The Potential of Selenium-Based Therapies for Ocular Oxidative Stress.

Oxidative stress plays a critical role in the development of chronic ocular conditions including cataracts, age-related macular degeneration, and diabetic retinopathy. There is a need to explore the potential of topical antioxidants to slow the progression of those conditions by mediating oxidative stress and maintaining ocular health. Selenium has attracted considerable attention because it is a component of selenoproteins and antioxidant enzymes. The application of selenium to a patient can increase selenoprotein expression, counteracting the effect of reactive oxygen species by increasing the presence of antioxidant enzymes, and thus slowing the progression of chronic ocular disorders. Oxidative stress effects at the biomolecular level for prevalent ocular conditions are described in this review along with some of the known defensive mechanisms, with a focus on selenoproteins. The importance of selenium in the eye is described, along with a discussion of selenium studies and uses. Selenium's antioxidant and anti-inflammatory qualities may prevent or delay eye diseases. Recent breakthroughs in drug delivery methods and nanotechnology for selenium-based ocular medication delivery are enumerated. Different types of selenium may be employed in formulations aimed at managing ocular oxidative stress conditions.

Amorphous solid dispersions of lidocaine and lidocaine HCl produced by ball milling with well-defined RAFT-synthesised methacrylic acid polymers.

This study focuses on the use of methacrylic acid polymers synthesised via the Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation method for the production of amorphous solid dispersions (ASDs) by ball milling, to kinetically solubilize a poorly water-soluble model drug. The solid-state characteristics and the physical stability of the formulations were investigated using X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. This was followed by dissolution studies in different media. It was discovered that the acidic polymers of methacrylic acid were capable of interacting with the weakly basic drug lidocaine and its hydrochloride salt form to produce ASDs when a polymer to drug ratio of 70:30 w/w was used. The ASDs remained amorphous following storage under accelerated aging conditions (40 °C and 75% relative humidity) over 8 months. Fast dissolution and increased lidocaine solubility in different media were obtained from the ASDs owing to the reduced microenvironment pH and enhanced solubilization of the drug caused by the presence of the acidic polymer in the formulation. Production of ASDs using well-defined RAFT-synthesised acidic polymers is a promising formulation strategy to enhance the pharmaceutical properties of basic poorly water-soluble drugs.

Extracellular vesicle-embedded materials.

Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in formulation due to their lack of immunogenicity and targeting capabilities. EVs have been shown to have similar regenerative and therapeutic effects to their parental cells and also have potential in disease diagnosis. To improve the therapeutic potential of EVs, researchers have developed various strategies for modifying them, including genetic engineering and chemical modifications which have been examined to confer target specificity and prevent rapid clearance after systematic injection. Formulation efforts have focused on utilising hydrogel and nano-formulation strategies to increase the persistence of EV localisation in a specific tissue or organ. Researchers have also used biomaterials or bioscaffolds to deliver EVs directly to disease sites and prolong EV release and exposure. This review provides an in-depth examination of the material design of EV delivery systems, highlighting the impact of the material properties on the molecular interactions and the maintenance of EV stability and function. The various characteristics of materials designed to regulate the stability, release rate and biodistribution of EVs are described. Other aspects of material design, including modification methods to improve the targeting of EVs, are also discussed. This review aims to offer an understanding of the strategies for designing EV delivery systems, and how they can be formulated to make the transition from laboratory research to clinical use.

Maillard Reaction Crosslinked Alginate-Albumin Scaffolds for Enhanced Fenofibrate Delivery to the Retina: A Promising Strategy to Treat RPE-Related Dysfunction.

