Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver.

Lipid-like nanoparticles (LNPs) have potential as non-viral delivery systems for mRNA therapies. However, repeated administrations of LNPs may lead to accumulation of delivery materials and associated toxicity. To address this challenge, we have developed biodegradable lipids which improve LNPs clearance and reduce toxicity. We modify the backbone structure of Dlin-MC3-DMA by introducing alkyne and ester groups into the lipid tails. We evaluate the performance of these lipids when co-formulated with other amine containing lipid-like materials. We demonstrate that these formulations synergistically facilitate robust mRNA delivery with improved tolerability after single and repeated administrations. We further identify albumin-associated macropinocytosis and endocytosis as an ApoE-independent LNP cellular uptake pathway in the liver. Separately, the inclusion of alkyne lipids significantly increases membrane fusion to enhance mRNA release, leading to synergistic improvement of mRNA delivery. We believe that the rational design of LNPs with multiple amine-lipids increases the material space for mRNA delivery.

Magnetic Retrieval of Encapsulated Beta Cell Transplants from Diabetic Mice Using Dual-Function MRI Visible and Retrievable Microcapsules.

Encapsulated beta cell transplantation offers a potential cure for a subset of diabetic patients. Once transplanted, beta cell grafts can help to restore glycemic control; however, locating and retrieving cells in the event of graft failure may pose a surgical challenge. Here, a dual-function nanoparticle-loaded hydrogel microcapsule is developed that enables graft retrieval under an applied magnetic field. Additionally, this system facilitates graft localization via magnetic resonance imaging (MRI), and graft isolation from the immune system. Iron oxide nanoparticles encapsulated within alginate hydrogel capsules containing viable islets are transplanted and the in vitro and in vivo retrieval of capsules containing nanoparticles functionalized with various ligands are compared. Capsules containing islets co-encapsulated with COOH-coated nanoparticles restore normal glycemia in immunocompetent diabetic mice for at least 6 weeks, can be visualized using MRI, and are retrievable in a magnetic field. Application of a magnetic field for 90 s via a magnetically assisted retrieval device facilitates rapid retrieval of up to 94% (±3.1%) of the transplant volume 24 h after surgical implantation. This strategy aids monitoring of cell-capsule locations in vivo, facilitates graft removal at the end of the transplant lifetime, and may be applicable to many encapsulated cell transplant systems.

Glucose-Responsive Nanoparticles for Rapid and Extended Self-Regulated Insulin Delivery.

To mimic native insulin activity, materials have been developed that encapsulate insulin, glucose oxidase, and catalase for glucose-responsive insulin delivery. A major challenge, however, has been achieving the desired kinetics of both rapid and extended release. Here, we tune insulin release profiles from polymeric nanoparticles by altering the degree of modification of acid-degradable, acetalated-dextran polymers. Nanoparticles synthesized from dextran with a high acyclic acetal content (94% of residues) show rapid release kinetics, while nanoparticles from dextran with a high cyclic acetal content (71% of residues) release insulin more slowly. Thus, coformulation of these two materials affords both rapid and extended glucose-responsive insulin delivery. In vivo analyses using both streptozotocin-induced type 1 diabetic and healthy mouse models indicate that this delivery system has the ability to respond to glucose on a therapeutically relevant time scale. Importantly, the concentration of human insulin in mouse serum is enhanced more than 3-fold with elevated glucose levels, providing direct evidence of glucose-responsiveness in animals. We further show that a single subcutaneous injection provides 16 h of glycemic control in diabetic mice. We believe the nanoparticle formulations developed here may provide a generalized strategy for the development of glucose-responsive insulin delivery systems.

Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation.

Therapeutic messenger RNA vaccines enable delivery of whole antigens, which can be advantageous over peptide vaccines. However, optimal efficacy requires both intracellular delivery, to allow antigen translation, and appropriate immune activation. Here, we developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation. Using a three-dimensional multi-component reaction system, we synthesized and evaluated the vaccine potential of over 1,000 lipid formulations. The top candidate formulations induced a robust immune response, and were able to inhibit tumor growth and prolong survival in melanoma and human papillomavirus E7 in vivo tumor models. The top-performing lipids share a common structure: an unsaturated lipid tail, a dihydroimidazole linker and cyclic amine head groups. These formulations induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway, rather than through Toll-like receptors, and result in limited systemic cytokine expression and enhanced anti-tumor efficacy.

mRNA Delivery for Therapeutic Anti-HER2 Antibody Expression In Vivo.

