Reduced Uptake of Oxidized Low-Density Lipoprotein by Macrophages Using Multiple Aptamer Combinations

Abstract

The accumulation of oxidized low-density lipoprotein (oxLDL) in macrophages leads to the formation of foam cells and atherosclerosis development. Reducing the uptake of oxLDL in macrophages decreases the incidence and progression of atherosclerosis. Four distinct single-strand DNA sequences, namely, AP07, AP11, AP25, and AP29, were selected that demonstrated specific binding to distinct regions of oxidized apolipoprotein B100 (apoB100; the protein component of oxLDL) with low HDOCK scores. These four DNA sequences were combined to generate aptamers that selectively bound to labeled Dil-oxLDL, and were subsequently added to murine RAW 264.7 macrophages to test their inhibitory effects using fluorescence spectrometry. The four combined aptamers at 10 μM reduced oxLDL uptake by 79 ± 4% compared to that of the untreated aptamer group. Flow cytometry data demonstrated that macrophages treated with aptamers reached only 32.6% of the Dil-oxLDL signal, a 50% reduction in fluorescence emission relative to that of the untreated group (64.4% Dil-oxLDL signal). Binding the four combined aptamers to the oxLDL surface disrupted the interaction between oxLDL and CD36 via cyclic voltammetry, effectively decreasing the level of uptake of oxLDL by macrophages. Results suggested that these aptamers could be used as alternative compounds to prevent the formation of foam cells, hence providing antiatherosclerosis activity.

Introduction

The World Heart Federation reported that cardiovascular diseases (CVD) constituted one-third of the total global mortality in 2021, as 20.5 million fatalities, with a consistent upward trend each year. The primary modifiable risk factors contributing to CVD are excessive levels of low-density lipoprotein (LDL) and cholesterol.¹ LDL in the bloodstream migrates to the coronary artery wall and undergoes oxidation due to the presence of free radicals generated by the blood vessel cell walls and macrophages including nitric oxide synthase, 15-lipoxygenase, or myeloperoxidase.² LDL particles are transformed to oxidized LDL (oxLDL), a pathogenic lipoprotein. The levels of oxLDL in the bloodstream serve as a critical signal for assessing the probability of developing CVD. Macrophages eliminate oxLDL by collecting oxLDL particles through scavenger receptors such as lectin-like oxidized LDL receptor-1 (LOX-1), scavenger receptor class B type I (SR-BI), and cluster of differentiation 36 (CD36).³⁻⁷ The lectin-like domain of LOX-1 comprises positively charged residues that promote interactions with the negatively charged oxidized apoB100 and oxidized phospholipids.⁸ The extracellular region of CD36 contains lipid-binding domains that serve as a hydrophobic pocket for the binding of oxidized phospholipids as well as oxidized apoB100.⁹ In contrast to LOX-1 and CD36, SR-BI selectively internalizes oxidized phospholipids and cholesteryl esters contained inside the oxLDL particle, rather than the entire particle itself.¹⁰ However, excessive uptake of oxLDL by macrophages leads to lipid accumulation and finally to foam cell formation. These foam cells stimulate the immune system to release inflammatory cytokines from lymphocytes. This generates an excessive number of smooth muscle cells that gather on the walls of blood vessels to develop fatty streaks, giving rise to atherosclerosis plaques.^11,12 Progressive accumulation of oxLDL leads to the rupture and subsequent death of foam cells, which act as markers for the early stages of atherosclerosis and also play a role in the overall atherosclerotic lesion process. Preventing oxLDL accumulation in foam cells significantly reduces both the occurrence and the advancement of atherosclerosis.

Preventive agents derived from both biological and chemical sources have demonstrated inhibitory effects on foam cell development. Several natural herb plant extracts such as Bacalein,¹³ Astragaloside IV,¹⁴ Isoborneol,¹⁵Borneolum syntheticum (Bingpian),¹⁶ and the extract product of coffee and green tea¹⁷ have an impact on treating fatty buildup. Chemical compounds such as metformin¹⁸ and atorvastatin¹⁹ have anti-inflammatory properties when used in statin therapy for CVD. Aptamers mimic biomaterials and have attracted attention for their potential use in diagnostic and therapeutic approaches. Aptamers are single-stranded DNA or RNA molecules selected through a process known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX) from combinatorial libraries of synthetic nucleic acids containing 60–100 nucleotides to obtain a specific sequence toward the target.²⁰ Their favorable characteristics include high stability, easy synthesis, low immunogenicity, low toxicity, and high affinity for their respective targets. Aptamers have already been studied in developing novel drugs and drug delivery systems such as AS1411 (a nucleolin-specific aptamer) for acute myeloid leukemia and breast cancer, ARC1779 (a von Willebrand factor-specific aptamer) for carotid artery disease, and NU172 (a thrombin-specific aptamer) for anticoagulation. They are currently undergoing clinical trials.²¹⁻²³ A Burkitt lymphoma-specific DNA aptamer-decorated artificial viral capsid was employed as the nanocarrier to deliver doxorubicin hydrochloride to Burkitt lymphoma cells.²⁴ A spherical cocktail of neutralizing aptamer-gold nanoparticles was created to inhibit the interaction between the host cell receptor and the binding domain of SARS-CoV-2.²⁵ Thus, aptamers show promise as alternative medications for some target disorders.

In our previous study, we identified a set of aptamers as promising candidates to exhibit specific binding ability toward oxLDL particles. A single aptamer can be utilized as the specific recognition element in an analytical system for determining oxLDL due to its high specificity and affinity.²⁶ LDL particles have a molecular weight of approximately 2,500 kDa. These complex aggregates consist of a single apolipoprotein B100 (apoB100) molecule and a large number of lipids.²⁷ The oxLDL particles undergo a variety of modifications, including oxidation of the lipids and apoB100. Aptamers targeting specific parts of oxLDL instead of targeting entire particles can improve selectivity. As previously reported, an entire oxLDL-based SELEX process produced a panel of ssDNA aptamers specific to oxLDL.²⁶ Aptamers specific to oxidized apoB100 on oxLDL were then screened.²⁸ Oxidized apoB100 was targeted because it interacts with scavenger receptors on macrophages, which is the first step in the uptake of oxLDL by these cells. The binding mechanism between each aptamer and oxidized apoB100 and its interaction was investigated by molecular docking. However, a single aptamer may not bind strongly and widely enough for protective strategies. In this article, aptamers that showed superior binding ability toward oxidized apoB100 were chosen to accurately predict the specific binding site on oxidized apoB100 using molecular docking modeling. The individual aptamers were then merged to enhance the likelihood of binding to various regions of oxidized apoB100. Finally, the combined aptamers were investigated as potential therapeutic agents that could bind directly to oxidized apoB100 of oxLDL, thereby reducing the uptake of oxLDL and the formation of foam cells in cell culture.

Experimental Section

Chemicals and Reagents

Murine RAW 264.7 macrophages were purchased from the ATCC Cell Bank (Biomedia, Bangkok, Thailand). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antibiotic-antimycotic (100×), and 0.25% trypsin-EDTA were provided by Gibco (Maryland, USA). FITC antimouse F4/80 antibody was purchased from Biolegend (California, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) was purchased from Thermo Fisher Scientific (Massachusetts, USA). 4′,6-Diamidino-2-phenylindole (DAPI) and thiazolyl blue tetrazolium bromide (MTT) were provided by PanReac AppliChem (Barcelona, Spain). RIPA cell lysis buffer was purchased from Energenesis Biomedical (Taipei, Taiwan). Isoborneol was purchased from Sigma-Aldrich (Missouri, USA). A colloidal solution of gold nanoparticles (AuNP) with a diameter of 25–30 nm was purchased from DCN Dx (California, USA). All other reagents used were analytical grades. The oxLDL-specific aptamers including AP07 (5′-CCATCACGGGGCAGGCGGACAAGGGGTAAGGGCCACATCA-3′), AP11 (5′-CTTCGATGTAGTTTTTGTATGGGGTGCCCTGGTTCCTGCA-3′), AP25 (5′-TCGATAGTTGAACATTGCCGATTTACCGGCTGGCGTCGTA-3′), AP29 (5′-TCCCATGCGCATCCAATCTTGCCTGATTTGAGAAGGACAC-3′)^26,28 and the LDL-specific aptamer (5′-ACCTCGATTTTATATTATTTCGCTTACCAACAACTGCAGA-3′)²⁹ were synthesized by Macrogen, Inc., South Korea. Deionized (DI) water with 18.2 MΩ resistance was obtained from a PURELAB flex3 ELGA LabWater (Buckinghamshire, UK) and used for all reagent preparations.

