Chitosan-Based Thermogelling System for Nose-to-Brain Donepezil Delivery: Optimising Formulation Properties and Nasal Deposition Profile.

Donepezil nasal delivery strategies are being continuously investigated for advancing therapy in Alzheimer's disease. The aim of this study was to develop a chitosan-based, donepezil-loaded thermogelling formulation tailored to meet all the requirements for efficient nose-to-brain delivery. A statistical design of the experiments was implemented for the optimisation of the formulation and/or administration parameters, with regard to formulation viscosity, gelling and spray properties, as well as its targeted nasal deposition within the 3D-printed nasal cavity model. The optimised formulation was further characterised in terms of stability, in vitro release, in vitro biocompatibility and permeability (using Calu-3 cells), ex vivo mucoadhesion (using porcine nasal mucosa), and in vivo irritability (using slug mucosal irritation assay). The applied research design resulted in the development of a sprayable donepezil delivery platform characterised by instant gelation at 34 °C and olfactory deposition reaching a remarkably high 71.8% of the applied dose. The optimised formulation showed prolonged drug release (t1/2 about 90 min), mucoadhesive behaviour, and reversible permeation enhancement, with a 20-fold increase in adhesion and a 1.5-fold increase in the apparent permeability coefficient in relation to the corresponding donepezil solution. The slug mucosal irritation assay demonstrated an acceptable irritability profile, indicating its potential for safe nasal delivery. It can be concluded that the developed thermogelling formulation showed great promise as an efficient donepezil brain-targeted delivery system. Furthermore, the formulation is worth investigating in vivo for final feasibility confirmation.

Towards the development of a biorelevant in vitro method for the prediction of nanoemulsion stability on the ocular surface.

Ophthalmic oil-in-water nanoemulsions (NEs) are a complex technological platform, representing an advancement in the treatment of dry eye disease. In addition to enabling the incorporation of poorly soluble active pharmaceutical ingredients (APIs), NEs provide prolonged residence time of APIs and other formulation components and consequent replenishment and stabilization of the compromised tear film. Ophthalmic NEs have been on the market for over 20 years, but considering their complexity, as well as the complex nature of the ocular surface, they are still a poorly understood advanced dosage form. The objective of this study was to develop a biorelevant in vitro method that would be able to predict the behavior of ophthalmic NEs after application. With that goal, NE formulations differing in critical material attributes and critical formulation variables were employed and subjected to simulated tear turnover and blinking. By gradually increasing the complexity of the in vitro method, we were able to detect key parameters influencing NE stability. The undertaken study presents a step forward in the development of in vitro tools that are fundamental to the reliable, cost and time-effective development of innovative and generic topical ophthalmic NEs.

Tailoring functional spray-dried powder platform for efficient donepezil nose-to-brain delivery.

Shortcomings of oral donepezil administration in the treatment of Alzheimer's disease have paved the way for ongoing investigations towards more efficient and safe donepezil nose-to-brain delivery. Herein we present the development of advantageous powder platform for donepezil nose-to-brain delivery, coupling careful design of chitosan and mannitol-based carrier matrix with spray-drying technology advantages and early consideration of adequate nasal administration mode, employing QbD approach. Unprecedentedly, ultrasonic nozzle was used to atomise the drying feed in response to size-related requirements for nasal aerosol particles. The optimised spray-drying process resulted in free-flowable dry powder with a great majority of particles larger than 10 µm, ensuring localised nasal deposition upon aerosolization, as evidenced by using 3D-printed nasal cavity model. QbD approach coupling formulation, process and administration parameters enabled optimisation of drug deposition profile reaching tremendously high 65.5 % of the applied dose deposited in the olfactory region. The leading formulation exhibited favourable swelling, mucoadhesion, drug release and permeation-enhancing properties, suiting the needs for efficient brain-targeted delivery. Results of in vitro biocompatibility and physico-chemical stability studies confirmed the leading formulation potential for safe and efficient donepezil nose-to-brain delivery. The obtained results encourage extending the study to an appropriate in vivo model needed for the final proof-of-concept.

