Sunitinib malate (SUM), widely used in cancer treatment for its anti-VEGF properties, has also been explored for ocular neovascular diseases. For ocular applications, sustained drug release is essential to reduce dosing frequency. Hyaluronic acid (HA)-based hydrogels are commonly used for controlled drug delivery, but their hydrophilicity leads to rapid drug diffusion, especially for water-soluble drugs like SUM. To address this, beta-cyclodextrin (beta-CD) polymers (2-300 kDa) were incorporated into tyramine-conjugated HA (HA-TA) (200-400 kDa) networks to extend drug release via the formation of host-guest inclusion complexes. SUM-CD intermolecular interactions were identified and characterised by 1H NMR and FTIR spectroscopies, and NOESY spectra further confirmed a 1 SUM: 2 beta-CD inclusion complex. beta-CD polymers (10 % w/v) were integrated into HA-TA (0.25, 0.5, 1 % w/v) networks enzymatically crosslinked using horseradish peroxidase and hydrogen peroxide, forming semi-interpenetrating polymer network hydrogels. These gels exhibited faster gelation, enhanced swelling behaviour, higher drug loading capacity, a denser matrix, and a longer SUM release duration compared to HA-TA hydrogels. In an in vitro flow model, post-gelation loading of SUM led to a longer release duration than pre-loading, with release continuing over 20 days. The HA-CD semi-IPN hydrogel therefore warrants further exploration for its potential ocular applications.

Hydrogels can provide a hydrated environment to encapsulate extracellular vesicles (EVs) while offering promising solutions to some of the challenges that limit their therapeutic potential, e.g. rapid clearance and propensity for enzymatic degradation and aggregation. This study explores the use of a hyaluronic acid-tyramine (HA-TA) hydrogel to prolong the delivery and enhance the stability of EVs. EVs were obtained from lentiviral-transduced HEK293T cells expressing luciferase and eGFP to enable easy quantification. Two encapsulation strategies were evaluated: (1) pre-loading, where EVs were mixed with HA-TA (2.58 % degree of substitution) precursor solution and subsequently crosslinked with 2 U/mL horseradish peroxidase (HRP) and 0.05 mM H2O2; and (2) post-loading, where EVs were soaked into pre-formed dehydrated hydrogels. Both methods improved EV stability over 7 days at 37 °C compared to free EVs. The pre-loading approach was ultimately selected due to its ability to give rapid in situ gelation within one minute. Controlled in vitro release of EVs from the pre-loaded hydrogels was observed to extend beyond 7 days, as determined by CD9 ELISA. The released EVs maintained their bioactivity, as evidenced by effective internalisation into ARPE-19 and H9c2 cell lines, with performance comparable to fresh EVs. The EV release profile could be varied by modifying the hydrogel concentration. These findings underscore the potential of HA-TA hydrogels for localised, sustained, EV delivery with preserved functionality.

Background/Objectives: Oxidative stress plays a critical role in the development of ocular diseases such as cataracts. Selenium nanoparticles (SeNPs) offer antioxidant benefits with low toxicity. This study aimed to evaluate the antioxidant activity of SeNPs coated with D-α-tocopheryl polyethylene glycol succinate (TPGS) in human lens epithelial (HLE) cells. Methods: SeNPs were synthesised by reducing sodium selenite with ascorbic acid in the presence of TPGS. Physicochemical characterisation was carried out using dynamic light scattering to assess size and surface charge. Antioxidant activity was measured by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Cytocompatibility was assessed on adult retinal pigment epithelial (ARPE-19) and HLE cells using PrestoBlue. Functional antioxidant performance was determined through enzymatic assays for glutathione peroxidase (GPx), thioredoxin reductase (TrxR), and glutathione (GSH), and lipid peroxidation was assessed using malondialdehyde (MDA) quantification. Catalase mimicry was evaluated under 3-amino-1,2,4-triazole (3-AT)-induced inhibition. Results: The optimal SeNP formulation had an average hydrodynamic diameter of 44 ± 3 nm, low PDI (<0.1), and a surface charge of −15 ± 3 mV. These TPGS-SeNPs demonstrated strong radical scavenging (EC50 ≈ 1.55 µg/mL) and were well tolerated by ARPE-19 cells (IC50 = 524 µg/mL), whereas HLE cells had a narrower biocompatibility window (≤0.4 µg/mL, IC50 = 2.2 µg/mL). Under oxidative stress, SeNPs significantly enhanced GPx and TrxR activity but did not affect GSH or MDA levels. No catalase-mimetic activity was observed. Conclusions: TPGS-SeNPs exhibit potent antioxidant enzyme modulation under stress conditions in HLE cells. Although not affecting all oxidative markers, these nanoparticles show promise for non-invasive strategies targeting lens-associated oxidative damage, including cataract prevention.

