The presentation will overview our on-going activities on laser ablative synthesis of novel biocompatible colloidal nanomaterials and their testing in biomedical tasks. Our original approach is based on ultra-short (fs) laser ablation from a solid target or already formed water-suspended colloids to fabricate “bare” (ligand-free) nanoparticles (NPs) with well-controlled size characteristics, as well as the ablation in the presence of functional molecules (dextran, PEG etc.) to properly coat NPs surface in order to minimize immune response. The presentation will describe different approaches to achieve appropriate characteristics of plasmonic (Au, Ag, TiN) and semiconductor (Si-based structures) nanomaterials and overview their biomedical applications. In particular, we show that bare laser-synthesized Au NPs can provide unique opportunities as SERS probes for identification of biological species such as yeast and bacteria (Listeria innocua and Escherichia coli) based on strong local electric field enhancement and exceptional purity of laser-synthesized NPs. We also show that bare metal nanoparticles synthesized by laser ablation can provide an order of magnitude better response in glucose oxidation tasks, which promises their use as elecrocatalysts in bioimplantable therapeutic devices, as well as overview applications of plasmonic nanomaterials in phototherapy and tissue engineering tasks. We finally overview applications of Si NPs, which exhibit a unique combination of biocompatibility and biodegradability options. In particular, we show that laser-synthesized NPs can provide a much better efficiency compared to chemically synthesized counterparts in a newly introduced method of mild cancer therapy using Si nanoparticles as sensitizers of radiofrequency radiation-based hyperthermia, as well as be efficient markers for bioimaging.
This paper investigates the preparation of composite metal-polymer nanostructures formed on the surface of polyethersulfone (PES) by an excimer laser beam. Conditions for laser beam modification varied with the laser fluence of 4-28 mJ·cm−2 and number of pulses up to 6000. The samples were further deposited with a layer of metals Ag, Au, Pd and Pt with a thickness of 5-20 nm and their surface morphology was examined by atomic force microscopy (AFM). Electrical properties of layers were also investigated and for optical properties, UV-Vis spectroscopy was used. Composites prepared by this approach were studied further for stability under laser modification. Surface morphology measurement revealed the creation of laser-induced periodic surface structures (LIPSS) in forms of nanoripples after modification with laser fluence value of 8 mJ·cm−2 and nanodots after modification with laser fluence value of 16 mJ·cm−2. It has been found that metal layer deposition of the surface leads to the formation of isolated clusters. Those metalized with the treatment of laser fluence 8 mJ∙cm-2, preferably aggregated on the ridge of the ripples and their electrical continuity was further examined. The nanostructured surfaces may be used as an antibacterial material, also with the potential to guide the specific types of cells, such are e.g. osteoblasts.
Polyurethane was treated by nitrogen ions with energy of 20 keV and fluence of 1е14 - 1е16 ions/cm2. Modified polyurethane was investigated by the IR FTIR and EPR spectroscopy, ASM microscopy. The contact angle and the out-of-contact energy of modified polyurethane are measured. Polyurethane after treatment have carbonized layer of 70 nm thickness and partly depolymerized layer below the carbonized layer. Carbonized layer of modified polyurethane contains highly active free radicals. It provides high hydrophilicity of modified polyurethane surface. Polyurethane after treatment have wave-like surface relief and cracks. High activity free radicals and hydrophilicity of modified polyurethane surface provides a saving of biological activity of the covalently attached proteins. As results, it provides to attachment and growing of endothelial cells on the polyurethane surface. The modified polyurethane can be applied for soft tissue medical implants.
TI6Al4V is a titanium alloy widely used in orthopedics. Nanostructure surface improves osseointegration of the implant (1). Incubation of simulated body fluid (SBF) forms hydroxyapatite-like layer on the exposed surfaces.
