Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

Abstract Background Rhodium (II) citrate (Rh2(H2cit)4) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh2(H2cit)4 as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh2(H2cit)4 and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh2(H2cit)4) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures. Results Treatment with free Rh2(H2cit)4 induced cytotoxicity that was dependent on dose, time, and cell line. The IC50 values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh2(H2cit)4-loaded maghemite nanoparticles (Magh-Rh2(H2cit)4) and Rh2(H2cit)4-loaded magnetoliposomes (Lip-Magh-Rh2(H2cit)4) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh2(H2cit)4, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh2(H2cit)4 induces cell death by apoptosis. Conclusions The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh2(H2cit)4 treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh2(H2cit)4 delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

Optical storage in azobenzene-containing epoxy polymers processed as Langmuir Blodgett films

In this study, azocopolymers containing different main-chain segments have been synthesized with diglycidyl ether of bisphenol A (DGEBA, DER 332, n=0.03) and the azochromophore Disperse Orange 3 (DO3) cured with twomonoamines, viz. benzylamine (BA) and m-toluidine (MT). The photoinduced birefringence was investigated in films produced with these azopolymers using the spin coating (SC) and Langmuir Blodgett (LB) techniques. In the LB films, birefringence increased with the content of azochromophore and the film thickness, as expected. The nanostructured nature of the LB films led to an enhanced birefringence and faster dynamics in the writing process, compared to the SC films. In summary, the combination of azocopolymers and the LBmethod may allow materials with tuned properties for various optical applications, including in biological systems were photoisomerization may be used to trigger actions such as drug delivery.

Effect of interaction with micelles on the excited-state optical properties of zinc porphyrins and J-aggregates formation

This work reports on the photophysical properties of zinc porphyrins meso-tetrakis methylpyridiniumyl (Zn2+TMPyP) and meso-tetrakis sulfonatophenyl (Zn2+TPPS) in homogeneous aqueous solutions and in the presence of sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide (CTAB) micelles. The excited-state dynamic was investigated with the Z-scan technique, UV-Vis absorption, and fluorescence spectroscopy. Photophysical parameters were obtained by analyzing the experimental data with a conventional five-energy-level diagram. The interaction of the charged side porphyrin groups with oppositely charged surfactants can reduce the electrostatic repulsion between porphyrin molecules leading to aggregation, which affected the porphyrin characteristics such as absorption cross-sections, lifetimes and quantum yields. The interaction between anionic ZnTPPS with cationic CTAB micelles induced the formation of porphyrin J-aggregates, while this effect was not observed in the interaction of ZnTMPyP with SDS micelles. This difference is, probably, due to the difference in electrostatic repulsion between the porphyrin molecules. The insights obtained by these results are important for the understanding of the photophysical behavior of porphyrins, regarding potential applications in pharmacokinetics as encapsulation of photosensitizer for drug delivery systems and in its interaction with cellular membrane.

Biomimetic Materials for Biomedical Applications

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Ingegnerizzazione e design di nanoparticelle core-shell di magnetite/silice

In the past decade the study of superparamagnetic nanoparticles has been intensively developed for many biomedical applications such as magnetically assisted drug delivery, MRI contrast agents, cells separation and hyperthermia therapy. All of these applications require nanoparticles with high magnetization, equipped also with a suitable surface coating which has to be non-toxic and biocompatible. In this master thesis, the silica coating of commercially available magnetic nanoparticles was investigated. Silica is a versatile material with many intrinsic features, such as hydrophilicity, low toxicity, proper design and derivatization yields particularly stable colloids even in physiological conditions. The coating process was applied to commercial magnetite particles dispersed in an aqueous solution. The formation of silica coated magnetite nanoparticles was performed following two main strategies: the Stöber process, in which the silica coating of the nanoparticle was directly formed by hydrolysis and condensation of suitable precursor in water-alcoholic mixtures; and the reverse microemulsions method in which inverse micelles were used to confine the hydrolysis and condensation reactions that bring to the nanoparticles formation. Between these two methods, the reverse microemulsions one resulted the most versatile and reliable because of the high control level upon monodispersity, silica shell thickness and overall particle size. Moving from low to high concentration, within the microemulsion region a gradual shift from larger particles to smaller one was detected. By increasing the amount of silica precursor the silica shell can also be tuned. Fluorescent dyes have also been incorporated within the silica shell by linking with the silica matrix. The structure of studied nanoparticles was investigated by using transmission electron microscope (TEM) and dynamic light scattering (DLS). These techniques have been used to monitor the syntetic procedures and for the final characterization of silica coated and silica dye doped nanoparticles. Finally, field dependent magnetization measurements showed the magnetic properties of core-shell nanoparticles were preserved. Due to a very well defined structure that combines magnetic and luminescent properties together with the possibility of further functionalization, these multifunctional nanoparticles are potentially useful platforms in biomedical fields such as labeling and imaging.

