Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulations.

The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.

Study of the antibacterial and cytotoxic activity of chitosan and its derivatives chemically modified with phthalic anhydride and ethylenediamine.

The insertion of hydrophobic and hydrophilic chains in the chitosan molecule can improve its antibacterial activity, expanding its range of application in several areas of medical-pharmaceutical sciences. Thus, this work aimed to increase the antibacterial activity of chitosan through the modification reaction with phthalic anhydride (QF) and subsequent reaction with ethylenediamine (QFE). The chitosan and derivatives obtained were characterized by elemental analysis, 13C Nuclear Magnetic Resonance (13C NMR), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Thermogravimetric Analysis (TG), where it was possible to prove the chemical modification. Both materials showed a greater antibacterial inhibitory effect against Gram-positive bacteria, Staphylococcus aureus, emphasizing antibacterial activity against Gram-negative bacteria, Escherichia coli, with values above 70 % of the inhibitory effect, which is a promising result. Assays with human fibroblast cells by the [3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium (MTT)] bromide reduction test did not indicate toxicity in the materials. Thus, the derived materials showed promise for biomedical applications since they combined excellent antibacterial activity against gram-positive and gram-negative strains and did not show cytotoxicity.

Lipid nanoparticles coated with chitosan using a one-step association method to target rifampicin to alveolar macrophages.

This work proposes the development and characterization of solid lipid nanoparticles (SLNs) loaded with rifampicin (RIF) aiming to enhance mucoadhesion of the SLNs and consequently internalization by the alveolar macrophages (AMs). The lipid nanoparticles (NPs) were characterized and the results showed that the NPs obtained present a spherical or a starry shape with diameter around 250-500 nm, a monodisperse population, with zeta potential between -31 mV for uncoated SLNs and +33 mV for coated SLNs. The drug EE was approximately 90 % and the loading capacity (LC) 4.5 %. The SLNs coated with chitosan by the association method (aC-SLNs) show an effective mucoadhesive profile, verified by the turdimetry and surface loading method, corroborated with the cellular assays. The presence of chitosan in the aC-SLNs promotes higher mucoadhesive properties to the NPs and permeability in A549, suggesting that the safe aC-SLNs-RIF can be used as a promising drug delivery system for improving tuberculosis treatment.

Drug Delivery Systems on Leprosy Therapy: Moving Towards Eradication?

Leprosy disease remains an important public health issue as it is still endemic in several countries. Mycobacterium leprae, the causative agent of leprosy, presents tropism for cells of the reticuloendothelial and peripheral nervous system. Current multidrug therapy consists of clofazimine, dapsone and rifampicin. Despite significant improvements in leprosy treatment, in most programs, successful completion of the therapy is still sub-optimal. Drug resistance has emerged in some countries. This review discusses the status of leprosy disease worldwide, providing information regarding infectious agents, clinical manifestations, diagnosis, actual treatment and future perspectives and strategies on targets for an efficient targeted delivery therapy.

pH-responsive chitosan based hydrogels affect the release of dapsone: Design, set-up, and physicochemical characterization.

Dapsone (DAP) is a bactericidal agent used in the treatment of leprosy, caused by Mycobacterium leprae. Despite its therapeutic potential, DAP has low solubility, which results in allow therapeutic index and a high microbial resistance. Recently, new approaches were used to increase the DAP solubility. In particular, the use of interpenetrating polymer network (IPN)-hydrogels based chitosan (CS) for the controlled release of DAP provides some advantages because they can modify their swelling properties and network structures as a response to environmental stimuli. The aim of this study was to synthesize and physicochemically characterize pH-responsive chitosan/polymer hydrogels to control the release of DAP. For this reason, different combination of polymers, such as polyvinyl pyrrolidone, polyethylene glycol and hydroxypropyl methylcellulose, and concentrations of the cross-linking agents (glutaraldehyde) were used and then blended to the CS. The resulting hydrogels were evaluated in terms of physicochemical and swelling properties, rheological analysis and in vitro release of DAP at different pHs (1.2-6.8). Hydrogels were further characterized by Fourier transformed infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) analysis. pH-responsive DAP-loaded hydrogels may represent the set-up for developing potential oral formulations for the treatment of leprosy caused by Mycobacterium leprae.

Mannosylated solid lipid nanoparticles for the selective delivery of rifampicin to macrophages.