In situ gelling nanosuspension as an advanced platform for fluticasone propionate nasal delivery.

In this work we present the development of in situ gelling nanosuspension as advanced form for fluticasone propionate nasal delivery. Drug nanocrystals were prepared by wet milling technique. Incorporation of drug nanocrystals into polymeric in situ gelling system with pectin and sodium hyaluronate as constitutive polymers was fine-tuned attaining appropriate formulation surface tension, viscosity and gelling ability. Drug nanonisation improved the release profile and enhanced formulation mucoadhesive properties. QbD approach combining formulation and administration parameters resulted in optimised nasal deposition profile, with 51.8% of the dose deposited in the middle meatus, the critical region in the treatment of rhinosinusitis and nasal polyposis. Results obtained in biocompatibility and physico-chemical stability studies confirmed the leading formulation potential for safe and efficient nasal corticosteroid delivery.

Resveratrol and its nanocrystals: A promising approach for cancer therapy?

There has been a significant research interest in nanocrystals as a promising technology for improving the therapeutic efficacy of poorly water-soluble drugs, such as resveratrol. Little is known about the interaction of nanocrystals with biological tissue. The aim of this study was to investigate the potential use of resveratrol (RSV) and its nanocrystals (NANO-RSV) as antitumor agents in Ehrlich ascites tumour (EAT)-bearing mice and the interaction of nanocrystals with biological tissue through biochemical and histological changes of kidney, liver and EAT cells. After intraperitoneal injection of 2.5 × 106 cells into the abdominal cavity of mice, treatment of animals was started next day by injecting RSV or NANO-RSV at a dose of either 25 or 50 mg/kg every other day for 14 days. The results show that the administration of resveratrol and its nanocrystals lead to significant reductions in the proliferation of tumour cells in the abdominal cavity, and a reduction of the number of blood vessels in the peritoneum, with low systemic toxicity. In histopathological examinations, greater hepatocellular necrosis and apoptosis, hepatic fibrosis around the central vein and degeneration with minor fatty change were observed with RSV than with NANO-RSV. Inflammation with proximal tubular necrosis and renal glomerulus swelling were also observed, together with slight elevation of several biochemical parameters in both the RSV and NANO-RSV groups. In order to increase the beneficial effects and reduce risks associated with resveratrol nanocrystals, additional factors such as dose, genetic factors, health status, and the nature of the target cells should also be considered.

A Dry Powder Platform for Nose-to-Brain Delivery of Dexamethasone: Formulation Development and Nasal Deposition Studies.

Nasal route of administration offers a unique opportunity of brain targeted drug delivery via olfactory and trigeminal pathway, providing effective CNS concentrations at lower doses and lower risk for adverse reactions compared to systemic drug administration. Therefore, it has been recently proposed as a route of choice for glucocorticoids to control neuroinflammation processes in patients with severe Covid-19. However, appropriate delivery systems tailored to enhance their efficacy yet need to emerge. In this work we present the development of sprayable brain targeting powder delivery platform of dexamethasone sodium phosphate (DSP). DSP-loaded microspheres, optimised employing Quality-by-Design approach, were blended with soluble inert carriers (mannitol or lactose monohydrate). Powder blends were characterized in terms of homogeneity, flow properties, sprayability, in vitro biocompatibility, permeability and mucoadhesion. Nasal deposition studies were performed using 3D printed nasal cavity model. Mannitol provided better powder blend flow properties compared to lactose. Microspheres blended with mannitol retained or enlarged their mucoadhesive properties and enhanced DSP permeability across epithelial model barrier. DSP dose fraction deposited in the olfactory region reached 17.0% revealing the potential of developed powder platform for targeted olfactory delivery. The observed impact of nasal cavity asymmetry highlighted the importance of individual approach when aiming olfactory region.

Efficacy and Safety of Azelaic Acid Nanocrystal-Loaded In Situ Hydrogel in the Treatment of Acne Vulgaris.

