Coding sequences of individual toll-like receptors (TLR) were amplified from cDNA of human and mouse macrophages using Phusion high-fidelity DNA polymerase (ThermoFisher) and cloned to the multiple-cloning site of the pAdTrack-CMV vector, which expresses GFP from an independent CMV promoter (Addgene). The coding sequence of the TLR chaperone protein UNC93B1 and co-receptor CD14 were similarly amplified from human macrophage cDNA and inserted to the pMSCV-neo and pMSCV-puro vectors, respectively (Clontech). The NanoLuc Reporter Vector with NF-kB Response Element (pNL3.2.NF-kB-RE) is commercially available (Promega). Transfections of plasmid DNA were performed using Lipofectamine 3000 (LifeTechnologies), per manufacturer’s instructions. In vivo grade nt-LNA (Cat# 339203, Qiagen) was purchased in individual lots of 100 mg, reconstituted in sterile saline, aliquoted, and stored at -20C. Single-stranded RNA oligonucleotides were synthesized with a phosphorothioate backbone (PS; Integrated DNA Technologies). Nucleoside analogues CL075 and Resiquimod (R848) were purchased commercially (Sigma). Bafilomycin A1 and EGTA-AM were purchased commercially (Sigma). For transfections, nt-LNA, ssRNA, and sRNA oligonucleotides were complexed with DOTAP (Sigma) according to manufacturer’s instructions. Ligands for TLR1/2 (Pam3CSK4), TLR2 (Heat-killed Listeria monocytogenes), TLR3 (Poly(I:C)), TLR4 (LPS Escherichia coli K12), TLR5 (Flagellin Salmonella typhimurium), TLR6/2 (FSL-1) and TLR9 (ODN2006) were purchased commercially (Invivogen) and diluted according to manufacturer’s recommendations. Antibodies used for immunoblotting and flow cytometry in this study are provided in Supplementary Table 10.
Animals and models of atherosclerosis
Wild-type (WT), Tlr7-/-, Ldlr-/- and Apoe-/- mice of the C57BL/6 background were purchased (The Jackson Laboratory). Animal protocols were approved by and performed according to the regulations of Vanderbilt University Medical Center’s Institutional Animal Care and Usage Committee (IACUC) and animal studies complied with all relevant ethical regulations for vertebrate animal research (#M1600252-01). Mice were maintained on a chow diet (NIH-31) or an atherogenic diet containing 42% kcal from milk fat and 0.2% cholesterol added (Envigo; TD.88137) in a 12h:12h light-dark cycle with unrestricted access to food and water. For atherosclerosis progression studies, 10-week-old female Apoe-/- mice were converted from a chow diet to a atherogenic diet for 4 weeks receiving weekly intraperitoneal injections of saline (n=10) or non-targeting locked-nucleic acid (nt-LNA; 30 mg/kg; n=10) that were randomized within cages to limit cage effects. For atherosclerotic regression studies, male and female Ldlr-/- mice were fed the atherogenic diet for 14-weeks to promote hypercholesterolemia and lesion development (n=25/sex). Ldlr-/- mice fed a chow diet for this time period were used as an age-matched control. After 14-weeks of atherogenic diet feeding, mice from each cage were randomly assigned to either a baseline group that was sacrificed at this time (n=7-8/sex), or one of two treatment groups that were converted to a chow diet for 4 additional weeks with weekly intraperitoneal injections of saline (n=9/sex) or nt-LNA (n=8-9/sex), as previously described40. Results of the regression study are pooled from two independent cohorts of mice staggered by 4 weeks. At sacrifice, blood was collected by cannulating the vena cava using an EDTA-coated syringe and plasma was obtained by centrifugation at 2,500 x g for 10 min. Isolated plasma was stored at -80oC prior to analysis. Unless otherwise indicated, tissues were flash frozen in liquid nitrogen and stored at -80oC.
Lipid analysis of mouse plasma
Plasma collected during animal sacrifice was assayed directly for total cholesterol and triglycerides using colorimetric kits (Pointe Scientific). For assessment of lipid/lipoprotein distribution, 200 µL of mouse plasma was diluted in SEC running buffer and injected in the ÄKTA SEC system composed of three tandem Superdex-200 Increase columns with collection of 1.5 mL fractions. Individual fractions were then assessed for total protein (BCA; Pierce) and lipids (Pointe Scientific).
