Anita Hafner

Nanotherapeutics in the EU: an overview on current state and future directions

The application of nanotechnology in areas of drug delivery and therapy (ie, nanotherapeutics) is envisioned to have a great impact on public health. The ability of nanotherapeutics to provide targeted drug delivery, improve drug solubility, extend drug half-life, improve a drug’s therapeutic index, and reduce a drug’s immunogenicity has resulted in the potential to revolutionize the treatment of many diseases. In this paper, we review the liposome-, nanocrystal-, virosome-, polymer therapeutic-, nanoemulsion-, and nanoparticle-based approaches to nanotherapeutics, which represent the most successful and commercialized categories within the field of nanomedicine. We discuss the regulatory pathway and initiatives endeavoring to ensure the safe and timely clinical translation of emerging nanotherapeutics and realization of health care benefits. Emerging trends are expected to confirm that this nano-concept can exert a macro-impact on patient benefits, treatment options, and the EU economy.

Melatonin-loaded lecithin/chitosan nanoparticles: Physicochemical characterisation and permeability through Caco-2 cell monolayers

In this study, the potential of lecithin/chitosan nanoparticles (NPs) as a mucoadhesive colloidal nanosystem for transmucosal delivery of melatonin was investigated. The size, zeta potential and melatonin loading of the lecithin/chitosan NPs were investigated as a function of lecithin type (Lipoid S45, S75 and S100) and chitosan content in the preparation. The NPs were characterised by mean diameter and zeta potential ranging between 121.6 and 347.5 nm, and 7.5 and 32.7 mV, respectively, and increasing with lecithin-negative charge and chitosan content in the preparation. Melatonin loadings were up to 7.1%. All NPs were characterised by prolonged release profiles with an initial burst (approximately 25%), followed by a slow release phase. Approximately 60-70% of melatonin was released in 4h. The permeability of melatonin was investigated using Caco-2 cells as an in vitro model of the epithelial barrier. Melatonin permeability from an NP suspension prepared with Lipoid S45 lecithin and a lecithin-to-chitosan weight ratio (L/C) of 20:1 (sample C2) was significantly improved compared to the permeability of melatonin from the solution (P<0.001) and from all other NPs investigated (P<0.05). The results obtained by the cell viability studies (MTT and LDH leakage assays) showed that C2 NP suspension did not induce plasma membrane damage or decrease cell viability and could be safely applied to Caco-2 cells in the concentration range tested (<400 microg/ml).

A Nonionic Surfactant/Chitosan Micelle System in an Innovative Eye Drop Formulation

Micelle systems composed of the polyoxyethylated nonionic surfactant Pluronic F127 (F127) and cationic polyelectrolyte chitosan (CH) were prepared with dexamethasone (DEX) as a hydrophobic model drug. The F127/CH micelles were characterised by their hydrodynamic diameter and a zeta-potential ranging between 25.4 and 28.9 nm and +9.3 and +17.6 mV, respectively. The DEX loading was between 0.48% and 0.56%, and no significant influence of CH on DEX loading was observed. All micelle systems were characterised by prolonged release profiles. The addition of CH significantly enhanced the in vitro DEX release rate and transport across Caco-2 cell monolayers, as compared to the CH-free F127 micelle system. This colloidal carrier was well tolerated in rabbit eyes, and no clinically abnormal signs in various ocular structures were observed. The increase in intraocular pressure (IOP) in rabbits was used to evaluate DEX ocular bioavailability. The AUC values showed a 1.7- and 2.4-fold increase in bioavailability with F127 and F127/0.015 (w/v) % CH micelle systems, respectively, as compared to a standard DEX suspension. These data indicate improved intraocular DEX absorption from the micelle systems, which can be ascribed to both F127 and CH corneal permeability enhancement.

Lecithin/chitosan nanoparticles for transdermal delivery of melatonin.

