mRNA-lipid nanoparticle COVID-19 vaccines : structure and stability

A drawback of the current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at (ultra)low temperatures. Understanding the root cause of the instability of these vaccines may help to rationally improve mRNA-LNP product stability and thereby ease the temperature conditions for storage. In this review we discuss proposed structures of mRNA-LNPs, factors that impact mRNA-LNP stability and strategies to optimize mRNA-LNP product stability. Analysis of mRNA-LNP structures reveals that mRNA, the ionizable cationic lipid and water are present in the LNP core. The neutral helper lipids are mainly positioned in the outer, encapsulating, wall. mRNA hydrolysis is the determining factor for mRNA-LNP instability. It is currently unclear how water in the LNP core interacts with the mRNA and to what extent the degradation prone sites of mRNA are protected through a coat of ionizable cationic lipids. To improve the stability of mRNA-LNP vaccines, optimization of the mRNA nucleotide composition should be prioritized. Secondly, a better understanding of the milieu the mRNA is exposed to in the core of LNPs may help to rationalize adjustments to the LNP structure to preserve mRNA integrity. Moreover, drying techniques, such as lyophilization, are promising options still to be explored

Dynamic fluorescence microscopy of cellular uptake of intercalating model drugs by ultrasound-activated microbubbles

The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. SYTOX Green, an intercalating fluorescent dye that displays > 500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 x 10(-3) to 2.2 x 10(-3) s(-1) for 1 and 20 mu M, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line

Ultrasound and microbubbles for the treatment of ocular diseases : From preclinical research towards clinical application

The unique anatomy of the eye and the presence of various biological barriers make efficacious ocular drug delivery challenging, particularly in the treatment of posterior eye diseases. This review focuses on the combination of ultrasound and microbubbles (USMB) as a minimally invasive method to improve the efficacy and targeting of ocular drug delivery. An extensive overview is given of the in vitro and in vivo studies investigating the mechanical effects of ultrasound-driven microbubbles aiming to: (i) temporarily disrupt the blood–retina barrier in order to enhance the delivery of systemically administered drugs into the eye, (ii) induce intracellular uptake of anticancer drugs and macromolecules and (iii) achieve targeted delivery of genes, for the treatment of ocular malignancies and degenerative diseases. Finally, the safety and tolerability aspects of USMB, essential for the translation of USMB to the clinic, are discussed.Peer reviewe

HIGHLY EFFECTIVE LUNG DELIVERY OF A FULLY HUMAN MONOCLONAL ANTIBODY TARGETING PSEUDOMONAS AERUGINOSA FOLLOWING INTRA-NASAL ADMINISTRATION

A time-course study was performed to assess lung concentrations and bioactivities of a fully human monoclonal antibody (mAb) targeting Pseudomonas aeruginosa (PA) flagella type b (LST-007) following intra-nasal administration. Intra-nasal administration of LST-007 (5 mg/kg) resulted in high mAb concentrations (11 µg/ml) within the bronchoalveolar lavage (BAL) fluid at 15 min post-administration, which decreased to 3 µg/ml at 4 hr. In marked contrast, LST-007 concentrations in blood were 110 and 11 fold lower at the same time points. Scrutinization of BAL fluid demonstrated the presence of both intact and immunoreactive LST-007 towards PAO1 bacteria, which impeded bacterial motility, underscoring the maintenance of biological activity. These compelling data lay credence that intra-nasal and thus potential inhalation modes of administration might represent bona fide routes for a targeted mAb delivery to the lung environment and establishment of therapeutically effective concentrations. Such delivery approaches could help combat life-threatening, pneumonia infections caused by PA. KEY WORDS:Monoclonal antibody, intra-nasal, bronchoalveolar lavage; Pseudomonas aeruginos

Lmx1a-Dependent Activation of miR-204/211 Controls the Timing of Nurr1-Mediated Dopaminergic Differentiation

The development of midbrain dopaminergic (DA) neurons requires a fine temporal and spatial regulation of a very specific gene expression program. Here, we report that during mouse brain development, the microRNA (miR-) 204/211 is present at a high level in a subset of DA precursors expressing the transcription factor Lmx1a, an early determinant for DA-commitment, but not in more mature neurons expressing Th or Pitx3. By combining different in vitro model systems of DA differentiation, we show that the levels of Lmx1a influence the expression of miR-204/211. Using published transcriptomic data, we found a significant enrichment of miR-204/211 target genes in midbrain dopaminergic neurons where Lmx1a was selectively deleted at embryonic stages. We further demonstrated that miR-204/211 controls the timing of the DA differentiation by directly downregulating the expression of Nurr1, a late DA differentiation master gene. Thus, our data indicate the Lmx1a-miR-204/211-Nurr1 axis as a key component in the cascade of events that ultimately lead to mature midbrain dopaminergic neurons differentiation and point to miR-204/211 as the molecular switch regulating the timing of Nurr1 expression