We have prepared nanostructured Ti6Al4V using anodic oxidation method at 30V. Subsequently, both nanostructured (N) and polished discs (Ti) were exposed to SBF (NH, TiH) for 7 days. The surfaces were evaluated using SEM. Human mesenchymal stem cells (hMSCs) were seeded on the discs at a density 12,000 cell/cm2 in a MEM alpha medium supplemented with 10% fetal bovine serum and penicillin/streptomycin, 40 µg/mL ascorbate-2-phosphate, 100 nM dexamethasone, and 10 mM glycerol-2-phosphate disodium salt for 21 days. The cells were tested for metabolic activity, dsDNA amount; type I collagen synthesis was evaluated using immunohistochemical staining and confocal microscopy analysis.
All samples supported cell adhesion and proliferation. No statistical differences were seen in MTS assay. Both Ti and TiH showed significantly higher DNA amount compared to both nanostructured samples and glass on day 1. On day 21, higher amount of DNA was found on Ti, TiH, NH compared to N. The highest collagen amount was found on N and TiH surfaces, low amount was found on Ti and glass.
SBF positively influenced growth and osteogenic differentiation of hMSCs.
ACKNOWLEDGEMENTS: The project was supported by the Czech Science Foundation project Nr. 16-14758S and by the Ministry of Education, Youth and Sports of the Czech Republic under NPU I: LO1508.
REFERENCE: [1] Filova E. et al. Int J Nanomedicine. 2015; 10:7145-63.
Synthetic biodegradable nanofibers are of great interest as materials for the development of scaffolds in tissue engineering. To ensure nanofiber biocompatibility, the recent research focuses on an increase of their hydrophilicity and surface charge promoting the cell adhesion. A promising way is the modification of nanofiber surfaces by the deposition of plasma polymers containing polar groups. The present work deals with the coating of polycaprolactone (PCL) nanofibrous mats by plasma enhanced chemical vapor deposition (PECVD) of cyclopropylamine (CPA) thin films introducing positively charged amine groups under various plasma conditions. The surface chemistry was studied by X-ray photoelectron spectroscopy (XPS) that revealed its dependence not only on the plasma parameters but also on the type of substrate and its position in the substrate holder. The content of NH2 groups in CPA coatings was determined by chemical derivatization combined with XPS. Scanning electron microscopy proved homogenous and reproducible CPA polymer cover of PCL nanofibers. Regardless of the plasma conditions, cell multiple tests showed an improvement in proliferation of mouse myoblasts and rat vascular smooth muscle cells cultivated on CPA modified PCL nanofibers compared to the untreated control. Besides, an unusual phenomenon was observed in case of mouse myoblasts: cells were extremely attached to the plasma coated PCL surfaces. Treatment with trypsin/ethylenediaminetetraacetic acid (EDTA), which allows cells to detach from the control surface in 10 minutes, was ineffective. Cells remained attached even after 30 minutes of trypsinisation. This phenotype is currently under study in order to reveal its mechanism.
Hierarchical plant surface structures show many characteristics that are an inspiration for humans. They are associated with the process of photosynthesis and provide a hydrophobic or a hydrophilic behaviour, an absorption of solar radiation, etc. Due to evaluation of synthetic analogical structures we are able to obtain important information about their potential applicability in a technical practice. The easiest way to prepare them is a two-stage replication process. This process involves the creation of the so-called first replica, that is the mold for the second replica - the “copy” of natural surface. Replicas can be made from various types of polymers. The aim of the paper is to compare the influence of different types of polymers on the character and quality of synthetic structures - replicas. A hierarchical structure of the pansy flower (Viola x wittrockiana) was selected as a replication pattern. The silicone elastomer President Light Body (Coltene Wahledent, Hamburg, Germany) was used for the first (negative) replicas preparation. Thermoset (EP), thermoplastic (PE), biodegradable polymers (PCL), as well as aqueous (PVA) and alcoholic polymer solutions (PVB) were used to create the second (positive) replicas. Replicas quality was evaluated by scanning electron microscopy (Carl Zeiss ULTRA Plus) and by contact angle measuring (See System). Different hierarchical surface structures and different contact angles were identified according to types of polymers (amorphous, semi-crystalline, chemical composition) and used technology of replication. An easy, a fast and a low cost replication process can be used for copying structures up to hundreds/tens of nanometers.
