Development and characterization of Poly(L-lactic acid) (PLLA) platforms for bone tissue engineering

The development of scaffolds based on biomaterials is a promising strategy for Tissue Engineering and cellular regeneration. This work focuses on Bone Tissue Engineering, the aim is to develop electrically tailored biomaterials with different crystalline and electric features, and study their impacts onto cell biological behavior, so as to predict the materials output in the enhancement of bone tissue regeneration. It is accepted that bone exhibits piezoelectricity, a property that has been proved to be involved in bone growth/repair mechanism regulation. In addition electrical stimulations have been proved to influence bone growth and repair. Piezoelectric materials are therefore widely investigated for a potential use in bone tissue engineering. The main goal is the development of novel strategies to produce and employ piezoelectric biomaterials, with detailed knowledge of mechanisms involved in cell-material interaction. In the current work, poly (L-lactic) acid (PLLA), a synthetic semi-crystalline polymer, exhibiting biodegradibility, biocompatibility and piezoelectricity is studied and proposed as a promoter of enhanced tissue regeneration. PLLA has already been approved for implantation in human body by the Food and Drug Administration (FDA), and at the moment it is being used in several clinical strategies. The present study consists of first preparing films with different degrees of crystallinity and characterizing these PLLA films, in terms of surface and structural properties, and subsequently assessing the behavior of cells in terms of viability, proliferation, morphology and mineralization for each PLLA configuration. PLLA films were prepared using the solvent cast technique and submitted to different thermal treatments in order to obtain different degrees of crystallinity. Those platforms were then electrically poled, positively and negatively, by corona discharge in order to tailor their electrical properties. The cellular assays were conducted by using two different osteoblast cell lines grown directly onto the PLLA films:Human osteoblast Hob, a primary cell culture and Human osteosarcoma MG-63 cell line. This thesis gives also a comprehensive introduction to the area of Bone Tissue Engineering and provides a review of the work done in this field in the past until today, in that same field, including the one related with bone’s piezoelectricity. Then the experimental part deals with the effects of the crystallinity degrees and of the polarization in terms of surface properties and cellular bio assays. Three different degrees of crystallinity, and three different polarization conditions were prepared; which results in 9 different configurations under investigation.

Genetic control of development in the parasitic nematode Haemonchus contortus by microRNAs

The parasitic nematode Haemonchus contortus has a major impact on the welfare and economic sustainability of small ruminant farming throughout the world. Increasing drug resistance requires the development of novel therapeutic agents. To further this process, we examined the fundamental biology of development in H. contortus, specifically, the potential role of microRNAs (miRNAs). miRNAs are short, non-coding RNA molecules that negatively regulate gene expression. In the free-living nematode Caenorhabditis elegans, miRNAs regulate a variety of genes including those involved in development. This thesis describes the expression patterns, potential targets and possible functions of miRNAs in H. contortus throughout development.

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

DEVELOPMENT AND REPRODUCIBILITY OF THE INSTRUMENTO DE AVALIAÇÃO DA PROMOÇÃO DA SAÚDE NA UNIVERSIDADE - IAPSU (ASSESSMENT TOOL FOR HEALTH PROMOTION AT THE UNIVERSITY)

Objective: To present the Instrumento de Avaliação da Promoção da Saúde na Universidade – IAPSU (Assessment Tool for Health Promotion at the University) and its reproducibility assessment process. Methods: Cross-sectional study conducted between May and July 2014 with 50 students from a university of Fortaleza, Ceará, which developed the IAPSU through the analysis of government documents and a systematic review of the literature on a potentially healthy university. The tool has 41 questions divided into five domains: physical activity, diet, environmental factors, psychosocial factors and alcohol and drug use, integrative and complementary practices. To assess the inter-examiner reproducibility, the students answered the IAPSU twice, applied by two different examiners; to assess the intraexaminer reproducibility, another application of the instrument was performed after seven days. Results: The study comprised 40 Nursing students and 10 Physical Therapy students, with a mean age of 25 ± 5.4 years; 88% were women and white individuals were predominant. In the reproducibility assessment, strong intraclass, intra- and inter-examiner correlation coefficients - above 0.8 - were observed in all the domains. Conclusion: The IAPSU is a reproducible and reliable instrument for assessing health promotion at the university.

