In Vitro and In Vivo Evaluation of a Folate-Targeted Copolymeric Submicrohydrogel Based on N-Isopropylacrylamide as 5-Fluorouracil Delivery System

Folate-targeted poly[(p-nitrophenyl acrylate)-co-(N-isopropylacrylamide)] nanohydrogel (F-SubMG) was loaded with 5-fluorouracil (5-FU) to obtain low (16.3 +/- 1.9 mu g 5-FU/mg F-SubMG) and high (46.8 +/- 3.8 mu g 5-FU/mg F-SubMG) load 5-FU-loaded F-SubMGs. The complete in vitro drug release took place in 8 h. The cytotoxicity of unloaded F-SubMGs in MCF7 and HeLa cells was low; although it increased for high F-SubMG concentration. The administration of 10 mu M 5-FU by 5-FU-loaded F-SubMGs was effective on both cellular types. Cell uptake of F-SubMGs took place in both cell types, but it was higher in HeLa cells because they are folate receptor positive. After subcutaneous administration (28 mg 5-FU/kg b.w.) in Wistar rats, F-SubMGs were detected at the site of injection under the skin. Histological studies indicated that the F-SubMGs were surrounded by connective tissue, without any signs of rejections, even 60 days after injection. Pharmacokinetic study showed an increase in MRT (mean residence time) of 5-FU when the drug was administered by drug-loaded F-SubMGs.

The effect of the drug colchicine on the early life of fish

The alkaloid drug colchicine is a mitotic inhibitor. The results of this study show that colchicine influence the normal functioning of the mitotic process in Sarotherodon galilaeus, S. melanotheron and the hybrid S. galilaeus, X S. melanotheron leading to the production of unusual chromosomal events such as anaphase bridges, laggards and polyploid cells. These unusual events could have serious genetic implications in the area of variability of the chromosome number. The use of colchicine also produces results with consistent karyotypes and better morphology as well as providing detailed information on the behaviour of the chromosome of the early life of fish. The knowledge of such information will be of great use in cytotaxonomy, fish breeding and in studying the effects of sub-lethal levels of water pollutants on fish

Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Model systems for the study of drug hypersensitivity

I. It was not possible to produce anti-tetracycline antibody in laboratory animals by any of the methods tried. Tetracycline protein conjugates were prepared and characterized. It was shown that previous reports of the detection of anti-tetracycline antibody by in vitro-methods were in error. Tetracycline precipitates non-specifically with serum proteins. The anaphylactic reaction reported was the result of misinterpretation, since the observations were inconsistent with the known mechanism of anaphylaxis and the supposed antibody would not sensitize guinea pig skin. The hemagglutination reaction was not reproducible and was extremely sensitive to minute amounts of microbial contamination. Both free tetracyclines and the conjugates were found to be poor antigens.

II. Anti-aspiryl antibodies were produced in rabbits using 3 protein carriers. The method of inhibition of precipitation was used to determine the specificity of the antibody produced. ε-Aminocaproate was found to be the most effective inhibitor of the haptens tested, indicating that the combining hapten of the protein is ε-aspiryl-lysyl. Free aspirin and salicylates were poor inhibitors and did not combine with the antibody to a significant extent. The ortho group was found to participate in the binding to antibody. The average binding constants were measured.

Normal rabbit serum was acetylated by aspirin under in vitro conditions, which are similar to physiological conditions. The extent of acetylation was determined by immunochemical tests. The acetylated serum proteins were shown to be potent antigens in rabbits. It was also shown that aspiryl proteins were partially acetylated. The relation of these results to human aspirin intolerance is discussed.

III. Aspirin did not induce contact sensitivity in guinea pigs when they were immunized by techniques that induce sensitivity with other reactive compounds. The acetylation mechanism is not relevant to this type of hypersensitivity, since sensitivity is not produced by potent acetylating agents like acetyl chloride and acetic anhydride. Aspiryl chloride, a totally artificial system, is a good sensitizer. Its specificity was examined.

IV. Protein conjugates were prepared with p-aminosalicylic acid and various carriers using azo, carbodiimide and mixed anhydride coupling. These antigens were injected into rabbits and guinea pigs and no anti-hapten IgG or IgM response was obtained. Delayed hypersensitivity was produced in guinea pigs by immunization with the conjugates, and its specificity was determined. Guinea pigs were not sensitized by either injections or topical application of p-amino-salicylic acid or p-aminosalicylate.

