The prognostic significance of lysosomal protective protein (cathepsin A) in breast ductal carcinoma in situ.

AIMS: Cathepsin A (CTSA) is a key regulatory enzyme for galactoside metabolism. Additionally, it has a distinct proteolytic activity and plays a role in tumour progression. CTSA is differentially expressed at the mRNA level between breast ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). In this study, we aimed to characterise CTSA protein expression in DCIS and evaluate its prognostic significance. METHODS AND RESULTS: A large cohort of DCIS [n = 776 for pure DCIS and n = 239 for DCIS associated with IBC (DCIS/IBC)] prepared as a tissue microarray was immunohistochemically stained for CTSA. High CTSA expression was observed in 48% of pure DCIS. High expression was associated with features of poor DCIS prognosis, including younger age at diagnosis (<50 years), higher nuclear grade, hormone receptor negativity, HER2 positivity, high proliferative index and high hypoxia inducible factor 1 alpha expression. High CTSA expression was associated with shorter recurrence-free interval (RFI) (P = 0.0001). In multivariate survival analysis for patients treated with breast conserving surgery, CTSA was an independent predictor of shorter RFI (P = 0.015). DCIS associated with IBC showed higher CTSA expression than pure DCIS (P = 0.04). In the DCIS/IBC cohort, CTSA expression was higher in the invasive component than the DCIS component (P < 0.0001). CONCLUSION: CTSA is not only associated with aggressive behaviour and poor outcome in DCIS but also a potential marker to predict co-existing invasion in DCIS.

Stable increased formulation atomization using a multi-tip nozzle device.

Electrohydrodynamic atomization (EHDA) is an emerging technique for the production of micron and nano-scaled particles. The process often involves Taylor cone enablement, which results in a fine spray yielding formulated droplets, which then undergo drying during deposition. In this work, novel multi-tip emitter (MTE) devices were designed, engineered and utilized for potential up-scaled EHDA, by comparison with a conventional single-needle system. To demonstrate this, the active ketoprofen (KETO) was formulated using polyvinylpyrrolidone (PVP) polymer as the matrix material. Here, PVP polymer (5% w/v) solution was prepared using ethanol and distilled water (80:20) as the vehicle. KETO was incorporated as 5% w/w of PVP. Physical properties of resulting solutions (viscosity, electrical conductivity, density and surface tension) were obtained. Formulations were electrosprayed through both single and novel MTEs under EHDA conditions at various flow rates (5-300 µl/min) and applied voltages (0-30 kV). The atomization process using MTEs and single nozzle was monitored at using various process parameters via a digital optical camera. Resulting particles were collected 200 mm below processing heads and were analyzed using differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Digital recordings confirmed stable MTE jetting at higher flow rates. Electron micrographs confirmed particle size variation arising due to nozzle head design and evidenced stable jetting derived greater near-uniform particles. DSC, XRD and TGA confirm KETO molecules were encapsulated and dispersed into PVP polymer particles. In conclusion, novel MTE devices enabled stable atomization even at higher flow rates when compared to conventional single-needle device. This indicates an exciting approach for scaling up (EHDA) in contrast to current efforts focusing on multiple-nozzle and pore-based processing outlets.

Electrosprayed mesoporous particles for improved aqueous solubility of a poorly water soluble anticancer agent: in vitro and ex vivo evaluation.

Encapsulation of poorly water-soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen-Schmidt condensation. KAZ3 was loaded into mesoporous (SBA-15 and MCM-41) and non-porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (~79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30-fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco-2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water-soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials.

Liposomal Drug Delivery Systems and Anticancer Drugs.

Cancer is a life-threatening disease contributing to ~3.4 million deaths worldwide. There are various causes of cancer, such as smoking, being overweight or obese, intake of processed meat, radiation, family history, stress, environmental factors, and chance. The first-line treatment of cancer is the surgical removal of solid tumours, radiation therapy, and chemotherapy. The systemic administration of the free drug is considered to be the main clinical failure of chemotherapy in cancer treatment, as limited drug concentration reaches the tumour site. Most of the active pharmaceutical ingredients (APIs) used in chemotherapy are highly cytotoxic to both cancer and normal cells. Accordingly, targeting the tumour vasculatures is essential for tumour treatment. In this context, encapsulation of anti-cancer drugs within the liposomal system offers secure platforms for the targeted delivery of anti-cancer drugs for the treatment of cancer. This, in turn, can be helpful for reducing the cytotoxic side effects of anti-cancer drugs on normal cells. This short-review focuses on the use of liposomes in anti-cancer drug delivery.

