A Synergistic Chemoimmunotherapy System Leveraging PD-L1 Blocking and Bioorthogonal Prodrug Activation.

Novel strategies to facilitate tumor-specific drug delivery and restore immune attacks remain challenging in overcoming the current limitations of chemoimmunotherapy. An antitumor chemoimmunotherapy system comprising bioorthogonal reaction-ready group tetrazine (TZ) modified with an anti-PD-L1 antibody (αPD-L1TZ) and TZ-activatable prodrug vinyl ether-doxorubicin (DOX-VE) for self-reinforced anti-tumor chemoimmunotherapy is proposed. The αPD-L1TZ effectively disrupts the PD-L1/PD-1 interaction and activates the DOX prodrug in situ through the bioorthogonal click reaction of TZ and VE. Conversely, the activated DOX upregulates PD-L1 on the surface of tumor cells, facilitating tumor accumulation of αPD-L1TZ and enhancing DOX-VE activation. Furthermore, the activated DOX-induced immunogenic cell death of tumor cells, substantially improving the response efficiency of αPD-L1 in an immune-suppressive tumor microenvironment. Thus, PD-L1 blocking and bioorthogonal in situ prodrug activation synergistically enhance the antitumor efficacy of the chemoimmunotherapy system. Therefore, the system significantly enhances αPD-L1 tumor accumulation and prodrug activation and induces a robust immunological memory effect to prevent tumor recurrence and metastasis. Thus, a feasible chemoimmunotherapy combination regimen is presented.

Cyclic amplification of intracellular ROS boosts enzymatic prodrug activation for enhanced chemo-immunotherapy.

Tumor-associated enzyme activated prodrug is a potential strategy to overcome the limitations of chemotherapeutic agents. However, the efficiency of enzymatic prodrug activation is limited by the inability to reach adequate enzyme levels in vivo. Herein, we report an intelligent nanoplatform with cyclic amplification of intracellular reactive oxygen species (ROS) that significantly up-regulates the expression of tumor-associated enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to efficiently activate the prodrug of doxorubicin (DOX) for enhanced chemo-immunotherapy. The nanoplatform termed as CF@NDOX was fabricated by self-assembly of the amphiphilic cinnamaldehyde (CA) containing poly(thioacetal) conjugated with ferrocene (Fc) and poly(ethylene glycol) (PEG) (TK-CA-Fc-PEG), which further encapsulated the NQO1 responsive prodrug of DOX (NDOX). After CF@NDOX accumulates in tumors, the TK-CA-Fc-PEG with ROS responsive thioacetal group responds to endogenous ROS in tumor to release CA, Fc or NDOX. CA induces mitochondria dysfunction and elevates the intracellular hydrogen peroxide (H2O2) levels, which react with Fc to generate highly oxidative hydroxyl radical (•OH) through Fenton reaction. The •OH not only promotes ROS cyclic amplification but also increase the expression of NQO1 through Keap1-Nrf2 pathway regulation, which further boost the prodrug activation of NDOX for enhanced chemo-immunotherapy. Overall, our well-designed intelligent nanoplatform provides a tactic to enhance the antitumor efficacy of tumor-associated enzyme activated prodrug. STATEMENT OF SIGNIFICANCE: In this work, a smart nanoplatform CF@NDOX with intracellular ROS cyclic amplification for continuous upregulation of NQO1 enzyme expression was innovatively designed. It could utilize Fenton reaction of Fc to increase the level of NQO1 enzyme and CA to increase the level of intracellular H2O2, thereby facilitating the continuous Fenton reaction. This design allowed for a sustained elevation of the NQO1 enzyme, and a more complete activation of the NQO1 enzyme in response to the prodrug NDOX. This smart nanoplatform can achieve a desirable anti-tumor effect with the combined therapy of chemotherapy and ICD effects.

Dual stimulus-triggered bioorthogonal nanosystem for spatiotemporally controlled prodrug activation and near-infrared fluorescence imaging.

In this study, we combined low pH and cathepsin B dual-stimulus-triggered delivery carriers with a bioorthogonal reaction-activated prodrug to achieve regulated activation of the prodrug. A workable method for precise tumor therapy and imaging is provided by the bioorthogonal reaction, which activates the prodrug and fluorescent probe.

