Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy.

BACKGROUND: Immunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency. METHODS: Herein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes. RESULTS: The SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu. CONCLUSIONS: Eventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.

Photo-induced crosslinked and anti-PD-L1 peptide incorporated liposomes to promote PD-L1 multivalent binding for effective immune checkpoint blockade therapy.

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 DC8,9PC with photo-polymerized diacetylenic moiety, 1,2-dipalmitoylphosphatidylcholine (DPPC) and anti-PD-L1 peptide (D-form NYSKPTDRQYHF)-conjugated DSPE-PEG2k (anti-PD-L1-DSPE-PEG2k) 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-PEG2k 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.

Fluorescence-Based Mono- and Multimodal Imaging for In Vivo Tracking of Mesenchymal Stem Cells.

The advancement of stem cell therapy has offered transformative therapeutic outcomes for a wide array of diseases over the past decades. Consequently, stem cell tracking has become significant in revealing the mechanisms of action and ensuring safe and effective treatments. Fluorescence stands out as a promising choice for stem cell tracking due to its myriad advantages, including high resolution, real-time monitoring, and multi-fluorescence detection. Furthermore, combining fluorescence with other tracking modalities-such as bioluminescence imaging (BLI), positron emission tomography (PET), photoacoustic (PA), computed tomography (CT), and magnetic resonance (MR)-can address the limitations of single fluorescence detection. This review initially introduces stem cell tracking using fluorescence imaging, detailing various labeling strategies such as green fluorescence protein (GFP) tagging, fluorescence dye labeling, and nanoparticle uptake. Subsequently, we present several combinations of strategies for efficient and precise detection.

Micro-syringe chip-guided intratumoral administration of lipid nanoparticles for targeted anticancer therapy.

BACKGROUND: Nano-sized drug delivery system has been widely studied as a potential technique to promote tumor-specific delivery of anticancer drugs due to its passive targeting property, but resulting in very restricted improvements in its systemic administration so far. There is a requirement for a different approach that dramatically increases the targeting efficiency of therapeutic agents at targeted tumor tissues. METHODS: To improve the tumor-specific accumulation of anticancer drugs and minimize their undesirable toxicity to normal tissues, a tumor-implantable micro-syringe chip (MSC) with a drug reservoir is fabricated. As a clinically established delivery system, six liposome nanoparticles (LNPs) with different compositions and surface chemistry are prepared and their physicochemical properties and cellular uptake are examined in vitro. Subsequently, MSC-guided intratumoral administration is studied to identify the most appropriate for the higher tumor targeting efficacy with a uniform intratumoral distribution. For efficient cancer treatment, pro-apoptotic anticancer prodrugs (SMAC-P-FRRG-DOX) are encapsulated to the optimal LNPs (SMAC-P-FRRG-DOX encapsulating LNPs; ApoLNPs), then the ApoLNPs are loaded into the 1 µL-volume drug reservoir of MSC to be delivered intratumorally for 9 h. The tumor accumulation and therapeutic effect of ApoLNPs administered via MSC guidance are evaluated and compared to those of intravenous and intratumoral administration of ApoLNP in 4T1 tumor-bearing mice. RESULTS: MSC is precisely fabricated to have a 0.5 × 4.5 mm needle and 1 µL-volume drug reservoir to achieve the uniform intratumoral distribution of LNPs in targeted tumor tissues. Six liposome nanoparticles with different compositions of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (PEG2000-DSPE) are prepared with average sizes of 100-120 nm and loaded into the 1 µL-volume drug reservoir in MSC. Importantly negatively charged 10 mol% of PS-containing LNPs are very slowly infused into the tumor tissue through the micro-syringe of the MSC over 6 h. The intratumoral targeting efficiency of MSC guidance is 93.5%, effectively assisting the homogeneous diffusion of LNPs throughout the tumor tissue at 3.8- and 2.7-fold higher concentrations compared to the intravenous and intratumoral administrations of LNPs, respectively. Among the six LNP candidates 10 mol% of PS-containing LNPs are finally selected for preparing pro-apoptotic SMAC-P-FRRG-DOX anticancer prodrug-encapsulated LNPs (ApoLNPs) due to their moderate endocytosis rate high tumor accumulation and homogenous intratumoral distribution. The ApoLNPs show a high therapeutic effect specifically to cathepsin B-overexpressing cancer cells with 6.6 µM of IC50 value while its IC50 against normal cells is 230.7 µM. The MSC-guided administration of ApoLNPs efficiently inhibits tumor growth wherein the size of the tumor is 4.7- and 2.2-fold smaller than those treated with saline and intratumoral ApoLNP without MSC, respectively. Moreover, the ApoLNPs remarkably reduce the inhibitor of apoptosis proteins (IAPs) level in tumor tissues confirming their efficacy even in cancers with high drug resistance. CONCLUSION: The MSC-guided administration of LNPs greatly enhances the therapeutic efficiency of anticancer drugs via the slow diffusion mechanism through micro-syringe to tumor tissues for 6 h, whereas they bypass most hurdles of systemic delivery including hepatic metabolism, rapid renal clearance, and interaction with blood components or other normal tissues, resulting in the minimum toxicity to normal tissues. The negatively charged ApoLNPs with cancer cell-specific pro-apoptotic prodrug (SMAC-P-FRRG-DOX) show the highest tumor-targeting efficacy when they are treated with the MSC guidance, compared to their intravenous or intratumoral administration in 4T1 tumor-bearing mice. The MSC-guided administration of anticancer drug-encapsulated LNPs is expected to be a potent platform system that facilitates overcoming the limitations of systemic drug administration with low delivery efficiency and serious side effects.

