Biopharmaceutical evaluation of epigallocatechin gallate-loaded cationic lipid nanoparticles (EGCG-LNs): In vivo, in vitro and ex vivo studies.

Cationic lipid nanoparticles (LNs) have been tested for sustained release and site-specific targeting of epigallocatechin gallate (EGCG), a potential polyphenol with improved pharmacological profile for the treatment of ocular pathologies, such as age-related macular edema, diabetic retinopathy, and inflammatory disorders. Cationic EGCG-LNs were produced by double-emulsion technique; the in vitro release study was performed in a dialysis bag, followed by the drug assay using a previously validated RP-HPLC method. In vitro HET-CAM study was carried out using chicken embryos to determine the potential risk of irritation of the developed formulations. Ex vivo permeation profile was assessed using rabbit cornea and sclera isolated and mounted in Franz diffusion cells. The results show that the use of cationic LNs provides a prolonged EGCG release, following a Boltzmann sigmoidal profile. In addition, EGCG was successfully quantified in both tested ocular tissues, demonstrating the ability of these formulations to reach both anterior and posterior segment of the eye. The pharmacokinetic study of the corneal permeation showed a first order kinetics for both cationic formulations, while EGCG-cetyltrimethylammonium bromide (CTAB) LNs followed a Boltzmann sigmoidal profile and EGCG-dimethyldioctadecylammonium bromide (DDAB) LNs a first order profile. Our studies also proved the safety and non-irritant nature of the developed LNs. Thus, loading EGCG in cationic LNs is recognised as a promising strategy for the treatment of ocular diseases related to anti-oxidant and anti-inflammatory pathways.

Design and elaboration of freeze-dried PLGA nanoparticles for the transcorneal permeation of carprofen: Ocular anti-inflammatory applications.

This work aimed the design and development of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) for the ocular delivery of Carprofen (CP) by a central rotatable composite design 2(3)+ star. NPs showed adequate size for ocular administration (189.50 ± 1.67 nm), low polydispersity (0.01 ± 0.01), negative charge surface (-22.80 ± 0.66 mV) and optimal entrapment efficiency (74.70 ± 0.95%). Physicochemical analysis confirmed that CP was dispersed inside the NPs. The drug release followed a first order kinetic model providing greater sustained CP release after lyophilization. Ex vivo permeation analysis through isolated rabbit cornea revealed that a sufficient amount of CP was retained in the tissue avoiding excessive permeation and thus, potential systemic levels. Ex vivo ocular tolerance results showed no signs of ocular irritancy, which was also confirmed by in vivo Draize test. In vivo ocular anti-inflammatory efficacy test confirmed an optimal efficacy of NPs and its potential application in eye surgery.

Physicochemical characterization of epigallocatechin gallate lipid nanoparticles (EGCG-LNs) for ocular instillation.

The encapsulation of epigallocatechin gallate (EGCG) in lipid nanoparticles (LNs) could be a suitable approach to avoid drug oxidation and epimerization, which are common processes that lead to low bioavailability of the drug limiting its therapeutic efficacy. The human health benefits of EGCG gained much interest in the pharmaceutical field, and so far there are no studies reporting its encapsulation in LNs. The purpose of this study has been the development of an innovative system for the ocular delivery of EGCG using LNs as carrier for the future treatment of several diseases, such as dry eye, age-related macular degeneration (AMD), glaucoma, diabetic retinopathy and macular oedema. LNs dispersions have been produced by multiple emulsion technique and previously optimized by a factorial design. In order to increase ocular retention time and mucoadhesion by electrostatic attraction, two distinct cationic lipids were used, namely, cetyltrimethylammonium bromide (CTAB) and dimethyldioctadecylammonium bromide (DDAB). EGCG has been successfully loaded in the LNs dispersions and the nanoparticles analysis over 30 days of storage time predicted a good physicochemical stability. The particles were found to be in the nanometer range (<300 nm) and all the evaluated parameters, namely pH, osmolarity and viscosity, were compatible to the ocular administration. The evaluation of the cationic lipid used was compared regarding physical and chemical parameters, lipid crystallization and polymorphism, and stability of dispersion during storage. The results show that different lipids lead to different characteristics mainly associated with the acyl chain composition, i.e. double lipid shows to have influence in the crystallization and stability. Despite the recorded differences between DTAB and DDAB, both cationic LNs seem to fit the parameters for ocular drug delivery.

Validation of a high performance liquid chromatography method for the stabilization of epigallocatechin gallate.