Quantification of Atherosclerosis:
For each experiment, mice were fasted for 4h at the end of the dark phase and sacrificed with isoflurane. Blood was collected by cannulating the vena cava and mice were perfused with 1X PBS until vasculature was clear. The heart, with aortic root attached was harvested, embedded with OCT and frozen on dry-ice. As detailed by Paigen et al.46, 30, 10µm sections were taken from the beginning of the aortic root (where valve-cusps first appear) using a Leica CM3050s cryostat by the same technician for all studies. For each root, every other slide for the 300-µm span was stained with Oil-Red-O (ORO) to identify the neutral lipid deposition. To quantify lesion area, ORO positive area was imaged using the KS300 imaging system (Kontron Elektronik GmbH), as previously described47–49, and reported as ORO/mm2. The area under the curve (AUC) was calculated from the lesion area of serial sections. All other stains were conducted on sections 40-60 mm distal of the aortic sinus. Masson’s Trichrome staining and immunohistochemistry for F4/80 (Novus Biologicals; secondary by Vector Laboratories) were performed by the Vanderbilt Translational Pathology Shared Resource core using 4 sections per animal and quantified using the KS300 imaging system applying methodologies previously described50. For immunofluorescence studies, frozen aortic root sections were dried at 55oC for 10 minutes and then fixed in formalin for 20 minutes. Slides then underwent 3x 30 dip rinses in deionized water. Slides then underwent antigen retrieval (H-3300, VectorLabs) in a pressure cooker for 10 seconds. Slides were then rinsed (30 dips) in 1x PBS 3 times. In a humidified chamber, slides were blocked using 1% BSA for 30 minutes and then aspirated and incubated overnight in primary antibody (Mac2, 1:10,000, Cedarlane). Slides were then rinsed (30 dips) 3X in 1X PBS and then probed with secondary antibody (Goat anti-rat AF488, 1:200, ThermoFisher) and sealed with a coverslip. Fluorescence microscopy was completed using a Nikon STORM scope and images were recorded for subsequent analysis in Image-J (Fiji).
Cell culture studies
HEK293T cells were maintained in DMEM supplemented with sodium pyruvate (110 mg/L), L-glutamine, 10% heat-inactivated FBS, and 1% penicillin-streptamycin (GIBCO). THP-1 cells were maintained in RPMI1640 supplemented with 25 mM HEPES, L-glutamine, 10% heat-inactivated FBS, and 1% penicillin-streptamycin (GIBCO) on suspension culture dishes (Corning). To generate THP-1 macrophages, THP-1 cells were suspended in fresh media spiked with 5 ng/mL 12-myristate 13-acetate (PMA) and transferred to non-tissue culture treated plates for 48h. After 48h, adherent cells were washed twice with PBS and given fresh media without PMA for 24h51. For siRNA experiments, non-differentiated THP-1 cells were concentrated 10-fold in Opti-MEM and incubated with 300 mM scrambled or siTLR8 siRNA (ON TARGETplus; Horizon Discovery), or without siRNA, and electroporated using program Y-001 on a Nucleofector 2b device (Lonza). Cells were immediately diluted 10-fold in RPMI containing 5 ng/mL PMA and differentiated, as above. Primary bone marrow-derived macrophages (BMDM) were generated by incubating total bone marrow aspirates isolated from the tibia and femur of C57BL/6 mice in DMEM containing sodium pyruvate (110 mg/L), L-glutamine, 10% heat-inactivated FBS, and 1% penicillin-streptamycin (GIBCO) supplemented with murine GM-CSF (50 ng/mL; Tonbo Bioscience) for 72h on non-treated tissue culture plates 52,53. After 72h, non-adherent cells were washed with PBS and provided additional fresh media containing GM-CSF with indicated treatments.
NF-κB Activity (Luciferase) Reporter Assay:
HEK293T cells were seeded in 24- or 96-well plates and allowed to attach overnight in full media. The following day, cells were transfected with 1 mg/mL plasmid DNA (35% pAdTrack-CMV, 30% pMSCV-puro-CD14, 30% pMSCV-neo-UNC93B1, 5% pNL3.2.NF-kB-RE) using Lipofectamine 3000. After 3h, transfection media was carefully aspirated and replaced with fresh media for 24h. The following day treatments were performed as indicated for 20-24h at which time media was removed and a passive lysis buffer (Nano-Glo Luciferase Assay Buffer; Promega) was added, and lysates were incubated on a horizontal shaker for 10 min at 25oC with 500 RPM. Lysates were then transferred to an opaque 96-well plate for measurement of GFP (co-expressed with GOI from pAdTrack-CMV) fluorescence as an indicator of cell density and transfection efficiency on a Synergy Mx plate-reader. Subsequently, the NanoLuc substrate Furimazine was diluted in lysis buffer, added to lysates with a multi-channel pipette, and incubated with agitation for 2 min at room temperature. Luminescence was then quantified using a Synergy Mx plate-reader. Luciferase activity was calculated as luminescence divided by fluorescence and reported as a relative fold change.