In this study, the potential of lecithin/chitosan nanoparticles (NPs) as colloidal nanosystem for transdermal melatonin delivery was investigated. Mean diameter and zeta-potential of NPs differing in lecithin type (Lipoid S45 and S100) and chitosan content ranged between 113.7 and 331.5 nm and 4.6 and 31.2 mV, respectively. Melatonin loadings were up to 7.2%. The potential of lecithin/chitosan NPs to enhance transdermal melatonin delivery was investigated by determining the drug flux across dermatomed porcine skin and its skin deposition. Lecithin/chitosan NPs provided 1.3–2.3-fold higher flux compared to melatonin solution. The highest flux, 9.0 ± 0.21 µg/cm2/h, was observed for S45 lecithin/chitosan NPs with lecithin/chitosan weight ratio of 20:1. NP possible cytotoxicity in vitro was evaluated using human skin keratinocytes and fibroblasts. It was demonstrated that lecithin/chitosan NPs can be applied to skin cells at concentrations up to 200 µg/mL without inducing plasma membrane damage or cell viability decrease.

Characterization of various deformable liposomes with metronidazole.

Objective: The aim of this study was to investigate various deformable liposomes for their potential application for the vaginal administration of metronidazole. Materials and methods: Deformable liposomes composed of egg phosphatidylcholine (EPC) and various surfactants [sodium deoxycholate (SDCh), Tween 80 or Span 80] and conventional liposomes consisting of EPC and egg phosphatidylglycerol-sodium (EPG-Na) were prepared with and without metronidazole. Additionally, a freeze-thaw method was applied to both classes of vesicles (liposomes) containing the drug to improve its trapping capacity. All of the liposomes prepared were characterized and compared in terms of size, polydispersity, zeta potential, entrapment efficiency and their permeability on a Caco-2 cell monolayer. Results and discussion: Conventional liposomes, both with and without metronidazole, were larger than the deformable vesicles. The presence of ethanol in the preparations of the elastic EPC/SDCh and EPC/Tween 80 liposomes was found to affect the particle size in terms of reducing this parameter. Different types of vesicles were compared for their trapping efficiency of metronidazole and the highest entrapment was observed with conventional liposomes. However, deformable EPC/SDCh liposomes were found to enhance the permeability of metronidazole more effectively than the conventional liposomes based on the in vitro model of the epithelial barrier. Conclusion: These preliminary data indicate that EPC/SDCh liposomes may have a promising future in vaginal delivery of metronidazole. Therefore, additional investigations on elastic vesicles and their incorporation in a suitable vehicle should be considered to further evaluate their applicability in vaginal drug delivery.

Melatonin-loaded chitosan/Pluronic® F127 microspheres as in situ forming hydrogel: An innovative antimicrobial wound dressing.

The aim of this study was to develop melatonin-loaded chitosan based microspheres as dry powder formulation suitable for wound dressing, rapidly forming hydrogel in contact with wound exudate. Microspheres were produced by spray-drying method. Fractional factorial design was employed to elucidate the effect of formulation and process parameters (feed flow rate, inlet air temperature, chitosan concentration, chitosan/melatonin ratio and chitosan/Pluronic® F127 ratio) on the product characteristics related to process applicability (production yield, entrapment efficiency and product moisture content) and microsphere performance in biological environment (microsphere mean diameter and surface charge). Appropriate formulation and process parameters for the establishment of efficient drying process resulting in fine-tuned chitosan and chitosan/Pluronic® F127 microspheres (efficient melatonin encapsulation, small diameter positive surface charge and low moisture content) were identified. Microspheres were characterized by appropriate flowability and high rate and extent of fluid uptake. Incorporation of Pluronic® F127 in microsphere matrix resulted in high melatonin amorphization and consequent higher melatonin release rate. Entrapment of melatonin in chitosan/Pluronic® F127 microspheres has potentiated chitosan antimicrobial activity against Staphylococcus aureus and five clinical isolates S. aureus MRSA strains. Microspheres were shown to be biocompatible with skin keratinocytes and fibroblasts at concentrations relevant for antimicrobial activity against planktonic bacteria.

