PH-Sensitive Multiligand Gold Nanoplatform Targeting Carbonic Anhydrase IX Enhances the Delivery of Doxorubicin to Hypoxic Tumor Spheroids and Overcomes the Hypoxia-Induced Chemoresistance.
ABSTRACT: Hypoxia is a common feature of solid tumors contributing to resistance to chemotherapy. Selective delivery of chemotherapeutic drugs to hypoxic tumor niche remains an unsolved issue. For this purpose, we constructed a gold nanoplatform targeting carbonic anhydrase IX (CA IX) epitope, which is overexpressed in hypoxic tumor cells versus in normal tissues. We designed compatible low-molecular weight carbonic anhydrase inhibitor (CAI) ligands and doxorubicin (Dox) ligands and optimized protocols for efficient decoration of gold nanoparticles (Au NPs) to achieve both good targeting ligand density and optimum drug loading, while preserving colloidal stability. The optimized Dox-HZN-DTDP@Au NPs-LA-PEG2000-CAI (THZN) nanoplatform was proved to be very efficient toward killing HT-29 tumor cells, especially under hypoxic conditions, as compared with the nontargeting nanoplatform. This also mediated the effective release of doxorubicin in the lysosomes following internalization, as revealed by confocal microscopy. Furthermore, using tumor spheroids as a representative model for hypoxic solid tumors, our THZN nanoplatform enhanced the selective delivery of doxorubicin up to 2.5 times and minimized chemoresistance, showing better tumor drug penetration as compared to that in free drug treatment. Our technology is the first CA IX-targeting gold nanoplatform for efficient delivery of doxorubicin to hypoxic tumors in a controlled fashion, with the perspective to improve the therapy of solid tumors and minimize chemoresistance.
Project description:Carbonic anhydrase (CA) IX overexpresses exclusively on cell membranes of hypoxic tumors, regulating the acidic tumor microenvironment. Small molecules of CA inhibitor modified with short peptide successfully achieve CA IX-targeted self-assembly that localizes CA inhibitors on hypoxic cancer cell surfaces and enhances their inhibition efficacy and selectivity. CA IX-related endocytosis also promotes selective intracellular uptake of these nanofibers under hypoxia, in which nanofiber structures increase in size with decreasing pH. This effect subsequently causes intracellular acid vesicle damage and blocks protective autophagy. The versatility of tunable nanostructures responding to cell milieu impressively provokes selective toxicities and provides strategic therapy for hypoxic tumors. Moreover, in vivo tests demonstrate considerable antimetastatic and antiangiogenesis effects in breast tumors, and particularly remarkable enhancement of antitumor efficacy in doxorubicin administration. With its biocompatible components and distinctive hypoxia therapies, this nanomaterial advances current chemotherapy, providing a new direction for hypoxic cancer therapy.
Project description:Drug resistance is one of the significant clinical burden in renal cell carcinoma (RCC). The development of drug resistance is attributed to many factors, including impairment of apoptosis, elevation of carbonic anhydrase IX (CA IX, a marker of tumor hypoxia), and infiltration of tumorigenic immune cells. To alleviate the drug resistance, we have used Sorafenib (Sor) in combination with tumor hypoxia directed nanoparticle (NP) loaded with a new class of apoptosis inducer, CFM 4.16 (C4.16), namely CA IX-C4.16. The NP is designed to selectively deliver the payload to the hypoxic tumor (core), provoke superior cell death in parental (WT) and Everolimus-resistant (Evr-res) RCC and selectively downmodulate tumorigenic M2-macrophage. Copper-free 'click' chemistry was utilized for conjugating SMA-TPGS with Acetazolamide (ATZ, a CA IX-specific targeting ligand). The NP was further tagged with a clinically approved NIR dye (S0456) for evaluating hypoxic tumor core penetration and organ distribution. Imaging of tumor spheroid treated with NIR dye-labeled CA IX-SMA-TPGS revealed remarkable tumor core penetration that was modulated by CA IX-mediated targeting in hypoxic-A498 RCC cells. The significant cell killing effect with synergistic combination index (CI) of CA IX-C4.16 and Sor treatment suggests efficient reversal of Evr-resistance in A498?cells. The CA IX directed nanoplatform in combination with Sor has shown multiple benefits in overcoming drug resistance through (i) inhibition of p-AKT, (ii) upregulation of tumoricidal M1 macrophages resulting in induction of caspase 3/7 mediated apoptosis of Evr-res A498?cells in macrophage-RCC co-culturing condition, (iii) significant in vitro and in vivo Evr-res A498 tumor growth inhibition as compared to individual therapy, and (iv) untraceable liver and kidney toxicity in mice. Near-infrared (NIR) imaging of CA IX-SMA-TPGS-S0456 in Evr-res A498 RCC model exhibited significant accumulation of CA IX-oligomer in tumor core with >3-fold higher tumor uptake as compared to control. In conclusion, this proof-of-concept study demonstrates versatile tumor hypoxia directed nanoplatform that can work in synergy with existing drugs for reversing drug-resistance in RCC accompanied with re-education of tumor-associated macrophages, that could be applied universally for several hypoxic tumors.
Project description:We have synthesized a new series of coumarin-based compounds demonstrating high selectivity and potent effects with low nanomolar affinity against the tumor associated carbonic anhydrase (CA, EC 22.214.171.124) isoforms hCA IX and XII. A number of these compounds were evaluated ex vivo against human prostate (PC3) and breast (MDA-MB-231) cancer cell lines. Compounds 4b and 15 revealed effective cytotoxic effects after 48 h of incubation in both normoxic and hypoxic conditions with PC3 cancer cell line. However, compound 3 showed selective cytotoxic effects against MDA-MB-231 in hypoxic condition. These results may be of particular importance for the choice of future drug candidates targeting hypoxic tumors and metastases, considering the fact that a selective carbonic anhydrase CA IX inhibitor (SLC-0111) is presently in phase II clinical trials.
Project description:Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.
Project description:Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
Project description:Proof-of-principle studies in ovarian, lung, and brain cancer patients have shown that fluorescence-guided surgery can enable removal of otherwise undetectable malignant lesions, decrease the number of cancer-positive margins, and permit identification of disease-containing lymph nodes that would have normally evaded resection. Unfortunately, the current arsenal of tumor-targeted fluorescent dyes does not permit identification of all cancers, raising the need to design new tumor-specific fluorescent dyes to illuminate the currently undetectable cancers. In an effort to design a more universal fluorescent cancer imaging agent, we have undertaken to synthesize a fluorophore that could label all hypoxic regions of tumors. We report here the synthesis, in vitro binding, and in vivo imaging of a near-infrared (NIR) fluorescent dye that is targeted to carbonic anhydrase IX (CA IX), i.e., a widely accepted marker of hypoxic tissues. The low molecular weight NIR probe, named Hypoxyfluor, is shown to bind CA IX with high affinity and accumulate rapidly and selectively in CA IX positive tumors. Because nearly all human cancers contain hypoxic regions that express CA IX abundantly, this NIR probe should facilitate surgical resection of a wide variety of solid tumors.
Project description:Tumor hypoxia is a well-recognized driver of resistance to traditional cancer therapies such as chemotherapy and radiation therapy. We describe development of a new nanoconstruct composed of gold nanorods (GNRs) conjugated to carbonic anhydrase IX (CAIX) antibody that specifically binds to CAIX, a biomarker of hypoxia, to facilitate targeting tumor hypoxic areas for focused photothermal ablation. Physicochemical characterization studies confirmed the size, shape, monodispersity, surface charge, and serum stability of the GNRs. Enzyme-linked immunosorbent assays and cellular binding and uptake studies confirmed successful conjugation of antibody to the GNRs and specificity for CAIX. Near-infrared irradiation of CAIX-overexpressing cells treated with GNR/anti-CAIX resulted in significantly higher cell death than cells treated with control GNRs. In vivo biodistribution studies using hyperspectral imaging and inductively coupled plasma mass spectrometry confirmed intravenous administration results not only in greater accumulation of GNR/anti-CAIX in tumors than control GNRs but also greater penetration into hypoxic areas of tumors. Near-infrared ablation of these tumors showed no tumor regression in the sham-treated group, regression but recurrence in the non-targeted-GNR group, and complete tumor regression in the targeted-GNR group. GNR/anti-CAIX nanoconstructs show promise as hypoxia targeting and photothermal ablation agents for cancer treatment.