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

A Delphi Process to Optimize Quality and Performance of Drug Evaluation in Neonates

Background: Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. The objective of this work was to identify the major criteria considered necessary for selecting neonatal intensive care units that are able to perform drug evaluations competently. Methodology and Main Findings: This Delphi process was conducted with an international multidisciplinary panel of 25 experts from 13 countries, selected to be part of two committees (a scientific committee and an expert committee), in order to validate criteria required to perform drug evaluation in neonates. Eighty six items were initially selected and classified under 7 headings: "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6). Sixty-one items were retained and headings were rearranged after the first round, 34 were selected after the second round. A third round was required to validate 13 additional items. The final set includes 47 items divided under 5 headings. Conclusion: A set of 47 relevant criteria will help to NICUs that want to implement, conduct or participate in drug trials within a neonatal network identify important issues to be aware of. Summary Points: 1) Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. 2) The present Delphi study was conducted with an international multidisciplinary panel of 25 experts from 13 countries and aims to identify the major criteria considered necessary for selecting neonatal intensive care units (NICUs) that are able to perform drug evaluations competently. 3) Of the 86 items initially selected and classified under 7 headings - "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6) - 47 items were selected following a three rounds Delphi process. 4) The present consensus will help NICUs to implement, conduct or participate in drug trials within a neonatal network.

Biofunctionalization of alginate microcapsules: advances in cell-based drug delivery systems

172 p.

Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.

Exploring new labelling strategies for boronated compounds: towards fast development and efficient assessment of BNCT drug candidates

208 p.

Avaliação da toxicidade da emodina e sua associação com radiação ultravioleta A em cepas de Escherichia coli e células da linhagem A549

A emodina é uma antraquinona estruturalmente semelhante à aloe-emodina e ambas tem sido apontadas como capazes de causar lesões oxidativas pela produção de ERO. Sua presença em produtos dermocosméticos e de higiene pessoal, associada às informações de que a fotoativação de antraquinonas levaria ao aumento de lesões oxidativas causadas por ERO, torna relevante o estudo da associação da emodina com a radiação UVA. O objetivo desse trabalho foi avaliar a citotoxicidade induzida pela associação da emodina com doses subletais de radiação UVA, em células de Escherichia coli (selvagem e cepas deficientes em enzimas do BER), através de ensaios de sobrevivência bacteriana (taxa de dose de UVA igual a 20J/m/s, totalizando 108kJ/m ao final de 90min de experimento), e em células da linhagem A549 pela exclusão do corante azul de tripan e sobrevivência clonogênica(taxa de dose de UVA igual a 20J/m/s, totalizando 36kJ/m ao final de 30min de experimento). Além disso, a genotoxicidade desses agentes foi estudada por eletroforese em gel de agarose de DNA plasmidial (taxa de dose de UVA igual a 16J/m/s, totalizando 57,6kJ/m ao final de 60min de experimento). De acordo com os resultados: i) Concentrações iguais ou abaixo de 5,55mM de emodina não alteraram a sobrevivência em nenhuma das cepas estudas; ii) As proteínas Xth e Fpg parecem ter um papel importante no reparo das lesões causadas pela emodina, em altas concentrações, sugerindo a participação do reparo por excisão de bases (BER) nesse processo; iii) A associação da emodina com a radiação UVA se mostrou citotóxica em todas as cepas de E. coli; iv) O gene nfo foi o mais importante na resistência bacteriana às lesões induzidas pela associação dos dois agentes, reforçando o envolvimento do BER e indicando uma possível participação do reparo por incisão de nucleotídeos (NIR); v) A emodina parece ter interagido com o DNA plasmidial, alterando seu padrão de migração no gel de agarose; vi) Em células da linhagem A549, a emodina causa efeitos tóxicos imediatos que parecem ser reparados ao longo do tempo. Porém, quando a droga permaneceu por 24 horas em contato com as células, houve uma diminuição na sobrevivência celular que parece ser dosedependente; vii) As concentrações de 10μM e 25μM de emodina, quando associadas ao UVA, se mostraram responsáveis pela redução de mais de 50% na sobrevivência nas células A549, chegando a 100% de morte quando a concentração de emodina foi de 50μM; viii) A radiação UVA potencializou os efeitos citotóxicos da emodina, nos 2 modelos experimentais do presente estudo, indicando que a interação da emodina com a radiação UVA seja genotóxica e portanto prejudicial à saúde.

Evaluación de la posible capacidad genotóxica de fármacos antihipertensivos: losartán e irbesartán

[es] La hipertensión arterial (AHT), estado patológico definido como la elevación persistente de la presión arterial, es considerada uno de los principales problemas de salud pública. El tratamiento de la patología se realiza preferentemente con fármacos antihipertensivos. Los pacientes se encuentran sometidos a una exposición larga e ininterrumpida a estos fármacos. Los fármacos antihipertensivos se clasifican en función del mecanismo de acción por el que logran su efecto. Una de las clases son los antagonistas de los receptores de angiotensina II (ARB). Los ARB son la última clase terapéutica incluida en la terapia antihipertensiva. En este Trabajo Fin de Grado, se ha evaluado la posible capacidad genotóxica in vitro, de dos tipos de fármacos ARB (losartán e irbesartán) mediante el empleo de uno de los métodos citogenéticos más utilizados, el ensayo de micronúcleos (MN). El índice de división nuclear (NDI) fue usado también como medida de genotoxicidad. El análisis se ha realizado en linfocitos de sangre periférica (PBL) de 10 individuos control mediante dos tipos de cultivo uno sin fármaco y otro añadiendo los fármacos a los cultivos en una concentración igual a la que se encuentra en el plasma de pacientes. Los resultados muestran un aumento estadísticamente significativo de la frecuencia de células binucleadas con micronúcleos (BNMN) pero no se observan diferencias estadísticamente significativas en el índice de división nuclear. Estos resultados sugieren un posible efecto genotóxico de los fármacos pero sería necesario llevar a cabo estudios en una población más amplia e in vivo con los mismos fármacos para confirmarlo.

Avaliação in vitro de duas resinas macias para reembasamento de próteses modificadas pela incorporação de clorexidina

Resinas macias para reembasamento de próteses são largamente utilizadas após cirurgias para estabilizarem a prótese e condicionarem o tecido, aguardando a completa cicatrização. É importante que o material não seja facilmente colonizado por biofilme oral e se possível, evite a contaminação do sítio cirúrgico. Objetivou-se avaliar o efeito da incorporação de clorexidina às resinas acrílicas macias para o reembasamento de próteses totais, através de análises de liberação, citotoxicidade e efeito inibitório de um biofilme de C. albicans. Foram confeccionados corpos de provas (CDPs) com as resinas Trusoft e Coe-soft, com incorporação de 0%, 0,5%, 1,0% e 2,0% de clorexidina, totalizando 8 grupos. A liberação de clorexidina foi avaliada através da mensuração da mudança na densidade óptica da solução de armazenamento, na qual ficaram imersos os CDPs, por espectrometria UV, a cada 48 horas, durante 40 dias. A citotoxicidade celular foi avaliada em fibroblastos (linhagem L929), que ficaram 24 horas em contato com meio de cultura no qual os CDPs ficaram previamente imersos, pela técnica de absorção de corante vermelho neutro após 24, 48 e 72 horas e semanalmente até o 28 dia. E, por fim, a atividade antifúngica contra a C. albicans (ATCC 10231) foi avaliada de duas maneiras: (1) teste de difusão em ágar, no qual os CDPs foram colocados em placas de BHI previamente inoculadas com C. albicans, com medição do halo de inibição após 48 horas de incubação a 37C; (2) a avaliação da inibição da formação de um biofilme de C. albicans sobre a superfície dos CDPs pela quantificação por metil tetrazólio (MTT) a cada 48 horas, durante 22 dias, com leitura feita em espectrofotômetro de UV. Os dados obtidos foram inseridos no programa SigmaStat (versão 3.1, USA) para realizar as análises estatísticas. As diferenças estatísticas foram determinadas por análises de variâncias do tipo ANOVA e todos os procedimentos para comparações múltiplas pareadas foram feitos utilizando-se o método Holm-Sidak, com nível de significância global igual a 0,05. A clorexidina adicionada às resinas testadas foi capaz de ser liberada para o meio de armazenagem, proporcionalmente à quantidade de clorexidina incorporada, porém com diferentes cinéticas de liberação entre as resinas, visto que a Trusoft libera até 71% do total de clorexidina liberada nas primeiras 48 horas e a Coe-soft, até 44%. Ambas as resinas com incorporação de clorexidina apresentaram efeito citotóxico adicional, se comparadas às resinas sem clorexidina, porém para a Coe-soft não houve diferença estatística dos valores, apenas para a Trusoft (p<0,001). Ocorreu formação de halo de inibição proporcionalmente às concentrações de resinas adicionadas, com maiores halos para a resina Trusoft (p<0,001), e sem formação de halo para as resinas sem clorexidina; a inibição da formação de biofilme, realizada somente com a resina Coe-soft, mostrou total inibição durante 8, 12 e 16 dias, para a incorporação de 0,5%, 1,0% e 2,0% respectivamente, sendo uma diminuição estatisticamente significativa (p<0,001) em relação à resina sem incorporação de clorexidina, que não apresentou inibição do biofilme.

Avaliação da ação antitumoral in vitro da pterocarpanoquinona LQB 118 e estudo de alguns mecanismos de ação

Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata.