Conventional Hyperthermia Research Articles

Quantitative analysis of radiosensitizing effect for magnetic hyperthermia-radiation combined therapy on prostate cancer cells.

Magnetic hyperthermia (MHT) has emerged as a promising therapeutic approach in the field of radiation oncology due to its superior precision in controlling temperature and managing the heating area compared to conventional hyperthermia. Recent studies have proposed solutions to address clinical safety concerns associated with MHT, which arise from the use of highly concentrated magnetic nanoparticles and the strong magnetic field needed to induce hyperthermic effects. Despite these efforts, challenges remain in quantifying therapeutic outcomes and developing treatment plan systems for combining MHTwith radiation therapy (RT). This study aims to quantitatively measure the therapeutic effect, including radiation dose enhancement (RDE) in the magnetic hyperthermia-radiation combined therapy (MHRT), using the equivalent radiation dose (EQD) estimation method. To conduct EQD estimation for MHRT, we compared the therapeutic effects between the conventional hyperthermia-radiation combined therapy (HTRT) and MHRT in human prostate cancer cell lines, PC3 and LNCaP. We adopted a clonogenic assay to validate RDE and the radiosensitizing effect induced by MHT. The data on survival fractions were analyzed using both the linear-quadradic model and Arrhenius model to estimate the biological parameters describing RDE and radiosensitizing effect of MHRT for both cell lines through maximum likelihood estimation. Based on these parameters, a new survival fraction model was suggested forEQD estimation of MHRT. The newly designed model describing the MHRT effect, effectively captures the variations in thermal and radiation dose for both cell lines (R2>0.95), and its suitability was confirmed through the normality test of residuals. This model appropriately describes the survival fractions up to 10Gy for PC3 cells and 8Gy for LNCaP cells under RT-only conditions. Furthermore, using the newly defined parameter r, the RDE effect was calculated as 29% in PC3 cells and 23% in LNCaP cells. EQDMHRT calculated through this model was 9.47Gy for PC3 and 4.71Gy for LNCaP when given 2Gy and MHT for 30 min. Compared to EQDHTRT, EQDMHRT showed a 26% increase for PC3 and a 20% increase for LNCaP. The proposed model effectively describes the changes of the survival fraction induced by MHRT in both cell lines and adequately represents actual data values through residual analysis. Newly suggested parameter r for RDE effect shows potential for quantitative comparisons between HTRT and MHRT, and optimizing therapeutic outcomes in MHRT for prostate cancer.

New application area for magnetocaloric materials: Hyperthermia method

Conventional Hyperthermia Method is still used for cancer treatment by heating whole body or specific parts of it. Current form of this method includes some possible negative consequences like destroying healthy cells. In this work, a new approach for Hyperthermia method based on magnetocaloric effect has been offered to solve these problems. In order to bring practical use of this new approach, appropriate properties of magnetocaloric material for Hyperthermia method have been determined. These parameters are magnetic entropy change, adiabatic temperature change, the Curie temperature and Full Width at Half Maximum. In accordance with these parameters, some example materials obtained from the literature are listed. Additional parameters also play a role in determining the material and possible ways to obtain new materials have been reported.

Relationship between Energy Dosage and Apoptotic Cell Death by Modulated Electro-Hyperthermia

