Joana F. Fangueiro

Current State-of-Art and New Trends on Lipid Nanoparticles (SLN and NLC) for Oral Drug Delivery

Lipids and lipid nanoparticles are extensively employed as oral-delivery systems for drugs and other active ingredients. These have been exploited for many features in the field of pharmaceutical technology. Lipids usually enhance drug absorption in the gastrointestinal tract (GIT), and when formulated as nanoparticles, these molecules improve mucosal adhesion due to small particle size and increasing their GIT residence time. In addition, lipid nanoparticles may also protect the loaded drugs from chemical and enzymatic degradation and gradually release drug molecules from the lipid matrix into blood, resulting in improved therapeutic profiles compared to free drug. Therefore, due to their physiological and biodegradable properties, lipid molecules may decrease adverse side effects and chronic toxicity of the drug-delivery systems when compared to other of polymeric nature. This paper highlights the importance of lipid nanoparticles to modify the release profile and the pharmacokinetic parameters of drugs when administrated through oral route.

Cross-linked chitosan microspheres for oral delivery of insulin: Taguchi design and in vivo testing

Insulin-loaded chitosan microspheres were engineered by emulsion cross-linking method using glutaraldehyde as cross-linker. Taguchi orthogonal method was applied to optimize the production time and reduce the number of experiments required to obtain an optimized formulation. Three variables were evaluated, i.e. chitosan and glutaraldehyde concentrations, and cross-linking time at three levels. The dependent variables were the mean particle size and the encapsulation efficiency. The optimal formulation was obtained with chitosan 3% (w/v), glutaraldehyde 3.5% (v/v), and cross-linking time of 5h, characterized by microspheres with a mean particle size of 29.5 μm, and insulin encapsulation efficiency of 71.6±1.3%. In vivo studies were carried out using male Wistar albino rats, revealing a significant reduction in blood glucose level after administration of the optimized formulation, in comparison to a subcutaneous insulin injection. Chitosan microspheres were superior in terms of sustaining protein release over conventional insulin therapy.

Tramadol hydrochloride: Pharmacokinetics, pharmacodynamics, adverse side effects, co-administration of drugs and new drug delivery systems

Tramadol hydrochloride (TrHC) is a synthetic analgesic drug exhibiting opioid and non-opioid properties, acting mainly on the central nervous system. It has been mostly used to treat pain, although its use to treat anxiety and depression has also been documented. These properties arise from the fact that they inhibit serotonin (5-HT) reuptake augmenting 5-HT concentration on the synaptic cleft. Despite this, TrHC has also been described to have several side effects which are mainly due to its fast metabolization and excretion which in turn requires multiple doses per day. To surpass this limitation, new pharmaceutical formulations are being developed intending the protection, target and sustained delivery as well as a reduction on daily dose aiming a reduction on the side effects. In the present work we have revised the efficacy, safety, biological and adverse effects of TrHC, and the added value of developing a novel drug delivery system for topical administration.

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.

Predictive modeling of insulin release profile from cross-linked chitosan microspheres

Insulin-loaded microspheres composed of chitosan 3% (w/v), and loading 120 IU insulin were produced by emulsion cross-linking method. Cross-linking time was 5 h and glutaraldehyde 3.5% (v/v) was used as cross-linker. Swelling ratio studies were evaluated to predict release of insulin from chitosan microspheres. Bacitracin and sodium taurocholate were incorporated in the formulations as proteolytic enzyme inhibitor and absorption enhancer, respectively. In vitro insulin release studies were performed in phosphate buffer pH 7.4 and also in HCl pH 2 with and without trypsin. Activity of bacitracin was also evaluated. In vitro release showed a controlled profile up to 12 h and the formulation containing 0.15% (w/v) of bacitracin revealed a maximum biological activity of about 49.1 ± 4.1%. Mathematical modeling using Higuchi and Korsmeyer-Peppas suggested a non-Fickian diffusion as the mechanism of insulin release. Insulin-loaded chitosan microspheres for oral delivery showed to be an innovative and reliable delivery system to overcome conventional insulin therapy.

