María José Cózar-Bernal

Insulin-loaded PLGA microparticles: flow focusing versus double emulsion/solvent evaporation

Context: Oral administration of insulin is severely limited by very low bioavailability. Biocompatible polymeric nanocarriers have been investigated to overcome this problem. Flow focusing (FF) has revolutionized current engineering of poly(D,L-lactide-co-glycolide) (PLGA) based micromedicines. This technique has never been used to formulate insulin-loaded PLGA microparticles. Objective: Investigation of the benefits rising from the synthesis of insulin-loaded PLGA microplatforms by FF, compared to double emulsion/solvent evaporation method. Materials and methods: Both synthesis methodologies were compared in terms of geometry, surface physicochemical properties and insulin vehiculization capabilities. The stability of the peptide during the formulation procedure was further analysed. Results: FF permitted the preparation of insulin-loaded microcarriers with better geometry and physicochemical properties for the oral route, along with greater insulin loading capabilities and sustained insulin release kinetics. Discussion and conclusion: Results have lead to the identification of the best formulation conditions for the engineering of insulin-loaded PLGA microparticles against diabetes.

Protein-loaded PLGA microparticles engineered by flow focusing: physicochemical characterization and protein detection by reversed-phase HPLC.

In the present study, a novel synthesis technique based on the flow focusing (FF) technology is investigated for the preparation of green fluorescent protein (GFP)-loaded poly(D,L-lactide-co-glycolide) (PLGA) microparticles. To our knowledge, this novel technique has never been applied to the formulation of proteins in polymeric systems. A simple, specific and rapid reversed-phase HPLC (RP-HPLC) method was validated for the determination of GFP in PLGA microparticles with the best chromatographic peak resolution, reduced run time and low cost of analysis. In order to achieve the finest GFP-loaded polymeric particles, experimental parameters mainly associated to the FF device were studied (liquid flow rate and pressure of the focusing air). Very high GFP encapsulation values (>90%) were obtained by this technique, and the electrokinetic characterization of these systems suggested that this protein was incorporated into the polymeric matrix. This study is intended to offer information on which to base the development of high molecular weight protein-loaded polymeric delivery systems prepared by FF.

Development and validation of a high performance chromatographic method for determining sumatriptan in niosomes.

In this paper, a novel, precise, specific, accurate and rapid reversed-phase high performance liquid chromatographic method was developed, optimized and validated for determining sumatriptan succinate in niosomes with the best chromatographic peak resolution, reduced run time and low cost of analysis. The formulation has been previously optimized in terms of composition and preparation technique to obtain a high drug encapsulation efficiency and adequate vesicle size distribution. This method showed the best resolution by using Spherisorb OSD2 C18 column (250 mm × 4.6 mm, 5 μm) using phosphate buffer (0.05 M):acetonitrile (80:20, v/v; pH adjusted to 6.0) as a mobile phase at a flow rate of 1 mL/min and wavelength of 214 nm. The main objective of this research was to demonstrate the robustness of the reversed-phase HPLC method development by applying the Taguchi robust methodology. The signal-to-noise ratio (S/N) was employed as a quality measurement. This tool permits to establish the influence of some selected factors (acetonitrile:phosphate ratio, pH buffer, oven temperature and flow rate) on two responses (peak areas and retention time). On the basis of the results obtained, we can conclude that this analytical method was robust for all the factors studies, as exception of the flow rate, where the higher quality was obtained for the fewer values (0.8 mL/min). Therefore, this parameter must be carefully controlled when this method was employed, to avoid any modification in the peak areas overall.

Calcium alginate microspheres containing metformin hydrochloride niosomes and chitosomes aimed for oral therapy of type 2 diabetes mellitus

Metformin is an oral hypoglycemic agent used in the type 2 diabetes, whose poor bioavailability and short half-life make the development of effective extended-release formulations highly desirable. Different metformin-loaded chitosomal and niosomal formulations were developed and suitably characterized, but were unable to provide the desired sustained release. The entrapment of both kinds of colloidal dispersions in calcium alginate beads enabled to strongly reduce the amount of drug released at gastric level (from 18 up to a maximum of 30%), and to obtain a sustained release in simulated intestinal fluid, which was properly tuned by varying the percentage of calcium alginate in the beads. In vivo studies on rats revealed a significant improvement of metformin hypoglycemic effect when orally administered as chitosomal and even more as niosomal dispersion entrapped in alginate beads, not only with respect to the drug as such, but also to the alginate beads loaded with the plain drug. The more intense and sustained therapeutic effect with time provided by the drug-in niosomes-in alginate bead formulation could be very profitable for maintaining tight blood glucose levels over prolonged period of time after oral administration, allowing a reduction of its dose and related collateral effects, and improving patient compliance.

Deformability properties of timolol-loaded transfersomes based on the extrusion mechanism. Statistical optimization of the process

Abstract The purpose of this work was to analyze the deformability properties of different timolol maleate (TM)-loaded transfersomes by extrusion. This was performed because elastic liposomes may contribute to the elevation of amount and rate of drug permeation through the corneal membrane. This paper describes the optimization of a transfersome formulation by use of Taguchi orthogonal experimental design and two different statistical analysis approaches were utilized. The amount of cholesterol (F1), the amount of edge-activator (F2), the distribution of the drug into the vesicle (F3), the addition of stearylamine (F4) and the type of edge-activator (F5) were selected as causal factors. The deformability index, the phosphorous recovery, the vesicle size, the polydispersity index, the zeta potential and percentage of drug entrapped were fixed as the dependent variables and these responses were evaluated for each formulation. Two different statistical analysis approaches were applied. The better statistical approach was determined by comparing their prediction errors, where regression analysis provided better optimized responses than marginal means. From the study, an optimized formulation of TM-loaded transfersomes was prepared and obtained for the proposed ophthalmic delivery for the treatment of open angle glaucoma. It was found that the lipid to surfactant ratio and type of surfactant are the main key factors for determining the flexibility of the bilayer of transfersomes. From in vitro permeation studies, we can conclude that TM-loaded transfersomes may enhance the corneal transmittance and improve the bioavailability of conventional TM delivery.

Artificial neural networks as alternative tool for minimizing error predictions in manufacturing ultradeformable nanoliposome formulations

Abstract This work was aimed at determining the feasibility of artificial neural networks (ANN) by implementing backpropagation algorithms with default settings to generate better predictive models than multiple linear regression (MLR) analysis. The study was hypothesized on timolol-loaded liposomes. As tutorial data for ANN, causal factors were used, which were fed into the computer program. The number of training cycles has been identified in order to optimize the performance of the ANN. The optimization was performed by minimizing the error between the predicted and real response values in the training step. The results showed that training was stopped at 10 000 training cycles with 80% of the pattern values, because at this point the ANN generalizes better. Minimum validation error was achieved at 12 hidden neurons in a single layer. MLR has great prediction ability, with errors between predicted and real values lower than 1% in some of the parameters evaluated. Thus, the performance of this model was compared to that of the MLR using a factorial design. Optimal formulations were identified by minimizing the distance among measured and theoretical parameters, by estimating the prediction errors. Results indicate that the ANN shows much better predictive ability than the MLR model. These findings demonstrate the increased efficiency of the combination of ANN and design of experiments, compared to the conventional MLR modeling techniques.

A comparative study of stabilising effect and antioxidant activity of different antioxidants on levodopa-loaded liposomes.

Abstract The aim of this study was to evaluate the stability of levodopa liposomes co-loaded with three different antioxidants (curcumin, ascorbic acid, and superoxide dismutase (SOD)). For this purpose, multilamellar liposomes were prepared. Curcumin was added into the lipid bilayer while ascorbic acid and SOD were placed into the aqueous phase. The influence of preparation technique and surface charge were also investigated. Vesicles were characterised and free radical scavenging potential was determined. From stability study, ascorbic acid showed better stabilising effect. These co-loaded liposomes also exhibited potential radical scavenging activity where ascorbic acid played a key role. From the study of different preparation techniques and charge, we concluded that cationic liposomes made by Thin Layer Evaporation following extrusion offered the best physicochemical and stability properties. A dual mechanism of these liposomes implies the chemical stabilisation of levodopa (dose reduction) and the antioxidant effect, with a preventive effect on Parkinson’s disease.

Ophthalmic administration of a 10-fold-lower dose of conventional nanoliposome formulations caused levels of intraocular pressure similar to those induced by marketed eye drops

ABSTRACT The purpose of this study was to compare the in vivo efficacy of several timolol (TM)‐loaded liposomal formulations with current TM antiglaucoma treatment (aqueous 0.5% w/v eye drops). In this study, conventional liposomes (CL) and deformable liposomes, without (DL1) and with ethanol (DL2) were prepared and characterized. In addition, in vitro release and permeation studies, as well as in vivo lowering intraocular pressure (IOP) and biocompatibility studies were performed. It was found that the quali and quantitative lipid bilayer composition played a significant role in modifying the physical properties of vesicles. The deformability study and electronic microscopy images revealed that membrane elasticity of DL1 and DL2 was much higher than CL. However, in vitro permeation results showed that the flux and permeability coefficient were significantly higher in CL compared to DL. The IOP study revealed that TM‐loaded CL showed the best pharmacological activity, in comparison to deformable vesicles. Compared to the eye drops, CL formulation could equally reduce the IOP but using a concentration 10‐fold lower, whereas the effective time was significantly longer. In addition, the formulations showed no irritant effects after instillation on the ocular surface. Graphical abstract Figure. No Caption available.

Surface functionalizing of a lipid nanosystem to promote brain targeting: step-by-step design and physico-chemical characterization

Abstract The use of lipid nanosystems as drug delivery to the central nervous system may be advantageous over the current strategies. The aim of this study was to develop and characterize functionalized liposomes for treatment of brain diseases. The covalent method of coupling IgG to liposomes via the derivatized lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide](MPB-PE) was investigated. Optimized coupling conditions are shown to result in the efficient conjugation of IgG to liposomes containing low concentrations of MPB-PE (3/1 SH:IgG). The qualitative analysis has shown that after the extrusion process, more homogeneous populations of vesicles have been obtained with a nanometric size suitable to be effective to further anchor the protein. Negative values of zeta potential demonstrate that they are stable systems. Lyophilization was used to maintain the stability of the formulation. These very interesting results encourage further investigations to formulate peptide- and protein-loaded immunoliposomes, making targeting of liposomes as an attractive approach for brain drug delivery.

Surface-Charged Vesicles for Penetration Enhancement

This chapter aims to summarize the current approaches employed in delivering drugs through the skin, focusing in vesicle systems. In recent years, several vesicles have been developed to improve the permeation of drugs through the skin. Different types of lipid vesicles are formulated depending on the additives used for preparation: Transfersome™, flexosomes, ethosomes, niosomes, vesosomes, invasomes, and polymerosomes. In addition, an exhaustive revision about different strategies to provide positive or negative charge to the liposomes has been reported. Also, the influence of physicochemical properties of drug on the permeation behavior from these vesicles has been analyzed. Finally, new strategies and advances in the use of charged liposomes have also included.