Mild magnetic hyperthermia is synergistic with an antibiotic treatment against dual species biofilms consisting of S. aureus and P. aeruginosa by enhancing metabolic activity.

OBJECTIVE: The wound biofilm infections that develop tolerance to standard-of-care antimicrobial treatment has been increasing. The objective of this study was to demonstrate a proof-of-concept of mild magnetic nanoparticle (MNP)/alternating magnetic field (AMF) hyperthermia as an anti-biofilm therapy against multispecies biofilm infections. METHODS: Using both an in vitro cell culture and in vivo murine model of wound infection, we investigated whether MNP/AMF hyperthermia applied at a mild thermal dosage would be synergistically effective against dual species biofilm infection consisting of S. aureus and P. aeruginosa when combined with a broad-spectrum antibiotic, ciprofloxacin (CIP). RESULTS: The combined treatment of MNP/AMF hyperthermia and CIP to the wounds of diabetic mice (db/db mice) significantly reduced the CFU number of S. aureus and P. aeruginosa by 2-log and 3-log, respectively, compared to the untreated control group, whereas either mild MNP/AMF hyperthermia or CIP treatment alone had little effect on the eradication of both bacteria. Our gene microarray data obtained from the culture of S. aureus biofilm suggest that mild MNP/AMF could shift the expression of genes for cellular respiration from anaerobic fermentation to an aerobic glycolytic/tricarboxylic acid cycle (TCA) pathway, implicating that the beneficial effect of mild MNP/AMF hyperthermia on the increased susceptibility of biofilm bacteria to an antibiotic treatment is associated with an increased metabolic activity. CONCLUSION: Our results support the translational potential of mild MNP/AMF as an adjunctive therapy that can be combined with a broad-spectrum antibiotic treatment for the management of wound biofilm infections associated with multispecies bacteria.

mNP hyperthermia and hypofractionated radiation activate similar immunogenetic and cytotoxic pathways.

OBJECTIVE: The goal of this study is to better understand the immunogenetic expression and related cytotoxic responses of moderate but clinically relevant doses of hypofractionated radiation (1x15 Gy and 3x8 Gy) and magnetic nanoparticle hyperthermia (mNPH, CEM43 30). METHODS: Genetic, protein, immunopathology and tumor growth delay assessments were used to determine the immune and cytotoxic responses following radiation and mNPH alone and in combination. Although the thermal dose used, 43 C°/30 min (CEM43 30), typically results in modest independent cytotoxicity, it has shown the ability to stimulate an immune response and enhance other cancer treatments. The radiation doses studied (15 Gy and 3x8 Gy) are commonly used in preclinical research and are effective in selected stereotactic and palliative treatment settings, however they are not commonly used as first-line primary tumor treatment regimens. RESULTS: Our RNA-based genetic results suggest that while many of the cytotoxic and immune gene and protein pathways for radiation and hyperthermia are similar, radiation, at the doses used, results in a more consistent and expansive anti-cancer immune/cytotoxic expression profile. These results were supported by immunohistochemistry based cytotoxic T-cell tumor infiltration and tumor growth delay studies. When used together radiation and hyperthermia led to greater immune and cytotoxic activity than either modality alone. CONCLUSION: This study clearly shows that modest, but commonly used hypofractionated radiation and hyperthermia doses share many important immune and cytotoxic pathways and that combining the treatments, as compared to either treatment alone, results in genetic and biological anti-cancer benefits.

Design and construction of a Maxwell-type induction coil for magnetic nanoparticle hyperthermia.

