Rozprawy doktorskie na temat „Bio-medical Application - Drug Delivery”

Nanoparticle mediated drug delivery approaches provide potential opportunities for targeting and killing of intracellular bacteria. Among them, the porous silica nanoparticles deserve special attention due to their multifunctional properties such as high drug loading, controlled drug release and targeting of organs/cells. A review of the functional requirements of an ideal drug delivery system is provided. A general comparison between different drug delivery carriers and key issues to be addressed for intracellular drug delivery is discussed. Acid catalyzed and acid-base catalyzed, sol-gel derived, silica xerogel systems were investigated for sustained release of an aminoglycosides antimicrobial against salmonella infection in a mouse model. The release of gentamicin from the inner hollow part of the carrier is delayed. Further, the higher porosity of the acidâ base catalyzed silica xerogel allows for high drug loading compared to the acid catalyzed silica xerogel system. Efficacy of these particles in killing intracellular bacteria (salmonella) was determined by administering three doses of porous silica loaded gentamicin. This proved to be useful in reducing the salmonella in the liver and spleen of infected mice. Furthermore, the presence of silanol groups provides the ability to functionalize the silica xerogel system with organic groups, poly (ethylene glycol) (PEG), to further increase the hydrophilicity of the silica xerogel matrix and to modify the drug release properties. Increase in the hydrophilicity of the matrix allows for faster drug release rate. In order to facilitate controlled drug release, magnetic porous silica xerogels were fabricated by incorporating iron particles within the porous silica. The particles were fabricated using an acid-base catalyzed sol-gel technique. The in-vitro drug release studies confirm that the release rate can be changed by the magnetic field "ON-OFF" mechanism. This novel drug release methodology combined with the property of high drug loading capacity proves to be influential in treating salmonella intracellular bacteria. The potential application of any drug delivery carrier relies on the ability to deliver the requisite drug without adversely affecting the cells over the long term. We have developed silica/calcium nanocomposites and evaluated their solubility behavior. The solubility of particles was characterized by particle size measurements for different periods of time. It was found that the solubility behaviour of the silica/calcium particles was dependent on their calcium content. The results obtained demonstrate the potential to use mesoporous silica/calcium nano-composites for drug delivery applications. The significant contribution of this research to drug delivery technology is on design and development of the novel porous core-shell silica nano-structures. This new core-shell nano-structure combines all the above mentioned properties (high drug loading, magnetic field controlled drug release, and solubility). The main aim of preparing these porous core-shell particles is to have a control over the solubility and drug release property, which is a significant phenomenon, which has not been achieved in any other drug delivery systems. The shell layer acts as a capping agent which dissolves at a controllable rate. The rate at which the shell layer dissolves depends on the composition of the particles. This shell prevents the drug â leakageâ from the particles before reaching the target site. The core layer drug loading and release rate was modified by application of a magnetic field. Additionally, inclusion of the calcium ions in the core layer destabilizes the silica network and allows the particles to dissolve at an appropriate rate (which can be controlled by the concentration of the calcium ions).
Ph. D.

Block copolymer nanoparticles (NPs) have gained great attention among researcher for various medical application mainly due to their extraordinary optical, chemical, and biological properties. The current thesis presents design of multifunctional polymeric NPs for imaging and drug delivery system (DDS) with an in-vitro study of their participation in drug release and cell viability. The NPs were synthesized using reversible addition chain fragmentation transfer (RAFT)-mediated emulsion polymerization via polymerization induce self-assembly (PISA) approach. The environment-friendly emulsion polymerization process of n-buytl acrylate (n-BA) in water is highly efficient. The process produced uniform NPs which would have control over the particle size and molecular weight of the compound. Herein we report a novel simultaneous encapsulation of camptothecin (CPT) and Nile red (NR) into poly(ethylene glycol) methyl ether methacrylate-co-N-hydroxyethyl acrylamide-b-poly n-buytlacrylate (PEGA-co-HEAA)-b-P(n-BA) during the particles formation with a small particle size of 66 nm, high conversion ~80% and encapsulation efficiency of ~50%. The In vitro drug release of the CPT from the NPs exhibited an initial burst (70-80%) within 6h. cell viability was evaluated for the NPs against RAW 264.7 cell line, which indicated the designed NPs are biocompatible and not toxic.

The idea of utilizing nanomaterials in bio-related applications has been extensively practiced during the recent decades. Magnetic nanoparticles (MPs), especially superparamagnetic iron oxide nanoparticles have been demonstrated as promising candidates for biomedicine. A protective coating process with biocompatible materials is commonly performed on MPs to further enhance their colloidal and chemical stability in the physiological environment. Mesoporous hollow silica is another class of important nanomaterials that are extensively studied in drug delivery area for their ability to carry significant amount of guest molecules and release in a controlled manner. In this study, different synthetic approaches that are able to produce hybrid nanomaterials, constituting both mesoporous hollow silica and magnetite nanoparticles, are described. In a two-step approach, pre-synthesized magnetite nanoparticles are either covalently conjugated to the surface of polystyrene beads and coated with silica or embedded/enclosed in the porous shell during a nanosized CaCO3 templated condensation of silica precursors, followed by acid dissolution to generate the hollow structure. It was demonstrated that the hollow interior is able to load large amount of hydrophobic drugs such as ibuprofen while the mesoporous shell is capable of prolonged drug. In order to simplify the fabrication procedure, a novel in-situ method is developed to coat silica surface with magnetite nanoparticles. By refluxing the iron precursor with mesoporous hollow silica nanospheres in polyamine/polyalcohol mixed media, one is able to directly form a high density layer of magnetite nanoparticles on silica surface during the synthesis, leaving reactive amine groups for further surface functionalization such as fluorescence conjugation. This approach provides a convenient synthesis for silica nanostructures with promising potential for drug delivery and multimodal imaging. In addition to nanoparticles, nanowires also benefit the research and development of instruments in clinical diagnosis. Semiconductive nanowires have demonstrated their advantage in the fabrication of lab-on-a-chip devices to detect many charge carrying molecules such as antibody and DNA. In our study, In2O3 and silicon nanowire based field effect transistors were fabricated through bottom-up and top-down approaches, respectively, for ultrasensitive bio- detection of toxins such as ricin. The specific binding and non-specific interaction of nanowires with antibodies were also investigated.

