Ayyoob Arpanaei

Immobilisation of living bacteria for AFM imaging under physiological conditions

Atomic force microscopy (AFM) holds great potential for studying the nanoscale surface structures of living cells, and to measure their interactions with abiotic surfaces, other cells, or specific biomolecules. However, the application of AFM in microbiology is challenging due to the difficulty of immobilising bacterial cells to a flat surface without changing the cell surface properties or cell viability. We have performed an extensive and thorough study of how to functionalise surfaces in order to immobilise living bacteria for AFM studies in liquid environments. Our aim was to develop a scheme which allows bacterial cells to be immobilised to a flat surface with sufficient strength to avoid detachment during the AFM scanning, and without affecting cell surface chemistry, structure, and viability. We compare and evaluate published methods, and present a new, reproducible, and generally applicable scheme for immobilising bacteria cells for an AFM imaging. Bacterial cells were immobilised to modified glass surfaces by physical confinement of cells in microwells, physisorption to positively charged surfaces, covalent binding to amine- or carboxyl-terminated surfaces, and adsorption to surfaces coated with highly adhesive polyphenolic proteins originating from the mussel Mytilus edulis. Living cells could be immobilised with all of these approaches, but many cells detached when immobilised by electrostatic interactions and imaged in buffers like PBS or MOPS. Cells were more firmly attached when immobilised by covalent binding, although some cells still detached during AFM imaging. The most successful method revealed was immobilisation by polyphenolic proteins, which facilitated firm immobilisation of the cells. Furthermore, the cell viability was not affected by this immobilisation scheme, and adhesive proteins thus provide a fast, reproducible, and generally applicable scheme for immobilising living bacteria for an AFM imaging.

Surface functionalisation of PLGA nanoparticles for gene silencing

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFalpha in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

Highly Ordered Mixed Protein Patterns Over Large Areas from Self‐Assembly of Binary Colloids

and fundamental studies of cell biology, [ 8 ] all require solidsupported proteins to function effi ciently. Proteins also adsorb non-specifi cally to surfaces limiting the long-term effectiveness of many devices. [ 9 , 10 ] Protein patterning has become a very useful approach for both improving our understanding of how proteins in 2D function and for enhancing the performance of devices. Numerous techniques have been adopted for generating patterns of biomolecules. [ 11–15 ] Some of the limitations associated with previously reported techniques include controlling the immobilization of more than one protein over large areas with high throughput, the requirement for multiple step preparations, high cost, use of harsh chemicals that may infl uence activity of protein molecules within pattern, and some of them require complex instrumentation with restricted availability to many laboratories. Thus, alternative techniques for decorating surfaces with multiple proteins with a high degree of control of the spacing, topography, order and concentration within a pattern is highly desirable. Highly ordered binary, and perhaps even more complex colloidal crystals, potentially offer an alternative, simple solution for generating protein patterns since it is possible to control the surface density, the crystal structure, size and surface chemistry of the particles. Several methods have also been proposed for the growth of binary colloidal crystals, among them evaporation based assembly is the easiest and a single step method. [ 16 ] Here, we take advantage of a simple evaporation based self-assembly method, and propose an alternative and rapid route to generate highly ordered multiple protein patterns with precise control over the spacing between the proteins and the pattern size over

Electrospun aligned PLGA and PLGA/gelatin nanofibers embedded with silica nanoparticles for tissue engineering

Aligned poly lactic-co-glycolic acid (PLGA) and PLGA/gelatin nanofibrous scaffolds embedded with mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. The mean diameters of nanofibers were 641±24 nm for the pure PLGA scaffolds vs 418±85 nm and 267±58 nm for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The contact angle measurement results (102°±6.7 for the pure PLGA scaffold vs 81°±6.8 and 18°±8.7 for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively) revealed enhanced hydrophilicity of scaffolds upon incorporation of gelatin and MSNPs. Besides, embedding the scaffolds with MSNPs resulted in improved tensile mechanical properties. Cultivation of PC12 cells on the scaffolds demonstrated that introduction of MSNPs into PLGA and PLGA/gelatin matrices leads to the improved cell attachment and proliferation as well as long cellular processes. DAPI staining results indicated that cell proliferations on the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds were strikingly (nearly 2.5 and 3 folds, respectively) higher than that on the aligned pure PLGA scaffolds. These results suggest superior properties of silica nanoparticles-incorporated PLGA/gelatin eletrospun nanofibrous scaffolds for the stem cell culture and tissue engineering applications.