Simulation of Molecular Docking and Interaction Analysis between the Aptamers and Oxidized apoB100

Sixteen DNA sequences containing 40 nucleotides each targeting oxLDL, namely AP02, AP06, AP07, AP09, AP11, AP14, AP16, AP20, AP21, AP22, AP25, AP26, AP27, AP29, AP31, and AP32, were obtained from our previous in vitro SELEX process, as shown in Table S1.^26,28 Molecular docking is a computational technique employed to forecast the interaction between a small (ligand) and a larger molecule (target) by assessing the binding strength through estimating their structural features. The HDOCK web server (http://hdock.phys.hust.edu.cn/) was utilized for conducting protein–DNA docking simulations.^30,31 First, the tertiary structure of oxidized apoB100 containing 954 amino acids was employed as a target molecule because it is present on the surface of the oxLDL particle.²⁸ Sixteen aptamer sequences were chosen as ligands to predict dock binding to targets. The HDOCK algorithm was used to determine the optimal binding position on the complex formed by oxidized apoB100 and aptamers, yielding a docking score. The docking position with the lowest HDOCK score is the most favorable for the interaction between oxidized apoB100 and the aptamer, indicating a stronger binding affinity. The interaction between the docking complex of combined aptamers and oxidized apoB100 was analyzed using the protein–ligand interaction profiler (PLIP) web server (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) and visualized as a three-dimensional structure using ChimeraX software.³²

Preparation of Combined Aptamers

The individual lyophilized aptamers, namely, AP07, AP11, AP25, and AP29, were dissolved in DI water to achieve a final concentration of 100 μM. A combination of the four aptamers (AP07/AP11/AP25/AP29) at a concentration of 100 μM was prepared by mixing 100 μM each of AP07, AP11, AP25, and AP29 in a sterile container at an equal ratio of 1:1:1:1. The combinations of two aptamers (AP11/AP29) and three aptamers (AP07/AP11/AP29) were also prepared by using the same method. All the desired concentrations of combined aptamers were achieved through dilution with DI water, heated at 95 °C for 5 min, and cooled on ice for 5 min to yield a single strand of the aptamer.

Preparation of Oxidized Low-Density Lipoprotein

A purified low-density lipoprotein (LDL) solution was obtained during our previous research project with permission from the Human Research Ethical Committee at the Faculty of Medical Technology, Prince of Songkla University (no. EC66–04). Oxidized low-density lipoprotein (oxLDL) was prepared from an LDL solution based on previous protocols.²⁶ Briefly, LDL at a protein concentration of 0.1 mg/mL was prepared in 1× PBS. Then, it was oxidized with 50 μM CuSO₄ in DI water and incubated at 37 °C for 24 h. The formation of malondialdehyde (MDA) as the oxLDL product was monitored by using the thiobarbituric acid reactive substances (TBARS) assay. After boiling the oxLDL solution with the TBARS reagent for 15 min, the mixed solution was placed on ice for 10 min to terminate the reaction. All samples were monitored for absorbance at 532 nm and compared to the standard MDA solution using a Multiskan microplate spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA).

Dil-Fluorescence Dye Labeling of oxLDL

Since oxLDL is normally colorless, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) fluorescent dye was used to stain the lipid moiety of oxLDL. This allowed for visualizing oxLDL particles as well as tracking dynamic oxLDL uptake by macrophages. Dil staining of oxLDL was performed as previously described.³³ Dil conjugated oxLDL (Dil-oxLDL) was prepared by adding 50 μL of 0.1 mg/mL Dil in 1× PBS to 1 mL of 4 μM oxLDL. The mixture was incubated at 37 °C for 8 h under light protection. To isolate the Dil-oxLDL, the mixture solution supernatant was collected after 60 min of centrifugation at 14 000 rpm to remove excess unbound Dil.

Binding Ability and Selectivity of Aptamers Using Aptamer/Gold Nanoparticle Aggregation-Based Colorimetric Assay

An aptamer/gold nanoparticle (AuNP) aggregation-based colorimetric assay was performed following our previous study.²⁶ The binding ability of individual aptamers and their combinations was examined, including AP07, AP11, AP25, and AP29, a combination of two aptamers (AP11/AP29 at a ratio of 1:1), a combination of three aptamers (AP07/AP11/AP29 at a ratio of 1:1:1), a combination of four aptamers (AP07/AP11/AP25/AP29 at a ratio of 1:1:1:1), and the LDL-specific aptamer. Under the optimal condition, individual aptamers or aptamer combinations at a concentration of 5 μM (25 μL) were mixed with 25 μL of 0.5 μM oxLDL and incubated at 25 °C for 5 min. Then, 50 μL of AuNP solution (OD 520 nm = 1) was added and left for 6 min followed by 25 μL of 80 mM NaCl solution for 8 min. Absorbance values at 520 and 630 nm were determined using a Multiskan microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The signal ratio at 630/520 nm was calculated.

The selectivities of individual aptamers, including AP07, AP11, AP25, and AP29, a combination of four aptamers (AP07/AP11/AP25/AP29 at a ratio of 1:1:1:1), and the LDL-specific aptamer were investigated by exposing them to oxLDL and compared to suspected interferences at physiologically normal concentrations. LDL (2.6 mM), high-density lipoprotein (HDL, 1.3 mM), human serum albumin (HSA, 0.8 mM), γ-globulin (IgG, 20 μM), and oxLDL (0.5 μM) served as the samples in the aptamer/AuNP aggregation-based colorimetric assay as previously described. The relative effect of all targets was analyzed in relation to the oxLDL reaction using the following formula: [absorbance ratios at 630/520 nm of the suspected interferences/absorbance ratios at 630/520 nm of the oxLDL] × 100.

Investigation of Zeta Potentials of oxLDL, Aptamers, and Their Complex Aggregates

To assess the zeta potential of each material, LDL with a protein concentration of 0.1 mg/mL in 1× PBS, 4 μM oxLDL in 1× PBS, 10 μM individual aptamers in DI water, 10 μM of the four combined aptamers in DI water, and the complex of oxLDL/aptamers at a ratio of 1:1 were introduced into capillary cells (model DTS 1070). The zeta potential was analyzed by electrophoretic light scattering (ELS) on a Zetasizer Ultra Instrument (Malvern Panalytical, Malvern, UK) at 25 °C.

Cell Culture

Macrophage cells (Raw264.7) were cultivated in a 25 cm³ tissue culture flask containing complete cell culture medium, comprising Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic-antimycotic solution. All incubations took place in an incubator (Thermo Fisher Scientific, Massachusetts, USA) under a humidified atmosphere of 5% carbon dioxide (CO₂) at 37 °C. The morphology of the macrophages was monitored by an Evos cell imaging system inverted microscope (Thermo Fisher Scientific, Massachusetts, USA) until they reached 80–90% cell confluency to be used in the experiments then.

Cell Viability Testing

This study utilized 4 μM oxLDL, 4 μM Dil-oxLDL, various concentrations of four combined aptamers, and 10 μM isoborneol as the test compounds in the cell culture. The optimal concentration of these compounds, which did not induce toxic effects or lead to cytotoxicity in macrophages, was determined through cell viability testing. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (MTT assay) was tested for the cytotoxicity of oxLDL, Dil-oxLDL, the four combined aptamers, and isoborneol. Isoborneol stock at a concentration of 100 mM was prepared in DMSO and subsequently diluted with 1× PBS to achieve a working concentration of 10 μM. Cells at a density of 3 × 10⁴ cells/well were cultured in a 96-well tissue culture plate for 24 h before performing the experiment. When reaching the stage of subconfluence, the cells were treated with 100 μL of 4 μM oxLDL, 4 μM Dil-oxLDL (negative control), 10 μM isoborneol (positive control), and the four combined aptamers at different concentrations (2, 5, 10, 15, and 20 μM), respectively. The cell control group was untreated macrophages. After 24 h of incubation, the medium was discarded, followed by adding 100 μL of 0.1 mg/mL MTT in 1× PBS and incubating for 4 h in the dark. The solution was then removed, and 300 μL of 100% DMSO was added to the lysate formazan crystals. The absorbance was measured at 570 nm. All of the experiments were performed in triplicate. The viability percentage was calculated as [Absorbance at 570 nm of sample/Absorbance at 570 nm of the control] × 100.

