Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model

Javier Bustamante Mamani; Bruna Souto Marinho; Gabriel Nery de Albuquerque Rego; Mariana Penteado Nucci; Fernando Alvieri; Ricardo Silva dos Santos; João Victor Matias Ferreira; Fernando Anselmo de Oliveira; Lionel Fernel Gamarra

doi:10.31744/einstein_journal/2020AO4954

. 2019 Dec 20;18:eAO4954. doi: 10.31744/einstein_journal/2020AO4954

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Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model

Javier Bustamante Mamani ^1,^✉, Bruna Souto Marinho ¹, Gabriel Nery de Albuquerque Rego ¹, Mariana Penteado Nucci ², Fernando Alvieri ¹, Ricardo Silva dos Santos ¹, João Victor Matias Ferreira ¹, Fernando Anselmo de Oliveira ¹, Lionel Fernel Gamarra ¹

PMCID: PMC6924828 PMID: 31939525

ABSTRACT

Objective:

To evaluate the magnetic hyperthermia therapy in glioblastoma tumor-on-a-Chip model using a microfluidics device.

Methods:

The magnetic nanoparticles coated with aminosilane were used for the therapy of magnetic hyperthermia, being evaluated the specific absorption rate of the magnetic nanoparticles at 300 Gauss and 305kHz. A preculture of C6 cells was performed before the 3D cells culture on the chip. The process of magnetic hyperthermia on the Chip was performed after administration of 20μL of magnetic nanoparticles (10mgFe/mL) using the parameters that generated the specific absorption rate value. The efficacy of magnetic hyperthermia therapy was evaluated by using the cell viability test through the following fluorescence staining: calcein acetoxymethyl ester (492/513nm), for live cells, and ethidium homodimer-1 (526/619nm) for dead cells dyes.

Results:

Magnetic nanoparticles when submitted to the alternating magnetic field (300 Gauss and 305kHz) produced a mean value of the specific absorption rate of 115.4±6.0W/g. The 3D culture of C6 cells evaluated by light field microscopy imaging showed the proliferation and morphology of the cells prior to the application of magnetic hyperthermia therapy. Fluorescence images showed decreased viability of cultured cells in organ-on-a-Chip by 20% and 100% after 10 and 30 minutes of the magnetic hyperthermia therapy application respectively.

Conclusion:

The study showed that the therapeutic process of magnetic hyperthermia in the glioblastoma on-a-chip model was effective to produce the total cell lise after 30 minutes of therapy.

Keywords: Glioblastoma/therapy, Hyperthermia, Nanoparticles, Microfluidics, C6 cells

INTRODUCTION

In the last decade, nanotechnology and nanomaterials has led to advances and introduced a new area in medicine, the nanomedicine.⁽¹^,²⁾ As a result of the studies developed in this area, a number of products have been commercialized and used in clinical medicine,⁽³^,⁴⁾ such as biocompatible nanoparticles applied both to therapy and to diagnosis of tumors.⁽⁵⁾ One of the techniques applied to treatment of tumors is hyperthermia that uses strategies based on radiofrequency waves, ultrasound and microwave, as well as laser light treatments associated with the use of nanoparticles with magnetic properties.⁽⁶^–⁸⁾ Magnetic nanoparticles (MNP), when submitted to alternating magnetic field (AMF), generate heat by transforming magnetic energy into thermal energy used in treatment of tumors, such as shown in figure 1. The increase of temperature, called hyperthermia, by using MNP, corresponds to what is called magnetic hyperthermia therapy (MHT). The MHT has been investigated for treatment of glioblastoma (GBM) tumors because of the fact that these primary malignant brain tumors are more lethal and they present resistance to treatment of chemotherapy and radiotherapy, an event after surgical intervention. Still, although a number of therapeutic advances have been achieved, the development of new studies about treatment of GBM is scarce.⁽⁹⁾

Treatment of GBM, using the MHT technique,⁽¹⁰^,¹¹⁾ consist of administration of MNP in tumor tissue and heating of tumor cells to temperature between 41 to 43°C, by application of AMF, depending on magnetic field (B) and frequency of oscillation (freq) as shown in figure 1A.

Glioblastoma tumors studies with MHT technique have been emphasized because they increase patient's survival compared with other treatments, in addition to not present relevant collateral effects.⁽¹²^,¹³⁾ The assessment of new therapeutic approach for treatment of GBM by using MHT technique was showed in vitro and murine models, however, this in vitro models did not respond clearly the reason why they are incapable of mimic tumor in an environment that the tumor is, and, in murine models, there are a difference between use xerographic or autologous models.⁽¹⁴⁾ However, in 3D cell culture model, which use technology of microfluidics devices (chip), cultivated tumor cells mimic the development of tumor tissue most near to reality,⁽¹⁴⁾ enabling, in this system, the assessment of therapeutic approaches, such as MHT, which does not occur in model in vitro 2D, which is not so efficient to visualize the behavior of cancer cells during coculture.⁽¹⁵^,¹⁶⁾

By using organ-on-a-chip modality, it is possible to mimic tumors of GBM and apply therapy of MHT after administration of dispersed MNP in aqueous medium in cavities of chip, such as shown in figure 1B. There are some appropriated geometrics for development of GBM model on-a-chip, such as adequate channels for development of tumor tissue, GBM on-a-chip, such as adequate channels for development of tumor tissue, correct administration of drugs/nanoparticles, change of fluids for cell maintenance, among other (figure 1C). Other advantage of development of tumor tissue and assessment of therapy of MHT on-a-chip is the adequacy to guidelines of 3Rs (Replacement, Reduction, and Refinement) reducing the use of animals.

