Claudia Zylberberg

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.

Bio-inspired cryo-ink preserves red blood cell phenotype and function during nanoliter vitrification.

Current red-blood-cell cryopreservation methods utilize bulk volumes, causing cryo-injury of cells, which results in irreversible disruption of cell morphology, mechanics, and function. An innovative approach to preserve human red-blood-cell morphology, mechanics, and function following vitrification in nanoliter volumes is developed using a novel cryo-ink integrated with a bioprinting approach.

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.

Current perspectives on the use of ancillary materials for the manufacture of cellular therapies

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly, there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore, AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such, AMs must be carefully selected and appropriately qualified during the cell therapy development process. However, the ongoing evolution of cell therapy research, limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition, compliance, and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry, with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety, users must work with their suppliers and regulators to qualify each AM to assess source, purity, identity, safety, and suitability in a given application.

Critical elements in the development of cell therapy potency assays for ischemic conditions

A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs.

Twenty years of the International Society for Cellular Therapies: the past, present and future of cellular therapy clinical development

Historical perspective Some 20 years ago, as the International Society for Cellular Therapies (ISCT) was being founded (1992), it was impossible to imagine all of the scientifi ca nd clinical advances about to occur in the subsequent two decades. As evidenced by the annual publication rate and manuscript focus, the field of stem cell biology and therapeutics has mushroomed and diversified over the last two decades: 1992 saw 3280 “stem cell” papers published (NCBI database). Given the pioneering work of E. Donall Thomas four decades earlier that led to Thomas and Joseph Murray receiving the Nobel Prize for Medicine in 1990, it is not surprising that the majority (51%) of these papers focused on hematopoietic cells (HSC) and HSC transplantation; 19% of the papers were directed to embryonic stem cell (ESC) biology, with only w1% each on mesenchymal biology or induced pluripotency biology. By 2012 (the last full year of record),

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.

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.