Gold Nanoparticles: Tunable Characteristics and Potential for Nasal Drug Delivery.

A general procedure to prepare gold nanourchins (GNUs) via a seed-mediated method was followed using dopamine hydrochloride as a reducing agent and silver nitrate salt (AgNO3) as a shape-directing agent. The novelty of this study comes from the successful incorporation of the prepared gold urchins as an aqueous suspension in a nasal pressurized metered dose inhaler (pMDI) formulation and the investigation of their potential for olfactory targeting for direct nose-to-brain drug delivery (NTBDD). The developed pMDI formulation was composed of 0.025% w/w GNUs, 2% w/w Milli-Q water, and 2% w/w EtOH, with the balance of the formulation being HFA134a propellant. Particle integrity and aerosolization performance were examined using an aerosol exposure system, whereas the nasal deposition profile was tested in a sectioned anatomical replica of human nasal airways. The compatibility of the gold dispersion with the nasal epithelial cell line RPMI 2650 was also investigated in this study. Colloidal gold was found to be stable following six-month storage at 4 °C and during the lyophilization process utilizing a pectin matrix for complete re-dispersibility in water. The GNUs were intact and discrete following atomization via a pMDI, and 13% of the delivered particles were detected beyond the nasal valve, the narrowest region in the nasal cavity, out of which 5.6% was recovered from the olfactory region. Moreover, the formulation was found to be compatible with the human nasal epithelium cell line RPMI 2650 and excellent cell viability was observed. The formulated GNU-HFA-based pMDI is a promising approach for intranasal drug delivery, including deposition in the olfactory region, which could be employed for NTBDD applications.

A Cell-Based Nasal Model for Screening the Deposition, Biocompatibility, and Transport of Aerosolized PLGA Nanoparticles.

The olfactory region of the nasal cavity directly links the brain to the external environment, presenting a potential direct route to the central nervous system (CNS). However, targeting drugs to the olfactory region is challenging and relies on a combination of drug formulation, delivery device, and administration technique to navigate human nasal anatomy. In addition, in vitro and in vivo models utilized to evaluate the performance of nasal formulations do not accurately reflect deposition and uptake in the human nasal cavity. The current study describes the development of a respirable poly(lactic-co-glycolic acid) nanoparticle (PLGA NP) formulation, delivered via a pressurized metered dose inhaler (pMDI), and a cell-containing three-dimensional (3D) human nasal cast model for deposition assessment of nasal formulations in the olfactory region. Fluorescent PLGA NPs (193 ± 3 nm by dynamic light scattering) were successfully formulated in an HFA134a-based pMDI and were collected intact following aerosolization. RPMI 2650 cells, widely employed as a nasal epithelial model, were grown at the air-liquid interface (ALI) for 14 days to develop a suitable barrier function prior to exposure to the aerosolized PLGA NPs in a glass deposition apparatus. Direct aerosol exposure was shown to have little effect on cell viability. Compared to an aqueous NP suspension, the transport rate of the aerosolized NPs across the RPMI 2650 barrier was higher at all time points indicating the potential advantages of delivery via aerosolization and the importance of employing ALI cellular models for testing respirable formulations. The PLGA NPs were then aerosolized into a 3D-printed human nasal cavity model with an insert of ALI RPMI 2650 cells positioned in the olfactory region. Cells remained highly viable, and there was significant deposition of the fluorescent NPs on the ALI cultures. This study is a proof of concept that pMDI delivery of NPs is a viable means of targeting the olfactory region for nose-to-brain drug delivery (NTBDD). The cell-based model allows not only maintenance under ALI culture conditions but also sampling from the basal chamber compartment; hence, this model could be adapted to assess drug deposition, uptake, and transport kinetics in parallel under real-life settings.

An in vitro and ex vivo wound infection model to test topical and systemic treatment with antibiotics.