Tuberculosis (TB) is still a devastating disease and more people have died of TB than any other infectious diseases throughout the history. The current therapy consists of a multidrug combination in a long-term treatment, being associated with the appearance of several adverse effects. Thus, solid lipid nanoparticles (SLNs) were developed using mannose as a lectin receptor ligand conjugate for macrophage targeting and to increase the therapeutic index of rifampicin (RIF). The developed SLNs were studied in terms of diameter, polydispersity index, zeta potential, encapsulation efficiency (EE) and loading capacity (LC). Morphology, in vitro drug release and differential scanning calorimetry studies, macrophage uptake studies, cell viability and storage stability studies were also performed. The diameter of the SLNs obtained was within the range of 160-250 nm and drug EE was above 75%. The biocompatibility of M-SLNs was verified and the internalization in macrophages was improved with the mannosylation. The overall results suggested that the developed mannosylated formulations are safe and a promising tool for TB therapy targeted for macrophages.

Overcoming clofazimine intrinsic toxicity: statistical modelling and characterization of solid lipid nanoparticles.

The aim of this work was to develop solid lipid nanoparticles (SLNs) loaded with clofazimine (CLZ) (SLNs-CLZ) to overcome its intrinsic toxicity and low water solubility, for oral drug delivery. A Box-Behnken design was constructed to unravel the relations between the independent variables in the selected responses. The optimized SLNs-CLZ exhibited the following properties: particle size ca 230 nm, zeta potential of -34.28 mV, association efficiency of 72% and drug loading of 2.4%, which are suitable for oral delivery. Further characterization included Fourier transformed infrared spectroscopy that confirmed the presence of the drug and the absence of chemical interactions. By differential scanning calorimetry was verified the amorphous state of CLZ. The storage stability studies ensured the stability of the systems over a period of 12 weeks at 4°C. In vitro cytotoxicity studies evidenced no effect of both drug-loaded and unloaded SLNs on MKN-28 gastric cells and on intestinal cells, namely Caco-2 and HT29-MTX cells up to 25 µg ml-1 in CLZ. Free CLZ solutions exhibited IC50 values of 16 and 20 µg ml-1 for Caco-2 and HT29-MTX cells, respectively. It can be concluded that the optimized system, designed considering important variables for the formulation of poorly soluble drugs, represents a promising platform for oral CLZ delivery.

Development of PLGA nanoparticles loaded with clofazimine for oral delivery: Assessment of formulation variables and intestinal permeability.

The use of polymeric nanoparticles as delivery systems is a promising tool to overcome drawbacks related to low aqueous solubility of drugs, which limit their in vivo bioavailability. The aim of this study was to decrease clofazimine (CLZ) toxicity using experimental design to formulate CLZ loaded in PLGA nanoparticles (NPs-CLZ) through a Plackett-Burman design (PBD). A screening PBD was constructed with twelve formulations involving six variables among process and formulation parameters and the selected responses were particle size, polydispersity index (PDI), association efficiency (AE) and drug loading (DL). The formulation was achieved based on the desirability tool, and the obtained NPs-CLZ formulation was characterized regarding morphology, physicochemical properties, in vitro release and cellular studies. Particle size, PDI, AE and DL were found to be 211±3nm, 0.211±0.009, 70±5% and 12±1%, respectively. Physicochemical studies confirmed the absence of chemical interactions between CLZ and other nanoparticles constituents and the amorphous state of CLZ, while morphological analysis revealed the spherical shape of the particles. In vitro release profile of CLZ from NPs-PLGA showed a slow pattern of drug release. Cell viability studies towards intestinal cells revealed that NPs-CLZ did not show CLZ toxicity on Caco-2 and HT29-MTX cells compared to free CLZ solutions. Moreover, CLZ could permeate Caco-2 monolayers substantially at the end of 8h. It can be concluded that the proposed NPs-CLZ represent a promising platform to the oral delivery of CLZ as they were able to decrease its intrinsic toxicity, with improved absorption.

Mucoadhesive chitosan-coated solid lipid nanoparticles for better management of tuberculosis.

Taking into consideration the potential mucoadhesion properties of systems in lung delivery, this paper describes the preparation and characterization of chitosan-coated solid lipid nanoparticles (C-SLNs) loaded with rifampicin (RIF) as anti-tuberculosis (anti-TB) drug. The process of development and characterization of the NPs in terms of size, surface charge, encapsulation efficiency (EE), morphology, in vitro drug release, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), in vitro assessment of mucoadhesive property, cell viability and permeability studies are documented. Results showed that the SLNs had a smooth spherical shape with a size of ca. 245-344 nm and with a zeta potential around -30 mV for SLNs and +40 mV for C-SLNs. The surface charge variation from negative to positive charge and FTIR analysis demonstrated the successful process of coating the nanoparticles (NPs) surface with chitosan. The DSC thermograms were in agreement with the nanostructure of the SLNs. The EE of drug was found to be higher than 90% and the loading capacity (LC) around 4.5%. C-SLNs show higher in vitro muchoadesive properties and a higher permeability in alveolar epithelial cells A549 than uncoated SLNs, indicating that the developed C-SLNs can be used as a promising carrier for sasfer and efficient management of TB.

