Applications of Cu2+-Loaded Silica Nanoparticles to Photothermal Therapy and Tumor-Specific Fluorescence Imaging.

Copper-based nanomaterials have been employed as therapeutic agents for cancer therapy and diagnosis. Nevertheless, persistent challenges, such as cellular toxicity, non-uniform sizes, and low photothermal efficiency, often constrain their applications. In this study, we present Cu2+-loaded silica nanoparticles fabricated through the chelation of Cu2+ ions by silanol groups. The integration of Cu2+ ions into uniformly sized silica nanoparticles imparts a photothermal therapy effect. Additionally, the amine functionalization of the silica coating facilitates the chemical conjugation of tumor-specific fluorescence probes. These probes are strategically designed to remain in an 'off' state through the Förster resonance energy transfer mechanism until exposed to cysteine enzymes in cancer cells, inducing the recovery of their fluorescence. Consequently, our Cu2+-loaded silica nanoparticles demonstrate an efficient photothermal therapy effect and selectively enable cancer imaging.

Applications of the Photocatalytic and Photoacoustic Properties of Gold Nanorods in Contrast-Enhanced Ultrasound and Photoacoustic Imaging.

The applications of ultrasound imaging are often limited due to low contrast, which arises from the comparable acoustic impedance of normal tissues and disease sites. To improve the low contrast, we propose a contrast agent called gas-generating laser-activatable nanorods for contrast enhancement (GLANCE), which enhances ultrasound imaging contrast in two ways. First, GLANCE absorbs near-infrared lasers and generates nitrogen gas bubbles through the photocatalytic function of gold nanorods and photolysis of azide compounds. These gas bubbles decrease the acoustic impedance and highlight the injection site from the surrounding tissues. Second, GLANCE exhibits photoacoustic properties owing to the gold nanorods that emit photoacoustic signals upon laser irradiation. Additionally, GLANCE offers several benefits for biomedical applications such as nanometer-scale size, adjustable optical absorption, and biocompatibility. These distinctive features of GLANCE would overcome the limitations of conventional ultrasound imaging and facilitate the accurate diagnosis of various diseases.

Recent Trend of Ultrasound-Mediated Nanoparticle Delivery for Brain Imaging and Treatment.

In view of the fact that the blood-brain barrier (BBB) prevents the transport of imaging probes and therapeutic agents to the brain and thus hinders the diagnosis and treatment of brain-related disorders, methods of circumventing this problem (e.g., ultrasound-mediated nanoparticle delivery) have drawn much attention. Among the related techniques, focused ultrasound (FUS) is a favorite means of enhancing drug delivery via transient BBB opening. Photoacoustic brain imaging relies on the conversion of light into heat and the detection of ultrasound signals from contrast agents, offering the benefits of high resolution and large penetration depth. The extensive versatility and adjustable physicochemical properties of nanoparticles make them promising therapeutic agents and imaging probes, allowing for successful brain imaging and treatment through the combined action of ultrasound and nanoparticulate agents. FUS-induced BBB opening enables nanoparticle-based drug delivery systems to efficiently access the brain. Moreover, photoacoustic brain imaging using nanoparticle-based contrast agents effectively visualizes brain morphologies or diseases. Herein, we review the progress in the simultaneous use of nanoparticles and ultrasound in brain research, revealing the potential of ultrasound-mediated nanoparticle delivery for the effective diagnosis and treatment of brain disorders.

Mesenchymal Stem Cell-Mediated Deep Tumor Delivery of Gold Nanorod for Photothermal Therapy.