The development of a drug delivery system (DDS) is essential to remedy the limitations of free drug molecules. The use of silica as a DDS over other systems (for example, liposomes) can be attributed to it being more robust and versatile. This thesis investigates bio-inspired silica (BIS) and compares it to mesoporous silica nanoparticles (MSN), which have received much attention for drug delivery applications. The BIS synthesis utilised amines to condense silica quicker than MSN, under benign conditions and without the use of hazardous chemicals. With this synthesis method drugs can be loaded in situ and there is potential for amines to have dual function of condensing silica and acting as functionalisation. BIS has also been shown to be more biocompatible than MSN. Due to these reasons it can be argued that BIS has the potential to be a more desirable silica DDS than MSN.Using ibuprofen as a model drug, reaction conditions (e.g. choice of amine additive, synthesis pH and maturation time) were systematically investigated to elucidate their effects upon drug loading and release. BIS synthesised with the amine poly(allylamine hydrochloride) (PAH) (which will henceforth referred to as BIS-PAH) was focused on, as this was the only amine system which released a significant proportion of loaded drug and achieved comparable or improved ibuprofen loading when compared to MCM-41. PAH plays an important role in facilitating the loading of ibuprofen, however if too much is present, release is inhibited greatly. The condensation rate of silica is also an important factor; when condensation rate was increased more drug was able to be released. This is likely due to less of the drug being entrapped within the silica particle and more being phys-adsorbed to the silica surface. Next the use of BIS to deliver hydrocortisone (HC) was investigated. Current treatments for adrenocorticoid insufficiency using hydrocortisone do no mimic the natural circadian variation in levels of blood cortisol. Firstly, the stability of HC during the in situ loading process was measured and data are presented that show that HC must be loaded post-synthesis, to avoid degradation in the reaction mixture. The efficiency of loading was largely unaffected by amine, however, only BIS-PAH allowed for drug release. Longer BIS-PAH maturation times gave lowered loading but the release was improved. Finally, biocompatibility of BIS was also investigated and it was found that, BIS was able to pass through the gut wall into the blood stream, and it was non-haemolytic when compared to MCM-41. There is a potential for bioaccumulation due to silica’s chemical stability. Although the use of BIS for delivery of hydrocortisone was unsuccessful, BIS does have several advantages over MCM-41 (such as quicker synthesis route, involving a one-pot synthesis and drug loading method, simple controllability, lack of hazardous chemicals and superior biocompatibility) and the results presented here show that BIS has similar or improved drug loading and release profiles to MCM-41 when using ibuprofen. With further drug and biocompatibility experiments, these benefits give BIS real potential as a viable DDS to be further investigated.

The goal of this project was to develop a series of nano platforms for single cell analysis and drug delivery. Nanoparticles are a promising option to improve our medical therapies by controlling biodistribution and pharmacokinetics of therapeutics. Nanosystems also offer significant opportunity to improve current imaging modalities. The systems developed during this thesis work can be foundations for developing advanced therapies for obesity and improving our fundamental understandings of single cell behavior. The first of the two systems we attempt to create was a drug delivery system that could selectively target adipose tissue to deliver uncoupling agents and drive browning of adipose tissue and associated weight loss. Protonophores have a history of significant toxic side effects in cardiac and neuronal tissues a recently discovered protonophore, but BAM-15, has been shown to have reduced cytotoxicity. We hypothesized that the altered biodistribution of BAM-15 encapsulated in a nanoparticle could provide systemic weight loss with minimized side effects. The second system developed utilized quantum dots to create a fluorescent barcode that could be repeatedly identified using quantitative fluorescent emission readings. This platform would allow for the tracking of individual cells, allowing repeat interrogation across time and space in complex multicellular environments. Ultimately this work demonstrates the process and complexity involved in developing nanoparticulate systems meant to interact with incredibly complex intracellular environments.

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Immunoglobulin (Ig) light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African Americans, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 years causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the characterization of AG10 , a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10 , combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy. The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life of therapeutic peptides. The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of a model peptide Gonadotropin Releasing Hormone (GnRH) and its analog GnRH-A without compromising their potency. Apart from GnRH, we have used other peptides to study their proteolytic stability in vitro . Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo . We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

A literature review of medical implant applications of mesoporous silica films was written, highlighting the advantages and limitations of different film synthesis methods. Both films synthesized through the EISA sol-gel method and particulate films, including those synthesized through the direct growth method, were reviewed and discussed. All films were found to have their strengths and weaknesses, however, the films synthesized through the direct growth method was found to be the most promising type for coating implants. In addition to the literature review, copper-doped mesoporous silica films were synthesized on titanium grade 2 substrates. SEM shows that particles grown on all the films and EDX elemental analysis confirms the presence of copper in the material. Nitrogen physisorption measurements show that particles with incorporated copper have a higher specific surface area, and pore volume compared to un-doped particles. No copper content could be confirmed through FTIR. The particles grown on titanium substrates were more rod-like compared to the ones grown on the silicon substrates as control.