Core-shell macromolecules with dendritic polyphenylene core and polymer shells

Core-shell macromolecules with dendritic polyphenylene core and polymer shell Zusammenfassung / Abstract Core-shell macromolecular structures have become of great interest in materials science because they gave an opportunity to combine a large variety of chemical and physical properties in the single molecule, by combination of different (in terms of chemistry and physics) cores and shells. The interest in such complex structures was provoked by their potential applications in the coating and painting industry (latexes), as supports for catalysts in polymer industry, or as nano-containers and transporters for genes or drug delivery. The aim of this study was the synthesis, characterization and further application of core-shell macromolecules possessing a hydrophobic stiff core (polyphenylene dendrimers) surrounded with a hydrophilic, soft, covalently bonded polymer shell (poly(ethylene oxide) and its copolymers). The requirements for such complex substances were that they should be well-defined in terms of molecular weight (narrow molecular weight distribution) and in molecular structure. The preparation of core-shell molecules containing dendrimer as a core was possible via two synthetic routs: “grafting-onto” and “grafting-from”. The resulting core-shell macromolecules possessed narrow polydispersity as guaranteed by the excellent structural and functional definition of the dendrimer and the narrow polydispersity of the PEO, PS-b-PEO and PI-b-PEO attached to the dendrimer surface. Additional investigation of the size of the particles indicated a relation between both the length and the number of the polymer chains and the hydrodynamic radius determined by Dynamic Light Scattering and Fluorescent Correlation Spectroscopy. Core-shell nano-particles were applied as metallocene supports in heterogeneous olefin polymerizations. Our results indicate that such catalyst systems, that have a size of at least one order of magnitude smaller than the used by now organic supports, could be very useful as model compounds for investigations on catalyst fragmentation and its influence on the product parameters.

Sintesi e caratterizzazione di sistemi nanostrutturati per il trattamento del Glioblastoma multiforme

Nanomedicine is a science based on the preparation of nanosystems for biomedical application. The drugs can be entrapped inside the nanocarriers to improve the drug concentration in the diseased issue through a drug delivery approach; polimeric materials as PLGA-b-PEG has been revealed good properties for this purpose. To improve the nanosystem efficiency it is possible to bind a targeting agent on the carrier surface. In this thesis work silver nanoparticles or drugs as Temsirolimus and Alisertib have been entrapped in PLGA-b-PEG carriers. Chlorotoxin has been linked on the carrier surface as a specific targeting agent for brain tumors. Citotoxicity in vitro of the nanosystems on Glioblastoma cells has been studied.

Synthese von amphiphilen Bürstenpolymeren mit Kern-Schale-Architektur und Untersuchungen zur Verkapselung von hydrophilen Modellsubstanzen

Die dieser Arbeit zugrundeliegenden Nanopartikel wurden mittels der Makromonomer-Strategie aus polymerisierbaren Polystyrol-b-Poly(2-vinylpyridin) Oligomeren dargestellt. Die Bürstenpolymere besitzen eine polare PS-Schale und einen polaren Kern (P2VP), dessen Polarität durch Quaternisierung deutlich erhöht werden kann. Die Bürstenpolymere weisen bei Molmassen um 400 - 800 kg/mol einen Teilchendurchmesser von ca. 15 - 20 nm auf. Die Nanopartikel eignen sich dazu, hydrophile Farbstoffe in unpolaren Lösungsmitteln zu solubilisieren. Durch spektroskopische Untersuchungen wurden in Abhängigkeit der chemischen Struktur und der Bürstenpolymere Beladungsgrade von über 1 g Farbstoff pro Gramm Polymer ermittelt. Die Beladung der Nanopartikel folgt hierbei einer nichttrivialen Kinetik, was möglicherweise durch eine wasserinduzierte Überstrukturbildung während der Beladung bedingt ist. Mittels isothermer Titrationskalorimetrie konnten die Wechselwirkungen zwischen polymeren Substrat und niedermolekularen Liganden genauer charakterisiert werden. Teilweise werden hierbei zweistufige Titrationsverläufe und "überstöchiometrische" Beladung der Bürstenpolymere beobachtet. Den Hauptbeitrag zur Wechselwirkung liefert hierbei die exotherme Wechselwirkung zwischen basischen Polymer und saurem Farbstoff. Die hohe Farbstoffbeladung führt zur deutlichen Vergrößerung der einzelnen Nanopartikel, was sowohl in Lösung durch Lichtstreu-Techniken als auch auf Oberflächen mit Hilfe des AFM zu beobachten ist. Durch Untersuchungen mit der analytischen Ultrazentrifuge konnte nachgewiesen werden, dass sich der eingelagerte Farbstoff in einem Polaritäts-abhängigen Gleichgewicht mit der Umgebung steht, er somit auch wieder aus den Nanopartikeln freigesetzt werden kann. Darüberhinaus wurden im Rahmen der Arbeit erste Erfolge bei der Synthese von wasserlöslichen Nanopartikeln mit Poly(2-vinylpyridin)-Kern erzielt. Als hierfür geeignet stellte sich eine Synthesestrategie heraus, bei der zunächst ein Bürstenpolymer mit P2VP-Seitenketten dargestellt und dieses anschließend mit geeignet funktionalisierten Polyethylenoxid-Ketten zum Kern-Schale Teilchen umgesetzt wurde. Neben Untersuchungen zum Mizellisierungsverhalten von PEO-b-P2VP Makromonomeren wurden deren Aggregate in Wasser hinsichtlich ihrer Zelltoxizität durch in-vitro Experimente an C26-Mäusekarzinom-Zellen charakterisiert. Die extrem geringe Toxizität macht das PEO-P2VP System zu einem potentiellen Kandidaten für drug-delivery Anwendungen. Besonders die pH-abhängige Löslichkeitsänderung des Poly(2-vinylpyridin) erscheint hierbei besonders interessant.