Nanoreinforced Hydrogels for Tissue Engineering: Biomaterials that are Compatible with Load-Bearing and Electroactive Tissues

Given their highly porous nature and excellent water retention, hydrogel-based biomaterials can mimic critical properties of the native cellular environment. However, their potential to emulate the electromechanical milieu of native tissues or conform well with the curved topology of human organs needs to be further explored to address a broad range of physiological demands of the body. In this regard, the incorporation of nanomaterials within hydrogels has shown great promise, as a simple one-step approach, to generate multifunctional scaffolds with previously unattainable biological, mechanical, and electrical properties. Here, recent advances in the fabrication and application of nanocomposite hydrogels in tissue engineering applications are described, with specific attention toward skeletal and electroactive tissues, such as cardiac, nerve, bone, cartilage, and skeletal muscle. Additionally, some potential uses of nanoreinforced hydrogels within the emerging disciplines of cyborganics, bionics, and soft biorobotics are highlighted.

3D Biomaterial Microarrays for Regenerative Medicine: Current State-of-the-Art, Emerging Directions and Future Trends

Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem cell differentiation into tissue-specific lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000-fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive for tissue engineering and drug screening applications.

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films

Aims:  Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel.

Incorporation of mesoporous silica nanoparticles into random electrospun PLGA and PLGA/gelatin nanofibrous scaffolds enhances mechanical and cell proliferation properties

Poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin random nanofibrous scaffolds embedded with different amounts of mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. To evaluate the effects of nanoparticles on the scaffolds, physical, chemical, and mechanical properties as well as in vitro degradation behavior of scaffolds were investigated. The mean diameters of nanofibers were 974±68nm for the pure PLGA scaffolds vs 832±70, 764±80, and 486±64 for the PLGA/gelatin, PLGA/10wt% MSNPs, and the PLGA/gelatin/10wt% MSNPs scaffolds, respectively. The results suggested that the incorporation of gelatin and MSNPs into PLGA-based scaffolds enhances the hydrophilicity of scaffolds due to an increase of hydrophilic functional groups on the surface of nanofibers. With porosity examination, it was concluded that the incorporation of MSNPs and gelatin decrease the porosity of scaffolds. Nanoparticles also improved the tensile mechanical properties of scaffolds. Using in vitro degradation analysis, it was shown that the addition of nanoparticles to the nanofibers matrix increases the weight loss percentage of PLGA-based samples, whereas it decreases the weight loss percentage in the PLGA/gelatin composites. Cultivation of rat pheochromocytoma cell line (PC12), as precursor cells of dopaminergic neural cells, on the scaffolds demonstrated that the introduction of MSNPs into PLGA and PLGA/gelatin matrix leads to improved cell attachment and proliferation and enhances cellular processes.

Large-Area Protein Patterns Generated by Ordered Binary Colloidal Assemblies as Templates

We demonstrate the use of binary colloidal assemblies as lithographic masks to generate tunable Au patterns on SiO(2) substrates with dimensions ranging from micrometers to nanometers. Such patterns can be modified with different chemistries to create patterns with well-defined sites for selective adsorption of proteins, where the pattern size and spacing is adjustable depending on particle choice. In our system, the binary colloidal assemblies contain large and small particles of similar or different material and are self-assembled from dilute dispersions with particle size ratios ranging from 0.10 to 0.50. This allows masks with variable morphology and thus production of chemical patterns of tunable geometry. Finally, the Au or SiO(2) regions of the pattern are surface modified with protein resistant oligoethyleneglycol self-assembled molecules, which facilitates site selective adsorption of proteins into the unmodified regions of the pattern. This we show with fluorescently labeled bovine serum albumin.

Study of Molecular Conformation and Activity-Related Properties of Lipase Immobilized onto Core–Shell Structured Polyacrylic Acid-Coated Magnetic Silica Nanocomposite Particles

A facile approach for the preparation of core-shell structured poly(acrylic acid) (PAA)-coated Fe3O4 cluster@SiO2 nanocomposite particles as the support materials for the lipase immobilization is reported. Low- or high-molecular-weight (1800 and 100,000, respectively) PAA molecules were covalently attached onto the surface of amine-functionalized magnetic silica nanoacomposite particles. The successful preparation of particles were verified by scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential measurement, and Fourier-transform infrared (FTIR) techniques. Once lipase is covalently immobilized onto the particles with an average diameter of 210 ± 50 nm, resulting from high binding sites concentrations on the low- and high-molecular-weight PAA-coated particles, high lipase immobilization efficiencies (86.2% and 89.9%, respectively), and loading capacities (786 and 816 mg g(-1), respectively) are obtained. Results from circular dichroism (CD) analysis and catalytic activity tests reveal an increase in the β-sheet content of lipase molecules upon immobilization, along with an enhancement in their activities and stabilities. The lipases immobilized onto the low- and high-molecular-weight PAA-coated particles show maximum activities at 55 and 50 °C, respectively, which are ∼28% and ∼15% higher than that of the free lipase at its own optimum temperature (40 °C), respectively. The immobilized lipases exhibit excellent performance at broader temperature and pH ranges and high thermal and storage stabilities, as well as superior reusability. These prepared magnetic nanocomposite particles can be offered as suitable support materials for efficient immobilization of enzymes and improvement of the immobilized enzymes properties.