Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties.

Hydrophobically Grafted Pullulan Nanocarriers for Percutaneous Delivery: Preparation and Preliminary In Vitro Characterisation.

Polymeric colloidal nanocarriers formulated from hydrophobically grafted carbohydrates have been the subject of intensive research due to their potential to increase the percutaneous penetration of hydrophilic actives. To this goal, a series of hydrophobically grafted pullulan (BMO-PUL) derivatives with varying degree of grafting (5-64%) was prepared through functionalisation with 2-(butoxymethyl)oxirane. The results demonstrated that monodispersed BMO-PUL nanocarriers (size range 125-185 nm) could be easily prepared via nanoprecipitation; they exhibit close-to-spherical morphology and adequate stability at physiologically relevant pH. The critical micellar concentration of BMO-PUL was found to be inversely proportional to their molecular weight (Mw) and degree of grafting (DG), with values of 60 mg/L and 40 mg/L for DG of 12.6% and 33.8%, respectively. The polymeric nanocarriers were loaded with the low Mw hydrophilic active α-arbutin (16% loading), and the release of this active was studied at varying pH values (5 and 7), with a slightly faster release observed in acidic conditions; the release profiles can be best described by a first-order kinetic model. In vitro investigations of BMO-PUL nanocarriers (concentration range 0.1-4 mg/mL) using immortalised skin human keratinocytes cells (HaCaT) evidenced their lack of toxicity, with more than 85% cell viability after 24 h. A four-fold enhance in arbutin permeation through HaCaT monolayers was recorded when the active was encapsulated within the BMO-PUL nanocarriers. Altogether, the results obtained from the in vitro studies highlighted the potential of BMO-PUL nanocarriers for percutaneous delivery applications, which would warrant further investigation in vivo.

Personalised 3D Printed Medicines: Optimising Material Properties for Successful Passive Diffusion Loading of Filaments for Fused Deposition Modelling of Solid Dosage Forms.

Although not readily accessible yet to many community and hospital pharmacists, fuse deposition modelling (FDM) is a 3D printing technique that can be used to create a 3D pharmaceutical dosage form by employing drug loaded filaments extruded via a nozzle, melted and deposited layer by layer. FDM requires printable filaments, which are commonly manufactured by hot melt extrusion, and identifying a suitable extrudable drug-excipient mixture can sometimes be challenging. We propose here the use of passive diffusion as an accessible loading method for filaments that can be printed using FDM technology to allow for the fabrication of oral personalised medicines in clinical settings. Utilising Hansen Solubility Parameters (HSP) and the concept of HSP distances (Ra) between drug, solvent, and filament, we have developed a facile pre-screening tool for the selection of the optimal combination that can provide a high drug loading (a high solvent-drug Ra, >10, and an intermediate solvent-filament Ra value, ~10). We have identified that other parameters such as surface roughness and stiffness also play a key role in enhancing passive diffusion of the drug into the filaments. A predictive model for drug loading was developed based on Support Vector Machine (SVM) regression and indicated a strong correlation between both Ra and filament stiffness and the diffusion capacity of a model BCS Class II drug, nifedipine (NFD), into the filaments. A drug loading, close to 3% w/w, was achieved. 3D printed tablets prepared using a PVA-derived filament (Hydrosupport, 3D Fuel) showed promising characteristics in terms of dissolution (with a sustained release over 24 h) and predicted chemical stability (>3 years at 25 °C/60% relative humidity), similar to commercially available NFD oral dosage forms. We believe FDM coupled with passive diffusion could be implemented easily in clinical settings for the manufacture of tailored personalised medicines, which can be stored over long periods of time (similar to industrially manufactured solid dosage forms).

Evaluation of Antibacterial and Cytotoxicity Properties of Silver Nanowires and Their Composites with Carbon Nanotubes for Biomedical Applications.

In this work, we prepared silver nanowires (AgNWs) via the polyol method in the presence or absence of single wall carbon nanotubes (CNTs) and tested their physicochemical, antibacterial and cytotoxic properties. Results showed that the introduction of CNTs lead to the formation of AgNWs at lower temperature, but the final product characteristics of AgNWs and AgNWs-CNT were not significantly different. AgNWs exhibited antibacterial properties against all the studied bacterial species via the formation of oxygen reactive species (ROS) and membrane damage. Furthermore, AgNWs exhibited a dose-dependent and time-dependent toxicity at concentrations ≥ 10 µg/mL. Fibroblasts appeared to be more resistant than human colorectal adenocarcinoma (Caco-2) and osteoblasts to the toxicity of AgNWs. The cytotoxicity of AgNWs was found to be related to the formation of ROS, but not to membrane damage. Overall, these results suggest that AgNWs are potential antibacterial agents against E. coli, S. aureus, MRSA and S. saprophyticus, but their dosage needs to be adjusted according to the route of administration.

