Dual enzyme responsive and targeted nanocapsules for intracellular delivery of anticancer agents

We report the fabrication of dual enzyme responsive hollow nanocapsules which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements, integrated in the nanocapsule walls, undergo degradation in the presence of either trypsin or hyaluronidase leading to the release of encapsulated drug molecules. These nanocapsules, which were crosslinked and functionalised with folic acid, showed minimal drug leakage when kept in pH 7.4 PBS buffer, but released the drug molecules at a rapid rate in the presence of either one of the triggering enzymes. Studies on cellular interactions of these nanocapsules revealed that doxorubicin loaded nanocapsules were taken up by cervical cancer cells via folic acid receptor medicated endocytosis. Interestingly the nanocapsules were able to disintegrate inside the cancer cells and release doxorubicin which then migrated into the nucleus to induce cell death. This study indicates that these nanocapsules fabricated from biopolymers can serve as an excellent platform for targeted intracellular drug delivery to cancer cells.

Anticancer Effects of Brominated Indole Alkaloid Eudistomin H from Marine Ascidian Eudistoma viride Against Cervical Cancer Cells (HeLa)

Marine invertebrates called ascidians are prolific producers of bioactive substances. The ascidian Eudistoma viride, distributed along the Southeast coast of India, was investigated for its in vitro cytotoxic activity against human cervical carcinoma (HeLa) cells by the MTT assay. The crude methanolic extract of E. viride, with an IC50 of 53 mu g/ml, was dose-dependently cytotoxic. It was more potent at 100 mu g/ml than cyclohexamide (1 mu g/ml), reducing cell viability to 9.2%. Among nine fractions separated by chromatography, ECF-8 exhibited prominent cytoxic activity at 10 mu g/ml. The HPLC fraction EHF-21 of ECF-8 was remarkably dose- and time-dependently cytotoxic, with 39.8% viable cells at 1 mu g/ml compared to 51% in cyclohexamide-treated cells at the same concentration; the IC50 was 0.49,mu g/ml. Hoechst staining of HeLa cells treated with EHF-2I at 0.5 mu g/ml revealed apoptotic events such an cell shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies. Cell size and granularity study showed changes in light scatter, indicating the characteristic feature of cells dying by apoptosis. The cell-cycle analysis of HeLa cells treated with fraction EHF-21 at 1 mu g/ml showed the marked arrest of cells in G(0)/G(1), S and G(2)/M phases and an increase in the sub G(0)/G(1) population indicated an increase in the apoptotic cell population. The statistical analysis of the sub-G(1) region showed a dose-dependent induction of apoptosis. DNA fragmentation was also observed in HeLa cells treated with EHF-21. The active EHF-2I fraction, a brominated indole alkaloid Eudistomin H, led to apoptotic death of HeLa cells.

Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity

Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. Here we report the engineering of personalized tumour ecosystems that contextually conserve the tumour heterogeneity, and phenocopy the tumour microenvironment using tumour explants maintained in defined tumour grade-matched matrix support and autologous patient serum. The functional response of tumour ecosystems, engineered from 109 patients, to anticancer drugs, together with the corresponding clinical outcomes, is used to train a machine learning algorithm; the learned model is then applied to predict the clinical response in an independent validation group of 55 patients, where we achieve 100% sensitivity in predictions while keeping specificity in a desired high range. The tumour ecosystem and algorithm, together termed the CANScript technology, can emerge as a powerful platform for enabling personalized medicine.

Pluronic copolymer encapsulated SCR7 as a potential anticancer agent

Nonhomologous end joining (NHEJ) of DNA double strand breaks (DSBs) inside cells can be selectively inhibited by 5,6-bis-(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) which possesses anticancer properties. The hydrophobicity of SCR7 decreases its bioavailability which is a major setback in the utilization of this compound as a therapeutic agent. In order to circumvent the drawback of SCR7, we prepared a polymer encapsulated form of SCR7. The physical interaction of SCR7 and Pluronic (R) copolymer is evident from different analytical techniques. The in vitro cytotoxicity of the drug formulations is established using the MTT assay.

Protamine-carboxymethyl cellulose magnetic nanocapsules for enhanced delivery of anticancer drugs against drug resistant cancers

Multidrug resistance is a major therapeutic challenge faced in the conventional chemotherapy. Nanocarriers are beneficial in the transport of chemotherapeutics by their ability to bypass the P-gp efflux in cancers. Most of the P-gp inhibitors under phase II clinical trial are facing failures and hence there is a need to develop a suitable carrier to address P-gp efflux in cancer therapy. Herein, we prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin against highly drug resistant HeLa cells. The experimental results revealed that improved cellular uptake, enhanced drug intensity profile with greater percentage of apoptotic cells was attained when doxorubicin loaded magnetic nanocapsules were used in the presence of external magnetic field. Hence, we conclude that this magnetic field assisted nanocapsule system can be used for delivery of chemotherapeutics for potential therapeutic efficacy at minimal dose in multidrug resistant cancers. From the Clinical Editor: Many cancer drugs fail when cancer cells become drug resistant. Indeed, multidrug resistance (MDR) is a major therapeutic challenge. One way that tumor cells attain MDR is by over expression of molecular pumps comprising of P-glycoprotein (P-gp) and multidrug resistant proteins (MRP), which can expel chemotherapeutic drugs out of the cells. In this study, the authors prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin. The results show that there was better drug delivery and efficacy even against MDR tumor cells. (C) 2015 Elsevier Inc. All rights reserved.

Synthesis and evaluation of the biological activity of N `-2-oxo-1,2 dihydro-3H-indol-3-ylidene] benzohydrazides as potential anticancer agents

New N'-2-oxo-1,2-dihydro-3H-indol-3-ylidene]benzohydrazide derivatives were synthesized and evaluated for their cytotoxic properties against murine leukemia, L1210, human leukemia, REH and K562, human T-cell leukemia, CEM and human cervix carcinoma, HeLa cells. Among the tested compounds, the 3,4,5-trimethoxy-N'-5-methyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene]ben zohydrazide derivative (5t) emerged as the most potent inhibitor against all the tumor cell lines evaluated. To investigate the mechanism of action, 5t was further studied by cell cycle analysis, mitochondrial membrane potential analysis, DNA fragmentation and Annexin V-FITC flow cytometric analysis, which suggested that 5t was able to induce apoptosis at submicromolar range.

Metal Complexes of Curcumin for Cellular Imaging, Targeting, and Photoinduced Anticancer Activity

CONSPECTUS: Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-kappa B besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and damaging the cancer cells on photoactivation in visible light while being minimally toxic in darkness. In this Account, we have made an attempt to review the current status of the chemistry of metal curcumin complexes and present results from our recent studies on curcumin complexes showing remarkable in vitro photocytotoxicity. The undesirable dark toxicity of the complexes can be reduced with suitable choice of the metal and the ancillary ligands in a ternary structure. The complexes can be directed to specific subcellular organelles. Selectivity by targeting cancer cells over normal cells can be achieved with suitable ligand design. We expect that this methodology is likely to provide an impetus toward developing curcumin-based photochemotherapeutics for anticancer treatment and cure.

Iron(III) salicylates of dipicolylamine bases showing photo-induced anticancer activity and cytosolic localization

An iron(III) salicylate having a dipicolylamine base (andpa) with a photoactive anthracenyl moiety is prepared, characterized, and studied for its photo-induced anticancer activity and cellular localization in HeLa and MCF-7 cells. Its phenyl analogue is structurally characterized by X-ray crystallography. The complex has a ternary structure in which the dipicolylamine ligand and salicylic acid in dianionic form (sal) display respective tridentate and bidentate mode of coordination in Fe(sal)(phdpa)Cl] (1). Complex Fe(sal)(andpa)Cl] (2) having a pendant anthracenyl moiety shows significant photocytotoxicity in visible light (400-700 nm) giving IC50 values of 8.6 +/- 0.7 and 3.4 +/- 0.9 mu M in HeLa and MCF-7 cells, while being essentially nontoxic in the dark (IC50 > 100 mu M). The complex shows cytosolic localization in the cancer cells. Formation of hydroxyl radicals ((OH)-O-center dot) as the reactive oxygen species is evidenced from the pUC19 DNA photocleavage studies. (C) 2015 Elsevier Ltd. All rights reserved.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Mitochondria-Targeting Iron(III) Catecholates for Photoactivated Anticancer Activity under Red Light

Iron(III) catecholates Fe(R-bpa)(R-dopa)Cl] (1, 2) with a triphenylphosphonium (TPP) moiety, where R-bpa is 2-(TPP-N,N-bis((pyridin-2-yl)methyl)ethanamine) chloride (TPPbpa) and R-dopa is 4-{2-(anthracen-9-yl)methylamino]ethyl}benzene-1,2-diol (andopa, 1) or 4-{2-(pyren-1-yl)-methylamino]ethyl}benzene-1,2-diol (pydopa, 2), were synthesized and their photocytotoxicity studied. Complexes 3 and 4 with phenyl-N,N-bis(pyridin-2-yl)methyl]methanamine (phbpa) were used as controls. The catecholate complexes showed an absorption band near 720 nm. The 5e(-) paramagnetic complexes showed a Fe-III/Fe-II irreversible response near -0.45 V and a quasi-reversible catechol/semiquinone couple near 0.5 V versus saturated calomel electrode (SCE) in DMF/0.1 M tetrabutylammonium perchlorate. They showed photocytotoxicity in red/visible light in HeLa, HaCaT, MCF-7, and A549 cells. Complexes 1 and 2 displayed mitochondrial localization, reactive oxygen species (ROS) generation under red light, and apoptotic cell death. Control complexes 3 and 4 exhibited uniform distribution throughout the cell. The complexes showed DNA photocleavage under red light (785 nm), forming hydroxyl radicals as the ROS.

Remarkable photo-induced anticancer activity of vitamin-S6 Schiff base copper(II) complexes of pyridyl bases

Vitamin-B6 (VB6) Schiff base (H2L) copper(II) complexes of pyridyl bases, viz. Cu(bpy)(L)] (1), Cu(phen)(L)] (2) and Cu(dppz)(L)] (3), where bpy is 2,2'-bipyridine, phen is 1,10-phenanthroline and dppz is dipyrido3,2-a:2',3'c]phenazine are synthesized, characterized and their phto-induced anticancer activity studied. The non-electrolytic one electron paramagnetic complexes exhibit a d-d band near 700 nm in DMF. The dppz complex intercalatively binds to calf-thymus DNA with binding constant (K-b) values of similar to 10(6) M-1. This complex exhibits low chemical nuclease activity but excellent DNA photocleavage activity when irradiated with red light of 705 nm forming (OH)-O-center dot radical. It displays remarkable photocytotoxicity in human cervical cancer cells (HeLa) giving IC50 value of 0.9 mu M in visible light (400-700 nm) while being less toxic in darkness (IC50 : 23 mu M). The cellular uptake of the complexes seems to be via VB6 transporting membrane carrier mediated diffusion pathway. Photo-induced cell death follows apoptotic pathway involving photo-generated intracellular reactive oxygen species.

Novel synergistic photocatalytic degradation of antibiotics and bacteria using V-N doped TiO2 under visible light: the state of nitrogen in V-doped TiO2

We report the synthesis of vanadium and nitrogen co-doped TiO2 for photocatalysis mainly emphasizing the state of nitrogen doping into TiO2 in the presence of vanadium ions. Considering the increase in antibiotic resistance developed by microbes due to the excess of pharmaceutical waste in the ecosystem, the photocatalytic activity was measured by degrading an antibiotic, chloramphenicol. A novel experiment was conducted by degrading the antibiotic and bacteria in each other's vicinity to focus on their synergistic photo-degradation by V-N co-doped TiO2. The catalysts were characterized using XRD, DRS, PL, TEM, BET and XPS analysis. Both interstitial and substitutional nitrogen doping were achieved with V-TiO2, showing high efficiency under visible light for antibiotic and bacterial degradation. In addition, the effect of doping concentration of nitrogen and vanadium in TiO2 and catalyst loading was studied thoroughly. Reusability experiments show that the prepared V-N co-doped TiO2 was stable for many cycles.

Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.