Chitosan and Its Structural Modifications for siRNA Delivery.

The use of RNA interference mechanism and small interfering RNA (siRNA) in cancer gene therapy is a very promising approach. However, the success of gene silencing is underpinned by the efficient delivery of intact siRNA into the targeted cell. Nowadays, chitosan is one of the most widely studied non-viral vectors for siRNA delivery, since it is a biodegradable, biocompatible and positively charged polymer able to bind to the negatively charged siRNA forming nanoparticles (NPs) that will act as siRNA delivery system. However, chitosan shows several limitations such as low transfection efficiency and low solubility at physiological pH. Therefore, a variety of chemical and non-chemical structural modifications of chitosan were investigated in the attempt to develop a chitosan derivative showing the features of an ideal siRNA carrier. In this review, the most recently proposed chemical modifications of chitosan are outlined. The type of modification, chemical structure, physicochemical properties, siRNA binding affinity and complexation efficiency of the modified chitosan are discussed. Moreover, the resulting NPs characteristics, cellular uptake, serum stability, cytotoxicity and gene transfection efficiency in vitro and/or in vivo are described and compared to the unmodified chitosan. Finally, a critical analysis of a selection of modifications is included, highlighting the most promising ones for this purpose in the future.

Chitosan Hydrogels Cross-Linked with Trimesic Acid for the Delivery of 5-Fluorouracil in Cancer Therapy.

Chitosan exhibits unique properties making it a suitable material for drug delivery. Considering the rising popularity of hydrogels in this field, this work offers a comprehensive study of hydrogels constituted by chitosan and cross-linked with 1,3,5-benzene tricarboxylic acid (BTC; also known as trimesic acid). Hydrogels were prepared by cross-linking chitosan with BTC in different concentrations. The nature of the gels was studied through oscillatory amplitude strain and frequency sweep tests within the linear viscoelastic region (LVE) limit. The flow curves of the gels revealed shear thinning behavior. High G' values imply strong cross-linking with improved stability. The rheological tests revealed that the strength of the hydrogel network increased with the cross-linking degree. Hardness, cohesiveness, adhesiveness, compressibility, and elasticity of the gels were determined using a texture analyzer. The scanning electron microscopy (SEM) data of the cross-linked hydrogels showed distinctive pores with a pore size increasing according to increasing concentrations (pore size range between 3-18 µm). Computational analysis was performed by docking simulations between chitosan and BTC. Drug release studies employing 5-fluorouracil (5-FU) yielded a more sustained release profile with 35 to 50% release among the formulations studied in a 3 h period. Overall, this work demonstrated that the presence of BTC as cross-linker leads to satisfactory mechanical properties of the chitosan hydrogel, suggesting potential applications in the sustained release of cancer therapeutics.

Advances in Chitosan-Based CRISPR/Cas9 Delivery Systems.

Clustered regularly interspaced short palindromic repeat (CRISPR) and the associated Cas endonuclease (Cas9) is a cutting-edge genome-editing technology that specifically targets DNA sequences by using short RNA molecules, helping the endonuclease Cas9 in the repairing of genes responsible for genetic diseases. However, the main issue regarding the application of this technique is the development of an efficient CRISPR/Cas9 delivery system. The consensus relies on the use of non-viral delivery systems represented by nanoparticles (NPs). Chitosan is a safe biopolymer widely used in the generation of NPs for several biomedical applications, especially gene delivery. Indeed, it shows several advantages in the context of gene delivery systems, for instance, the presence of positively charged amino groups on its backbone can establish electrostatic interactions with the negatively charged nucleic acid forming stable nanocomplexes. However, its main limitations include poor solubility in physiological pH and limited buffering ability, which can be overcome by functionalising its chemical structure. This review offers a critical analysis of the different approaches for the generation of chitosan-based CRISPR/Cas9 delivery systems and suggestions for future developments.

Development of drug alone and carrier-based GLP-1 dry powder inhaler formulations.