Engineering butylglyceryl-modified polysaccharides towards nanomedicines for brain drug delivery.

Colloidal systems prepared from carbohydrates are subject of intense research due to their potential to enhance drug permeability through biological membranes, however their characteristics and performance are never compared directly. Here we report the results of a comparative investigation of a series of butylglyceryl-modified polysaccharides (chitosan, guar gum, and pullulan) that were formulated into nanoparticles and loaded with a range of model actives (Doxorubicin, Rhodamine B, Angiotensin II). Butylglyceryl-modified guar gum and corresponding pullulan nanocarriers were more stable at physiological pH compared to those obtained from modified chitosan, and studies of the in-vitro interactions with mouse brain endothelial cells (bEnd3) indicated an increased biological membrane permeability and lack of toxicity at application-relevant concentrations. No significant haemolytic effect was observed, and confocal microscopy and flow cytometry studies confirmed the efficient cellular uptake and cytoplasmic localisation of NPs. Most promising characteristics for brain drug delivery applications were demonstrated by butylglyceryl pullulan nanocarriers.

Antibacterial PMMA Composite Cements with Tunable Thermal and Mechanical Properties.

PMMA-based cements are the most used bone cements in vertebroplasty and total hip arthroplasty. However, they present several drawbacks, including susceptibility to bacterial infection, monomer leakage toxicity, and high polymerization temperature, which can all lead to damage to the surrounding tissues and their failure. In the present study, silver nanowires (AgNWs) have been introduced to bestow antibacterial properties; chitosan (CS) to promote porosity and to reduce the polymerization temperature, without negatively affecting the mechanical performance; and methacryloyl chitosan (CSMCC) to promote cross-linking with methyl methacrylate (MMA) and reduce the quantity of monomer required for polymerization. Novel PMMA cements were formulated containing AgNWs (0 and 1% w/w) and CS or CSMCC at various concentrations (0, 10, 20, and 30% w/w), testing two different ratios of powder and MMA (P/L). Mechanical, thermal, antibacterial, and cytotoxic properties of the resulting composite cements were tested. Cements with concentrations of CS > 10% presented a significantly reduced polymerization temperature. The mechanical performances were affected for concentrations > 20% with a P/L concentration equal to 2:1. Concentrations of AgNWs as low as 1% w/w conferred antimicrobial activity against S. aureus, whereas biofilm formation on the surface of the cements was increased when CS was included in the preparation. The combination of CS and AgNWs allowed a higher concentration of Ag+ to be released over time with enhanced antimicrobial activity. Inclusion of AgNWs did not affect cell viability on the scaffolds. In conclusion, a combination of CS and AgNWs may be beneficial for reducing both polymerization temperature and biofilm formation, without significantly affecting mesenchymal stem cell proliferation on the scaffolds. No advantages have been noticed as a result of the reducing P/L ratio or using CSMCC instead of CS.

Butylglyceryl Pectin Nanoparticles: Synthesis, Formulation and Characterization.

Pectin is a polysaccharide with very good gel forming properties that traditionally has found important applications in foods and pharmaceutical industries. Although less studied, chemical modifications of pectin leading to a decrease in its hydrophilicity can be useful for the development of novel drug carriers. To this aim, butylglyceryl pectins (P-OX4) were synthesized via functionalization with n-butylglycidyl ether and subsequently formed into nanoparticles. Chromatographic, spectroscopic, and thermal analytical methods were employed to characterize the novel butylglyceryl pectins (P-OX4) obtained, prior to their formulation into nanoparticles via nanoprecipitation. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectroscopy confirmed a degree of modification in these materials in the range 10.4-13.6%, and thermal stability studies indicated an increase in both the thermal decomposition onset and glass transition temperature values (compared to those of the original pectin). An increase in the molecular weight and a decrease in the viscosity of P-OX4, when compared to the starting material, were also observed. The resulting nanoformulations were investigated in terms of particle morphology, size and stability, and it was found that particles were roughly spherical, with their size below 300 nm, and a negative zeta potential (-20 to -26 mV, indicating good stability). Having demonstrated the ability to load Doxorubicin at the level of 10%, their potential in drug delivery applications warrants further investigations.

