Phosphonium polymethacrylates for siRNA delivery: effect of polymer and RNA structural parameters on polyplex assembly and gene knockdown

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates –and their corresponding ammonium analogues– were synthesized by RAFT polymerization. Both the nature of the charged heteroatom (N vs. P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs. trioxyethylene) were systematically varied. Polymers efficiently bound siRNA at N+/P- or P+/P- ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. Whilst siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalised by GFP-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when short interfering RNA (siRNA) was replaced with novel, multimerised long interfering liRNA (liRNA) in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing

Mechanism of mucosal permeability enhancement of CriticalSorb® (Solutol® HS15) investigated In Vitro in cell cultures.

Purpose CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. Methods Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2–10. Results This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a ‘classical’ tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. Conclusion Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Enhanced permeation by amphiphilic surfactant is spatially heterogenous at membrane and cell level

In the context of increased interest in permeability enhancement technologies to achieve mucosal delivery of drugs and biologics, we report our study on effects of the amphiphilic surfactant at cell membrane and cell population levels. Our results show that modulation in membrane order and fluidity initially occurs on insertion of individual surfactant molecules into the outer leaflet of membrane lipid bilayer; a process occurring at concentrations below surfactant's critical micellar concentration. The surfactant insertion, and consequent increase in membrane fluidity, are observed to be spatially heterogenous, i.e. manifested as ‘patches’ of increased membrane fluidity. At the cell population level, spatially heterogeneous activity of surfactant is also manifested, with certain cells displaying high permeability amongst a ‘background’ population. We propose that this heterogeneity is further manifested in a broad profile of intracellular and nuclear exposure levels to a model drug (doxorubicin) observed in cell population. The study points to heterogeneous nature of surfactant effects at cell membrane and cells in population levels

Evaluation of calcium depletion as a strategy for enhancement of mucosal absorption of macromolecules

Extracellular calcium is crucial for functioning of the epithelial barrier. Compounds that bind calcium, reducing its extracellular levels, have therefore been investigated as mucosal absorption enhancers. However, the conditions under which calcium reduction sufficiently modulates the epithelial barrier to result in meaningful improvements in mucosal drug absorption are unclear. Present work investigated the settings in which calcium depletion leads to optimal epithelial barrier-modulating effects. Using Calu-3 and Caco-2 cell layers and inducing calcium depletion site-specifically (apically, basolaterally or on both sides) we demonstrate that apical calcium removal produces a modest effect on the tight junctions (the extent of the effect being dependent on the duration of apical calcium unavailability), whilst basolateral calcium exhaustion leads to a prominent effect on the epithelial barrier. However, using polyacrylic acid as an example, we show that polymeric calcium-binding agents proposed as mucosal absorption-enhancing excipients alter calcium levels exclusively on the apical side of the epithelium, which explains their modest effect on epithelial barrier modulation (also demonstrated in our work). Therefore the use of calcium-depleting agents, especially those based on macromolecular polymers, is a relatively inefficacious strategy to promote mucosal absorption of macromolecules

Epithelial toxicity of alkylglycoside surfactants

Alkylglycoside surfactants have been proposed as drug delivery excipients with the potential to enhance mucosal drug absorption of therapeutic macromolecules. Previous work reported their drug absorption-promoting potential by demonstrating that several compounds within this class of surfactants improve mucosal absorption of peptides, proteins and other macromolecules. However, detailed investigation of their toxicity has not been conducted. Using Calu-3 epithelial cell layers as a model of the airway mucosa, and liposomes as models of cell membranes, this work investigates the cytotoxicity of dodecylmaltoside, tridecylmaltoside and tetradecylmaltoside, as representative alkylglycosides. A combination of different toxicity assays and other tests indicating cell membrane disruption were used to assess cytotoxicity. The alkylglycosides tested induced a dramatic reduction in cell viability, cell membrane and liposome-disruptive effects, as well as abrogation of transepithelial electrical resistance that did not recover completely. Importantly, these phenomena were noted at concentrations markedly lower than those typically used in the literature studies demonstrating the absorption-enhancing properties of alkylglycosides. This work therefore demonstrates that alkylglycosides exhibit significant toxicity towards airway epithelial cells, most likely resulting from a membrane-damaging effect, highlighting a need for further evaluation of their safety as absorption-enhancing excipients

A novel combined strategy for the physical PEGylation of polypeptides

Poly(ethylene glycol) (PEG) may be covalently conjugated to peptide drugs to overcome their rapid clearance but in doing so their potency can be lost. Here, a non-covalent approach was used to conjugate PEG bearing a terminal cholanic moiety (mPEG(5kDa)-cholane) to a 28 amino acid peptide, vasoactive intestinal peptide (VIP). Palmitoylation of the peptide was essential to facilitate physical interaction via a single binding site involving two mPEG(5kDa)-cholane molecules with an affinity constant of similar to 3.10(4) M-1; these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12 nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH 7.4 (4 mu g/mL) and markedly increased peptide solution stability at 25 degrees C over 30 days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an similar to 10% decrease in solubility. The predominantly a-helical conformation of the peptide alone at pH 5-6.5 was lost at pH 7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an similar to 10% loss of a-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1 mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG(5kDa)-cholane dramatically increased peptide concentration in the systemic circulation. This is the first demonstration of non-covalent PEGylation of acylated peptides, an important biologic class, which improves in vitro and in vivo properties, and thereby may prove an alternative to covalent PEGylation strategies

The Expanding Therapeutic Utility of Botulinum Neurotoxins

Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products