Modulated electro-hyperthermia (mEHT) is a form of mild hyperthermia (HT) used for cancer treatment. The principle utility of HT is the ability not only to increase cell temperature, but also to increase blood flow and associated pO2 to the microenvironment. While investigational evidence has shown the unique ability of mEHT to elicit apoptosis in cancer cells, in vivo and in vitro, the same trait has not been observed with conventional HT. There is dissension as to what allows mEHT to elicit apoptosis despite heating to only mild temperatures, with the predominant opinion in favor of increased temperature at a cellular level as the driving force. For this study, we hypothesized that in addition to temperature, the amount of electrical energy delivered is a major factor in induction of apoptosis by mEHT. To evaluate the impact of electrical energy on apoptosis, we divided generally practiced mEHT treatment into 3 phases: Phase I (treatment start to 10 min. mark): escalation from 25 °C to 37 °C Phase II (10 min. mark to 15 min. mark): escalation from 37 °C to 42 °C Phase III (15 min. mark to 45 min. mark): maintenance at 42 °C Combinations of mEHT at 18 W power, mEHT at 7.5 W power, water bath, and incubator were applied to each of the three phases. Power output was recorded per second and calculated as average power per second. Total number of corresponding Joules emitted per each experiment was also recorded. The biological effect of apoptotic cell death was assayed by annexin-V assay. In group where mEHT was applied for all three phases, apoptosis rate was measured at 31.18 ± 1.47%. In group where mEHT was only applied in Phases II and III, apoptosis rate dropped to 20.2 ± 2.1%. Where mEHT was only applied in Phase III, apoptosis was 6.4 ± 1.7%. Interestingly, when mEHT was applied in Phases I and II, whether Phase III was conducted in either water bath at 42 °C or incubator at 37 °C, resulted in nearly identical apoptosis rates, 26 ± 4.4% and 25.9 ± 3.1%, respectively. These results showed that accumulation of mEHT at high-powered setting (18 W/sec) during temperature escalation (Phase I and Phase II), significantly increased apoptosis of tested cancer cells. The data also showed that whereas apoptosis rate was significantly increased during temperature escalation by higher power (18 W/sec), apoptosis was limited during temperature maintenance with lower power (7.5 W/sec). This presents that neither maintenance of 42 °C nor accumulation of Joules by mEHT has immediate correlating effect on apoptosis rate. These findings may offer a basis for direction of clinical application of mEHT treatment.

Modulated electro‐hyperthermia suppresses H19, a tumor promoting long non coding RNA, in a triple‐negative breast cancer model

IntroductionModulated electro‐hyperthermia (mEHT) is a complementary antitumor therapy, based on selective tumor cell killing by a 13.56 MHz radiofrequency induced electric field. The H19 long non coding RNA (lncRNA) is involved in tumor progression and metastasis. It’s overexpression is associated with poor prognosis and therapy resistance in breast cancer. We observed previously significant tumor inhibitory effects of mEHT.AimsOur aim was to investigate the hypothesis, that mEHT effects are related to H19 lncRNA inhibition in triple negative breast cancer (TNBC) spheroids and in a TNBC bearing mouse model.Methods4T1 spheroids were embedded in Matrigel® and treated with a single mEHT, conventional hyperthermia (cHT) or normothermia (Ctr) for 30 minutes. Spheroids were collected 24 hours after treatment, RNA was isolated and processed for Real‐Time PCR (RT‐PCR). TNBC cells (highly aggressive 4T1 or less aggressive 4T07) were inoculated orthotopically in female BALB/c mice. Tumor growth was monitored in vivo by digital caliper and ultrasound (Phillips Sonos 5500), mice were randomized into two groups based on tumor size. Mice were treated with mEHT in monotherapy or in combination with methotrexate (MTX) 2 or 3 times for 30 minutes with 0.7±0.3W power to achieve 40°C skin temperature above the tumor. At the end of the experiments mice were euthanized, the tumors were dissected and processed for molecular biologic techniques. H19 expression was measured with RT‐PCR, results were normalized to GAPDH.ResultsSingle mEHT or cHT treatment of 4T1 spheroids reduced significantly H19 expression compared to a normothermic control (Ctr:0.004±0.0004, cHT:0.001±0.0001 mEHT:0.0006±0.0002, p<0.0001). mEHT decreased H19 expression more effectively, than conventional HT (cHT:0.001±0.0001 mEHT:0.0006±0.0002, p<0.01). There was a significant decrease in H19 expression of 4T1 tumors in vivo after two (sham:0.068±0.044, mEHT:0.033±0.024, p<0.05) and three mEHT treatments (sham:0.097±0.059 vs mEHT:0.050±0.030, p<0.05) compared to the sham group. In case of combination treatments H19 expression was significantly lower in the mEHT+MTX group compared to MTX only (MTX:0.104±0,038 vs mEHT+MTX:0.056±0.025, p<0.01). The basic expression of H19 was significantly lower in 4T07 tumors compared to the more aggressive 4T1 tumors (4T07:0.006±0.004 vs 4T1:0.399±0.071, p<0.0001). In 4T07 tumors H19 expression did not change after three mEHT treatments (sham:0.404±0.334 vs mEHT:1.391±1.840, p>0.10) compared to the sham group.ConclusionOur results demonstrate, that modulated electro‐hyperthermia can reduce the expression of tumor promoting H19 lncRNA in vitro and in vivo both in monotherapy and in combination with chemotherapy. Our findings suggest, that mEHT as an alternative complementary treatment could promote antitumor therapy by inhibiting the tumor progression mediating H19 lncRNA expression. More effective therapy against the more aggressive 4T1 line may be related to higher baseline expression and more significant effect on H19.Support or Funding InformationGrantNVKP_16‐1‐2016‐0042, EFOP‐3.6.3‐VEKOP‐16‐2017‐00009

