Sol-gel derived B2O3-CaO borate bioactive glasses with hemostatic, antibacterial and pro-angiogenic activities.

Sol-gel borate bioactive glasses (BGs) are promising ion-releasing biomaterials for wound healing applications. Here, we report the synthesis of a series of binary B2O3-CaO borate BGs (CaO ranging from 50 to 90 mol%) using a sol-gel-based method. The influence of CaO content in B2O3-CaO borate BG on morphology, structure and ion release behavior was investigated in detail. Reduced dissolution (ion release) and crystallization could be observed in borate BGs when CaO content increased, while the morphology was not significantly altered by increasing CaO content. Our results evidenced that the ion release behavior of borate BGs could be tailored by tuning the B2O3/CaO molar ratio. We also evaluated the in vitro cytotoxicity, hemostatic, antibacterial and angiogenic activities of borate BGs. Cytocompatibility was validated for all borate BGs. However, borate BGs exhibited composition-dependent hemostatic, antibacterial and angiogenic activities. Generally, higher contents of Ca in borate BGs facilitated hemostatic activity, while higher contents of B2O3 were beneficial for pro-angiogenic activity. The synthesized sol-gel-derived borate BGs are promising materials for developing advanced wound healing dressings, given their fast ion release behavior and favorable hemostatic, antibacterial and angiogenic activities.

An in silico model of the capturing of magnetic nanoparticles in tumour spheroids in the presence of flow.

One of the main challenges in improving the efficacy of conventional chemotherapeutic drugs is that they do not reach the cancer cells at sufficiently high doses while at the same time affecting healthy tissue and causing significant side effects and suffering in cancer patients. To overcome this deficiency, magnetic nanoparticles as transporter systems have emerged as a promising approach to achieve more specific tumour targeting. Drug-loaded magnetic nanoparticles can be directed to the target tissue by applying an external magnetic field. However, the magnetic forces exerted on the nanoparticles fall off rapidly with distance, making the tumour targeting challenging, even more so in the presence of flowing blood or interstitial fluid. We therefore present a computational model of the capturing of magnetic nanoparticles in a test setup: our model includes the flow around the tumour, the magnetic forces that guide the nanoparticles, and the transport within the tumour. We show how a model for the transport of magnetic nanoparticles in an external magnetic field can be integrated with a multiphase tumour model based on the theory of porous media. Our approach based on the underlying physical mechanisms can provide crucial insights into mechanisms that cannot be studied conclusively in experimental research alone. Such a computational model enables an efficient and systematic exploration of the nanoparticle design space, first in a controlled test setup and then in more complex in vivo scenarios. As an effective tool for minimising costly trial-and-error design methods, it expedites translation into clinical practice to improve therapeutic outcomes and limit adverse effects for cancer patients.

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality.

Background: Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. Methods: In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs. Results: SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. Conclusion: In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

Magnetic Removal of Candida albicans Using Salivary Peptide-Functionalized SPIONs.

Purpose: Magnetic separation of microbes can be an effective tool for pathogen identification and diagnostic applications to reduce the time needed for sample preparation. After peptide functionalization of superparamagnetic iron oxide nanoparticles (SPIONs) with an appropriate interface, they can be used for the separation of sepsis-associated yeasts like Candida albicans. Due to their magnetic properties, the magnetic extraction of the particles in the presence of an external magnetic field ensures the accumulation of the targeted yeast. Materials and Methods: In this study, we used SPIONs coated with 3-aminopropyltriethoxysilane (APTES) and functionalized with a peptide originating from GP340 (SPION-APTES-Pep). For the first time, we investigate whether this system is suitable for the separation and enrichment of Candida albicans, we investigated its physicochemical properties and by thermogravimetric analysis we determined the amount of peptide on the SPIONs. Further, the toxicological profile was evaluated by recording cell cycle and DNA degradation. The separation efficiency was investigated using Candida albicans in different experimental settings, and regrowth experiments were carried out to show the use of SPION-APTES-Pep as a sample preparation method for the identification of fungal infections. Conclusion: SPION-APTES-Pep can magnetically remove more than 80% of the microorganism and with a high selective host-pathogen distinction Candida albicans from water-based media and about 55% in blood after 8 minutes processing without compromising effects on the cell cycle of human blood cells. Moreover, the separated fungal cells could be regrown without any restrictions.

