This PDF file contains the front matter associated with SPIE Proceedings Volume 11659, including the Title Page, Copyright information, and Table of Contents.

Proton radiography is a promising imaging technique that can be used to improve the treatment plan quality for proton therapy, by providing accurate estimates of proton stopping power. While a proton radiograph has accurate information about proton stopping power, it also has an inherently low tissue contrast for diagnostic purposes, as compared to X-ray imaging. The nature of energetic, massive protons as a radiographic probe is that they require a high-Z tracer to provide sufficient proton scatter in order to delineate target structures. Gold nanoparticles could be that ideal tracer due to a Z = 79, and their biocompatibility. Here the detection thresholds for gold-nanoparticle targeted tumors are evaluated using instantaneous, 800-MeV proton radiography, at the Los Alamos Neutron Science Center. Data is compared against MRI data in pre-clinical mouse models with 4T1 tumors directly injected with gold nanoparticle solution. The proton radiography system is then optimized using novel collimation schemes, including a dark field proton radiographic setup, that aimed to increase sensitivity and reduce dose. Results evaluated here are extrapolated to 211-MeV proton radiographic energy, to compare against expectations at clinical treatment energies. At that lower energy, proton radiography is more sensitive to the multiple Coulomb scattering introduced by a high-Z tracer.

Pancreatic ductal adenocarcinoma (PDAC) is a deep seated and aggressive tumour which resists most current therapeutics resulting in only 5% survival rate. Gemcitabine, although the first line of treatment still has limited therapeutic value due to severe side effects. New treatment modalities are urgently required which efficiently deliver drugs to the target tumour area without causing adverse off-target side effects. We aimed to combine two clinical treatments for cancer, chemotherapy and RadioDynamic Therapy (RDT), to overcome current treatment for PDAC. In this project, specially designed nanoparticles (NP) were utilised as nanodrug carriers. Our poly(lactic-co-glycolide) (PLGA) nanoparticles were successfully loaded with both chemotherapeutic agent Gemcitabine and RDT agent Verteporfin. Addition surface conjugation with GE11 peptide allowed to precisely target Panc-1 cancer cell lines overexpressing EGFR receptors. Our results show PLGA was successfully fine-tuned with desirable physicochemical properties. Gemcitabine and Verteporfin were sufficiently loaded into the PLGA NP and surface conjugation with GE11 peptide was confirmed using absorbance spectroscopy. The resulting GE11-PLGA-VP-GEM NPs showed size 86.38±15 nm, polydispersity 0.57±0.1 and zeta potential measurements of -2.87±1 mV. Encapsulation efficiency (EE) was determined to be 20% for Verteporfin and 24% for Gemcitabine. This combination offers a multimodal treatment system whereby Gemcitabine is able to be effectively delivered to Panc-1 cells and, simultaneously, Verteporfin, induced by Xray radiation generates ROS to cause cell death. Cellular uptake studies were evaluated and the treatment of live Panc-1 cells with GE11-PLGA-VP-GEM NP induced cell death. Furthermore, Verteporfin, excited at clinically relevant X-ray dosages generated ROS.

Diatoms are microalgae which have their unique cell encased into a nanostructured silica shell called frustule. The silica shells of diatoms can be envisioned as micro/nano structures suitable to further chemical modification yielding smart functional nanomaterials. Differently from the chemical production of silica, the biosynthesis of natural SiO2 occurs in mild conditions and it does not require the use of toxic precursors or reagents. Biosilica from diatoms features interesting properties such as high surface area, mechanical resistance and nanotexturization, which makes it appealing for applications in photonics, sensing, optoelectronics, biomaterial science and biomedicine. In addition, frustules’ biosilica can be easily chemically modified to add new functions. This can be done by simple surface functionalization, and/or in vivo by adding specific molecules to the culture medium. We have shown applications of chemically modified frustules for bone cells growth. In particular, we have demonstrated that in vivo functionalization of diatom biosilica with sodium alendronate results in osteoactive material. We have also demonstrated the production of functional structures by coating living diatoms with biomimetic organic polymers, like polydopamine (PDA). The resulting living heterostructures turn out to be intriguing platforms for additional chemical modifications, such as anchoring enzymes, affording multifunctional materials for biological applications. Finally, we have also shown that photonic microstructures can be produced by in vivo incorporation of tailored light emitting molecules in living Thalassiosira weissflogii diatoms. With a similar approach, biosilica has been doped with phosphorescent Ir complexes. Overall, our studies point out intriguing biotechnological routes to multifunctional nanomaterials for biomedicine and nanotechnology starting from unicellular algae.

We present a method of high resolution, non-invasive, in vivo vascular imaging obtained using watersoluble and bright SWIR-emitting gold nanoclusters presenting an anisotropic surface charge combined with SWIR detection and Monte Carlo processing of the images. We applied this approach to quantify vessel complexity in mice presenting vascular disorders.

Here we investigate Förster Resonant Energy Transfer (FRET) occurring between luminescent colloidal semiconductor quantum dots (QDs) and fluorescent streptavidin molecules (FSA), chemically coupled thanks to N-hydroxysuccinimide (NHS) and biotin. In some conditions, QDs are known to form agglomerates throughout their functionalization by NHS and biotin molecules. Thus, we wondered how this collective aggregation could influence the efficiency of FRET. Interestingly, this proves to enhance the energy transfer from QDs to FSA. In terms of detection threshold, aggregated-QD-based systems lead to a limit down to 5 nM, while it is up to 80 nM for non-aggregated ones. Therefore, these unexpected results evidence our ability to exploit QDs aggregation for the design of biosensing systems with lower and lower molecular detection thresholds. Unlike the common beliefs, QD agglomeration is an asset that we can benefit from in order to improve the performance of QD-based biosensors. As a counterpart, this requires a fine monitoring of the emission spectrum of QDs while they are aggregating. This is why we provide a complete characterization of the QD fluorescence throughout their chemical funtionalization with NHS and biotin, supporting that such precautions are mandatory. Further, we show it is necessary to distinguish hetero-FRET (between QDs and FSA) from homo-FRET (between QDs within a same aggregate) in order to avoid misleading interpretations.

There is a growing interest in the three-photon luminescence (3PL) of plasmonic nanoparticles. The 3PL from gold nanoparticle has been applied to deep-tissue imaging previously, but the efficiency of the process is not yet properly quantified. In this work, we look into confirming three-photon luminescence in gold nanorods at various wavelengths and measure its three-photon action cross-sections. We performed this analysis by investigating the non-linear emission from gold nanorods at 1500 nm by a custom-built scanning laser confocal microscope, as well as the bulk non-linear absorption is also measured by the z-scan technique.