Review of the Clinical Evidences of Modulated Electro-Hyperthermia (mEHT) Method: An Update for the Practicing Oncologist.

Background: Modulated electro-hyperthermia (mEHT) is a variation of the conventional hyperthermia which selectively targets the malignant cell membranes in order to heat the malignant tissue and sensitize the tissue to oncology treatments. Although widely applied, the formulation of guidelines for the use thereof is still in progress for many tumors.Aim: In this paper we review the literature on the effects of mEHT in cancer patients on local disease control and survival.Methodology: Our review on data presents the collected experience with capacitive hyperthermia treatments with the EHY-2000+ device (OncoTherm Ltd., Germany). A literature search was conducted in Pubmed and articles were grouped and discussed according to: trial type, animal studies, in vitro studies, and reviews. Search results from Conference s; Trial Registries; Thesis and Dissertations and the Oncothermia Journal were included in the discussions.Results: Modulated electro-hyperthermia is a safe form of hyperthermia which has shown to effectively sensitizes deep tumors, regardless of the thickness of the adipose layers. The technology has demonstrated equal benefits compared to other forms of hyperthermia for a variety of tumors. Given the effective heating ability to moderate temperatures, the improved tumor perfusion, and ability to increase drug absorption, mEHT is a safe and effective heating technology which can be easily applied to sensitize tumors which have demonstrated benefits with the addition of hyperthermia. Modulated electro-hyperthermia also appears to improve local control and survival rates and appears to induce an abscopal (systemic) response to ionizing radiation.Conclusion: Based on clinical studies, the method mEHT is a feasible hyperthermia technology for oncological applications. Concomitant utilization of mEHT is supported by the preclinical and clinical data.

Oncological hyperthermia: The correct dosing in clinical applications.

The problem with the application of conventional hyperthermia in oncology is firmly connected to the dose definition, which conventionally uses the concept of the homogeneous (isothermal) temperature of the target. Its imprecise control and complex evaluation is the primary barrier to the extensive clinical applications. The aim of this study was to show the basis of the problems of the misleading dose concept. A clear clarification of the proper dose concept must begin with the description of the limitations of the present doses in conventional hyperthermia applications. The surmounting of the limits the dose of oncologic hyperthermia has to be based on the applicability of the Eyring transition state theory on thermal effects. In order to avoid the countereffects of thermal homeostasis, the use of precise heating on the nanoscale with highly efficient energy delivery is recommended. The nano-scale heating allows for an energy-based dose to control the process. The main aspects of the method are the following: i) It is not isothermal (no homogeneous heating); ii) malignant cells are heated selectively; and iii) it employs high heating efficacy, with less energy loss. The applied rigorous thermodynamical considerations show the proper terminology and dose concept of hyperthermia, which is based on the energy-absorption (such as in the case of ionizing radiation) instead of the temperature-based ideas. On the whole, according to the present study, the appropriate dose in oncological hyperthermia must use an energy-based concept, as it is well-known in all the ionizing radiation therapies. We propose the use of Gy (J/kg) in cases of non-ionizing radiation (hyperthermia) as well.

Abstract 196: The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth

Abstract The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10. This study was supported by NVKP 16-1- 2016-0042 grant. Citation Format: Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 196.

