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The recent discovery of cuproptosis, a novel copper-ion-induced cell death pathway, has suggested the novel therapeutic potential for treating heterogeneous and drug-resistant cancers. Currently, copper ionophore-based therapeutics have been designed to treat cancers, utilizing copper ions as a strategic tool to impede tumor proliferation and promote cellular demise. However, limitations of copper ionophore-based therapies include nontargeted delivery of copper ions, low tumor accumulation, and short half-life. Strategies to enhance specificity involve targeting intracellular cuproptosis mechanisms using nanotechnology-based drugs. Additionally, the importance of exploring combination therapies cannot be overstated, as they are a key strategy in improving the efficacy of cancer treatments. Recent studies have reported the anticancer effects of nanomedicines that can induce cuproptosis of cancer both in vitro and in vivo. These cuproptosis-targeted nanomedicines could improve delivery efficiency with the pharmacokinetic properties of copper ion, resulting in increasing cuproptosis-based anticancer effects. This review will summarize the intricate nexus between copper ion and carcinogenesis, examining the pivotal roles of copper homeostasis and its dysregulation in cancer progression and fatality. Furthermore, we will introduce the latest advances in cuproptosis-targeted nanomedicines for cancer treatment. Finally, the challenges in cuproptosis-based nanomedicines will be discussed for future development directions., Competing Interests: Competing interests: The authors declare that they have no competing interests., (Copyright © 2024 Dahye Noh et al.)

While proteolysis-targeting chimeras (PROTACs) hold great potential for persistently reprogramming the immunosuppressive tumor microenvironment via targeted protein degradation, precisely activating them in tumor tissues and preventing uncontrolled proteolysis at off-target sites remain challenging. Herein, a light-triggered PROTAC nanoassembly (LPN) for photodynamic indoleamine 2,3-dioxygenase (IDO) proteolysis is reported. The LPN is derived from the self-assembly of prodrug conjugates, which comprise a PROTAC, cathepsin B-specific cleavable peptide linker, and photosensitizer, without any additional carrier materials. In colon tumor models, intravenously injected LPNs initially silence the activity of PROTACs and accumulate significantly in targeted tumor tissues due to an enhanced permeability and retention effect. Subsequently, the cancer biomarker cathepsin B begins to trigger the release of active PROTACs from the LPNs through enzymatic cleavage of the linkers. Upon light irradiation, tumor cells undergo immunogenic cell death induced by photodynamic therapy to promote the activation of effector T cells, while the continuous IDO degradation of PROTAC simultaneously blocks tryptophan metabolite-regulated regulatory-T-cell-mediated immunosuppression. Such LPN-mediated combinatorial photodynamic IDO proteolysis effectively inhibits tumor growth, metastasis, and recurrence. Collectively, this study presents a promising nanomedicine, designed to synergize PROTACs with other immunotherapeutic modalities, for more effective and safer cancer immunotherapy., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

A series of successes in RNA interference (RNAi) therapies for liver diseases using lipid nanoparticles and N -acetylgalactosamine have heralded a current era of RNA therapeutics. However, alternative delivery strategies are required to take RNAi out of the comfort zone of hepatocytes. Here we report SIRPα IgV/anti-CD47 siRNA (vS-siCD47) conjugates that selectively and persistently disrupt the antiphagocytic CD47/SIRPα axis in solid tumors. Conjugation of the SIRPα IgV domain protein to siRNAs enables tumor dash through CD47-mediated erythrocyte piggyback, primarily blocking the physical interaction between CD47 on cancer cells and SIRPα on phagocytes. After internalization of the vS-siCD47 conjugates within cancer cells, the detached free-standing anti-CD47 siRNAs subsequently attack CD47 through the RNAi mechanism. The dual-action approach of the vS-siCD47 conjugate effectively overcomes the "don't eat me" barrier and stimulates phagocyte-mediated tumor destruction, demonstrating a highly selective and potent CD47-blocking immunotherapy. This delivery strategy, employing IgV domain protein-siRNA conjugates with a dual mode of target suppression, holds promise for expanding RNAi applications beyond hepatocytes and advancing RNAi-based cancer immunotherapies for solid tumors.

Cancer immunotherapy represents a revolutionary strategy, leveraging the patient's immune system to inhibit tumor growth and alleviate the immunosuppressive effects of the tumor microenvironment (TME). The recent emergence of immune checkpoint blockade (ICB) therapies, particularly following the first approval of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors like ipilimumab, has led to significant growth in cancer immunotherapy. The extensive explorations on diverse immune checkpoint antibodies have broadened the therapeutic scope for various malignancies. However, the clinical response to these antibody-based ICB therapies remains limited, with less than 15% responsiveness and notable adverse effects in some patients. This review introduces the emerging strategies to overcome current limitations of antibody-based ICB therapies, mainly focusing on the development of small interfering ribonucleic acid (siRNA)-based ICB therapies and innovative delivery systems. We firstly highlight the diverse target immune checkpoint genes for siRNA-based ICB therapies, incorporating silencing of multiple genes to boost anti-tumor immune responses. Subsequently, we discuss improvements in siRNA delivery systems, enhanced by various nanocarriers, aimed at overcoming siRNA's clinical challenges such as vulnerability to enzymatic degradation, inadequate pharmacokinetics, and possible unintended target interactions. Additionally, the review presents various combination therapies that integrate chemotherapy, phototherapy, stimulatory checkpoints, ICB antibodies, and cancer vaccines. The important point is that when used in combination with siRNA-based ICB therapy, the synergistic effect of traditional therapies is strengthened, improving host immune surveillance and therapeutic outcomes. Conclusively, we discuss the insights into innovative and effective cancer immunotherapeutic strategies based on RNA interference (RNAi) technology utilizing siRNA and nanocarriers as a novel approach in ICB cancer immunotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Immune checkpoint blockade (ICB) therapy targeting PD-L1 via monoclonal antibody (mAb) has shown extensive clinical benefits in the diverse types of advanced malignancies. However, most patients are completely refractory to ICB therapy owing to the PD-L1 recycling mechanism. Herein, we propose photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes (immune checkpoint blockade liposomes; ICB-LPs) to promote PD-L1 multivalent binding for inducing lysosomal degradation of PD-L1 in tumor cells. The ICB-LPs are prepared by formulation of DC 8,9 PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG 2k (anti-PD-L1-DSPE-PEG 2k ) in a molar ratio of 45:45:10, followed by cross-linking of liposomal bilayer upon UV irradiation. The 10 mol% anti-PD-L1-DSPE-PEG 2k incorporated ICB-LPs have a nano-sized lipid bilayer structure with an average diameter of 137.7 ± 1.04 nm, showing a high stability in serum condition. Importantly, the ICB-LPs efficiently promote the multivalent binding with PD-L1 on the tumor cell membrane, which are endocytosed with aim to deliver PD-L1 to the lysosomes, wherein the durable PD-L1 degradation is observed for 72 h, in contrast to anti PD-L1 mAbs showing the rapid PD-L1 recycling within 9 h. The in vitro co-culture experiments with CD8 + T cells show that ICB-LPs effectively enhance the T cell-mediated antitumor immune responses against tumor cells by blocking the PD-L1/PD-1 axis. When ICB-LPs are intravenously injected into colon tumor-bearing mice, they efficiently accumulate within the targeted tumor tissues via both passive and active tumor targeting, inducing a potent T cell-mediated antitumor immune response by effective and durable PD-L1 degradation. Collectively, this study demonstrates the superior antitumor efficacy of crosslinked and anti-PD-L1 peptide incorporated liposome formulation that promotes PD-L1 multivalent binding for trafficking of PD-L1 toward the lysosomes instead of the recycling endosomes., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors.)

