This PDF file contains the front matter associated with SPIE Proceedings Volume 10507, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Lanthanide-based upconversion nanoparticles (UCNPs) offer new strategies for luminescence-based sensing and imaging. One of the best studied materials are hexagonal 𝛽-NaYF4 UCNPs doped with 20 % Yb3+ and 2 % Er3+, which efficiently convert 976 nm light to photons emitted at 540 nm, 655 nm, and 845 nm, respectively, reveal long luminescence lifetimes (> 100 μs), and are very photostable and chemically inert.[1,2] The properties of their upconversion (UC) luminescence (UCL) are, however, strongly influenced by particle size, concentration and spatial arrangement of dopant ions, surface chemistry, and microenvironment.[3,4] In addition, the multiphotonic absorption processes responsible for UCL render UCL dependent on excitation power density (𝑃). The rational design of brighter UCNPs particle architectures encouraged us to assess systematically the influence of these parameters on UCL for differently doped UCNPs relying on the commonly used -NaYF4 matrix using steady state and time resolved fluorometry as well as integrating sphere spectroscopy for P varied over almost three orders of magnitude. This includes comprehensive studies of the influence of size and shell, Yb3+ and Er3+ dopand concentrations, and energy transfer processes from UCNPs to surface-bound organic dyes or vice versa [5]. Our results underline the need for really quantitative luminescence studies for mechanistic insights, the potential of high P to compensate for UCL surface quenching, and the matrix- and P-dependence of the optimum dopand concentration. Key words: upconverting nanoparticles, size, FRET, fluorescence, absolute fluorescence quantum yield, fluorescence decay kinetics, power density dependence References. [1] Haase, M.; Schafer, H., Angew. Chem. Int. Ed. 2011, 50, 5808-5829. [2] Liu, G. K., Chem. Soc. Rev. 2015, 44, 1635-1652. [3] Würth, C.; Kaiser, M.; Wilhelm, S.; Grauel, B.; Hirsch, T., Resch-Genger, U.; Nanoscale 2017, 9, 4283-4294. [4] Kaiser, M.; Würth, C.; Hyppanen I.; Soukka T;, Resch-Genger, Nanoscale 2017, on line (10.1039/C7NR02449E). [5] Kraft, M.; Würth, C.; Muhr, V.; Hirsch, T.; Resch-Genger, U., Nanoscale, submitted.

Quantum dots (QDs) emitting in the visible are of interest for many biomedical applications, including bioimaging, biosensing, drug targeting, and photodynamic therapy. However, a significant limitation is that QDs typically contain cadmium, which makes prospects for their FDA approval very unlikely. Previous work has focused on InP and ZnO as alternative semiconductor materials for QDs. However, these nanoparticles have also been shown to be cytotoxic. High-efficiency luminescent ZnTe-based QDs could be a reasonable alternative to Cd-containing QDs. In this paper, we present preliminary results of our recent studies of ZnTe-based QDs, including their synthesis, structural characterization, and optical properties.

Bright-field thermal imaging at the microscopic level was proved to be possible by quantitatively measuring the transmitted phase with a Quadriwave Lateral Shearing Interferometry (QWLSI). This approach, introduced by G. Baffou in collaboration with PHASICS SA [1], is able to measure temperature fields from temperature-induced refractive index changes of the medium surrounding heat sources. These heat sources are generated by a continuous plane laser wave source illuminating nano-particles at their plasmonic resonance. Heat maps and temperature fields are measured in parallel thanks to a QWLSI SID4BIO camera, developed by PHASICS. The quantitative phase signal is deconvolved to obtain the temperature variation and heat map values. With this set-up, a resolution of 1°C has been achieved. We present here a highly-resolution thermal imaging system applied to the detection and characterization of isolated single nano-particles by measuring their cross-section size. Because single nano-particles induce low temperature signals, we implement a noise-reduction modality based on synchronous detection. To do so, we use a modulated laser source locked-in with the SID4Bio camera. Since the heating process is modulated in time, this configuration improves the signal-to-noise ratio of the temperature measurement as we get rid of most of low-frequency noise which is a main contribution of the noise signal. The carried out experimental measurements presented will be analyzed along with the theoretical calculations introduced in [1]. References [1] Baffou, G., Bon, P., Savatier, J., Polleux, J., Zhu, M., Merlin, M., ... & Monneret, S. (2012). Thermal imaging of nanostructures by quantitative optical phase analysis. ACS nano, 6(3), 2452-2458.

A two-step high temperature colloidal doping technique has been used to synthesis Co-Ni co-doped CdS/ZnS core/shell nanocrystals in organic phase. Co-Ni ions were incorporated into the CdS quantum dots core pursued by ZnS shell (six monolayers) which was developed on top of it. The co-doped nanocrystals were further characterized usingtransmission electron microscopy (TEM),X-ray diffraction (XRD), ultraviolet-visible (UV-VIS) spectroscopy and photoluminescence (PL) spectroscopy measurements. The co-doped CdS/ZnS nanocrystals show two peaks; the sharp emission peak is due to CdS quantum dot at 440 nm while the broad emission peak (520–650nm) is due typical to Co-Ni ions as compared to undoped CdS/ZnS nanocrystals.