RNA Isolation and Real-time PCR:
For cells, total RNA was isolated using Total RNA Purification kits (Norgen), as per manufacturer’s instructions. For tissue, total RNA was isolated using miRNeasy Mini kits (Qiagen), as per manufacturer’s instructions. For lipoproteins, total RNA was isolated using miRNeasy Mini kits with slight modifications to the manufacturer’s instructions. Briefly, Qiazol was added at 10x initial volume of lipoprotein concentrate, vortexed for 30-60 s and allowed to incubate for 5 minat room temperature prior to addition of 0.2 volumes of chloroform. After phase separation, the aqueous phase was mixed with an equal volume of 100% ethanol, vortexed, and stored overnight at -80oC. The following day, the RNA sample was applied to the mini column per manufacturer’s instructions. For targeted isolation of human small RNAs, 1x107 THP-1 cells were pelleted by centrifugation and lysed in Qiazol and processed according to Appendix A of manufacturer’s instructions (Qiagen). For targeted isolation of bacterial sRNA, an overnight culture of E. coli (DH5a; Invitrogen, Cat# 18265-017) was pelleted at 4,000 x g, washed 2X with PBS, and incubated with 1 µg/mL lysozyme for 5 min at room temperature with agitation. Qiazol was then added and the partial cell lysate was pulverized with Zirconium beads (Sigma) in an oscillating tissue homogenizer for 30 s. Subsequently, the sample was processed following the Appendix A protocol of the miRNeasy Mini kit (Qiagen) for the separate isolation of long and small RNAs. Total RNA, sRNA and LDL-sRNA were assessed by PicoChip on a 2100 Bioanalyzer instrument (Agilent). cDNA was generated from total RNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher) for mRNA targets. For sRNA targets, the miRCURY LNA RT kit was used for cDNA-synthesis (Qiagen). Real-time PCR was performed for targeted transcripts and sRNAs using the Power SYBR 2X Master Mix (ThermoFisher) on QuantStudio 6 or QuantStudio 12 instruments (LifeTechnologies). RPOLII served as a housekeeper for human cells and Rplp01 (36b4) served as a housekeeper for murine cells and tissues For LDL-sRNAs, an arbitrary Ct of 35 was used for RQV calculations. All primer sequences are available upon request. Relative quantitative values (RQV) were calculated by the delta-Ct method and reported as fold changes.
High-throughput RNA Sequencing:
Individual total RNA sequencing libraries were generated using Ovation Human FFPE RNA-Seq Multiplex System (NuGen) kits, per manufacturer’s instructions. Individual libraries were assessed for quality using the Agilent 2100 Bioanalyzer and quantified with a Qubit Fluorometer. Paired-end sequencing of multiplexed libraries was performed on the NovaSeq6000 (Illumina) platform by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core (Vanderbilt University, Nashville, TN). Reads were trimmed to remove adapter sequences using Cutadapt v1.16 54 and aligned to the human hg19 genome using STAR v2.5.3a55. GENCODE human v19 gene annotations were provided to STAR to improve the accuracy of mapping. Quality control on both raw reads and adaptor-trimmed reads was performed using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc). featureCounts v1.15.2 56 was used to count the number of mapped reads to each gene. Significantly differential expressed genes with FDR-adjusted p-value < 0.05 and absolute fold change > 2.0 were detected by DESeq2 (v1.18.1) 57. Gene ontology and transcription factor analyses were assessed from differentially expressed mRNAs using the MetaCore software suite (Clarivate Analytics).