Gold nanoparticles (AuNPs) with various sizes and morphologies have been extensively investigated for effective photothermal therapy (PTT) against multiple cancer types. However, a highly dynamic and complex tumor microenvironment (TME) considerably reduces the efficacy of PTT by limiting deep tumor penetration of AuNPs. Herein, we propose a mesenchymal stem cell (MSC)-mediated deep tumor delivery of gold nanorod (AuNR) for a potent PTT. First, MSCs are treated with tetraacylated N-azidomannosamine (Ac4ManNAz) to introduce modifiable azide (N3) groups on the cell surface via metabolic glycoengineering. Then, AuNRs modified with bio-orthogonal click molecules of bicyclo[6.1.0]nonyne (AuNR@BCN) are chemically conjugated to the N3 groups on the MSC surface by copper-free click chemistry reaction, resulting in AuNR@MSCs. In cultured MSCs, the appropriate condition to incorporate the AuNR into the MSCs is optimized; in addition, the photothermal efficiency of AuNR-MSCs under light irradiation are assessed, showing efficient heat generation in vitro. In colon tumor-bearing mice, intravenously injected AuNR@MSCs efficiently accumulate within the tumor tissues by allowing deep tissue penetration owing to the tumor homing effect by natural tumor tropism of AuNR@MSCs. Upon localized light irradiation, the AuNR@MSCs significantly inhibit colon tumor growth by the enhanced photothermal effect compared to conventional AuNRs. Collectively, this study shows a promising approach of MSCs-mediated deep tumor delivery of AuNR for effective PTT.

Light-triggered photodynamic nanomedicines for overcoming localized therapeutic efficacy in cancer treatment.

Photodynamic nanomedicines have significantly enhanced the therapeutic efficacy of photosensitizers (PSs) by overcoming critical limitations of PSs such as poor water solubility and low tumor accumulation. Furthermore, functional photodynamic nanomedicines have enabled overcoming oxygen depletion during photodynamic therapy (PDT) and tissue light penetration limitation by supplying oxygen or upconverting light in targeted tumor tissues, resulting in providing the potential to overcome biological therapeutic barriers of PDT. Nevertheless, their localized therapeutic effects still remain a huddle for the effective treatment of metastatic- or recurrent tumors. Recently, newly designed photodynamic nanomedicines and their combination chemo- or immune checkpoint inhibitor therapy enable the systemic treatment of various metastatic tumors by eliciting antitumor immune responses via immunogenic cell death (ICD). This review introduces recent advances in photodynamic nanomedicines and their applications, focusing on overcoming current limitations. Finally, the challenges and future perspectives of the clinical translation of photodynamic nanomedicines in cancer PDT are discussed.

How Did Conventional Nanoparticle-Mediated Photothermal Therapy Become "Hot" in Combination with Cancer Immunotherapy?

One of the promising cancer treatment methods is photothermal therapy (PTT), which has achieved good therapeutic efficiency through nanoparticle-based photoabsorbers. Because of the various functions of nanoparticles, such as targeting properties, high light-to-heat conversion, and photostability, nanoparticle-mediated PTT successfully induces photothermal damage in tumor tissues with minimal side effects on surrounding healthy tissues. The therapeutic efficacy of PTT originates from cell membrane disruption, protein denaturation, and DNA damage by light-induced heat, but these biological impacts only influence localized tumor areas. This conventional nanoparticle-mediated PTT still attracts attention as a novel cancer immunotherapy, because PTT causes immune responses against cancer. PTT-induced immunogenic cell death activates immune cells for systemic anti-cancer effect. Additionally, the excellent compatibility of PTT with other treatment methods (e.g., chemotherapy and immune checkpoint blockade therapy) reinforces the therapeutic efficacy of PTT as combined immunotherapy. In this review, we investigate various PTT agents of nanoparticles and compare their applications to reveal how nanoparticle-mediated PTT undergoes a transition from thermotherapy to immunotherapy.

Tumor-activated carrier-free prodrug nanoparticles for targeted cancer Immunotherapy: Preclinical evidence for safe and effective drug delivery.

As immunogenic cell death (ICD) inducers initiating antitumor immune responses, certain chemotherapeutic drugs have shown considerable potential to reverse the immunosuppressive tumor microenvironment (ITM) into immune-responsive tumors. The application of these drugs in nanomedicine provides a more enhanced therapeutic index by improving unfavorable pharmacokinetic (PK) profiles and inefficient tumor targeting. However, the clinical translation of conventional nanoparticles is restricted by fundamental problems, such as risks of immunogenicity and potential toxicity by carrier materials, premature drug leakage in off-target sites during circulation, low drug loading contents, and complex structure and synthetic processes that hinder quality control (QC) and scale-up industrial production. To address these limitations, tumor-activated carrier-free prodrug nanoparticles (PDNPs), constructed only by the self-assembly of prodrugs without any additional carrier materials, have been widely investigated with distinct advantages for safe and more effective drug delivery. In addition, combination immunotherapy based on PDNPs with other diverse modalities has efficiently reversed the ITM to immune-responsive tumors, potentiating the response to immune checkpoint blockade (ICB) therapy. In this review, the trends and advances in PDNPs are outlined, and each self-assembly mechanism is discussed. In addition, various combination immunotherapies based on PDNPs are reviewed. Finally, a physical tumor microenvironment remodeling strategy to maximize the potential of PDNPs, and key considerations for clinical translation are highlighted.

Thiol-Responsive Gold Nanodot Swarm with Glycol Chitosan for Photothermal Cancer Therapy.

Photothermal therapy (PTT) is one of the most promising cancer treatment methods because hyperthermal effects and immunogenic cell death via PTT are destructive to cancer. However, PTT requires photoabsorbers that absorb near-infrared (NIR) light with deeper penetration depth in the body and effectively convert light into heat. Gold nanoparticles have various unique properties which are suitable for photoabsorbers, e.g., controllable optical properties and easy surface modification. We developed gold nanodot swarms (AuNSw) by creating small gold nanoparticles (sGNPs) in the presence of hydrophobically-modified glycol chitosan. The sGNPs assembled with each other through their interaction with amine groups of glycol chitosan. AuNSw absorbed 808-nm laser and increased temperature to 55 °C. In contrast, AuNSw lost its particle structure upon exposure to thiolated molecules and did not convert NIR light into heat. In vitro studies demonstrated the photothermal effect and immunogenic cell death after PTT with AuNSW. After intratumoral injection of AuNSw with laser irradiation, tumor growth of xenograft mouse models was depressed. We found hyperthermal damage and immunogenic cell death in tumor tissues through histological and biochemical analyses. Thiol-responsive AuNSw showed feasibility for PTT, with advanced functionality in the tumor microenvironment.

Theragnostic Glycol Chitosan-Conjugated Gold Nanoparticles for Photoacoustic Imaging of Regional Lymph Nodes and Delivering Tumor Antigen to Lymph Nodes.

Lymph node mapping is important in cancer immunotherapy because the morphology of lymph nodes is one of the crucial evaluation criteria of immune responses. We developed new theragnostic glycol-chitosan-coated gold nanoparticles (GC-AuNPs), which highlighted lymph nodes in ultrasound-guided photoacoustic (US/PA) imaging. Moreover, the ovalbumin epitope was conjugated GC-AuNPs (OVA-GC-AuNPs) for delivering tumor antigen to lymph node resident macrophage. In vitro studies proved the vigorous endocytosis activity of J774A.1 macrophage and consequent strong photoacoustic signals from them. The macrophages also presented a tumor antigen when OVA-GC-AuNPs were used for cellular uptake. After the lingual injection of GC-AuNPs into healthy mice, cervical lymph nodes were visible in a US/PA imaging system with high contrast. Three-dimensional analysis of lymph nodes revealed that the accumulation of GC-AuNPs in the lymph node increased as the post-injection time passed. Histological analysis showed GC-AuNPs or OVA-GC-AuNPs located in subcapsular and medullar sinuses where macrophages are abundant. Our new theragnostic GC-AuNPs present a superior performance in US/PA imaging of lymph nodes without targeting moieties or complex surface modification. Simultaneously, GC-AuNPs were able to deliver tumor antigens to cause macrophages to present the OVA epitope at targeted lymph nodes, which would be valuable for cancer immunotherapy.

