Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Drug delivery systems based on gold nanomaterials are regarded as one of the most effective methods for the treatment of cancer. In this work, we developed a nanocarrier based on a thiol-organofunctionalized chitosan and gold nanoparticles (AuNPs)for docetaxel, an antineoplastic drug used for the treatment of lung, prostate, leukemia and malignant melanoma tumors. Initially, chitosan (CS) was modified with cysteine (CYS) by coupling them using N,N′-Dicyclohexylcarbodiimide (DCC). The presence of thiols groups in CS was confirmed by FTIR spectroscopy. Subsequently, AuNPs were synthesized and passivated by sodium citrate. From UV-vis spectroscopy analysis, it was possible to obtain a strong absorbance band at 520 nm, which is related to the localized surface plasmon resonance (LSPR). The size distribution of the nanoparticles was obtained by TEM, showing an average size of around 18nm. Then, a study using UV-vis spectroscopy, DLS, zeta potential and SEM was performed to understand the formation of the nanocomposite AuNPs/CS-SH. In this study, it was observed that the nanoparticles systematically aggregate in the presence of the polymeric matrix, changing their optical and morphological properties through time and amount of CS-SH. The release profile of the DTX with CS-SH was twice as much as the free drug, as well as, the material gave a controlled release up to 48 hours and constant up to 120 hours, unlike the free drug. After that time, the absorbance values remained constant until approximately 80 hours, unlike the free drug. The use of the zero order kinetic model allowed a better understanding of the drug release profile with the material. / Com o advento da nanotecnologia, sistemas avançados de entrega de fármacos têm sido eficazes no tratamento do câncer. Nesse trabalho, desenvolvemos um carreador para o fármaco antineoplásico docetaxel (DTX) a partir do sistema de liberação controlada formado por uma quitosana organofuncionalizada com grupos tióis e nanopartículas de ouro (AuNPs). Para a produção do carreador, a quitosana (CS) foi modificada com cisteína e acoplada por dicicloexilcarbodiimida (DCC). A presença dos grupos tióis na CS foi confirmada pelos espectros obtidos por FTIR. Posteriormente, as AuNPs foram sintetizadas e passivadas pelo método do citrato de sódio e, a partir da espectroscopia de absorção na região do UV-vis, foi possível obter uma banda de absorbância intensa na região de 520 nm. Os diâmetros médios das nanopartículas foram observados por TEM, obtendo um tamanho médio de aproximadamente 18 nm. Na sequência, realizamos um estudo cinético para entendermos melhor o comportamento entre o material produzido e as AuNPs, comprovando a interação destes a partir das medidas de FTIR, essa técnica também foi importante para comprovação da interação entre o fármaco e o material produzido. Complementar a este estudo, as técnicas de DLS, potencial Zeta e MEV foram utilizadas, sendo fundamentais para definirmos a massa ideal da quitosana modificada (CS-SH) nos testes de liberação. O perfil de liberação do DTX com o material foi o dobro em relação ao fármaco livre, bem como, o material proporcionou uma liberação mais lenta, nas primeiras 8 horas e continuou liberando até aproximadamente as 48 horas. Após esse tempo, os valores de absorbâncias permaneceram constantes até aproximadamente as 80 horas, diferentemente do fármaco livre. A utilização do modelo cinético de ordem zero possibilitou um melhor entendimento do perfil de liberação do fármaco com o material. / São Cristóvão, SE

No description available.

No / This work explored the role of skin appendages (shunt route) in passive and iontophoretic drug and liposome penetration. The technique used an epidermis and stratum corneum sandwich from the same skin donor with the additional stratum corneum forming the top layer of the sandwich. Penetration was monitored during occluded passive and iontophoretic (0.5 mA cm-2) delivery of mannitol and estradiol solutions, and ultradeformable liposomes containing estradiol. The shunt route had a significant role during passive penetration of mannitol (hydrophilic compound), but was negligible during penetration of estradiol (lipophilic drug) and liposomes. In iontophoresis, the shunt route significantly contributed to the overall flux of all preparations, being highest for mannitol. However, shunts were not the only pathway for iontophoretic drug delivery and evidence was observed for the creation of new aqueous pathways via disorganization of the intercellular lipid domain of stratum corneum. The skin sandwich technique should prove valuable for general studies on routes of skin penetration.

Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

In der vorliegenden Arbeit wurden zwei neuartige biodegradierbare Nanopartikel (NP)-Typen definierter Größe auf ihre Eignung als Wirkstofftransporter für die Verwendung in der Photodynamischen Therapie (PDT) untersucht. Die Verwendung biodegradierbarer NP als Wirkstofftransporter in der PDT ist ein vielversprechender neuer Ansatz, der im Rahmen dieser Arbeit erstmalig untersucht wurde. Die in dieser Arbeit untersuchten NP bestehen aus humanem Serumalbumin (HSA), beziehungsweise Polylactid-co-Glycolid (PLGA) und wurden mit den Photosensibilisatoren (PS) Phäophorbid a (Pheo), Meta-Tetra (Hydroxy-Phenyl)- Porphyrin (mTHPP) oder Meta-Tetra (Hydroxy-Phenyl)-Chlorin (mTHPC) beladen. Es wurden die endozytotische Aufnahme, der lysosomale Abbau der NP und die intrazelluläre Freisetzung der PS in Abhängigkeit von der Inkubationszeit in vitro an humanen Krebszellen untersucht. Um die Effizienz der Photosensibilisierung durch die mit PS beladenen NP zu bestimmen, wurden die Phototoxizität und die intrazelluläre Singulettsauerstoffgenerierung bestimmt. Es konnte gezeigt werden, dass sowohl HSA- als auch PLGA-NP das Potential besitzen als Wirkstofftransporter in der PDT Verwendung zu finden. Insbesondere mTHPC-beladene NP wirken durch starke intrazelluläre Singulettsauerstoffgenerierung sehr phototoxisch. Die Experimente zeigen, dass die intrazelluläre PS-Konzentration geringeren Einfluss auf die Effizienz der Photosensibilisierung hat als die Freisetzung der PS und deren intrazelluläre Lokalisation. Die Biodegradierbarkeit von HSA und PLGA ermöglicht einen schnellen Abbau. Dadurch kann bereits bei sehr geringen intrazellulären PS-Konzentrationen hohe Phototoxizität erreicht werden. Der Fokus bei der Optimierung sollte einerseits in einer stabilen Verbindung zwischen PS und NP liegen, andererseits in einer effizienten Freisetzung nach der zellulären Aufnahme. Deshalb sind biodegradierbare Polymere sehr vielversprechende Materialien für die Entwicklung von PS-NP-Transportsystemen. / In the present study, two novel biodegradable nanoparticle (NP) types with a defined size were examined for their suitability as drug delivery systems for use in photodynamic therapy (PDT). NP drug transporters already found a successful application in chemotherapy but the use of biodegradable NP in PDT is a new promising challenge. The investigated NP consist of human serum albumin (HSA) and poly(lactic-co-glycolic acid) (PLGA) and were loaded with the photosensitizers (PS) pheophorbide a (Pheo), meta-tetra(hydroxy-phenyl)-porphyrin (mTHPP) or meta- tetra(hydroxy-phenyl)-chlorin(mTHPC). The endocytotic intracellular uptake and the time dependent drug release caused by decomposition of the biodegradable PS loaded nanoparticles were studied in vitro on Jurkat cells in suspension. The phototoxicity as well as the intracellular singlet oxygen generation was investigated for different incubation times. It was shown that both, HSA and PLGA NPs are promising carriers for PDT agents regarding uptake and phototoxicity. Especially the mTHPC loaded NPs show a very efficient phototoxicity caused by a very high singlet oxygen generation after the release of the PSs. The experiments show that the overall intracellular PS concentration is of less importance for the efficiency of the photosensitization compared to the amount of intracellular drug release and the intracellular localisation of the PS molecules. The biodegradability of the HSA and PLGA nanoparticles enables a fast intracellular drug release that causes high phototoxicity even for very low intracellular PS concentrations. Thus, the strategy for efficient PS loaded NP carriers is not a maximum loading. The main challenge is to create carriers with highly stable PS NP bonding to prevent any drug release before intracellular uptake combined with the ability of a complete drug release inside the target cells. Thus, biodegradable polymers are very promising materials for the design of NP-PS delivery systems

