Silver nanoparticles - From the synthesis to the biological application

The main topic of this doctoral thesis was the synthesis and characterization of defined silver nanoparticles (Ag NPs). These particles were synthesized with different sizes and modified surface chemistry. Two core sizes were synthesized. One, very small particle (~2 nm core) with hydrophilic ligands at the surface and second, bigger particles (~4.2 nm core) with different kinds of coatings were prepared. Additionally, the potential of these particles were tested for their use in biological systems. For this, they had to be stable in aqueous media. To assess this, their stability against different salt concentration was measured. Another important point, especially for silver nanoparticles, was its cytotoxicity. The cytotoxicity of the silver particles is a very important research topic due to the antimicrobial effect observed in silver NPs. Here, the toxicity of the different particles was measured in two different cell lines. The very small particles (~2 nm), so called nanoclusters (NCs), were prepared in two steps. After the reduction of the silver precursor first an etching step and afterwards a ligand exchange reaction were done. This ligand exchange also allowed the particles to be stable in aqueous media. During the whole synthesis the size and the distribution of the particles changed. At the beginning the particles showed a broad distribution and sizes up to almost 30 nm. After the etching the distribution decreased a lot and the sizes shrunk to 5 nm. The final core size of about 2 nm with a narrow distribution was reached after the ligand exchange being the particles stable in an aqueous suspension. A big advantage of these nanoclusters was that they showed fluorescence in the red and did not need to be labeled with an additional dye for in vitro experiments. The second type of particles showed a core size of around 4.2 nm and was synthesized in a simple reduction reaction. For this reaction first a precursor of the stabilizing ligand had to be synthesized. After the synthesis the particles were stable as a gray powder and showed a hydrophobic surface. To get the particles into aqueous phase two different methods were used. First a ligand exchange reaction with a hydrophilic ligand and second the coating of the particles with an amphiphilic polymer. One advantage of the coating process was the easy modification of the surface afterwards. This was shown by the modification of the particles with a dye and/or polyethylene glycol (PEG) chains. A further advantage was that the toxicity of the particles was highly reduced by this process. This reduced toxicity was on one hand due to the increased stability of the particles and on the other hand due to the reduced uptake of the particles when their surface was saturated with PEG. An increase of the stability against sodium chloride could also been shown for commercial gold particles using the same coating process. The commercial gold nanoparticles were stabilized by citrate molecules at the beginning and so first a ligand exchange including a phase transfer to the organic phase had to be done to reach the same surface like the silver nanoparticles. Nevertheless, the Ag NPs showed a cytotoxic effect, which was due to the release of Ag(I) ions. This release was measured under different pH conditions. Under neutral pH values neither the Ag NPs stabilized by hydrophilic ligand molecules nor the once stabilized by the polymer coating showed a release of more than 0.1% of ions up to 14 days. Under acidic conditions (pH 3) all the particles showed a release of about 1% of ions already after 7 days. In comparison of the total amount of silver with a silver salt the concentration of silver ions from the particles are low but they are “more toxic” because of the better uptake respectively their release inside the cell. To summarize it can be said that a defined synthesis and modification of different Ag NPs could be done but although they showed a very high stability they never lost their cytotoxicity

Diverse Applications of Nanomedicine

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic. \ua9 2017 American Chemical Society

Gold Nanoparticles: Synthesis, Surface Modification and Functionalization for Biomedical Applications

The nanoparticles (NPs) surface plays an important role in the interaction of the NPs with surrounding environments, defines their fate in the biological media and it can be engineered to provide a large number of functional groups for different applications. The main topic of this thesis is the synthesis, the surface modification and the characterization of gold NPs (GNPs). The NPs were prepared with different sizes (up to 100 nm) and shapes (spherical and rods). The employed NPs were prepared originally in aqueous medium, stabilized by citric ions in case of spherical GNPs (SGNPs) and by hexadecyltrimethylammonium bromide (CTAB) in case of gold nanorods (GNRs). These ligand molecules are weakly bound to the NPs surface and thus, they are unsuitable for biomedical applications. Stabilizing of these NPs was the target of this work and then, it was achieved by exchange these ligands by other molecules with higher affinity and finally wrapping the NPs with an amphiphilic polymer (poly(isobutylene-alt-maleic anhydride) dodecylamine grafted, (PMA)). The polymer coating technique has been used over the past years for coating of NPs, which are synthesized originally, and only dispersible in organic media. The NPs obtained using this methodology are highly stable in physiological media. Aiming to use this technique in water-soluble NPs, a new round-trip process was developed using a phase transfer step before the polymer coating. The NPs were stabilized with α-metoxi-ω-thiol-poly-(ethyleneglycol)- (PEG) chains (mPEG-SH (Mw= 750 Da)) and then transferred from water to chloroform using dodecylamine (DDA). The DDA-capped NPs were coated with a modified amphiphilic polymer due to the hydrophobic interaction between the hydrophobic ligands (carbon chains) on the surface of the NPs and the hydrophobic side chains of the used polymer. The resulted polymer coated NPs were cleaned and characterized using different techniques, such as agarose gel electrophoresis, UV-Vis spectroscopy, dynamic light scattering (DLS), laser Doppler anemometry (LDA) and transmission electron microscopy (TEM). Furthermore, the surface of the polymer-coated NPs was modified using different molecular weight of PEG to provide them with higher colloidal stability and prevent the formation of the so-called protein corona. The colloidal stability of all particles was assayed against different biological media via UV-Vis spectroscopy and DLS. The toxicity of these NPs was tested in cancer and non-cancer cells lines showing no-toxicity up to 1 mg/mL concentration levels. Additionally, the effect of NPs size, shape, and surface coating on their interaction with plasma proteins and blood cells was studied

An Investigation of Gene Delivery Barriers for Poly(beta-amino ester)s and Hybrid Gold-Polymeric Nanoparticles

Gene therapy is promising because nearly all inheritable diseases and cancer has an underlying genetic component. However, the development of a gene vector capable of delivering nucleic acids efficiently while remaining safe has been a challenge. Generally speaking, viral vectors are highly adept at delivering nucleic acids intracellularly. Viral vectors, however, have been known to be associated with insertional mutagenesis and immunogenicity and in severe cases, death. Furthermore, viral vectors are difficult to chemically modify for optimization and to mass produce. To date, despite that there have been more than 2000 gene therapy clinical trials worldwide, the U.S. FDA has yet to approve a gene therapy application. The lack of gene therapy approval by regulatory bodies delineates the need for safer and more effective vectors. A safer alternative to viral-based vectors are cationic, amine-containing polymeric vectors. Cationic polymers are capable of ionically complexing nucleic acid and forming nanoparticles on the order of 100-250 nm. This particle diameter is capable of extravasating through mal-formed tumor vasculature. The polymers can be engineered to have a buffering ability that allows the nucleic acid cargo to escape the lysosomal degradation pathway post-endocytosis. Ester-containing backbone polymers are capable of hydrolytic degradation, which helps mitigate toxicity. However, much is unknown regarding how polymer structure affects gene delivery. To date, the polymer vectors which work well have been discovered through empirical methods. Understanding how polymer structure affects gene delivery function would allow for a more rational approach to designing new vectors for gene delivery. The objective of this work has been two-fold: the first objective has been to elucidate non-viral gene delivery barriers – in particular investigating polymer structure-function relationships for polymeric vectors; the second objective has been to develop a polymeric/inorganic hybrid, theranostic-enabling nanoparticle platform technology capable of co-delivering DNA and siRNA and modulating temporal release. More specifically, this work details how polymer structure of poly(β-amino ester)s (PBAEs) affects polymer-DNA binding and how binding affects transfection levels, viability, and nanoparticle physical properties (zeta potential and diameter). We found transfection levels are biphasic with respect to binding in two human cancer cell lines and that binding constants in the range of (1-6)x104 M-1 were necessary but not sufficient for optimal transfection. We also investigated the comparative binding strengths of branched and linear polyethyleneimine, poly(L-lysine) and PBAEs with plasmid DNA and found PBAEs have the weakest binding. This work also details new bioassays including a more high throughput method for assessing cellular and nuclear uptake rates using flow cytometry. This method may be used for elucidating structure-function relationships in various cell types. An auto-fitting, first order mass-action kinetic model was developed in MatLab to quantify the intracellular delivery rate constants for comparing delivery bottlenecks of various polymer structures in various cell lines. This model was used to assess rate differences between polymers which do not transfect well, tansfect mediocre, and transfect well in primary human glioblastoma in vitro. The model recapitulated the experimental data with good agreement without needing to extrapolate data from literature. Principal component analysis is a method to look at large data sets with unknown variable correlations and to quantify how each variable is correlated with another, as well as which and to what degree each variable may drive another. Principal component analysis was utilized to look at 27 physico-chemical properties and cell gene delivery outcomes (i.e., uptake, transfection levels, and viability). We found that certain key parameters, such as hydrophobicity, drove uptake and transfection. The development of a theranostic-enabling platform technology involved gold nanoparticles and a layer-by-layer coating process to create polymer-inorganic hybrids. Gold nanoparticles are relatively biocompatible, are monodisperse, have interesting optical properties, have photothermal capabilities, and are easily chemically modified via thiol bonds. Gold nanoparticles can be synthesized to absorb a desired wavelength of electromagnetic radiation and can be imaged through various modalities such as photoacoustics or X-rays for diagnostic purposes. The theranostic-enabling technology was capable of co-delivering DNA and siRNA as well as delivering two layers of DNA with two different expression time profiles. Co-delivering DNA and siRNA could allow for the knockdown of a dysfunctional aberrant protein while replacing it with a functional protein. The ability to express proteins with different time profiles could have multiple applications such as controlling stem cell differentiation