There are limited treatments currently available for retinal diseases such as age-related macular degeneration (AMD). Cell-based therapy holds great promise in treating these degenerative diseases. Three-dimensional (3D) polymeric scaffolds have gained attention for tissue restoration by mimicking the native extracellular matrix (ECM). The scaffolds can deliver therapeutic agents to the retina, potentially overcoming current treatment limitations and minimizing secondary complications. In the present study, 3D scaffolds made up of alginate and bovine serum albumin (BSA) containing fenofibrate (FNB) were prepared by freeze-drying technique. The incorporation of BSA enhanced the scaffold porosity due to its foamability, and the Maillard reaction increased crosslinking degree between ALG with BSA resulting in a robust scaffold with thicker pore walls with a compression modulus of 13.08 KPa suitable for retinal regeneration. Compared with ALG and ALG-BSA physical mixture scaffolds, ALG-BSA conjugated scaffolds had higher FNB loading capacity, slower release of FNB in the simulated vitreous humour and less swelling in water and buffers, and better cell viability and distribution when tested with ARPE-19 cells. These results suggest that ALG-BSA MR conjugate scaffolds may be a promising option for implantable scaffolds for drug delivery and retinal disease treatment.

Real-Time Monitoring Platform for Ocular Drug Delivery.

Real-time measurement is important in modern dissolution testing to aid in parallel drug characterisation and quality control (QC). The development of a real-time monitoring platform (microfluidic system, a novel eye movement platform with temperature sensors and accelerometers and a concentration probe setup) in conjunction with an in vitro model of the human eye (PK-Eye™) is reported. The importance of surface membrane permeability when modelling the PK-Eye™ was determined with a "pursing model" (a simplified setup of the hyaloid membrane). Parallel microfluidic control of PK-Eye™ models from a single source of pressure was performed with a ratio of 1:6 (pressure source:models) demonstrating scalability and reproducibility of pressure-flow data. Pore size and exposed surface area helped obtain a physiological range of intraocular pressure (IOP) within the models, demonstrating the need to reproduce in vitro dimensions as closely as possible to the real eye. Variation of aqueous humour flow rate throughout the day was demonstrated with a developed circadian rhythm program. Capabilities of different eye movements were programmed and achieved with an in-house eye movement platform. A concentration probe recorded the real-time concentration monitoring of injected albumin-conjugated Alexa Fluor 488 (Alexa albumin), which displayed constant release profiles. These results demonstrate the possibility of real-time monitoring of a pharmaceutical model for preclinical testing of ocular formulations.

Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations.

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day-1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L-1 h-1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of -1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7-9 °C, relative to those at 1-4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14-18% at 25-100 mg/mL, relative to those at 1-4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh.

Local Ugandan Production of Stable 0.2% Chlorhexidine Eye Drops.

Purpose: The purpose of this study was to develop a protocol to prepare buffered chlorhexidine (CHX) eye drops (0.2% w/v) in the United Kingdom that can be reproduced at a production facility in Uganda. Buffered CHX eye drops can prevent CHX degradation and improve ocular tolerability during the treatment of fungal keratitis. Methods: Buffered CHX eye drops in amber glass containers were prepared using sodium acetate buffer at pH 5.90 to 6.75. Two commercial CHX solutions and CHX in water were used as controls. Eye drops were stored at 40°C (70% humidity, 21 months) in the United Kingdom and at ambient temperature in Uganda (30 months). High-performance liquid chromatography was used to determine CHX stability over time, and pH was monitored. Sterility was achieved using an autoclave (121°C, 15 minutes) and water bath (100°C, 30 minutes). Results: The pH of acetate-buffered CHX eye drops did not change over 21 months at 40°C or at ambient temperature (30 months), whereas the pH of the unbuffered aqueous CHX displayed significant fluctuations, with an increase in acidity. The CHX concentration remained the same in both buffered and unbuffered eye-drop solutions. Eye drops sterilization was successful using an autoclave and a water bath. Conclusions: Stable, sterile, buffered CHX eye drops (pH 6.75) were successfully prepared first in the United Kingdom and then reproducibly in Uganda. This eye drops can be prepared in a hospital or pharmacy setting with limited resources, thus providing a cost-effective treatment for fungal keratitis. Translational Relevance: A protocol has been developed to prepare buffered CHX eye drops in low- and middle-income countries to treat fungal keratitis.