Electrokinetics of Temperature for Development and Treatment of Effusions

Introduction: Hyperthermia is a complementary therapy in oncology having various pros and contras for its application. Ascites, pleural effusion, edema and other electrolyte accumulations are frequently excluded from the treatability of the patients with heating locally or systemically. The special gathering of electrolytes is sometimes contraindicated, at times not mentioned in the clinical protocols. However, it is certainly challenging in the oncology where micro and macro edemas, as well as larger electrolyte accumulations (e.g. ascites, pleural effusion), are very frequent. Methods: Excluding patients with accumulation of free electrolytes limits the applications of hyperthermia. To find a solution we are studying the microvasculature and fluid dynamism together with the electric field effects, including the injury currents. The hyperthermia method which we investigate is the modulated electro-hyperthermia (mEHT). We use the Starling’s equation and the injury current in the frame of non-equilibrium thermodynamics and in connection with the biologically closed electric circuits. Results: It is shown that mEHT, unlike the conventional hyperthermia, is applicable for patients who have edema and other free-electrolytes in the volume which is targeted. The heterogeneous heating (unlike the homogeneous, isothermal conventional hyperthermia) promotes the development of tumor-specific immune actions, and so has less adverse-effects, and longer survival time for patients in advanced, metastatic cancers too. Conclusion: mEHT is well applicable in cases of ascites, pleural effusion, edema and other electrolyte accumulations when a patient is treated in complex (complementary) oncological therapy.

What is on the Horizon for Hyperthermic Cancer Therapy?

Cancer is a complex disease. Malignancy can often obscure the localization of the original tumor, which can mislead the patient and clinicians. Cancer means that the tumor is not benign; rather, it is malignant and thus systemic, even when dissemination or micro-metastases have not been discovered yet. Conventional hyperthermia induces tumor cell necrosis, but with isothermal heating and poor depth selection, it has multiple drawbacks. The new trend is mild hyperthermia, with support from complementary treatments. Furthermore, the trend for loco-regional treatment is developing toward immune modulation, especially toward tumor-specific immune reactions. Our objective is to present a review and discuss the trends regarding hyperthermia in oncology.

Measurements of nanoparticle-enhanced heating from 1 MHz ultrasound in solution and in mice bearing CT26 colon tumors

Hyperthermia is considered as a new approach for cancer therapy. Non-selectivity of tissue heating in conventional hyperthermia methods results in collateral damages to healthy tissues and this is the greatest obstacle against hyperthermia in clinic. Herein, to promote the efficiency of conventional hyperthermia methods, nanoparticle-enhanced heating from 1MHz ultrasound was investigated in vitro and in vivo.The experiments were conducted on two mediums; (1) various colloidal nano-solutions (in vitro section) and (2) CT26 mouse colon carcinoma tumor loaded by various nanoparticles (in vivo section). Experiments in this study were designed to evaluate and compare the sonosensitizing potentials of gold nanoparticles (AuNPs), iron oxide nanoparticles (IONPs), and nano-graphene oxide (NGO) in enhancement of ultrasound-induced heat generation. The temperature profile of the solutions and the animal tumors containing nanoparticles were recorded during sonication.An increased heating rate during sonication was observed for both in vitro and in vivo mediums when the nanoparticles were present. Our in vitro experiments revealed that percentages of increases in temperature elevation rates were 12.5%, 20.4%, and 37.5% for IONPs, NGO, and AuNPs, respectively. Compared to the nanoparticles-free tumors, direct injection of AuNPs, NGO and IONPs into the tumors and subsequent sonication for 10min caused an increased temperature elevation rate of 37.5%, 24.1% and 16.1%, respectively.AuNPs, IONPs and NGO are proposed as ultrasound responsive nanomaterials with the potential of focusing the energy of acoustic waves on the tumor and inducing localized hyperthermia.

Comparison of biological effects of modulated electro-hyperthermia and conventional heat treatment in human lymphoma U937 cells.