DNA block copolymers - synthesis, morphologies and applications

The last decades have witnessed significant and rapid progress in polymer chemistry and molecular biology. The invention of PCR and advances in automated solid phase synthesis of DNA have made this biological entity broadly available to all researchers across biological and chemical sciences. Thanks to the development of a variety of polymerization techniques, macromolecules can be synthesized with predetermined molecular weights and excellent structural control. In recent years these two exciting areas of research converged to generate a new type of nucleic acid hybrid material, consisting of oligodeoxynucleotides and organic polymers. By conjugating these two classes of materials, DNA block copolymers are generated exhibiting engineered material properties that cannot be realized with polymers or nucleic acids alone. Different synthetic strategies based on grafting onto routes in solution or on solid support were developed which afforded DNA block copolymers with hydrophilic, hydrophobic and thermoresponsive organic polymers in good yields. Beside the preparation of DNA block copolymers with a relative short DNA-segment, it was also demonstrated how these bioorganic polymers can be synthesized exhibiting large DNA blocks (>1000 bases) applying the polymerase chain reaction. Amphiphilic DNA block copolymers, which were synthesized fully automated in a DNA synthesizer, self-assemble into well-defined nanoparticles. Hybridization of spherical micelles with long DNA templates that encode several times the sequence of the micelle corona induced a transformation into rod-like micelles. The Watson-Crick motif aligned the hydrophobic polymer segments along the DNA double helix, which resulted in selective dimer formation. Even the length of the resulting nanostructures could be precisely adjusted by the number of nucleotides of the templates. In addition to changing the structural properties of DNA-b-PPO micelles, these materials were applied as 3D nanoscopic scaffolds for organic reactions. The DNA strands of the corona were organized by hydrophobic interactions of the organic polymer segments in such a fashion that several DNA-templated organic reactions proceeded in a sequence specific manner; either at the surface of the micelles or at the interface between the biological and the organic polymer blocks. The yields of reactions employing the micellar template were equivalent or better than existing template architectures. Aside from its physical properties and the morphologies achieved, an important requirement for a new biomaterial is its biocompatibility and interaction with living systems, i.e. human cells. The toxicity of the nanoparticles was analyzed by a cell proliferation assay. Motivated by the non-toxic nature of the amphiphilic DNA block copolymers, these nanoobjects were employed as drug delivery vehicles to target the anticancer drug to a tumor tissue. The micelles obtained from DNA block copolymers were easily functionalized with targeting units by hybridization. This facile route allowed studying the effect of the amount of targeting units on the targeting efficacy. By varying the site of functionalization, i.e. 5’ or 3’, the outcome of having the targeting unit at the periphery of the micelle or in the core of the micelle was studied. Additionally, these micelles were loaded with an anticancer drug, doxorubicin, and then applied to tumor cells. The viability of the cells was calculated in the presence and absence of targeting unit. It was demonstrated that the tumor cells bearing folate receptors showed a high mortality when the targeting unit was attached to the nanocarrier.

De Novo Design of Foldamers for Preparation of Nanostructured Materials

This thesis deals with the synthesis and the conformation analysis of hybrid foldamers containing the 4-carboxyoxazolidin-2-one unit or related molecules, in which an imido-type function is obtained by coupling the nitrogen of the heterocycle with the carboxylic acid moiety of the next unit. The imide group is characterized by a nitrogen atom connected to an endocyclic and an exocyclic carbonyl, which tend always to adopt the trans conformation. As a consequence of this locally constrained disposition effect, these imide-type oligomers are forced to fold in ordered conformations. The synthetic approach is highly tuneable with endless variations, so, simply by changing the design and the synthesis, a wide variety of foldamers with the required properties may be prepared “on demand”. Thus a wide variety of unusual secondary structures and interesting supramolecular materials may be obtained with hybrid foldamers. The behaviour in the solid state of some of these compounds has been analyzed in detail, thus showing the formation of different kinds of supramolecular materials that may be used for several applications. A winning example is the production of a bolaamphiphilic gelators that may also be doped with small amounts of dansyl containing compounds, needed to show the cellular uptake into IGROV-1 cells, by confocal laser scanning microscopy. These gels are readily internalized by cells and are biologically inactive, making them very good candidates in the promising field of drug delivery. In the last part of the thesis, a particular attention was directed to the search of new scaffolds that behave as constrained amino acid mimetics, showing that tetramic acids derivatives could be good candidates for the synthesis and applications of molecules having an ordered secondary structure.

Electrostatically self-assembled nanoparticles based on biomolecules

In this work, two different systems were investigated to develop fundamental understanding of the self-assembly behavior of polyelectrolytes and small organic counterions with a certain geometry. Complexes formed were characterized by light scattering in solution, as well as UV-Vis spectroscopy, analytical ultracentrifugation, gel electrophoresis, zeta potential and IR spectroscopy. The morphologies of the aggregates were observed by AFM in dried state on surface. The charge ratio, the valence and the structure of the counterion were shown to represent key parameters in the complexation. The influence of polyelectrolyte type and molecular weights was also determined for the structure formed.rnrnOne system was mainly focused on the association of double-strand DNA with non-intercalating divalent and tetravalent organic counterions. The other model system involved linear NaPSS and oligolysines. In addition, various influences on the morphology of the charged self-assembly complexes in AFM studies were discussed. It was shown that electrostatic self-assembly of DNA and non-intercalating counterions as well as of a linear synthetic polyelectrolyte with oligolysine counterions that can build mutual hydrogen bonds can yield supramolecular aggregates of a defined size. Various morphologies (flower-like, rod-like, toroidal and spherical) of the assemblies were obtained for different combinations of polyelectrolyte and counterions. Results presented in this work are of importance for the fundamental understanding of the association behavior of various polyelectrolytes and organic counterions. The selection of biopolymers for the study may give an opportunity to transfer the basic research results into biological applications, such as gene therapy or drug delivery.rn