RNA vaccines have demonstrated their ability to solve the issues posed by the COVID-19 pandemic. This success has led to the renaissance of research into mRNA and their nanoformulations as potential therapeutic modalities for various diseases. The potential of mRNA as a template for synthesizing proteins and protein fragments for cancer immunotherapy is now being explored. Despite the promise, the use of mRNA in cancer immunotherapy is limited by challenges, such as low stability against extracellular RNases, poor delivery efficiency to the target organs and cells, short circulatory half-life, variable expression levels and duration. This review highlights recent advances in chemical modification and advanced delivery systems that are helping to address these challenges and unlock the biological and pharmacological potential of mRNA therapeutics in cancer immunotherapy. The review concludes by discussing future perspectives for mRNA-based cancer immunotherapy, which holds great promise as a next-generation therapeutic modality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Synergistic immunotherapy of immune checkpoint blockade (ICB) and immunogenic cell death (ICD) has shown remarkable therapeutic efficacy in various cancers. However, patients show low response rates and undesirable outcomes to these combination therapies owing to the recycling mechanism of programmed death-ligand 1 (PD-L1) and the systemic toxicity of ICD-inducing chemotherapeutic drugs. Herein, we propose all-in-one glycol chitosan nanoparticles (CNPs) that can deliver anti-PD-L1 peptide (PP) and doxorubicin (DOX) to targeted tumor tissues for a safe and more effective synergistic immunotherapy. The PP-CNPs, which are prepared by conjugating ᴅ-form PP (NYSKPTDRQYHF) to CNPs, form stable nanoparticles that promote multivalent binding with PD-L1 proteins on the targeted tumor cell surface, resulting in effective lysosomal PD-L1 degradation in contrast with anti-PD-L1 antibody, which induces recycling of endocytosed PD-L1. Consequently, PP-CNPs prevent subcellular PD-L1 recycling and eventually destruct immune escape mechanism in CT26 colon tumor-bearing mice. Moreover, the ICD inducer, DOX is loaded into PP-CNPs (DOX-PP-CNPs) for synergistic ICD and ICB therapy, inducing a large number of damage-associated molecular patterns (DAMPs) in targeted tumor tissues with minimal toxicity in normal tissues. When the DOX-PP-CNPs are intravenously injected into CT26 colon tumor-bearing mice, PP and DOX are efficiently delivered to the tumor tissues via nanoparticle-derived passive and active targeting, which eventually induce both lysosomal PD-L1 degradation and substantial ICD, resulting in a high rate of complete tumor regression (CR: 60%) by a strong antitumor immune response. Collectively, this study demonstrates the superior efficacy of synergistic immunotherapy using all-in-one nanoparticles to deliver PP and DOX to targeted tumor tissues., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Background: Claudin-4 (CLDN4), a tight junction protein, is overexpressed in several types of cancer, and is considered a biomarker for cancer-targeted treatment. CLDN4 is not exposed in normal cells, but becomes accessible in cancer cells, in which tight junctions are weakened. Notably, surface-exposed CLDN4 has recently been found to act as a receptor for Clostridium perfringens enterotoxin (CPE) and fragment of CPE (CPE17) that binds to the second domain of CLDN4., Methods: Here, we sought to develop a CPE17-containing liposome that targets pancreatic cancers through binding to exposed CLDN4., Results: Doxorubicin (Dox)-loaded, CPE17-conjugated liposomes (D@C-LPs) preferentially targeted CLDN4-expressing cell lines, as evidenced by greater uptake and cytotoxicity compared with CLDN4-negative cell lines, whereas uptake and cytotoxicity of Dox-loaded liposomes lacking CPE17 (D@LPs) was similar for both CLDN4-positive and negative cell lines. Notably, D@C-LPs showed greater accumulation in targeted pancreatic tumor tissues compared with normal pancreas tissue; in contrast, Dox-loaded liposomes lacking CPE17 (D@LPs) showed little accumulation in pancreatic tumor tissues. Consistent with this, D@C-LPs showed greater anticancer efficacy compared with other liposome formulations and significantly extended survival., Conclusions: We expect our findings will aid in the prevention and treatment of pancreatic cancer and provide a framework for identifying cancer-specific strategies that target exposed receptors., (© 2023. The Author(s).)

Adoptive cell transfer (ACT) has shown remarkable therapeutic efficacy against blood cancers such as leukemia and lymphomas, but its effect is still limited due to the lack of well-defined antigens expressed by aberrant cells within tumors, the insufficient trafficking of administered T cells to the tumor sites, as well as immunosuppression induced by the tumor microenvironment (TME). In this study, we propose the adoptive transfer of photosensitizer (PS)-loaded cytotoxic T cells for a combinational photodynamic and cancer immunotherapy. Temoporfin (Foscan ® ), a clinically applicable porphyrin derivative, was loaded into OT-1 cells (PS-OT-1 cells). The PS-OT-1 cells efficiently produced a large amount of reactive oxygen species (ROS) under visible light irradiation in a culture; importantly, the combinational photodynamic therapy (PDT) and ACT with PS-OT-1 cells induced significant cytotoxicity compared to ACT alone with unloaded OT-1 cells. In murine lymphoma models, intravenously injected PS-OT-1 cells significantly inhibited tumor growth compared to unloaded OT-1 cells when the tumor tissues were locally irradiated with visible light. Collectively, this study suggests that combinational PDT and ACT mediated by PS-OT-1 cells provides a new approach for effective cancer immunotherapy.

Background: Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs., Results: The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5β-cholanic acid and the amphiphilic glycol chitosan-5β-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36-288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs (liver, lung, spleen, kidney and heart) after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs (90 mg/kg, three times) induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction., Conclusions: This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings., (© 2023. The Author(s).)

