Reconsidering freeze-induced protein aggregation: Air bubbles as the root cause of ice-water interface stress.

Freeze-induced stress causing aggregation of proteins has typically been primarily attributed to the ice-water interface. However, we hypothesize that the underlying observed and perceived detrimental effect of ice is, to some extent, attributed to air bubbles expelled from ice crystal lattices or to nanobubbles existing prior to freezing. The reduction of dissolved air was achieved via a deaeration process by placing samples in a reduced pressure chamber, while the reduction of nanobubbles was achieved by filtering samples via a syringe filter.

Thin-film freeze-drying of an influenza virus hemagglutinin mRNA vaccine in unilamellar lipid nanoparticles with blebs.

Messenger RNA (mRNA) vaccines have revolutionized the fight against infectious diseases and are poised to transform other therapeutic areas. Lipid nanoparticles (LNP) represent the most successful delivery system for mRNA. While the mRNA-LNP products currently in clinics are stored as frozen suspensions, there is evidence that freeze-drying mRNA-LNP into dry powders can potentially enable their storage and handling at non-freezing temperatures.

Functional dry powders of connexin-containing extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising drug delivery system. Connectosomes are a specialized type of EVs that contain connexins in their membranes. Connexin is a surface transmembrane protein that forms connexin hemichannels.

Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.

Purpose: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs).

Methods: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure.

Effect of lipid composition on RNA-Lipid nanoparticle properties and their sensitivity to thin-film freezing and drying.

A library of 16 lipid nanoparticle (LNP) formulations with orthogonally varying lipid molar ratios was designed and synthesized, using polyadenylic acid [poly(A)] as a model for mRNA, to explore the effect of lipid composition in LNPs on (i) the initial size of the resultant LNPs and encapsulation efficiency of RNA and (ii) the sensitivity of the LNPs to various conditions including cold storage, freezing (slow vs. rapid) and thawing, and drying. Least Absolute Shrinkage and Selection Operator (LASSO) regression was employed to identify the optimal lipid molar ratios and interactions that favorably affect the physical properties of the LNPs and enhance their stability in various stress conditions.

Serological and Molecular Epidemiology of Chikungunya Virus Infection in Vietnam, 2017-2019.

Chikungunya fever is an acute febrile illness caused by the chikungunya virus (CHIKV), which is transmitted by mosquitoes. Since 1965, only a few studies with limited scope have been conducted on CHIKV in Vietnam. Thus, this study aimed to determine the seroprevalence and molecular epidemiology of CHIKV infection among febrile patients in Vietnam from 2017 to 2019.

Effect of the amount of cationic lipid used to complex siRNA on the cytotoxicity and proinflammatory activity of siRNA-solid lipid nanoparticles.

When preparing siRNA-encapsulated solid lipid nanoparticles (siRNA-SLNs), cationic lipids are commonly included to condense and lipophilize the siRNA and thus increase its encapsulation in the SLNs. Unfortunately, cationic lipids also contribute significantly to the cytotoxicity and proinflammatory activity of the SLNs. Previously, our group developed a TNF-α siRNA-SLN formulation that showed strong activity against rheumatoid arthritis unresponsive to methotrexate in a mouse model.

Aerosolizable Plasmid DNA Dry Powders Engineered by Thin-film Freezing.

Purpose: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery.

Methods: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e.

Entrapment of air microbubbles by ice crystals during freezing exacerbates freeze-induced denaturation of proteins.

Freezing techniques are an essential part of biologics manufacturing processes, yet the formation of ice/water interfaces can impart detrimental effects on proteins. However, the absence of chemical and structural differences between ice and liquid water poses the question as to why ice can destabilize proteins. We hypothesize that the destabilizing stress of the ice-liquid water interface does not originate from the ice-water system itself but rather from the air microbubbles present during the freezing process.

Aggregation of Lactoferrin Caused by Droplet Atomization Process via a Two-Fluid Nozzle: The Detrimental Effect of Air-Water Interfaces.

Biological macromolecules, especially therapeutic proteins, are delicate and highly sensitive to denaturation from stresses encountered during the manufacture of dosage forms. Thin-film freeze-drying (TFFD) and spray freeze-drying (SFD) are two processes used to convert liquid forms of protein into dry powders. In the production of inhalable dry powders that contain proteins, these potential stressors fall into three categories based on their occurrence during the primary steps of the process: (1) droplet formation (e.

Safety and immunogenicity of Nanocovax, a SARS-CoV-2 recombinant spike protein vaccine: Interim results of a double-blind, randomised controlled phase 1 and 2 trial.

Background: Nanocovax is a recombinant severe acute respiratory syndrome coronavirus 2 subunit vaccine composed of full-length prefusion stabilized recombinant SARS-CoV-2 spike glycoproteins (S-2P) and aluminium hydroxide adjuvant.

Methods: We conducted a dose-escalation, open label trial (phase 1) and a randomized, double-blind, placebo-controlled trial (phase 2) to evaluate the safety and immunogenicity of the Nanocovax vaccine (in 25 mcg, 50 mcg, and 75 mcg doses, aluminium hydroxide adjuvanted (0·5 mg/dose) in 2-dose regime, 28 days apart (ClinicalTrials.gov number, NCT04683484).

Insight into hydroxyl radical-mediated cleavage of caged methylene blue: the role of Fenton's catalyst for antimalarial hybrid drug activation.

Methylene blue with a 10-N carbamoyl linkage was reported to be a hydroxyl radical triggered cleavable ligand. Probed by this platform, hemoproteins were demonstrated to be a much more efficient Fenton's catalyst than commonly used inorganic Fe(ii) salts. The applicability of this ligand was demonstrated through the capability of being triggered by elevated reactive oxygen species levels at diseased tissue, with malaria-parasitized erythrocytes as an in vitro model.

Methylene blue as a far-red light-mediated photocleavable multifunctional ligand.

Methylene blue (MB) with a 10-N-carbamoyl linkage was discovered and developed as a multifunctional far-red (660 nm) photocleavable ligand capable of rendering a series of MB-conjugated compounds with off-to-on fluorescence switch properties through the controlled release of MB.

Hemopressin-Based pH-Sensitive Hydrogel: A Potential Bioactive Platform for Drug Delivery.

Peptides with proper sequences are capable of self-assembling into well-defined nanostructures, which can subsequently grow and entangle into three-dimensional nanomatrices. In this study, hemopressin, a cannabinoid receptor-modulating peptide derived from the α-chain of hemoglobin known to self-assemble into nanofibrils, was examined for its potential applicability as a gelator. The results indicated that hemopressin's gel formation was dependent on pH and salt concentration.