Epigallocatechin gallate (EGCG) is a green tea catechin with potential health benefits, such as anti-oxidant, anti-carcinogenic and anti-inflammatory effects. In general, EGCG is highly susceptible to degradation, therefore presenting stability problems. The present paper was focused on the study of EGCG stability in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) medium regarding the pH dependency, storage temperature and in the presence of ascorbic acid a reducing agent. The evaluation of EGCG in HEPES buffer has demonstrated that this molecule is not able of maintaining its physicochemical properties and potential beneficial effects, since it is partially or completely degraded, depending on the EGCG concentration. The storage temperature of EGCG most suitable to maintain its structure was shown to be the lower values (4 or -20 °C). The pH 3.5 was able to provide greater stability than pH 7.4. However, the presence of a reducing agent (i.e., ascorbic acid) was shown to provide greater protection against degradation of EGCG. A validation method based on RP-HPLC with UV-vis detection was carried out for two media: water and a biocompatible physiological medium composed of Transcutol®P, ethanol and ascorbic acid. The quantification of EGCG for purposes, using pure EGCG, requires a validated HPLC method which could be possible to apply in pharmacokinetic and pharmacodynamics studies.

Design of nanosuspensions and freeze-dried PLGA nanoparticles as a novel approach for ophthalmic delivery of pranoprofen.

Pranoprofen (PF)-loaded poly (lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) were optimized and characterized as a means of exploring novel formulations to improve the biopharmaceutical profile of this drug. These systems were prepared using the solvent displacement technique, with polyvinyl alcohol (PVA) as a stabilizer. A factorial design was applied to study the influence of several factors (the pH of the aqueous phase and the stabilizer, polymer and drug concentrations) on the physicochemical properties of the NPs. After optimization, the study was performed at two different aqueous phase pH values (4.50 and 5.50), two concentrations of PF (1.00 and 1.50 mg/mL), three of PVA (5, 10, and 25 mg/mL), and two of PLGA (9.00 and 9.50 mg/mL). These conditions produced NPs of a size appropriate particle size for ocular administration (around 350 nm) and high entrapment efficiency (80%). To improve their stability, the optimized NPs were lyophilized. X-ray, FTIR, and differential scanning calorimetry analysis confirmed the drug was dispersed inside the particles. The release profiles of PF from the primary nanosuspensions and rehydrated freeze-dried NPs were similar and exhibited a sustained drug delivery pattern. The ocular tolerance was assessed by an HET-CAM test. No signs of ocular irritancy were detected (score 0).

Design of cationic lipid nanoparticles for ocular delivery: development, characterization and cytotoxicity.

In the present study we have developed lipid nanoparticle (LN) dispersions based on a multiple emulsion technique for encapsulation of hydrophilic drugs or/and proteins by a full factorial design. In order to increase ocular retention time and mucoadhesion by electrostatic attraction, a cationic lipid, namely cetyltrimethylammonium bromide (CTAB), was added in the lipid matrix of the optimal LN dispersion obtained from the factorial design. There are a limited number of studies reporting the ideal concentration of cationic agents in LN for drug delivery. This paper suggests that the choice of the concentration of a cationic agent is critical when formulating a safe and stable LN. CTAB was included in the lipid matrix of LN, testing four different concentrations (0.25%, 0.5%, 0.75%, or 1.0%wt) and how composition affects LN behavior regarding physical and chemical parameters, lipid crystallization and polymorphism, and stability of dispersion during storage. In order to develop a safe and compatible system for ocular delivery, CTAB-LN dispersions were exposed to Human retinoblastoma cell line Y-79. The toxicity testing of the CTAB-LN dispersions was a fundamental tool to find the best CTAB concentration for development of these cationic LN, which was found to be 0.5 wt% of CTAB.

[Endogenous endophthalmitis: case report and brief review of a serious ocular disease]. / Endoftalmitis endógenas: revisión de una serie de casos y revaloración de una entidad ocular grave.

INTRODUCTION: Endogenous endophthalmitis (EE) is a prevalent but serious disease. Our aim was to describe cases of EE, with emphasis in the risk factors and the improvement of the prognosis. METHODS: A review of EE cases was done between 1996-2011 in a secondary care hospital in Spain. The reported variables were: comorbidities, isolated microorganisms, susceptibility to antimicrobial treatment and visual prognosis. RESULTS: 9 cases of EE were analyzed. All had some underlying disease, diabetes mellitus being the most frequent. Seven of the nine cases had a history of eye injury. Extraocular source of infection was identified in 7 cases, with predominantly gastrointestinal disease. Most microorganisms were isolated from blood cultures. The visual prognosis was unfavorable in five patients and was associated with virulent microorganisms and delayed treatment. CONCLUSIONS: EE is a rare disease that involve immunocompromised patients with ophthalmic disease. To improve prognosis, appropriate diagnosis and early treatment is require. Therefore, we recommend funduscopy examination in patients with sepsis, risk factors and prior history of ocular disease.

