Surface engineering of silica nanoparticles for oral insulin delivery: characterization and cell toxicity studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dually fluorescent silica nanoparticles

The cationic dyes 9-aminoacridine (9AA) and safranine (Sf) were entrapped into silica spheres of about 0.2 mu m diameter prepared by modified Stober method. The fluorescent materials are investigated by steady-state and time-resolved emission, in addition of confocal fluorescence microscopy. Silica particles containing 9-aminoacridine (SP9AA) and safranine (SPSf or both dyes (SPSf9AA) are emissive particles. When both dyes are present in the same particle but loaded in sequential stages 9AA emission is quenched as a consequence of energy transfer from 9AA (donor) to Sf (acceptor). This result suggests that particle growing processes where the acceptor is incorporated first into the core do not prevent donor/acceptor pairs to be close due to an overlay of the concentration gradients of both dyes in a radial core-shell like distribution. (C) 2010 Elsevier B.V. All rights reserved.

Dye doped, core / shell silica nanoparticles: synthesis, characterization, & biotechnological applications

The aim of this thesis was to design, synthesize and develop a nanoparticle based system to be used as a chemosensor or as a label in bioanalytical applications. A versatile fluorescent functionalizable nanoarchitecture has been effectively produced based on the hydrolysis and condensation of TEOS in direct micelles of Pluronic® F 127, obtaining highly monodisperse silica - core / PEG - shell nanoparticles with a diameter of about 20 nm. Surface functionalized nanoparticles have been obtained in a one-pot procedure by chemical modification of the hydroxyl terminal groups of the surfactant. To make them fluorescent, a whole library of triethoxysilane fluorophores (mainly BODIPY based), encompassing the whole visible spectrum has been synthesized: this derivatization allows a high degree of doping, but the close proximity of the molecules inside the silica matrix leads to the development of self - quenching processes at high doping levels, with the concomitant fall of the fluorescence signal intensity. In order to bypass this parasite phenomenon, multichromophoric systems have been prepared, where highly efficient FRET processes occur, showing that this energy pathway is faster than self - quenching, recovering the fluorescence signal. The FRET efficiency remains very high even four dye nanoparticles, increasing the pseudo Stokes shift of the system, attractive feature for multiplexing analysis. These optimized nanoparticles have been successfully exploited in molecular imaging applications such as in vitro, in vivo and ex vivo imaging, proving themselves superior to conventional molecular fluorophores as signaling units.

Design and Characterization of Luminescent Silica Nanoparticles

The aim of this thesis was to design, synthesize and characterize dye-doped silica nanoparticles (DDSNPs) to be used as chemosensors or labels in bioanalytical applications. DDSNPs represent one of the most versatile and useful components in nanomedicine displaying important features such as high colloid stability in water, low toxicity, one-pot inexpensive synthesis and tunable fluorescence emission. Starting from the one-pot and highly reproducible synthesis of “silica-core/PEG shell” DDSNPs based on the use of micelles of Pluronic F127, in which take place both hydrolysis and condensation of the silica precursor and of the dyes functionalized with a triethoxysilane group, we developed DDSNPs suitable for optical and optoacustic imaging, drug loading and chemical sensing obtaining very interesting results for the further development of nanomedicine.

Multiscale Characterization of Acrylic Bone Cement Modified with Functionalized Mesoporous Silica Nanoparticles

