Liposome production and concurrent loading of drug simulants by microfluidic hydrodynamic focusing.

Liposomes are spherical vesicles enclosed by phospholipid bilayers. Nanoscale liposomes are widely employed for drug delivery in the pharmaceutical industry. In this study, nanoscale liposomes are fabricated using the microfluidic hydrodynamic focusing (MHF) approach, and the effects of flow rate ratio (FRR) on liposome size and drug loading efficiency are studied. Fluorescein isothiocyanate modified dextran is used as a hydrophilic drug simulant and Nile red is used as a hydrophobic drug simulant. The experiment results show that hydrophilic drug simulant loading efficiency increases as FRR increases and eventually plateaues at around 90% loading efficiency. The hydrophobic drug simulant loading efficiency and FRR have a positive linear correlation when FRR varies from 10 to 50. Concurrent loading of both hydrophilic and hydrophobic drug simulants maintains the same loading efficiencies as those of loading each drug simulant alone. A negative correlation between liposome size and FRR is also confirmed. Unloaded liposomes and hydrophilic drug-loaded liposomes are of the same sizes, and are smaller than the ones loaded with the hydrophobic drug simulants alone or combined. The results suggest tunable liposome size and drug loading efficiency with the MHF technique. This provides evidence to encourage further studies of microfluidic liposome fabrication in the pharmaceutical industry.

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Establishment of a trimodality analytical platform for tracing, imaging and quantification of gold nanoparticles in animals by radiotracer techniques.

This study aims to establish a (198)Au-radiotracer technique for in vivo tracing, rapid quantification, and ex vivo visualization of PEGylated gold nanoparticles (GNPs) in animals, organs and tissue dissections. The advantages of GNPs lie in its superior optical property, biocompatibility and versatile conjugation chemistry, which are promising to develop diagnostic probes and drug delivery systems. (198)Au is used as a radiotracer because it simultaneously emits beta and gamma radiations with proper energy and half-life; therefore, (198)Au can be used for bioanalytical purposes. The (198)Au-tagged radioactive gold nanoparticles ((198)Au-GNPs) were prepared simply by irradiating the GNPs in a nuclear reactor through the (197)Au(n,γ)(198)Au reaction and subsequently the (198)Au-GNPs were subjected to surface modification with polyethylene glycol to form PEGylated (198)Au-GNPs. The (198)Au-GNPs retained physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition studies were performed by intravenously injecting three types of (198)Au-GNPs with or without PEGylation into mice; the γ radiation in blood specimens and dissected organs was then measured. The (198)Au-radiotracer technique enables rapid quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of (198)Au-GNPs in living animals. Moreover, autoradiography, which recorded beta particles from (198)Au, enabled visualizing the heterogeneous biodisposition of (198)Au-GNPs in different microenvironments and tissues. In this study, the (198)Au-radiotracer technique facilitated creating a trimodality analytical platform for tracing, quantifying and imaging GNPs in animals.

Tip-enhanced Raman spectroscopic imaging shows segregation within binary self-assembled thiol monolayers at ambient conditions.

Phase segregation of coadsorbed thiol molecules on a gold surface was investigated with nanoscale chemical imaging using tip-enhanced Raman spectroscopy (TERS). Samples were prepared using mixed solutions containing thiophenol (PhS) and an oligomeric phenylene-ethynylene (OPE) thiol, with 10:1, 2:1, and 1:1 molar ratios. Phase segregation into domains with sizes from ≈30 to 240 nm is observed with these molar ratios. A comparison of TERS images with different pixel sizes indicates that a pixel size bigger than 15 nm is not reliable in defining nanodomains, because of undersampling. In this study, the formation of nanodomains was clearly evident based on the molecular fingerprints provided by TERS, while ambient scanning tunneling microscopy (STM) was not capable of discerning individual domains via their apparent height difference. TERS therefore allows to image nanodomains in binary self-assembled monolayers, which are invisible to methods solely relying on topographic or electron density characteristics of self-assembled monolayers. Moreover, TERS mapping provides statistical data to describe the distribution of molecules on the sample surface in a well-defined manner. Peak ratio histograms of selected TERS signals from samples prepared with different mixing ratios give a better understanding of the adsorption preference of the thiols studied, and the relationship of their mixing ratio in solution and adsorbed on the surface.