Silver nanoparticles (NPs) exhibit significant antimicrobial activity against a broad range of bacteria and fungi at concentrations ranging from a few ppm to tens of ppm that are not cytotoxic to human cells. Silver NPs also strongly enhance antibacterial activity against multiresistant, β-lactamase and carbapenemase-producing Enterobacteriaceae when combined with antibiotics such as cefotaxime, ceftazidime, meropenem, ciprofloxacin and gentamicin. As a result, silver NPs have already been successfully applied in various biomedical and antimicrobial technologies and products used in every-day life as an alternative to conventional antimicrobials. While bacterial resistance to antibiotics has been discussed extensively in the literature, the possible development of resistance to silver NPs after repeated long-term exposure has not been fully explored. We report that the Gram-negative bacteria can develop resistance to silver NPs after prolonged exposure. The observed resistance stems from the production of the adhesive flagellum protein flagellin, which triggers the aggregation of silver NPs and eliminates their antibacterial effects. The resistance mechanism cannot be overcome by stabilization of silver NPs by polymers or surfactants. However, it is possible to suppress it by inhibiting flagellin production with pomegranate rind extract.
ACKNOWLEDGEMENTS: The authors gratefully acknowledge the support provided by project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic, the Czech Science Foundation (project No.15-22248S), the Ministry of Health of the Czech Republic (AZV VES 15-27726A) and the Internal Grants of Palacky University in Olomouc (IGA_PrF_2018_020 and IGA_LF_2018_019).
Surface functionalization of 2D MoS2 materials offers a convenient way to tune their properties and facilitate their application in the field of bio-sensing and nanomedicine. In this study, three different liquid phase exfoliation (LPE) methods were utilized for MoS2 exfoliation: LPE in 45% ethanol solution, LPE in ultrapure water and amino acid intercalation assisted LPE. The three products were bio-conjugated with PEG molecules and through a biotin-avidin-biotin bridge they were functionalised with specific antibody M75 having exclusive affinity towards cancer cells expressing CAIX protein on their membrane. We are aiming to achieve high selectivity towards cancerous cells utilizing an antigen-antibody based cancer detection scheme. This study uses CAIX antigen tagged MDCK cells simulating the cancerous environment and pure MDCK cells as control. Cellular intake studies were performed by confocal Raman microscopy imaging and confocal laser scanning microscopy before and after binding the specific antibody. The preliminary results indicate higher density of internalised nanoplatforms in the case of MDCK cells expressing CAIX compared to the control cells. However, further studies are required to validate these results.
Currently used xenegeneic biological heart valve prostheses are decellularized and crosslinked with glutaraldehyde. They usually undergo degeneration and calcification. Pericardium-based heart valve prostheses, re-seeded with autologous cells, i.e. adipose tissue-derived cells (ASCs) and endothelial cells could have longer durability and biocompatibility. In order to improve cell adhesion, ingrowth into decellularized pericardium, various fibrin (Fb) layers were developed, i.e. Fb, Fb with covalently bound heparin (H), Fb with either VEGF or FGF2 in different concentrations (1 ng/ml, 10 ng/ml, 100 ng/ml) or with both VEGF and FGF2 (100 ng/ml). Growth factors were attached onto Fb via heparin or were adsorbed. ASCs were seeded on theses layers in DMEM supplemented with 2% FS, TGFβ1 and BMP-4 (both 2.5 ng/ml), and with ascorbic acid. Cell adhesion and growth/viability was assessed by counted cell number/MTS evaluation. ASCs were stained for differentiation markers of smooth muscle cells, such as alpha-actin, calponin, caldesmon, and myosin heavy chain. On day 7, ASCs on Fb_H_VEGF layers produced both calponin and alpha actin. An increased FGF concentration caused reduced calponin staining of ASCs. Lack of heparin in fibrin assemblies inhibited production of both alpha actin and calponin in ASCs. The highest ASCs density/viability was found on Fb_H_VEGF_FGF layer. The proper formulation of fibrin coatings could be favourable for ASCs growth and differentiation and could subsequently support endothelialization of cardiovascular prostheses with endothelial cells.