In recent years, a number of reports have focused on the use of polyesters in drug delivery due to their intrinsic biocompatibility and biodegradability. In this thesis, aliphatic polyesters were synthesized by polycondensation reaction and ring opening polymerization reactions. The properties of the polymers and drug delivery potential of the resultant materials were evaluated. In the polycondensation reactions, a series of aliphatic polyesters of similar molecular weight were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effects of phenyl group side-chain substitution on polymer properties was investigated. A solvent-free melt polycondensation method using scandium (III) triflate as catalyst at an industrially relevant temperature (120 °C) was used. As the phenyl content increased, the polymers changed from semicrystalline to amorphous in state. The loading capability of polymers was checked by formulating nanoparticles containing coumarin 6 as a fluorescent dye analogue of active drugs. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid showed the highest dye loading among the set of materials synthesised. This polymer was found to be degradable over time under selected experimental conditions. Amphiphilic block co-polymers from the PluronicTM class were used to stabilize, in PBS, nanoparticles formed from these polyesters by nanoprecipitation routes. The metabolic activity, cell membrane integrity and lysosomal functions of C3A cells dosed with the polymers were determined to observe the cytocompatibility of the highest dye-loaded nanoparticles. Activity relative to undosed C3A cells was retained at more than 80% in the all of the assays. Imaging of Pluronic coated and uncoated nanoparticles in C3A cells suggested that both types of the nanoparticles were endocytosed in the early stage of the study (within 10 min). The internalization of nanoparticles was increased progressively over the study time. These results indicated the possible utility of the selected polymers in diagnostic and delivery applications. Ring opening polymerization (ROP) reactions were used for the synthesis of a diblock (mPEG-b-PεDL) and a triblock (PεDL-b-PEG-b-PεDL) copolymer from a seven membered ε-decalactone (ε-DL) monomer obtained from renewable sources. A diblock (mPEG-b-PεDL) copolymer was compared with structurally similar mPEG-b-PCL copolymer synthesized via ROP of ε-caprolactone (ε-CL) monomer, which can be considered as a non-renewable monomer. A six membered δ-decalactone (δ-DL) was also used for the synthesis of a diblock copolymer (mPEG-b-PδDL) to compare the reaction kinetics and properties of the copolymers. The copolymers were prepared via bulk polymerisation using 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) as a metal-free catalyst to replace the conventionally used stannous octoate [Sn(Oct)2]. A higher polymerization efficiency was achived with TBD compared to Sn(Oct)2 catalyst. However, a notable difference in the reaction temperature required for ε-DL and δ-DL polymerization was observed. The comparison with a structural analogue, i.e. ε-CL, demonstrated that the ε-DL polymerization was inhibited due to the presence of the alkyl chain of ε-DL monomer. However, a higher reaction time (12 h for TBD and 24 h for Sn(Oct)2) in CROP of ε-DL was addressed by using microwave based ring opening polymerization (MROP) reaction. The MROP was adopted as a ‘green’ and cheap heating method alternative to conventional heating (CROP) for the synthesis of mPEG-b-PεDL diblock copolymers using TBD as a catalyst. All the reactions were conducted in bulk. The MROP was designed based on the dielectric properties of all the reacting materials, as it was found that ε-DL monomers showed good absorption of MW radiation (tanδ>0.5). Accordingly, MROP resulted in a higher rate of ε-DL polymerization compared to CROP but comparison of the synthesis of mPEG-b-PCL copolymer by MROP indicated that the presence of the alkyl chain in ε-DL monomer significantly reduced the rate of polymerization. The synthesized mPEG-b-PεDL copolymer was investigated as a potential drug delivery vehicle for solubilization and controlled delivery of indomethacin. The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous core) was compared against well-established mPEG-b-PCL micelles (semicrystalline core). The drug-polymer compatibility was also determined through a predictive computational approach to access the drug solubilisation (or drug loading) into hydrated micelles. The micelles were prepared by solvent evaporation method and characterized for size, morphology, indomethacin (IND) loading and release. Both of the micelle formulations showed a uniform distribution of spherical micelles with size <60 nm. However, a significantly higher size of empty mPEG-b- PεDL micelle was observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted with PCL core as determined by modified Flory-Huggins interaction parameters (sp) using the Hanson solubility parameter (HSP) approach. The compatibility of the drug was determined for both of the segments (hydrophilic and hydrophobic) of the copolymers and found to be in the order of sp (PεDL)> sp (mPEG)> sp (PCL). The predictions suggested that more IND should encapsulate within the micelles with PCL core compared to PDL core, but the IND loading experiments revealed an overall higher loading in PεDL core (6.55 wt%) compared to PCL core (5.39 wt%) (P < 0.05, unpaired student’s t-test). However, consideration of the IND loading per unit volume of the micelles revealed that the PCL cored micelles was able to load 1.5 times more compared to the PεDL cored micelles. This result illustrated the higher compatibility of the IND with PCL core in accordance with the solubility parameter calculations. These data also suggested that the overall higher IND loading in PεDL core was attributable to the amorphous nature of the core which increased the core volume by 1.81 times compared to the PCL core. Drug release studies showed the sustained release pattern from both of the micelle systems although the semicrystalline PCL core (80% drug release in 110 h) was able to release the drug for a longer period compared to PεDL core (80% drug release in 72 h). Cell viability tests demonstrated the cytocompatibility of the mPEG-b-PεDL polymer. The micelles were internalized effectively in the early stages of the study and progressively increased with time. The results of the present thesis suggested that novel aliphatic polyester can be good candidates for the drug delivery applications and further studies can explore the possible applications of these polymers in the biomedical field.