Antibody-based drugs are a leading class of biologics used to treat a variety of diseases, including cancer. However, wide antibody implementation is hindered by manufacturing challenges and high production cost. Use of in-vitro-transcribed mRNA (IVT-mRNA) for endogenous protein expression has the potential to circumvent many of the shortcomings of antibody production and therapeutic application. Here, we describe the development of an IVT-mRNA system for in vivo delivery of a humanized anti-HER2 (also known as ERBB2) antibody, trastuzumab, and demonstrate its anticancer activity. We engineered the IVT-mRNA sequence to maximize expression, then formulated the IVT-mRNA into lipid-based nanoparticles (LNPs) to protect the mRNA from degradation and enable efficient in vivo delivery. Systemic delivery of the optimized IVT-mRNA loaded into LNPs resulted in antibody serum concentrations of 45 ± 8.6 µg/mL for 14 days after LNP injection. Further studies demonstrated an improved pharmacokinetic profile of the produced protein compared to injection of trastuzumab protein. Finally, treatment of tumor-bearing mice with trastuzumab IVT-mRNA LNPs selectively reduced the volume of HER2-positive tumors and improved animal survival. Taken together, the results of our study demonstrate that using IVT-mRNA LNPs to express full-size therapeutic antibodies in the liver can provide an effective strategy for cancer treatment and offers an alternative to protein administration.

Drug delivery across length scales.

Over the last century, there has been a dramatic change in the nature of therapeutic, biologically active molecules available to treat disease. Therapies have evolved from extracted natural products towards rationally designed biomolecules, including small molecules, engineered proteins and nucleic acids. The use of potent drugs which target specific organs, cells or biochemical pathways, necessitates new tools which can enable controlled delivery and dosing of these therapeutics to their biological targets. Here, we review the miniaturisation of drug delivery systems from the macro to nano-scale, focussing on controlled dosing and controlled targeting as two key parameters in drug delivery device design. We describe how the miniaturisation of these devices enables the move from repeated, systemic dosing, to on-demand, targeted delivery of therapeutic drugs and highlight areas of focus for the future.

Improved delivery of PLGA microparticles and microparticle-cell scaffolds in clinical needle gauges using modified viscosity formulations.

Polymer microparticles are widely used as acellular drug delivery platforms in regenerative medicine, and have emerging potential as cellular scaffolds for therapeutic cell delivery. In the clinic, PLGA microparticles are typically administered intramuscularly or subcutaneously, with the clinician and clinical application site determining the precise needle gauge used for delivery. Here, we explored the role of needle diameter in microparticle delivery yield, and develop a modified viscosity formulation to improve microparticle delivery across a range of clinically relevant needle diameters. We have identified an optimal biocompatible formulation containing 0.25% pluronic F127 and 0.25% carboxymethyl cellulose, which can increase delivery payload to 520% across needle gauges 21-30G, and note that needle diameter impacts delivery efficacy. We use this formulation to increase the delivery yield of PLGA microparticles, and separately, PLGA-cell scaffolds supporting viable mesenchymal stem cells (MSCs), demonstrating the first in vitro delivery of this cell scaffold system. Together, these results highlight an optimal formulation for the delivery of microparticle and microparticle-cell scaffolds, and illustrate how careful choice of delivery formulation and needle size can dramatically impact delivery payload.

T cell immunoengineering with advanced biomaterials.

Recent advances in biomaterials design offer the potential to actively control immune cell activation and behaviour. Many human diseases, such as infections, cancer, and autoimmune disorders, are partly mediated by inappropriate or insufficient activation of the immune system. T cells play a central role in the host immune response to these diseases, and so constitute a promising cell type for manipulation. In vivo, T cells are stimulated by antigen presenting cells (APC), therefore to design immunoengineering biomaterials that control T cell behaviour, artificial interfaces that mimic the natural APC-T cell interaction are required. This review draws together research in the design and fabrication of such biomaterial interfaces, and highlights efforts to elucidate key parameters in T cell activation, such as substrate mechanical properties and spatial organization of receptors, illustrating how they can be manipulated by bioengineering approaches to alter T cell function.

Mechanisms of Polymer-Templated Nanoparticle Synthesis: Contrasting ZnS and Au.

We combine solution small-angle X-ray scattering (SAXS) and high-resolution analytical transmission electron microscopy (ATEM) to gain a full mechanistic understanding of substructure formation in nanoparticles templated by block copolymer reverse micelles, specifically poly(styrene)-block-poly(2-vinylpyridine). We report a novel substructure for micelle-templated ZnS nanoparticles, in which small crystallites (∼4 nm) exist within a larger (∼20 nm) amorphous organic-inorganic hybrid matrix. The formation of this complex structure is explained via SAXS measurements that characterize in situ for the first time the intermediate state of the metal-loaded micelle core: Zn(2+) ions are distributed throughout the micelle core, which solidifies as a unit on sulfidation. The nanoparticle size is thus determined by the radius of the metal-loaded core, rather than the quantity of available metal ions. This mechanism leads to particle size counterintuitively decreasing with increasing metal content, based on the modified interactions of the metal-complexed monomers in direct contrast to gold nanoparticles templated by the same polymer.

The early career researcher's toolkit: translating tissue engineering, regenerative medicine and cell therapy products.

Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization.

Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.