In vitro and in vivo antimicrobial activity of sodium colistimethate and amikacin-loaded nanostructured lipid carriers (NLC).

Sodium colistimethate (SCM) and amikacin (AMK) are among the few antibiotics effective against resistant P. aeruginosa, K. pneumoniae and A. baumannii; however, their toxicity severely limits their use. Enclosing antibiotics into nanostructured lipid carriers (NLC) might decrease drug toxicity and improve antibiotic disposition. In this work, SCM or AMK was loaded into different NLC formulations, through high pressure homogenization, and their in vitro and in vivo effectiveness was analyzed. The encapsulation process did not reduce drug effectiveness since in vitro SCM-NLC and AMK-NLC drug activity was equal to that of the free drugs. As cryoprotectant, trehalose showed better properties than dextran. Instead, positive chitosan coating was discarded due to its limited cost-efficiency. Finally, the in vivo study in acute pneumonia model revealed that intraperitoneal administration was superior to the intramuscular route and confirmed that (-) SCM-NLC with trehalose, was the most suitable formulation against an extensively drug-resistant A. baumannii strain.

Design and characterization of calcium alginate microparticles coated with polycations as protein delivery system.

Bovine serum albumin (BSA) loaded calcium alginate microparticles (MPs) produced in this study by a w/o emulsification and external gelation method exhibited spherical and fairly smooth and porous morphology with 1.052 ± 0.057 µm modal particle size. The high permeability of the calcium alginate hydrogel lead to a potent burst effect and too fast protein release. To overcome these problems, MPs were coated with polycations, such as chitosan, poly-L-lysine and DEAE-dextran. Our results demonstrated that coated MPs showed slower release and were able to significantly reduce the release of BSA in the first hour. Therefore, this method can be applied to prepare coated alginate MPs which could be an optimal system for the controlled release of biotherapeutic molecules. Nevertheless, further studies are needed to optimize delivery properties which could provide a sustained release of proteins.

Preclinical safety of topically administered nanostructured lipid carriers (NLC) for wound healing application: biodistribution and toxicity studies.

Re-activation of the healing process is a major challenge in the field of chronic wound treatment. For that purpose, lipid-nanoparticles, especially nanostructured lipid carriers (NLC), possess extremely useful characteristics such as biodegradability, biocompatibility and long-term stability, besides being suitable for drug delivery. Moreover, they maintain wound moisture due to their occlusive properties, which have been associated with increased healing rates. In the light of above, NLC have been extensively used topically for wound healing; but to date, there are no safety-preclinical studies concerning such type of application. Thus, in this work, biodistribution studies were performed in rats with the NLC previously developed by our research group, using technetium-99 m (99mTc-NLC) as radiomarker, topically administered on a wound. 99mTc-NLC remained on the wound for 24 h and systemic absorption was not observed after administration. In addition, toxicological studies were performed to assess NLC safety after topical administration. The results obtained demonstrated that NLC were non-cytotoxic, non-sensitizing and non-irritant/corrosive. Overall, it might be concluded that developed NLC remained at the administration area, potentially exerting a local effect, and were safe after topical administration on wounds.

Morphological Changes in a Severe Model of Parkinson's Disease and Its Suitability to Test the Therapeutic Effects of Microencapsulated Neurotrophic Factors.

The unilateral 6-hydroxydopamine (6-OHDA) lesion of medial forebrain bundle (MFB) in rats affords us to study the advanced stages of Parkinson's disease (PD). Numerous evidences suggest synergic effects when various neurotrophic factors are administered in experimental models of PD. The aim of the present work was to assess the morphological changes along the rostro-caudal axis of caudo-putamen complex and substantia nigra (SN) in the referred model in order to test the suitability of a severe model to evaluate new neurorestorative therapies. Administration of 6-OHDA into MFB in addition to a remarkable depletion of dopamine in the nigrostriatal system induced an increase of glial fibrillary acidic protein (GFAP)-positive cells in SN and an intense immunoreactivity for OX-42, vascular endothelial growth factor (VEGF), and Lycopersycum esculentum agglutinin (LEA) in striatum and SN. Tyrosine hydroxylase (TH) immunostaining revealed a significant decrease of the TH-immunopositive striatal volume in 6-OHDA group from rostral to caudal one. The loss of TH-immunoreactive (TH-ir) neurons and axodendritic network (ADN) was higher in caudal sections. Morphological recovery after the implantation of microspheres loaded with VEGF and glial cell line-derived neurotrophic factor (GDNF) in parkinsonized rats was related to the preservation of the TH-ir cell number and ADN in the caudal region of the SN. In addition, these findings support the neurorestorative role of VEGF+GDNF in the dopaminergic system and the synergistic effect between both factors. On the other hand, a topological distribution of the dopaminergic system was noticeable in the severe model, showing a selective vulnerability to 6-OHDA and recovering after treatment.

