IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNß and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Heparin-coated iron oxide nanoparticles: application as a liver contrast agent, toxicity and pharmacokinetics.

The prepared heparin-coated iron oxide nanoparticles (Hep-IONPs) contrasted cholangioma tumors in the liver in T2 MRI. The NPs were not toxic to rats and rabbits after 14 days of consecutive IV injections as observed from the monitoring of the body weight and biochemical and hematological parameters. No embryotoxic or immunotoxic side effects of the material were detected. However, we observed mutagenicity of iron oxide NPs in the Ames test and micronucleus assay. The pharmacokinetic studies showed that Hep-IONPs circulated in the blood for 14 days after IV injection. The liver iron level reached its maximum after 6 hours and slowly decreased within 30 days. Altogether, these results suggest that the synthesized Hep-IONPs are promising for use as the MRI contrast agent to identify liver malignancies.

Hafnium Oxide-Based Nanoplatform for Combined Chemoradiotherapy.

Recently, the combined therapy has become one of the main approaches in cancer treatment. Combining different approaches may provide a significant outcome by triggering several death mechanisms or causing increased damage of tumor cells without hurting healthy ones. The supramolecular nanoplatform based on a high-Z metal reported here is a suitable system for the targeted delivery of chemotherapeutic compounds, imaging, and an enhanced radiotherapy outcome. HfO2 nanoparticles coated with oleic acid and a monomethoxypoly(ethylene glycol)-poly(ε-caprolactone) copolymer shell (nanoplatform) are able to accumulate inside cancer cells and release doxorubicin (DOX) under specific conditions. Neither uncoated nor coated nanoparticles show any cytotoxicity in vitro. DOX loaded onto a nanoplatform demonstrates a lower IC50 value than pure DOX. X-ray irradiation of cancer cells loaded with a nanoplatform shows a higher death rate than that for cells without nanoparticles. These results provide an important foundation for the development of complex nanoscale systems for combined cancer treatment.

Cationic Magnetite Nanoparticles for Increasing siRNA Hybridization Rates.

An investigation of the interaction principles of nucleic acids and nanoparticles is a priority for the development of theoretical and methodological approaches to creating bionanocomposite structures, which determines the area and boundaries of biomedical use of developed nanoscale devices. «Nucleic acid-magnetic nanoparticle¼ type constructs are being developed to carry out the highly efficient detection of pathogens, create express systems for genotyping and sequencing, and detect siRNA. However, the data available on the impact of nanoparticles on the behavior of siRNA are insufficient. In this work, using nanoparticles of two classical oxides of inorganic chemistry (magnetite (Fe3O4) and silica (SiO2) nanoparticles), and widely used gold nanoparticles, we show their effect on the rate of siRNA hybridization. It has been determined that magnetite nanoparticles with a positive charge on the surface increase the rate of siRNA hybridization, while negatively charged magnetite and silica nanoparticles, or positively charged gold nanoparticles, do not affect hybridization rates (HR).

Effects of Metal Oxide Nanoparticles on Toll-Like Receptor mRNAs in Human Monocytes.

For the widespread application of nanotechnology in biomedicine, it is necessary to obtain information about their safety. A critical problem is presented by the host immune responses to nanomaterials. It is assumed that the innate immune system plays a crucial role in the interaction of nanomaterials with the host organism. However, there are only fragmented data on the activation of innate immune system factors, such as toll-like receptors (TLRs), by some nanoparticles (NPs). In this study, we investigated TLRs' activation by clinically relevant and promising NPs, such as Fe3O4, TiO2, ZnO, CuO, Ag2O, and AlOOH. Cytotoxicity and effects on innate immunity factors were studied in THP-1(Tohoku Hospital Pediatrics-1) cell culture. NPs caused an increase of TLR-4 and -6 expression, which was comparable with the LPS-induced level. This suggests that the studied NPs can stimulate the innate immune system response inside the host. The data obtained should be taken into account in future research and to create safe-by-design biomedical nanomaterials.

Deoxyribozyme-Based DNA Machines for Cancer Therapy.

Soon after their discovery, RNA-cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ-based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano-associations that can address some of these problems. Herein we discuss the possibility of building RCDZ-based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ-based RNA-cleavage functions. An important advantage of such "nanomachines" is the possibility to cleave a housekeeping gene mRNA in a cancer-cell-specific manner. The proposed design could become a starting point for building sophisticated DNA-based nanodevices for cancer treatment.

Optically Active Hybrid Materials Based on Natural Spider Silk.