The recent progresses offered by nanotechnology in the manipulation of matter lead to the development of several nanoparticles (NPs) and nanodevices for medical applications. In oncology, nanosized objects are particularly attractive as drug delivery systems since it is expected that engineered nanovehicles of appropriate size and functionalised with specific ligands/antibodies will improve the efficacy and selectivity of cancer therapies by exploiting both the passive and active mechanism of tumour targeting. The use of delivery systems is particularly appealing in those therapies in which the administration of the drug in aqueous formulations leads to drug aggregation with decreased activity or scarce bioavailability and tumour selectivity. This is the case of most of the photosensitizers used in photodynamic therapy (PDT), which display hydrophobicity and poor selective accumulation in malignant tissues. In the last decades, PDT is emerging as a promising cancer treatment modality in alternative to conventional therapies, which often demonstrate systemic drug toxicity and multidrug-resistance phenomena. PDT is based on the administration of a photosensitizer (PS) that accumulates in the tumour and after activation with light of appropriate wavelengths, reacts with surrounding molecular oxygen leading to the formation of cytotoxic reactive oxygen species (ROS) with consequent cellular and vasculature damages. In this PhD thesis, three different nanosystems, namely, liposomes, poly-(D,L-lactide-co-glycolide) nanoparticles (PLGA NPs) and ORganically Modified SILica nanoparticles (ORMOSIL NPs) were considered for the delivery of the second generation PS meta-tetra(hydroxyphenyl)chlorin (m-THPC, Temoporfin) to cancer cells in vitro. In particular, drug delivery efficiency, dark and phototoxicity of the m-THPC nanoparticle-based formulations were evaluated. To improve m-THPC bioavailability and tumour selectivity, in the design of the nanovehicles PEGylation and targeting of NPs were considered as essential strategies in order to prolong NP circulation in the bloodstream and exploit active mechanisms of tumour targeting. For the delivery of m-THPC using unilamellar liposomes, four different PEGylated liposomal formulations (trade name Fospeg®, provided by Biolitec Research) in which the length (PEG750, PEG2000, PEG5000) and the density (2%, 8%) of PEG were varied, were tested in vitro in normal lung fibroblasts CCD-34Lu and in cancer A549 lung epithelial cells. Compared to drug delivered in the standard solvent (Foscan®, ethanol/PEG 400/water (20:30:50, by vol.)), liposomal m-THPC showed a decreased intracellular uptake in both cell lines, but the presence of the delivery system highly reduced the dark cytotoxicity of the drug. The reduction of the PS dark toxicity increased with the increasing of PEG density on liposome surface, while the length of PEG chains did not affect significantly the toxic effect of m-THPC in the dark. However, photo-toxicity measured in A549 cells was only slightly affected by the reduced uptake of m-THPC delivered by Fospeg®, and the efficiency of PDT-induced cell killing was comparable among the different liposomal formulations. Interestingly, the intracellular localization of m-THPC delivered as Fospeg® or Foscan® was the same (Golgi apparatus and endoplasmic reticulum) suggesting drug release from liposomes, especially in the presence of the serum proteins, being m-THPC only physically entrapped within liposomes. m-THPC release was confirmed by the fact that liposomes covalently labelled with rhodamine were effectively were taken up by cells but, differently from m-THPC, localized in the acidic compartments of the cells. In spite of m-THPC release from liposomes, the Fospeg® formulation was exploited to target actively cancer cells by liposome conjugation with folic acid (FA), being FA-receptors (FRs) over-expressed in several human tumours. Thus, specific uptake and photo-toxicity of FA-targeted liposomes (FA-Fospeg) with respect to liposomes of the same composition but lacking FA (un-targeted Fospeg) was evaluated in KB (FR-positive) and in A549 (FR-negative) cells. The uptake of m-THPC delivered as FA-Fospeg was twice that of un-targeted Fospeg in KB cells; however only a modest fraction (~ 15%) of the targeted vehicle was effectively internalized by FR-mediated endocytosis while nonspecific internalization remained the prevailing mechanism of liposomes uptake in both cell lines. The improved m-THPC uptake obtained with FA-Fospeg in FR over-expressing cells translated into a 1.5 higher photo-induced toxicity. A novel formulation of bare and PEGylated PLGA NPs in which m-THPC was physically entrapped were synthesized (Prof J. Kos, University of Ljubljana) and evaluated in vitro and in vivo for phototherapy and fluorescence-based tumour imaging applications. In vitro studies carried out on A549, MCF10A neo T (breast cancer cells) and U937 (lymphoma derived pro-monocytic cells) cell lines, showed reduced uptake of PEGylated NPs with respect to non PEGylated NPs. As for Fospeg®, the use of the delivery system led to a significant reduction of m-THPC dark toxicity.. As expected for PEGylated NPs, the efficiency of cell internalization of m-THPC entrapped in PEG PLGA was reduced by 50% with respect to that in the standard solvent, but surprisingly cytotoxicity induced in irradiated A549 cells was quite comparable. At 24 h post-injection in vivo biodistribution of bare and PEGylated PLGA NPs compared to Foscan® was assessed in mice, showing very similar drug accumulation in the major organs but reduced skin uptake for both NP formulations. Thus, even if m-THPC release in the presence of serum proteins was measured in vitro, PEGylated PLGA NPs appeared potentially useful as stealth and biodegradable PS delivery systems. The premature release of the PS from the delivery system was completely avoided with the covalent link of m-THPC to the silane matrix of highly PEGylated ORMOSIL NPs (Prof. F. Mancin, University of Padova). This type of NPs exhibited a very low extent of cell internalization in vitro due to their high degree of PEGylation, making NP targeting an essential prerequisite to enhance intracellular drug delivery. In addition to FA, the RGD peptide and the antibody Cetuximab, which bind respectively the integrin α5ß3 receptor and epidermal growth factor receptor (EGFR), were exploited as targeting agents for ORMOSIL NPs and the specific uptake and photo-toxicity of m-THPC delivered by conjugated NPs were evaluated in vitro. The study revealed how the characteristics of the targeting agents are of crucial importance in determining the performances of targeted PEGylated nanosystems. In fact, the hydrophobic FA was very likely buried in the PEG layer and was unable to drive the selective uptake of ORMOSIL NPs while RGD peptide and Cetuximab antibody displayed some selectivity toward cells over-expressing their receptors (HUVEC cells over-expressing integrin α5ß3 receptors and A431 cells over-expressing EGFR). Unfortunately, the enhanced and selective uptake of m-THPC obtained by the two latter targeted ORMOSIL NPs was not accompanied by efficient and selective photo-induced cytotoxicity; it appeared that the selectivity of NP uptake was achieved in scarce drug cell loading conditions, determining only low PDT efficacy. The assessment of the biocompatibility of NPs is of fundamental importance for their safe use in nanomedicine. Since ORMOSIL NPs are not well characterised from this point of view, a toxicological characterization of empty ORMOSIL NPs were carried out in vitro in normal (CCD-34Lu) and cancer (A549, NCIH-2347) lung cells. The study included traditional cell viability and cytotoxicity tests (MTS test, LDH release assay, ROS production, cell membrane permeabilization measurements and electron microscopy analyses) in combination with a genome-wide analysis of gene expression profiles of cells exposed to NPs. The results pointed out that different types of cells respond quite differently to NPs and PEGylation of NPs highly affected the cytotoxicity profiles. PEGylation of ORMOSIL NPs completely abolished the toxicity of the nanosystem in CCD-34Lu and NCIH-2347 cells. On the contrary PEG ORMOSIL NPs induced necrotic cell death of A549 by increasing the permeability of the plasma membrane. At sub-lethal concentrations alteration of gene expression and inflammation were measured in A549 cells exposed to. The different response to PEG NPs is very likely explained considering the peculiarity of the cell type and the particular interaction of NPs with cell and internalization mechanisms. In fact, it was shown clearly that NPs internalized in A549 cells localized in and affected the morphology and the functioning of pulmonary surfactant containing lamellar bodies, peculiar of alveolar type II cells of which A459 cells represents an in vitro models.
