Orientador: Luiz Shiguero Matsubara / Banca: Clovis Frim / Banca: Márcia Koike / Banca: Ana Lúcia dos Anjos / Banca: Paula Schmidt Azevedo Gaiolla / Resumo: A doxorrubicina, ou adriamicina, é uma droga utilizada como agente antineoplásico no tratamento de tumores sólidos e neoplasias hematológicas, principalmente. Apesar de seu amplo uso, apresenta como efeito colateral mais importante a cardiotoxicidade. A toxicidade crônica é bastante conhecida e estudada e cursa com miocardiopatia dilatada e quadro clínico clássico de insuficiência cardíaca. A toxicidade aguda, por cursar com quadro clínico pouco exuberante e manifestar-se através de alterações eletrocardiográficas, é muito pouco diagnosticada e seu mecanismo fisiopatológico não é totalmente conhecido. Os mecanismos envolvidos na toxicidade cardíaca são distintos dos mecanismos de ação da droga e são múltiplos: aumento do estresse oxidativo, aumento da apoptose e alteração na dinâmica intracelular do cálcio. Nossa hipótese é que ocorra lesão estrutural e funcional cardíaca, precocemente, após infusão da doxorrubicina. O objetivo do presente estudo foi avaliar a função ventricular esquerda agudamente após administração de doxorrubicina, a expressão gênica das proteínas reguladoras do trânsito de cálcio, a atividade de metaloproteinases 2 e 9 no miocárdio e alterações das citocinas inflamatórias no miocárdio de ratos tratados com a droga. Para isso, foram utilizados ratos Wistar machos adultos (n=35), que foram submetidos à infusão intraperitoneal de dose única de doxorrubicina de 20 mg/Kg ou volume equivalente de salina (grupo controle). Os animais foram eutanasiados 48 horas após injeção da droga. Todos os ratos foram submetidos ao ecocardiograma antes e 48 horas após a injeção da doxorrubicina. Além da avaliação da função cardíaca in vivo pelo ecocardiograma, a função ventricular esquerda foi avaliada in vitro através de estudo do coração isolado, segundo preparação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Doxorubicin, or adriamycin, is a drug used as an antineoplastic agent in the treatment of solid tumors and hematologic malignancies. Despite of its use, there are several side effects, and the most important is cardiotoxicity. Chronic toxicity is well known and studied. It presented with dilated cardiomyopathy and clinical features of heart failure. The acute toxicity has mild clinical signs manifesting usually as electrocardiographic changes. Probably, this acute effect is underdiagnosed and the pathophysiological mechanism is not fully understood. The mechanisms involved in cardiac toxicity are multiple, and include increased oxidative stress, increased apoptosis and alteration in intracellular calcium dynamics. Our hypothesis is that structural and functional damage occurs in the heart early after infusion of doxorubicin. The purpose of this study was to evaluate left ventricular function acutely after doxorubicin administration. In addition, gene expression of calcium regulatory proteins, activity of metalloproteinases 2 and 9, and inflammatory cytokines in the myocardium of rats treated with this drug will also be evaluated. Thus, we used adult male Wistar rats (n = 35) who received a single-dose, by intraperitoneal infusion, of doxorubicin (20 mg / kg) or equivalent volume of saline (control group). After 48 hours of drug injection the rats were euthanized. All animals were submitted to echocardiography before drug infusion and immediately before euthanasia. Besides in vivo cardiac function evaluation by echocardiography. In vitro left ventricular function was assessed by isolated perfusion heart study, according to Langendorff preparation. We evaluated interstitial collagen and myocyte hypertrophy by light microscopy, and cardiac tissue metalloproteinases 2 and 9 activity was assessed by zymography. In addition... (Complete abstract click electronic access below) / Doutor

Abstract Samson Dada and Dr. Hua Mei, Department of Chemistry, College of Arts and Sciences, East Tennessee State University, Johnson City, TN Our overall research goal is to synthesize a water-soluble, bio-absorbable theranostic nanoparticle (NP) that will improve diagnostic and therapeutic efficacy for cancer. Such theranostic nanoparticles are composed of carbon dots (CDs), conjugated with a targeting agent through a non-cleavable peptide bond; and an anticancer drug Doxorubicin (DOX) using an acid-labile hydrazine linkage for targeted delivery and bio-imaging functions. Recent studies have shown that Carbon dots (CDs) are of interest in biological applications due to their unique properties such as inherent fluorescence, extremely high biocompatibility, and facile synthetic route. The large surface area and multiple surface functionalities make CDs versatile platforms to conjugate with other moieties, including therapeutic agents or targeting agents. The target agents, such as folic acid (FA), are proposed to be permanently linked with CDs to improve the target specificity of the tumor cells. Folic acid is used as a targeting agent as it is a water-soluble, low molecular weight vitamin as it plays an essential role in cell survival and binds with high affinity to the folate receptor (FR) – a membrane-anchored protein that is a cancer biomarker. The multimodal nano-platforms of CDs can also facilitate the delivery the anticancer drugs. The anticancer drug is attached by a cleavable linker that can release the drug inside the tumor cell. We will use the cytotoxic chemotherapeutic agent doxorubicin (DOX) as an example. One series of CDs, FA-CD and FA-CD-DOX, are successfully prepared in the lab. The UV-vis and Fluorescence spectra of the sample was investigated and compared. The concentration of each part in nanoparticles are calculated. The final Drug Load Content (DLC) and Drug Load Efficiency (DLE) are also calculated and compared with the literature. Another series of FA-CD-DOX will be prepared and compared. The characterization of the diagnostic and therapeutic potential of the NP particles will be carried out in the pharmaceutical department.

