11 research outputs found
Prognostic and predictive value of TP53 mutations in node-positive breast cancer patients treated with anthracycline- or anthracycline/taxane-based adjuvant therapy : results from the BIG 02-98 phase III trial
No full textIntroduction: Pre-clinical data suggest p53-dependent anthracycline-induced apoptosis and p53-independent taxane activity. However, dedicated clinical research has not defined a predictive role for TP53 gene mutations. The aim of the current study was to retrospectively explore the prognosis and predictive values of TP53 somatic mutations in the BIG 02-98 randomized phase III trial in which women with node-positive breast cancer were treated with adjuvant doxorubicin-based chemotherapy with or without docetaxel. Methods: The prognostic and predictive values of TP53 were analyzed in tumor samples by gene sequencing within exons 5 to 8. Patients were classified according to p53 protein status predicted from TP53 gene sequence, as wild-type (no TP53 variation or TP53 variations which are predicted not to modify p53 protein sequence) or mutant (p53 nonsynonymous mutations). Mutations were subcategorized according to missense or truncating mutations. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Results: TP53 gene status was determined for 18% (520 of 2887) of the women enrolled in BIG 02-98. TP53 gene variations were found in 17% (90 of 520). Nonsynonymous p53 mutations, found in 16.3% (85 of 520), were associated with older age, ductal morphology, higher grade and hormone-receptor negativity. Of the nonsynonymous mutations, 12.3% (64 of 520) were missense and 3.6% were truncating (19 of 520). Only truncating mutations showed significant independent prognostic value, with an increased recurrence risk compared to patients with non-modified p53 protein (hazard ratio = 3.21, 95% confidence interval = 1.740 to 5.935, P = 0.0002). p53 status had no significant predictive value for response to docetaxel. Conclusions: p53 truncating mutations were uncommon but associated with poor prognosis. No significant predictive role for p53 status was detected
Avaliação de polimorfismos nos genes EGF e EGFR e a susceptibilidade à pré-eclâmpsia severa
No full textCerca de 10-15% das causas de mortalidade e morbidade materna em países desenvolvidos e 37% das causas de morte obstétricas diretas no Brasil podem ser associadas à pré-eclâmpsia. A pré-eclâmpsia é uma patologia multissistêmica definida por uma hipertensão associada a uma proteinúria, após a 20ª semana de gestação. As manifestações clínicas desta doença podem se apresentar como uma síndrome materna ou fetal e de acordo com a gravidade podem ser classificadas em leve ou severa e de início precoce ou tardio. Apesar do conhecimento limitado sobre esta patologia, existem fortes evidências de envolvimento do componente genético na etiologia da pré-eclâmpsia. O fator de crescimento epidérmico (EGF) desempenha um papel importante na regulação do crescimento, proliferação e diferenciação celular, através da ligação ao seu receptor, o EGFR. Acredita-se que este fator esteja relacionado com a regulação do crescimento e da função placentária durante a gestação. Variações na sequência do DNA desses genes podem levar a uma alteração nos níveis de transcrição gênica e, como consequência, ser responsável por mudanças nos níveis de produção e/ou atividade desses fatores. O polimorfismo EGF +61 G>A está associado com a produção in vitro da proteína EGF e os polimorfismos EGFR -216 G>T e -191 C>A estão correlacionados a mudanças na atividade do promotor e na expressão de RNAm desse gene. O objetivo geral do nosso estudo foi avaliar uma possível associação entre polimorfismos funcionais nos genes EGF (+61 G>A) e EGFR (-216 G>T e -191 C>A) e a susceptibilidade à pré-eclâmpsia severa na população de gestantes do Estado do Rio de Janeiro, através de um estudo caso-controle. Como objetivos específicos, além de analisarmos uma possível interação entre os polimorfismos no desenvolvimento da pré-eclâmpsia severa, buscamos associar os polimorfismos ao histórico familiar da doença. O estudo foi composto por dois grupos, pareados por etnia: um grupo caso composto por 98 mulheres com pré-eclâmpsia severa e um grupo controle com 98 mulheres saudáveis. Os polimorfismos EGF (+61 G>A) e EGFR (-216 G>T e -191 C>A) foram avaliados pela reação em cadeia da polimerase seguida por análise