Intravital electrochemical nanosensor as a tool for the measurement of reactive oxygen/nitrogen species in liver diseases.

Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein-based sensors, which have several disadvantages for in vivo use. Intravital electrochemical nanosensors can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allow dynamic ROS/RNS measurements in vivo. Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl4, partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical detection that any exposure to the peritoneum in vivo leads to an increase in total ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the total ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice 24 h after HE and in sham-operated mice. We quantified dose-dependent ROS/RNS production in CCl4-induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.

Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma.

Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.

Variation in tumor pH affects pH-triggered delivery of peptide-modified magnetic nanoparticles.

Acidification of the extracellular matrix, an intrinsic characteristic of many solid tumors, is widely exploited for physiologically triggered delivery of contrast agents, drugs, and nanoparticles to tumor. However, pH of tumor microenvironment shows intra- and inter-tumor variation. Herein, we investigate the impact of this variation on pH-triggered delivery of magnetic nanoparticles (MNPs) modified with pH-(low)-insertion peptide (pHLIP). Fluorescent flow cytometry, laser confocal scanning microscopy and transmission electron microscopy data proved that pHLIP-conjugated MNPs interacted with 4T1 cells in two-dimensional culture and in spheroids more effectively at pH 6.4 than at pH 7.2, and entered the cell via clathrin-independent endocytosis. The accumulation efficiency of pHLIP-conjugated MNPs in 4T1 tumors after their intravenous injection, monitored in vivo by magnetic resonance imaging, showed variation. Analysis of the tumor pH profiles recorded with implementation of original nanoprobe pH sensor, revealed obvious correlation between pH measured in the tumor with the amount of accumulated MNPs.

Temperature-controlled magnetic nanoparticles hyperthermia inhibits primary tumor growth and metastases dissemination.

Magnetic hyperthermia (MHT) is a promising approach for cancer therapy. However, a systematic MHT characterization as function of temperature on the therapeutic efficiency is barely analyzed. Here, we first perform comparative temperature-dependent analysis of the cobalt ferrite nanoparticles-mediated MHT effectiveness in two murine tumors models - breast (4T1) and colon (CT26) cancer in vitro and in vivo. The overall MHT killing capacity in vitro increased with the temperature and CT26 cells were more sensitive than 4T1 when heated to 43 °C. Well in line with the in vitro data, such heating cured non-metastatic CT26 tumors in vivo, while only inhibiting metastatic 4T1 tumor growth without improving the overall survival. High-temperature MHT (>47 °C) resulted in complete 4T1 primary tumor clearance, 25-40% long-term survival rates, and, importantly, more effective prevention of metastasis comparing to surgical extraction. Thus, the specific MHT temperature must be defined for each tumor individually to ensure a successful antitumor therapy.

Neutrophils in viral infection.

Neutrophils are the first wave of recruited immune cells to sites of injury or infection and are crucial players in controlling bacterial and fungal infections. Although the role of neutrophils during bacterial or fungal infections is well understood, their impact on antiviral immunity is much less studied. Furthermore, neutrophil function in tumor pathogenesis and cancer treatment has recently received much attention, particularly within the context of oncolytic virus infection where neutrophils produce antitumor cytokines and enhance oncolysis. In this review, multiple functions of neutrophils in viral infections and immunity are discussed. Understanding the role of neutrophils during viral infection may provide insight into the pathogenesis of virus infections and the outcome of virus-based therapies.

Multimodal doxorubicin loaded magnetic nanoparticles for VEGF targeted theranostics of breast cancer.

In presented paper we have developed new system for cancer theranostics based on vascular endothelial growth factor (VEGF) targeted magnetic nanoparticles. Conjugation of anti-VEGF antibodies with bovine serum albumin coated PEGylated magnetic nanoparticles allows for improved binding with murine breast adenocarcinoma 4T1 cell line and facilitates doxorubicin delivery to tumor cells. It was shown that intravenous injection of doxorubicin loaded VEGF targeted nanoparticles increases median survival rate of mice bearing 4T1 tumors up to 50%. On the other hand magnetic resonance imaging (MRI) of 4T1 tumors 24 h after intravenous injection showed accumulation of nanoparticles in tumors, thus allowing simultaneous cancer therapy and diagnostics.

Herpes simplex virus inoculation in murine rete testis results in irreversible testicular damage.

This study aimed to establish the influence of herpes simplex virus (HSV) on testis morphology and germ cell development using a model of ascending urogenital HSV infection in mice. Adult C57BL/6J mice were inoculated with 100 plaque-forming units of HSV1 in rete testis. Viral proteins and HSV DNA were detected from 3 days postinoculation (DPI), while capsids and virions could be visualized at 6 DPI. Infectious activity of HSV was revealed by rapid culture method in testes from 3 to 14 DPI, and virus DNA by PCR - from 3 to 100 DPI. Germ and Sertoli cells were infected during the early stages of the infection, whereas interstitial cells only occasionally contained the virus at 21 and 45 DPI. Microscopic analysis revealed severe degeneration of the germinal epithelium in the infected testes. By 21 DPI, testes became atrophic and most Sertoli cells were destroyed. No testicular regeneration and no spermatozoa in the epididymis were observed at 45 and 100 DPI. From 3 DPI, inflammatory cells accumulated in the interstitium between damaged tubules; a significant increase in the number of CD4(+), CD8(+) T lymphocytes and F4/80(+) cells was observed in the infected testes. This study shows that in the case of HSV retrograde ascent into seminiferous tubules, the acute viral infection results in irreversible atrophy of the germinal epithelium, orchitis and infertility. These results may be used to further study viral orchitis and the influence of HSV on spermatogenesis and male fertility.

The need for paradigm shift: prognostic significance and implications of standard therapy-related systemic immunosuppression in glioblastoma for immunotherapy and oncolytic virotherapy.

Despite significant advances in our knowledge regarding the genetics and molecular biology of gliomas over the past two decades and hundreds of clinical trials, no effective therapeutic approach has been identified for adult patients with newly diagnosed glioblastoma, and overall survival remains dismal. Great hopes are now placed on combination immunotherapy. In clinical trials, immunotherapeutics are generally tested after standard therapy (radiation, temozolomide, and steroid dexamethasone) or concurrently with temozolomide and/or steroids. Only a minor subset of patients with progressive/recurrent glioblastoma have benefited from immunotherapies. In this review, we comprehensively discuss standard therapy-related systemic immunosuppression and lymphopenia, their prognostic significance, and the implications for immunotherapy/oncolytic virotherapy. The effectiveness of immunotherapy and oncolytic virotherapy (viro-immunotherapy) critically depends on the activity of the host immune cells. The absolute counts, ratios, and functional states of different circulating and tumor-infiltrating immune cell subsets determine the net immune fitness of patients with cancer and may have various effects on tumor progression, therapeutic response, and survival outcomes. Although different immunosuppressive mechanisms operate in patients with glioblastoma/gliomas at presentation, the immunological competence of patients may be significantly compromised by standard therapy, exacerbating tumor-related systemic immunosuppression. Standard therapy affects diverse immune cell subsets, including dendritic, CD4+, CD8+, natural killer (NK), NKT, macrophage, neutrophil, and myeloid-derived suppressor cell (MDSC). Systemic immunosuppression and lymphopenia limit the immune system's ability to target glioblastoma. Changes in the standard therapy are required to increase the success of immunotherapies. Steroid use, high neutrophil-to-lymphocyte ratio (NLR), and low post-treatment total lymphocyte count (TLC) are significant prognostic factors for shorter survival in patients with glioblastoma in retrospective studies; however, these clinically relevant variables are rarely reported and correlated with response and survival in immunotherapy studies (e.g., immune checkpoint inhibitors, vaccines, and oncolytic viruses). Our analysis should help in the development of a more rational clinical trial design and decision-making regarding the treatment to potentially improve the efficacy of immunotherapy or oncolytic virotherapy.

Neutrophil as a Carrier for Cancer Nanotherapeutics: A Comparative Study of Liposome, PLGA, and Magnetic Nanoparticles Delivery to Tumors.

Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types-liposomes, PLGA, and magnetite nanoparticles-was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application.

Oncolytic Efficacy of a Recombinant Vaccinia Virus Strain Expressing Bacterial Flagellin in Solid Tumor Models.

Oncolytic viral therapy is a promising novel approach to cancer treatment. Oncolytic viruses cause tumor regression through direct cytolysis on the one hand and recruiting and activating immune cells on the other. In this study, to enhance the antitumor efficacy of the thymidine kinase-deficient vaccinia virus (VV, Lister strain), recombinant variants encoding bacterial flagellin (subunit B) of Vibrio vulnificus (LIVP-FlaB-RFP), firefly luciferase (LIVP-Fluc-RFP) or red fluorescent protein (LIVP-RFP) were developed. The LIVP-FLuc-RFP strain demonstrated exceptional onco-specificity in tumor-bearing mice, detected by the in vivo imaging system (IVIS). The antitumor efficacy of these variants was explored in syngeneic murine tumor models (B16 melanoma, CT26 colon cancer and 4T1 breast cancer). After intravenous treatment with LIVP-FlaB-RFP or LIVP-RFP, all mice tumor models exhibited tumor regression, with a prolonged survival rate in comparison with the control mice. However, superior oncolytic activity was observed in the B16 melanoma models treated with LIVP-FlaB-RFP. Tumor-infiltrated lymphocytes and the cytokine analysis of the serum and tumor samples from the melanoma-xenografted mice treated with these virus variants demonstrated activation of the host's immune response. Thus, the expression of bacterial flagellin by VV can enhance its oncolytic efficacy against immunosuppressive solid tumors.

Infection of non-cancer cells: A barrier or support for oncolytic virotherapy?

Oncolytic viruses are designed to specifically target cancer cells, sparing normal cells. Although numerous studies demonstrate the ability of oncolytic viruses to infect a wide range of non-tumor cells, the significance of this phenomenon for cancer virotherapy is poorly understood. To fill the gap, we summarize the data on infection of non-cancer targets by oncolytic viruses with a special focus on tumor microenvironment and secondary lymphoid tissues. The review aims to address two major questions: how do attenuated viruses manage to infect normal cells, and whether it is of importance for oncolytic virotherapy.

Repeated dosing improves oncolytic rhabdovirus therapy in mice via interactions with intravascular monocytes.

There is debate in the field of oncolytic virus (OV) therapy, whether a single viral dose, or multiple administrations, is better for tumor control. Using intravital microscopy, we describe the fate of vesicular stomatitis virus (VSV) delivered systemically as a first or a second dose. Following primary administration, VSV binds to the endothelium, initiates tumor infection and activates a proinflammatory response. This initial OV dose induces neutrophil migration into the tumor and limits viral replication. OV administered as a second dose fails to infect the tumor and is captured by intravascular monocytes. Despite a lack of direct infection, this second viral dose, in a monocyte-dependent fashion, enhances and sustains infection by the first viral dose, promotes CD8 T cell recruitment, delays tumor growth and improves survival in multi-dosing OV therapy. Thus, repeated VSV dosing engages monocytes to post-condition the tumor microenvironment for improved infection and anticancer T cell responses. Understanding the complex interactions between the subsequent viral doses is crucial for improving the efficiency of OV therapy and virus-based vaccines.

In Vivo Tracking for Oncolytic Adenovirus Interactions with Liver Cells.

Hepatotoxicity remains an as yet unsolved problem for adenovirus (Ad) cancer therapy. The toxic effects originate both from rapid Kupffer cell (KCs) death (early phase) and hepatocyte transduction (late phase). Several host factors and capsid components are known to contribute to hepatotoxicity, however, the complex interplay between Ad and liver cells is not fully understood. Here, by using intravital microscopy, we aimed to follow the infection and immune response in mouse liver from the first minutes up to 72 h post intravenous injection of three Ads carrying delta-24 modification (Ad5-RGD, Ad5/3, and Ad5/35). At 15-30 min following the infusion of Ad5-RGD and Ad5/3 (but not Ad5/35), the virus-bound macrophages demonstrated signs of zeiosis: the formation of long-extended protrusions and dynamic membrane blebbing with the virus release into the blood in the membrane-associated vesicles. Although real-time imaging revealed interactions between the neutrophils and virus-bound KCs within minutes after treatment, and long-term contacts of CD8+ T cells with transduced hepatocytes at 24-72 h, depletion of neutrophils and CD8+ T cells affected neither rate nor dynamics of liver infection. Ad5-RGD failed to complete replicative cycle in hepatocytes, and transduced cells remained impermeable for propidium iodide, with a small fraction undergoing spontaneous apoptosis. In Ad5-RGD-immune mice, the virus neither killed KCs nor transduced hepatocytes, while in the setting of hepatic regeneration, Ad5-RGD enhanced liver transduction. The clinical and biochemical signs of hepatotoxicity correlated well with KC death, but not hepatocyte transduction. Real-time in vivo tracking for dynamic interactions between virus and host cells provides a better understanding of mechanisms underlying Ad-related hepatotoxicity.

Neutrophil and Nanoparticles Delivery to Tumor: Is It Going to Carry That Weight?

