Prostate cancer radiotherapy: potential applications of metal nanoparticles for imaging and therapy

Prostate cancer (CaP) is the most commonly diagnosed cancer in males. There have been dramatic technical advances in radiotherapy delivery, enabling higher doses of radiotherapy to primary cancer, involved lymph nodes and oligometastases with acceptable normal tissue toxicity. Despite this, many patients relapse following primary radical therapy, and novel treatment approaches are required. Metal nanoparticles are agents that promise to improve diagnostic imaging and image-guided radiotherapy and to selectively enhance radiotherapy effectiveness in CaP. We summarize current radiotherapy treatment approaches for CaP and consider pre-clinical and clinical evidence for metal nanoparticles in this condition.<br/><br/>Prostate cancer (CaP) is the most commonly diagnosed cancer in males and is responsible for more than 10,000 deaths each year in the UK.1 Technical advances in radiotherapy delivery, including image-guided intensity-modulated radiotherapy (IG-IMRT), have enabled the delivery of higher radiation dose to the prostate, which has led to improved biochemical control. Further improvements in cancer imaging during radiotherapy are being developed with the advent of MRI simulators and MRI linear accelerators.2���4<br/><br/>Nanotechnology promises to deliver significant advancements across numerous disciplines.5 The widest scope of applications are from the biomedical field including exogenous gene/drug delivery systems, advanced biosensors, targeted contrast agents for diagnostic applications and as direct therapeutic agents used in combination with existing treatment modalities.6���11 This diversity of application is especially evident within cancer research, with a myriad of experimental anticancer strategies currently under investigation.<br/><br/>This review will focus specifically on the potential of metal-based nanoparticles to augment the efficacy of radiotherapy in CaP, a disease where radiotherapy constitutes a major curative treatment modality.12 Furthermore, we will also address the clinical state of the art for CaP radiotherapy and consider how these treatments could be best combined with nanotherapeutics to improve cancer outcomes.

Multifunctional nanoparticles for MR and fluorescence imaging

In the past few years a new generation of multifunctional nanoparticles (NPs) has been proposed for biomedical applications, whose structure is more complex than the structure of their predecessor monofunctional counterparts. The development of these novel NPs aims at enabling or improving the performance in imaging, diagnosis and therapeutic applications. The structure of such NPs comprises several components exhibiting various functionalities that enable the nanoparticles to perform multiple tasks simultaneously, such as active targeting of certain cells or compartmentalization, imaging and delivery of active drugs. This thesis presents two types of bimodal bio-imaging probes and describes their physical and chemical properties, namely their texture, structure, and 1H dynamics and relaxometry, in order to evaluate their potential as MRI contrast agents. The photoluminescence properties of these probes are studied, aiming at assessing their interest as optical contrast agents. These materials combine the properties of the trivalent lanthanide (Ln3+) complexes and nanoparticles, offering an excellent solution for bimodal imaging. The designed T1- type contrast agent are SiO2@APS/DTPA:Gd:Ln or SiO2@APS/PMN:Gd:Ln (Ln= Eu or Tb) systems, bearing the active magnetic center (Gd3+) and the optically-active ions (Eu3+ and Tb3+) on the surface of silica NPs. Concerning the relaxometry properties, moderate r1 increases and significant r2 increases are observed in the NPs presence, especially at high magnetic fields, due to susceptibility effects on r2. The Eu3+ ions reside in a single low-symmetry site, and the photoluminescence emission is not influenced by the simultaneous presence of Gd3+ and Eu3+. The presence of Tb3+, rather than Eu3+ ion, further increases r1 but decreases r2. The uptake of these NPs by living cells is fast and results in an intensity increase in the T1-weighted MRI images. The optical features of the NPs in cellular pellets are also studied and confirm the potential of these new nanoprobes as bimodal imaging agents. This thesis further reports on a T2 contrast agent consisting of core-shell NPs with a silica shell surrounding an iron oxide core. The thickness of this silica shell has a significant impact on the r2 and r2* relaxivities, and a tentative model is proposed to explain this finding. The cell viability and the mitochondrial dehydrogenase expression given by the microglial cells are also evaluated.