In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

Applied evolution: An integrated approach to studying life history traits in response to drug selection

The use of chemical control measures to reduce the impact of parasite and pest species has frequently resulted in the development of resistance. Thus, resistance management has become a key concern in human and veterinary medicine, and in agricultural production. Although it is known that factors such as gene flow between susceptible and resistant populations, drug type, application methods, and costs of resistance can affect the rate of resistance evolution, less is known about the impacts of density-dependent eco-evolutionary processes that could be altered by drug-induced mortality. The overall aim of this thesis was to take an experimental evolution approach to assess how life history traits respond to drug selection, using a free-living dioecious worm (Caenorhabditis remanei) as a model. In Chapter 2, I defined the relationship between C. remanei survival and Ivermectin dose over a range of concentrations, in order to control the intensity of selection used in the selection experiment described in Chapter 4. The dose-response data were also used to appraise curve-fitting methods, using Akaike Information Criterion (AIC) model selection to compare a series of nonlinear models. The type of model fitted to the dose response data had a significant effect on the estimates of LD50 and LD99, suggesting that failure to fit an appropriate model could give misleading estimates of resistance status. In addition, simulated data were used to establish that a potential cost of resistance could be predicted by comparing survival at the upper asymptote of dose-response curves for resistant and susceptible populations, even when differences were as low as 4%. This approach to dose-response modeling ensures that the maximum amount of useful information relating to resistance is gathered in one study. In Chapter 3, I asked how simulations could be used to inform important design choices used in selection experiments. Specifically, I focused on the effects of both within- and between-line variation on estimated power, when detecting small, medium and large effect sizes. Using mixed-effect models on simulated data, I demonstrated that commonly used designs with realistic levels of variation could be underpowered for substantial effect sizes. Thus, use of simulation-based power analysis provides an effective way to avoid under or overpowering a study designs incorporating variation due to random effects. In Chapter 4, I 3 investigated how Ivermectin dosage and changes in population density affect the rate of resistance evolution. I exposed replicate lines of C. remanei to two doses of Ivermectin (high and low) to assess relative survival of lines selected in drug-treated environments compared to untreated controls over 10 generations. Additionally, I maintained lines where mortality was imposed randomly to control for differences in density between drug treatments and to distinguish between the evolutionary consequences of drug treatment versus ecological processes affected by changes in density-dependent feedback. Intriguingly, both drug-selected and random-mortality lines showed an increase in survivorship when challenged with Ivermectin; the magnitude of this increase varied with the intensity of selection and life-history stage. The results suggest that interactions between density-dependent processes and life history may mediate evolved changes in susceptibility to control measures, which could result in misleading conclusions about the evolution of heritable resistance following drug treatment. In Chapter 5, I investigated whether the apparent changes in drug susceptibility found in Chapter 4 were related to evolved changes in life-history of C. remanei populations after selection in drug-treated and random-mortality environments. Rapid passage of lines in the drug-free environment had no effect on the measured life-history traits. In the drug-free environment, adult size and fecundity of drug-selected lines increased compared to the controls but drug selection did not affect lifespan. In the treated environment, drug-selected lines showed increased lifespan and fecundity relative to controls. Adult size of randomly culled lines responded in a similar way to drug-selected lines in the drug-free environment, but no change in fecundity or lifespan was observed in either environment. The results suggest that life histories of nematodes can respond to selection as a result of the application of control measures. Failure to take these responses into account when applying control measures could result in adverse outcomes, such as larger and more fecund parasites, as well as over-estimation of the development of genetically controlled resistance. In conclusion, my thesis shows that there may be a complex relationship between drug selection, density-dependent regulatory processes and life history of populations challenged with control measures. This relationship could have implications for how resistance is monitored and managed if life histories of parasitic species show such eco-evolutionary responses to drug application.

