Extent and Effect of Covering Laryngeal Structures with Synthetic Laryngeal Mucus via Two Different Administration Techniques.

OBJECTIVE: The first goal of this study was to investigate the coverage of laryngeal structures using two potential administration techniques for synthetic mucus: inhalation and lozenge ingestion. As a second research question, the study investigated the potential effects of these techniques on standardized voice assessment parameters. METHODS: Fluorescein was added to throat lozenges and to an inhalation solution to visualize the coverage of laryngeal structures through blue light imaging. The study included 70 vocally healthy subjects. Fifty subjects underwent administration via lozenge ingestion and 20 subjects performed the inhalation process. For the first research question, the recordings from the blue light imaging system were categorized to compare the extent of coverage on individual laryngeal structures objectively. Secondly, a standardized voice evaluation protocol was performed before and after each administration to determine any measurable effects of typical voice parameters. RESULTS: The administration via inhalation demonstrated complete coverage of all laryngeal structures, including the vocal folds, ventricular folds, and arytenoid cartilages, as visualized by the fluorescent dye. In contrast, the application of the lozenge predominantly covered the pharynx and laryngeal surface toward the aryepiglottic fold, but not the inferior structures. All in all, the comparison before and after administration showed no clear effect, although a minor deterioration of the acoustic signal was noted in the shimmer and cepstral peak prominence after the inhalation. CONCLUSIONS: Our findings indicate that the inhalation process is a more effective technique for covering deeper laryngeal structures such as the vocal folds and ventricular folds with synthetic mucus. This knowledge enables further in vivo studies on the role of laryngeal mucus in phonation in general, and how it can be substituted or supplemented for patients with reduced glandular activity as well as for heavy voice users.

Long-term performance assessment of fully automatic biomedical glottis segmentation at the point of care.

Deep Learning has a large impact on medical image analysis and lately has been adopted for clinical use at the point of care. However, there is only a small number of reports of long-term studies that show the performance of deep neural networks (DNNs) in such an environment. In this study, we measured the long-term performance of a clinically optimized DNN for laryngeal glottis segmentation. We have collected the video footage for two years from an AI-powered laryngeal high-speed videoendoscopy imaging system and found that the footage image quality is stable across time. Next, we determined the DNN segmentation performance on lossy and lossless compressed data revealing that only 9% of recordings contain segmentation artifacts. We found that lossy and lossless compression is on par for glottis segmentation, however, lossless compression provides significantly superior image quality. Lastly, we employed continual learning strategies to continuously incorporate new data into the DNN to remove the aforementioned segmentation artifacts. With modest manual intervention, we were able to largely alleviate these segmentation artifacts by up to 81%. We believe that our suggested deep learning-enhanced laryngeal imaging platform consistently provides clinically sound results, and together with our proposed continual learning scheme will have a long-lasting impact on the future of laryngeal imaging.

A single latent channel is sufficient for biomedical glottis segmentation.

Glottis segmentation is a crucial step to quantify endoscopic footage in laryngeal high-speed videoendoscopy. Recent advances in deep neural networks for glottis segmentation allow for a fully automatic workflow. However, exact knowledge of integral parts of these deep segmentation networks remains unknown, and understanding the inner workings is crucial for acceptance in clinical practice. Here, we show that a single latent channel as a bottleneck layer is sufficient for glottal area segmentation using systematic ablations. We further demonstrate that the latent space is an abstraction of the glottal area segmentation relying on three spatially defined pixel subtypes allowing for a transparent interpretation. We further provide evidence that the latent space is highly correlated with the glottal area waveform, can be encoded with four bits, and decoded using lean decoders while maintaining a high reconstruction accuracy. Our findings suggest that glottis segmentation is a task that can be highly optimized to gain very efficient and explainable deep neural networks, important for application in the clinic. In the future, we believe that online deep learning-assisted monitoring is a game-changer in laryngeal examinations.

[Salvage laryngectomy after primary radio- and radiochemotherapy : A retrospective study. German version]. / Salvage-Laryngektomie nach primärer Radio- und Radiochemotherapie : Eine retrospektive Fallzusammenfassung.