Acne vulgaris is a common, multifactorial, inflammatory skin disease affecting the pilosebaceous unit. Topical therapy is the first choice in the treatment of mild to moderate acne, and azelaic acid (AZA) is one of the most commonly used drugs. The aim of this study was to evaluate the safety and efficacy of a low-dose azelaic acid nanocrystal (AZA-NC) hydrogel in the treatment of mild to moderate facial acne. The study was designed as a double-blind, randomized controlled trial. Patients were randomized to treatment with AZA-NC hydrogel, 10%, or AZA cream, 20%, administered in quantities of approximately 1 g twice daily for 8 weeks. Efficacy of therapy was measured by the number of lesions and safety by the frequency and severity of adverse events. At week 8, the success rate of treatment with AZA-NC hydrogel, 10%, was 36.51% (p < 0.001) versus 30.37% (p < 0.001) with AZA cream. At week 8, treatment with AZA-NC hydrogel, 10%, resulted in a significant reduction in total inflammatory lesions from baseline of 39.15% (p < 0.001) versus 33.76% (p < 0.001) with AZA cream, and a reduction in non-inflammatory lesions from baseline of 34.58% (p < 0.001) versus 27.96% (p < 0.001) with AZA cream, respectively. The adverse event rate was low and mostly mild.

Functional ibuprofen-loaded cationic nanoemulsion: Development and optimization for dry eye disease treatment.

Inflammation plays a key role in dry eye disease (DED) affecting millions of people worldwide. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used topically to act on the inflammatory component of DED, but their limited aqueous solubility raises formulation issues. The aim of this study was development and optimization of functional cationic nanoemulsions (NEs) for DED treatment, as a formulation approach to circumvent solubility problems, prolong drug residence at the ocular surface and stabilize the tear film. Ibuprofen was employed as the model NSAID, chitosan as the cationic agent, and lecithin as the anionic surfactant enabling chitosan incorporation. Moreover, lecithin is a mixture of phospholipids including phosphatidylcholine and phosphatidylethanolamine, two constituents of the natural tear film important for its stability. NEs were characterized in terms of droplet size, polydispersity index, zeta-potential, pH, viscosity, osmolarity, surface tension, entrapment efficiency, stability, sterilizability and in vitro release. NEs mucoadhesive properties were tested rheologically after mixing with mucin dispersion. Biocompatibility was assessed employing 3D HCE-T cell-based model and ex vivo model using porcine corneas. The results of our study pointed out the NE formulation with 0.05% (w/w) chitosan as the lead formulation with physicochemical properties adequate for ophthalmic application, mucoadhesive character and excellent biocompatibility.

In vitro evaluation of stearylamine cationic nanoemulsions for improved ocular drug delivery.

Oil-in-water nanoemulsions (NEs) represent one of the formulation approaches to improve eye-related bio-availability of lipophilic drugs. The potential of cationic NEs is pronounced due to the electrostatic interaction of positively charged droplets with negatively charged mucins present in the tear film, providing prolonged formulation residence at the ocular surface. The aim of this study was to develop a cationic ophthalmic NE with cationic lipid stearylamine (SA) as a carrier of a positive charge. The addition of a nonionic surfactant provided the dual electro-steric stabilization of NEs and enabled tuning of SA concentration to achieve an optimal balance between its interaction with mucins and biocompatibility. Physicochemical characterization, stability profile, in vitro mucoadhesion study and biocompatibility study employing 3D HCE-T cell-based model of corneal epithelium pointed out the NE with 0.05 % (m/m) SA as the leading formulation. Minimizing SA content while retaining droplet/mucin interactions is of great importance for efficacy and safety of future ophthalmic drug products.

Cryocornea - toward enhancing the capacity and throughput of ex vivo corneal model.