OBJECTIVE

To evaluate efficiency of magnetic hyperthermia therapy in glioblastoma on-a-chip.

METHODS

The study was carried out in an Experimental Research Center and Experimental and Surgical Training Center of the Teaching and Research Institute at Hospital Israelita Albert Einstein, Sao Paulo, Brazil.

Magnetic nanoparticles

Dispersed MNP in aqueous medium forming one ferrofluid, available commercially as fluidMAG-Amine (Chemicell, Berlin, Germany), have used in therapy of MHT in GBM on-a-chip model. The MNP has nucleus with crystalline phase of magnetite (Fe₃O₄), being coated with aminosilane, which turns it biocompatible. The hydrodynamic diameter of MNP is 100nm with number of nanoparticles ~1.8×10¹⁵/g and density of ~1.25g/cm³.

Description of magnetic hyperthermia equipment

Therapy of MHT in microfluidic device was applied using system of magnetic heating composed by: DM100 applicator (nB nanoScale Biomagnetics, Zaragoza, Spain) of adjustable magnetic field (50-300 Gauss) in a variety of modes of frequencies (305, 557, 715 and 874kHz); and controller module DMC1 (nB nanoScale Biomagnetics, Zaragoza, Spain), which enabled to conduct program of trials, monitoring of measures and analysis of results. The monitoring of temperature was taken using fiber optic temperature measurement sensors (Luxtron 3204, Luxtron Corporation, Northwestern Parkway, CA, USA). The system was controlled by software MaNiaC (nB nanoScale Biomagnétics, Zaragoza, Spain), which enabled program and data processing.

Determination of specific absorption rate of magnetic nanoparticles of iron oxide

Determination of specific absorption rate (SAR) of MNP (10mgFe/mL) was conducted by submitting these to AMF (300 Gauss, 305kHz), registering ranging temperature over time that, for statistical purposes, four measurements were done. Calculations of SAR was done using MaNiaC software, using the relation

S A R (W / g) = \frac{m_{N P} c_{N P} + m_{1} c_{1}}{m_{N P}} (d T / d t) m a x,

in which m_NP is the mass of MNP, m₁ the mass of water (1000kg/m³), c_NP the specific heat of magnetite (0.16kCal/kg°C), c₁ the specific heat of water (1.0kCal/kg°C) and (dT/dt)max is maximal ranging of heating curve of MNP.

Cell pre-culture of C6 lineage

In this study, we used C6 cells of glioma, a GBM lineage multiform of rats (Cell bank of Rio de Janeiro - BCRJ, code: 0057), which has the ability to form in vivo tumors and share a number of malignant characteristics with GBM human.⁽¹⁷^,¹⁸⁾

These C6 cells were cultivated using RPMI (GIBCO^® Invitrogen Corporation, CA, USA), supplemented with 10% of fetal bovine serum (FBS) (GIBCO^® Invitrogen Corporation, CA, USA), 1% of penicillin- streptomycin (GIBCO^® Invitrogen Corporation, CA, USA) and 1% of L-glutamine (GIBCO^® Invitrogen Corporation, CA, USA) at 37°C (5% CO₂), in bottles of cell culture of 75cm² (Corning, USA). When achieve cell confluence of 85%, cells were trypsinized using 0.25% trypsin (GIBCO^® Ivitrogen Corporation, CA, USA), collected, centrifuged at 800rpm for 5 minutes, resuspended in culture medium in cell concentration of 10⁷cells/mL and keep refrigerated on ice bath.

3D cell culture of C6 cells on-a-chip

To mimic tumor tissue of GBM, we used microfluidic device from SynVivo Inc (Huntsville, AL, US). This chip was formed by two compartments, one central and the other external, separated by porous interface, aiming the culture of the C6 cells in 3D in central cavity, being prepared as described in SynVivo protocol.⁽¹⁹⁾ Basically, 15μL of Matrigel^® (40mg/cm²) (EMD Milipore, Billerica MA) were injected in central compartment, using a syringe and sterile Tygon tubing (0.02″ ID × 0.06″ OD) SynVivo Inc, Huntsville, AL, US) and kept on the fridge at 5°C for a period of 2 hours. Culture medium non-supplemented RPMI was injected for washing compartment, and C6 cells (10⁷cells/mL), with flow rate of 2μL/minute, using bomb 11 Elite Nanomite (Harvard Apparatus, Holliston, MA). External compartment was supplemented RPMI with 10% SFB to 5μL/minute and maintained during all period of culture. The chip was placed in 5% carbon dioxide oven and 37°C for 3D cell culture of C6 cells, with change of culture mean of cavity every 4 hours, during 48 hours.