Intranasal Administration of chitosan-Coated Nanostructured Lipid Carriers Loaded with GDNF Improves Behavioral and Histological Recovery in a Partial Lesion Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder, but current therapies are only symptomatic. A promising alternative to address the neurodegenerative process is the use of neurotrophic factors, such as the glial cell-derived neurotrophic factor (GDNF). However, its clinical use has been limited due to its short half-life and rapid degradation after in vivo administration, in addition to difficulties in crossing the blood-brain barrier (BBB). This barrier is a limiting factor in brain drug development, making the future progression of neurotherapeutics difficult. In the past few years, intranasal drug delivery has appeared as an alternative non-invasive administration route to bypass the BBB and target drugs directly to the CNS. Thus, the aim of this work was to study the in vivo neuroprotective effect of intranasally administered GDNF, encapsulated in chitosan-coated nanostructured lipid carrier (CS-NLC-GDNF), in a 6-OHDA partially lesioned rat model. The developed CS-NLC-GDNF showed a particle size of approximately 130 nm and high encapsulation efficiency. The in vitro study in PC-12 cells demonstrated the ability of the encapsulated GDNF to protect these cells against 6-OHDA toxin. After two weeks of daily intranasal administration of treatments, the administration of CS-NLC-GDNF achieved a behavioral improvement in rats, as well as a significant improvement in both the density of TH+ fibres in the striatum and the TH+ neuronal density in the SN. Thus, it can be concluded that the nose-to-brain delivery of CS-NLC-GDNF could be a promising therapy for the treatment of PD.

Enhanced Hippocampal Neurogenesis in APP/Ps1 Mouse Model of Alzheimer's Disease After Implantation of VEGF-loaded PLGA Nanospheres.

During adult life, hippocampus is an important brain region involved in neurogenesis. The generation and cell death of newly generated neuronal cells in this region have critical roles in brain maintenance and alterations in these processes are seen in Alzheimer's disease (AD). For the purpose of carrying out a neuroregenerative strategy, we propose a novel approach based on the encapsulation of vascular endothelial growth factor (VEGF) in poly (lactic co-glycolic acid) (PLGA) biodegradable nanospheres (NS) administered by craniotomy to stimulate the proliferation of neuronal precursors in a transgenic mouse model of AD. VEGF loaded nanospheres were prepared by double emulsion solvent evaporation technique, obtaining 200 nm nanospheres with a biphasic release profile. After demonstrating their efficacy in the proliferation and differentiation of neuronal cell cultures, in vivo studies were carried out. 3 months after VEGF-NS were implanted directly into the cerebral cortex of APP/Ps1 mice, the determination of BrdU(+) cells in the whole hippocampal region and specifically in the dentate gyrus, demonstrated a significantly enhanced cellular proliferation in VEGF-NS treated group. These results were also confirmed showing an increased number of DCX(+) and NeuN(+) cells. Hence, PLGA-VEGF nanospheres may be a potential strategy to modulate proliferative neuronal progenitors in the hippocampal region, and therefore, provide new insight for future therapeutic approaches in AD.

Development and in vitro evaluation of lipid nanoparticle-based dressings for topical treatment of chronic wounds.