Development of nanostructured hydrogel for spatial and temporal controlled release of active compounds

L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures.

Improving drug discovery using hybrid softcomputing methods

Virtual screening (VS) methods can considerably aid clinical research, predicting how ligands interact with drug targets. Most VS methods suppose a unique binding site for the target, but it has been demonstrated that diverse ligands interact with unrelated parts of the target and many VS methods do not take into account this relevant fact. This problem is circumvented by a novel VS methodology named BINDSURF that scans the whole protein surface in order to find new hotspots, where ligands might potentially interact with, and which is implemented in last generation massively parallel GPU hardware, allowing fast processing of large ligand databases. BINDSURF can thus be used in drug discovery, drug design, drug repurposing and therefore helps considerably in clinical research. However, the accuracy of most VS methods and concretely BINDSURF is constrained by limitations in the scoring function that describes biomolecular interactions, and even nowadays these uncertainties are not completely understood. In order to improve accuracy of the scoring functions used in BINDSURF we propose a hybrid novel approach where neural networks (NNET) and support vector machines (SVM) methods are trained with databases of known active (drugs) and inactive compounds, being this information exploited afterwards to improve BINDSURF VS predictions.

Improving drug discovery using a neural networks based parallel scoring function

Virtual Screening (VS) methods can considerably aid clinical research, predicting how ligands interact with drug targets. Most VS methods suppose a unique binding site for the target, but it has been demonstrated that diverse ligands interact with unrelated parts of the target and many VS methods do not take into account this relevant fact. This problem is circumvented by a novel VS methodology named BINDSURF that scans the whole protein surface to find new hotspots, where ligands might potentially interact with, and which is implemented in massively parallel Graphics Processing Units, allowing fast processing of large ligand databases. BINDSURF can thus be used in drug discovery, drug design, drug repurposing and therefore helps considerably in clinical research. However, the accuracy of most VS methods is constrained by limitations in the scoring function that describes biomolecular interactions, and even nowadays these uncertainties are not completely understood. In order to solve this problem, we propose a novel approach where neural networks are trained with databases of known active (drugs) and inactive compounds, and later used to improve VS predictions.

Development of multifunctional lipid nanocapsules for the co-delivery of paclitaxel and CpG-ODN in the treatment of glioblastoma

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Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-beta-cyclodextrin inclusion complex

Ropivacaine (RVC) is an enantiomerically pure local anesthetic (LA) largely used in surgical procedures, which presents physico-chemical and therapeutic properties similar to those of bupivacaine (BPV), but associated to less systemic toxicity This study focuses on the development and pharmacological evaluation of a RVC in 2-hydroxypropyl-beta-cyclodextrin (HP-P-CD) inclusion complex. Phase-solubility diagrams allowed the determination of the association constant between RVC and HP-beta-CD (9.46 M-1) and showed an increase on RVC solubility upon complexation. Release kinetics revealed a decrease on RVC release rate and reduced hemolytic effects after complexation. (onset at 3.7 mM and 11.2 mM for RVC and RVCHP-beta-CD, respectively) were observed. Differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and X-ray analysis (X-ray) showed the formation and the morphology of the complex. Nuclear magnetic resonance (NMR) and job-plot experiments afforded data regarding inclusion complex stoichiometry (1:1) and topology. Sciatic nerve blockade studies showed that RVCHP-beta-CD was able to reduce the latency without increasing the duration of motor blockade, but prolonging the duration and intensity of the sensory blockade (p < 0.001) induced by the LA in mice. These results identify the RVCHP-beta-CD complex as an effective novel approach to enhance the pharmacological effects of RVC, presenting it as a promising new anesthetic formulation. (c) 2007 Elsevier B.V All rights reserved.