Small RNA Sequencing:
Individual small RNA libraries were generated from LDL-sRNA using the NEXTFlex Small RNA Library Preparation Kits v3 for Illumina Platforms (Perkin-Elmer), per manufacturer’s instructions with the following modifications: 1) 3’- and 5’-adapters were diluted 1:8, 2) 3’-adapter ligation was performed overnight in multiple steps – 25oC for 2h, 20oC for 4h and 16oC overnight, 3) following cDNA synthesis and prior to barcoding PCR, the supplementary modified step F protocol was followed (i.e. no size selection protocol), 4) PCR amplification was 20 cycles. Following PCR amplification, individual libraries were size-selected (136-200 bp product) using pre-cast 3% agarose gel cassettes with a Pippin Prep (Sage Sciences) and quantified on a Qubit Fluorometer. Single-end sequencing (SE-75) of equimolar multiplexed libraries was performed on the NextSeq500 (Illumina) platform by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core (Vanderbilt University, Nashville, TN). Bioinformatic processing was performed using in-house data analysis pipeline (TIGER), previously described in detail34. Briefly, Cutadapt54 was used to trim 3’ adapters for raw reads. All reads with less than 16 nucleotides (nts) were designated as “too short” and discarded. The adaptor-trimmed reads were mapped to the hg19 genome, with additional rRNA and tRNA reference sequences, by Bowtie1 (v1.1.2) 58 allowing only one mismatch (1MM). Reads <20 nts that failed to be annotated as sRNA without perfect alignment (PM) to human genome were discarded. Remaining unmapped reads were then mapped in parallel to exogenous structural RNA databases and curated microbial genome databases allowing no mismatches (PM). Reads that failed to align to any strategy were categorized as “unknown”.
Single Cell RNA Sequencing:
After collection of the heart and aortic root, as described above (n=8/treatment), the aortic arch and thoracic aorta were removed, cleaned, and placed into 1 mL of RPMI 1640 containing 10% FBS on ice, as previously described59. Once all aortas were harvested, aortas were minced to small pieces using vigorous scissor action and digested by shaking at 37oC for 30 min in 1 mL of RPMI 1640 containing 2.6 U/mL Liberase TM (Sigma), 60 U/mL hyaluronidase (Sigma), 0.1 mg/ml DNase I (Sigma), 1 µg/mL actinomycin D (Sigma), 0.744 U/mL Elastase (Worthington Biochemical). Digested Samples were placed in fresh tubes and spun at 500xg for 10 min at 4oC. Pellets were pooled and filtered through a 70 mm cell strainer using RPMI + 10% FBS. Fc receptors were blocked with anti-mouse CD16/32 (BioLegend) for 10 min at 4oC prior to surface marker staining. Cells were stained with Pacific Blue anti-CD45 (BioLegend); FITC anti-Ter119/RBC (BD Biosciences); and PerCP-Cy5.5 anti-CD3 (BD Pharmingen) for 30 min at 4oC. Cells were washed 2X with RPMI + 10% FBS, filtered, and sorted using a 4-laser FACSAria III by the VUMC Flow Cytometry Shared Research Core. Dead cells and red blood cells were excluded and CD45+CD3- cells were collected using a 100 mm nozzle into RPMI 1640 + 10% FBS. Each sample (saline = 13,055 cells; nt-LNA = 19,980 cells) was processed for single cell 3' RNA sequencing utilizing the 10X Chromium system through the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core (Vanderbilt University, Nashville, TN). Libraries were prepared using P/N 1000006, 1000080, and 1000020 following the manufacturer's protocol. The libraries were sequenced using the NovaSeq6000 with 150 bp paired-end reads (PE-150). RTA (version 2.4.11; Illumina) was used for base calling and analysis was completed using 10X Genomics Cell Ranger software v2.1.1. scRNABatchQC software package was used for assessment of scSEQ data quality control, as previously described60. Clustering analysis was generated by Seurat61. Initial cell type classification (i.e. myeloid/macrophage, B cells, T cells, NK cells) of complete datasets was based on cell activity database62. Refined annotation of clusters was conducted manually following recommendations of a meta-analysis of similar sequencing approaches17; however, discrepancies between predicted clusters based on the previously reported meta-analysis and our data were observed. These differences were likely attributable to the early atherosclerosis model employed in our study. Hence, we observed two resident macrophage clusters distinguishable by the expression of B-cell markers Cd79a, Ms4a1, Fcamr and Ighm (C1 vs C4), which may be the result of pre/pro-B cell differentiation to macrophages63. Aortic intimal resident macrophages (Mac-AIR; C9) were categorized by their expression of Cx3cr1 and Itgax, as well as genes in lipid metabolism (Abcg1, Scarb1, Pparg, Cebpb)64. Interestingly, this population also had selective expression of Cd300e, as well as enriched expression of Nr4a1, a transcription factor associated with resident macrophage differentiation65. Repair macrophages (C14) were categorized by their expression of Mlxipl (ChREBP), Alox15 and Fn1 (Fibronectin)66. Transitioning cells (C5) showed expression of proliferation genes (Top2a, Mki67, Pclaf), as well as differentiation modulators Celf1 and Nr4a1. EdgeR software was used for differential expression analysis between treatments and within cell-types67. Gene ontology and transcription factor analyses were assessed from differentially expressed mRNAs using the MetaCore software suite (Clarivate Analytics).