Made available in DSpace on 2015-08-19T13:52:55Z (GMT). No. of bitstreams: 2 6.pdf: 1663850 bytes, checksum: 9af5d15cc4767f2da9ac347b8aa5a350 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / Esta dissertação propõe a habilitação operacional de qualidade, além da habilitação legal, do serviço de transporte de medicamentos para atendimento a uma empresa farmacêutica pública, ainda na etapa do edital de contratação. A empresa transportadora, participante do edital de contratação, deverá comprovar a capacidade de execução do serviço de transporte atendendo às normas legais de contratação pública e de qualidade sanitária, vigentes no território nacional.Este estudo apresenta os quesitos para a habilitação operacional de qualidade em um edital de contratação de transporte de medicamentos, compondo a etapa de qualificação. Os quesitos solicitados estão estruturados em especificações e conceitos de qualidade para a documentação legal, para a documentação técnica e para as operações a serem executadas, como controles e documentos pertinentes.As atividades realizadas durante o transporte estão descritas em formato de fluxogramas e, conforme registrado nestes fluxogramas, as etapas críticas estão avaliadas através de análise de riscos. Para controle dos riscos analisados estão propostos documentos de acompanhamento e registros pertinentes para confirmar a segurança da execução.Ainda para a habilitação operacional de qualidade e para acompanhamento da prestação do serviço de transporte, este estudo organiza um roteiro de auditoria. Este roteiro pode ser aplicado na etapa de habilitação para a contratação e na continuidade do contrato de prestação do serviço de transporte de medicamentos. O roteiro de auditoria detalha os riscos mais graves e deficiências de maior incidência nas atividades executadas no transporte de medicamentos. / This dissertation proposes the quality operational accreditation, as well as the legal certification, of drug transportation services when assisting public pharmaceutical companies still during the public notice process. Transportation companies, who are participating on the public notice process, must prove the hability of executing the transportation services, at testing to legal regulations of public procurement and sanitary surveillance in effect on national territory. This study introduces the requirements for the quality operational accreditation in a public notice for the hiring of medication transportion, composing the evaluation stage. The necessary requirements are structured by specifications and quality concepts for the legal documentation, for the technical documentation, and for the activities to be performed, with the relevant monitoring and documents. The activities performed during the transportation are described in flow charts and, as registered in these flow charts, the critical stages are assessed through risk analysis. In order to monitor the analyzed risks are proposed accompanying documents and relevant registrations to ensure the safe execution of the activity. Still for the purpose of quality operational accreditation and overseeing the transportation services, this study organizes an audit script. This script can be used on the qualification stage for the hiring and during the continuity of the drug transportation service contract. The audit scripts details the most serious risks and the deficiencies with the highest recurrence rates in the activities performed during the transportation of medications.

