Igor Nabiev

Molecular Interaction of Proteins and Peptides with Nanoparticles

The interaction of proteins in living cells is one of the key processes in the maintenance of their homeostasis. Introduction of additional agents into the chain of these interactions may influence homeostatic processes. Recent advances in nanotechnologies have led to a wide use of nanoparticles (NPs) in industrial and biomedical applications. NPs are small enough to enter almost all compartments of the body, including cells and organelles, and to complicate the pattern of protein interactions. In some cases, interaction of nanoscale objects with proteins leads to hazardous consequences, such as abnormal conformational changes leading to exposure of cryptic peptide epitopes or the appearance of abnormal functions caused by structural modifications. In addition, the high local protein concentration resulting from protein adsorption on NPs may provoke avidity effects arising from close spatial repetition of the same protein. Finally, the interaction of NPs with proteins is known to induce cooperative effects, such as promotion or inhibition of protein fibrillation or self-assembling of NPs on macromolecules serving as a template. It is obvious that better understanding of the molecular mechanisms of nano-bio interactions is crucial for further advances in all nanotechnological applications. This review summarizes recent progress in understanding the molecular mechanisms of the interactions between proteins or peptides and NPs in order to predict the structural, functional, and/or nanotoxic consequences of these interactions.

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy.

A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10−10M) solutions of doxorubicin or adriamycin (DOX), and 4′O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.

Highly Stable Fluorescent Nanocrystals as a Novel Class of Labels for Immunohistochemical Analysis of Paraffin-Embedded Tissue Sections

Highly Stable Fluorescent Nanocrystals as a Novel Class of Labels for Immunohistochemical Analysis of Paraffin-Embedded Tissue Sections

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes

UNLABELLED Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.

Fluorescent Quantum Dots as Artificial Antennas for Enhanced Light Harvesting and Energy Transfer to Photosynthetic Reaction Centers

The development of artificial photosynthetic systems that utilize solar energy is one of the most challenging goals of chemistry and material sciences. The straightforward way to construct an artificial photosynthetic device for practical solar fuel production for the practical use of solar energy is to mimic the structural and functional organization of the natural photosynthetic machinery. In photosynthetic organisms, light is initially absorbed by antenna protein–pigment complexes in which it induces an excited electronic state (exciton), and then excitons (or electron–hole pairs) are transferred by means of F rster resonance energy transfer (FRET) to specialist chlorophyll cofactors in specialized reaction centers (RCs); here, excitons dissociate into their constituent carriers which are used in chemical transformations for the synthesis of high-energy molecules that fuel the organism. An artificial device that mimics this process for solar energy conversion should include, among other components, an efficient light-harvesting antenna capable of transferring the excitation energy to the RC. Based on the principle of photosynthesis, a variety of artificial antenna systems have been developed using supramolecular chemistry in which dendrimers incorporate porphyrins or other organic fluorophores or organometallic complexes. Although efficient excitation-energy transfer was obtained in such systems, the use of organic fluorophores in light-harvesting systems is rather limited because of their narrow spectral windows for light-collecting and lack of photostability. Recently it was suggested that inorganic nanocrystals, which are able to collect light over a wide spectral window, may achieve significantly greater absorption than natural photosystems, thus enhancing and could thus be used to enhance the light-harvesting process. Simultaneously, these nanocrystals may also be very efficient in excitationenergy transfer. This has led us to contemplate the development of hybrid materials in which light energy harvested by the nanocrystals in the optical region may be transferred to the RC in order to enhance the efficiency of the photosynthetic process. The simplest and best understood photosynthetic RC is that found in purple bacteria (Rhodobacter sphaeroides, for example). Although RCs from different photosynthetic organisms vary in their structure and composition, they are always composed of complexes of pigments and proteins, and RC fromRb. sphaeroides is known to be a good model of all the photosynthetic RCs. Here, we demonstrate that photoluminescent quantum dots (QDs) of these selected photoluminescence (PL) wavelengths may be tagged with the RC of Rh. sphaeroides in such a way that FRET from the QD to the RC is realized (Figure 1). A nearly threefold increase in the rate of generation of excitons in the RC is demonstrated, and theoretical estimates predict even stronger enhancements, thus indicating that further optimization is possible. Advances in inorganic synthesis have resulted in the production of monodispersed QDs such as highly photoluminescent CdSe/ZnS core/shell and CdTe nanocrystals. The light absorption by these QDs appears as a quasicontinuous superposition of peaks with extinction coefficients orders of magnitude higher than those of organic molecules. QDs are ultrastable against photobleaching, and the quantum [*] Prof. I. Nabiev CIC NanoGUNE Consolider, 20018 San Sebastian (Spain) and EA n83798, Universit de Reims Champagne-Ardenne 51100 Reims (France) and Ikerbasque, Basque Foundation for Science 48011 Bilbao (Spain) Fax: (+34)943-574-001 E-mail: i.nabiev@nanogune.eu

Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery

Arguably targeting is one of the biggest problems for controlled drug delivery. In the case that drugs can be directed with high efficiency to the target tissue, side effects of medication are drastically reduced. Colloidal inorganic nanoparticles (NPs) have been proposed and described in the last 10years as new platforms for in vivo delivery. However, though NPs can introduce plentiful functional properties (such as controlled destruction of tissue by local heating or local generation of free radicals), targeting remains an issue of intense research efforts. While passive targeting of NPs has been reported (the so-called enhanced permeation and retention, EPR effect), still improved active targeting would be highly desirable. One classical approach for active targeting is mediated by molecular recognition via capture molecules, i.e. antibodies (Abs) specific for the target. In order to apply this strategy for NPs, they need to be conjugated with Abs against specific biomarkers. Though many approaches have been reported in this direction, the controlled bioconjugation of NPs is still a challenge. In this article the strategies of controlled bioconjugation of NPs will be reviewed giving particular emphasis to the following questions: 1) how can the number of capture molecules per NP be precisely adjusted, and 2) how can the Abs be attached to NP surfaces in an oriented way. Solution of both questions is a cornerstone in controlled targeting of the inorganic NPs bioconjugates.

Quantum dot surface chemistry and functionalization for cell targeting and imaging.

Quantum 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.

Probing Cell‐Type‐Specific Intracellular Nanoscale Barriers Using Size‐Tuned Quantum Dots

The compartmentalization of size-tuned luminescent semiconductor nanocrystal quantum dots (QDs) in four distinctive cell lines, which would be representative of the most likely environmental exposure routes to nanoparticles in humans, is studied. The cells are fixed and permeabilized prior to the addition of the QDs, thus eliminating any cell-membrane-associated effects due to active QD uptake mechanisms or to specificity of signaling routes in different cell types, but leaving intact the putative physical subcellular barriers. All quantitative assays are performed using a high content analysis (HCA) platform, thereby obtaining robust data on large cell populations. While smaller QDs 2.1 nm in diameter enter the nuclei and localize to the nucleoli in all cell types, the rate and dynamics of their passage vary depending on the cell origin. As the QD size is increased to 4.4 nm, penetration into the cell is reduced but each cell line displays its own cutoff size thresholds reflecting cell-type-determined cytoplasmic and nuclear pore penetration specificity. These results give rise to important considerations regarding the differential compartmentalization and susceptibility of organs, tissues, and cells to nanoparticles, and may be of prime importance for biomedical imaging and drug-delivery research employing nanoparticle-based probes and systems.

Basic Principles and Current Trends in Colloidal Synthesis of Highly Luminescent Semiconductor Nanocrystals

The principal methods for the synthesis of highly luminescent core-shell colloidal quantum dots (QDs) of the most widely used CdSe, CdS, ZnSe, and other A(II) B(VI) nanocrystals are reviewed. One-pot versus multistage core synthesis approaches are discussed. The noninjection one-pot method ensures slow, controllable growth of core nanocrystals starting from magic-size seed recrystallization, which yields defect-free cores with strictly specified sizes and shapes and a high monodispersity. Subsequent injection of shell precursors allows the formation of gradient core-shell QDs with a smooth potential barrier for electrons and holes, without strains or interfacial defects, and, as a consequence, a luminescence quantum yield (QY) approaching 100%. These general approaches can also be applied to semiconductor core-shell QDs other than A(II) B(VI) ones to cover the broad spectral range from the near-UV to IR regions of the optical spectrum, thus displacing fluorescent organic dyes from their application areas.

Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts

New antitumor 12-alkoxy-benzo[c]phenanthridinium derivatives were obtained in high yields through multistep syntheses. Analysis of DNA binding and human DNA topoisomerase I inhibitory activities demonstrates that new compounds, combining 2, 6, and 12 substitutions, interact strongly with DNA and exhibit important topoisomerase I inhibition. The cytotoxicities against solid tumor cell lines are also determined and compared with those for fagaronine and ethoxidine.