Spatial specific delivery of combinational chemotherapeutics to combat intratumoral heterogeneity.

Hypoxia-induced intratumoral heterogeneity poses a major challenge in tumor therapy due to the varying susceptibility to chemotherapy. Moreover, the spatial distribution patterns of hypoxic and normoxic tissues makes conventional combination therapy less effective. In this study, a tumor-acidity and bioorthogonal chemistry mediated in situ size transformable nanocarrier (NP@DOXDBCO plus iCPPAN3) was developed to spatially deliver two combinational chemotherapeutic drugs (doxorubicin (DOX) and PR104A) to combat hypoxia-induced intratumoral heterogeneity. DOX is highly toxic to tumor cells in normoxia state but less toxic in hypoxia state due to the hypoxia-induced chemoresistance. Meanwhile, PR104A is a hypoxia-activated prodrug has less toxic in normoxia state. Two nanocarriers, NP@DOXDBCO and iCPPAN3, can cross-link near the blood vessel extravasation sites through tumor acidity responsive bioorthogonal click chemistry to enhance the retention of DOX in tumor normoxia. Moreover, PR104A conjugated to the small-sized dendritic polyamidoamine (PAMAM) released under tumor acidity can penetrate deep tumor tissues for hypoxic tumor cell killing. Our study has demonstrated that this site-specific combination chemotherapy is better than the traditional combination chemotherapy. Therefore, spatial specific delivery of combinational therapeutics via in situ size transformable nanocarrier addresses the challenges of hypoxia induced intratumoral heterogeneity and provides insights into the combination therapy.

Corrigendum: Efficacy and Safety of Shenqu Xiaoshi Oral Liquid Compared With Domperidone Syrup in Children With Functional Dyspepsia.

[This corrects the article DOI: 10.3389/fphar.2022.831912.].

Bioorthogonal chemistry and illumination controlled programmed size-changeable nanomedicine for synergistic photodynamic and hypoxia-activated therapy.

Photodynamic therapy (PDT) can aggravate the hypoxia aggravation and be further utilized for the activation of hypoxia-activated prodrug (HAP). Ideally, photosensitizers (PSs) are mainly administrated to tumor vasculatures adjacent to regions with high oxygen to effectively generate reactive oxygen species (ROS) effectively and further aggravate tumor hypoxia, while the HAP is delivered to the inner tumor as far as possible for efficient activation. However, a delivery system capable of transporting PSs and HAP to the desired region respectively for the optimum effect is urgently needed. Here, we developed a bioorthogonal click chemistry and illumination controlled programmed size-changeable nanomedicine for synergistic photodynamic and hypoxia-activated therapy. It utilized tumor acidity responsive bioorthogonal click reaction for crosslinking nanoparticles to construct a drug depot with tumor vasculatures adjacent region retention for PDT in normoxia. Under laser illumination, cleavage of the ROS-responsive thioketal (TK) crosslinker to release small sized poly(amidoamine) (PAMAM) dendrimer conjugated with HAP for enhanced tumor penetration into the hypoxic region. Therefore, this strategy could differentially deliver PSs and HAP in desired spatial distribution, eventually achieving the enhanced synergistic enhancement in the combined PDT and hypoxia-activated therapy.

Efficacy and Safety of Shenqu Xiaoshi Oral Liquid Compared With Domperidone Syrup in Children With Functional Dyspepsia.