Gold nanostars are well-known as effective substrates for applications in which near field enhancements are sought, owing to their uniquely sharp protruding spikes. In particular, we have shown how they can be employed to build sensing platforms for the direct identification of small molecule analytes via surface enhanced Raman spectroscopy (SERS) achieving femtomolar limits of detection. We have also demonstrated how they can be conjugated to peptides for the identification of cancerous cells, and to aptamers for targeting and recognition of prostate cancer cells, with reduced cytotoxicity compared to other colloidal counterparts. Herein, we will show how gold nanostars can be uniquely employed for the identification of influenza virus mutations at the single cell levels and how silica coating layers can be selectively employed to increase stability and biocompatibility and tune the electric field enhancement. However, what is also well known about these nanoparticles is that they are severely impacted by synthetic protocols with low batch-to-batch reproducibility and substantial sample polydispersity, which limit their applicability and scale up. We have addressed this issue by developing a novel bottom-up seed-mediated protocol that produces nanostars with tunable spike lengths and intense localized surface plasmon resonance (LSPR) bands that fall well into the IR region of the spectrum. The unique plasmonic properties of these nanostars, which are also characterized by multiple, well-separated higher harmonics of the fundamental LSPR mode, promise to render them quite advantageous for biomedical applications.

Enhancement in enzymatic activity after attachment to nanoparticle surfaces has been observed in numerous enzyme systems, although the underlying mechanism for these enhancements remains largely unknown. This work explores the utility of a model based on a reaction scheme that takes into account some of the many interactions between substrate, product, and nanoparticle that can occur. This model was utilized to make predictions about the type of behavior that should manifest itself with quantum dots peripherally displayed around beta-galactosidase (&beta-gal) and confirmed empirically. &beta-gal is a homotetrameric enzyme which at ~465 kDa is significantly larger than the 4.2 nm diameter green emitting quantum dots utilized to decorate its periphery. Because &beta-gal operates near the diffusion limit, this provides an opportunity to selectively investigate certain aspects of enzyme enhancement when attached to a nanoparticle with minimal perturbation to the native enzyme structure. Enzymatic assays were performed with both free enzyme and quantum dot-decorated enzymes in a side-by-side format where kinetic processes were challenged by increasing viscosity with glycerol and competitive inhibitors such as lactose. The results from this model suggest it is possible to achieve significant enhancements in a diffusion limited enzyme’s catalytic rate (< i>k< sub>cat< sub>< i>) after NP attachment without substantial changes to the enzyme’s structure or function. Because cell free synthetic biology is gaining importance, this approach will yield insights on how enzymes can be utilized ex vivo and how being attached to NP scaffolds yields kinetic enhancement, possibly through enhanced product dissociation.

In this paper we investigate how the density of oligonucleotides around spherical 15 nm gold nanoparticles (AuNPs) influences their stability against degradation by DNase I. We show that a significant decrease in the number of oligonucleotides attached to the AuNP surface is directly correlated with an increase in the DNA degradation by DNase I. Our experimental observations suggest that a close packing of oligonucleotides into a 3d arrangement at the surface of AuNPs endows the probes with the necessary stability required for their use in intracellular applications.

Advancements in nanotechnology sensors have aided in the detection of subtle, but significant cellular deviations that may mark certain stages of diseases. Gold nanorods (GNRs) are often studied for this purpose due to their tunable optical properties and ease in surface functionalization. The absorption properties of GNRs are governed by the localized surface plasmon resonance (LSPR), which strongly depends on the GNR’s aspect ratio and on interparticle interactions. By controlling the coupling of nearby rods, a sensor can be created to respond to temperature fluctuations in the local environment. Here, we fabricated thermo-responsive gold nanorod assemblies by conjugating GNRs in end-to-end or side-by-side configurations using Poly(N-isopropyl acrylamide) (PNIPAM). End-to-end assemblies were fabricated through mixture of GNRs and PNIPAM in DI water. GNRs and PNIPAM were combined in DI water and dimethylformamide (DMF) under sonication to achieve side-by-side configuration. The optical absorption of the assemblies was measured by UV-Visible spectroscopy at different temperatures. As the temperature increased, the polymer contracted and initiated plasmon coupling between the GNRs. The optical spectrum experienced a blue- or red-shift for side-by-side or end-to-end configurations, respectively. Spectral tunability reversal was observed when cooled. Experimental results were verified by finite-difference time-domain (FDTD) calculations, which demonstrated spectral shifts under similar parameters. We present methods for fabrication of thermo-responsive gold nanorods for use as a local thermal nanosensor.

Ultrasmall fluorescent gold nanoclusters (Au NCs) have drawn considerable research interest owing to their molecular like properties such as d-sp and sp-sp transitions, and intense fluorescence. Fluorescent Au NCs have especial attraction in biological system owing to their biocompatibility and high photostability. Recently, several strategies have been adapted to design an artificial light-harvesting system using Au NCs for potential applications. Here, we have designed Au nanoclusters based dsDNA (double stranded deoxyribonucleic acid) nano assemblies where the Au nanocluster is covalently attached with Alexa Fluor 488 (A488) dye tagged dsDNA. Investigation reveals that the incorporation of Ag⁺ into dsDNA enhances the rate of energy transfer from A488 to Au NCs. In addition cadmium telluride quantum dot (CdTe QDs) based Au NCs hybrid material shows the significant enhancement of energy transfer 35% to 83% with changing the capping ligand of Au NCs from glutathione (GSH) to bovine serum albumin (BSA) protein. Another hybrid system is developed using carbon dots and dye encapsulated BSA-protein capped Au NCs for efficient light harvesting system with 83% energy transfer efficiency. Thus, Au NCs base nano bio assemblies may open up new possibilities for the construction of artificial light harvesting system.

The emerging field of remote enzyme activation, or the ability to remotely turn thermophilic increase enzyme activity, could be a valuable tool for understanding cellular processes. Through exploitation of the temperature dependence of enzymatic processes and high thermal stability of thermophilic enzymes these experiments utilize nanoparticles as ‘antennae’ that convert radiofrequency (RF) radiation into local heat, increasing activity of the enzymes without increasing the temperature of the surrounding bulk solution. To investigate this possible tool, thermolysin, a metalloprotease was covalently conjugated to 4nm gold coated magnetite particles via peptide bond formation with the protecting ligand shell. RF stimulated protease activity at 17.76 MHz in a solenoid shaped antenna, utilizing both electric and magnetic field interactions was investigated. On average 40 percent higher protease activity was observed in the radio frequency fields then when bulk heating the sample to the same temperature. This is attributed to electrophoretic motion of the nanoparticle enzyme conjugates and local regions of heat generated by the relaxation of the magnetite cores with the oscillating field. Radio frequency local heating of nanoparticles conjugated to enzymes as demonstrated could be useful in the activation of specific enzymes in complex cellular environments.

We review here different methods to measure the bulk viscosity of complex fluids using micron-sized magnetic wires. The wires are characterized by length of a few microns and diameter of a few hundreds of nanometers. We first draw analogies between cone-and-plate rheometry and wire-based microrheology. In particular we highlight that magnetic wires can be operated as stress-controlled rheometers for two types of testing, the creep-recovery and steady shear experiments. In the context of biophysical applications, the cytoplasm of different cell lines including fibroblasts, epithelial and tumor cells is studied. It reveals that the interior of living cells can be described as a viscoelastic liquid with a static viscosity comprised between 10 and 100 Pas. We extend the previous approaches and show that the proposed technique can also provide time resolved viscosity data, which for cells display strong temporal fluctuations. The present work demonstrates the high potential of the magnetic wires for quantitative rheometry in confined espaces.

The human mouth is a host of a large gamut of bacteria species, with over 700 of different bacteria strains identified. Most of these bacterial species are harmless, some are beneficial (such as probiotics assisting in food digestion), but some are responsible for various diseases, primarily tooth decay and gum diseases such as gingivitis and periodontitis. Dental plaque has a complicated structure that varies from patient to patient, but a common factor in most cases is the single species of bacterium acting as a secondary colonizer, namely Fusobacterium nucleatum, while the actual disease is caused by a variety of tertiary colonizers. We hypothesize that destruction of a compound biofilm containing Fusobacterium nucleatum will prevent tertiary colonizers (oral pathogens) from establishing a biofilm, and thus will protect the patient from developing gingivitis and periodontitis. In this paper, we report on the effects of exposure of compound biofilms of a primary colonizer Streptococcus gordonii combined with Fusobacterium nucleatum to iron oxide nanoparticles as possible bactericidal agent.

Metallic nanoparticles are very interesting because of their potential use in microelectronics, optical devices, biomedical and sensing applications. Their individual electromagnetic response is highly dependent on the specific size, shape, and surrounding environment. Nowadays, there are different methods which allow us to fine tune these parameters and thus, the materials properties. On the other hand, surface enhance Raman scattering (SERS) spectroscopy is a powerful ultra-sensitive technique which allows detection down to single molecule levels. However, the production of effective and highly active SERS substrates is mainly based on the generation of hot spots created by plasmonic nanoparticles. Nonetheless, the lack of capability to form reproducible organized structures at large scales is still a very important challenge to solve in order to produce substrates with homogeneous and reproducible SERS intensities over large areas. In this work, we report novel methods to produce arrays of nanoparticles, either as continuous or as isolated super-crystals made with plasmonic nanoparticles.[1] These macro-scale organizations were created via confinement controlled drying and spin coating. SERS studies on the super-crystals shape and size were conducted. Moreover, the produced structures were effectively use for SERS sensing showing very good reproducibility among big areas. This fact, make them perfect candidates as ultrasensitive substrates for SERS due to the controlled formation of hot spots. Which provide high and uniform SERS enhancement over extended areas. [1] Nanoscale. 2016, 8 12702

Nanoparticle (NP)-mediated drug delivery offers the potential to overcome limitations of systemic delivery, including the ability to specifically target cargo and control release of NP-associated drug cargo. Doxorubicin (DOX) is a widely used FDA-approved cancer therapeutic; however, multiple side effects limit its utility. Thus, there is wide interest in modulating toxicity after cell delivery. Our goal here was to realize a NP-based DOX-delivery system that can modulate drug toxicity by controlling the release kinetics of DOX from the surface of a hard NP carrier. To achieve this, we employed a quantum dot (QD) as a central scaffold which DOX was appended via three different peptidyl linkages (ester, disulfide, hydrazone) that are cleavable in response to various intracellular conditions. Attachment of a cell penetrating peptide (CPP) containing a positively charged polyarginine sequence facilitates endocytosis of the ensemble. Polyhistidine-driven metal affinity coordination was used to self-assemble both peptides to the QD surface, allowing for fine control over both the ratio of peptides attached to the QD as well as DOX dose delivered to cells. Microplate-based Förster resonance energy transfer assays confirmed the successful ratiometric assembly of the conjugates and functionality of the linkages. Cell delivery experiments and cytotoxicity assays were performed to compare the various cleavable linkages to a control peptide where DOX is attached through an amide bond. The role played by various attachment chemistries used in QD-peptide-drug assemblies and their implications for the rationale in design of NPbased constructs for drug delivery is described here.

Nanoparticles are extensively used for drug delivery applications. One crucial parameter is the nanoparticle degradation rate, which governs the targeted release of drugs and pharmacokinetics. Optimizing the rate of degradation is a crucial challenge that requires accurate nano-characterization methods. Here we present on-chip microscopy-based techniques to quantify nanoparticle degradation. The lensfree holographic on-chip microscope that was used for this purpose consists of a set of individually-controlled fiber-coupled LEDs, a band-pass filter, a CMOS imager and a temperature controlled polyethylene-glycol reservoir. This mobile microscope captures a set of sub-pixel shifted holograms by turning on/off individual LEDs, where each nanoparticle hologram is significantly enhanced using self-assembled polyethylene-glycol nanolenses. These shifted holograms are used to synthesize a pixel super-resolved hologram, which is then reconstructed to obtain the phase image of the nanoparticles, containing their size information with a sizing accuracy of ±11 nm. Using this technique we quantified three types of growth-factor loaded nanoparticles, i.e., degradable, partially-degradable and non-degradable. By sampling a subset of the particles undergoing degradation at discrete time points, we observed a drastic reduction in the size of the degradable nanoparticles in <10 min, whereas the others did not show any major change till ~24 hours. As an alternative approach, we also created of a sandwich assay by placing a protease-coated coverslip on the nanoparticle/nanolens complex and monitored the particle degradation in real-time. These approaches provide a cost-effective and high-throughput platform to monitor the degradation process of nanoparticles using a sample volume that is >3 orders-of-magnitude smaller than other techniques.

Aedes aegypti is one of the mosquito species with major epidemiological importance in Brazil, involved with the transmission of the arboviruses such as Dengue, Chikungunya and Zika. Since the mosquito is well adapted to the urban environments, where there are large amounts of breeding sites for larvae and pupae, its control has become increasingly difficult. Since 1996, the usual control is made by using chemical larvicides, but the continued use of some of these compounds has led to the selection of A. aegypti resistant populations. Therefore, the search for new insecticidal substances is necessary to guarantee the control of this specie. Our goal is to establish a new larvicide with high toxicity without the drawback of resistance. For this, we developed a low-cost green synthesized silver nanoparticles with a poly(methacrylic acid) outer layer, catalyzed with ultra violet light. We tested nanoparticles samples produced from different batches in laboratory bioassays against fourth-instar larvae. The results showed a desired toxic activity, presenting 10% to 100% of mortalities in concentrations ranging from 0.01 to 1.1 ppm, and from the bioassay we have obtained LC₅₀ = 0.027 ppm and LC₉₀ = 0.044 ppm, after 24 hours of exposure. Imaging the threated larvae by optical microscopy and optical coherence tomography helped to clarify the potential larvae death mechanism. These results associated with the simplicity and low cost of production of these silver nanoparticles, reveal their great potential for the development of products to control of A. aegypti larvae.

Amphiphilic macromolecules with multiple borondipyrromethene (BODIPY) chromophores appended to a common poly(methacrylate) backbone were synthesized by the random co-polymerization of appropriate methacrylate monomers. The resulting polymers incorporate also hydrophilic oligo(ethylene glycol) and hydrophobic decyl side chains designed to impose aqueous solubility and insulate the chromophoric components from each other respectively. The presence of multiple chromophores translates into a significant enhancement in molar absorption coefficient, relative to a model BODIPY monomer. The effective insulation of the fluorophores minimizes interchromophoric interactions and mitigates depressive effects on the fluorescence quantum yield. The overall result is a 6-fold enhancement in brightness, relative to the model monomer. These macromolecular probes can be injected into live Caenorhabditis elegans to allow their visualization with a 4-fold increase in signal intensity, relative to the model system. Furthermore, they can be conjugated to secondary antibodies, under standard amide-coupling conditions, with negligible influence on the binding affinity of the biomoleucles to allow the implementation of immunolabeling protocols.

Using a few solution phase NMR spectroscopy techniques, including 1H NMR and 31P NMR, we have characterized the organic shell on CdSe-ZnS core-shell quantum dots and tracked changes in its composition when the QD dispersions are subjected to varying degrees of purification. Combining solution phase NMR with diffusion ordered spectroscopy (DOSY), we were able to distinguish between freely diffusing ligands in the sample from those bound on the surfaces. Additionally, matrix assisted laser desorption ionization (MALDI) and FTIR measurements were used to provide complementary and supporting information on the organic ligand coating for these nanocrystals. We found that the organic shell is dominated by monomeric or oligomeric n-hexylphosphonic acid (HPA), along with small portion of 1-hexadecyl amine (HDA). The presence of TOP/TOPO (tri-n-octylphosphine / tri-noctylphosphine oxide) molecules is much smaller, even though large excess of TOP/TOPO were used during the QD growth. These results indicate that HPA (alkyl phosphonate) exhibits the strongest coordination affinity to ZnS-rich QD surfaces grown using the high temperature injection route.

The unique photophysical properties of gold nanomaterials combined with progress in developing effective surfacefunctionalization strategies has motivated researchers to employ them as tools for use in biomedical imaging, biosensing, diagnostics, photothermal therapy, and as drug and gene delivery vehicles. However, a major challenge limiting these advancements has been the unavailability of effective strategies to deliver these and other nanocrystals into the cytoplasm of live cells. In this study, we demonstrate that the use of a chemically-synthesized anti-microbial peptide, SVS-1, can promote non-endocytic uptake of both small size gold nanoparticles (AuNPs) and larger size gold nanorods (AuNRs) into mammalian cells. For this, colloidally stable AuNP and AuNRs, surface ligated with an amine-functionalized polymer, His-PIMA-PEG-OCH₃/NH₂ were prepared. The amine groups allow dual, covalent attachment of cysteine terminated SVS-1 (via a thioether linkage) and NHS-ester-Texas-Red dye onto the nanocrystal surfaces. We use fluorescence microscopy to demonstrate nanocrystal staining throughout the cytoplasmic volume of the cells incubated with these conjugates. More importantly, we have conducted additional endocytosis inhibition experiments where cells were incubated with the conjugates at 4°C. Here too, the imaging data have shown significant levels of nanocrystal uptake, further verifying that physical translocation of these conjugates takes place through the cell membrane independent of endocytosis. These findings are promising and can provide critical support for the widespread applications of nanomaterials in the field of biology.

A cell spheroid can be as large as 1 mm in diameter, containing more than 300,000 cells. It can be used for simulating a cancer tumor in cell treatment study. In this study, the cell uptake behaviors in cell spheroids are investigated. In particular, the penetration depths of Au nanoring, Au nanorod, and photosensitizer (AlPcS) in cell spheroids are evaluated based on a newly proposed technique of using confocal fluorescent microscopy. By using a geometric computation process, we can estimate the width of a fluorescent belt region, which corresponds to the uptake depth. Based on such evaluations, we can understand whether the quiescent viable cells in the middle layer of a cell spheroid. Also, by using a cell culture insert, we can estimate the uptake Au nanoparticle number per cell through mass spectrometry measurement. It is found that after 48-hour incubation time, the Au nanoparticles and photosensitizer all have the penetration depths in the range of 110-140 m. Such uptake penetration depths control the cell damage depths under laser illuminations. For observing the uptake depths, we use different fluorescent dyes to incubate the cell spheroids. It is found that the penetration depth of a dye also relies on its molecular weight. The smaller uptake penetration depths of Au nanoparticles into a cell spheroid can be attributed to the settlement of Au nanoparticles in the incubation solutions such that the upper portion of a cell spheroid has no access to the Au nanoparticles.

In this work we investigated the nature of quenching between different types of 2D materials (WS₂, MoS₂ and graphene oxide) and oligonucleotide coated-upconversion nanoparticles. This study contributes towards the efficient design of biosensors based on 2D materials and DNA-coated upconversion nanoparticles.

The physiological parameters are often dynamically changing during disease progression, such as cancer, infections, and degenerative diseases, rendering them not only attractive biomarkers for disease diagnosis, but also potential tools for self healing. With the deep understanding of cancer, masses of bioresponsive smart systems were designed for precision cancer medications, engaging biological triggers or pathological abnormalities such as pH, redox, enzyme, biomolecules, shear force, hypoxia, etc. In our work, we have chosen the endogenous microRNA as specific triggers and biomarkers for cancer diagnosis and treatment. To address the issue of low copy numbers of microRNA in single cells, we developed programmable nanodevices consisting of gold nanorods and MNAzymes to realize stringent recognition and in situ amplification of intracellular miRNAs for multiplexed detection and controlled drug release. Furthermore, smart nanocarriers were prepared to achieve specific and efficient release of Dox and siRNAs in tumor cells via enzyme-free cascade amplification with endogenous microRNA as trigger and ATP as fuel through toehold-mediated strand displacement. On the other hand, to maximize specific tumor uptake and minimize off-target internalization in in vivo study, the surface of the nanocarriers were engineered by using temperature sensitive polymers. The in vivo results showed that a low dosage of nanocarriers could precisely enter the tumor under the NIR laser guidance, releasing the cargoes and inducing severe cell apoptosis, though they only brought minimum damage to normal organs. The combination of nanomaterials, polymers, and DNA nanomachines provided a promising tool for designing smart nanodevices for precise disease diagnosis and treatment.

Recent developments in nanotechnology has provided new tools for cancer therapy and diagnosis. Among other nanomaterial systems, gold nanoparticles are being used as radiation dose enhancers and anticancer drug carriers in cancer therapy. Fate of gold nanoparticles within biological tissues can be probed using techniques such as TEM (transmission electron microscopy) and SEM (Scanning Electron Microscopy) due to their high electron density. We have shown for the first time that cancer drug loaded gold nanoparticles can reach the nucleus (or the brain) of cancer cells enhancing the therapeutic effect dramatically. Nucleus of the cancer cells are the most desirable target in cancer therapy. In chemotherapy, smart delivery of highly toxic anticancer drugs through packaging using nanoparticles will reduce the side effects and improve the quality and care of cancer patients. In radiation therapy, use of gold nanoparticles as radiation dose enhancer is very promising due to enhanced localized dose within the cancer tissue. Recent advancement in nanomaterial characterization techniques will facilitate mapping of nanomaterial distribution within biological specimens to correlate the radiobiological effects due to treatment. Hence, gold nanoparticle mediated combined chemoradiation would provide promising tools to achieve personalized and tailored cancer treatments in the near future.

Combined photothermo-chemotherapy is a new strategy for cancer treatment which improves the therapeutic outcome by synergistic effects of both therapies. Here, we presented a multifunctional gold nanoshell that exhibited excellent photothermal conversion and delivered the hydrophobic chemotherapy drug, SN-38. The positively charged SN-38-loaded PDMA-PCL micelles were decorated with a gold layer by in situ reduction of chloroauric acid on the surface of micelles. Scanning and transmission electron microscopy images proved micelles were successfully decorated and the resulting gold nanoshells had a spherical morphology with a narrow size distribution. The synthesized gold nanoshells displayed a broad surface plasmon resonance peak in the near-infrared wavelength region and a great photothermal conversion ability. After pegylation, gold nanoshells were stable in biological media and appeared highly biocompatible in the absence of laser irradiation. Upon near-infrared laser irradiation, incident energy was converted into heat by gold nanoshells on SN-38-loaded micelles (SN-38@pGNS), which causes local temperature increase and triggers the release of encapsulated drug. Compared to SN-38, SN-38-loaded micelles, or laser with drug-free gold nanoshells alone, combined photothermo-chemotherapy using SN-38@pGNS with laser irradiation killed colorectal cancer cells with higher efficacy in vitro and demonstrated significant tumor suppression in vivo, suggesting that gold nanoshells on drug-loaded micelles delivered SN-38 and photothermal therapy in synergistic actions and might be a potential candidate for future colorectal cancer therapy.

Photosensitizer can be taken up directly by cells or through the linkage with gold nanoparticles (NPs). When gold NPs linked with a photosensitizer are taken up by cells, both photodynamic (PDT) and photothermal effects for damaging cells can be generated under the illumination of a light source of an appropriate wavelength. The gold NPs and photosensitizer can escape from cells through an exocytosis process such that the efficiency of cell damage may decay with time. In this study, we investigate the dependencies of cell damage efficiency, gold nanoring (NRI) and photosensitizer amounts stayed in cells on the delay time. In particular, we differentiate the two possible escape pathways of the photosensitizer, AlPcS, i.e., AlPcS escape alone after its linkage with Au NRI is broken and AlPcS escape together with the linked Au NRI. Also, we intend to understand the dependence of the aforementioned AlPcS escape pathway on the location of Au NRIs inside a cell after they are taken up. It is found that generally, the PDT-induced cell damage efficiency decays more slowly when AlPcS is taken up together with linked Au NRI, compared with the case that AlPcS is taken up alone by cells. This trend indicates that the photosensitizer is more stable when it is linked with an Au NP. This result is confirmed by the observations of Au NRI and photosensitizer escapes through mass spectrometry, flow cytometry, and confocal fluorescent microscopy.

The main principle of photodynamic therapy (PDT) is to kill malignant cells by generation of reactive oxygen species (ROS). PDT appeared highly effective when ROS can be produced in subcellular location such as plasma membrane. The plasma membrane maintains the structural integrity of the cell and regulates multiple important cellular processes, such as endocytosis, trafficking, and apoptotic pathways, could be one of the best points to kill the cancer cells. Previously, we have developed a plasma membrane-targeted liquid crystal nanoparticle (LCNP) formulation that can be loaded with dyes or drugs. Here we highlight the utility of this LCNP for membrane targeted delivery and imaging for a photosensitizer (PS) for PDT applications.

Flavonoids are one of the important naturally available small molecules found in our daily diets. They have been considered as potential therapeutic agents for anticancer therapy. Despite their anti-cancer properties, their therapeutic application is very limited due to poor water solubility, which results in poor bioavailability to the diseased cells. Hence, to overcome this limitation of Flavonoids, Quercetin (Qct), the most extensively studied flavonoid, prompted us to encapsulate it within nanoparticles. We have successfully encapsulated Qct within cationic polymer based nanoparticles using simple two-step self-assembly fabrication method and studied its effect on absorption and emission properties of Qct. This study was aimed at Qct encapsulation and its effect on the optical properties of Qct for the diagnostic applications. Our results indicate that Qct was efficiently encapsulated within the polymeric nanoparticles. This resulted into ~17 times increase in fluorescence emission of encapsulated Qct (Qct-NPs) in comparison with its aqueous suspension. Thus, Qct-NPs can be utilized as a fluorescent probe for various biomedical applications. These probes will have multiple functions integrated into a single nanostructure, enabling the Qct nanoparticles for imaging and therapy. This is the first report on the effect of nanoencapsulation on optical properties of Qct. Thus, Qct-NPs can be harnessed as an effective theranostic agent, and that will not only allow to image and but also treat the cancer in a single clinical procedure.

Cadmium-free near infrared (NIR) emitting quantum dots (QDs) have significant potential for multiplexed tissue-depth imaging applications in the first optical tissue window (i.e., 650 – 900 nm). Indium phosphide (InP) chemistry provides one of the more promising cadmium-free options for biomedical imaging, but the full tunability of this material has not yet been achieved. Specifically, InP QD emission has been tuned from 480 – 730 nm in previous literature reports, but examples of samples emitting from 730 nm to the InP bulk bandgap limit of 925 nm are lacking. We hypothesize that by generating inverted structures comprising ZnSe/InP/ZnS in a core/shell/shell heterostructure, optical emission from the InP shell can be tuned by changing the InP shell thickness, including pushing deeper into the NIR than current InP QDs. Colloidal synthesis methods including hot injection precipitation of the ZnSe core and a modified successive ion layer adsorption and reaction (SILAR) method for stepwise shell deposition were used to promote growth of core/shell/shell materials with varying thicknesses of the InP shell. By controlling the number of injections of indium and phosphorous precursor material, the emission peak was tuned from 515 nm to 845 nm (2.41 – 1.47 eV) with consistent full width half maximum (FWHM) values of the emission peak ~0.32 eV. To confer water solubility, the nanoparticles were encapsulated in PEGylated phospholipid micelles, and multiplexing of NIR-emitting InP QDs was demonstrated using an IVIS imaging system. These materials show potential for multiplexed imaging of targeted QD contrast agents in the first optical tissue window.

Thermosensitive photothermal agents (AuNRs@pNIPAAm) for accurate temperature control were prepared by covalent binding pNIPAAm to gold nanorods. Stretched pNIPAAm collapses when temperature increases around low critical solution temperature, inducing a redshift of LSPR wavelength. To obtain a high-throughput LSPR scattering spectral mapping of AuNRs@pNIPAAm particles, we demonstrate a spatiotemporal resolution plasmonic spectroscopy (SRPS) which provides the LSPR wavelength of multiple AuNRs@ pNIPAAm particles simultaneously at millisecond level (limited by camera frame rate). SRPS is performed on a home-made two-channel highly inclined and laminated optical (HILO) sheet dark-field microscope. Nanoparticles with LSPR wavelength of ~750 nm are incubated with cells. Two continuous lasers (with wavelength of 730 and 785 nm, avoid the broadband scattering background from biological environment) are combined and modulated in order to generate a pulse beam. This beam is then directed onto the cover-slide with an appropriate incident angle to generate a HILO sheet, which is suitable for high spatial resolution imaging of nanoparticles inside cell samples. Another 808 nm laser is focused on the sample to heat nanoparticles. Scattering light of nanoparticles is collected by the same objective, which is recorded by a CMOS camera. CMOS camera and the pulse laser are synchronized in order to capture the dark-field images from two wavelength lasers periodically, which generates a two-channel image sequence. With this configuration, we measure the scattering intensity of nanoparticles illuminated by two lasers simultaneously, which can be further converted to LSPR wavelength image.

Fluorescent gold nanoclusters (AuNCs) are novel promising nanomaterials for biomedical applications because of their unique physiochemical properties. We use AuNCs stabilized with mercaptoundecanoic acid (MUA-AuNCs) which show a fluorescence emission that peaks at a wavelength around 525 nm, which is in the same wavelength region as the autofluorescence of a biological cell. However, the fluorescence decay time of the MUA-AuNCs (100 ns) is much longer in comparison to the autofluorescence (3 ns). Fluorescence lifetime imaging microscopy (FLIM) is a powerful method to discriminate between emitters of different fluorescence lifetimes. We biofunctionalize these MUA-AuNCs with biomolecules that specifically bind to the receptor expressed on the cell's membrane. To get an image of the whole cell and the bound and internalized AuNCs we use cross-sectional FLIM scans in axial direction at different heights through the cell. We distinguish between specifically bound and internalized MUA-AuNCs on and in cells by means of different FLIM methods supported by element-specific scanning electron microscopy and semi-empirical simulations.

The use of silver nanoparticles as optical clearing agent (OCA) enhancers to improve caries diagnostic by optical coherence tomography (OCT) is demonstrated here. Five molars with no evident cavitation were selected. The OCAs were based on aqueous solution of silver nanoparticles (AgNP, 1.18x 10¹⁴ particles/mL, ø ≈ 10nm) and its dilution at 10% in glycerol. Teeth were placed on a platform with a micrometric screw, and after applying the OCAs, they were scanned with a Callisto SD-OCT system operating ate 930nm central wavelength. The occlusal surfaces were scanned by OCT, capturing crosssectional images with 8 mm transversal scanning, generating numerical matrices (2000x512). The OCT images had their transverse dimension preserved. AgNP-OCAs promoted image stretching due to the modification in the light optical path caused by AgNP-OCAs refractive indices close to that of the enamel. AgNP-OCAs evidenced the enamel birefringence and highlighted initial demineralization areas, that presented defined margins with higher contrast between sound and demineralized regions, with higher OCT signal intensity in those areas.

This ex vivo study evaluated the efficiency of nano-silver fluoride (NSF) to enamel remineralization of deciduous teeth submitted to a high cariogenic challenge. 33 specimens of sound enamel were distributed into three groups: treated with sodium fluoride, with NSF, and deionized water. All specimens were submitted to microhardness test before chemical caries induction; post-induction of caries; and post-cariogenic challenge. The high cariogenic challenge was carried out in 14 daily cycles. There was no significant statistical difference (p=0.958) of enamel microhardness between NSFtreated and NaF-treated specimens. However, it presented significant difference between water-treated specimens and both NSF-treated and NaF-treated specimens (p=0.003).Furthermore, optical coherence tomography was employed for caries diagnosis, as it can be used in clinical environment.

One of the drugs used to treat ovarian cancer is cisplatin. However, cisplatin kills normal surrounding tissue in addition to cancer cells. To improve tumor targeting efficiency, our lab uses neural stem cells (NSCs), which migrate directly to ovarian tumors. If free cisplatin is loaded into NSCs for targeted drug delivery, it will kill the NSCs. To prevent the drug cisplatin from killing both the NSCs and normal surrounding tissue, our lab synthesizes silica nanoparticles (SiNPs) that act as a protective carrier. The big picture here is to maximize efficiency of tumor targeting using NSCs and minimize toxicity to these NSCs using SiNPs. The goal of this project is to optimize the stability of SiNPs, which is important for efficient drug loading.

To do this, the concentration of tetraethyl orthosilicate (TEOS), one of the main components of SiNPs, was varied. We hypothesized that more TEOS equates to more stable SiNPs because TEOS contributes carbon to SiNPs, and thus a tightly-packed chemical structure results in a stable particle. Then, the stability of the SiNPs were checked in cell media and phosphate buffered saline (PBS). Lastly, the SiNPs were analyzed for their porosity using the transmission electron microscope (TEM).

TEM imaging showed white spots in the 200-800 μL TEOS batches and no white spots in the 1000-1800 μL TEOS batches. The white spots were pores, which indicate instability.

We concluded that the ultimate factor that determines the stability of SiNPs (100 nm) is the concentration of organic substance.