Fluorescence Spectrometry, Flluorescence Microscopy, and Flow Cytometry to Investigate Aptamer Influence on the Uptake of oxLDL by Macrophages

The effects of aptamers and isoborneol on the fluorescence intensity of Dil-oxLDL were first examined to ensure that they did not interfere with the fluorescence intensity of Dil-oxLDL. The fluorescence intensity of each sample, including 1× PBS, 10 μM isoborneol, 10 μM of the four combined aptamers, 4 μM oxLDL, 4 μM Dil-oxLDL, 10 μM isoborneol/4 μM Dil-oxLDL complex, and 10 μM of the four combined aptamers/4 μM Dil-oxLDL complex, was measured using a fluorescence spectrophotometer (F-7100, Hitachi, Japan) with 549 nm excitation and 565 nm emission filters. All of the measurements were conducted in triplicate. The relative impacts of all the samples were compared to the fluorescence intensity of Dil-oxLDL using the following formula: [Fluorescence intensity at 565 nm of sample/Fluorescence intensity at 565 nm of Dil-oxLDL] × 100.

Investigation of the Influence of Individual and Combined Aptamers on the Uptake of oxLDL by Macrophages

Isoborneol at 10 μM was utilized as a positive control because it can inhibit the accumulation of lipids and the development of macrophage foam cells.¹⁵ To determine the efficacy of individual aptamers and the combined aptamers as inhibitors of oxLDL internalization by macrophages, the uptake of Dil-oxLDL was determined by fluorescence spectrometry. Macrophages at a density of 5 × 10⁴ cells/well containing 500 μL of complete cell culture medium were cultured in a 24-well tissue culture plate for 24 h before performing the experiment. When reaching the stage of subconfluence, the macrophages were treated with 4 μM Dil-oxLDL (negative control), a mixture of 10 μM isoborneol and 4 μM Dil-oxLDL (positive control), a mixture of 10 μM oxLDL-nonspecific aptamer (LDL-specific aptamer) and 4 μM Dil-oxLDL, a mixture of 10 μM individual aptamers (AP07, AP11, AP25, and AP29) and 4 μM Dil-oxLDL, and a mixture of the combination of two aptamers (AP11/AP29, 10 μM), three aptamers (AP07/AP11/AP29, 10 μM), and four aptamers (AP07/AP11/AP25/AP29, 10 μM) and 4 μM Dil-oxLDL. The treated materials and Dil-oxLDL were mixed at a ratio of 1:1. Untreated macrophages served as the cell control while the four combined aptamers alone-treated macrophages served as the aptamer-treated cell control. Then, 500 μL of the complete medium was added and incubated at 5% CO₂ and 37 °C for 24 h. The amount of internalized Dil-oxLDL in the macrophages was determined by using fluorescence spectrometry. In brief, the cells were rinsed with a 1× PBS solution and then lysed using 500 μL of 1 × RIPA cell lysis buffer to break the cell membrane and retrieve the internalized Dil-oxLDL in the solution, following the manufacturer’s protocol. The lysate solution was then transferred to a centrifuge tube and spun for 15 min at 18 630 × g to remove any remaining cell debris. The supernatant was collected to measure Dil-oxLDL by using a fluorescence spectrophotometer with 549 nm excitation and 565 nm emission filters. All of the measurements were repeated in triplicate. The relative impact of fluorescence intensity was calculated using the formula: [Fluorescence intensity at 565 nm of sample/Fluorescence intensity at 565 nm of the negative control (Dil-oxLDL-treated group)] × 100.

Investigation of the Influence of Combined Aptamer Concentrations on the Uptake of oxLDL by Macrophages

Macrophages at a density of 5 × 10⁴ cells/well in a 24-well plate were treated with a mixture of the four combined aptamers (at concentrations of 2, 5, 8, 10, and 13 μM) and 4 μM Dil-oxLDL. Additional groups, comprising cell control, negative control, and positive control, were conducted using the previously described method. The morphology of internalized Dil-oxLDL macrophages was also investigated by fluorescence microscopy. The cells were rinsed with 1× PBS solution, fixed with 1 mL of absolute methanol for 10 min, and washed with 1× PBS solution. Then, 500 μL of 0.9 nM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining in 1× PBS was added to stain the macrophage nucleus.³⁴ The cells were incubated at 5% CO₂ and 37 °C for 10 min under light protection. Then, the solution was removed, washed with 1× PBS solution twice, and observed under a Zeiss fluorescence microscope (Carl Zeiss AG, Germany).

Flow cytometry was used to determine the uptake of Dil-oxLDL by macrophages. First, the tested group of cells as mentioned earlier were removed from the well by adding 200 μL of 0.25% trypsin-EDTA solution, incubating at 37 °C for 10 min, and adding 400 μL of complete medium to stop the reaction. Then, the solution was transferred to a centrifuge tube and spun at 18,630 × g for 5 min to collect the cell pellet. The F4/80-FITC antibody was employed to specifically identify the macrophages. To stain macrophages with the F4/80-FITC antibody, 50 μL of cell pellet was mixed with 5 μL of 0.5 mg/mL F4/80-FITC and incubated under light protection on ice for 20 min. The cells were rinsed twice with 1× PBS, mixed with 300 μL of 1× PBS, and examined by flow cytometry (BD Accuri C6 Plus, Biosciences, USA).

Investigation of the Influence of Aptamers on the Uptake of oxLDL by Macrophages and Foam Cell Formation by Oil Red O Staining

Oil red O staining is a technique that specifically stains triglycerides and cholesteryl oleate, facilitating the visualization of lipid accumulation within cells. It is widely employed to examine foam cell formation in macrophages.³⁵ To evaluate intracellular lipid accumulation in macrophages, an oil red O dye was used to stain the lipid contents of oxLDL. A stock oil red O solution at a concentration of 0.7 g/dL was prepared by dissolving 0.7 g of oil red O powder in 100 mL of 100% isopropanol. The working solution was then prepared by diluting the stock solution with DI water in a ratio of 3:2 and filtering through a 0.22 μm filter before use. The same tested group of cells with individual aptamers and combined aptamers, as mentioned earlier, were examined using 4 μM oxLDL instead of 4 μM Dil-oxLDL. All the treated groups were incubated for 24 h. The accumulation of intracellular lipids in macrophages was then determined using oil red O staining. In brief, macrophages were rinsed twice with 1× PBS and then fixed with 10% formaldehyde (100 μL) for 30 min. Then, the cells were rinsed twice with 60% isopropanol, added with 200 μL of working oil red O solution, and incubated at 25 °C for 10 min. The cells were then rinsed ten times with DI water and observed using an inverted microscope. Then, the oil red O within the cells was dissolved with 100% isopropanol (300 μL) before the absorbance was measured at 520 nm by using a microplate spectrophotometer. The relative impact of absorbance was calculated using the formula: [Absorbance at 520 nm of sample/Absorbance at 520 nm of the negative control (oxLDL-treated group)] × 100.

Investigation of the Ability of Aptamers to Reduce oxLDL Binding to CD36 by Cyclic Voltammetry

Cyclic voltammetry (CV) was performed to investigate the electrochemical behavior of the interaction between CD36 and oxLDL. The experiments utilized commercially available screen-printed gold electrodes (model 220AT, Metrohm Inula GmbH, Vienna, Austria), employing Ag/AgCl as the reference electrode and platinum as the counter electrode. CD36 was cross-linked onto the gold working electrode through cysteamine/glutaraldehyde functionalization. The gold working electrodes were functionalized with a 2.5 mM aqueous cysteamine solution and incubated at 25 °C for 10 h to introduce amine (−NH₂) functionalities. The electrodes were then rinsed with DI water and dried with an argon gas stream. A 5 mM glutaraldehyde solution was then added and incubated for 2 h to introduce aldehyde (−CHO) functionalities, followed by washing with DI water and drying with argon gas. Then, 0.03 mg/mL CD36 containing – NH₂ groups was immobilized onto the CHO-functionalized gold electrode surface and incubated at 4 °C for 10 h. The unreacted aldehyde groups were blocked with a 0.5 mM ethanolamine solution for 20 min, washed with DI water, and stored in the refrigerator until measurement. CV analysis was conducted using a DropSens μStat-i 400 BiPotentiostat/Galvanostat/Impedance analyzer (EIS) (Metrohm Inula GmbH, Vienna, Austria) and a 5 mM potassium ferricyanide (K₃Fe(CN)₆) as the redox probe. A 4 μM oxLDL, a mixture of 4 μM oxLDL with 10 μM of the individual aptamers (AP07, AP11, AP25, and AP29), and a mixture of 4 μM oxLDL with 10 μM of the four combined aptamers (AP07/AP11/AP25/AP29) were utilized as the test samples for CV analysis. In brief, 5 μL of each sample was introduced on the CD36-immobilized working electrode and incubated at 25 °C for 30 min. Then, the electrode was rinsed with DI water, followed by adding 100 μL of 5 mM K₃Fe(CN)_6. The scan range was from −0.5 V to +0.5 V and the voltammograms were recorded at a scan rate of 50 mV/s.

