Nano-sensitizer with self-amplified drug release and hypoxia normalization properties potentiates efficient chemoradiotherapy of pancreatic cancer.

The hypoxic nature of pancreatic cancer, one of the most lethal malignancies worldwide, significantly impedes the effectiveness of chemoradiotherapy. Although the development of oxygen carriers and hypoxic sensitizers has shown promise in overcoming tumor hypoxia. The heterogeneity of hypoxia-primarily caused by limited oxygen penetration-has posed challenges. In this study, we designed a hypoxia-responsive nano-sensitizer by co-loading tirapazamine (TPZ), KP372-1, and MK-2206 in a metronidazole-modified polymeric vesicle. This nano-sensitizer relies on efficient endogenous NAD(P)H quinone oxidoreductase 1-mediated redox cycling induced by KP372-1, continuously consuming periphery oxygen and achieving evenly distributed hypoxia. Consequently, the normalized tumor microenvironment facilitates the self-amplified release and activation of TPZ without requiring deep penetration. The activated TPZ and metronidazole further sensitize radiotherapy, significantly reducing the radiation dose needed for extensive cell damage. Additionally, the coloaded MK-2206 complements inhibition of therapeutic resistance caused by Akt activation, synergistically enhancing the hypoxic chemoradiotherapy. This successful hypoxia normalization strategy not only overcomes hypoxia resistance in pancreatic cancer but also provides a potential universal approach to sensitize hypoxic tumor chemoradiotherapy by reshaping the hypoxic distribution.

Sonodynamic and sonomechanical effect on cellular stemness and extracellular physicochemical environment to potentiate chemotherapy.

BACKGROUND: Hypoxia-activated prodrug (HAP) is a promising candidate for highly tumor-specific chemotherapy. However, the oxygenation heterogeneity and dense extracellular matrix (ECM) of tumor, as well as the potential resistance to chemotherapy, have severely impeded the resulting overall efficacy of HAP. RESULTS: A HAP potentiating strategy is proposed based on ultrasound responsive nanodroplets (PTP@PLGA), which is composed of protoporphyrin (PpIX), perfluoropropane (PFP) and a typical HAP, tirapazamine (TPZ). The intense vaporization of PFP upon ultrasound irradiation can magnify the sonomechanical effect, which loosens the ECM to promote the penetration of TPZ into the deep hypoxic region. Meanwhile, the PpIX enabled sonodynamic effect can further reduce the oxygen level, thus activating the TPZ in the relatively normoxic region as well. Surprisingly, abovementioned ultrasound effect also results in the downregulation of the stemness of cancer cells, which is highly associated with drug-refractoriness. CONCLUSIONS: This work manifests an ideal example of ultrasound-based nanotechnology for potentiating HAP and also reveals the potential acoustic effect of intervening cancer stem-like cells.

Novel carbamodithioate regulates cellular hypoxia through chemical activation of prolyl hydroxylase-2 for breast cancer chemoprevention.

Inhibition of prolylhydroxylase-2 (PHD-2) in both normoxic and hypoxic cells is a critical component of solid tumours. The present study aimed to identify small molecules with PHD-2 activation potential. Virtually screening 4342 chemical compounds for structural similarity to R59949 and docking with PHD-2. To find the best drug candidate, hits were assessed for drug likeliness, antihypoxic and antineoplastic potential. The selected drug candidate's PHD-2 activation, cytotoxic and apoptotic potentials were assessed using 2-oxoglutarate, MTT, AO/EtBr and JC-1 staining. The drug candidate was also tested for its in-vivo chemopreventive efficacy against DMBA-induced mammary gland cancer alone and in combination with Tirapazamine (TPZ). Virtual screening and 2-oxoglutarate assay showed BBAP-6 as lead compound. BBAP-6 exhibited cytotoxic and apoptotic activity against ER+ MCF-7. In carmine staining and histology, BBAP-6 alone or in combination with TPZ restored normal surface morphology of the mammary gland after DMBA produced malignant alterations. Immunoblotting revealed that BBAP-6 reduced NF-κB expression, activated PHD-2 and induced intrinsic apoptotic pathway. Serum metabolomics conducted with 1H NMR confirmed that BBAP-6 prevented HIF-1α and NF-κB-induced metabolic changes in DMBA mammary gland cancer model. In a nutshell, it can be concluded that BBAP-6 activates PHD-2 and exhibits anticancer potential.

