Microscopic Vesicles Research Articles

Advancements and Technical Considerations for Extracellular Vesicle Isolation and Biomarker Identification in Glioblastoma.

Extracellular vesicles (EVs) are membrane-bound particles released by all cells. Previous research has found that these microscopic vesicles contribute to intercellular signaling and communication. EVs carry a variety of cargo, including nucleic acids, proteins, metabolites, and lipids. The composition of EVs varies based on cell of origin. Therefore, EVs can serve as an important biomarker in the diagnosis and treatment of various cancers. EVs derived from glioblastoma (GBM) cells carry biomarkers, which could serve as the basis for a potential diagnostic strategy known as liquid biopsy. Multiple EV isolation techniques exist, including ultrafiltration, size exclusion chromatography, flow field-flow fractionation, sequential filtration, differential ultracentrifugation, and density-gradient ultracentrifugation. Recent and ongoing work aims to identify cellular markers to distinguish GBM-derived EVs from those released by noncancerous cells. Strategies include proteomic analysis of GBM EVs, identification of GBM-specific metabolites, and use of Food and Drug Administration-approved 5-aminolevulinic acid-an oral agent that causes fluorescence of GBM cells-to recognize GBM EVs in a patient's blood. In addition, accurately and precisely monitoring changes in EV cargo concentrations could help differentiate between pseudoprogression and GBM recurrence, thus preventing unnecessary surgical interventions.

Investigating the mechanochemistry of membrane area asymmetry generation: A combined mass-action and micromechanical study using methyl-beta-cyclodextrin.

Membrane asymmetry is important for cell signaling and is coupled with membrane morphological remodeling protein conformational regulation, sorting, and function. With respect to the lipids composing a bilayer, membrane asymmetry is divided into two contributions including 1) area asymmetry- that is, unequal monolayer surface areas that two apposed leaflets would tend to favor were they uncoupled and 2) compositional asymmetry- that is, different lipid compositions between the leaflets. For in vitro research on membrane asymmetry, methyl-β-cyclodextrin (mbCD) has been previously applied as a lipid-exchange catalyst to tune the compositional asymmetry of synthetic membranes. Inspired by this approach, we opted to study the physical chemistry of 1-component vesicle systems where mbCD was present not as a catalyst but as a reactant. In reaction mixtures at ambient conditions containing nanoscale vesicles, we performed a fluoresence-quenching assay to see that mbCD extracts phospholipids from only the outer leaflets of vesicles and, thereby, must generate area asymmetry in such mixtures. For certain molar ratios, mbCD can fully solubilize all phospholipid and we used 90o light scattering to map this behavior on a two-dimensional phase diagram at ambient temperature. Through temperature-cycling experiments, we showed an irreversibility in mbCD-phospholipid phase behavior. This granted access to stable solutions containing no self-assembled lipid structures with larger molar ratios of phospholipid to mbCD than could be achieved by isothermal reaction conditions. By adjusting the molar ratios of these solutions, we generated both negative and positive curvature in microscopic vesicles (i.e., inward and outward bending, respectively). Tunability of the curvature is consistent with mass-action considerations relating to net flow of phospholipid out of/into vesicles. We believe our approach using mbCD pre-complexed with phospholipid will enhance experimental research on the biophysical consequences of area asymmetry.

Surface Modification of Liposomes Using Folic Acid.

Liposomes are usually defined as spherically shaped microscopic vesicles that consist of one or more phospholipid bilayer membranes. They are widely used in drug delivery due to their biocompatibility, biodegradability, and stability. In recent years, a growing body of research shows that folic acid (FA)-modified liposomes can be targeted to deliver therapeutics to tumor and inflammation sites via receptor-mediated endocytosis between FA and folate receptor (FR). Taking this advantage, FA-modified liposomes are usually used in the targeted treatment of cancer, atherosclerosis, and arthrosis. In this chapter, we provided a classical thin-film hydration method to prepare FA-modified liposomes. We expect that our strategies would provide new opportunities for the development of FA-modified liposomes for research and clinical uses.