Microbiota-mediated metabolic perturbations in the gut and brain of mice after microplastic exposure.

Microplastics (MPs), emerging environmental toxicants, have drawn attention because of their wide distribution in the environment. Exposure to MPs induces gut microbiota dysbiosis, intestinal barrier dysfunction, metabolic perturbations, and neurotoxicity in different rodents. However, the relationship between MPs, gut microbiota, and the metabolome of the gut and brain in mice remains unclear. In this study, female C57BL/6 mice were orally gavaged with vehicle, 200 nm MP, and 800 nm MP three times per week for four weeks. Cecal contents were collected for gut microbiota analysis using 16S rRNA gene sequencing. Intestinal and brain tissues from mice were used to determine metabolic profiles using liquid chromatography-mass spectrometry (LC-MS). The results showed that MP altered microbiota composition, accompanied by metabolic perturbations in the mouse gut and brain. Specifically, Firmicutes and Bacteroidetes were suggested to be important phyla for MP exposure, partially dominating further metabolite alterations. Simultaneously, MP-induced metabolic profiles were associated with energy homeostasis and bile acid, nucleotide, and carnitine metabolic pathways. The results of the mediation analysis further revealed an MP-microbiota-metabolite relationship. Our results indicate that MPs can induce gut dysbiosis and disturb metabolic dysfunction in the mouse brain and/or intestine. Integrative omics approaches have the potential to monitor MP-induced molecular responses in various organs and systematically elucidate the complex mechanisms of human health effects.

3D-printed microfluidic device for high-throughput production of lipid nanoparticles incorporating SARS-CoV-2 spike protein mRNA.

Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL min-1 range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL min-1) of LNPs compared to commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (>90%). The 3D-printed device provides a cost-effective and easily prepared solution for high-throughput LNP production.

Identification of an 85-kb Heterozygous 4p Microdeletion With Full Genome Analysis in Autosomal Dominant Charcot-Marie-Tooth Disease.

Background and Objectives: Charcot-Marie-Tooth disease (CMT) is a syndrome of a hereditary neurodegenerative condition affecting the peripheral nervous system and is a single gene disorder. Deep phenotyping coupled with advanced genetic techniques is critical in discovering new genetic defects of rare genetic disorders such as CMT. Methods: We applied multidisciplinary investigations to examine the neurophysiology and nerve pathology in a family that fulfilled the diagnosis of CMT2. When phenotype-guided first-tier genetic tests and whole-exome sequencing did not yield a molecular diagnosis, we conducted full genome analysis by examining phased whole-genome sequencing and whole-genome optical mapping data to search for the causal variation. We then performed a systematic review to compare the reported patients with interstitial microdeletion in the short arm of chromosome 4. Results: In this family with CMT2, we reported the discovery of a heterozygous 85-kb microdeletion in the short arm of chromosome 4 (4p16.3)[NC_000004.12:g.1733926_1819031del] spanning 3 genes [TACC3 (intron 6-exon 16), FGFR3 (total deletion), and LETM1 (intron 10-exon14)] that cosegregated with disease phenotypes in family members. The clinical features of peripheral nerve degeneration in our family are distinct from the well-known 4p microdeletion syndrome of Wolf-Hirschhorn syndrome, in which brain involvement is the major phenotype. Discussion: In summary, we used the full genome analysis approach to discover a new microdeletion in a family with CMT2. The deleted segment contains 3 genes (TACC3, FGFR3, and LETM1) that likely play a role in the pathogenesis of nerve degeneration.

Transoral Tongue Suspension for Obstructive Sleep Apnea-A Preliminary Study.