Abstract Our overall research goal is to synthesize a water-soluble, bio-absorbable theranostic nanoparticle (NP) that will improve diagnostic and therapeutic efficacy for cancer. Such theranostic nanoparticles are composed of carbon dots (CDs), conjugated with a targeting agent through a non-cleavable peptide bond; and an anticancer drug Doxorubicin (DOX) using an acid-labile hydrazine linkage for targeted delivery and bio-imaging functions. Recent studies have shown that Carbon dots (CDs) are of interest in biological applications due to their unique properties such as inherent fluorescence, extremely high biocompatibility, and facile synthetic route. The large surface area and multiple surface functionalities make CDs versatile platforms to conjugate with other moieties, including therapeutic agents or targeting agents. The target agents, such as folic acid (FA), are proposed to be permanently linked with CDs to improve the target specificity of the tumor cells. Folic acid is used as a targeting agent as it is a water-soluble, low molecular weight vitamin as it plays an essential role in cell survival and binds with high affinity to the folate receptor (FR) – a membrane-anchored protein that is a cancer biomarker. The multimodal nano-platforms of CDs can also facilitate the delivery the anticancer drugs. The anticancer drug is attached by a cleavable linker that can release the drug inside the tumor cell. We will use the cytotoxic chemotherapeutic agent doxorubicin (DOX) as an example. One series of CDs, FA-CD and FA-CD-DOX, are successfully prepared in the lab. The UV-vis and Fluorescence spectra of the sample was investigated and compared. The concentration of each part in nanoparticles are calculated. The final Drug Load Content (DLC) and Drug Load Efficiency (DLE) are also calculated and compared with the literature. Another series of FA-CD-DOX will be prepared and compared. The characterization of the diagnostic and therapeutic potential of the NP particles will be carried out in the pharmaceutical department.

No / Chemotherapy has severe side effects in normal rapidly proliferating organs, such as hair follicles, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin (DXR), and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in DXR-treated hair follicles versus controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL (tumor necrosis factor–related apoptosisinducing ligand) receptors 1/2), as well as of a large number of keratin-associated protein genes, were seen after DXR treatment. Hair follicle apoptosis induced by DXR was significantly inhibited by either TRAIL-neutralizing antibody or caspase-8 inhibitor, thus suggesting a previously unreported role for TRAIL receptor signaling in mediating DXR-induced hair loss. These data demonstrate that the early phase of the hair follicle response to DXR includes upregulation of apoptosis-associated markers, as well as substantial reorganization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies toward the design of effective approaches for the management of chemotherapy-induced hair loss.

Yes / Phenylacetyl-peptide amphiphiles were designed, which upon cleavage by a disease-associated enzyme reconfigure from micellar aggregates to fibres. Upon this morphological change, a doxorubicin payload could be retained in the fibres formed, which makes them valuable carriers for localised formation of nanofibre depots for slow release of hydrophobic anticancer drugs.