Co-delivery of Plinabulin and Tirapazamine boosts anti-tumor efficacy by simultaneously destroying tumor blood vessels and killing tumor cells.

It is imperative to optimize chemotherapy for heightened anti-tumor therapeutic efficacy. Unrestrained tumor cell proliferation and sustained angiogenesis are pivotal for cancer progression. Plinabulin, a vascular disrupting agent, selectively destroys tumor blood vessels. Tirapazamine (TPZ), a hypoxia-activated prodrug, intensifies cytotoxicity in diminishing oxygen levels within tumor cells. Despite completing Phase III clinical trials, both agents exhibited modest treatment efficiency due to dose-limiting toxicity. In this study, we employed methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-b-PDLLA) to co-deliver Plinabulin and TPZ to the tumor site, concurrently disrupting blood vessels and eliminating tumor cells, addressing both symptoms and the root cause of tumor progression. Plinabulin was converted into a prodrug with esterase response (PSM), and TPZ was synthesized into a hexyl chain-containing derivative (TPZHex) for effective co-delivery. PSM and TPZHex were co-encapsulated with mPEG-b-PDLLA, forming nanodrugs (PT-NPs). At the tumor site, PT-NPs responded to esterase overexpression, releasing Plinabulin, disrupting blood vessels, and causing nutritional and oxygen deficiency. TPZHex was activated in response to increased hypoxia, killing tumor cells. In treating 4T1 tumors, PT-NPs demonstrated enhanced therapeutic efficacy, achieving a 92.9 % tumor suppression rate and a 20 % cure rate. This research presented an innovative strategy to enhance synergistic efficacy and reduce toxicity in combination chemotherapy.

H2S-driven chemotherapy and mild photothermal therapy induced mitochondrial reprogramming to promote cuproptosis.

The elevated level of hydrogen sulfide (H2S) in colon cancer hinders complete cure with a single therapy. However, excessive H2S also offers a treatment target. A multifunctional cascade bioreactor based on the H2S-responsive mesoporous Cu2Cl(OH)3-loaded hypoxic prodrug tirapazamine (TPZ), in which the outer layer was coated with hyaluronic acid (HA) to form TPZ@Cu2Cl(OH)3-HA (TCuH) nanoparticles (NPs), demonstrated a synergistic antitumor effect through combining the H2S-driven cuproptosis and mild photothermal therapy. The HA coating endowed the NPs with targeting delivery to enhance drug accumulation in the tumor tissue. The presence of both the high level of H2S and the near-infrared II (NIR II) irradiation achieved the in situ generation of photothermic agent copper sulfide (Cu9S8) from the TCuH, followed with the release of TPZ. The depletion of H2S stimulated consumption of oxygen, resulting in hypoxic state and mitochondrial reprogramming. The hypoxic state activated prodrug TPZ to activated TPZ (TPZ-ed) for chemotherapy in turn. Furthermore, the exacerbated hypoxia inhibited the synthesis of adenosine triphosphate, decreasing expression of heat shock proteins and subsequently improving the photothermal therapy. The enriched Cu2+ induced not only cuproptosis by promoting lipoacylated dihydrolipoamide S-acetyltransferase (DLAT) heteromerization but also performed chemodynamic therapy though catalyzing H2O2 to produce highly toxic hydroxyl radicals ·OH. Therefore, the nanoparticles TCuH offer a versatile platform to exert copper-related synergistic antitumor therapy.

Glutathione-Responsive Polymersome with Continuous Glutathione Depletion for Enhanced Photodynamic Therapy and Hypoxia-Activated Chemotherapy.