Cell Viability and Immunogenic Function of T Cells Loaded with Nanoparticles for Spatial Guidance in Magnetic Fields.

Immune cell therapies, such as adoptive T cell therapies, are an innovative and powerful treatment option for previously non-treatable diseases. Although immune cell therapies are thought to be very specific, there is still the danger of developing severe to life-threatening side effects due to the unspecific distribution of the cells throughout the body (on-target/off-tumor effects). A possible solution for the reduction of these side effects and the improvement of tumor infiltration is the specific targeting of the effector cells (e.g., T cells) to the desired destination (e.g., tumor region). This can be achieved by the magnetization of cells with superparamagnetic iron oxide nanoparticles (SPIONs) for spatial guidance via external magnetic fields. A prerequisite for the use of SPION-loaded T cells in adoptive T cell therapies is that cell viability and functionality after nanoparticle loading are preserved. Here, we demonstrate a protocol to analyze cell viability and functionality such as activation, proliferation, cytokine release, and differentiation at a single cell level using flow cytometry.

Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids.

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-ß pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Comparative in vitro and in vivo Evaluation of Different Iron Oxide-Based Contrast Agents to Promote Clinical Translation in Compliance with Patient Safety.

Introduction: One of the major challenges in the clinical translation of nanoparticles is the development of formulations combining favorable efficacy and optimal safety. In the past, iron oxide nanoparticles have been introduced as an alternative for gadolinium-containing contrast agents; however, candidates available at the time were not free from adverse effects. Methods: Following the development of a potent iron oxide-based contrast agent SPIONDex, we now performed a systematic comparison of this formulation with the conventional contrast agent ferucarbotran and with ferumoxytol, taking into consideration their physicochemical characteristics, bio- and hemocompatibility in vitro and in vivo, as well as their liver imaging properties in rats. Results: The results demonstrated superior in vitro cyto-, hemo- and immunocompatibility of SPIONDex in comparison to the other two formulations. Intravenous administration of ferucarbotran or ferumoxytol induced strong complement activation-related pseudoallergy in pigs. In contrast, SPIONDex did not elicit any hypersensitivity reactions in the experimental animals. In a rat model, comparable liver imaging properties, but a faster clearance was demonstrated for SPIONDex. Conclusion: The results indicate that SPIONDex possess an exceptional safety compared to the other two formulations, making them a promising candidate for further clinical translation.

In Vitro Setup for Determination of Nanoparticle-Mediated Magnetic Cell and Drug Accumulation in Tumor Spheroids under Flow Conditions.

Superparamagnetic iron oxide nanoparticles (SPIONs) are used in nanomedicine as transporter systems for therapeutic cargos, or to magnetize cells to make them magnetically guidable. In cancer treatment, the site-directed delivery of chemotherapeutics or immune effector cells to the tumor can increase the therapeutic efficacy in the target region, and simultaneously reduce toxic side-effects in the rest of the body. To enable the transfer of new methods, such as the nanoparticle-mediated transport from bench to bedside, suitable experimental setups must be developed. In vivo, the SPIONs or SPION-loaded cells must be applied into the blood stream, to finally reach the tumor: consequently, targeting and treatment efficacy should be analyzed under conditions which are as close to in vivo as possible. Here, we established an in vitro method, including tumor spheroids placed in a chamber system under the influence of a magnetic field, and adapted to a peristaltic pump, to mimic the blood flow. This enabled us to analyze the magnetic capture and antitumor effects of magnetically targeted mitoxantrone and immune cells under dynamic conditions. We showed that the magnetic nanoparticle-mediated accumulation increased the anti-tumor effects, and reduced the unspecific distribution of both mitoxantrone and cells. Especially for nanomedical research, investigation of the site-specific targeting of particles, cells or drugs under circulation is important. We conclude that our in vitro setup improves the screening process of nanomedical candidates for cancer treatment.

Optical Microscopy Systems for the Detection of Unlabeled Nanoparticles.

Label-free detection of nanoparticles is essential for a thorough evaluation of their cellular effects. In particular, nanoparticles intended for medical applications must be carefully analyzed in terms of their interactions with cells, tissues, and organs. Since the labeling causes a strong change in the physicochemical properties and thus also alters the interactions of the particles with the surrounding tissue, the use of fluorescently labeled particles is inadequate to characterize the effects of unlabeled particles. Further, labeling may affect cellular uptake and biocompatibility of nanoparticles. Thus, label-free techniques have been recently developed and implemented to ensure a reliable characterization of nanoparticles. This review provides an overview of frequently used label-free visualization techniques and highlights recent studies on the development and usage of microscopy systems based on reflectance, darkfield, differential interference contrast, optical coherence, photothermal, holographic, photoacoustic, total internal reflection, surface plasmon resonance, Rayleigh light scattering, hyperspectral and reflectance structured illumination imaging. Using these imaging modalities, there is a strong enhancement in the reliability of experiments concerning cellular uptake and biocompatibility of nanoparticles, which is crucial for preclinical evaluations and future medical applications.

Scavenging of bacteria or bacterial products by magnetic particles functionalized with a broad-spectrum pathogen recognition receptor motif offers diagnostic and therapeutic applications.

Sepsis is a dysregulated host response of severe bloodstream infections, and given its frequency of occurrence and high mortality rate, therapeutic improvements are imperative. A reliable biomimetic strategy for the targeting and separation of bacterial pathogens in bloodstream infections involves the use of the broad-spectrum binding motif of human GP-340, a pattern-recognition receptor of the scavenger receptor cysteine rich (SRCR) superfamily that is expressed on epithelial surfaces but not found in blood. Here we show that these peptides, when conjugated to superparamagnetic iron oxide nanoparticles (SPIONs), can separate various bacterial endotoxins and intact microbes (E. coli, S. aureus, P. aeruginosa and S. marcescens) with high efficiency, especially at low and thus clinically relevant concentrations. This is accompanied by a subsequent strong depletion in cytokine release (TNF, IL-6, IL-1ß, Il-10 and IFN-γ), which could have a direct therapeutic impact since escalating immune responses complicates severe bloodstream infections and sepsis courses. SPIONs are coated with aminoalkylsilane and capture peptides are orthogonally ligated to this surface. The particles behave fully cyto- and hemocompatible and do not interfere with host structures. Thus, this approach additionally aims to dramatically reduce diagnostic times for patients with suspected bloodstream infections and accelerate targeted antibiotic therapy. STATEMENT OF SIGNIFICANCE: Sepsis is often associated with excessive release of cytokines. This aspect and slow diagnostic procedures are the major therapeutic obstacles. The use of magnetic particles conjugated with small peptides derived from the binding motif of a broad-spectrum mucosal pathogen recognition protein GP-340 provides a highly efficient scavenging platform. These peptides are not found in blood and therefore are not subject to inhibitory mechanisms like in other concepts (mannose binding lectine, aptamers, antibodies). In this work, data are shown on the broad bacterial binding spectrum, highly efficient toxin depletion, which directly reduces the release of cytokines. Host cells are not affected and antibiotics not adsorbed. The particle bound microbes can be recultured without restriction and thus be used directly for diagnostics.

Biocompatibility of Dextran-Coated 30 nm and 80 nm Sized SPIONs towards Monocytes, Dendritic Cells and Lymphocytes.

Dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) of various sizes can be used as contrast agents in magnetic resonance imaging (MRI) of different tissues, e.g., liver or atherosclerotic plaques, after intravenous injection. In previous studies, the blood compatibility and the absence of immunogenicity of SPIONDex was demonstrated. The investigation of the interference of SPIONDex with stimulated immune cell activation is the aim of this study. For this purpose, sterile and endotoxin-free SPIONDex with different hydrodynamic sizes (30 and 80 nm) were investigated for their effect on monocytes, dendritic cells (DC) and lymphocytes in concentrations up to 200 µg/mL, which would be administered for use as an imaging agent. The cells were analyzed using flow cytometry and brightfield microscopy. We found that SPIONDex were hardly taken up by THP-1 monocytes and did not reduce cell viability. In the presence of SPIONDex, the phagocytosis of zymosan and E. coli by THP-1 was dose-dependently reduced. SPIONDex neither induced the maturation of DCs nor interfered with their stimulated maturation. The particles did not induce lymphocyte proliferation or interfere with lymphocyte proliferation after stimulation. Since SPIONDex rapidly distribute via the blood circulation in vivo, high concentrations were only reached locally at the injection site immediately after application and only for a very limited time. Thus, SPIONDex can be considered immune compatible in doses required for use as an MRI contrast agent.