Il recente progresso apportato dalla nanotecnologia nella manipolazione della materia ha portato al conseguente sviluppo di diversi tipi di nanoparticelle e nanodevices per applicazioni biomediche. In campo oncologico, oggetti dalle dimensioni nanometriche si sono dimostrati particolarmente interessanti in qualità di sistemi per la veicolazione di farmaci, poiché si presume che l’ingegnerizzazione dei nanoveicoli e la loro funzionalizzazione con specifici ligandi/anticorpi possa portare ad un miglioramento dell’efficacia e della selettività delle terapie antitumorali sfruttando meccanismi di targeting del tumore sia passivi che attivi. L’utilizzo di sistemi di veicolazione è particolarmente importante nel caso di terapie nelle quali la somministrazione dei farmaci in formulazioni acquose conduce a fenomeni di aggregazione con conseguente diminuzione di attività e di disponibilità nel circolo sanguineo, o nel caso di farmaci con scarsa selettività per il tumore. Appartengono a queste categorie la maggior parte dei fotosensibilizzanti utilizzati in terapia fotodinamica (PDT), poiché farmaci di natura idrofobica e con scarsa selettività di accumulo nei tessuti maligni. Negli ultimi decenni, la PDT si è dimostrata una promettente tecnica di trattamento del cancro in alternativa alle terapie convenzionali che invece generalmente dimostrano alta tossicità sistemica e fenomeni di farmaco-resistenza. La PDT si basa sulla somministrazione di un fotosensibilizzante (PS) che accumulatosi nel tumore, e dopo essere stato attivato con opportune lunghezze d’onda di luce, è in grado di reagire con l’ossigeno molecolare che lo circonda generando specie reattive dell’ossigeno (ROS) altamente citotossiche con conseguente danno cellulare e vascolare. In questa tesi di dottorato, tre diversi nanosistemi quali liposomi, nanoparticelle PLGA (poly-(D,L-lactide-co-glycolide)) e nanoparticelle di silice organicamente modificata (ORMOSIL), sono stati presi in considerazione per la veicolazione del fotosensibilizzante di seconda generazione meta-tetra(hydroxyphenyl)chlorin (m-THPC, Temoporfin) in cellule tumorali in vitro. In particolare, sono state valutate l’efficienza di veicolazione del farmaco, la tossicità buia e fotoindotta delle diverse formulazioni di m-THPC. Per migliorare la biodisponibilità e la selettività per il tumore della m-THPC, nella progettazione dei nanoveicoli sono state considerate quali strategie essenziali la pegilazione e il targeting delle particelle, in modo da prolungare la circolazione dei nanosistemi nel flusso sanguineo e in modo da sfruttare meccanismi attivi di targeting del tumore. Per la veicolazione della m-THPC utilizzando liposomi unilamellari sono state saggiate in vitro quattro diverse formulazioni liposomiali pegilate (Fospeg®, fornito dalla ditta Biolitec Research) con lunghezza (PEG750, PEG2000, PEG5000) e densità del PEG (2%, 8%) variabili, utilizzando come linee cellulari fibroblasti di polmone normali (CCD-34Lu) e cellule tumorali di epitelio polmonare (A549). Se paragonate al farmaco somministrato in forma libera in soluzione (Foscan®, etanolo/PEG 400/acqua (20:30:50, vol/vol)), le formulazioni liposomiali di m-THPC hanno mostrato una ridotta internalizzazione in entrambe le linee cellulari, ma nello stesso tempo la presenza del sistema di veicolazione ha portato alla significativa riduzione della tossicità buia del farmaco. La riduzione della tossicità buia del farmaco è risultata proporzionale all’aumento della densità di PEG sulla superficie del liposoma mentre la lunghezza delle catene di PEG sembra essere ininfluente nel limitare l’effetto tossico della m-THPC al buio. Comunque, la ridotta internalizzazione della m-THPC veicolata tramite Fospeg® influenza in modo solo parziale la fototossicità misurata in cellule A549, mentre l’efficienza d’induzione di mortalità in seguito a trattamento fotodinamico è risultata paragonabile tra le diverse formulazioni saggiate. Indipendentemente dalla veicolazione tramite Fospeg® o Foscan®, è stata riscontrata la medesima localizzazione intracellulare della m-THPC (apparato del Golgi e reticolo endoplasmatico) suggerendo il possibile rilascio del farmaco dalla formulazione liposomiale in presenza di proteine del siero, essendo la m-THPC solamente fisicamente intrappolata all’interno dei liposomi. Il rilascio della m-THPC è stato confermato dal fatto che liposomi nei quali viene legata covalentemente rodamina vengono effettivamente internalizzati dalle cellule e, differentemente dalla m-THPC, si accumulano nei compartimenti acidi intracellulari. Nonostante il rilascio del fotosensibilizzante dai liposomi, la formulazione Fospeg® è comunque stata utilizzata per veicolare selettivamente la m-THPC in cellule cancerose tramite la coniugazione dei liposomi con acido folico, essendo i recettori del folato sovraespressi in diversi tumori umani. Quindi sono state valutate l’internalizzazione specifica e la fototossicità di liposomi coniugati con folato (liposomi folato) rispetto a liposomi della stessa composizione ma privi di acido folico (liposomi non coniugati) in cellule KB e A549, rispettivamente positive e negative per l’espressione di recettori del folato. In cellule KB, l’internalizzazione della m-THPC si è rivelata doppia in caso di veicolazione con liposomi folato, malgrado solo una modesta parte (~15%) dei nanosistemi coniugati con folato siano effettivamente internalizzati tramite meccanismi di endocitosi mediata da recettore, essendo invece un’internalizzazione di tipo aspecifico il meccanismo prevalente per l’internalizzazione dei liposomi in entrambe le linee cellulari saggiate. In ogni caso, all’aumentato accumulo di m-THPC ottenuto tramite la veicolazione con Fospeg coniugato con folato in cellule che sovra esprimono il recettore, ne è conseguita una tossicità dopo irradiamento aumentata di circa 1.5 volte. Riguardo invece la veicolazione di m-THPC tramite particelle PLGA, formulazioni nude o pegilate sono state sintetizzate (Prof. J. Kos, Università di Lubiana) e saggiate sia in vitro che in vivo per la loro potenziale applicazione in fototerapia o in diagnosi dei tumori, sfruttando la fluorescenza del fotosensibilizzante fisicamente intrappolato all’interno delle particelle. Studi in vitro condotti su cellule A549, MCF10A neo T (derivate da tumore del seno) e U937 (cellule pro-monocitiche derivate da linfoma), hanno mostrato una ridotta internalizzazione della formulazione di m-THPC pegilata rispetto a quella nuda. Anche con particelle PLGA e come già visto per il Fospeg®, l’utilizzo di un sistema di veicolazione porta alla significativa riduzione della citotossicità buia della m-THPC. L’efficienza d’internalizzazione del fotosensibilizzante veicolato tramite particelle PLGA pegilate viene ridotta circa del 50% rispetto alla sua veicolazione nella formulazione standard ma sorprendentemente l’effetto citotossico indotto in cellule A549 irradiate è quasi paragonabile. La biodistribuzione della m-THPC (veicolata tramite nanoparticelle PLGA nude o pegilate o nella formulazione standard) è stata valutata 24 ore dopo la sua iniezione in topi, mostrando una simile distribuzione nei vari organi ma una significativa riduzione dell’accumulo a livello epidermico per entrambe le formulazioni nanoparticellari. Quindi, nonostante anche per le particelle PLGA pegilate sia stato misurato il rilascio di m-THPC in presenza di proteine sieriche, esse appaiono un buon sistema di veicolazione di fotosensibilizzanti soprattutto per le loro caratteristiche ‘stealth’ e per la loro biodegradabilità. Il rilascio prematuro del fotosensibilizzante è stato invece completamente limitato con il legame covalente della m-THPC alla matrice silanica di particelle ORMOSIL altamente pegilate (Prof. F. Mancin, Università di Padova). Tuttavia questo tipo di particelle ha mostrato un’internalizzazione intracellulare estremamente bassa derivata dall’elevato grado di pegilazione, ponendo come requisito essenziale il targeting delle particelle. In qualità di agenti di targeting per le particelle ORMOSIL pegilate sono stati valutati, oltre al folato, anche il peptide ciclico RGD e l’anticorpo Cetuximab, essendo questi ultimi in grado di legarsi rispettivamente ad integrine α5ß3 e al recettore del fattore di crescite dell’epidermide (EGFR). L’internalizzazione selettiva e la fototossicità della m-THPC veicolata tramite le tre diverse nanoparticelle funzionalizzate sono state valutate in vitro in opportuni sistemi cellulari. Tale studio ha mostrato come le caratteristiche dell’agente di targeting influenzino in modo sostanziale la selettività di tali nanosistemi pegilati. Infatti, mentre il folato altamente idrofobico si ripiega verosimilmente verso la corona di PEG rendendosi inefficace nel guidare selettivamente le particelle ORMOSIL, il peptide RGD e l’anticorpo Cetuximab hanno mostrato una certa selettività nei confronti di cellule sovraesprimenti i rispettivi recettori (cellule HUVEC sovraesprimenti recettori per le integrine α5ß3 e cellule A431 sovraesperimenti EGFR). Tuttavia, l’aumentato accumulo selettivo della m-THPC ottenuto tramite la coniugazione delle nanoparticelle con RGD e Cetuximab non ha portato ad una conseguente aumentata efficienza e selettività nell’induzione di citotossicità in seguito ad irradiamento. Tale risultato è verosimilmente imputabile al fatto che la selettività di accumulo delle nanoparticelle viene raggiunta in condizioni nelle quali la disponibilità del farmaco nelle cellule è troppo bassa, con conseguente scarsa efficacia dopo trattamento fotodinamico. La valutazione della biocompatibilità delle nanoparticelle risulta di fondamentale importanza per un’applicazione sicura della nanotecnologia in campo medico. Quindi, poiché le nanoparticelle ORMOSIL non sono ancora state ben caratterizzate da tale punto di vista, un loro profilo tossicologico è stato tracciato in vitro in cellule polmonari normali (CCD-34Lu) e tumorali (A549, NCIH-2347). Nello studio sono stati combinati esperimenti tradizionali di valutazione della vitalità cellulare e della citotossicità (test MTS, saggio del rilascio di LDH, valutazione della produzione di ROS, misure di permeabilizzazione di membrana, analisi di microscopia elettronica) con un’analisi dei profili di espressione genica estesa all’intero genoma di cellule esposte alle nanoparticelle. I risultati hanno mostrato come diversi tipi di cellule rispondono in modo abbastanza differente all’esposizione alle nanoparticelle e come la pegilazione influisce fortemente sui profili di citotossicità. Infatti, la pegilazione delle particelle ORMOSIL è in grado di abolire completamente la tossicità dei nanosistemi in cellule CCD-34Lu e NCIH-2347 mentre le stesse particelle pegilate inducono morte per necrosi in cellule A549, aumentandone la permeabilità di membrana. Inoltre nelle medesime cellule, concentrazioni sub-letali di nanoparticelle inducono infiammazione e alterazione dell’espressione genica. La differente risposta all’esposizione alle nanoparticelle pegilate delle cellule A549 è spiegabile considerando la peculiarità di questo tipo cellulare, e in particolare l’interazione delle particelle stesse con le cellule e il loro meccanismo d’internalizzazione. Infatti, è stato mostrato in modo chiaro che le nanoparticelle vengono internalizzate in corpi lamellari contenenti il surfattante polmonare, peculiari di cellule alveolari di tipo II, delle quali le cellule A549 rappresentano un modello in vitro. Tale accumulo delle nanoparticelle nei corpi lamellari porta alla modifica della morfologia degli stessi e una pesante alterazione della loro funzionalità.

