List of Abstracts 2016: SESSION C - BIONANOTECHNOLOGY, NANOMATERIALS IN MEDICINE

The integration of multiple functionalities in a single object with nanometric size is crucial for the development of efficient tools for nanomedicine applications. In particular, gold nanoparticles (Au NPs) have been intensively exploited as the starting component for the realization of sophisticated nanomedicine agents, where the desired functionalities are conferred by surface conjugation of various substances such as hydrophilic polymers, proteins for selective targeting, organic molecules with specific spectroscopic fingerprints or stimuli-responsive drugs. However, the space available on Au NPs surface is limited and only a finite number of the desired new functions can be hosted.
We recently investigated a different approach to the design of Au NPs-based nanomedicines, in which new physical properties are added by doping the gold crystalline lattice to obtain an alloy. This approach, which is usually complicated by thermodynamic constraints and synthetic challenges, becomes accessible by laser ablation synthesis in liquid solution (LAL). LAL operates out of thermodynamic equilibrium because NPs are obtained by laser ablation of a solid target immersed in a liquid solution.
Here, some examples of gold-based multifunctional structures will be presented, with special focus on binary metal alloys in which the plasmonic properties of gold are combined with the magnetic properties of iron. By acting on the composition of these nanoalloys, one can obtain several properties desirable for a nanomedicine agent, such as simultaneous contrast ability for magnetic resonance imaging, x-ray absorption computed tomography and Raman imaging, or superior plasmon absorption for photothermal applications, or even biodegradability. Besides, these alloys retain the surface chemistry of gold, which is fully exploitable for conjugation with thiolated molecules.
Overall, the LAL approach can be exploited for the realization of a library of multifunctional doped Au NPs and other nanoalloys of interest for nanomedicine applications.

It was the aim of this study to develop SEDDS for oral enoxaparin administration and evaluate it in vitro and in vivo.
The emulsifying properties of SEDDS composed of long chain lipids (LC-SEDDS), medium chain lipids (MC-SEDDS), short chain lipids (SC-SEDDS) and no lipids (NL-SEDDS) were evaluated. Thereafter, enoxaparin was incorporated via hydrophobic ion pairing in chosen SEDDS, which were investigated regarding their mucus permeating properties, stability towards pancreatic lipase, cytotoxicity and drug release profile. Finally, in vivo performance of SEDDS was evaluated.
The average droplet size of chosen LC-SEDDS, MC-SEDDS and NL-SEDDS ranged between 30 - 40 nm. MC-SEEDS containing 30% Captex 8000, 30% Capmul MCM, 30% Cremophor EL and 10% propylene glycol and NL-SEDDS containing 31.5% Labrafil 1944, 22.5% Capmul PG-8, 9% propylene glycol, 27% Cremophor EL and 10% DMSO exhibited 2-fold higher mucus diffusion than LC-SEDDS and were chosen further studies. The enoxaparin-dodecylamine complex (ENOX/DOA) was incorporated in a payload of 2% (w/v) into MC-SEDDS and NL-SEDDS. After 90 min 97% of MC-SEDDS and 5% of NL-SEDDS were degraded by pancreatic lipase. Both MC-SEDDS and NL-SEDDS showed sustained in vitro enoxaparin release. Furthermore, enoxaparin loaded yielded an absolute enoxaparin bioavailability of 2.02% and 2.25%, respectively.
According to the above mentioned findings, SEDDS could be considered as a potential oral LMWH delivery system.

Immunosensors with highly sensitive and rapid detection capabilities of various biomolecules are of a great demand in the field of biomedicine and environmental control. The two mostly employed transducer principles are either optical surface plasmon resonance (SPR) and mass sensitive quartz crystal microbalance (QCM). The first task encountered in the immunosensor development is the preparation of a coating matrix suitable for the immobilization of antibody on the golden layer of SPR or QCM. The most popular approaches are based on wet chemical treatments such as self-assembled monolayers (SAMs) of alkanethiols, disulfides, or polyethylenimine (PEI). However, these approaches suffer from several drawbacks such as a poor stability, long time preparation, unstable baseline or a high level of noise. As an alternative to SAMs, the deposition of thin functional coatings by plasma polymerization can be employed. The plasma polymerization has already been successfully applied to the deposition of thin films containing carboxyl, amine, anhydride groups. It is known that the essential chemical and thickness stability of the plasma layers can be achieved by tuning the plasma parameters. In this work, pulsed plasma polymerization of cyclopropylamine is employed to deposit stable amine-rich thin films on the surfaces of QCM and SPR chips. The AL01 antibody, specific to human serum albumin (HSA), was attached to the QCM and SPR chips surfaces via glutaraldehyde activation. For the developed immunosensors the stable baseline was recorded. Selective and high response was achieved during the reaction with the solution of antigen. The results confirmed that the introduced methodology have great potential for biosensing applications.

Delivery of anti-cancer therapeutic materials specifically to tumor locations is highly needed. Specifically, poor solubility, narrow therapeutic window, and intensive cytotoxicity to normal tissues are the key factors influencing success or failure of cancer therapy.
The nanofibers are innovative materials with specific properties such as high surface area and porousness which make them promising candidates for anti-cancer remedy. The paper propose and study the use of nanofibre-based drug delivery system specifically designed for the cancer therapy. The nanofibers of the drug delivery system was constructed by electrospun polycaprolactone (PCL) nanofibrous layer which served as a reservoir for anticancer drug paclitaxel (PTX). The proposed nanofiber layer allows creation of a fibrous scaffold being able to entrap drugs with a high loading capacity and high encapsulation efficiency thanks to their low weight and inherent high surface to-volume ratio.
The porousness of morphology of PCL nanofibers was observed by scanning electron microscopy. The PTX release profiles of the nanofibre-based carriers with different thickness in PBS solution were analyzed by UV–vis spectrophotometer. In vitro pancreatic cancer cells activities of the nanofibers were examined by WST-1 assay.
Cell culture results showed that the anticancer drug-loaded nanofibers were good in prohibiting the cell attachment and proliferation. The obtained results strongly suggested that PCL nanofibre-based carrier had the effect of controlled release of the PTX and may be promising candidate for delivering anticancer drugs, especially in postoperative local chemotherapy via surgical implantation of the scaffold. ACKNOWLEDGEMENTS: This work was supported by the following grants: (i) the Ministry of Education, Youth and Sports in the framework of National Program for Sustainability I, the OPR & DI project LO1201, Centre for Nanomaterials, Advanced Technologies and Innovation (CZ.1.05/2.1.00/01.0005), (ii) the program EEA and Norway Grants - programme CZ07.

Electrospinning is controlled by wide spectrum of parameters, which include not only process parameters (initial voltage, temperature, air flow, electrode distance) but also the influence of solution composition cannot be excluded. To perform electrospinning of pure hyaluronic acid is very difficult. Therefore the electrospinable support polymer as polyethylene oxide is usually used for such electrospinning process. In our experiments there were prepared solutions for electrospinning with the same tradition composition exhibiting different properties and different process parameters should be used for electrospinning. A pressure to find out the reason of this irregularity appeared.
As the only source of hyaluronic acid was used there was proposed an experiment leading to comparison of electrospinning processes of polyethylene oxide with same molecular weight but different polymer source. The blend of polyethylene oxide and hyaluronic acid was electrospun by patented 4SPIN technology. Experiments consisted of solution preparation, measurement of solution parameters and electrospinning. Finally the step of sterilization by water steam was added.
The results exhibited, that blends significantly differ in solution parameters which is increased by the sterilization step and resulted in changes in electrospinning potential. The viscosity, conductivity and surface tension were decreased. Specific reason was studied by various method as IR spectrometry, TGA, DSC.