Assay of magnetic hyperthermia process in glioblastoma on-a-chip

After 48 hours of 3D cell culture of C6 cells on-a-chip, we performed the MHT assay. For this reason, the chip was removed from the incubator, and central cavity was washed with RPMI (0%FBS); subsequently, we injected 20μL of MNP, in concentration of 10mgFe/mL in the same local using infusion pump. The chip was taken up to the MHT equipment and placed on applicator, as shown in figure 2. The experiment was planned for application of AMF (300 Gauss, 305kHz) on chip for period of 30 minutes. Aiming to have temperature control in therapeutic range from 41 to 43°C, we used the amount colloidal suspension of MNP (200μL contained in eppendorf in concentration of 10mgFe/mL) as referential sample that was submitted to same AMF together with chip and the temperature was monitored by fiber optic system.

Evaluation of efficiency of magnetic hyperthermia process in glioblastoma on-a-chip

The assessment of efficiency of therapy of MHT in GBM on-a-chip was conducted using LIVE/DEAD^® kit (Molecular Probes, Eugene, Oregon, USA) of assay of viability and/or cell toxicity, through image of fluorescence, in which were used 4mM of Calcein acetoxymethyl ester (Ca-AM) and 12mM of ethidium homodimer-1 (EthD-1). The fluorescence of both staining occur to interact with live cells (for Ca-AM-excitation/emission: 492/513nm) or dead cells (for EthD-1 – excitation/emission: 526/619nm). Green fluorescence of acetoxymethyl ester indicates the activity of intracellular esterases of viable cells, and red fluorescence of EthD-1 indicates loss of integrity of plasmatic membrane. The analysis of cell viability before and after the MHT therapy in tumor cells on-a-chip was conducted by injecting 15μL of solution formed by Ca-AM and EthD-1 in central cavity of chip and, subsequently, we registered fluorescence images, using inverted microscopy Nikon Eclipse Ti-E (Tokio, Japan). The counting of live (green) and dead (red) cells were done in two regions of organ-on-a-chip (region I and II, bifurcation of entrance of fluids and central cavity of chip, respectively). The experiment since the 3D cell cultivation up to assessment of cell viability was repeated three times.

RESULTS

Assessment of specific absortion rate of iron oxide magnetic nanoparticles

Capacity of heating of MNP was characterized for application in MHT. The heating curve of MNP is shown in figure 3, and indicates rapid increase of temperature in period of 60 seconds. The inset of figure 3 (box plot) shows the assessment of distribution of values of SAR with mean values of 115.4±6.0W/g.

3D cell culture of C6 cells on-a-chip

3D cell culture of C6 cells was conducted in central cavity of chip and evaluated through images of microscopy of clear field. Figure 4, shows images of C6 cells in culture after 4 and 48 hours of sowing (Figures 4A and 4B), respectively. For this reason, morphology of C6 cells and their proliferation (Figures 4C and 4D) in central region of the chip.

Images of figure 4, we could observe the beginning of C6 cells growth in isolated regions forming islands, with cell proliferation within islands, beginning the 3D cell cultivation over the Matrigel^® structure mimicking the formation of GBM tumor tissue.

Assessment of efficiency of magnetic hyperthermia therapy in glioblastoma on-a-chip

After growth of tumor tissue on the chip, we applied the therapy of MHT to conduct assay of viability of C6 cells, such as shown in figure 5. The microfluidic device in figure 5A shows regions of evaluation of cell viability indicated by frames in blue (region I, showing the bifurcation of fluid entrance) and red (region II, showing the central cavity). Figures 5B and 5C show images of microscopy of clear field of C6 cells cultivation in regions I and II, respectively. Figures 5D and 5E include images of fluorescence that correspond to live C6 cells reacting to Ca-AM before therapy of MHT in regions I and II of Chip, respectively. In figure 5F and 5G, we observed fluorescence image of region I and II with live C6 cells (green) and dead cells (red) that react to Ca-AM and EthD-1, respectively, after 10 minutes of therapy of MHT in GBM on-a-chip. In figure 5H and 5J, we observed by images of fluorescence of regions I and II, respectively, only dead C6 cells (red) that reacted with EthD-1 staining, after 30 minutes of MHT therapy.

Fluorescence assay of figure 5 showed that MNP with SAR value (115.4±6.0W/g) was adequate for heating of tumor tissue up to therapeutic temperature, when submitted to one AMF with magnetic field of 300 Gauss and frequency of 305kHz. Hyperthermia treatment showed a reduction of cell viability in 20%, after 10 minutes, and in 100% after 30 minutes of MHT, through the use of kit of cell viability (LIVE/DEAD^®).