Loco-regional hyperthermia treatment has long history in oncology. Modulated electro-hyperthermia (mEHT, trade name: oncothermia) is an emerging curative treatment method in this field due to its highly selective actions. The impedance-matched, capacitive-coupled modulated radiofrequency (RF) current is selectively focused in the malignant cell membrane of the cancer cells. Our objective is studying the cell-death process and comparing the cellular effects of conventional water-bath hyperthermia treatment to mEHT. The U937 human histiocytic lymphoma cell line was used for the experiments. In the case of conventional hyperthermia treatment, cells were immersed in a thermoregulated water bath, whereas in the case of mEHT, the cells were treated using a special RF generator (LabEHY, Oncotherm) and an applicator. The heating dynamics, the maximum temperature reached (42 °C) and the treatment duration (30 min) were exactly the same in both cases. Cell samples were analysed using different flow cytometric methods as well as microarray gene expression assay and western blot analysis was also used to reveal the molecular basis of the induced effects. Definite difference was observed in the biological response to different heat treatments. At 42 °C, only mEHT induced significant apoptotic cell death. The GeneChip analysis revealed a whole cluster of genes, which are highly up-regulated in case of only RF heating, but not in conventional heating. The Fas, c-Jun N-terminal kinases (JNK) and ERK signalling pathway was the dominant factor to induce apoptotic cell death in mEHT, whereas the cell-protective mechanisms dominated in case of conventional heating. This study has clearly shown that conventional hyperthermia and RF mEHT can result in different biological responses at the same temperature. The reason for the difference is the distinct, non-homogenous energy distribution on the cell membrane, which activates cell death-related signalling pathways in mEHT treatment but not in conventional heat treatment.

Improving immunological tumor microenvironment using electro-hyperthermia followed by dendritic cell immunotherapy

BackgroundThe treatment of intratumoral dentritic cells (DCs) commonly fails because it cannot evoke immunity in a poor tumor microenvironment (TME). Modulated electro-hyperthermia (mEHT, trade-name: oncothermia) represents a significant technological advancement in the hyperthermia field, allowing the autofocusing of electromagnetic power on a cell membrane to generate massive apoptosis. This approach turns local immunogenic cancer cell death (apoptosis) into a systemic anti-tumor immune response and may be implemented by treatment with intratumoral DCs.MethodsThe CT26 murine colorectal cancer model was used in this investigation. The inhibition of growth of the tumor and the systemic anti-tumor immune response were measured. The tumor was heated to a core temperature of 42 °C for 30 min. The matured synergetic DCs were intratumorally injected 24 h following mEHT was applied.ResultsmEHT induced significant apoptosis and enhanced the release of heat shock protein70 (Hsp70) in CT26 tumors. Treatment with mEHT-DCs significantly inhibited CT26 tumor growth, relative to DCs alone or mEHT alone. The secondary tumor protection effect upon rechallenging was observed in mice that were treated with mEHT-DCs. Immunohistochemical staining of CD45 and F4/80 revealed that mEHT-DC treatment increased the number of leukocytes and macrophages. Most interestingly, mEHT also induced infiltrations of eosinophil, which has recently been reported to be an orchestrator of a specific T cell response. Cytotoxic T cell assay and ELISpot assay revealed a tumor-specific T cell activity.ConclusionsThis study demonstrated that mEHT induces tumor cell apoptosis and enhances the release of Hsp70 from heated tumor cells, unlike conventional hyperthermia. mEHT can create a favorable tumor microenvironment for an immunological chain reaction that improves the success rate of intratumoral DC immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1690-2) contains supplementary material, which is available to authorized users.

Abstract 3609: In vitro comparison of conventional hyperthermia and electro-hyperthemia