Effects of Growth Hormone on Pancreatic Cancer Derived Exosomes

In 2020, the National Cancer Institute (NCI) estimates 57,600 new cases and 47,050 deaths in the US due to pancreatic ductal adenocarcinoma (PDAC). A dismal 10% five-year overall survival rate in PDAC is attributed to late diagnosis, limited treatment options, a remarkably high metastasis rate, and resistance of this cancer to available therapies. Therefore, a better understanding of the mechanisms of how PDAC tumors acquire drug resistance and spread to distal parts of the body are necessary for developing novel therapeutic approaches. Exosomes, microscopic vesicles released from most cells (both tumor and non-tumor) have been recently established to play a significant role in cell to cell communication. Exosomes modulate their target cell responses systematically depending on the nature of exosomal cargoes (nucleic acids, proteins, and lipids). PDAC derived exosomes have been implicated to promote metastasis via forming a pre-metastatic niche of cells as well as enhancing drug resistance. Growth hormone (GH) secreted primarily by the pituitary gland promotes metastasis and drug resistance as shown by plethora of studies. No study has directly assessed the effect of GH on exosomal cargoes in terms of promoting metastases and drug resistance. In this report, we show that GH modulates various pancreatic cancer cell exosomal cargoes which in turn potentially amplifies tumor invasion and metastases. Our data shows that GH treatment on human and mouse PDAC cells increases the exosomal protein levels of TGFβ - a critical inducer of epithelial-to-mesenchymal transition (EMT, a process leading to metastasis). In addition, GH treatment also increases extracellular matrix-degrading enzymes, MMP2 and 9, as well as multi-drug efflux pump ABCC1, ABCB1, and ABCG2 in PDAC cells. These results strongly implicate GH action in driving EMT and chemoresistance via exosomes in pancreatic cancer. Exosomes have a crucial impact especially in the areas of diagnostics and therapeutics. This report is the first to show that GH modulates the effects of exosomes secreted by pancreatic cancer cells. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute.

Light-Induced Reversible Hierarchical Self-Assembly of Amphiphilic Diblock Copolymers into Microscopic Vesicles with Tunable Optical and Nanocarrier Properties.

In contrast to the conventional hierarchical self-assembly process, effective methods to enable reversible hierarchical self-assembly of block copolymers are comparatively few and limited in scope. Herein, we report, for the first time, a simple yet robust strategy for light-induced reversible hierarchical self-assembly of an amphiphilic diblock copolymer, poly(4-vinylpyridine)-block-poly[6-[4-(4-butyloxyphenylazo)phenoxy]hexyl methacrylate] (denoted P4VP-b-PAzoMA). The hierarchical structures are constructed via a two-step self-assembly process (first-level reverse micelles, second-level compound micelles, and rearrangement into micrometer-sized vesicles) driven by use of solvent. Intriguingly, because of reversible photoinduced trans-to-cis isomerization of azobenzene moieties in PAzoMA, the vesicles could disassemble into subunits upon UV light and then recover the nearly identical vesicular morphology upon visible light. Such a reversible hierarchical self-assembly process is accompanied by reversible fluorescence, encapsulation, and controlled release of dyes and can be used as a template for the synthesis of nanoparticles. Clearly, the ability to render the light-enabled reversible hierarchical self-assembly provides a unique platform for smart delivery vehicles and templates for nanomaterials.

Food ingrdient encapsulation using nanoliposome technology: a review study

The nanoliposome is a microscopic vesicle containing a phospholipid bilayer that surrounds a fluid space. The thickness of this bilayer lipid is usually between 3 and 6 nm, but the liposomes formed from them can have a diameter between 50 nm to 50 μm. Their unique properties have led to numerous applications in several scientific and technological fields. Nanoliposomes can cause controlled release of various biologically active agents, including food components and nutraceuticals at the right place and time. Therefore, nano-liposomes can increase the cellular uptake of encapsulated materials and increase their effectiveness. Food additives such as vitamins, enzymes, synthetic and natural antioxidants, synthetic and natural antibacterial compounds etc. can be converted to stable materials using nanoliposomes. As a result, this article reviews various aspects of nanoliposomes, including existing preparation techniques, and their various applications in food technology.