de polimorfismos por tamanho de fragmentos de restrição (PCR-RFLP). As variáveis categóricas, frequências alélicas e genotípicas foram comparadas através do teste do exato de Fisher, e o teste t de Student foi utilizado para comparação das variáveis contínuas em cada grupo. Os resultados demonstram que o alelo A do polimorfismo -191 do gene EGFR está associado com a susceptibilidade à pré-eclâmpsia severa (pA e EGFR -216 G>T) e a susceptibilidade à pré-eclâmpsia severa (p>0,05), assim como também não foi encontrada relação entre a interação dos polimorfismos, histórico familiar e o desenvolvimento da pré-eclâmpsia severa. Além desses resultados, também foram encontradas diferenças significativas ao avaliarmos as características demográficas e clínicas entre os grupos. Este é o primeiro estudo a avaliar associações entre pré-eclâmpsia severa e os polimorfismos -216 G>T e -191C>A do gene EGFR e o primeiro estudo na população brasileira a investigar a associação do polimorfismo EGF +61 G>A e a doença. Com esse achado, podemos sugerir que o polimorfismo, o -191C>A do gene EGFR, possa ser o responsável por alguma regulação na produção do EGFR, e que através dessa regulação possa desempenhar algum papel importante na susceptibilidade à pré-eclâmpsia severa em mulheres do Estado do Rio de Janeiro.Conselho Nacional de Desenvolvimento Científico e TecnológicoFundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de JaneiroCoordenação de Aperfeiçoamento de Pessoal de Nível SuperiorAbout 10-15% of maternal deaths in development countries and approximately 37% of direct obstetrics deaths in Brazil can be assigned to preeclampsia. Preeclampsia is a multisystem disorder that usually occurs after 20 week of pregnancy and it is determined by the presence of hypertension associated with proteinuria. The clinical findings of preeclampsia can manifest as either a maternal syndrome or fetal syndrome. In addition, the preeclampsia can be classified as mild to severe, and in early or late-onset preeclampsia. Despite the limited knowledge of this pathology, there is a strong evidence of involvement of the genetic component in the etiology of preeclampsia. The epidermal growth factor (EGF) plays an important role in regulating cell growth, proliferation and differentiation, through binding its receptor, EGFR. Evidences suggest that this growth factor and its receptor are involved in growth regulation of placental function during the pregnancy. Variations in the DNA sequence in the EGF and EGFR genes can lead to an altered gene transcription and consequently can be responsible for changes in production and/or activity of these factors. The EGF +61 G>A polymorphism is significantly associated with in-vitro EGF protein production and the EGFR -216 G>T and -191 C>A polymorphisms are correlated with changes in promoter activity and expression of EGFR mRNA. The aim of this study was to verify the association between EGF +61 G>A, EGFR -216 G>T and -191 C>A polymorphisms and susceptibility to severe preeclampsia in the population of Rio de Janeiro through a case-control design. The specific objectives were to assess the association between these polymorphisms and the history family of preeclampsia, and also to analyze a possible interaction among these polymorphisms on the development of severe preeclampsia. The study was composed by two groups matched by ethnicity: the case group with 98 women with severe preeclampsia and the control group with 98 healthy women. Polymerase chain reaction restriction fragment length polymorphism analyses (PCR-RFLP) were performed to genotype EGF +61 G>A, EGFR -216 G>T and -191 C>A polymorphisms. Categorical variables, allelic and genotype frequencies were compared in each group applying Fisher´s exact test and a Student t test was used for continuous variables. The results showed that the A allele of the -191 C>A polymorphism of the EGFR gene is associated with susceptibility to severe preeclampsia (PA EGF and -216 G>T EGFR polymorphisms (P>0,05), as well as no correlation was found between the interaction of these polymorphisms, history family and the development of severe preeclampsia. We also found differences when we evaluated demographic and clinical characteristics between the two groups. This is the first study to assess the associations between -191 C>A and -216 G>T EGFR genetics polymorphisms and severe preeclampsia and the first study in Brazilian population to investigate the association between +61 G>A EGF polymorphism and severe preeclampsia. These findings suggest that the polymorphism-191C>A of the EGFR gene may be responsible for some regulation in the production of the EGFR, and that through this regulation this polymorphism might play an important role in the susceptibility to severe preeclampsia in women from Rio de Janeiro
Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR
No full textOBJECTIVE: The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature. MATERIAL AND METHODS: A group of volunteers was recruited to collect buccal cells using a mouthwash solution. The collected solution was divided into 3 tubes, one tube were used for immediate extraction and the remaining received ethanol and were kept at room temperature for 4 and 8 days followed by dna extraction. The concentration, purity and integrity of the dna were determined using spectrophotometry and electrophoresis. DNA quality differences among the three incubation times were also evaluated for genotyping EGF +61 a/g (rs 4444903) polymorphism by PCR-RFLP and for IRF6 polymorphism (rs 17015215) using real-time PCR. RESULTS: There was no significant difference of dna yield (p=0.75) and purity (p=0.86) among the three different incubation times. DNA obtained from different incubation times presented high-molecular weight. The PCR-RFLP and real time pcr reactions were successfully performed for all DNA samples, even those extracted after 8 days of incubation. All samples genotyped by real-time pcr presented c allele for irf6 gene polymorphism (homozygous: cc; heterozygous: Ct) and the C allele was used as a reference for Ct values. The samples presented the same genotype for the different times in both techniques. CONCLUSION: We demonstrated that the method described herein is simple and low cost, and that DNA can be extracted and pcr amplified after storage in mouthwash solution at room temperature
MMP13 polymorphism decreases risk for dental caries.
No full textRecent evidence suggests that genetic studies may contribute to a better understanding of individual susceptibility to caries. Matrix metalloproteinases (MMPs) and their tissue inhibitors have been suggested to be involved in the caries process. The purpose of this study was to determine if polymorphisms in MMP2 (rs243865), MMP9 (rs17576), MMP13 (rs2252070), and TIMP2 (rs7501477) were associated with caries. Eligible unrelated children and adolescents were evaluated using a cross-sectional design. Data on oral health habits was obtained through a questionnaire and caries data was collected by clinical examination. Genotyping of the selected polymorphisms was carried out by real-time PCR. Allele and genotype frequencies were compared between individuals with and without caries experience. Of 505 subjects, 212 were caries-free and most subjects (61.2%) had mixed dentition. Allele frequency of MMP2, MMP13 and TIMP2 was different between caries-affected and caries-free individuals, with significant association for MMP13 (p = 0.004). Mutant allele carriers for MMP13 demonstrated a significantly decreased risk for caries (OR = 0.538, 95% CI 0.313-0.926); this result remained significant after adjustment for candidate genes, type of dentition and dietary factors. Allelic and genotype frequencies of the polymorphism in MMP9 were similar in caries-affected and caries-free individuals. Genetic variations in MMP13 may contribute to individual differences in caries susceptibility. Our findings reinforce that susceptibility to caries results from gene-environment interactions.\ud
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The functional EGF+61 polymorphism and nonsyndromic oral clefts susceptibility in a Brazilian population
