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.

Protein encapsulation by electrospinning and electrospraying.

Given the increasing interest in the use of peptide- and protein-based agents in therapeutic strategies, it is fundamental to develop delivery systems capable of preserving the biological activity of these molecules upon administration, and which can provide tuneable release profiles. Electrohydrodynamic (EHD) techniques, encompassing electrospinning and electrospraying, allow the generation of fibres and particles with high surface area-to-volume ratios, versatile architectures, and highly controllable release profiles. This review is focused on exploring the potential of different EHD methods (including blend, emulsion, and co-/multi-axial electrospinning and electrospraying) for the development of peptide and protein delivery systems. An overview of the principles of each technique is first presented, followed by a survey of the literature on the encapsulation of enzymes, growth factors, antibodies, hormones, and vaccine antigens using EHD approaches. The possibility for localised delivery using stimuli-responsive systems is also explored. Finally, the advantages and challenges with each EHD method are summarised, and the necessary steps for clinical translation and scaled-up production of electrospun and electrosprayed protein delivery systems are discussed.

The Effect of Solvent Vapor Annealing on Drug-Loaded Electrospun Polymer Fibers.

Electrospinning has emerged as a powerful strategy to develop controlled release drug delivery systems but the effects of post-fabrication solvent vapor annealing on drug-loaded electrospun fibers have not been explored to date. In this work, electrospun poly(ԑ-caprolactone) (PCL) fibers loaded with the hydrophobic small-molecule spironolactone (SPL) were explored. Immediately after fabrication, the fibers are smooth and cylindrical. However, during storage the PCL crystallinity in the fibers is observed to increase, demonstrating a lack of stability. When freshly-prepared fibers are annealed with acetone vapor, the amorphous PCL chains recrystallize, resulting in the fiber surfaces becoming wrinkled and yielding shish-kebab like structures. This effect does not arise after the fibers have been aged. SPL is found to be amorphously dispersed in the PCL matrix both immediately after electrospinning and after annealing. In vitro dissolution studies revealed that while the fresh fibers show a rapid burst of SPL release, after annealing more extended release profiles are observed. Both the rate and extent of release can be varied through changing the annealing time. Further, the annealed formulations are shown to be stable upon storage.

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.

The effect of collection substrate on electrospun ciprofloxacin-loaded poly(vinylpyrrolidone) and ethyl cellulose nanofibers as potential wound dressing materials.

In this work, nanofibers based on hydrophilic poly(vinylpyrrolidone) (PVP) and hydrophobic ethyl cellulose (EC) were generated via electrospinning. A model antibiotic, ciprofloxacin (CIF), was also incorporated into the fibers. Fibers were collected on both a foil substrate and a commercial gauze, the latter in the interests of developing a smart fabric. Electron microscopy images revealed that the fibers collected on both foil and fabric were homogeneous and cylindrical. Infrared spectroscopy, X-ray diffraction and differential scanning calorimetry demonstrated that CIF was successfully loaded into the fibers and present in the amorphous physical form. In vitro drug release tests were conducted to simulate drug release from the formulations into a wound site, and as expected the hydrophilic fibers showed much faster release than their hydrophobic analogues. CIF was released through a combined mechanism of polymer erosion and drug diffusion, and the EC nanofibers displayed close to zero-order release over three days. Fibroblast cells are able to grow and proliferate on the fibers. Finally, inhibition zone assays revealed that the growth of both Gram positive and Gram negative bacteria could be effectively inhibited as a result of the presence of CIF in the fibers. There were no marked differences between the fibers collected on foil and on gauze, and electrospinning can be performed directly onto a gauze substrate to prepare a smart fabric.

In situ antibody-loaded hydrogel for intravitreal delivery.

Therapeutic protein medicines have transformed the treatment of blinding diseases (e.g. age-related macular degeneration, AMD) during the last 1-2 decades. Many blinding conditions such as AMD are chronic; and require multiple intravitreal injections over a long period to achieve a high and reproducible dose needed for clinical benefit. Prolonging the duration of action of ophthalmic drugs is critical to reduce the frequency of injections. Thermoresponsive hydrogels (e.g. N-isopropylacrylamide, NIPAAM) that collapse in physiological conditions can entrap and sustain the release of a therapeutic protein. However, most NIPAAM hydrogels are not biodegradable and often requires invasive surgery to remove the depot. Here, we report the preparation of a hydrogel derived from NIPAAM and acrylated hyaluronic acid (Ac-HA) as a biodegradable, macromolecular crosslinker. Ac-HA was prepared by the acrylation of hyaluronic acid (HA). Antibody (infliximab (INF), 5.0 mg/mL or bevacizumab (BEVA), 12.5 mg/mL), NIPAAM (0.35 mmol) and Ac-HA (2.0-10.0 mg/mL, 40.0-200.0 nmol) were first mixed prior to redox polymerisation to ensure maximal protein mixing and to shorten the burst release. Hydrogels with lower amounts of Ac-HA (2.0-4.0 mg/mL, 40.0-80.0 nmol) showed favourable lower critical solution temperature (LCST) values and injectability (27-29G) than higher amounts of Ac-HA (>4.0 mg/mL, >80.0 nmol). These hydrogels were further characterised (swelling ratio (SR), water retention (WR) and rheology). All hydrogels degraded in presence of bovine testes hyaluronidase (0-50 U/mL, 37 °C, 100 rpm). Release studies of BEVA-loaded hydrogels were investigated in vitro using the PK-Eye™ model, which estimates the human clearance times of proteins from the back of the eye. Phosphate buffered saline (PBS, pH 7.4, 37 °C) was used rather than simulated vitreous to more effectively map trends between the formulations. A zero-order release profile was observed between days 5 to 50 with 43.3 ±â€¯9.5% protein released at day 50. Determining protein binding and functionality from a formulation is crucial to determine the optimal formulation prior to more detailed studies that might be necessary. BEVA showed binding to human vascular growth endothelial factor (VEGF165) throughout the study (two months) while still maintaining a therapeutic dose (123.5 ±â€¯45.6 ng) in the posterior cavity of the PK-Eye™ model. These encouraging results suggest that extended release of proteins in the vitreous can be achieved using injectable hydrogels derived from NIPAAM and HA.

