Times Higher Cytotoxicity Research Articles

Stimuli-Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood-to-Brain Trafficking and Tumor Targeting.

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis-driven stimuli-responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood-brain barrier (BBB) trafficking and lack of GBM targeting-two major hurdles for anti-GBM therapies. NPs are dual-surface tailored with a i) brain-targeted acid-responsive Angiopep-2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L-Histidine moiety that provides NP preferential accumulation into GBM cells post-BBB crossing. In tumor invasive margin patient cells, the stimuli-responsive multifunctional NPs target GBM cells, enhance cell uptake by 12-fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long-term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli-responsive multifunctional NPs as an effective anti-GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment.

Multi-endpoint assays reveal more severe toxicity induced by chloraminated effluent organic matter than chloraminated natural organic matter

Disinfection by chloramination produces toxic byproducts and the difference in toxicity of reclaimed and drinking water treated by chloramination remains unclear. This study investigated cytotoxic effects at the same concentrations of dissolved organic matter and showed that chloraminated effluent organic matter (EfOM) induced 1.7 times higher cytotoxicity than chloraminated natural organic matter (NOM) applied to simulate drinking water. Chloraminated EfOM induced more reactive nitrogen species than chloraminated NOM, and chloraminated EfOM and NOM induced similar and higher levels of reactive oxygen species than the negative control, respectively. Consequently, intracellular macromolecule damage indicated by DNA/RNA damage marker 8‑hydroxy-(deoxy)guanosine and the intracellular protein carbonyl concentration induced by chloraminated EfOM was higher and slightly more than that induced by chloraminated NOM, respectively. These data were consistent with the effects on cell physiological processes. Cell cycle arrest mainly occurred in G2 phase by chloraminated EfOM and NOM. Early apoptotic cells, which could return to normal, increased upon exposure to high concentrations of chloraminated EfOM and NOM. Moreover, necrotic cells were significantly increased from 0.5% to 2.5% when the concentration increased from 20- to 60-fold chloraminated EfOM, but were not obviously changed by chloraminated NOM. These results indicated that the comprehensive intracellular changes induced by toxic substances in chloraminated EfOM were more irreversible and induced more cell death than chloraminated NOM.

Anti-Proliferative Potential of Quercetin Loaded Polymeric Mixed Micelles on Rat C6 and Human U87MG Glioma Cells.

Quercetin (Qu) is a natural flavonoid present in many commonly consumed food items and is also identified as a potential anticancer agent. The present study evaluates the Qu-loaded polymeric mixed micelles (Qu-PMMs) against C6 and U87MG glioma cell lines. The Box–Behnken Design (BBD) was employed to study the influence of independent variables such as Soluplus, Vitamin-E polyethyleneglycol-1000 succinate (E-TPGS), and poloxamer 407 concentrations on dependent variables including particle size (PS), polydispersity index (PDI), and percentage entrapment efficiency (%EE) of the prepared Qu-PMMs. The Qu-PMMs were further characterized by Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Scanning Electron Microscope (SEM), and were assessed for in vitro drug release, effect on cell viability, migration, cellular uptake, and apoptosis assays. The PS, PDI, and % EE of the optimized PMMs were 107.16 ± 1.06 nm, 0.236 ± 0.053, and 77.46 ± 1.94%, respectively. The FTIR and XRD revealed that the Qu was completely entrapped inside the PMMs. The SEM analysis confirmed the spherical shape of micelles. The in vitro cell viability study showed that the Qu-PMMs had 1.7 times higher cytotoxicity against C6 and U87MG cells than Qu pure drug (Qu-PD). Furthermore, Qu-PMMs demonstrated superior cellular uptake, inhibited migration, and induced apoptosis when tested against C6 and U87MG cells than pure Qu. Thus, the polymeric mixed micelle (PMMs) enhanced the therapeutic effect of Qu and can be considered an effective therapeutic strategy to treat Glioma.