The study aimed to develop two types of dry powder inhaler (DPI) formulations containing glucagon-like peptide-1(7-36) amide (GLP-1): carrier-free (drug alone, no excipients) and carrier-based DPI formulations for pulmonary delivery of GLP-1. This is the first study focusing on the development of excipient free GLP-1 DPI formulations for inhaled therapy in Type 2 diabetes. The aerosolisation performance of both DPI formulations was studied using a next generation impactor and a DPI device (Handihaler®) at flow rate of 30 L min-1. Carriers employed were either a 10% w/w glycine-mannitol prepared by spray freeze drying or commercial mannitol. Spray freeze dried (SFD) carrier was spherical and porous whereas commercial mannitol carrier exhibited elongated particles (non-porous). GLP-1 powder without excipients for inhalation was prepared using spray drying and characterised for morphology including size, thermal behaviour, and moisture content. Spray dried (SD) GLP-1 powders showed indented/dimpled particles in the particle size range of 1-5 µm (also mass median aerodynamic diameter, MMAD: <5 µm) suitable for pulmonary delivery. Across formulations investigated, carrier-free DPI formulation showed the highest fine particle fraction (FPF: 90.73% ± 1.76%, mean ± standard deviation) and the smallest MMAD (1.96 µm ± 0.07 µm), however, low GLP-1 delivered dose (32.88% ± 7.00%, total GLP-1 deposition on throat and all impactor stages). GLP-1 delivered dose was improved by the addition of SFD 10% glycine-mannitol carrier to the DPI formulation (32.88% ± 7.00%-45.92% ± 5.84%). The results suggest that engineered carrier-based DPI formulations could be a feasible approach to enhance the delivery efficiency of GLP-1. The feasibility of systemic pulmonary delivery of SD GLP-1 for Type 2 diabetes therapy can be further investigated in animal models.

Overcoming the protein corona in chitosan-based nanoparticles.

Numerous properties of chitosan have led to its extensive use in the formulation of nanomaterials for drug delivery. However, the cationic surface of chitosan-based nanoparticles adsorbs proteins upon exposure to biological fluids, forming a phenomenon known as 'protein corona'. This causes several effects such as decreased bioavailability and limited in vivo clinical applications of chitosan nanoparticles. Understanding and overcoming the effects of protein adsorption on chitosan nanoparticles is key for drug delivery purposes. This review focuses on the strategies implemented to increase the stability of chitosan nanoparticles in the systemic circulation by averting the formation of protein corona and the limitations of PEGylation.

Biomedical and Pharmacological Uses of Fluorescein Isothiocyanate Chitosan-Based Nanocarriers.

Chitosan-based nanocarriers (ChNCs) are considered suitable drug carriers due to their ability to encapsulate a variety of drugs and cross biological barriers to deliver the cargo to their target site. Fluorescein isothiocyanate-labeled chitosan-based NCs (FITC@ChNCs) are used extensively in biomedical and pharmacological applications. The main advantage of using FITC@ChNCs consists of the ability to track their fate both intra and extracellularly. This journey is strictly dependent on the physico-chemical properties of the carrier and the cell types under investigation. Other applications make use of fluorescent ChNCs in cell labeling for the detection of disorders in vivo and controlling of living cells in situ. This review describes the use of FITC@ChNCs in the various applications with a focus on understanding their usefulness in labeled drug-delivery systems.

Overcoming the Blood-Brain Barrier: Functionalised Chitosan Nanocarriers.

The major impediment to the delivery of therapeutics to the brain is the presence of the blood-brain barrier (BBB). The BBB allows for the entrance of essential nutrients while excluding harmful substances, including most therapeutic agents; hence, brain disorders, especially tumours, are very difficult to treat. Chitosan is a well-researched polymer that offers advantageous biological and chemical properties, such as mucoadhesion and the ease of functionalisation. Chitosan-based nanocarriers (CsNCs) establish ionic interactions with the endothelial cells, facilitating the crossing of drugs through the BBB by adsorptive mediated transcytosis. This process is further enhanced by modifications of the structure of chitosan, owing to the presence of reactive amino and hydroxyl groups. Finally, by permanently binding ligands or molecules, such as antibodies or lipids, CsNCs have showed a boosted passage through the BBB, in both in vivo and in vitro studies which will be discussed in this review.

Fluorescein Isothiocyanate Chitosan Nanoparticles in Oral Drug Delivery Studies.

Oral administration of drugs is one of the most patient-friendly drug delivery routes. However, drug bioavailability via the oral route remains poor due to the harsh gastrointestinal environment. In recent years, many nanocarriers have been designed to overcome this limitation. Among those, chitosan nanoparticles (ChNPs) have proved to be a quite popular choice. Here, we highlight the use of fluorescein isothiocyanate-tagged ChNPs as an invaluable tool to monitor the fate of ChNPs encapsulating oral drugs, leading to an in-depth understanding of drug biodistribution and, in turn, shedding light on ways to improve bioavailability.

1H NMR quantification of spray dried and spray freeze-dried saccharide carriers in dry powder inhaler formulations.