Part I. Following a general historical review of the development of drug targeting, critical evaluations were made of current targeted drug delivery systems. Based on the results shown by previous studies, magnetic albumin microspheres containing adriamycin is one of the most promising targetable delivery systems for the treatment of solid tumors. It was also apparent that the pharmacokinetics of drugs associated with magnetic albumin microspheres had not been determined. A systematic study of the multiple variables involved in albumin microsphere preparation was completed to identify to what extent these variables affected the microsphere size distribution. The results of this investigation led to an optimal method of microsphere preparation. Information obtained from the above studies was applied to the production of magnetic albumin microspheres containing adriamycin suitable for in vivo use. The problems of separation and quantitation of adriamycin and adriamycinol in biological matrices were investigated using ion-pairing high pressure liquid chromatography. An optimized chromatographic system was presented for the analysis of these compounds in rat serum and tissues. The disposition of adriamycin following administration as magnetic albumin microspheres and as a solution was studied by monitoring adriamycin concentrations in multiple rat tissues for forty-eight hours after administration. The magnetic dosage form was targeted to a predefined tail segment with a magnetic field strength of 8000 G applied for 30 min after dosing. A physiological pharmacokinetic model was used to describe the disposition of adriamycin after both dosage forms. The model developed following adriamycin administration as a solution served as the foundation for the model for adriamycin when it was administered as the magnetic dosage form. Part II. The present investigation was designed to characterize the kinetics of ibuprofen in plasma and synovial fluid, which in the past, has been flawed by inadequate study protocols. After administration of a single dose and at steady-state, ibuprofen concentrations were measured simultaneously in plasma and synovial fluid obtained from eight patients with rheumatoid arthritis. The extent of accumulation of ibuprofen in each fluid was determined. The degree of ibuprofen protein binding in plasma and synovial fluid was also determined and related to its kinetic behavior.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the most effective antineoplastic drug utilized in oncologic practice today. However, its clinical use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced quality of life, irreversible heart failure and death. The mechanisms involved in the pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen species, the maintenance of calcium homeostasis and are the most extensively damaged by DOX, they have become the main focus of novel therapeutic interventions. The morphology and function of these dynamic organelles are regulated in part by mitochondrial fission and fusion events, as well as mitochondrial quality control systems. Since mitochondrial morphology is often associated with crucial cellular functions, this study aimed to investigate the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and both play a role in maintaining calcium homeostasis, this study further assessed the effects of chronic DOX treatment on ER function and calcium status. Materials and Methods: In order to fully establish the effect of chronic DOX treatment in vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived Girardi heart cells were cultured under standard culture conditions until ± 70-80% confluency was reached, where after treatment commenced. Cells were treated daily with 0.2 and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER stress were determined with Western blotting. Organelle morphology was visualized with fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load. In order to determine the oxidative capacity, stress and status within the cells following treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally, intracellular and mitochondrial calcium was assessed and quantified with superresolution structured illumination microscopy (SR-SIM) and flow cytometry respectively. Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro cardiac cell models. This study further demonstrated that the expression of mitochondrial fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was clearly evident from the morphology of these organelles, which displayed short, highly fragmented mitochondria with a dispersed network following treatment. Chronic DOX also downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were highly upregulated following treatment, indicating activation of the UPP and mitophagy. Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation. Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both concentrations of DOX, an increase in oxidative stress status was shown following DOX treatment. In this regard lipid peroxidation significantly increased, while redox status of the endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated. Discussion: Results from this study indicate that chronic DOX treatment disrupts the balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial fragmentation is mediated by the downregulation of fusion proteins regulated by the E3 ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium. Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell against damaged mitochondria. This study also indicates that during chronic DOX-induced cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics and mitochondrial quality control systems. Further investigations targeted at limiting mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme. Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW) te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase, het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en kalsiumstatus ondersoek. Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse. Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC, TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde illuminasie mikroskopie (SR-SIM) en vloeisitomerie. Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α, afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die UPW gestimuleer. Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase. Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer. Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)

A funcionalização de superfície nas nanocápsulas contendo doxorrubicina com o peptídeo RGD é uma estratégia promissora devido a ligação preferencial na integrina αvβ3 expressa em células tumorais. Este estudo objetivou o desenvolvimento, caracterização e estudos biológicos de nanocápsulas multiparedes com doxorrubicina e funcionalizadas com RGD. Para isso, na primeira etapa do trabalho foi realizada a síntese do peptídeo RGD. Os produtos obtidos foram caracterizados por análises de infravermelho e RMN de 1H. Na segunda etapa foram desenvolvidas formulações de nanocápsulas com doxorrubicina ou cloridrato de doxorrubicina, e, nanocápsulas multiparedes revestidas com quitosana, íons zinco, RGD ou fenilalanina. Essas suspensões foram caracterizadas através da determinação do pH, diâmetro de partícula por diferentes técnicas, potencial zeta, eficiência de encapsulação e eficiência de associação do RGD na superfície da nanopartícula. Na terceira etapa, foram realizados ensaios de viabilidade celular por MTT após 24 e 72h com as formulações desenvolvidas em células de câncer de mama (MCF7) e glioblastoma humano (U87MG). As formulações apresentaram diferentes valores de citotoxicidade e, utilizando o Gráfico de Pareto foi possível determinar os fatores que exercem maior influencia. Em células MCF7 foi a concentração de fármaco e tempo de tratamento e, nas células U87MG além desses fatores, a funcionalização mostrou-se determinante. Além disso, foi avaliada a captação das nanocápsulas funcionalizadas com RGD e fenilalanina após 24h nas células tumorais e células de queratinócitos humanos (HaCat), com diferentes níveis de expressão da integrina αvβ3. O estudo mostrou menores valores de captação nas células HaCat (sem expressão de integrina αvβ3) para as duas formulações testadas. Finalmente as nanocápsulas funcionalizadas com RGD apresentaram maior captação em células U87MG com maior expressão da integrina αvβ3. / The surface functionalization in nanocapsules containing doxorubicin with RGD peptide is a promising strategy due to preferential binding in the αvβ3 integrin expressed on tumor cells. This study aimed the development, characterization, and biological studies of multiwall nanocapsules containing doxorubicin and functionalized with RGD. For this reason, in the first stage of this study the synthesis of RGD peptide was performed and the products characterized by infrared analysis and 1H NMR. Besides, nanocapsules formulations were developed containing doxorubicin or doxorubicin hydrochloride, and multiwall nanocapsules coated with chitosan, zinc ions, RGD or phenylalanine. These suspensions were characterized by pH determination, particle diameter by different techniques, zeta potential, encapsulation efficiency, and association efficiency of RGD on the surface of the nanoparticle. Additionally, it was performed cell viability assays by MTT after 24 and 72 hours with formulations developed in breast cancer (MCF7) and human glioblastoma cells (U87MG). Formulations showed different cytotoxicity values. The Pareto chart was possible to determine factors that have more influence. In MCF7 cells was drug concentration and treatment time, and U87MG cells, besides these factors, the functionalization was decisive. Furthermore, it was performed the cellular uptake of nanocapsules functionalized with RGD or phenylalanine after 24 hours in tumor cells and human keratinocyte cells (HaCaT), with different levels of expression αvβ3 integrin. The study showed less uptake in HaCaT cells (without expression αvβ3 integrin) for the two formulations applied, and the nanocapsules functionalized with RGD showed more uptake in U87MG cells, with higher expression of integrin αvβ3.