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles.

The introduction of chirality into aqueous self-assemblies by employing isotactic block copolymers (BCPs) is an emerging field of interest as it promises special membrane properties of polymersomes not accessible by atactic BCPs. However, isotactic BCPs typically exhibit crystalline behaviour, inducing high membrane stiffness and limiting their applicability in systems involving membrane proteins or sensitive cargo. In this study, an isotactic yet fully amorphous BCP is introduced which overcomes these limitations. Three BCPs composed of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG), differing solely in their tacticities (R/S, R and S), were synthesised and characterised regarding their structural, optical and thermal properties. Their self-assembly into homogenous phases of nanoscopic polymersomes (referred to as small unilamellar vesicles, SUVs) was analysed, revealing stability differences between SUVs composed of the different BCPs. Additionally, microscopic giant unilamellar vesicles (GUVs) were prepared by double emulsion microfluidics. Only the atactic BCP formed GUVs which were stable over several hours, whereas GUVs composed of isotactic BCPs ruptured within several minutes after formation. The ability of atactic PBO-b-PG to form microreactors was elucidated by reconstituting the membrane protein OmpF in the GUV membrane by microfluidics and performing an enzyme reaction inside its lumen. The system presented here serves as platform to design versatile vesicles with flexible membranes composed of atactic or isotactic BCPs. Hence, they allow for the introduction of chirality into nano- or microreactors which is a yet unstudied field and could enable special biotechonological applications.

Role of exosomes in pathogenesis of pulmonary diseases (review)

The article presents modern data on exosomes - microscopic extracellular vesicles with a diameter of 30-180 nanometers, released into the intercellular space by cells of the respiratory organs. The cells of the body’s respiratory system secrete exosomes into the intercellular space in a normal state, as well as during the development of the disease. The concentration of exosomes depends on the type of cell and includes mRNA, miRNAs, DNA and signaling proteins. Some exosomal proteins, such as CD63, CD81, CD9, CD24 and heat shock protein (Hsp70) are universal and they are usually used as exosomal markers. In respiratory diseases, in particular in patients with chronic obstructive pulmonary disease, IL-1P and miRNAs such as miR-15b, miR-223, miR-1274a, miR-424, mir-210 are significantly increased; miR-21 is the most common miRNA isolated from lung tissue, increased expression of this RNA is associated with symptoms of asthma, idiopathic pulmonary fibrosis and lung cancer. Exosome analysis makes it possible to distinguish between pulmonary and extrapulmonary forms of tuberculosis based on exosomal markers such as MPT64. Circulating exosomes are stable in biological fluids; therefore, analysis of exosomal microRNAs may indicate the state of the human respiratory system. This review opens up the possibility of using new diagnostic and therapeutic targets for various diseases of the respiratory system.

Biomarker bolsters cancer nanosensor

Arizona State University’s Ye (Tony) Hu and his colleagues have delivered what they hope will be a double dose of good news for detecting pancreatic cancer. Pancreatic cancer is one of the leading causes of cancer deaths because it often goes undetected in early stages, according to the Mayo Clinic. The researchers report a rapid and inexpensive nanoparticle-based diagnostic fueled, in part, by a biomarker on the surface of vesicles released by pancreatic tumors (Nat. Biomed. Eng. 2017, DOI: 10.1038/s41551-016-0021). As tumors develop, they release microscopic vesicles into a person’s blood. If biosensors could isolate and identify these vesicles, researchers could potentially catch cancer early by pricking a person’s finger and analyzing blood droplets. This process would be easier and less costly than more conventional biopsies or positron emission tomography scans, Hu says. But all cells release vesicles, and determining whether those vesicles come from healthy cells or cancerous tissue

