Photoluminescent Nanomaterials for Medical Biotechnology.

Creation of various photoluminescent nanomaterials has significantly expanded the arsenal of approaches used in modern biomedicine. Their unique photophysical properties can significantly improve the sensitivity and specificity of diagnostic methods, increase therapy effectiveness, and make a theranostic approach to treatment possible through the application of nanoparticle conjugates with functional macromolecules. The most widely used nanomaterials to date are semiconductor quantum dots; gold nanoclusters; carbon dots; nanodiamonds; semiconductor porous silicon; and up-conversion nanoparticles. This paper considers the promising groups of photoluminescent nanomaterials that can be used in medical biotechnology: in particular, for devising agents for optical diagnostic methods, sensorics, and various types of therapy.

Multifunctional Complexes Based on Photoluminescent Upconversion Nanoparticles for Theranostics of the HER2-Positive Tumors.

Combining diagnostic and therapeutic functions in one agent is a promising strategy in the development of personalized approaches to the treatment of cancer. The opportunity to combine diagnostics and therapy appeared with the development of nanobiotechnologies and was realized in the concept of theranostics. To date, a number of promising agents based on nanomaterials capable of diagnosing, targeted therapeutic effects, and monitoring the response of tumor cells were obtained within the approach of theranostics. In this work, a new type of theranostic complexes based on upconversion nanoparticles coated with polyethylene glycol and functionalized with the DARPin-LoPE recombinant targeted toxin was developed. Selective binding of complexes to human breast adenocarcinoma cells overexpressing the HER2 receptor and specific toxicity to them were shown.

Ultraviolet phototoxicity of upconversion nanoparticles illuminated with near-infrared light.

Recently introduced upconversion nanoparticles (UCNPs) have pushed the depth of photodynamic therapy (PDT) treatment to the centimetre range by converting deeply-penetrating near-infrared (NIR) radiation to visible radiation for photoexcitation of PDT drugs. Here we demonstrate that the direct exposure of the cancer tissue to phototoxic ultraviolet radiation generated by NIR-photoexcited UCNPs enabled successful PDT. To this aim, core/shell UCNPs of the formula NaYF4:Yb3+Tm3+/NaYF4 featuring an enhanced band in the ultraviolet UV-A and UV-B spectral bands were rationally designed and synthesised. Coupling UCNPs to the recombinant modules of the Designed Ankyrin Repeat Protein (DARPin) fused to a fluorescent protein mCherry allowed the target delivery of DARPin-mCherry/UCNP to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2/neu receptors, as confirmed by fluorescence microscopy. DARPin-mCherry/UCNPs were demonstrated to be phototoxic to SK-BR-3 cells under 975 nm laser irradiation at a dose of 900 J cm-2 due to the UV photoexcitation of endogenous photosensitizers and concomitant generation of reactive oxygen species. The Lewis lung cancer mouse model was employed to demonstrate the feasibility of PDT using UCNP-mediated UV excitation of endogenous photosensitizers in the tumor tissue at a NIR dose of 1200 J cm-2. This study paves the way for exploring and harnessing UV photoexcitation processes in deep tissues in vivo.

Bioreactor-Based Tumor Tissue Engineering.

This review focuses on modeling of cancer tumors using tissue engineering technology. Tumor tissue engineering (TTE) is a new method of three-dimensional (3D) simulation of malignant neoplasms. Design and development of complex tissue engineering constructs (TECs) that include cancer cells, cell-bearing scaffolds acting as the extracellular matrix, and other components of the tumor microenvironment is at the core of this approach. Although TECs can be transplanted into laboratory animals, the specific aim of TTE is the most realistic reproduction and long-term maintenance of the simulated tumor properties in vitro for cancer biology research and for the development of new methods of diagnosis and treatment of malignant neoplasms. Successful implementation of this challenging idea depends on bioreactor technology, which will enable optimization of culture conditions and control of tumor TECs development. In this review, we analyze the most popular bioreactor types in TTE and the emerging applications.

Riboflavin photoactivation by upconversion nanoparticles for cancer treatment.