Overview of Antibody Drug Delivery.

Monoclonal antibodies (mAbs) are one of the most important classes of therapeutic proteins, which are used to treat a wide number of diseases (e.g., oncology, inflammation and autoimmune diseases). Monoclonal antibody technologies are continuing to evolve to develop medicines with increasingly improved safety profiles, with the identification of new drug targets being one key barrier for new antibody development. There are many opportunities for developing antibody formulations for better patient compliance, cost savings and lifecycle management, e.g., subcutaneous formulations. However, mAb-based medicines also have limitations that impact their clinical use; the most prominent challenges are their short pharmacokinetic properties and stability issues during manufacturing, transport and storage that can lead to aggregation and protein denaturation. The development of long acting protein formulations must maintain protein stability and be able to deliver a large enough dose over a prolonged period. Many strategies are being pursued to improve the formulation and dosage forms of antibodies to improve efficacy and to increase the range of applications for the clinical use of mAbs.

Electrospun formulations of bevacizumab for sustained release in the eye.

Medicines based on vascular endothelial growth factor (VEGF) neutralising antibodies such as bevacizumab have revolutionized the treatment of age related macular degeneration (AMD), a common blinding disease, and have great potential in preventing scarring after surgery or accelerating the healing of corneal injuries. However, at present frequent invasive injections are required to deliver these antibodies. Such administration is uncomfortable for patients and expensive for health service providers. Much effort is thus focused on developing dosage forms that can be administered less frequently. Here we use electrospinning to prepare a solid form of bevacizumab designed for prolonged release while maintaining antibody stability. Electrospun fibers were prepared with bevacizumab encapsulated in the core, surrounded by a poly-ε-caprolactone sheath. The fibers were generated using aqueous bevacizumab solutions buffered at two different pH values: 6.2 (the pH of the commercial product; Fbeva) and 8.3 (the isoelectric point of bevacizumab; FbevaP). The fibers had smooth and cylindrical morphologies, with diameters of ca. 500nm. Both sets of bevacizumab loaded fibers gave sustained release profiles in an in vitro model of the subconjunctival space of the eye. Fbeva displayed first order kinetics with t1/2 of 11.4±4.4 days, while FbevaP comprises a zero-order reservoir type release system with t1/2 of 52.9±14.8 days. Both SDS-PAGE and surface plasmon resonance demonstrate that the bevacizumab in FbevaP did not undergo degradation during fiber fabrication or release. In contrast, the antibody released from Fbeva had degraded, and failed to bind to VEGF. Our results demonstrate that pH control is crucial to maintain antibody stability during the fabrication of core/shell fibers and ensure release of functional protein. STATEMENT OF SIGNIFICANCE: Bevacizumab is a potent protein drug which is highly effective in the treatment of degenerative conditions in the eye. To be effective, frequent injections into the eye are required, which is deeply unpleasant for patients and expensive for healthcare providers. Alternative methods of administration are thus highly sought after. In our work, we use the electrospinning technique to prepare fiber-based formulations loaded with bevacizumab. By careful control of the experimental parameters we are able to stabilize the protein during processing and ensure a constant rate of release over more than two months in vitro. These fibers could thus be used to reduce the frequency of dosing required, reducing cost and improving patient outcomes.

Solid-state protein formulations.

When formulated as liquid dosage forms, therapeutic proteins and peptides often show instability during handling as a result of chemical degradation. Solid formulations are frequently required to maintain protein stability during storage, transport and upon administration. Herein we highlight current strategies used to formulate pharmaceutical proteins in the solid form. An overview of the physical instabilities which can arise with proteins is first described. The key solidification techniques of crystallization, freeze-drying and particle forming technologies are then discussed. Examples of current commercial products that are formulated in the solid state are provided and include neutral protamine Hagedorn - insulin crystal suspensions, freeze-dried monoclonal antibodies and leuproride polylactide-co-glycolide microparticles. Finally, future perspectives in solid-state protein formulation are described.