Dependence of Quantum Dot Toxicity In Vitro on Their Size, Chemical Composition, and Surface Charge.

Semiconductor nanocrystals known as quantum dots (QDs) are of great interest for researchers and have potential use in various applications in biomedicine, such as in vitro diagnostics, molecular tracking, in vivo imaging, and drug delivery. Systematic analysis of potential hazardous effects of QDs is necessary to ensure their safe use. In this study, we obtained water-soluble core/shell QDs differing in size, surface charge, and chemical composition of the core. All the synthesized QDs were modified with polyethylene glycol derivatives to obtain outer organic shells protecting them from degradation. The physical and chemical parameters were fully characterized. In vitro cytotoxicity of the QDs was estimated in both normal and tumor cell lines. We demonstrated that QDs with the smallest size had the highest in vitro cytotoxicity. The most toxic QDs were characterized by a low negative surface charge, while positively charged QDs were less cytotoxic, and QDs with a greater negative charge were the least toxic. In contrast, the chemical composition of the QD core did not noticeably affect the cytotoxicity in vitro. This study provides a better understanding of the influence of the QD parameters on their cytotoxicity and can be used to improve the design of QDs.

Nanoparticles With a Specific Size and Surface Charge Promote Disruption of the Secondary Structure and Amyloid-Like Fibrillation of Human Insulin Under Physiological Conditions.

Nanoparticles attract much interest as fluorescent labels for diagnostic and therapeutic tools, although their applications are often hindered by size- and shape-dependent cytotoxicity. This cytotoxicity is related not only to the leak of toxic metals from nanoparticles into a biological solution, but also to molecular cytotoxicity effects determined by the formation of a protein corona, appearance of an altered protein conformation leading to exposure of cryptic epitopes and cooperative effects involved in the interaction of proteins and peptides with nanoparticles. In the last case, nanoparticles may serve, depending on their nature, as centers of self-association or fibrillation of proteins and peptides, provoking amyloid-like proteinopathies, or as inhibitors of self-association of proteins, or they can self-assemble on biopolymers as on templates. In this study, human insulin protein was used to analyze nanoparticle-induced proteinopathy in physiological conditions. It is known that human insulin may form amyloid fibers, but only under extreme experimental conditions (very low pH and high temperatures). Here, we have shown that the quantum dots (QDs) may induce amyloid-like fibrillation of human insulin under physiological conditions through a complex process strongly dependent on the size and surface charge of QDs. The insulin molecular structure and fibril morphology have been shown to be modified at different stages of its fibrillation, which has been proved by comparative analysis of the data obtained using circular dichroism, dynamic light scattering, amyloid-specific thioflavin T (ThT) assay, transmission electron microscopy, and high-speed atomic force microscopy. We have found important roles of the QD size and surface charge in the destabilization of the insulin structure and the subsequent fibrillation. Remodeling of the insulin secondary structure accompanied by remarkable increase in the rate of formation of amyloid-like fibrils under physiologically normal conditions was observed when the protein was incubated with QDs of exact specific diameter coated with slightly negative specific polyethylene glycol (PEG) derivatives. Strongly negatively or slightly positively charged PEG-modified QDs of the same specific diameter or QDs of bigger or smaller diameters had no effect on insulin fibrillation. The observed effects pave the way to the control of amyloidosis proteinopathy by varying the nanoparticle size and surface charge.

Single- and two-photon imaging of human micrometastases and disseminated tumour cells with conjugates of nanobodies and quantum dots.

Early detection of malignant tumours and, especially, micrometastases and disseminated tumour cells is still a challenge. In order to implement highly sensitive diagnostic tools we demonstrate the use of nanoprobes engineered from nanobodies (single-domain antibodies, sdAbs) and fluorescent quantum dots (QDs) for single- and two-photon detection and imaging of human micrometastases and disseminated tumour cells in ex vivo biological samples of breast and pancreatic metastatic tumour mouse models expressing human epidermal growth factor receptor 2 (HER2) or carcinoembryonic antigen (CEA). By staining thin (5-10 µm) paraffin and thick (50 µm) agarose tissue sections, we detected HER2- and CEA-positive human tumour cells infiltrating the surrounding tissues or metastasizing to different organs, including the brain, testis, lung, liver, and lymph nodes. Compared to conventional fluorescently labelled antibodies the sdAb-HER2-QD and sdAb-CEA-QD nanoprobes are superior in detecting micrometastases in tissue sections by lower photobleaching and higher brightness of fluorescence signals ensuring much better discrimination of positive signals versus background. Very high two-photon absorption cross-sections of QDs and small size of the nanoprobes ensure efficient imaging of thick tissue sections unattainable with conventional fluorescent probes. The nanobody-QD probes will help to improve early cancer diagnosis and prognosis of progression by assessing metastasis.

Dependence of Nanoparticle Toxicity on Their Physical and Chemical Properties.

Studies on the methods of nanoparticle (NP) synthesis, analysis of their characteristics, and exploration of new fields of their applications are at the forefront of modern nanotechnology. The possibility of engineering water-soluble NPs has paved the way to their use in various basic and applied biomedical researches. At present, NPs are used in diagnosis for imaging of numerous molecular markers of genetic and autoimmune diseases, malignant tumors, and many other disorders. NPs are also used for targeted delivery of drugs to tissues and organs, with controllable parameters of drug release and accumulation. In addition, there are examples of the use of NPs as active components, e.g., photosensitizers in photodynamic therapy and in hyperthermic tumor destruction through NP incorporation and heating. However, a high toxicity of NPs for living organisms is a strong limiting factor that hinders their use in vivo. Current studies on toxic effects of NPs aimed at identifying the targets and mechanisms of their harmful effects are carried out in cell culture models; studies on the patterns of NP transport, accumulation, degradation, and elimination, in animal models. This review systematizes and summarizes available data on how the mechanisms of NP toxicity for living systems are related to their physical and chemical properties.

IL-13 but not IL-4 signaling via IL-4Rα protects mice from papilloma formation during DMBA/TPA two-step skin carcinogenesis.

Interleukin 4 (IL-4) was shown to be tumor-promoting in full carcinogenesis studies using 3-methylcholanthrene (MCA). Because heretofore the role of IL-4 in DMBA/TPA (9,10-dimethyl-1,2-benz-anthracene/12-O-tetradecanoylphorbol-13-acetate) two-stage carcinogenesis was not studied, we performed such experiments using either IL-4(-/-) or IL-4Rα(-/-) mice. We found that IL-4Rα(-/-) but not IL-4(-/-) mice have enhanced papilloma formation, suggesting that IL-13 may be involved. Indeed, IL-13(-/-) mice developed more papillomas after exposure to DMBA/TPA than their heterozygous IL-13-competent littermate controls. However, when tested in a full carcinogenesis experiment, exposure of mice to 25 µg of MCA, both IL-13(-/-) and IL-13(+/-) mice led to the same incidence of tumors. While IL-4 enhances MCA carcinogenesis, it does not play a measurable role in our DMBA/TPA carcinogenesis experiments. Conversely, IL-13 does not affect MCA carcinogenesis but protects mice from DMBA/TPA carcinogenesis. One possible explanation is that IL-4 and IL-13, although they share a common IL-4Rα chain, regulate signaling in target cells differently by employing distinct JAK/STAT-mediated signaling pathways downstream of IL-13 or IL-4 receptor complexes, resulting in different inflammatory transcriptional programs. Taken together, our results indicate that the course of DMBA/TPA- and MCA-induced carcinogenesis is affected differently by IL-4 versus IL-13-mediated inflammatory cascades.