Wachstumsfaktoren sind wichtige Steuerelemente des Knochenzellmetabolismus. Im Verlauf der Frakturheilung kommt es zur Ausschüttung von zahlreichen Wachstumsfaktoren, Zytokinen und Botenstoffen im und um den Bereich des Frakturspalts, die systemisch oder lokal, endokrin, parakrin oder autokrin wirksam werden können. Für verschiedene Wachstumsfaktoren konnten in zahlreichen Studien osteoinduktive und die Frakturheilung positiv beeinflussende Wirkungen nachgewiesen werden. In vitro und in vivo Studien belegen, dass einige dieser Faktoren wie Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta1 (TGF-beta1) und Bone morphogenetic protein-2 (BMP-2) einen stimulierenden Effekt auf osteo- und chondrogene Zellen aufweisen und somit die Knochenheilung stimulieren. Der genaue Wirkmechanismus dieses positiven Effektes der Wachstumsfaktoren und ihre Interaktion im Verlauf der Frakturheilung ist nicht bekannt. Die lokale Applikation der Faktoren für einen therapeutischen Einsatz bei der Frakturheilung stellte bisher jedoch ein Problem dar. Mit der entwickelten biodegradierbaren Poly(D,L-Laktid)-Beschichtung von Implantaten können eingearbeitete Wachstumsfaktoren kontrolliert und lokal direkt an der Fraktur freigesetzt werden. Das beschichtete Implantat dient dabei der Stabilisation der Fraktur und gleichzeitig als Wirkstoffträger. Die Beschichtung weist eine hohe mechanische Stabilität auf. Die eingearbeiteten Wachstumsfaktoren behalten ihre biologische Aktivität in der Beschichtung und werden kontrolliert lokal freigesetzt. Um den Effekt lokal applizierter Wachstumsfaktoren auf die Frakturheilung zu untersuchen, wurde ein standardisiertes geschlossenes Frakturmodell entwickelt, das der klinischen Situation möglichst nahe ist und reproduzierbar durchgeführt werden kann. Untersucht wurde der Effekt der Wachstumsfaktoren IGF-I, TGF-beta1 und BMP-2 und des Trägermaterials PDLLA sowie lokale und systemische unerwünschte Wirkungen. Die Ergebnisse zeigten einen signifikant grösseren stimulierenden Effekt von IGF-I auf die Frakturheilung im Vergleich zur TGF-beta1 Applikation. Die kombinierte Gabe beider Faktoren ergab einen signifikant grösseren Effekt auf die torsionale Stabilität und die Kallusreifung im Vergleich zur Einzelapplikation. Beide Faktoren scheinen einen synergistischen Effekt auf die Frakturheilung zu haben. Die lokale Applikation von BMP-2 beschleunigte ebenso, wie die lokale Freisetzung von IGF-I und TGF-beta1 die Frakturheilung signifikant. Deutliche Unterschiede zwischen IGF-I / TGF-beta1 und BMP-2 konnten nicht festgestellt werden.Allerdings zeigte sich bei der Verwendung von BMP-2 auch ausserhalb der Frakturzone eine grössere Mineralisation der Kortikalis, die bei IGF-I / TGF-beta1 nicht zu beobachten ist. Auch im Grosstiermodell bestätigte sich die Wirksamkeit dieser bioaktiven Oberflächen-beschichtung auf die Osteotomieheilung. Die PDLLA-Beschichtung alleine, ohne eingearbeitete Wachstumsfaktoren, zeigte bereits einen positiven Effekt auf die Frakturheilung. Die Untersuchungen belegen, dass die lokale Freisetzung von Wachstumsfaktoren aus einer biodegradierbaren PDLLA-Beschichtung von Implantaten die Frakturheilung signifikant beschleunigt, wobei keine unerwünschten lokalen oder systemischen Wirkungen beobachtet werden konnten. Bei dem Vergleich lokaler (durch Wachstumsfaktoren) mit systemischer Stimulationsmöglichkeit (durch Wachstumshormon) der Frakturheilung lässt sich zusammenfassend feststellen, dass die kombinierte Anwendung beider Stimulationsmöglichkeiten zu keiner weiteren Steigerung der Heilungsvorgänge führte. Weitere Untersuchungen wurden hinsichtlich der genauen Rolle und Interaktion der Wachstumsfaktoren durchgeführt. Vor allem die Frühphase scheint hierbei eine entscheidende Rolle bei der Frakturheilung einzunehmen. Es zeigte sich hierbei eine deutliche Stimulation der Osteoblastendifferenzierung mit einer Erhöhung der Kollagen-1 Produktion in vitro sowie eine Steigerung der Proliferationsrate und Angiogenese mit einem schnelleren Ablauf der Phasen der Frakturheilung in vivo durch lokal appliziertes IGF-I und TGF-beta1. Weitere Anwendungen der entwickelten Beschichtungstechnologie stellen die lokale Applikation von Wachstumsfaktoren von beschichteten PDLLA-Cages bei der intervertebralen Spondylodese sowie die lokale Applikation von Antibiotika aus einer PDLLA-Beschichtung von Implantaten zur Prophylaxe der Implantat-assoziierten Osteomyelitis dar.Basierend auf diesen Ergebnissen steht der Einsatz PDLLA-Gentamicin beschichteter intramedullärer Tibianägel kurz vor der klinischen Anwendung.Eine Zulassung durch die entsprechenden Behörden ist erfolgt.Die klinische Anwendung Wachstumsfaktoren-beschichteter Implantate ist bereits in der Vorbereitung. / Growth factors are important regulators of bone metabolism. During fracture healing many growth factors or cytokines were locally released at the facture site. In several studies, different growth factors demonstrated osteoinductive and fracture stimulating properties. In vitro and in vivo studies showed a stimulating effect of Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta1 (TGF-beta1) and Bone morphogenetic protein-2 (BMP-2) on osteo- and chondrogenetic cells. The exact effectiveness and the interaction of these growth factors during fracture healing is not known so far. Further, the local application of these factors for therapeutically use in fracture treatment is still a problem. The developed biodegradable poly(D,L-lactide)-coating of implants allows the local and controlled release of incorporated growth factors directly at the fracture site. The coated implant serves on the one hand for fracture stabilization and on the other hand as a drug delivery system. The coating has a high mechanical stability. The incorporated growths factors remain biologically active in the coating and were released in a sustained and controlled manner. To investigate the effect of locally released growth factors IGF-I, TGF-beta1 and BMP-2 and the carrier PDLLA on fracture healing, standardised closed fracture models were developed with a close relationship to clinical situation. Further, possible local and systemic side effects were analysed. The results demonstrated a significantly higher stimulating effect of IGF-I on fracture healing compared to TGF-beta1. The combined application of both growth factors showed a synergistic effect on the mechanical stability and callus remodeling compared to single treatment. The local release of BMP-2 also enhanced fracture healing significantly - comparable to combination of IGF-I and TGF-beta1. However, a higher rate of mineralisation was measurable outside the fracture region using BMP-2 in a rat fracture model. Using a large animal model on pigs with a 1 mm osteotomy gap, the effectiveness of locally released growths factors could be confirmed. Further, the PDLLA-coating without any incorporated growth factors demonstrated a significantly effect on healing processes in both models. These investigations showed, that the local release of growth factors from PDLLA coated implants significantly stimulate fracture healing without any local or systemic side effects. Comparing systemic with local stimulation techniques, we found an improvement of fracture healing by systemic administration of growth hormone and local application of IGF-I and TGF-beta1. However, the combined use of both simulation techniques did not lead to a further increase of healing processes. Investigations on the effectiveness and the interaction of growth factors during fracture healing demonstrated an dramatic effect in the early phases of healing processes. The growth factors stimulate the differentiation of osteoblasts with a higher production of collagen I in vitro and increase osteogenesis and vascularisation of the fracture callus in vivo. Further applications of the coating technology are the use of PDLLA and growth factor coated cages for the stimulation of intervertebral fusion and the use of PDLLA and Gentamicin coated implants in order to prevent implant associated infections. The clinical use of antibiotic and growth factor coated implants are in preparation.

The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology. / Ph. D.