Potential of Magnetic Hyperthermia to Stimulate Localized Immune Activation.

Magnetic hyperthermia (MH) harnesses the heat-releasing properties of superparamagnetic iron oxide nanoparticles (SPIONs) and has potential to stimulate immune activation in the tumor microenvironment whilst sparing surrounding normal tissues. To assess feasibility of localized MH in vivo, SPIONs are injected intratumorally and their fate tracked by Zirconium-89-positron emission tomography, histological analysis, and electron microscopy. Experiments show that an average of 49% (21-87%, n = 9) of SPIONs are retained within the tumor or immediately surrounding tissue. In situ heating is subsequently generated by exposure to an externally applied alternating magnetic field and monitored by thermal imaging. Tissue response to hyperthermia, measured by immunohistochemical image analysis, reveals specific and localized heat-shock protein expression following treatment. Tumor growth inhibition is also observed. To evaluate the potential effects of MH on the immune landscape, flow cytometry is used to characterize immune cells from excised tumors and draining lymph nodes. Results show an influx of activated cytotoxic T cells, alongside an increase in proliferating regulatory T cells, following treatment. Complementary changes are found in draining lymph nodes. In conclusion, results indicate that biologically reactive MH is achievable in vivo and can generate localized changes consistent with an anti-tumor immune response.

Monitoring Intracranial Cerebral Hemorrhage Using Multicontrast Real-Time Magnetic Particle Imaging.

Magnetic particle imaging (MPI) is an innovative radiation-free tomographic imaging method providing excellent temporal resolution, contrast, sensitivity, and safety. Mobile human MPI prototypes suitable for continuous bedside monitoring of whole-brain perfusion have been developed. However, for the clinical translation of MPI, a crucial gap in knowledge still remains: while MPI can visualize the reduction in blood flow and tissue perfusion in cerebral ischemia, it is unclear whether MPI works in intracranial hemorrhage. Our objective was to investigate the capability of MPI to detect intracranial hemorrhage in a murine model. Intracranial hemorrhage was induced through the injection of collagenase into the striatum of C57BL/6 mice. After the intravenous infusion of a long-circulating MPI-tailored tracer consisting of superparamagnetic iron oxides, we detected the intracranial hemorrhage in less than 3 min and could monitor hematoma expansion in real time. Multicontrast MPI can distinguish tracers based on their physical characteristics, core size, temperature, and viscosity. By employing in vivo multicontrast MPI, we were able to differentiate areas of liquid and coagulated blood within the hematoma, which could provide valuable information in surgical decision making. Multicontrast MPI also enabled simultaneous imaging of hemorrhage and cerebral perfusion, which is essential in the care of critically ill patients with increased intracranial pressure. We conclude that MPI can be used for real-time diagnosis of intracranial hemorrhage. This work is an essential step toward achieving the clinical translation of MPI for point-of-care monitoring of different stroke subtypes.

Nanoparticle interactions with immune cells dominate tumor retention and induce T cell-mediated tumor suppression in models of breast cancer.

The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8+ T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy.

Physical characterization and in vivo organ distribution of coated iron oxide nanoparticles.

Citrate-stabilized iron oxide magnetic nanoparticles (MNPs) were coated with one of carboxymethyl dextran (CM-dextran), polyethylene glycol-polyethylene imine (PEG-PEI), methoxy-PEG-phosphate+rutin, or dextran. They were characterized for size, zeta potential, hysteresis heating in an alternating magnetic field, dynamic magnetic susceptibility, and examined for their distribution in mouse organs following intravenous delivery. Except for PEG-PEI-coated nanoparticles, all coated nanoparticles had a negative zeta potential at physiological pH. Nanoparticle sizing by dynamic light scattering revealed an increased nanoparticle hydrodynamic diameter upon coating. Magnetic hysteresis heating changed little with coating; however, the larger particles demonstrated significant shifts of the peak of complex magnetic susceptibility to lower frequency. 48 hours following intravenous injection of nanoparticles, mice were sacrificed and tissues were collected to measure iron concentration. Iron deposition from nanoparticles possessing a negative surface potential was observed to have highest accumulation in livers and spleens. In contrast, iron deposition from positively charged PEG-PEI-coated nanoparticles was observed to have highest concentration in lungs. These preliminary results suggest a complex interplay between nanoparticle size and charge determines organ distribution of systemically-delivered iron oxide magnetic nanoparticles.

