Acoustic stimulation of the human round window by laser-induced nonlinear optoacoustics.

The feasibility of low frequency pure tone generation in the inner ear by laser-induced nonlinear optoacoustic effect at the round window was demonstrated in three human cadaveric temporal bones (TB) using an integral pulse density modulation (IPDM). Nanosecond laser pulses with a wavelength in the near-infrared (NIR) region were delivered to the round window niche by an optical fiber with two spherical lenses glued to the end and a viscous gel at the site of the laser focus. Using IPDM, acoustic tones with frequencies between 20 Hz and 1 kHz were generated in the inner ear. The sound pressures in scala tympani and vestibuli were recorded and the intracochlear pressure difference (ICPD) was used to calculate the equivalent sound pressure level (eq. dB SPL) as an equivalent for perceived loudness. The results demonstrate that the optoacoustic effect produced sound pressure levels ranging from 140 eq. dB SPL at low frequencies ≤ 200 Hz to 90 eq. dB SPL at 1 kHz. Therefore, the produced sound pressure level is potentially sufficient for patients requiring acoustic low frequency stimulation. Hence, the presented method offers a potentially viable solution in the future to provide the acoustic stimulus component in combined electro-acoustic stimulation with a cochlear implant.

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery.

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids.

Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids.

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24 h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4 h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

Optoacoustic tones generated by nanosecond laser pulses can cover the entire human hearing range.

The aim of this work is to generate defined tones that cover the human hearing range in aqueous media for a later application in middle or inner ear implants. In our experiments, we investigated the characteristics of single laser pulses and pulse trains with different laser repetition rates of nanosecond laser pulses that were focused into aqueous media in a small volume. The frequency of the generated tones was limited by the spectral properties of the single acoustic pulses, which depended on the medium. Tones with fundamental frequencies above 8 kHz were generated using laser pulses focused into water. By replacing water with gel, tones between 500 Hz and 20 kHz could be produced. The generation of tones in the low-frequency range was only possible when laser pulse trains with pulse density modulated pulse patterns were applied in gel. This enabled the generation of tones between 20 Hz and 2 kHz. Consequently, the combination of different pulse patterns for the different frequency ranges allows generating optoacoustic tones between 20 Hz and 20 kHz in gel. Thus, we can cover the complete range of human hearing through optoacoustically generated tones.

Techniques for In Vivo Assessment of Corneal Biomechanics: Brillouin Spectroscopy and Hydration State - Quo Vadis? / Technologien zur In-vivo-Untersuchung der Biomechanik der Hornhaut: Brillouin-Spektroskopie und Hydratationszustand ­ quo vadis?

To assess the structural integrity of the cornea, non-invasive methods are needed for the local measurement of its mechanical properties. Among a number of established techniques and their associated advantages and disadvantages, Brillouin spectroscopy is still a relatively new technique, capable of determining the compressive modulus of biological tissue, specifically the cornea, in vivo. In the present paper, these various existing and developing technologies for corneal biomechanics are discussed and correlated.

Mechanical Stimulation Induces Vasa Vasorum Capillary Alignment in a Fibrin-Based Tunica Adventitia.

Generation of bioartificial blood vessels with a physiological three-layered wall architecture is a long pursued goal in vascular tissue engineering. While considerable advances have been made to resemble the physiological tunica intima and media morphology and function in bioartificial vessels, only very few studies have targeted the generation of a tunica adventitia, including its characteristic vascular network known as the vasa vasorum, which are essential for graft nutrition and integration. In healthy native blood vessels, capillary vasa vasorum are aligned longitudinally to the vessel axis. Thus, inducing longitudinal alignment of capillary tubes to generate a physiological tunica adventitia morphology and function may be advantageous in bioengineered vessels as well. In this study, we investigated the effect of two biomechanical stimulation parameters, longitudinal tension and physiological cyclic stretch, on tube alignment in capillary networks formed by self-assembly of human umbilical vein endothelial cells in tunica adventitia-equivalents of fibrin-based bioartificial blood vessels. Moreover, the effect of changes of the biomechanical environment on network remodeling after initial tube formation was analyzed. Both, longitudinal tension and cyclic stretch by pulsatile perfusion induced physiological capillary tube alignment parallel to the longitudinal vessel axis. This effect was even more pronounced when both biomechanical factors were applied simultaneously, which resulted in an alignment of 57.2 ± 5.2% within 5° of the main vessel axis. Opposed to that, a random tube orientation was observed in vessels incubated statically. Scanning electron microscopy showed that longitudinal tension also resulted in longitudinal alignment of fibrin fibrils, which may function as a guidance structure for directed capillary tube formation. Moreover, existing microvascular networks showed distinct remodeling in response to addition or withdrawal of mechanical stimulation with corresponding increase or decrease of the degree of alignment. With longitudinal tension and cyclic stretch, we identified two mechanical stimuli that facilitate the generation of a prevascularized tunica adventitia-equivalent with physiological tube alignment in bioartificial vascular grafts. Impact statement Fibrin-based bioartificial vessels represent a promising regenerative approach to generate vascular grafts with superior biocompatibility and hemocompatibility compared to currently available synthetic graft materials. Precapillarization of bioartificial vascular grafts may improve nutrition of the vessel wall and integration of the graft into the target organism's microvasculature. In native vessels, physiological vasa vasorum alignment is pivotal for proper function of the tunica adventitia. Thus, it is necessary to induce longitudinal capillary alignment in the tunica adventitia of bioengineered vessels as well to secure long-term graft patency and function. This alignment can be reliably achieved by controlled biomechanical stimulation in vitro.