This research addresses the development and in vitro evaluation of lipid nanoparticle (NP)-based dressings to optimize the delivery of human recombinant epidermal growth factor (rhEGF) for the topical treatment of chronic wounds. The systems investigated were rhEGF-loaded solid lipid nanoparticles (rhEGF-SLN) and rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) formulated in wound dressings comprising either semi-solid hydrogels or fibrin-based solid scaffolds. Following detailed characterisation of the NP, in vitro diffusion cell experiments (coupled with dermatopharmacokinetic measurements), together with confocal microscopic imaging, conducted on both intact skin samples, and those from which the barrier (the stratum corneum) had been removed, revealed that (a) the particles remained essentially superficially located for at least up to 48h post-application, (b) rhEGF released on the surface of intact skin was unable to penetrate to the deeper, viable layers, and (c) sustained release of growth factor from the NP "drug reservoirs" into barrier-compromised skin was observed. There were no significant differences between the in vitro performance of rhEGF-SLN and rhEGF-NLC, irrespective of the formulation employed. It is concluded that, because of their potentially longer-term stability, the fibrin-based scaffolds may be the most suitable approach to formulate rhEGF-loaded lipid nanoparticles.

Topographical Distribution of Morphological Changes in a Partial Model of Parkinson's Disease--Effects of Nanoencapsulated Neurotrophic Factors Administration.

Administration of various neurotrophic factors is a promising strategy against Parkinson's disease (PD). An intrastriatal infusion of 6-hydroxidopamine (6-OHDA) in rats is a suitable model to study PD. This work aims to describe stereological parameters regarding rostro-caudal gradient, in order to characterize the model and verify its suitability for elucidating the benefits of therapeutic strategies. Administration of 6-OHDA induced a reduction in tyrosine hidroxylase (TH) reactivity in the dorsolateral part of the striatum, being higher in the caudal section than in the rostral one. Loss of TH-positive neurons and axodendritic network was highly significant in the external third of substantia nigra (e-SN) in the 6-OHDA group versus the saline one. After the administration of nanospheres loaded with neurotrophic factors (NTF: vascular endothelial growth factor (VEGF) + glial cell line-derived neurotrophic factor (GDNF)), parkinsonized rats showed more TH-positive fibers than those of control groups; this recovery taking place chiefly in the rostral sections. Neuronal density and axodendritic network in e-SN was more significant than in the entire SN; the topographical analysis showed that the highest difference between NTF versus control group was attained in the middle section. A high number of bromodeoxyuridine (BrdU)-positive cells were found in sub- and periventricular areas in the group receiving NTF, where most of them co-expressed doublecortin. Measurements on the e-SN achieved more specific and significant results than in the entire SN. This difference in rostro-caudal gradients underpins the usefulness of a topological approach to the assessment of the lesion and therapeutic strategies. Findings confirmed the neurorestorative, neurogenic, and synergistic effects of VEGF+GDNF administration.

Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates.

The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres.

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freund's complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.

Survival of different cell lines in alginate-agarose microcapsules.

Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

Development and optimisation of alginate-PMCG-alginate microcapsules for cell immobilisation.

Mechanical stability, uniformity of size, complete encapsulation of cells and optimal microenvironment are major challenges in the design and development of microcapsules for cell immobilisation purposes. In this work, a novel microcapsule chemistry based on polyelectrolyte complexation between alginate and poly(methylene-co-guanidine) (PMCG) is presented. We have characterised the effect of PMCG concentration and time of exposure on microcapsule diameter and membrane thickness, selecting a PMCG concentration of 0.5% (v/v) and an exposure time of 1 min as optimal parameters for a correct coating. Afterwards, the mechanically most resistant alginate-PMCG-alginate (A-PMCG-A) microcapsule type was chosen according to two different stability studies. Beads with a solid core and an inhomogeneous internal configuration resulted in stronger microcapsules. Further, the selected A-PMCG-A beads presented both an increased stability compared to classical Ca(2+)/alginate and alginate-poly-L-lysine-alginate (APA) microcapsules, and had an adequate microenvironment for cell viability. This new chemistry allows the controlled adjustment of microcapsule size and wall thickness, offering new alternatives for cell transplantation.

Immune response after oral administration of the encapsulated malaria synthetic peptide SPf66.