A few imaging applications using quantum dots (QDs) will be introduced, which will cover from visible to infrared(IR) for the wavelengths and from cellular super-resolution to whole body in vivo imaging for the object scale. (1) QDs were conjugated to tumor-specific antibodies(Abs) with zwitterionic surface coating to reduce nonspecific bindings. The Ab-QD probes were used to diagnose tumors for sectioned mouse tissues, fresh mouse colons stained ex vivo and in vivo, and also for fresh human colon adenoma tissues. The probes successfully detected not only cancers that are readily discernible by bare eyes but also hyperplasia and adenoma regions. Multiplexed QD, spray-and-wash, and endoscopy approach provided a significant advantage for detecting small or flat tumors that may be missed by conventional endoscopic examinations. QD-Ab probe was also used in conjunction with a ratiometric fluorescent molecular probe, cresyl violet–glutamic acid derivative, that ratiometrically switches between two fluorescent colors in response to the enzyme activity of λ-glutamyltranspeptidase. Co-application of the two kinds of probes, QD-Abs and the ratiometric molecular probe, afforded accurate visualization of carcinomas, hyperplasia and adenoma regions. (2) Amphiphilic polyethyleneimine derivatives (amPEIs) were synthesized and used to encapsulate dozens of QDs. The QD-amPEI showed very efficient QD cellular fluorescent labeling. Co-encapsulation of QDs and oxygen sensing phosphorescence Ru dyes allowed accurate and reversible oxygen sensing capability by the ratiometric signals, which was successfully applied to cellular and spheroid models. (3) Photo-modulating QDs were designed by conjugating crystal violets (CVs) on QD surface. The QD CV conjugates (QD-CVs) shows a single cycle of emission burst as go through the stage of initially quenched off to photo-activated on and back to photo-darkened off stage. The QD-CV probes were introduced into cells, and the visible light excitation yielded photo-modulation nearly ten folds in intracellular environment. Exploiting the stochastic PL burst of QD-CVs and multiplexing capability of QDs, simultaneous multi-color super-resolution localizations were demonstrated. (4) Recent technological advances have expanded fluorescence imaging into the second near-infrared region (NIR-II; wavelength = 1000–1700 nm), providing high spatial resolution through deep tissues. However, bright and compact fluorophores are rare in this region, and sophisticated control over NIR-II probes has not been achieved yet. We showcase an enzyme-activatable NIR-II probe that exhibits turn-on fluorescence upon matrix metalloprotease activity in tumor microenvironment. ​

Sensitive and specific detection of biomaterials packaged in exosomes and related extracellular vesicles (EVs) has the potential to revolutionize cancer diagnosis and monitoring. Yet current methods cannot readily distinguish tumor-associated EVs. Surface-enhanced Raman spectroscopy (SERS) represents a promising tool to address current limitations, but are challenging to implement in whole biofluids. Here we outline a simple SERS assay combining nanoparticles with biofluids purified to various extent. We measure variation between clinical samples of head and neck cancer and demonstrate that there is a trade-off between useful molecular information from purified EVs versus the time, cost, and difficulty of isolation procedures.

The unique optoelectronic properties of semiconductor nanocrystals (quantum dots, QDs), have led to many advances in optoelectronic devices, bioimaging, and biosensing. Recent studies have shown that atomically defined, zero-dimensional magic-size clusters (MSCs) play a crucial role during the nucleation and growth of QDs. A major challenge is the preparation of the MSCs in single-ensemble form without coexistence of other-size QDs. Here we present a heat-up one-pot synthesis approach for the preparation of single-sized ZnSe MSCs. By using the thiol ligand 1-dodecanethiol, we could obtain new insights into the complex interplay of precursors and ligands during MSCs formation.

Biocompatible colloidal nanomaterials are of great interest in the biomedical field due to their ability to modulate redox reactions that translates into antioxidant aides. Common wet syntheses processes utilized to obtain chalcogens nanoparticles have limitations such as low yield, high cost and use environmentally unfriendly chemical precursors and solvents. Pulsed laser ablation in liquids (PLAL) has shown to be an affordable, clean and rapid technique to produce chalcogen nanoparticles. Among the chalcogens, selenium (Se) has well-known capabilities of regulating the glutathione to reduced glutathione (GSH/GSSG) ratio, an established marker of ROS antioxidant activity in eukaryotic cells. Recently there has been an interest to include heavier chalcogens, e.g., tellurium (Te), in biological enzymatic interactions; however, due to its relative cytotoxicity, use of Te nanoparticles as an alternative to reduce glutathione, has not been fully investigated. In this work, we introduce the synthesis and characterization of a selenium-tellurium (SeTe) nano-alloy by PLAL using a deep eutectic solvent (DES), water and acetone as the liquid phase to exploit DES’s biocompatible composition and its influence on the PLAL synthesis kinetics that result in production of polycrystalline, sub-100nm nanoparticles. To investigate the formation of nano-alloy, we compare the features and properties of colloidal nanoparticles produced by PLAL at three wavelengths, 1064, 532 and 355 nm, respectively. We test bioactivity of SeTe nano-alloys, using A-375 (malignant melanoma) and C-33A (epithelial retinoblastoma) cells through assessing viability and proliferation to determine their capabilities towards use as anticancer treatments.

The integration of biosensors into the clinic can transform the ability to monitor personal and public health. High sensitivity and specificity have been realized leveraging nanostructured sensors and surface enhanced Raman spectroscopy (SERS), which can be further integrated by fluorescence to push the boundaries of sensitivity, selectivity, and multiplexing. However, to ensure accurate and reliable applicability, the nanostructured probes need to be designed, synthesized, and characterized very rigorously. In my talk, I will report on our development of intracellular probes for the detection of viral pathogens, providing tips and guidelines to reduce opsonization, increase stability, reduce cytotoxicity, and improve performance.

The use of plasmonic nanoparticles for biomedical applications has been widely explored, resulting in significant advances in the construction of optical biosensors. The shape and size of AuNPs determines the spectral signature of their Localized Surface Plasmon Resonance (LSPR) and, therefore, the features of their plasmonic band can be used to monitor surface changes such as those related to protein binding or nanoparticle aggregation. In this work, gold nanoparticles (AuNPs) were produced based on a green and sustainable methodology using tea leaves. The phytochemicals present in tea act as reducing and stabilizing agents. To optimize the AuNPs deposition (nanomaterial proximity, homogenization and substrate coverage), ITO surfaces were modified with different materials, namely sol-gel matrices (e.g. (3-aminopropyl) triethoxysilane (APTES)), cross-linking agents (e.g., glutaraldehyde) and biopolymers (e.g., Bovine Serum Albumin (BSA)). The produced AuNPs were deposited directly onto ITO surfaces functionalized with APTES or in a mixture of BSA and glutaraldehyde; these matrices are transparent and thus suitable for optical applications. The functionalization procedure of ITO surfaces with the referred materials was performed by two methodologies: i) direct deposition of the matrix solution using a micropipette and ii) ultrasound irradiation. The resulting functionalized ITO surfaces were compared and characterized by light transmission spectroscopy. Accordingly, the tea-AuNPs deposited in the presence of BSA and glutaraldehyde provided the best plasmonic response, being the most promising ones for the development of an optical immunosensor.

Colloidal semiconductor quantum dots (QDs) constitute zero-dimension excitonic materials characterized by quantized states and able to emit fluorescence. QDs are promising materials for catalysis, molecular recognition and biosensing. In this context, our work consists in the design of a new class of biochemical sensors based on QD-grafted chips to benefit from their high opto-electronic activity in the visible range. In order to achieve this, we functionalize silica and CaF2 susbtrates with 4 nm-diameter CdTe QDs and organic molecules (e.g. phenylamine). The originality of our work then lies in two-colour sum-frequency generation nonlinear optical spectroscopy, mixing Raman and IR spectroscopies. This technique enables to probe and to quantify the coupling between the excitonic properties of the QDs and the vibrational response of their molecular environment: two tunable visible and IR laser beams are mixed on the QD-grafted chips to electronically excite the QDs while the vibrational spectroscopy of the surrounding organic species is performed. Through this approach, we clearly demonstrated a correlation between QDs and molecules. Especially, the vibrational response of the molecules is maximized when the first excitonic state of the CdTe QDs is pumped by the visible beam, which means it is possible to enhance the detection of given biomolecules thanks to QDs. Considering that confined excitons transfer their energy to molecules through dipolar interaction, the model we developed accounts indeed for such a behaviour.

In this paper we introduce DNA origami as a tool to study protein-protein interactions, including ligand-receptor interactions of the tumor necrosis superfamily. Additionally, we introduce DNA origami-templated biomineralization, which allows for the formation of super-stable inorganic nanoconstructs with programmable shape and size for potential downstream biomedical applications.

CRISPR-Cas9 is an efficient and reliable tool to cleave targeted portions of DNA. Once the DNA been cut by the tool, genes are ready to be cut, inserted or modified as desired. Conventionally, CRISPR is carried by a virus into cells. However, potential safety issues exists, as the cell can physiologically respond to viral invasion, or its genome can be inserted with unwanted virus genes. Here, we show that gold nanoparticles serves as preferable successful vectors of CRISPR-Cas9, with extra benefit to allow stable imaging. This toxicity-free alternative method avoids the aforementioned issues and introduces the CRISPR-Cas9 complex safely

CRISPR-Cas9 is a gene editing tool which has promise for the treatment and prevention of many complex diseases.  Together with a short guide RNA, CRISPR-Cas9 can recognize and cut the corresponding DNA at a targeted location. Viruses are traditionally used as carriers of CRISPR-Cas9 into target cells. However, viruses may cause immunogenic complications. Alternatively, gold nanoparticles can be utilized as carriers of CRISPR-Cas9. Gold nanoparticles in the near infrared regime exhibit unique optical properties. In this work, we show gold nanoparticles can be stably and efficiently crosslinked to the CRISPR complex.These alternative carriers are toxicity free to and allow effective and efficient introduction of the CRISPR system.

Cold storage can be challenging and expensive for the transportation and storage of biologics. We are developing a new processing technique, light-assisted drying (LAD), to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigeration. DNA and RNA have emerged as building blocks for versatile biological drugs, called therapeutic nucleic acids (TNAs). NANPs have been developed to simultaneously deliver multiple TNAs and to conditionally activate TNAs and control their immunorecognition. The structural and chemical instability of NANPs over long-term storage at ambient temperatures is a challenge that may hamper broad use of this promising technology. In this work we apply the LAD technique to NANPs. NANPs suspended in a droplet of trehalose solution are irradiated with a near-IR laser to accelerate drying. As water is removed, the trehalose forms a protective matrix. The laser allows for careful control of sample temperature during processing. This is important as NANPs are thermally sensitive. In this study, RNA cubes (a type of NANP) were LAD processed and then stored for 1 month. Damage to LAD-processed NANPs was assessed after storage using gel electrophoresis and compared to unprocessed controls stored at 4°C. The thermal histories of samples were monitored during processing to determine the importance of temperature excursions on NANP viability after processing. The trehalose matrix was characterized using polarized light imaging to determine if crystallization occurred during storage, damaging embedded NANPs. These preliminary studies indicate that LAD processing can stabilize NANPs for dry-state storage at room temperatures.

Pancreatic cancer is one of the deadliest with a 5-year survival of 6 to 9%. Nanotechnology offers paradigm-changing opportunity to treat such cancers. Successful integration of nanotechnology into the current paradigm of cancer therapy requires proper understanding of the interface between nanoparticles (NPs) and cancer cells, as well as other key components within the tumor microenvironment (TME), such as normal fibroblasts (FBs) and cancer-associated FBs (CAFs). So far, much focus has been on cancer cells, but FBs and CAFs also play a critical role: FBs suppress the tumor growth while CAFs promote it. It is not yet known how NPs interact with FBs and CAFs compared to cancer cells. Hence, our goal was to elucidate the extent of NP uptake and retention in cancer cells, FBs, and CAFs of pancreatic origin to further understand the fate of NPs in a real tumor-like environment. We used gold nanoparticles as our model NP system due to their numerous applications in cancer therapy, including radiotherapy and chemotherapy. Our NP uptake studies revealed that both cancer cells and CAFs take up 50% more NPs compared to NFs. We also looked at the potential of NP retention. Cancer cells and CAFs were still managed retain between 70 to 80% of NPs over a 24 hour time period. Higher uptake and retention of NPs in cancer cells and CAFs vs FBs is very important in promoting NP-based applications in cancer therapy. Our results show potential in modulating uptake and retention of GNPs among key components of TME, in an effort to develop NP-based strategies to suppress the tumor growth. An ideal NP-based platform would eradicate tumor cells, protect FBs, and deactivate CAFs. Therefore, this study lays a road map to exploit the TME for the advancement of “smart” nanomedicines that would constitute the next generation of cancer therapeutics to treat pancreatic cancer.