Purpose: We describe a modified Helmholtz induction coil, or Maxwell coil, that generates alternating magnetic fields (AMF) having field uniformity (≤10%) within a = 3000 cm3 volume of interest for magnetic hyperthermia research.Materials and methods: Two-dimensional finite element analysis (2D-FEA) was used for electromagnetic design of the induction coil set and to develop specifications for the required matching network. The matching network and induction coil set were fabricated using best available practices and connected to a 120 kW industrial induction heating power supply. System performance was evaluated by magnetic field mapping with a magnetic field probe, and tests were performed using gel phantoms.Results: Tests verified that the system generated a target peak AMF amplitude along the coil axis of ∼35 kA/m (peak) at a frequency of 150 ± 10 kHz while maintaining field uniformity to >90% of peak for a volume of ∼3000 cm3.Conclusions: The induction coil apparatus comprising three independent loops, i.e., Maxwell-type improves upon the performance of simple solenoid and Helmholtz coils by providing homogeneous flux density fields within a large volume while minimizing demands on power and stray fields. Experiments with gel phantoms and analytical calculations show that future translational research efforts should be devoted to developing strategies to reduce the impact of nonspecific tissue heating from eddy currents; and, that an inductor producing a homogeneous field has significant clinical potential for deep-tissue magnetic fluid hyperthermia.

Immunogenetic effects of low dose (CEM43 30) magnetic nanoparticle hyperthermia and radiation in melanoma cells.

Objective: In this in vitro study we have used an RNA quantification technique, nanoString, and a conventional protein analysis technique (Western Blot) to assess the genetic and protein expression of B16 murine melanoma cells following a modest magnetic nanoparticle hyperthermia (mNPH) dose equivalent to 30 minutes @ 43°C (CEM43 30) and/or a clinically relevant 8 Gy radiation dose.Methods: Melanoma cells with mNPs(2.5 µg Fe/106 cells) were pelleted and exposed to an alternating magnetic field (AMF) to generate the targeted thermal dose. Thermal dose was accurately monitored by a fiber optic probe and automatically maintained at CEM43 30. All cells were harvested 24 hours after treatment.Results: The mNPH dose demonstrated notable elevations in the thermotolerance/immunogenic HSP70 gene and a number of chemoattractant and toll-like receptor gene pathways. The 8 Gy dose also upregulated a number of important immune and cytotoxic genetic and protein pathways. However, the mNPH/radiation combination was the most effective stimulator of a wide variety of immune and cytotoxic genes including HSP70, cancer regulating chemokines CXCL10, CXCL11, the T-cell trafficking chemokine CXCR3, innate immune activators TLR3, TLR4, the MDM2 and mTOR negative regulator of p53, the pro-apoptotic protein PUMA, and the cell death receptor Fas. Importantly a number of the genetic changes were accurately validated by protein expression changes, i.e., HSP70, p-mTOR, p-MDM2.Conclusion: These results not only show that low dose mNPH and radiation independently increase the expression of important immune and cytotoxic genes but that the effect is greatly enhanced when they are used in combination.

Enhancing the abscopal effect of radiation and immune checkpoint inhibitor therapies with magnetic nanoparticle hyperthermia in a model of metastatic breast cancer.

Purpose: Enhancing immune responses in triple negative breast cancers (TNBCs) remains a challenge. Our study aimed to determine whether magnetic iron oxide nanoparticle (MION) hyperthermia (HT) can enhance abscopal effects with radiotherapy (RT) and immune checkpoint inhibitors (IT) in a metastatic TNBC model.Methods: One week after implanting 4T1-luc cells into the mammary glands of BALB/c mice, tumors were treated with RT (3 × 8 Gy)±local HT, mild (HTM, 43 °C/20 min) or partially ablative (HTAbl, 45 °C/5 min plus 43 °C/15 min),±IT with anti-PD-1 and anti-CTLA-4 antibodies (both 4 × 10 mg/kg, i.p.). Tumor growth was measured daily. Two weeks after treatment, lungs and livers were harvested for histopathology evaluation of metastases.Results: Compared to untreated controls, all treatment groups demonstrated a decreased tumor volume; however, when compared against surgical resection, only RT + HTM+IT, RT + HTAbl+IT and RT + HTAbl had similar or smaller tumors. These cohorts showed more infiltration of CD3+ T-lymphocytes into the primary tumor. Tumor growth effects were partially reversed with T-cell depletion. Combinations that proved most effective for primary tumors generated modest reductions in numbers of lung metastases. Conversely, numbers of lung metastases showed potential to increase following HT + IT treatment, particularly when compared to RT. Compared to untreated controls, there was no improvement in survival with any treatment.Conclusions: Single-fraction MION HT added to RT + IT improved local tumor control and recruitment of CD3+ T-lymphocytes, with only a modest effect to reduce lung metastases and no improvement in overall survival. HT + IT showed potential to increase metastatic dissemination to lungs.