Statistical Analysis

Quantitative data were presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad software. An unpaired t test was conducted using the mean, SD, and number of independent experiments. P-values below 0.05 were considered significant and depicted as *P-values < 0.05.

Results and Discussion

Molecular Docking Simulation between Combined Aptamers and Oxidized apoB100

As mentioned, our previous research on solid-phase-based SELEX methodology yielded 16 ssDNA candidate sequences that showed potential binding affinity toward oxLDL.²⁶ Each of the 16 aptamer-oxidized apoB100 interactions was investigated by molecular docking to identify the aptamers with superior binding characteristics. The tertiary structures of the aptamers were predicted via loop-free-energy decomposition and employed as ligands for molecular docking. The oxidized apoB100 tertiary structure (amino acid residues: 1–954) was chosen as the protein target due to its external positioning on the surface of oxLDL and its ability to interact with the aptamers. Figure S1 depicts four groups obtained from the 16 aptamers showing their closely aligned binding sites on oxidized apoB100. These groups were categorized as group A including AP07, AP14, AP27, and AP32; group B AP11 and AP26; group C AP06, AP09, AP16, AP20, AP22, and AP25; and group D AP02, AP21, AP29, and AP31. The aptamers with the lowest HDOCK scores in each group, namely, AP07, AP11, AP25, and AP29 were chosen for interaction analysis due to their superior binding ability toward oxidized apoB100.

Figure 1(A-D) demonstrates the optimal configuration for the interaction position between aptamers (AP07 (blue strains), AP11 (green strains), AP25 (orange strains), and AP29 (yellow strains)) and oxidized apoB100 (purple-colored molecules) with the lowest HDOCK score of each aptamer. Figure 1 (E) displays the integrated docking locations of four aptamers targeting distinct regions of oxidized apoB100. As previously stated, individual aptamers may not bind strongly or widely enough to provide effective protection. Therefore, the individual aptamers were merged to enhance the likelihood of binding to various regions of oxidized apoB100.

Figure 1.

Three-dimensional structures of the stimulation complex between AP07 (A; blue strains), AP11 (B; green strains), AP25 (C; orange strains), AP29 (D; yellow strains), and four combined aptamers (E) and oxidized apoB100 (purple-colored molecules) in the presence of molecular stimulations.

Protein–ligand interaction profiler (PLIP) analysis revealed additional interactions between aptamers (orange licorice) and oxidized apoB100 (blue licorice), including hydrophobic interactions, hydrogen bonds, Π-cation interactions, and salt bridges, as depicted in Figure 2(A-D). Table S2 presents the specific amino acid residues that interacted by PLIP analysis between each aptamer and the oxidized apoB100. The AP07 complex contains 3 hydrophobic interactions, 14 hydrogen bonds, and 7 salt bridges. The complex between AP11 and oxidized apoB100 consists of 1 hydrophobic interaction, 13 hydrogen bonds, and 1 salt bridge, while the AP25 complex has 14 hydrogen bonds, 1 Π-Cation interactions, and 2 salt bridges. By contrast, the AP29 complex comprises 7 hydrophobic interactions, 14 hydrogen bonds, 1 Π-Cation interaction, and 3 salt bridges. The results indicate that each aptamer exhibited strong binding ability to oxidized apoB100. Hydrogen bonds and hydrophobic interactions are primary contributors to the binding affinity between aptamers and protein.³⁶ A salt bridge, formed by combining hydrogen bonding and ionic interaction, enhances the stability of proteins by forming strong electrostatic interactions to maintain the proper conformation of the protein and restrict the occurrence of low-energy conformations in a complex.^37,38

Figure 2.

All interacting residues by PLIP analysis between AP07 (A), AP11 (B), AP25 (C), and AP29 (D) (orange lines) with the oxidized apoB100 (blue lines) including hydrophobic interactions, hydrogen bonds, and salt bridges.

OxLDL comprises a complex structure of cholesteryl ester and triglycerides enclosed in a molecular surface that includes a blend of phospholipids, free cholesterol, and apoB100. ApoB100 has attracted interest because of its role as a receptor ligand, and free radicals can directly interact with its amino acid residues. Molecular docking studies can predict the specific binding site of aptamer on oxidized apoB100. However, the binding ability of these aptamers and the lipid membrane of the oxLDL surface depends on the limitations of the current software and computer processing capabilities. These aptamers can also bind to the lipid part of the oxLDL surface, as well as to the oxidized apoB100.

Binding Ability and Selectivity of Aptamers

The aptamer capacity to bind to the “entire” oxLDL was then investigated through the aptamer/gold nanoparticle (AuNP) aggregation-based colorimetric test. AuNPs are typically stabilized by the adsorption of negatively charged ions from a citrate solution, which prevents strong van der Waals interactions and maintains their red color, as shown in Figure 3(A). The addition of 80 mM NaCl induced AuNP aggregation, resulting in a color shift to purple. To establish a negative control for each aptamer, the aptamers were incubated with the AuNP, followed by the addition of NaCl solution. The negatively charged aptamers bound to the positively charged surface of the AuNPs, leading to an overall increase in the surface negative charge. As a result, the solution maintained its red color, even in the presence of high salt concentrations. In the presence of oxLDL, the aptamer selectively binds to oxLDL, thereby reducing the capacity of the AuNPs to resist aggregation induced by salt. This interaction leads to a color change in the solution, transitioning from red to purple, with the extent of the color shift being directly proportional to the amount of oxLDL present.^26,28Figure 3(B) shows the absorbance ratios at 630 and 520 nm, which are associated with the purple reaction color that indicates the binding capacity of each aptamer when adding the sample without oxLDL (negative control, blue column) and the sample with oxLDL (orange column). Binding factors are also represented as the signal ratio of the sample with oxLDL to the sample without oxLDL (gray columns). The binding factors of individual aptamers, ranked from highest to lowest, were AP11 (2.94 ± 0.15), AP29 (2.88 ± 0.14), AP07 (2.71 ± 0.14), and AP25 (2.41 ± 0.12). The two aptamers with the highest binding factors, AP11 and AP29, were combined, yielding a binding factor of 2.82 ± 0.14, comparable to each individual. Combining three aptamers, AP07/AP11/AP29, increased the binding factor to 2.97 ± 0.15. By contrast, the combination of four aptamers, AP07/AP11/AP25/AP29, significantly increased the binding factor to 3.54 ± 0.17, demonstrating that combining four aptamers provided superior binding ability compared to individual or combinations of two or three aptamers. The LDL-specific aptamer was also tested, yielding a binding factor of less than 1 (0.70 ± 0.04), indicating its incapacity to bind to oxLDL. The LDL-specific aptamer was employed as a negative DNA control in subsequent experiments.

Figure 3.

Binding ability between aptamers and oxLDL using the AuNP aggregation-based colorimetric assay. (A) Colorimetric changes of the AuNP among the eight groups, and (B) the absorbance ratio at 630/520 nm (left-hand axis) of the negative control (blue columns) and oxLDL sample (orange columns). The right-hand axis displays the corresponding binding factor (gray columns).

Figure 4 shows the selectivity of individual aptamers, four combined aptamers, and the LDL aptamer in their reactions with oxLDL, LDL, HDL, HSA, and IgG. Figure 4(A) shows the color of the reaction mixture after the addition of each analyte. Both the individual and combined aptamers specifically bound to oxLDL, resulting in a significant purple color shift while exhibiting minimal interaction with other suspected interferences. The LDL-aptamer specifically bound to LDL resulting in a purple color shift and had a low interaction with oxLDL and other potential interferences. Figure 4(B) presents the relative effect of exposure to different (lipo) proteins compared to the oxLDL response. All individual aptamers and the four combined aptamers responded to LDL, HDL, HSA, and IgG at less than 10%. Therefore, AP07, AP11, AP25, and AP29 and their combination strongly favored oxLDL, indicating sufficient selectivity for further experiments. The selectivity of the four combined aptamers was not markedly enhanced, because each aptamer offered good selectivity.