No full textAbstractNonsyndromic oral clefts are considered a problem of public health in Brazil, presenting a multifactorial etiology that involves genetic and environmental components, such as maternal alcohol consumption. Several candidate genes have been investigated to identify some association with nonsyndromic clefts risk. The epidermal growth factor (EGF) gene is implicated in the normal craniofacial development and its functional +61 A>G polymorphism has been related to cancer susceptibility. It has been suggested that cancer and oral clefts may share the same molecular pathways.Objective Our goal was to evaluate the association between the EGF+61 A>G polymorphism and nonsyndromic oral clefts susceptibility.Material and Methods The case-control study included 218 cleft cases and 253 controls from Brazil. The control group was comprised of individuals without congenital malformations, dental anomalies and family history of clefts. The cleft phenotypes and subphenotypes were determined based on clinical examination. Genomic DNA was extracted from oral mucosa cells obtained by mouthwash. The EGF+61 A>G polymorphism genotype was determined by polymerase chain reaction-restriction fragment length polymorphism.Results We noticed the association between maternal alcohol consumption during pregnancy and cleft occurrence. The A allele and AA genotype were over-represented in cleft cases compared with control group when we considered the bilateral cleft lip with or without cleft palate (CL±P) cases, cleft cases with tooth agenesis and cleft cases presenting family history of cleft, but the differences were not statistically significant. Contradictorily, the G allele was higher in cleft palate only (CP) cases than in control group, showing a borderline p value. Comparing the different cleft phenotypes, we observed statistical differences between CP and CL±P cases. Our data suggest the EGF+61 A>G polymorphism was not related with nonsyndromic oral clefts susceptibility in a Brazilian population, but supported the different genetic background between CL±P and CP. Moreover, we confirmed the potential effect of maternal alcohol intake on cleft risk in our population
Challenges on the toxicological predictions of engineered nanoparticles
No full textThe perceived enormous potential of nanotechnology in contributing to sustainable innovation has led to the growth of investments into new industrial applications and consumer products. However, the lack of tools that are needed to generate early knowledge about the potential adverse effects, combined with the uncertainties regarding the health and safety risks of engineered nanoparticles (ENPs), are a potential threat to the acceptability by society of the nanotechnology innovations, due to the rising societal concerns that are based on generic worries. In order to tackle these issues, it has been necessary to adopt a more proactive approach into nanotechnology safety assessments. Multiple projects have been initiated around the world in order to understand how ENPs interact with living organisms, but the validation of most of the emerging knowledge may take years. This is while robust risk assessment results are urgently needed, in order to support timely regulatory decisions and risk management actions. The goal of this paper has been to review the present knowledge on the physicochemical characteristics of ENPs, focusing on titanium dioxide (TiO2), gold (Au), copper oxide (CuO), and zinc oxide (ZnO), as well as on their biological interactions. In addition, the paper has been aimed at the identification of the main challenges on the current toxicological characterisation of these ENPs. Focus will also be given in this article to those ENPs that have been described by the Consumer Product Inventory as having prevalent nanomaterials present in consumer products, but also, with those having therapeutic and diagnostic applications, due to their physical (ex: confined plasmon resonances) and biological (biocompatibility and antimicrobial) properties.Bundesministerium für Bildung und FrauenFundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Postgraduate Program on Translational Biomedicine Grande Rio University, Rua Professor José de Souza Herdy, 1216 - Jardim Vinte e Cinco de AgostoBrazilian Branch of the Institute of Biotribocorrosion and Nanomedicine (IBTN-Br)Laboratory of Bioengineering and In Vitro Toxicology Directory of Metrology Applied to Life Sciences (Dimav) National Institute of Metrology Quality and Technology (INMETRO)European Centre for the Sustainable Impact of Nanotechnology ECSIN LABNational Research Centre for the Working Environment, Lerso Parkallé 105Physics Department São Paulo State University – UNESP, Av. Eng. Luiz Edmundo Carrijo CoubeHelmholtz-Centre for Environmental Research –UFZ, Permoserstraße 15University Ca' Foscari Venice, Dorsoduro, 3246Fluminense Federal University Dental School, R. Miguel de Frias, 9 - IcaraíLaboratory of Ultrastructure and Cellular Biology Hertha Meyer Institute of Biophysics Carlos Chagas Filho Federal University of Rio de JaneiroDepartment of Chemistry University of Illinois at Urbana-ChampaignPhysics Department São Paulo State University – UNESP, Av. Eng. Luiz Edmundo Carrijo CoubeBundesministerium für Bildung und Frauen: 03X013
Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel
No full textThe scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p<0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p<0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost)
Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications
No full textTitanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação para a Ciência e a Tecnologia (FCT)Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Diagnóstico e Cirurgia, Faculdade de Odontologia de Araraquara, Araraquara, RUA HUMAITÁ Nº 1680, CENTRO, CEP 14801903, SP, BrasilDepartment of Periodontology, Araraquara Dental School, University Estadual Paulista, Rua Humaitá 1680, 14801-903 Araraquara, São Paulo, BrazilDirectory of Metrology Applied to Life Science, National Institute of Metrology Quality and Technology, Av. N. S. das Graças 50, Xerém, Duque de Caxias, Rio de Janeiro, BrazilBrazilian Branch of Institute of Biomaterials, Tribocorrosion and Nanomedicine (IBTN/Br), BrazilCentre for Mechanical and Materials Technologies, University of Minho, Campus de Azurém, 4800-058 Guimarães, PortugalClinical Research Unit, Antonio Pedro Hospital, Fluminense Federal University, Niterói, BrazilPhysics Department, Federal University of Juiz de Fora (UFJF), Rua José Lourenço Kelmer, 36036-900 Minas Gerais, BrazilMetrology Materials Division, National Institute of Metrology Quality and Technology, Av. N. S. das Graças 50, Xerém, Duque de Caxias, Rio de Janeiro, BrazilDental School, Fluminense Federal University, Niterói, BrazilUniversidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Diagnóstico e Cirurgia, Faculdade de Odontologia de Araraquara, Araraquara, RUA HUMAITÁ Nº 1680, CENTRO, CEP 14801903, SP, BrasilDepartment of Periodontology, Araraquara Dental School, University Estadual Paulista, Rua Humaitá 1680, 14801-903 Araraquara, São Paulo, BrazilDepartamento de Química e Bioquímica, Universidade Estadual Paulista — UNESP, Distrito de Rubião Junior S/N, 18618-970 Botucatu, São Paulo, BrazilDepartamento de Física, Universidade Estadual Paulista — UNESP, Av. Eng. Luiz Edmundo Carrijo Coube, 14-01 Bauru, São Paulo, BrazilFAPESP: 2012/03633-1CNPq: 490761/2013-5CNPq/PROMETRO: 563165/2010-3FSFRH/BD: 70857/2010PTDC/CTM: 68160/2006PTDC/CTM: 67500/2006CAPES/PVE: 1317/11-
Investigating the impact of gold nanoparticles on cells: from transcription to behavior
No full textGold nanoparticles (Au NPs) are being research extensively for various biomedical applications; their applicability arises from a unique set of optical and physical properties brought on by their nanoscale dimensions.1 Furthermore, facile and scalable synthetic and surface functionalization strategies for Au NPs make these properties highly tunable.1 These areas of research are still relatively new and the number of publications per year referring to gold nanomaterials has skyrocketed from 11 in 1990, to 673 in 2000, to more than 31,400 in 2015 (Web of Science database, topic search = “gold nano*”, accessed March 2, 2016). The potential application of Au NPs for disease detection, diagnosis and therapy has motivated numerous analyses of their interactions with biomolecules, cells, animals, humans and the environment.1,2 A vast majority of studies aimed at gaining a better understanding of how cells interact with and are influenced by Au NPs have focused mainly on measuring cytotoxicity and simple cell processes like proliferation or NP uptake. While Au NPs larger than 4-5 nm in diameter (with appropriate, non-toxic surface coatings) have been shown to be largely non-cytotoxic, there can be subtle non-toxic effects induced by Au NPs.3 The adsorption of soluble proteins onto NP surfaces (the protein corona) is highly studied, but little attention has been paid to how those interactions perturb gene expression of cells or to understanding NP interactions with other types of biomolecules. This thesis aims to look deeper into how molecular level effects of NPs in cells and cellular environments can lead to down-stream changes to cell gene expression and behavior.