Preparation and characterization of PEG-coated silica nanoparticles for oral insulin delivery

The present study reports the production and characterization of PEG-coated silica nanoparticles (SiNP-PEG) containing insulin for oral administration. High (PEG 20,000) and low (PEG 6000) PEG molecular weights were used in the preparations. SiNP were produced by sol-gel technology followed by PEG adsorption and characterized for in vitro release by Franz diffusion cells. In vitro permeation profile was assessed using everted rat intestine. HPLC method has been validated for the determination of insulin released and permeated. Insulin secondary structure was performed by circular dichroism (CD). Uncoated SiNP allowed slower insulin release in comparison to SiNP-PEG. The coating with high molecular weight PEG did not significantly (p> 0.05) alter insulin release. The slow insulin release is attributed to the affinity of insulin for silanol groups at silica surface. Drug release followed second order kinetics for uncoated and SiNP-PEG at pH 2.0. On the other hand, at pH 6.8, the best fitting was first-order for SiNP-PEG, except for SiNP which showed a Boltzmann behavior. Comparing the values of half-live, SiNP-PEG 20,000 showed a faster diffusion followed by Si-PEG 6000 and SiNP. CD studies showed no conformational changes occurring after protein release from the nanoparticles under gastrointestinal simulated conditions.

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.

Nanoemulsions (NEs), liposomes (LPs) and solid lipid nanoparticles (SLNs) for retinyl palmitate: Effect on skin permeation

The aim of this study was to develop biocompatible lipid-based nanocarriers for retinyl palmitate (RP) to improve its skin delivery, photostability and biocompatibility, and to avoid undesirable topical side effects. RP loaded nanoemulsions (NEs), liposomes (LPs) and solid lipid nanoparticles (SLNs) were characterized in terms of size, surface electrical charge, pH, drug encapsulation efficiency and morphology. Spherical-shaped nanocarriers with a negatively charged surface (>|40|mV) and mean size lower than 275 nm were produced with adequate skin compatibility. The rheological properties showed that aqueous dispersions of SLNs followed a non-Newtonian behavior, pseudoplastic fluid adjusted to Herschel-Bulkley equation, whereas LPs and NEs exhibited a Newtonian behavior. SLNs offered significantly better photoprotection than LPs and NEs for RP. The cumulative amount of drug permeated through human skin at the end of 38 h was 6.67 ± 1.58 μg, 4.36 ± 0.21 μg and 3.64 ± 0.28 μg for NEs, LPs and SLNs, respectively. NEs flux was significantly higher than SLNs and LPs: NEs (0.37 ± 0.12 μg/h) > LPs (0.15 ± 0.09 μg/h) > SLNs (0.10 ± 0.05 μg/h). LPs offered significant higher skin retention than NEs and SLNs. Finally, even though all developed nanocarriers were found to be biocompatible, according to histological studies, NE was the system that most disrupted the skin. These encouraging findings can guide in proper selection of topical carriers among the diversity of available lipid-based nanocarriers, especially when a dermatologic or cosmetic purpose is desired.

A novel lipid nanocarrier for insulin delivery: production, characterization and toxicity testing.

A novel nanocarrier based on solid lipid nanoparticles (SLNs) was developed for insulin delivery using a novel double emulsion method. Physical stability of particles was assessed by size analysis using dynamic light scattering (DLS), matrix crystallinity by differential scanning calorimetry (DSC) and toxicity analysis by Drosophila melanogaster testing. Insulin-SLNs were composed of Softisan®100 1.25% wt, Lutrol®F68 1% wt, soybean lecithin 0.125% wt, and loaded with 0.73–0.58 mg/mL peptide. Placebo-SLNs (insulin-free) also contained 0.025% wt Tween®80. Mean particle sizes of placebo-SLN and insulin-SLN were 958 ± 9.5 and 978 ± 8.3 nm, respectively. The polydispersity index (PI) was 0.28 ± 0.018 and 0.29 ± 0.013, respectively. Polarized light microscopy analysis depicted no aggregation of developed particles. DSC analysis allowed characterizing SLN with 43–51% matrix crystallinity. Using Drosophila melanogaster test, no toxicity was reported for SLN and for the bulk lipid. This study shows that SLNs are promising and helpful to overcome conventional insulin therapy, in particular for their lack of toxicity for oral delivery.

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.