Electrically atomised formulations of timolol maleate for direct and on-demand ocular lens coatings.

Advances in nanotechnology have enabled solutions for challenging drug delivery targets. While the eye presents numerous emerging opportunities for delivery, analysis and sensing; issues persist for conventional applications. This includes liquid phase formulation localisation on the ocular surface once administered as formulated eye-drops; with the vast majority of dosage (>90%) escaping from the administered site due to tear production and various drainage mechanisms. The work presented here demonstrates a single needle electrohydrodynamic (EHD) engineering process to nano-coat (as an on demand and controllable fiber depositing method) the surface of multiple contact lenses rendering formulations to be stationary on the lens and at the bio-interface. The coating process was operational based on ejected droplet charge and glaucoma drug timolol maleate (TM) was used to demonstrate surface coating optimisation, bio-surface permeation properties (flux, using a bovine model) and various kinetic models thereafter. Polymers PVP, PNIPAM and PVP:PNIPAM (50:50%w/w) were used to encapsulate the active. Nano-fibrous and particulate samples were characterised using SEM, FTIR, DSC and TGA to confirm structural and thermal stability of surface coated formulations. More than 52% of nano-structured coatings (for all formulations) were <200nm in diameter. In vitro studies show coatings to exhibit biphasic release profiles; an initial burst release followed by sustained release; with TM-loaded PNIPAM coating releasing most drug after 24h (89.8%). Kinetic modelling (Higuchi, Korsmeyer-Peppas) was indicative of quasi-Fickian diffusion whilst biological evaluation demonstrates adequate ocular tolerability. Results from permeation studies indicate coated lenses are ideal to reduce dosing regimen, which in turn will reduce systemic drug absorption. Florescent microscopy demonstrated probe and probe embedded coating behaviour from lens surface in vitro. The multiple lens surface coating method demonstrates sustained drug release yielding promising results; suggesting both novel device and method to enhance drug activity at the eyes surface which will reduce formulation drainage.

Approaches in topical ocular drug delivery and developments in the use of contact lenses as drug-delivery devices.

Drug-delivery approaches have diversified over the last two decades with the emergence of nanotechnologies, smart polymeric systems and multimodal functionalities. The intended target for specific treatment of disease is the key defining developing parameter. One such area which has undergone significant advancements relates to ocular delivery. This has been expedited by the development of material advancement, mechanistic concepts and through the deployment of advanced process technologies. This review will focus on the developments within lens-based drug delivery while touching on conventional and current methods of topical ocular drug delivery. A summary table will provide quick reference to note the key findings in this area. In addition, the review also elucidates current theranostic and diagnostic approaches based on ocular lenses.

Pharmaceutical and biomaterial engineering via electrohydrodynamic atomization technologies.

Complex micro- and nano-structures enable crucial developments in the healthcare remit (e.g., pharmaceutical and biomaterial sciences). In recent times, several technologies have been developed and explored to address key healthcare challenges (e.g., advanced chemotherapy, biomedical diagnostics and tissue regeneration). Electrohydrodynamic atomization (EHDA) technologies are rapidly emerging as promising candidates to address these issues. The fundamental principle driving EHDA engineering relates to the action of an electric force (field) on flowing conducting medium (formulation) giving rise to a stable Taylor cone. Through careful optimization of process parameters, material properties and selection, nozzle and needle design, and collection substrate method, complex active micro- and nano-structures are engineered. This short review focuses on key selected recent and established advances in the field of pharmaceutical and biomaterial applications.

Influences of copolymers (Copovidone, Eudragit RL PO and Kollicoat MAE 30 DP) on stability and bioactivity of spray-dried and freeze-dried lysozyme.

