SESSION C - BIONANOTECHNOLOGY, NANOMATERIALS IN MEDICINE

In this presentation I will present 2 groups of nanoparticles: Magnetic nanoparticles and gold nanocrystals, they have applications in their own right and yet are intertwined for more advanced combination of magnetic and optical properties. High magnetic moment nanoparticles will have many potential applications in sensing, separation, diagnosis and treatment of diseases such as cancer, which is still a leading cause of disease worldwide. Magnetic nanoparticles offers an exciting new alternative treatment, with local heating of affected areas allows to eliminate the side effect of current radio-or chemotherapy. The most cutting edge research has recently focused on the combination of heat treatment with traditional cancer drugs. That synergistic effect with enhance the treatment efficacy of both hyperthermia and drugs. While gold nanoparticles could be used for sensing and diagnosis.
REFERENCES:
I. Robinson, S. Zacchini, L.D. Tung, S. Maenosono, N.T.K. Thanh (2009). Synthesis and Characterization of Magnetic Nanoalloys from Bimetallic Carbonyl Clusters, Chem. Mater. 13: 3021.// 3. L. T. Lu, L. D. Tung, J. Long, D.G. Fernig and N.T.K. Thanh (2009) Facile and Green Synthesis of Stable, Water-soluble Magnetic CoPt Hollow Nanostructures Assisted by Multi-thiol Ligands, Journal of Material Chemistry, 19: 6023// 4. Blanco-Andujar, C., Ortega, D., Pankhurst, Q. A., Thanh, N. T. K.* (2012) Elucidating the morphological and structural evolution of iron oxide nanoparticles formed by sodium carbonate in aqueous medium. J.Mat. Chem., 12498-12506. //5. Blanco-Andujar, C., Southern, P., Ortega, D., Nesbitt, S.A., Pankhurst, Q.A., Thanh, N.T.K.* (2015) High performance multi-core iron oxide nanoparticles for magnetic hyperthermia: microwave synthesis, and the role of core-to-core interactions. Nanoscale. 7: 1768-1775.// 6. R. M. Pallares, S. L. Kong, H. R. Tan, Thanh, N.T.K *, Y. Lu and X. Su (2015) A plasmonic nanosensor with inverse sensitivity for circulating cell-free DNA quantification. ChemComm. 2015, DOI: 10.1039/C5CC05331E

Proteome modifications in a biological system can potentially mirror the occurrence of pathologies, even if the individuation of the proteome fingerprint correlated to a specific disease represents a very complex task. When a nanomaterial is introduced in a biological fluid, protein compete for the formation of a protein corona on the nanoparticle surface, thus, depending on the specific proteome, different patterns of proteins will form the final protein corona shell, depending on their affinity for the nanoparticle surface. Novel surface active maghemite nanoparticles (SAMNs) display a remarkable selectivity toward protein corona formation, thus they are able to concentrate proteins and peptides presenting high affinity for their surface even if present in very low amount. Among 3000 proteins present in fetal calf serum, SAMNs lead to the formation of a self-assembled corona shell with 22 selected proteins, representing minor plasma proteins, conversely bovine serum albumin (BSA), representing 80% of the total serum proteins, shows negligible absorption. Moreover, SAMNs were introduced in milk samples from healthy cows and from animals affected by mastitis, and the selectively bound protein corona shell was easily analyzed and quantified by gel electrophoresis and characterized by mass spectrometry. Upon incubation in mastitic milk, SAMNs were able to selectively bind αs2-casein fragments containing the FALPQYLK sequence, which were not present in healthy samples. The present report proposes protein competition for SAMN protein corona formation as a mean for mirroring proteome modifications, thus the selected protein shell on nanoparticles can result in a fingerprint of a specific pathology.