Recent Studies and Progress in the Intratumoral Administration of Nano-Sized Drug Delivery Systems.

Over the last 30 years, diverse types of nano-sized drug delivery systems (nanoDDSs) have been intensively explored for cancer therapy, exploiting their passive tumor targetability with an enhanced permeability and retention effect. However, their systemic administration has aroused some unavoidable complications, including insufficient tumor-targeting efficiency, side effects due to their undesirable biodistribution, and carrier-associated toxicity. In this review, the recent studies and advancements in intratumoral nanoDDS administration are generally summarized. After identifying the factors to be considered to enhance the therapeutic efficacy of intratumoral nanoDDS administration, the experimental results on the application of intratumoral nanoDDS administration to various types of cancer therapies are discussed. Subsequently, the reports on clinical studies of intratumoral nanoDDS administration are addressed in short. Intratumoral nanoDDS administration is proven with its versatility to enhance the tumor-specific accumulation and retention of therapeutic agents for various therapeutic modalities. Specifically, it can improve the efficacy of therapeutic agents with poor bioavailability by increasing their intratumoral concentration, while minimizing the side effect of highly toxic agents by restricting their delivery to normal tissues. Intratumoral administration of nanoDDS is considered to expand its application area due to its potent ability to improve therapeutic effects and relieve the systemic toxicities of nanoDDSs.

All-in-one glycol chitosan nanoparticles for co-delivery of doxorubicin and anti-PD-L1 peptide in cancer immunotherapy.

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.

Perspectives for Improving the Tumor Targeting of Nanomedicine via the EPR Effect in Clinical Tumors.

Over the past few decades, the enhanced permeability and retention (EPR) effect of nanomedicine has been a crucial phenomenon in targeted cancer therapy. Specifically, understanding the EPR effect has been a significant aspect of delivering anticancer agents efficiently to targeted tumors. Although the therapeutic effect has been demonstrated in experimental models using mouse xenografts, the clinical translation of the EPR effect of nanomedicine faces several challenges due to dense extracellular matrix (ECM), high interstitial fluid pressure (IFP) levels, and other factors that arise from tumor heterogeneity and complexity. Therefore, understanding the mechanism of the EPR effect of nanomedicine in clinics is essential to overcome the hurdles of the clinical translation of nanomedicine. This paper introduces the basic mechanism of the EPR effect of nanomedicine, the recently discussed challenges of the EPR effect of nanomedicine, and various strategies of recent nanomedicine to overcome the limitations expected from the patients' tumor microenvironments.

Thermally Managed, Injectable Optoelectronic Probe with Heat Dissipation Guide for Photodynamic Therapy.

The development of fabrication technologies and appearance of new materials has resulted in dramatic increase in the performance of electronic devices, while the overall size has decreased. Recent electronic devices made of micro/nano-size components show high efficiency and outstanding performance with compact size, but these devices have revealed several fatal problems. In particular, the isolated heat that is generated by numerous components concentrated in a limited small area at high density, such as bio-integrated devices, is an issue that needs to be urgently addressed, because it is closely related to the performance and lifetime of electronic devices. To solve these problems, the microscale light emitting diode (µLED)-based neural probe is introduced on an injectable heat dissipation guide. The heat dissipation guide is made of boron nitride (BN) nanomaterials with high thermal conductivity. The heat management noticeably improves the optical output performance of the µLEDs, in which BN effectively dissipates heat, and allows enhanced lighting from the LEDs to be transmitted through brain tissue without thermal damage. Moreover, it shows remarkable improvement in the therapeutic effect of photodynamic therapy of mouse cancer cells.