The high glutathione (GSH) level of the tumor microenvironment severely affects the efficacy of photodynamic therapy (PDT). The current GSH depletion strategies have difficulty meeting the dual needs of security and efficiency. In this study, we report a photosensitizer Chlorin e6 (Ce6) and hypoxia-activated prodrug tirapazamine (TPZ) coloaded cross-linked multifunctional polymersome (TPZ/Ce6@SSPS) with GSH-triggered continuous GSH depletion for enhanced photodynamic therapy and hypoxia-activated chemotherapy. At tumor sites, the disulfide bonds of TPZ/Ce6@SSPS react with GSH to realize decross-linking for on-demand drug release. Meanwhile, the generated highly reactive quinone methide (QM) can further deplete GSH. This continuous GSH depletion will amplify tumor oxidative stress, enhancing the PDT effect of Ce6. Aggravated tumor hypoxia induced by PDT activates the prodrug TPZ, resulting in an enhanced combination of PDT and hypoxia-activated chemotherapy. Both in vitro and in vivo results demonstrate the efficient GSH depletion and potent antitumor activities by TPZ/Ce6@SSPS. This work provides a strategy for the design of a continuous GSH depletion platform, which holds great promise for enhanced combination tumor therapy.

Dissolving Microneedle-Based Cascade-Activation Nanoplatform for Enhanced Photodynamic Therapy of Skin Cancer.

Purpose: Photodynamic therapy (PDT) has been an attractive strategy for skin tumor treatment. However, the hypoxic microenvironment of solid tumors and further O2 consumption during PDT would diminish its therapeutic effect. Herein, we developed a strategy using the combination of PDT and hypoxia-activated bioreductive drug tirapazamine (TPZ). Methods: TPZ was linked to DSPE-PEG-NHS forming DSPE-PEG-TPZ to solve leakage of water-soluble TPZ and serve as an antitumor agent and monomer molecule further forming the micellar. Chlorin e6 (Ce6) was loaded in DSPE-PEG-TPZ forming DSPE-PEG-TPZ@Ce6 (DPTC). To further improve tumor infiltration and accumulation, hyaluronic acid was adopted to make DPTC-containing microneedles (DPTC-MNs). Results: Both in vitro and in vivo studies consistently demonstrated the synergistic antitumor effect of photodynamic therapy and TPZ achieved by DPTC-MNs. With laser irradiation, overexpressions of PDT tolerance factors NQO1 and HIF-1α were inhibited by this PDT process. Conclusion: The synergistic effect of PDT and TPZ significantly improved the performance of DPTC-MNs in the treatment of melanoma and cutaneous squamous cell carcinoma and has good biocompatibility.

Developing Hypoxia-Sensitive System via Designing Tumor-Targeted Fullerene-Based Photosensitizer for Multimodal Therapy of Deep Tumor.

Photodynamic therapy (PDT) has been approved for clinic. However, powerless efficiency for deep hypoxic tumor therapy remains an enormous challenge for PDT. Herein, a hypoxia-sensitive nanotherapeutic system (FTCD-SRGD) based on fullerene (C70) and anoxic activating chemical prodrug tirapazamine (TPZ) is rationally designed for multimodal therapy of deep hypoxic tumors. To enhance the accumulation and achieve specific drug release in tumor, the FTCD-SRGD is modified with cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGDfK) peptide and disulfide bonds. With the exacerbated hypoxic microenvironment created by C70 consuming O2 for generating reactive oxygen species (ROS), TPZ is activated to produce toxic radical species to ablate deep tumors, which achieves a synergistic treatment of C70-mediated PDT and hypoxia-enhanced chemotherapy. Additionally, given this hypoxia-sensitive system-induced immunogenic cell death (ICD) activating anticancer cytotoxic T lymphocyte to result in more susceptible tumor to immunotherapy, FTCD-SRGD plus immune checkpoint inhibitor (anti-PD-L1) fully inhibit deep hypoxic tumors by promoting infiltration of effector T cells in tumors. Collectively, it is the first time to develop a multimodal therapy system with fullerene-based hypoxia-sensitive PS for deep tumors. The powerful multimodal nanotherapeutic system for combining hypoxia-enhanced PDT and immunotherapy to massacre deep hypoxic tumors can provide a paradigm to combat the present bottleneck of tumor therapy.

Sorafenib-Encapsulated Liposomes to Activate Hypoxia-Sensitive Tirapazamine for Synergistic Chemotherapy of Hepatocellular Carcinoma.

