Noninvasive In Vivo Imaging of T-Cells during Cancer Immunotherapy Using Rare-Earth Nanoparticles.

Only a minority of patients respond positively to cancer immunotherapy, and addressing this variability is an active area of immunotherapy research. Infiltration of tumors by immune cells is one of the most significant prognostic indicators of response and disease-free survival. However, the ability to noninvasively sample the tumor microenvironment for immune cells remains limited. Imaging in the near-infrared-II region using rare-earth nanocrystals is emerging as a powerful imaging tool for high-resolution deep-tissue imaging. In this paper, we demonstrate that these nanoparticles can be used for noninvasive in vivo imaging of tumor-infiltrating T-cells in a highly aggressive melanoma tumor model. We present nanoparticle synthesis and surface modification strategies for the generation of small, ultrabright, and biocompatible rare-earth nanocrystals necessary for deep tissue imaging of rare cell types. The ability to noninvasively monitor the immune contexture of a tumor during immunotherapy could lead to early identification of nonresponding patients in real time, leading to earlier interventions and better outcomes.

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.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

Engineering a Two-Component Hemostat for the Treatment of Internal Bleeding through Wound-Targeted Crosslinking.

Primary hemostasis (platelet plug formation) and secondary hemostasis (fibrin clot formation) are intertwined processes that occur upon vascular injury. Researchers have sought to target wounds by leveraging cues specific to these processes, such as using peptides that bind activated platelets or fibrin. While these materials have shown success in various injury models, they are commonly designed for the purpose of treating solely primary or secondary hemostasis. In this work, a two-component system consisting of a targeting component (azide/GRGDS PEG-PLGA nanoparticles) and a crosslinking component (multifunctional DBCO) is developed to treat internal bleeding. The system leverages increased injury accumulation to achieve crosslinking above a critical concentration, addressing both primary and secondary hemostasis by amplifying platelet recruitment and mitigating plasminolysis for greater clot stability. Nanoparticle aggregation is measured to validate concentration-dependent crosslinking, while a 1:3 azide/GRGDS ratio is found to increase platelet recruitment, decrease clot degradation in hemodiluted environments, and decrease complement activation. Finally, this approach significantly increases survival relative to the particle-only control in a liver resection model. In light of prior successes with the particle-only system, these results emphasize the potential of this technology in aiding hemostasis and the importance of a holistic approach in engineering new treatments for hemorrhage.

A new label-free optical imaging method for the lymphatic system enhanced by deep learning.

Our understanding of the lymphatic vascular system lags far behind that of the blood vascular system, limited by available imaging technologies. We present a label-free optical imaging method that visualizes the lymphatic system with high contrast. We developed an orthogonal polarization imaging (OPI) in the shortwave infrared range (SWIR) and imaged both lymph nodes and lymphatic vessels of mice and rats in vivo through intact skin, as well as human mesenteric lymph nodes in colectomy specimens. By integrating SWIR-OPI with U-Net, a deep learning image segmentation algorithm, we automated the lymph node size measurement process. Changes in lymph nodes in response to cancer progression were monitored in two separate mouse cancer models, through which we obtained insights into pre-metastatic niches and correlation between lymph node masses and many important biomarkers. In a human pilot study, we demonstrated the effectiveness of SWIR-OPI to detect human lymph nodes in real time with clinical colectomy specimens. One Sentence Summary: We develop a real-time high contrast optical technique for imaging the lymphatic system, and apply it to anatomical pathology gross examination in a clinical setting, as well as real-time monitoring of tumor microenvironment in animal studies.

Phage Particles of Controlled Length and Genome for In Vivo Targeted Glioblastoma Imaging and Therapeutic Delivery.

M13 bacteriophage (phage) are versatile, genetically tunable nanocarriers that have been recently adapted for use as diagnostic and therapeutic platforms. Applying p3 capsid chlorotoxin fusion with the "inho" circular single-stranded DNA (cssDNA) gene packaging system, we produced miniature chlorotoxin inho (CTX-inho) phage particles with a minimum length of 50 nm that can target intracranial orthotopic patient-derived GBM22 glioblastoma tumors in the brains of mice. Systemically administered indocyanine green conjugated CTX-inho phage accumulated in brain tumors, facilitating shortwave infrared detection. Furthermore, we show that our inho phage can carry cssDNA that are transcriptionally active when delivered to GBM22 glioma cells in vitro. The ability to modulate the capsid display, surface loading, phage length, and cssDNA gene content makes the recombinant M13 phage particle an ideal delivery platform.

Surface Plasmon Enhanced Upconversion Fluorescence in Short-Wave Infrared for In Vivo Imaging of Ovarian Cancer.

Short-wave infrared (SWIR; 850-1700 nm) upconversion fluorescence enables "autofluorescence-free" imaging with minimal tissue scattering, yet it is rarely explored due to the lack of strongly emissive SWIR upconversion fluorophores. In this work, we apply SWIR upconversion fluorescence for in vivo imaging with exceptional image contrast. Gold nanorods (AuNRs) are used to enhance the SWIR upconversion emission of small organic dyes, forming a AuNR-dye nanocomposite (NC). A maximal enhancement factor of ∼1320, contributed by both excitation and radiative decay rate enhancement, is achieved by varying the dye-to-AuNR ratio. In addition, the upconversion emission intensity of both free dyes and AuNR-dye NCs depends linearly on the excitation power, indicating that the upconversion emission mechanism remains unchanged upon enhancement, and it involves one-photon absorption. Moreover, the SWIR upconversion emission shows a significantly higher signal contrast than downconversion emission in the same emission window in a nonscattering medium. Finally, we apply the surface plasmon enhanced SWIR upconversion fluorescence for in vivo imaging of ovarian cancer, demonstrating high image contrast and low required dosage due to the suppressed autofluorescence.

Peptide-Based Cancer Vaccine Delivery via the STINGΔTM-cGAMP Complex.

With the advent of bioinformatic tools in efficiently predicting neo-antigens, peptide vaccines have gained tremendous attention in cancer immunotherapy. However, the delivery of peptide vaccines remains a major challenge, primarily due to ineffective transport to lymph nodes and low immunogenicity. Here, a strategy for peptide vaccine delivery is reported by first fusing the peptide to the cytosolic domain of the stimulator of interferon genes protein (STINGΔTM), then complexing the peptide-STINGΔTM protein with STING agonist 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The process results in the formation of self-assembled cGAMP-peptide-STINGΔTM tetramers, which enables efficient lymphatic trafficking of the peptide. Moreover, the cGAMP-STINGΔTM complex acts not only as a protein carrier for the peptide, but also as a potent adjuvant capable of triggering STING signaling independent of endogenous STING protein-an especially important attribute considering that certain cancer cells epigenetically silence their endogenous STING expression. With model antigen SIINFEKL, it is demonstrated that the platform elicits effective STING signaling in vitro, draining lymph node targeting in vivo, effective T cell priming in vivo as well as antitumoral immune response in a mouse colon carcinoma model, providing a versatile solution to the challenges faced in peptide vaccine delivery.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

Structural ceramic batteries using an earth-abundant inorganic waterglass binder.

Sodium trisilicate waterglass is an earth-abundant inorganic adhesive which binds to diverse materials and exhibits extreme chemical and temperature stability. Here we demonstrate the use of this material as an electrode binder in a lay-up based manufacturing system to produce structural batteries. While conventional binders for structural batteries exhibit a trade-off between mechanical and electrochemical performance, the waterglass binder is rigid, adhesive, and facilitates ion transport. The bulk binder maintains a Young's modulus of >50 GPa in the presence of electrolyte solvent while waterglass-based electrodes have high rate capability and stable discharge capacity over hundreds of electrochemical cycles. The temperature stability of the binder enables heat treatment of the full cell stack following lay-up shaping in order to produce a rigid, load-bearing part. The resulting structural batteries exhibit impressive multifunctional performance with a package free cell stack-level energy density of 93.9 Wh/kg greatly surpassing previously published structural battery materials, and a tensile modulus of 1.4 GPa.

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.

Simulating selective binding of a biological template to a nanoscale architecture: a core concept of a clamp-based binding-pocket-favored N-terminal-domain assembly.

The biological template and its mutants have vital significance in next generation remediation, electrochemical, photovoltaic, catalytic, sensing and digital memory devices. However, a microscopic model describing the biotemplating process is generally lacking on account of modelling complexity, which has prevented widespread commercial use of biotemplates. Here, we demonstrate M13-biotemplating kinetics in atomic resolution by leveraging large-scale molecular dynamics (MD) simulations. The model reveals the assembly of gold nanoparticles on two experimentally-based M13 phage types using full M13-capsid structural models and with polarizable gold nanoparticles in explicit solvent. Both mechanistic and structural insights into the selective binding affinity of the M13 phage to gold nanoparticles are obtained based on a previously unconsidered clamp-based binding-pocket-favored N-terminal-domain assembly and also on surface-peptide flexibility. These results provide a deeper level of understanding of protein sequence-based affinity and open the route for genetically engineering a wide range of 3D electrodes for high-density low-cost device integration.

Designing yeast as plant-like hyperaccumulators for heavy metals.

Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr90. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.

Virus-Templated Nickel Phosphide Nanofoams as Additive-Free, Thin-Film Li-Ion Microbattery Anodes.

Transition metal phosphides are a new class of materials generating interest as alternative negative electrodes in lithium-ion batteries. However, metal phosphide syntheses remain underdeveloped in terms of simultaneous control over phase composition and 3D nanostructure. Herein, M13 bacteriophage is employed as a biological scaffold to develop 3D nickel phosphide nanofoams with control over a range of phase compositions and structural elements. Virus-templated Ni5 P4 nanofoams are then integrated as thin-film negative electrodes in lithium-ion microbatteries, demonstrating a discharge capacity of 677 mAh g-1 (677 mAh cm-3 ) and an 80% capacity retention over more than 100 cycles. This strong electrochemical performance is attributed to the virus-templated, nanostructured morphology, which remains electronically conductive throughout cycling, thereby sidestepping the need for conductive additives. When accounting for the mass of additional binder materials, virus-templated Ni5 P4 nanofoams demonstrate the highest practical capacity reported thus far for Ni5 P4 electrodes. Looking forward, this synthesis method is generalizable and can enable precise control over the 3D nanostructure and phase composition in other metal phosphides, such as cobalt and copper.

Thermally robust solvent-free biofluids of M13 bacteriophage engineered for high compatibility with anhydrous ionic liquids.

Here, we demonstrate a chemical modification strategy to create biomaterials of the M13 bacteriophage with extraordinary thermal stability, and high compatibility with non-aqueous ionic liquids. The results provide a blueprint for developing soft materials with well-defined architectures that may find broad applicability in the next generation of flexible devices.

Creating fluorescent quantum defects in carbon nanotubes using hypochlorite and light.

Covalent doping of single-walled carbon nanotubes (SWCNTs) can modify their optical properties, enabling applications as single-photon emitters and bio-imaging agents. We report here a simple, quick, and controllable method for preparing oxygen-doped SWCNTs with desirable emission spectra. Aqueous nanotube dispersions are treated at room temperature with NaClO (bleach) and then UV-irradiated for less than one minute to achieve optimized O-doping. The doping efficiency is controlled by varying surfactant concentration and type, NaClO concentration, and irradiation dose. Photochemical action spectra indicate that doping involves reaction of SWCNT sidewalls with oxygen atoms formed by photolysis of ClO- ions. Variance spectroscopy of products reveals that most individual nanotubes in optimally treated samples show both pristine and doped emission. A continuous flow reactor is described that allows efficient preparation of milligram quantities of O-doped SWCNTs. Finally, we demonstrate a bio-imaging application that gives high contrast short-wavelength infrared fluorescence images of vasculature and lymphatic structures in mice injected with only ~100 ng of the doped nanotubes.

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.