Sustained Release from Injectable Composite Gels Loaded with Silver Nanowires Designed to Combat Bacterial Resistance in Bone Regeneration Applications.

One-dimensional nanostructures, such as silver nanowires (AgNWs), have attracted considerable attention owing to their outstanding electrical, thermal and antimicrobial properties. However, their application in the prevention of infections linked to bone tissue regeneration intervention has not yet been explored. Here we report on the development of an innovative scaffold prepared from chitosan, composite hydroxyapatite and AgNWs (CS-HACS-AgNWs) having both bioactive and antibacterial properties. In vitro results highlighted the antibacterial potential of AgNWs against both gram-positive and gram-negative bacteria. The CS-HACS-AgNWs composite scaffold demonstrated suitable Ca/P deposition, improved gel strength, reduced gelation time, and sustained Ag⁺ release within therapeutic concentrations. Antibacterial studies showed that the composite formulation was capable of inhibiting bacterial growth in suspension, and able to completely prevent biofilm formation on the scaffold in the presence of resistant strains. The hydrogels were also shown to be biocompatible, allowing cell proliferation. In summary, the developed CS-HACS-AgNWs composite hydrogels demonstrated significant potential as a scaffold material to be employed in bone regenerative medicine, as they present enhanced mechanical strength combined with the ability to allow calcium salts deposition, while efficiently decreasing the risk of infections. The results presented justify further investigations into the potential clinical applications of these materials.

Towards carborane-functionalised structures for the treatment of brain cancer.

Boron neutron capture therapy (BNCT) is a promising targeted chemoradiotherapeutic technique for the management of invasive brain tumors, such as glioblastoma multiforme (GBM). A prerequisite for effective BNCT is the selective targeting of tumour cells with 10B-rich therapeutic moieties. To this end, polyhedral boranes, especially carboranes, have received considerable attention because they combine a high boron content with relative low toxicity and metabolic inertness. Here, we review progress in the molecular design of recently investigated carborane derivatives in light of the widely accepted performance requirements for effective BNCT.

Investigations of octylglyceryl dextran-graft-poly(lactic acid) nanoparticles for peptide delivery to the brain.

AIM: Develop modified dextran nanoparticles showing potential to assist with drug permeation across the blood-brain barrier for the delivery of neuropeptides. METHODS: Nanoparticles loaded by emulsification with model macromolecular actives were characterized in terms of stability, cytotoxicity and drug-release behavior. Peptide-loaded nanoformulations were tested in an in vivo trout model and in food-deprived mice. RESULTS: Nanoformulations loaded with model peptides showed good stability and appeared nontoxic in low concentration against human brain endothelial cells. They were found to preserve the bioactivity of loaded peptides (angiotensin II) as demonstrated in vivo using a trout model, and to induce a transient reduction of food consumption in mice when loaded with an anorexigenic octaneuropeptide. CONCLUSION: Octylglyceryl dextran-graft-poly(lactic acid) nanoparticles formulated by emulsification demonstrate potential for peptide delivery.

Artemisinins as potential anticancer agents: uptake detection in erythrocytes using Fourier transform infrared spectroscopy and cytotoxicity against bladder cancer cells.

AIMS: Semisynthetic derivatives of the antimalarial drug artemisinin may also possess anticancer properties. The ability to detect artemisinin uptake and distribution in cells would facilitate live cell imaging without labelling. This study describes mid-range infrared absorption spectra for three artemisinin variants and attempts to detect their presence in a simple cell model (erythrocytes). Cytotoxicity assays assess potential anticancer properties against bladder cancer cells. METHODS: Mid-range Fourier transform infrared spectra were obtained from dry preparations of dihydroartemisinin (DHA), artesunate (ART) and artemether (ARTE). Erythrocytes were prepared from normal blood and incubated for 30 min at 37°C with the three artemisinin derivatives. Cytospin preparations were prepared on aluminium foil for spectroscopy. Potential for growth inhibition in the RT112 bladder carcinoma cell line was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide residual viable biomass method. RESULTS: Spectra were obtained from the three native compounds. Repeat scans after 8 weeks showed ART and ARTE to be stable, stored under manufacturer's recommendations. DHA exhibited marked changes over the same period. It was possible by subtraction to detect DHA in cytospins, but not ART or ARTE. The fit between the subtraction spectrum and that of the native compound was >80%. DHA and ART showed strong cytotoxic potential against RT112 cells. CONCLUSIONS: The artemisinin derivatives tested exhibit unique mid-range infrared absorption spectra which can be used to monitor degradation and, for DHA, can be detected by subtraction in loaded erythrocytes rendering future imaging studies feasible. Its cytotoxic efficacy against RT112 cells suggests bladder cancer as a possible target disease.

