Transforming ovarian cancer care by targeting minimal residual disease.

Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences.

A Machine Learning-optimized system for on demand, pulsatile, photo- and chemo-therapeutic treatment using near-infrared responsive MoS 2 -based microparticles in a breast cancer model.

Cancer therapy research is of high interest because of the persistence and mortality of the disease and the side effects of traditional therapeutic methods, while often multimodal treatments are necessary based on the patient's needs. The development of less invasive modalities for recurring treatment cycles is thus of critical significance. Herein, a light-activatable microparticle system was developed for localized, pulsatile delivery of anticancer drugs with simultaneous thermal ablation, by applying controlled ON-OFF thermal cycles using near-infrared laser irradiation. The system is composed of poly(caprolactone) microparticles of 200 µm size with incorporated molybdenum disulfide (MoS 2 ) nanosheets as the photothermal agent and hydrophilic doxorubicin or hydrophobic violacein, as model drugs. Upon irradiation the nanosheets heat up to ≥50 °C leading to polymer matrix melting and release of the drug. MoS 2 nanosheets exhibit high photothermal conversion efficiency and allow for application of low power laser irradiation for the system activation. A Machine Learning algorithm was applied to acquire optimal laser operation conditions; 0.4 W/cm 2 laser power at 808 nm, 3-cycle irradiation, for 3 cumulative minutes. In a mouse subcutaneous model of 4T1 triple-negative breast cancer, 25 microparticles were intratumorally administered and after 3-cycle laser treatment the system conferred synergistic phototherapeutic and chemotherapeutic effect. Our on-demand, pulsatile synergistic treatment resulted in increased median survival up to 40 days post start of treatment compared to untreated mice, with complete eradication of the tumors at the primary site. Such a system could have potential for patients in need of recurring cycles of treatment on subcutaneous tumors.

Surface Plasmon-Enhanced Short-Wave Infrared Fluorescence for Detecting Sub-Millimeter-Sized Tumors.

Short-wave infrared (SWIR, 900-1700 nm) enables in vivo imaging with high spatiotemporal resolution and penetration depth due to the reduced tissue autofluorescence and decreased photon scattering at long wavelengths. Although small organic SWIR dye molecules have excellent biocompatibility, they have been rarely exploited as compared to their inorganic counterparts, mainly due to their low quantum yield. To increase their brightness, in this work, the SWIR dye molecules are placed in close proximity to gold nanorods (AuNRs) for surface plasmon-enhanced emission. The fluorescence enhancement is optimized by controlling the dye-to-AuNR number ratio and up to ≈45-fold enhancement factor is achieved. In addition, the results indicate that the highest dye-to-AuNR number ratio gives the highest emission intensity per weight and this is used for synthesizing SWIR imaging probes using layer-by-layer (LbL) technique with polymer coating protection. Then, the SWIR imaging probes are applied for in vivo imaging of ovarian cancer and the surface coating effect on intratumor distribution of the imaging probes is investigated in two orthotopic ovarian cancer models. Lastly, it is demonstrated that the plasmon-enhanced SWIR imaging probe has great potential for fluorescence imaging-guided surgery by showing its capability to detect sub-millimeter-sized tumors.

M13 Virus-Based Framework for High Fluorescence Enhancement.

Fluorescence imaging is a powerful tool for studying biologically relevant macromolecules, but its applicability is often limited by the fluorescent probe, which must demonstrate both high site-specificity and emission efficiency. In this regard, M13 virus, a versatile biological scaffold, has previously been used to both assemble fluorophores on its viral capsid with molecular precision and to also target a variety of cells. Although M13-fluorophore systems are highly selective, these complexes typically suffer from poor molecular detection limits due to low absorption cross-sections and moderate quantum yields. To overcome these challenges, a coassembly of the M13 virus, cyanine 3 dye, and silver nanoparticles is developed to create a fluorescent tag capable of binding with molecular precision with high emissivity. Enhanced emission of cyanine 3 of up to 24-fold is achieved by varying nanoparticle size and particle-fluorophore separation. In addition, it is found that the fluorescence enhancement increases with increasing dye surface density on the viral capsid. Finally, this highly fluorescent probe is applied for in vitro staining of E. coli. These results demonstrate an inexpensive framework for achieving tuned fluorescence enhancements. The methodology developed in this work is potentially amendable to fluorescent detection of a wide range of M13/cell combinations.

Real-Time Single-Walled Carbon Nanotube-Based Fluorescence Imaging Improves Survival after Debulking Surgery in an Ovarian Cancer Model.

Improved cytoreductive surgery for advanced stage ovarian cancer (OC) represents a critical challenge in the treatment of the disease. Optimal debulking reaching no evidence of macroscopic disease is the primary surgical end point with a demonstrated survival advantage. Targeted molecule-based fluorescence imaging offers complete tumor resection down to the microscopic scale. We used a custom-built reflectance/fluorescence imaging system with an orthotopic OC mouse model to both quantify tumor detectability and evaluate the effect of fluorescence image-guided surgery on post-operative survival. The contrast agent is an intraperitoneal injectable nanomolecular probe, composed of single-walled carbon nanotubes, coupled to an M13 bacteriophage carrying a modified peptide binding to the SPARC protein, an extracellular protein overexpressed in OC. The imaging system is capable of detecting a second near-infrared window fluorescence (1000-1700 nm) and can display real-time video imagery to guide intraoperative tumor debulking. We observed high microscopic tumor detection with a pixel-limited resolution of 200 µm. Moreover, in a survival-surgery orthotopic OC mouse model, we demonstrated an increased survival benefit for animals treated with fluorescence image-guided surgical resection compared to standard surgery.