Objectives: To evaluate the safety and efficacy of a novel technique for transoral tongue suspension (TOTS) in obstructive sleep apnea (OSA) patients. Material and Methods: The retrospective study enrolled 24 consecutive OSA patients (21 males; average age, 43 years; average apnea−hypopnea index (AHI), 42.2 event/h; average body mass index (BMI), 25.7 kg/m2) with tongue obstruction confirmed by drug-induced sleep endoscopy. All patients received TOTS as the main procedure in conjunction with uvulopalatopharyngoplasty (UPPP). Key procedures of TOTS included a transoral sublabial approach, drilling two holes on the mandible, passing the polypropylene through the hole to the tongue base using a suture passer and returning the polypropylene through loop traction, and tying the polypropylene to the mandible. Lingual tonsil ablation (n = 8) was also implemented in hypertrophic lingual tonsils (grades III and IV). Results: The operation time for TOTS was around 30 min. No wound bleeding or airway compromise occurred throughout the postoperative period. Minor complications were temporary and included swelling of the tongue, numbness of the lower incisor, and sublabial wound dehiscence (n = 2). The quality of life improved significantly in the patients' subjective daytime sleepiness according to the Epworth Sleepiness Scale (11.4 ± 3.2 vs. 5.7 ± 1.6, p < 0.001). The objective parameters of OSA also improved significantly in the apnea/hypopnea index (42.2 ± 21.8 vs. 19.5 ± 16.2, p < 0.001), minimal oxygen saturation (77.1 ± 12.2 vs. 81.7 ± 8.1, p = 0.026), and snoring index (207 ± 141 vs. 101 ± 91, p = 0.03). Conclusions: The demonstrated TOTS showed its advantage in low morbidity with a scarless exterior and easy performance with free availability in treating adult OSA patients with tongue obstruction. TOTS combined with UPPP significantly improved AHI and daytime sleepiness. TOTS can be implemented with lingual tonsillectomy to achieve both stabilization of the tongue and widening of hypopharyngeal airway.

LigaSure small jaw versus conventional neck dissection: a systematic review and meta-analysis.

BACKGROUND: Neck dissection has a central role in the management of head and neck cancers. This systematic review aimed to compare the intraoperative and postoperative parameters between conventional and LigaSure Small Jaw (LSJ)-assisted neck dissection. METHODS: PubMed (MEDLINE), Embase, and the Cochrane Library were searched. independently by two authors for relevant articles comparing the outcomes of conventional and LSJ-assisted neck dissection. Data from each study were extracted, and a random-effects model was used in the pooled analysis. RESULTS: Compared with conventional techniques, LSJ-assisted neck dissection was associated with a significantly reduced operative time. The rates of postoperative hematoma, infection, amount of intraoperative blood loss, the length of hospital stay and the drainage amount showed no significant intergroup differences. CONCLUSIONS: The meta-analysis provides evidence that properly using LSJ may reduce the operative time compared with that of conventional techniques. Surgeons may consider using LSJ in neck dissection according to personal experiences.

AFM for structure and dynamics of biomembranes.

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.

Tongue imaging during drug-induced sleep ultrasound in obstructive sleep apnea patients.

OBJECTIVES: The aims of this study are to examine the changes of tongue thickness and distance of two lingual arteries through drug-induced sleep ultrasound, and explore the relationship between sonographic measurements and clinical data. MATERIALS AND METHODS: A total of 26 confirmed obstructive sleep apnea patients were recruited in this one-year study. All patients received ultrasound examination twice (wakefulness and drug-induced sleep) in sleep center under level 1 polysomnographic monitor. Drug-induced sleep was performed by administration of one Stilnox (Zolpidem, 2 mg/tablet) and ultrasound procedure commenced once stage 2 sleep shown in polysomnography. Ultrasound imaging was implemented via submental approach with transducer position at the sagittal midline of the submental area (sagittal view) to measure thickness of the tongue. Transducer was then moved at a transverse midpoint between the inferior border of the mandible and the hyoid bone (transverse view) to measure the distance between 2 lingual arteries. RESULTS: The distance between 2 lingual arteries elongated significantly (p < .001) and thickness of tongue muscle became thinner during drug-induced sleep. The distance between 2 lingual arteries (sleep) had positive correlation with apnea/hypopnea index (AHI, r = 0.51, p = .008) and body mass index (BMI, r = 0.46, p = .018). CONCLUSION: Drug-induced sleep ultrasound is feasible to measure changes of tongue in OSA patients. Ultrasound imaging showed that tongue muscle became thinner in conjunction with significant widening in distance between two lingual arteries during hypnotic-induced sleep and that was positively correlated with AHI and BMI. Drug-induced sleep ultrasound may be helpful to enhance safety in tongue surgery for OSA patients.