BACKGROUND: Recurrent and residual laryngeal cancer after organ-preserving radio- or radiochemotherapy is associated with a poor prognosis. Salvage surgery is the most important therapeutic option in these cases. OBJECTIVE: The study assessed rates of recurrence and residual tumor as well as survival and complication rates after salvage laryngectomy at the authors' academic cancer center. MATERIALS AND METHODS: A retrospective examination of all patients receiving laryngectomy between 2001 and 2019 due to tumor residuals or recurrence after primary radio- and radiochemotherapy was conducted. RESULTS: A total of 33 salvage procedures were performed. Defect reconstruction was performed by free flap surgery in 30.3% (n = 10) and regional flap surgery in 15.2% (n = 5) . One patient received regional flap surgery and free flap surgery simultaneously. Overall survival after 1, 2, and 5 years was 68.7, 47.9, and 24.2%, and disease-free survival was 81.6, 47.8, and 24.2%, respectively, with 48.5% (n = 16) postoperative tumor recurrences overall. Disease-free survival was significantly shorter for tumor extension into or onto the hypopharynx (p = 0.041). Postoperatively, 72.7% of patients developed a pharyngocutaneous fistula, of which 24.2% required surgical treatment. The hospital stay was 28.0 ± 16.1 days. CONCLUSION: Salvage laryngectomy is associated with a high rate of treatable complications and high morbidity. Nevertheless, considering the advanced tumor stages treated, it allows for respectable oncological results.

OpenHSV: an open platform for laryngeal high-speed videoendoscopy.

High-speed videoendoscopy is an important tool to study laryngeal dynamics, to quantify vocal fold oscillations, to diagnose voice impairments at laryngeal level and to monitor treatment progress. However, there is a significant lack of an open source, expandable research tool that features latest hardware and data analysis. In this work, we propose an open research platform termed OpenHSV that is based on state-of-the-art, commercially available equipment and features a fully automatic data analysis pipeline. A publicly available, user-friendly graphical user interface implemented in Python is used to interface the hardware. Video and audio data are recorded in synchrony and are subsequently fully automatically analyzed. Video segmentation of the glottal area is performed using efficient deep neural networks to derive glottal area waveform and glottal midline. Established quantitative, clinically relevant video and audio parameters were implemented and computed. In a preliminary clinical study, we recorded video and audio data from 28 healthy subjects. Analyzing these data in terms of image quality and derived quantitative parameters, we show the applicability, performance and usefulness of OpenHSV. Therefore, OpenHSV provides a valid, standardized access to high-speed videoendoscopy data acquisition and analysis for voice scientists, highlighting its use as a valuable research tool in understanding voice physiology. We envision that OpenHSV serves as basis for the next generation of clinical HSV systems.

A Deep Learning Enhanced Novel Software Tool for Laryngeal Dynamics Analysis.

Purpose High-speed videoendoscopy (HSV) is an emerging, but barely used, endoscopy technique in the clinic to assess and diagnose voice disorders because of the lack of dedicated software to analyze the data. HSV allows to quantify the vocal fold oscillations by segmenting the glottal area. This challenging task has been tackled by various studies; however, the proposed approaches are mostly limited and not suitable for daily clinical routine. Method We developed a user-friendly software in C# that allows the editing, motion correction, segmentation, and quantitative analysis of HSV data. We further provide pretrained deep neural networks for fully automatic glottis segmentation. Results We freely provide our software Glottis Analysis Tools (GAT). Using GAT, we provide a general threshold-based region growing platform that enables the user to analyze data from various sources, such as in vivo recordings, ex vivo recordings, and high-speed footage of artificial vocal folds. Additionally, especially for in vivo recordings, we provide three robust neural networks at various speed and quality settings to allow a fully automatic glottis segmentation needed for application by untrained personnel. GAT further evaluates video and audio data in parallel and is able to extract various features from the video data, among others the glottal area waveform, that is, the changing glottal area over time. In total, GAT provides 79 unique quantitative analysis parameters for video- and audio-based signals. Many of these parameters have already been shown to reflect voice disorders, highlighting the clinical importance and usefulness of the GAT software. Conclusion GAT is a unique tool to process HSV and audio data to determine quantitative, clinically relevant parameters for research, diagnosis, and treatment of laryngeal disorders. Supplemental Material https://doi.org/10.23641/asha.14575533.

Salvage laryngectomy after primary radio- and radiochemotherapy : A retrospective study.