Rediscovery of nanoparticle-based therapeutics: boosting immunogenic cell death for potential application in cancer immunotherapy.

Immunogenic cell death (ICD) occurring by chemical and physical stimuli has shown the potential to activate an adaptive immune response in the immune-competent living body through the release of danger-associated molecular patterns (DAMPs) into the tumor microenvironment (TME). However, limitations to the long-term immune responses and systemic toxicity of conventional ICD inducers have led to unsatisfactory therapeutic efficacy in ICD-based cancer immunotherapy. Until now, various nanoparticle-based ICD-inducers have been developed to induce an antitumor immune response without severe toxicity, and to efficiently elicit an anticancer immune response against target cancer cells. In this review, we introduce a recent advance in the designs and applications of nanoparticle-based therapeutics to elicit ICD for effective cancer immunotherapy. In particular, combination strategies of nanoparticle-based ICD inducers with typical theranostic modalities are introduced intensively. Subsequently, we discuss the expected challenges and future direction of nanoparticle-based ICD inducers to provide strategies for boosting ICD in cancer immunotherapy. These versatile designs and applications of nanoparticle-based therapeutics for ICD can provide advantages to improve the therapeutic efficacy of cancer immunotherapy.

Tumor-Targeting Glycol Chitosan Nanoparticles for Cancer Heterogeneity.

Nanomedicine is extensively employed for cancer treatment owing to its unique advantages over conventional drugs and imaging agents. This increased attention to nanomedicine, however, has not fully translated into clinical utilization and patient benefits due to issues associated with reticuloendothelial system clearance, tumor heterogeneity, and complexity of the tumor microenvironment. To address these challenges, efforts are being made to modify the design of nanomedicines, including optimization of their physiochemical properties, active targeting, and response to stimuli, but these studies are often performed independently. Combining favorable nanomedicine designs from individual studies may improve therapeutic outcomes, but, this is difficult to achieve as the effects of different designs are interconnected and often conflicting. Glycol chitosan nanoparticles (CNPs) are shown to accumulate in tumors, suggesting that this type of nanoparticle may constitute a good basis for the additional modification of nanoparticles. Here, multifunctional glycol CNPs designed to overcome multiple obstacles to their use are described and key factors influencing in vivo targeted delivery, targeting strategies, and interesting stimulus-responsive designs for improving cancer nanomedicine are discussed.

Recent Trends in In Situ Enzyme-Activatable Prodrugs for Targeted Cancer Therapy.

Enzyme-activatable anticancer prodrugs are modified medications that are composed of an anticancer drug, cleavable linker, and functional moiety. The purpose of such a prodrug structure is to generate multipurpose functions that traditional drugs cannot perform and to reduce the toxicity of conventional anticancer drugs by the mask of the cleavable linker. Once the cleavable linker is degraded via a specific chemical reaction in the cancer microenvironment, the cytotoxicity of the degraded prodrugs is selectively recovered. Among many factors that cleave the linker, we focus on the overexpressed enzymes in cancer. Because of the selective enzymatic degradation of the cleavable linker and the high local concentration of specific enzymes in cancer, the enzyme-activatable prodrugs could show low toxicity in normal tissues, while showing comparable anticancer effect in tumors. In addition, some prodrugs provide additional features, such as cancer imaging, drug release monitoring, tumor targeting, and enhanced stability, which conventional anticancer drugs cannot possess. In this review, we summarize currently developed enzyme-activatable prodrugs according to their activating enzymes, and categorize them by their additional functions, e.g. targeting, imaging, and delivery. This summary of enzyme-activatable prodrugs may help in the design of anticancer prodrugs, and in the establishment of a personalized cancer treatment strategy.