Abstract Purpose: Deep heating hypertermia methods are a promising cancer treatment modalities used to enhance conventional treatment. This could be achieved through radiofrequency field en-bloc heating by Thermotron RF-8 and selective tumor heating by Oncotherm-Labehy, respectively. This is the first study to evaluate the different cytotoxic effect and action mechanism on cancer cells after mild hyperthermia (MHT) by Thermotron RF-8 or Oncothermia-Labehy. Experimental Design: Hepatoma HepG2 cells were used for MHT treatment (42°C for 30minute). Either by Oncotherm-Labehy or Thermotron RF-8 or water bath treatment were compared. Apoptosis evaluated 24h after MTH as measured by Annexin-V assay and cell cycle analysis. Mitochondria-targeted damage as measured by ROS production with or without Mito-TEMPO treatment and membrane-targeted damage as measured by caspase-8 expression, adhesion molecule expression, heat shock protein (HSP) release and calcium channel disturbance were evaluated and compared. Results: MHT resulted in similar cell cycle arrest on G2/M. Oncothermia treatment resulted in higher apoptosis rate (38.87±6.58) than RF-8 (25.13±2.26), water bath (18.77±0.35) and control (14.93±0.76). The expression of Caspase-8 is higher by oncothermia treatment, while caspase-3 and 9 levels were similar between 3 types of treatment. HSPs were upregulated by MHT, however a marked HSP release outside cell and E-cadherin overexpression on the membrane were only observed after oncothermia treatment. Conclusion: In this report, we found that different hyperthermia devices may induce different mechanisms on their cytotoxic effects and cell responses to heat. Oncothermia may selectively target on cell membrane before targeting on mitochondria, while RF-8 has no membrane targeting effect. These results may provide rational basis for future clinical application of hyperthermia treatment. Citation Format: Kwan-Hwa Chi, Yu-Shan Samuel Wang, Cheng-Chung Huang, Chao-Chun Chang, Mau-Shin Chi, Kai-Lin Yang, Yuk-Wah Tsang, Gabor Andocs. In vitro comparison of conventional hyperthermia and electro-hyperthemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3609. doi:10.1158/1538-7445.AM2015-3609

Nanoheating without Artificial Nanoparticles Part II. Experimental Support of the Nanoheating Concept of the Modulated Electro-Hyperthermia Method, Using U937 Cell Suspension Model

There are intensive debates about the effects and mechanisms of radiofrequency (RF) hyperthermia in oncology. We theoretically modelled the mechanism of the nanoheating effect of the RF current at the cellular and subcellular level. Then, we experimentally investigated the mechanism of heating in comparison with selective modulated electrohyperthermia and water-bath heating conventional hyperthermia (WHT) using the U937 suspension cell line model. The two heating-processes resulted in different distributions of energy-absorption, causing different mechanisms of the thermal processes. Both of the mechanisms are thermal (fit to Arrhenius plot) but the selectively absorbed energy by the plasma membrane rafts and the cell-cell contacts of the cells results in earlier cell-destruction than in case of unselective homogeneous heating. This thermal effect is used for the characterisation of selective heating. The experimental results clearly support the previous theoretical considerations; the cell killing effect can be realised at lower temperature ranges in the case of the modulated electro-hyperthermia (mEHT, trade-name: oncothermia) method than with WHT.

The effect of repetitive mild hyperthermia on body temperature, the autonomic nervous system, and innate and adaptive immunity.

The effect of repetitive mild hyperthermia on body temperature, the autonomic nervous system, and innate and adaptive immunity was investigated using a new hyperthermia treatment system, nanomist sauna (NMS). Six healthy volunteers participated and the concentration of catecholamines and cortisol, and the frequency and function of leukocytes in the peripheral blood were investigated before and after successive 7 days of hyperthermia treatment (20 min/day, 40°C, 100% relative humidity). After treatment, the blood level of adrenaline and cortisol on the 7th day was decreased compared with the 1st day, indicating the suppression of the sympathetic nervous system activity. Moreover, the frequency of CD56(+)NK, CD56(+)NKT and B cells on the 7th day tended to be increased compared with the 1st day. The frequency of HLA-DR-positive NK and NKT cells and expression of HLA-DR on B and T cells increased. The cytotoxicity of NK cells and proliferative response of B cells were also elevated. The results indicate that repetitive mild hyperthermia treatment might suppress excessive sympathetic dominance and modify immunity. Additionally, because it can provide the same effects as conventional hyperthermia treatments with minimal burden to the body, NMS may be a novel patient- and elderly-friendly hyperthermia treatment for health promotion.

Alternating magnetic field-induced hyperthermia increases iron oxide nanoparticle cell association/uptake and flux in blood-brain barrier models.

