Vladimir A. Semchishen

Feasibility study of the optical imaging of a breast cancer lesion labeled with upconversion nanoparticle biocomplexes

Abstract. Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications.

Quantitative Imaging of Single Upconversion Nanoparticles in Biological Tissue

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.

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.

PEG-modified upconversion nanoparticles for in vivo optical imaging of tumors

A novel surface modification approach of brightly luminescent upconversion nanoparticles (UCNPs) is reported. Inorganic core@shell UCNPs (core – NaYF4 co-doped with Yb3+ and Tm3+ ions, shell – NaYF4) were modified by intercalation with amphiphilic copolymer poly(maleic anhydride-alt-1-octadecene) followed by cross-linking with poly(ethylene glycol) diglycidyl ether (PEG-DGE). The proposed approach enables preparation of UCNPs with an outmost PEG-containing layer, which provides steric stabilization and low non-specific protein adsorption. Intravenous injection of PEG-functionalized UCNPs into the mice results in extension of the UCNP blood circulation time up to 1 hour. In vivo epi-luminescence imaging of the mouse model with Lewis lung carcinoma is ensured by the high quantum yield of the modified UCNPs and passive targeting associated with efficient UCNP accumulation in solid tumors.

Enhanced spatial resolution in optical imaging of biotissues labelled with upconversion nanoparticles using a fibre-optic probe scanning technique

The new generation of synthetic nanomaterials, upconversion nanoparticles (UCNPs), have the potential for high-contrast optical imaging of biological tissue by virtue of their unique luminescent properties which enable the autofluorescence and excitation signals to be completely suppressed and avoid biotissue absorption. The potential for deep tissue imaging, such as whole animal imaging, is demonstrated in this report on a comparative study of two epiluminescent imaging methods suitable for the localization of a UCNP-labelled pathology site buried in highly scattering biological tissue modelled by an optical tissue phantom. The lateral resolution exhibited in scanning imaging by an illumination-collection fibre-optic probe appeared to be almost 1.73 times better than that shown by the wide-field CCD commonly used in diffuse optical tomography systems. We attribute this improved lateral resolution to the enhanced angular selectivity of the illumination-collection regime and to the nonlinear dependence of the UCNP luminescence on the excitation intensity.

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.

Light beam shaping and homogenization (LSBH) by irregular microlens structure for medical applications

Purpose: The increasing interest in a homogeneous Gaussian light beam profile for applications in ophthalmology e.g. photorefractive keratectomy (PRK) requests simple optical systems with low energy losses. Therefore, we developed the Light Shaping Beam Homogenizer (LSBH) working from UV up to mid-IR. Method: The irregular microlenses structure on a quartz surface was fabricated by using photolithography, chemical etching and chemical polishing processes. This created a three dimensional structure on the quartz substrate characterized in case of a Gaussian beam by random law distribution of individual irregularities tilts. The LSBH was realized for the 193 nm and the 2.94 micrometer wavelengths. Simulation results obtained by 3-D analysis for an arbitrary incident light beam were compared to experimental results. Results: The correlation to a numerical Gaussian fit is better than 94% with high uniformity for an incident beam with an intensity modulation of nearly 100%. In the far field the cross section of the beam shows always rotation symmetry. Transmittance and damage threshold of the LSBH are only dependent on the substrate characteristics. Conclusions: considering our experimental and simulation results it is possible to control the angular distribution of the beam intensity after LSBH with higher efficiency compared to diffraction or holographic optical elements.

Model for Optimization of the UV-A/Riboflavin Strengthening (cross-linking) of the Cornea: Percolation Threshold

To achieve the maximum level of collagen strengthening within the shortest treatment time possible, we have developed a mathematical model which is used to optimize the process of corneal cross‐linking. This model is able to predict the temporal and spatial distribution of generated cross‐links within the corneal stroma and hence the increase in the elasticity modulus. Theory predicts corneal strengthening at low radiation intensities and the absence of the strengthening effect at radiation intensities above the threshold level, which agrees with the experimental results. The model takes account of the initial riboflavin concentration and bleaching, light intensity and time of illumination.

Biocompatible upconversion ink for hidden anticounterfeit labeling

Ink for rapid the application of anticounterfeit labels by means of standard printing devices has been developed based on upconversion nanoparticles (nanophosphors). Printing ink is made of an aqueous dispersion of nanoparticles with a NaYF4:YbTm/NaYF4 core–shell structure at a concentration of 0.5 mg/mL. The surface of nanoparticles is modified with amphiphilic polymers. The biosafety of ink is demonstrated in primary cultures of human fibroblasts. The hidden labeling, which is invisible in ambient lighting, is performed by the method of inkjet printing on paper. A printed image is visualized by IR laser irradiation at a wavelength of 975 nm. It is demonstrated that additional modalities of protection can be obtained by encoding the spectra and intensities of the anti-Stokes luminescence lines when combining the dopant lanthanides in nanoparticles.

Application of semiconductor and upconversion nanomaterials in cosmetics, coatings, and phantoms

In this paper, an overview of selected applications of semiconductor (TiO2 and ZnO) and upconversion nanoparticles is presented. Depending on the size, the former are used as scattering and absorbing compounds in sunscreens and tissuemimicking phantoms; and in combination with gypsum – also as an antibacterial coating for indoor premises, while the latter, especially in combination with optical clearing – as a promising component for deep-biotissue imaging both in vitro and in vivo.