Combination therapy with the synergistic effect is an effective way in cancer chemotherapy. Herein, an antiangiogenic sorafenib (SOR) and hypoxia-activated prodrug tirapazamine (TPZ)-coencapsulated liposome (LipTPZ/SOR) is prepared for chemotherapy of hepatocellular carcinoma (HCC). SOR is a multi-target tyrosine kinase inhibitor that can inhibit tumor cell proliferation and angiogenesis. The antiangiogenesis effect of SOR can reduce oxygen supply and aggravate tumor hypoxia, which is able to activate hypoxia-sensitive prodrug TPZ, exhibiting the synergistic antitumor effect. LipTPZ/SOR at different molar ratios of TPZ and SOR can significantly inhibit the proliferation of hepatocellular carcinoma cells. The mole ratio of TPZ and SOR was optimized to 2:1, which exhibited the best synergetic antitumor effect. The synergistic antitumor mechanism of SOR and TPZ was also investigated in vivo. After treated with SOR, the number of vessels was decreased, and the degree of hypoxia was aggravated in tumor tissues. What is more, in the presence of SOR, TPZ could be activated to inhibit tumor growth. The combination of TPZ and SOR exhibited an excellent synergistic antitumor effect. This research not only provides an innovative strategy to aggravate tumor hypoxia to promote TPZ activation but also paints a blueprint about a new nanochemotherapy regimen for the synergistic chemotherapy of HCC, which has excellent biosafety and bright clinical application prospects.

Photoresponsive metal-organic framework with combined photodynamic therapy and hypoxia-activated chemotherapy for the targeted treatment of rheumatoid arthritis.

Activated M1-type macrophages, which produce inflammatory factors that exacerbate rheumatoid arthritis (RA), represent crucial target cells for inhibiting the disease process. In this study, we developed a novel photoresponsive targeted drug delivery system (TPNPs-HA) that can effectively deliver the hypoxia-activated prodrug tirapazamine (TPZ) specifically to activated macrophages. After administration, this metal-organic framework, PCN-224, constructed uing the photosensitizer porphyrin, exhibits the ability to generate excessive toxic reactive oxygen species (ROS) when exposed to near-infrared light. Additionally, the oxygen-consumed hypoxic environment further activates the chemotherapeutic effect of TPZ, thus creating a synergistic combination of photodynamic therapy (PDT) and hypoxia-activated chemotherapy (HaCT) to promote the elimination of activated M1-type macrophages. The results highlight the significantly potential of this photoresponsive nano-delivery system in providing substantial relief for RA. Furthermore, these findings support its effectiveness in inhibiting the disease process of RA, thereby offering new possibilities for the development of precise and accurate strategies for RA.

A two-stage exacerbated hypoxia nanoengineering strategy induced amplifying activation of tirapazamine for microwave hyperthermia-chemotherapy of breast cancer.

Microwave hyperthermia (MH) is an emerging treatment for solid tumors, such as breast cancer, due to its advantages of minimally invasive and deep tissue penetration. However, MH induced tumor hypoxia is still an obstacle to breast tumor treatment failure. Therefore, an original nanoengineering strategy was proposed to exacerbate hypoxia in two stages, thereby amplifying the efficiency of activating tirapazamine (TPZ). And a novel microwave-sensitized nanomaterial (GdEuMOF@TPZ, GEMT) is designed. GdEuMOF (GEM) nanoparticles are certified excellent microwave (MW) sensitization performance, thus improving tumor selectivity to achieve MH. Meanwhile MW can aggravate the generation of thrombus and caused local circulatory disturbance of tumor, resulting in the Stage I exacerbated hypoxia environment passively. Due to tumor heterogeneity and uneven hypoxia, GEMT nanoparticles under microwave could actively deplete residual oxygen through the chemical reaction, exacerbating hypoxia level more evenly, thus forming the Stage II of exacerbated hypoxia environment. Consequently, a two-stage exacerbated hypoxia GEMT nanoparticles realize amplifying activation of TPZ, significantly enhance the efficacy of microwave hyperthermia and chemotherapy, and effectively inhibit breast cancer. This research provides insights into the development of progressive nanoengineering strategies for effective breast tumor therapy.

Tirapazamine combined with photodynamic therapy improves the efficacy of ABZSO nanoparticles on Echinococcosis granulosus via further enhancing "breaking-then-curing".