Spray dried microparticles for controlled delivery of mupirocin calcium: Process–tailored modulation of drug release

Spray dried microparticles containing mupirocin calcium were designed as acrylic matrix carriers with modulated drug release for efficient local drug delivery at minimum daily dose. Particle generation in spray drying and its effect on release performance were assessed by varying drug : polymer ratios with consequently altered initial saturations. Narrow-sized microparticles with mean diameters of 1.7–2.5 µm were obtained. Properties of the generated solid dispersions were examined by X-ray, thermal (thermogravimetric analysis, modulated differential scanning calorimetry) and spectroscopic (Fourier transformed infrared, Fourier transformed Raman) methods and correlated with drug loading and in vitro release. The best control over mupirocin release was achieved for 2 : 1 (w/w) drug : polymer ratio and found to be strongly process-dependent. For a particular ratio, increased feed concentration (>4%) boosted while increased inlet temperature (≥100°C) reduced drug release. Antimicrobial activity testing confirmed that encapsulated drug preserved its antibacterial effectiveness. Conclusively, spray drying was proven as a suitable method for preparing structured microparticles which can control drug release even at exceptionally high drug loadings.

Development and In Vitro Characterization of Chitosan-based Microspheres for Nasal Delivery of Promethazine

ABSTRACT Conventional and composed promethazine-loaded microspheres were prepared by spray drying of chitosan solution systems and double water-in-oil-in-water (W/O/W) emulsion systems, respectively. Double emulsions were prepared in two different feed concentrations, with chitosan dissolved in both water phases, and ethylcellulose dissolved in oil phase. Swelling and bioadhesive properties of the microspheres depended on the chitosan content, type and the feed concentration of spray-dried system. Results obtained suggested that better ethylcellulose microcapsules with promethazine in the chitosan matrix were formed when less concentrated emulsion systems were spray-dried. Thus, in case of such a system, with ethylcellulose/chitosan weight ratio of 1:2, prolonged promethazine release was obtained.

Evaluation of cationic nanosystems with melatonin using an eye-related bioavailability prediction model.

In this study, two types of nanosystems, namely lecithin/chitosan nanoparticles and Pluronic® F127/chitosan micelles, have been prepared and evaluated for their potential for the ocular delivery of melatonin, which is known to exert an ocular hypotensive effect. The melatonin content, particle size, zeta potential and in vitro drug release properties were studied as a function of the presence of chitosan in the nanosystem. Lecithin/chitosan nanoparticles were evaluated in terms of the mucoadhesive properties by a newly established method based on HCE-T cells, also used in in vitro biocompatibility and permeability studies. Lecithin/chitosan nanoparticles were significantly larger than the corresponding F127/chitosan micelles (mean diameter of 241.8 vs. 20.7nm, respectively) and characterised by a higher surface charge (22.7 vs. 4.3mV, respectively). The HCE-T cell viability assay did not show significant toxic effects of nanosystems investigated at the (relevant) chitosan concentration tested. The permeability study results confirmed the permeation enhancing effect of F127, which was hindered in the presence of chitosan. Lecithin/chitosan nanoparticles were characterised by prominent mucoadhesive properties and prolonged melatonin release, which was shown to control melatonin permeation across an in vitro corneal epithelial model. Such properties demonstrate the potential for nanoparticles to provide an extended pre-corneal residence time of melatonin, ensuring higher eye-related bioavailability and extended intraocular pressure reduction compared to melatonin in both aqueous and micelle solutions.

Nanoparticle-mediated interplay of chitosan and melatonin for improved wound epithelialisation.

Herein, we propose an innovative approach to improving wound healing. Our strategy is to deliver melatonin locally at the wound site by means of lecithin/chitosan nanoparticles. We used four types of chitosan that differed in terms of molecular weight and/or deacetylation degree. Melatonin encapsulation efficiency, nanoparticle size, zeta potential, biocompatibility and in vitro drug release were studied as a function of the type of chitosan used in preparation. The nanoparticles were evaluated in terms of their potential to promote wound epithelialisation via an in vitro scratch assay using a human keratinocyte (HaCaT) monolayer. The model wounds were treated with nanoparticle suspensions at a chitosan concentration of 5μgml(-1), which was based on preceding cell biocompatibility studies. Nanoparticles prepared with different types of chitosan showed similar effect on the keratinocyte proliferation/migration. Nanoparticle-mediated interplay of chitosan and melatonin was shown to be crucial for improved wound epithelialisation.