Hydroxytyrosol oleate: A promising neuroprotective nanocarrier delivery system of oleuropein and derivatives.

Olive Phenols (OPs) are known to be potent antioxidants and possess various bioactivities and health benefits. Epidemiological studies suggested that consumption of olive oil reduces the risk of different diseases exerting a protective effect against certain malignant tumors (prostate, breast, digestive tract, endothelium, etc.). However, extremely low absorption rate of olive phenolic compounds restricts their bioactivity. In this context, solid lipid nanoparticles (SLNs) are a promising solution because they provide higher drug stability and can incorporate both lipophilic and hydrophilic drugs. Interesting experimental results have been obtained using hydroxytyrosol oleate (HtyOle) as a main component of a nanoparticle delivery system containing oleuropein (OL), oleuropein aglycone (3,4-DHPEA-EA), or hydroxytyrosol itself (Hty). In this work, hydroxytyrosol oleate (HtyOle) and hydroxytyrosol oleate (HtyOle)-based solid lipid nanoparticles were prepared and characterized. In addition, we evaluatedin vitro their antioxidant activity by DPPH assays and by ROS formation using the SH-SY5Y cell line.

Stable Dried Catalase Particles Prepared by Electrospraying.

Therapeutic proteins and peptides are clinically important, offering potency while reducing the potential for off-target effects. Research interest in developing therapeutic polypeptides has grown significantly during the last four decades. However, despite the growing research effort, maintaining the stability of polypeptides throughout their life cycle remains a challenge. Electrohydrodynamic (EHD) techniques have been widely explored for encapsulation and delivery of many biopharmaceuticals. In this work, we explored monoaxial electrospraying for encapsulation of bovine liver catalase, investigating the effects of the different components of the electrospraying solution on the integrity and bioactivity of the enzyme. The catalase was successfully encapsulated within polymeric particles made of polyvinylpyrrolidone (PVP), dextran, and polysucrose. The polysorbate 20 content within the electrospraying solution (50 mM citrate buffer, pH 5.4) affected the catalase loading-increasing the polysorbate 20 concentration to 500 µg/mL resulted in full protein encapsulation but did not prevent loss in activity. The addition of ethanol (20% v/v) to a fully aqueous solution improves the electrospraying process by reducing surface tension, without loss of catalase activity. The polymer type was shown to have the greatest impact on preserving catalase activity within the electrosprayed particles. When PVP was the carrier there was no loss in activity compared with fresh aqueous solutions of catalase. The optimum particles were obtained from a 20% w/v PVP or 30% w/v PVP-trehalose (1:1 w/w) solution. The addition of trehalose confers stability advantages to the catalase particles. When trehalose-PVP particles were stored at 5 °C, enzymatic activity was maintained over 3 months, whereas for the PVP-only analogue a 50% reduction in activity was seen. This demonstrates that processing catalase by monoaxial electrospraying can, under optimised conditions, result in stable polymeric particles with no loss of activity.

Computational and Experimental Evaluation of the Stability of a GLP-1-like Peptide in Ethanol-Water Mixtures.

Aggregation resulting from the self-association of peptide molecules remains a major challenge during preformulation. Whereas certain organic solvents are known to promote aggregation, ethanol (EtOH) is capable of disrupting interactions between peptide molecules. It is unclear whether it is beneficial or counterproductive to include EtOH in formulations of short peptides. Here, we employed molecular dynamics simulations using the DAFT protocol and MARTINI force field to predict the formation of self-associated dimers and to estimate the stability of a GLP-1-like peptide (G48) in 0-80% aqueous EtOH solutions. Both simulation and experimental data reveal that EtOH leads to a remarkable increase in the conformational stability of the peptide when stored over 15 days at 27 °C. In the absence of EtOH, dimerisation and subsequent loss in conformational stability (α-helix → random coil) were observed. EtOH improved conformational stability by reducing peptide-peptide interactions. The data suggest that a more nuanced approach may be applied in formulation decision making and, if the native state of the peptide is an α-helix organic solvent, such as EtOH, may enhance stability and improve prospects of long-term storage.