Graphene-Induced Hyperthermia (GIHT) Combined With Radiotherapy Fosters Immunogenic Cell Death.

Radiotherapy and chemotherapy are the standard interventions for cancer patients, although cancer cells often develop radio- and/or chemoresistance. Hyperthermia reduces tumor resistance and induces immune responses resulting in a better prognosis. We have previously described a method to induce tumor cell death by local hyperthermia employing pegylated reduced graphene oxide nanosheets and near infrared light (graphene-induced hyperthermia, GIHT). The spatiotemporal exposure/release of heat shock proteins (HSP), high group mobility box 1 protein (HMGB1), and adenosine triphosphate (ATP) are reported key inducers of immunogenic cell death (ICD). We hypothesize that GIHT decisively contributes to induce ICD in irradiated melanoma B16F10 cells, especially in combination with radiotherapy. Therefore, we investigated the immunogenicity of GIHT alone or in combination with radiotherapy in melanoma B16F10 cells. Tumor cell death in vitro revealed features of apoptosis that is progressing fast into secondary necrosis. Both HSP70 and HMGB1/DNA complexes were detected 18 hours post GIHT treatment, whereas the simultaneous release of ATP and HMGB1/DNA was observed only 24 hours post combined treatment. We further confirmed the adjuvant potential of these released DAMPs by immunization/challenge experiments. The inoculation of supernatants of cells exposed to sole GIHT resulted in tumor growth at the site of inoculation. The immunization with cells exposed to sole radiotherapy rather fostered the growth of secondary tumors in vivo. Contrarily, a discreet reduction of secondary tumor volumes was observed in mice immunized with a single dose of cells and supernatants treated with the combination of GIHT and irradiation. We propose the simultaneous release of several DAMPs as a potential mechanism fostering anti-tumor immunity against previously irradiated cancer cells.

Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles Enable a Stable Non-Spilling Loading of T Cells and Their Magnetic Accumulation.

T cell infiltration into a tumor is associated with a good clinical prognosis of the patient and adoptive T cell therapy can increase anti-tumor immune responses. However, immune cells are often excluded from tumor infiltration and can lack activation due to the immune-suppressive tumor microenvironment. To make T cells controllable by external forces, we loaded primary human CD3+ T cells with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs). Since the efficacy of magnetic targeting depends on the amount of SPION loading, we investigated how experimental conditions influence nanoparticle uptake and viability of cells. We found that loading in the presence of serum improved both the colloidal stability of SPIONs and viability of T cells, whereas stimulation with CD3/CD28/CD2 and IL-2 did not influence nanoparticle uptake. Furthermore, SPION loading did not impair cytokine secretion after polyclonal stimulation. We finally achieved 1.4 pg iron loading per cell, which was both located intracellularly in vesicles and bound to the plasma membrane. Importantly, nanoparticles did not spill over to non-loaded cells. Since SPION-loading enabled efficient magnetic accumulation of T cells in vitro under dynamic conditions, we conclude that this might be a good starting point for the investigation of in vivo delivery of immune cells.

An Endoplasmic Reticulum Specific Pro-amplifier of Reactive Oxygen Species in Cancer Cells.

The folding and export of proteins and hydrolysis of unfolded proteins are disbalanced in the endoplasmic reticulum (ER) of cancer cells, leading to so-called ER stress. Agents further augmenting this effect are used as anticancer drugs including clinically approved proteasome inhibitors bortezomib and carfilzomib. However, these drugs can affect normal cells, which also rely strongly on ER functions, leading, for example, to accumulation of reactive oxygen species (ROS). To address this problem, we have developed ER-targeted prodrugs activated only in cancer cells in the presence of elevated ROS amounts. These compounds are conjugates of cholic acid with N-alkylaminoferrocene-based prodrugs. We confirmed their accumulation in the ER of cancer cells, their anticancer efficacy, and cancer cell specificity. These prodrugs induce ER stress, attenuate mitochondrial membrane potential, and generate mitochondrial ROS leading to cell death via necrosis. We also demonstrated that the new prodrugs are activated in vivo in Nemeth-Kellner lymphoma (NK/Ly) murine model.