AIMS: This study aimed to develop a wound infection model that could be used to test antibiotic-loaded electrospun matrices for the topical treatment of infected skin and compare the effectiveness of this treatment to systemically applied antibiotics. METHODS AND RESULTS: 3D-printed flow chambers were made in which Staphylococcus aureus biofilms were grown either on a polycarbonate membrane or explanted porcine skin. The biofilms were then treated either topically, by placing antibiotic-loaded electrospun matrices on top of the biofilms, or systemically by the addition of antibiotics in the growth medium that flowed underneath the membrane or skin. The medium that was used was either a rich medium or an artificial wound fluid. The results showed that microbial viability in the biofilms was reduced to a greater extent with the topical electrospun matrices when compared to systemic treatment. CONCLUSIONS: An ex vivo infection model was developed that is flexible and can be used to test both topical and systemic treatment of wound infections. It represents a significant improvement over previous in vitro models that we have used to test electrospun membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of a relatively simple wound infection model in which different delivery methods and dosage regimes can be tested is beneficial for the development of improved treatments for wound infections.

Disposable Coverslip for Rapid Throughput Screening of Malaria Using Attenuated Total Reflection Spectroscopy.

Malaria is considered to be one of the most catastrophic health issues in the whole world. Vibrational spectroscopy is a rapid, robust, label-free, inexpensive, highly sensitive, nonperturbative, and nondestructive technique with high diagnostic potential for the early detection of disease agents. In particular, the fingerprinting capability of attenuated total reflection spectroscopy is promising as a point-of-care diagnostic tool in resource-limited areas. However, improvements are required to expedite the measurements of biofluids, including the drying procedure and subsequent cleaning of the internal reflection element to enable high throughput successive measurements. As an alternative, we propose using an inexpensive coverslip to reduce the sample preparation time by enabling multiple samples to be collectively dried together under the same temperature and conditions. In conjunction with partial least squares regression, attenuated total reflection spectroscopy was able to detect and quantify the parasitemia with root mean square error of cross-validation and R2 values of 0.177 and 0.985, respectively. Here, we characterize an inexpensive, disposable coverslip for the high throughput screening of malaria parasitic infections and thus demonstrate an alternative approach to direct deposition of the sample onto the internal reflection element.

Intestinal Transcytosis of a Protein Cargo and Nanoparticles Mediated by a Non-Toxic Form of Pseudomonas aeruginosa Exotoxin A.

The low permeability of nanoparticles (NPs) across the intestinal epithelium remains a major challenge for their application of delivering macromolecular therapeutic agents via the oral route. Previous studies have demonstrated the epithelial transcytosis capacity of a non-toxic version of Pseudomonas aeruginosa exotoxin A (ntPE). Here, we show that ntPE can be used to deliver the protein cargo green fluorescent protein (GFP) or human growth hormone (hGH), as genetic fusions, across intact rat jejunum in a model where the material is administered by direct intra-luminal injection (ILI) in vivo in a transcytosis process that required less than 15 min. Next, ntPE chemically coupled onto biodegradable alginate/chitosan condensate nanoparticles (AC NPs-ntPE) were shown to transport similarly to ntPE-GFP and ntPE-hGH across rat jejunum. Finally, AC NPs-ntPE loaded with GFP as a model cargo were demonstrated to undergo a similar transcytosis process that resulted in GFP being colocalized with CD11c+ cells in the lamina propria after 30 min. Control NP preparations, not decorated with ntPE, were not observed within polarized epithelial cells or within the cells of the lamina propria. These studies demonstrate the capacity of ntPE to facilitate the transcytosis of a covalently associated protein cargo as well as a biodegradable NP that can undergo transcytosis across the intestinal epithelium to deliver a noncovalently associated protein cargo. In sum, these studies support the potential applications of ntPE to facilitate the oral delivery of macromolecular therapeutics under conditions of covalent or non-covalent association.

In Vitro Evaluation of Nasal Aerosol Depositions: An Insight for Direct Nose to Brain Drug Delivery.