In this work, activated carbon particles (CPs) were functionalized with amide-amine groups varying in alkyl chain lengths and the number of amine groups. Four types of composite scaffolds were prepared via sputtering CPs into electrospun polycaprolactone (PCL) micro-nanofibres: three of them with three types of functionalized CPs and one with plain activated CPs. Plain PCL nanofibres and the composite micro-nanofibrous scaffold with plain activated CPs were used as comparative samples. The structure of the materials was studied using scanning electron microscopy. The specific surface area of the particles and scaffolds was measured via nitrogen and krypton adsorption and calculated from Brunauer-Emmet-Teller equation. Cytocompatibility of the materials was tested using 3T3 mouse fibroblasts. Cell viability and proliferation was measured by MTT assay on days 1, 3, 8 and 14 after cell seeding. The samples were then stained using fluorescent dyes and examined via fluorescence microscopy (FM). During the FM analyses, all scaffolds containing CPs underwent degradation when irradiated with either green or blue light, which could be used for selective degradation. However, the mechanism of the degradation has not been fully explained yet. The scaffolds with functionalised CPs showed better cytocompatibility than the scaffold with plain CPs.

Zinc oxide is known as an inorganic antibacterial agent and exhibits strong both antibacterial and antifungal activity. ZnO antibacterial efficiency strongly depends on its particles size, composition, defective structure, and, subsequently, the method of ZnO nanoparticles preparation plays an important role in their activity. In the present work pulsed laser ablation (PLA) of metallic Zn target was used to prepare ZnO nanoparticles dispersions in liquids. This method does not require using chemical additives (stabilizing agents, surfactants) and leads to the formation of “pure” nanoparticles. Utilizing such prepared dispersions and powders allowed to investigate antibacterial efficiency of ZnO nanoparticles without influence of additive agents, and to study a mechanism of their activity. It was found, that ZnO nanoparticles obtained by PLA shown higher antibacterial activity than that of silver nanoparticles.
ACKNOWLEDGEMENTS:
This study was supported by The Tomsk State University Academic D.I. Mendeleev Fund Program (research grant # 8.2.57.2015).

Multifunctional Nanoparticles Technology MFNPT is considered worldwide as the next ‘big revolution. In particular, knowledge based nanocolloid growth, nanoparticles surface engineering along with Up-scaling count to the most challenging activities. However, future highly competitive nanobiomarkets will desire a rapid screening of best performing nanocolloidal candidates and their conversion into customized prototypes ready for commercial uses. All that will only be possible by introducing the key enabling technologies eliminating the unwanted human and laboratory artifacts. This contribution gives a brief overview of current approaches to tackle the aforementioned goals.
In the first part, selected critical parameters will be discussed which control the nanoparticles quality reflected in superconcentrated non-aggregated colloidal state as well as in tunable size range of electronic quantum confinement and thermodynamic melting point depression. The second part of this talk highlights the most progressive key enabling technologies in wet chemical nanomaterials design: (1) WANDA platform (Work station for Automated Nanomaterials Development and Analysis) for differently sized and shaped nanocolloids ; (2) fs-Laser TPA nm/µm/mm-printing platform allows rapid perfectly programmed manufacturing of artificial structure-tuned materials (Metamaterials), which bring many new interesting (and yet unexploited) potential applications, controlled not only by chemical compositions but mainly by the designed architecture per se.

The interaction of cells with materials designed for body implantation depends on physicochemical surface properties of these materials. In this study, we investigated the behavior of human osteoblast-like MG-63 cells in cultures on Ti-6Al-4V alloy, stainless steel AISI 316 LWM, polyetheretherketon (PEEK) and ultra-high molecular weight polyethylene (UHMWPE) modified by shot blasting, polishing, coating with amorphous hydrogenated carbon (a-C:H) and combinations of these treatments.
On days 1 after seeding, the highest cell number and spreading were obtained on shot-blasted and a-C:H-coated Ti-6Al-4V. These cells also contained the highest concentration of beta1-integrin adhesion receptors.
On day 3, the highest cell numbers were obtained on polished and a-C:H-coated Ti-6Al-4V. A relatively high cell number was also obtained on polished stainless steel. The cells on both types of samples contained the highest concentrations of alphav-integrins, vinculin and osteocalcin.
On day 8, the highest cell numbers were obtained on shot-blasted and a-C:H-coated Ti-6Al-4V and on non-modified PEEK. The cells on non-modified PEEK contained a relatively high concentration of talin, and on polished PEEK, the highest concentration of osteopontin. The lowest cell number was obtained on polished and relatively highly hydrophobic UHMWPE.
Therefore, the shot-blasted and a-C:H-coated Ti-6Al-4V, polished stainless steel and PEEK seem to be the most appropriate materials for constructing bone-anchoring parts of joint prostheses, while polished UHMWPE can be used for creation of articular surfaces, where the colonization with cells is not desirable.
ACKNOWLEDGEMENTS:
Supported by the GACR (grant No. P107/12/1025) and the Ministry of Health of the Czech Republic (grant No. 15-32497A).