Objective: The objective of this study was to investigate the effects of the concentration of two intracellular (i.e. propylene glycol and glycerol) and four extracellular (i.e. dextran, hydroxypropyl methylcellulose, polyvinylpyrolidone, trehalose) cryoprotective agents as well as the effects of freeze-thawing procedures on the corneal cryoprotection.Significance: The corneal cryopreservation may possibly become the long-term storage technique of choice for collection of animal corneas suitable for ex vivo drug testing.Methods: The integrity of corneal barrier was evaluated by measurements of transepithelial electrical resistance.Results: Under the investigated experimental conditions the best result was obtained for slow freezing (2 h at -20 °C followed by 46 h at -70 °C) and rapid thawing (0.25 h at 34 °C) procedure where 20% (w/V) trehalose in Krebs Ringer buffer solution was used as extracellular cryoprotective agent.Conclusions: The selection of corneal freeze-thawing protocol as well as the optimal type and concentration of a cryoprotective agent allows the cryostorage of porcine corneal tissues with suitable TEER properties (cryocornea).

Preparation of in situ hydrogels loaded with azelaic acid nanocrystals and their dermal application performance study.

Azelaic acid (AZA) is a dicarboxylic acid that is topically used in the treatment of acne and rosacea since it possesses antibacterial and keratolytic activity. The primary objective of this study was to develop an AZA nanocrystal suspension. It is expected that improved solubility and dissolution rate will result in advanced biopharmaceutical properties, primarily the dermal bioavailability. Furthermore, a topical nanocrystal AZA-loaded hydrogels composed of Pluronic® F127 and hyaluronic acid mixture that are able to deliver AZA into the stratum corneum and deeper skin layers were considered. This study was conducted in order to: 1) determine the effect of non-ionic Polysorbate 60 on the stabilization and particle size of the AZA nanocrystals, as well as the effect of Pluronic® F127, used as an in situ gelation agent, and hyaluronic acid on the viscoelastic properties and the drug release of composed hydrogels, 2) determine the relationship between the rheological properties of the gels and the penetration of AZA into the stratum corneum. The composed hydrogels revealed pseudoplastic flow behaviour. The increase in Pluronic® F127 concentration induced a domination of elastic over viscous behaviour of the gels. The gel containing 15% of Pluronic® F127, 1% of hyaluronic acid and lyophilised 10% nanocrystal AZA suspension was considered to be an optimal formulation, since it possessed the rheological and drug delivery properties desirable for an in situ gelling platform for dermal application.

Innovative sprayable in situ gelling fluticasone suspension: Development and optimization of nasal deposition.

The aim of this study was to develop an innovative in situ gelling suspension for effective nasal delivery of fluticasone. Pectin, gellan gum and sodium hyaluronate were used as gelling/thickening agents, and Tween 80 as a suspending agent. The influence of the formulation and/or administration parameters on formulation sprayability and nasal deposition was explored with an appropriate experimental design with the range for parameters in the design obtained from previous research and domain knowledge. All formulations exhibited appropriate sprayability and instant gelation upon mixing with simulated nasal fluid exhibiting weak gel properties convenient for nasal delivery. Targeted turbinate deposition depended on administration and formulation parameters, including their interactions. Decrease in the administration angle from horizontal plane, increase in inspiratory flow and presence of sodium hyaluronate significantly increased deposition in turbinate region. Parameters in interactions included concentration of polymers, surfactant and fluticasone, as well as administration angle. Selected formulations with high turbinate deposition exhibited significant increase in viscosity upon gelation, showing potential to prolong the drug retention at the nasal mucosa. The highest effect on the gel viscosity, strength and fluticasone release profile was observed for gellan gum, thus recognised as crucial parameter for the optimisation of overall therapeutic effect.

Biopharmaceutical evaluation of surface active ophthalmic excipients using in vitro and ex vivo corneal models.