The nasal cavity is an attractive route for both local and systemic drug delivery and holds great potential for access to the brain via the olfactory region, an area where the blood-brain barrier (BBB) is effectively absent. However, the olfactory region is located at the roof of the nasal cavity and only represents ~5-7% of the epithelial surface area, presenting significant challenges for the deposition of drug molecules for nose to brain drug delivery (NTBDD). Aerosolized particles have the potential to be directed to the olfactory region, but their specific deposition within this area is confounded by a complex combination of factors, which include the properties of the formulation, the delivery device and how it is used, and differences in inter-patient physiology. In this review, an in-depth examination of these different factors is provided in relation to both in vitro and in vivo studies and how advances in the fabrication of nasal cast models and analysis of aerosol deposition can be utilized to predict in vivo outcomes more accurately. The challenges faced in assessing the nasal deposition of aerosolized particles within the paediatric population are specifically considered, representing an unmet need for nasal and NTBDD to treat CNS disorders.

Decellularized grass as a sustainable scaffold for skeletal muscle tissue engineering.

Scaffold materials suitable for the scale-up and subsequent commercialization of tissue engineered products should ideally be cost effective and accessible. For the in vitro culture of certain adherent cells, synthetic fabrication techniques are often employed to produce micro- or nano-patterned substrates to influence cell attachment, morphology, and alignment via the mechanism of contact guidance. Here we present a natural scaffold, in the form of decellularized amenity grass, which retains its natural striated topography and supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts, without the need for additional functionalization. This presents an inexpensive, sustainable scaffold material and structure for tissue engineering applications capable of influencing cell alignment, a desired property for the culture of skeletal muscle and other anisotropic tissues.

Silk fibroin/gelatin microcarriers as scaffolds for bone tissue engineering.

Microcarrier cell scaffolds have potential as injectable cell delivery vehicles or as building blocks for tissue engineering. The use of small cell carriers allows for a 'bottom up' approach to tissue assembly when moulding microparticles into larger structures, which can facilitate the introduction of hierarchy by layering different matrices and cell types, while evenly distributing cells through the structure. In this work, silk fibroin (SF), purified from Bombyx mori cocoons, was blended with gelatin (G) to produce materials composed of varying ratios of the two components (SF: G 25:75, 50:50, and 75:25). Cell compatibility to these materials was first confirmed in two-dimensional culture and found to be equivalent to standard tissue culture plastic, and better than SF or G alone. The mechanical properties of the blends were investigated and the blended materials were found to have increased Young's moduli over SF alone. Microcarriers of SF/G blends with defined diameters were generated in a reproducible manner through the use of an axisymmetric flow focussing device, constructed from off-the-shelf parts and fittings. These SF/G microcarriers supported adhesion of rat mesenchymal stem cells with high degrees of efficiency under dynamic culture conditions and, after culturing in osteogenic differentiation medium, cells were shown to have characteristics typical of osteoblasts. This work illustrates that microcarriers composed of SF/G blends are promising building blocks for osteogenic tissue engineering.

The effect of ozone gas sterilization on the properties and cell compatibility of electrospun polycaprolactone scaffolds.

The growing area of tissue engineering has the potential to alleviate the shortage of tissues and organs for transplantation, and electrospun biomaterial scaffolds are extremely promising devices for translating engineered tissues into a clinical setting. However, to be utilized in this capacity, these medical devices need to be sterile. Traditional methods of sterilization are not always suitable for biomaterials, especially as many commonly used biomedical polymers are sensitive to chemical-, thermal- or radiation-induced damage. Therefore, the objective of this study was to evaluate the suitability of ozone gas for sterilizing electrospun scaffolds of polycaprolactone (PCL), a polymer widely utilized in tissue engineering and regenerative medicine applications, by evaluating if scaffolds composed of either nanofibres or microfibres were differently affected by the sterilization method. The sterility, morphology, mechanical properties, physicochemical properties, and response of cells to nanofibrous and microfibrous PCL scaffolds were assessed after ozone gas sterilization. The sterilization process successfully sterilized the scaffolds and preserved most of their initial attributes, except for mechanical properties. However, although the scaffolds became weaker after sterilization, they were still robust enough to use as tissue engineering scaffolds and this treatment increased the proliferation of L929 fibroblasts while maintaining cell viability, suggesting that ozone gas treatment may be a suitable technique for the sterilization of polymer scaffolds which are significantly damaged by other methods.

Dual crosslinked pectin-alginate network as sustained release hydrophilic matrix for repaglinide.

Repaglinide, an oral antidiabetic agent, has a rapid onset of action and short half-life of approximately 1h. Developing a controlled and prolonged release delivery system is required to maintain its therapeutic plasma concentration and to eliminate its adverse effects particularly hypoglycemia. The present study aimed to develop controlled release repaglinide loaded beads using sodium alginate and pectin with dual cross-linking for effective control of drug release. The prepared beads were characterized for size, percentage drug entrapment efficiency, in vitro drug release and the morphological examination using scanning electron microscope. For the comparative study, the release profile of a marketed conventional tablet of repaglinide (Prandin® tablets 2mg, Novo Nordisk) was determined by the same procedure as followed for beads. The particle size of beads was in the range of 698±2.34-769±1.43µm. The drug entrapment efficiency varied between 55.24±4.61 to 82.29±3.42%. The FTIR results suggest that there was no interaction between repaglinide and excipients. The XRD and DSC results suggest partial molecular dispersion and amorphization of the drug throughout the system. These results suggest that repaglinide did not dissolve completely in the polymer composition and seems not to be involved in the cross-linking reaction. The percent drug release was decreased with higher polymer concentrations. In conclusion, the developed beads could enhance drug entrapment efficiency, prolong the drug release and enhance bioavailability for better control of diabetes.

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

Ozone Gas as a Benign Sterilization Treatment for PLGA Nanofiber Scaffolds.

The use of electrospun nanofibers for tissue engineering and regenerative medicine applications is a growing trend as they provide improved support for cell proliferation and survival due, in part, to their morphology mimicking that of the extracellular matrix. Sterilization is a critical step in the fabrication process of implantable biomaterial scaffolds for clinical use, but many of the existing methods used to date can negatively affect scaffold properties and performance. Poly(lactic-co-glycolic acid) (PLGA) has been widely used as a biodegradable polymer for 3D scaffolds and can be significantly affected by current sterilization techniques. The aim of this study was to investigate pulsed ozone gas as an alternative method for sterilizing PLGA nanofibers. The morphology, mechanical properties, physicochemical properties, and response of cells to PLGA nanofiber scaffolds were assessed following different degrees of ozone gas sterilization. This treatment killed Geobacillus stearothermophilus spores, the most common biological indicator used for validation of sterilization processes. In addition, the method preserved all of the characteristics of nonsterilized PLGA nanofibers at all degrees of sterilization tested. These findings suggest that ozone gas can be applied as an alternative method for sterilizing electrospun PLGA nanofiber scaffolds without detrimental effects.

Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and are Compatible with Human Skin Cells.

PURPOSE: To investigate the destruction of clinically-relevant bacteria within biofilms via the sustained release of the antibiotic tetracycline from zein-based electrospun polymeric fibrous matrices and to demonstrate the compatibility of such wound dressing matrices with human skin cells. METHODS: Zein/PCL triple layered fibrous dressings with entrapped tetracycline were electrospun. The successful entrapment of tetracycline in these dressings was validated. The successful release of bioactive tetracycline, the destruction of preformed biofilms, and the viability of fibroblast (FEK4) cells were investigated. RESULTS: The sustained release of tetracycline from these matrices led to the efficient destruction of preformed biofilms from Staphylococcus aureus MRSA252 in vitro, and of MRSA252 and ATCC 25923 bacteria in an ex vivo pig skin model using 1 × 1 cm square matrices containing tetracycline (30 µg). Human FEK4 cells grew normally in the presence of these matrices. CONCLUSIONS: The ability of the zein-based matrices to destroy bacteria within increasingly complex in vitro biofilm models was clearly established. An ex vivo pig skin assay showed that these matrices, with entrapped tetracycline, efficiently kill bacteria and this, combined with their compatibility with a human skin cell line suggest these matrices are well suited for applications in wound healing and infection control.

An investigation into the stability of commercial versus MG63-derived hepatocyte growth factor under flow cultivation conditions.

