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.

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.

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.

Study of Cytotoxicity and Internalization of Redox-Responsive Iron Oxide Nanoparticles on PC-3 and 4T1 Cancer Cell Lines.

Redox-responsive and magnetic nanomaterials are widely used in tumor treatment separately, and while the application of their combined functionalities is perspective, exactly how such synergistic effects can be implemented is still unclear. This report investigates the internalization dynamics of magnetic redox-responsive nanoparticles (MNP-SS) and their cytotoxicity toward PC-3 and 4T1 cell lines. It is shown that MNP-SS synthesized by covalent grafting of polyethylene glycol (PEG) on the magnetic nanoparticle (MNP) surface via SS-bonds lose their colloidal stability and aggregate fully in a solution containing DTT, and partially in conditioned media, whereas the PEGylated MNP (MNP-PEG) without S-S linker control remains stable under the same conditions. Internalized MNP-SS lose the PEG shell more quickly, causing enhanced magnetic core dissolution and thus increased toxicity. This was confirmed by fluorescence microscopy using MNP-SS dual-labeled by Cy3 via labile disulfide, and Cy5 via a rigid linker. The dyes demonstrated a significant difference in fluorescence dynamics and intensity. Additionally, MNP-SS demonstrate quicker cellular uptake compared to MNP-PEG, as confirmed by TEM analysis. The combination of disulfide bonds, leading to faster dissolution of the iron oxide core, and the high-oxidative potential Fe3+ ions can synergically enhance oxidative stress in comparison with more stable coating without SS-bonds in the case of MNP-PEG. It decreases the cancer cell viability, especially for the 4T1, which is known for being sensitive to ferroptosis-triggering factors. In this work, we have shown the effect of redox-responsive grafting of the MNP surface as a key factor affecting MNP-internalization rate and dissolution with the release of iron ions inside cancer cells. This kind of synergistic effect is described for the first time and can be used not only in combination with drug delivery, but also in treatment of tumors responsive to ferroptosis.

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.

Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action.

Cell therapy is an emerging approach to stroke treatment with a potential to limit brain damage and enhance its restoration after the acute phase of the disease. In this study we tested directly reprogrammed neural precursor cells (drNPC) derived from adult human bone marrow cells in the rat middle cerebral artery occlusion (MCAO) model of acute ischemic stroke using human placenta mesenchymal stem cells (pMSC) as a positive control with previously confirmed efficacy. Cells were infused into the ipsilateral (right) internal carotid artery of male Wistar rats 24 h after MCAO. The main goal of this work was to evaluate real-time distribution and subsequent homing of transplanted cells in the brain. This was achieved by performing intra-arterial infusion directly inside the MRI scanner and allowed transplanted cells tracing starting from their first pass through the brain vessels. Immediately after transplantation, cells were observed in the periphery of the infarct zone and in the brain stem, 15 min later small numbers of cells could be discovered deep in the infarct core and in the contralateral hemisphere, where drNPC were seen earlier and in greater numbers than pMSC. Transplanted cells in both groups could no longer be detected in the rat brain 48-72 h after infusion. Histological and histochemical analysis demonstrated that both the drNPC and pMSC were localized inside blood vessels in close contact with the vascular wall. No passage of labeled cells through the blood brain barrier was observed. Additionally, the therapeutic effects of drNPC and pMSC were compared. Both drNPC and pMSC induced substantial attenuation of neurological deficits evaluated at the 7th and 14th day after transplantation using the modified neurological severity score (mNSS). Some of the effects of drNPC and pMSC, such as the influence on the infarct volume and the survival rate of animals, differed. The results suggest a paracrine mechanism of the positive therapeutic effects of IA drNPC and pMSC infusion, potentially enhanced by the cell-cell interactions. Our data also indicate that the long-term homing of transplanted cells in the brain is not necessary for the brain's functional recovery.

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.

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.

Toxicity of iron oxide nanoparticles: Size and coating effects.

Toxicological research of novel nanomaterials is a major developmental step of their clinical approval. Since iron oxide magnetic nanoparticles have a great potential in cancer treatment and diagnostics, the investigation of their toxic properties is very topical. In this paper we synthesized bovine serum albumin-coated iron oxide nanoparticles with different sizes and their polyethylene glycol derivative. To prove high biocompatibility of obtained nanoparticles the number of in vitro toxicological tests on human fibroblasts and U251 glioblastoma cells was performed. It was shown that albumin nanoparticles' coating provides a stable and biocompatible shell and prevents cytotoxicity of magnetite core. On long exposure times (48 hours), cytotoxicity of iron oxide nanoparticles takes place due to free radical production, but this toxic effect may be neutralized by using polyethylene glycol modification.

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.