Acrylic bone cement is widely used to anchor orthopedic implants to bone and mechanical failure of the cement mantle surrounding an implant can contribute to aseptic loosening. In an effort to enhance the mechanical properties of bone cement, a variety of nanoparticles and fibers can be incorporated into the cement matrix. Mesoporous silica nanoparticles (MSNs) are a class of particles that display high potential for use as reinforcement within bone cement. Therefore, the purpose of this study was to quantify the impact of modifying an acrylic cement with various low-loadings of mesoporous silica. Three types of MSNs (one plain variety and two modified with functional groups) at two loading ratios (0.1 and 0.2 wt/wt) were incorporated into a commercially available bone cement. The mechanical properties were characterized using four-point bending, microindentation and nanoindentation (static, stress relaxation, and creep) while material properties were assessed through dynamic mechanical analysis, differential scanning calorimetry, thermogravimetric analysis, FTIR spectroscopy, and scanning electron microscopy. Four-point flexural testing and nanoindentation revealed minimal impact on the properties of the cements, except for several changes in the nano-level static mechanical properties. Conversely, microindentation testing demonstrated that the addition of MSNs significantly increased the microhardness. The stress relaxation and creep properties of the cements measured with nanoindentation displayed no effect resulting from the addition of MSNs. The measured material properties were consistent among all cements. Analysis of scanning electron micrographs images revealed that surface functionalization enhanced particle dispersion within the cement matrix and resulted in fewer particle agglomerates. These results suggest that the loading ratios of mesoporous silica used in this study were not an effective reinforcement material. Future work should be conducted to determine the impact of higher MSN loading ratios and alternative functional groups. (C) 2014 Elsevier Ltd. All rights reserved.

An in vitro toxicity evaluation of gold-, PLLA- and PCL-coated silica nanoparticles in neuronal cells for nanoparticle-assisted laser-tissue soldering.

The uptake of silica (Si) and gold (Au) nanoparticles (NPs) engineered for laser-tissue soldering in the brain was investigated using microglial cells and undifferentiated and differentiated SH-SY5Y cells. It is not known what effects NPs elicit once entering the brain. Cellular uptake, cytotoxicity, apoptosis, and the potential induction of oxidative stress by means of depletion of glutathione levels were determined after NP exposure at concentrations of 10(3) and 10(9)NPs/ml. Au-, silica poly (ε-caprolactone) (Si-PCL-) and silica poly-L-lactide (Si-PLLA)-NPs were taken up by all cells investigated. Aggregates and single NPs were found in membrane-surrounded vacuoles and the cytoplasm, but not in the nucleus. Both NP concentrations investigated did not result in cytotoxicity or apoptosis, but reduced glutathione (GSH) levels predominantly at 6 and 24h, but not after 12 h of NP exposure in the microglial cells. NP exposure-induced GSH depletion was concentration-dependent in both cell lines. Si-PCL-NPs induced the strongest effect of GSH depletion followed by Si-PLLA-NPs and Au-NPs. NP size seems to be an important characteristic for this effect. Overall, Au-NPs are most promising for laser-assisted vascular soldering in the brain. Further studies are necessary to further evaluate possible effects of these NPs in neuronal cells.

Protocol optimization for the mild detemplation of mesoporous silica nanoparticles resulting in enhanced texture and colloidal stability

Porosity development of mesostructured colloidal silica nanoparticles is related to the removal of the organic templates and co-templates which is often carried out by calcination at high temperatures, 500-600 °C. In this study a mild detemplation method based on the oxidative Fenton chemistry has been investigated. The Fenton reaction involves the generation of OH radicals following a redox Fe3+/Fe2+ cycle that is used as catalyst and H2O2 as oxidant source. Improved material properties are anticipated since the Fenton chemistry comprises milder conditions than calcination. However, the general application of this methodology is not straightforward due to limitations in the hydrothermal stability of the particular system under study. The objective of this work is three-fold: 1) reducing the residual Fe in the resulting solid as this can be detrimental for the application of the material, 2) shortening the reaction time by optimizing the reaction temperature to minimize possible particle agglomeration, and finally 3) investigating the structural and textural properties of the resulting material in comparison to the calcined counterparts. It appears that the Fenton detemplation can be optimized by shortening the reaction time significantly at low Fe concentration. The milder conditions of detemplation give rise to enhanced properties in terms of surface area, pore volume, structural preservation, low Fe residue and high degree of surface hydroxylation; the colloidal particles are stable during storage. A relative particle size increase, expressed as 0.11%·h-1, has been determined.

Detemplation of soft mesoporous silica nanoparticles with structural preservation

A mild protocol that allows the template removal of soft un-aged silica nanoparticles was investigated. After oxidizing the organic template by Fenton chemistry, a good structural preservation is only achieved when the material is equilibrated and dried in a low-surface tension solvent. This avoids excessive capillary stress induced by the high surface tension of water, a major component in the Fenton reaction medium. The Fenton reaction should be carried out under mild conditions as well; otherwise the sample deteriorates by extensive hydrolysis, and capillary stress, and the structural ordering diminishes severely. We propose employing 10 ppm Fe concentration at 70 °C for 24 h for the cetyltrimethylammonium bromide template. The proposed protocol involves 2 steps resulting in an overall significantly higher pore volume attributed to the wider pores and limited particle agglomeration, while the calcined counterpart evidences aggregation and loss of the hexagonal ordering. n-BuOH exchange is unnecessary when the mesophase is stabilized by ageing, as the structure resists the water capillary stress. © The Royal Society of Chemistry 2013.

Phytochemical-loaded mesoporous silica nanoparticles for nose-to-brain olfactory drug delivery

Central nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11-14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2%±2.2% over 24h and 9.4±0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100μM) demonstrating a decrease in cell viability to 58.2±8.5% and curcumin an IC50 of 33±0.18μM. Furthermore confocal microscopy demonstrated nanoparticles of <500nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.

Physicochemical characteristics of silica nanoparticles tailored for nanomedicine

Multifunktionella kiseldioxidnanopartiklar har en bred tillämpning inom nanomedicin. En attraktiv kombination är samtidig läkemedelstillförsel och spårning av nanopartiklarna, vilket är känt som ”teranostik”. Genom kontrollerad design av kiseldioxidnanopartiklarna kan terapi och diagnostik på detta sätt kombineras i samma partikel. För en lyckad användning av nanopartiklar inom nanomedicin måste deras fysikalisk-kemiska egenskaper vara välkontrollerade för att kunna förutspå deras beteende i biologiska system. I denna studie tillverkades kiseldioxidnanopartiklar med varierande storlek, form, yta och sammansättning med fokus på att utrusta nanopartiklarna med multifunktionalitet och på så sätt främja deras användning inom biomedicinska tillämpningar. Kiseldioxidnanopartiklar med sfäriska och stav-liknande former, porösa, icke-porösa och ihåliga strukturer tillverkades. De erhållna resultaten visade att nanopartiklarnas form har större inverkan på upptaget i celler jämfört med effekten av deras ytladdning. Nanopartiklarnas dispersionsstabilitet är en annan viktig aspekt för både diagnostiska och terapeutiska tillämpningar. Genom att ändra kiseldioxidnanopartiklarnas fysikalisk-kemiska ytegenskaper med ytfunktionalisering, bedömdes dispersionstabiliteten av nanopartiklarna. Nanopartiklarnas ytsammansättning justerades och skillnader i dispergerbarhet och dispersionsstabilitet undersöktes i biologiskt medium. Inom terapeutiska tillämpningar är målsökande läkemedelsfyllda nanopartiklar en lovande taktik som medför lägre läkemedelsdoser och minskar sidoeffekterna. I denna avhandling designades mesoporösa kiseldioxidnanopartiklar för användning som målsökande läkemedelsbärare. Dessa partiklar laddades med ett potentiellt anti-cancer läkemedel. En klart högre apoptotisk effekt kunde påvisas med de läkemedelsfyllda partiklarna jämfört med fri drog in vitro. En viktig egenskap för sådana multifunktionella nanopartiklar är också att kunna spåra dem under läkemedelsfrisättningen i biologisk miljö med olika bildåtergivningsmetoder. Detta kan uppnås genom att infoga en markör i kiseldioxidnätverket. Kiseldioxidnanopartiklarnas bildåtergivningsförmåga modifierades på olika sätt och inverkan på detekterbarheten analyserades med fluorescerande metoder och med magnetisk resonanstomografi. Denna avhandling lyfter fram de kritiska parametrarna vid syntes av kiseldioxidnanopartiklar för teranostiska tillämpningar. Olika metoder undersöktes för att erhålla skräddarsydda nanopartiklar. Detta arbete bidrar med en djup insikt i hur nanopartiklarnas fysikalisk-kemiska egenskaper påverkar deras beteende i biologisk miljö, och arbetet kan därför fungera som riktlinje för att designa säkra och effektiva nanomediciner.

Gold coating of silica and zinc oxide nanoparticles by the surface reduction of gold(I) chloride

The possibility of a surface inner sphere electron transfer mechanism leading to the coating of gold via the surface reduction of gold(I) chloride on metal and semi-metal oxide nanoparticles was investigated. Silica and zinc oxide nanoparticles are known to have very different surface chemistry, potentially leading to a new class of gold coated nanoparticles. Monodisperse silica nanoparticles were synthesised by the well known Stöber protocol in conjunction with sonication. The nanoparticle size was regulated solely by varying the amount of ammonia solution added. The presence of surface hydroxyl groups was investigated by liquid proton NMR. The resultant nanoparticle size was directly measured by the use of TEM. The synthesised silica nanoparticles were dispersed in acetonitrile (MeCN) and added to a bis acetonitrile gold(I) co-ordination complex [Au(MeCN)2]+ in MeCN. The silica hydroxyl groups were deprotonated in the presence of MeCN generating a formal negative charge on the siloxy groups. This allowed the [Au(MeCN)2]+ complex to undergo ligand exchange with the silica nanoparticles, which formed a surface co-ordination complex with reduction to gold(0), that proceeded by a surface inner sphere electron transfer mechanism. The residual [Au(MeCN)2]+ complex was allowed to react with water, disproportionating into gold(0) and gold(III) respectively, with gold(0) being added to the reduced gold already bound on the silica surface. The so-formed metallic gold seed surface was found to be suitable for the conventional reduction of gold(III) to gold(0) by ascorbic acid. This process generated a thin and uniform gold coating on the silica nanoparticles. This process was modified to include uniformly gold coated composite zinc oxide nanoparticles (Au@ZnO NPs) using surface co-ordination chemistry. AuCl dissolved in acetonitrile (MeCN) supplied chloride ions which were adsorbed onto ZnO NPs. The co-ordinated gold(I) was reduced on the ZnO surface to gold(0) by the inner sphere electron transfer mechanism. Addition of water disproportionated the remaining gold(I) to gold(0) and gold(III). Gold(0) bonded to gold(0) on the NP surface with gold(III) was reduced to gold(0) by ascorbic acid (ASC), which completed the gold coating process. This gold coating process of Au@ZnO NPs was modified to incorporate iodide instead of chloride. ZnO NPs were synthesised by the use of sodium oxide, zinc iodide and potassium iodide in refluxing basic ethanol with iodide controlling the presence of chemisorbed oxygen. These ZnO NPs were treated by the addition of gold(I) chloride dissolved in acetonitrile leaving chloride anions co-ordinated on the ZnO NP surface. This allowed acetonitrile ligands in the added [Au(MeCN)2]+ complex to surface exchange with adsorbed chloride from the dissolved AuCl on the ZnO NP surface. Gold(I) was then reduced by the surface inner sphere electron transfer mechanism. The presence of the reduced gold on the ZnO NPs allowed adsorption of iodide to generate a uniform deposition of gold onto the ZnO NP surface without the use of additional reducing agents or heat.

Mesoporous silica-chondroitin sulphate hybrid nanoparticles for targeted and bio-responsive drug delivery

This work proposes the fabrication of a novel targeted drug delivery system based on mesoporous silica-biopolymer hybrids that can release drugs in response to biological stimuli present in cancer cells. The proposed system utilizes mesoporous silica nanoparticles as a carrier to host the drug molecules. A bio-polymer cap is attached onto these particles which serves the multiple functions of drug retention, targeting and bio-responsive drug release. The biopolymer chondroitin sulphate used here is a glycosaminoglycan that can specifically bind to receptors over-expressed in cancer cells. This molecule also possesses the property of disintegrating upon exposure to enzymes over-expressed in cancer cells. When these particles interact with cancer cells, the chondroitin sulphate present on the surface recognizes and attaches onto the CD44 receptors facilitating the uptake of these particles. The phagocytised particles are then exposed to the degradative enzymes, such as hyaluronidase present inside the cancer cells, which degrade the cap resulting in drug release. By utilizing a cervical cancer cell line we have demonstrated the targetability and intracellular delivery of hydrophobic drugs encapsulated in these particles. It was observed that the system was capable of enhancing the anticancer activity of the hydrophobic drug curcumin. Overall, we believe that this system might prove to be a valuable candidate for targeted and bioresponsive drug delivery.

Layer-by-layer assembly of functional silica and Au nanoparticles for fabricating electrogenerated chemiluminescence sensor

We described the use of silica nanoparticles as building blocks for the immobilization of electrogenerated chemiluminescence (ECL) reagent Ru(bpy)3" and the fabrication of layer-by-layer assembly film by alternating the deposition of the Ru(bpy)3 2'-doped silica nanoparticles and Au nanoparticles.

Organosilylated complex [Eu(TTA)3(Bpy-Si)]: A bifunctional moiety for the engeneering of luminescent silica-based nanoparticles for bioimaging

A new highly luminescent europium complex with the formula [Eu(TTA) 3(Bpy-Si)], where TTA stands for the thenoyltrifluoroacetone, (C 4H3S)COCH2COCF3, chelating ligand and Bpy-Si, Bpy-CH2NH(CH2)3Si(OEt)3, is an organosilyldipyridine ligand displaying a triethoxysilyl group as a grafting function has been synthesized and fully characterized. This bifunctional complex has been grafted onto the surface of dense silica nanoparticles (NPs) and on mesoporous silica microparticles as well. The covalent bonding of [Eu(TTA)3(Bpy-Si)] inside uniform Stöber silica nanoparticles was also achieved. The general methodology proposed could be applied to any silica matrix, allowed high grafting ratios that overcome chelate release and the tendency to agglomerate. Luminescent silica-based nanoparticles SiO2-[Eu(TTA)3(Bpy-Si)], with a diameter of 28 ± 2 nm, were successfully tested as a luminescent labels for the imaging of Pseudomonas aeruginosa biofilms. They were also functionalized by a specific monoclonal antibody and subsequently employed for the selective imaging of Escherichia coli bacteria. © 2013 American Chemical Society.

Indocyanine Green Loaded Biocompatible Nanoparticles: Stabilisation of Indocyanine Green (ICG) Using Biocompatible Silica-Poly (&epsilon-Caprolactone) Grafted Nanocomposites

Indocyanine green (ICG) is a chemically labile compound which needs to be stabilized in aqueous media to be used in biomedical applications. In the present study, poly(ε-caprolactone) (PCL), a semi-crystalline polyester, was used to encapsulate and stabilize ICG in a hydrophobic environment. A hydrophobic and biocompatible nanocomposite was obtained by the process of encapsulating inorganic silica. ICG was embedded in the hydrophobic polymer coating by starting from a well-defined silica (Si) core of either 80 nm or 120 nm diameter, which served as a template for a ‘grafting from’ approach using ε-caprolactone. The obtained nanocomposite Si grafted PCL/ICG was based on silica nanoparticles grafted with PCL, in which ICG was adsorbed. The nanoparticles were characterized by IR spectroscopy, thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The change in the surface charge and the colloidal stability of the nanoparticles was followed by zeta potential measurements. This approach of synthesizing nanocomposite-based ICG demonstrates a new route to stabilize ICG. We synthesized biocompatible nanoparticles containing a high ICG concentration and exhibiting excellent stability to aqueous decomposition.