Liposomes for Drug Delivery

Liposomes are a novel drug delivery system (NDDS), they are vesicular structures consisting of bilalyers which form spontaneously when phospholipids are dispersed in water. They are microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane composed of lipid bilayers. NDDS aims to deliver the drug at a rate directed by the needs of the body during the period of treatment and direct the place of action. Liposomes are colloidal spheres of cholesterol non-toxic surfactants, sphingolipids, glycolipids, long chain fatty acids and even membrane proteins and drug molecules or it is also called vesicular system. It differs in size, composition and charge and drug carrier loaded with variety of molecules such as small drug molecules, proteins, nucleotides or plasmids etc. Few drugs are formulated as liposomes to improve their therapeutic index. Hence a number of vesicular drug delivery systems such as liposomes, niosomes, transfersomes and pharmacosomes are developed. The focus of this chapter is to the various method of preparation, characterization of liposomes, advantages and applications, etc.

Proliposomes as Pharmaceutical Drug Delivery System: A Brief Review

Liposomes, the microscopic vesicles, are composed of a bilayer of phospholipids and a novel drug delivery system that provides drug at predetermined rate decided according to need, pharmacological aspects, drug profile, physiological conditions of body etc. But liposomes exhibit poor stability problem and this leads to their storage problem. To circumvent this problem Pro-liposomes (PLs) were discovered in 1986. Pro-liposomes are free flowing granular products composed of drug and phospholipid precursors which on hydration lead to liposomes. This paper reviews various features regarding pro-liposomes, their method of preparation, evaluation, applications and pointing out its potential to be employed for different routes of administration.

Simulation of Osmotic Swelling by the Stochastic Immersed Boundary Method

We develop computational methods for the simulation of osmotic swelling phenomena relevant to microscopic vesicles containing transformable solute molecules. We introduce stochastic immersed boundary methods (SIBMs) that can capture osmotically driven fluid transport through semipermeable elastic membranes subject to thermal fluctuations. We also develop numerical methods to handle within SIBMs an elastic shell model for a neo-Hookean material. Our extended SIBM allows for capturing osmotic swelling phenomena driven by concentration changes and interactions between a discrete collection of confined particles while accounting for the thermal fluctuations of the semipermeable membrane and the hydrodynamic transport of solvent. We use our computational methods to investigate osmotic phenomena in regimes that go beyond the classical Van't Hoff theory. We develop statistical mechanics theories for osmotic swelling of vesicles when there are significant interactions between particles that can transform over time. We validate our theoretical results against detailed computational simulations. Our methods are expected to be useful for a wide class of applications allowing for the simulation of osmotically driven flows, thermally fluctuating semipermeable elastic structures, and solute interactions.

ROLE OF LIPOSOME IN NOVEL DRUG DELIVERY SYSTEM

Liposome was found by Alec Bangham of Babraham Institute in Cambridge, England in 1965.In 1990; drugs with liposome and Amphotericin B were approved by Ireland. In 1995 America F.D.A approved liposor doxodubicin. In 1965s, it was well recognized that microscopic lipid vesicles, known as liposomes, could be utilized to encapsulate drugs and dyes for the purpose of systemic administration and drug targeting.Considerable progress was made in 1980s, in engeneering liposomes to circulate longer in blood and remain intact while doing so. The liposome a microscopic spherical particle formed by a lipid bilayer enclosing an aqueous compartment.An artificial microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipid layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs. Liposome was discovered about 40 years ago by Bangham and coworkersand was defined as microscopic spherical vesicles that form when phospholipids are hydrated or exposed to anaqueous environment.Liposomes are microscopic vesicles composed of a bilayer of phospholipids or any similar amphipathic lipids. They can encapsulate and effectively deliver both hydrophilic and lipophilic substances2‐3 and may be used as a non‐toxic vehicle for insoluble drugs. Liposomes are composed of small vesicles of phospholipids encapsulating an aqueous space ranging from about 0.03 to 10 µm in diameter. The membrane of liposome is made of phospholipids, which have phosphoric acid sides to form the liposome bilayers.Liposomes can be manufacturing in different lipid comopsitions or by different method show variation in par. Size , size distribution, surface electrical potential, no. of lamella, encapsulation efficacy,Surface modification showed great advantage to produce liposomes of different mechanisims, kinetic properties and biodistribution