Superparamagnetic iron oxide nanoparticles (IONPs) are being investigated for brain cancer therapy because alternating magnetic field (AMF) activates them to produce hyperthermia. For central nervous system applications, brain entry of diagnostic and therapeutic agents is usually essential. We hypothesized that AMF-induced hyperthermia significantly increases IONP blood-brain barrier (BBB) association/uptake and flux. Cross-linked nanoassemblies loaded with IONPs (CNA-IONPs) and conventional citrate-coated IONPs (citrate-IONPs) were synthesized and characterized in house. CNA-IONP and citrate-IONP BBB cell association/uptake and flux were studied using two BBB Transwell(®) models (bEnd.3 and MDCKII cells) after conventional and AMF-induced hyperthermia exposure. AMF-induced hyperthermia for 0.5 h did not alter CNA-IONP size but accelerated citrate-IONP agglomeration. AMF-induced hyperthermia for 0.5 h enhanced CNA-IONP and citrate-IONP BBB cell association/uptake. It also enhanced the flux of CNA-IONPs across the two in vitro BBB models compared to conventional hyperthermia and normothermia, in the absence of cell death. Citrate-IONP flux was not observed under these conditions. AMF-induced hyperthermia also significantly enhanced paracellular pathway flux. The mechanism appears to involve more than the increased temperature surrounding the CNA-IONPs. Hyperthermia induced by AMF activation of CNA-IONPs has potential to increase the BBB permeability of therapeutics for the diagnosis and therapy of various brain diseases.

The effects of non-invasive radiofrequency treatment and hyperthermia on malignant and nonmalignant cells.

Background: Exposure of biological subjects to electromagnetic fields with a high frequency is associated with temperature elevation. In our recent studies, we reported that non-invasive radiofrequency (RF) treatment at 13.56 MHz with the field ranging from 1 KeV to 20 KeV/m2 inhibits tumor progression in animals with abdominal tumor xenografts and enhances the anticancer effect of chemotherapy. The RF treatment was followed by temperature elevation in tumors to approximately 46 °C during 10 min of exposure. In contrast, the temperature of normal tissues remained within a normal range at approximately 37 °C. Whether all biological effects of RF treatment are limited to its hyperthermic property remains unclear. Here, we compared how RF and hyperthermia (HT) treatments change the proliferation rate, oxygen consumption and autophagy in malignant and nonmalignant cells. Methods: In the current study, cancer and nonmalignant cells of pancreatic origin were exposed to the RF field or to conventional HT at 46 °C, which was chosen based on our previous in vivo studies of the tumor-specific RF-induced hyperthermia. Results: Only RF treatment caused declines in cancer cell viability and proliferation. RF treatment also affected mitochondrial function in cancer cells more than HT treatment did and, unlike HT treatment, was followed by the elevation of autophagosomes in the cytoplasm of cancer cells. Importantly, the effects of RF treatment were negligible in nonmalignant cells. Conclusion: The obtained data indicate that the effects of RF treatment are specific to cancer cells and are not limited to its hyperthermic property.

Hollow microspheres based on – Folic acid modified – Hydroxypropyl Cellulose and synthetic multi-responsive bio-copolymer for targeted cancer therapy: Controlled release of daunorubicin, in vitro and in vivo studies

