Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

An Update on Self-Amplifying mRNA Vaccine Development.

This review will explore the four major pillars required for design and development of an saRNA vaccine: Antigen design, vector design, non-viral delivery systems, and manufacturing (both saRNA and lipid nanoparticles (LNP)). We report on the major innovations, preclinical and clinical data reported in the last five years and will discuss future prospects.

Robust Microfluidic Technology and New Lipid Composition for Fabrication of Curcumin-Loaded Liposomes: Effect on the Anticancer Activity and Safety of Cisplatin.

Curcumin exhibits potent anticancer activity via various mechanisms, but its in vivo efficacy has been hampered by poor solubility. Nanotechnology has been employed to deliver curcumin, but most of the reported systems suffered from low drug loading capacity and poor stability. Here, we report the development and optimization of a liposomal formulation for curcumin (Lipo-Cur) using an automated microfluidic technology. Lipo-Cur exhibited a mean diameter of 120 nm with a low polydispersity index (<0.2) and superior loading capacity (17 wt %) compared to other reported liposomal systems. Lipo-Cur increased the water solubility of curcumin by 700-fold, leading to 8-20-fold increased systemic exposure compared to the standard curcumin suspension formulation. When coadministered with cisplatin to tumor-bearing mice, Lipo-Cur augmented the antitumor efficacy of cisplatin in multiple mouse tumor models and decreased the nephrotoxicity. This is the first report demonstrating the dual effects of curcumin enabled by a nanoformulation in enhancing the efficacy and reducing the toxicity of a chemo-drug in animal models under a single and low dose administration.

Dual-Mode Dark Field and Surface-Enhanced Raman Scattering Liposomes for Lymphoma and Leukemia Cell Imaging.

Multifunctional probes are needed to characterize individual cells simultaneously by different techniques to provide complementary information. A preparative method and an in vitro demonstration of function are presented for a dual-function dark field microscopy/surface-enhanced Raman scattering (SERS) liposome probe for cancer. Liposomes composed of zwitterionic lipids are valuable both to limit biofouling and to serve as a modular matrix to incorporate a variety of functional molecules and hence are used here as vehicles for SERS-active materials. Dark field microscopy and SERS represent new combined functionalities for targeted liposomal probes. Two methods of antibody conjugation to SERS liposomes are demonstrated: (i) direct conjugation to functional groups on the SERS liposome surface and (ii) postinsertion of lipid-functionalized antibody fragments (Fabs) into preformed SERS liposomes. In vitro experiments targeting both lymphoma cell line LY10 and primary human chronic lymphocytic leukemia (CLL) cells demonstrate the usefulness of these probes as optical contrast agents in both dark field and Raman microscopy.

Microfluidic Production and Application of Lipid Nanoparticles for Nucleic Acid Transfection.

Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic acids while maintaining the particle characteristics that have made LNPs successful in later stages of genetic medicine development. Additionally, this method and the resulting LNPs are seamlessly scalable to quantities appropriate for in vivo models and development of nucleic acid therapeutics.The present work describes the methodology for preparing LNPs loaded with siRNA, mRNA or plasmids using a commercially available microfluidic instrument and an accompanying transfection kit. Guidelines for application to cultured cells in a well-plate format are also provided.

Microfluidic Assembly To Synthesize Dual Enzyme/Oxidation-Responsive Polyester-Based Nanoparticulates with Controlled Sizes for Drug Delivery.

Controlling the size and narrow size distribution of polymer-based nanocarriers for targeted drug delivery is an important parameter that significantly influences their colloidal stability, biodistribution, and targeting ability. Herein, we report a high-throughput microfluidic process to fabricate colloidally stable aqueous nanoparticulate colloids with tunable sizes at 50-150 nm and narrow size distribution. The nanoparticulates are designed with different molecular weight polyesters having both ester bonds (responsive to esterase) and sulfide linkages (to oxidative reaction) on the backbones, thus exhibiting dual esterase/oxidation responses, causing the destabilization of the nanoparticulates to lead to the controlled release of encapsulated therapeutics. The systematic investigation on both microfluidic and formulation parameters enables to control their properties as allowing for decreasing nanoparticulate sizes as well as improving colloidal stability and cytotoxicity. Further to such control over smaller size and narrow size distribution, dual stimuli-responsive degradation and excellent cellular uptake could suggest that the microfluidic nanoparticulates stabilized with polymeric stabilizers could offer the versatility toward dual smart drug delivery exhibiting enhanced release kinetics.

Microfluidics: a transformational tool for nanomedicine development and production.

Microfluidic devices are mircoscale fluidic circuits used to manipulate liquids at the nanoliter scale. The ability to control the mixing of fluids and the continuous nature of the process make it apt for solvent/antisolvent precipitation of drug-delivery nanoparticles. This review describes the use of numerous microfluidic designs for the formulation and production of lipid nanoparticles, liposomes and polymer nanoparticles to encapsulate and deliver small molecule or genetic payloads. The advantages of microfluidics are illustrated through examples from literature comparing conventional processes such as beaker and T-tube mixing to microfluidic approaches. Particular emphasis is placed on examples of microfluidic nanoparticle formulations that have been tested in vitro and in vivo. Fine control of process parameters afforded by microfluidics, allows unprecedented optimization of nanoparticle quality and encapsulation efficiency. Automation improves the reproducibility and optimization of formulations. Furthermore, the continuous nature of the microfluidic process is inherently scalable, allowing optimization at low volumes, which is advantageous with scarce or costly materials, as well as scale-up through process parallelization. Given these advantages, microfluidics is poised to become the new paradigm for nanomedicine formulation and production.