Investigation of Colonic Regeneration via Precise Damage Application Using Femtosecond Laser-Based Nanosurgery.

Organoids represent the cellular composition of natural tissue. So called colonoids, organoids derived from colon tissue, are a good model for understanding regeneration. However, next to the cellular composition, the surrounding matrix, the cell-cell interactions, and environmental factors have to be considered. This requires new approaches for the manipulation of a colonoid. Of key interest is the precise application of localized damage and the following cellular reaction. We have established multiphoton imaging in combination with femtosecond laser-based cellular nanosurgery in colonoids to ablate single cells in the colonoids' crypts, the proliferative zones, and the differentiated zones. We observed that half of the colonoids recovered within six hours after manipulation. An invagination of the damaged cell and closing of the structure was observed. In about a third of the cases of targeted crypt damage, it caused a stop in crypt proliferation. In the majority of colonoids ablated in the crypt, the damage led to an increase in Wnt signalling, indicated via a fluorescent lentiviral biosensor. qRT-PCR analysis showed increased expression of various proliferation and Wnt-associated genes in response to damage. Our new model of probing colonoid regeneration paves the way to better understand organoid dynamics on a single cell level.

How Localized Z-Disc Damage Affects Force Generation and Gene Expression in Cardiomyocytes.

The proper function of cardiomyocytes (CMs) is highly related to the Z-disc, which has a pivotal role in orchestrating the sarcomeric cytoskeletal function. To better understand Z-disc related cardiomyopathies, novel models of Z-disc damage have to be developed. Human pluripotent stem cell (hPSC)-derived CMs can serve as an in vitro model to better understand the sarcomeric cytoskeleton. A femtosecond laser system can be applied for localized and defined damage application within cells as single Z-discs can be removed. We have investigated the changes in force generation via traction force microscopy, and in gene expression after Z-disc manipulation in hPSC-derived CMs. We observed a significant weakening of force generation after removal of a Z-disc. However, no significant changes of the number of contractions after manipulation were detected. The stress related gene NF-kB was significantly upregulated. Additionally, α-actinin (ACTN2) and filamin-C (FLNc) were upregulated, pointing to remodeling of the Z-disc and the sarcomeric cytoskeleton. Ultimately, cardiac troponin I (TNNI3) and cardiac muscle troponin T (TNNT2) were significantly downregulated. Our results allow a better understanding of transcriptional coupling of Z-disc damage and the relation of damage to force generation and can therefore finally pave the way to novel therapies of sarcomeric disorders.

Intracellular Cargo Delivery Induced by Irradiating Polymer Substrates with Nanosecond-Pulsed Lasers.

There is a great need in the biomedical field to efficiently, and cost-effectively, deliver membrane-impermeable molecules into the cellular cytoplasm. However, the cell membrane is a selectively permeable barrier, and large molecules often cannot pass through the phospholipid bilayer. We show that nanosecond laser-activated polymer surfaces of commercial polyvinyl tape and black polystyrene Petri dishes can transiently permeabilize cells for high-throughput, diverse cargo delivery of sizes of up to 150 kDa. The polymer surfaces are biocompatible and support normal cell growth of adherent cells. We determine the optimal irradiation conditions for poration, influx of fluorescent molecules into the cell, and post-treatment viability of the cells. The simple and low-cost substrates we use have no thin-metal structures, do not require cleanroom fabrication, and provide spatial selectivity and scalability for biomedical applications.

Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation.

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development.

Evaluation of laser induced sarcomere micro-damage: Role of damage extent and location in cardiomyocytes.

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laser-based nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6-7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms.

High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling.

To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

Vascular Network Formation on Macroporous Polydioxanone Scaffolds.

In this study, microvascular network structures for tissue engineering were generated on newly developed macroporous polydioxanone (PDO) scaffolds. PDO represents a polymer biodegradable within months and offers optimal material properties such as elasticity and nontoxic degradation products. PDO scaffolds prepared by porogen leaching and cryo-dried to achieve pore sizes of 326 ± 149.67 µm remained stable with equivalent values for Young's modulus after 4 weeks. Scaffolds were coated with fibrin for optimal cell adherence. To exclude interindividual differences, autologous fibrin was prepared out of human plasma-derived fibrinogen and proved a comparable quality to nonautologous commercially available fibrinogen. Fibrin-coated scaffolds were seeded with recombinant human umbilical vein endothelial cells expressing GFP (GFP-HUVECs) in coculture with adipose tissue-derived mesenchymal stem cells (AD-hMSCs) to form vascular networks. The growth factor content in culture media was optimized according its effect on network formation, quantified and assessed by AngioTool®. A ratio of 2:3 GFP-HUVECs/AD-hMSCs in medium enriched with 20 ng/mL vascular endothelial growth factor, basic fibroblast growth factor, and hydrocortisone was found to be optimal. Network structures appeared after 2 days of cultivation and stabilized until day 7. The resulting networks were lumenized that could be verified by dextran staining. This new approach might be suitable for microvascular tissue patches as a useful template to be used in diverse vascularized tissue constructs. Impact statement We consider this work as important for the current research in the field of tissue engineering and the development of new and functional tissue. The approach for the production of vascularized tissue patches, consisting of the biodegradable synthetic polymer polydioxanone and of the physiological, autologous, and patient-specific polymer fibrin, and seeded with endothelial cells and mesenchymal stem cells, displayed within this work, could be useful for the sustaining development of diverse and more complex tissue constructs. Therefore, these scaffolds could be used as a cornerstone for future tissue engineering approaches.

Longitudinal imaging and femtosecond laser manipulation of the liver: How to generate and trace single-cell-resolved micro-damage in vivo.

The liver is known to possess extensive regenerative capabilities, the processes and pathways of which are not fully understood. A necessary step towards a better understanding involves the analysis of regeneration on the microscopic level in the in vivo environment. We developed an evaluation method combining longitudinal imaging analysis in vivo with simultaneous manipulation on single cell level. An abdominal imaging window was implanted in vivo in Balb/C mice for recurrent imaging after implantation. Intravenous injection of Fluorescein Isothiocyanate (FITC)-Dextran was used for labelling of vessels and Rhodamine 6G for hepatocytes. Minimal cell injury was induced via ablation with a femtosecond laser system during simultaneous visualisation of targeted cells using multiphoton microscopy. High-resolution imaging in vivo on single cell level including re-localisation of ablated regions in follow-up measurements after 2-7 days was feasible. Targeted single cell manipulation using femtosecond laser pulses at peak intensities of 3-6.6 µJ led to enhancement of FITC-Dextran in the surrounding tissue. These reactions reached their maxima 5-15 minutes after ablation and were no longer detectable after 24 hours. The procedures were well tolerated by all animals. Multiphoton microscopy in vivo, combined with a femtosecond laser system for single cell manipulation provides a refined procedure for longitudinal evaluation of liver micro-regeneration in the same region of interest. Immediate reactions after cell ablation and tissue regeneration can be analysed.

Characterization of Tissue Engineered Endothelial Cell Networks in Composite Collagen-Agarose Hydrogels.

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either MatrigelTM and rat tail collagen I (MatrigelTM/rCOL) or human collagen (hCOL) with SeaPlaqueTM agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in MatrigelTM/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of MatrigelTM/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported.

Establishment of a guided, in vivo, multi-channel, abdominal, tissue imaging approach.