BACKGROUND: Photodynamic therapy (PDT) has a promising application prospect in Echinococcus granulosus (Egs), however, the hypoxic environment of Egs and the hypoxia associated with PDT will greatly limit its effects. As a hypoxic-activated pre-chemotherapeutic drug, tirapazamine (TPZ) can be only activated and produce cytotoxicity under hypoxia environment. Albendazole sulfoxide (ABZSO) is the first choice for the treatment of Egs. This study aimed to explore the effects of ABZSO nanoparticles (ABZSO NPs), TPZ combined with PDT on the activity of Egs in vitro and in vivo. METHODS: The Egs were divided into control, ABZSO NPs, ABZSO NPs + PDT, and ABZSO NPs + TPZ + PDT groups, and the viability of Egs was determined using methylene blue staining. Then, the ROS, LDH and ATP levels were measured using their corresponding assay kit, and H2AX and TopoI protein expression was detected by western blot. The morphology of Egs with different treatments was observed using hematoxylin eosin (HE) staining and scanning electron microscopy (SEM). After that, the in vivo efficacy of ABZSO NPs, TPZ and PDT on Egs was determined in a Egs infected mouse model. RESULTS: In vitro experiments showed that the combined treatment of TPZ, ABZSO NPs and PDT significantly inhibited Egs viability; and significantly increased ROS levels and LDH contents, while decreased ATP contents in Egs; as well as up-regulated H2AX and down-regulated TopoI protein expression. HE staining and SEM results showed that breaking-then-curing treatment seriously damaged the Egs wall. Additionally, in vivo experiments found that the combination of ABZSO NPs, PDT and TPZ had more serious calcification and damage of the wall structure of cysts. CONCLUSIONS: ABZSO NPs combined with TPZ and PDT has a better inhibitory effect on the growth of Egs in vitro and in vivo based on the strategy of "breaking-then-curing".

The Development of Nonthermal Plasma and Tirapazamine as a Novel Combination Therapy to Treat Melanoma In Situ.

Although melanoma accounts for only 5.3% of skin cancer, it results in >75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ.

Repurposing High-Throughput Screening Identifies Unconventional Drugs with Antibacterial and Antibiofilm Activities against Pseudomonas aeruginosa under Experimental Conditions Relevant to Cystic Fibrosis.

Pseudomonas aeruginosa is the most common pathogen infecting cystic fibrosis (CF) lungs, causing acute and chronic infections. Intrinsic and acquired antibiotic resistance allow P. aeruginosa to colonize and persist despite antibiotic treatment, making new therapeutic approaches necessary. Combining high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses for drugs. This study screened a drug library of 3,386 drugs, mostly FDA approved, to identify antimicrobials against P. aeruginosa under physicochemical conditions relevant to CF-infected lungs. Based on the antibacterial activity, assessed spectrophotometrically against the prototype RP73 strain and 10 other CF virulent strains, and the toxic potential evaluated toward CF IB3-1 bronchial epithelial cells, five potential hits were selected for further analysis: the anti-inflammatory and antioxidant ebselen, the anticancer drugs tirapazamine, carmofur, and 5-fluorouracil, and the antifungal tavaborole. A time-kill assay showed that ebselen has the potential to cause rapid and dose-dependent bactericidal activity. The antibiofilm activity was evaluated by viable cell count and crystal violet assays, revealing carmofur and 5-fluorouracil as the most active drugs in preventing biofilm formation regardless of the concentration. In contrast, tirapazamine and tavaborole were the only drugs actively dispersing preformed biofilms. Tavaborole was the most active drug against CF pathogens other than P. aeruginosa, especially against Burkholderia cepacia and Acinetobacter baumannii, while carmofur, ebselen, and tirapazamine were particularly active against Staphylococcus aureus and B. cepacia. Electron microscopy and propidium iodide uptake assay revealed that ebselen, carmofur, and tirapazamine significantly damage cell membranes, with leakage and cytoplasm loss, by increasing membrane permeability. IMPORTANCE Antibiotic resistance makes it urgent to design new strategies for treating pulmonary infections in CF patients. The repurposing approach accelerates drug discovery and development, as the drugs' general pharmacological, pharmacokinetic, and toxicological properties are already well known. In the present study, for the first time, a high-throughput compound library screening was performed under experimental conditions relevant to CF-infected lungs. Among 3,386 drugs screened, the clinically used drugs from outside infection treatment ebselen, tirapazamine, carmofur, 5-fluorouracil, and tavaborole showed, although to different extents, anti-P. aeruginosa activity against planktonic and biofilm cells and broad-spectrum activity against other CF pathogens at concentrations not toxic to bronchial epithelial cells. The mode-of-action studies revealed ebselen, carmofur, and tirapazamine targeted the cell membrane, increasing its permeability with subsequent cell lysis. These drugs are strong candidates for repurposing for treating CF lung P. aeruginosa infections.