DISCUSSION

Microfluidics have provided a large development in tissue engineering, aiming to understand biologic processes in vitro studies.⁽²⁰⁾ The development of these microfluidic systems to mimic tumors are on use in a number of therapies, raising the interest of scientific community, in order to replace the use of murine models.⁽²¹^–²³⁾ One of these therapies is applied by hyperthermia treatments, such as MHT in tumors.

Studies of MHT in tumor cells using MNP show a large potential in treatment for tumors of GBM, however, because of variety of parameters of application of MHT and the use of different types of tumor cells, it has been difficult to evaluate which are the best parameters of this therapy in tumor treatment, as well as this constitutes a barrier for application of this modality as standard in treatment of GBM.⁽²⁴⁾ This can be observed in review of Gupta et al.,⁽²⁵⁾ which describe different parameters of applications of MHT in models of tumor in vitro and characteristics of MNP used. In the study by Hanini et al.,⁽²⁶⁾ an evaluation was conducted of MHT in glioma cells (U87-MG) treated with MNP of γ-Fe₂O₃ coated with polyol, with diameter of 10nm, in concentration of 50μgFe/mL, submitted to AMF with frequency of oscillation of 700kHz and magnetic field of 289.67Oe, with time of application of 60 minutes, keeping the therapeutic temperature of 42°C and showing reduction cell viability of 50%. In other study, we used cell of glioma T-9 and MNP of magnetite with diameter of 35nm in concentration of 7.2mgFe/mL, applying AMF with 118kHz and 383.72Oe, achieving cell lyse of 100% in 60 minutes.⁽²⁷⁾ By using the same tumor cell used in our study, Gupta et al.,⁽²⁸⁾ evaluated C6 glioma cells of rats and NIH3T3 fibroblast of mice, using MNP of Fe₂O₃ coated with steviosides, of 4.62nm of diameter, in concentration of 100μgFe/mL, applying AMF with 405kHz and 168Oe for 30 minutes, achieving therapeutic temperature of 43°C, and showing a decrease in cell viability of 40% and, after 4 hours of coculture, an additional reduction of 34%. The enhancement of nanomaterial for this therapeutic approach has also been the focus of this study to obtain the best SAR that reflects in efficiency of MHT technique.

However, in vivo studies, such the one conducted by Jordan et al.,⁽²⁹⁾ the efficacy of therapy of MHT was evaluated in brain tumor of Fisher's rats induced by RG-2 cells, with two types of MNP - one coated with aminosilane and other dextran. Results showed that application of AMF (100kHz and 225.72Oe) with MNP coated with aminosilane was more efficient in reduction of rate of cell proliferation than when coated with dextran. This study had adequate values of SAR, on the range of 10 to 100W/g – values considered typical of SAR for this type of application.⁽³⁰⁾ In our study, the MNP of iron oxide (magnetite) were also coated with aminosilane and mean value of SAR of these MNP was 115.4±6.0W/g. Clinical studies published in the literature have also reported⁽³¹^–³³⁾ the used of this type of MNP coated with aminosilane, and parameters of application of MHT are similar to those used in our GBM study on-a-chip. These similar characteristic, also use the C6 tumor cells that mimic the human GBM, assist in transposition of data for human model, allowing better evaluation of altered therapeutic approach, such as the MHT, combined or not with other techniques in high-severity illness and low response to conventional treatments such as the GBM.

Currently, the organ on-a-chip model of GBM has been used to evaluate the ability to model progression of hyper cellular regions of GBM, observed in patients, and mimicking the obstruction of blood vessels, modeling the delivery of nutrients, and gradients of oxygen during the evolution of GBM;⁽³⁴⁾ screening of high performance drugs and prolonged drug delivery;⁽³⁵^–³⁷⁾ to evaluate vascular compartment that present a network of vessels in communication with solid 3D tumors mimicking microenvironment of tumor, including the knowledge as Enhanced Permeability and Retention (EPR),⁽³⁸⁾ among others.

For this reason, the optimization of therapy of MHT in microfluidic device that mimic the characteristics of GBM is presented as potential application for translational in humans.

The models of GBM on-a-chip of our study provides basis for implementation of this method technique of MHT, aiming to evaluate its potential therapeutic in GBM, temporally, although our model has presented one limiting factor, which was the lack of vascular network associated with tumor tissue, but this must be implemented in future studies.

CONCLUSION

Our study showed efficiency of magnetic hyperthermia therapy for treatment of glioblastoma on-a-chip with lise of all tumor cells after 30 minutes of magnetic hyperthermia using nanoparticles of iron oxide coated with aminosilane, which is used in clinical trials. In addition, specific absorption rate was often used in therapy assays of magnetic hyperthermia in tumors of human glioblastoma.

ACKNOWLEDGEMENTS

This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (400856/2016-6) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2016/21470-3; 2014/50983-3).