Thesis (Ph. D.)--State University of New York at Binghamton, Thomas J. Watson School of Engineering and Applied Science, Department of Systems Science and Industrial Engineering, 2009..
Includes bibliographical references.

Carbon dots (CDs) are a novel carbon-based nanomaterial that draws a great attention in the last decade. The easy way of synthesis and the cheapest nature of the precursors used, stimulate a lot of scientist to propose new way of synthesis and applications. Notwithstanding the number of paper published, a systematic and reproducible way of synthesis is not yet been achieved, as well as a common definition of the resulting nanomaterials and their properties. In this thesis, the synthetic protocols and the characterization procedures used for the CDs will be deeply investigated in order to apply the nanomaterials in different applications. In particular, carbon dots synthesized from fully biocompatible precursors such as glucose, fructose and ascorbic acid, are characterized and employed for drug loading applications. The study reveals that the choice of the precursors is a crucial step because it affects the structural properties of the nanomaterials and the biological properties, revealing an unexpected toxicity for the fructose derived CDs, ascribable to its thermal degradation pathway. Furthermore, the drug loading capabilities were found to be correlated to the morphology of the nanoparticles, revealing the crucial role of the π-π interactions in achieving a loading up to 28 %wt for the glucose-based CDs. A second study was conduct on citric acid based CDs, in order to determine the best synthetic approach for photocatalytic applications. The nanoparticles synthesized both by hydrothermal and pyrolysis treatment, from sole citric acid and in combination with a nitrogen doping agent, were deeply characterized. The analysis reveals the difference in chemical, structural, and optical properties between the two synthetic methods. The photoreduction of methylviologen (MV) was used as model reaction to study the photocatalytic ability of the CDs, and the results reveal a relationship between the synthetic methods employed, the structural and optical properties of the CDs and their ability to act as a sensitizer. The amorphous nitrogen doped CDs reveals to be the best choice for this application with an initial rate conversion comparable to other reported in literature. The same CDs were also tested for the photocatalytic cleavage of C-O bonds in activated esters without the use of metals. The study reveals that CDs can successfully catalyze the reaction, with complete conversion and almost total selectivity. In addition, the CDs employed, shows different reactivity depending on the precursors and synthetic methods employed for their synthesis, with the same trend displayed for the MV photoreduction. This study highlights the ability of CDs to act as photosensitizer, without the addition of metals, in an organic reaction, opening a new scenario in the use of this nanomaterial in photocatalytic applications.

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.

Breast cancer is considered the second most commonly diagnosed type of cancer across the world. The common modes of treatment are limited by severe side-effects that hinder the efficacy of the drugs, compromise the patients' quality of life and often lead to other disorders. One of the main focuses of nanobiotechnology research is to develop novel anti-cancer drug delivery systems that improve the drug efficacy, limit harmful side effects and also allow for the delivery of developing therapeutics that are rapidly degraded in circulation, such as small interfering RNA (siRNA). Nano-carriers are helpful particularly in anti-cancer drug delivery due to the Enhanced Permeability and Retention (EPR) effect. In the current research study, we developed and investigated the use of surface modified HSA nanoparticles for the delivery of anti-cancer therapeutics in breast cancer applications. Results showed formation of modified HSA nanoparticles of sizes below 150 nm and contained a positive surface charge. The cellular uptake of the nanoparticles was higher in coated particles (average: ~70%) than uncoated particles. Furthermore, the cytotoxicity assessment of modified HSA nanoparticles suggested that empty particles are biocompatible and non-toxic to cells. Therefore, the presented PEI-enhanced and TAT-coated HSA nanoparticles form an appealing delivery system for anti-cancer therapeutics with a potential for clinical application.
Le cancer du sein est considéré comme le deuxième type de cancer le plus couramment diagnostiqué à travers le monde. La plupart des traitements sont characterisés par des effets secondaires nocifs qui limitent l'efficacité des médicaments, compromettent la qualité de vie des patients et conduisent souvent à d'autres troubles nocifs. L'un des principaux axes de recherche en nanobiotechnologie est de développer un nouveaux système de délivrance qui permet d'améliorer l'efficacité du médicament, de limiter les effets secondaires nocifs et aussi de permettre la livraison de molecules qui sont rapidement dégradées dans la circulation, tels que les petits ARN interférents (siRNA). Les nano-transporteurs sont utiles en particulier dans l'administration de médicaments anticancerigenes en raison de leur perméabilité accrue et de leur conservation (EPR). Dans l'étude de la recherche actuelle, nous avons développé et étudié l'utilisation de nanoparticules HSA à surface modifiée pour la livraison de médicaments anticancéreux dans les applications de cancer du sein. Les résultats ont montré la formation de nanoparticules HSA de tailles modifiées en dessous de 150 nm contenant une charge de surface positive. L'absorption cellulaire des nanoparticules est plus élevée dans les particules enrobées (moyenne: ~ 70%) que les particules non enrobée. Par ailleurs, l'évaluation de la cytotoxicité des nanoparticules HSA modifiées a suggéré que les particules vides sont biocompatibles et non toxiques pour les cellules. Par conséquent, les nanoparticules HSA revêtues de TAT et PEI-améliorée forment un système de prestation idéale pour les thérapies anti-cancereuses avec un potentiel d'application clinique.