The effect of sodium chloride on ethanolic fractionation of dilute gelatin solutions

Gelatin is the denaturation product of the protein collagen, the main constituent of animal skin, bone, and connective tissue. The conversion of collagen to gelatin results in a heterogeneous product with a broad molecular weight profile (MWP), which is important in determining the behaviour of the protein in solution. Addition of successive increments of a non-solvent, such as ethanol, to gelatin solutions causes the Florey- Huggins solvent-protein interaction parameter, χ, of the system to successively exceed the critical value for the different molecular weight fractions, causing progressive desolvation of the polymer. When sufficient solvent molecules are removed, the gelatin molecules begin to aggregate, resulting in phase separation, and forming a coacervate or, if sufficient desolvation occurs, a precipitate. Modification of the net charge of the protein molecules, by adjusting the solution pH to values ranging about the iso-electric point (IEP), influences the degree of interaction between the different molecular weight fractions, and hence the response of the protein to non-solvent.1 It can be hypothesised that alteration of the molecular charge intensity by changes in the ionic strength of the solution would affect the overall response of the protein. The objective of this work was to determine the effect of dilute NaCl concentrations on the response of B225 and B75 gelatins to the non-solvent ethanol at different pH’s.peer-reviewe

Quality by design optimisation of active pharmaceutical ingredient dispersions based on physicochemical parameters

Current information lacks richness in systematic methods that may give an indication as to the effectiveness of a specific set of dispersing conditions prior to their application, particularly for dispersions in hydrophobic media. This study addressed the possibility of constructing a predictive model that may indicate the effectiveness of a specific set of dispersion conditions prior to their application, particularly for dispersions in hydrophobic media. In particular, the objectives were to determine the physicochemical characteristics of the dispersing media that are potentially influential in the dispersability of powders, to characterise the properties of the active pharmaceutical ingredients that could contribute to the ability to form stable dispersions, and to characterise the dispersions of the active pharmaceutical ingredients (APIs) in the dispersing media or dispersing media-surfactant mixtures.peer-reviewe

Refractory Stage M Ganglioneuroblastoma With Bone Metastases and a Favorable, Chronic Course of Disease:Description of a Patient Cohort

Refractory stage M neuroblastoma (NB) is associated with a poor prognosis and a progressive course of disease. Here, we describe a unique group of patients with a discrepant clinical course. Seven histologically confirmed ganglioneuroblastoma (GNB) (n=6) and differentiating NB (n=1) patients were identified who were diagnosed with stage M disease based on iodine-123-metaiodobenzylguanidine avid bone metastases. Six patients started on high-risk treatment, without tumor response (stable disease). Treatment was discontinued before the start of consolidation treatment because of refractory response in all patients. Unexpectedly, after cessation of treatment no progression of disease occurred. In 2 patients, the primary tumors expanded (>25%) very slowly during 1.5 and 3 years, and remained stable thereafter. Metabolically, a slow decrease of urinary homovanillic acid and vanillylmandelic acid levels and iodine-123-metaiodobenzylguanidine avidity was observed. All patients are alive with presence of metastatic disease after a median follow-up of 17 years (range: 6.7 to 27 y). Interestingly, at diagnosis, 6 patients were asymptomatic, 6 patients had GNB morphology, and 5 patients had meningeal metastases. These are all features seen in only a small minority of stage M patients. This GNB entity illustrates the clinical heterogeneity of neuroblastic tumors and can be used to further study the developmental origin of different NB subtypes

Structure and Dynamics of Thermosensitive pDNA Polyplexes Studied by Time-Resolved Fluorescence Spectroscopy

Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 degrees C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.Peer reviewe

Influence of pH and gelatin concentration on the size and zeta potential of gelatin colloidal dispersions

Gelatin is a heterogeneous mixture of water-soluble proteins. Its broad molecular weight profile may be responsible for the variation in the experimental conditions required for nanoparticle formation. Since this may hinder the application of gelatin nanoparticles, it is important to determine the effect of several factors on the properties and stability of primary dispersions. The phase behaviour of gelatin in ethanol-water binary solvent systems has already been studied using transmittance values. The objective of this project was to use dynamic light scattering and laser Doppler electrophoresis to increase the understanding of the effects of pH and various dilute gelatin concentrations on the size, polydispersity and zeta potential of dispersions of B225 bovine skin gelatin in hydroalcoholic solutions.peer-reviewe