Adoptive Transfer of Photosensitizer-Loaded Cytotoxic T Cells for Combinational Photodynamic Therapy and Cancer Immuno-Therapy.

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.

In vivo toxicity evaluation of tumor targeted glycol chitosan nanoparticles in healthy mice: repeated high-dose of glycol chitosan nanoparticles potentially induce cardiotoxicity.

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 5ß-cholanic acid and the amphiphilic glycol chitosan-5ß-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.

In situ albumin-binding and esterase-specifically cleaved BRD4-degrading PROTAC for targeted cancer therapy.

Proteolysis-targeting chimeras (PROTACs) have recently been of great interest in cancer therapy. However, the bioavailability of PROTACs is considerably restricted due to their high hydrophobicity, poor cell permeability, and thereby low tumor targeting ability. Herein, esterase-cleavable maleimide linker (ECMal)-conjugated bromodomain 4 (BRD4)-degrading PROTAC (ECMal-PROTAC) is newly synthesized to exploit plasma albumin as an 'innate drug carrier' that can be accumulated in targeted tumor tissues. The BRD4-degrading ECMal-PROTAC is spontaneously bound to albumins via the thiol-maleimide click chemistry and its esterase-specific cleavage of ECMal-PROTAC is characterized in physiological conditions. The albumin-bound ECMal-PROTACs (Alb-ECMal-PROTACs) have an average size of 6.99 ± 1.38 nm, which is similar to that of free albumins without denaturation or aggregation. When Alb-ECMal-PROTACs are treated to 4T1 tumor cells, they are actively endocytosed and reach their highest intracellular level within 12 h. Furthermore, the maleimide linkers of Alb-ECMal-PROTACs are cleaved by the esterase to release free BRD-4 degrading PROTACs and the cell-internalized PROTACs successfully catalyze the selective degradation of BRD4 proteins, resulting in BRD4 deficiency-related apoptosis. When ECMal-PROTACs are intravenously injected into tumor-bearing mice, they exhibit a 16.3-fold higher tumor accumulation than free BRD4-PROTAC, due to the shuttling effect of albumin for tumor targeting. Finally, ECMal-PROTACs show 5.3-fold enhanced antitumor efficacy compared to free BRD4-PROTAC, without provoking any severe systemic toxicity. The expression of Bcl-2 and c-Myc, the downstream oncogenic proteins of BRD4, are also effectively suppressed. In summary, the in situ albumin binding of ECMal-PROTAC is proven as a promising strategy that effectively modulates its pharmacokinetics and therapeutic performance with high applicability to other types of PROTACs.

Cancer-Specific Delivery of Proteolysis-Targeting Chimeras (PROTACs) and Their Application to Cancer Immunotherapy.

Proteolysis-targeting chimeras (PROTACs) are rapidly emerging as a potential therapeutic strategy for cancer therapy by inducing the degradation of tumor-overexpressing oncogenic proteins. They can specifically catalyze the degradation of target oncogenic proteins by recruiting E3 ligases and utilizing the ubiquitin-proteasome pathway. Since their mode of action is universal, irreversible, recyclable, long-lasting, and applicable to 'undruggable' proteins, PROTACs are gradually replacing the role of conventional small molecular inhibitors. Moreover, their application areas are being expanded to cancer immunotherapy as various types of oncogenic proteins that are involved in immunosuppressive tumor microenvironments. However, poor water solubility and low cell permeability considerably restrict the pharmacokinetic (PK) property, which necessitates the use of appropriate delivery systems for cancer immunotherapy. In this review, the general characteristics, developmental status, and PK of PROTACs are first briefly covered. Next, recent studies on the application of various types of passive or active targeting delivery systems for PROTACs are introduced, and their effects on the PK and tumor-targeting ability of PROTACs are described. Finally, recent drug delivery systems of PROTACs for cancer immunotherapy are summarized. The adoption of an adequate delivery system for PROTAC is expected to accelerate the clinical translation of PROTACs, as well as improve its efficacy for cancer therapy.

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.

Enhanced delivery of the phototherapeutic nanoparticles via hepatocyte overload.

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.

Intracellular Glucose-Depriving Polymer Micelles for Antiglycolytic Cancer Treatment.