BACKGROUND: Recurrent and residual laryngeal cancer after organ-preserving radio- or radiochemotherapy is associated with a poor prognosis. Salvage surgery is the most important therapeutic option in these cases. OBJECTIVE: The study assessed rates of recurrence and residual tumor as well as survival and complication rates after salvage laryngectomy at the authors' academic cancer center. MATERIALS AND METHODS: A retrospective examination of all patients receiving laryngectomy between 2001 and 2019 due to tumor residuals or recurrence after primary radio- and radiochemotherapy was conducted. RESULTS: A total of 33 salvage procedures were performed. Defect reconstruction was performed by free flap surgery in 30.3% (n = 10) and regional flap surgery in 15.2% (n = 5) . One patient received regional flap surgery and free flap surgery simultaneously. Overall survival after 1, 2, and 5 years was 68.7, 47.9, and 24.2%, and disease-free survival was 81.6, 47.8, and 24.2%, respectively, with 48.5% (n = 16) postoperative tumor recurrences overall. Disease-free survival was significantly shorter for tumor extension into or onto the hypopharynx (p = 0.041). Postoperatively, 72.7% of patients developed a pharyngocutaneous fistula, of which 24.2% required surgical treatment. The hospital stay was 28.0 ± 16.1 days. CONCLUSION: Salvage laryngectomy is associated with a high rate of treatable complications and high morbidity. Nevertheless, considering the advanced tumor stages treated, it allows for respectable oncological results.

Interdependencies between acoustic and high-speed videoendoscopy parameters.

In voice research, uncovering relations between the oscillating vocal folds, being the sound source of phonation, and the resulting perceived acoustic signal are of great interest. This is especially the case in the context of voice disorders, such as functional dysphonia (FD). We investigated 250 high-speed videoendoscopy (HSV) recordings with simultaneously recorded acoustic signals (124 healthy females, 60 FD females, 44 healthy males, 22 FD males). 35 glottal area waveform (GAW) parameters and 14 acoustic parameters were calculated for each recording. Linear and non-linear relations between GAW and acoustic parameters were investigated using Pearson correlation coefficients (PCC) and distance correlation coefficients (DCC). Further, norm values for parameters obtained from 250 ms long sustained phonation data (vowel /i/) were provided. 26 PCCs in females (5.3%) and 8 in males (1.6%) were found to be statistically significant (|corr.| ≥ 0.3). Only minor differences were found between PCCs and DCCs, indicating presence of weak non-linear dependencies between parameters. Fundamental frequency was involved in the majority of all relevant PCCs between GAW and acoustic parameters (19 in females and 7 in males). The most distinct difference between correlations in females and males was found for the parameter Period Variability Index. The study shows only weak relations between investigated acoustic and GAW-parameters. This indicates that the reduction of the complex 3D glottal dynamics to the 1D-GAW may erase laryngeal dynamic characteristics that are reflected within the acoustic signal. Hence, other GAW parameters, 2D-, 3D-laryngeal dynamics and vocal tract parameters should be further investigated towards potential correlations to the acoustic signal.

Machine learning based identification of relevant parameters for functional voice disorders derived from endoscopic high-speed recordings.

In voice research and clinical assessment, many objective parameters are in use. However, there is no commonly used set of parameters that reflect certain voice disorders, such as functional dysphonia (FD); i.e. disorders with no visible anatomical changes. Hence, 358 high-speed videoendoscopy (HSV) recordings (159 normal females (NF), 101 FD females (FDF), 66 normal males (NM), 32 FD males (FDM)) were analyzed. We investigated 91 quantitative HSV parameters towards their significance. First, 25 highly correlated parameters were discarded. Second, further 54 parameters were discarded by using a LogitBoost decision stumps approach. This yielded a subset of 12 parameters sufficient to reflect functional dysphonia. These parameters separated groups NF vs. FDF and NM vs. FDM with fair accuracy of 0.745 or 0.768, respectively. Parameters solely computed from the changing glottal area waveform (1D-function called GAW) between the vocal folds were less important than parameters describing the oscillation characteristics along the vocal folds (2D-function called Phonovibrogram). Regularity of GAW phases and peak shape, harmonic structure and Phonovibrogram-based vocal fold open and closing angles were mainly important. This study showed the high degree of redundancy of HSV-voice-parameters but also affirms the need of multidimensional based assessment of clinical data.

Determination of Clinical Parameters Sensitive to Functional Voice Disorders Applying Boosted Decision Stumps.

