Sandro Matosevic

Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line.

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore.

Layer-by-layer cell membrane assembly

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion of both purified and in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Pharmaceutical liposomal drug delivery: a review of new delivery systems and a look at the regulatory landscape

Abstract Liposomes were the first nanoscale drug to be approved for clinical use in 1995. Since then, the technology has grown considerably, and pioneering recent work in liposome-based delivery systems has brought about remarkable developments with significant clinical implications. This includes long-circulating liposomes, stimuli-responsive liposomes, nebulized liposomes, elastic liposomes for topical, oral and transdermal delivery and covalent lipid-drug complexes for improved drug plasma membrane crossing and targeting to specific organelles. While the regulatory bodies’ opinion on liposomes is well-documented, current guidance that address new delivery systems are not. This review describes, in depth, the current state-of-the-art of these new liposomal delivery systems and provides a critical overview of the current regulatory landscape surrounding commercialization efforts of higher-level complexity systems, the expected requirements and the hurdles faced by companies seeking to bring novel liposome-based systems for clinical use to market.

Engineering liposomal nanoparticles for targeted gene therapy

Recent mechanistic studies have attempted to deepen our understanding of the process by which liposome-mediated delivery of genetic material occurs. Understanding the interactions between lipid nanoparticles and cells is still largely elusive. Liposome-mediated delivery of genetic material faces systemic obstacles alongside entry into the cell, endosomal escape, lysosomal degradation and nuclear uptake. Rational design approaches for targeted delivery have been developed to reduce off-target effects and enhance transfection. These strategies, which have included the modification of lipid nanoparticles with target-specific ligands to enhance intracellular uptake, have shown significant promise at the proof-of-concept stage. Control of physical and chemical specifications of liposome composition, which includes lipid-to-DNA charge, size, presence of ester bonds, chain length and nature of ligand complexation, is integral to the performance of targeted liposomes as genetic delivery agents. Clinical advances are expected to rely on such systems in the therapeutic application of liposome nanoparticle-based gene therapy. Here, we discuss the latest breakthroughs in the development of targeted liposome-based agents for the delivery of genetic material, paying particular attention to new ligand and cationic lipid design as well as recent in vivo advances.

Natural killer-92 cells maintain cytotoxic activity after long-term cryopreservation in novel DMSO-free media

Natural killer (NK) cells are a critical part of the innate immune system, and have emerged as attractive targets for immunotherapies for various malignancies. Alongside the need for expansion of NK cells to reach clinically useful numbers, a critical component in the availability of NK cells for allogeneic therapy is cryopreservation. While a continuously-growing cell line such as NK-92 can avoid issues associated with isolating, activating, expanding, and manufacturing large numbers of peripheral blood-derived NKs, cryopreservation of these cells has not made much progress. NK cells are highly sensitive to freezing and thawing, while the use of DMSO during cryopreservation raises serious safety concerns. In this work, we evaluated a number of cryoprotectants that do not contain DMSO for their capacity to cryopreserve NK-92 cells over long-term while retaining their cytotoxic activity and viability, with the aim of identifying potential replacements to DMSO for safe clinical use of these cells.

Matrix metalloproteinases as reagents for cell isolation.

Cell isolation methods for therapeutic purposes have seen little advancement over the years. The original methods of stem cell and islet isolation using bacterial collagenases were developed in the early 1980s and are still used today. Bacterial collagenases are subject to autodegradation, and isolates obtained with these enzymes may be contaminated with endotoxins, reducing cell viability and contributing to toxicity in downstream applications. Here we describe a novel method for isolation of mesenchymal stem cells from adipose tissue (ADSC) utilizing recombinantly produced matrix metalloproteases (MMPs). The ADSCs isolated by MMPs displayed essentially identical morphological and phenotypical characteristics to cells isolated by bacterially-derived collagenase I and Liberase™. Samples isolated with MMPs and Liberase™ had comparable levels of CD73, CD90, and CD105. The adipogenic and osteogenic potential of the ADSCs isolated by MMPs was retained as compared to cells isolated with Liberase™. However, ADSCs isolated by Liberase™ displayed 6% contamination with other cells as per negative markers revealed by PE staining, as opposed to<1% for all MMP-treated samples. MMP-based cell isolation may contribute to optimization of transplantation technology.

Regenerative Medicine in the State of Florida: Letter Outlining the Florida Organization for Regenerative Medicine