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Einstein (Sao Paulo). 2019 Dec 20;18:eAO4954. [Article in Portuguese]

Terapia de magneto-hipertermia no modelo de tumor de glioblastoma on-a-Chip

RESUMO

Objetivo:

Avaliar a terapia de magneto-hipertermia em modelo de tumor de glioblastoma on-a-Chip.

Métodos:

As nanopartículas magnéticas recobertas com aminosilana foram utilizadas para a terapia da magneto-hipertermia, sendo avaliada a taxa de absorção específica das nanopartículas magnéticas em 300 Gauss e 305kHz. Uma pré-cultura de células C6 foi realizada e, seguidamente, foi feito o cultivo das células 3D no chip. O processo de magneto-hipertermia no chip foi realizado após administração de 20μL de nanopartículas magnéticas (10mgFe/mL), utilizando os parâmetros que geraram o valor da taxa de absorção específica. A eficácia da terapia de magneto-hipertermia foi avaliada pela viabilidade celular por meio dos corantes fluorescentes acetoximetiléster de calceína (492/513nm), para células vivas, e etídio homodímero-1 (526/619nm), para células mortas.

Resultados:

As nanopartículas magnéticas, quando submetidas ao campo magnético alternado (300 Gauss e 305kHz), produziram um valor médio da taxa de absorção específica de 115,4±6,0W/g. A cultura 3D das células C6 avaliada por imagem de microscopia de campo claro mostrou a proliferação e a morfologia das células antes da aplicação da terapia de magneto-hipertermia. As imagens de fluorescência mostraram diminuição da viabilidade das células cultivadas no organ-on-a-Chip em 20% e 100% após 10 e 30 minutos, respectivamente, da aplicação da terapia de magneto-hipertermia.

Conclusão:

O processo terapêutico da magneto-hipertermia no modelo de tumor glioblastoma on-a-chip foi eficaz para produzir lise total das células após 30 minutos de terapia.

Descritores: Glioblastoma/terapia, Hipertermia, Nanopartículas, Microfluídica, Células C6

INTRODUÇÃO

Na última década, a nanotecnologia e os nanomateriais deram lugar a uma nova área na medicina, a denominada “nanomedicina”.⁽¹^,²⁾ Como resultado das pesquisas desenvolvidas nesta área, diversos produtos já estão sendo comercializados e usados na clínica médica,⁽³^,⁴⁾ como as nanopartículas biocompatíveis aplicadas tanto na terapia como no diagnóstico de tumores.⁽⁵⁾ Uma das técnicas aplicadas no tratamento de tumores é a terapia hipertérmica, que utiliza estratégias baseadas em ondas de radiofrequência, ultrassom e microndas, bem como tratamentos com laser associado ao uso de nanopartículas com propriedades magnéticas.⁽⁶^–⁸⁾ As nanopartículas magnéticas (NPM), quando submetidas a um campo magnético alternado (CMA), geram aquecimento, mediante a transformação de energia magnética em energia térmica usada no tratamento de tumores, como mostrado na figura 1. O incremento da temperatura, denominando de hipertermia, usando NPM, corresponde ao que é chamado de terapia de magneto-hipertermia (MHT). A MHT tem sido alvo de pesquisas no tratamento de tumores de glioblastoma (GBM) devido ao fato de ser um dos tumores cerebrais malignos primários mais letais e que apresentam resistência ao tratamento de quimioterapia e radioterapia, mesmo após intervenção cirúrgica. Ainda que existam muitos avanços terapêuticos, o desenvolvimento de pesquisas sobre o tratamento do GBM é incipiente.⁽⁹⁾

O tratamento do GBM, mediante a técnica de MHT,⁽¹⁰^,¹¹⁾ consiste na administração de NPM no tecido tumoral e no aquecimento das células tumorais a uma temperatura entre 41 a 43°C, mediante aplicação do CMA, dependente do campo magnético (B) e frequência de oscilação (freq) como é mostrado na figura 1A.

Estudos de GBM com a técnica da MHT vêm se destacando por apresentarem aumento da sobrevida do paciente quando comparado a outros tratamentos, além de não apresentar efeitos colaterais relevantes.⁽¹²^,¹³⁾

A avaliação de novas abordagens terapêuticas visando ao tratamento do GBM por meio da técnica da MHT foi demonstrada nos modelos in vitro e murinos, mas os modelos in vitro não respondem de forma clara, porque não são capazes de mimetizar o tumor no ambiente no qual ele se encontra, e, nos modelos murinos, existe uma diferença entre usar modelos xenográficos ou autólogos.⁽¹⁴⁾ Entretanto, no modelo de cultura celular 3D, que utiliza a tecnologia dos dispositivos microfluídicos (chip), as células tumorais cultivadas mimetizam o desenvolvimento do tecido tumoral de maneira mais próxima do real,⁽¹⁴⁾ possibilitando, nesse sistema, a avaliação de abordagens terapêuticas, como da MHT, o que não acontece no modelo 2D in vitro, o qual não se mostra tão eficiente para visualizar o comportamento de células cancerígenas durante a cocultura.⁽¹⁵^,¹⁶⁾

