ signal, PS is essential for the recognition and phagocytosis of apoptotic cells in efferocytosis.28 Activating PS receptors by administrating PS-containing liposomes has been shown to restore the efferocytosis capacity of macrophages in several in vivo studies.36 In the present study, when macrophages were co-incubated with sHDLs and apoptotic cells, increasing POPS percentages led to decreased efferocytosis of bait cells, which may be attributed to the competition of PS receptors.22 However, when macrophages were pre-treated with sHDLs, slightly higher efferocytosis efficiency was shown on macrophages treated with 25%- or 50%-POPS/POPC-sHDLs. The net effects of POPS-containing sHDLs on efferocytosis in vivo may depend on various factors such as dose and pharmacokinetic profiles.

Recognition of apoptosis cells by PS receptors has been shown to lead to a rapid increase in ABCA-1 expression and enhanced cholesterol efflux.37–38 Thus, the hypothesis that increasing PS exposure may enhance cholesterol efflux was tested.18 However, in vitro studies showed little difference in cholesterol efflux capacity between POPC-sHDLs and POPS/POPC-sHDLs. This result is consistent with previous studies by Darabi et al., where PS containing recombinant HDLs did not increase ABCA-1 expression or in vitro cholesterol efflux.22 Interestingly, in the in vivo pharmacodynamic study, POPS-containing sHDLs showed a lightly higher cholesterol mobilization capacity while presenting similar pharmacokinetic profiles to POPC-sHDLs. Since reverse cholesterol transport is a complex process, such difference in cholesteterol mobilization may be attributed to other steps in reverse cholesterol transport such as HDL maturation and liver cholesterol clearance. For example, as a ligand of SR-B1, PS incorporation might change the interaction between HDLs and hepatic cells, affecting the cholesterol clearance efficiency.39 The enrichment of PS to sHDLs may also alter the interaction of sHDL with lecithin:cholesterol acyltransferase and lipid transfer proteins. The mechanisms underlying the inconsistentin vivo and in vitro results would warrant further investigation.

It was reported that incorporating PS into liposomes could accelerate the elimination of liposomes due to the increased uptake of liposomes in the liver.40 As the liver is the major elimination organ for sHDLs, there was a concern that introducing PS may negatively affect the pharmacokinetic profiles of sHDLs. However, there was little difference in serum phospholipid levels between the POPC-sHDL group and the two POPS/POPC-sHDL groups, suggesting a limited impact of PS to sHDL pharmacokinetic profiles. The different effects of PS may be explained by different elimination mechanisms of liposomes and sHDLs. Typically, liposomes are mainly eliminated by the reticuloendothelial system such as Kupffer cells in an opsonization-dependent manner.41 However, with HDL-mimicking structures and small particle sizes, sHDLs are generally considered to be able to evade the reticuloendothelial system.42–43 Such difference was exemplified in an early pharmacokinetic study of PS-containing liposomes in rats. Following an initial rapid elimination attributed to elimination by the reticuloendothelial system, PS component in liposomes was found to be incorporated into HDL and eliminated at a slower rate with a similar half-life as rat HDL.44 Such observation may reconcile the discrepancy in the effects of PS on pharmacokinetic properties between liposomes and sHDLs.

5. Conclusion

In this study, a series of sHDLs were prepared with ApoA-1 mimetic peptide, POPC, and POPS. Increasing PS content in sHDL particles improved particle stability and significantly increased the anti-inflammatory effects of sHDLs. Limited impacts were found on cholesterol efflux capacity and pharmacokinetic profiles of sHDLs. Such results suggest the therapeutic potential of PS-containing sHDLs on inflammation resolution in atherosclerosis and other inflammatory diseases.

Supplementary Material

Supplementary Materials

Acknowledgment

The authors would like to acknowledge Dr. Anatol Kontush and Dr. Maryam Darabi for their advice on this research project. The research was supported by the National Institutes of Health (R01 GM113832 and R21 NS111191 to A.S.). M.Y was supported by American Heart Association Predoctoral Fellowship (19PRE34400017). K.H.D is supported by Pharmacological Sciences Training Program (T32 GM007767), Translational Cardiovascular Research and Entrepreneurship Training Program (T32 HL125242), and American Foundation for Pharmaceutical Education Pre-Doctoral Fellowship. T.H. was supported by the Pharmaceutical Research and Manufacturers of America Foundation as a pre-doctoral fellow in drug discovery.