Protein stability is the most crucial factor in protein pharmaceutical preparations. Various techniques were applied for producing stable protein formulations such as spray-drying and freeze-drying. However, heating and freezing stresses are disadvantages for proteins using these methods, respectively. Accordingly, excipients have been used to preserve therapeutic effects of proteins during processing and for long period of time. Therefore, influences of Copovidone, Eudragit® RL-PO and Kollicoat® MAE-30 DP (as excipients) on stability and integrity of lysozyme (as a model protein) in spray-dried and freeze-dried forms were investigated. Protein formulations in both dried forms were prepared without and with the addition of mentioned excipients at different concentrations. Protein formulations were characterized for yield determination, morphology using scanning electron microscopic (SEM), thermal analysis by Differential Scanning Calorimetry (DSC), secondary structure stability using Fourier transform infrared (FT-IR) spectroscopy and biological activity. All protein formulations were subjected to a stability study as solid protein formulations for 3 weeks at 24 °C/76% relative humidity and aqueous protein samples were stored at 50 °C for 30 min in a water bath. Results showed that Copovidone successfully preserved integrity and biological activity of lysozyme before and after storage in both spray-dried and freeze-dried forms with more advantage for using higher concentration of the same excipient. Smooth spheres of spray-dried lysozyme formulations with Copovidone were smaller than spray-dried lysozyme without and with Kollicoat® MAE-30 DP, which affected %yield produced. Copovidone has demonstrated valuable protection ability for lysozyme.

Formulation and evaluation of anti-rheumatic dexibuprofen transdermal patches: a quality-by-design approach.

Dexibuprofen (DXIBN) transdermal patches were formulated using various concentrations of selected polymeric excipients (matrix material; ethyl cellulose and polyvinylpyrrolidone, plasticizer (di-N-butyl phthalate), and a conventional permeation enhancer (almond oil)). Initial patch formulations were evaluated for their physiochemical properties (thickness, moisture uptake, final moisture content, and DXIBN content). Also, impact of patch components on resulting tensile strength and in vitro permeation were used to predict an optimal patch formulation using a quality-by-design (QbD) approach, which was subsequently evaluated and further compared with a commercial oral tablet dosage form for in vitro and in vivo release (rabbit model). Initially formulated patches demonstrated uniform thickness (0.44 ± 0.02 cm), relatively low moisture uptake (7.87 ± 1.11 w/w %), and highly acceptable drug loading values (100.0 ± 0.026%). The tensile strength of patches increased significantly with matrix polymer concentration and to a lesser degree with increase in plasticizer and permeation enhancer content, although these affected the permeation of DXIBN. Predicted properties (tensile strength and DXIBN steady-state flux) for the QbD-optimized formulation were in close agreement to experimental results. The QbD optimal patch formulation behavior differed significantly from the commercial tablet formulation in vivo. Such model-based predictions (QbD approach) will reduce cost and time in formulation development sciences.

Microneedle Coating Techniques for Transdermal Drug Delivery.

Drug administration via the transdermal route is an evolving field that provides an alternative to oral and parenteral routes of therapy. Several microneedle (MN) based approaches have been developed. Among these, coated MNs (typically where drug is deposited on MN tips) are a minimally invasive method to deliver drugs and vaccines through the skin. In this review, we describe several processes to coat MNs. These include dip coating, gas jet drying, spray coating, electrohydrodynamic atomisation (EHDA) based processes and piezoelectric inkjet printing. Examples of process mechanisms, conditions and tested formulations are provided. As these processes are independent techniques, modifications to facilitate MN coatings are elucidated. In summary, the outcomes and potential value for each technique provides opportunities to overcome formulation or dosage form limitations. While there are significant developments in solid degradable MNs, coated MNs (through the various techniques described) have potential to be utilized in personalized drug delivery via controlled deposition onto MN templates.

EHDA Spraying: A Multi-Material Nano-Engineering Route.

Electrohydrodynamic atomization (EHDA) enabling platform technologies have gathered significant momentum over the last two decades. Utilisation of the underpinning jetting process in tandem with desired materials (including polymers, ceramics, metals and even naturally occurring compounds such as peptides, DNA and cells) provides the basis for novel engineered therapies. Through EHDA processes, the generation of a variety of nano-meter and micro-meter scaled structures with control on surface and encapsulation features is attainable in a single step. While a host of adaptable EHDA techniques have evolved (e.g. printing and template patterning), there are two main processes that continue to dominate: electrospraying (ESy) and electrospinning (ESp). Although ESp has drawn considerable researcher interest for nanofibre applications, ESy is an important and timely process for nano- and micro-particle fabrication. Thus, an appropriate evaluation of ESy is vital. This short review focuses on key developments in the ESy field in relation to nanotechnologies with potential healthcare applications using metals, polymers and ceramics. An insight into the process of particle formation (during EHDA spraying or ESy), process parameters and materials specifications, is provided. Emerging biomedical and other healthcare research through nanotechnologies are highlighted.

In vitro characterisation of Span 65 niosomal formulations containing proteins.

Proteins can be encapsulated in niosomes as they are known to protect proteins against the surrounding environment. Niosomes of Span 65/cholesterol/pluronic F -127 were prepared by thin film hydration method. Insulin and lysozyme were chosen as model proteins. Niosomes were characterised for morphology by Transmission Electron Microscopy (TEM) and vesicles size using Dynamic Light Scattering. The entrapment efficiency of protein in niosomes was determined by complete vesicle disruption using 50:50% isopropanol:buf fer, followed by analysis of the resulting solutions by HPLC method. Thermal behaviour of the niosomes was investigated using Differential Scanning Calorimetry (DSC). Protection of proteins against simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were also assessed. The results showed that niosomes prepared with different molar ratios % of Span 65, cholesterol and pluronic F-127 successfully produced with insulin and lysozyme. For insulin containing niosomes, the ratio % of 64.7 (Span 65): 32.3 (cholesterol): 3.0 (pluronic F - 127) produced the highest protein encapsulation efficiency and the smallest vesicle size of 653.6 nm. For lysozyme containing niosomes, the maximum protein encapsulation was found in 72.75/24.25/3.00% molar ratio of Span 65/cholesterol/pluronic F -127 niosomes with vesicle size of 627.3 nm. The release study of proteins from the niosomal preparations in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) revealed that insulin and lysozyme efflux from the niosomes is a biphasic process. The results indicate that Span 65 niosomes could be developed as controlled release dosage forms for delivery of peptides and proteins such as insulin and lysozyme.

Compare and contrast the effects of surfactants (PluronicF-127 and CremophorEL) and sugars (ß-cyclodextrin and inulin) on properties of spray dried and crystallised lysozyme.

The stabilisation of proteins using different excipients in dried forms for possible therapeutic use is extensively studied. However, the effects of excipients on proteins in crystallised forms are sparsely documented. Therefore, the influences of PluronicF-127 and CremophorEL (as surfactants) and ß-cyclodextrin and inulin (as sugars) on stability and biological activity of lysozyme, a model protein, in spray dried and crystallised forms were investigated. Spray dried and crystallised lysozyme were prepared in absence and presence of the mentioned excipients in a concentration of 0.05% w/v. The protein formulations were characterised in both solution state (using biological assay, particle size analysis and protein concentration determination) and solid state (employing yield determination, scanning electron microscopic (SEM) examination, Fourier transform infrared (FT-IR) spectroscopy for secondary structure analysis and Differential Scanning Calorimetry (DSC) for thermal study). Also, protein samples were assayed for their biological activities after exposing to storage stability study for 20 weeks in solid states at 24 °C/76% relative humidity (RH) and in aqueous states at 24 °C. The results showed that lysozyme crystals with CremophorEL, PluronicF-127, ß-cyclodextrin and inulin maintained protein thermal stability (as indicated by DSC) to greater extent compared with spray dried protein formulations. Also, PluronicF-127 was competent to recover 100% lysozyme from crystallisation protein solutions (as confirmed by yield determination); this surfactant was able to prevent aggregate formation within spray dried lysozyme (as demonstrated by particle size analysis). The presence of PluronicF-127, ß-cyclodextrin and inulin preserved the protein biological activity in freshly prepared spray dried and crystallised samples. PluronicF-127 was competent to protect lysozyme in both spray dried and crystallised forms after storage. PluronicF-127 has proved to be a promising protectant of proteins. The improved stability of the spray dried and crystallised protein containing PluronicF-127 shows promise for delivery of proteins via inhalation (in a spray dried form which has particle size range suitable for inhalation as revealed by particle size analysis and SEM) and injectable routes (in spray dried and crystallised forms). The way excipients react with proteins is different in the case of spray drying and crystallisation techniques, hence the choice of the additives and the processing techniques play a great role in controlling protein properties, activity and stability as shown in this study.