Nanosystems as modulators of intestinal dapsone and clofazimine delivery.

The aim of this work was to assess the feasibility of drug nanosystems combination for oral therapy of multibacillary leprosy. The anti-leprotic drugs dapsone (DAP) and clofazimine (CLZ) were incorporated within polymeric nanosystems and studied per se and in combination. DAP was loaded in Eudragit L100 nanoparticles (NPs-DAP) while CLZ was loaded in (poly(lactic-co-glycolic acid) (NPs-CLZ). The nanosystems exhibited around 200 nm in size and a drug loading of 12% for each drug. In vitro cytotoxicity on intestinal Caco-2 cells revealed that after 8 h incubation, DAP alone and within NPs were not toxic up to 100 µg mL-1, while CLZ per se was toxic, reducing cell viability to 30% at 50 µg mL-1. Caco-2 exposed to the combination of NPs-DAP (100 µg mL-1) and NPs-CLZ (50 µg mL-1) exhibited 80% of viability. Caco-2 monolayer permeability assays revealed that DAP and CLZ in the nanosystems per se or in NPs-DAP/ NPs-CLZ combination crossed the intestinal barrier. No significant differences were observed between the single nanosystems or in combination with the apparent permeability values and the amount of permeated drug. Thus, the NPs-DAP/NPs-CLZ combination seems to be a promising platform to deliver both drugs in association, representing an important step towards the improvement of multibacillary leprosy therapy.

Folate-targeted nanostructured lipid carriers for enhanced oral delivery of epigallocatechin-3-gallate.

The well-known pleiotropic health benefits of green tea are mainly attributed to epigallocatechin-3-gallate (EGCG), a polyphenolic compound from the group of catechins. EGCG's poor stability and intestinal permeability, however, can strongly impair its biological activities. In this work, EGCG-loaded nanostructured lipid carriers (NLC) functionalized with folic acid were optimized through a Box-Behnken design intended to provide an enhanced oral absorption and increased bioavailability of EGCG. Size, zeta potential and encapsulation efficiency (EE) of the produced spherical nanoparticles were evaluated. NLC were further characterized by Differential Scanning Calorimetry (DSC). An in vitro release study in simulated gastric and intestinal fluids was conducted and the storage stability of the nanoparticles was evaluated over a period of 8weeks. The overall results demonstrated the suitability of the developed formulation for the oral delivery of EGCG and its potential for applications in food industry.

pH-sensitive nanoparticles for improved oral delivery of dapsone: risk assessment, design, optimization and characterization.

AIM: To optimize the production of pH-sensitive dapsone (DAP) nanoparticles based on Eugradit L100 (NPs-EL100-DAP) for oral delivery. MATERIALS & METHODS: NPs-EL100-DAP were optimized using a Plackett-Burman design and a Box-Behnken design. The physicochemical properties of the obtained nanoparticles were monitored by microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, differential scanning calorimetry, in vitro release assays, and examined for cytotoxicity and permeation across intestinal barrier. RESULTS: The in vitro release assay of NPs-EL100-DAP confirmed the nanoparticles' pH sensitivity and the ability to deliver DAP at intestinal environment. NPs-EL100-DAP demonstrated enhanced intestinal interactions in comparison to free DAP, across Caco-2 monolayers. CONCLUSION: These studies demonstrate the potential of NPs-EL100-DAP as a therapeutic platform for oral treatment of leprosy.

Design and statistical modeling of mannose-decorated dapsone-containing nanoparticles as a strategy of targeting intestinal M-cells.

The aim of the present work was to develop and optimize surface-functionalized solid lipid nanoparticles (SLNs) for improvement of the therapeutic index of dapsone (DAP), with the application of a design of experiments. The formulation was designed to target intestinal microfold (M-cells) as a strategy to increase internalization of the drug by the infected macrophages. DAP-loaded SLNs and mannosylated SLNs (M-SLNs) were successfully developed by hot ultrasonication method employing a three-level, three-factor Box-Behnken design, after the preformulation study was carried out with different lipids. All the formulations were systematically characterized regarding their diameter, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, and loading capacity. They were also subjected to morphological studies using transmission electron microscopy, in vitro release study, infrared analysis (Fourier transform infrared spectroscopy), calorimetry studies (differential scanning calorimetry), and stability studies. The diameter of SLNs, SLN-DAP, M-SLNs, and M-SLN-DAP was approximately 300 nm and the obtained PDI was <0.2, confirming uniform populations. Entrapment efficiency and loading capacity were approximately 50% and 12%, respectively. Transmission electron microscopy showed spherical shape and nonaggregated nanoparticles. Fourier transform infrared spectroscopy was used to confirm the success of mannose coating process though Schiff's base formation. The variation of the ZP between uncoated (approximately -30 mV) and mannosylated formulations (approximately +60 mV) also confirmed the successful coating process. A decrease in the enthalpy and broadening of the lipid melting peaks of the differential scanning calorimetry thermograms are consistent with the nanostructure of the SLNs. Moreover, the drug release was pH-sensitive, with a faster drug release at acidic pH than at neutral pH. Storage stability for the formulations for at least 8 weeks is expected, since they maintain the original characteristics of diameter, PDI, and ZP. These results pose a strong argument that the developed formulations can be explored as a promising carrier for treating leprosy with an innovative approach to target DAP directly to M-cells.

Optimization of nanostructured lipid carriers loaded with methotrexate: A tool for inflammatory and cancer therapy.

The aim of this study was to optimize and assess the potential of nanostructured lipid carriers (NLC), prepared by the hot ultrasonication method, as carrier for methotrexate (MTX), highlighting the application of factorial design. Preliminary screening drug/lipid solubility, allowed us to select Witepsol(®) E85 as the solid lipid and Mygliol(®) 812 as liquid lipid for the NLC loaded with MTX. Then, a 3-level, 3-factor Box-Behnken design and validated by ANOVA analysis; the correspondence between the predicted values and those measured experimentally confirmed the robustness of the design. Properties of optimized MTX-loaded NLCs such as morphology, size, zeta potential, entrapment efficiency, storage stability, in vitro drug release and cytotoxicity were investigated. NLCs loaded with MTX exhibited spherical shape with 252-nm, a polydispersity of 0.06±0.02, zeta potential of -14 mV and an entrapment efficiency of 87%. In vitro release studies revealed a fast initial release followed by a prolonged release of MTX from the NLC up to 24-h. The release kinetics of the optimized NLC best fitted the Peppas-Korsmeyer model for physiological and inflammatory environments and the Hixson-Crowell model skin simulation conditions. No toxicity was observed in fibroblasts. Thus, the optimized MTX-loaded NLC have the potential to be exploited as delivery system.

Rational and precise development of amorphous polymeric systems with dapsone by response surface methodology.

This work aimed to design dapsone (DAP) amorphous Polymeric Dispersions (PD) using design of experiments (DoE) and response surface methodology (RSM) as optimization tools in order to tailor the biopharmaceutical properties toward its oral delivery. A two-factor, three-level (3(2)) statistical design was implemented to study the influence of input variables (amount of PVP K30 and Pluronic F68) on the equilibrium solubility of DAP of the physical mixture (PM), kneaded (KN) and freeze dried (FD) PDs. Through the analysis, it was found that equilibrium solubility of DAP was improved with increasing of PVP K30, mainly for FD PDs, but decreased with increasing Pluronic F68 concentration. XRD and FTIR spectrum revealed the amorphous characteristic of FD PDs and SEM confirmed the homogeneity of the system leading to enhanced surface area and consequent dissolution rate. The in vitro dissolution rate of PDs was significantly faster compared to DAP and PM, and all the similarity factors (f2) were below 50, demonstrating the differences on the dissolution profiles. The results established the effectiveness of PDs for improvement of dissolution and solubility of DAP and the success in the implementation of DoE and RSM as QbD tools in the design of PDs.

Quality by design: discussing and assessing the solid dispersions risk.

The poor water solubility tops the list of undesirable physicochemical properties in the drug discovery and Solid Dispersions (SDs) has been frequently used to enhance dissolution of such compounds. Although, some challenges limit the studies of SD commercial application. During recent years, the Quality by Design (QbD) approach has begun to change drug development, and focus on pharmaceutical production, which shifted from an univariate empirical understanding for a systematic multivariate process. In this review, some possible variables during the development process, formulation and production of SDs were defined, introducing and applying the QbD concept. The proposed work presented important definitions as well as its application in the pharmaceutical product and process design, especially the challenges encountered during the development of formulations of poorly soluble drugs. In this aspect, the SD technique was deeply discussed, in which some important parameters during SD design and production were mentioned as method of production, polymers commonly used, methods for characterization and stability evaluation, in addition of biopharmaceutical considerations. Finally, a specific risk assessment for the design and production of SD and critical points were discussed, which was a positive evolution and may lead to better understanding of SD for a rational formulation.