Magnetic nanoparticle (MNP) is of high practicle interest in the domain of biomedicine and biomedical application e.g., drug delivery, magnetic fluid hyperthermia, magnetic resonance imaging, biosensing. The MNPs are explored due to its high tunability of its physical and magnetic properties. A considerable effort has been devoted for the synthesis of MNP e.g., ball milling, arc-melting, co-precipitation, microwave and microfluidics techniques. However, hydrogels can offer a new platform for in situ synthesis of biomineralized MNP and magnetic hydrogels (MH), because of its an advantageous controlled network structure and biodegradable property that can be considered as eco-friendly material. In this work, PVP-CMC hydrogel is used as matrix for synthesis of biomineralized MNP, where the unique porous networks structure exhibited within swollen/wet PVP-CMC hydrogel. Here, PVP-CMC hydrogel function as a chemical reactor, where inorganic salts (FeCl2 and FeCl3) are reacting with precipitating agents ammonium hydro-oxide (NH4OH) to generate biomineralized MNP. This method prevents from aggregation and shows a narrow particle size distribution. X-ray diffraction studies reveal the formation of magnetite phase. Structural and morphological analysis using transmission electron microscopy shows the spherical shaped MNP with size of 10 ± 2 nm. The saturation magnetization of the MNP was 78 emu/g and 12 emu/g for MH, respectively. This synthesis process can be used to synthesize “bare” MNP, MH and extended to other oxides by changing the salts for the biomedical applications. ACKNOWLEDGEMENTS: The work was supported by MŠMT ČR-USA Kontakt II (LH14050) and NPUI(LO1504) Ministry of Education, Youth and Sports of the Czech Republic.

Renal clearable ultrasmall nanoparticles (sub-10 nm size) have attracted increasing attention for medicinal applications [1, 2]. In this direction, ultrasmall silicon nanoparticles (Si-NPs) are gaining in importance, especially in the imaging of cancer cells [3]. Si-NPs display tuneable photoluminescence, high resistance against photo-bleaching, chemical stability after functionalization and biocompatibility. Covalent modification of the surface with red-emitting dyes for optical imaging, bifunctional chelator agents for radiometals enabling SPECT or PET, and simultaneously targeting vector molecules opens the avenue for the development of new targeted dual imaging agents. In vivo imaging with fast clearing agents possessing numerous surface groups is very attractive. However, defined surface functionalization, especially with suitable targeting molecules, is fairly challenging. Here we report on the surface functionalization of amine-terminated Si-NPs (< 5 nm) with a defined quantity of red-emitting dyes and vector molecules, in order to design targeting optical imaging agents. The modification of these Si-NPs with appropriate chelators for radiometals, such as 64Cu, allows for performing positron emission tomography. ACKNOWLEDGEMENTS: Work financially supported by Helmholtz Virtual Institute “NanoTracking”, Agreement No. VH-VI-421. REFERENCES: [1] B. H. Kim, M. J. Hackett, J. Park, T. Hyeon, Chemistry of Materials 2014, 26, 59-71.; [2] K. Zarschler, L. Rocks, N. Licciardello, L. Boselli, E. Polo, K. P. Garcia, L. De Cola, H. Stephan, K. A. Dawson, Nanomedicine: Nanotechnology, Biology and Medicine 2016, 12, 1663-1701.; [3] C. Tu, X. Ma, A. House, S. M. Kauzlarich, A. Y. Louie, ACS Medicinal Chemistry Letters 2011, 2, 285-288.

Polymer pen lithography (PPL) is a powerful method to generate patterns of bioactive substances with features in the nano- to microrange and over large surface areas (square centimeters). Especially, it allows for the generation of multiplexed pattern arrays (i.e. more than one component within near vicinity or within one sub-pattern). This makes the technology very interest for applications in the biomedical field ranging from sensing, to screening purposes and cell culture experiments. The multiplexing capability allows to present more than one component within an underlying pattern to a single cell, offering many options for polarization, differentiation and activation experiments. This paper will present recent progress in the application of multiplexed PPL in regard to covalent immobilization by click-chemistry approaches and introduction of versatile binding motifs in form of oligo-nucleotides for DNA-directed immobilization (DDI). The generated patterns can be encapsulated within microfluidic chips for easier handling and enhanced stability and reproducibility in use. Two examples of biomedical applications are presented in the form of mast cell activation studies and the capture of circulating tumor cells (CTCs) in such systems. As example for applications in biosensing, the use of PPL generated ink pads for the multiplexed functionalization of arrays of goblet shaped whispering gallery mode (WGM) sensors is demonstrated.