Spider silk is a natural material possessing unique properties such as biocompatibility, regenerative and antimicrobial activity, and biodegradability. It is broadly considered an attractive matrix for tissue regeneration applications. Optical monitoring and potential control over tissue regrowth are attractive tools for monitoring of this process. In this work, we show upconversion modification of natural spider silk fibers with inorganic nanoparticles. To achieve upconversion, metal oxide nanoparticles were doped with low concentrations of rare-earth elements, producing potentially biocompatible luminescent nanomaterials. The suggested approach to spider silk modification is efficient and easy to perform, opening up sensing and imaging possibilities of biomaterials in a noninvasive and real-time manner in bio-integration approaches.

Sol-gel derived boehmite nanostructures is a versatile nanoplatform for biomedical applications.

Alumina is one of the most promising carriers for drug delivery due to the long history of its usage as a vaccine adjuvant. Sol-gel synthesis provides excellent conditions for entrapment of biomolecules within an inorganic cage providing stabilization of proteins under the extremal conditions. In this paper, we show in vitro investigation of monodisperse alumina xerogel nanocontainers (AXNCs) using bovine serum albumin as a model protein entrapped in sol-gel alumina building blocks. Particularly, dose and cell-type dependent cytotoxicity in HeLa and A549 cancer cell lines were employed as well as investigation of antibacterial effect and stability of AXNCs in different biological media. It was shown, that the release of entrapped protein could be provided only in low pH buffer (as in cancer cell cytoplasm). This property could be applied for anticancer drug development. We also discovered boehmite nanoparticles effect on horizontal gene transfer and observed the appearance of antibiotic resistance by means of exchanging of the corresponding plasmid between two different E. coli strains. The present work may help to understand better the influence of AXNCs on various biological systems, such as prokaryotic and eukaryotic cells, and the activity of AXNCs in different biological media.

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA.

Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.

Toxicity Patterns of Clinically Relevant Metal Oxide Nanoparticles.

Nanostructured drugs are being approved for clinical use, although there is a serious deficit of systematic studies of these materials. Data on toxicity of nanoparticles (NPs) can vary due to different methods of preparation, size, and shape. We investigated the toxicity against cultured human cells, the acute toxicity in mice, and the influence on conjugative transfer of antibiotic resistance genes of clinically relevant NPs such as TiO2, ZrO2, HfO2, Ta2O5, Fe3O4, and AlOOH. NPs were synthesized as aqueous sols by the same method in aqueous solution, with almost identical size 2-10 nm. None of these NPs was cytotoxic at concentrations compatible with water solubility. Furthermore, TiO2, HfO2, Ta2O5, Fe3O4, and AlOOH were not toxic to mice after oral administration. However, ZrO2 showed rather high toxicity, with LD50 2277.8 mg/kg. Experiments with plasmid transfer between bacteria demonstrated that AlOOH NPs were the most hazardous since this material promoted the emergence of resistance to antibiotics. Thus, although our metal oxide NPs are largely non-toxic, their properties may differ in specific biological situations.

Urokinase-Conjugated Magnetite Nanoparticles as a Promising Drug Delivery System for Targeted Thrombolysis: Synthesis and Preclinical Evaluation.

Mortality and disabilities as outcomes of cardiovascular diseases are primarily related to blood clotting. Optimization of thrombolytic drugs is aimed at the prevention of side effects (in particular, bleeding) associated with a disbalance between coagulation and anticoagulation caused by systemically administered agents. Minimally invasive and efficient approaches to deliver the thrombolytic agent to the site of clot formation are needed. Herein, we report a novel nanocomposite prepared by heparin-mediated cross-linking of urokinase with magnetite nanoparticles (MNPs@uPA). We showed that heparin within the composition evoked no inhibitory effects on urokinase activity. Importantly, the magneto-control further increased the thrombolytic efficacy of the composition. Using our nanocomposition, we demonstrated efficient lysis of experimental clots in vitro and in animal vessels followed by complete restoration of blood flow. No sustained toxicity or hemorrhagic complications were registered in rats and rabbits after single bolus i.v. injection of therapeutic doses of MNPs@uPA. We conclude that MNPs@uPA is a prototype of easy-to-prepare, inexpensive, biocompatible, and noninvasive thrombolytic nanomedicines potentially useful in the treatment of blood clotting.

Magnetite Nanocontainers: Toward Injectable Highly Magnetic Materials for Targeted Drug Delivery.

Nanocontainers based solely on magnetite NPs have been synthesized by indirect gelation of stable magnetite hydrosol at ambient temperature using the microemulsion-assisted sol-gel method. Containers synthesized have adjustable size and consist of ∼10 nm magnetite nanoparticles linked by Fe-O-Fe interparticle bonds. The material demonstrates high magnetization values up to 60 emu/g and low cytotoxicity against both HeLa and postnatal human fibroblast (up to 260 µg/mL). The systems developed are perspective as a drug depot, particularly for magnetically controlled thrombolysis.

Thrombin@Fe3O4 nanoparticles for use as a hemostatic agent in internal bleeding.

Bleeding remains one of the main causes of premature mortality at present, with internal bleeding being the most dangerous case. In this paper, magnetic hemostatic nanoparticles are shown for the first time to assist in minimally invasive treatment of internal bleeding, implying the introduction directly into the circulatory system followed by localization in the bleeding zone due to the application of an external magnetic field. Nanoparticles were produced by entrapping human thrombin (THR) into a sol-gel derived magnetite matrix followed by grinding to sizes below 200 nm and subsequent colloidization. Prepared colloids show protrombotic activity and cause plasma coagulation in in vitro experiments. We also show here using a model blood vessel that the THR@ferria composite does not cause systematic thrombosis due to low activity, but being concentrated by an external magnetic field with simultaneous fibrinogen injection accelerates local hemostasis and stops the bleeding. For instance, a model vessel system with circulating blood at the puncture of the vessel wall and the application of a permanent magnetic field yielded a hemostasis time by a factor of 6.5 shorter than that observed for the control sample. Biocompatibility of composites was tested on HELF and HeLa cells and revealed no toxic effects.

A pure magnetite hydrogel: synthesis, properties and possible applications.

A magnetite-only hydrogel was prepared for the first time by weak base mediated gelation of stable magnetite hydrosols at room temperature. The hydrogel consists of 10 nm magnetite nanoparticles linked by interparticle Fe-O-Fe bonds and has the appearance of a dark-brown viscous thixotropic material. The water content in the hydrogel could be up to 93.6% by mass while volume fraction reaches 99%. The material shows excellent biocompatibility and minor cytotoxic effects at concentrations up to 207 µg mL-1. The gel shows excellent sorption capacity for heavy metal adsorption such as chrome and lead ions, which is 225% more than the adsorption capacity of magnetite nanoparticles. Due to thixotropic nature, the gel demonstrates mechanical stimuli-responsive release behavior with up to 98% release triggered by ultrasound irradiation. The material shows superparamagnetic behavior with a coercivity of 65 emu g-1 at 6000 Oe. The magnetite gels prepared could be used for the production of magnetite aerogels, magnetic drug delivery systems with controlled release and highly efficient sorbents for hydrometallurgy.

The controllable destabilization route for synthesis of low cytotoxic magnetic nanospheres with photonic response.

We present a new approach for obtaining magnetic nanospheres with tunable size and high magnetization. The method is implemented via controllable destabilization of a stable magnetite hydrosol with glycerol, leading to the formation of aggregates followed by their stabilization with the citrate shell. This inexpensive, simple and easily scalable approach required no special equipment. The obtained samples were characterized by high stability and magnetization over 80 emu/g. Effects of synthetic conditions on physicochemical properties of nanospheres were monitored by hydrodynamic size, zeta potential, and polydispersity of magnetite aggregates. The size of the resulting aggregates varied between 650 nm and 40 nm, and the zeta potential from +30 mV to -43 mV by changing the ratio of the reagents. Under optimal conditions the clusters with a diameter of 80 nm were produced with a narrow size distribution ±3 nm. These characteristics allowed for optical response to the external magnetic field, thereby producing a magnetic photon liquid. Due to biocompatibility of the reagents used in the synthesis the nanospheres evoked a negligible cytotoxicity for human non-malignant and tumor cell lines. These results make new materials valuable in photonics and biomedicine.

Synthesis of a rare-earth doped hafnia hydrosol: Towards injectable luminescent nanocolloids.

A major obstacle in the introduction of luminescent nanoparticles (NPs) for medical applications is that quantum dots, the most widely studied luminescent materials, despite being biologically safe after coating with a bioshell, still contain a toxic core mostly consisting of semi-conductor NPs, which are not approved by regulatory agencies. Here we point to a potential solution of this problem by using rare-earth (RE) doped hafnia NPs. Hafnia is approved for medical injections as an effective means for the treatment of radiosensitive and radioresistant tumors and can significantly decrease potential toxicity of RE ions. As a step towards the achievement of this goal we describe the development of a bio-friendly method for the preparation of a stable doped hafnia hydrosol with an isoelectric point (IEP) of 8.2, which shows high fluorescence and biocompatibility in regular coagulant tests and cytotoxic assays.