Orientadores: Eneida de Paula, Cíntia Maria Saia Cereda / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T18:07:47Z (GMT). No. of bitstreams: 1 Queiroz_VivianeAparecida_M.pdf: 5281811 bytes, checksum: 4084a85b3806be04ca8c3375db357399 (MD5) Previous issue date: 2012 / Resumo: Dentre os fármacos usados para prevenir e aliviar a dor encontram-se os anestésicos locais (AL). A bupivacaína (BVC) é um dos AL do tipo amino-amida mais utilizado na terapia da dor aguda e crônica. Sua combinação com opióides lipofílicos tornou-se uma prática comum para analgesia em procedimentos cirúrgicos, com o objetivo de reduzir a dose necessária de AL sem comprometer a qualidade de analgesia. Contudo, a BVC é conhecida por apresentar uma acentuada toxicidade para o sistema nervoso central e sistema cardiovascular. Uma alternativa para diminuir os efeitos tóxicos, bem como aumentar o efeito terapêutico é a utilização de sistemas de liberação modificada de fármacos, que empregam carreadores como as ciclodextrinas. Neste trabalho avaliamos a eficácia anestésica e o perfil farmacocinético da BVC (na forma do complexo BVC hidroxipropil-beta-ciclodextrina - BVC:HP--CD, na razão molar de 1:1) em associação com o opióide sufentanil (SUF), em modelos animais. Foram realizados também ensaios de citotoxicidade in vitro, em cultura de células de fibroblastos 3T3 e de neurogliomas NG97, sendo a viabilidade celular determinada através do teste de redução do MTT. Para avaliação da atividade analgésica, realizou-se o teste pressão na pata em ratos da linhagem Unib: Wistar; os animais foram tratados com injeção intratecal de: BVC e BVC:HP-?-CD nas concentrações de 4, 8 e 16 mM (0,125; 0,25 e 0,5%, respectivamente), associadas ou não com 38,8 ?M de SUF; o bloqueio motor induzido por essas formulações foi também avaliado. Para determinação da concentração plasmática e do perfil farmacocinético das formulações descritas, foram utilizados coelhos albinos da raça Nova Zelândia, que receberam tratamento por via intratecal, com BVC livre ou complexada (16 mM), associada ou não com SUF (38,8 ?M); as amostras foram analisadas por espectrometria de massas. Nos testes de avaliação de toxicidade in vitro, células 3T3 e NG97 foram tratadas com as formulações acima, mas em concentrações que variaram de 0,2 a 4 mM, para a BVC livre ou complexada, em associação ou não com SUF em concentração proporcionalmente correspondentes à dos testes in vivo (9,5 ?M). Nos resultados de atividade analgésica, o tratamento com BVC:HP-?-CD+SUF prolongou o efeito analgésico, em comparação com BVC+SUF, em 1,9 e 1,8 vezes nas concentrações de BVC de 4 e 8 mM, respectivamente. Além disso, não foram verificadas diferenças estatísticas entre os grupos quanto à função motora dos animais. Em relação ao perfil farmacocinético da BVC, o grupo BVC:HP-?-CD apresentou características de lenta liberação, com baixos níveis plasmáticos, que não foram alteradas após a associação ao coadjuvante SUF. Esses resultados evidenciam uma potencial aplicação clínica da associação medicamentosa BVC:HP-?-CD e SUF, para reduzir a frequência de administração e também a concentração necessária de BVC para promover analgesia em procedimentos cirúrgicos / Abstract: Local anesthetics (LA) are among the most important medicines used to prevent and alleviate pain. Bupivacaine (BVC) is the drug of choice in the treatment of acute and cronic pain, worldwide. It has been used in association with lipophilic opioids in surgical procedures, reducing the required amount of anesthetic without loss of the analgesic level achieved. Bupivacaine is also known to be toxic for the Central Nervous and Cardiovascular systems. The development of drug delivery systems, with carriers such as cyclodextrins, is an interesting approach to reduce toxic side effects and to enhance the therapeutic index of biological active compounds. Here we evaluated the anesthetic activity as well as the pharmacokinetic profile of BVC (free and complexed with hydroxypropyl-beta-cyclodextrins - BVC:HP--CD in a1:1 molar ratio) in association with the opioid sufentanil (SUF) in animal models. The cytotoxicity of such formulations against 3T3 fibroblasts and NG97 neuroglioma cells in culture were also investigated by the use of MTT reduction tests. Sensorial (paw pressure test) and motor blockage was evaluated after intrathecal administration of BVC and BVC:HP-?-CD at concentrations = 4, 8 and 16mM (0.125; 0.25 and 0,5%, respectively), associated or not with 38.8 ?M SUF, to Unib WH rats. BVC plasma levels and pharmacokinetic profile were evaluated through mass spectroscopy in New Zealand rabbits, intrathecally administrated with 16 mM BVC (free or complexed with HP--CD), associated of not with 38.8 ?M SUF. For the in vitro tests 3T3 and NG97 cells were treated with the formulations at concentrations that ranged from 0.2 to 4 mM BVC (free or complexed) plus 9.5 ?M SUF, i.e. proportionally equivalent to the concentration used at the in vivo tests. Treatment with BVC:HP-?-CD+SUF was able to improve the antinonciceptive effect of bupivacaine 1.8-1.9 times (4 and 8 mM, respectively), in comparison to BVC+SUF. No statistical differences were observed for the motor blockade among the groups. Regarding the pharmacokinetic profile of BVC, the BVC:HP-?-CD group showed slow release characteristics with low plasma levels, with no changes after the association to the SUF supporting. Altogether these results point toward a potential application for the pharmaceutical association of BVC:HP-?-CD with SUF, reducing the concentrations and the administration frequency required for analgesia effect in surgical procedure / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1(-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1(nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1(nmf164)/Npc1(nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1(nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-beta-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs. These findings indicate that in Npc1(nmf164) homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.