Conflicts of Interest

Dr. Schwendeman declares financial interests for board membership, as a paid consultant, for research funding, and as equity holder in EVOQ Therapeutics. The University of Michigan has a financial interest in EVOQ Therapeutics.

Footnotes

Supporting Information

LPS binding capacity and anti-inflammatory effects of different sHDLs with pretreatment-activation experimental setup.

Reference

  • 1.Kontush A; Lindahl M; Lhomme M; Calabresi L; Chapman MJ; Davidson WS, Structure of HDL: particle subclasses and molecular components. Handbook of experimental pharmacology 2015, 224, 3–51. [DOI] [PubMed] [Google Scholar]
  • 2.Dergunov AD; Baserova VB, Different pathways of cellular cholesterol efflux. Cell Biochemistry and Biophysics 2022, 80 (3), 471–481. [DOI] [PubMed] [Google Scholar]
  • 3.Trakaki A; Marsche G, Current understanding of the immunomodulatory activities of high-density lipoproteins. Biomedicines 2021, 9 (6), 587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Primer KR; Psaltis PJ; Tan JTM; Bursill CA, The Role of High-Density Lipoproteins in Endothelial Cell Metabolism and Diabetes-Impaired Angiogenesis. Int J Mol Sci 2020, 21 (10), 3633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Karlsson H; Kontush A; James RW, Functionality of HDL: antioxidation and detoxifying effects. Handbook of experimental pharmacology 2015, 224, 207–228. [DOI] [PubMed] [Google Scholar]
  • 6.Favari E; Thomas MJ; Sorci-Thomas MG, High-density lipoprotein functionality as a new pharmacological target on cardiovascular disease: unifying mechanism that explains high-density lipoprotein protection toward the progression of atherosclerosis. Journal of Cardiovascular Pharmacology 2018, 71 (6), 325–331. [DOI] [PubMed] [Google Scholar]
  • 7.Kim SY; Yu M; Morin EE; Kang J; Kaplan MJ; Schwendeman A, High-Density Lipoprotein in Lupus: Disease Biomarkers and Potential Therapeutic Strategy. Arthritis Rheumatol 2020, 72 (1), 20–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Begue F; Tanaka S; Mouktadi Z; Rondeau P; Veeren B; Diotel N; Tran-Dinh A; Robert T; Velia E; Mavingui P; Lagrange-Xelot M; Montravers P; Couret D; Meilhac O, Altered high-density lipoprotein composition and functions during severe COVID-19. Sci Rep 2021, 11 (1), 2291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Zakiev E; Feng M; Sukhorukov V; Kontush A, HDL-Targeting Therapeutics: Past, Present and Future. Curr Pharm Des 2017, 23 (8), 1207–1215. [DOI] [PubMed] [Google Scholar]
  • 10.Capodanno D; Mehran R; Gibson CM; Angiolillo DJ, CSL112, a reconstituted, infusible, plasma-derived apolipoprotein A-I: safety and tolerability profiles and implications for management in patients with myocardial infarction. Expert Opin Investig Drugs 2018, 27 (12), 997–1005. [DOI] [PubMed] [Google Scholar]
  • 11.Keyserling CH; Barbaras R; Benghozi R; Dasseux JL, Development of CER-001: Preclinical Dose Selection Through to Phase I Clinical Findings. Clin Drug Invest 2017, 37 (5), 483–491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dasseux J-L, ETC-642, a Peptide/phospholipids complex that mimics the structure and functions of high density lipoproteins (HDL). 2004; p presented at TIDES Conference. [Google Scholar]
  • 13.Gille A; Easton R; D’Andrea D; Wright SD; Shear CL, CSL112 enhances biomarkers of reverse cholesterol transport after single and multiple infusions in healthy subjects. Arterioscler Thromb Vasc Biol 2014, 34 (9), 2106–2114. [DOI] [PubMed] [Google Scholar]
  • 14.Keyserling CH; Hunt TL; Klepp HM; Scott RA; Barbaras R; Schwendeman A; Lalwani N; Dasseux JL, CER-001, a Synthetic HDL-Mimetic, Safely Mobilizes Cholesterol in Healthy Dyslipidemic Volunteers. Circulation 2011, 124 (21), A15525. [Google Scholar]