Nonaqueous sol-gel routes to doped metal oxide nanoparticles: synthesis, characterization, assembly and properties

The results presented in this thesis have been achieved under the Ph.D. project entitled ���Nonaqueous Sol-Gel routes to doped metal oxide nanoparticles: Synthesis, characterization, assembly and properties���. The purpose of this study is the investigation of metal oxide nanostructures doped with metals of a diverse nature, leading to different type of applications. The easier control over the reaction kinetics in solvothermal routes, compared to aqueous methods, allows to better match the reactivity between metal oxide precursors, paving the way to a facile and low temperature production of doped oxides. In this manuscript diverse examples of the exploitation of the ���Benzyl Alcohol Route��� are discussed. Such a powerful pathway was utilized for the synthesis of transition metal doped zirconia, hafnia and various perovskites, and the study of their magnetic properties, as well as the synthesis of rare earth doped zirconium oxide. A further extension, proving the solidity of the synthetic method, is shown for the preparation of Li4Ti5O12 nanocrystals carrying excellent electrochemical properties for lithium-ion battery applications. Finally, the effect of doping and other reaction parameters on the assembly of the nanocrystals is discussed. These studies were carried out principally at the University of Aveiro, as well as at the University of Montpellier II and at the Seoul National University due to complementary available expertises and equipments.

Synthesis and functionalization of hydroxyapatite nanoparticles

Gra��as aos desenvolvimentos na ��rea da s��ntese de nanomaterais e ��s potentes t��cnicas de caracteriza����o �� nanoescala conseguimos hoje visualizar uma nanopart��cula (NP) como um dispositivo de elevado potencial terap��utico. A melhoria da sua efectividade terap��utica requer no entanto o aprofundamento e sistematiza����o de conhecimentos, ainda muito incipientes, sobre toxicidade, selectividade, efeitos colaterais e sua depend��ncia das pr��prias caracter��sticas f��sico-qu��micas da NP em an��lise. O presente trabalho, elegendo como alvo de estudo uma subst��ncia considerada biocompat��vel e n��o t��xica, a hidroxiapatite (Hap), pretende dar um contributo para esta ��rea do conhecimento. Definiram-se como metas orientadoras deste trabalho (i) estudar a s��ntese de nanoparticulas de Hap (Hap NP), e a modifica����o das caracter��sticas f��sico-qu��micas e morfol��gicas das mesmas atrav��s da manipula����o das condi����es de s��ntese; (ii) estudar a funcionaliza����o das Hap NP com nanoestruturas de ouro e com ��cido f��lico, para lhes conferir capacidades acrescidas de imagiologia e terap��uticas, particularmente interessantes em aplica����es como o tratamento do cancro (iii) estudar a resposta celular a materiais nanom��tricos, com propriedades f��sico-qu��micas diversificadas. No que se refere �� s��ntese de Hap NP, comparam-se dois m��todos de s��ntese qu��mica distintos, a precipita����o qu��mica a temperatura fisiol��gica (WCS) e a s��ntese hidrot��rmica (HS), em meios aditivados com i��o citrato. A s��ntese WCS originou part��culas de tamanho nanom��trico, com uma morfologia de agulha, pouco cristalinas e elevada ��rea superficial especifica. A s��ntese HS �� temperatura de 180��C permitiu obter part��culas de dimens��es tamb��m nanom��tricas mas com ��rea espec��fica inferior, com morfologia de bastonete prism��tico com sec����o recta hexagonal e elevada cristalinidade. Com o objectivo de aprofundar o papel de algumas vari��veis experimentais na defini����o das caracter��sticas finais das part��culas de hidroxiapatite, designadamente o papel do i��o citrato (Cit), variou-se a raz��o molar [Cit/Ca] da solu����o reagente e o tempo de s��ntese. Demonstrou-se que o i��o citrato e outras esp��cies qu��micas resultantes da sua decomposi����o nas condi����es t��rmicas (180��C) de s��ntese tem um papel preponderante na velocidade de nuclea����o e de crescimento dessas mesmas part��culas e por conseguinte nas caracter��sticas f��sico-qu��micas das mesmas. Elevadas raz��es [Cit/Ca] originam part��culas de dimens��o microm��trica cuja morfologia �� discutida no contexto do crescimento com agrega����o. Com o objectivo de avaliar a citotoxicidade in vitro das nanopart��culas sintetizadas procedeu-se �� esteriliza����o das mesmas. O m��todo de esteriliza����o escolhido foi a autoclavagem a 121�� C. Avaliou-se o impacto do processo de esteriliza����o nas caracter��sticas das part��culas, verificando-se contrariamente ��s part��culas WCS, que as part��culas HS n��o sofrem altera����es significativas de morfologia, o que se coaduna com as condi����es de s��ntese das mesmas, que s��o mais severas do que as de esteriliza����o. As part��culas WCS sofrem processos de dissolu����o e recristaliza����o que se reflectem em altera����es significativas de morfologia. Este estudo demonstrou que a etapa de esteriliza����o de nanopart��culas para aplica����es biom��dicas, por autoclavagem, pode alterar substancialmente as propriedades das mesmas, sendo pois criticamente importante caracterizar os materiais ap��s esteriliza����o. Os estudos citotoxicol��gicos para dois tipos de part��culas esterilizadas (HSster e WCSster) revelaram que ambas apresentam baixa toxicidade e possuem potencial para a modela����o do comportamento de c��lulas osteobl��sticas. Tendo em vista a funcionaliza����o da superf��cie das Hap NP para multifun����es de diagn��stico e terapia exploraram-se condi����es experimentais que viabilizassem o acoplamento de nanopart��culas de ouro �� superf��cie das nanopart��culas de Hidroxiapatite (Hap-AuNP). Tirando partido da presen��a de grupos carbox��licos adsorvidos na superf��cie das nanopart��culas de Hap foi poss��vel precipitar part��culas nanom��tricas de ouro (1,5 a 2,5 nm) na superf��cie das mesmas adaptando o m��todo descrito por Turkevich. No presente trabalho as nanopart��culas de Hap funcionaram assim como um template redutor do ouro i��nico de solu����o, propiciando localmente, na superf��cie das pr��prias nanopart��culas de Hap, a sua redu����o a ouro met��lico. A nuclea����o do ouro �� assim contextualizada pelo papel redutor das esp��cies qu��micas adsorvidas, designadamente os grupos carbox��licos derivados de grupos citratos que presidiram �� s��ntese das pr��prias nanopart��culas de Hap. Estudou-se tamb��m a funcionaliza����o das Hap NP com ��cido f��lico (FA), uma mol��cula biologicamente interessante por ser de f��cil reconhecimento pelos receptores existentes em c��lulas cancer��genas. Os resultados confirmaram a liga����o do ��cido f��lico �� superf��cie das diferentes part��culas produzidas HS e Hap-AuNPs. Gra��as ��s propriedades ��pticas do ouro nanom��trico (efeito plasm��o) avaliadas por espectroscopia vis-UV e ��s potencialidades de hipertermia local por convers��o fotot��rmica, as nanoestruturas Hap-AuNPs produzidas apresentam-se com elevado interesse enquanto nanodispositivos capazes de integrar fun����es de quimio e terapia t��rmica do cancro e imagiologia. O estudo da resposta celular aos diversos materiais sintetizados no presente trabalho foi alvo de an��lise na tentativa de se caracterizar a toxicidade dos mesmos bem como avaliar o seu desempenho em aplica����es terap��uticas. Demonstrou-se que as Hap NP n��o afectam a prolifera����o das c��lulas para concentra����es at�� 500 ���g/ml, observando-se um aumento na express��o gen��tica da BMP-2 e da fosfatase alcalina. Verificou-se tamb��m que as Hap NP s��o suscept��veis de internaliza����o por c��lulas osteobl��sticas MG63, apresentando uma velocidade de dissolu����o intracelular relativamente reduzida. A resposta celular ��s Hap-AuNP confirmou a n��o citotoxicidade destas part��culas e revelou que a presen��a do ouro na superf��cie das Hap NP aumenta a taxa prolifera����o celular, bem como a express��o de par��metros osteog��nicos. No seu conjunto os resultados sugerem que os v��rios tipos de part��culas sintetizadas no presente estudo apresentam tamb��m comportamentos interessantes para aplica����es em engenharia de tecido ��sseo.

