Reactive oxygen species-responsive dual-targeted nanosystem promoted immunogenic cell death against breast cancer.

The development of an optimal treatment modality to improve the therapeutic outcome of breast cancer patients is still difficult. Poor antigen presentation to T cells is a major challenge in cancer immunotherapy. In this study, a synergistic immunotherapy strategy for breast cancer incorporating immune cell infiltration, immunogenic cell death (ICD), and dendritic cell (DC) maturation through a reactive oxygen species (ROS)-responsive dual-targeted smart nanosystem (anti-PD-L1-TKNP) for the simultaneous release of DOX, R848, and MIP-3α in the tumor microenvironment is reported. Following local injection, anti-PD-L1-DOX-R848-MIP-3α/thioketal nanoparticle (TKNP) converts tumor cells to a vaccine owing to the combinatorial effect of DOX-induced ICD, R848-mediated immunostimulatory properties, and MIP-3α-induced immune cell recruitment in the tumor microenvironment. Intratumoral injection of anti-PD-L1-DOX-R848-MIP-3α/TKNP caused significant regression of breast cancer. Mechanistic studies reveal that anti-PD-L1-DOX-R848-MIP-3α/TKNP specifically targets tumor tissue, resulting in maximum exposure of calreticulin (CRT) and HMGB1 in tumors, and significantly enhances intratumoral infiltration of CD4+ and CD8+ T cells in tumors. Therefore, a combined strategy using dual-targeted ROS-responsive TKNP highlights the significant application of nanoparticles in modulating the tumor microenvironment and could be a clinical treatment strategy for effective breast cancer management.

Dual receptor specific nanoparticles targeting EGFR and PD-L1 for enhanced delivery of docetaxel in cancer therapy.

Dual-receptor targeted (DRT) nanoparticles which contain two distinct targeting agents may exhibit higher cell selectivity, cellular uptake, and cytotoxicity toward cancer cells than single-ligand targeted nanoparticle systems without additional functionality. The purpose of this study is to prepare DRT poly(lactic-co-glycolic acid) (PLGA) nanoparticles for targeting the delivery of docetaxel (DTX) to the EGFR and PD-L1 receptor positive cancer cells such as human glioblastoma multiform (U87-MG) and human non-small cell lung cancer (A549) cell lines. Anti-EGFR and anti-PD-L1 antibody were decorated on DTX loaded PLGA nanoparticles to prepare DRT-DTX-PLGA via. single emulsion solvent evaporation method. Physicochemical characterizations of DRT-DTX-PLGA, such as particle size, zeta-potential, morphology, and in vitro DTX release were also evaluated. The average particle size of DRT-DTX-PLGA was 124.2 ± 1.1 nm with spherical and smooth morphology. In the cellular uptake study, the DRT-DTX-PLGA endocytosed by the U87-MG and A549 cells was single ligand targeting nanoparticle. From the in vitro cell cytotoxicity, and apoptosis studies, we reported that DRT-DTX-PLGA exhibited high cytotoxicity and enhanced the apoptotic cell compared to the single ligand-targeted nanoparticle. The dual receptor mediated endocytosis of DRT-DTX-PLGA showed a high binding affinity effect that leads to high intracellular DTX concentration and exhibited high cytotoxic properties. Thus, DRT nanoparticles have the potential to improve cancer therapy by providing selectivity over single-ligand-targeted nanoparticles.

T-cell engaging poly(lactic-co-glycolic acid) nanoparticles as a modular platform to induce a potent cytotoxic immunogenic response against PD-L1 overexpressing cancer.

Bispecific nanoparticles (NPs) are conjugated with two antibodies that enhance T cell cytotoxicity by sequentially targeting CD3 and tumor-specific proteins. This interaction redirects T cells to specific tumor antigens and activates them to lyse tumor cells by blocking two different signaling pathways simultaneously. This study developed NP-based bispecific T-cell engagers (nanoBiTEs), which are R848-loaded bispecific poly(lactic-co-glycolic acid) NPs decorated with anti-CD3 antibody targeting T cells and anti-PD-L1 antibody targeting PD-L1 ligands (bis-R848-PLGA-NPs). Bis-R848-PLGA-NPs enhance the immunogenic response in destroying cancer cells by restoring the T cell effector functions. These interactions allow T cells to come in close proximity to the tumor cells. Finally, the release of R848 from PLGA-NPs activates dendritic cells, enhancing T cell activation. In vitro results show maximum internalization of bis-R848-PLGA-NPs in SK-OV3 and B16F10 cell lines, attributed to high PD-L1 expression in both cells. Furthermore, bis-R848-PLGA-NPs-treated CD8+ T cells exhibit a significantly increased total amount of CD8+/CD25+, CD8+/CD69+, and cytokine expression that leads to the robust inhibition of PD-L1 expressed cancer cells. Additionally, tumor growth is significantly inhibited by bis-R848-PLGA-NPs in the B16F10 xenograft mouse model and significantly enhanced intratumoral infiltration of CD4+ and CD8+ T cells, as well as tumor-infiltrated cytokines.