Endoftalmitis endógenas: revisión de una serie de casos y revaloración de una entidad ocular grave / Endogenous endophthalmitis: case report and brief review of a serious ocular disease

Endogenous endophthalmitis (EE) is a prevalent but serious disease. Our aim was to describe cases of EE, with emphasis in the risk factors and the improvement of the prognosis. Methods: A review of EE cases was done between 1996-2011 in a secondary care hospital in Spain. The reported variables were: comorbidities, isolated microorganisms, susceptibility to antimicrobial treatment and visual prognosis. Results: 9 cases of EE were analyzed. All had some underlying disease, diabetes mellitus being the most frequent. Seven of the nine cases had a history of eye injury. Extraocular source of infection was identified in 7 cases, with predominantly gastrointestinal disease. Most microorganisms were isolated from blood cultures. The visual prognosis was unfavorable in five patients and was associated with virulent microorganisms and delayed treatment. Conclusions: EE is a rare disease that involve immunocompromised patients with ophthalmic disease. To improve prognosis, appropriate diagnosis and early treatment is require. Therefore, we recommend funduscopy examination in patients with sepsis, risk factors and prior history of ocular disease.

La endoftalmitis endógena (EE) es una patología poco prevalente aunque grave. Nuestro objetivo es describir los casos de EE diagnosticados en un hospital secundario español, con particular atención a los factores de riesgo y la posible mejora del pronóstico. Material y Métodos: Revisamos las historias clínicas de los pacientes diagnosticados de EE entre 1996-2011. Las variables recogidas fueron: co-morbilidades, microorganismo/s aislados y su susceptibilidad a los antimicrobianos, tratamiento administrado y pronóstico visual. Resultados: Se estudiaron nueve casos de EE. Todos presentaban alguna enfermedad de base, siendo diabetes mellitus la más frecuente. Siete de los nueve casos presentaron antecedentes de lesión ocular. La probable fuente extraocular se identificó en siete casos, predominando el foco gastrointestinal. La mayoría de microorganismos se aisló de hemocultivos. El pronóstico visual fue desfavorable en cinco pacientes, asociándose a microorganismos virulentos y al retraso terapéutico. Conclusiones: La EE es una enfermedad inusual que afecta a pacientes con inmunidad disminuida y antecedentes oftalmológicos. Para mejorar el pronóstico se requiere un diagnóstico acertado y un tratamiento precoz, todo un reto para médicos clínicos y microbiólogos. Por ello, recomendamos realizar un fondo de ojo a los pacientes con sepsis, factores de riesgo de EE y antecedentes de patología ocular.

Experimental factorial design applied to mucoadhesive lipid nanoparticles via multiple emulsion process.

Solid lipid nanoparticles (SLNs) produced from multiple emulsions technology theoretically enclose an inner aqueous compartment suitable for hydrophilic biomolecules. This paper reports a 3(3) full factorial design study to optimize SLNs formulations for hydrophilic biomolecules. The concentrations of solid lipid, lipophilic and hydrophilic emulsifiers were set as the 3 independent variables. Mean particle size (Z-Ave), polydispersity index (PI) and zeta potential (ZP) were set as the dependent variables. The selected optimized parameters were set as 1.0 wt% of solid lipid, 0.25 wt% of lipophilic emulsifier and 1.5 wt% of hydrophilic emulsifier. The coating of SLNs with sodium alginate was found to improve the ZP of the lipid particles and these results suggest that the ideal concentration was 0.75 wt%. The influence of low pH (i.e., about 2-3) in the inner aqueous phase was stronger than higher pH values, contributing for the production of larger droplet sizes. Nevertheless, these systems can be useful for the incorporation of biomolecules requiring a pH ranging between 4 and 10. SLNs based on multiple emulsions technology were found to be a promising approach for the incorporation of several hydrophilic drugs, such as proteins and peptides.

Nanostructured lipid carriers for triamcinolone acetonide delivery to the posterior segment of the eye.

Triamcinolone acetonide (TA) is a corticosteroid drug currently administered by intravitreal injection for a broad spectrum of inflammatory, edematous and angiogenic ocular diseases. To increase the drug's bioavailability by ocular instillation, TA was encapsulated in nanostructured lipid carriers (NLC), previously optimized by our group using a factorial design approach. In the present paper, nanometric (∼200 nm), unimodal and negatively charged NLC loaded with the fluorescent lipid marker Nile red (NR-NLC) and drug (TA-NLC) were produced by high pressure homogenization. Based on the selected formulations, in vivo tests were carried out by eye-drop instillation of NR-NLC in mice, revealing the systems' ability of delivering lipophilic actives to the posterior segment of the eye via the corneal and non-corneal pathways. Short and long-term stability of TA-NLC was assessed by high performance stability analysis using the Turbiscan®. The results showed a backscattering of less than 1.5% and during a period of 6 months, anticipated the low tendency of these particles for aggregation during shelf life when stored at room temperature.