Doctor of Philosophy
Department of Biochemistry and Molecular Biophysics
John M. Tomich
Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.

Trans-2-Aminocyclohexanol (TACH) is a promising model for pH-triggerable molecular switches with a variety of potential applications. In particular, such a switch, when incorporated into cationic liposomes, provides a novel design of the pH-sensitive helper lipids for gene delivery. Protonation of TACH molecules results in a strong intramolecular hydrogen bond between the amino and its neighboring hydroxyl groups, which triggers a conformational flip, and forces changes of the relative position of other substituents on the ring. In this work, a library of TACH-lipids has been designed and built based on structural modifications of both hydrophilic headgroups and hydrophobic tails, and their conformational behavior has been studied by 1 H NMR. NMR-titration has been done to quantitatively monitor the conformational switch for TACH derivatives. It was discovered that conformational behavior of TACH-lipids is independent from the length or shape of their hydrophobic tails. Therefore, a simplified model was suggested based on TACH with diethyl groups instead of hydrocarbon tails. Conformational study of these models has demonstrated that the position of equilibrium shift A [special characters omitted] BH + can be effectively changed by altering structure of NR 2 R 3 group. Furthermore, the pH-induced conformational flip occurs in a certain pH range that mostly depends on the basicity of group NR 2 R 3 , allowing a broad tuning of the pH-sensitivity of TACH-based conformational switches in a wide range of acidity. The hydrophilic OH group was also modified to influence the conformational equilibrium. External stimuli including addition of acid, change of solvent and of the solution ionic strength also showed impact on conformation equilibrium to different extents. To explore the potential to serve as pH-sensitive helper lipids in gene delivery, a variety of TACH-lipids were incorporated into lipoplexes together with the cationic lipid DOTAP to mediate DNA transfection in Bl6F1 and HeLa cancer cell lines. The lipoplex comprising TACH-lipid 3o (R 1 = C 19 H 37 ; R 2 R 3 = CF 3 CH 2 NH) exhibited one to two orders of magnitude better transfection efficiency than the one with the conventional helper lipid DOPE while only inducing slight higher cytotoxicity. Thus, the lipid can be suggested as a novel helper lipid for efficient gene transfection with low cytotoxicity.

Functionalized gold nanoparticles (AuNPs) provide an excellent scaffold for numerous biological applications. In these systems, the gold core imparts stability to the assembly, while the monolayer allows tuning of surface characteristics such as charge and hydrophobicity. The nano-scale size and tunable surface properties have made them an ideal candidate for manipulating protein-protein/protein-nucleic acid interactions, and delivery of therapeutics. In this thesis work, it has been demonstrated how the surface coating plays an important role in achieving a desired goal. Using organic synthesis as a tool, the monolayer was tailored to afford useful particles with biocompatibility and the ability to respond in the cellular environment. The recognition units present on the periphery of particles dictates/controls their interactions with biomolecular or cell surfaces. As described here, these engineered particles exhibited a number of bio-applications, including folding of a peptide into an α-helix, binding with DNA, and cellular delivery of genes and proteins.

Device-associated infections are a major healthcare challenge as they are responsible for high disease burden in critically ill patients. In this doctoral thesis project, we have developed drug-eluting antibacterial catheters to prevent catheter-related infections. Niclosamide (NIC), originally an antiparasitic drug with anti-staphylococcal activity, was incorporated into the polymeric matrix of thermoplastic polyurethane (TPU) via solvent casting, and catheters were fabricated using hot-melt extrusion technology. Similarly, based on Ink jet printing technology, polymeric inks containing niclosamide, were developed to coat polyethylene catheters. Both antibacterial approaches will release niclosamide in a controlled and sustained way, thus exerting its antimicrobial effect leading to the prevention device-associated infections. The mechanical and physicochemical properties of the devices ere studied. Moreover, the antibacterial efficacy of NIC-loaded catheters was validated with an in vivo biomaterial-associated infection model using a methicillin-susceptible and methicillin-resistant strain of S. aureus. The released NIC from the produced catheters reduced bacterial colonization of the catheter as well as of the surrounding tissue. In summary, the NIC-releasing catheters prevented implant colonization and reduced the bacterial colonization of peri-catheter tissue by methicillin sensitive as well as resistant S. aureus in a biomaterial-associated infection mouse model and has good prospects for preclinical development.