Deep-tissue optical imaging of near cellular-sized features.

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has ~125 cells), out of ~37 trillion cells in the human body. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000-1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, "Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared" (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without a priori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.

Enhanced Cell Capture on Functionalized Graphene Oxide Nanosheets through Oxygen Clustering.

With the global rise in incidence of cancer and infectious diseases, there is a need for the development of techniques to diagnose, treat, and monitor these conditions. The ability to efficiently capture and isolate cells and other biomolecules from peripheral whole blood for downstream analyses is a necessary requirement. Graphene oxide (GO) is an attractive template nanomaterial for such biosensing applications. Favorable properties include its two-dimensional architecture and wide range of functionalization chemistries, offering significant potential to tailor affinity toward aromatic functional groups expressed in biomolecules of interest. However, a limitation of current techniques is that as-synthesized GO nanosheets are used directly in sensing applications, and the benefits of their structural modification on the device performance have remained unexplored. Here, we report a microfluidic-free, sensitive, planar device on treated GO substrates to enable quick and efficient capture of Class-II MHC-positive cells from murine whole blood. We achieve this by using a mild thermal annealing treatment on the GO substrates, which drives a phase transformation through oxygen clustering. Using a combination of experimental observations and MD simulations, we demonstrate that this process leads to improved reactivity and density of functionalization of cell capture agents, resulting in an enhanced cell capture efficiency of 92 ± 7% at room temperature, almost double the efficiency afforded by devices made using as-synthesized GO (54 ± 3%). Our work highlights a scalable, cost-effective, general approach to improve the functionalization of GO, which creates diverse opportunities for various next-generation device applications.

Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells.

Peripheral blood can provide valuable information on an individual's immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.1 Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single-domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (∼30 µL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.

Carbon nanotubes as in vivo bacterial probes.

With the rise in antibiotic-resistant infections, non-invasive sensing of infectious diseases is increasingly important. Optical imaging, although safer and simpler, is less developed than other modalities such as radioimaging, due to low availability of target-specific molecular probes. Here we report carbon nanotubes (SWNTs) as bacterial probes for fluorescence imaging of pathogenic infections. We demonstrate that SWNTs functionalized using M13 bacteriophage (M13-SWNT) can distinguish between F'-positive and F'-negative bacterial strains. Moreover, through one-step modification, we attach an anti-bacterial antibody on M13-SWNT, making it easily tunable for sensing specific F'-negative bacteria. We illustrate detection of Staphylococcus aureus intramuscular infections, with ~3.4 × enhancement in fluorescence intensity over background. SWNT imaging presents lower signal spread ~0.08 × and higher signal amplification ~1.4 × , compared with conventional dyes. We show the probe offers greater ~5.7 × enhancement in imaging of S. aureus infective endocarditis. These biologically functionalized, aqueous-dispersed, actively targeted, modularly tunable SWNT probes offer new avenues for exploration of deeply buried infections.

Scalable enhancement of graphene oxide properties by thermally driven phase transformation.

Chemical functionalization of graphene is promising for a variety of next-generation technologies. Although graphene oxide (GO) is a versatile material in this direction, its use is limited by the production of metastable, chemically inhomogeneous and spatially disordered GO structures under current synthetic protocols, which results in poor optoelectronic properties. Here, we present a mild thermal annealing procedure, with no chemical treatments involved, to manipulate as-synthesized GO on a large scale to enhance sheet properties with the oxygen content preserved. Using experiments supported by atomistic calculations, we demonstrate that GO structures undergo a phase transformation into prominent oxidized and graphitic domains by temperature-driven oxygen diffusion. Consequently, as-synthesized GO that absorbs mainly in the ultraviolet region becomes strongly absorbing in the visible region, photoluminescence is blue shifted and electronic conductivity increases by up to four orders of magnitude. Our thermal processing method offers a suitable way to tune and enhance the properties of GO, which creates opportunities for various applications.

M13 virus based detection of bacterial infections in living hosts.

We report a first method for using M13 bacteriophage as a multifunctional scaffold for optically imaging bacterial infections in vivo. We demonstrate that M13 virus conjugated with hundreds of dye molecules (M13-Dye) can target and distinguish pathogenic infections of F-pili expressing and F-negative strains of E. coli. Further, in order to tune this M13-Dye complex suitable for targeting other strains of bacteria, we have used a 1-step reaction for creating an anti-bacterial antibody-M13-Dye probe. As an example, we show anti-S. aureus-M13-Dye able to target and image infections of S. aureus in living hosts, with a 3.7× increase in fluorescence over background.