Novel tools in humane animal research should benefit the animal as well as the experimentally obtained data. Imaging technologies have proven to be versatile and also in accordance with the demands of the 3 R principle. However, most imaging technologies are either limited by the target organs, number of repetitive imaging sessions, or the maximal resolution. We present a technique-, which enables multicolor abdominal imaging on a tissue level. It is based on a small imaging fiber endoscope, which is guided by a second commercial endoscope. The imaging fiber endoscope allows the distinction of four different fluorescence channels. It has a size of less than 1 mm and can approximately resolve single cells. The imaging fiber was successfully tested on cells in vitro, excised organ tissue, and in mice in vivo. Combined with neural networks for image restauration, high quality images from various abdominal organs of interest were realized. The second endoscope ensured a precise placement of the imaging fiber in vivo. Our approach of guided tissue imaging in vivo, combined with neuronal networks for image restauration, permits the acquisition of fluorescence-microscope like images with minimal invasive surgery in vivo. Therefore, it is possible to extend our approach to repetitive imaging sessions. The cost below 30 thousand euros allows an establishment of this approach in various scenarios.

Myoblast adhesion and proliferation on biodegradable polymer films with femtosecond laser-fabricated micro through-holes.

Controlling cell adhesion and cell differentiation is necessary to fabricate a tissue with arbitrary properties for tissue engineering applications. A substrate with a porous structure as a cell scaffold allows the diffusion of the cell culture medium through the scaffold. In this work, we show that the femtosecond laser fabricated micro through-holes in biodegradable polymer films, enhance myoblast adhesion, and accelerates proliferation and differentiation. ChR2-C2C12 and UT-C2C12 cells were seeded on the films with micro through-holes each fabricated by a single femtosecond laser pulse. Cell adhesion was enhanced on films with holes fabricated by laser irradiation. In addition, cell proliferation was accelerated on films with micro through-holes that penetrate the film, compared to on films with micro craters that do not penetrate the film. On films with arrays consisting of micro through-holes, cells aligned along the arrays and cell fusion was enhanced, indicating the acceleration of cell differentiation.

CD14 and ALPK1 Affect Expression of Tight Junction Components and Proinflammatory Mediators upon Bacterial Stimulation in a Colonic 3D Organoid Model.

Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10 -/-, Cd14 -/-, and Alpk1 -/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14 -/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1 -/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.

Biodistribution, biocompatibility and targeted accumulation of magnetic nanoporous silica nanoparticles as drug carrier in orthopedics.

BACKGROUND: In orthopedics, the treatment of implant-associated infections represents a high challenge. Especially, potent antibacterial effects at implant surfaces can only be achieved by the use of high doses of antibiotics, and still often fail. Drug-loaded magnetic nanoparticles are very promising for local selective therapy, enabling lower systemic antibiotic doses and reducing adverse side effects. The idea of the following study was the local accumulation of such nanoparticles by an externally applied magnetic field combined with a magnetizable implant. The examination of the biodistribution of the nanoparticles, their effective accumulation at the implant and possible adverse side effects were the focus. In a BALB/c mouse model (n = 50) ferritic steel 1.4521 and Ti90Al6V4 (control) implants were inserted subcutaneously at the hindlimbs. Afterwards, magnetic nanoporous silica nanoparticles (MNPSNPs), modified with rhodamine B isothiocyanate and polyethylene glycol-silane (PEG), were administered intravenously. Directly/1/7/21/42 day(s) after subsequent application of a magnetic field gradient produced by an electromagnet, the nanoparticle biodistribution was evaluated by smear samples, histology and multiphoton microscopy of organs. Additionally, a pathohistological examination was performed. Accumulation on and around implants was evaluated by droplet samples and histology. RESULTS: Clinical and histological examinations showed no MNPSNP-associated changes in mice at all investigated time points. Although PEGylated, MNPSNPs were mainly trapped in lung, liver, and spleen. Over time, they showed two distributional patterns: early significant drops in blood, lung, and kidney and slow decreases in liver and spleen. The accumulation of MNPSNPs on the magnetizable implant and in its area was very low with no significant differences towards the control. CONCLUSION: Despite massive nanoparticle capture by the mononuclear phagocyte system, no significant pathomorphological alterations were found in affected organs. This shows good biocompatibility of MNPSNPs after intravenous administration. The organ uptake led to insufficient availability of MNPSNPs in the implant region. For that reason, among others, the nanoparticles did not achieve targeted accumulation in the desired way, manifesting future research need. However, with different conditions and dimensions in humans and further modifications of the nanoparticles, this principle should enable reaching magnetizable implant surfaces at any time in any body region for a therapeutic reason.

Parameters for Optoperforation-Induced Killing of Cancer Cells Using Gold Nanoparticles Functionalized With the C-terminal Fragment of Clostridium Perfringens Enterotoxin.

Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10-11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells.