At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting. The present work encompasses the large-scale production of colloidally stable nanoruby particles, the demonstration of their biofunctionality and negligible cytotoxicity, as well as the validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to those of state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an example, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to μ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single-particle sensitivity.

Radiofrequency ablation (RFA) and doxorubicin (Dox) chemotherapy are separately approved for liver cancer therapy; however, both have limited success in the clinic due to suboptimal/nonuniform heating and systemic side effects, respectively. Here, we report a biodegradable nanoparticle (NP) system showing excellent RF hyperthermic response together with the ability to locally deliver Dox in the liver under RF trigger and control. The nanosystem was prepared by doping a clinically permissible dose (∼4.3 wt %, 0.03 ppm) of stannous ions in alginate nanoparticles (∼100 nm) coloaded with Dox at ∼13.4 wt % concentration and surface conjugated with galactose for targeting asialo-glycoprotein receptors in liver tumors. Targeted NP-uptake and increased cytotoxicity when combined with RF exposure was demonstrated in HEPG2 liver cancer cells. Following in vitro (chicken liver phantom) demonstration of locally augmented RF thermal response, in vivo scintigraphic imaging of 99Tc-labeled NPs was performed to optimize liver localization in Sprague–Dawley (SD) rats. RF ablation was performed in vivo using a cooled-tip probe, and uniformly enhanced (∼44%) thermal ablation was demonstrated with magnetic resonance imaging along with RF-controlled Dox release. In orthotopic rat liver tumor models, real-time infrared imaging revealed significantly higher (∼20 °C) RF thermal response at the tumor site, resulting in uniform augmented ablation (∼80%) even at a low RF power exposure of 15 W for just 1 min duration. Being a clinically acceptable, biodegradable material, alginate nanoparticles hold strong translational potential for augmented RF hyperthermia combined with triggered drug release.

Protein-protein interactions at the plasma membrane mediate transmembrane signaling. Dual-channel fluorescence cross-correlation spectroscopy (dc-FCCS) is a method with which these interactions can be quantified in a cellular context. However, factors such as incomplete maturation of fluorescent proteins, spectral crosstalk, and fluorescence resonance energy transfer (FRET) affect quantification. Some of these can be corrected or accounted for during data analysis and/or interpretation. Here, we experimentally and analytically demonstrate that it is difficult to correct the error caused due to FRET when applying dc-FCCS to measure binding affinity or bound molecular concentrations. Additionally, the presence of dark fluorescent proteins due to incomplete maturation introduces further errors, which too cannot be corrected in the presence of FRET. Based on simulations, we find that modalities such as pulse-interleaved excitation FCCS do not eliminate FRET-induced errors. Finally, we demonstrate that the detrimental effect of FRET can be eliminated with careful experimental design when applying dc-FCCS to quantify protein-protein interactions at the plasma membrane of living cells.

Background: Enhanced T cell performance and fitness are imperative for the success of adoptive T cell-based therapies. Beyond the types of genetic modifications to CAR/TCR T cells, there is a growing body of literature demonstrating that relatively simple preconditioning protocols can also be used to improve T cell fitness/function. We studied, the impact that preconditioning in elevated concentrations of leucine, glutamine, and arginine has on the killing efficacy and bioenergetics of MART-1-specific TCR T cells. Methods: Using the Agilent xCELLigence RTCA eSight and Seahorse we assessed the killing efficiency and bioenergetics of engineered T-cells after Arginine, Glutamine, and Leucine pre-conditioning using MART-1 specific TCR T cells. CD3+ T-cells (Hemacare, Seattle, WA) were transduced with retrovirus SAMEN-DMF5 with a CD34 marker gene, against MART-1. The T cells were pre-conditioned in a range of concentrations varying between 0-6mM for 7 days, followed by a killing assay using MART-1 expressing melanoma cell line as target cells (624.38) engineered to express a red-fluorescent nuclear protein. The comparison was made with transduced T-cells grown in RPMI (no added amino acid supplementation denoted as RPMI_TCR), RPMI supplemented with Arginine (Arg_TCR) and non-transduced T cells. The T cell killing was measured using impedance/imaging-based assays. CD34 assessment was performed using Novocyte and SRC (spare respiratory capacity) and oxygen consumption rate (OCR) were measured using seahorse assays. Results: Whereas supplementing the growth medium with 6 mM Arginine increased killing efficacy dramatically (up to ∼6-fold), elevated leucine and glutamine concentrations were found to have minimal impact on MART-1 TCR T cell killing of melanoma cells. Arginine (6 mM) supplementation increased basal respiration, ATP linked OCR, and maximal respiration compared to the RPMI control. SRC of Arg_TCR T cells was significantly higher than RPMI preconditioned T cells, a parameter previously correlated with T cell persistence. To check the effect of a shortened pre-conditioning period, 2, and 4 days of pre-conditioning were done along with the 7 days method. After a preconditioning step of only 2 days, Arginine preconditioned T cells acquired a killing efficacy that is >2x higher compared to their counterparts RPMI_TCR T cells. Extending the duration of preconditioning from 2 to 4 days has minimal impact on the RPMI control T cells but more than doubles the killing efficacy of the high Arg grown T cells. Conclusions: In conclusion, Arginine pre-conditioning significantly improved T cell potency and mitochondrial respiration through metabolic rewiring. Citation Format: Rashmi R. Pillai, Xiaoyu Zhang, Yama Abassi, Brandon Lamarche, Mark M. Garner. Arginine pre-conditioning improves T-cell potency and metabolic fitness measured by real-time impedance and seahorse assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB094.

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