Fucoidan Prolongs the Circulation Time of Dextran-Coated Iron Oxide Nanoparticles.

The magnetic properties and safety of dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) have facilitated their clinical use as MRI contrast agents and stimulated research on applications for SPIONs in particle imaging and magnetic hyperthermia. The wider clinical potential of SPIONs, however, has been limited by their rapid removal from circulation via the reticuloendothelial system (RES). We explored the possibility of extending SPION circulatory time using fucoidan, a seaweed-derived food supplement, to inhibit RES uptake. The effects of fucoidan on SPION biodistribution were evaluated using ferucarbotran, which in its pharmaceutical formulation (Resovist) targets the RES. Ferucarbotran was radiolabeled at the iron oxide core with technetium-99m (99mTc; t1/2 = 6 h) or zirconium-89 (89Zr; t1/2 = 3.3 days). Results obtained with 99mTc-ferucarbotran demonstrated that administration of fucoidan led to a 4-fold increase in the circulatory half-life (t1/2 slow) from 37.4 to 150 min (n = 4; P < 0.0001). To investigate whether a longer circulatory half-life could lead to concomitant increased tumor uptake, the effects of fucoidan were tested with 89Zr-ferucarbotran in mice bearing syngeneic subcutaneous (GL261) tumors. In this model, the longer circulatory half-life achieved with fucoidan was associated with a doubling in tumor SPION uptake (n = 5; P < 0.001). Fucoidan was also effective in significantly increasing the circulatory half-life of perimag-COOH, a commercially available SPION with a larger hydrodynamic size (130 nm) than ferucarbotran (65 nm). These findings indicate successful diversion of SPIONs away from the hepatic RES and show realistic potential for future clinical applications.

An optimised spectrophotometric assay for convenient and accurate quantitation of intracellular iron from iron oxide nanoparticles.

We report the development and optimisation of an assay for quantitating iron from iron oxide nanoparticles in biological matrices by using ferene-s, a chromogenic compound. The method is accurate, reliable and can be performed with basic equipment common to many laboratories making it convenient and inexpensive. The assay we have developed is suited for quantitation of iron in cell culture studies with iron oxide nanoparticles, which tend to manifest low levels of iron. The assay was validated with standard reference materials and with inductively coupled plasma-mass spectrometry (ICP-MS) to accurately measure iron concentrations ∼1 × 10-6 g in about 1 × 106 cells (∼1 × 10-12 g Fe per cell). The assay requires preparation and use of a working solution to which samples can be directly added without further processing. After overnight incubation, the absorbance can be measured with a standard UV/Vis spectrophotometer to provide iron concentration. Alternatively, for expedited processing, samples can be digested with concentrated nitric acid before addition to the working solution. Optimization studies demonstrated significant deviations accompany variable digestion times, highlighting the importance to ensure complete iron ion liberation from the nanoparticle or sample matrix to avoid underestimating iron concentration. When performed correctly, this method yields reliable iron ion concentration measurements to ∼2 × 10-6 M (1 × 10-7 g/ml sample).

Image-guided thermal therapy with a dual-contrast magnetic nanoparticle formulation: A feasibility study.