Discovering Novel Small Molecule Compound for Prevention of Monoclonal Antibody Self-Association.

Designing an antibody with the desired affinity to the antigen is challenging, often achieved by lengthening the hydrophobic CDRs, which can lead to aggregation and cause major hindrance to the development of successful biopharmaceutical products. Aggregation can cause immunogenicity, viscosity and stability issues affecting both the safety and quality of the product. As the hydrophobic residues on the CDR are required for direct binding to antigens, it is not always possible to substitute these residues for aggregation-reduction purposes. Therefore, discovery of specific excipients to prevent aggregation is highly desirable for formulation development. Here, we used a combination of in silico screening methods to identify aggregation-prone regions on an aggregation-prone therapeutic antibody. The most aggregation-prone region on the antibody was selected to conduct virtual screening of compounds that can bind to such regions and act as an aggregation breaker. The most promising excipient candidate was further studied alongside plain buffer formulations and formulations with trehalose using coarse-grained molecular dynamics (CGMD) simulations with MARTINI force field. Mean interaction value between two antibody molecules in each formulation was calculated based on 1024 replicates of 512 ns of such CGMD simulations. Corresponding formulations with an excipient:antibody ratio of 1:5 were compared experimentally by measuring the diffusion interaction parameter kD and accelerated stability studies. Although the compound with the highest affinity score did not show any additional protective effects compared with trehalose, this study proved using a combination of in silico tools can aid excipient design and formulation development.

Effects of Flow Hydrodynamics and Eye Movements on Intraocular Drug Clearance.

New in vitro prototypes (PK-Eye™) were tested with and without eye movement to understand diffusion and convection effects on intraocular clearance. Port placement in front ((i) ciliary inflow model) and behind the model lens ((ii) posterior inflow model) was used to study bevacizumab (1.25 mg/50 µL) and dexamethasone (0.1 mg/100 µL) in phosphate-buffered saline (PBS, pH 7.4) and simulated vitreal fluid (SVF). Dexamethasone was studied in a (iii) retinal-choroid-sclera (RCS) outflow model (with ciliary inflow and two outflow pathways). Ciliary vs. posterior inflow placement did not affect the half-life for dexamethasone at 2.0 µL/min using PBS (4.7 days vs. 4.8 days) and SVF (4.9 days with ciliary inflow), but it did decrease the half-life for bevacizumab in PBS (20.4 days vs. 2.4 days) and SVF (19.2 days vs. 10.8 days). Eye movement only affected the half-life of dexamethasone in both media. Dexamethasone in the RCS model showed approximately 20% and 75% clearance from the RCS and anterior outflows, respectively. The half-life of the protein was comparable to human data in the posterior inflow model. Shorter half-life values for a protein in a ciliary inflow model can be achieved with other eye movements. The RCS flow model with eye movement was comparable to human half-life data for dexamethasone.

3D Printed Punctal Plugs for Controlled Ocular Drug Delivery.

Dry eye disease is a common ocular disorder that is characterised by tear deficiency or excessive tear evaporation. Current treatment involves the use of eye drops; however, therapeutic efficacy is limited because of poor ocular bioavailability of topically applied formulations. In this study, digital light processing (DLP) 3D printing was employed to develop dexamethasone-loaded punctal plugs. Punctal plugs with different drug loadings were fabricated using polyethylene glycol diacrylate (PEGDA) and polyethylene glycol 400 (PEG 400) to create a semi-interpenetrating network (semi-IPN). Drug-loaded punctal plugs were characterised in terms of physical characteristics (XRD and DSC), potential drug-photopolymer interactions (FTIR), drug release profile, and cytocompatibility. In vitro release kinetics of the punctal plugs were evaluated using an in-house flow rig model that mimics the subconjunctival space. The results showed sustained release of dexamethasone for up to 7 days from punctal plugs made with 20% w/w PEG 400 and 80% w/w PEGDA, while punctal plugs made with 100% PEGDA exhibited prolonged releases for more than 21 days. Herein, our study demonstrates that DLP 3D printing represents a potential manufacturing platform for fabricating personalised drug-loaded punctal plugs with extended release characteristics for ocular administration.