Advances in regenerative medicine are generating transformative solutions to many of today’s incurable medical conditions. These advances have resulted from interdisciplinary efforts across the biological sciences, medicine, and engineering, changes in the regulatory environment—including the passage of the 21st Century Cures Act—and ongoing efforts to drive industry standardization. Increasingly, these advances have been catalyzed by regional initiatives around the U.S. and the world aimed at expediting the discovery, translation, and commercialization of transformative treatments. The proliferation of new models like the Centre for Commercialization of Regenerative Medicine (CCRM) in Canada, the California Institute for Regenerative Medicine (CIRM), and the Advanced Regenerative Manufacturing Institute (ARMI) in New Hampshire, among others, has inspired the creation of the Florida Organization for Regenerative Medicine (FORM), a nonprofit organization with a mission to facilitate translational research, commercialization, education, and therapeutic validation in the area of regenerative medicine with the ultimate aim of improving patient outcomes, creating high-quality jobs, and accelerating innovation through collaboration in the State of Florida and beyond. FORM is comprised ofmultiple strong and complementary clinical and research groups within Florida, including (from North to South) theMayo Clinic’s Center for RegenerativeMedicine; the University of Florida’s Center for Regenerative Medicine, which operates across the university and is based in the College of Medicine (CoM), and Institute for Cell and Tissue Science and Engineering (ICTSE) in the College of Engineering (CoE); Nova Southeastern University’s Cell Therapy Institute (CTI); Florida Atlantic University’s Center for Molecular Biology and Biotechnology (CMBB); and the University of Miami’s Diabetes Research Institute (DRI), Cell Transplant Center (CTC), and Interdisciplinary Stem Cell Institute (ISCI). Furthermore, FORM aims to convene nonprofit organizations and for-profit companies integral to the state’s regenerative medicine industry, including 42Bio, LLC, Akron Biotechnology, LLC, AxoGen, Corp, BioFlorida, Inc., Brammer Bio, LLC, CytoSen Therapeutics, Inc., Iovance Biotherapeutics, Inc., Lifelink Foundation, Inc., Leidos Health (subsidiary of Leidos, Inc.), Longeveron, LLC, RTI Surgical, Inc., and Vestion, Inc., among others. FORMaims to leverage state, federal, and philanthropic funding to foster strong and integrative collaboration among academic, government and private sectors in order to accelerate activities necessary to establish the State of Florida as one of the hubs of this nascent and important industry. These activities include supporting translational research, facilitating the commercialization of therapeutics and engineered tissues, creating and driving workforce development initiatives, and validating novel treatments to support the safe and efficacious uptake of regenerativemedicine. The State of Florida is home to various institutions and companies active in the regenerative medicine space. Given the strength of its research and clinical institutions, the dynamism of its industrial base, and its size and demographics, the state can and should be a key driver in the emergence of this new paradigm in human health. FORM endeavors to convene the state’s leading institutions and companies to drive economic growth, accelerate innovation, and improve patient outcomes through the development and commercialization of regenerative therapies.

Adenosinergic signaling as a target for natural killer cell immunotherapy

Purinergic signaling through adenosine plays a key role in immune regulation. Hypoxia-driven accumulation of extracellular adenosine results in the generation of an immunosuppressive niche that fuels tumor development. Such immunometabolic modulation has shown to be a promising therapeutic target through blockade of adenosine receptors which mediate adenosine’s immunosuppressive function, or cancer-associated ectonucleotidases CD39 and CD73 that catalyze the synthesis of adenosine. Adenosinergic signaling heavily implicates natural killer cells through both direct and indirect effects on their cytolytic activity, expression of cytotoxic granules, interferon-γ, and activating receptors. Continuing work has uncovered multiple checkpoints linked to adenosine within the purinergic signaling cascade as contributing to immune evasion from NK cell effector function. Here, we discuss these checkpoints and the recent body of work that focuses on adenosinergic signaling as a target for natural killer cell of cancer.

Viral and Nonviral Engineering of Natural Killer Cells as Emerging Adoptive Cancer Immunotherapies

Natural killer (NK) cells are powerful immune effectors whose antitumor activity is regulated through a sophisticated network of activating and inhibitory receptors. As effectors of cancer immunotherapy, NK cells are attractive as they do not attack healthy self-tissues nor do they induce T cell-driven inflammatory cytokine storm, enabling their use as allogeneic adoptive cellular therapies. Clinical responses to adoptive NK-based immunotherapy have been thwarted, however, by the profound immunosuppression induced by the tumor microenvironment, particularly severe in the context of solid tumors. In addition, the short postinfusion persistence of NK cells in vivo has limited their clinical efficacy. Enhancing the antitumor immunity of NK cells through genetic engineering has been fueled by the promise that impaired cytotoxic functionality can be restored or augmented with the use of synthetic genetic approaches. Alongside expressing chimeric antigen receptors to overcome immune escape by cancer cells, enhance their recognition, and mediate their killing, NK cells have been genetically modified to enhance their persistence in vivo by the expression of cytokines such as IL-15, avoid functional and metabolic tumor microenvironment suppression, or improve their homing ability, enabling enhanced targeting of solid tumors. However, NK cells are notoriously adverse to endogenous gene uptake, resulting in low gene uptake and transgene expression with many vector systems. Though viral vectors have achieved the highest gene transfer efficiencies with NK cells, nonviral vectors and gene transfer approaches—electroporation, lipofection, nanoparticles, and trogocytosis—are emerging. And while the use of NK cell lines has achieved improved gene transfer efficiencies particularly with viral vectors, challenges with primary NK cells remain. Here, we discuss the genetic engineering of NK cells as they relate to NK immunobiology within the context of cancer immunotherapy, highlighting the most recent breakthroughs in viral vectors and nonviral approaches aimed at genetic reprogramming of NK cells for improved adoptive immunotherapy of cancer, and, finally, address their clinical status.

Bioengineered liposome–scaffold composites as therapeutic delivery systems

The therapeutic potential of liposomes can be amplified when combined with biomaterial scaffolds. Such configurations overcome the convergent demands of therapies by enabling enhanced delivery, environmental responsiveness and potency. Liposomes benefit from the increased physical and mechanical strength, favorable rheological properties and natural environment conducive to improved tissue formation that scaffolds provide, while enabling biocompatible delivery of hydrophilic and lipophilic compounds that can be further functionalized to achieve targeted delivery. Topical, ocular, oral, nasal and vaginal applications have been explored using various polymer- or nanofiber-based scaffolds. Mechanistic and rheological findings on complexation between biomaterials, liposomes and cargo have led to multimodal systems with tremendous clinical potential. A review of the key developments in bioengineered liposome-scaffold composites is presented in this manuscript.