Development of an Accelerated Rotator-based Drug Release Method for the Evaluation of Bupivacaine Multivesicular Liposomes.

PURPOSE: A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. METHODS: Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24 h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. RESULTS: The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24 h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. CONCLUSIONS: An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.

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

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

Apolipoprotein-mimetic nanodiscs reduce lipid accumulation and improve liver function in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a severe lipid storage disorder caused by the diminished activity of the acid sphingomyelinase enzyme. ASMD is characterized by the accumulation of sphingomyelin in late endosomes and lysosomes leading to progressive neurological dysfunction and hepatosplenomegaly. Our objective was to investigate the utility of synthetic apolipoprotein A-I (ApoA-I) mimetics designed to act as lipid scavengers for the treatment of ASMD. We determined the lead peptide, 22A, could reduce sphingomyelin accumulation in ASMD patient skin fibroblasts in a dose dependent manner. Intraperitoneal administration of 22A formulated as a synthetic high-density lipoprotein (sHDL) nanodisc mobilized sphingomyelin from peripheral tissues into circulation and improved liver function in a mouse model of ASMD. Together, our data demonstrates that apolipoprotein mimetics could serve as a novel therapeutic strategy for modulating the pathology observed in ASMD.

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways.

Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293.

Mechanistic evaluation of the initial burst release of leuprolide from spray-dried PLGA microspheres.

Spray-dried poly(lactic-co-glycolic acid) (PLGA) peptide-loaded microspheres have demonstrated similar long-term in vitro release kinetics compared to those produced by the solvent evaporation method and commercial products. However, the difficult-to-control initial burst release over the first 24 h after administration presents an obstacle to product development and establishing bioequivalence. Currently, detailed information about underlying mechanisms of the initial burst release from microspheres is limited. We investigated the mechanism and extent of initial burst release using 16 previously developed spray-dried microsphere formulations of the hormone drug, leuprolide acetate, with similar composition to the commercial 1-month Lupron Depot® (LD). The burst release kinetics was measured with a previously validated continuous monitoring system as well as traditional sample-and-separate methods. The changes in pore structure and polymer permeability were investigated by SEM imaging and the uptake of a bodipy-dextran probe. In vitro results were compared to pharmacokinetics in rats over the same interval. High-burst, spray-dried microspheres were differentiated in the well-mixed continuous monitoring system but reached an upper limit when measured by the sample-and-separate method. Pore-like occlusions observed by confocal microscopy in some formulations indicated that particle swelling may have contributed to probe diffusion through the polymer phase and showed the extensive internal pore structure of spray-dried particles. Continuous monitoring revealed a rapid primary (1°) phase followed by a constant-rate secondary (2°) release phase, which comprised ∼80% and 20% of the 24-hr release, respectively. The ratio of 1° phase duration (t1°) and the characteristic probe diffusion time (τ) was highly correlated to 1° phase release for spray dried particles. Of the four spray-dried formulations administered in vivo, three spray-dried microspheres with similar polymer density showed nearly ideal linear correlation between in vivo absorption and well-mixed in vitro release kinetics over the first 24 h. By contrast, the more structurally dense LD and a more-dense in-house formulation showed a slight lag phase in vivo relative to in vitro. Furthermore, in vitro dimensionless times (tburst/τ) were highly correlated with pharmacokinetic parameters for spray-dried microspheres but not for LD. While the correlation of increases in effective probe diffusion and 1° phase release strongly suggests diffusion through the polymer matrix as a major release mechanism both in vitro and in vivo, a fixed lower limit for this release fraction implies an alternative release mechanism. Overall, continuous monitoring release and probe diffusion appears to have potential in differentiating between leuprolide formulations and establishing relationships between in vitro release and in vivo absorption during the initial burst period.

Quantitative chemometric phenotyping of three-dimensional liver organoids by Raman spectral imaging.

Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

Natural polysaccharides and their derivatives as potential medical materials and drug delivery systems for the treatment of peripheral nerve injuries.

Peripheral nerve repair following injury is one of the most serious problems in neurosurgery. Clinical outcomes are often unsatisfactory and associated with a huge socioeconomic burden. Several studies have revealed the great potential of biodegradable polysaccharides for improving nerve regeneration. We review here the promising therapeutic strategies involving different types of polysaccharides and their bio-active composites for promoting nerve regeneration. Within this context, polysaccharide materials widely used for nerve repair in different forms are highlighted, including nerve guidance conduits, hydrogels, nanofibers and films. While nerve guidance conduits and hydrogels were used as main structural scaffolds, the other forms including nanofibers and films were generally used as additional supporting materials. We also discuss the issues of ease of therapeutic implementation, drug release properties and therapeutic outcomes, together with potential future directions of research.

Characterization of exparel bupivacaine multivesicular liposomes.

Exparel is a bupivacaine multivesicular liposomes (MVLs) formulation developed based on the DepoFoam technology. The complex composition and the unique structure of MVLs pose challenges to the development and assessment of generic versions. In the present work, we developed a panel of analytical methods to characterize Exparel with respect to particle size, drug and lipid content, residual solvents, and pH. In addition, an accelerated in vitro drug release assay was developed using a rotator-facilitated, sample-and-separate experimental setup. The proposed method could achieve over 80% of bupivacaine release within 24 h, which could potentially be used for formulation comparison and quality control purposes. The batch-to-batch variability of Exparel was examined by the established analytical methods. Four different batches of Exparel showed good batch-to-batch consistency in drug content, particle size, pH, and in vitro drug release kinetics. However, slight variation in lipid contents were observed.

Gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Regenerative endodontics represents a paradigm shift in dental pulp therapy for necrotic young permanent teeth. However, there are still challenges associated with attaining maximum root canal disinfection while supporting angiogenesis and preserving resident stem cells viability and differentiation capacity. Here, we developed a hydrogel system by incorporating antibiotic-eluting fiber-based microparticles in gelatin methacryloyl (GelMA) hydrogel to gather antimicrobial and angiogenic properties while prompting minimum cell toxicity. Minocycline (MINO) or clindamycin (CLIN) was introduced into a polymer solution and electrospun into fibers, which were further cryomilled to attain MINO- or CLIN-eluting fibrous microparticles. To obtain hydrogels with multi-therapeutic effects, MINO- or CLIN-eluting microparticles were suspended in GelMA at distinct concentrations. The engineered hydrogels demonstrated antibiotic-dependent swelling and degradability while inhibiting bacterial growth with minimum toxicity in dental-derived stem cells. Notably, compared to MINO, CLIN hydrogels enhanced the formation of capillary-like networks of endothelial cells in vitro and the presence of widespread vascularization with functioning blood vessels in vivo. Our data shed new light onto the clinical potential of antibiotic-eluting gelatin methacryloyl hydrogel as an injectable scaffold with multi-therapeutic effects to promote antimicrobial disinfection and angiogenesis for regenerative endodontics.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

Regulatory utility of mechanistic modeling to support alternative bioequivalence approaches: A workshop overview.

On September 30 and October 1, 2021, the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics cosponsored a live virtual workshop titled "Regulatory Utility of Mechanistic Modeling to Support Alternative Bioequivalence Approaches." The overall aims of the workshop included (i) engaging the generic drug industry and other involved stakeholders regarding how mechanistic modeling and simulation can support their product development and regulatory submissions; (ii) sharing the current state of mechanistic modeling for bioequivalence (BE) assessment through case studies; (iii) establishing a consensus on best practices for using mechanistic modeling approaches, such as physiologically based pharmacokinetic modeling and computational fluid dynamics modeling, for BE assessment; and (iv) introducing the concept of a Model Master File to improve model sharing between model developers, industry, and the FDA. More than 1500 people registered for the workshop. Based on a postworkshop survey, the majority of participants reported that their fundamental scientific understanding of mechanistic models was enhanced, there was greater consensus on model validation and verification, and regulatory expectations for mechanistic modeling submitted in abbreviated new drug applications were clarified by the workshop.