The demand for miniaturization and parallelization of analytical and diagnostic tests for DNA, proteins, peptides, glycans, the detection of small molecule and screening of polymer properties has resulted in the development of microarray based technologies with spots sizes in the micrometer range. Different support formats are used for these applications as slides, wafers, microplates, microfluidic chips and biosensors. Further development focuses on new components and methods as nanostructured substrates, probe deposition techniques, strategies of probe immobilization and signal amplification using nanoparticles to improve sensitivity, reproducibility and to minimize material consumption.
The various support formats results in different technical challenges for the development and manufacturing phase. Supports with relatively large surfaces as slides or wafers require a highest degree of homogeneity in the biofunctionalization process. Microfluidic chips are made of special polymer materials with complex geometries. In many cases regions of different wettabilities and surface functionality are desirable for fluidic function, reagent storage and capture probe immobilization. Printing of probes on biosensors requires highly exactly spotting at predefined positions based on sophisticated optical systems to enable online target alignment.
We will present new developments in spot based analytics that involve the use of nanomaterials and application of the Scienion picoliter printing technology for reagent deposition and nanomaterial research.

Development of methods allowing determination of even ultralow levels of proteins, including tumor markers, in complex clinical samples is a scientific challenge. Early diagnosis of cancer is crucial in current clinical oncology practice. Levels of tumor markers are usually determined in serum, tissues or rarely in urine. Immunoassays or immunohistochemistry based methods are usually employed and are considered as golden standards. However, their utilization could be, due to their sample requirements, limited in several cases.
Here presented approach utilizes surface enhanced Raman spectroscopy (SERS), with many recognized advantages, including simplicity of use, direct control of the analytic process and ultra-low limits of detection. Developed approach enabled to determine two important tumor markers (HER2 and EpCAM) using a single nanomaterial based sensor at mg/L concentration levels.
Such method could find application not only in research area but also in clinical practice because of its simplicity and ultralow limits of detection.

Ionic interactions between oppositely charged polyelectrolytes and surfactants can - under proper conditions - lead to formation of physical gels. Surfactants form micellar structures enabling to solubilize hydrophobic substances in the otherwise aqueous environment of the gel. Resulting materials can find use in the formulation of delivery systems, e.g. hydrophobic drugs especially in topical applications. We report on the techniques of preparation of such materials from positively charged polysaccharides (chitosan, cationized dextran) and negatively charged surfactants and on their rheological properties in dependence of their composition.

Noninvasive diagnostics provides in situ insight to the structural and functional features of the investigated systems and organs at their original place in the living organism. The use of magnetic and optical techniques is emerging for these purposes. Improved sensing in tissues, which in turn can differentiate normal tissue from diseased one, can be achieved by using magnetic contrast agents (CA) and optical emissive probes. f-Block elements (rare Earths) are outstanding candidates for this, since their chelates combine unique magnetic and luminescent characteristics.
The precipitation of lanthanide complexes from water miscible organic solvents to polyelectrolyte-containing water solutions under effective stirring is an example of easy and effective synthetic pathway to embed large amount of lanthanide functional complexes to the nanoparticle. The synthesis of europium-doped luminescent polyelectrolyte colloids was first described four years ago [1] and later was applied to various terbium [2], and gadolinum [3] complexes.
Polyelectrolyte-coated nanoparticles consisting of terbium and gadolinium complexes with calix [4] arene tetra-diketone ligand were first synthesized. An antenna effect of the ligand on Tb(III) green luminescence and presence of water molecules in the coordination sphere of Gd(III) bring strong luminescent and magnetic performance to core-shell nanoparticles. The size and the structure of the colloids were studied using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The correlation between photophysical and magnetic properties of the nanoparticles and their core composition is highlighted. The core composition was optimized for the longitudinal relaxivity being greater than that of the commercial magnetic resonance imaging (MRI) contrast agents together with high level of Tb(III)-centered luminescence. The easy synthesis of the bifunctional species provides the tuning of both magnetic and luminescent output of nanoparticles via simple variation of lanthanide chelates concentrations in the initial synthetic solution. The exposure by the nanoparticles results in negligible effect on cell viability of periphery human blood lymphocytes, decreased platelet aggregation and bright coloring of pheochromocytoma 12 (PC 12) tumor cells, indicating the nanoparticles as promising candidates for dual magneto-fluorescent bioimaging. REFERENCES: 1. Mustafina, A., et al., Coll. Surf. B: Biointerfaces, 88(1): p. 490-496, 2011.; 2. Shamsutdinova, N., et al., New Journal of Chemistry, 38(9): p. 4130-4140, 2014.; 3. Shamsutdinova, N., et al., Chemistry Select, 1, pp. 1377-1383, 2016.