The objective of this study was to systematically investigate the effects of surface active ophthalmic excipients on the corneal permeation of ophthalmic drugs using in vitro (HCE-T cell-based model) and ex vivo (freshly excised porcine cornea) models. The permeation of four ophthalmic drugs (i.e., timolol maleate, chloramphenicol, diclofenac sodium and dexamethasone) across in vitro and ex vivo corneal models was evaluated in the absence and presence of four commonly used surface active ophthalmic excipients (i.e., Polysorbate 80, Tyloxapol, Cremophor® EL and Pluronic® F68). The concentration and self-aggregation-dependent effects of surface active ophthalmic excipients on ophthalmic drug permeability were studied from the concentration region where only dissolved monomer molecules of surface active ophthalmic excipients exist, as well as the concentration region in which aggregates of variable size and dispersion are spontaneously formed. Neither the surface active ophthalmic excipients nor the ophthalmic drugs at all concentrations that were tested significantly affected the barrier properties of both corneal models, as assessed by transepithelial electrical resistance (TEER) monitoring during the permeability experiments. The lowest concentration of all investigated surface active ophthalmic excipients did not significantly affect the ophthalmic drug permeability across both of the corneal models that were used. For three ophthalmic drugs (i.e., chloramphenicol, diclofenac sodium and dexamethasone), depressed in vitro and ex vivo permeability were observed in the concentration range of either Polysorbate 80, Tyloxapol, Cremophor® EL or Pluronic® F68, at which self-aggregation is detected. The effect was the most pronounced for Cremophor® EL (1 and 2%, w/V) and was the least pronounced for Pluronic® F68 (1%, w/V). However, all surface active ophthalmic excipients over the entire concentration range that was tested did not significantly affect the in vitro and ex vivo permeability of timolol maleate, which is the most hydrophilic ophthalmic drug that was investigated. The results of the dynamic light scattering measurements point to the association of ophthalmic drugs with self-aggregates of surface active ophthalmic excipients as the potential mechanism of the observed permeability-depressing effect of surface active ophthalmic excipients. A strong and statistically significant correlation was observed between in vitro and ex vivo permeability of ophthalmic drugs in the presence of surface active ophthalmic excipients, which indicates that the observed permeability-altering effects of surface active ophthalmic excipients were comparable and were mediated by the same mechanism in both corneal models.

D-Optimal Design in the Development of Rheologically Improved In Situ Forming Ophthalmic Gel.

In situ forming ophthalmic gels need to be fine tuned considering all the biopharmaceutical challenges of the front of the eye in order to increase drug residence time at the application site resulting in its improved bioavailability and efficacy. The aim of this study was to develop in situ forming ophthalmic poloxamer P407/poloxamer P188/chitosan gel fine tuned in terms of polymer content, temperature of gelation, and viscosity. Minimizing the total polymer content while retaining the advantageous rheological properties has been achieved by means of D-optimal statistical design. The optimal in situ forming gel was selected based on minimal polymer content (P407, P188, and chitosan concentration of 14.2%, 1.7%, and 0.25% w/w, respectively), favorable rheological characteristics, and in vitro resistance to tear dilution. The optimal in situ forming gel was proved to be robust against entrapment of active pharmaceutical ingredients making it a suitable platform for ophthalmic delivery of active pharmaceutical ingredients with diverse physicochemical properties.

An overview of in vitro dissolution/release methods for novel mucosal drug delivery systems.

In vitro dissolution/release tests are an important tool in the drug product development phase as well as in its quality control and the regulatory approval process. Mucosal drug delivery systems are aimed to provide both local and systemic drug action via mucosal surfaces of the body and exhibit significant differences in formulation design, as well as in their physicochemical and release characteristics. Therefore it is not possible to devise a single test system which would be suitable for release testing of such complex dosage forms. This article is aimed to provide a comprehensive review of both compendial and noncompendial methods used for in vitro dissolution/release testing of novel mucosal drug delivery systems aimed for ocular, nasal, oromucosal, vaginal and rectal administration.

Lipid/alginate nanoparticle-loaded in situ gelling system tailored for dexamethasone nasal delivery.