Drug-Eluting Porous Embolic Microspheres for Trans-Arterial Delivery of Dual Synergistic Anticancer Therapy for the Treatment of Liver Cancer.

Blockage of blood supply while administering chemotherapy to tumors, using trans-arterial chemoembolization (TACE), is the most common treatment for intermediate and advanced-stage unresectable Hepatocellular carcinoma (HCC). However, HCC is characterized by a poor prognosis and high recurrence rates (≈30%), partly due to a hypoxic pro-angiogenic and pro-cancerous microenvironment. This study investigates how modifying tissue stress while improving drug exposure in target organs may maximize the therapeutic outcomes. Porous degradable polymeric microspheres (MS) are designed to obtain a gradual occlusion of the hepatic artery that nourishes the liver, while enabling efficient drug perfusion to the tumor site. The fabricated porous MS are introduced intrahepatically and designed to release a combination therapy of Doxorubicin (DOX) and Tirapazamine (TPZ), which is a hypoxia-activated prodrug. Liver cancer cell lines that are treated with the combination therapy under hypoxia reveal a synergic anti-proliferation effect. An orthotopic liver cancer model, based on N1-S1 hepatoma in rats, is used for the efficacy, biodistribution, and safety studies. Porous DOX-TPZ MS are very effective in suppressing tumor growth in rats, and induction tissue necrosis is associated with high intratumor drug concentrations. Porous particles without drugs show some advantages over nonporous particles, suggesting that morphology may affect the treatment outcomes.

Sandwich-Structured Implants to Obstruct Multipath Energy Supply and Trigger Self-Enhanced Hypoxia-Initiated Chemotherapy Against Postsurgical Tumor Recurrence and Metastasis.

As a currently common strategy to treat cancer, surgical resection may cause tumor recurrence and metastasis due to residual postoperative tumors. Herein, an implantable sandwich-structured dual-drug depot is developed to trigger a self-intensified starvation therapy and hypoxia-induced chemotherapy sequentially. The two outer layers are 3D-printed using a calcium-crosslinked mixture ink containing soy protein isolate, polyvinyl alcohol, sodium alginate, and combretastatin A4 phosphate (CA4P). The inner layer is one patch of poly (lactic-co-glycolic acid)-based electrospun fibers loaded with tirapazamine (TPZ). The preferentially released CA4P destroys the preexisting blood vessels and prevents neovascularization, which obstructs the external energy supply to cancer cells but aggravates hypoxic condition. The subsequently released TPZ is bioreduced to cytotoxic benzotriazinyl under hypoxia, further damaging DNA, generating reactive oxygen species, disrupting mitochondria, and downregulating hypoxia-inducible factor 1α, vascular endothelial growth factor, and matrix metalloproteinase 9. Together these processes induce apoptosis, block the intracellular energy supply, counteract the disadvantage of CA4P in favoring intratumor angiogenesis, and suppress tumor metastasis. The in vivo and in vitro results and the transcriptome analysis demonstrate that the postsurgical adjuvant treatment with the dual-drug-loaded sandwich-like implants efficiently inhibits tumor recurrence and metastasis, showing great potential for clinical translation.

Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.

Tirapazamine-loaded UiO-66/Cu for ultrasound-mediated promotion of chemodynamic therapy cascade hypoxia-activated anticancer therapy.

Chemodynamic therapy (CDT), an emerging oncology treatment, has received considerable attention owing to its high selectivity, less aggressiveness, and endogenous stimulation. However, the complex intra-tumor environment limits the therapeutic effect. In this study, Cu+ was directly doped into the structure of the UiO-66 matrix using an in situ one-pot oil bath method. The as-formed UiO-66/Cu possessed a large surface area, making it feasible to modify folic acid (FA) and carry more chemotherapeutic agents like tirapazamine (TPZ), thus forming UiO-66/Cu-FA-TPZ nanoplatforms. For CDT, the nanoplatform catalyzed the cyclic generation of the highly oxidizing hydroxyl radical (·OH) from H2O2. Particularly, low-frequency ultrasound enhanced the curative effect. Notably, in a tumor, a severe hypoxic environment and ultrasound can activate more TPZ for safe and efficient chemotherapy, achieving synergistic and hypoxia-activated tumor treatment with a low risk of side effects. Moreover, the nanoplatform exhibits computed tomography imaging functions for combined diagnosis and treatment. Our designed nanoplatform overcomes the dilemma of insufficient efficacy from conventional therapy attributed to a hypoxic environment, expecting to guide the design of future treatment regimens for hypoxic tumors.