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A Atresia de esÃfago (AE) à uma anomalia estrutural que acontece em fetos quando nÃo ocorre a septaÃÃo completa do septo esofagotraqueal. Estudou-se a repercussÃo da AE induzida por Doxorrubicina (Doxo) na contratilidade in vitro do esÃfago distal e fundo de estÃmago. 26 Ratas wistar (267 g), com acasalamento controlado, nos dias 8 e 9 de gestaÃÃo receberam 2,2 mg/Kg de Doxo intraperitonealmente, enquanto 13 ratas controle receberam o mesmo volume de NaCl 0,9%. No dia 21,5 as ratas foram submetidas a cesariana, anÃlise dos fetos para confirmaÃÃo da AE e divisÃo em 3 grupos: controle, cujas mÃes receberam apenas NaCl 0,9%; Doxo sem AE, cujas mÃes receberam Doxo, mas nÃo desenvolveram AE; e Doxo com AE, os que desenvolveram AE. Foram montados em sistema para banho de ÃrgÃo isolado os anÃis de fundo de estÃmago dos fetos e em sistema miÃgrafo de agulha os segmentos de esÃfago distal, ambos contendo soluÃÃo fisiolÃgica Tyrode a 37 ÂC, pH 7,4 e oxigenaÃÃo constante, tensÃo basal de 1 g para estÃmago e 8 mN para esÃfago. Realizou-se curva concentraÃÃo-efeito ao agonista colinÃrgico Carbacol (CCh) (0,01 â 300 &#956;M) em ambos os tecidos nos 3 grupos. Em seguida, agora apenas nos segmentos de esÃfago isolado, foi construÃda uma curva concentraÃÃo-efeito ao KCl (10 â 100 mM), em que a contraÃÃo se deve prioritariamente à entrada de cÃlcio do meio extracelular por meio de canais operados por voltagem (VOC). A anÃlise estatÃstica foi determinada utilizando two-way anÃlise de variÃncia (ANOVA) e a significÃncia foi testada pelo Student-Newman-Keuls test. No fundo de estÃmago nÃo houve diferenÃa estatÃstica entre os grupos na resposta contrÃtil ao CCh (p>0,05, ANOVA), os valores da CE50 dos animais controle foram 2,17 [1,03 â 4,58] &#956;M e Emax 0,084  0,016 g/mg de tecido (n=7); Doxo sem AE 1,47 [0,83 â 2,61] &#956;M e 0,068  0,006 g/mg de tecido (n=12); Doxo com AE 3,26 [1,90 â 5,60] &#956;M e 0,070  0,022 g/mg de tecido (n=6). No esÃfago, animais controle com Emax 5,97  0,58 mN (n=11) foram estatisticamente diferentes (p<0,05, ANOVA) dos grupos Doxo sem AE 4,48  0,34 mN (n=11) e Doxo com AE 4,42  0,68 mN (n=8), enquanto a CE50 nÃo apresentou diferenÃa estatÃstica significativa entre os grupos (p>0,05, ANOVA) controle 190 [96 â 379] nM, Doxo sem AE 228 [125 â 418] nM e Doxo com AE 439 [206 â 936] nM, quanto a resposta contrÃtil ao CCh. Na resposta ao KCl houve incremento de tensÃo inferior ao observado com CCh sem diferenÃa entre os trÃs grupos (p>0,05, ANOVA), 8 valores de Emax foram no controle 1,31  0,14 mN (n=5), Doxo sem AE 1,27  0,42 mN (n=7) e Doxo com AE 1,21  0,20 mN (n=7). Concluiu-se que o tratamento de ratas com Doxo durante o perÃodo gestacional leva a uma diminuiÃÃo da contratilidade de esÃfago isolado de seus fetos, independente do desenvolvimento de AE. Aparentemente, essa diminuiÃÃo nÃo se deve a uma menor funcionalidade dos canais VOC. O fundo de estÃmago isolado nÃo apresentou alteraÃÃes da resposta contrÃtil. / Esophageal atresia (EA) is a structural anomaly that results from an incomplete esophago-traqueal septation in the fetus during intrauterine development. The in vitro contractility of the distal esophagus and gastric fundus of fetuses with esophageal atresia induced by Doxorubicin (Doxo) was studied. 26 Female Wistar rats (267 g), were subjected to date-controlled mating, subsequently receiving 2.2 mg/kg Doxo intraperitoneally on days 8 and 9 of pregnancy, while a controlled group of 13 rats received the same volume of 0.9% NaCl. On day 21.5 the pregnant rats were submitted to a cesarean surgery, with the fetuses analysed to confirm EA and thereafter divided into 3 groups: control, whose mothers received only 0.9% NaCl; Doxo without EA, whose mothers received Doxo but not developed EA; Doxo with EA, who developed EA. After being sacrificed, ring-strips of the gastric fundus were obtained from the fetuses and mounted in isolated organ bath, while the distal esophageal strips were mounted in wire myograph system; both strips contained a standard Tyrode solution maintained at 37 ÂC, pH 7.4, in addition to constant oxygenation and a basal tension of 1 g for the fundic strips and 8 mN for the esophagus. For each set up, we carried out a cholinergic-agonist concentration- effect curve with Carbachol (CCh) (0.01 â 300 &#956;M) in both tissue in the three groups. The