Evaluation of Salivary Exosomal Chimeric GOLM1-NAA35 RNA as a Potential Biomarker in Esophageal Carcinoma.

PURPOSE: Transcriptionally induced chimeric RNAs are an important emerging area of research into molecular signatures for biomarker and therapeutic target development. Salivary exosomes represent a relatively unexplored, but convenient, and noninvasive area of cancer biomarker discovery. However, the potential of cancer-derived exosomal chimeric RNAs in saliva as biomarkers is unknown. Here, we explore the potential clinical utility of salivary exosomal GOLM1-NAA35 chimeric RNA (seG-NchiRNA) in esophageal squamous cell carcinoma (ESCC). EXPERIMENTAL DESIGN: In a retrospective study, the prognostic significance of G-NchiRNA was determined in ESCC tissues. The correlation between seG-NchiRNA and circulating exosomal or tumoral G-NchiRNA was ascertained in cultured cells and mice. In multiple prospective cohorts of patients with ESCC, seG-NchiRNA was measured by qRT-PCR and analyzed for diagnostic accuracy, longitudinal monitoring of treatment response, and prediction of progression-free survival (PFS). RESULTS: Exosomal G-NchiRNA was readily detectable in ESCC cells and nude mouse ESCC xenografts. SeG-NchiRNA levels reflected tumor burden in vivo and correlated with tumor G-NchiRNA levels. In prospective studies of a training cohort (n = 220) and a validation cohort (n = 102), seG-NchiRNA levels were substantially reduced after ESCC resection. Moreover, seG-NchiRNA was successfully used to evaluate chemoradiation responsiveness, as well as to detect disease progression earlier than imaging studies. Changes in seG-NchiRNA levels also predicted PFS of patients after chemoradiation. CONCLUSIONS: SeG-NchiRNA constitutes an effective candidate noninvasive biomarker for the convenient, reliable assessment of therapeutic response, recurrence, and early detection.

Polymer-based open-channel blockers for the acetylcholine receptor: the effect of spacer length on blockade kinetics.

Blocking open ion channels provides a promising way to modulate synaptic transmission. Using the muscle-type acetylcholine receptor (AChR) as a test system, we seek to develop blockers that have blockade kinetics tunable via structural modifications. Here we investigate whether the blockade kinetics can be modulated by specifying the length of a poly(ethylene glycol) (PEG) spacer incorporated into the blocker. Single-channel electrophysiological experiments show that simple bis(trimethylammonium) compounds ( 1a- 3) both activate the AChR and block the open channel. The blockade kinetics are found to depend on spacer length: both the association and dissociation rate constants decrease with increasing spacer length. The decrease in the association rate constant can be quantitatively explained by the entropic cost of polymer confinement in the transmembrane lumen, while the decrease in the dissociation rate constant is consistent with weak, additive noncovalent interactions between the channel and the spacer. These results provide useful insights into the future design of kinetically tunable open-channel blockers for the AChR.

Tumortropic monocyte-mediated delivery of echogenic polymer bubbles and therapeutic vesicles for chemotherapy of tumor hypoxia.