The synthetic peptide SPf66 adsorbed on alum is one of the few Plasmodium falciparum vaccines which have been tested in field trials. We previously reported that subcutaneous administration of SPf66 loaded PLGA microparticles (MP) enhances the antibody response to this antigen compared to the conventional alum formulation. We now evaluate the suitability of polymeric formulations to obtain systemic immune responses by gastric intubation of Balb/c mice. Formulations composed of 1:1 mixtures of PLGA 50:50 and 75:25 (lactic:glycolic) microparticles were administered by the oral route, and when animals were boosted 3 weeks later significant systemic IgG antibody responses were elicited, comparable to alum triple shot and superior to the aqueous vaccine given by the oral route. The finding of IgG2a isotype for PLGA-vaccinated mice compared to the absent levels of this isotype for the alum-vaccinated group could be interpreted as a sign of Th1-like immune response and cellular immune response activation. Our results confirm that using the appropriate schedule the oral administration of PLGA particles is suitable to obtain systemic immune responses to the carried antigen.

Development and characterization of solid lipid nanoparticles loaded with magnetite.

This paper describes the preparation of colloidal lipid particles containing magnetite from warm emulsions. A two step method was used to obtain the nanoparticles: (i) formulation of a transparent phase by heating a O/W emulsion (aqueous surfactant solution melted with a lipid phase, containing the ethyl oleate and soybean lecithin) in which modified lipophilic magnetite is incorporated, and (ii) preparation of the nanoparticles by dispersing the warm transparent phase in cold water (7 degrees C) under mechanical stirring. The latter method gives spherical nanoparticles of a mean size of 62 nm measured by Photon Correlation Spectroscopy and Transmission Electronic Microscopy. The magnetite entrapment efficiency was determined by use of a magnetophoretic sedimentation method.

Determination of salbutamol enantiomers by high-performance capillary electrophoresis and its application to dissolution assays.

Capillary zone electrophoresis was successfully applied to the chiral separation of salbutamol after addition of a suitable cyclodextrin chiral selector to the electrophoresis buffer. Parameters important in achieving enantiomeric separation are cyclodextrin type, mobile phase pH and applied field strength. In our study, salbutamol enantiomeric separation was obtained with the following conditions: heptakis (2,6-di-O-methyl)-beta-cyclodextrin in 40 mM Tris (pH 2.5) and at 15 kV, obtaining a 3.09 resolution with migration times of 13.74 min for (R)-salbutamol and 13.98 min for (S)-salbutamol. Linearity, limit of quantitation, precision and accuracy were established using this method. The calibration curve was linear in a range of 1-40 micrograms ml-1 of racemic salbutamol (0.5-20 micrograms ml-1 of each enantiomer). This method was applied to evaluate the enantioselective release of salbutamol and taking into account the hypothesis that one enantiomer of a chiral drug would be released faster than the other from a pharmaceutical dosage form containing a racemic drug and a chiral excipient. For this purpose, matrix tablets formed by chiral excipients such as hydroxypropylmethylcellulose (HPMC) were considered. The release of the enantiomers of salbutamol from the formulations containing HPMC was found to be equivalent, with constant dissolution values (K) of 1.187 +/- 0.223% min-n for (R)-salbutamol and 1.076 +/- 0.268% min-n for (S)-salbutamol.

Influence of shaking and surfactants on the release of bsa from plga microspheres.

The aim of the present work was to study the release of a model protein, bovine serum albumin (BSA) encapsulated within biodegradable poly (D,L-lactide-co-glycolide) (PLGA) microspheres prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface adsorbed protein, encapsulation efficiency and release kinetics. Two types of in vitro assays were developed to evaluate the influence of shaking and the addition of surfactants on the release profile of encapsulated protein. Scanning electron microscopy (SEM) observation showed spherical and smooth surface particles, with a mean particle size of 20 microm and an encapsulation efficiency of 81%. Surface associated protein was about 25%. The in vitro release profile showed a biphasic pattern described by means of a biexponential equation. There was an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release obtained profiles in static and dynamic assays showed statistically significant differences in the amount of the released protein, whereas the release rate was not affected. The burst effect was 28.30+/-1.63% and 35.20+/-1.50% of the total encapsulated protein for the static and dynamic assays respectively. The addition of surfactants (SDS) to the release medium increased the rate and the amount of drug released. In both assays the value of the slow release rate constant, beta, was 0.029+/-0.002 days(-1) when the surfactant was added, and 0.017+/-0.0014 days(-1) in the samples without surfactant. It is believed that the surfactant leads to an increase in the microsphere surface polarity which allows channel and pore formation inside the polymer through which the protein diffuses easily.