Com o uso da modalidade organ-on-a-chip, é possível mimetizar tumores de GBM e aplicação da terapia da MHT após a administração das NPM dispersas no meio aquoso nas cavidades do chip, como mostrado na figura 1B. Existem algumas geometrias apropriadas para o desenvolvimento do modelo de GBM on-a-chip, como canais adequados para o desenvolvimento do tecido tumoral, correta administração de drogas/nanopartículas, troca de fluidos para a manutenção celular, entre outras (Figura 1C). Outra vantagem de desenvolver o tecido tumoral e avaliar a terapia de MHT on-a-chip está na adequação às diretrizes dos 3Rs (Redução, refinamento e substituição - replacement, reduction, e refinement) reduzindo o uso de animais.

OBJETIVO

Avaliar a eficiência da terapia de magneto-hipertermia em tumores de glioblastoma on-a-chip.

MÉTODOS

O estudo foi realizado no Centro de Pesquisa Experimental e no Centro de Experimentação e Treinamento em Cirurgia do Instituto Israelita de Ensino e Pesquisa do Hospital Albert Einstein, São Paulo, Brasil.

Nanopartículas magnéticas

As NPM dispersas em meio aquoso formando um ferrofluido, disponíveis comercialmente como fluidMAG-Amine (Chemicell, Berlin, Germany), foram usadas na terapia da MHT no modelo GBM on-a-chip. As NPM possuem núcleo com fase cristalina da magnetita (Fe₃O₄), sendo cobertas com aminosilana, que as torna biocompatíveis. O diâmetro hidrodinâmico das NPM é de 100nm, com número de nanopartículas ~1,8×10¹⁵/g e densidade de ~1,25g/cm³.

Descrição do equipamento de magneto-hipertermia

A terapia de MHT no dispositivo microfluídico foi aplicada usando sistema de aquecimento magnético composto por: aplicador DM100 (nB nanoScale Biomagnétics, Zaragoza, Spain) de campo magnético ajustável (50-300 Gauss) em diversos modos de frequências (305, 557, 715 e 874kHz); e um módulo controlador DMC1 (nB nanoScale Biomagnétics, Zaragoza, Spain), que permitiu realizar a programação dos ensaios, monitorização das medições e análise dos resultados. A monitorização da temperatura foi realizada mediante sonda de temperatura de fibra ótica (Luxtron 3204, Luxtron Corporation, Northwestern Parkway, CA, USA). O sistema foi controlado pelo software MaNiaC (nB nanoScale Biomagnétics, Zaragoza, Spain), que facilitou a programação e o processamento de dados.

Determinação da taxa de absorção específica das nanopartículas magnéticas de óxido de ferro

A determinação da taxa de absorção específica (SAR - specific absorption rate) das NPM (10mgFe/mL) foi realizada submetendo estas ao CMA (300 Gauss, 305kHz), registrando a variação da temperatura no tempo e que, para fins estatísticos, foram realizadas em quatro medidas. O cálculo da SAR foi realizado mediante o software MaNiaC, utilizando a relação

S A R (W / g) = \frac{m_{N P} c_{N P} + m_{1} c_{1}}{m_{N P}} (d T / d t) m a x,

onde m_NP é a massa das NPM, m₁ massa da água (1000kg/m³), c_NP o calor específico da magnetita (0,16kCal/kg°C), c₁ o calor específico da água (1,0kCal/kg°C) e (dT/dt)max é máxima variação da curva de aquecimento das NPM.

Pré-cultura celular da linhagem C6

Foram utilizadas, neste estudo, células C6 de glioma, uma linhagem de GBM multiforme de ratos (Banco de Células de Rio de Janeiro – BCRJ, código: 0057), que tem a capacidade de formar tumores in vivo e compartilhar várias características malignas com o GBM humano.⁽¹⁷^,¹⁸⁾ Estas células C6 foram cultivadas em meio RPMI (GIBCO^® Invitrogen Corporation, CA, USA), suplementado com 10% de soro fetal bovino (SFB) (GIBCO^® Invitrogen Corporation, CA, USA), 1% de penicilina-estreptomicina (GIBCO^® Invitrogen Corporation, CA, USA) e 1% de L-glutamina (GIBCO^® Invitrogen Corporation, CA, USA) a 37°C (5% CO₂), em garrafas de cultura celular de 75cm² (Corning, USA). Ao atingir confluência celular de 85%, as células foram tripsinizadas utilizando 0,25% tripsina (GIBCO^® Ivitrogen Corporation, CA, USA), coletadas, centrifugadas a 800rpm por 5 minutos, ressuspensas em meio de cultura na concentração celular de 10⁷ células/mL e mantidas resfriadas em banho de gelo.