Overcoming limitations often experienced in nanomedicine delivery toward hypoxia regions of malignant tumors remains a great challenge. In this study, a promising modality for active hypoxia drug delivery was developed by adopting tumortropic monocytes/macrophages as a cellular vehicle for co-delivery of echogenic polymer/C5F12 bubbles and doxorubicin-loaded polymer vesicles. Through the remote-controlled focused ultrasound (FUS)-triggered drug liberation, therapeutic monocytes show prominent capability of inducing apoptosis of cancer cells. The in vivo and ex vivo fluorescence imaging shows appreciable accumulation of cell-mediated therapeutics in tumor as compared to the nanoparticle counterpart residing mostly in liver. Inhibition of tumor recurrence with γ-ray pre-irradiated Tramp-C1-bearing mice receiving therapeutic monocytes intravenously alongside the FUS activation at tumor site was significantly observed. Immunohistochemical examination of tumor sections confirms successful cellular transport of therapeutic payloads to hypoxic regions and pronounced cytotoxic action against hypoxic cells. Following the intravenous administration, the cellular-mediated therapeutics can penetrate easily to a depth beyond 150 µm from the nearest blood vessels within pre-irradiated tumor while nanoparticles are severely limited to a depth of ca 10-15 µm. This work demonstrates the great promise of cellular delivery to carry therapeutic payloads for improving chemotherapy in hypoxia by combining external trigger for drug release.

Poly(ethylene glycol) as a scaffold for high-affinity open-channel blockers of the mouse nicotinic acetylcholine receptor.

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1-5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1-5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.

Systemic and local reactions of a water-soluble copolymer bone on a bony defect of rabbit model.

BACKGROUND: Ostene, a synthetic water-soluble bone hemostatic agent, is commercially available. In the current study, we evaluated the systemic and local effects of this copolymer in a rabbit model. METHODS: Eighteen rabbits underwent creation of a bony defect at right iliac crest. These rabbits were then evenly divided into 3 groups. In group 1, the defect surfaces were treated with bone wax; in group 2, the defect surfaces were treated with Ostene; in group 3, the defect surfaces were not treated with anything. Then, the animals underwent blood examinations, including WBC count, CRP, and ESR at 0, 1, 3, and 6 weeks, and were killed at 6 weeks for histologic examination. Another 6 rabbits (group 4) underwent the same surgical treatment of group 2 animals but had blood examinations of BUN and creatinine. RESULTS: The blood examinations showed that the WBC count, CRP, and ESR of all the animals in the first 3 groups were within normal limits in the postoperative periods. Microscopic examinations demonstrated residual bone wax and fibrotic tissue at the defect surfaces in group 1 animals. However, there was no Ostene at the defect surfaces in group 2 animals. The groups 2 and 3 animals showed no fibrotic tissue at the defect surfaces. The group 4 animals showed normal serum levels of BUN and creatinine in the postoperative periods. CONCLUSION: Ostene is absorbable and induces no systemic inflammation (including acute renal damage) and local inflammation in animal bodies.

Effect of support corrugation on silica xerogel--supported phase-separated lipid bilayers.

Lipid bilayers supported by substrates with nanometer-scale surface corrugations hold interest in understanding both nanoparticle-membrane interactions and the challenges of constructing models of cell membranes on surfaces with desirable properties, e.g., porosity. Here, we successfully form a two-phase (gel-fluid) lipid bilayer supported by nanoporous silica xerogel. Surface topology, lateral diffusion coefficient, and lipid density in comparison to mica-supported lipid bilayers were characterized by atomic force microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and quantitative fluorescence microscopy, respectively. We found that the two-phase lipid bilayer follows the silica xerogel surface contours. The corrugation imparted on the lipid bilayer results in a lipid density that is twice that on a flat mica surface in the fluid regions. In direct agreement with the doubling of actual bilayer area in a projected area, we find that the lateral diffusion coefficient (D) of fluid lipids on silica xerogel (approximately 1.7 microm2/s) is lower than on mica (approximately 3.9 microm2/s) by both FRAP and FCS techniques. Furthermore, the gel-phase domains on silica xerogel compared to mica were larger and less numerous. Overall, our results suggest the presence of a relatively defect-free continuous two-phase lipid bilayer that penetrates approximately midway into the first layer of approximately 50 nm silica xerogel beads.

Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol.

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed approximately 2.5, approximately 10, and approximately 20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at approximately 10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.