PURPOSE/OBJECTIVE: The aim of this study was to develop and investigate the properties of a magnetic iron oxide nanoparticle-ethiodised oil formulation for image-guided thermal therapy of liver cancer. MATERIALS AND METHODS: The formulation comprises bionised nano-ferrite (BNF) nanoparticles suspended in ethiodised oil, emulsified with polysorbate 20 (BNF-lip). Nanoparticle size was measured via photon correlation spectroscopy and transmission electron microscopy. In vivo thermal therapy capability was tested in two groups of male Foxn1(nu) mice bearing subcutaneous HepG2 xenograft tumours. Group I (n = 12) was used to screen conditions for group II (n = 48). In group II, mice received one of BNF-lip (n = 18), BNF alone (n = 16), or PBS (n = 14), followed by alternating magnetic field (AMF) hyperthermia, with either varied duration (15 or 20 min) or amplitude (0, 16, 20, or 24 kA/m). Image-guided fluoroscopic intra-arterial injection of BNF-lip was tested in New Zealand white rabbits (n = 10), bearing liver VX2 tumours. The animals were subsequently imaged with CT and 3 T MRI, up to 7 days post-injection. The tumours were histopathologically evaluated for distribution of BNF-lip. RESULTS: The BNF showed larger aggregate diameters when suspended in BNF-lip, compared to clear solution. The BNF-lip formulation produced maximum tumour temperatures with AMF >20 kA/m and showed positive X-ray visibility and substantial shortening of T1 and T2 relaxation time, with sustained intratumoural retention up to 7 days post-injection. On pathology, intratumoural BNF-lip distribution correlated well with CT imaging of intratumoural BNF-lip distribution. CONCLUSION: The BNF-lip formulation has favourable thermal and dual imaging capabilities for image-guided thermal therapy of liver cancer, suggesting further exploration for clinical applications.

Monoclonal antibody conjugated magnetic nanoparticles could target MUC-1-positive cells in vitro but not in vivo.

MUC1 antigen is recognized as a high-molecular-weight glycoprotein that is unexpectedly over-expressed in human breast and other carcinomas. In contrast, C595 a monoclonal antibody (mAb) against the protein core of the human urinary epithelial machine, is commonly expressed in breast carcinomas. The aim of this study was to conjugate ultra-small super paramagnetic iron oxide nanoparticles (USPIO) with C595 mAb, in order to detect in vivo MUC1 expression. A dual contrast agent (the C595 antibody-conjugated USPIO labeled with 99mTc) was prepared for targeted imaging and therapy of anti-MUC1-expressing cancers. The C595 antibody-conjugated USPIO had good stability and reactivity in the presence of blood plasma at 37 °C. No significant differences were observed in immunoreactivity results between conjugated and nonconjugated nanoparticles. The T1 and T2 measurements show >79 and 29% increments (for 0.02 mg/ml iron concentrations) in T1 and T2 values for USPIO-C595 in comparison with USPIO, respectively. The nanoprobes showed the interesting targeting capability of finding the MUC1-positive cell line in vitro. However, we found disappointing in vivo results (i.e. very low accumulation of nanoprobes in the targeted site while >80% of the injected dose per gram was taken up by the liver and spleen), not only due to the coverage of targeting site by protein corona but also because of absorption of opsonin-based proteins at the surface of nanoprobes.

Functionalized magnetic nanoparticles for the detection and quantitative analysis of cell surface antigen.

Cell surface antigens as biomarkers offer tremendous potential for early diagnosis, prognosis, and therapeutic response in a variety of diseases such as cancers. In this research, a simple, rapid, accurate, inexpensive, and easily available in vitro assay based on magnetic nanoparticles and magnetic cell separation principle was applied to identify and quantitatively analyze the cell surface antigen expression in the case of prostate cancer cells. Comparing the capability of the assay with flow cytometry as a gold standard method showed similar results. The results showed that the antigen-specific magnetic cell separation with antibody-coated magnetic nanoparticles has high potential for quantitative cell surface antigen detection and analysis.

The effect of cell cluster size on intracellular nanoparticle-mediated hyperthermia: is it possible to treat microscopic tumors?