Riboflavin (Rf) is a vitamin and endogenous photosensitizer capable to generate reactive oxygen species (ROS) under UV-blue irradiation and kill cancer cells, which are characterized by the enhanced uptake of Rf. We confirmed its phototoxicity on human breast adenocarcinoma cells SK-BR-3 preincubated with 30-µM Rf and irradiated with ultraviolet light, and proved that such Rf concentrations (60 µM) are attainable in vivo in tumour site by systemic intravascular injection. In order to extend the Rf photosensitization depth in cancer tissue to 6 mm in depth, we purpose-designed core/shell upconversion nanoparticles (UCNPs, NaYF4:Yb3+:Tm3+/NaYF4) capable to convert 2% of the deeply-penetrating excitation at 975 nm to ultraviolet-blue power. This power was expended to photosensitise Rf and kill SK-BR-3 cells preincubated with UCNPs and Rf, where the UCNP-Rf energy transfer was photon-mediated with ~14% Förster process contribution. SK-BR-3 xenograft regression in mice was observed for 50 days, following the Rf-UCNPs peritumoural injection and near-infrared light photodynamic treatment of the lesions.

Submicron polyacrolein particles in situ embedded with upconversion nanoparticles for bioassay.

We report a new surface modification approach of upconversion nanoparticles (UCNPs) structured as inorganic hosts NaYF4 codoped with Yb(3+) and Er(3+) based on their encapsulation in a two-stage process of precipitation polymerization of acrolein under alkaline conditions in the presence of UCNPs. The use of tetramethylammonium hydroxide both as an initiator of acrolein polymerization and as an agent for UCNP hydrophilization made it possible to increase the polyacrolein yield up to 90%. This approach enabled the facile, lossless embedment of UCNPs into the polymer particles suitable for bioassay. These particles are readily dispersible in aqueous and physiological buffers, exhibiting excellent photoluminescence properties, chemical stability, and also allow the control of particle diameters. The feasibility of the as-produced photoluminescent polymer particles mean-sized 260 nm for in vivo optical whole-animal imaging was also demonstrated using a home-built epi-luminescence imaging system.

Large-scale production and characterization of biocompatible colloidal nanoalumina.

The rapid uptake of nanomaterials in life sciences calls for the development of universal, high-yield techniques for their production and interfacing with biomolecules. Top-down methods take advantage of the existing variety of bulk and thin-film solid-state materials for improved prediction and control of the resultant nanomaterial properties. We demonstrate the power of this approach using high-energy ball milling (HEBM) of alumina (Al2O3). Nanoalumina particles with a mean size of 25 nm in their most stable α-crystallographic phase were produced in gram quantities, suitable for biological and biomedical applications. Nanomaterial contamination from zirconia balls used in HEBM was reduced from 19 to 2% using a selective acid etching procedure. The biocompatibility of the milled nanomaterial was demonstrated by forming stable colloids in water and physiological buffers, corroborated by zeta potentials of +40 mV and -40 mV and characterized by in vitro cytotoxicity assays. Finally, the feasibility of a milled nanoalumina surface in anchoring a host of functional groups and biomolecules was demonstrated by the functionalization of their surface using facile silane chemistry, resulting in the decoration of the nanoparticle surface with amino groups suitable for further conjugation of biomolecules.

Visualization of upconverting nanoparticles in strongly scattering media.

Optical visualization systems are needed in medical applications for determining the localization of deep-seated luminescent markers in biotissues. The spatial resolution of such systems is limited by the scattering of the tissues. We present a novel epi-luminescent technique, which allows a 1.8-fold increase in the lateral spatial resolution in determining the localization of markers lying deep in a scattering medium compared to the traditional visualization techniques. This goal is attained by using NaYF4:Yb(3+)Tm(3+)@NaYF4 core/shell nanoparticles and special optical fiber probe with combined channels for the excitation and detection of anti-Stokes luminescence signals.

Specific visualization of tumor cells using upconversion nanophosphors.

The development of targeted constructs on the basis of photoluminescent nanoparticles with a high photo- and chemical stability and absorption/emission spectra in the "transparency window" of biological tissues is an important focus area of present-day medical diagnostics. In this work, a targeted two-component construct on the basis of upconversion nanophosphors (UCNPs) and anti-tumor 4D5 scFv was developed for selective labeling of tumor cells overexpressing the HER2 tumor marker characteristic of a number of human malignant tumors. A high affinity barnase : barstar (Bn : Bs) protein pair, which exhibits high stability in a wide range of pH and temperatures, was exploited as a molecular adapter providing self-assembly of the two-component construct. High selectivity for the binding of the two-component 4D5 scFv-Bn : UCNP-Bs construct to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2 was demonstrated. This approach provides an opportunity to produce similar constructs for the visualization of different specific markers in pathogenic tissues, including malignant tumors.