Cationic α-helical antimicrobial peptides (AMPs) hold promise for treatment of the raising multi-drug resistant microbial infections, due to their broad spectrum of activity and membrane-perturbing mechanism of action. Compared to conventional antibiotics, these features make them newsworthy molecules that hardly induce microorganisms to acquire resistance to them. Among these pathogens, Pseudomonas aeruginosa is the most clinically relevant Gram-negative bacterium known to cause serious human infections, e.g. pneumoniae, especially in immune-compromised patients, such as cystic fibrosis (CF) sufferers and keratitis, associated to contact lens (CL) wear. This is due to the unique ability of this pathogen to adhere to different types of inert materials or biological tissues, and to grow in a more resistant and dangerous sessile life form, called biofilm. Recently, two Esculentin-1a-derived antimicrobial peptides i.e. Esc(1-21) and its D-amino acids containing Esc(1-21)-1c, [Esc peptides], have been fully characterized for their powerful antipseudomonal activity against both planktonic and biofilm forms. The diastereomer showed a higher bactericidal activity than the all-L isomer against the more dangerous Pseudomonas biofilm phenotype; a lower cytotoxicity and higher biostability. However, when tested in vitro against the free-living form of this pathogen, it displayed a weaker bactericidal effect. Here, to investigate the reason accounting for this discrepancy, mechanistic studies on intact bacterial cells were initially carried out. Then to further understand the effect of packing parameters, i.e. composition, charge, shape and negative intrinsic curvature of membrane phospholipids in the membrane-permeabilizing activity of Esc peptides, leakage assays and circular dichroism spectroscopy analysis were carried out. Our results have suggested that the weaker in vitro antibacterial activity of Esc(1-21)-1c on the planktonic phenotype of the Gram-negative bacterium P. aeruginosa is mainly correlated to a slighter ability in permeabilizing both outer and inner bacterial membranes. Notably, experiments with lipid vesicles have suggested that if electrostatic interactions between negatively-charged membrane phospholipids and positively-charged peptide molecules do play a crucial role in the peptides’ membrane perturbing activity, this latter is hampered by the bilayer structure packing parameters including hydrogen bonding and intrinsic curvature, associated to phosphatidylserine (PE), especially for the diastereomer compared to all-L parent peptide. In parallel, we explored the molecular mechanism underlying the biofilm inhibition activity of Esc peptides when used at dosages below the minimal growth inhibitory concentration (1/8 MIC), by studying the peptides’ effect on the expression of key genes involved in the bacterial virulence and motility, as well as the peptide’ interaction with the bacterial signaling nucleotide ppGpp. Our findings revealed that the two D-amino acids containing Esc(1-21)-1c, confer the peptide the ability to downregulate the expression of biofilm-associated genes, likely as a result of increased peptide stability and prolonged binding to ppGpp compared to the all-L peptide. Furthermore, we reported two different applicative strategies to ameliorate the biological properties of these two AMPs: (i) encapsulation in poly(lactide-co-glycolide) (PLGA) nanoparticles; and (ii) covalent conjugation to soft CLs. In the first case, to enhance the peptides’ bioavailability and to optimize their translocation to the target infectious site, Esc peptides were loaded into PLGA nanoparticles (NPs) engineered with polyvinyl alcohol (PVA). The peptides-loaded NPs were found to be more efficient in diffusing through artificial CF mucus and simulated bacterial extracellular matrix compared to the free peptides. Moreover, they were more efficient in inhibiting P. aeruginosa growth under both in vitro and in vivo conditions at long term. In the second case, Esc peptides were covalently immobilized to hydrogel soft CLs and tested for their ability to reduce bacterial colonization. The antimicrobial CLs were able to cause more than four log reduction in the number of bacterial cells within 20 min and to reduce bacterial adhesion to their surface in 24 hours. Finally, the ability of both peptides to limit the onset of microbial resistance was also evaluated by exposing Pseudomonas strains to multiple cycles of treatment at sub-MIC dosages. Interestingly, in contrast with conventional antibiotics, Esc peptides did not induce resistance in P. aeruginosa cells. Overall, besides providing knowledges on the molecular mode(s) of action the two esculentin-derived AMPs, our data suggest that Esc peptides, particularly Esc(1-21)-1c, have great potential to be developed as novel drugs for treatment and prevention of P. aeruginosa pneumonia and keratitis.

博士
國立交通大學
應用化學系碩博士班
105
The prime objective of this thesis is the design and synthesis of chemosensor probes for applications in bio-imaging and redox stimuli-responsive nanocarriers based on MSNPs for targeted drug delivery in cancer therapy. Chapter 1 describes the significance of chemosensors and mechanism of signal transduction of several organic fluorescent probes for metal ions and ROS & RNS. Chapter 2 discusses a fluorescent probe HCTe for rapid detection of hypochlorous acid based on the specific HOCl-promoted oxidation of diphenyl telluride. The reaction is accompanied by an 82-fold increase in the fluorescence quantum yield (from 0.009 to 0.75). The fluorescence turn-on mechanism is achieved by the suppression of photoinduced electron transfer (PET) from the diphenyl telluride group to BODIPY. The fluorescence intensity of the reaction between HOCl and HCTe is linear in the HOCl concentration range of 1 to 10 μM with a detection limit of 41.3 nM (S/N = 3). In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrated that HCTe could be an efficient fluorescent probe for HOCl detection in living cells. Chapter 3 describes the pyrene-based fluorescent sensor for Cu(II) detection. It demonstrated high selectivity towards Cu2+ ions via PET based fluorescence enhancement. However, the metal ions Ag+, Ca2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+ produced no changes in the fluorescence emission of the system. The binding constant (Ka) of Cu2+ binding to PHP was found to be 1.00 X 104 M-1. The 1: 1 binding ratio of PHP-Cu2+ complex was determined from the Job plot. The maximum fluorescence enhancement caused by Cu2+ was observed between the pH ranges 5.0–10. Additionally, the PHP-Cu2+ complex reversibility with addition of EDTA was observed. Confocal fluorescence microscopy imaging using RAW264.7 cells showed that PHP can be used as an effective fluorescent probe for detecting Cu2+ in living cells. Chapter 4 describes the probe RhoSe for selective detection of mercury ions (Hg2+). RhoSe shows colorimetric and fluorescent turn-on responses towards Hg2+. The sensor probe RhoSe exhibited a fast response for Hg2+ with excellent sensitivity and the detection limits was found to be 12nM. The binding ratio of RhoSe-Hg2+ was determined by Job plots as a 1:1 ratio, and the effective pH range for Hg2+ detection was 4.0–10. Importantly, the reversibility of the RhoSe-Hg2+ complex was observed through the addition of Na2S. For practical applications, the strip method was utilized to detect Hg2+ in water. In addition, cell imaging experiments demonstrated that RhoSe is an effective fluorescent probe for Hg2+ detection in vitro and in vivo. Chapter 5 reports, a rhodamine based fluorescent probe RhoTe for Hg2+ detection. The fluorescent probe RhoTe displayed a selective response to mercury ions over other metal ions and ROS. Additionally, experiments with confocal fluorescence microscopy imaging using Hela cells and zebrafish showed that RhoTe can be used as an effective fluorescent probe for detecting Hg2+ in living cells and organism. To the best our knowledge, we first designed an incorporating of ‘dual lock’ rhodamine based fluorescent probe RhoTe receptors provides an alternative route for Hg2+ sensing. Chapter 6 discusses the mesoporous silica nanoparticle (MSNPs) for tumor-triggered targeting drug delivery to cancerous cells. Transferrin was covalently anchored on the surface of mesoporous silica nanoparticles via disulfide linking for glutathione-induced intracellular drug release. In this case, tumor-targeting agent enhances drug accumulation at the tumor site and transferrin functions as the gatekeeper to control the drug release. The successful functionalization of redox responsive MSNPs was confirmed by using BET / BJH, TEM, TGA, NMR and FT-IR respectively. More importantly, we demonstrated that the nanoparticles enter the cancer cell through the recognition of Tf receptor and the Tf gatekeeper is removed by the cleavage of the disulfide bond using an endogenous glutathione stimulus.