ACKNOWLEDGEMENTS: Supported by Grant Agency of the Czech Republic (Centre of Excelence P108/12/G108) and Ministry of Health of the Czech Republic (Grant No: 15-29153A).
The experiments in the present work describe, the synthesis and preparation, rheological, conductometric and titration calorimetry based characterization, and drug dissolution of different hyaluronic acid (HyA)-based colloidal drug delivery systems. During this investigations the hydrophobic ketoprofen (KP), one of the most used non-steroidal anti-inflammatory drug (NSAID) has been used as model drug.For the moderation of the highly hydrophilic character of HyA and encapsulation of the hydrophobic drug the cross-linked derivatives at cross-linking ratio of 50, 75 and 100 % have been synthesized. In addition, to achieve the increasing the encapsulation efficiency, the hydrophobized variants of HyA have also been prepared in aqueous medium by modifying the structure with positively charged cetyltrimethylammonium bromide (CTAB) surfactant at different HyA monomer/CTAB mass ratios. It was established that the original coherent gel-like structure of the hydrophilic and well solvated polymer chains change into an incoherent colloidal system due to modifications that were verified by rheological investigations. The cross-linking results in the formation of polydispersed nanoparticles (NPs) in the range of ca. 40-110 nm, while an average diameter of ~ 100-120 nm was obtained for surfactant neutralized colloids. Taking into account the dissolution profiles of the covalent and ionic systems it can be concluded that nearly 70% of the encapsulated KP molecules dissolve from the totally cross-linked HyA systems after 8h, while the dissolved amount of KP is about 20% from the surfactant modified colloidal system at HyA monomer/CTAB 1:0.8 mass ratio. It was also established that increasing the amount of surfactant the release of KP turns to diffusion and erosion controlled way. It has been confirmed that the ionically modified HyA may be a potential candidate for controlled drug release of KP.
The development of new and effective drug delivery strategies with enhanced therapeutic abilities is actively pursued. Two-dimensional nanomaterials such as graphene oxide (GO) has been vastly explored by many in drug delivery strategies. In fact, several studies have confirmed that GO has promising potential in targeted delivery of anticancer drugs as few-layered nanomaterials, such as GO, allow higher adsorptive capacity and can easily be internalized into cells. Despite the vast research on GO as nanocarriers, different preparation methods of GO can exhibit different influence on its properties. Hence, we have successfully synthesized graphene oxide nanoplatelets (GONPs) from well-defined and commercially available stacked graphite nanofibers (SGNF). Cytotoxicity assessments revealed negligible toxicity of the as-synthesized GONP. Administration of the chemotherapeutic drug, cisplatin, with GONP on the other hand have proven to increase the toxicity of cisplatin. This results suggest that the presence of GONP enhanced the anticancer property of cisplatin. In addition, X-Ray Photoelectron Spectroscopy (XPS) and Energy Dispersive X-ray Spectroscopy (EDS) of GONP-CP showed possible interaction of cisplatin with GONP which suggests binding and have potential as drug carriers in future drug delivery and cancer therapy applications.