Hollow-layered nanoparticles for therapeutic delivery of peptide prepared using electrospraying.

The viability of single and coaxial electrospray techniques to encapsulate model peptide-angiotensin II into near mono-dispersed spherical, nanocarriers comprising N-octyl-O-sulphate chitosan and tristearin, respectively, was explored. The stability of peptide under controlled electric fields (during particle generation) was evaluated. Resulting nanocarriers were analysed using dynamic light scattering and electron microscopy. Cell toxicity assays were used to determine optimal peptide loading concentration (~1 mg/ml). A trout model was used to assess particle behaviour in vivo. A processing limit of 20 kV was determined. A range of electrosprayed nanoparticles were formed (between 100 and 300 nm) and these demonstrated encapsulation efficiencies of ~92 ± 1.8%. For the single needle process, particles were in matrix form and for the coaxial format particles demonstrated a clear core-shell encapsulation of peptide. The outcomes of in vitro experiments demonstrated triphasic activity. This included an initial slow activity period, followed by a rapid and finally a conventional diffusive phase. This was in contrast to results from in vivo cardiovascular activity in the trout model. The results are indicative of the substantial potential for single/coaxial electrospray techniques. The results also clearly indicate the need to investigate both in vitro and in vivo models for emerging drug delivery systems.

Peptide Self-Assemblies for Drug Delivery.

Peptide amphiphiles (PAs) are novel engineered biomaterials able to self-assemble into supramolecular systems that have shown significant promise in drug delivery across the cell membane and across challenging biological barriers showing promise in the field of brain diseases, regenerative medicine and cancer. PAs are amino-acid block co-polymers, with a peptide backbone composed usually of 8-30 amino acids, a hydrophilic block formed by polar amino acids, a hydrophobic block which usually entails either non-polar or aromatic amino acids and alkyl, acyl or aryl lipidic tails and in some cases a spacer or a conjugated targeting moiety. Finely tuning the balance between the hydrophilic and hydrophobic blocks results in a range of supramolecular structures that are usually stabilised by hydrophobic, electrostatic, ß-sheet hydrogen bonds and π-π stacking interactions. In an aqueous environment, the final size, shape and interfacial curvature of the PA is a result of the complex interplay of all these interactions. Lanreotide is the first PA to be licensed for the treatment of acromegaly and neuroendocrine tumours as a hydrogel administered subcutaneously, while a number of other PAs are undergoing preclinical development. This review discusses PAs architecture fundamentals that govern their self-assembly into supramolecular systems for applications in drug delivery.

Nanoparticles of alkylglyceryl-dextran-graft-poly(lactic acid) for drug delivery to the brain: Preparation and in vitro investigation.

Poly(lactic acid), which has an inherent tendency to form colloidal systems of low polydispersity, and alkylglyceryl-modified dextran - a material designed to combine the non-immunogenic and stabilising properties of dextran with the demonstrated permeation enhancing ability of alkylglycerols - have been combined for the development of nanoparticulate, blood-brain barrier-permeating, non-viral vectors. To this end, dextran, that had been functionalised via treatment with epoxide precursors of alkylglycerol, was covalently linked to poly(lactic acid) using a carbodiimide cross-linker to form alkylglyceryl-modified dextran-graft-poly(lactic acid). Solvent displacement and electrospray methods allowed the formulation of these materials into nanoparticles having a unimodal size distribution profile of about 100-200nm and good stability at physiologically relevant pH (7.4). The nanoparticles were characterised in terms of hydrodynamic size (by Dynamic Light Scattering and Nanoparticle Tracking Analysis), morphology (by Scanning Electron Microscopy and Atomic Force Microscopy) and zeta potential, and their toxicity was evaluated using MTT and PrestoBlue assays. Cellular uptake was evidenced by confocal microscopy employing nanoparticles that had been loaded with the easy-to-detect Rhodamine B fluorescent marker. Transwell-model experiments employing mouse (bEnd3) and human (hCMEC/D3) brain endothelial cells revealed enhanced permeation (statistically significant for hCMEC/D3) of the fluorescent markers in the presence of the nanoparticles. Results of studies using Electric Cell Substrate Impedance Sensing suggested a transient decrease of the barrier function in an in vitro blood-brain barrier model following incubation with these nanoformulations. An in ovo study using 3-day chicken embryos indicated the absence of whole-organism acute toxicity effects. The collective in vitro data suggest that these alkylglyceryl-modified dextran-graft-poly(lactic acid) nanoparticles are promising candidates for in vivo evaluations that would test their capability to transport therapeutic actives to the brain.