Synthesis, characterization, and antitumor biological evaluation of novel fluorine‐containing platinum (IV) complexes

A series of antitumor platinum (IV) complexes based on ammonia and fluoropyridine carrier groups with biotin and chloride ions as axial groups have been designed and synthesized in this paper. The structures of platinum (IV) complexes were fully characterized by 1H NMR, ESI‐MS, and IR. Such Pt (IV) complexes have certain tumor targeting properties, and the Pt (II) complexes released in the reducing microenvironment could combine with DNA. Compared with cisplatin, complexes S3 and S4 exhibited similar cytotoxicity against SW480, HCT116, and HepG2 cancer cell lines according to in vitro antitumor activity evaluation. Particularly, Complex S2 showed 1.5–3.0 times higher cytotoxicity than cisplatin and picoplatin against selected cell lines. According to the structure–activity relationship, the introduction of fluorine atoms and axial chloride has an important influence on the strong cytotoxic activity of Pt (IV) complex. In addition, the axial biotin molecule increased the targeting of the complex to tumor cells. Overall, the novel fluorine‐containing Pt (IV) complex S2 is an attractive antitumor lead compound with further research value.

Hydrophilic Polyhedral Oligomeric Silsesquioxane, POSS(OH)32, as a Complexing Nanocarrier for Doxorubicin and Daunorubicin.

A novel strategy, recently developed by us, to use polyhedral oligomeric silsesquioxanes (POSS) as an anti-cancer drug carrier is presented. Anthracycline:POSS complexes were prepared by simple co-addition of doxorubicin (DOX) or daunorubicin (DAU) with hydrophilic POSS(OH)32. Co-delivery of POSS and anthracyclines led to higher anti-cancer activity towards HeLa (cervical cancer endothelial) and MCF-7 (human breast adenocarcinoma) cell lines. The obtained supramolecular hybrid complexes were characterised by nuclear magnetic resonance (NMR) spectroscopy (nuclear Overhauser effect spectroscopy [NOESY] and homonuclear correlation spectroscopy [COSY]), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). The two-dimensional (2D) NOESY spectra of the complexes showed the cross-correlation peaks for hydroxyl groups of POSS (~4.3–4.8 ppm) with OH groups of DOX and DAU. FTIR showed that hydroxyl group of POSS can interact with amine and hydroxyl groups of DOX and DAU. The viability of HeLa and MCF-7 was analysed with the MTT assay to evaluate the cytotoxicity of free DOX and DAU and the relevant complexes with POSS at different molar ratios. At a low DOX concentration (2.5 µM), for molar ratios 1:1, 1:4, and 1:8 (POSS:DOX), the complexes showed two and three times higher cytotoxicity towards HeLa and MCF-7 cells, respectively, than DOX itself after both 24- and 48-h incubation. The 1 µM concentration for a 1:4 POSS:DOX molecular ratio and the 2.5 µM concentration for all complexes were more toxic towards MCF-7 cells than free DOX after 48-h incubation. In the case of POSS:DAU complexes, there was higher toxicity than that of free drug after 48-h incubation. It can be concluded that the formation of non-covalent complexes increases toxicity of anthracycline drugs towards Hela and MCF-7 cells. The novel complexes are inexpensive to prepare and more effective than free drugs at low systemic toxicity.

Design, synthesis and SAR study of 3rd-generation taxoids bearing 3-CH3, 3-CF3O and 3-CHF2O groups at the C2-benzoate position.

It has been shown that inclusion of CF3O and CHF2O groups to drug candidates often improve their pharmacological properties, especially metabolic stability, membrane permeability and PK profile. Moreover, the unique non-spherical structure of the OCHF2 group can provide interesting and beneficial characteristics. Accordingly, new 3rd-generation taxoids, bearing 3-OCF3 or 3-OCF2H (and 3-CH3 for comparison) at the C2 benzoate moiety, were synthesized and their potencies against drug-sensitive and drug-resistant cancer cell lines examined. In this study, our previous SAR studies on 3rd-generation taxoids were expanded to disclose that CH3, CF3O and CHF2O groups are well tolerated at this position and enhance potency, especially against MDR-cancer cell lines so that these taxoids can virtually overcome MDR. These new taxoids exhibit up to 7 times higher cytotoxicity (IC50) than paclitaxel against drug-sensitive cancer cell lines (MCF7 and LCC6-WT) and 2-3 orders of magnitude higher potency than paclitaxel against drug-resistant ovarian, breast and colon cancer cell lines with MDR-phenotype (NCI/ADR, LCC6-MDR and LDL-1), as well as pancreatic cancer cell line, CFPAC-1. Since it has been shown that a bulky group at this position reduces potency, it is noteworthy that rather bulky CF3O and CHF2O groups are well tolerated. Molecular modeling analysis indicated the favorable van der Waals interactions of CF3O and CHF2O groups in the binding site. It is also worthy of note that new taxoids, bearing a CHF2O group at the C2 benzoate position (1-06 series), exhibited the highest potencies against MDR-cancer cell lines and cancer stem cell (CSC)-enriched cancer cell lines. These new 3rd-generation taxoids are promising candidates for highly potent chemotherapeutic agents, as well as payloads for tumor-targeting drug conjugates such as antibody-drug conjugates (ADCs).

Synthesis and Biological Evaluation of Novel Triple-Modified Colchicine Derivatives as Potent Tubulin-Targeting Anticancer Agents.

Specific modifications of colchicine followed by synthesis of its analogues have been tested in vitro with the objective of lowering colchicine toxicity. Our previous studies have clearly shown the anticancer potential of double-modified colchicine derivatives in C-7 and C-10 positions. Here, a series of novel triple-modified colchicine derivatives is reported. They have been obtained following a four-step strategy. In vitro cytotoxicity of these compounds has been evaluated against four human tumor cell lines (A549, MCF-7, LoVo, and LoVo/DX). Additionally, the mode of binding of the synthesized compounds was evaluated in silico using molecular docking to a 3D structure of β-tubulin based on crystallographic data from the Protein Data Bank and homology methodology. Binding free energy estimates, binding poses, and MlogP values of the compounds were obtained. All triple-modified colchicine derivatives were shown to be active at nanomolar concentrations against three of the investigated cancer cell lines (A549, MCF-7, LoVo). Four of them also showed higher potency against tumor cells over normal cells as confirmed by their high selectivity index values. A vast majority of the synthesized derivatives exhibited several times higher cytotoxicity than colchicine, doxorubicin, and cisplatin.

Pentapeptides for the treatment of small cell lung cancer: Optimisation by Nind-alkyl modification of the tryptophan side chain

The pentapeptide, tert-Prenyl4th-NH2 (DMePhe-DTrp-Phe-DTrp(N-tert-prenyl)-Leu-NH2), has recently been reported by our group to exhibit properties of substance P (SP) antagonist G against small cell lung cancer (SCLC). In this study, we undertook a systematic structure activity investigation to optimise this lead compound to improve its in vitro anti-tumour activity and biocompatibility. A series of d-tryptophan (D-Trp) derivatives were synthesised, with a range of aliphatic N-alkyl chains (methyl to pentyl) on the indole nitrogen (Nind). These were incorporated into the pentapeptide sequence by substitution of the Nind-tert-prenylated D-Trp 4th residue with the Nind-alkylated D-Trp derivatives. These pentapeptides were significantly more potent than tert-Prenyl4th-NH2, with the Nind-butyl modification generating the most cytotoxic peptides. Compared to tert-Prenyl4th-NH2, a single butyl modification on the 4th D-Trp residue (Butyl4th-NH2) showed a ∼3 fold enhancement in cytotoxicity in either the chemo-naive H69 or the DMS79 (originating from a patient treated with chemotherapeutics and radiation therapy) SCLC cell lines. In addition, the di-butylated sequence on the 2nd and 4th D-Trp residues (Butyl2nd,4th-NH2) gave ∼4.5 times higher cytotoxicity against the H69 cell line and a ∼2 fold increase against the DMS79 cell line, compared to tert-Prenyl4th-NH2. The favoured position for butyl modification was the 4th D-Trp residue, as the Butyl2nd-NH2 peptide gave lower cytotoxicity on both cell lines. Butylated peptide sequences, when exposed to neat mouse plasma for 24 h at 37 °C, were found to resist degradation with >80% remaining intact compared to ∼58% for tert-Prenyl4th-NH2. The degradation pathway in plasma occurs via de-amidation of the C-terminus, confirmed by mass spectrometry and RP-HPLC analysis. The butyl modification also conferred resistance to metabolism when tested using S9 liver fraction from mouse. The optimum analogue responsive against the DMS79 cell line was the Butyl4th-NH2 pentapeptide, which revealed a concentration dependent increase in apoptosis: the level of late apoptotic cells rose from ∼36% at 2 μM to ∼96% at 6 μM, as determined by flow cytometry, compared to the unmodified peptide that showed no such effect. Concluding, the butyl substitutions offered the best perspective for high cytotoxicity, induction of apoptosis and metabolic compatibility thereby comprising an improved broad spectrum SP antagonist candidate for treatment of SCLC.

High oral bioavailability of 2-methoxyestradiol in PEG-PLGA micelles-microspheres for cancer therapy

The development of effective oral preparations of 2-methoxyestradiol (2-ME) has been a worldwide problem. In this study, breakthrough progress has been made in oral delivery of 2-ME by encapsulating 2-ME micelles in pH-responsive microspheres. 2-ME micelles with small particle size (58nm) and high drug loading (7.94±0.23%) were successfully prepared and showed slower release characteristics and 12.8 times higher cytotoxicity on 4T1 cells than free 2-ME. The pH-sensitive microspheres could slowly release 82% of 2-ME micelles for 10h in PBS (7.4). After oral administration to rats, 2-ME-micelles-microspheres compared to 2-ME micelles could prolong significantly blood retention time (MRT) of 2-ME by 12 times and enhance oral absolute bioavailability from 49.99% to 121.68%. In vivo imaging of 4T1 tumor-bearing mice showed that retention time and intensity of the fluorescence signal in each 2-ME preparation group all were consistent with the pharmacokinetic results and intact 2-ME micelles in the microspheres could be absorbed in intestine to blood to target to tumor. Furthermore, after the 4T1 tumor-bearing mice were treated with oral 2-ME-micelles-microspheres for 21days, the tumor growth inhibition rate achieved 94.93% and the mice weight gained significantly, indicating their high antitumor activity and low toxicity. While oral administration of 2-ME micelles and 2-ME microspheres all were ineffective under the same conditions. Therefore, micelles-microspheres might be a promising candidate for oral administration of 2-ME for cancer therapy.

Cytotoxicity of copper(II)-complexes with some S-alkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear copper(II)-complex with S-ethyl derivative of thiosalicylic acid

The spectroscopically predicted structure of the obtained copper(II)-complex with S-ethyl derivative of thiosalicylic acid was confirmed by X-ray structural study and compared to previously reported crystal structure of the Cu complex with S-methyl derivative. Single crystals suitable for X-ray measurements were obtained by slow crystallization from a water solution. Cytotoxic effects of S-alkyl (R = benzyl (L1), methyl (L2), ethyl (L3), propyl (L4) and butyl (L5)) derivatives of thiosalicylic acid and the corresponding binuclear copper(II)-complexes on murine colon carcinoma cell lines, CT26 and CT26.CL25 and human colon carcinoma cell line HCT-116 were reported here. The analysis of cancer cell viability showed that all the tested complexes had low cytotoxic effect on murine colon carcinoma cell lines, but several times higher cytotoxicity on normal human colon carcinoma cells.

In vitro delivery of curcumin with cholesterol-based cationic liposomes

A new cholesterol-based cationic lipid was synthesized; liposomes prepared on its basis were evaluated as drug delivery vehicles for curcumin. Free and liposome-encapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED50 values ranging from 2.5-10 microM exhibited 2-8 times higher cytotoxicity than free curcumin. The synthetic cholesterol-based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED50 values of 90-210 microM.

Hyaluronic Acid-Based Nanogel–Drug Conjugates with Enhanced Anticancer Activity Designed for the Targeting of CD44-Positive and Drug-Resistant Tumors

Many drug-resistant tumors and cancer stem cells (CSC) express elevated levels of CD44 receptor, a cellular glycoprotein binding hyaluronic acid (HA). Here, we report the synthesis of nanogel-drug conjugates based on membranotropic cholesteryl-HA (CHA) for efficient targeting and suppression of drug-resistant tumors. These conjugates significantly increased the bioavailability of poorly soluble drugs with previously reported activity against CSC, such as etoposide, salinomycin, and curcumin. The small nanogel particles (diameter 20-40 nm) with a hydrophobic core and high drug loads (up to 20%) formed after ultrasonication and demonstrated a sustained drug release following the hydrolysis of biodegradable ester linkage. Importantly, CHA-drug nanogels demonstrated 2-7 times higher cytotoxicity in CD44-expressing drug-resistant human breast and pancreatic adenocarcinoma cells compared to that of free drugs and nonmodified HA-drug conjugates. These nanogels were efficiently internalized via CD44 receptor-mediated endocytosis and simultaneous interaction with the cancer cell membrane. Anchoring by cholesterol moieties in the cellular membrane after nanogel unfolding evidently caused more efficient drug accumulation in cancer cells compared to that in nonmodified HA-drug conjugates. CHA-drug nanogels were able to penetrate multicellular cancer spheroids and displayed a higher cytotoxic effect in the system modeling tumor environment than both free drugs and HA-drug conjugates. In conclusion, the proposed design of nanogel-drug conjugates allowed us to significantly enhance drug bioavailability, cancer cell targeting, and the treatment efficacy against drug-resistant cancer cells and multicellular spheroids.

In vitroDelivery of Curcumin with Cholesterol-Based Cationic Liposomes

A new cholesterolbased cationic lipid was synthe sized; liposomes prepared on its basis were eval� uated as drug delivery vehicles for curcumin. Free and liposomeencapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED50 values ranging from 2.5-10 µM exhibited 2-8 times higher cytotoxicity than free curcumin. The synthetic cholesterol� based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED50 values of 90-210 µM.

Hyaluronate Tethered, “Smart” Multiwalled Carbon Nanotubes for Tumor-Targeted Delivery of Doxorubicin

The present study reports the optimized synthesis, physicochemical characterization, and biological evaluation of a novel, multiwalled carbon nanotube-hyaluronic acid (MWCNT-HA) conjugate, complexed with an anticancer agent, Doxorubicin (DOX) via π-π stacking interaction. The therapeutic conjugate was concomitantly labeled with a near-infrared fluorescent dye, Alexa-Flour-647 (AF-647), and radiotracer Technetium-99m ((99m)Tc) to track its whereabouts both in vitro and in vivo via optical and scintigraphic imaging techniques. Covalent functionalization of MWCNTs with HA facilitated their internalization into human lung adenocarcinoma, A549 cells via hyaluronan receptors (HR) mediated endocytosis. Internalized nanotubes showed lysosomal trafficking, followed by low pH-triggered DOX release under endolysosomal conditions. Consequently, DOX-loaded HA-MWCNTs exhibited 3.2 times higher cytotoxicity and increased apoptotic activity than free DOX in equivalent concentrations. Organ distribution studies in Ehlrich ascites tumor (EAT) bearing mice model indicated that tumor specific localization of (99m)Tc-MWCNT-HA-DOX is significantly higher than both free drug and nontargeted MWCNTs. Pharmacodynamic studies in chemically breast-cancer-induced rats showed that the tumor-growth inhibitory effect of HA-MWCNT-DOX was 5 times higher than free DOX in equivalent concentration. DOX delivered through HA-MWCNTs was devoid of any detectable cardiotoxity, hepatotoxicity, or nephrotoxicity. All these promising attributes make HA-MWCNTs a "smart" platform for tumor-targeted delivery of anticancer agents.

Preparation and cytotoxic activity of poly(ethylene glycol)-modified poly(amidoamine) dendrimers bearing adriamycin

We have developed poly(amidoamine) (PAMAM) dendrimers that have poly(ethylene glycol) (PEG) grafts at all dendrimer chain ends. To obtain PEG-modified dendrimers with sites for conjugation of anticancer drugs for this study, we prepared PAMAM G4 dendrimers that have a glutamic acid (Glu) residue at every chain end of dendrimer; PEG chains were attached to amino groups of Glu residues. We then combined the anticancer drug adriamycin to side chains of the Glu residues using an amide bond, [PEG–Glu(ADR)-G4], or hydrazone bond, [PEG–Glu(NHN–ADR)-G4]. For the dendrimers bearing adriamycin through amide linkage, adriamycin was released only slightly at pH 7.4 and 5.5. Although a negligible level of release occurred at pH 7.4 for dendrimers with adriamycin via hydrazone linkage, a remarkable extent of adriamycin release was induced at pH 5.5, which corresponds to the pH of late endosome. These adriamycin-bearing dendrimers showed much lower toxicity to HeLa cells than did free adriamycin. However, compared to PEG–Glu(ADR)-G4, PEG–Glu(NHN–ADR)-G4 exhibited 7 times higher cytotoxicity, suggesting the importance of pH-sensitive hydrazone linkage for high cytotoxicity. Furthermore, the PEG-modified dendrimers exhibited an equivalent level of toxicity to that of adriamycin-resistant SBC-3/ADR100 cells and their parent adriamycin-sensitive SBC-3 cells.