Gas-generating nanoparticles for contrast-enhanced ultrasound imaging.

We present gas-generating solid nanoparticles as a new concept of an ultrasound contrast agent. The developed nanoparticles are sufficiently small (less than 100 nm in diameter) to escape vasculature and yet, upon external pulsed laser light activation, release nitrogen gas for enhanced contrast in ultrasound imaging. The gas-generating nanoconstructs combine the photocatalytic function of gold nanoparticles and photolysis of azide compounds. Using ultrasound imaging, we demonstrate the controlled, on-demand generation of nitrogen gas from nanoparticles due to the decomposition of azide groups triggered by pulsed laser irradiation. The resulting gas forms bubbles that cause backscattered ultrasound signals and, therefore, modulate the contrast in ultrasound imaging.

Photoacoustic imaging of cancer cells with glycol-chitosan-coated gold nanoparticles as contrast agents.

Utility of glycol-chitosan-coated gold nanoparticles (GC-AuNPs) as a photoacoustic contrast agent for cancer cell imaging was demonstrated. Through the synergistic effect of glycol chitosan and gold nanoparticles, GC-AuNPs showed cellular uptake in breast cancer cells and resulted in strong photoacoustic signals in tissue-mimicking cell phantoms. The performance of GC-AuNPs as contrast agents was established with photoacoustic imaging and confirmed with dark-field microscopy. The cell phantoms displayed strong photoacoustic signals if cells were incubated more than 3 h with GC-AuNPs, compared with PEG-AuNPs that showed no photoacoustic signal increase. The enhanced photoacoustic signals originated from the plasmon coupling effect of GC-AuNPs after the cellular uptake in cancer cells. Importantly, photoacoustic imaging of cancer cells was achieved with GC-AuNPs­contrast agents that did not require antibodies or complex surface modification. The endocytosis of GC-AuNPs was also confirmed with dark-field microscopy. The results show that GC-AuNPs have potential as a photoacoustic contrast agent for cellular imaging including tumor tissue imaging.

Ultrasound-guided immunofunctional photoacoustic imaging for diagnosis of lymph node metastases.

Metastases, rather than primary tumors, determine mortality in the majority of cancer patients. A non-invasive immunofunctional imaging method was developed to detect sentinel lymph node (SLN) metastases using ultrasound-guided photoacoustic (USPA) imaging combined with glycol-chitosan-coated gold nanoparticles (GC-AuNPs) as an imaging contrast agent. GC-AuNPs, injected peritumorally into breast tumor-bearing mice, were taken up by immune cells, and subsequently transported to the SLN. Two-dimensional and three-dimensional USPA imaging was used to isolate the signal from GC-AuNP-tagged cells. Volumetric analysis was used to quantify GC-AuNP accumulation in the SLN after cellular uptake and transport by immune cells. The results show that the spatio-temporal distribution of GC-AuNPs in the SLN was affected by the presence of metastases. The parameter describing the spatial distribution of GC-AuNP-tagged cells within the SLN was more than 2-fold lower in metastatic lymph nodes compared with non-metastatic controls. Histological analysis confirmed that the distribution of GC-AuNP-tagged immune cells is changed by the presence of metastatic cells. The USPA immunofunctional imaging successfully distinguished metastatic from non-metastatic lymph nodes using biocompatible nanoparticles. This method could aid physicians in the detection of micrometastases, thus guiding SLN biopsy and avoiding unnecessary biopsy procedures.

Direct Imaging of Cerebral Thromboemboli Using Computed Tomography and Fibrin-targeted Gold Nanoparticles.