An investigation of alkaline phosphatase enzymatic activity after electrospinning and electrospraying.

The high target specificity and multifunctionality of proteins has led to great interest in their clinical use. To this end, the development of delivery systems capable of preserving their bioactivity and improving bioavailability is pivotal to achieve high effectiveness and satisfactory therapeutic outcomes. Electrohydrodynamic (EHD) techniques, namely electrospinning and electrospraying, have been widely explored for protein encapsulation and delivery. In this work, monoaxial and coaxial electrospinning and electrospraying were used to encapsulate alkaline phosphatase (ALP) into poly(ethylene oxide) fibres and particles, respectively, and the effects of the processing techniques on the integrity and bioactivity of the enzyme were assessed. A full morphological and physicochemical characterisation of the blend and core-shell products was performed. ALP was successfully encapsulated within monolithic and core-shell electrospun fibres and electrosprayed particles, with drug loadings and encapsulation efficiencies of up to 21% and 99%, respectively. Monoaxial and coaxial electrospinning were equally effective in preserving ALP function, leading to no activity loss compared to fresh aqueous solutions of the enzyme. While the same result was observed for monoaxial electrospraying, coaxial electrospraying of ALP caused a 40% reduction in its bioactivity, which was attributed to the high voltage (22.5 kV) used during processing. This demonstrates that choosing between blend and coaxial EHD processing for protein encapsulation is not always straightforward, being highly dependent on the chosen therapeutic agent and the effects of the processing conditions on its bioactivity.

A simple route to functionalising electrospun polymer scaffolds with surface biomolecules.

Surface functionalisation of polymeric electrospun scaffolds with therapeutic biomolecules is often explored in regenerative medicine and tissue engineering. However, the bioconjugation method must be carefully selected to prevent partial or full loss of activity of the biomolecule following chemical manipulation. Perfluorophenyl azide bearing a N-hydroxysuccinimide (PFPA-NHS) active ester group is a versatile tool for UV-initiated covalent coupling of amine-containing molecules to hydrocarbon-based polymers, such as polydioxanone or polycaprolactone (PCL). This study therefore explored the feasibility of PFPA-NHS functionalisation of electrospun PCL scaffolds with model biomolecules. Protein conjugation was extensively explored using fluorescence staining and attachment studies, confirming the retention of amine coupling capability following photografting of PFPA-NHS to the PCL surface. The effect of the washing method used to remove unreacted PFPA was explored in Caco-2 cell viability studies, and it was determined that sonication washing is required to avoid cell death. A model enzyme, catalase, was then successfully attached to the surface of PCL scaffolds for potential applications in oncological photodynamic therapy. Catalase retained its enzymatic activity following attachment to the fibres and the majority of the enzyme (~60%) remained bound to the fibre after incubation in an aqueous environment for six days. The anticipated prolonged presentation and sustained release of proteins as a result of PFPA-NHS conjugation could be advantageous in progressing protein-based therapies.

Dual-acting therapeutic proteins for intraocular use.

Intravitreally injected antibody-based medicines have revolutionised the treatment of retinal disease. Bispecific and dual-functional antibodies and therapeutic proteins have the potential to further increase the efficacy of intraocular medicines.

Injectables and Depots to Prolong Drug Action of Proteins and Peptides.