Background: Treatment of functional dyspepsia (FD) in children is generally symptomatic and unsatisfactory. Traditional Chinese medicines, such as Shenqu Xiaoshi Oral Liquid (SXOL), have been recommended to alleviate dyspeptic symptoms. However, evidence of their safety and efficacy remains limited to date. AIM: To assess whether 2 weeks of therapy with SXOL was non-inferior to domperidone syrup in children with FD. Methods: In this randomized, double-blind, double-simulated, non-inferiority, multi-center clinical trial, we recruited children (3-14 years) with FD according to the Rome IV criteria from 17 tertiary medical centers across China. Patients were randomly allocated (1:1) to receive SXOL or domperidone syrup for 2 weeks. We compared the participants' clinical scores from both groups based on the severity and frequency of dyspepsia symptoms according to Rome IV criteria (0, 1, 2, and 4 weeks after randomization). The primary endpoint was the total response rate, which was defined as the proportion of patients with a decrease of 30% or more in the FD symptoms clinical score from baseline, at the end of the 2-weeks treatment. A non-inferiority margin of -10% was set. Secondary endpoints and adverse events were assessed. This trial is registered with www.Chictr.org.cn, number ChiCTR1900022654. Results: Between February 2019 and March 2021, a total of 373 patients were assessed for eligibility, and 356 patients were enrolled and randomized. The clinical response rate at week two was similar for SXOL [118 (83.10%) of 142] and domperidone [128 (81.01%) of 158]; difference 2.09; 95% CI -6.74 to 10.71, thereby establishing non-inferiority. The total FD symptom scores were significantly improved in the two groups at 1-, 2-, and 4-weeks follow-up periods (p < 0.005). The decrease in symptom score compared with the baseline were similar between these two groups. Over the total study period, 10 patients experienced at least one treatment-related adverse event [six (3.37%)] in the SXOL group, four [(2.25%) in the domperidone group], although no serious adverse event was noted. Conclusion: Treatment with SXOL effectively improves dyspeptic symptoms and is well tolerated. In addition, it is not inferior to domperidone syrup and leads to sustained improvement in Chinese children with FD.

Tumor-Acidity and Bioorthogonal Chemistry-Mediated On-Site Size Transformation Clustered Nanosystem to Overcome Hypoxic Resistance and Enhance Chemoimmunotherapy.

Hypoxia, a common feature of most solid tumors, causes severe tumor resistance to chemotherapy and immunotherapy. Herein, a tumor-acidity and bioorthogonal chemistry-mediated on-site size transformation clustered nanosystem is designed to overcome hypoxic resistance and enhance chemoimmunotherapy. The nanosystem utilized the tumor-acidity responsive group poly(2-azepane ethyl methacrylate) with a rapid response rate and highly efficient bioorthogonal click chemistry to form large-sized aggregates in tumor tissue to enhance accumulation and retention. Subsequently, another tumor-acidity responsive group of the maleic acid amide with a slow response rate was cleaved allowing the aggregates to slowly dissociate into ultrasmall nanoparticles with better tumor penetration ability for the delivery of doxorubicin (DOX) and nitric oxide (NO) to a hypoxic tumor tissue. NO can reverse a hypoxia-induced DOX resistance and boost the antitumor immune response through a reprogrammed tumor immune microenvironment. This tumor-acidity and bioorthogonal chemistry-mediated on-site size transformation clustered nanosystem not only helps to counteract a hypoxia-induced chemoresistance and enhance antitumor immune responses but also provides a general drug delivery strategy for enhanced tumor accumulation and penetration.

Tumor-acidity and bioorthogonal chemistry-mediated construction and deconstruction of drug depots for ferroptosis under normoxia and hypoxia.

Mounting evidence shows that tumor hypoxia stress promotes tumor invasion and metastasis and induces therapeutic resistance. Oxygen-independent Fenton reaction, which refers to the iron-catalyzed conversion of endogenous hydrogen peroxide (H2O2) to hydroxyl radical (·OH), has been designed for ferroptosis therapy. Nevertheless, the treatment efficiency is compromised by limited H2O2 content and limited tumor retention and penetration of nanoparticles. Herein, we designed a tumor-acidity and bioorthogonal chemistry mediated construction and deconstruction of drug depots for tumor ferroptosis under normoxia and hypoxia. Briefly, the dendritic poly(amidoamine) (PAMAM, G4) was modified using cinnamaldehyde (CA) to deplete GSH and increase H2O2 levels, and ferrocene (Ferr) served as Fenton reaction catalyst to generate PFC. Subsequently, PFC was modified with maleic acid amide with slow pH-response rate and poly(2-azepane ethyl methacrylate) (PAEMA) with rapid pH-response rate, accompanied with highly efficient bioorthogonal chemistry to construct and deconstruct drug depots for enhanced tumor retention and penetration. The small-sized PFC potentially induced H2O2 self-supplied ferroptosis under normoxia and hypoxia. In sum, this work utilizes two tumoral acidity-responsive groups with different response rates and highly efficient bioorthogonal click chemistry, which paves a way for ferroptosis and provides a general drug delivery strategy with enhanced tumor retention and penetration. STATEMENT OF SIGNIFICANCE: Oxygen independent Fenton reaction refers to the conversion of endogenous H2O2 to ·OH which has been designed for ferroptosis therapy. Nevertheless, limited H2O2 level and abundant GSH in tumor cells could both compromise the treatment efficiency. Herein, we developed a tumor-acidity and bioorthogonal chemistry mediated construction and deconstruction of drug depots, which elevate the intracellular H2O2 level and deplete GSH for tumor ferroptosis under normoxia and hypoxia microenvironment. This work utilizes two tumoral acidity response groups with different response rates and highly efficient bioorthogonal click reactions, which paves a way for tumor cell ferroptosis and provides a general drug delivery strategy for enhanced tumor accumulation and penetration.