Charged pullulan derivatives for the development of nanocarriers by polyelectrolyte complexation

Pullulan, a neutral polysaccharide, was chemically modified in order to obtain two charged derivatives: reaction with SO3(.)DMF complex afforded a sulfate derivative (SP), while reaction with glycidyltrimethylammonium chloride gave a quaternary ammonium salt (AP). The presence of the charged groups was confirmed by FTIR. Assessment of the positions where the reaction took place was based on (1)H- and (13)C NMR (COSY, HSQC-TOCSY, HSQC-DEPT, and HMBC) experiments. Estimation of the degree of substitution (DS) was made from elemental analysis data, and further confirmed by NMR peak areas in the case of AP. These new derivatives showed the capability to condense with each other, forming nanoparticles with the ability to associate a model protein (BSA) and displaying adequate size for drug delivery applications, therefore making them good candidates for the production of pullulan-based nanocarriers by polyelectrolyte complexation.

Development and optimization of fluoxetine orally disintegrating tablets using Box-Behnken design

Purpose: To develop and optimise some variables that influence fluoxetine orally disintegrating tablets (ODTs) formulation. Methods: Fluoxetine ODTs tablets were prepared using direct compression method. Three-factor, 3- level Box-Behnken design was used to optimize and develop fluoxetine ODT formulation. The design suggested 15 formulations of different lubricant concentration (X1), lubricant mixing time (X2), and compression force (X3) and then their effect was monitored on tablet weight (Y1), thickness (Y2), hardness (Y3), % friability (Y4), and disintegration time (Y5). Results: All powder blends showed acceptable flow properties, ranging from good to excellent. The disintegration time (Y5) was affected directly by lubricant concentration (X1). Lubricant mixing time (X2) had a direct effect on tablet thickness (Y2) and hardness (Y3), while compression force (X3) had a direct impact on tablet hardness (Y3), % friability (Y4) and disintegration time (Y5). Accordingly, Box-Behnken design suggested an optimized formula of 0.86 mg (X1), 15.3 min (X2), and 10.6 KN (X3). Finally, the prediction error percentage responses of Y1, Y2, Y3, Y4, and Y5 were 0.31, 0.52, 2.13, 3.92 and 3.75 %, respectively. Formula 4 and 8 achieved 90 % of drug release within the first 5 min of dissolution test. Conclusion: Fluoxetine ODT formulation has been developed and optimized successfully using Box- Behnken design and has also been manufactured efficiently using direct compression technique.

Development of paclitaxel-loaded liposomal systems with anti-her2 antibody for targeted therapy

Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.

Development of docetaxel and alendronate-loaded chitosan-conjugated polylactide-co-glycolide nanoparticles: In vitro characterization in osteosarcoma cells

Purpose: To develop docetaxel (DTX)- and alendronate (ALN)-loaded, chitosan (CS)-conjugated polylactide- co-glycolide (PLGA) nanoparticles (NPs) to increase therapeutic efficacy in osteosarcoma cells. Methods: Drug-loaded PLGA NPs were prepared by nanoprecipitation and chemically conjugated by the carboxylic group of PLGA to the amine-bearing CS polymer. The nanocarrier was characterized by dynamic light scattering, transmission electron microscopy, scanning electron microscopy, and differential scanning calorimetry as well as by in vitro drug release and cell culture studies. Results: NP size was within the tumour targeting range (~200 nm) with an effective positive charge (20 mV), thus increasing cellular uptake efficiency. Morphological analysis revealed clear spherical particles with uniform dispersion. The NPs exhibited identical sustained release kinetics for both DTX and ALN. CS-conjugated PLGA with dual-drug-loaded (DTX and AL) NPs showed typical time-dependent cellular uptake and also displayed superior cytotoxicity in MG-63 cells compared with blank NPs, which were safe and biocompatible. Conclusion: Combined loading of DTX and ALN in NPs increased the therapeutic efficacy of the formulation for osteosarcoma treatment, thus indicating the potential benefit of a combinatorial drug regimen using nanocarriers for effective treatment of osteosarcoma.