Phototherapeutic nanoparticles (NPs) were prepared with methylene blue (MB), indocyanine green (ICG), and Solutol through self-assembly. Generation of reactive oxygen species and elevation of temperature were observed that verify the photodynamic/photothermal effects of the NPs. Morphology and size distribution of the NPs were examined by transmittance electron microscopy and dynamic light scattering. The biodistribution of the NPs and their antitumor efficacy were examined using tumor-bearing mice to understand the phototherapeutic effect of the NPs on tumors. To enhance targetability with enhanced therapeutic efficacy, empty NPs (Solutol nanoparticles without MB and ICG) at different concentrations were injected along with the phototherapeutic NPs. Enhanced delivery of the phototherapeutic NPs at the tumor site was examined based on hepatocyte overload., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Many preclinically tested nanoparticles in existing animal models fail to be directly translated into clinical applications because of their poor resemblance to human cancer. Herein, the enhanced permeation and retention (EPR) effect of glycol chitosan nanoparticles (CNPs) in different tumor microenvironments (TMEs) was compared using different pancreatic tumor models, including pancreatic cancer cell line (BxPC3), patient-derived cancer cell (PDC), and patient-derived xenograft (PDX) models. CNPs were intravenously injected into different tumor models, and their accumulation efficiency was evaluated using non-invasive near-infrared fluorescence (NIRF) imaging. In particular, differences in angiogenic vessel density, collagen matrix, and hyaluronic acid content in tumor tissues of the BxPC3, PDC, and PDX models greatly affected the tumor-targeting efficiency of CNPs. In addition, different PDX models were established using different tumor tissues of patients to predict the clinical EPR effect of CNPs in inter-patient TMEs, wherein the gene expression levels of PECAM1, COL4A1, and HAS1 in human tumor tissues were observed to be closely related to the EPR effect of CNPs in PDX models. The results suggested that the PDX models could mimic inter-patient TMEs with different blood vessel structures and extracellular matrix (ECM) content that critically affect the tumor-targeting ability of CNPs in different pancreatic PDX models. This study provides a better understanding of the heterogeneity and complexity of inter-patient TMEs that can predict the response of various nanoparticles in individual tumors for personalized cancer therapy., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

RNA interference (RNAi) is a major cellular mechanism regulating gene expression in which short double-stranded RNA molecules called small interfering RNA (siRNA) mediate sequence-specific mRNA degradation. RNAi technology has recently emerged as a promising therapeutic platform for the effective treatment of various diseases caused by inappropriate gene activity, such as cancer. However, the clinical translation of siRNA therapeutics has been hampered by the major hurdles associated with biological instability and limited delivery efficiency. Based on the various efforts, recent siRNA delivery strategies using cationic lipids and polymers allowed to enhance pharmacokinetics and delivery efficiency, resulting in potent and liver-targeted RNAi therapy. However, non-specific protein adsorption, high liver accumulation, and severe toxicity of cationic nanocarriers still limit the possibility of transfer of siRNA therapeutics from the laboratory to the clinic. One of the promising delivery strategies to overcome the limitations of siRNA therapeutics is carrier-free bioconjugation which is chemically modified and connected with biocompatible molecules such as lipids, peptides, antibodies, aptamers, and polymers. These molecularly engineered siRNA conjugates can be utilized for RNAi delivery to tissues beyond the liver, providing opportunities for clinical translation. This review focused on introducing the recent progress in molecularly engineered siRNA conjugates and their applications toward overcoming the limitations of siRNA for tumor-targeted delivery and therapy., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

Immune checkpoint blockade (ICB) has shown remarkable therapeutic efficacy in a variety of cancers. However, patients exhibit unexpectedly low response rates to ICB therapy owing to the unwanted recycling and cellular abundance of PD-L1. Herein, rational design of PD-L1 multivalent binding liposome is investigated through PEGylated liposomes incorporating different ratios of PD-L1 binding peptide. Liposomes incorporating 10 mol% PD-L1 binding peptides (10-PD-L1-Lipo) promote the multivalent binding with PD-L1 on tumor cell surface, which is endocytosed for its trafficking toward the lysosomes instead of the recycling endosomes. Thereby, 10-PD-L1-Lipo leads to a significant PD-L1 degradation that prevents its recycling and cellular abundance compared to anti-PD-L1 antibody, disrupting immune escape mechanism of tumor cells and enhancing T cell-mediated antitumor immunity. Moreover, a clinically applicable doxorubicin (DOX) liposomal formulation is established via drug encapsulation into 10-PD-L1-Lipo. The resulting DOX-PD-L1-Lipo primes tumors via immunogenic chemotherapy by preferential DOX accumulation by the EPR effect and overcomes PD-L1 abundance induced following chemotherapy through multivalent binding-mediated PD-L1 degradation. As a result, the synergistic immunogenic chemotherapy and multivalent binding-mediated PD-L1 degradation by DOX-PD-L1-Lipo show significantly enhanced antitumor efficacy and immune responses in colon tumor models. Collectively, this study suggests the rationally designed PEGylated liposomes to promote PD-L1 multivalent binding providing a new route for safe and more effective ICB therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A prodrug is bioreversible medication that is specifically converted to the active drugs by enzymes overexpressed in the tumor microenvironment, which can considerably reduce the chemotherapy-induced side effects. However, prodrug strategies usually have low antitumor efficacy compared to free drugs by delayed drug release. This is because they need time to be activated by enzymatic cleavage and they also cannot be fully recovered to the active drugs. Therefore, highly potent anticancer drug should be considered to expect a sufficient antitumor efficacy. Herein, we propose tumor-specific monomethyl auristatin E (MMAE) prodrug nanoparticles for safe and effective chemotherapy. The cathepsin B-specific cleavable FRRG peptide and MMAE are chemically conjugated via one-step simple synthetic chemistry. The resulting FRRG-MMAE molecules form stable nanoparticles without any additional carrier materials by hydrophobic interaction-derived aggregations. The FRRG-MMAE nanoparticles efficiently accumulate within the tumor tissues owing to the enhanced permeability and retention (EPR) effect and inhibit the tubulin polymerization by releasing free MMAE in the cathepsin B-overexpressed tumor cells. In contrast, FRRG-MMAE nanoparticles maintain a non-toxic inactive state in the normal tissues owing to innately low cathepsin B expression, thereby reducing MMAE-related severe toxicity. Collectively, this study provides a promising approach for safe and effective chemotherapy via MMAE-based prodrug nanoparticles, which may open new avenues for advanced drug design for translational nanomedicine.

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 (Ac 4 ManNAz) to introduce modifiable azide (N 3 ) 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 N 3 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., Competing Interests: The authors declare no conflict of interest.

Various immunotherapeutic agents that can elicit antitumor immune responses have recently been developed with the potential for improved efficacy in treating cancer. However, insufficient delivery efficiency at the tumor site, along with severe side effects after systemic administration of these anticancer agents, have hindered their therapeutic application in cancer immunotherapy. Hydrogels that can be directly injected into tumor sites have been developed to help modulate or elicit antitumor responses. Based on the biocompatibility, degradability, and controllable mechanochemical properties of these injectable hydrogels, various types of immunotherapeutic agents, such as hydrophobic anticancer drugs, cytokines, antigens, and adjuvants, have been easily and effectively encapsulated, resulting in the successful elicitation of antitumor immune responses and the retention of long-term immunotherapeutic efficacy following administration. This review summarizes recent advances in combination immunotherapy involving injectable hydrogel-based chemoimmunotherapy, photoimmunotherapy, and radioimmunotherapy. Finally, we briefly discuss the current limitations and future perspectives on injectable hydrogels for the effective combination immunotherapy of tumors.