Lipoprotein Isolation and Preparation of Reconstituted LDL:
Human whole blood was collected from fasted male and female subjects age 18 or older (IRB#170046) in K2EDTA tubes after informed written consent was obtained. Aliquoted plasma was either used fresh or stored at -80oC and used after one freeze-thaw. Sequential density-gradient ultracentrifugation (DGUC) using a Beckman-Coulter Optima XPN-80 Ultracentrifuge with either SW32Ti or SW41Ti swinging bucket rotors was used to isolate VLDL (1.006-1.018), LDL (1.019-1.063), and HDL (1.064-1.021) by adjusting sample density with potassium bromide, as previously described68. Isolated lipoproteins were immediately dialyzed in 1X PBS for at least 4 changes of 200-fold excess buffer. After (up to) 48h dialysis, lipoproteins were concentrated using Amicon Ultra Centrifugal Filters (3 kDa molecular weight cutoff; Millipore) and then immediately sterile-filtered (0.22µm) by syringe. Total protein concentration was measured by BCA Protein Assay Kit (Pierce). Isolated lipoproteins were stored in the dark at 4oC and monitored for aggregation and loss of color. Best results were obtained from freshly collected plasma and newly processed nLDL. As quality control, LDL isolated and stored under these conditions for up to one month were assayed for oxidation using the TBARs method, and compared against matched LDL oxidized by indicated concentrations and exposure time to copper sulfate69. Reconstituted LDL (rLDL) was prepared as previously described with minimal modifications35. Briefly, de-salted nLDL was mixed with potato starch (Sigma) by vortexing and flash frozen in liquid nitrogen prior to lyophilization for 6h. Neutral lipids were selectively removed by sequential heptane extractions at -10oC, prior to reconstitution with cholesteryl linoleate (NuChek-Prep) spiked with a 1:20 ratio of fluorescent 18:2 TopFluor cholesteryl ester (Avanti Polar Lipids) solubilized in heptane. This mixture was evaporated under a nitrogen stream until powder dry. Formation of rLDL particles was solubilizated in 10 mM tricine, pH 8.4 for 108h at 4oC. Insoluble starch was pelleted by centrifugation at 2000xg and solubilized rLDL was extracted and sterile-filtered. In parallel, nLDL and rLDL samples were analyzed by size-exclusion chromatography (SEC) in running buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.2% NaN3) using two tandem Superose-6 gel filtration columns (Cytiva) on an ÄKTA SEC system (Cytiva). The distribution of fluorescent cholesteryl ester was measured by fluorometer using the Synergy Mx plate-reader. Total protein and lipid composition of LDL samples were determined by colorimetric kits: total cholesterol (Pointe Scientific), triglycerides (Pointe Scientific), Phospholipid C (Wako) and protein by BCA (Pierce).
Cell lysates were collected on ice in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) containing Halt Protease and Phosphatase Inhibitors Cocktail and rotated at 4oC for 1h. Insoluble cell debris was pelleted at 12,000xg for 10 min in a pre-chilled centrifuge (4oC), and soluble protein lysates were transferred to new tubes on ice. For immunoblots of cells, 12 µL of cell lysate (180 µL / well of a 6-well plate) was mixed with 4 µL of 4x Protein Sample Loading Buffer (LI-COR), denatured at 70oC for 5 minutes or 48oC for 10 min and immediately placed on ice. For immunoblots of mouse plasma, 1.5 mL of plasma was mixed with 10.5 mL RIPA buffer containing Halt Protease Inhibitors Cocktail (ThermoFisher) and 4 mL of 4X Protein Sample Loading Buffer (LI-COR), denatured at 70oC for 5 min and immediately placed on ice. Denatured protein samples were then run on NuPAGE 4-12% Bis-Tris or 3-8% Tris-Acetate gels (Life Technologies) at 125V on ice with Precision Plus Protein Kaleidoscope Pre-Stained Protein Standards (BioRad) or HiMark Pre-Stained Protein Standards (Life Technologies) as molecular weight references. After electrophoresis, gels were transferred to Nitrocellulose membranes using the iBlot system (Life Technologies). Blots were then blocked in Intercept TBS Blocking Buffer (LI-COR) for 30 min prior to dilution of primary antibody. Primary antibodies were incubated for at least 1h at room temperature or overnight at 4oC, washed 3X with tris-buffered saline containing 0.05% Tween-10 for 10 min. Subsequently, this process was repeated for secondary antibodies. Blots were assayed using the LI-COR Odyssey Infrared Imaging system, analyzed using Image Studio Lite and Image J software suites.