HypothesisConventional chemotherapy drugs such as anthracyclines show no specific activity. They destroy cancer cells but also and the healthy ones, and for that reason exhibit high toxicity. In order to alleviate the toxic effects of chemotherapeutic drugs, the administration dose is being minimized, while their reactivity against tumor cells is lessened. This problem can be overcome or at least reduced by using nanoscale drug delivery systems to target the pathogenic area. The present work deals with the synthesis, characterization and biological evaluation of multi-responsive hollow microspheres coated with Hydroxypropyl Cellulose (HPC)-a biocompatible and thermosensitive polysaccharide-conjugated with folic acid as well promising drug vehicles for targeted cancer therapy. ExperimentsThe synthetic route consists of two steps. In the first step, a single layer of sensitive copolymers is ((Methacrylic acid (MAA), N-(2-Hydroxypropyl) methacrylamide (HPMA) and N,N′-(disulfanediylbis(ethane-2,1-diyl))bis(2-methylacrylamide) (DSBMA)) fabricated on a sacrificial template of SiO2 and in the second step, an additional layer of the folic acid modified HPC coat the microspheres’ surface. The layers fabrication is performed through a combination of distillation precipitation co-polymerization and chemical deposition method. The loading capacity (% LC) and encapsulation efficiency (% EE) percentages of the chemotherapeutic agent daunorubicin (DNR) in the fabricated microspheres were calculated through the standard curve methodology. In addition, the releasing properties of the resulting spheres are investigated, using the above mentioned methodology. It is worth mentioning that, spheres release the entrapped drug under combined conditions such acidic and reductive environment along with conventional hyperthermia. Cytotoxic activity of the synthesized spheres was investigated by using the well-established method of MTT assay in MCF-7 (breast cancer), HeLa (cervical cancer) and HEK 293 (Human Embryonic Kidney healthy cells) cell lines. Confocal and fluorescence microscopy were used to confirm the in vitro targeted ability of folic acid modified drug loaded microspheres in HeLa, to that overexpress folate receptors, MCF-7 and 3T3 cells, as negative folate cell substrate. Finally, radiolabelling of the spheres is performed, with a gamma emitting radionuclide (99mTc), to assess their in vivo profile by means of scintigraphic imaging and biodistribution studies. FindingsHollow spheres release the encapsulated drug under acidic environment, conventional hyperthermia or in the presence of glutathione (reductive environment). The ability of modified drug carriers to target the HeLa cells, was confirmed by confocal and fluorescence microscopy. The resulting spheres are observed to be promising drug-carriers for cancer treatment due to their releasing properties under tumor’s environment and high concentration in HeLa cells via endocytosis. In addition, the empty vehicles have no toxicity in healthy cells and present antimicrobial activity.

Enhancing therapeutic efficacy through designed aggregation of nanoparticles

Particle size is a key determinant of biological performance of sub-micron size delivery systems. Previous studies investigating the effect of particle size have primarily focused on well-dispersed nanoparticles. However, inorganic nanoparticles are prone to aggregation in biological environments. In our studies, we examined the consequence of aggregation on superparamagnetic iron oxide (SPIO) nanoparticle-induced magnetic hyperthermia. Here we show that the extent and mechanism of hyperthermia-induced cell kill is highly dependent on the aggregation state of SPIO nanoparticles. Well-dispersed nanoparticles induced apoptosis, similar to that observed with conventional hyperthermia. Sub-micron size aggregates, on the other hand, induced temperature-dependent autophagy through generation of oxidative stress. Micron size aggregates caused rapid membrane damage, resulting in acute cell kill. Overall, this work highlights the potential for developing highly effective anticancer therapeutics through designed aggregation of nano delivery systems.

High-Intensity Focused Ultrasound-Induced, Localized Mild Hyperthermia to Enhance Anti-cancer Efficacy of Systemic Doxorubicin: An Experimental Study

The aim of this study was to evaluate the enhancement of the efficacy of systemic doxorubicin by pulsed high-intensity focused ultrasound (HIFU)-induced, localized mild hyperthermia. For the in vitro study, the intranuclear uptake of doxorubicin by squamous cell carcinoma (SCC)-7 cells incubated at different temperatures was compared. For the in vivo study, mice with SCC-7 tumors were assigned to either the control, conventional hyperthermia, HIFU hyperthermia, doxorubicin-alone, conventional hyperthermia + doxorubicin or HIFU hyperthermia + doxorubicin group. Conventional hyperthermia was induced by immersing the tumor in warm water (42.5°C), and HIFU hyperthermia was induced by HIFU after optimizing the parameters with direct temperature measurements (frequency = 1 MHz, pulse repetition frequency = 5 Hz, power = 12 W, duty cycle = 50%). In the in vitro study, fluorescence was more intense at 42°C than at 37°C and was time dependent. In the in vivo study, tumor growth in the HIFU hyperthermia + doxorubicin group was most prominently suppressed with the highest apoptotic index compared with all other groups (p < 0.05). Pulsed HIFU-induced localized mild hyperthermia enhanced the anti-cancer efficacy of systemic doxorubicin more than conventional mild hyperthermia.