Despite advances in cancer therapy, the discovery of effective cancer treatments remains challenging. In this study, a simple method was developed to increase the efficiency of doxorubicin (DOX) delivery in a lung metastasis model. This method comprises a simple configuration to increase the delivery efficiency via precise engineering of the size, shape, loading content, and biodegradability of the drug delivery system. This system had a 3 μm discoidal shape and exerted approximately 90% burst release of the drug within the first 24 h. There was no cytotoxicity of the drug carrier up to a concentration of 1 mg ml -1 , and DOX from the carrier was delivered into the cancer cells, exhibiting an anticancer effect comparable to that of the free drug. The ex vivo results revealed a strong correlation between the location of cancer cells in the lung and the location of DOX delivered by this drug delivery system. These drug carriers were confirmed to intensively deliver DOX to cancer cells in the lung, with minimal off-target effects. These findings indicate that this delivery system can be a new approach to improving the survival rate and reducing the side effects caused by anticancer drugs without the use of targeting ligands and polyethylene glycol.

Heart rate variability (HRV) is closely related to changes in the autonomic nervous system (ANS) associated with stress and pain. In this study, we investigated whether HRV could be used to assess cancer pain in mice with peritoneal metastases. At 12 days after cancer induction, positive indicators of pain such as physiological characteristics, appearance, posture, and activity were observed, and time- and frequency-domain HRV parameters such as mean R-R interval, square root of the mean squared differences of successive R-R intervals, and percentage of successive R-R interval differences greater than 5 ms, low frequency (LF), high frequency (HF), and ratio of LF and HF power, were found to be significantly decreased. These parameters returned to normal after analgesic administration. Our results indicate that overall ANS activity was decreased by cancer pain and that HRV could be a useful tool for assessing pain.

Traditionally, organic chemical reactions require organic solvents, toxic catalysts, heat, or high pressure. However, copper-free click chemistry has been shown to have favorable reaction rates and orthogonality in water, buffer solutions, and physiological conditions without toxic catalysts. Strain-promoted azide-alkyne cycloaddition and inverse electron-demand Diels-Alder reactions are representative of copper-free click chemistry. Artificial chemical reactions via click chemistry can also be used outside of the laboratory in a controllable manner on live cell surfaces, in the cytosol, and in living bodies. Consequently, copper-free click chemistry has many features that are of interest in biomedical research, and various new materials and strategies for its use have been proposed. Herein, recent remarkable trials that have used copper-free click chemistry are described, focusing on their applications in molecular imaging and therapy. The research is categorized as nanoparticles for drug delivery, imaging agents for cell tracking, and hydrogels for tissue engineering, which are rapidly advancing fields based on click chemistry. The content is based primarily on the experience with click chemistry-based biomaterials over the last 10 years., (© 2022 Wiley-VCH GmbH.)

Immunogenic cell death (ICD) is a powerful trigger eliciting strong immune responses against tumors. However, traditional chemoimmunotherapy (CIT) does not last long enough to induce sufficient ICD, and also does not guarantee the safety of chemotherapeutics. To overcome the disadvantages of the conventional approach, we used doxorubicin (DOX) as an ICD inducer, and poly(lactic-co-glycolic acid) (PLGA)-based nanomedicine platform for controlled release of DOX. The diameter of 138.7 nm of DOX-loaded PLGA nanoparticles (DP-NPs) were stable for 14 days in phosphate-buffered saline (PBS, pH 7.4) at 37 °C. Furthermore, DOX was continuously released for 14 days, successfully inducing ICD and reducing cell viability in vitro. Directly injected DP-NPs enabled the remaining of DOX in the tumor site for 14 days. In addition, repeated local treatment of DP-NPs actually lasted long enough to maintain the enhanced antitumor immunity, leading to increased tumor growth inhibition with minimal toxicities. Notably, DP-NPs treated tumor tissues showed significantly increased maturated dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) population, showing enhanced antitumor immune responses. Finally, the therapeutic efficacy of DP-NPs was maximized in combination with an anti-programmed death-ligand 1 (PD-L1) antibody (Ab). Therefore, we expect therapeutic efficacies of cancer CIT can be maximized by the combination of DP-NPs with immune checkpoint blockade (ICB) by achieving proper therapeutic window and continuously inducing ICD, with minimal toxicities.

Drug delivery to tumor sites using nanotechnology has been demonstrated to overcome the drawbacks of conventional anticancer drugs. Altering the surface shape and geometry of nanocomposites alters their chemical properties, which can confer multiple attributes to nanocarriers for the treatment of cancer and their use as imaging agents for cancer diagnosis. However, heterogeneity and blood flow in human cancer limit the distribution of nanoparticles at the site of tumor tisues. For targeted delivery and controlled release of drug molecules in harsh tumor microenvironments, smart nanocarriers combined with various stimuli-responsive materials have been developed. In this review, we describe nanomaterials for smart anticancer therapy as well as their pharmaceutical aspects including pharmaceutical process, formulation, controlled drug release, drug targetability, and pharmacokinetic or pharmacodynamic profiles of smart nanocarriers. Inorganic or organic-inorganic hybrid nanoplatforms and the electrospinning process have also been briefly described here.

The gold nanorods (GNRs) embedded alginate-chitosan (scaffold), which was designed and fabricated to produce efficient handling of the cell proliferations. Scaffold embedded GNR (SGNR) and NIR (near infrared) irradiations are developing into an interesting medical prognosis tool for rabbit chondrocyte (RC) proliferation. SGNR contained a pattern of uniform pores. Biocompatibility and cellular proliferation achieved by disclosures to NIR irradiations, providing high cell survival. SGNR and NIR irradiations could produce mechanical and biochemical cues for regulating RCs proliferations. To determine the thermal stress, it exposed RCs to 39-42 °C for 0-240 min at the start point of the cell culture cycle. It produced photothermal stress in cellular surrounding (cells located adjacent to and within scaffold) and it deals with the proliferation behavior of RC. All the processes were modeled with experimental criteria and time evolution process. Our system could help the cell proliferation by generating heat for cells. Hence, the present strategy could be implemented for supporting cell therapeutics after transplantation. This implementation would open new design techniques for integrating the interfaces between NIR irradiated and non-irradiated tissues., (© 2021. The Author(s).)

Immune checkpoint blockade is a promising approach for cancer immunotherapy, but many patients do not respond due to the immunosuppressive tumor microenvironment (ITM). Herein, we propose visible-light-triggered prodrug nanoparticles (LT-NPs) for reversing ITM into high immunogenic tumors to potentiate checkpoint blockade immunotherapy. The photosensitizer (verteporfin; VPF), cathepin B-specific cleavable peptide (FRRG), and doxorubicin (DOX) conjugates are self-assembled into LT-NPs without any additional carrier material. The LT-NPs are specifically cleaved to VPF and DOX in cathepsin B-overexpressing cancer cells, thereby inducing cancer-specific cytotoxicity and immunogenic cell death (ICD) upon visible light irradiation. In tumor models, LT-NPs highly accumulate within tumors via the enhanced permeability and retention effect, and photochemotherapy of VPF and DOX induces effective ICD and maturation of dendritic cells to stimulate cross-presentation of cancer-antigens to T cells. Furthermore, LT-NPs with PD-L1 blockade greatly inhibit tumor growth, tumor recurrence, and lung metastasis by initiating a strong antitumor immune response. The photochemotherapy by LT-NPs provides a promising strategy for effective checkpoint blockade immunotherapy.

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.

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.