Towards boron neutron capture therapy: the formulation and preliminary in vitro evaluation of liposomal vehicles for the therapeutic delivery of the dequalinium salt of bis-nido-carborane.

Liposomes of phosphatidylcholine or of dimyristoylphosphatidylcholine that incorporate bis-nido-carborane dequalinium salt are stable in physiologically relevant media and have in vitro toxicity profiles that appear to be compatible with potential therapeutic applications. These features render the structures suitable candidate boron-delivery vehicles for evaluation in the boron neutron capture therapy of cancer.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

The formulation of polyhedral boranes for the boron neutron capture therapy of cancer.

The early promise of boron neutron capture therapy as a method for the treatment of cancer has been inhibited by the inherent toxicity associated with therapeutically useful doses of ¹°B-containing pharmacophores, the need for target-tissue specificity and the challenges imposed by biological barriers. Although developments in the synthetic chemistry of polyhedral boranes have addressed issues of toxicity to a considerable extent, the optimisation of the transport and the delivery of boronated agents to the site of action--the subject of this review--is a challenge that is addressed by the development of innovative formulation strategies.

Multifunctional poly(alkyl methacrylate) films for dental care.

Towards the evaluation of non-permanent dental coatings for their capacity to impart dental-care benefits, thin films of a homologous series of comb-like poly(alkyl methacrylate)s (ethyl to octadecyl) have been deposited, from aqueous latex formulations, onto dentally relevant substrates. AFM studies have shown that the thickness (40-300 nm) and surface roughness (8-12 nm) of coherent polymer films are influenced by the degree of polymerization and by the length of the pendant chain. Of the polymers under consideration, poly(butyl methacrylate) formed a close-packed film that conferred to dental substrates a high degree of inhibition to acid-mediated erosion (about 27%), as evaluated by released-phosphate determinations. The potential utility of the coatings to act as anti-sensitivity barriers has been evaluated by determining the hydraulic conductance of coated bovine-dentine substrates; single treatments of dentine discs with poly(butyl methacrylate) or with poly(ethyl methacrylate) effected mean respective reductions in fluid flow of about 23% with respect to water-treated controls; repeated applications of the poly(butyl methacrylate) latex led to mean reductions in fluid flow of about 80%. Chromometric measurements have shown that pellicle-coated hydroxyapatite discs treated with poly(butyl methacrylate), poly(hexyl methacrylate) or poly(lauryl methacrylate) exhibit significant resistance to staining by food chromogens.

Toward drug delivery into the brain: synthesis, characterization, and preliminary in vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles.

A series of O-substituted alkylglyceryl chitosans with systematically varied degrees of grafting was prepared through synthetic steps that involved the protection of amino moieties via phthaloylation and employed for the formulation of aqueous nanoparticulate systems that may be capable of delivering drugs to the brain. Dynamic light scattering studies have shown that nanoparticles with physiologically relevant aqueous stabilities may be prepared following the partial quaternization of these alkylglyceryl-modified chitosans. Preliminary in vitro tests using a mouse-brain endothelial cell model have indicated the efficient cellular uptake of these nanoparticles and identified butylglyceryl chitosan and butylglyceryl N,N,N-trimethyl chitosan as promising materials for the formulation of colloidal systems that could act as drug carriers into the brain.

Hybrid polymeric hydrogels for ocular drug delivery: nanoparticulate systems from copolymers of acrylic acid-functionalized chitosan and N-isopropylacrylamide or 2-hydroxyethyl methacrylate.

Nanoparticulate hybrid polymeric hydrogels (10-70 nm) have been obtained via the radical-induced co-polymerization of acrylic acid-functionalized chitosan with either N-isopropylacrylamide or 2-hydroxyethyl methacrylate, and the materials have been investigated for their ability to act as controlled release vehicles in ophthalmic drug delivery. Studies on the effects of network structure upon swelling properties, adhesiveness to substrates that mimic mucosal surfaces and biodegradability, coupled with in vitro drug release investigations employing ophthalmic drugs with differing aqueous solubilities, have identified nanoparticle compositions for each of the candidate drug molecules. The hybrid nanoparticles combine the temperature sensitivity of N-isopropylacrylamide or the good swelling characteristics of 2-hydroxyethyl methacrylate with the susceptibility of chitosan to lysozyme-induced biodegradation.