  • 15.Karalis I; Jukema JW, HDL Mimetics Infusion and Regression of Atherosclerosis: Is It Still Considered a Valid Therapeutic Option? Curr Cardiol Rep 2018, 20 (8), 66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kontush A; Lhomme M; Chapman MJ, Thematic Review Series: High Density Lipoprotein Structure, Function, and Metabolism Unraveling the complexities of the HDL lipidome. Journal of lipid research 2013, 54 (11), 2950–2963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Camont L; Lhomme M; Rached F; Le Goff W; Negre-Salvayre A; Salvayre R; Calzada C; Lagarde M; Chapman MJ; Kontush A, Small, dense high-density lipoprotein-3 particles are enriched in negatively charged phospholipids: relevance to cellular cholesterol efflux, antioxidative, antithrombotic, anti-inflammatory, and antiapoptotic functionalities. Arterioscler Thromb Vasc Biol 2013, 33 (12), 2715–2723. [DOI] [PubMed] [Google Scholar]
  • 18.Darabi M; Kontush A, Phosphatidylserine in atherosclerosis. Curr Opin Lipidol 2016, 27 (4), 414–420. [DOI] [PubMed] [Google Scholar]
  • 19.Glassman FY; Schneider JL; Ramakrishnan R; Dingman RK; Ramanathan M; Bankert RB; Balu-Iyer SV, Phosphatidylserine Is Not Just a Cleanup Crew but Also a Well-Meaning Teacher. J Pharm Sci 2018, 107 (8), 2048–2054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Naeini MB; Bianconi V; Pirro M; Sahebkar A, The role of phosphatidylserine recognition receptors in multiple biological functions. Cell Mol Biol Lett 2020, 25 (1), 1–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Darabi M; Kontush A, Can phosphatidylserine enhance atheroprotective activities of high-density lipoprotein? Biochimie 2016, 120, 81–86. [DOI] [PubMed] [Google Scholar]
  • 22.Darabi M; Lhomme M; Dahik VD; Guillas I; Frisdal E; Tubeuf E; Poupel L; Patel M; Gautier EL; Huby T; Guerin M; Rye KA; Lesnik P; Le Goff W; Kontush A, Phosphatidylserine enhances anti-inflammatory effects of reconstituted HDL in macrophages via distinct intracellular pathways. FASEB J 2022, 36 (5), e22274. [DOI] [PubMed] [Google Scholar]
  • 23.Tang J; Kuai R; Yuan W; Drake L; Moon JJ; Schwendeman A, Effect of size and pegylation of liposomes and peptide-based synthetic lipoproteins on tumor targeting. Nanomedicine 2017, 13 (6), 1869–1878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Yuan W; Ernst K; Kuai R; Morin EE; Yu M; Sviridov DO; Tang J; Mei L; Li D; Ackermann R; Remaley AT; Schwendeman A, Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo. Nanomedicine 2023, 48, 102646. [DOI] [PubMed] [Google Scholar]
  • 25.Guo L; Morin EE; Yu MZ; Mei L; Fawaz MV; Wang Q; Yuan YX; Zhan CG; Standiford TJ; Schwendeman A; Li XA, Replenishing HDL with synthetic HDL has multiple protective effects against sepsis in mice. Science Signaling 2022, 15 (725), eabl9322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Patel H; Ding B; Ernst K; Shen L; Yuan W; Tang J; Drake LR; Kang J; Li Y; Chen Z; Schwendeman A, Characterization of apolipoprotein A-I peptide phospholipid interaction and its effect on HDL nanodisc assembly. Int J Nanomedicine 2019, 14, 3069–3086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Schwendeman A; Sviridov DO; Yuan W; Guo Y; Morin EE; Yuan Y; Stonik J; Freeman L; Ossoli A; Thacker S; Killion S; Pryor M; Chen YE; Turner S; Remaley AT, The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties. J Lipid Res 2015, 56 (9), 1727–1737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Birge RB; Boeltz S; Kumar S; Carlson J; Wanderley J; Calianese D; Barcinski M; Brekken RA; Huang X; Hutchins JT; Freimark B; Empig C; Mercer J; Schroit AJ; Schett G; Herrmann M, Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ 2016, 23 (6), 962–978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Fawaz MV; Kim SY; Li D; Ming R; Xia Z; Olsen K; Pogozheva ID; Tesmer JJG; Schwendeman A, Phospholipid Component Defines Pharmacokinetic and Pharmacodynamic Properties of Synthetic High-Density Lipoproteins. J Pharmacol Exp Ther 2020, 372 (2), 193–204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Li D; Fawaz MV; Morin EE; Ming R; Sviridov D; Tang J; Ackermann R; Olsen K; Remaley AT; Schwendeman A, Effect of Synthetic High Density Lipoproteins Modification with Polyethylene Glycol on Pharmacokinetics and Pharmacodynamics. Mol Pharm 2018, 15 (1), 83–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Darabi M; Guillas-Baudouin I; Le Goff W; Chapman MJ; Kontush A, Therapeutic applications of reconstituted HDL: When structure meets function. Pharmacology & therapeutics 2016, 157, 28–42. [DOI] [PubMed] [Google Scholar]
  • 32.Toita R; Kang JH; Tsuchiya A, Phosphatidylserine liposome multilayers mediate the M1-to-M2 macrophage polarization to enhance bone tissue regeneration. Acta Biomater 2022, 154, 583–596. [DOI] [PubMed] [Google Scholar]
  • 33.Doran AC; Yurdagul A Jr.; Tabas I, Efferocytosis in health and disease. Nat Rev Immunol 2020, 20 (4), 254–267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Abdolmaleki F; Farahani N; Gheibi Hayat SM; Pirro M; Bianconi V; Barreto GE; Sahebkar A, The Role of Efferocytosis in Autoimmune Diseases. Front Immunol 2018, 9, 1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Yurdagul A Jr.; Doran AC; Cai B; Fredman G; Tabas IA, Mechanisms and Consequences of Defective Efferocytosis in Atherosclerosis. Front Cardiovasc Med 2017, 4, 86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Fernandez-Boyanapalli RF; Frasch SC; McPhillips K; Vandivier RW; Harry BL; Riches DW; Henson PM; Bratton DL, Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4. Blood 2009, 113 (9), 2047–2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Fond AM; Lee CS; Schulman IG; Kiss RS; Ravichandran KS, Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1. J Clin Invest 2015, 125 (7), 2748–2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kiss RS; Elliott MR; Ma Z; Marcel YL; Ravichandran KS, Apoptotic cells induce a phosphatidylserine-dependent homeostatic response from phagocytes. Curr Biol 2006, 16 (22), 2252–2258. [DOI] [PubMed] [Google Scholar]
  • 39.Fluiter K; van Berkel TJ, Scavenger receptor B1 (SR-B1) substrates inhibit the selective uptake of high-density-lipoprotein cholesteryl esters by rat parenchymal liver cells. Biochem J 1997, 326 (2), 515–519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Kamps JA; Morselt HW; Scherphof GL, Uptake of liposomes containing phosphatidylserine by liver cells in vivo and by sinusoidal liver cells in primary culture: in vivo-in vitro differences. Biochem Biophys Res Commun 1999, 256 (1), 57–62. [DOI] [PubMed] [Google Scholar]
  • 41.Liu D; Liu F; Song YK, Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin. Biochim Biophys Acta 1995, 1235 (1), 140–146. [DOI] [PubMed] [Google Scholar]
  • 42.Kuai R; Li D; Chen YE; Moon JJ; Schwendeman A, High-Density Lipoproteins: Nature’s Multifunctional Nanoparticles. ACS Nano 2016, 10 (3), 3015–3041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Raut S; Mooberry L; Sabnis N; Garud A; Dossou AS; Lacko A, Reconstituted HDL: Drug Delivery Platform for Overcoming Biological Barriers to Cancer Therapy. Front Pharmacol 2018, 9, 1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Palatini P; Viola G; Bigon E; Menegus AM; Bruni A, Pharmacokinetic characterization of phosphatidylserine liposomes in the rat. Br J Pharmacol 1991, 102 (2), 345–350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials
NIHMS1969019-supplement-Supplementary_Materials.docx (93.1KB, docx)

RESOURCES