Extracellular vesicles (EVs) are essential mediators in intercellular communication that have emerged as natural therapeutic nanomedicines for the treatment of intractable diseases. Their therapeutic applications, however, have been limited by unpredictable in vivo biodistribution after systemic administration. To control the in vivo fate of EVs, their surfaces should be properly edited, depending on the target site of action. Herein, based on bioorthogonal copper-free click chemistry (BCC), surface-edited EVs were prepared by using metabolically glycoengineered cells. First, the exogenous azide group was generated on the cellular surface through metabolic glycoengineering (MGE) using the precursor. Next, PEGylated hyaluronic acid, capable of binding specifically to the CD44-expressing cells, was labelled as the representative targeting moiety onto the cell surface by BCC. The surface-edited EVs effectively accumulated into the target tissues of the animal models with rheumatoid arthritis and tumour, primarily owing to prolonged circulation in the bloodstream and the active targeting mechanism. Overall, these results suggest that BCC combined with MGE is highly useful as a simple and safe approach for the surface modification of EVs to modulate their in vivo fate., Competing Interests: The authors declare the following competing interests: Y. W. Cho is the chief executive officer of ExoStemTech Inc. D.‐G. Jo and J. H. Park are stockholders of ExoStemTech Inc. The other authors declare no competing financial interests., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Nanoparticles have been used for effectively delivering imaging agents and therapeutic drugs into stem cells. However, nanoparticles are not sufficiently internalized into stem cells; thus, new delivery method of nanoparticles into stem cells is urgently needed. Herein, we develop bicyclo[6.1.0]nonyne (BCN)-conjugated gold nanoparticles (BCN-AuNPs), which can be bioorthogonally conjugated to azide (-N 3 ) groups on the surface of metabolically engineered stem cells via bioorthogonal click chemistry. For incorporating azide groups on the cell surface, first, human adipose-derived mesenchymal stem cells (hMSCs) were metabolically engineered with N-azidoacetylmannosamine-tetraacylated (Ac 4 ManNAz). Second, clickable BCN-AuNPs were bioorthogonally conjugated to azide groups on Ac 4 ManNAz-treated hMSCs. Importantly, a large amount of BCN-AuNPs was specifically conjugated to metabolically engineered hMSCs and then internalized rapidly into stem cells through membrane turnover mechanism, compared to the conventional nanoparticle-derived endocytosis mechanism. Furthermore, BCN-AuNPs entrapped in endosomal/lysosomal compartment could escape efficiently to the cytoplasm of metabolically engineered stem cells. Finally, BCN-AuNPs in stem cells were very safe, and they did not affect stem cell functions, such as self-renewal and differentiation capacity. These bioorthogonally conjugated nanoparticles on metabolically engineered stem cells can enhance the cellular uptake of nanoparticles via bioorthogonal conjugation mechanism.

Non-invasive tracking of T-cells may help to predict the patient responsiveness and therapeutic outcome. Herein, we developed bioorthogonal T-cell labeling and tracking strategy using bioorthogonal click chemistry. First, ovalbumin (OVA) antigen-specific cytotoxic T-cells (CTLs) were incubated with N-azidoacetyl-D-mannosamine-tetraacylated (Ac 4 ManNAz) for incorporating azide (N 3 ) groups on the surface of CTLs via metabolic glycoengineering. Subsequently, azide groups on the CTLs were chemically labeled with near infrared fluorescence (NIRF) dye, Cy5.5, conjugated dibenzylcyclooctyne (DBCO-Cy5.5) via bioorthogonal click chemistry, resulting in Cy5.5-labeled CTLs (Cy5.5-CTLs). The labeling efficiency of Cy5.5-CTLs could be readily controlled by changing concentrations of Ac 4 ManNAz and DBCO-Cy5.5 in cultured cells. Importantly, Cy5.5-CTLs presented the strong NIRF signals in vitro and they showed no significant changes in the functional properties, such as cell viability, proliferation, and antigen-specific cytolytic activity. In ovalbumin (OVA)-expressing E.G-7 tumor-bearing immune-deficient mice, intravenously injected Cy5.5-CTLs were clearly observed at targeted solid tumors via non-invasive NIRF imaging. Moreover, tumor growth inhibition of E.G-7 tumors was closely correlated with the intensity of NIRF signals from Cy5.5-CTLs at tumors after 2-3 days post-injection. The Cy5.5-CTLs showed different therapeutic responses in E.G-7 tumor-bearing immune-competent mice, in which they were divided by their tumor growth efficacy as 'high therapeutic response (TR (+))' and 'low therapeutic response (TR (-))'. These different therapeutic responses of Cy5.5-CTLs were highly correlated with the NIRF signals of Cy5.5-CTLs at targeted tumor tissues in the early stage. Therefore, non-invasive tracking of T-cells can be able to predict and elicit therapeutic responses in the adoptive T-cell therapy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac 4 ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as 'condensed round shape' and 'spread shape' in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine., (Copyright © 2020. Published by Elsevier Ltd.)

Chemotherapy has shown remarkable therapeutic efficacy for various types of cancer. However, drug resistance reduces the effectiveness and sensitivity of chemotherapy, leading treatment failure and cancer relapse in many clinical indications. Herein, we propose cancer-specific drug-drug nanoparticles (DD-NPs) that improve the therapeutic efficacy of chemotherapy against drug-resistant cancer. Cancer-specific and pro-apoptotic drug-drug conjugate was prepared by conjugating the pro-apoptotic peptide drug (SMAC; Ala-Val-Pro-Ile-Ala-Gln, AVPIAQ) and cathepsin B-cleavable peptide (Phe-Arg-Arg-Gly, FRRG) to a doxorubicin (DOX), resulting in SMAC-FRRG-DOX that allows self-assembled into nanoparticles. The resulting DD-NPs were specifically cleaved to pro-apoptotic SMAC and cytotoxic DOX only in cathepsin B-overexpressing cancer cells, inducing a synergy of the pro-apoptotic activity with the chemotherapy. In MCF-7 breast tumor-bearing mice, intravenously injected DD-NPs highly accumulated at targeted tumor tissues via enhanced permeability and retention (EPR) effect, releasing SMAC and DOX, which showed a synergetic pro-apoptotic/chemotherapy. Furthermore, DD-NPs greatly suppressed tumor growth and improved overall survival in a metastatic lung cancer model. Collectively, these cancer-specific drug-drug nanoparticles may be a promising strategy to treat drug-resistant cancers with high cancer cell-specificity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2020 Wiley-VCH GmbH.)