Quantitative analysis using proton NMR (1H qNMR) has been employed in various areas such as pharmaceutical analysis (e.g., dissolution study), vaccines, natural products analysis, metabolites, and macrolide antibiotics in agriculture industry. However, it is not routinely used in the quantification of saccharides in dry powder inhaler (DPI) formulations. The aim of this study was to develop a 1H NMR method for the quantification of saccharides employed in DPI formulations. Dry powders as DPI carriers were prepared by spray drying (SD) and spray freeze drying (SFD) using three saccharides: namely D-mannitol, D-sorbitol and D-(+)-sucrose. The calibration curves constructed for all three saccharides demonstrated linearity with R2 value of 1. The 1H qNMR method produced accurate (relative error %: 0.184-3.697) and precise data with high repeatability (RSD %: 0.517-3.126) within the calibration curve concentration range. The 1H qNMR method also demonstrated significant sensitivity with low values of limit of detection (0.058 mM for D-mannitol, 0.045 mM for D-(+)-sucrose, and 0.056 mM for D-sorbitol) and limit of quantitation (0.175 mM for D-mannitol, 0.135 mM for D-(+)-sucrose, and 0.168 mM for D-sorbitol). Pulmonary deposition via impaction experiments of the three saccharides was quantified using the developed method. It was found that SFD D-mannitol (68.99%) and SFD D-(+)-sucrose (66.62%) exhibited better delivered dose (total saccharide deposition in throat and all impactor stages) than SD D-mannitol (49.03%) and SD D-(+)-sucrose (57.70%) (p < 0.05). The developed 1H qNMR methodology can be routinely used as an analytical method to assess pulmonary deposition in impaction experiments of saccharides employed as carriers in DPI formulations.

Liposome formulations of o-carborane for the boron neutron capture therapy of cancer.

Based on the promise of liposomes as convenient vehicles for the transport of boronated agents for the boron neutron capture therapy (BCNT) of cancer, this paper reports a method for the formulation and characterisation of stable o-carborane-loaded liposomes (ca. 80-100 nm) of dipalmitoyl-phosphatidylcholine (DPPC) or 1,2-distearol-sn-glycerol-3-phosphocholine (DSPC). Preliminary pharmaceutical characterisation experiments have demonstrated the integrity of both DPPC and DSPC liposomal membranes in serum and in PBS and also indicate that these o-carborane-loaded liposomes are candidate carrier vehicles for further evaluation with a view to exploitation in BNCT.

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.

Labelling of electronic cigarettes: regulations and current practice.

BACKGROUND: Over the past decade e-cigarettes have established themselves in the global market. E-cigarettes triggered much interest in relation to their content and efficacy as smoking cessation tools, but less attention has been paid to users and environmental safety warnings and guidance. Several regulations have been introduced to promote their safe handling and disposal. From May 2016, liquids and cartridges will be regulated by European Community Directives (ECDs) 2001/83/EC and 93/42/EEC, or 2014/40/EU if marketed as tobacco-related products. Currently, manufacturers and distributors must abide by the Chemical (Hazard Information and Packaging for Supply) Regulations 2009 (CHIP) or Classification, Labelling and Packaging Regulations (CLP), the latter replacing CHIP in June 2015. OBJECTIVE: In this work, the compliance of marketed e-liquids and e-cigarettes with current European Union and UK legislations is assessed. RESULTS: E-liquids and e-cigarettes (21 and 9 brands, respectively) were evaluated. Evidence of non-compliance was found in relation to the CHIP/CLP toxic (13%) and environmental (37%) pictograms, tactile warning (23%), nominal amount of solution (30%), supplier contact telephone number and address (40%). None of the evaluated e-cigarettes displayed information on the correct disposal/recycling of batteries in line with the ECD 2006/66/EC. CONCLUSIONS: More stringent enforcement of regulations is needed to ensure not only the user's safety and awareness, but also the safeguarding of the environment.

Correction: Boron-containing delocalised lipophilic cations for the selective targeting of cancer cells.

[This corrects the article DOI: 10.1039/C6MD00383D.].

Pharmacological Development of Target-Specific Delocalized Lipophilic Cation-Functionalized Carboranes for Cancer Therapy.