Single-plex and Multiplex ELISA:
Media supernatants of cell culture experiments were assayed for indicated cytokine concentrations by MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore) on the Luminex MAGPIX System by the Vanderbilt Hormone Assay and Analytical Services Core, per manufacturer’s recommendations. For individual assays, media cytokine concentrations were assayed by conventional ELISA for human and mouse IL-6 and TNF-a (DuoSet; R&D Systems).
Formulation of PLGA-CU-CPT9a microparticles:
The selective TLR8 inhibitor CU-CPT9a is not water soluble, requiring alternative methods for effective drug delivery in cell culture systems. Hence, drug-loaded microparticles (MPs) were formulated using a conventional oil-in-water (O/W) emulsion technique. Poly(lactide-co-glycolide) (PLGA, 50:50 lactide:glycolide, 10 kDa, Sigma) was dissolved at 100 mg/ml in dichloromethane (DCM) with vortexing and brief sonication. CU-CPT9a (Sigma) was resuspended at 10 mg/ml in DCM. 0.5 mL of drug solution was added to 0.5 mL PLGA solution, at which point the drug fully dissolved. The polymer/drug O phase was added dropwise to 5 mL 1.5% poly(vinyl alcohol) (PVA, 30-70 kDa, Sigma) in water and homogenized for 30 s at 10,000 rpm with an IKA T18 Digital Ultra-Turrax homogenizer. The O/W emulsion was transferred to a 100 mL round bottom flask, and DCM was removed via rotary evaporation to complete MP hardening. The aqueous MP suspension was centrifuged at 7,500xg for 10 min and resuspended in deionized water to remove excess PVA, and the resuspended MPs were aliquoted for lyophilization and subsequently stored at -20oC. Immediately prior to experiments, lyophilized MP were equilibrated to room temperature and re-suspended with PBS to 4 mg/ml prior to dilution at indicated doses. Resulting particles were deposited on a glass coverslip, imaged on a Nikon Eclipse Ti inverted scope and diameters measured using Nikon Ti software. Drug loading was quantified by dissolving in dimethylsulfoxide and measuring the ultraviolet-visible absorbance spectra relative to free CU-CPT9a on a Varian UV-VIS spectrophotometer. Resulting particles were 3.39 +/- 1.93 microns in diameter with 84.8 microgram drug per mg MP (encapsulation efficiency = 84.8%).
Statistics, Reproducibility and Rigor:
Data shown for in vitro experiments are representative experiments performed at least three times with independent donors/isolations of LDL. In vitro experiments were performed with technical replicates of 3-4. Unless otherwise specified data are mean +/- SEM. For animal studies, all treatments were randomized within individual cages to limit cage effects. For atherosclerosis studies, quantification of lesion area, fibrosis, immunohistochemistry and immunofluorescence were assessed in a blinded fashion by at least two researchers. Harvest and preparation of heart and aortas for histology was also performed blinded by a single technician. Where two treatments were compared with a sample size >3 for each treatment a Mann-Whitney test (two-sided) was performed with alpha = 0.05. Where three or more groups were compared a One-Way ANOVA was performed using Benjamini, Krieger and Yekutieli post-hoc test to control the false discovery rate (Q=0.05; Fig. 1 and Fig. 4) or Sidak’s post-hoc test to control Type I errors (Fig. 5). Where more than one test was performed for three or more groups a Two-Way ANOVA was performed using Benjamini, Krieger and Yekutieli post-hoc test to control the false discovery rate (Q=0.05). To determine whether differences existed between treatments in dose-response curves a non-linear regression with extra sum-of-squares F test was performed. Schematics and aspects of some figures were created with BioRender.com, Tableau and R Studio. GraphPad Prism 8 (GraphPad Software) was used to create graphs and calculate statistical significances. Images were assembled in Affinity Designer (Serif) to generate figures.