Figure 4.

Selectivity of the individual aptamers, the four combined aptamers, and the LDL-specific aptamer. (A) Color of the reaction in the presence of oxLDL and suspected interferences (LDL, HDL, HSA, and IgG) and (B) relative effect of exposure to suspected interference compared to the oxLDL response of oxLDL-aptamers and the LDL response of the LDL-specific aptamer.

Zeta Potentials of oxLDL, Aptamers, and Aptamer/oxLDL Complex

Native LDL generally exhibits a negative surface potential attributed to the presence of phospholipids, free cholesterol, and apoB100 on its surface, which reveal its overall anionic properties.³⁹ During oxidation, lipid- and protein-oxidation processes alter the LDL surface by increasing the negative surface potential. Lipid oxidation influenced oxidized phospholipids and formed aldehydes, whereas protein oxidation resulted in a reduction of the positive charge of apoB100 lysine amino groups due to the formation of dienes, resulting in a higher net negative charge of oxLDL compared to LDL. Figure 5 shows the zeta potentials of LDL and oxLDL (gray columns), aptamers (blue columns), and the aptamer/lipoprotein complex (orange columns). The zeta potential of oxLDL (−38.21 ± −0.68 mV) was higher, indicating a more negative charge compared to LDL (−10.62 ± −1.27 mV). The DNA aptamers exhibited a negative charge attributed to the phosphate groups in the DNA backbone. The zeta potential of individual aptamers, AP07 (−17.65 ± −0.80 mV), AP11 (−23.28 ± −0.34 mV), AP25 (−17.47 ± −0.73 mV), AP29 (−16.25 ± −1.94 mV), and the LDL aptamer (−14.61 ± −1.10 mV) exhibited slightly different zeta potentials to the four combined aptamers (−18.77 ± −1.08 mV). Results indicated that the combination of aptamers did not influence the zeta potential compared to each aptamer. The less negatively charged aptamers bound to the more negatively charged oxLDL, leading to an overall decrease in the negative charge of oxLDL. Hence, all aptamer/oxLDL complexes exhibited decreased negative charge compared to oxLDL. The zeta potentials exhibited no significant differences among the individual aptamers/oxLDL complexes (AP07/oxLDL complex = −27.70 ± −1.66 mV, AP11/oxLDL complex = −27.08 ± −0.37 mV, AP25/oxLDL complex = −30.85 ± −1.48 mV, and AP25/oxLDL complex = −27.01 ± −0.90 mV). The LDL aptamer/oxLDL mixture (−34.32 ± −1.82 mV) exhibited a higher negative charge than the oxLDL aptamer/oxLDL complex, indicating that the LDL aptamer had a lower binding ability for oxLDL compared to the oxLDL aptamer. By contrast, the four combined aptamers/oxLDL complex resulted in a significant decrease in negative charge to −23.15 ± −1.85 mV. A decrease in the negative charge of the zeta potential of oxLDL following aptamer binding indicated that the aptamers successfully adhered to the surface of oxLDL, thereby demonstrating their ability to bind to oxLDL. The interaction of the four combined aptamers with oxLDL resulted in a decrease of negative charge compared to individual aptamers, attributed to their enhanced binding ability. This finding aligned with the results from the AuNP aggregation-based colorimetric assay, demonstrating the ability of the four combined aptamers to bind at different locations on oxLDL, as indicated by the molecular docking data.

Figure 5.

Average zeta potential of LDL and oxLDL (gray columns), the aptamers (blue columns), and the aptamer/oxLDL complexes (orange columns).

The molecular docking models showed that each aptamer could bind to distinct regions of oxidized apoB100 through different interactions. Results from the AuNP aggregation-based colorimetric assay and zeta potential data indicated that the four combined aptamers enhanced the binding ability of oxLDL compared to that of the individual aptamers. Thus, the four combined aptamers were employed as an agent to specifically bind to oxLDL and their efficacy in reducing oxLDL uptake by macrophages was investigated through cell culture experiments.

Effects of Aptamers and Isoborneol on the Fluorescence Intensity of Dil-oxLDL

Figure 6 shows the fluorescence intensity spectra, scanned from 540 to 690 nm (as depicted in the inset). The fluorescence intensity of 1× PBS (black line), 10 μM isoborneol (orange line), and 10 μM of the four combined aptamers (blue line) exhibited no increase, while the fluorescence intensity of oxLDL (purple line) slight increased. The relative fluorescence intensities of 1× PBS, isoborneol, the four combined aptamers, and oxLDL compared to Dil-oxLDL were 1.06 ± 0.02%, 1.30 ± 0.01%, 1.10 ± 0.01%, and 7.89 ± 0.09%, respectively indicating that they did not impact the fluorescence emission at 565 nm. By contrast, the 4 μM Dil-oxLDL (red line) showed a strong fluorescence emission peak at 565 nm, corresponding to the excitation of Dil-fluorescence at 549 nm. The complex of Dil-oxLDL and isoborneol (green line, relative intensity = 96.84% ± 0.68%) and the four combined aptamers (dark blue line, relative intensity = 101.44% ± 2.40%) displayed slightly different fluorescence intensities compared to the fluorescence emission of Dil-oxLDL. Therefore, 10 μM isoborneol and 10 μM of the four combined aptamers did not significantly influence the fluorescence intensity of 4 μM Dil-oxLDL, indicating that they could be effectively utilized with Dil-oxLDL for further experiments.

Figure 6.

Fluorescence spectra (inset) and relative impact of fluorescence intensity exposure from the seven sample groups: 1× PBS (black line), 10 μM isoborneol (orange line), 10 μM of the four combined aptamers (blue line), 4 μM oxLDL (purple line), 4 μM Dil-oxLDL (red line), the complex of 10 μM isoborneol and 4 μM Dil-oxLDL (green line), and the complex of 10 μM of the four combined aptamers and 4 μM Dil-oxLDL (dark blue line).

Cytotoxicity Testing

Cell cytotoxicity was demonstrated by the MTT assay after incubating macrophages with different compounds for 24 h. When compared to the untreated group (100% viability), the viability percentages of the treated macrophages with 10 μM isoborneol, 4 μM oxLDL, 4 μM Dil-oxLDL, and the combined aptamers at 2, 5, 10, 15, and 20 μM were 83 ± 3%, 91 ± 6%, 86 ± 5%, 103 ± 3%, 96 ± 7%, 102 ± 10%, 90 ± 4%, and 81 ± 6%, respectively, as shown in Figure 7. OxLDL-treated cells appeared to increase cytotoxicity compared to the untreated cells due to the formation of foam cells, which can trigger cellular toxicity through the release of pro-inflammatory cytokines. However, the viability of cells treated with oxLDL showed no significant differences when compared to untreated cells (P-values >0.05). Aptamers, which are single-stranded DNA, typically exhibit low toxicity to cells. However, aptamers at high concentrations of 20 μM can induce toxicity compared to the untreated cells (P-values <0.05). ISO 10993–5 recommends that cell viability percentages exceeding 80% are classified as noncytotoxic, while those between 80% and 60% are considered mild and moderate cytotoxicity, and below 40% as strong cytotoxicity.⁴⁰ Macrophages treated with 10 μM isoborneol, 4 μM oxLDL, 4 μM Dil-oxLDL, and 2–15 μM of the four combined aptamers exhibited vitality levels consistently over 80%, suggesting the absence of significant cytotoxic effects and demonstrating that most of the cell population maintained its functionality. However, the higher concentration of aptamers at 20 μM led to a cell viability below 80%, suggesting that raising the concentration of aptamers to 20 μM may induce toxicity in macrophages. Therefore, 10 μM isoborneol, 4 μM oxLDL, 4 μM Dil-oxLDL, and 2–15 μM of the four combined aptamers were used in subsequent experiments.

Figure 7.

Cell viability percentages of treated macrophages with 10 μM isoborneol, 4 μM oxLDL, 4 μM Dil-oxLDL, and 2–20 μM of the four combined aptamers compared to untreated macrophages (control group). Values are shown as the mean ± SD (n = 3).