PURPOSE: Tumor cell heterogeneity and microenvironment represent major hindering factors in the clinical setting toward achieving the desired selectivity and specificity to malignant tissues for molecularly targeted cancer therapeutics. In this study, the cellular and molecular evaluation of several delocalized lipophilic cation (DLC)-functionalized carborane compounds as innovative anticancer agents is presented. METHODS: The anticancer potential assessment of the DLC-carboranes was performed in established normal (MRC-5, Vero), cancer (U-87 MG, HSC-3) and primary glioblastoma cancer stem (EGFR(pos), EGFR(neg)) cultures. Moreover, the molecular mechanism of action underlying their pharmacological response is also analyzed. RESULTS: The pharmacological anticancer profile of DLC-functionalized carboranes is characterized by: a) a marked in vitro selectivity, due to lower concentration range needed (ca. 10 fold) to exert their cell growth-arrest effect on U-87 MG and HSC-3, as compared with that on MRC-5 and Vero; b) a similar selective growth inhibition behavior towards EGFR(pos) and EGFR(neg) cultures (>10 fold difference in potency) without, however, the activation of apoptosis in cultures; c) notably, in marked contrast to cancer cells, normal cells are capable of recapitulating their full proliferation potential following exposure to DLC-carboranes; and, d) such pharmacological effects of DLC-carboranes has been unveiled to be elicited at the molecular level through activation of the p53/p21 axis. CONCLUSIONS: Overall, the data presented in this work indicates the potential of the DLC-functionalized carboranes to act as new selective anticancer therapeutics that may be used autonomously or in therapies involving radiation with thermal neutrons. Importantly, such bifunctional capacity may be beneficial in cancer therapy.

Survey of community pharmacists' perception of electronic cigarettes in London.

OBJECTIVES: To seek community pharmacists' perception on use, safety and possible effectiveness of e-cigarettes as quit smoking tools, and their future regulation. SETTING: A survey of a sample of 154 community pharmacies across London, UK. CONTEXT: E-cigarettes have exclusively established themselves in the market through consumers-led demand. To date, e-cigarettes still remain unregulated and can be easily purchased in shops, over the internet, but more controversially also in pharmacies in the UK. Pharmacists find themselves with a shortage of information on their safety and efficacy, and may experience an ethical dilemma when consulted by patients/customers. KEY FINDINGS: Response rate: 60% (n=92). Independent pharmacies accounted for 90% of the sample. The majority of participants (73%) sell e-cigarettes. A minority of participants (20%) have been presented with adverse effects such as cough and dry mouth. As possible reasons for their use, pharmacists ranked 'aid in stop smoking' as the most important (56%), with 'cheaper alternative' (43%) and 'social/recreational use' (31%) being the least important ones. Safety issues were raised as statements such as 'e-liquid in cartridges may be toxic' were agreed by 52% of respondents. The majority of pharmacists (97%) were supportive of e-cigarettes being regulated, expressing current concerns regarding excipients (42%) and nicotine content (34%). Participants indicated that they would require training in the form of information packs (88%), online tutorials (67%), continuous professional development (CPD) workshops (43%) to cover safety, counselling, dosage instructions, adverse effects and role in the smoking cessation care pathway in the future. CONCLUSIONS: Pharmacists expressed concerns about the safety of e-cigarettes, especially regarding the amounts of excipients and nicotine as these still remain unregulated. Currently, there are no guidelines for pharmacists regarding e-cigarettes. Community pharmacists look forward to regulations so to conduct their duties in a more confident and legislated fashion.

Analysis of 2-oxothiazolidine-4-carboxylic acid by hydrophilic interaction liquid chromatography: application for ocular delivery using chitosan nanoparticles.

Oxidative damage due to low levels of glutathione (GSH) is one of the main causes of cataract formation. It has been reported that 2-oxothiazolidine-4-carboxylic acid (OTZ), a cysteine prodrug, can increase the cellular level of GSH. Currently, there is no analytical method to separate and quantify OTZ from aqueous humour samples for cataract research. The present study aims to develop and validate a hydrophilic interaction liquid chromatography (HILIC) method for the quantification of OTZ in simulated aqueous humour (SAH). The developed method was validated according to FDA guidelines. Accuracy, precision, selectivity, sensitivity, linearity, lower limit of quantification (LLOQ), lower limit of detection (LLOD) and stability were the parameters assessed in the method validation. The developed method was found to be accurate and precise with LLOQ and LLOD of 200 and 100 ng/mL, respectively; method selectivity was confirmed by the absence of any matrix interference with the analyte peak. The constructed calibration curve was linear in the range of 0.2-10 µg/mL, with a regression coefficient of 0.999. In addition, the OTZ was found to be stable in SAH after three freeze/thaw cycles. Chitosan nanoparticles loaded with OTZ were formulated by the ionic gelation method. The nanoparticles were found to be uniform in shape and well dispersed with average size of 153 nm. The in vitro release of OTZ from the nanoparticles was quantified using the developed analytical method over 96 h. Permeation of OTZ through excised bovine cornea was measured using HILIC. The lag time and the flux were 0.2 h and 3.05 µg/cm(2) h, respectively.

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.

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.