Electrospraying is a processing technique that has gained much interest to prepare polymeric particles. The technique operates at ambient temperature, thereby avoiding heat induced degradation of labile therapeutics (e. g. peptides and proteins). Exposure to organic solvents can be minimised by co-axial electrospraying through separation of core (aqueous) and shell (organic) solvents. However, aqueous solutions are often difficult to electrospray due to high surface tension. Immiscibility between the core-shell solvents creates a further process challenge. Herein, we describe for the first time the use of hydrophobic ion pairing (HIP) to encapsulate a polypeptide into polymeric particles prepared by co-axial electrospraying. Peptide ion pairs were prepared to incorporate a model peptide - teriparatide - into an organic solvent, permitting facile electrospraying while also protecting the peptide from denaturation. Teriparatide loaded PLGA particles were generated by electrospraying from aqueous or ethanolic peptide solutions (core). A PLGA solution in chloroform (with and without cosolvents) was employed as the shell solution. The aqueous core solution led to a teriparatide encapsulation efficiency of 79.2 f 19.8 %, which was not significantly different from the ethanolic core (57.1 f 14.5 %). When aqueous solutions were used the process lacked reproducibility, resulting in low process yields (61.3 f 4.0 %). In contrast, when an organic core was used a dry powder bed was achieved with a yield of 102.2 f 8.8 %. The peptide's integrity and biological functionality were retained after electrospraying as ion pairs, as evidenced in a cell-based PTH1 receptor binding assay.

The design of stable formulations remains a major challenge for protein therapeutics, particularly the need to minimize aggregation. Experimental formulation screens are typically based on thermal transition midpoints (T m), and forced degradation studies at elevated temperatures. Both approaches give limited predictions of long-term storage stability, particularly at low temperatures. Better understanding of the mechanisms of action for formulation of excipients and buffers could lead to improved strategies for formulation design. Here, we identified a complex impact of glycine concentration on the experimentally determined stability of an antibody Fab fragment and then used molecular dynamics simulations to reveal mechanisms that underpin these complex behaviors. T m values increased monotonically with glycine concentration, but associated ΔS vh measurements revealed more complex changes in the native ensemble dynamics, which reached a maximum at 30 mg/mL. The aggregation kinetics at 65 °C were similar at 0 and 20 mg/mL glycine, but then significantly slower at 50 mg/mL. These complex behaviors indicated changes in the dominant stabilizing mechanisms as the glycine concentration was increased. MD revealed a complex balance of glycine self-interaction, and differentially preferred interactions of glycine with the Fab as it displaced hydration-shell water, and surface-bound water and citrate buffer molecules. As a result, glycine binding to the Fab surface had different effects at different concentrations, and led from preferential interactions at low concentrations to preferential exclusion at higher concentrations. During preferential interaction, glycine displaced water from the Fab hydration shell, and a small number of water and citrate molecules from the Fab surface, which reduced the protein dynamics as measured by root-mean-square fluctuation (RMSF) on the short time scales of MD. By contrast, the native ensemble dynamics increased according to ΔS vh, suggesting increased conformational changes on longer time scales. The aggregation kinetics did not change at low glycine concentrations, and so the opposing dynamics effects either canceled out or were not directly relevant to aggregation. During preferential exclusion at higher glycine concentrations, glycine could only bind to the Fab surface through the displacement of citrate buffer molecules already favorably bound on the Fab surface. Displacement of citrate increased the flexibility (RMSF) of the Fab, as glycine formed fewer bridging hydrogen bonds to the Fab surface. Overall, the slowing of aggregation kinetics coincided with reduced flexibility in the Fab ensemble at the very highest glycine concentrations, as determined by both RMSF and ΔS vh, and occurred at a point where glycine binding displaced neither water nor citrate. These final interactions with the Fab surface were driven by mass action and were the least favorable, leading to a macromolecular crowding effect under the regime of preferential exclusion that stabilized the dynamics of Fab.

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.

Teriparatide (and analogue peptides) are the only FDA approved anabolic treatments for osteoporosis. Current therapies are administered as a daily subcutaneous injection, which limits patient adherence and clinical efficacy. To achieve the desired anabolic effect, a controlled delivery system must ensure a pulsatile release profile over a prolonged period. Thermo-responsive formulations (e.g. liposomes) can undergo a temperature-related phase-transition which can allow active control of drug release. Herein, thermo-responsive liposomes were developed to permit precise control over the teriparatide release rate through modulation of temperature. Entrapment of hydrophilic molecules, including peptides within liposomes remains challenging due to the large volume of hydration. In this work, hydrophobic ion pairing was employed for the first time to enhance peptide entrapment within liposomes. The method resulted in a hydrophobic complex that achieved high teriparatide entrapment (>75%) in sub-200 nm, monodispersed liposomes. Hydrophobic ion pairing outperformed other entrapment approaches. Several liposomal formulations with transition temperatures between 38 – 50 °C were obtained by modulation of the phospholipid composition. In vitro release assays demonstrated temperature-dependent release kinetics with faster rates of release observed at/above the transition temperature. The maintenance of biological activity of released teriparatide was demonstrated in a cell-based assay utilising the PTH1R receptor. Overall, this provides the first proof-of-concept of the suitability of thermo-responsive systems for pulsatile delivery of teriparatide and similar peptides.