Computed tomography (CT) is the current standard for time-critical decision-making in stroke patients, informing decisions on thrombolytic therapy with tissue plasminogen activator (tPA), which has a narrow therapeutic index. We aimed to develop a CT-based method to directly visualize cerebrovascular thrombi and guide thrombolytic therapy. Glycol-chitosan-coated gold nanoparticles (GC-AuNPs) were synthesized and conjugated to fibrin-targeting peptides, forming fib-GC-AuNP. This targeted imaging agent and non-targeted control agent were characterized in vitro and in vivo in C57Bl/6 mice (n = 107) with FeCl3-induced carotid thrombosis and/or embolic ischemic stroke. Fibrin-binding capacity was superior with fib-GC-AuNPs compared to GC-AuNPs, with thrombi visualized as high density on microCT (mCT). mCT imaging using fib-GC-AuNP allowed the prompt detection and quantification of cerebral thrombi, and monitoring of tPA-mediated thrombolytic effect, which reflected histological stroke outcome. Furthermore, recurrent thrombosis could be diagnosed by mCT without further nanoparticle administration for up to 3 weeks. fib-GC-AuNP-based direct cerebral thrombus imaging greatly enhance the value and information obtainable by regular CT, has multiple uses in basic / translational vascular research, and will likely allow personalized thrombolytic therapy in clinic by a) optimizing tPA-dosing to match thrombus burden, b) enabling the rational triage of patients to more radical therapies such as endovascular clot-retrieval, and c) potentially serving as a theranostic platform for targeted delivery of concurrent thrombolysis.

The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells.

There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1 µM and varying sizes of 15, 30, 50, 75 and 100 nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1 week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30 nm and 50 nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50 nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50 nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

Fluorescent dye labeled iron oxide/silica core/shell nanoparticle as a multimodal imaging probe.

PURPOSE: To develop an MRI/optical multimodal imaging probe based on dye-conjugated iron oxide/silica core/shell nanoparticle, and investigate the distance-dependent fluorescence quenching through careful control of the distance between the iron oxide core and fluorescent dyes. METHODS: Different size of core/shell nanoparticles were prepared by varying the silica shell width. PEGylation on the surface of silica shell was followed to improve the stability of particles in the physiological condition. In vitro cytotoxicity was evaluated by the MTT assay on a HeLa cell line and in vivo imaging of subcutaneous SCC7 xenografted mice was performed using MRI/optical imaging modalities. RESULTS: Diameter and ζ-potential of the nanoparticles were measured, and TEM images demonstrated the mono-disperse nature of the particles. Quenching efficiency of the dyes on the surface was nearly 100% in the smallest nanoparticle, while almost no quenching effect was observed for the largest nanoparticle. In vitro cytotoxicity showed nearly 90% cell viability at 0.15 Fe mg/mL, a comparable concentration for clinical use. The tumor area was significantly darkened after the nanoparticle injection due to the high transverse relaxivity value of the nanoparticles. Fluorescence signal was affected by the particle size due to the distance-dependent quenching/dequenching behaviour.

Biocompatible glycol chitosan-coated gold nanoparticles for tumor-targeting CT imaging.

PURPOSE: The application of gold nanoparticles (AuNPs) in biomedical field was limited due to the low stability in the biological condition. Herein, to enhance stability and tumor targeting ability of AuNPs, their surface was modified with biocompatible glycol chitosan (GC) and the in vivo biodistribution of GC coated AuNPs (GC-AuNPs) were studied through computed tomography (CT). METHODS: Polymer-coated gold nanoparticles were produced using GC as a reducing agent and a stabilizer. Their feasibility in biomedical application was explored through CT in tumor-bearing mice. RESULTS: Stability of gold nanoparticles increased in the physiological condition due to the GC coating layer on the surface. Tomographic images of tumor were successfully obtained in the tumor-xenografted animal model when the GC-AuNPs were used as a CT contrast agent. The tumor targeting property of the gold nanoparticles was due to the properties of GC because GC-AuNPs were accumulated in the tumor, while most of heparin-coated nanoparticles were found in the liver and spleen. CONCLUSIONS: The polymer properties on the surface played an important role in the behavior of gold nanoparticles in the biological condition and the enhanced stability and tumor targeting property of nanoparticles were inherited from GC on the surface.