Interaction of the heart and its close and distant neighbours: report of the Meeting of the ESC Working Groups Myocardial Function and Cellular Biology

Intercellular and inter-organ communication is required to maintain homoeostasis in multicellular organisms. Given its life-supporting function, the heart is at the centre of a heavily studied network of interactions. Despite the long-recognized ability to receive and relay inputs to and from far and close neighbours, surprising new networks of interactions as well as new communication factors and mechanisms are currently emerging. The Myocardial Function and the Cellular Biology of the Heart Working groups of the European Society of Cardiology convened during 2–5 May 2013 to discuss emerging data and ongoing studies on intercellular and inter-organ communication, in health and disease. Cells interact through either direct contact or secreted agonists. The signal transduction machinery inside cells eventually translates interactions with limited numbers of agonists into a cellular response. This process of scanty molecules being pinpointed by high-affinity receptors is now flanked by other emerging mechanisms of communication that involve highly abundant messengers binding to relatively low-affinity detectors. Two novel intercellular communication paths are currently arising: the flooding of cells with secreted vesicles, the so-called exosomes, with various but information-rich contents, and the sensing of different species of circulating RNA, such as miRNAs that, once captured, elicit post-transcriptional regulation of gene expression, and the extracellular RNAs (eRNA) that, from the outside of cells, trigger membrane enzyme activation and generation of classical receptor agonists. ### 2.1 Exosomes and miRNA An exciting new concept in the field of intercellular and inter-organ communication is the idea that microscopic vesicles may ferry proteins and RNA through interstitial fluid and blood. Building on this concept, miRNA and proteins from plasma microvesicles and exosomes may offer a great potential as biomarkers for cardiovascular risk and cellular injury. Specific miRNAs act as master regulators of angiogenesis, cardiomyocyte proliferation, and regulation of cell survival. In particular, the importance of miR-199, which seems to regulate a …

Profile of Ira Mellman

Nestled in an industrial sprawl at the foot of California’s San Bruno Mountain, the biotechnology firm Genentech faces the steel-gray waters of the San Francisco Bay. From his perch in the postcard-worthy campus, National Academy of Sciences member Ira Mellman has presided over a small cadre of cancer biologists for nearly half a decade. When Mellman became vice president of research oncology at the firm in 2007, he was already a household name among the world’s leading cell biologists thanks to his discovery of microscopic vesicles called endosomes, which shuttle proteins between compartments within cells. Over the years, he has become something of a scientific decathlete, adding entire dimensions to our understanding of the sorting of proteins in cells, the workings of the immune system, and the drivers of cancer. Since his appointment at Genentech, Mellman has helped usher a handful of cancer drugs into clinical trials, including antibodies against proteins implicated in a range of cancers. Ira Mellman. The South San Francisco campus of Genentech. A native New Yorker, Mellman moved to the American Midwest to study music at Oberlin College in Ohio. His father, a sometime teacher at Yale University, had long urged him to turn his gift for playing musical instruments into a lifelong pursuit, convinced that a musical career held more allure than one in chemistry. But an undergraduate biology laboratory course marked a turning point in his career, steering him away from music. Four decades later, he recalls the decision to pursue science with vindicated hindsight. “The more time I spent doing research, the more I became interested. Science was a promising choice, and has proven to be a fulfilling one,” he says. Mellman’s foray into biology began through an acquaintance with Oberlin professor and plant biochemist David Miller, who had been trying to deconstruct the cell walls of algae. Together with Miller, Mellman found that nearly one-third of the cell wall of a species of Chlamydomonas algae was composed of a sugar-rich protein named extensin. The finding led to an auspicious report in the Journal of Cell Biology (1); 30 years later, Mellman became the journal’s editor-in-chief.

Nano-sized cosmetic formulations or solid nanoparticles in sunscreens: A risk to human health?