BACKGROUND: Various voice assessment tools, such as questionnaires and aerodynamic voice characteristics, can be used to assess vocal function of individuals. However, not much is known about the best combinations of these parameters in identification of functional dysphonia in clinical settings. METHODS: This study investigated six scores from clinically commonly used questionnaires and seven acoustic parameters. 514 females and 277 males were analyzed. The subjects were divided into three groups: one healthy group (N01) (49 females, 50 males) and two disordered groups with perceptually hoarse (FD23) (220 females, 96 males) and perceptually not hoarse (FD01) (245 females, 131 males) sounding voices. A tree stumps Adaboost approach was applied to find the subset of parameters that best separates the groups. Subsequently, it was determined if this parameter subset reflects treatment outcome for 120 female and 51 male patients by pairwise pre- and post-treatment comparisons of parameters. RESULTS: The questionnaire "Voice-related-quality-of-Life" and three objective parameters ("maximum fundamental frequency", "maximum Intensity" and "Jitter Percent") were sufficient to separate the groups (accuracy ranging from 0.690 (FD01 vs. FD23, females) to 0.961 (N01 vs. FD23, females)). Our study suggests that a reduced parameter subset (4 out of 13) is sufficient to separate these three groups. All parameters reflected treatment outcome for patients with hoarse voices, Voice-related-quality-of-Life showed improvement for the not hoarse group (FD01). CONCLUSION: Results show that single parameters are insufficient to separate voice disorders but a set of several well-chosen parameters is. These findings will help to optimize and reduce clinical assessment time.

BAGLS, a multihospital Benchmark for Automatic Glottis Segmentation.

Laryngeal videoendoscopy is one of the main tools in clinical examinations for voice disorders and voice research. Using high-speed videoendoscopy, it is possible to fully capture the vocal fold oscillations, however, processing the recordings typically involves a time-consuming segmentation of the glottal area by trained experts. Even though automatic methods have been proposed and the task is particularly suited for deep learning methods, there are no public datasets and benchmarks available to compare methods and to allow training of generalizing deep learning models. In an international collaboration of researchers from seven institutions from the EU and USA, we have created BAGLS, a large, multihospital dataset of 59,250 high-speed videoendoscopy frames with individually annotated segmentation masks. The frames are based on 640 recordings of healthy and disordered subjects that were recorded with varying technical equipment by numerous clinicians. The BAGLS dataset will allow an objective comparison of glottis segmentation methods and will enable interested researchers to train their own models and compare their methods.

Secondary Tracheoesophageal Puncture After Laryngectomy Increases Complications With Shunt and Voice Prosthesis.

OBJECTIVES/HYPOTHESIS: To evaluate the demographics, clinical features, management, and prognostic indicators of tracheoesophageal puncture complications in patients undergoing placement of voice prosthesis following cancer treatment. STUDY DESIGN: Retrospective analysis. METHODS: A retrospective analysis was conducted of cases from a tertiary referral center diagnosed between 1996 and 2015. Multivariate logistic regression was used to determine factors associated with tracheoesophageal puncture (TEP) and voice prostheses-complication-free survival (TEP/VP-CFS). RESULTS: One hundred fourteen cases were identified. Most patients were males (92.9%) with pT3 (26.8%) or pT4 (58.1%) N+ (53.6%) tumors. All patients received laryngectomy as the primary treatment, with 75% of patients receiving adjuvant radiation therapy or chemoradiotherapy. Complications with TEP were common (65.2%). The most frequent problem was salivary leakage (50.0%), which at the same time was the most common reason for changing the prosthesis. On univariate regression analysis, prosthesis placement time after adjuvant radiotherapy (hazard ratio [HR]: 4.17, 95% confidence interval [CI]: 2-8.69), secondary prosthesis placement after primary surgery (HR: 3.97, 95% CI: 1.99-7.9), and laryngectomy with flap reconstruction (HR: 1.96, 95% CI: 0.99-3.89) were significant prognosticators for complications. Multivariate regression analysis revealed secondary prosthesis placement after adjuvant radiotherapy (HR: 3.66, 95% CI: 1.39-9.68) or after primary surgery (HR: 2.57, 95% CI: 0.92-7.2) to be the strongest predictors of reduced TEP/VP-CFS. CONCLUSIONS: Secondary prosthesis placement after primary surgery, placement after previous irradiation, and laryngectomy with flap reconstruction are predictors of poor TEP/VP-CFS. Planned adjuvant radiotherapy is not a contraindication for TEP with prosthetic placement, but it is very important to place the prosthesis during the primary surgery or at least before scheduled radiotherapy. LEVEL OF EVIDENCE: 4 Laryngoscope, 2020.