The scale-up of tissue engineering cell culture must ensure that conditions are maintained while also being cost effective. Here we analyse the stability of hepatocyte growth factor (HGF) to investigate whether concentrations change under dynamic conditions, and compare commercial recombinant human HGF as an additive in 'standard medium', to HGF secreted by the osteosarcoma cell line MG63 as a 'preconditioned medium'. After 3 h under flow conditions, HGF in the standard medium degraded to 40% of its original concentration but HGF in the preconditioned medium remained at 100%. The concentration of secreted HGF was 10 times greater than the working concentration of commercially-available HGF. Thus HGF within this medium has increased stability; MG63-derived HGF should therefore be investigated as a cost-effective alternative to current lyophilised powders for use in in vitro models. Furthermore, we recommend that those intending to use HGF (or other growth factors) should consider similar stability testing before embarking on experiments with media flow.

A hydrazide-anchored dendron scaffold for chemoselective ligation strategies.

Chemoselective ligation, including "click" chemistry, has found wide utility in general synthetic strategies and the specific modification of polymers and biomolecules. This has resulted in a number of applications of such approaches, particularly in the biomedical area, including diagnostic imaging and drug delivery. However, tools to chemoselectively decorate target molecules with multiple copies of a particular drug, ligand or label are lacking. We describe the design and synthesis of a hydrazide-anchored dendron scaffold for chemoselective ligation to carbonyl moieties, and demonstrate its use in the modification of aldehyde-rich surfaces with the RGD integrin-binding ligand.

Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

Multicellular aggregation of maltol-modified cells triggered by Fe(3+) ions.

The synthesis of a maltol-derived hydrazide is described which, once attached to a cell surface, induces rapid multicellular aggregation selectively in the presence of Fe(3+) ions. Heterocellular aggregates are also reported.

Killing bacteria within biofilms by sustained release of tetracycline from triple-layered electrospun micro/nanofibre matrices of polycaprolactone and poly(ethylene-co-vinyl acetate).

We report the controlled release of the antibiotic tetracycline (tet) HCl from a triple-layered electrospun matrix consisting of a central layer of poly(ethylene-co-vinyl acetate (PEVA) sandwiched between outer layers of poly-ε-caprolactone (PCL). These micro/nanofibre layers with tet successfully encapsulated (essentially quantitatively at 3 and 5 % w/w) in each layer, efficiently inhibited the growth of a panel of bacteria, including clinical isolates, as shown by a modified Kirby-Bauer disc assay. Furthermore, they demonstrated high biological activity in increasingly complex models of biofilm formation (models that are moving closer to the situation in a wound) by stopping biofilm formation, by killing preformed biofilms and killing mature, dense biofilm colonies of Staphylococcus aureus MRSA252. Tet is clinically useful with potential applications in wound healing and especially in complicated skin and skin-structure infections; electrospinning provides good encapsulation efficiency of tet within PCL/PEVA/PCL polymers in micro/nanofibre layers which display sustained antibiotic release in formulations that are anti-biofilm.

Zein/polycaprolactone electrospun matrices for localised controlled delivery of tetracycline.

We report the controlled release of the antibiotic tetracycline (Tet) from triple-layered (3L) electrospun matrices consisting of zein or a zein/PCL blend, where the drug was loaded into the central layer with the two outer layers acting as diffusion barriers. These fibrous matrices successfully encapsulated Tet and efficiently inhibited the growth of a clinical isolate, the methicillin-resistant Staphylococcus aureus strain MRSA252, as demonstrated in a modified Kirby-Bauer disc assay over 5 days. Whilst untreated zein fibres are unstable in an aqueous environment, rapidly shrinking due to plasticisation and film formation, blending zein with PCL stabilised the electrospun matrices and prevented them from shrinking. These 3L formulations display sustained antibiotic release and provide a proof of concept for zein-based polymeric matrices as wound dressings to treat or prevent bacterial infection. This is the first demonstration of the controlled release of a clinically used antibiotic from electrospun zein-based matrices.

Simple pyrazoline and pyrazole "turn on" fluorescent sensors selective for Cd2+ and Zn2+ in MeCN.

An efficient two-step synthesis of pyrazoline ligand is described which is an effective "turn on" fluorescent sensor for Cd(2+) in MeCN. Oxidation to the corresponding pyrazole ligand creates a "turn on" fluorescent sensor now selective for Zn(2+) and able to distinguish it from Cd(2+).