Raloxifene is a highly insoluble, highly metabolized serum estrogen receptor modulator approved for use in the treatment of osteoporosis. Hydroxybutenyl-beta-cyclodextrin (HBenBCD) is a novel solubility enhancer previously demonstrated to increase the oral bioavailability of tamoxifen, letrozole, and itraconazole. The current study evaluated the pharmacokinetics of raloxifene in oral and intravenous formulations with HBenBCD in male Wistar-Hannover rats. Analytical methodology to measure raloxifene and its metabolites was developed by measuring raloxifene metabolism in vitro. Formulation with HBenBCD significantly increased raloxifene oral bioavailability. Mean ± S.D. oral bioavailabilities were 2.6 ± 0.4% for raloxifene formulated with microcrystalline cellulose, 7.7 ± 2.1% for a solid capsule formulation of raloxifene:HBenBCD complex, and 5.7 ± 1.3% for a liquid-filled capsule formulation containing raloxifene:HBenBCD/PEG400/H2O. Relative to raloxifene/microcrystalline filled capsules, the presence of HBenBCD in the solid capsule formulation afforded: (i) a decrease in raloxifene Tmax (2.5 ± 0.5 h versus 4.0 ± 0.5 h); (ii) a two-fold increase in raloxifene Cmax and a three-fold increase in raloxifene AUC; and (iii) a 12-fold increase in raloxifene glucuronide Cmax and a 6.5-fold increase in raloxifene glucuronide AUC. Hence, these studies demonstrate that raloxifene formulations containing HBenBCD significantly increased the oral bioavailability in rats relative to formulations that did not contain HBenBCD.

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise -cyclodextrinmodified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116p53 /PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Active substances can be stabilized to be protected from undesirable reactions, aggregation, and leaking, which would keep the intended functions of the active substances without premature degradation. Among such active substances are paraffin-based organic phase change materials (PCMs) and essential oils (EOs), which feature attractive characteristics, e.g., high latent heat of fusion and inherent antimicrobial activity. However, their high volatility requires an effective stabilization strategy. Petroleum-based synthetic polymers have often been employed to stabilize PCMs and EOs by encapsulation and complexation pathways. Despite their proven effectiveness, these polymers are from non-renewable resources, and non-degradable and often toxic, which has prompted a need to develop a substitute arising from natural polymers that are environmentally benign, biodegradable, and sustainable. Valorization of biomass in this regard would add extra value to biomass otherwise burned or wasted. This dissertation will present the development of microcapsules and inclusion complexes consisting of a hydrophobic active substance core and a polysaccharidic shell originating from biomass. The first two chapters will explain the introduction and experimental details. Chapter 3 will present the microencapsulation of n-hexadecane as PCM via oil-in-water (O/W) Pickering emulsions stabilized by unmodified cellulose nanofibrils (CNFs) through a sonochemical technique. Chapter 4 will investigate the incorporation of the PCM-CNF microcapsules into TEMPO-oxidized CNF films for building application. Finally, Chapter 5 will show the synthesis of EOs-beta cyclodextrin (βCD) inclusion complexes as a guest-host system through a sonochemical technique.