Physicochemical characterization and functionality comparison of Humira®(adalimumab), Remicade®(infliximab) and Simponi Aria®(golimumab).

FDA-approved anti-TNFα biopharmaceuticals are successful in treating a range of autoimmune diseases. However, not all anti-TNFα products are identical in their patient outcomes, suggesting that there may be product-specific differences stemming from protein structural differences, doses and routes of administration. In this work, we focus only on structural and functional differences across three full-length anti-TNFα mAbs (Humira®, Remicade®, and Simponi Aria®) to better understand the implications of such differences on the products' efficacy. For structural characterization, we quantified N-glycans using mass spectrometry and fluorescence labeling. From these studies, we observed that Remicade® had the highest percent of afucosylated glycans (15.5 ± 1.3 %) and the largest number of unique glycans, 28. While Humira® had the fewest unique glycans, 15, and 11.4 ± 0.8 % of afucosylated, high-mannose glycans. For the functional studies we tested TNFα binding via ELISA, FcγRIIIa binding via AlphaLISA and effector function using an ADCC bioreporter assay. Humira® had a significantly lower EC50 (1.9 ± 0.1 pM) for ELISA and IC50 (10.5 ± 1.1 nM) for AlphaLISA, suggesting that Humira® has higher TNFα and FcγRIIIa binding affinity than Remicade® and Simponi Aria®. Humira® was also the most potent in the bioreporter assay with an EC50 value of 0.55 ± 0.03 nM compared to Remicade® (0.64 ± 0.04 nM) and Simponi Aria® (0.67 ± 0.03 nM). This comparison is significant as it highlights functional differences between mAbs with shared mechanisms of action when examined in a single laboratory and under one set of conditions.

Establishing the suitability of model-integrated evidence to demonstrate bioequivalence for long-acting injectable and implantable drug products: Summary of workshop.

On November 30, 2021, the US Food and Drug administration (FDA) and the Center for Research on Complex Generics (CRCG) hosted a virtual public workshop titled "Establishing the Suitability of Model-Integrated Evidence (MIE) to Demonstrate Bioequivalence for Long-Acting Injectable and Implantable (LAI) Drug Products." This workshop brought relevant parties from the industry, academia, and the FDA in the field of modeling and simulation to explore, identify, and recommend best practices on utilizing MIE for bioequivalence (BE) assessment of LAI products. This report summerized presentations and panel discussions for topics including challenges and opportunities in development and assessment of generic LAI products, current status of utilizing MIE, recent research progress of utilizing MIE in generic LAI products, alternative designs for BE studies of LAI products, and model validation/verification strategies associated with different types of MIE approaches.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Phospholipids impact the protective effects of HDL-mimetic nanodiscs against lipopolysaccharide-induced inflammation.

Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment.

Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature.

Electrospun Azithromycin-Laden Gelatin Methacryloyl Fibers for Endodontic Infection Control.

This study was aimed at engineering photocrosslinkable azithromycin (AZ)-laden gelatin methacryloyl fibers via electrospinning to serve as a localized and biodegradable drug delivery system for endodontic infection control. AZ at three distinct amounts was mixed with solubilized gelatin methacryloyl and the photoinitiator to obtain the following fibers: GelMA+5%AZ, GelMA+10%AZ, and GelMA+15%AZ. Fiber morphology, diameter, AZ incorporation, mechanical properties, degradation profile, and antimicrobial action against Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii were also studied. In vitro compatibility with human-derived dental pulp stem cells and inflammatory response in vivo using a subcutaneous rat model were also determined. A bead-free fibrous microstructure with interconnected pores was observed for all groups. GelMA and GelMA+10%AZ had the highest fiber diameter means. The tensile strength of the GelMA-based fibers was reduced upon AZ addition. A similar pattern was observed for the degradation profile in vitro. GelMA+15%AZ fibers led to the highest bacterial inhibition. The presence of AZ, regardless of the concentration, did not pose significant toxicity. In vivo findings indicated higher blood vessel formation, mild inflammation, and mature and thick well-oriented collagen fibers interweaving with the engineered fibers. Altogether, AZ-laden photocrosslinkable GelMA fibers had adequate mechanical and degradation properties, with 15%AZ displaying significant antimicrobial activity without compromising biocompatibility.