Direct local delivery of immunogenic cell death (ICD) inducers to a tumor site is an attractive approach for leading ICD effectively, due to enabling the concentrated delivery of ICD inducers to the tumor site. Herein, we prepared doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) using different molecular weight PLGA (7000 g/mol and 12,000 g/mol), showing different drug release kinetics. The different release kinetics of DOX might differently stimulate a tumor cell-specific immune response by releasing damage-associated molecular patterns (DAMPs), resulting in showing a different antitumor response in the living body. DOX-PLGA 7K NPs showed faster DOX release kinetics than DOX-PLGA 12K NPs in the physiological condition. DOX-PLGA 7K NPs and DOX-PLGA 12K NPs were successfully taken up by the CT-26 tumor cells, subsequently showing different DOX localization times at the nucleus. Released DOX successfully lead to cytotoxicity and HMGB1 release in vitro. Although the DOX-PLGA 7K NPs and DOX-PLGA 12K NPs showed different sustained DOX release kinetics in vitro, tumor growth of the CT-26 tumor was similarly inhibited for 28 days post-direct tumor injection. Furthermore, the immunological memory effect was successfully established by the ICD-based tumor-specific immune responses, including DC maturation and tumor infiltration of cytotoxic T lymphocytes (CTLs). We expect that the controlled release of ICD-inducible chemotherapeutic agents, using different types of nanomedicines, can provide potential in precision cancer immunotherapy by controlling the tumor-specific immune responses, thus improving the therapeutic efficacy.

The dense extracellular matrix (ECM) in heterogeneous tumor tissues can prevent the deep tumor penetration of drug-loaded nanoparticles, resulting in a limited therapeutic efficacy in cancer treatment. Herein, we suggest that the deep tumor penetration of doxorubicin (DOX)-loaded glycol chitosan nanoparticles (CNPs) can be improved using high-intensity focused ultrasound (HIFU) technology. Firstly, we prepared amphiphilic glycol chitosan-5β-cholanic acid conjugates that can self-assemble to form stable nanoparticles with an average of 283.7 ± 5.3 nm. Next, the anticancer drug DOX was simply loaded into the CNPs via a dialysis method. DOX-loaded CNPs (DOX-CNPs) had stable nanoparticle structures with an average size of 265.9 ± 35.5 nm in aqueous condition. In cultured cells, HIFU-treated DOX-CNPs showed rapid drug release and enhanced cellular uptake in A549 cells, resulting in increased cytotoxicity, compared to untreated DOX-CNPs. In ECM-rich A549 tumor-bearing mice, the tumor-targeting efficacy of intravenously injected DOX-CNPs with HIFU treatment was 1.84 times higher than that of untreated DOX-CNPs. Furthermore, the deep tumor penetration of HIFU-treated DOX-CNPs was clearly observed at targeted tumor tissues, due to the destruction of the ECM structure via HIFU treatment. Finally, HIFU-treated DOX-CNPs greatly increased the therapeutic efficacy at ECM-rich A549 tumor-bearing mice, compared to free DOX and untreated DOX-CNPs. This deep penetration of drug-loaded nanoparticles via HIFU treatment is a promising strategy to treat heterogeneous tumors with dense ECM structures., Competing Interests: The authors declare no conflict of interest.

The development of heat-generating magnetic nanostructures is critical for the effective management of tumors using magnetic hyperthermia. Herein, we demonstrate that polyethylene glycol (PEG)-coated iron oxide (magnetite, Fe 3 O 4 ) multigranule nanoclusters (PEG-MGNCs) can enhance the efficiency of hyperthermia-based tumor suppression in vitro and in vivo . MGNCs consisting of granules (crystallites) measuring 22.9 nm in diameter were prepared via the hydrothermal polyol method, followed by the surface modification of MGNCs with PEG-dopamine. The freshly prepared PEG-MGNCs exhibit 145.9 ± 10.2 nm diameter on average under aqueous conditions. The three-dimensional structures of PEG-MGNCs enhance the hyperthermic efficacy compared with PEGylated single iron-oxide nanoparticles (NPs), resulting in severe heat damage to tumor cells in vitro . In the SCC7 tumor-bearing mice, near-infrared fluorescence dye (Cy5.5)-labeled PEG-MGNCs are successfully accumulated in the tumor tissues because of NP-derived enhanced permeation and retention effect. Finally, the tumor growth is significantly suppressed in PEG-MGNC-treated mice after two-times heat generation by using a longitudinal solenoid, which can generate an alternating magnetic field under high-frequency (19.5 kA/m, 389 kHz) induction. This study shows for the first time that the PEG-MGNCs greatly enhance the hyperthermic efficacy of tumor treatment both in vitro and in vivo .

Theranostic nanoparticles can deliver therapeutic agents as well as diverse imaging agents to tumors. The enhanced permeation and retention (EPR) effect is regarded as a crucial mechanism for the tumor-targeted delivery of nanoparticles. Although a large number of studies of the EPR effect of theranostic nanoparticles have been performed, the effect of the change in the body size of the host on the EPR effect is not fully understood. In this regard, comparative research is needed on the behavior of nanoparticles in large animals for developing the nanoparticles to the clinical stage. In this study, we prepared fluorophore (indocyanine green (ICG) or cyanine 5.5 (Cy5.5))-conjugated glycol chitosan nanoparticles (CNPs) for comparing the tumor-targeting efficacy in VX2 tumor-bearing mouse and rabbit models. As expected, the CNPs formed nano-sized spherical nanoparticles and were stable for 8 days under aqueous conditions. The CNPs also exhibited dose-dependent cellular uptake into VX2 tumor cells without cytotoxicity. The half-life of the near-infrared fluorescence (NIRF) signals in the blood were 3.25 h and 4.73 h when the CNPs were injected into mice and rabbits, respectively. Importantly, the CNPs showed excellent tumor accumulation and prolonged biodistribution profiles in both the VX2 tumor-bearing mouse and rabbit models, wherein the tumor accumulation was maximized at 48 h and 72 h, respectively. Based on the excellent tumor accumulation of the CNPs, finally, the CNPs were used in the image-guided surgery of the rabbit orthotopic VX2 lung tumor model. The lung tumor tissue was successfully removed based on the NIRF signal from the CNPs in the tumor tissue. This study shows that CNPs can be potentially used for tumor theragnosis in small animals and large animals.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N -azidoacetyl-d-mannosamine (Ac 4 ManNAz) to generate unnatural azide groups (-N 3 ) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

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.

Various types of albumin-binding molecules have been conjugated to anticancer drugs, and these modified prodrugs could be effective in cancer treatments compared to free anticancer drugs. However, the tumor targeting of albumin-binding prodrugs has not been clearly investigated. Herein, we examined the in vitro and in vivo tumor-targeting efficiency of three different albumin-binding molecules including albumin-binding peptide (DICLPRWGCLW: PEP), fatty acid (palmitic acid: PA), and maleimide (MI), respectively. In order to characterize the different targeting efficiency of albumin-binding molecules, PEP, PA, or MI was chemically labeled with near-infrared fluorescence (NIRF) dye, Cy5.5, in resulting PEP-Cy5.5, PA-Cy5.5, and MI-Cy5.5. These NIRF dye-labeled albumin-binding molecules were physically or chemically bound to albumin via gentle incubation in aqueous conditions in vitro. Notably, PA-Cy5.5 with reversible and multivalent binding affinities formed stable albumin complexes, compared to PEP-Cy5.5 and MI-Cy5.5, confirmed via surface plasmon resonance measurement, gel electrophoresis assay, and albumin-bound column-binding test. In tumor-bearing mice model, the different albumin-binding affinities of PA-Cy5.5, PEP-Cy5.5, and MI-Cy5.5 greatly contributed to their tumor-targeting ability. Even though the binding affinity of PEP-Cy5.5 and MI-Cy5.5 to albumin is higher than that of PA-Cy5.5 in vitro, intravenous PA-Cy5.5 showed a higher tumor-targeting efficiency in tumor-bearing mice compared to that of PEP-Cy5.5 and MI-Cy5.5. The reversible and multivalent affinities of albumin-binding molecules to native serum albumin greatly increased the pharmacokinetics and tumor-targeting efficiency in vivo.