Wood-based panel products (WBPs) are considered highly sustainable due to their carbon neutrality, low embodied energy, and ability to be produced from various low-grade wood species. However, WBPs exhibit limited resistance to biological degradation, which can deteriorate their mechanical performance and shorten their service life. Additionally, WBPs may not be fully compatible with traditional wood preservation chemicals. This dissertation demonstrated a facile method of synthesizing essential oil-based natural preservatives compatible with WBPs. Four essential oils (EOs), namely thymol (THY), trans-cinnamaldehyde (tCN), carvacrol (CRV), and eugenol (EUG), were complexed with beta-cyclodextrin (beta-CD) to form beta-CD-EOs inclusion complexes through a sonochemical process using a high-power ultrasonicator (750W). The sonochemical variables, such as a molar ratio between beta-CD and EO, ultrasonication power, and centrifugation speed and time, were optimized to improve the inclusion yield of EOs in the complex and facilitate the large-scale production of beta-CD-EOs. Attenuated Fourier transform infrared spectroscopy (AT-FTIR) and X-ray diffraction analysis confirmed the formation of inclusion complexes at the different production scales. The inclusion yield of EOs in the complexes was estimated by ultraviolet-visible spectrophotometry (UV-vis) as well as thermogravimetric analysis (TGA), which was capable of differentiating unreacted EOs and loosely encapsulated EO molecules from the estimation of the inclusion yield. Differential scanning calorimetry (DSC) revealed that beta-CD-tCN did not adversely affect the curing properties of polymeric methylene diisocyanate (pMDI) used as a glue for plywood. The influence of beta-CD-tCN addition on the bonding strength of the wood composite was assessed by a lap shear strength test using 3-ply SYP plywood, following ASTM D906-20. Statistical analysis using the Tukey test at p<=0.05 indicated no significant difference between the bonding strength of pMDI and beta-CD-tCN as an additive, indicating that beta-CD-tCN could be successfully implemented in the manufacturing process of wood composite products as natural preservatives.

An inclusion complex between β-cyclodextrin and insoluble guest compounds has been reported by several researchers. The main purpose of forming an inclusion complex between β-CD and sparingly soluble guests is to enhance the guest’s solubility and mask its undesirable properties. Preliminary studies have shown that when conjugated with target-specific moieties, these inclusion complexes can be used in pharmaceutical applications for drug delivery. β-Sitosterol, a plant sterol, has been well documented to reduce tumor cell growth and migration as well as exhibit apoptotic characteristics. An issue with this plant sterol and most pharmaceutical compounds is their lack of solubility. In this study, we propose that an inclusion complex will enhance the solubility of this sterol and change the physicochemical properties of the sparingly soluble guest. We first prepared the β-CD:β-Sitosterol inclusion complex and characterized the samples in both solid and solution state. The complex was characterized using FT-IR, DSC, SEM and NMR. IR studies of the inclusion complex and physical mixture revealed changes in the characteristic peaks of the inclusion complex suggestive of the formation of a new compound. 1HNMR studies revealed an upfield resonance shift of β-CD internal protons (H3 and H5) as an equal molar ratio of β-Sitosterol is introduced into the β-CD mixture. 2D NOESY NMR studies suggest that the initial sites of interaction of β-CD and β-Sitosterol occur between the aliphatic tail of β-Sitosterol and H3 of β-CD. 2D ROESY NMR reveals that the cyclic head of β-Sitosterol also interacts with the cavity of β-CD suggesting that β-Sitosterol may be completely encapsulated inside β-CD’s cavity. From these initial studies, we hypothesize that the β-CD-PEG-FA will facilitate absorption of β-Sitosterol and increase the drug delivery vehicle’s solubility as a whole. Since most tumor cells over-express folic acid, inclusion of folic acid in the construct of the vehicle will direct these sterols to tumor sites. β-cyclodextrin-PEG, a precursor to the bio-conjugate for antitumor delivery of sterols, was synthesized and characterized.

A controlled assembly of natural beta-cyclodextrin modified Au NPs mediated by dopamine is demonstrated. Furthermore, a simple and sensitive colorimetric detection for dopamine is established by the concentration-dependent assembly.

A controlled assembly of natural beta-cyclodextrin modified Au NPs mediated by dopamine is demonstrated. Furthermore, a simple and sensitive colorimetric detection for dopamine is established by the concentration-dependent assembly.

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