Novel drug delivery systems for releasing growth factors to the CNS: focus on Alzheimer's and Parkinson's diseases.

Alzheimer's disease (AD) and Parkinson's disease (PD) represent the most common neurodegenerative disorders and affect more than 35 million people. Due to the limited effectiveness of available treatments in halting the neurodegenerative process, new therapies, such therapies based on growth factors (GFs), have been investigated. Nevertheless, the efficacies of these new treatments depend not only on the application of neurotrophins but also on the approaches used to deliver these proteins such that they can reach the brain. This review summarises the most widely used drug delivery systems (DDSs) for releasing GFs as possible treatments for AD and PD.

Encapsulation of Aß(1-15) in PLGA microparticles enhances serum antibody response in mice immunized by subcutaneous and intranasal routes.

The aim of the present work was to develop an easy, safe and effective vaccine in Balb/c mice using the Aß(1-15) peptide as immunogen entrapped in PLGA microparticles to reduce the risk of an adverse T cell-mediated response. Aß(1-15,) which contains the N-terminal antibody epitope of the full Aß(1-42) peptide was encapsulated in PLGA by a modified solvent evaporation/extraction technique using a double emulsion system. Microparticles were characterized in terms of size distribution (1.22±0.28 µm), encapsulation efficiency (75.05±4.17%), surface associated peptide (59.81±0.96%) and "in vitro" release profile. Balb/c mice were immunized by subcutaneous and intranasal routes with three 30 µg doses of the peptide microencapsulated in PLGA. A solution of the peptide alone and an emulsion in the Freund's adjuvant were administered subcutaneously as control groups. Antibody levels elicited against the toxic Aß(1-40) fraction in the serum of PLGA microparticles treated groups were higher than that of the peptide alone groups. Our initial results indicate that immunotherapy with Aß(1-15) loaded PLGA microparticles could be a promising approach for the future development of a safe vaccine against Alzheimer's disease.

Novel extended-release formulation of lovastatin by one-step melt granulation: in vitro and in vivo evaluation.

The objective of this study was to apply a one-step melt granulation method to develop an extended-release formulation of lovastatin (LOV-ER). We prepared a formulation using PEG 6000 as binder agent in a laboratory scale high-shear mixer. In vitro dissolution studies showed that the release of the drug from the new formulation followed a zero-order kinetic with no differences in the release profile with either the pH media or the agitation rate. The pharmacokinetic of lovastatin and its metabolite lovastatin acid was evaluated after the administration of the new formulation to Beagle dogs in fasted conditions and after a high-fat meal, and compared to the marketed formulation Altoprev®. After the administration of LOV-ER, extended plasma profiles of lovastatin and its active metabolite were achieved in both fasted conditions and after the high-fat meal. Plasma levels of lovastatin and lovastatin acid were always higher when the LOV-ER formulation was administered with the high-fat meal. A high variability in plasma levels and pharmacokinetic parameters was obtained, being this variability higher when the formulation was administered under fasting conditions. Our results suggest that there is an increase in lovastatin bioavailability when the formulation is administered after the high-fat meal. When we compare LOV-ER and Altoprev®, both administered after the high-fat meal, we found significant differences (p<0.05) in C(max) of lovastatin and in AUC(0-∞) and MRT of lovastatin acid. No differences were detected between both formulations in fasting conditions. In this regard, the high-fat meal seems to increase the absorption extent of lovastatin from LOV-ER formulation and to delay the absorption rate of the drug from Altoprev®. In conclusion, we developed a lovastatin formulation that provided extended plasma levels that confirm that one-step melt granulation in high-shear mixer could be an easy and cost-effective technique for extended-release formulation development.