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

Quantum dots induce charge-specific amyloid-like fibrillation of insulin at physiological conditions

International audienceAgglomeration of some proteins may give rise to aggregates that have been identified as the main cause of amyloid diseases. For example, fibrillation of insulin is related to diabetes mellitus. Quantum dots (QDs) are of special interest as tagging agents for diagnostic and therapeutic studies due to their broad absorption spectra, narrow emission spectra, and high photostability. In this study, PEGylated CdSe/ZnS QDs have been shown to induce the formation of amyloid-like fibrils of human insulin under physiological conditions, this process being dependent on the variation of the surface charge of the nanoparticles (NPs) used. Circular dichroism (CD), protein secondary structure analysis, thioflavin T (ThT) fluorescence assay, and the dynamic light scattering (DLS) technique have been used for comparative analysis of different stages of the fibrillation process. In particular, insulin secondary structure remodelling accompanied by a considerable increase in the rate of amyloid fiber formation have been observed after insulin was mixed with PEGylated QDs. Nanoparticles may significantly influence the rate of protein fibrillation and induce new mechanisms of amyloid diseases, as well as offer opportunities for their treatment

Label-Free Flow Multiplex Biosensing via Photonic Crystal Surface Mode Detection

International audienceCirculating cancer markers are metabolic products found in body fluids of cancer patients, which are specific for a certain type of malignant tumors. Cancer marker detection plays a key role in cancer diagnosis, treatment, and disease monitoring. The growing need for early cancer diagnosis requires quick and sensitive analytical approaches to detection of cancer markers. The approach based on the photonic crystal surface mode (PC SM) detection has been developed as a label-free high-precision biosensing technique. It allows real-time monitoring of molecular and cellular interactions using independent recording of the total internal reflection angle and the excitation angle of the PC surface wave. We used the PC SM technique for simultaneous detection of the ovarian cancer marker cancer antigen 125 and two breast cancer markers, human epidermal growth factor receptor 2 and cancer antigen 15-3. The new assay is based on the real-time flow detection of specific interaction between the antigens and capture antibodies. Its particular advantage is the possibility of multichannel recording with the same chip, which can be used for multiplexed detection of several cancer markers in a single experiment. The developed approach demonstrates high specificity and sensitivity for detection of all three biomarkers

Quantum Dot Surface Chemistry and Functionalization for Cell Targeting and Imaging

International audienceQuantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay

Structure–function relationships in polymeric multilayer capsules designed for cancer drug delivery

International audienceThe targeted delivery of cancer drugs to tumor-specific molecular targets represents a major challenge in modern personalized cancer medicine. Engineering of micron and submicron polymeric multilayer capsules allows the obtaining of multifunctional theranostic systems serving as controllable stimulus-responsive tools with a high clinical potential to be used in cancer therapy and detection. The functionalities of such theranostic systems are determined by the design and structural properties of the capsules. This review (1) describes the current issues in designing cancer cell–targeting polymeric multilayer capsules, (2) analyzes the effects of the interactions of the capsules with the cellular and molecular constituents of biological fluids, and (3) presents the key structural parameters determining the effectiveness of capsule targeting. The influence of the morphological and physicochemical parameters and the origin of the structural components and surface ligands on the functional activity of polymeric multilayer capsules at the molecular, cellular, and whole-body levels are summarized. The basic structural and functional principles determining the future trends of theranostic capsule development are established and discussed

Photonic Crystal Surface Mode Imaging for Multiplexed Real-Time Detection of Antibodies, Oligonucleotides, and DNA Repair Proteins

Sensors based on photonic crystal (PC) surface mode imaging are promising tools for label-free drug screening and discovery, diagnostics, and analysis of ligand–receptor interactions. Imaging of PC surface modes has been demonstrated to allow simultaneous real-time detection of multiple events at the sensor surface. Here, we report the engineering of a lateral-flow microfluidic assay where PC surface mode imaging is used for multiplexed detection of biomolecular targets (antibodies, oligonucleotides, and a DNA repair protein), as well as kinetic data on their interactions obtained without additional labelling or signal amplification. Our data demonstrate the suitability of the biosensing platform designed for ultrasensitive, quick, and low-cost detection and monitoring of interactions between different biomolecules

Designing Functionalized Polyelectrolyte Microcapsules for Cancer Treatment

International audienceThe engineering of delivery systems for drugs and contrasting labels ensuring the simultaneous imaging and treatment of malignant tumors is an important hurdle in developing new tools for cancer therapy and diagnosis. Polyelectrolyte microcapsules (MCs), formed by nanosized interpolymer complexes, represent a promising platform for the designing of multipurpose agents, functionalized with various components, including high- and low-molecular-weight substances, metal nanoparticles, and organic fluorescent dyes. Here, we have developed size-homogenous MCs with different structures (core/shell and shell types) and microbeads containing doxorubicin (DOX) as a model anticancer drug, and fluorescent semiconductor nanocrystals (quantum dots, QDs) as fluorescent nanolabels. In this study, we suggest approaches to the encapsulation of DOX at different stages of the MC synthesis and describe the optimal conditions for the optical encoding of MCs with water-soluble QDs. The results of primary characterization of the designed microcarriers, including particle analysis, the efficacy of DOX and QDs encapsulation, and the drug release kinetics are reported. The polyelectrolyte MCs developed here ensure a modified (prolonged) release of DOX, under conditions close to normal and tumor tissues; they possess a bright fluorescence that paves the way to their exploitation for the delivery of antitumor drugs and fluorescence imaging

Dependence of Nanoparticle Toxicity on Their Physical and Chemical Properties

Abstract 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

Oriented Conjugates of Single-domain Antibodies and Fluorescent Quantum Dots for Highly Sensitive Detection of Tumor-associated Biomarkers in Cells and Tissues

International audienceOur recent results in the field of engineering and application of highly oriented conjugates of single-domain antibodies and fluorescent quantum dots are summarized. These novel conjugates proved to be excellent nanoprobes for immunolabeling of tumor-associated biomarkers on cells and tissue specimens detectable by means of flow cytometry or one- or two-photon confocal microscopy. The results are discussed in terms of the most promising future applications of these conjugates and further developments in this field

Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase IIα, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas

International audienceHuman DNA topoisomerase I (topo I) is the molecular target ofthe camptothecin group of anticancer drugs. Laboratory studies haveshown that the cellular response to topo I–targeted drugs depends onthe topo I expression and DNA replication rate and the apoptoticpathway activity. In this study, we tested potential indicators of thesensitivity of topo I–targeted drugs in 36 cases of oral squamous cellcarcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sec-tions were immunostained with monoclonal antibodies against Ki-67,p53, and topo I, and with polyclonal antibodies against DNA topo-isomerase II-alpha (topo II-alpha). These markers were also tested in 18epithelial hyperplastic lesions and 18 mild dysplasias. Immunostain-ing was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei werecounted per case for each antibody. The results support the possi-bility of using topo II-alpha staining for assessing the proliferative activ-ity. High expression of topo II-and topo I in OSCCs suggests thatthey may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostain-ing was found to be uninformative. Analysis of the relationshipbetween immunohistochemical results and clinical and pathologicparameters (the T and N stages and differentiation) showed that onlythe differentiation parameter correlated with the topo I expressionrate. Thus, significant increase in the topo I expression in the poorlydifferentiated OSCCs suggests their higher sensitivity to drugtreatment