Light-Responsive Pt(IV) Prodrugs with Controlled Photoactivation and Low Dark Toxicity.

Light-induced release of cisplatin from Pt(IV) prodrugs represents a promising approach for precise control over the antiproliferative activity of Pt-based chemotherapeutic drugs. This method has the potential to overcome crucial drawbacks of conventional cisplatin therapy, such as high general toxicity toward healthy organs and tissues. Herein, we report two Pt(IV) prodrugs with BODIPY-based photoactive ligands Pt-1 and Pt-2, which were designed using carbamate and triazole linkers, respectively. Both prodrugs demonstrated the ability to release cisplatin under blue light irradiation without the requirement of an external reducing agent. Dicarboxylated Pt-2 prodrug turned out to be more stable in the dark and more sensitive to light than its monocarbamate Pt-1 counterpart; these observations were explained using DFT calculations. The investigation of the photoreduction mechanism of Pt-1 and Pt-2 prodrugs using DFT modeling and ΔG0 PET estimation suggests that the photoinduced electron transfer from the singlet excited state of the BODIPY axial ligand to the Pt(IV) center is the key step in the light-induced release of cisplatin from the complexes. Cytotoxicity studies demonstrated that both prodrugs were nontoxic in the dark and toxic to MCF-7 cells under low-dose irradiation with blue light, and the observed effect was solely due to the cisplatin release from the Pt(IV) prodrugs. Our research presents an elegant synthetic approach to light-activated Pt(IV) prodrugs and presents findings that may contribute to the future rational design of photoactivatable Pt(IV) prodrugs.

Electrochemical Nanopipette Sensor for In Vitro/In Vivo Detection of Cu2+ Ions.

In vitro/in vivo detection of copper ions is a challenging task but one which is important in the development of new approaches to the diagnosis and treatment of cancer and hereditary diseases such as Alzheimer's, Wilson's, etc. In this paper, we present a nanopipette sensor capable of measuring Cu2+ ions with a linear range from 0.1 to 10 µM in vitro and in vivo. Using the gold-modified nanopipette sensor with a copper chelating ligand, we evaluated the accumulation ability of the liposomal form of an anticancer Cu-containing complex at three levels of biological organization. First, we detected Cu2+ ions in a single cell model of human breast adenocarcinoma MCF-7 and in murine melanoma B16 cells. The insertion of the nanoelectrode did not result in leakage of the cell membrane. We then evaluated the distribution of the Cu-complex in MCF-7 tumor spheroids and found that the diffusion-limited accumulation was a function of the depth, typical for 3D culture. Finally, we demonstrated the use of the sensor for Cu2+ ion detection in the brain of an APP/PS1 transgenic mouse model of Alzheimer's disease and tumor-bearing mice in response to injection (2 mg kg-1) of the liposomal form of the anticancer Cu-containing complex. Enhanced stability and selectivity, as well as distinct copper oxidation peaks, confirmed that the developed sensor is a promising tool for testing various types of biological systems. In summary, this research has demonstrated a minimally invasive electrochemical technique with high temporal resolution that can be used for the study of metabolism of copper or copper-based drugs in vitro and in vivo.

Upconversion Nanoparticles Intercalated in Large Polymer Micelles for Tumor Imaging and Chemo/Photothermal Therapy.