Influence of Individual and Combined Aptamers on the Uptake of oxLDL by Macrophages

The uptake of Dil-oxLDL by macrophages was quantified by measuring the fluorescence emission at 565 nm, with results expressed as a percentage of the relative effects compared with the group treated with Dil-oxLDL. Figure 8 shows the relative impact of each aptamer, as well as their combined influence on the uptake of Dil-oxLDL by macrophages. The cells treated with 4 μM Dil-oxLDL served as the negative control, which was used to evaluate all relative impacts of fluorescence intensity. Untreated macrophages and macrophages treated with aptamers alone served as the cell control and aptamer-treated cell control, respectively exhibiting minimal relative impact compared to the negative control. The positive control, indicated by treated macrophages with isoborneol/Dil-oxLDL, exhibited a fluorescence intensity of 39 ± 3%, correlating to a 61 ± 3% reduction in the uptake of Dil-oxLDL, and attributed to the properties of isoborneol in decreasing the uptake of oxLDL into cells.¹⁵ The influence of each aptamer, including AP07, AP11, AP25, AP29, and the LDL-specific aptamer was clarified on the uptake of Dil-oxLDL by macrophages. A concentration of 10 μM of all aptamers was selected for the first study due to their demonstrated high response in cell viability assays. The reduced percentages in the uptake of Dil-oxLDL by cells for individual aptamers, listed from highest to lowest, were AP11 (28 ± 7%), AP29 (26 ± 5%), AP07 (25 ± 5%), and AP25 (16 ± 3%), corresponding to the ranking of the binding ability determined by the AuNP-aggregation colorimetric experiment. A statistical analysis comparing these individual aptamers with the negative control demonstrated substantial differences (*: P-values <0.05), suggesting that the individual aptamers decreased Dil-oxLDL absorption in macrophages. However, the reduction abilities did not exhibit significant differences across the individual aptamers (P-values >0.05). By contrast, cells treated with the LDL-specific aptamer demonstrated Dil-oxLDL uptake comparable to the negative control, indicating that this nonspecific aptamer did not reduce oxLDL uptake. The combinations of two, three, and four aptamers, namely AP11/AP29, AP07/AP11/AP29, and AP07/AP11/AP25/AP29, were also evaluated, resulting in percentage reductions in Dil-oxLDL uptake of 50 ± 3%, 61 ± 6%, and 74 ± 3%, respectively. The reduction abilities of the combined aptamers were significantly higher than the individual aptamers (**: P-values <0.05). The four combined aptamers (AP07/AP11/AP25/AP29) significantly enhanced the reduction of Dil-oxLDL uptake by macrophages compared to the combinations of two and three aptamers. Therefore, the four combined aptamers were utilized in further experiments.

Figure 8.

Relative impact of fluorescence intensity exposure from 12 groups; the untreated macrophages, aptamer alone, Dil-oxLDL, Isoborneol/Dil-oxLDL, LDL aptamer/Dil-oxLDL, AP07/Dil-oxLDL, AP11/Dil-oxLDL, AP25/Dil-oxLDL, AP29/Dil-oxLDL, (AP11/AP29)/Dil-oxLDL, (AP07/AP11/AP29)/Dil-oxLDL, and (AP07/AP11/AP25/AP29)/Dil-oxLDL. Values are shown as the mean ± SD (n = 3). Values with statistical significance in each comparison with Dil-oxLDL-treated macrophages (*: P-values <0.05) and with individual aptamers (**: P-values <0.05).

Influence of Combined Aptamer Concentrations on the Uptake of oxLDL by Macrophages

As previously mentioned, the reduction abilities did not show significant differences among the individual aptamers. Therefore, AP07 was chosen as the representative individual aptamer for comparison with the four combined aptamers. Figure 9 presents the relative impact of fluorescence intensity of Dil-oxLDL of the cell lysate obtained from macrophages treated with 10 μM AP07/Dil-oxLDL and with (2–13 μM) four combined aptamers/Dil-oxLDL compared to Dil-oxLDL-treated macrophages, respectively. Isoborneol served as the positive control. The four combined aptamers at concentrations ranging from 2 to 13 μM demonstrated a significant decrease (*: P-values <0.05) in intracellular lipid accumulation compared to the group treated with Dil-oxLDL. AP07 at 10 μM reduced the uptake of Dil-oxLDL by 20 ± 5% compared to the group treated with Dil-oxLDL alone. By contrast, the mixture of four aptamers at a low concentration of 2 μM resulted in a significant reduction in the uptake of Dil-oxLDL, by 51 ± 11%. This result demonstrated that the four combined aptamers protected macrophages significantly better than the individual aptamers, even at lower concentrations. Increasing the concentration of the four combined aptamers to 5 μM resulted in a further significant decrease in oxLDL uptake (78 ± 1%) compared to 2 μM of the four combined aptamers (**: P-values <0.05). No significant differences in the decrease in oxLDL uptake were recorded for the four combined aptamers at concentrations of 8, 10, and 13 μM (75 ± 5%, 79 ± 4%, and 81 ± 6%, respectively) compared to 5 μM. This result suggested that increasing aptamer concentrations beyond 5 μM did not enhance their ability to reduce oxLDL uptake because the 5 μM aptamer concentration reached the equivalence state of binding to oxLDL particles at the concentration of 4 μM. Therefore, the excess aptamers could not bind significantly. Thus, increasing aptamer concentrations to 13 μM did not enhance their ability to reduce oxLDL uptake. Therefore, the one-time excess concentration of the four combined aptamers at 10 μM with noncytotoxic effects was chosen for subsequent experiments to ensure complete binding.

Figure 9.

Relative impact of fluorescence intensity exposure from the five sample groups: untreated macrophages, Dil-oxLDL-treated macrophages, isoborneol/Dil-oxLDL-treated macrophages, 10 μM AP07/Dil-oxLDL-treated macrophages, and 2–13 μM four combined aptamers/Dil-oxLDL-treated macrophages. Values are shown as the mean ± SD (n = 3). Values with statistical significance in each comparison with Dil-oxLDL-treated macrophages (*: P-values <0.05) and with 2 μM of the four combined aptamers (**: P-values <0.05).

Internalization of Dil-oxLDL and the shape of macrophages were characterized by using fluorescence microscopy. The cell nucleus and lipid content of oxLDL were stained using DAPI and Dil, resulting in blue and red colors, respectively. Figure 10 depicts the fluorescence microscopy bright field images, fluorescence images, and a combination of bright and fluorescence images. Figure 10(A) shows the spherical shape of untreated macrophages (control group) containing only DAPI-stained blue nuclei. Following a 24 h treatment of 4 μM Dil-oxLDL with macrophages, the cell shape changed, including the formation of pseudopodia and an intracellular lipid accumulation in the cytoplasm, as a result of phagocytosis and the buildup of Dil-oxLDL within the cells. The morphology of the cells displayed a variety of phenotypes, such as dendritic and spindle-shaped macrophages performing phagocytic functions as well as giant round cells that resembled foam cells. The cytoplasm contained red spots representing internalized oxLDL, as depicted in Figure 10(B). Figure 10(C) shows macrophages treated with 4 μM Dil-oxLDL and 10 μM isoborneol. The number of lipid-internalized macrophages was reduced because adding isoborneol decreased the uptake of oxLDL by macrophages. Figure 10(D) shows the outcome of administering 10 μM of the four combined aptamers to 4 μM of the Dil-oxLDL-treated macrophages. This treatment reduced the number of red spots of oxLDL accumulation within the cells compared to that of Dil-oxLDL-treated macrophages. Cells treated with aptamers/oxLDL retained intracellular lipids, although less than the oxLDL-treated group, resulting in a spindle-shaped cell morphology. Therefore, the binding of the four combined aptamers to Dil-oxLDL led to a decrease in the uptake of Dil-oxLDL by macrophages.

Figure 10.

Macrophage characteristics under a fluorescence microscope: (A) untreated macrophages, (B) Dil-oxLDL-treated macrophages, (C) isoborneol/Dil-oxLDL-treated macrophages, and (D) four combined aptamers/Dil-oxLDL-treated macrophages.

The macrophages containing oxLDL and the cell alterations following the four combined aptamer treatments were confirmed by quantifying the fluorochrome-labeled Dil-oxLDL and F4/80-FITC-labeled macrophages using fluorescence-activated cell sorting (FACS). Figure 11 illustrates the flow cytometry analysis results performed with FlowJo (version 10.10.0). Figure 11(A1–A4) depicts the results of the flow cytometry investigation utilizing both Dil-oxLDL and F4/80-FITC antibody labeling. Quadrant 1 (Q1) displays the sorted cell population that contained solely the F4/80-FITC, suggesting the presence of macrophages. Q2 contained cells that had both Dil-oxLDL and F4/80-FITC, indicating the presence of macrophages with oxLDL. Figure 11(A1) demonstrates that the untreated macrophages had a 1.95% presence of Dil-oxLDL while the Dil-oxLDL-treated group in Figure 11(A2) exhibited a significantly higher percentage of 70.3% positive macrophages containing oxLDL compared to all macrophages. Figure 11(A3) presents the results of the positive control (isoborneol/Dil-oxLDL-treated macrophages). The number of macrophages containing oxLDL decreased to 54.1%. Macrophages containing oxLDL reduced to 33.4% following treatment with 10 μM of the four combined aptamers, as illustrated in Figure 11(A4).

Figure 11.

Flow cytometry analysis of the four samples: (1) untreated cells, (2) Dil-oxLDL-treated macrophages, (3) isoborneol/Dil-oxLDL-treated macrophages, and (4) four combined aptamers/Dil-oxLDL-treated macrophages. A1–A4: flow cytometry study of F4/80-FITC antibody and Dil-oxLDL; B1–B4: flow cytometry study of Dil-oxLDL inside the macrophages (PE-A+).

The quantity of intracellular Dil-oxLDL in macrophages (PE-A+) was examined, as shown in Figure 11(B1–B4). Figure 11(B1) presents untreated macrophages that exhibited a relatively low signal of Dil-oxLDL (1.25% of PE-A+). By contrast, the Dil-oxLDL-treated group had a significantly higher positive signal of Dil-oxLDL (64.4% PE-A+), as shown in Figure 11(B2). The Dil-oxLDL signal reduced by 48.5% of PE-A+ in the isoborneol/Dil-oxLDL-treated macrophages, while the signal of the four combined aptamers/Dil-oxLDL-treated macrophages also decreased to 32.6% of PE-A+, as illustrated in Figure 11(B3) and Figure 11(B4), respectively. The four combined aptamers (10 μM) exhibited superior efficacy in reducing oxLDL uptake by macrophages compared with the positive control agent (10 μM) at a similar concentration.

Influence of Aptamers on the Uptake of oxLDL by Macrophages Using Oil Red O Staining

The effects of aptamers on the uptake of oxLDL by macrophages and foam cell morphology were assessed using oil red O staining. Figure 12 presents the oil red O staining results across different groups of the treated macrophages. The oil red O staining of untreated macrophages (Figure 12(A)) and aptamer-treated macrophages (Figure 12(B)) revealed the absence of lipid accumulation in the cells. Untreated macrophages showed a uniform size and displayed mixed morphologies, including round and dendritic/spindle shapes. The morphologies of macrophages showed no significant changes following treatment with the four combined aptamers, suggesting that these aptamers alone did not induce morphological alterations in macrophages. Macrophages treated with oxLDL (Figure 12(C), a negative control) displayed a significant accumulation of intracellular lipids in the cytoplasm, as evidenced by the significant red staining. The cell body exhibited an increase in size coupled with the formation of pseudopodia, resulting in a dendritic and spindle-shaped cell morphology. The number of cells exhibiting dendritic/spindle changes in the isoborneol/oxLDL (Figure 12(D)) and aptamers/oxLDL (Figure 12(F-L)) groups was significantly higher compared to the untreated group, attributable to the activation of the oxLDL aggregate. For the influence of reducing oxLDL internalization, the LDL-specific aptamer/oxLDL-treated cells (Figure 12(E)) exhibited lipid buildup comparable to the negative control, suggesting that the nonspecific aptamer could not bind to oxLDL or reduce its internalization. By contrast, isoborneol/oxLDL and aptamers/oxLDL showed partial oxLDL uptakes into macrophages. The four combined aptamers exhibited the greatest efficacy in reducing the lipid accumulation.

Figure 12.

Oil red O staining of lipid accumulation under an inverted microscope: (A) untreated macrophages, (B) combined aptamers (AP07/AP11/AP25/AP29), (C) oxLDL, (D) isoborneol/oxLDL, (E) LDL aptamer/oxLDL, (F) AP07/oxLDL, (G) AP11/oxLDL, (H) AP25/oxLDL, (I) AP29/oxLDL, (J) (AP11/29)/oxLDL, (K) (AP07/AP11/AP29)/oxLDL, and (L) (AP07/AP11/AP25/AP29)/oxLDL.

Figure 13 presents the relative impact of absorbance of oil red O-stained lipids in treated cells compared to the negative control. Macrophages treated with 10 μM isoborneol/oxLDL exhibited a significant decrease in lipid accumulation of 58 ± 7% while 10 μM individual aptamers, AP07, AP11, AP25, and AP29, reduced by 31 ± 4%, 32 ± 5%, 27 ± 8%, and 31 ± 3%, respectively compared to the negative control (*: P < 0.05). The combination of two, three, and four aptamers yielded 55 ± 7%, 61 ± 3%, and 68 ± 3%, respectively. The four combined aptamers exhibited a significant decrease in oxLDL uptake compared to the two and three combined aptamers (**: P < 0.05). All quantitative results of oil red O staining corresponded to the fluorescence-based technique. The percentage of oxLDL uptake reduction varied based on the inter-experimental assay conducted. Results indicated a significant potential for using the four combined aptamers (AP07/AP11/AP25/AP29) to decrease the uptake of oxLDL by macrophages, emphasizing their prospective therapeutic efficacy. In addition, further investigation of the phenotypic alterations in macrophages, which are crucial within plaques, is necessary to elucidate the underlying mechanisms of in vivo antiatherosclerosis activity.

Figure 13.

Relative impact of oil red O staining of macrophages in untreated macrophages, combined aptamer (AP07/AP11/AP25/AP29) alone, oxLDL, Isoborneol/oxLDL, LDL aptamer/oxLDL, AP07/oxLDL, AP11/oxLDL, AP25/oxLDL, AP29/oxLDL, (AP11/29)/oxLDL, (AP07/AP11/AP29)/oxLDL, and (AP07/AP11/AP25/AP29)/oxLDL. Values are shown as the mean ± SD (n = 3). Values with statistical significance in each comparison with oxLDL-treated macrophages (*: P-values <0.05) and with two and three combined aptamers (**: P-values <0.05).

Influence of Aptamers on Reducing the Binding Interaction between oxLDL and CD36

The cluster of differentiation 36 (CD36), an 88-kDa transmembrane glycoprotein expressed on macrophages as a scavenger receptor, exhibits high-affinity binding to oxLDL. The scavenger receptor functions of CD36 have been thoroughly studied because of its significance in the development of atherosclerosis.⁹ The influence of aptamers on reducing the binding interaction between oxLDL and CD36 was investigated by cyclic voltammetry. CD36 was first immobilized on an electrode surface to simulate its presentation on the macrophage surface. The binding ability of CD36 to oxLDL was assessed by exposing oxLDL to the sensor system, resulting in a reduction of electron transfer corresponding to a decrease in the anodic peak current. The assay utilized indirect cyclic voltammetry due to the non-electroactive nature of the CD36. The K₃Fe(CN)₆ buffer played a crucial role in facilitating electron transfer at the electrode surface, allowing for measurement of the electrochemical response. Figure 14 presents a cyclic voltammogram of seven different sample groups utilizing CD36 as the selective layer on the sensor electrode. First, the 1× PBS (sample without oxLDL, black line) was introduced, yielding a peak current of 65 μA, which served as the baseline response of CD36 in the absence of oxLDL interaction. Then, the sample containing oxLDL (red line) was added, resulting in a decrease in electron transfer to 52 μA, attributed to binding between oxLDL and CD36. With the addition of individual aptamers (AP07 (orange line), AP11 (brown line), AP25 (blue line), and AP29 (purple line)/oxLDL complexes, peak currents were presented at 60, 60, 57, and 59 μA, respectively, indicating that binding of each aptamer to the oxLDL surface disrupted the interaction between oxLDL and CD36. When the four combined aptamers/oxLDL complexes (green line) were added, the peak current was 63 μA, indicating a slight difference compared to the blank. The four combined aptamers exhibited the most significant effect, further reducing the interaction between oxLDL and CD36. In addition, the influence of aptamers on reducing the binding interaction between oxLDL and other types of scavenger receptors (i.e., LOX-1 and SR-BI) should be investigated further.

Figure 14.

Cyclic voltammogram of seven different sample groups using CD36 as the selective layer of sensor electrode: Sample without oxLDL (blank, black line), four combined aptamers/oxLDL (green line), AP07/oxLDL (orange line), AP11/oxLDL (brown line), AP25/oxLDL (blue line), AP11/oxLDL (purple line), and sample with oxLDL (red line).