Cancer stem cell antigen nanodisc cocktail elicits anti-tumor immune responses in melanoma.

One of the major reasons for poor cancer outcomes is the existence of cancer stem cells (CSCs). CSCs are a small subpopulation of tumor cells that can self-renew, differentiate into the majority of tumor cells, and maintain tumorigenicity. As CSCs are resistant to traditional chemotherapy and radiation, they contribute to metastasis and relapse. Thus, new approaches are needed to target and eliminate CSCs. Here, we sought to target and reduce the frequency of CSCs in melanoma by therapeutic vaccination against CSC-associated transcription factors, such as Sox2 and Nanog, and aldehyde dehydrogenase (ALDH). Toward this goal, we have identified novel immunogenic peptide epitopes derived from CSC-associated Sox2 and Nanog and synthesized synthetic high-density lipoprotein (sHDL) nanodisc vaccine formulated with Sox2, Nanog, and ALDH antigen peptides together with CpG, a Toll-like receptor 9 agonist. Vaccination with nanodiscs containing six CSC antigen peptides elicited robust T cell responses against CSC-associated antigens and promoted intratumoral infiltration of CD8+ T cells, while reducing the frequency of CSCs and CD4+ regulatory T cells within melanoma tumors. Nanodisc vaccination effectively reduced tumor growth and significantly extended animal survival without toxicity toward normal stem cells. Overall, our therapeutic strategy against CSCs represents a cost-effective, safe, and versatile approach that may be applied to melanoma and other cancer types, as well as serve as a critical component in combined therapies to target and eliminate CSCs.

Development of activated endothelial targeted high-density lipoprotein nanoparticles.

Endothelial inflammation is an important pathophysiological driving force in various acute and chronic inflammatory diseases. High-density lipoproteins (HDLs) play critical roles in regulating endothelial functions and resolving endothelial inflammation. In the present study, we developed synthetic HDLs (sHDLs) which actively target inflamed endothelium through conjugating vascular cell adhesion protein 1 (VCAM-1) specific VHPK peptide. The active targeting of VHPK-sHDLs was confirmed in vitro on TNF-α activated endothelial cells. VHPK-sHDLs presented potent anti-inflammatory efficacies in vitro through the reduction of proinflammatory cytokine production and inhibition of leukocyte adhesion to activated endothelium. VHPK-sHDLs showed increased binding on inflamed vessels and alleviated LPS-induced lung inflammation in vivo. The activated endothelium-targeted sHDLs may be further optimized to resolve endothelial inflammation in various inflammatory diseases.

Effect of Lipid Composition on the Atheroprotective Properties of HDL-Mimicking Micelles.

Atherosclerosis progression is driven by an imbalance of cholesterol and unresolved local inflammation in the arteries. The administration of recombinant apolipoprotein A-I (ApoA-I)-based high-density lipoprotein (HDL) nanoparticles has been used to reduce the size of atheroma and rescue inflammatory response in clinical studies. Because of the difficulty in producing large quantities of recombinant ApoA-I, here, we describe the preparation of phospholipid-based, ApoA-I-free micelles that structurally and functionally resemble HDL nanoparticles. Micelles were prepared using various phosphatidylcholine (PC) lipids combined with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2k) to form nanoparticles of 15-30 nm in diameter. The impacts of PC composition and PEGylation on the anti-inflammatory activity, cholesterol efflux capacity, and cholesterol crystal dissolution potential of micelles were investigated in vitro. The effects of micelle composition on pharmacokinetics and cholesterol mobilization ability were evaluated in vivo in Sprague Dawley rats. The study shows that the composition of HDL-mimicking micelles impacts their overall atheroprotective properties and supports further investigation of micelles as a therapeutic for the treatment of atherosclerosis.