Firstly, the impact of Au nanorods (Au NRs) on 3D cancer cell migration via interactions between Au NRs and extracellular matrix (ECM) structural proteins was examined.4 While experiments on the influence of NPs on cell behaviors exist, nearly all of these studies neglect the impact of the ECM. In vivo cells exist in complex, fibrous 3D environments and series of intricate biochemical, cell-cell and cell-ECM interactions govern behaviors such as migration. Cancer cell migration allows tumor cells to spread and metastasize to new areas of the body, but little is known about how Au NR interaction with the ECM after injection and targeting to tumors may affect this process. The inevitable contact of in vivo Au NRs with the ECM presents a possible source of unintended side effects. In order to study how gold nanoparticles can influence ECM properties and cell-ECM interactions, we have created a nested-gel type I collagen matrix for measuring whether Au NRs alter the migration of MDA-MB-231 human breast cancer cells in 3D collagen environments. In contrast to the few studies of Au NR-induced migration changes in 2D environments, our results show that Au NRs in a model ECM increase the frequency of spontaneous cellular migration. The presence of negatively-charged polyelectrolyte-coated Au NRs during the collagen self-assembly process was shown to induce mechanical and structural changes, to alter molecular diffusion, and to affect cellular adhesion, morphology, locomotion strategy and protease expression. The results demonstrate the indirect impact nanoparticles can exert on cell behaviors within three-dimensional ECMs.
The shape and surface chemistry of Au NPs was also investigated in terms of the role of these factors in cellular transcriptomic (gene expression) responses after both short- and long-term exposures.5 Respectively, human dermal fibroblasts (HDF) and prostate cancer (PC3) cells were exposed to 0.1 nM (24 hours) and 1.0 nM (48 hours) concentrations of Au NPs of four different, but related surface chemistries. A combination of microarray gene expression analysis techniques and typical cellular characterization was used to learn more about how the nature of the Au NP surface coating influences cells on a molecular level. Identity, charge and lability of surface coatings (and cell type and dosing parameters) were all implicated as important factors to consider in order to predict gene expression responses. In a separate study, HDF cells were exposed to 0.1 nM (low-dose) Au NPs of different shapes and surface coatings for 20 weeks. The long-term effects of acute (24 hour) and chronic (20 weeks) exposure were measured by viability, proliferation, NP uptake, and morphology studies combined with gene expression analysis of genes related to stress and toxicity pathways. It is rare to find chronic exposure studies, especially with Au NPs, and these experiments showed that acute, sub-cytotoxic doses of NPs may induce long-term stress on cells. These cells were found to react very differently to acute versus chronic doses of the same NPs after 20 weeks. Additionally, surface coating was shown to have a much larger impact on determining NP-cell interactions than shape of Au NPs. In all, we have expanded the collective understanding of the molecular interactions Au NPs experience inside cells based on surface chemistry, shape, dosage and exposure time and parameters.
References
1. Dreaden, E.C.; Alkilany, A.M.; Huang, X.; Murphy, C.J.; El-Sayed, M.A. The Golden Age: Gold Nanoparticles for Biomedicine. Chem. Soc. Rev. 2012, 41, 2740–2779.
2. Yang, X.; Yang, M.; Pang, B.; Vara, M.; Xia, Y. Gold Nanomaterials at Work in Biomedicine. Chem. Rev. 2015, 115, 10410–10488.
3. Alkilany, A.M.; Lohse, S.E.; Murphy, C.J. The Gold Standard: Gold Nanoparticle Libraries to Understand the Nano-Bio Interface. Acc. Chem. Res. 2013, 46, 650–661.
4. Grzincic, E.M.; Murphy, C.J. Gold Nanorods Indirectly Promote Migration of Metastatic Human Breast Cancer Cells in Three-Dimensional Cultures. ACS Nano 2015, 9, 6801–6816.
5. Grzincic, E.M.; Yang, J.A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C.J. Global Transcriptomic Analysis of Model Human Cell Lines Exposed to Surface-Modified Gold Nanoparticles: The Effect of Surface Chemistry. Nanoscale 2015, 7, 1349–1362.Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2018-05-01The student, Elissa Grzincic, accepted the attached license on 2016-04-14 at 19:38.The student, Elissa Grzincic, submitted this Dissertation for approval on 2016-04-14 at 19:46.This Dissertation was approved for publication on 2016-04-19 at 08:16.DSpace SAF Submission Ingestion Package generated from Vireo submission #9234 on 2016-07-07 at 14:16:45Made available in DSpace on 2016-07-07T21:14:44Z (GMT). No. of bitstreams: 3
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Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts
No full textBackground: Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce.Methods: We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits.Results: Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts.Conclusions: These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies
