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.

Inhalable dry powders of a monoclonal antibody against SARS-CoV-2 virus made by thin-film freeze-drying.

Monoclonal antibodies (mAbs) represent a promising modality for the prevention and treatment of viral infections. For infections that initiate from the respiratory tract, direct administration of specific neutralizing mAbs into lungs has advantages over systemic injection of the same mAbs. Herein, using AUG-3387, a human-derived mAb with high affinity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its various variants, as a model mAb, we formulated the mAb into dry powders by thin-film freeze-drying, confirmed that the AUG-3387 mAb reconstituted from the dry powders retained their integrity, high affinity to the SARS-CoV-2 spike protein receptor binding domain (RBD), as well as ability to neutralize RBD-expressing pseudoviruses.

Targeted treatment for biofilm-based infections using PEGylated tobramycin.

Chronic infections often involve biofilm-based bacteria, in which the biofilm results in significant resistance against antimicrobial agents and prevents eradication of the infection. The physicochemical barrier presented by the biofilm matrix is a major impediment to the delivery of many antibiotics. Previously, PEGylation has been shown to improve antibiotic penetration into biofilms in vitro.

Mannosylated polyethylenimine-cholesterol-based nanoparticles for targeted delivery of minicircle DNA vaccine against COVID-19 to antigen-presenting cells.

DNA vaccines can be a potential solution to protect global health, triggering both humoral and cellular immune responses. DNA vaccines are valuable in preventing intracellular pathogen infections, and therefore can be explored against coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2). This work explored different systems based on polyethylenimine (PEI), functionalized for the first time with both cholesterol (CHOL) and mannose (MAN) to deliver parental plasmid (PP) and minicircle DNA (mcDNA) vectors encoding the receptor-binding domain (RBD) of SARS-CoV-2 to antigen-presenting cells (APCs).

Intranasal delivery of thin-film freeze-dried monoclonal antibodies using a powder nasal spray system.

Monoclonal antibodies (mAbs) administered intranasally as dry powders can be potentially applied for the treatment or pre-exposure prevention of viral infections in the upper respiratory tract. However, a method to transform the mAbs from liquid to dry powders suitable for intranasal administration and a device that can spray the dry powders to the desired region of the nasal cavity are needed to fully realize the potentials of the mAbs. Herein, we report that thin-film freeze-dried mAb powders can be sprayed into the posterior nasal cavity using Aptar Pharma's Unidose (UDS) Powder Nasal Spray System.

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.

Connexin-Containing Vesicles for Drug Delivery.

Connexin is a transmembrane protein present on the cell membrane of most cell types. Connexins assemble into a hexameric hemichannel known as connexon that pairs with another hemichannel present on a neighboring cell to form gap junction that acts as a channel or pore for the transport of ions and small molecules between the cytoplasm of the two cells. Extracellular vesicles released from connexin-expressing cells could carry connexin hemichannels on their surface and couple with another connexin hemichannel on a distant recipient cell to allow the transfer of the intravesicular content directly into the cytoplasm.

Inhalable dry powders of microRNA-laden extracellular vesicles prepared by thin-film freeze-drying.

Extracellular vesicles (EVs) are endogenous vesicles that comprise a variety of submicron vesicular structures. Among these, exosomes have been widely investigated as delivery systems for small and large molecules. Herein, the thin-film freeze-drying technology was utilized to engineer aerosolizable dry powders of miR-335-laden induced EVs (iEV-335) generated in B cells for potential delivery into the lung to treat primary lung cancer and/or pulmonary metastases.

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.

Viability of Lactobacillus acidophilus in Thin-Film Freeze-Dried Powders Filled in Delayed-Release Vegetarian Capsules in a Simulated Gastric Fluid.

Previously, we have shown that thin-film freeze-drying can be applied to prepare dry powders of bacteria such as Lactobacillus acidophilus. Herein, we tested the viability of L. acidophilus in thin-film freeze-dried powders (TFF powders) filled in delayed-release vegetarian capsules in a simulated gastric fluid (SGF) consisting of 0.

Effect of nanoparticle size on their distribution and retention in chronic inflammation sites.

Nanomedicines are increasingly researched and used for the treatment of chronic inflammatory diseases. Herein, the effect of the size of nanoparticles on their distribution and retention in chronic inflammatory sites, as compared to healthy tissues, was studied in a mouse model with chronic inflammation in one of the hind footpads. Using PEGylated gold nanoparticles of 2, 10, 100, and 200 nm, we found that although the smaller nanoparticles of 2 and 10 nm showed greater distribution and slower clearance in the inflamed footpad than the relatively larger nanoparticles of 100 and 200 nm, the larger nanoparticles of 100 and 200 nm were more selectively distributed in the inflamed hind footpad than in the healthy hind footpad in the same mouse.

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.

Bioprinting: A focus on improving bioink printability and cell performance based on different process parameters.

Three-dimensional (3D) bioprinting is an emerging biofabrication technique that shows great potential in the field of tissue engineering, regenerative medicine and advanced drug delivery. Despite the current advancement of bioprinting technology, it faces several obstacles such as the challenge of optimizing the printing resolution of 3D constructs while retaining cell viability before, during, and after bioprinting. Therefore, it is of great significance to fully understand factors that influence the shape fidelity of printed structures and the performance of cells encapsulated in bioinks.

Feasibility of intranasal delivery of thin-film freeze-dried, mucoadhesive vaccine powders.

Intranasal vaccination by directly applying a vaccine dry powder is appealing. However, a method that can be used to transform a vaccine from a liquid to a dry powder and a device that can be used to administer the powder to the desired region(s) of the nasal cavity are critical for successful intranasal vaccination. In the present study, using a model vaccine that contains liposomal monophosphoryl lipid A and QS-21 adjuvant (AdjLMQ) and ovalbumin (OVA) as a model antigen, it was shown that thin-film freeze-drying can be applied to convert the liquid vaccine containing sucrose at a sucrose to lipid ratio of 15:1 (w/w) into dry powders, in the presence or absence of carboxymethyl cellulose sodium salt (CMC) as a mucoadhesive agent.

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.

Targeted treatment of triple-negative-breast cancer through pH-triggered tumour associated macrophages using smart theranostic nanoformulations.

Triple-negative breast cancer (TNBC) represents 15-25 % of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting.

Accelerated water removal from frozen thin films containing bacteria.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids that contain temperature-sensitive materials. Herein, using Escherichia coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate from the frozen thin films generated by thin-film freezing (TFF) is significantly faster than from the bulk frozen solids in conventional shelf freeze-drying. This is likely because the loosely stacked frozen thin films provided a larger solid-air interface and the low thickness of the thin films provided a low mass transfer resistance.

Pharmaceutical dry powders of small molecules prepared by thin-film freezing and their applications - A focus on the physical and aerosol properties of the powders.

Thin-film freeze-drying is a dry powder engineering technology that involves in a rapid thin-film freezing (TFF) process followed by lyophilization to remove solvent. TFF process has been successfully used to produce pharmaceutical powders of the small and large molecule drugs as well as live organisms. This paper provides a comprehensive review of the pharmaceutical applications of TFF powders of small molecule drugs intended for pulmonary delivery or oral administration.

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.

Formulation of dry powders of vaccines containing MF59 or AddaVax by Thin-Film Freeze-Drying: Towards a dry powder universal flu vaccine.

MF59® is an oil-in-water (O/W) nanoemulsion-based vaccine adjuvant that is often used in seasonal and pandemic influenza vaccines. We explored the feasibility of developing dry powders of vaccines adjuvanted with MF59 or AddaVax™, a preclinical grade equivalent of MF59 with the same composition and droplet size as MF59, by thin-film freeze-drying (TFFD). Liquid AddaVax alone was successfully converted to a dry powder by TFFD using trehalose as a stabilizing agent while maintaining the droplet size distribution of AddaVax after it was reconstituted.