Time-programmed activation of dual polyprodrugs for synergistic cascade oxidation-chemotherapy.

Combination therapy using multiple drugs with time-programmed administration is promising for enhanced cancer treatment. However, it is still challenging to achieve time-programmed drug release from a single nanocarrier. Here, dual polyprodrugs of hemicyanine dye (CyNH2) and doxorubicin (DOX) are developed to achieve time-programmed prodrug activation for synergistic cascade oxidation therapy and chemotherapy. The polyprodrug NPDOX/Cy, composed of CyNH2, is modified with a glutathione (GSH)-responsive disulfate group, while DOX is modified with a reactive oxygen species (ROS)-response thioketal (TK) group. Upon uptake by cancer cells overexpressing GSH, CyNH2 can be activated quickly and accumulate in the mitochondria to induce mitochondrial damage and ROS upregulation, thus achieving subsequent burst activation of DOX through the ROS-triggered cleavage of the TK linker. The early activation of CyNH2 makes the cancer cells more sensitive to subsequent DOX treatment for a synergistic effect of from oxidation therapy and chemotherapy. Therefore, the polyprodrug with time-programmed drug activation developed in this work provides a promising strategy for synergistic cancer therapy.

Dual-locking nanoprobe based on hemicyanine for orthogonal stimuli-triggered precise cancer imaging and therapy.

Currently, stimulus-responsive nanomedicines are usually activated by a single cancer-associated biomarker and utilize different image/therapeutic agents for cancer imaging/therapy, which restricts the specificity of nanomedicine and complicates their design. Herein, we report a novel dual-locking theranostic nanoprobe (DL-P) based on near-infrared (NIR) hemicyanine CyNH2 with two orthogonal stimuli of cancer cell lysosomal pH (first "lock")- and lysosome-overexpressed cathepsin B (CTB, second "lock")-triggered NIR fluorescence turn-on and drug activation to improve the specificity of cancer imaging and therapy. The fluorescence of CyNH2 was initially quenched due to intramolecular charge transfer (ICT) but could be selectively activated under the dual-key stimulation of lysosomal pH and CTB to liberate CyNH2, resulting in strong NIR fluorescence turn-on for cancer imaging. Moreover, CyNH2 caused mitochondrial dysfunction to inhibit cancer cell proliferation in the absence of laser irradiation, which can be used in cancer therapy. Compared with previously reported probes that respond to a single stimulus, this dual-locking nanoprobe that is responsive to two orthogonal stimuli triggers with integrated imaging and therapy function in a single agent exhibits increased selectivity and specificity, which provides a prospective strategy for precise cancer imaging and therapy.

Theranostic Heterodimeric Prodrug with Dual-Channel Fluorescence Turn-On and Dual-Prodrug Activation for Synergistic Cancer Therapy.

Theranostic prodrugs that can precisely monitor drug activation with synergistic therapeutic effects are highly desirable for personalized medicine. In this study, a theranostic heterodimeric prodrug, CyNH-SS-DOX, with synchronous and independent dual-channel fluorescence turn-on and dual-prodrug activation for synergistic cancer therapy is developed. A hemicyanine fluorescent drug, CyNH2 , with good therapeutic effects found in this work, is conjugated to doxorubicin (DOX) through a disulfide linker to form CyNH-SS-DOX. Before activation, both the fluorescence of DOX and CyNH2 are in the off state and the toxicity is low. In the presence of intracellular glutathione, both the fluorescence of DOX and CyNH2 at different channels are turned on. Meanwhile, DOX and CyNH2 are activated in a synergistic anticancer effect. It is believed that CyNH-SS-DOX is promising for monitoring prodrug activation in dual-fluorescence channels and for enhancing therapeutic efficacy with few side effects.

Amplification of tumor oxidative stresses by Poly(disulfide acetal) for multidrug resistance reversal.

Discovering new strategies to overcome multidrug resistance (MDR) is still urgently needed. MDR is associated with the overexpression of transmembrane efflux pumps, and adenosine triphosphate (ATP) is indispensable for its function. Herein, we developed a pH- and glutathione (GSH)-responsive amphiphilic poly(disulfide acetal) (PCS) containing cinnamaldehyde (CA) and disulfide groups that amplify oxidative stress for anticancer drug delivery and simultaneously overcome drug resistance in cancer cells. Reactive oxygen species (ROS)-generating CA and the disulfide groups to deplete GSH and synergize to amplify oxidative stress in cancer cells by oxidizing nicotinamide adenine dinucleotide with hydrogen (NADH) to nicotinamide adenine dinucleotide (NAD+). The production of ATP is preferentially inhibited, leading to the malfunction of efflux pumps due to the lack of ATP and making resistant cells more impressionable to anticancer drugs. The in vitro and in vivo experiments confirmed that PCS could induce amplified oxidative stress and efficiently overcome MDR in cancer cells. We believe that the polymer with amplified oxidative stress in cancer cells holds great promise in developing polymer-based drug delivery systems to reverse MDR for cancer therapy.

An NIR-Fluorophore-Based Theranostic for Selective Initiation of Tumor Pyroptosis-Induced Immunotherapy.

Pyroptosis is an inflammatory form of programmed cell death that can effectively eliminate malignant cells and boost anticancer immunity. However, most of the current pyroptosis inducers lack cell selectivity, which may cause severe side effects for cancer therapy. In this work, for the first time, the authors discovered that the commonly used near-infrared (NIR) fluorogenic hemicyanine (CyNH2 ) induces pyrolysis to kill cancer cells and boost antitumor immunity. Cancer cells overexpressing the NAD(P)H: quinone oxidoreductase isozyme 1 (NQO1)-responsive theranostic (NCyNH2 ) are designed for selective cell pyroptosis and are nonfluorescent with low toxicity before activation. In the presence of NQO1, the fluorescence of CyNH2 is restored and can selectively initiate pyroptosis of cancer cells and further lead to systemic antitumor immunity activation for solid tumor therapy. Thus, this fluorogenic NIR dye may represent a novel theranostic agent for the selective initiation of tumor pyroptosis.

Dual-Drug Backboned Polyprodrug with a Predefined Drug Combination for Synergistic Chemotherapy.

The codelivery of drugs at specific optimal ratios to cancer cells is vital for combination chemotherapy. However, most of the current strategies are unable to coordinate the loading and release of drug combinations to acquire precise and controllable synergistic ratios. In this work, we designed an innovative dual-drug backboned and reduction-sensitive polyprodrug PEG-P(MTO-ss-CUR) containing the anticancer drugs mitoxantrone (MTO) and curcumin (CUR) at an optimal synergistic ratio to reverse drug resistance. Due to synchronous drug activation and polymer backbone degradation, drug release at the predefined ratio with a synergistic anticancer effect was demonstrated by in vitro and in vivo experiments. Therefore, the dual-drug delivery system developed in this work provides a novel and efficient strategy for combination chemotherapy.

Pig-to-baboon heterotopic heart transplantation--exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens.

BACKGROUND: Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb. METHODS: All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n=4) expressed additional CD55, and those into Group B (n=3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n=2) or abatacept (Regimen 2: n=2) or anti-CD40mAb+belatacept (Regimen 3: n=2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy. RESULTS: Group A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2. CONCLUSIONS: The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.