participation of voltage-operated channels (VOCs) was studied; a KCl- concentration-effect curve (10 â 100 mM) was conducted on isolated esophageal strips. Collected data was subjected to two-way analysis of variance (ANOVA) and the significance was tested using Student-Newman-Keuls test. There was not significant statistical difference in fundic stripsâ contractility in response to CCh (p>0.05, ANOVA), the EC50 values of the control animals were 2.17 [1.03 â 4.58] &#956;M and Emax 0.084  0.016 g/mg tissue (n=7); Doxo without EA 1.47 [0.83 â 2.61] &#956;M and 0.068  0.006 g/mg tissue (n=12); Doxo with EA 3.26 [1.90 â 5.60] &#956;M and 0.070  0.022 g/mg tissue (n=6). However, significant statistical difference was noted (p<0.05, ANOVA), in esophageal stripsâ contractility in response to CCh in the Emax value of control 5.97  0.58 mN (n=11), vs Doxo without EA 4.48  0.34 mN (n=11) and Doxo with EA 4.42  0.68 mN (n=8), while there was not significant statistical difference (p>0.05, ANOVA) in the EC50 value of control 190 [96 â 379] nM, Doxo without EA 228 [125 â 418] nM and Doxo with EA 439 [206 â 936] nM. Tensional response to KCl were present in all groups, though lower than that seen in response &#65532;to CCh, however not statistically different when comparing all the three groups (p>0.05, ANOVA), Emax of control was 1.31  0.14 mN (n=5), Doxo without EA 1.27  0.42 mN (n=7) and Doxo with EA 1.21  0.20 mN (n=7). It is possible to conclude that the treatment of rats with Doxo during pregnancy leads to decrease contractility of isolated esophagus of their fetuses, independent of the development of EA. Apparently, such a decrease is not due to a lower functionality of VOC channels. The isolated gastric fundus strips showed no change in contractile response.

Background: The molecular and cellular mechanisms corresponding to the compensatory and maladaptive hypertrophy and remodeling of the left ventricle with chronic doxorubicin (DOX) treatment are currently unclear. Non-invasive methods of determining these changes are still deficient. To investigate these changes, 8 groups of rats in 4 different studies including a control saline group of the same age, gender and strain were evaluated for cardiac morphology and function including: (1) DOX dose response using a cumulative dose of 7.5mg/kg, and 15mg/kg in 8-10 week old female Sprague-Dawley (SD) rats, (2) strain differences were investigated in response to a cumulative dose of 15mg/kg in 8-10 week old female Fisher (F344) rats compared to the SD rats treated with same dose, (3) the role of gender and aging were studied in response to DOX at a cumulative dose of 3mg/kg in male and female neonates, and (4) combined losartan and a cumulative dose of 15mg/kg of DOX in 8-10 week old female SD rats compared to controls of saline and 15mg/kg treated SD rats. Method: Onset of cardiac toxicity was assessed by echocardiography and the rat model of heart failure was developed when the fractional shortening declined ≤ 40%. The mean arterial pressure and single-photon-emission computer tomography scanning and Tc-99m-HYNIC-Annexin V were performed at week 10 to analyze blood pressure and quantify apoptosis, respectively. All rats were euthanized at week 10 except for the neonates and two of the 7.5mg/kg-treated SD rats that were left alive for study of long -term cardiac side effects. The heart and kidney tissues were harvested for protein isolation and histopathological studies. Blood samples were collected for hematological and lipid profile analysis in all the rats. Results: A dose- and time-dependent increase in LVmass coincided with a parallel increase in MAP, kidney damage, expression of myocardial erbB2, heat shock protein 90 Akt, mTOR, GSK-3β, TGF-β, pSMAD2, and cardiomyocyte apoptosis in SD rats treated with 7.5mg/kg and 15mg/kg of DOX at week 10. The 7.5 kg/kg treatment showed adaptive hypertrophy whereas the 15mg/kg treatment group showed maladaptive hypertrophy. However decompensation was apparent by week 14 in other rats treated with 7.5mg/kg. LVmass, FS, MAP, kidney damage, red blood cells and blood lipid levels were not significantly altered in the F344 rats compared to the 15 mg/kg-treated SD rats. Losartan supplementation reduced the left ventricular hypertrophy, improved myocardial contractility, and reduced TGF-β expression compared to the DOX-treated SD rats. The 3mg/kg of DOX in neonates induced cardiac toxicity and deaths in about 60% of males 50 weeks after treatment; the females instead developed mammary tumors. Conclusion: The results of this study suggest that age, gender, and strain differences are risks factors for doxorubicin-induced harmful reno-cardiovascular toxicity. The inhibition of TGF-β expression by losartan can be used in prevention of chronic doxorubicin-induced cardiac toxicity without interfering with its anti-tumor activities.