Project description:Hypoxic tumors have been identified as appropriate indicators of tumor malignancy. However, no convenient plasma marker for hypoxic tumors has been described. Therefore, to identify a novel, convenient plasma marker for hypoxic tumors, we used microarray analysis to compare gene expression profiles of normoxic and hypoxic tumor tissues of mice bearing melanomas. Among the upregulated genes detected in hypoxic tumors, we chose to study the secretory protein lipocalin2 (LCN2) as a marker for hypoxic tumors. LCN2 protein levels in the plasma of mice bearing hypoxic tumors were significantly increased compared with those in mice bearing normoxic tumors. Interestingly, LCN2 mRNA levels were 17-fold higher in HIF-1?-positive hypoxic tumors than in HIF-1?-negative normoxic tumors. Furthermore, LCN2 mRNA levels were significantly higher in the B16-F1 cells and various human tumor cells cultured under hypoxic conditions than in cells cultured under normoxic conditions, while no changes in mRNA expression were observed in nontumor NIH-3T3 cells, even under hypoxic conditions. In cultured cells, the expression pattern of LCN2 was mostly consistent with that of HIF-1?, whereas that of a conventional hypoxic marker, carbonic anhydrase IX, was not. Collectively, our data suggested that LCN2 was a useful plasma marker for hypoxic tumors.
Project description:Carbonic anhydrase XII (CAXII) is a membrane enzyme that maintains pH homeostasis and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Here, we investigated a panel of eight CAXII inhibitors (compounds 1-8), for their potential to reverse Pgp mediated tumor cell chemoresistance. Inhibitors (5 nM) were screened in human and murine cancer cells (colon, lung, breast, bone) with different expression levels of CAXII and Pgp. We identified three CAXII inhibitors (compounds 1, 2 and 4) that significantly (? 2 fold) increased the intracellular retention of the Pgp-substrate and chemotherapeutic doxorubicin, and restored its cytotoxic activity. The inhibitors lowered intracellular pH to indirectly impair Pgp activity. Ca12-knockout assays confirmed that the chemosensitizing property of the compounds was dependent on active CAXII. Furthermore, in a preclinical model of drug-resistant breast tumors compound 1 (1900 ng/kg) restored the efficacy of doxorubicin to the same extent as the direct Pgp inhibitor tariquidar. The expression of carbonic anhydrase IX had no effect on the intracellular doxorubicin accumulation. Our work provides strong evidence that CAXII inhibitors are effective chemosensitizer agents in CAXII-positive and Pgp-positive cancer cells. The use of CAXII inhibitors may represent a turning point in combinatorial chemotherapeutic schemes to treat multidrug-resistant tumors.
Project description:Hypoxic cells dynamically translocate during tumor growth and after radiotherapy. The most desirable direction for therapy targeting hypoxic cells is combining imaging and therapy (theranostics), which may help realize personalized medicine. Here, we conducted cancer radiotheranostics targeting carbonic anhydrase-IX (CA-IX), which is overexpressed in many kinds of hypoxic cancer cells, using low-molecular-weight 111In and 90Y complexes with a bivalent ureidosulfonamide scaffold as the CA-IX-binding moiety ([111In/90Y]US2). Methods: The targeting ability of [111In]US2 was evaluated by in vivo biodistribution study in CA-IX high-expressing (HT-29) tumor-bearing mice. In vivo imaging of HT-29 tumors was carried out using single photon emission computed tomography (SPECT). [90Y]US2 was administered to HT-29 tumor-bearing mice to evaluate cancer therapeutic effects. Results: [111In]US2 highly and selectively accumulated within HT-29 tumors (4.57% injected dose/g tumor at 1 h postinjection), was rapidly cleared from the blood pool and muscle after 4 h based on a biodistribution study, and visualized HT-29 tumor xenografts in mice at 4 h postinjection with SPECT. Radionuclide-based therapy with [90Y]US2 significantly delayed HT-29 tumor growth compared with that of untreated mice (P = 0.02 on day 28, Student's t-test), without any critical hematological toxicity due to its rapid pharmacokinetics. Conclusion: These results indicate that cancer radiotheranostics with [111In/90Y]US2 provides a novel strategy of theranostics for cancer hypoxia.