The application of cell carriers for transporting nanodrugs to the tumor draws much attention as the alternative to the passive drug delivery. In this concept, the neutrophil (NΦ) is of special interest as this cell is able to uptake nanoparticles (NPs) and cross the vascular barrier in response to tumor signaling. There is a growing body of literature describing NP-NΦ interactions in vitro and in vivo that demonstrates the opportunity of using these cells to improve the efficacy of cancer therapy. However, a number of conceptual and technical issues need to be resolved for translating the technology into clinics. The current review summarizes the recent advances and challenges associated with NP-NΦ interactions, with the special focus on the complex interplay between the NP internalization pathways and the modulation of NΦ activity, and its potential consequences for nanodrug delivery.

Cobalt Ferrite Nanoparticles for Tumor Therapy: Effective Heating versus Possible Toxicity.

Magnetic nanoparticles (MNPs) are widely considered for cancer treatment, in particular for magnetic hyperthermia (MHT). Thereby, MNPs are still being optimized for lowest possible toxicity on organisms while the magnetic properties are matched for best heating capabilities. In this study, the biocompatibility of 12 nm cobalt ferrite MNPs, functionalized with citrate ions, in different dosages on mice and rats of both sexes was investigated for 30 days after intraperitoneal injection. The animals' weight, behavior, and blood cells changes, as well as blood biochemical parameters are correlated to histological examination of organs revealing that cobalt ferrite MNPs do not have toxic effects at concentrations close to those used previously for efficient MHT. Moreover, these MNPs demonstrated high specific loss power (SLP) of about 400 W g-1. Importantly the MNPs retained their magnetic properties inside tumor tissue after intratumoral administration for several MHT cycles within three days. Thus, cobalt ferrite MNPs represent a perspective platform for tumor therapy by MHT due to their ability to provide effective heating without exerting a toxic effect on the organism. This opens up new avenues for smaller MNPs sizes while their heating efficiency is maintained.

Platelet-Mediated NET Release Amplifies Coagulopathy and Drives Lung Pathology During Severe Influenza Infection.

The influenza A virus (IAV) causes a respiratory tract infection with approximately 10% of the population infected by the virus each year. Severe IAV infection is characterized by excessive inflammation and tissue pathology in the lungs. Platelet and neutrophil recruitment to the lung are involved in the pathogenesis of IAV, but the specific mechanisms involved have not been clarified. Using confocal intravital microscopy in a mouse model of IAV infection, we observed profound neutrophil recruitment, platelet aggregation, neutrophil extracellular trap (NET) production and thrombin activation within the lung microvasculature in vivo. Importantly, deficiency or antagonism of the protease-activated receptor 4 (PAR4) reduced platelet aggregation, NET production, and neutrophil recruitment. Critically, inhibition of thrombin or PAR4 protected mice from virus-induced lung tissue damage and edema. Together, these data imply thrombin-stimulated platelets play a critical role in the activation/recruitment of neutrophils, NET release and directly contribute to IAV pathogenesis in the lung.

Intravital imaging of liposome behavior upon repeated administration: A step towards the development of liposomal companion diagnostic for cancer nanotherapy.

Accumulation of liposomal drugs into human tumors has substantial variability influencing the probability of positive response to the therapy. Therefore, it becomes very important to identify the eligibility of patients for various treatment options. The existing strategies of tumor stratification using companion diagnostics are based on the assumption that the initial and subsequent doses of nanoparticles (NP) behave in a sufficiently similar manner to enable a valuable prognosis. Here, we use a combination of in vivo imaging techniques to validate the applicability of magnetic liposomes (ML) as a reliable tool to predict whether or not the tumor would respond to nanomedicine therapy. The results demonstrated that liposome biodistribution, interactions with immune cells, and extravasation behavior in tumors were not affected by the pretreatment with liposomes 24 h prior to the repeat dosing. Co-administration of liposomal doxorubicin (DXR) and liposomes loaded with maghemite NP resulted in a high colocalization rate between two nanomedicines in tumors suggesting that neither contrast agent, nor chemotherapeutics altered biodistribution of liposomes. Based on magnetic resonance imaging of 4T1 tumors performed before and 6 h after ML treatment, animals were classified into high and low accumulation subgroups. Higher ML deposition in tumors was associated with a reduction in lesion size and enhanced survival in animals treated with liposomal DXR, but not with DXR alone. Given that liposomes are the most numerous class of clinically approved nanomedicines the development of safe and cost-effective liposomal companion diagnostic suitable for non-invasive imaging is of paramount importance for improving the efficacy of cancer therapy.

Neutrophil-mediated transport is crucial for delivery of short-circulating magnetic nanoparticles to tumors.

Recently neutrophil-based nanoparticles (NPs) drug delivery systems have gained considerable attention in cancer therapy. Numerous studies have been conducted to identify optimal NPs parameters for passive tumor targeting, while there is a fundamental dearth of knowledge about the factors governing cell-mediated delivery. Here, by using intravital microscopy and magnetic resonance imaging, we describe accumulation dynamics of 140 nm magnetic cubes and clusters in murine breast cancer (4T1) and colon cancer (CT26) models. Notwithstanding rapid clearance from the blood flow, NPs readily accumulated in tumors at later time points. Both NPs types were captured mostly by intravascular neutrophils immediately after injection, and transmigration of NPs-bound neutrophils through the vessel wall was first shown in real-time. A dramatic drop in NPs accumulation upon Ly6G and Gr1 depletion further confirmed the role of neutrophils as a biocarrier for targeting tumors. Of note, for shorter circulating NPs, a cell-dependent delivery route was more impactful, while the accumulation of longer circulating counterpart was less compromised by neutrophil depletion. Neutrophil-mediated transport was also shown to depend on tumor type, with more efficiency noted in neutrophil-rich tumors. Revealing NPs characteristics and host factors influencing the neutrophil-based tumor targeting will help to rationally design drug delivery systems for improved cancer treatment. STATEMENT OF SIGNIFICANCE: Utilizing host cells as trojan horses for delivery nanodrugs to tumor site is a promising approach for cancer therapy. However, it is not clear yet how nanoparticles characteristics and tumor properties affect the efficiency of cell-based nanoparticles transport. Here, we compare neutrophil-based delivery of different-shaped magnetic nanoparticles (cubes and clusters) in two tumor models. The results suggest that neutrophil-mediated route is more impactful for rapidly cleared cubes, than for longer circulating clusters. The efficiency of cell-based accumulation also correlated with the level of neutrophils recruitment to different tumor types. These finding are important for rationale design of nanocarriers and predicting the efficiency of neutrophil-mediated drug delivery between patients and tumor types.

Extravasating Neutrophils Open Vascular Barrier and Improve Liposomes Delivery to Tumors.

Liposomes are the most extensively used nanocarriers in cancer therapy. Despite the advantages these vehicles provide over free drugs, there are still limitations with regards to the efficiency of liposomes delivery to tumors and off-target accumulation. A better understanding of nanodrugs extravasation mechanisms in different tumor types and normal vessels is needed to improve their antitumor activity. We used intravital microscopy to track for fluorescent liposomes behavior in xenograft tumor models (murine breast cancer 4T1 and melanoma B16, human prostate cancer 22Rv1) and normal skin and identified two distinct extravasation patterns. Microleakage, a local perivascular nanoparticle deposition, was found both in malignant and healthy tissues. This type of liposomes leakage does not provide access to tumor cells and is presumably responsible for drug deposition in normal tissues. In contrast, macroleakage penetrated deep into tissues and localized predominantly on the tumor-host interface. Although neutrophils did not uptake liposomes, their extravasation appeared to initiate both micro- and macroleakages. Based on neutrophils and liposomes extravasation dynamics, we hypothesized that microleakage and macroleakage are subsequent steps of the extravasation process corresponding to liposomes transport through endothelial and subendothelial barriers. Of note, extravasation spots were detected more often in the proximity of neutrophils, and across studied tumor types, neutrophils counts correlated with leakage frequencies. Reduced liposomes accumulation in 4T1 tumors upon Ly6G depletion further corroborated neutrophils role in nanoparticles delivery. Elucidating liposomes extravasation routes has a potential to help improve existing strategies and develop effective nanodrugs for cancer therapy.

Intravital microscopy reveals a novel mechanism of nanoparticles excretion in kidney.

Developing nanocarriers that accumulate in targeted organs and are harmlessly eliminated still remains a big challenge. Nanoparticles (NP) biodistribution is governed by their size, composition, surface charge and coverage. The current thinking in bionanotechnology is that renal clearance is limited by glomerular basement membrane pore size (≈6 nm), although there is a growing evidence that NP exceeding the threshold can also be excreted with urine. Here we compare biodistribution of PEGylated 140 nm iron oxide cubes and clusters with a special focus on renal accumulation and excretion. Atomic emission spectroscopy, fluorescent microscopy and magnetic resonance imaging revealed rapid and transient accumulation of magnetic NP in kidney. Using intravital microscopy we tracked in real time NP translocation from peritubular capillaries to basal compartment of tubular cells and subsequent excretion to the lumen within 60 min after systemic administration. Transmission electron microscopy revealed persistence of intact full-sized NP in urine 2 h post injection. The results suggest that translocation through peritubular endothelium to tubular epithelial cells is an alternative mechanism of renal clearance enabling excretion of NP above glomerular cut-off size.