Thermoresponsive CuS based nanocomposites Mimicking Red Blood Cells for Combinatorial Therapy and Photoacoustic Imaging

Abstract

Non-small cell lung cancer (NSCLC) remains a major public health threat in the US, even with successful early-stage surgeries. Recurrence and advanced stages present complex diagnostic and therapeutic challenges. Traditional treatments, such as chemotherapy and targeted therapies like tyrosine kinase inhibitors (TKIs), face is- sues including non-specific targeting, toxicity, poor absorption, and the development of resistance. Overcoming resistance due to genetic mutations and tumor hetero- geneity is crucial, highlighting the need to identify novel vulnerabilities in NSCLC for continued treatment progress. Also, combination therapy is being widely explored to overcome this problem. Photothermal therapy (PTT) is a promising emerging cancer treatment. PTT uses photothermal transduction agents (PTAs) to convert light energy into heat, inducing cancer cell death. PTT’s advantages include precise tumor targeting through adjustable laser irradiation, minimizing damage to healthy tissues, and being a highly effective, non-invasive treatment. Effective PTAs should have high photothermal conversion efficiency (PCE), non-overlapping absorption with tumor background, and strong tumor accumulation. Moreover, PTT agents can serve as exogenous contrast agents for photoacoustic imaging (PAI), enabling light-based theranostics. PAI is a non-invasive imaging tech- nique that uses light and sound waves to produce high-resolution optical images of internal tissues. Combined with PTT, PAI can track the delivery of light-absorbing PTT agents to the target site and monitor the treatment in real-time, minimizing damage to healthy tissues. This approach reduces side effects and increases treatment effec- tiveness. Nano PTAs, which leverage the enhanced permeability and retention (EPR) effect and active targeting, show particular promise. Past decades have seen advancement in nanoparticle-based drug delivery plat- forms. However, advancements in nano-drug delivery platforms often face challenges in penetrating deep into the tumor microenvironment due to their size and design. Smaller nanoparticles can penetrate deeply but are easily washed away, while larger ones remain at the tumor site but fail to penetrate deeply. Modifying nanoparticle size, surface chemistry, and other properties can improve circulation time, tumor targeting, and penetration. Innovative designs that change size upon reaching the tumor site can enhance circulation and effective tumor distribution. Addressing these challenges and exploring new strategies can maximize the potential of nanoparticle drug delivery for improved cancer treatment outcomes. To address the current gaps in the field, we have explored the loading of CuS nanoparticles (5-20 nm) into smart thermoresponsive nanogels (200-225 nm) along- side an FDA-approved TKI for lung cancer, targeting the EGFR to achieve stimuli- responsive combinatorial therapy. This innovative multi-stage approach has aimed to enhance deep tumor targeting. Upon encountering specific stimuli, the nanogel has been designed to degrade in the tumor environment, facilitating the targeted release of the drug into the tumor microenvironment. Additionally, we have aimed to investigate the potential of coating this loaded nanogel with RBC membrane, thereby mimicking its behavior to avoid opsonization. Combining CuS nanoparticles, a targeted drug, and a stimuli-responsive release mechanism within a nanogel system, this study should offer a promising strategy for achieving more effective and precise deep tumor theranostics for lung cancer. The first task was to prepare CuS nanoparticles with appropriate photothermal ef- ficiency, along with suitable particle size and molecular targeting capability. We syn- thesized CuS nanoparticles with PVP as a stabilizing agent, producing PC10, PCK30, and PC40 variants. We investigated how different PVP molecular weights affect their crystalline properties and photoacoustic/photothermal responses. All nanoparticles showed a crystalline structure, were <10 nm, and exhibited absorbance peaks at 1020 nm, indicating strong photoacoustic and photothermal capabilities. PC10 stood out with superior photoacoustic properties and achieved a remarkable photothermal efficiency of 51%. Cytotoxicity assays confirmed cell compatibility and enhanced apoptosis rates. These results highlight the potential of PVP-stabilized CuS nanoparticles for advanced cancer theranostics. The system’s crucial limitation was the inability to achieve tar- geted functionality without complex ring-opening reactions. We then investigated an alternative system aiming for both optimal photothermal efficiency and targeted deliv- ery. Prior studies have shown anti-cancer properties of functionalized gold (Au) nanoparticles, leading to the exploration of CuS/Au nanohybrids for imaging and therapy. This study focuses on developing ultra-small CuS-Au (TSP-CA) nanohybrids using seed- mediated galvanic reduction with tannic acid (TA) and sodium citrate (SC) as reducing agents. PVP-CuS nanoparticles, prepared from previous research, were used, and TSP-CA nanohybrids averaging 8 nm in size were formed, confirmed by X-ray