As previously studied, macrophages tend to internalize negatively charged materials, such as nanostructured DNA⁴¹ and DNA-functionalized gold nanoparticles⁴² through scavenger receptors. Scavenger receptors bind negatively charged ligands through their clusters of conserved positively charged residues.⁴³ During oxidation, the positive charge of apoB100 lysine amino groups decreases due to the formation of dienes, resulting in an increased net negative charge of oxLDL compared to LDL. Therefore, the increased surface negative charge of oxLDL enhanced scavenger receptor (e.g., LOX-1, CD36) binding. As previously mentioned, the decreased negative charge in the zeta potential of oxLDL following specific binding by the four combined aptamers indicated that the aptamers successfully adhered to the surface of oxLDL. The mechanism of the combined aptamers in reducing the uptake of oxLDL may also involve binding to oxLDL particles. The decreased surface negative charge of the aptamers/oxLDL complex reduced scavenger receptor binding due to the increase in repulsive charges on the scavenger receptor ligand binding surface, leading to lower cellular uptake of the aptamers/oxLDL complex. The four combined aptamers bound to the surrounding proteins at different locations and enclosed or covered a portion of the oxidized apoB100 on oxLDL. This interaction decreased the exposed surface area of oxidized apoB100, which, in turn, limited its interaction with receptors, leading to a decreased uptake of oxLDL by macrophages. Aptamers can selectively bind to target molecules, potentially inhibiting their interactions with cell surface receptors. This mechanism effectively hinders the entry of ligands or pathogens into the cell through receptor-mediated endocytosis.⁴⁴ Aptamers also act as competitive inhibitors by binding to identical locations as the target ligand, thus reducing the ability of the ligand to bind to its receptor and undergo internalization.^45,46

Table 1 shows preventive agents derived from biological and chemical sources that exhibited reductions in the level of oxLDL uptake and foam cell formation. Monoclonal autoantibodies (EO6 and EO3) bind specifically to oxidized phospholipids of oxLDL, reducing the interaction between oxLDL and scavenger receptors.⁴⁷ Antibodies provide high binding affinity and high specificity but high cost and complexity of production. Isoborneol and Bingpian metabolites have multitarget therapeutic potential and show promise in regulating complex biological pathways. However, their mechanism of action remains unknown, and clinical validation is required to confirm their efficacy. Nanomaterials provide significant advantages as drug delivery strategies for targeted medication delivery, drug encapsulation, and controlled release. Black phosphorus nanosheets were successfully developed and effectively scavenged reactive oxygen species and suppressed pro-inflammatory cytokine production in lesional macrophages.⁴⁸ Nanoscale amphiphilic polymers showed a superior ability to inhibit oxLDL internalization.⁴⁹ Nanomaterials exhibit biocompatibility and responsive systems; however, they face challenges including manufacturing complexity, instability under physiological conditions, limited long-term degradation, toxicity, and the potential for immune responses. Aptamers provide superior selectivity to the target, easy synthesis, low immunogenicity, and low toxicity, but they are highly susceptible to chemical and enzymatic degradation. The presence of nucleases in physiological environments decreases the performance of aptamers, which restricts their use in some applications. Aptamers in therapeutic applications are susceptible to nuclease degradation and rapid renal excretion, which considerably limits their in vivo applications.⁵⁰ Conventional aptamers have undergone modifications to address these limitations, thereby enhancing their binding affinity to the target, improving stability, and preventing degradation by in vivo nucleases. For instance, the modification of the aptamer was conducted using heterocycles, hydrophobic groups, phenyl, large naphthyl, and a more complex indole to substitute the dT base in the DNA library with dU modified at the 5′ position of the base.⁵¹ Nuclease-resistant circular aptamers enhance the metabolic stability of aptamers in serum and biological fluids. Modifications of 5′ and 3′ terminals in aptamer is additionally protected against exonuclease degradation.⁵² The integration of aptamers with drug delivery systems to enhance resistance in harsh physiological environments and extend their half-life should also be considered.

Table 1. Comparison of Preventive Agents Exhibiting Reductions in oxLDL Uptake and Foam Cell Formation.

Name	Antibody47,53	Isoborneol/Bingpian metabolites15,16	Black phosphorus nanosheets (BPNSs)48	Amphiphilic polymers (AMs)49	Four combined aptamers (ssDNA) (this work)
Source	Biological component	Biological component	Chemical component	Chemical component	Synthetic biological component
Mechanism	Bind to oxLDL, reducing the interaction between oxLDL and scavenger receptors	Bind to oxLDL, reducing the interaction between oxLDL and scavenger receptors	ROS-responsive, carriers resolve in D1 to macrophages	Bind to oxLDL, reducing the interaction between oxLDL and scavenger receptors	Bind to oxLDL, reducing the interaction between oxLDL and scavenger receptors
Capacity for reduction	Inhibit phagocytosis by 80% at 10 μg/mL E06/EO12	Reduce oxLDL uptake by 80% at 20 μM isoborneol	Reduce ROS and reactive nitrogen species	Reduce oxLDL uptake by 80% at 10–6 M AMs	Reduce oxLDL uptake by 79 ± 4% at 10 μM four combined aptamers
Advantage	- Widely distributed technologies	- Multitarget effects	- Drug encapsulation and controlled release	- Drug encapsulation and controlled release	- High affinity
			- Biocompatibility	- Biocompatibility	- High specificity
	- Long half-life in vivo (less susceptible to serum degradation and renal filtration)	- Less susceptible to nuclease degradation	- Photothermal properties	- Responsive systems	- Less immunogenic- Low toxicity
			- Surface functionalization	- High stability	- Low cost and commercially available synthesis
			- Less susceptible to nuclease degradation	- Less susceptible to nuclease degradation	- Low batch-to-batch variation
Limitation	- High cost of synthesis	- Action mechanism uncertainty	- Toxicity concerns	- Manufacturing complexity	- Susceptible to chemical and enzymatic degradation
	- High batch-to-batch variation	- Variability in composition	- Manufacturing complexity	- Often immunogenic	- Short half-life
	- Often immunogenic	- Limited clinical evidence	- Often immunogenic		- Challenges in delivery to target
	- Thermally unstable	- High batch-to-batch variation

Conclusions

A molecular docking model was used to explore how the combined aptamers attached to certain areas of oxidized apoB100 through various interactions. The ability of the combined aptamers to reduce the uptake of oxLDL by macrophages was also investigated by using a cell culture experiment. The cytotoxicity test results indicated that the four combined aptamers (AP07/AP11/AP25/AP29) at concentrations of up to 15 μM did not exhibit any significant toxicity in the macrophages. The cell culture results, involving Dil-oxLDL staining and determination of fluorescence intensity, strongly suggested that introducing 10 μM concentrations of the four combined aptamers attached to oxLDL reduced the uptake of oxLDL by macrophages by 79 ± 4% compared to that of oxLDL alone. Flow cytometry demonstrated Dil-oxLDL absorption by the macrophage group without the four combined aptamers of 64.4% PE-A+, while the combined aptamers reduced Dil-oxLDL uptake by 32.6% of PE-A+. Oil red O also indicated the potential of utilizing the combination of four combined aptamers to enhance the reduction of oxLDL uptake by macrophages, consequently decreasing foam cell formation. Cyclic voltammetry demonstrated that the four aptamers bound to the oxLDL surface, thereby reducing the interaction between CD36 and oxLDL. The four combined aptamers bound to oxLDL and decreased the uptake by macrophages, leading to reduced development of foam cells and showing potential as future therapeutic agents in the treatment of atherosclerosis.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.4c01432.

• Table S1, sequences of the 16 aptamers targeting oxLDL obtained from the in vitro SELEX process; Figure S1, three-dimensional structure of the stimulation complex between each aptamer (colored strains) and oxidized apoB100 (purple-colored molecules) with HDOCK score; Table S2, interaction analysis from PLIP between combined aptamers and oxidized apoB100 (PDF)

Author Contributions

Soemwit Khongwichit: Methodology, Investigation, Validation, and Writing - Original Draft, Review & Editing. Piyawut Swangphon: Conceptualization, Methodology, Investigation, Validation, Resources, Review & Editing. Aekkaraj Nualla-ong and Napat Prompat: Methodology, Investigation, and Writing - Review & Editing. Maliwan Amatatongchai and Peter A. Lieberzeit: Investigation and Writing - Review & Editing. Suticha Chunta: Conceptualization, Methodology, Investigation, Validation, Resources, Supervision, Project administration, Funding acquisition, and Writing - Original Draft, Review & Editing.

The authors declare no competing financial interest.