AIM: To compare the measured surface temperature of variable size ensembles of cells heated by intracellular magnetic fluid hyperthermia with heat diffusion model predictions. MATERIALS & METHODS: Starch-coated Bionized NanoFerrite (Micromod Partikeltechnologie GmbH, Rostock, Germany) iron oxide magnetic nanoparticles were loaded into cultured DU145 prostate cancer cells. Cell pellets of variable size were treated with alternating magnetic fields. The surface temperature of the pellets was measured in situ and the associated cytotoxicity was determined by clonogenic survival assay. RESULTS & CONCLUSION: For a given intracellular nanoparticle concentration, a critical minimum number of cells was required for cytotoxic hyperthermia. Above this threshold, cytotoxicity increased with increasing cell number. The measured surface temperatures were consistent with those predicted by a heat diffusion model that ignores intercellular thermal barriers. These results suggest a minimum tumor volume threshold of approximately 1 mm(3), below which nanoparticle-mediated heating is unlikely to be effective as the sole cytotoxic agent.

Preliminary study of injury from heating systemically delivered, nontargeted dextran-superparamagnetic iron oxide nanoparticles in mice.

AIM: To assess the potential for injury to normal tissues in mice due to heating systemically delivered magnetic nanoparticles in an alternating magnetic field (AMF). MATERIALS & METHODS: Twenty three male nude mice received intravenous injections of dextran-superparamagnetic iron oxide nanoparticles on days 1-3. On day 6, they were exposed to AMF. On day 7, blood, liver and spleen were harvested and analyzed. RESULTS: Iron deposits were detected in the liver and spleen. Mice that had received a high-particle dose and a high AMF experienced increased mortality, elevated liver enzymes and significant liver and spleen necrosis. Mice treated with low-dose superparamagnetic iron oxide nanoparticles and a low AMF survived, but had elevated enzyme levels and local necrosis in the spleen. CONCLUSION: Magnetic nanoparticles producing only modest heat output can cause damage, and even death, when sequestered in sufficient concentrations. Dextran-superparamagnetic iron oxide nanoparticles are deposited in the liver and spleen, making these the sites of potential toxicity. Original submitted 16 August 2011; Revised submitted 21 March 2012; Published online 26 July 2012.

Short communication: nanoparticle thermotherapy and external beam radiation therapy for human prostate cancer cells.

UNLABELLED: Nanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here. RESULTS: NPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher. CONCLUSIONS: In human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.

Development of multivalent radioimmunonanoparticles for cancer imaging and therapy.

BACKGROUND: Noninvasive, focused hyperthermia can be achieved by using an externally applied alternating magnetic field (AMF) if effective concentrations of nanoparticles can be delivered to the target cancer cells. Targeting agents, for example, monoclonal antibodies or peptides, linked to magnetic iron oxide nanoparticles (NP), represent a promising strategy to target cancer cells and hyperthermia. METHODS: We have developed a new radioconjugate NP ((111)In-DOTA-di-scFv-NP), using recombinantly generated antibody fragments, di-scFv-c, for the imaging and therapy of anti-MUC-1-expressing cancers, because aberrant MUC-1 is abundantly expressed on the majority of human epithelial cancers. Anti-MUC-1 di-scFv-c (50 kDa) were engineered, generated, and selected to link maleimide functionalized nanoparticles (NP-M). DOTA chelate was conjugated with di-scFv-c for radionuclide chelation to trace the radioimmunonanoparticles (RINPs) in vivo. RESULTS: Heat-inducing NP-M were prepared with maleimide density >15 per particle for site-specific thiolation. The specific activity of the RINP was 4-5 microCi (111)In/mg with >10 molecules of di-scFv per NP. We characterized the RINP by polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, size-exclusion chromatography, and tumor-cell binding. RINP had a >90% di-scFv conjugated to NP and an immunoreactivity >80% relative to unmodified di-scFv-c on HBT 3477 and DU145 tumor cells. Pharmacokinetics and whole-body autoradiography studies demonstrated that a 5% injected dose was targeted in tumor after 24 hours. CONCLUSIONS: Further development of this new preparation of RINP may provide uniquely high tumor-targeting NP for AMF-driven tumor hyperthermia with less spleen and kidney accumulation.

Thermal dosimetry predictive of efficacy of 111In-ChL6 nanoparticle AMF--induced thermoablative therapy for human breast cancer in mice.