Engineering tiny nano-bots that actively and directly transport payloads to specific locations is envisioned to be part of future nano-bio-technology. Mimicking biomotors, scientists used catalytic reaction to power artificial nano-bots. Self-powered micro-nano-bots can be fabricated from multiple materials, shapes and by various methods, and have demonstrated several proof-of-concept applications in robotics, Lab-chip biosensing, nanomedicine, and environmental field [1].
Nanomotors demonstrated the transport of drugs [2] micro-objects [3] and cells [4] with wireless magnetic guidance [5], temperature [6], and light [7]. Furthermore, they can act collectively reacting to external stimuli like chemotactic behaviour [8] and are capable of cleaning polluted water [9].
Here, I will present our recent developments in this fascinating field. We fabricate nano-bots from mesoporous silica nanoparticles, microspheres and rolled-up thin films into microtubular jets. Very recently, we have found that hybrid Micro-bio-bots combine the best from the two worlds, biology and nanomaterials providing very promising bio-related applications.
REFERENCES:
[1] S. Sanchez, Ll. Soler and J. Katuri. Angew.Chem.Int.Edit. 54,1414-1444 (2015)
[2] X. Ma, K. Hahn and S. Sanchez. J. Am. Chem.Soc. 137 (15), 4976–4979 (2015)
[3] A. A. Solovev et al, Adv. Funct. Mater., 20, 2430 (2010); Solovev A. A. et al., ACS Nano, 6, 1751. (2012)
[4] S. Sanchez et al, Chem. Commun., 47, 698. (2011)
[5] Khalil, App. Phys. Lett. 103, 172404 (2013)
[6] S. Sanchez et al, J. Am. Chem. Soc., 133, 4860 (2011); Soler et al. LabChip 13, 4299 (2013)
[7] Solovev A.A. et al, Angew. Chem. Int. Ed., 50, 10875. (2011);
[8] Baraban, L. et al., Angew. Chem. Int. Ed., 52, 5552. (2013)
[10] Soler, Ll. et al., ACS Nano, 7, 9611 (2013); Gao, W., Wang, J., ACS Nano, 8, 3170 (2014); Soler, L. Sanchez, S. Nanoscale, 6, 7175 (2014)

Covalent modification of graphene by attachment of suitable heteroatoms represents an attractive way to tailor its physical, chemical and sensing properties. In this context, development of new graphene derivatives for electrochemical sensing might result in fabrication of many new high-performing analytical devices based on different strategies reflecting their particular chemical and/or physical properties. Such biosensors can be afterwards used for detection of specific base sequences, which is important for the rapid diagnosis and treatment of various diseases.
In this work, we present two examples of graphene derivatives: thiofluorographene and low fluorinated graphene. Thiofluorographene, obtained by simple nucleophilic substitution of fluorine atoms in a polar solvent, was used as a low cost biosensor for impedimetric detection of DNA hybridization. Fluorinated graphene prepared in control way by fluorination in F2 stream at defined pressure and temperature was used as platform for electrochemical sensing of different biomolecules, i.e. NADH, ascorbic acid and dopamine.

In the last two decades functionalized gold nanoparticles (AuNPs) have been widely used in optical affinity biosensors for the detection of molecules at low concentrations. Typically, functionalized AuNPs are applied in the last step of the sandwich assay after the target molecules have been captured on the surface of a biosensor chip. This approach allows an increase in the sensitivity of biosensors by several orders of magnitude. Despite this immense potential of functionalized AuNPs, however, experimentally observed enhancements are often lower.
In this contribution we will present and discuss the use of functionalized AuNPs with a surface plasmon resonance (SPR) biosensor for the detection of cancer biomarker carcinoembryonic antigen (CEA). We have established a sandwich assay using AuNPs functionalized with proteins such as antibodies or streptavidin molecules. Specifically, we will discuss how a variety of the experimental aspects affect the enhancement of the SPR signal, including the size of the AuNPs, the detection assay, and the surface functionalization of the AuNPs. Finally, we will demonstrate that for an optimized experimental arrangement we were able to detect CEA marker in 50 % human blood plasma samples at levels as low as 100 pg/mL, which is well below the physiological level of CEA.