Non-specific cellular uptake of surface-functionalized quantum dots.

We report a systematic empirical study of nanoparticle internalization into cells via non-specific pathways. The nanoparticles were comprised of commercial quantum dots (QDs) that were highly visible under a fluorescence confocal microscope. Surface-modified QDs with basic biologically significant moieties, e.g. carboxyl, amino, and streptavidin, were used, in combination with surface derivatization with polyethylene glycol (PEG) for a range of immortalized cell lines. Internalization rates were derived from image analysis and a detailed discussion about the effect of nanoparticle size, charge and surface groups is presented. We find that PEG derivatization dramatically suppresses the non-specific uptake while PEG-free carboxyl and amine functional groups promote QD internalization. These uptake variations displayed a remarkable consistency across different cell types. The reported results are important for experiments concerned with cellular uptake of surface-functionalized nanomaterials, both when non-specific internalization is undesirable and when it is intended for material to be internalized as efficiently as possible.

Observation and control of blinking nitrogen-vacancy centres in discrete nanodiamonds.

Nitrogen-vacancy colour centres in diamond can undergo strong, spin-sensitive optical transitions under ambient conditions, which makes them attractive for applications in quantum optics, nanoscale magnetometry and biolabelling. Although nitrogen-vacancy centres have been observed in aggregated detonation nanodiamonds and milled nanodiamonds, they have not been observed in very small isolated nanodiamonds. Here, we report the first direct observation of nitrogen-vacancy centres in discrete 5-nm nanodiamonds at room temperature, including evidence for intermittency in the luminescence (blinking) from the nanodiamonds. We also show that it is possible to control this blinking by modifying the surface of the nanodiamonds.

Dialysis-assisted fiber optic spectroscopy for in situ biomedical sensing.

A miniature fiber optic spectrometer enclosed within a semipermeable (dialysis) membrane is proposed for in vivo interstitial sensing applications. The semipermeable membrane acts as a molecular filter, allowing only small molecules to pass through to the sampling volume. This filtering, in principle, should enable continuous in vivo drug sensing, removing the necessity for complex microdialysis systems. We use a biological phantom to examine the reliable detection of a fluorescence signal from small dye molecules in the presence of large fluorophores and scatterers. We find that spectral artefacts arising from scatterers and large fluorophores are substantially suppressed, simplifying the spectral analysis. In addition, the measured sampling rate of 157 s is superior to existing in vivo tissue assaying techniques such as microdialysis, which can take tens of minutes.

Achromatic optical phase shifter-modulator.

We propose and demonstrate, theoretically and experimentally, a novel achromatic optical phase shifter-modulator based on a frequency-domain optical delay line configured to maintain zero group delay as variable phase delay is generated by means of tilting a mirror. Compared with previously reported phase shifter-modulators, e.g., based on the Pancharatnam (geometric) phase, our device is high speed and polarization insensitive and produces a large, bounded phase delay that, uniquely, is one-to-one mapped to a measurable parameter, the tilt angle.

Semiconductor line source for low-coherence interferometry.

The suitability for low-coherence interferometry of a high-power, semiconductor laser line source operated at a forward bias current below threshold is demonstrated. Measurements of the important characteristics of the source are presented. For example, the source produces an output power of 1.3 mW and a spatially uniform coherence length of 16 mum at a bias current of 86% of threshold (250 mA) at 20 degrees C. The usefulness of the source is verified by measurement of the line profile of a contact lens.

Real-time detection technique for Doppler optical coherence tomography.

We propose and demonstrate a novel detection technique, based on a modified electronic phase-locked loop, for Doppler optical coherence tomography. The technique permits real-time simultaneous reflectivity and continuous, bidirectional velocity mapping in turbid media over a wide velocity range with minimal sensitivity penalty compared with conventional optical coherence tomography, which is a major advance over current postprocessing and discrete parallel detection techniques.

Near-field optical microscope image formation: a theoretical and experimental study.

A quantitative comparison between theory and experiment has been carried out for a collection-mode near-field optical microscope. A 30-nm (lateral dimension) cylindrical dielectric sample was imaged. This image was compared with the result of theoretical calculations that used a classical macroscopic nonglobal model based on the excitation theorem. Good agreement was obtained, with image inversion, edge enhancement, and edge asymmetry correctly predicted.