KEYWORDS: 3D image processing, 3D modeling, Tissue optics, Neuroimaging, Brain, Optical imaging, 3D surface sensing, Optical sensing, Animal model studies, Brain cancer
Successful clinical translation of optical techniques and therapies that advance the detection and treatment of high-grade brain cancer, glioblastoma multiforme (GBM), needs controlled, ethical and practical GBM models that accurately represent the biological reality. However, the available test-beds are not biologically accurate (artificial phantoms); are hindered by complex physiology and ethical concerns (animal models); or involve practical complexity due to rapid biological degradation of the samples ex vivo (surgical biopsies). Here, we present the development and validation of an in vitro, biologically accurate, 3-dimensional living GBM tumour model produced by tissue engineering techniques.
Our 3D living equivalents of GBM tumour tissue are in the millimeter size range, consist of brain-specific extracellular matrix and living cells, and exhibit the relevant (often unfavorable) tissue optical properties such as scattering and tissue auto-fluorescence. The model also reproduces essential challenges in translational neurophotonics that are due to uneven tissue surface topography, variation in structural, optical and biochemical properties of matrix, heterogeneous cellular phenotypes and uneven distribution of exogenous contrast and therapeutic agents.
We will show results of depth-resolved and wide-field imaging of the living GBM-equivalents in laboratory microscopic and theatre-based imaging systems under normal and fluorescence-guided surgery conditions using the typical 5-ALA to fluorescent PpIX conversion by GBM cells, in addition to 3D mapping of exogenous contrast agents such as fluorescent cell viability markers. These results illustrate the versatility of our 3D-engineered GBM model as macroscopic test-bed for the development of optical tools to improve the detection and treatment of brain cancer.
Background: Upconverting nanoparticles (UCNPs) represent a unique class of nanomaterials, able to convert infrared excitation light into long lifetime visible and infrared photoluminescence, within the “optical transparency window” of biological tissues. This makes UCNPs an attractive contrast agent for background-free bioimaging. However, assynthesized UCNPs are hydrophobic and need additional surface coating for stability in water-based solutions and further functionalization. Polyethylenimine (PEI), a polycationic amphiphilic polymer, is a well-known transfection agent for gene delivery and a popular material for UCNPs surface hydrophilization. Combining the functional properties of UCNPs and PEI is extremely useful for precise visualization of genetic manipulations and intracellular drug delivery. At the same time, PEI is toxic to cells, while the photoluminescent properties of UCNPs are very sensitive to surface chemistry and environment. Then, creation of hydrophilic, biocompatible and simultaneously bright UCNPs, modified by PEI (UCNP-PEI), is a challenging task.
Objectives: To analyze the effects of multilayer shielding coatings on cytotoxicity, cellular uptake and photoluminescent properties of UCNP-PEI.
Methods and results: UCNP-PEI were modified with additional two or three layers of various polymers and characterized by size, surface charge and photophysical properties. HaCaT keratinocytes were exposed to the particles for 24 or 120 h to study the cytotoxicity and cellular uptake. The results show that onion-like coatings of UCNP-PEI simultaneously decrease cytotoxicity and relative luminescence of the particles, depending on structure and method of formation of multilayer coating.
Conclusions: Rational design of UCNP-PEI using extra coatings layers can help to keep acceptable levels of biocompatibility and photoluminescence intensity.
Nanoparticle-based delivery of drugs and contrast agents holds great promise in cancer research, because of the increased delivery efficiency compared to ‘free’ drugs and dyes. A versatile platform to investigate nanotechnology is the chick embryo chorioallantoic membrane tumour model, due to its availability (easy, cheap) and accessibility (interventions, imaging). In our group, we developed this model using several tumour cell lines (e.g. breast cancer, colon cancer). In addition, we have synthesized in-house silica coated photoluminescent upconversion nanoparticles with several functional groups (COOH, NH2, PEG). In this work we will present the systematic assessment of their in vivo blood circulation times. To this end, we injected chick embryos grown ex ovo with the functionalized UCNPs and obtained a small amount of blood at several time points after injection to create blood smears The UCNP signal from the blood smears was quantified using a modified inverted microscope imaging set-up. The results of this systematic study are valuable to optimize biochemistry protocols and guide nanomedicine advancement in the versatile chick embryo tumour model.
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.
Widespread applications of nanotechnology materials have raised safety concerns due to their possible penetration through skin and concomitant uptake in the organism. This calls for systematic study of nanoparticle transport kinetics in skin, where high-resolution optical imaging approaches are often preferred. We report on application of emerging luminescence nanomaterial, called upconversion nanoparticles (UCNPs), to optical imaging in skin that results in complete suppression of background due to the excitation light back-scattering and biological tissue autofluorescence. Freshly excised intact and microneedle-treated human skin samples were topically coated with oil formulation of UCNPs and optically imaged. In the first case, 8- and 32-nm UCNPs stayed at the topmost layer of the intact skin, stratum corneum. In the second case, 8-nm nanoparticles were found localized at indentations made by the microneedle spreading in dermis very slowly (estimated diffusion coefficient, Dnp=3-7×10−12 cm2·s−1). The maximum possible UCNP-imaging contrast was attained by suppressing the background level to that of the electronic noise, which was estimated to be superior in comparison with the existing optical labels.
We report about preliminary results of using upconversion luminophores (UCLs) for tissue imaging. We manufactured
luminophores particles of different sizes and tissue-mimicking phantoms for this study. Results of our experiments on
imaging correspond well with our Monte Carlo simulations of luminescence detection from an imbedded vessel filled
with UCLs.
A method is proposed for determining blood oxygen saturation in frozen tissue. The method is based on a spectral
camera system equipped with an Acoustic-Optical-Tuneable-Filter. The HSI-setup is validated by measuring series of
unfrozen and frozen samples of a hemoglobin-solution, a hemoglobin-intralipid mixture and whole blood with varying
oxygen saturation. The theoretically predicted linear relation between oxygen saturation and absorbance was observed in
both the frozen sample series and the unfrozen series. In a final proof of principal, frozen myocardial tissue was
measured. Higher saturation values were recorded for ventricle and atria tissue compared to the septum and connective
tissue. These results are not validated by measurements with another method. The formation of methemoglobin during
freezing and the presence of myoglobin in the tissue turned out to be possible sources of error.
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