Targeting Cancer Stem Cells: Therapeutic and diagnostic strategies by the virtue of nanoparticles.

Cancer stem cells (CSCs) are the subpopulation of cells present within a tumor with the properties of self-renewing, differentiating, and proliferating. Owing to the presence of ATP-binding cassette drug pumps and increased expression of anti-apoptotic proteins, the conventional chemotherapeutic agents have failed to eliminate CSCs resulting in relapse and resistance of cancer. Therefore, to obtain long-lasting clinical responses and avoid the recurrence of cancer, it is crucial to develop an efficient strategy targeting CSCs by either employing a differentiation therapy or specifically delivering drugs to CSCs. Several intracellular and extracellular cancer specific biomarkers are overexpressed by CSCs and are utilized as targets for the development of new approaches in the diagnosis and treatment of CSCs. Moreover, several nanostructured particles, alone or in combination with current treatment approaches, have been used to improve the detection, imaging, and targeting of CSCs, thus addressing the limitations of cancer therapies. Targeting CSC surface markers, stemness-related signaling pathways, and tumor microenvironmental signals has improved the detection and eradication of CSCs and, therefore, tumor diagnosis and treatment. This review summarizes a variety of promising nanoparticles targeting the surface biomarkers of CSCs for the detection and eradication of tumor-initiating stem cells, used in combination with other treatment regimens.

Recent progress in stimuli-responsive nanosystems for inducing immunogenic cell death.

Low immunogenicity and immunosuppressive tumor microenvironments are major hurdles in the application of cancer immunotherapy. To date, several immunogenic cell death (ICD) inducers have been reported to boost cancer immunotherapy by triggering ICD. ICD is characterized by the release of proinflammatory cytokines, danger-associated molecular patterns (DAMPs) and tumor associated antigens which will generate anticancer immunity by triggering adaptive immune cells. However, application of ICD inducers is limited due to severe toxicity issues and inefficient localization in the tumor microenvironment. To circumvent these challenges, stimuli-responsive nanoparticles have been exploited for improving cancer immunotherapy by limiting its toxicity. The combination of stimuli-responsive nanoparticles with an ICD inducer serves as a promising strategy for increasing the clinical applications of ICD induction in cancer immunotherapy. Here, we outline recent advances in ICD mediated by stimuli-responsive nanoparticles that may be near-infrared (NIR)-responsive, pH-responsive, redox responsive, pH and enzyme responsive, or pH and redox responsive, and evaluate their significant potential for successful clinical translation in cancer immunotherapy.

Hypoxia-Mediated ROS Amplification Triggers Mitochondria-Mediated Apoptotic Cell Death via PD-L1/ROS-Responsive, Dual-Targeted, Drug-Laden Thioketal Nanoparticles.

Amalgamation of the reactive oxygen species (ROS)-responsive stimulus with nanoparticles has gained considerable interest owing to their high tumor specificity. Hypoxia plays a pivotal role in the acceleration of intracellular ROS production. Herein, we report the construction of a cancer cell (PD-L1)- and ROS-responsive, dual-targeted, temozolomide (TMZ)-laden nanosystem which offers a better anticancer effect in a hypoxic tumor microenvironment. A dual-targeted system boosted permeation in the cancer cells. Hypoxic conditions elevating the high ROS level accelerated the in situ release of TMZ from anti-PD-L1-TKNPs. Hyperaccumulated ROS engendered from TMZ caused oxidative damage leading to mitochondria-mediated apoptosis. TMZ fabricated in the multifunctional nanosystem (anti-PD-L1-TMZ-TKNPs) provided excellent tumor accumulation and retarded tumor growth under in vivo conditions. The elevated apoptosis effect with the activation of an apoptotic marker, DNA double-strand breakage marker, and downregulation of the angiogenesis marker in the tumor tissue following treatment with anti-PD-L1-TMZ-TKNPs exerts robust anticancer effect. Collectively, the nanoconstruct offers deep tumor permeation and high drug release and broadens the application of the ROS-responsive nanosystem for a successful anticancer effect.