Personal care products (PCP) often contain micron- or nano-sized formulation components, such as nanoemulsions or microscopic vesicles. A large number of studies suggest that such vesicles do not penetrate human skin beyond the superficial layers of the stratum corneum. Nano-sized PCP formulations may enhance or reduce skin absorption of ingredients, albeit at a limited scale. Modern sunscreens contain insoluble titanium dioxide (TiO₂) or zinc oxide (ZnO) nanoparticles (NP), which are efficient filters of UV light. A large number of studies suggest that insoluble NP do not penetrate into or through human skin. A number of in vivo toxicity tests, including in vivo intravenous studies, showed that TiO₂ and ZnO NP are non-toxic and have an excellent skin tolerance. Cytotoxicity, genotoxicity, photo-genotoxicity, general toxicity and carcinogenicity studies on TiO₂ and ZnO NP found no difference in the safety profile of micro- or nano-sized materials, all of which were found to be non-toxic. Although some published in vitro studies on insoluble nano- or micron-sized particles suggested cell uptake, oxidative cell damage or genotoxicity, these data are consistent with those from micron-sized particles and should be interpreted with caution. Data on insoluble NP, such as surgical implant-derived wear debris particles or intravenously administered magnetic resonance contrast agents suggest that toxicity of small particles is generally related to their chemistry rather than their particle size. Overall, the weight of scientific evidence suggests that insoluble NP used in sunscreens pose no or negligible risk to human health, but offer large health benefits, such as the protection of human skin against UV-induced skin ageing and cancer.

Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design

We report on a continuous droplet interface crossing encapsulation (cDICE) technique for the high-yield production of microscopic vesicles tunable in size and content. Overcoming some of the current technique limitations, such a simple setup has a great potential in various fields from encapsulation to the design of biomimetic cells and artificial tissues.

Constraining the dynamics of volcanic eruptions by characterization of pumice textures

We have characterized the textures of pumice clasts from Phlegraean Fields to gain insights into the conduit flow-dynamics of alkaline explosive eruptions. Vesicularities, vesicle number densities, and vesicle sizes and shapes were measured to obtain the bulk and groundmass properties of the juvenile fraction of Campanian Ignimbrite (CI) and Agnano Monte Spina (AMS) eruptions. The results report the coexistence of three end-member pumice types in the deposits of both eruptions, 1) microvesicular, 2) tube and 3) expanded, which differ according to clast morphology and the macro- to microscopic vesicle texture. Vesicularities (0.85-0.94 for CI, 0.51-0.91 for AMS) and vesicle number densities (2-4×105 cm-2 in CI, 3×105-106 cm-2 in AMS) span quite a wide range in all the three pumice types. Overall, tube pumices exhibit the highest bulk (0.89) and groundmass (CI 0.85, AMS 0.82) average vesicle volume fractions but the lowest average vesicle number densities (CI 2×105, AMS 4×105 cm-2). Comparison with textures of calc-alkaline pumices has revealed many similarities and points to a common origin and distribution of the products from both magma compositions within the volcanic conduit. In addition, the results of the textural analysis were interpreted in the light of the conduit flow modeling of Phlegraean Fields eruptions. The comparison of textural observations with results from simulations of conduit magma ascent has exhibited a good agreement between measured and numerically calculated vesicularities for both compositions, helping to constrain the overall dynamics of alkaline versus calc-alkaline eruptions.

Encapsulation of Food Ingredients Using Nanoliposome Technology

Nanoliposomes are microscopic vesicles composed of phospholipid bilayers entrapping one or more aqueous compartments. Their unique properties have triggered numerous applications in several scientific and technological fields. Nanoliposomes can provide controlled release of various bioactive agents, including food ingredients and nutraceuticals, at the right place and the right time. Therefore, they increase the effectiveness and cellular uptake of the encapsulated material. Reactive, sensitive, or volatile additives (vitamins, enzymes, antioxidants, slimming agents, etc.) can be turned into stable ingredients using nanoliposomes. This article reviews various aspects of nanoliposomes including currently available preparation methods, and their application in food technology.

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95±0.02°C/min), medium (73±3°C/min) and fast (265±12°C/min) cooling rates and storage in liquid nitrogen, followed by a 37°C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%±5% vs 29%±4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.