A Multifunctional Nano-Delivery System Against Rheumatoid Arthritis by Combined Phototherapy, Hypoxia-Activated Chemotherapy, and RNA Interference.

Purpose: Effective therapy for rheumatoid arthritis (RA) keeps a challenge due to the complex pathogenesis of RA. It is not enough to completely inhibit the process of RA with any single therapy method. The purpose of the research is to compensate for the insufficiency of monotherapy using multiple treatment regimens with different mechanisms. Material and Methods: In this study, we developed a new method to synthesize mesoporous silica nanoparticles hybridized with photosensitizer PCPDTBT (HNs). Branched polyethyleneimine-folic acid (PEI-FA) could be coated on the surface of HNs through electrostatic interactions. It simultaneously blocked the hypoxia-activated prodrug tirapazamine loaded into the mesopores and binded with Mcl-1 siRNA (siMcl-1) that interfered with the expression of the anti-apoptotic protein Mcl-1. Released from the co-delivery nanoparticles (PFHNs/TM) Tirapazamine and siMcl-1 upon exposure to acidic conditions of endosomes/lysosomes in activated macrophages. Under NIR irradiation, photothermal therapy and photodynamic therapy derived from PCPDTBT, hypoxia-activated chemotherapy derived from tirapazamine, and RNAi derived from siMcl-1 were used for the combined treatment for RA by killing activated macrophages. PEI-FA-coated PFHNs/TM exhibited activated macrophage-targeting characteristics, thereby enhancing the in vitro and in vivo NIR-induced combined treatment of RA. Results: The prepared PFHNs/TM have high blood compatibility (far below 5% of hemolysis) and ideal in vitro phototherapy effect while controlling the TPZ release and binding siMcl-1. We prove that PEI-FA-coated PFHNs/TM not only protect the bound siRNA but also are selectively uptaked by activated macrophages through FA receptor-ligand-mediated endocytosis, and effectively silence the target anti-apoptotic protein by siMcl-1 transfection. In vivo, PFHNs/TM have also been revealed to be selectively enriched at the inflammatory site of RA, exhibiting NIR-induced anti-RA efficacy. Conclusion: Overall, these FA-functionalized, pH-responsive PFHNs/TM represent a promising platform for the co-delivery of chemical drugs and nucleic acids for the treatment of RA cooperating with NIR-induced phototherapy.

A dual-response drug delivery system with X-ray and ROS to boost the anti-tumor efficiency of TPZ via enhancement of tumor hypoxia levels.

The selective anti-tumor activity and less toxic nature of hypoxia-activated prodrugs including tirapazamine (TPZ) are harbored by hypoxia levels in tumors, the inadequacy of which leads to failure in clinical trials. Thus, the development of effective clinical applications of TPZ requires advanced strategies to intensify hypoxia levels in tumors effectively and safely. In this study, we designed and fabricated a paclitaxel (PTX)-loaded dual-response delivery system with a low dose (e.g., 2 Gy) of X-ray and reactive oxygen species on the basis of diselenide block copolymers. Upon the external X-ray stimulus, the system accurately released encapsulated PTX at tumor sites and remarkably improved tumor hypoxia levels by causing severe damage to tumor blood vessels. Subsequently, these enhanced tumor hypoxia levels effectively activated the reduction of TPZ into benzotriazinyl free radicals, which significantly improved the antitumor efficacy of our system against 4T1 breast cancer cells with an initial tumor volume of 500 mm3. Moreover, the dual-stimulus coordinated and controlled release of PTX was found to largely avoid the off-target effects of PTX on normal cells while exhibiting very limited side effects in experimental mice. The current novel strategy for regulating tumor hypoxia levels offers an effective and safe way to activate TPZ for the treatment of large solid tumors.