Redox/photo dual-responsive, self-targeted, and photosensitizer-laden bismuth sulfide nanourchins for combination therapy in cancer.

Targeted and stimuli-sensitive nanobombs for the release of therapeutic agents after laser irradiation of the tumor site are gaining widespread attention as personalized anticancer regimens. In this study, redox and photo dual-responsive, folate receptor-targeted nanourchin carriers for chemo-, photodynamic, and photothermal therapy were constructed by the amalgamation of an outer layer of polyethylene glycol (PEG)-S-S-methotrexate (MTX) and an inner core of indocyanine green (ICG)-loaded bismuth sulfide (Bi2S3) nanoparticles for cancer treatment. MTX introduces the carrier to folate receptors resulting in the internalization of nanoparticles into cancer cells, specifically and increasingly. In the reducing environment inside cancer cells, MTX was cleaved, resulting in a burst release that effectively inhibited tumor growth. Simultaneously, the fusion of Bi2S3 and ICG in the inner core absorbed energy from a near-infrared radiation (NIR) laser to generate heat and reactive oxygen species, which further ablated the tumors and synergistically enhanced the anticancer activity of MTX. These results indicate the successful preparation of combined nanourchins (NUs) showing GSH-induced and laser-responsive release of MTX and ICG, accompanied by hyperthermia via Bi2S3 and ICG. Effective in vitro cellular internalization, cellular cytotoxicity, and pro-apoptotic behavior of the nanosystem were achieved through a targeting, redox, and NIR-responsive combination strategy. In vivo biodistribution and photothermal imaging also revealed tumor-selective and -retentive, as well as thermally responsive attributes. Ultimately, this in vivo antitumor study shows an effective tumor ablation by these nanourchins without affecting the vital organs. Our findings indicate that using these targeted redox- and laser-responsive combination therapeutic carriers can be a promising strategy in folate receptor-expressing tumors.

Localized therapy using anti-PD-L1 anchored and NIR-responsive hollow gold nanoshell (HGNS) loaded with doxorubicin (DOX) for the treatment of locally advanced melanoma.

Drug resistance and inefficient localization of chemotherapeutic agent limit the current treatment strategy in locally advanced melanoma (MEL), accounting to the 10-year survival rate from 24% to 68%. In this study we constructed anti-PD-L1 conjugated and doxorubicin loaded hollow gold nanoshell (T-HGNS-DOX) for targeted and localized chemo-photothermal therapy of MEL by the conjugation of LA-PEG-anti-PD-L1 antibody and short PEG chain on the surface of HGNS-DOX. Near infrared (NIR) as well as pH dependent drug release profile was observed. Significant uptake of DOX following NIR due to high PD-L1 receptors resulted in pronounced anticancer effect of T-HGNS-DOX. Following intratumoral administration, maximum nanoparticles retention with the significant reduction in tumor growth was observed as a result of elevated apoptosis marker (cleaved caspase-3, cleaved PARP) as well as downregulation of proliferative (Ki-67) and angiogenesis marker (CD31). Cumulatively, our system avoids the systemic toxicities of the nanosystem thereby providing maximum chemotherapeutic retention in tumor.

Macrophage-Membrane-Camouflaged Disintegrable and Excretable Nanoconstruct for Deep Tumor Penetration.

The consolidation of nanovectors with biological membranes has recently been a subject of interest owing to the prolonged systemic circulation time and delayed clearance by the reticuloendothelial system of such systems. Among the different biomembranes, the macrophage membrane has a similar systemic circulation time, with an additional chemotactic aptitude, targeting integrin proteins. In this study, we aimed to establish a laser-activated, disintegrable, and deeply tumor-penetrative nanoplatform. We used a highly tumor-ablative and laser-responsive disintegrable copper sulfide nanoparticle, loaded it with paclitaxel, and camouflaged it with the macrophage membrane for the fabrication of PTX@CuS@MMNPs. The in vitro paclitaxel release profile was favorable for release in the tumor microenvironment, and the release was accelerated after laser exposure. Cellular internalization was improved by membrane encapsulation. Cellular uptake, cytotoxicity, reactive oxygen species generation, and apoptosis induction of PTX@CuS@MMNPs were further improved upon laser exposure, and boosted permeation was achieved by co-administration of the tumor-penetrating peptide iRGD. In vivo tumor accumulation, tumor inhibition rate, and apoptotic marker expression induced by PTX@CuS@MMNPs were significantly improved by laser irradiation and iRGD co-administration. PTX@CuS@MMNPs induced downregulation of cellular proliferation and angiogenic markers but no significant changes in body weight, survival, or significant toxicities in vital organs after laser exposure, suggesting their biocompatibility. The disintegrability of the nanosystem, accredited to biodegradability, favored efficient elimination from the body. In conclusion, PTX@CuS@MMNPs showed promising traits in combination therapies for excellent tumor eradication.

Enhanced Caspase-Mediated Abrogation of Autophagy by Temozolomide-Loaded and Panitumumab-Conjugated Poly(lactic-co-glycolic acid) Nanoparticles in Epidermal Growth Factor Receptor Overexpressing Glioblastoma Cells.

The mechanism of cell death has attracted a great deal of research interest in the design of antitumor therapy in recent days. Several attempts have been carried out in this direction and in our study also, we studied this phenomenon with the design of panitumumab (PmAb)-conjugated and temozolomide (TMZ)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs), termed PmAb-TMZ-PLGA-NPs. First, PmAb was functionalized on the surface of TMZ-PLGA-NPs using ethyl(dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS) chemistry. Targeted PLGA-NPs significantly enhanced the cellular uptake of nanoparticles in the U-87 MG cell line as a result of the high epidermal growth factor receptor (EGFR) expression, compared to the LN229 cell line. Our study demonstrated that following the treatment of PmAb-TMZ-PLGA-NPs, a more pronounced anticancer effect was noticed in comparison with free TMZ and TMZ-PLGA-NPs. Further, a more pronounced cytotoxic effect of PmAb-TMZ-PLGA-NPs was observed in the high EGFR-overexpressed glioblastoma multiforme (GBM) model (U-87 MG) cell line compared to the low EGFR GBM model (LN229). Our study demonstrated that the treatment of PmAb-TMZ-PLGA-NPs in GBM tried to adopt the autophagic pathway of the cell survival mechanism with the elevated level of autophagic marker (Beclin-1 and LC3B) at 24 h time point, thereby suppressing the expression of caspase-9 and PARP. However, at the 48 h time point, the elevated expression of caspase-9 and PARP with the downregulation of Beclin-1 and LC3B, following the treatment of PmAb-TMZ-PLGA-NPs in the GBM model, suggested that apoptotic cell death was superior over autophagic cell survival. It was also noteworthy the activation of caspase-9 was correlated with the continuous overproduction of reactive oxygen species up to a 48 h time point after the treatment of PmAb-TMZ-PLGA-NPs. This result sheds light on the biological effect of targeted chemotherapy and illustrates that PmAb-TMZ-PLGA-NPs could be applied for EGFR-overexpressed different cancer models.

Hyaluronic acid wreathed, trio-stimuli receptive and on-demand triggerable nanoconstruct for anchored combinatorial cancer therapy.

Hyaluronic acid (HA) assisted effective internalization into CD44 receptor-overexpressing cancer cells, which could offer an excellent cytotoxic profile and tumor alterations. In this study, duo-photothermal agents (copper sulfide (CuS) and graphene oxide (GO)), chemotherapeutic drug (doxorubicin (DOX)), and targeting moiety (HA) were incorporated into a complexed nanoconstruct for trio-responsive chemo-phototherapy. The nanosystem (CuS(DOX)-GO-HA) was demonstrating its responsive drug release and escalated photothermal behavior. The hyperthermia and photodynamic effect were observed along with efficient ROS generation in the presence of dual photosensitizers. The in vivo biodistribution and photothermal profile reflected a high accumulation and retention of the nanoconstruct in the tumor. Importantly, nanoconstructs effectively inhibit tumor growth based on tumor volume analysis and the altered expression of apoptosis, cell proliferation, and angiogenesis markers. Collectively, these findings suggest that this nanoconstruct has excellent antitumor effects in CD44 overexpressed cells showing the potential for clinical translation in the future.

Immunoadjuvants for cancer immunotherapy: A review of recent developments.

Cancer immunotherapy evolved as a new treatment modality to eradicate tumor cells and has gained in popularity after its successful clinical transition. By activating antigen-presenting cells (APCs), and thus, inducing innate or adaptive immune responses, immunoadjuvants have become promising tools for cancer immunotherapy. Different types of immunoadjuvants such as toll-like receptor (TLR) agonists, exosomes, and metallic and plant-derived immunoadjuvants have been studied for their immunological effects. However, the clinical use of immunoadjuvants is limited by short response rates and various side-effects. The rapid progress made in the development of nanoparticle systems as immunoadjuvant carrier vehicles has provided potential carriers for cancer immunotherapy. In this review article, we describe different types of immunoadjuvants, their limitations, modes of action, and the reasons for their clinical adoption. In addition, we review recent progress made in the nanoparticle-based immunoadjuvant field and on the combined use of nanoparticle-based immunoadjuvants and chemotherapy, phototherapy, radiation therapy, and immune checkpoint inhibitor-based therapy. STATEMENT OF SIGNIFICANCE: Cancer immunotherapy emerged as a new hope for treating malignant tumors. Different types of immunoadjuvants serve as an important tool for cancer immunotherapy by activating an innate or adaptive immune response. Limitation of free immunoadjuvant has paved the path for the development of nanoparticle-based immunoadjuvant therapy with the hope of prolonging the therapeutic efficacy. This review highlights the recent advancement made in nanoparticle-based immunoadjuvant therapy in modulating the adaptive and innate immune system. The application of the combinatorial approach of chemotherapy, phototherapy, radiation therapy adds synergy in nanoparticle-based immunoadjuvant therapy. It will broaden the reader's understanding on the recent progress made in immunotherapy with the aid of immunoadjuvant-based nanosystem.

Polydopamine-tailored paclitaxel-loaded polymeric microspheres with adhered NIR-controllable gold nanoparticles for chemo-phototherapy of pancreatic cancer.

Chemotherapeutic drugs often used as a first-line treatment of pancreatic cancer (PC) exhibit challenges due to resistance development, lack of selectivity, and tumor heterogeneity. Currently, combination chemo-photothermal therapy is known to enhance the therapeutic efficacy of chemotherapeutic drugs in PC. In this study, we develop adherent gold nanoparticles (GNPs) and paclitaxel (PTX)-loaded PLGA microspheres for the treatment of PC. Polydopamine (pD) was used as a linker to adhere GNPs to the surface of PLGA-Ms and characterized using TEM. Short-term cytotoxicity of GNPs-pD-PTX-PLGA-Ms with or without NIR treatment was evaluated using CCK-8 assays. ROS and western blot assay were performed to determine the intensity of ROS following the treatment of GNPs-pD-PTX-PLGA-Ms with or without NIR in Panc-1 cell line. Successful adhesion of GNPs on the microspheres was confirmed by TEM. CCK-8 assay revealed that GNPs-pD-PTX-PLGA-Ms with NIR showed three-fold higher cytotoxicity, compared to the group without NIR. Furthermore, ROS and western blot assay suggest that GNPs-pD-PTX-PLGA-Ms with NIR showed more ROS generation, followed by downregulation of the expression levels of antioxidant enzyme (SOD2 and CATALASE). These results suggest that the GNPs-pD-PTX-PLGA-Ms in combination with NIR irradiation can provide a synergistic chemo-photothermal therapy for the treatment of PC.

Doxorubicin and Anti-PD-L1 Antibody Conjugated Gold Nanoparticles for Colorectal Cancer Photochemotherapy.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. The prognosis and overall survival of CRC are known to be significantly correlated with the overexpression of PD-L1. Since combination therapies can significantly improve therapeutic efficacy, we constructed doxorubicin (DOX) conjugated and anti-PD-L1 targeting gold nanoparticles (PD-L1-AuNP-DOX) for the targeted chemo-photothermal therapy of CRC. DOX and anti-PD-L1 antibody were conjugated to the α-terminal end group of lipoic acid polyethylene glycol N-hydroxysuccinimide (LA-PEG-NHS) using an amide linkage, and PD-L1-AuNP-DOX was constructed by linking LA-PEG-DOX, LA-PEG-PD-L1, and a short PEG chain on the surface of AuNP using thiol-Au covalent bonds. Physicochemical characterizations and biological studies of PD-L1-AuNP-DOX were performed in the presence of near-infrared (NIR) irradiation (biologic studies were conducted using cellular uptake, apoptosis, and cell cycle assays in CT-26 cells). PD-L1-AuNP-DOX (40.0 ± 3.1 nm) was successfully constructed and facilitated the efficient intracellular uptake of DOX as evidenced by pronounced apoptotic effects (66.0%) in CT-26 cells. PD-L1-AuNP-DOX treatment plus NIR irradiation significantly and synergistically suppressed the in vitro proliferation of CT-26 cells by increasing apoptosis and cell cycle arrest. The study demonstrates that PD-L1-AuNP-DOX in combination with synergistic targeted chemo-photothermal therapy has a considerable potential for the treatment of localized CRC.