Surface plasmon resonance (SPR) is a modern method for the analysis of interactions of biological objects with different types of ultra-thin coatings. Some of them provide adsorption of biomolecules in their correct spatial orientation, thereby increasing the likelihood of obtaining a reliable signal in the study of further interactions, such as "antigen-antibody". In the present work the use of electrochemically synthesized films of polyaniline (PANI/HCl) and its complexes with polyaminosulfo acids (PANI/PSSA and PANI /PAMPSA) is proposed as sensing elements of SPR chips. The properties of these films in a wide range of pH and their interaction with various proteins and influenza virus A were studied. It is shown that the SPR responses of PANI/PSSA and PANI / PAMPSA are changed significantly in the pH range of 5-7, which is probably related to occurring structural changes. These results correlate with the spectrophotometry data. It is found that the surface charge of the films is changed by the introduction of various acids during the electrochemical synthesis. It is reflected in ability of the films to adsorb proteins with different pI. It was found that the influenza virus A/Texas/50/2012 adsorbed reversibly on the surface of PANI/HCl. However there is irreversible adsorption of antibodies to the virus in the correct spatial orientation that enables subsequent "antigen-antibody" reaction. It has been found that introduction of the influenza virus into the system with adsorbed antibodies leads to a brief increase in SPR signal. Then the signal falls due to complete desorption of immunocomplexes from the biosensor surface. It is shown that the influenza virus adsorbed irreversibly on the surface of interpolymer complexes in a wide concentration range. The findings offer the prospect of the use of these films for the creation of highly sensitive diagnostic systems.
This work was partially supported by the RFBR grant No. 14-03-31817_mol_a

Recent trends in medicine accompanied with a substantial progress in the fields of molecular biology and physiology increase demands on a development of fast and reliable analytical procedures aimed on the analysis of molecular targets frequently present in complex matrixes at ultra-trace concentration levels. One of interesting alternatives to already established procedures is Magnetically Assisted Surface Enhanced Raman spectroscopy (MA-SERS) (1,2).
MA-SERS utilizes surface functionalized nanocomposites, composed of magnetic and noble metal nanoparticles, which allow a simple magnetic separation of selected targets and their consecutive analysis using an effect of a surface enhanced Raman scattering. In this work, several cases will be shown. The developed MA-SERS analytical platform was utilized in the analysis of physiologically active compounds including dopamine(3) and immunoglobulin G(4).
REFERENCES:
(1) Shi, Z.; Wang, T.; Lin, H.; Wang, X.; Ding, J.; Shao, M. Nanoscale 2013, 5 (20), 10029–10033.
(2) Song, J.; Duan, B.; Wang, C.; Zhou, J.; Pu, L.; Fang, Z.; Wang, P.; Lim, T. T.; Duan, H. J. Am. Chem. Soc. 2014, 136 (19), 6838–6841.
(3) Ranc, V.; Markova, Z.; Hajduch, M.; Prucek, R.; Kvitek, L.; Kaslik, J.; Safarova, K.; Zboril, R. Anal. Chem. 2014, 86 (6), 2939–2946.
(4) Balzerova, A.; Fargasova, A.; Markova, Z.; Ranc, V.; Zboril, R. Anal. Chem. 2014, 86 (22), 11107–11114.

Bacterial pathogens are especially dangerous when airborne. Active cells in aerosolized water or dry spores can induce the rapid transmission of diseases. In such cases, counter-epidemic measures must be based on rapid diagnostics of the evolving pathogen.
Immunochemical assays demand the supply of antibodies, which make the rapid detection of multiple pathogens very complex and expensive. Modern genomic methods of bacteria identification can provide the reliable identification of vast majority of known bacteria and their spores, but suffer from the high analysis cost. Thus, rapid detection of bacterial pathogens has to be the antibody-independent chemical assay.
Raman spectroscopy of bacteria provides the fingerprint-quality spectra that can be used for the identification of bacterial genera [DOI:10.1039/b705973f, DOI:10.1039/c2an35448a]. However, normal Raman spectroscopy of bacteria is complicated by the fact that bacterial cells and spores are very weak Raman scatterers, and significant enhancement of bacterial spectra and spectra processing are needed for the reliable identification [DOI:10.1366/10-06173, DOI:10.1111/j.1745-4581.2008.00131.x].
In our work, we obtain SERS spectra of Gram(+) and Gram(-) bacteria either on reference aluminum foil substrates or on aluminosilicate substrates based on halloysite ceramic nanotubes. Aluminosilicate filtering materials could be exploited for the accumulation of biomaterial form air or water for the subsequent SERS analysis. Spectra of bacterial active cells and spores obtained on the Raman peak-free substrates could be processed automatically, thus opening the possibility of automated rapid detection of airborne pathogens.
ACKNOWLEDGEMENTS:
This work is supported by Ministry of Science and Education of Russian Federation (project 16.1812.2014/K).

Beside the development of novel low-molecular-weight anti-cancer agents, new formulations of "classic" cytostatic drugs, so called drug delivery systems (DDS), including their encapsulation into liposomes and nanoparticles or covalent binding to water-soluble polymers and micelles, appear to be a very promising strategy. Described micellar and star polymer-drug conjugates are high-molecular-weight (HMW) drug conjugates designed for enhanced passive tumor accumulation and release of drug in the acidic milieu of a tumor.
The therapeutic efficacy of the conjugates is based on three mechanisms of selectivity toward solid tumors: I) drug accumulation in tumors driven by enhanced permeability and retention (EPR) effect, which results in almost 100 times higher concentration of drug in the solid tumor than in normal tissue, II) pH-dependent release of drug from polymer-drug conjugate, which releases free drug more efficiently at a lower pH in tumors, and III) significant overcoming of drug resistance. The tumor-to-blood and tumor-to-muscle ratio for star and micellar polymer-doxorubicin conjugates increased with time, demonstrating that the conjugates passively accumulate within a tumor mass due to the EPR effect. The anti-tumor activities of linear, micellar and star copolymers containing Dox were compared using a well-defined model of experimental malignant tumor, mouse EL4 T cell lymphoma or various human xenograph models, inoculated in conventional mice or athymic nude mice. The highest efficacy, highest percentage of LTS, was achieved during the treatment with micellar and star conjugates. Moreover, we observed that the pH-sensitive polymer-drug conjugates have the potential to induce excellent antitumor effect without apparent adverse effects in treatment of highly chemotherapy resistant tumor models.
ACKNOWLEDGEMENTS:
This work was supported by the project ‘‘BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University’’ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund and by the Czech Science Foundation (project No. 15-02986S).

Context: Intraoral drug delivery as mucosal delivery pathway provides a huge platform in the pharmaceutical field.
Objective: Combining mucoadhesiveness and controlled release of thio-poly acrylic acid as advanced excipient for buccal drug delivery.
Materials and methods: Mediated by carbodiimide cysteine was covalently attached to poly acrylic acid. This thiomer was assessed with regard to cytotoxicity, stability, mucoadhesion, and rheology as well as release behavior of Lidocaine.
Results: Stability assays of thio-poly acrylic acid was complying with United States Pharmacopeia (USP) requirements. Mucoadhesion assay such as tensile (TWA), bioadhesion, rotating cylinder revealed as this thiomer was superior in comparison to non-thiolated poly acrylic acid with 7.61-fold, 2.8-fold, 5.61-fold improvement , respectively without any toxic effect. Lidocaine release showed 1.98-fold more controlled release over 3 h in comparison to unmodified poly acrylic acid.
Conclusion: Taken the findings in consideration, thio-poly acrylic acid provides excellent stability, controlled release and superior mucoadhesive features. The prolonged residence time of thio-poly acrylic acid represents a pillar in the buccal drug delivery.

New nanofibre-based mucoadhesive film was designed and prepared for oromucosal administration of nanocarriers for delivery of drug and vaccines. The mucoadhesive film is constructed from electrospun nanofibrous layer, mucoadhesive film layer and protective backing layer. While mucoadhesive film layer is responsible for tight adhesion of whole system to the oral mucosa after the application, the electrospun nanofibrous layer serves as a reservoir for therapeutic nanoparticles such as polymeric nanoparticles, liposomes, and potentially broad spectrum of nanopaticles as lipoplexes, virosomes, virus-like particles, dendrimers as well as therapeutic macromolecular drugs, antigens and allergens. The extremely large surface and its functionalization together with high porosity of electrospun nanofibres allow high quantities of nanoparticles be loaded, thus providing a high particle to nanofibre mass ratio. Nanoparticles can either be reversibly adsorbed or linked by non-covalent bonds (e.g. metallochelating or electrostatic interactions) to the surface of nanofibers or can be deposited in the pores in between the nanofibers. Nanofibrous reservoir allows fast release of nanoparticles and the depo at the mucosal surface is formed for prolonged period after the application. Mucoadhesive film was thoroughly studied by TEM, SEM, AFM and confocal microscopy, (další techniky dopsat) and adsobtion of varoious nanoparticles like liposomes, viruses, pDNA, PLGA nanoparticles and bacterial ghosts was demonstrated. The designed system is applicable to oral mucosa, especially to sublingual and buccal tissues and enables trans-/intramucosal delivery of nanoparticles. As proof of concept, the mucosal and lymph node delivery of PLGA-PEG nanoparticles and liposomes was demonstrated in porcine model. The main potential application of the system is sublingual immunisation and development of “printed vaccine technology”.
ACKNOWLEDGEMENTS:
Support: project CENATOX, Grant Agency of Czech Republic, GAP503/12/G147 (JT); the Ministry of Education, Youth and Sports CZ.1.07/2.3.00/20.0164 (JT), MZE0002716202 Czech Ministry of Agriculture; postdoc position of Štěpán Koudelka was supported by the European Social Fund and the State Budget of the Czech Republic – Project FNUSA-ICRC, Support for Neurological Research and Development Teams through Postdoc Position Formations CZ.1.07/2.3.00/30.0043.
PCT application supported by Global Acorn.
The team headed by Jaroslav Turánek, Milan Raška and Andrew D. Miller are obligated to the Ministry of Education, Youth and Sports for supporting of the project “FIT” (Pharmacology, Immunotherapy, nanoToxicology).

In this work chitosan based nanoparticles were prepared and modified by a star-shaped carboxy-terminated polylactide (S-PLA) in order to reduce the doxorubicin burst effect in physiological solution.
S-PLA was prepared by polycondensation reaction using pentetic acid as a core molecule and methansulfonic acid as catalyst and linked to chitosan backbone through coupling reaction. Afterwards, chitosan-(S-PLA) modified nanoparticles loaded with doxorubicin were prepared by polyelectrolytes complexation method using dextran sulfate sodium salt as polyanion.
Results show nanoparticles with diameter in the range 100-200 nm, spherical shape and -potential between 27-35 mV. The presence of (S-PLA) side chain does not influence the encapsulation efficiency of the drug but strongly reduce the initial burst effect compared with unmodified chitosan.

Hyaluronic acid in its natural form is a water-soluble polymer and its dissolving proceeds immediately after coming into a contact with moisture. Its extreme hydrophilic character prevents from several biomedical applications such as implants or anti-adhesive membranes, which must be designed to persist in an organism for defined period of time. Therefore it is necessary to increase water-stability of HA as well as to retain all its biological properties,
HA can be chemically modified to prolong its half-life in vivo and to extend the usage possibilities thereof.
The problems mentioned above are solved by the preparation of cinnamoyl hyaluronan. The nanofibers prepared from this photocurable derivative can be easily stabilized onto a surface by the solid-state photolight irradiation - mediated crosslinking. Such photochemical stabilization is achieved by the esterification of HA hydroxyls with anhydrides of trans-cinnamic acid as a photosensitive group which self-dimerizes under the UV irradiation. Several tie-points are formed onto a surface which results in increased hydrolytic stability of HA nanofibers.
Obtained derivatives were tested in electrospinning process in nanofiber layer preparationon 4 SPIN Technology. As the behaviour under aqueous conditions is limiting for biomedical applications the swelling capacity and water solubility of nanofiber layers made of cinnamoyl HA were tested. Moreover a single nanofiber was prepared and analysed by AFM and force spectroscopy to get the precise information about swelling, stiffness and solubility.