Polymer-Coated Inorganic Nanoparticles: Nanotools for Life Science Applications

This dissertaion focus on the synthesis, surface modification and characterization of inorganic nanoparticles(NPs), including magnetic, plasmonic and semiconductor NPs. With controlling the reaction conditions during the synthesis, different particle diameters in the range of 4 nm to 30 nm can be synthesized. Afterwards, polymer coating process was successfully applied to different materials by overcoating the NPs with an amphiphoilic polymer, which can make the particle water soluble. This work aimed to produce the polymer-­ coated nanoparticles,analyze and compare their physico-­‐chemical properties based on different materials,and further, to test their potential for different biological applications

Biodegradable Magneto-Luminescent Mesoporous Nanoparticles As New Nanobiomedical Tools In Cancer Treatment

Colorectal cancer (CRC) is the 3rd most common type of cancer worldwide and 2nd in mortality rate, accounting to 1.93 million cases and 916 000 deaths just in 2020. The development of CRC targeted therapies has been a hot topic in recent medical forums and among researchers, with the demand for non-invasive approaches, like oral ones, capable of preserving their therapeutic properties from administration to local action drastically increasing in the last decade. Its body location makes CRC a usually silent disease, whose commonly later detection and action usually lead to severe aggravations and even death. Targetability, drugs' resistance and toxicity of the said therapies are thus three of the key points for the development of effective therapies. On the other hand, the presence of cancerassociated bacterial infections and inflammations has been positively correlated with low treatment success rates and associated complications, with bacterial infections being particularly damaging in gastric and colorectal cancer. Some of the metabolites/toxins of these opportunistic bacteria have been linked to tumorigenesis and CRC drug-resistance. A conjoint therapy capable of addressing both CRC and associated bacteria, where drug preservation, null secondary toxicity and targetability are ensured. In light of the above-mentioned, herein we cover the synthesis of a new magnetofluorescent nanotherapy against both CRC and associated bacterial strains, going from the synthesis of sustainable, biocompatible, biodegradable and cheap nanocomposite particles (NANO3) to the testing of its dual drug cargo and delivery capacities, magneto-fluorescent properties and later assessment of its combined antibacterial and anti-tumor activity. The successful synthesis of new anti-CA IX targeting inhibitor as potential directing agent is also covered as a cheaper alternative to the commonly used and expensive antibodies. From an antimicrobial point of view, NANO3 showed significant activity against CRC-related bacterial strains, with their intrinsic magnetic properties potentiating a hyperthermia-assisted pH responsive release and antimicrobial therapy. The combined release of an antibiotic (i.e., ofloxacin) and an anti-tumor drug (i.e., doxorubicin) showed no hinderance on the activity of both active drugs. Accordingly, NANO3 anti-tumoral activity, arising solely from the loaded doxorubicin, was conserved and competitive to that of the free model drug. Targetability was conferred by successfully functionalizing of NANO3 with anti-EGFR and and/or anti-CA IX, the later favoring higher NANO3 uptake in HCT-116 cell line. A proof-of-concept oral delivery was also simulated, where a dual Eudragit® enteric coating successfully protected NANO3 formulations from stomachal and small intestine conditions, selectively dispensing it in colon environment and potentiating their use in a future anti-CRC oral therapy. In this thesis an optimization of a luminescent antimicrobial activity assay specific for nanoparticles was also carried out along with the evaluation of other cyanine-based antimicrobial scaffolds for a potential biomedical and food-based application.O cancro colorrectal (CRC) é o 3º tipo de cancro mais comum a nível mundial e o 2º na taxa de mortalidade, contabilizando 1,93 milhões de casos e 916 000 mortes apenas em 2020. O desenvolvimento de terapias orientadas para CRC tem sido um tema recorrente no meio médico e entre investigadores, tendo a procura de abordagens não invasivas, tais como as orais, capazes de preservar as suas propriedades terapêuticas desde a administração até à acção local, aumentado drasticamente na última década. A sua localização corporal faz do CRC uma doença geralmente silenciosa, cuja detecção e acção tardias conduzem a agravamentos do quadro clinico ou mesmo à morte. A capacidade de carga, a resistência dos medicamentos e a toxicidade das referidas terapias são assim três dos pontos-chave para o desenvolvimento de terapias eficazes. Por outro lado, a presença de infecções e inflamações bacterianas associadas ao cancro têm sido positivamente correlacionadas com baixas taxas de sucesso de tratamento e ao aumento de complicações, com particular impacto prejudicial em situações de cancro gástrico e colorrectal. Alguns dos metabolitos/toxinas destas bactérias oportunistas têm sido ligados à tumorigenese e à resistência aos medicamentos em CRC. Uma terapia conjunta capaz de tratar tanto CRC como as bactérias associadas, onde a preservação do medicamento, a toxicidade secundária nula e a capacidade de carga são asseguradas. Tendo em conta o acima mencionado, na presente tese detalha-se a síntese de uma nova nanoterapia magneto-fluorescente tanto contra CRC como contra estirpes bacterianas associadas. Indo assim, desde a síntese de partículas nanocompósitas sustentáveis, biocompatíveis, biodegradáveis e baratas (NANO3) até à dupla carga e entrega de medicamentos; bem como da avaliação das suas propriedades magneto-fluorescentes até à sua actividade antibacteriana e antitumoral combinada. A síntese bem-sucedida do novo inibidor de CA IX como potencial agente dirigente é também coberta como uma alternativa mais barata aos anticorpos normalmente utilizados. De um ponto de vista antimicrobiano, NANO3 mostrou uma actividade significativa contra estirpes bacterianas relacionadas com CRC, com as suas propriedades magnéticas intrínsecas potenciando uma libertação pHresponsiva, assistida por hipertérmia, e por fim a uma terapia antimicrobiana. A libertação combinada de um antibiótico (i.e.,, ofloxacina) e de um medicamento anti-tumoral (i.e., doxorubicina) não levou a qualquer redução da actividade de ambos os medicamentos. Assim, a actividade anti-tumoral de NANO3, proveniente unicamente da doxorubicina carregada, foi conservada e competitiva em relação à do medicamento livre. A capacidade de atuar especificamente sobre células de CRC foi conferida pela funcionalização das NANO3 com anti-EGFR e / ou anti-CA IX, o último dos quais favorecendo uma maior internalização de NANO3 na linha celular HCT-116. Foi também simulada uma prova de conceito de uma toma oral, onde um revestimento entérico duplo de Eudragit® protegeu com sucesso as formulações de NANO3 das condições estomacais e do intestino delgado, dispensando-o selectivamente em ambiente de cólon e potenciando a sua utilização numa futura terapia oral anti-CRC. Na presente tese foi também realizada uma optimização de um ensaio de actividade antimicrobiana luminescente específico para nanopartículas, juntamente com a avaliação de outros substratos antimicrobianos à base de cianina para uma potencial aplicação biomédica e alimentar

Plasma in Cancer Treatment

In the last decade, research on cold atmospheric plasma (CAP) has significantly advanced our understanding of the effect of CAP on cancer cells and their potential for cancer treatment. This effect is due to the reactive oxygen and nitrogen species (RONS) created by plasma. This has been demonstrated for different cancer cell lines and the first clinical trials showed promising results. In addition, plasma could be combined with other treatments—such as immunotherapy—to boost its anticancer activity. The addition of new research tools to study the response of cancer cells to CAP—such as 3D in vitro, in ovo, and in vivo models and in silico approaches—as well as the use of -OMICS technologies could aid in unravelling the underlying mechanisms of CAP in cancer treatment. In order to progress towards widespread clinical application of CAP, an integrated study of the multidimensional effect of CAP in cancer treatment is essential. In this book, reviews and original research papers are published that provide new insights into the mechanisms of cold atmospheric plasma in cancer treatment, based on in vitro and in vivo experiments, clinical studies, as well as computer modeling

APPLICATION OF CARBON NANOTUBES IN DRUG DELIVERY: TARGETING MITOCHONDRIA FOR CANCER THERAPY

Ph.DDOCTOR OF PHILOSOPH