Identification of infusion strategy for achieving repeatable nanoparticle distribution and quantification of thermal dosage using micro-CT Hounsfield unit in magnetic nanoparticle hyperthermia.

OBJECTIVES: The objective of this study was to identify an injection strategy leading to repeatable nanoparticle deposition patterns in tumours and to quantify volumetric heat generation rate distribution based on micro-CT Hounsfield unit (HU) in magnetic nanoparticle hyperthermia. METHODS: In vivo animal experiments were performed on graft prostatic cancer (PC3) tumours in immunodeficient mice to investigate whether lowering ferrofluid infusion rate improves control of the distribution of magnetic nanoparticles in tumour tissue. Nanoparticle distribution volume obtained from micro-CT scan was used to evaluate spreading of the nanoparticles from the injection site in tumours. Heating experiments were performed to quantify relationships among micro-CT HU values, local nanoparticle concentrations in the tumours, and the ferrofluid-induced volumetric heat generation rate (q(MNH)) when nanoparticles were subject to an alternating magnetic field. RESULTS: An infusion rate of 3 µL/min was identified to result in the most repeatable nanoparticle distribution in PC3 tumours. Linear relationships have been obtained to first convert micro-CT greyscale values to HU values, then to local nanoparticle concentrations, and finally to nanoparticle-induced q(MNH) values. The total energy deposition rate in tumours was calculated and the observed similarity in total energy deposition rates in all three infusion rate groups suggests improvement in minimising nanoparticle leakage from the tumours. The results of this study demonstrate that micro-CT generated q(MNH) distribution and tumour physical models improve predicting capability of heat transfer simulation for designing reliable treatment protocols using magnetic nanoparticle hyperthermia.

MicroCT image based simulation to design heating protocols in magnetic nanoparticle hyperthermia for cancer treatment.

OBJECTIVES: The objective is to design heating protocols to completely damage PC3 tumors after a single magnetic nanoparticle hyperthermia session with minimal collateral thermal damage, based on microCT image generated tumor and mouse models. METHODS: Tumor geometries and volumetric heat generation rate distributions that are generated from microCT scans in our previous study are imported into COMSOL 4.3® multiphysics for heat transfer simulations and heating protocol design using the Arrhenius damage model. Then, parametric studies are performed to evaluate how significantly the infusion rate affects the protocol design and its resulted collateral thermal damage. RESULTS: The simulated temperature field in the generated tumor geometry and volumetric heat generation rate distribution are reasonable and correlates well with the amount of the total thermal energy deposited into the tumors. The time needed for complete thermal damage is determined to be approximately 12min or 25min if one uses the Arrhenius integral Ω equal to 1 or 4 as the damage threshold, when the infusion rate is 3µL/min. The heating time increases 26% or 91% in the higher infusion rate groups of 4 or 5µL/min, respectively. Collateral thermal damage to the surrounding tissue is also assessed. Although the two larger infusion rate groups can still cause thermal damage to the entire tumor, the collateral thermal damage would have exceeded the design criterion of 5%, while the assessment criterion is acceptable only in the infusion rate group of 3µL/min. Based on the results of this study, we identify an injection strategy and heating protocols to be implemented in future animal experiments to evaluate treatment efficacy for model validation.

A Validated Methodological Approach to Prove the Safety of Clinical Electromagnetic Induction Systems in Magnetic Hyperthermia.

The present study focuses on the development of a methodology for evaluating the safety of MNH systems, through the numerical prediction of the induced temperature rise in superficial skin layers due to eddy currents heating under an alternating magnetic field (AMF). The methodology is supported and validated through experimental measurements of the AMF's distribution, as well as temperature data from the torsos of six patients who participated in a clinical trial study. The simulations involved a computational model of the actual coil, a computational model of the cooling system used for the cooling of the patients during treatment, and a detailed human anatomical model from the Virtual Population family. The numerical predictions exhibit strong agreement with the experimental measurements, and the deviations are below the estimated combined uncertainties, confirming the accuracy of computational modeling. This study highlights the crucial role of simulations for translational medicine and paves the way for personalized treatment planning.

E. coli Phagelysate: A Primer to Enhance Nanoparticles and Drug Deliveries in Tumor.

The tumor microenvironment (TME), where cancer cells reside, plays a crucial role in cancer progression and metastasis. It maintains an immunosuppressive state in many tumors and regulates the differentiation of precursor monocytes into M1 (anti-tumor)- and M2 (pro-tumor)-polarized macrophages, and greatly reduces anticancer drug and nanoparticle delivery. As a result, the effectiveness of recently developed chemo- and/or nanotechnology-mediated immune and magnetic nanoparticle hyperthermia (mNPH) therapies is inhibited significantly. One of the ways to overcome this limitation is to use E. coli phagelysate as a primer to modify the tumor microenvironment by switching tumor-associated M2 macrophages to anti-tumor M1 macrophages, and initiate the infiltration of tumor-associated macrophages (TAMs). Recently, bacteriophages and phage-induced lysed bacteria (bacterial phagelysates-BPLs) have been shown to be capable of modifying the tumor-associated environment. Phage/BPL-coated proteins tend to elicit strong anti-tumor responses from the innate immune system, prompting phagocytosis and cytokine release. It has also been reported that the microenvironments of bacteriophage- and BPL-treated tumors facilitate the conversion of M2-polarized TAMS to a more M1-polarized (tumoricidal) environment post-phage treatment. This paper demonstrates the feasibility and enhanced efficacy of combining E. coli phagelysate (EcPHL) and mNPH, a promising technology for treating cancers, in a rodent model. Specifically, we illustrate the EcPHL vaccination effect on the TME and mNP distribution in Ehrlich adenocarcinoma tumors by providing the tumor growth dynamics and histology (H&E and Prussian blue) distribution of mNP in tumor and normal tissue.

Evaluation of a Balloon Implant for Simultaneous Magnetic Nanoparticle Hyperthermia and High-Dose-Rate Brachytherapy of Brain Tumor Resection Cavities.

Previous work has reported the design of a novel thermobrachytherapy (TBT) balloon implant to deliver magnetic nanoparticle (MNP) hyperthermia and high-dose-rate (HDR) brachytherapy simultaneously after brain tumor resection, thereby maximizing their synergistic effect. This paper presents an evaluation of the robustness of the balloon device, compatibility of its heat and radiation delivery components, as well as thermal and radiation dosimetry of the TBT balloon. TBT balloon devices with 1 and 3 cm diameter were evaluated when placed in an external magnetic field with a maximal strength of 8.1 kA/m at 133 kHz. The MNP solution (nanofluid) in the balloon absorbs energy, thereby generating heat, while an HDR source travels to the center of the balloon via a catheter to deliver the radiation dose. A 3D-printed human skull model was filled with brain-tissue-equivalent gel for in-phantom heating and radiation measurements around four 3 cm balloons. For the in vivo experiments, a 1 cm diameter balloon was surgically implanted in the brains of three living pigs (40-50 kg). The durability and robustness of TBT balloon implants, as well as the compatibility of their heat and radiation delivery components, were demonstrated in laboratory studies. The presence of the nanofluid, magnetic field, and heating up to 77 °C did not affect the radiation dose significantly. Thermal mapping and 2D infrared images demonstrated spherically symmetric heating in phantom as well as in brain tissue. In vivo pig experiments showed the ability to heat well-perfused brain tissue to hyperthermic levels (≥40 °C) at a 5 mm distance from the 60 °C balloon surface.

Cancer therapy with iron oxide nanoparticles: Agents of thermal and immune therapies.

Significant research and preclinical investment in cancer nanomedicine has produced several products, which have improved cancer care. Nevertheless, there exists a perception that cancer nanomedicine 'has not lived up to its promise' because the number of approved products and their clinical performance are modest. Many of these analyses do not consider the long clinical history and many clinical products developed from iron oxide nanoparticles. Iron oxide nanoparticles have enjoyed clinical use for about nine decades demonstrating safety, and considerable clinical utility and versatility. FDA-approved applications of iron oxide nanoparticles include cancer diagnosis, cancer hyperthermia therapy, and iron deficiency anemia. For cancer nanomedicine, this wealth of clinical experience is invaluable to provide key lessons and highlight pitfalls in the pursuit of nanotechnology-based cancer therapeutics. We review the clinical experience with systemic liposomal drug delivery and parenteral therapy of iron deficiency anemia (IDA) with iron oxide nanoparticles. We note that the clinical success of injectable iron exploits the inherent interaction between nanoparticles and the (innate) immune system, which designers of liposomal drug delivery seek to avoid. Magnetic fluid hyperthermia, a cancer therapy that harnesses magnetic hysteresis heating is approved for treating humans only with iron oxide nanoparticles. Despite its successful demonstration to enhance overall survival in clinical trials, this nanotechnology-based thermal medicine struggles to establish a clinical presence. We review the physical and biological attributes of this approach, and suggest reasons for barriers to its acceptance. Finally, despite the extensive clinical experience with iron oxide nanoparticles new and exciting research points to surprising immune-modulating potential. Recent data demonstrate the interactions between immune cells and iron oxide nanoparticles can induce anti-tumor immune responses. These present new and exciting opportunities to explore additional applications with this venerable technology. Clinical applications of iron oxide nanoparticles present poignant case studies of the opportunities, complexities, and challenges in cancer nanomedicine. They also illustrate the need for revised paradigms and multidisciplinary approaches to develop and translate nanomedicines into clinical cancer care.

Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions.

Oxygen microenvironment affects the uptake of nanoparticles in head and neck tumor cells.

Survival of head and neck cancer patients has not improved in several decades despite advances in diagnostic and therapeutic techniques. Tumor hypoxia in head and neck cancers is a critical factor that leads to poor prognosis, resistance to radiation and chemotherapies, and increased metastatic potential. Magnetic nanoparticle hyperthermia (mNPHT) is a promising therapy for hypoxic tumors because nanoparticles (NP) can be directly injected into, or targeted to, hypoxic tumor cells and exposed to alternating magnetic fields (AMF) to induce hyperthermia. Magnetic NPHT can improve therapeutic effectiveness by two modes of action: 1) direct killing of hypoxic tumor cells; and 2) increase in tumor oxygenation, which has the potential to make the tumor more susceptible to adjuvant therapies such as radiation and chemotherapy. Prior studies in breast cancer cells demonstrated that a hypoxic microenvironment diminished NP uptake in vitro; however, mNPHT with intratumoral NP injection in hypoxic tumors increased tumor oxygenation and delayed tumor growth. In this study, head and neck squamous cell carcinoma (HNSCC) cell lines were incubated in normoxic, hypoxic, and hyperoxic conditions with iron oxide NP for 4-72 hours. After incubation, the cells were analyzed for iron uptake by mass spectrometry, Prussian blue staining, and electron microscopy. In contrast to breast cancer cells, uptake of NPs was increased in hypoxic microenvironments as compared to normoxic conditions in HNSCC cells. In future studies, we will confirm the effect of the oxygen microenvironment on NP uptake and efficacy of mNPHT both in vitro and in vivo.