Proteins and peptides have emerged in recent years to treat a wide range of multifaceted diseases such as cancer, diabetes and inflammation. The emergence of polypeptides has yielded advancements in the fields of biopharmaceutical production and formulation. Polypeptides often display poor pharmacokinetics, limited permeability across biological barriers, suboptimal biodistribution, and some proclivity for immunogenicity. Frequent administration of polypeptides is generally required to maintain adequate therapeutic levels, which can limit efficacy and compliance while increasing adverse reactions. Many strategies to increase the duration of action of therapeutic polypeptides have been described with many clinical products having been developed. This review describes approaches to optimise polypeptide delivery organised by the commonly used routes of administration. Future innovations in formulation may hold the key to the continued successful development of proteins and peptides with optimal clinical properties.

Preclinical challenges for developing long acting intravitreal medicines.

The majority of blinding conditions arise due to chronic pathologies in the retina. During the last two decades, antibody-based medicines administered by intravitreal injection directly into the back of the eye have revolutionised the treatment of chronic retinal diseases characterised by uncontrolled blood vessel growth, e.g. wet age-related macular degeneration (wAMD), diabetic retinopathy (DR) and choroidal neovascularisation (CNV). Although intravitreal injections have become a commonly performed ophthalmic procedure that provides a reproducible dose to maximise drug exposure in the back of the eye, there is a need to minimise the frequency and cumulative number of intravitreal injections. Developing longer acting intraocular therapies is one key strategy that is being pursued. Pharmaceutical preclinical development of intraocular medicines is heavily reliant on the use of animal models to determine ocular tolerability, pharmacokinetics, biodistribution and drug stability. Animal eyes are different from human eyes, such as the anatomy, organisation of vitreous macromolecular structure, aqueous outflow and immune response; all which impacts the ability to translate preclinical data into a clinical product. The development of longer acting protein formulations using animals is also limited because animals reject human proteins. Preclinical strategies also do not account for differences in the vitreous due to ageing and whether a vitrectomy has been performed. Intraocular formulations must reside and clear from the vitreous body, so there is a need for the formulation scientist to have knowledge about vitreous structure and physiology to facilitate preclinical development strategies. Preclinical pharmaceutical development paradigms used to create therapies for other routes of administration (e.g. oral, subcutaneous, pulmonary and intravenous) are grounded on the use of preclinical in vitro models. Analogous pharmaceutical strategies with appropriately designed in vitro models that can account for intraocular mass transfer to estimate pharmacokinetic profiles can be used to develop in vitro-in vivo correlations (IVIVCs) to accelerate the preclinical optimisation of long-acting intraocular formulations. Data obtained can then inform preclinical in vivo and clinical studies. With the now widespread use of intravitreal injections, it is also important during early preclinical studies to ensure there is a viable regulatory pathway for new therapies. Knowledge of the physiological, pharmaceutical and regulatory factors will help in the development of long-acting intravitreal medicines, which is rapidly evolving into a distinct pharmaceutical discipline.

A Novel Transdermal Protein Delivery Strategy via Electrohydrodynamic Coating of PLGA Microparticles onto Microneedles.

Transdermal delivery of biological therapeutics is emerging as a potent alternative to intravenous or subcutaneous injections. The latter possess major challenges including patient discomfort, the necessity for trained personnel, specialized sharps disposal, and risk of infection. The microneedle (MN) technology circumvents many of the abovementioned challenges, delivering biological materials directly into the skin and allowing sustained release of the active ingredient both in animal models and in humans. This study describes the use of electrohydrodynamic atomization (EHDA) to coat ovalbumin (OVA)-loaded PLGA nanoparticles onto hydrogel-forming MN arrays. The particles showed extended release of OVA over ca. 28 days. Microscopic analysis demonstrated that EHDA could generate a uniform particle coating on the MNs, with 30% coating efficiency. Furthermore, the coated MN array manifested similar mechanical characteristics and insertion properties to the uncoated system, suggesting that the coating should have no detrimental effects on the application of the MNs. The coated MNs resulted in no significant increase in anti-OVA-specific IgG titres in C57BL/6 mice in vivo as compared to the untreated mice (paired t-test, p > 0.05), indicating that the formulations are nonimmunogenic. The approach of using EHDA to coat an MN array thus appears to have potential as a novel noninvasive protein delivery strategy.