Rational design for the controlled aggregation of gold nanorods via phospholipid encapsulation for enhanced Raman scattering.

This study describes a procedure that found a balance between the ability of polymer-stabilized nanorods (NRs) to self-assemble and the creation of narrow gaps to make reproducibly bright surface-enhanced Raman scattering (SERS) nanorod dimers. NRs were end-functionalized with polymers, which enabled end-to-end self-assembly of NR chains and control over inter-rod separation through polymer molecular weight (MW). We found a way to quench the self-assembly, by phospholipid encapsulation, reducing the polydispersity of the aggregates while rendering them water-soluble. This reduction in polydispersity and preferential isolation of short-chain nanorod species is important for maximizing SERS enhancement from nanorod chains. We prepared NR aggregates that exhibit ∼5-50 times greater SERS intensity than isolated rods (and ∼750× greater than bare dye) depending on inter-rod separation, when using Oxazine 725 reporter molecules. Colloidal stability of NR aggregates and temporal stability of the SERS signal in water were observed for 110 days. With enhanced SERS intensity, water solubility, and stability, these NR aggregates are promising optical probes for future biological applications.

Surface-enhanced Raman scattering dye-labeled Au nanoparticles for triplexed detection of leukemia and lymphoma cells and SERS flow cytometry.

The labeling of cell surface receptors by fluorescent markers is an established method for the identification of cell phenotype in both research and clinical settings. Fluorescence dye labeling has inherent constraints, most notably the upper limit of labels per cell that may be probed using a single excitation source, in addition to a physical limit to the number of broad emission spectra that can be distinctly collected within the visible wavelength region. SERS labeling has the potential to mitigate these shortfalls. Herein, antibody-targeted, PEG-coated surface-enhanced Raman scattering (SERS) Au nanoparticles are used simultaneously to label three cell surface markers of interest on malignant B cells from the LY10 lymphoma cell line. The SERS probes were characterized by multiple methods to confirm their monodispersity and functionalization with both PEG and monoclonal antibodies. The specificity of the particles' cell labeling was demonstrated on both primary chronic lymphocytic leukemia and LY10 cells using SERS from cell suspensions and confocal Raman mapping, respectively. Fluorescence flow cytometry was employed to confirm the binding of SERS probes to LY10 over large cell populations, and the particles' SERS was collected directly from labeled cells using a commercial flow cytometer. To the best of our knowledge, this is the first demonstration of SERS flow cytometry from cells tagged with targeted SERS probes.

Phospholipid membrane encapsulation of nanoparticles for surface-enhanced Raman scattering.

Lipid-encapsulated surface-enhanced Raman scattering (SERS) nanoparticles, with promising applications in biomedical diagnostics, were produced. Gold nanoparticles, 60 nm in diameter, were coated with a ternary mixture of DOPC, sphingomyelin, and cholesterol. The lipid layer is versatile for engineering the chemical and optical properties of the particles. The stability of the lipid-encapsulated particles is demonstrated over a period of weeks. The versatility of the layer is demonstrated by the incorporation of three different Raman-active species using three different strategies. The lipid layer was directly observed by TEM, and the SERS spectrum of the three dye species was confirmed by Raman spectroscopy. UV-vis absorption and dynamic light scattering provide additional evidence of lipid encapsulation. The encapsulation is achieved in aqueous solution, avoiding phase transfer and possible contamination from organic solvents. Furthermore, when fluorescent dye-labeled lipids were employed in the encapsulant, the fluorescence and SERS activity of the particles were controlled by the use of dissolved ions in the preparation solution.

Phase segregation of untethered zwitterionic model lipid bilayers observed on mercaptoundecanoic-acid-modified gold by AFM imaging and force mapping.

Planar supported lipid bilayers (SLBs) are often studied as model cell membranes because they are accessible to a variety of surface-analytic techniques. Specifically, recent studies of lipid phase coexistence in model systems suggest that membrane lateral organization is important to a range of cellular functions and diseases. We report the formation of phase-segregated dioleoylphosphatidylcholine (DOPC)/sphingomyelin/cholesterol bilayers on mercaptoundecanoic-acid-modified (111) gold by spontaneous fusion of unilamellar vesicles, without the use of charged or chemically modified headgroups. The liquid-ordered (l(o)) and liquid-disordered (l(d)) domains are observed by atomic force microscopy (AFM) height and phase imaging. Furthermore, the mechanical properties of the bilayer were characterized by force-indentation maps. Fits of force indentation to Sneddon mechanics yields average apparent Young's moduli of the l(o) and l(d) phases of 100 +/- 2 and 59.8 +/- 0.9 MPa, respectively. The results were compared to the same lipid membrane system formed on mica with good agreement, though modulus values on mica appeared higher. Semiquantitative comparisons suggest that the mechanical properties of the l(o) phase are dominated by intermolecular van der Waals forces, while those of the fluid l(d) phase, with relatively weak van der Waals forces, are influenced appreciably by differences in surface charge density between the two substrates, which manifests as a difference in apparent Poisson ratios.

Field localization in very small aperture lasers studied by apertureless near-field microscopy.

Localized surface plasmon polaritons (SSPs) have been observed on very small aperture lasers using apertureless near-field microscopy. Fields around multiple apertures are shown to result from interferences of SPP point sources at each aperture and optical fields. The near-field optical pattern around a single aperture indicates the interference of SPPs with their scattered counterparts. Near-field measurements also confirmed a preferred orientation of the rectangular aperture waveguide for the signal localization in very small aperture lasers.