Scale-up criteria of laser ablation in the liquid phase with nanosecond pulses is studied for efficient generation of pure ceramic nanoparticles in an aqueous environment. Besides high laser fluence and low height of the applied liquid layer, specific pulse overlap and defined laser repetition rate are required for significant enhancement in nanoparticle productivity. The ablation rate increases by 350% by reducing the liquid film from 8 mm to 2.5 mm owing to reduced absorption and scattering of the incident laser beam by previously ablated nanoparticles. The controlled interpulse distance yields a further increase in material removal rate by another 300% compared to machining in the pulse overlap mode. The residual cavitation bubble from the previous laser pulse and the dispersed nanoparticle interaction with the following laser pulse and optimized temperature gradient in the lattice of the target are assumed to alter productivity. This hypothesis is confirmed by varying the repetition rate with equal laser fluence and pulse overlap, which causes a drastic rise in nanoparticle productivity by a factor of 65. A maximum corundum nanoparticle productivity of 1.3 g/h with Feret particle size of 30 nm is gained by 18.5 W of focused laser power at 4 kHz of repetition rate, providing 125 µm interpulse distance and liquid flow.
Surface pre-endothelialization is a promising approach to improve the hemocompatibility of implants, medical devices, and artificial organs. To promote the adhesive property of thermoplastic polyurethane (TPU) for endothelial cells (ECs), up to 1 wt % of gold (Au) or platinum (Pt) nanoparticles, fabricated by pulsed laser ablation in polymer solution, were embedded into the polymer matrix. The analysis of these nanocomposites showed a homogenous dispersion of the nanoparticles, with average diameters of 7 nm for Au or 9 nm for Pt. A dose-dependent effect was found when ECs were seeded onto nanocomposites comprising different nanoparticle concentrations, resulting in a fivefold improvement of proliferation at 0.1 wt % nanoparticle load. This effect was associated with a nanoparticle concentration-dependent hydrophilicity and negative charge of the nanocomposite. In dynamic flow tests, nanocomposites containing 0.1 wt % Au or Pt nanoparticles allowed for the generation of a confluent and resistant EC layer. Real-time polymerase chain reaction quantification of specific markers for EC activation indicated that ECs cultivated on nanocomposites remain in an inactivated, nonthrombogenic and noninflammatory state; however, maintain the ability to trigger an inflammatory response upon stimulation. These findings were confirmed by a platelet and leukocyte adhesion assay. The results of this study suggest the possible applicability of TPU nanocomposites, containing 0.1 wt % Au or Pt nanoparticles, for the generation of pre-endothelialized surfaces of medical devices.
A novel approach for nanofabricating protein-functionalized luminescent silicon nanoparticles based on infrared ultrafast laser ablation of silicon in an aqueous solution of Staphylococcus aureus protein A is reported. It is demonstrated that 8 nm protein A-capped silicon quantum dots with blue-green photoemissive properties are generated. The conjugation efficiency studies reveal a high percentage of protein A attached to the Si nanoparticle surface through physical adsorption phenomena during the in situ laser process. The biological functionality of laser-generated Staphylococcus aureus protein A-capped Si nanoparticles is investigated. Confocal and electron microscopy together with energy dispersive x-ray spectroscopy analysis show that these Si-based bio-nanostructures selectively bind IgG in the cells. Cell viability studies reveal that these protein A-capped Si nanoparticles are suitable for biological applications, demonstrating their potential as universal secondary biomarkers for in vivo applications such as long-term, real-time cell labeling, cell staining and controlled drug delivery.
Fast ex situ functionalization of gold nanoparticles with fluorophore-labeled cell-penetrating peptides is investigated with a novel liquid flow cascade injection system. Successful conjugation is proved by various methods, such as UV-vis spectrometry and electron microscopy, whereas nanoparticle size-quenching is clearly observed. By variation of the peptide concentration introduced promptly after particle generation, gold nanoparticle bioconjugates with different degrees of cluster formation and/or aggregation and different peptide surface coverage values are obtained. The sizes of synthesized inorganic-organic gold nanoparticle bioconjugate show obvious correlation with time-delayed conjugation, giving evidence that laser-generated nanoparticles continue growing outside the cavitation bubble in the multisecond time scale until achieving their final size. Introducing 6.6 μM bioactive ligands, the highest conjugation efficiency of 93% and ζ potential of 27.5 mV is reached at the shortest delay time (200 ms), resulting in 20 nm average sized bioconjugates. Finally, in a preliminary biological application, laser scanning confocal microscopy clearly revealed an amplified cellular uptake using HIV-1 transactivator peptide-conjugated gold nanoparticles compared with nonconjugated entities within embryonic fibroblasts after a short coincubation time of 1 h. The generation of high amounts of highly pure cell-penetrating nanomarkers by the nanosecond laser-assisted fast ex situ conjugation is thus a promising method to probe biological activities such as nanodrug internalization mechanisms.
Spermatozoa are not only essential for animal reproduction they also represent important tools for the manipulation of animal genetics. For instance, the genetic labeling and analysis of spermatozoa could provide a prospective complementation of pre-fertilization diagnosis and could help to prevent the inheritance of defective alleles during artificial insemination or to select beneficial traits in livestock. Spermatozoa feature extremely specialized membrane organization and restricted transport mechanisms making the labeling of genetically interesting DNA-sequences, e.g., with gold nanoparticles, a particular challenge. Here, we present a systematic study on the size-related internalization of ligand-free, monovalent and bivalent polydisperse gold nanoparticles, depending on spermatozoa membrane status. While monovalent conjugates were coupled solely to either negatively-charged oligonucleotides or positively-charged cell-penetrating peptides, bivalent conjugates were functionalized with both molecules simultaneously. The results clearly indicate that the cell membrane of acrosome-intact, bovine spermatozoa was neither permeable to ligand-free or oligonucleotide-conjugated nanoparticles, nor responsive to the mechanisms of cell-penetrating peptides. Interestingly, after acrosome reaction, which comprises major changes in sperm membrane composition, fluidity and charge, high numbers of monovalent and bivalent nanoparticles were found in the postequatorial segment, depicting a close and complex correlation between particle internalization and membrane organization. Additionally, depending on the applied peptide and for nanoparticle sizes < 10 nm even a successive nuclear penetration was observed, making the bivalent conjugates promising for future genetic delivery and sorting issues.
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