Lanthanide oxide and phosphate nanoparticles for thermometry and bimodal imaging

Nesta tese relatam-se estudos de fotoluminesc��ncia de nanopart��culas de ��xidos e fosfatos dopados com i��es trivalentes de lantan��deos, respectivamente, nanobastonetes de (Gd,Eu)2O3 e (Gd,Yb,Er)2O3 e nanocristais de (Gd,Yb,Tb)PO4, demonstrando-se tamb��m aplica����es destes materiais em revestimentos inteligentes, sensores de temperatura e bioimagem. Estuda-se a transfer��ncia de energia entre os s��tios de Eu3+ C2 e S6 dos nanobastonetes Gd2O3. A contribui����o dos mecanismos de transfer��ncia de energia entre s��tios para o tempo de subida 5D0(C2) �� descartada a favor da relaxa����o directa 5D1(C2) 5D0(C2) (i.e., transfer��ncia de energia entre n��veis). O maior tempo de decaimento do n��vel 5D0(C2) nos nanobastonetes, relativamente ao valor medido para o mesmo material na forma de microcristais, �� atribu��do, quer �� exist��ncia de espa��os livres entre nanobastonetes pr��ximos (factor de enchimento ou frac����o vol��mica), quer �� varia����o do ��ndice de refrac����o efectivo do meio em torno dos i��es Eu3+. A dispers��o de nanobastonetes de (Gd,Eu)2O3 em tr��s resinas epoxi comerciais atrav��s da cura por UV permite obter nanocomp��sitos epoxi- (Gd,Eu)2O3. Relatam-se estudos cin��ticos e das propriedades t��rmicas e de fotoluminesc��ncia destes nanocomp��sitos. Estes, preservam as t��picas propriedades de emiss��o do Eu3+, mostrando o potencial do m��todo de cura por UV para obter revistimentos inteligentes e fotoactivos. Considera-se um avan��o significativo a realiza����o de uma nanoplataforma ��ptica, incorporando aquecedor e term��metro e capaz de medir uma ampla gama de temperaturas (300-2000 K) �� escala nano, baseada em nanobastonetes de (Gd,Yb,Er)2O3 (term��metros) cuja superf��cie se encontra revestida com nanopart��culas de ouro. A temperature local �� calculada usando, quer a distribui����o de Boltzmann (300-1050 K) do r��cio de intensidades da convers��o ascendente 2H11=2!4I15=2/4S3=2!4I15=2, quer a lei de Planck (1200-2000 K) para uma emiss��o de luz branca atribu��da �� radia����o do corpo negro. Finalmente, estudam-se as propriedades de fotoluminesc��ncia correspondentes ��s convers��es ascendente e descendente de energia em nanocristais de (Gd,Yb,Tb)PO4 sintetizados por via hidrot��rmica. A relaxividade (resson��ncia magn��tica) do 1H destes materiais s��o investigadas, tendo em vista poss��veis aplica����es em imagem bimodal (luminesc��ncia e resson��ncia magn��tica nuclear).

Silver nanoparticles flow in an aquatic trophic chain

Silver nanoparticles (AgNP) have been produced and applied in a variety of products ranging from personal care products to food package containers, clothing and medicine utilities. The antimicrobial function of AgNP makes it very useful to be applied for such purposes. Silver (Ag) is a non-essential metal for organisms, and it has been historically present in the environment at low concentrations. Those concentrations of silver increased in the last century due to the use of Ag in the photographic industry and lately are expected to increase due to the use of AgNPs in consumer products. The presence of AgNP in the aquatic environment may pose a risk for aquatic species, and the effects can vary from lethal to sublethal effects. Moreover, the contact of aquatic organisms with AgNP may not cause immediately the death of individuals but it can be accumulated inside the animals and consequently transferred within the food chain. Considering this, the objective of this work was to study the transfer of silver nanoparticles in comparison to silver ions, which was used as silver nitrate, within an aquatic food chain model. To achieve this goal, this study was divided into four steps: the toxicity assessment of AgNP and AgNO3 to aquatic test-species, the bioaccumulation assessment of AgNP and AgNO3 by Pseudokirchneriella subcapitata and Daphnia magna under different exposure scenarios, and finally the evaluation of the trophic transfer of Ag through an experimental design that included the goldfish Carassius auratus in a model trophic chain in which all the species were exposed to the worse-case scenario. We observed that the bioconcentration of Ag by P. subcapitata is mainly driven by ionic silver, and that algae cannot internalize these AgNPs, but it does internalizes dissolved Ag. Daphnia magna was exposed to AgNP and AgNO3 through different exposure routes: water, food and both water and food. The worse-case scenario for Daphnia Ag bioaccumulation was by the joint exposure of contaminated water and food, showing that Ag body burdens were higher for AgNPs than for AgNO3. Finally, by exposing C. auratus for 10 days through contaminated water and food (supplied as D. magna), with another 7 days of depuration phase, it was concluded that the 10 days of exposure were not enough for fish to reach a plateau on Ag internal concentration, and neither the 7 days of elimination were sufficient to cause total depuration of the accumulated Ag. Moreover, a higher concentration of Ag was found in the intestine of fish when compared with other organs, and the elimination rate constant of AgNP in the intestine was very low. Although a potential for trophic transfer of AgNP cannot be suggested based in the data acquired in this study, there is still a potential environmental risk for aquatic species.

Effect assessment of nanoparticles toxicity in the terrestrial compartment

Over 11 million tons of nanomaterials (NMs) have been produced in 2012 and predictions point the increase in production. Despite predictions and extended usage via consumer products and industry, the understanding of the potential impact of these materials on the environment is virtually absent. The main aim of this thesis is to understand how a selected group of nanomaterials (metal based particles) may impact soil invertebrates, with special focus on the mechanisms of response. Since a case-by-case Environmental Risk Assessment (ERA) of all the emerging contaminants (particularly NMs) is impossible, among others due to time and cost reasons, to gain understanding on the mechanism of action and response is very important to reach a common paradigm. Understanding the modes of action provides predictive characters in cross particle extrapolation. Besides, it also provides insight for the production of new and sustainable materials. Overall, the effects of the selected NMs (Copper and Silver, Titanium and Zirconium oxides) and the respective salt forms, were investigated at the gene expression (using high-throughput tools, microarray and qPCR technology), biochemical (using enzymatic assays for analysis of oxidative stress markers) and organism (survival and reproduction as in OECD test guidelines) levels, this using standard soil species (Enchytraeus albidus, Enchytraeus crypticus, Eisenia fetida). Gene expression analysis provided valuable information on the mechanisms affected by each of the NMs. The gene expression profile highlighted a (nano)material signature and the effect of the duration of exposure. The functional analyses integrated with the biochemical and organism data, revealed a good understanding power. The biochemical parameters (oxidative stress related) were distinct across the materials and also influenced by duration of exposure and concentration. The standardized organismal responses differed the least between the various materials. The overall outcome is that, in this context of NMs effect assessment, gene expression and enzymatic assays introduced a very important knowledge gap, which could not had been achieved by the standard organismal effects alone. A reoccurring issue with some metal based NMs is the possible dissolution and subsequent release of ions that then causes toxicity e.g. Cu-NPs or Ag-NPs release Cu2+ or Ag+. The oxidation state of the particles was investigated, although this was not the focus of the thesis. The study of fate, e.g. dissolution of NPs, is also only in its beginning and the appropriate techniques are currently being developed. The results showed a specific nanoparticle effect. The UV exposure with titanium dioxide nanoparticles increased its effect.

Preparation and in vivo evaluation of insulin-loaded biodegradable nanoparticles prepared from diblock copolymers of PLGA and PEG

The aim of this study was to design a controlled release vehicle for insulin to preserve its stability and biological activity during fabrication and release. A modified, double emulsion, solvent evaporation, technique using homogenisation force optimised entrapment efficiency of insulin into biodegradable nanoparticles (NP) prepared from poly (dl-lactic-co-glycolic acid) (PLGA) and its PEGylated diblock copolymers. Formulation parameters (type of polymer and its concentration, stabiliser concentration and volume of internal aqueous phase) and physicochemical characteristics (size, zeta potential, encapsulation efficiency, in vitro release profiles and in vitro stability) were investigated. In vivo insulin sensitivity was tested by dietinduced type II diabetic mice. Bioactivity of insulin was studied using Swiss TO mice with streptozotocin-induced type I diabetic profile. Insulin-loaded NP were spherical and negatively charged with an average diameter of 200���400 nm. Insulin encapsulation efficiency increased significantly with increasing ratio of co-polymeric PEG. The internal aqueous phase volume had a significant impact on encapsulation efficiency, initial burst release and NP size. Optimised insulin NP formulated from 10% PEG-PLGA retained insulin integrity in vitro, insulin sensitivity in vivo and induced a sustained hypoglycaemic effect from 3 hours to 6 days in type I diabetic mice.

Effects of nanoparticles exposure in the mussel Mytilus Galloprovincialis

Tese de dout., Ci��ncias do Mar, Terra e Ambiente (Ecotoxicologia), Faculdade de Ci��ncias e Tecnologia, Univ. do Algarve, 2012

Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N���-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.

Effects of mixture of nanoparticles under different salinity in the clams Ruditapes philippinarum and Ruditapes decussatus

Disserta����o de mestrado, Biologia Marinha, Faculdade de Ci��ncias e Tecnologia, Universidadde do Algarve, 2015

Development of a melanoma therapeutic vaccine candidate by the coencapsulation of melanoma antigen peptides and toll-like receptor ligands as adjuvants into polymeric nanoparticles

Tese de doutoramento, Farm��cia (Tecnologia Farmac��utica), Universidade de Lisboa, Faculdade de Farm��cia, 2014

Control of endosomal Toll-like receptor (TLR) signaling by nanoparticles and applications in cancer therapy

Thesis (Ph.D.)--University of Washington, 2014