Synthesis and surface modification of silver and gold nanoparticles. Nanomedicine applications against Glioblastoma Multiforme.

In the last decades noble metal nanoparticles (NPs) arose as one of the most powerful tools for applications in nanomedicine field and cancer treatment. Glioblastoma multiforme (GBM), in particular, is one of the most aggressive malignant brain tumors that nowadays still presents a dramatic scenario concerning median survival. Gold nanorods (GNRs) and silver nanoparticles (AgNPs) could find applications such as diagnostic imaging, hyperthermia and glioblastoma therapy. During these three years, both GNRs and AgNPs were synthesized with the “salt reduction” method and, through a novel double phase transfer process, using specifically designed thiol-based ligands, lipophilic GNRs and AgNPs were obtained and separately entrapped into biocompatible and biodegradable PEG-based polymeric nanoparticles (PNPs) suitable for drug delivery within the body. Moreover, a synergistic effect of AgNPs with the Alisertib drug, were investigated thanks to the simultaneous entrapment of these two moieties into PNPs. In addition, Chlorotoxin (Cltx), a peptide that specifically recognize brain cancer cells, was conjugated onto the external surface of PNPs. The so-obtained novel nanosystems were evaluated for in vitro and in vivo applications against glioblastoma multiforme. In particular, for GNRs-PNPs, their safety, their suitability as optoacoustic contrast agents, their selective laser-induced cells death and finally, a high tumor retention were all demonstrated. Concerning AgNPs-PNPs, promising tumor toxicity and a strong synergistic effect with Alisertib was observed (IC50 10 nM), as well as good in vivo biodistribution, high tumor uptake and significative tumor reduction in tumor bearing mice. Finally, the two nanostructures were linked together, through an organic framework, exploiting the click chemistry azido-alkyne Huisgen cycloaddition, between two ligands previously attached to the NPs surface; this multifunctional complex nanosystem was successfully entrapped into PNPs with nanoparticles’ properties maintenance, obtaining in this way a powerful and promising tool for cancer fight and defeat.

Strategie formulative per la veicolazione nasale di farmaci

Microparticelle a base di complessi polielettrolitici di Chitosano/Pectina per il rilascio nasale di Tacrina cloridrato. Lo scopo di questo studio è stata la ricerca di nuove formulazioni solide per la somministrazione nasale di Tacrina cloridrato allo scopo di ridurre l’eccessivo effetto di primo passaggio epatico ed aumentarne la biodisponibilità a livello del Sistema Nervoso Centrale. La Tacrina è stata incapsulata in microparticelle mucoadesive a base di complessi elettrolitici di chitosano e pectina. Le microparticelle sono state preparate mediante due diversi approcci tecnologici (spray-drying e spray-drying/liofilizzazione) e analizzate in termini di caratteristiche dimensionali, morfologiche e chimico-fisiche. Nanoparticelle di Chitosano reticolate con Sodio Cromoglicato per il trattamento della rinite allergica. Il Sodio Cromoglicato è uno dei farmaci utilizzati per il trattamento della rinite allergica. Come noto, la clearance mucociliare provoca una rapida rimozione dei farmaci in soluzione dalla cavità nasale, aumentando così il numero di somministrazioni giornaliere e, di conseguenza, riducendo la compliance del paziente. Per ovviare a tale problema, si è pensato di includere il sodio cromoglicato in nanoparticelle di chitosano, un polimero capace di aderire alla mucosa nasale, prolungare il contatto della formulazione con il sito di applicazione e ridurre il numero di somministrazioni giornaliere. Le nanoparticelle ottenute sono state caratterizzate in termini di dimensioni, resa, efficienza di incapsulazione e caricamento del farmaco, potenziale zeta e caratteristiche mucoadesive. Analisi quantitativa di Budesonide amorfa tramite calorimetria a scansione differenziale. È stato sviluppato un nuovo metodo quantitativo allo stato solido basato sulla Calorimetria a Scansione Differenziale (DSC) in grado di quantificare in modo selettivo e accurato la quantità di Budesonide amorfa presente in una miscela solida. Durante lo sviluppo del metodo sono stati affrontati problemi relativi alla convalida di metodi analitici su campioni solidi quali la miscelazione di polveri solide per la preparazione di miscele standard e il calcolo della precisione.