Investigation of phonatory characteristics using ex vivo rabbit larynges.

Quantitative analysis of phonatory characteristics of rabbits has been widely neglected. However, preliminary studies established the rabbit larynx as a potential model of human phonation. This study reports quantitative data on phonation using ex vivo rabbit larynx models to achieve more insight into dependencies of three main components of the phonation process, including airflow, vocal fold dynamics, and the acoustic output. Sustained phonation was induced in 11 ex vivo rabbit larynges. For 414 phonatory conditions, vocal fold vibrations, acoustic, and aerodynamic parameters were analyzed as functions of longitudinal vocal fold pre-stress, applied air flow, and glottal closure insufficiency. Dimensions of the vocal folds were measured and histological data were analyzed. Glottal closure characteristics improved for increasing longitudinal pre-stress and applied airflow. For the subglottal pressure signal only the cepstral peak prominence showed dependency on glottal closure. In contrast, vibrational, acoustic, and aerodynamic parameters were found to be highly dependent on the degree of glottal closure: The more complete the glottal closure during phonation, the better the aerodynamic and acoustic characteristics. Hence, complete or at least partial glottal closure appears to enhance acoustic signal quality. Finally, results validate the ex vivo rabbit larynx as an effective model for analyzing the phonatory process.

Impact of Superparamagnetic Iron Oxide Nanoparticles on Vocal Fold Fibroblasts: Cell Behavior and Cellular Iron Kinetics.

PURPOSE: The voice is the most important instrument of communication. Tissue defects in the vocal fold (VF) area lead to serious reduction in quality of life, but thus far, no satisfactory VF implant exists. Therefore, we aim to establish a functional VF implant in a rabbit model by magnetic tissue engineering (MTE) using superparamagnetic iron oxide nanoparticles (SPION). Hence, iron quantification over time as well as cell behavior studies upon SPION treatment are of great importance. METHODS: Rabbit VF fibroblasts (VFF) were treated with different concentrations of SPIONs (20, 40, and 80 µg/cm2), and iron content was examined for up to 40 days using microwave plasma-atom emission spectroscopy. The effects of SPION treatment on VFF (adhesion, spreading, and migration), which are important for the formation of 3D structures, were tested. RESULTS: Cellular SPION quantification revealed that there was no residual iron remaining in VFFs after 40 days. SPIONs had a dose-dependent effect on cell adhesion, with good tolerability observed up to 20 µg/cm2. Migration and spreading were not significantly influenced by SPION treatment up to 80 µg/cm2. DISCUSSION AND CONCLUSION: To develop 3D structures, cell behavior should not be affected by SPION uptake. After 40 days, cells were free of iron as a result of metabolism or rarefication during cell division. Cell functions including adhesion, spreading, and migration were proven to be intact in a dose-dependent manner after SPION treatment, suggesting a safe usage of MTE for voice rehabilitation. Our results thus constitute a solid basis for a successful transfer of this technique into 3D constructs, in order to provide an individual and personalized human VF implant in the future.

Optical π phase shift created with a single-photon pulse.

A deterministic photon-photon quantum logic gate is a long-standing goal. Building such a gate becomes possible if a light pulse containing only one photon imprints a phase shift of π onto another light field. We experimentally demonstrate the generation of such a π phase shift with a single-photon pulse. A first light pulse containing less than one photon on average is stored in an atomic gas. Rydberg blockade combined with electromagnetically induced transparency creates a phase shift for a second light pulse, which propagates through the medium. We measure the π phase shift of the second pulse when we postselect the data upon the detection of a retrieved photon from the first pulse. This demonstrates a crucial step toward a photon-photon gate and offers a variety of applications in the field of quantum information processing.

Magnetic Tissue Engineering for Voice Rehabilitation - First Steps in a Promising Field.

