This PDF file contains the front matter associated with SPIE Proceedings Volume 11255, including the title page, copyright information, table of contents, and author and conference committee lists.

Endosomal escape remains the most prominent bottleneck at the intracellular level for non-viral vectors today. Gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation. Depending on the energy of nanosecond laser pulses, this can be achieved by either endosomal rupture by the mechanical energy from water vapor nanobubbles (VNBs) that emerge around the AuNP, or permeabilization of the endosomal membrane by heat diffusion. Here, we designed a siRNA/AuNP drug delivery system, to address the open question of how both photothermal effects influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. We found that, contrary to heat-mediated endosomal escape, VNB generation produced excellent transfection levels independent of the cell type, without inducing long-term changes in cell homeostasis.

CdSe_xS_1-x/ZnS quantum dots (QDs) can cover a broader spectral range than the commonly used CdSe/ZnS QDs and are potentially useful as biomarkers for tagging cell lines such as HeLa, A549, and MCF-7 due to their high photoluminescence intensity and stability in solution. So far, there have been few studies of colloidal CdSe_xS_1-x/ZnS QDs that would simultaneously investigate changes in a) the molar composition of QD cores, and b) the shell thickness, as well as the effects of these changes on the photoluminescence and quantum yield properties of the QDs. CdSe_yS_1-y QDs and CdSe_xS_1-x/ZnS core/shell QDs were synthesized via a previously reported and modified hot-injection procedure and via a telescoping one-pot synthesis based on the modified hot-injection procedure. Size, morphology, composition, and colloidal stability of these QD core/shell systems is reported with data obtained from TEM, XRD, TGA, DSC, DLS, and zeta potential techniques. Optical characterization is described using data collected from UV-Vis absorption spectrophotometry and photoluminescence spectroscopy.

Optical imaging for biomedical applications holds much promise, particularly at near infrared I and II windows. However, the few NIR dyes are often toxic and are prone to photobleaching, while there are barely any NIR-II dyes. Semiconductor quantum dots (QDs) have tunable bandgaps into the NIR-II window, resist photobleaching, and have high quantum yields; however, QDs are traditionally made of cadmium, lead, or other toxic components. Furthermore, these QDs accumulate in vital organs and are cleared on the scale of months to years, limiting clinical relevance. We have recently developed biodegradable, non-toxic QD platform composed of earth-abundant materials that can be cleared in under 1 month from all essential organs. Furthermore, this material exhibits a tunable bandgap out to 0.9 eV, reaching the NIR-II window. We demonstrate the degradability of this material in vitro and in vivo, as well as demonstrate its biocompatibility in a murine model.

Colloidal quantum dots (QDs) emitting in the near-infrared (NIR) spectrum are of interest for many biomedical applications, including bioimaging, biosensing, drug delivery, and photodynamic therapy. However, a significant limitation is that QDs are typically highly cytotoxic, containing materials such as indium arsenide (InAs), cadmium, or lead, which makes prospects for their FDA approval for human treatment very unlikely. Previous work on QDs in the NIR has focused on indium arsenide or cadmium chalcogenide cores coated with cadmium sulfide shells or zinc sulfide shells. Lead-based nanoparticles, such as lead selenide (PbSe) or lead sulfide (PbS) are also popular materials used for NIR emission. However, these nanoparticles have also been shown to be cytotoxic. Coating these Pb-based QDs with a biocompatible shell consisting of tin chalcogenides, such as tin sulfide (SnS) or tin selenide (TnSe), could be a reasonable alternative to improve their biocompatibility and reducing their cytotoxicity. In this paper, we report on our recent studies of PbSe-core QDs with Sn-containing shells, including synthesis, structural characterization, and investigation of optical properties. Characteristics of these QDs synthesized under different conditions are described. We conclude that their synthesis is challenging and still requires further work to avoid shell oxidation.

DNA-directed assembly of gold nanoparticles into precise two- and three-dimensional patterns has enabled bold advances in probing their optical properties such as the local enhancement in their surface plasmon resonance. DNA nanostructures synthesized using the principles of DNA origami have been programmed to contain unique capture sites for positioning metal nanoparticles in diverse geometries for applications in biosensing, therapy, and miniature electronics. However, to enable scalability beyond simple 2-3 nanoparticle architectures, it is important to understand the requirement for orthogonal capture sequences for attaching more than a single gold nanoparticle on a DNA nanostructure. In this work, we sought to assemble an angular gold nanorod-nanosphere-nanorod pattern on a DNA origami triangle with multiple capture sites utilizing a common capture sequence. Results indicate that gold nanospheres preferentially bound to all the capture sites on the DNA origami triangle and prevented attachment of gold nanorods. This suggests that requirement for orthogonal capture sites is correlated with the physical properties of the individual nanoparticle such as shape and size.

Artificial proteins are created that offer a universal and biocompatible approach to control the morphology of metal nanocrystals. First, we select, from a large library of artificial proteins (alpha-Repins), individuals that show specific binding for Au(111) facets. Gold nanocrystals synthesized with such -Rep are exclusively terminated by Au(111) planes. The protein-coated nanocrystals are then functionalized to perform biomolecule-driven self-assembly and on-surface enzymatic catalysis. Alternatively, growth templates with a complex and rigid shape could be constructed from two artificial proteins selected to act as the supercoil unit and the staple that spontaneously self-assemble into helical nanotubular templates observed by cryo-electron microscopy.

The inherent ability of gold nanoparticles (AuNPs) to transduce light energy into heat, coupled with their ease of bioconjugation has made them a powerful tool potentially capable of controlling biological activity. When combined with ultra-short pulses of light and the proper experimental conditions, AuNPs are capable of heating their local environment without increasing the bulk solution temperature. Gene therapy and siRNA delivery have emerged as promising applications for localized heating of AuNPs and as such, a number of different groups have used light to trigger the release of nucleic acids from the surface of AuNPs. While successful nucleic acid release is universally demonstrated in the literature, the mechanism of release varies between reports. Specifically, the reported release mechanism is either: 1) the thermal denaturing of a nucleic acid duplex and release of a “single stranded” nucleic acid into solution; 2) the cleavage of the prototypical gold-thiol bond used to tether the nucleic acid duplex to the surface, resulting in the release of the complete nucleic acid duplex; or 3) a combination of both. Due to the complex parameter space in these experimental systems (AuNP size/shape/composition, laser energy density/repetition rate/pulse width) it is not surprising that the reported release mechanisms differ. Here, we utilize examples from the literature in order to identify the key parameters that dictate the release mechanism of nucleic acids on AuNPs in an attempt to further a comprehensive understanding of this process.

Shape control of metal nanocrystals with engineered proteins is promising for integrated plasmonics, biosensing, and catalysis applications. We present artificial thermo-stable proteins used as inhibitors during gold nanocrystal growth in order to control the shape and crystallographic facet expression. This synthesis leads to (111)-terminated gold nanocrystals in high yield. The shape and size distribution are tuned towards nanoplates by adjusting synthetic conditions. When biotinylated alpha-Reps are used, the morphosynthetic properties are preserved and streptavidin sensing is observed through a marked 7-nm redshift in dark-field spectra. Single nanoplate streptavidin sensing experiments show high sensitivity (300 nm/RIU).

Plasmonic nanoparticles are expected to impact various fields, such as chemical analysis, superresolution imaging, and therapeutics. However, since plasmonic nanoparticles are known to be prone to aggregation, widespread use of plasmonic nanoparticles is still limited. While many methods have been proposed to enhance colloidal stability, a universal method to comprehensively detect stability is still lacking. We present a new comprehensive stability parameter (CSP) as a robust and universal method to quantify stability. Unlike other methods, CSP utilizes the entire UV-Vis spectrum to evaluate the aggregation and to avoid potential biasing issues by only looking at a single parameter. We quantitatively analyzed the colloidal stability of plasmonic particles with different surface coatings using CSP. This work establishes a standardized quantification method which can be used in the future nanoparticle applications.

The ability of DNA functionalised gold nanoparticles (AuNPs) to detect specific targets in vitro and in vivo has led to their development as suitable tools for sensing applications. However, endosomal entrapment is a common barrier in various nanoparticle delivery approaches. In this work, we present a new design strategy with the aim to enhance endosomal escape of DNA-coated AuNPs via the incorporation of a peptide that has been found to promote effective escape within cells. AuNPs are firstly modified with thiol terminated DNA strands followed by further surface functionalisation with cysteine terminated peptides. We show that optimized loading of peptides following DNA nanoparticle functionalisation of nanoparticles is feasible. DNA-peptide-coated AuNP hybrids show similar stability towards degradation by endocellular enzymes and similar specificity towards the detection of specific mRNA targets.

In the last few years, zwitterionic polymers have been developed as antifouling surface coatings. However, their ability to completely suppress protein adsorption at the surface of nanoparticles (NPs) in complex biological media remains undemonstrated. We have developed several polymeric zwitterionic nanoparticle ligands and investigated the formation of hard (irreversible) and soft (reversible) protein corona around fluorescent quantum dots (QDs) as model nanoparticles coated with sulfobetaine (SB), phosphorylcholine (PC) and carboxybetaine (CB) in model albumin solutions and in whole serum. We show for the first time a complete absence of protein corona around SB-coated NPs, while PC- CB-or PEG-coated NPs undergo reversible adsorption or partial aggregation. Single NP tracking in the cytoplasm of live cells corroborate these in vitro observations. This absence of protein adsorption was also tested on other nanomaterials, including iron oxide and gold NPs.

A large number of health problems, such as diabetes, hearing loss and retinal degeneration, can be cured by stimulation of neurons. One of the effective strategies for neural stimulation is through light-induced photoactive surfaces owing to the non-invasive and remotely accessible characteristics of light. Quantum dots are suitable candidate for such applications due to their absorption of visible light, bandgap tunability through quantum confinement effect and ease of integration into device structures due to their nanoscale size. In this study, we show that proper engineering of quantum dot nanostructure and band alignment of optoelectronic biointerface allow for bidirectional optical stimulation of neurons with high level control on stimuli strength.

Biointerfaces based on photovoltaic substrates has gained substantial attention for neural photostimulation[1, 2]. The control of Faradaic and capacitive charge transfer mechanisms by these substrates is important for effective and safe photostimulation of neurons. Faradaic mechanism uses charge transfer between electrode and electrolyte with oxidation and reduction reactions, and capacitive mechanism electrostatically perturb local ion concentration in the electrode/electrolyte interface[3, 4]. In this study, we show the control of light-activated Faradaic and capacitive charge transfer mechanisms by bulk heterojunction photovoltaic biointerfaces. We tuned the strength of the mechanisms via spatial control of the photogenerated electrons and holes in the biointerface architecture. For that, we explored three different architectures (Fig. 1) using intermediate ZnO and MoO_x layers and without any intermediate layer between the transparent metal oxide (ITO) and photoactive layer. The photoactive layer is composed of organic-inorganic ternary blend of poly(3-hexylthiophene-2,5-diyl (P3HT), [6,6]-Phenyl-C61-butyric acid methyl ester (PCBM) and PbS quantum dots that lead to strong displacement currents. Moreover, we observed that integration of PbS quantum dots in the active blend of P3HT:PCBM enhances both capacitive and faradaic photocurrents due to well-aligned energy diagram, stronger absorption and better surface morphology. To characterize the photostimulation, we seeded SHSY-5Y cells on our biointerfaces and recorded the membrane depolarization of single cells. The results are also supported by two-domain stimulation model[4, 5].

Enzyme amplified colorimetric sensing methods provide visual readouts without the need of instrumentation. However, these methods have issues due to the enzymes high cost, instability, extraction, and purification. Recently, it has been observed that metal oxide NPs mimic many natural enzymes. Fe3O4 magnetic NPs are now known to mimic the horseradish peroxidase enzyme that oxidizes chromogenic substrates such as TMB, ABTS, OPD into colored products. In this regard, we have designed and developed dopamine-functionalized iron oxide (Dop-Fe3O4) nanoparticles and exploited their enzyme mimicking ability to develop a simple colorimetric bacterial sensing strategy. These nanoparticles catalyse the oxidation of a chromogenic substrate in the presence of H2O2 into a green colored product. The catalytic activity of the nanoparticles is inhibited in the presence of bacteria, providing naked eye detection of bacteria at 104 cfu mL−1 and by spectrophotometric detection down to 102 cfu mL−1.

Semiconductor quantum dots (QDs) have great potential for multiplexed imaging and biosensing applications. Due to the quantum confinement effect, spectral tuning of the emission color of these nanocrystals is made possible through changing their size. However, QDs of different emission colors are dissimilar in their brightness values, defined as the product of molar extinction coefficient (ε) and quantum yield (QY). These differences arise from extinction coefficients which are coupled to the number of atoms and bonds constituting the QD. As a consequence, the relative brightness of QDs can be orders of magnitude higher for larger, red emitting QDs compared to their smaller blue/green emitting counterparts even with comparable QYs. This study addresses this problem by drawing a quantitative comparison of absorption properties of different type-I InP QDs, aiming to make these heterostructures suitable for accurate imaging and sensing applications. Tuning of the absorption cross-section and extinction coefficients, along with brightness tuning of the QDs has been performed through synthesizing a series of QDs with a combination of core sizes, shell thicknesses, and compositions.

Coating inorganic nanocrystals (e.g., quantum dots and gold nanoparticles) with polymer ligands presenting many lipoic acid (LA) anchoring groups provides them with excellent colloidal stability in aqueous media. Here we exploit the natural swelling of polymer macromolecules, which imposes a configuration that leaves a fraction of the anchors on the polymerstabilized nanocolloids free or uncoordinated and target them for conjugation using thiol-to-maleimide chemistry. This allows easy surface functionalization of the nanocrystals, without the need to introduce additional reactive groups. We apply a photoligation strategy to coat QDs and AuNPs, followed by coupling with maleimide-modified dyes. We then use optical absorption and resonance energy transfer measurements, to extract estimates for the fraction of accessible LAs per nanocrystal. To further prove the effectiveness of this approach, we construct a ratiometric pH sensing probe made of QDSNARF conjugates. The combination of the multi-coordinating ligand design and in-situ photoligation yields colloidally stable nanocrystals, presenting several thiol reactive sites. Our results are promising and could advance the integration of nanomaterials in biological sensing and imaging applications.

Phase-changing nanodroplets are sub-micron constructs with perfluorocarbon liquid cores that can be remotely vaporized with optical or acoustic energy to form gaseous microbubbles and expel their contents. This process can generate shock waves, shear forces, and microstreams in the interstitial tumor space, enhancing intratumoral drug delivery¹. Furthermore, the resulting microbubbles can act as ultrasound contrast agents, enabling imaging with high sensitivity to guide the delivery of therapeutics. In order to leverage these phenomena for drug co-delivery, we developed lipid-shell nanodroplets (NDs) with a liquid perfluoropentane core by a double emulsion technique. The droplets were loaded with cisplatin and paclitaxel as active agents, as well as indocyanine-green dye to allow for light-triggered drug release and enable photoacoustic imaging². Both the composition of the shell and of the core emulsion were optimized in order to develop nanoparticles with sizes lower than 300 nm (mean diameter 153 ± 77 nm) to enhance accumulation in tumors. The triggered release of both drugs from the nanodroplets was assessed by laser activation with NIR-light (750 nm). The viability of FaDu cells was used to measure in vitro drug delivery, showcasing the laser triggered release of drugs. The double drug formulation outperformed empty nanodroplets (p= 0.0006), PTX-loaded NDs (p < 0.0001), as well as combinations of paclitaxel and cisplatin in concentrations equivalent to the loaded droplets’ in terms of cell cytotoxicity effects. With these in mind, our long-term goal is to employ the nanodroplets to quantify delivered drug dose, while at the same time minimizing off-target effects.

Spectral tuning of UV luminescence of upconverting nanoparticles (UNPs) enables development of modern applications of upconversion such as infrared-driven photochemistry. We investigated spectral tuning of luminescence of upconverting nanoparticles composed of NaYF₄: Yb³⁺ , Tm³⁺ (69.5:30:0.5). We demonstrated spectral control of luminescence of these UNPs by adding lanthanide ion that acts as a quenching agent for selected wavelengths of luminescence of Tm³⁺. Erbium (Er³⁺), which contains an energy level closely matching that of the ultra-violet (UV) energy level of Tm³⁺ ( 1D2), was selected as the quenching ion in this study. First, we demonstrated energy transfer between the luminescent ion (Tm³⁺) and the quenching ion (Er ³⁺). Subsequently, we demonstrated tuning of the UV luminescence intensity of these UNPs. The desired spectral output of the UNP is dependent on two factors, the selection of the quenching agent and its doping ratio in UNP composition.