Teriparatide (and analogue peptides) are the only FDA approved anabolic treatments for osteoporosis. Current therapies are administered as a daily subcutaneous injection, which limits patient adherence and clinical efficacy. To achieve the desired anabolic effect, a controlled delivery system must ensure a pulsatile release profile over a prolonged period. Thermo-responsive formulations (e.g. liposomes) can undergo a temperature-related phase-transition which can allow active control of drug release. Herein, thermo-responsive liposomes were developed to permit control over teriparatide release rate through modulation of temperature. Entrapment of hydrophilic molecules, including peptides, within liposomes remains challenging due to the large volume of hydration. In this work, hydrophobic ion pairing was employed for the first time to enhance peptide entrapment within liposomes. The method resulted in a hydrophobic complex that achieved high teriparatide entrapment (>75 %) in sub-200 nm monodispersed liposomes. Hydrophobic ion pairing outperformed other entrapment approaches. Several liposomal formulations with transition temperatures between 38 and 50 °C were obtained by modulation of the phospholipid composition. In vitro assays demonstrated temperature-dependent release kinetics with faster rates of release observed at/above the transition temperature. The maintenance of biological activity of released teriparatide was demonstrated in a cell-based assay utilising the PTH1 receptor. Overall, this provides the first proof-of-concept of the suitability of thermo-responsive systems for pulsatile delivery of teriparatide and similar peptides.

We previously described FpFs 1̲ (Fab-PEG-Fab) as binding mimetics of IgGs. FpFs are prepared with di(bis-sulfone) conjugation reagents 3̲ that undergo disulfide rebridging conjugation with the accessible disulfide of each Fab (Scheme 1). We have now prepared bispecific FpFs 2̲ (bsFpF and Fab1-PEG-Fab2) as potential bispecific antibody mimetics with the intent that bsFpFs could be used in preclinical antibody development since sourcing bispecific antibodies may be challenging during preclinical research. The di(bis-sulfone) reagent 3̲ was first used to prepare a bsFpF 2̲ by the sequential conjugation of a first Fab and then a second Fab to another target (Scheme 2). Seeking to improve bsFpF synthesis, the asymmetric conjugation reagent, bis-sulfone bis-sulfide 1̲6̲, with different thiol conjugation reactivities at each terminus (Scheme 4) was examined and the bsFpFs appeared to be formed at similar conversion to the di(bis-sulfone) reagent 3̲. To explore the advantages of using common intermediates in the preparation of bsFpF families, we investigated bsFpF synthesis with a protein conjugation-ligation approach (Scheme 5). Reagents with a bis-sulfone moiety for conjugation on one PEG terminus and a ligation moiety on the other terminus were examined. Bis-sulfone PEG trans-cyclooctene (TCO) 2̲8̲ and bis-sulfone PEG tetrazine (Tz) 3̲0̲ were used to prepare several bsFpFs targeting various therapeutic targets (TNF-α, IL6R, IL17, and VEGF) and tissue affinity targets (hyaluronic acid and collagen II). Surface plasmon resonance (SPR) binding studies indicated that there was little difference between the dissociation rate constant (k d) for the unmodified Fab, mono-conjugated PEG-Fab and the corresponding Fab in a bsFpF. The Fab association rate (k a) in the bsFpF was slower than for PEG-Fab, which may be because of mass differences that influence SPR results. These observations suggest that each Fab will bind to its target independently of the other Fab and that bsFpF binding profiles can be estimated using the corresponding PEG-Fab conjugates.

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.

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.

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.

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.

Polymers can be used in nanoparticle associated formulations to encapsulate cytotoxic drugs (e.g., paclitaxel). Polyelectrolyte complexes (PECs) that form drug associated colloids also have potential to form particulate associated formulations. We used RAFT polymerisation to prepare small families of narrow molecular weight distributed (i) methacrylate block co-polymers comprised of oligomeric ethylene glycol, poly(ethylene glycol) methyl ether methacrylate (PEGMA), and dimethyl amino pendent chains, 2-(dimethylamino) ethyl methacrylate (DMAEMA), and (ii) poly(methacrylic acid), PMAA. These polymers were examined for their ability to form PECs capable of drug encapsulation. Optimal control in RAFT polymerisation was confirmed by the linear increase of molecular weight and the narrow dispersity of the polymers (<1.2) as determined by 1H nuclear magnetic resonance and gel permeation chromatography. Dynamic light scattering and transmission electron microscopy showed formation of well-defined monodispersed nanoparticles with a hydrodynamic diameter of 25 +/- 3 nm upon self-assembly of poly(PEGMA0.23-b-DMAEMA0.77)99 and PMAA75. These PECs are highly haemocompatible. Thin film hydration was used to encapsulate two hydrophobic drugs, paclitaxel and carmofur, into spherical nanoparticles. The results show that carmofur was encapsulated markedly more effectively than paclitaxel (72 vs 1.5%).

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.

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.

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.

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.

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.