The therapeutic efficacy of photodynamic therapy (PDT) in cancer treatment is attributed to the conversion of tumor oxygen into reactive singlet oxygen ( 1 O 2 ) using photosensitizers. However, poor tissue penetration and rapid oxygen depletion have limited the effectiveness of PDT. Therefore, we have developed visible light-induced apoptosis activatable nanoparticles of the photosensitizer (Ce6)-caspase 3 cleavable peptide (Asp-Glu-Val-Asp, DEVD)-anticancer drug monomethyl auristatin E (MMAE) conjugate, resulting in Ce6-DEVD-MMAE nanoparticles. The average size of self-assembled Ce6-DEVD-MMAE nanoparticles was 90.8 ± 18.9 nm. Compared with conventional PDT based on high-energy irradiation, the new therapy uses lower-energy irradiation to induce apoptosis of cancer cells, and activation of caspase 3 to successfully cleave the anticancer drug MMAE from the Ce6-DEVD-MMAE nanoparticles, resulting in strong cytotoxic effects in cancer cells. Notably, the one-time activation of MMAE in the Ce6-DEVD-MMAE nanoparticles further amplified the cytotoxic effect resulting in additional cell death in the absence of visible light irradiation. Furthermore, Ce6-DEVD-MMAE nanoparticles passively accumulated in the targeted tumor tissues via enhanced permeation and retention (EPR) effect in mice with squamous cell carcinoma (SCC7). The high levels of toxicity were retained after exposure to lower-energy irradiation. However, Ce6-DEVD-MMAE nanoparticles did not show any toxicity in the absence of exposure to visible light irradiation, in contrast to the toxicity of free MMAE (1-10 nM). Thus, the light-induced therapeutic strategy based on apoptotic activation of Ce6-DEVD-MMAE nanoparticles can be used to treat solid tumors inaccessible to conventional PDT., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Noninvasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which is closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye Cy5.5 was chemically conjugated to the BCN-NPs, and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe 3 O 4 NPs) were encapsulated into BCN-NPs, resulting in Cy5.5-labeled and OA-Fe 3 O 4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), first, hMSCs were treated with tetra-acetylated N -azidoacetyl-d-mannosamine (Ac 4 ManNAz) for generating azide (-N 3 ) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro . This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked by their migration from the implanted site to the brain stroke lesion using NIRF/ T 2 -weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo .

Cancer immunotherapy is an attractive treatment option under clinical settings. However, the major challenges of immunotherapy include limited patient response, limited tumor specificity, immune-related adverse events, and immunosuppressive tumor microenvironment. Therefore, nanoparticle (NP)-based drug delivery has been used to not only increase the efficacy of immunotherapeutic agents, but it also significantly reduces the toxicity. In particular, NP-based drug delivery systems alter the pharmacokinetic (PK) profile of encapsulated or conjugated immunotherapeutic agents to targeted cancer cells or immune cells and facilitate the delivery of multiple therapeutic combinations to targeted cells using single NPs. Recently, advanced NP-based drug delivery systems were effectively utilized in cancer immunotherapy to reduce the toxic side effects and immune-related adverse events. Repurposing these NPs as delivery systems of immunotherapeutic agents may overcome the limitations of current cancer immunotherapy. In this review, we focus on recent advances in NP-based immunotherapeutic delivery systems, such as immunogenic cell death (ICD)-inducing drugs, cytokines and adjuvants for promising cancer immunotherapy. Finally, we discuss the challenges facing current NP-based drug delivery systems that need to be addressed for successful clinical application., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Theranostic biomaterials have been creating great new opportunities in developing precision medicine in cancer treatment. Moreover, there exist up-coming potentials to find a new paradigm change in cancer treatment when theranostic biomaterials can precisely image a biological process at the exact tumor sites. With aid of molecular imaging technology, theranostic biomaterials can visualize targets, monitor efficiency of delivery carriers as well as therapeutic responses at tumor site. Major obstacles in designing theranostic biomaterials might be what providing target-specificity to biomaterials for improving therapeutic efficacy as well as visualization in cancer treatment. By which, we can find ways to minimize unwanted side effects to normal tissues in cancer therapy. This review focuses on how theranostic biomaterials can be designed with tumor-specific fluorescence imaging probes, especially tumor-specific activatable imaging probes, which in turn can be switched to therapeutic design with identical principle. Highlights such as designing by characteristics of cancer, finding ways to monitor therapeutic efficacy, potential benefits in theranostic biomaterials are also discussed., (Copyright © 2019. Published by Elsevier Ltd.)

Introduction : In the past decade, the glycol chitosan nanocarriers (GCNCs) have been widely used for tumor-targeted delivery of anticancer agents such as chemo drugs, peptides, and genes due to their biocompatibility, biodegradability, and easy fabrication. In particular, GCNCs can effectively solubilize water-insoluble chemo drugs as well as improve the delivery efficiency of chemo drugs to the tumor, resulting in maximizing therapeutic efficacy and minimizing side effect. In this review, we introduce the various applications of GCNCs for cancer treatment and these GCNCs demonstrate the great potential in overcoming challenges of chemotherapeutics. Areas covered : Various designs of GC nanocarriers have been reviewed. The current state of GC nanocarriers for delivering chemotherapeutics with a focus on their physicochemical properties including solubilization of anti-cancer drugs, sustained release, and tumor-selectivity. Furthermore, state of the art in delivery and therapeutic strategy using GC nanocarriers also introduced for overcoming challenges of chemotherapeutics. Expert opinion : Based on the reviewed literature, physicochemical properties of GC nanocarriers will have a great potential to overcome challenges posed by chemotherapeutics.

Cancer nanomedicine using nanoparticle-based delivery systems has shown outstanding promise in recent decades for improving anticancer treatment. However, limited targeting efficiency, low drug loading efficiency and innate toxicity of nanoparticles have caused severe problems, leaving only a few available in the clinic. Here, we newly developed carrier-free nanoparticles of cathepsin B-cleavable peptide (Phe-Arg-Arg-Gly; FRRG)-conjugated doxorubicin (DOX) prodrug (FRRG-DOX) that formed a stable nanoparticle structure with an average diameter of 213 nm in aqueous condition. The carrier-free nanoparticles of FRRG-DOX induced cytotoxicity against cathepsin B-overexpressed tumor cells whereas the toxicity was minimized in normal cells. In particular, the FRRG-DOX nanoparticles showed the successful tumor-targeting ability and enhanced therapeutic efficiency in human colon adenocarcinoma (HT-29) tumor-bearing mice via enhanced permeation and retention (EPR) effect. Furthermore, FRRG-DOX nanoparticles did not present any severe toxicity, such as non-specific cell death and cardiac toxicity, in normal tissues due to minimal expression of cathepsin B. This carrier-free nanoparticles of FRRG-DOX can solve the unavoidable problems of current nanomedicine, such as lower targeting efficiency, toxicity of nanoparticles themselves, and difficulty in mass production that are fatally caused by natural and synthetic nano-sized carriers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the past few years, there have been many efforts underway to develop effective wound healing treatments for traumatic injuries. In particular, wound-healing peptides (WHPs) and peptide-grafted dressings hold great promise for novel therapeutic strategies for wound management. This study reports a topical formulation of a new synthetic WHP (REGRT, REG) embedded in a hyaluronic acid (HA)-based hydrogel dressing for the enhancement of acute excisional wound repair. The copper-free click chemistry is utilized to form biocompatible HA hydrogels by cross-linking dibenzocyclooctyl-functionalized HA with 4-arm poly(ethylene glycol) (PEG) azide. The HA hydrogels are grafted with the REG peptide, a functional derivative of erythroid differentiation regulator1, displaying potent cell motility-stimulating ability, thus sustainably releasing physiologically active peptides for a prolonged period. Combined with the traditional wound healing benefits of HA, the HA hydrogel embedded REG (REG-HAgel) accelerates re-epithelialization in skin wound healing, particularly by promoting migration of fibroblasts, keratinocytes, and endothelial cells. REG-HAgels improve not only rate, but quality of wound healing with higher collagen deposition and more microvascular formation while being nontoxic. The peptide-grafted HA hydrogel system can be considered as a promising new wound dressing formulation strategy for the treatment of different types of wounds with combinations of various natural and synthetic WHPs.

Cancer immunotherapy has been emerging in recent years, due to the inherent nature of the immune system. Although recent successes of immunotherapeutics in clinical application have attracted development of a novel immunotherapeutics, the off-target side effect and low immunogenicity of them remain challenges for the effective cancer immunotherapy. Theranostic nanoparticle system may one of key technology to address these issues by offering targeted delivery of various types of immunotherapeutics, resulting in significant improvements in the tumor immunotherapy. However, appropriate design or engineering of nanoparticles will be needed to improve delivery efficiency of antigen, adjuvant and therapeutics, resulting in eliciting antitumor immunity. Here, we review the current state of the art of cancer immunotherapeutic strategies, mainly based on nanoparticles (NPs). This includes NP-based antigen/adjuvant delivery vehicles to draining lymph nodes, and tumor antigen-specific T-lymphocytes for cancer immunotherapy. Several NP-based examples are shown for immune checkpoint modulation and immunogenic cell death. These overall studies demonstrate the great potential of NPs in cancer immunotherapy. Finally, engineering NP strategies will provide great opportunities to improve therapeutic effects as well as optimization of treatment processes, allowing to meet the individual needs in the cancer immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although protein nanoparticles (PNPs) (e.g., viral capsids) capable of delivering a broad range of drug agents have shown distinctive advantages over synthetic nanomaterials, PNPs have an intrinsic drawback that hampers their clinical application, that is, potential immunogenicity. Here, a novel method for resolving the immunogenicity problem of PNPs, which is based on the genetic presentation of albumin-binding peptides (ABPs) on the surface of PNP, is reported. ABPs are inserted into the surface of a viral capsid (hepatitis B virus capsid/HBVC) while preserving the native self-assembly function of HBVC. The ABPs effectively gather human serum albumins around HBVC and significantly reduce both inflammatory response and immunoglobulin titer in live mice compared to ABP-free HBVC. Furthermore, ABP-conjugated HBVCs remain within tumors for a longer period than HBVCs conjugated to tumor cell receptor-bindingpeptides, indicating that the ABPs are also capable of enhancing tumor-targeting performance. Although applied to HBVC for proof of concept, this novel approach may provide a general platform for resolving immunogenicity and cancer-targeting problems of PNPs, which enables the development of a variety of PNP-based drug delivery carriers with high safety and efficacy.

Thrombosis is an important pathophysiologic phenomenon in various cardiovascular diseases, which can lead to oxygen deprivation and infarction of tissues by generation of a thrombus. Thus, direct thrombus imaging can provide beneficial in diagnosis and therapy of thrombosis. Herein, we developed thrombin-activatable fluorescent peptide (TAP) incorporated silica-coated gold nanoparticles (TAP-SiO 2 @AuNPs) for direct imaging of thrombus by dual near-infrared fluorescence (NIRF) and micro-computed tomography (micro-CT) imaging, wherein TAP molecules were used as targeted thrombin-activatable peptide probes for thrombin-specific NIRF imaging. The freshly prepared TAP-SiO 2 @AuNPs had an average diameter of 39.8 ± 2.55 nm and they showed the quenched NIRF signal in aqueous condition, due to the excellent quenching effect of TAP molecules on the silica-gold nanoparticle surface. However, 30.31-fold higher NIRF intensity was rapidly recovered in the presence of thrombin in vitro, due to the thrombin-specific cleavage of quenched TAP molecules on the gold particle surface. Furthermore, TAP-SiO 2 @AuNPs were successfully accumulated in thrombus by their particle size-dependent capturing property, and they presented a potential X-ray absorption property in a dose-dependent manner. Finally, thrombotic lesion was clearly distinguished from peripheral tissues by dual NIRF/micro-CT imaging after intravenous injection of TAP-SiO 2 @AuNPs in the in situ thrombotic mouse model, simultaneously. This study showed that thrombin-activatable fluorescent peptide incorporated silica-coated gold nanoparticles can be potentially used as a dual imaging probe for direct thrombus imaging and therapy in clinical applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Development of biopolymer-based imaging agents which can access rapidly and provide detailed information about the diseases has received much attention as an alternative to conventional imaging agents. However, development of biopolymer-based nanomaterials for tumor imaging still remains challenging due to their low sensitivity and image resolution. To surmount of these limitations, multimodal imaging agents have been developed, and they were widely utilized for theranostic applications. Herein, iodine containing echogenic glycol chitosan nanoparticles are developed for x-ray computed tomography (CT) and ultrasound (US) imaging of tumor diagnosis. X-ray CT/US dual-modal imaging probe was prepared by following below two steps. First, iodine-contained diatrizoic acid (DTA) was chemically conjugated to the glycol chitosan (GC) for the CT imaging. DTA conjugated GC (GC-DTA NPs) formed stable nanoparticles with an average diameter of 315 nm. Second, perfluoropentane (PFP), a US imaging agent, was physically encapsulated into GC-DTA NPs by O/W emulsion method yielding GC-DTA-PFP nanoparticles (GC-DTA-PFP NPs). The GC-DTA-PFP NPs formed nanoparticles in physiological condition, and they presented the strong x-ray CT, and US signals in phantom test in vitro. Importantly, GC-DTA-PFP NPs were effectively accumulated on the tumor site by enhanced permeation and retention (EPR) effects. Moreover, GC-DTA-PFP NPs showed x-ray CT, and US signals in tumor tissues after intratumoral and intravenous injection, respectively. Therefore, GC-DTA-PFP NPs indicated that x-ray CT/US dual-modal imaging using iodinated echogenic nanoparticles could be provided more comprehensive and accurate diagnostic information to diagnosis of tumor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.