Frontiers in theranostics are driving the demand for multifunctional nanoagents. Upconversion nanoparticle (UCNP)-based systems activated by near-infrared (NIR) light deeply penetrating biotissue are a powerful tool for the simultaneous diagnosis and therapy of cancer. The intercalation into large polymer micelles of poly(maleic anhydride-alt-1-octadecene) provided the creation of biocompatible UCNPs. The intrinsic properties of UCNPs (core@shell structure NaYF4:Yb3+/Tm3+@NaYF4) embedded in micelles delivered NIR-to-NIR visualization, photothermal therapy, and high drug capacity. Further surface modification of micelles with a thermosensitive polymer (poly-N-vinylcaprolactam) exhibiting a conformation transition provided gradual drug (doxorubicin) release. In addition, the decoration of UCNP micelles with Ag nanoparticles (Ag NPs) synthesized in situ by silver ion reduction enhanced the cytotoxicity of micelles at cell growth temperature. Cell viability assessment on Sk-Br-3, MDA-MB-231, and WI-26 cell lines confirmed this effect. The efficiency of the prepared UCNP complex was evaluated in vivo by Sk-Br-3 xenograft regression in mice for 25 days after peritumoral injection and photoactivation of the lesions with NIR light. The designed polymer micelles hold promise as a photoactivated theranostic agent with quattro-functionalities (NIR absorption, photothermal effect, Ag NP cytotoxicity, and Dox loading) that provides imaging along with chemo- and photothermal therapy enhanced with Ag NPs.

Photoinduced Reduction of Novel Dual-Action Riboplatin Pt(IV) Prodrug.

Controlled photoreduction of Pt(IV) prodrugs is a challenging task due to the possibility of targeted light-controlled activation of anticancer agents without affecting healthy tissues. Also, a conjugation of photosensitizers and clinically used platinum drugs into one Pt(IV) prodrug allows combining photodynamic therapy and chemotherapy approaches into one molecule. Herein, we designed the cisplatin-based Pt(IV) prodrug Riboplatin with tetraacetylriboflavin in the axial position. A novel Pt(IV) prodrug is able to act both as a photodynamic therapy (PDT) agent through the conversion of ground-state 3O2 to excited-state 1O2 and as an agent of photoactivated chemotherapy (PACT) through releasing of cisplatin under gentle blue light irradiation, without the requirement of a reducing agent. The light-induced behavior of Riboplatin was investigated using an electrochemical sensor in MCF-7 tumor spheroids. Photocontrolled cisplatin release and ROS generation were detected electrochemically in real time. This appears to be the first confirmation of simultaneous photoactivated release of anticancer drug cisplatin and ROS from a dual-action Pt(IV) prodrug observed from the inside of living tumor spheroids.

Anticancer Nanotherapeutics in Clinical Trials: The Work behind Clinical Translation of Nanomedicine.

The ultimate goal of nanomedicine has always been the generation of translational technologies that can ameliorate current therapies. Cancer disease represented the primary target of nanotechnology applied to medicine, since its clinical management is characterized by very toxic therapeutics. In this effort, nanomedicine showed the potential to improve the targeting of different drugs by improving their pharmacokinetics properties and to provide the means to generate new concept of treatments based on physical treatments and biologics. In this review, we considered different platforms that reached the clinical trial investigation, providing an objective analysis about their physical and chemical properties and the working mechanism at the basis of their tumoritr opic properties. With this review, we aim to help other scientists in the field in conceiving their delivering platforms for clinical translation by providing solid examples of technologies that eventually were tested and sometimes approved for human therapy.

Electrochemical Detection of a Novel Pt(IV) Prodrug with the Metronidazole Axial Ligand in the Hypoxic Area.

We report herein a Pt(IV) prodrug with metronidazole in axial positions Pt-Mnz. The nitroaromatic axial ligand was conjugated with a cisplatin scaffold to irreversibly reduce under hypoxic conditions, thereby retaining the Pt(IV) prodrug in the area of hypoxia. X-ray near-edge adsorption spectroscopy (XANES) on dried drug-preincubated tumor cell samples revealed a gradual release of cisplatin from the Pt-Mnz prodrug instead of rapid intracellular degradation. The ability of the prodrug to penetrate into three-dimensional (3D) spheroid cellular cultures was evaluated by a novel electrochemical assay via a platinum-coated carbon nanoelectrode, capable of single-cell measurements. Using a unique technique of electrochemical measurements in single tumor spheroids, we were able to both detect the real-time response of the axial ligand to hypoxia and establish the depth of penetration of the drug into the tumor model.

Pt(IV) Prodrugs with Non-Steroidal Anti-inflammatory Drugs in the Axial Position.

We report herein the design, synthesis, and biological investigation of a series of novel Pt(IV) prodrugs with non-steroidal anti-inflammatory drugs naproxen, diclofenac, and flurbiprofen, as well as these with stearic acid in the axial position. Six Pt(IV) prodrugs 5-10 were designed, which showed superior antiproliferative activity compared to cisplatin as well as an ability to overcome tumor cell line resistance to cisplatin. By tuning the drug lipophilicity via variation of the axial ligands, the most potent Pt(IV) prodrug 7 was obtained, with an enhanced cellular accumulation of up to 153-fold that of cisplatin and nanomolar cytotoxicity both in 2D and 3D cell cultures. Pt2+ species were detected at different depths of MCF-7 spheroids after incubation with Pt(IV) prodrugs using a Pt-coated carbon nanoelectrode. Cisplatin accumulation in vivo in the murine mammary EMT6 tumor tissue of BALB/c mice after Pt(IV) prodrug injection was proved electrochemically as well. The drug tolerance study on BALB/c mice showed good tolerance of 7 in doses up to 8 mg/kg.

New Cysteine-Containing PEG-Glycerolipid Increases the Bloodstream Circulation Time of Upconverting Nanoparticles.

Upconverting nanoparticles have unique spectral and photophysical properties that make them suitable for development of theranostics for imaging and treating large and deep-seated tumors. Nanoparticles based on NaYF4 crystals doped with lanthanides Yb3+ and Er3+ were obtained by the high-temperature decomposition of trifluoroacetates in oleic acid and 1-octadecene. Such particles have pronounced hydrophobic properties. Therefore, to obtain stable dispersions in aqueous media for the study of their properties in vivo and in vitro, the polyethylene glycol (PEG)-glycerolipids of various structures were obtained. To increase the circulation time of PEG-lipid coated nanoparticles in the bloodstream, long-chain substituents are needed to be attached to the glycerol backbone using ether bonds. To prevent nanoparticle aggregation, an L-cysteine-derived negatively charged carboxy group should be included in the lipid molecule.

Biomimetic approaches for targeting tumor-promoting inflammation.

With the ultimate goal of increasing tumor accumulation of therapeutics, various nanocarriers have been designed to overcome biological barriers encountered at each stage, from drug administration to the cancerous lesion. Stabilizing circulation and functionalization of the targeting surface impart high tumor accumulation properties to nanocarriers. However, various cells can recognize and infiltrate the tumor microenvironment more efficiently than synthetic carriers via overexpression of adhesive ligands, particularly in inflamed stroma of tumors. Thus, a new field of nanomedicine, called biomimicry, has evolved to generate nanoparticles with the same biological characteristics as cells that naturally infiltrate tumors. Revolutionary synthetic processes have been developed to transfer the cell membrane of leukocytes and mesenchymal cells to synthetic carriers. In addition, cells can generate their own "nanocarriers," known as exosomes, to transport molecular messages to distant sites, while biomimicry of viral and bacterial agents allows high targeting efficiency towards inflammatory immune cells. Alterations in the protein expression in cancer cells caused by inflammation can also be exploited for drug delivery. Finally, new developments in biomimetic drug delivery focus on turning the infiltrating cells into microcarriers that can actively perfuse the tumor and eventually release their therapeutic payload. In this review, we summarize recent developments in biomimetic drug delivery with a particular focus on targeting the tumor inflammatory microenvironment.

In Vitro/In Vivo Electrochemical Detection of Pt(II) Species.

The biodistribution of chemotherapy compounds within tumor tissue is one of the main challenges in the development of antineoplastic drugs, and techniques for simple, inexpensive, sensitive, and selective detection of various analytes in tumors are of great importance. In this paper we propose the use of platinized carbon nanoelectrodes (PtNEs) for the electrochemical detection of platinum-based drugs in various biological models, including single cells and tumor spheroids in vitro and inside solid tumors in vivo. We have demonstrated the quantitative direct detection of Pt(II) in breast adenocarcinoma MCF-7 cells treated with cisplatin and a cisplatin-based DNP prodrug. To realize the potential of this technique in advanced tumor models, we measured Pt(II) in 3D tumor spheroids in vitro and in tumor-bearing mice in vivo. The concentration gradient of Pt(II) species correlated with the distance from the sample surface in MCF-7 tumor spheroids. We then performed the detection of Pt(II) species in tumor-bearing mice treated intravenously with cisplatin and DNP. We found that there was deeper penetration of DNP in comparison to cisplatin. This research demonstrates a minimally invasive, real-time electrochemical technique for the study of platinum-based drugs.

Nanocurcumin-Loaded UCNPs for Cancer Theranostics: Physicochemical Properties, In Vitro Toxicity, and In Vivo Imaging Studies.

Formulation of promising anticancer herbal drug curcumin as a nanoscale-sized curcumin (nanocurcumin) improved its delivery to cells and organisms both in vitro and in vivo. We report on coupling nanocurcumin with upconversion nanoparticles (UCNPs) using Poly (lactic-co-glycolic Acid) (PLGA) to endow visualisation in the near-infrared transparency window. Nanocurcumin was prepared by solvent-antisolvent method. NaYF4:Yb,Er (UCNP1) and NaYF4:Yb,Tm (UCNP2) nanoparticles were synthesised by reverse microemulsion method and then functionalized it with PLGA to form UCNP-PLGA nanocarrier followed up by loading with the solvent-antisolvent process synthesized herbal nanocurcumin. The UCNP samples were extensively characterised with XRD, Raman, FTIR, DSC, TGA, UV-VIS-NIR spectrophotometer, Upconversion spectrofluorometer, HRSEM, EDAX and Zeta Potential analyses. UCNP1-PLGA-nanocurcumin exhibited emission at 520, 540, 660 nm and UCNP2-PLGA-nanocurmin showed emission at 480 and 800 nm spectral bands. UCNP-PLGA-nanocurcumin incubated with rat glioblastoma cells demonstrated moderate cytotoxicity, 60-80% cell viability at 0.12-0.02 mg/mL marginally suitable for therapeutic applications. The cytotoxicity of UCNPs evaluated in tumour spheroids models confirmed UCNP-PLGA-nanocurcumin therapeutic potential. As-synthesised curcumin-loaded nanocomplexes were administered in tumour-bearing laboratory animals (Lewis lung cancer model) and showed adequate contrast to enable in vivo and ex vivo study of UCNP-PLGA-nanocurcumin bio distribution in organs, with dominant distribution in the liver and lungs. Our studies demonstrate promise of nanocurcumin-loaded upconversion nanoparticles for theranostics applications.

Alternative mechanism of action of the DNP PtIV prodrug: intracellular cisplatin release and the mitochondria-mediated apoptotic pathway.

In a recent research paper Dr. Suxing Jin et al. reported two multispecific PtIV complexes DNP and NP with non-steroidal anti-inflammatory drug naproxen (NPX) as the axial ligand(s). Herein, we clarify the mechanism of action of DNP, its therapeutic target and intracellular redox-status.

Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Rapamycin synergizes the cytotoxic effects of MEK inhibitor binimetinib and overcomes acquired resistance to therapy in melanoma cell lines in vitro.

Objective The problem of drug resistance to BRAF-targeted therapy often occurs in melanoma treatment. Activation of PI3K/AKT/mTOR signaling pathway is one of the mechanisms of acquired resistance and a potential target for treatment. In the current research, we investigated that dual inhibition of mTOR and MEK synergistically reduced the viability of melanoma cells in vitro. Methods A combination of rapamycin (a macrolide immunosuppressant, mTOR inhibitor) and binimetinib (an anti-cancer small molecule, selective inhibitor of MEK) was studied using a panel of melanoma cell lines, including patient-derived cells. Results It was found, that combinatorial therapy of rapamycin (250 nM) and binimetinib (2 µM) resulted in 25% of cell viability compared to either rapamycin (85%) or binimetinib alone (50%) for A375 and vemurafenib-resistant Mel IL/R cells. The suppressed activation of mTOR and MEK by combined rapamycin and binimetinib treatment was confirmed using Western blot assay. Cell death occured via the apoptosis pathway; however, the combination treatment significantly increased the apoptosis only for Mel IL/R cells. The enhanced cytotoxic effect was also associated with enhanced cell cycle arrest in the G0/G1 phase. Conclusion In general, we provide the evidence that dual inhibition of mTOR and MEK could be promising for further preclinical investigations.

Fabrication and evaluation of nanocontainers for lipophilic anticancer drug delivery in 3D in vitro model.

Presently, most of anticancer drugs are high toxic for normal cells and, and as a result, they have severe side effects. Moreover, most of the formulations are lipophilic and have poor selectivity. To overcome these limitations, various drug delivery systems could be proposed. The aim of the current study was to fabricate novel polysaccharide nanocontainers (NC) by one-step ultrasonication technique and to evaluate their accumulation efficacy and cytotoxicity in 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. NC with mean sizes in a range of 340-420 nm with the core-shell structure are synthetized and characterized. The NC shell is composed from diethylaminoethyl dextran/xanthan gum polyelectrolyte complex, while the hydrophobic core was loaded with the lipophilic anticancer drug thymoquinone. To enhance NC accumulation in human breast adenocarcinoma MCF-7 cells, the NC surface was modified with poly-L-lysine (PLL) or polyethylene glycol. Cell uptake of the NC loaded with Nile Red into the tumor cells was investigated by laser scanning confocal microscopy, fluorescent flow cytometry and fluorimetry. Modification of the NC with PLL allowed to obtain the optimal drug delivery system with maximal cytotoxicity, which was tested by MTT-test. The developed NC are promising for lipophilic anticancer drug delivery.

Polymer-Scaffolded Synthesis of Periodic Mesoporous Organosilica Nanomaterials for Delivery Systems in Cancer Cells.

We developed four types of para-phenylene-bridged periodic mesoporous organosilica NPs (p-P PMO NPs) with tailored physical parameters including size, morphology, porosity, and surface area using a new polymer-scaffolding approach. The particles have been formulated to facilitate the codelivery of small-molecule hydrophobic/hydrophilic cargos such as model anticancer drugs (i.e., doxorubicin hydrochloride (DOX) and O6-benzylguanine) and model fluorescent dyes (i.e., rhodamine 6G and Nile red). p-P PMO NPs were synthesized via a cetyltrimethylammonium bromide (CTAB)-directed sol-gel process using two different organic solvents and in the presence of polymeric scaffolding constituents that led to morphologically distinct PMO NPs despite using the same organosilane precursors. After the formulation process, the polymeric scaffolding agent was conveniently washed away from the PMO NPs. Extensive analyses were used to characterize the physicochemical attributes of the PMO NPs such as their chemical composition, morphologies, etc. Spherical and rod-shaped PMOs of diameters ranging between 79 and 342 nm, surface areas between 770 and 1060 m2/g, and pore volumes between 0.79 and 1.37 cm3/g were prepared using the polymer-scaffolding approach. The performance of these materials toward drug-loading capacity, cytotoxicity, and cancer cell internalization was evaluated. Interestingly, the designed particles exhibited significantly high payloads of drugs and dyes (up to 78 and 94%, respectively). Cellular studies also demonstrated exceptional biocompatibility and marked internalization into both human breast cancer MCF-7 and glioblastoma U-87 MG cells. Further, DOX also possessed a noticeable release from particles and accumulation in cell nuclei with increased incubation time in vitro. Ultimately, this work validates the controlled design and synthesis of PMO NPs using a polymer-scaffolding approach and highlights the potential of these materials as excellent delivery systems for combination therapy with high loading capability to improve the therapeutic index for cancers.

Novel Copper-Containing Cytotoxic Agents Based on 2-Thioxoimidazolones.

A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the structure-activity relationship within a series of analogues with different substituents at the N(3) position, which govern the redox potentials of the Cu+2/Cu+1 redox couples, ROS generation ability, and intracellular accumulation. Possible cytotoxicity mechanisms, such as DNA damage, DNA intercalation, telomerase inhibition, and apoptosis induction, have been investigated. ROS formation in MCF-7 cells and three-dimensional (3D) spheroids was proven using the Pt-nanoelectrode. Drug accumulation and ROS formation at 40-60 µm spheroid depths were found to be the key factors for the drug efficacy in the 3D tumor model, governed by the Cu+2/Cu+1 redox potential. A nontoxic in vivo single-dose evaluation for two binuclear mixed-valence Cu+1/Cu+2 redox-active coordination compounds, 72k and 61k, was conducted.

Novel bexarotene derivatives: Synthesis and cytotoxicity evaluation for glioma cells in 2D and 3D in vitro models.

Glioblastoma (GBM) is an aggressive and lethal form of brain cancer with a high invasion capacity and a lack of effective chemotherapeutics. Retinoid bexarotene (BXR) inhibits the neurospheroidal colony formation and migration of primary glioblastoma cells but has side effects. To enhance the BXR glioblastoma selectivity and cytotoxicity, we chemically modified it at the carboxyl group with either nitroethanolamine (NEA) bearing a NO-donating group (a well-known bioactivity enhancer; BXR-NEA) or with a dopamine (DA) moiety (to represent the highly toxic for various tumor cells N-acyldopamine family; BXR-DA). These two novel compounds were tested in the 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Both BXR-DA and BXR-NEA were found to be more toxic for rat C6 and human U-87MG glioma cells than the initial BXR. After 24 h incubation of the cells (monolayer culture) with the drugs, the IC50 values were in the range of 28-42, and 122-152 µM for BXR derivatives and BXR, respectively. The cell death occurred via apoptosis according to the annexin staining and caspase activation. The tumor spheroids demonstrated higher resistance to the treatment compared to that one of the monolayer cultures. BXR-DA and BXR-NEA were more specific against tumor cells than the parental drug, in particular the selectivity index was 1.8-2.7 vs. 1.3-1.5, respectively. Moreover, they inhibited cell migration more effectively than parental BXR according to a scratch assay. Cell spreading from the tumor spheroids was also inhibited. Thus, the obtained BXR derivatives could be promising for glioblastoma treatment.

Local Overheating of Biotissue Labeled With Upconversion Nanoparticles Under Yb3+ Resonance Excitation.

Local overheating of biotissue is a critical step for biomedical applications, such as photothermal therapy, enhancement of vascular permeability, remote control of drug release, and so on. Overheating of biological tissue when exposed to light is usually realized by utilizing the materials with a high-absorption cross section (gold, silica, carbon nanoparticles, etc.). Here, we demonstrate core/shell NaYF4:Yb3+, Tm3+/NaYF4 upconversion nanoparticles (UCNPs) commonly used for bioimaging as promising near-infrared (NIR) absorbers for local overheating of biotissue. We assume that achievable temperature of tissue labeled with nanoparticles is high enough because of Yb3+ resonance absorption of NIR radiation, whereas the use of auxiliary light-absorbing materials or shells is optional for photothermal therapy. For this purpose, a computational model of tissue heating based on the energy balance equations was developed and verified with the experimentally obtained thermal-graphic maps of a mouse in response to the 975-nm laser irradiation. Labeling of biotissue with UCNPs was found to increase the local temperature up to 2°C compared to that of the non-labeled area under the laser intensity lower than 1 W/cm2. The cellular response to the UCNP-initiated hyperthermia at subcritical ablation temperatures (lower than 42°C) was demonstrated by measuring the heat shock protein overexpression. This indicates that the absorption cross section of Yb3+ in UCNPs is relatively large, and microscopic temperature of nanoparticles exceeds the integral tissue temperature. In summary, a new approach based on the use of UCNP without any additional NIR absorbers was used to demonstrate a simple approach in the development of photoluminescent probes for simultaneous bioimaging and local hyperthermia.