UNLABELLED: Antibody (mAb)-linked iron oxide nanoparticles (bioprobes) provide the opportunity to develop tumor specific thermal therapy (Rx) for metastatic cancer when inductively heated by an externally applied alternating magnetic field (AMF). To evaluate the potential of this Rx, in vivo tumor targeting, efficacy, and predictive radionuclide-based heat dosimetry were studied using (111)In-ChL6 bioprobes (ChL6 is chimeric L6) in a human breast cancer xenograft model. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 (DOTA is dodecanetetraacetic acid) was conjugated to polyethylene glycol-iron oxide-impregnated dextran 20-nm particles and purified as (111)In-bioprobes. (111)In doses of 740-1,110 kBq (20-30 muCi) (2.2 mg of bioprobes) were injected intravenously into mice bearing HBT3477 human breast cancer xenografts. Pharmacokinetic (PK) data were obtained at 1, 2, 3, and 5 d. AMF was delivered 72 h after bioprobe injection at amplitudes of 1,410 (113 kA/m), 1,300 (104 kA/m), and 700 (56 kA/m) oersteds (Oe) at 30%, 60%, and 90% "on" time (duty), respectively, and at 1,050 Oe (84 kA/m) at 50% and 70% duty over the 20-min treatment. Treated and control mice were monitored for 90 d. Tumor total heat dose (THD) from activated tumor bioprobes was calculated for each Rx group using (111)In-bioprobe tumor concentration and premeasured particle heat response to AMF amplitudes. Tumor growth delay was analyzed by Wilcoxon rank sum comparison of time to double, triple, and quintuple tumor volume in each group, and all groups were compared with the controls. RESULTS: Mean tumor concentration of (111)In-bioprobes at 48 h was 14 +/- 2 percentage injected dose per gram; this concentration 24 h before AMF treatment was used to calculate THD. No particle-related toxicity was observed. Toxicity was observed at the highest AMF amplitude-duty combination of 1,300 Oe and 60% over 20 min; 6 of 10 mice died acutely. Tumor growth delay occurred in all of the other groups, correlated with heat dose and, except for the lowest heat dose group, was statistically significant when compared with the untreated group. Electron microscopy showed (111)In-bioprobes on tumor cells and cell death by necrosis at 24 and 48 h after AMF. CONCLUSION: mAb-guided bioprobes (iron oxide nanoparticles) effectively targeted human breast cancer xenografts in mice. THD, calculated using empirically observed (111)In-bioprobe tumor concentration and in vitro nanoparticle heat induction by AMF, correlated with tumor growth delay.

Development of tumor targeting bioprobes ((111)In-chimeric L6 monoclonal antibody nanoparticles) for alternating magnetic field cancer therapy.

OBJECTIVES: (111)In-chimeric L6 (ChL6) monoclonal antibody (mAb)-linked iron oxide nanoparticle (bioprobes) pharmacokinetics, tumor uptake, and the therapeutic effect of inductively heating these bioprobes by externally applied alternating magnetic field (AMF) were studied in athymic mice bearing human breast cancer HBT 3477 xenografts. Tumor cell radioimmunotargeting of the bioprobes and therapeutic and toxic responses were determined. METHODS: Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, (111)In-7,10-tetra-azacyclododecane-N, N',N'',N'''-tetraacetic acid-ChL6 was conjugated to the carboxylated polyethylene glycol on dextran-coated iron oxide 20 nm particles, one to two mAbs per nanoparticle. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using (111)In-ChL6 bioprobe doses (20 ng/2.2 mg ChL6/ bioprobe), i.v. with 50 microg ChL6 in athymic mice bearing HBT 3477; a 153 kHz AMF was given 72 hours postinjection for therapy with amplitudes of 1,300, 1,000, or 700 Oe. Weights, blood counts, and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. RESULTS: (111)In-ChL6 bioprobe binding in vitro to HBT 3477 cells was 50% to 70% of that of (111)In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptakes of the (111)In-ChL6 bioprobes were not different from that observed in prior studies of (111)In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were shown with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT 3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. CONCLUSION: This study shows that mAb-conjugated nanoparticles (bioprobes), when given i.v., escape into the extravascular space and bind to cancer cell membrane antigen, so that bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.