BACKGROUND/AIM: The voice is one of the most important instruments of communication between humans. It is the product of intact and well-working vocal folds. A defect of these structures causes dysphonia, associated with a clear reduction of quality of life. Tissue engineering of the vocal folds utilizing magnetic cell levitation after nanoparticle loading might be a technique to overcome this challenging problem. MATERIALS AND METHODS: Vocal fold fibroblasts (VFFs) were isolated from rabbit larynges and cultured. For magnetization, cells were incubated with superparamagnetic iron oxide nanoparticles (SPION) and the loading efficiency was determined by Prussian blue staining. Biocompatibility was analyzed in flow cytometry by staining with annexin V-fluorescein isothiocyanate propidium iodide, 1,1',3,3,3',3'-hexamethylindodicarbo-cyanine iodide [DiIC1(5)] and propidium idodide-Triton X-100 to monitor phosphatidylserine exposure, plasma membrane integrity, mitochondrial membrane potential and DNA degradation. RESULTS: Isolated VFFs can be successfully loaded with SPION, and optimal iron loading associated with minimized cytotoxicity represents a balancing act in magnetic tissue engineering. CONCLUSION: Our data are a firm basis for the next steps of investigations. Magnetic tissue engineering using magnetic nanoparticle-loaded cells which form three-dimensional structures in a magnetic field will be a promising approach in the future.

Effect of BSA-coated Superparamagnetic Iron Oxide Nanoparticles on Granulosa Cells.

BACKGROUND: Since superparamagnetic iron oxide nanoparticles (SPION) possess unique features, they provide a huge platform for medical applications, especially for cancer diagnosis and therapy (e.g. imaging, and drug targeting). However, heterogeneous effects on mammalian cells with regard to reproductive tissue are described. An experimental study was carried out to study the effects of SPIONs on both the expression of steroid hormone receptor and viability of granulosa cells, which play a key role in ovarian health and fertility. MATERIALS AND METHODS: Human granulosa cells were cultured in vitro and incubated with different concentrations of SPIONs. After 48 h, steroid receptor expression and cell viability were evaluated. RESULTS: Treatment of granulosa cells with SPIONs did not affect estrogen receptor ß1 or progesterone receptor-A expression and had no significant effect on cell viability. CONCLUSION: Nanoparticles precoated with bovine serum albumin (BSA) do not alter granulosa cell phenotype, whereas literature suggests that other nanoparticles induce apoptosis and reduce steroid receptor expression. Our data indicate an overall better outcome using SPIONs coated with BSA.

Magnetic nanoparticle-based drug delivery for cancer therapy.

Nanoparticles have belonged to various fields of biomedical research for quite some time. A promising site-directed application in the field of nanomedicine is drug targeting using magnetic nanoparticles which are directed at the target tissue by means of an external magnetic field. Materials most commonly used for magnetic drug delivery contain metal or metal oxide nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs). SPIONs consist of an iron oxide core, often coated with organic materials such as fatty acids, polysaccharides or polymers to improve colloidal stability and to prevent separation into particles and carrier medium [1]. In general, magnetite and maghemite particles are those most commonly used in medicine and are, as a rule, well-tolerated. The magnetic properties of SPIONs allow the remote control of their accumulation by means of an external magnetic field. Conjugation of SPIONs with drugs, in combination with an external magnetic field to target the nanoparticles (so-called "magnetic drug targeting", MDT), has additionally emerged as a promising strategy of drug delivery. Magnetic nanoparticle-based drug delivery is a sophisticated overall concept and a multitude of magnetic delivery vehicles have been developed. Targeting mechanism-exploiting, tumor-specific attributes are becoming more and more sophisticated. The same is true for controlled-release strategies for the diseased site. As it is nearly impossible to record every magnetic nanoparticle system developed so far, this review summarizes interesting approaches which have recently emerged in the field of targeted drug delivery for cancer therapy based on magnetic nanoparticles.

Boron containing magnetic nanoparticles for neutron capture therapy--an innovative approach for specifically targeting tumors.

The selective delivery of (10)B into the tumor tissue remains to be further improved for successful and reliable Boron Neutron Capture Therapy applications. Magnetic Drug Targeting using intraarterially administered superparamagnetic nanoparticles and external magnetic fields already exhibited convincing results in terms of highly efficient and selective drug deposition. Using the same technique for the targeted (10)B delivery is a promising new approach. Here, systematic irradiation experiments of phantom cubes containing different concentrations of boron and nanoparticles as well as varying three-dimensional arrangements have been performed.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods.

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEON(LA)) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEON(LA) with an additional protein corona formed by bovine serum albumin (SEON(LA-BSA)) and commercially available Rienso(®) particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.