The explosion in the number of newly available functional nanomaterials capable to sensitively change their conductance in the proximity of charged analytes in parallel with the advances in nanofabrication and nanoimaging techniques have made available and cost-efficient a realm of novel strategies to exponentially increase the selectivity and sensitivity boundaries of the electrical detection of nanoobjects. Highly personalized diseases diagnostics and ubiquitous environmental monitoring are only two possible applications which could address a number of societal challenges ahead related to human life in a strongly globalized world. Nanoparticles, nanowires, 2D materials are dominated by quantum effects and employing them as active elements in transducers for novel devices opens up enormous perspectives for innovative sensor systems. Such devices bear the potential not only to outperform conventional sensor technology with respect to speed, sensitivity, long time stability and signal reliability but even to define completely new application fields in platforms that are cost efficient, flexible and portable. After an introduction of the fundamental sensing mechanisms of nanomaterials-based devices, I will present the innovative design and fabrication strategies for our sensor elements. The close interaction of simulation and experiment allows us to elaborate tailored, but also transferable, technological functionalization strategies for different analytes to cover a wide range of application scenarios. In a strongly interdisciplinary approach, we face the need for sophisticated integration and packaging solutions of the sensors into versatile lab-on-a-chip systems. Integrating the latter in microfluidic setups to provide large numbers of different assays just-in-time directs our technology in direction of highly automatized analytic procedures. The results of our research prove the vast potential of our sensing approach but also show the enormous space for further fundamental developments to boost the concept of nanomaterials-based sensor technology to a powerful and smart analytic tool capable of multiplexed studies, providing and evaluating rich and robust statistical output data. Recent results on artificial neuron behavior in our sensor devices will help shedding new light on the role of nanoarchitectures for a true neuromorphic computing.
The significant costs of newly developed pharmacological products are the toxicity examination on animals in pre-clinical trials. Our ultimate goal is to develop a biosensor array to determine acute cardiotoxicity. It capitalizes on the fact that heart muscle cells (cardiomyocytes) show beating in vitro. Based on the beating characteristics their pathophysiological status can be deducted. In addition to microscopic imaging, the method of monitoring the action and field potential of synchronically beating cardiomyocyte syncytia on multielectrode arrays (MEA) appears to be a suitable method for the investigation of the cardiac cells condition. For this purpose, MEA on the basis of organic electrochemical transistors (OECTs) have been developed. The strict biocompatibility demand on surfaces in contact with cultivating media and living cardiac cells organic polymer PEDOT:PSS were fulfilled. The MEA consisted of a microplate of 12x8 chimney - well array with transistors on the bottom for samples cultivation. For the fabrication, a screen printing method, primarily a contact field and an organic polymer, was used. The device was tested in simulated mode for the response of the drain current to the voltage pulses applied to the gate electrode up to 100 µV. The response to such gate voltage renders the device to be sensitive enough to detect the electrical signal of beating cardiomyocytes. Further development is directed towards higher time resolution by means of miniaturization of the transistors’ channel.
While nanoparticles based tumor therapy shows promise, its application is still limited by low nanoparticles (NPs) uptake by tumor tissues, indicating strategies to improve drug delivery are needed. Developing these will require greater understanding of NPs extravasation, diffusion and interaction with tumor and stroma cells. To help achieve this, we have established a technique for high-resolution imaging of NPs-host interactions within intact tissues of live mice using intravital microscopy (IVM). We used magnetic nanocubes (MNCbs), magnetic nanoclusters (MNCls) or magnetic liposomes (MNLs) to study NPs delivery to malignancies in 4T1, B16 and 22Rv1 tumor models. Three different extravasation routes were identified by IVM for studied NPs - foci of vast leakage, multiple local invaginations and neutrophil mediated transport. Intravascular monocytes were also capturing MNLs immediately after injection, but these cells did not extravasate into tissues. Vascular leakage was more profound in orthotopic 4T1 and B16 tumors rather than in 22RV-1 and heterotopic 4T1 models. Interestingly NPs accumulation in tumor microenvironment was mostly associated with non-tumor cells (monocytes, neutrophils, macrophages and pericytes). Preferential extravasation route and target cells was an NP type dependent phenomenon with MNCbs and MNCls mainly extravasating through foci of vast leakage and captured by macrophages, and MNLs preferentially leaking through small invagination and accumulating in pericytes. Understanding the mechanisms of NPs extravasation and potential role of host cells in NPs delivery is essential for optimizing nanodrug targeted delivery. ACKNOWLEDGEMENTS: The work was supported by the Ministry of Education and Science of the Russian Federation (RFMEFI57517X0147).