A new anticancer strategy to exploit abnormal metabolism of cancer cells rather than to merely control the drug release or rearrange the tumor microenvironment is reported. An antiglycolytic amphiphilic polymer, designed considering the unique metabolism of cancer cells (Warburg effect) and aimed at the regulation of glucose metabolism, is synthesized through chemical conjugation between glycol chitosan (GC) and phenylboronic acid (PBA). GC-PBA derivatives form stable micellar structures under physiological conditions and respond to changes in glucose concentration. Once the micelles accumulate at the tumor site, intracellular glucose capture occurs, and the resultant energy deprivation through the inhibition of aerobic glycolysis remarkably suppresses tumor growth without significant side effects in vivo. This strategy highlights the need to develop safe and effective cancer treatment without the use of conventional anticancer drugs.

Tumor-Specific Monomethyl Auristatin E (MMAE) Prodrug Nanoparticles for Safe and Effective Chemotherapy.

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.

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.

Preclinical development of carrier-free prodrug nanoparticles for enhanced antitumor therapeutic potential with less toxicity.

BACKGROUND: Nanomedicine has emerged as a promising strategy for cancer treatment. The most representative nanomedicine used in clinic is PEGylated liposomal doxorubicin DOXIL®, which is first FDA-approved nanomedicine. However, several shortcomings, such as low drug loading capacity, low tumor targeting, difficulty in mass production and potential toxicity of carrier materials, have hindered the successful clinical translation of nanomedicines. In this study, we report a preclinical development process of the carrier-free prodrug nanoparticles designed as an alternative formulation to overcome limitations of conventional nanomedicines in the terms of technical- and industrial-aspects. RESULTS: The carrier-free prodrug nanoparticles (F68-FDOX) are prepared by self-assembly of cathepsin B-specific cleavable peptide (FRRG) and doxorubicin (DOX) conjugates without any additional carrier materials, and further stabilized with Pluronic F68, resulting in high drug loading (> 50%). The precise and concise structure allow mass production with easily controllable quality control (QC), and its lyophilized powder form has a great long-term storage stability at different temperatures (- 4, 37 and 60 °C). With high cathepsin B-specificity, F68-FDOX induce a potent cytotoxicity preferentially in cancer cells, whereas their cytotoxicity is greatly minimized in normal cells with innately low cathepsin B expression. In tumor models, F68-FDOX efficiently accumulates within tumor tissues owing to enhanced permeability and retention (EPR) effect and subsequently release toxic DOX molecules by cathepsin B-specific cleavage mechanism, showing a broad therapeutic spectrum with significant antitumor activity in three types of colon, breast and pancreatic cancers. Finally, the safety of F68-FDOX treatment is investigated after single-/multi-dosage into mice, showing greatly minimized DOX-related toxicity, compared to free DOX in normal mice. CONCLUSIONS: Collectively, these results provide potential preclinical development process of an alternative approach, new formulation of carrier-free prodrug nanoparticles, for clinical translation of nanomedicines.

Liposome-mediated PD-L1 multivalent binding promotes the lysosomal degradation of PD-L1 for T cell-mediated antitumor immunity.

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.

Implantable micro-scale LED device guided photodynamic therapy to potentiate antitumor immunity with mild visible light.

BACKGROUND: Photodynamic therapy (PDT) is a promising strategy to promote antitumor immunity by inducing immunogenic cell death (ICD) in tumor cells. However, practical PDT uses an intense visible light owing to the shallow penetration depth of the light, resulting in immunosuppression at the tumor tissues. METHODS: Herein, we propose an implantable micro-scale light-emitting diode device (micro-LED) guided PDT that enables the on-demand light activation of photosensitizers deep in the body to potentiate antitumor immunity with mild visible light. RESULTS: The micro-LED is prepared by stacking one to four micro-scale LEDs (100 µm) on a needle-shape photonic device, which can be directly implanted into the core part of the tumor tissue. The photonic device with four LEDs efficiently elicits sufficient light output powers without thermal degradation and promotes reactive oxygen species (ROS) from a photosensitizer (verteporfin; VPF). After the intravenous injection of VPF in colon tumor-bearing mice, the tumor tissues are irradiated with optimal light intensity using an implanted micro-LED. While tumor tissues under intense visible light causes immunosuppression by severe inflammatory responses and regulatory T cell activation, mild visible light elicits potent ICD in tumor cells, which promotes dendritic cell (DC) maturation and T cell activation. The enhanced therapeutic efficacy and antitumor immunity by micro-LED guided PDT with mild visible light are assessed in colon tumor models. Finally, micro-LED guided PDT in combination with immune checkpoint blockade leads to 100% complete tumor regression and also establishes systemic immunological memory to prevent the recurrence of tumors. CONCLUSION: Collectively, this study demonstrates that micro-LED guided PDT with mild visible light is a promising strategy for cancer immunotherapy.