In this study, we suggest the development of nanoparticle loaded in situ gelling system suitable for corticosteroid nasal delivery. We propose lipid/alginate nanoparticles (size 252.3±2.4nm, polydispersity index 0.241, zeta-potential -31.7±1.0mV, dexamethasone (Dex) content 255±7µgml-1) dispersed in pectin solution (5mgml-1) that undergoes a sol-gel phase transition triggered by Ca2+ present in nasal mucosa. The viscoelasticity of gel obtained by mixing nanoparticle suspension in pectin continuous phase with simulated nasal fluid (1:1V/V) is characterised by a log-linear shear thinning viscosity behaviour. Observed viscosity corresponds to the range of viscosities of nasal mucus at physiological as well as under disease conditions. Nanoparticle-loaded gel was biocompatible with the selected epithelial cell model and, in comparison to dexamethasone solution, provided reduction in Dex release (t50% 2.1h and 0.6h, respectively) and moderated transepithelial permeation in vitro (Papp 7.88±0.15 and 9.73±0.57×10-6cms-1, respectively). In conclusion, this study showed the potential of the proposed system to provide local therapeutic effect upon administration of a lower corticosteroid dose and minimize the possibility for adverse effects as it can be easily sprayed as solution and delivered beyond nasal valve, ensure prolonged contact time with nasal mucosa upon gelation, and moderate corticosteroid release and permeation.

HCE-T cell-based permeability model: A well-maintained or a highly variable barrier phenotype?

The most extensively characterized human-derived cell line used in transcorneal permeability studies, in terms of passive transcellular and paracellular transport, transporter expression and metabolic enzymes, is the immortalized human corneal epithelial cell line (HCE-T). The purpose of this study is to describe the changes in the HCE-T barrier phenotype in vitro when valid cultivation conditions, in accordance with the standardized HCE-T cell-based model protocol, were employed. Evaluation of the structural and functional barrier properties revealed two different HCE-T barrier phenotypes, depending on the polycarbonate membrane pore size. Model I (pore size 0.4µm) was characterized by a multilayered HCE-T epithelium at the apical side and a weak barrier function (70-115Ω×cm2), whereas Model II (pore size 3µm) consisted of an apical lipophilic HCE-T monolayer and a basolateral lipophilic monolayer of migrated HCE-T cells that showed improved barrier properties (1700-2600Ω×cm2) compared with Model I. Considering the permeation of ophthalmic compounds and in vitro/ex vivo correlation, Model II was better able to predict transcorneal drug permeation. This study highlights the important aspects of HCE-T barrier phenotype variability that should be continuously monitored in the routine application of HCE-T cell-based models across both academic and pharmaceutical industry research laboratories.

Tear fluid-eye drops compatibility assessment using surface tension.

OBJECTIVE: To evaluate the compatibility of commercially available eye drop surface tension with the tear film physiological range and to characterize commonly used ophthalmic excipients in terms of their surface activity under eye-biorelevant conditions. SIGNIFICANCE: There are a number of quality requirements for the eye drops (e.g. tonicity, pH, viscosity, refractive index) that needs to comply with the physiological parameters of the eye surface. However, the adjustment of surface tension properties of the eye drops to the normal range of surface tension at the air/tear fluid interface (40-46 mN/m) has received rather less attention thus far. Yet, the surface tension at the air/tear fluid interface is of vital importance for the normal function of the eye surface. METHODS: The surface tension compatibility of the isotonic aqueous solutions of commonly used ophthalmic excipients as well as 18 approved eye drops with the tear fluid have been evaluated using surface tension method. RESULTS: Each ophthalmic ingredient including the preservatives, solubilizing agents and thickening agents can influence the surface tension of the final formulation. In case of complex ophthalmic formulations one should also consider the possible interactions among excipients and consequent impact on overall surface activity. Out of 18 evaluated eye drops, three samples were within, 12 samples were below and three samples were above the physiological range of the tear fluid surface tension. CONCLUSIONS: Our results provide a rationale for clinical studies aiming to assess the correlation between the eye drops surface tension and the tear film (in)stability.

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.

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.