photo- electron spectroscopy (XPS) showing the presence of Cu and Au. Photoacoustic (PA) experiments showed a higher signal-to-noise ratio, with photothermal therapy (PTT) efficiency estimated at 25%, demonstrating good photostability. In vitro experiments indicated ROS-mediated anticancer effects on lung cancer cells. Ex vivo studies using chicken breast confirmed PTT and PA signal generation at depths of 1 cm and 1.5 cm, with PA signals measured using a 2.25 MHz ultrasound transducer and a 960 nm laser at 1 cm depth. ROS and apoptosis studies revealed enhanced anticancer effects of the hybrid nanoparticles compared to individual nanoparticles. However, this system showed a considerable decrease in photothermal efficiency compared to our previous study. This decrease would compel us to use higher concentrations of particles to achieve the desired anti-cancer effect. We moved on to exploring other materials for CuS synthesis, which could offer the desired properties. While the interaction between mucin and cancer cells is still under exploration, mucin shows promise for developing nanoparticles for theranostics due to its stability- enhancing properties. However, mucin has not yet been utilized to synthesize copper sulfide (CuS) nanoparticles. This study investigates using mucin as a capping agent for CuS (Mu@CuS) nanoparticles to enhance stability for optical theranostic applications. The resulting nanoparticles, less than 10 nm in size, were crystalline and exhibited absorbance in the NIR II region, demonstrating optimal photothermal efficiency (38%) and photoacoustic contrast abilities. To explore targeting ability and cellular uptake, Rhodamine B was conjugated to the mucin-coated CuS nanoparticles (Mu@CuS-Rh), exploiting the negative charges on cancer cell membranes for mitochondrial delivery. Experiments on 2D cell culture and 3D spheroid models showed deep tumor pene- tration and effective photothermal therapeutic effects. Since the particles exhibited all the desirable attributes, they were tested for deep tumor penetration ability in vivo. In vivo, Multispectral optoacoustic imaging analysis of Mu@CuS-Rh nanoparticles re- vealed significant tumor uptake and retention for up to 7 hours, with notable accumulation in hypoxic tumor regions. Interestingly, these particles underscore the potential of Mu@CuS nanoparticles for hypoxic tumor imaging and targeted therapy as well. A thermoresponsive nanogel was developed using NIPAM and MAA to ensure ma- terial delivery only upon thermal stimulation. Various NIPAM and MAA ratios were tested to achieve a sol-gel transition temperature above body temperature. At 8% MAA, the transition temperature rose to 35°C, with most of the transition occurring above 40°C, indicating potential for temperature-triggered drug release. To address gaps in the field, CuS nanoparticles were loaded into these nanogels along with the FDA- approved tyrosine kinase inhibitor Erlotinib, targeting EGFR receptors in lung cancer. Mu@CuS was incorporated in situ during polymerization, while Erlotinib was loaded post-synthesis. TEM and SEM characterization confirmed successful loading. Combi- nation anti-cancer studies on lung cancer cell lines showed that the concentrations of Mu@CuS and Erlotinib could be reduced when used together, indicating a synergistic effect. Furthermore, the loaded nanogel was wrapped with an RBC membrane to form a composite (R@Ng-MuC/Er) for combinatorial theranostics. The photothermal evalu- ation showed that the composite nanoparticles could increase the temperature to 50°C when 1 mg was dispersed in PBS. Extensive in vitro and in vivo studies demonstrated the composite’s superior efficiency over Mu@CuS and Erlotinib alone. In vivo studies indicated significant tumor reduction in mice treated with R@Ng-MuC/Er, without toxic effects on major organs, as suggested by histology. Also, multispectral optoacoustic tomography suggested appropriate composite accumulation in the tumor microenvi- ronment, further explored for imaging and therapy. A biodistribution study suggested major clearance of particles by the liver. The diagnostic properties were evaluated using MSOT, confirming the composite’s potential for precise tumor theranostics. In conclusion, the developed system successfully achieved all the objectives of our study. Among various CuS nanoparticles, mucin-coated CuS nanoparticles (Mu@CuS) exhibited the most promising therapeutic potential. These nanoparticles showed en- hanced interaction with tumor cells, even in hypoxic conditions. Combined with Erlotinib and loaded into thermoresponsive nanogels, they provided a synergistic effect for com- binatorial chemotherapy and photothermal therapy, ensuring on-demand release and minimizing unnecessary interactions with normal cells. The potential of these nanoparticles for photoacoustic tomography further supports their use in image-guided cancer therapy. Their similarity to red blood cells suggests significant potential to evade im- mune responses, though further characterization is needed. In vivo experiments re- vealed substantial tumor regression with tumor imaging properties, highlighting their promising potential for lung cancer treatment. This system warrants further evaluation to advance towards clinical translation.