Uptake of gold nanoparticles in microvascular endothelial cells

The physicochemical properties of nanoparticles make them suitable for biomedical applications. Due to their ‘straight-forward’ synthesis, their known biocompatibility, their strong optical properties, their ability for targeted drug delivery and their uptake potential into cells gold nanoparticles are highly interesting for biomedical applications. In particular, the therapy of brain diseases (neurodegenerative diseases, ischemic stroke) is a challenge for contemporary medicine and gold nanoparticles are currently being studied in the hope of improving drug delivery to the brain.rnIn this thesis three major conclusions from the generated data are emphasized.rn1. After improvement of the isolation protocol and culture conditions, the formation of a monolayer of porcine brain endothelial cells on transwell filters lead to a reproducible and tight in vitro monoculture which exhibited in vivo blood brain barrier (BBB) characteristics. The transport of nanoparticles across the barrier was studied using this model.rn2. Although gold nanoparticles are known to be relatively bioinert, contaminants of the nanoparticle synthesis (i.e. CTAB or sodium citrate) increased the cytotoxicity of gold nanoparticles, as shown by various publications. The results presented in this thesis demonstrate that contaminants of the nanoparticle synthesis such as sodium citrate increased the cytotoxicity of the gold nanoparticles in endothelial cells but in a more dramatic manner in epithelial cells. Considering the increased uptake of these particles by epithelial cells compared to endothelial cells it was demonstrated that the observed decrease of cell viability appeared to be related to the amount of internalized gold nanoparticles in combination with the presence of the contaminant.rn3. Systematically synthesized gold nanoparticles of different sizes with a variety of surface modifications (different chemical groups and net charges) were investigated for their uptake behaviour and functional impairment of endothelial cells, one of the major cell types making up the BBB. The targeting of these different nanoparticles to endothelial cells from different parts of the body was investigated in a comparative study of human microvascular dermal and cerebral endothelial cells. In these experiments it was demonstrated that different properties of the nanoparticles resulted in a variety of uptake patterns into cells. Positively charged gold nanoparticles were internalized in high amounts, while PEGylated nanoparticles were not taken up by both cell types. Differences in the uptake behavior were also demonstrated for neutrally charged particles of different sizes, coated with hydroxypropylamine or glucosamine. Endothelial cells of the brain specifically internalized 35nm neutrally charged hydroxypropylamine-coated gold nanoparticles in larger amounts compared to dermal microvascular endothelial cells, indicating a "targeting" for brain endothelial cells. Co-localization studies with flotillin-1 and flotillin-2 showed that the gold nanoparticles were internalized by endocytotic pathways. Furthermore, these nanoparticles exhibited transcytosis across the endothelial cell barrier in an in vitro BBB model generated with primary porcine brain endothelial cells (1.). In conclusion, gold nanoparticles with different sizes and surface characteristics showed different uptake patterns in dermal and cerebral endothelial cells. In addition, gold nanoparticles with a specific size and defined surface modification were able to cross the blood-brain barrier in a porcine in vitro model and may thus be useful for controlled delivery of drugs to the brain.