Cultura celular 3D das células C6 on-a-chip

Para mimetizar o tecido tumoral de GBM, foi utilizada o dispositivo microfluídico da SynVivo Inc (Huntsville, AL, US). Este chip era formado por dois compartimentos, um central e outro externo, separados por interface porosa, visando à cultura de células C6 em 3D na cavidade central, sendo preparado como descrito no protocolo da SynVivo.⁽¹⁹⁾ Basicamente, 15μL de Matrigel^® (40mg/cm²) (EMD Milipore, Billerica MA) foram injetados no compartimento central, utilizando seringa e tubulação estéril de Tygon (0,02″ ID × 0,06″ OD) (SynVivo Inc, Huntsville, AL, US) e mantidos na geladeira a 5^oC por um período de 2 horas. Meio de cultura RPMI não suplementado foi injetado para lavagem do compartimento, e as células C6 (10⁷celúlas/mL) foram semeadas na cavidade central, a uma vazão de 2μL/minuto, utilizando bomba 11 Elite Nanomite (Harvard Apparatus, Holliston, MA). No compartimento externo, foi escoado RPMI suplementado com 10% SFB a 5μL/minuto e mantido durante todo o período de cultura. O chip foi colocado em estufa de dióxido de carbono a 5% e 37°C para a cultura 3D das células C6, com troca do meio de cultura da cavidade a cada 4 horas, durante 48 horas.

Ensaio do processo de magneto-hipertermia no glioblastoma on-a-chip

Após 48 horas de cultura 3D das células C6 on-a-chip, foi realizado o ensaio de MHT. Para tal finalidade, o chip foi retirado da estufa, e a cavidade central foi lavada com RPMI (0%SFB); seguidamente, foram injetados 20μL de NPM, na concentração de 10mgFe/mL no mesmo local, utilizando a bomba de infusão. O chip foi levado ao equipamento de MHT e colocado no aplicador, como mostrado na figura 2. O experimento foi planejado para aplicação do CMA (300 Gauss, 305kHz) no chip por um período de 30 minutos. Com a finalidade de ter o controle da temperatura na faixa terapêutica de 41 a 43^oC, foi utilizada quantidade da suspensão coloidal de NPM (200μL contidos em um eppendorf na concentração de 10mgFe/mL) como amostra referencial, que foi submetida ao mesmo CMA, em conjunto com o chip e a temperatura monitorada mediante o sistema de fibra ótica.

Avaliação da eficiência do processo de magneto-hipertermia no glioblastoma on-a-chip

A avaliação da eficiência da terapia de MHT no GBM on-a-chip foi realizada usando o kit LIVE/DEAD^® (Molecular Probes, Eugene, Oregon, USA) de ensaio de viabilidade e/ou toxicidade celular, mediante imagem de fluorescência, no qual foram usados 4mM de acetoximetiléster de calceína (Ca-AM) e 12mM de etídio homodímero-1 (EthD-1). A fluorescência de ambos os corantes ocorre ao interagir com células vivas (para Ca-AM – excitação/emissão: 492/513nm) ou mortas (para EthD-1 – excitação/emissão: 526/619nm). O verde fluorescente da acetoximetiléster indica a atividade de esterase intracelular de células viáveis, e o vermelho fluorescente do EthD-1 indica perda de integridade da membrana plasmática. A análise da viabilidade celular antes e após a terapia de MHT nas células tumorais on-a-chip foi realizada injetando 15μL da solução formada por Ca-AM e EthD-1 na cavidade central do chip e, seguidamente, foram registradas imagens de fluorescência, utilizando um microscópio invertido Nikon Eclipse Ti-E (Tokio, Japan). As contagens de células vivas (verde) e mortas (vermelhas) foram feitas nas duas regiões do organ-on-a-chip (região I e II, a bifurcação da entrada dos fluídos e a cavidade central do chip, respectivamente). O experimento desde o cultivo celular 3D até a avaliação da viabilidade celular foi repetido três vezes.

RESULTADOS

Avaliação da taxa de absorção específica das nanopartículas magnéticas de óxido de ferro

A capacidade de aquecimento das NPM foi caracterizada para aplicação na terapia da MHT. A curva de aquecimento das NPM é mostrada na figura 3, indicando rápido incremento da temperatura em um período de 60 segundos. O inset da figura 3 (box plot) mostra a avalição da distribuição dos valores da SAR com valor médio de 115,4±6,0W/g.

Cultura celular 3D das células C6 on-a-chip

A cultura 3D das células C6 foi realizada na cavidade central do chip e avaliada mediante imagens de microscopia de campo claro. A figura 4, mostra imagens de células C6 em cultura após 4 e 48 horas da semeação (Figuras 4A e 4B), respectivamente, assim como a morfologia das células C6 e sua proliferação (Figuras 4C e 4D) respectivamente, na região central do chip.

Nas imagens da figura 4, pode-se observar o início do crescimento das células C6 em regiões isoladas formando ilhas, com a proliferação celular nas ilhas, dando início ao cultivo celular em 3D sobre a estrutura de Matrigel^® mimetizando a formação de um tecido tumoral de GBM.

Avaliação da eficiência da terapia da magneto-hipertermia no tumor de glioblastoma on-a-chip

Após crescimento do tecido tumoral no chip, foi aplicada a terapia de MHT para realização do ensaio de viabilidade das células C6, como mostrado na figura 5. O dispositivo microfluídico na figura 5A mostra as regiões de avaliação da viabilidade celular indicadas por quadros nas cores azul (região I, mostrando a bifurcação da entrada de fluídos) e vermelho (região II, mostrando a cavidade central). As figuras 5B e 5C mostram imagens de microscopia de campo claro do cultivo de células C6 nas regiões I e II, respectivamente. Nas figuras 5D e 5E, são apresentadas imagens de fluorescência que correspondem às células C6 vivas reagentes a Ca-AM antes da terapia de MHT, nas regiões I e II do Chip, respectivamente. Na figura 5F e 5G, observamos a imagem de fluorescência da região I e II com células C6 vivas (verde) e mortas (vermelho) que reagiram ao Ca-AM e EthD-1, respectivamente, após 10 minutos da terapia de MHT no GBM on-a-chip. Na figura 5H e 5J, verificamos, por meio das imagens de fluorescência das regiões I e II, respectivamente, somente as células C6 mortas (vermelho) que reagiram com o corante EthD-1, após 30 minutos de terapia de MHT.

Os ensaios de fluorescência da figura 5 mostraram que NPM com valor de SAR (115,4±6,0W/g) foram adequadas para o aquecimento do tecido tumoral até a temperatura terapêutica, quando submetidas a um CMA com campo magnético de 300 Gauss e frequência de 305kHz. O tratamento hipertérmico mostrou redução da viabilidade celular em 20%, após 10 minutos, e em 100% após 30 minutos de MHT, mediante o uso do kit de viabilidade celular (LIVE/DEAD^®).

DISCUSSÃO

A microfluídica tem proporcionado grande desenvolvimento na engenharia de tecidos, visando ao entendimento de processos biológicos em estudos in vitro.⁽²⁰⁾ O desenvolvimento destes sistemas microfluídicos para mimetizar tumores está sendo utilizado em diversas terapias, despertando o interesse na comunidade científica, afim de substituir o uso de modelos murinos.⁽²¹^–²³⁾ Uma destas terapias é a aplicação de tratamentos hipertérmicos, como o MHT em tumores.

Estudos de MHT em células tumorais utilizando NPM mostram grande potencial no tratamento para tumores de GBM, porém, devido à variedade de parâmetros da aplicação de MHT e ao uso de diferentes tipos de células tumorais, tem sido difícil a avaliação de quais são os melhores parâmetros desta terapêutica no tratamento tumoral, bem como configura-se um obstáculo para aplicação desta modalidade como padrão no tratamento de GBM.⁽²⁴⁾ Isto pode ser verificado na revisão de Gupta et al.,⁽²⁵⁾ que descreveram os diferentes parâmetros de aplicação do MHT em modelos de tumor in vitro e as características da NPM utilizadas. No estudo de Hanini et al.,⁽²⁶⁾ foi realizada a avaliação do MHT em células de glioma (U87-MG) tratadas com NPM de γ-Fe₂O₃ recobertas com poliol, com diâmetro de 10nm, na concentração de 50μgFe/mL, submetidas ao CMA com frequência de oscilação de 700kHz e campo magnético de 289,67Oe, com tempo de aplicação de 60 minutos mantendo-se a uma temperatura terapêutica de 42°C e mostrando diminuição na viabilidade celular de 50%. Em outro estudo, foram utlizadas células de glioma T-9 e NPM à base de magnetita com diâmetro de 35nm na concentração de 7,2mgFe/mL, aplicando CMA com 118kHz e 383,72Oe, alcançando lise celular de 100%, em 60 mimutos.⁽²⁷⁾ Já com a mesma célula tumoral utilizada em nosso estudo, Gupta et al.,⁽²⁸⁾ avaliaram as células de glioma C6 de rato e fibroblasto de camundongo NIH3T3, utilizando NPM de Fe₂O₃ revestidas com esteviosídeo, de 4,62nm de diâmetro, na concentração de 100μgFe/mL, aplicando CMA com 405kHz e 168Oe durante 30 minutos, alcançando temperatura terapêutica de 43°C, e mostrando decréscimo na viabilidade celular de 40% e, após 4 horas de cocultura, diminuição adicional de 34%. O aprimoramento dos nanomateriais para esta abordagem terapêutica também tem sido foco de estudo para a obtenção da melhor SAR, que reflete na eficiência da técnica de MHT.

Já em estudos in vivo, como de Jordan et al.,⁽²⁹⁾ foi avaliada a eficácia da terapia da MHT, em tumor cerebral de ratos Fisher induzido com células RG-2, com dois tipos de NPM, uma recoberta com aminosilana e outra com dextrana, mostrando que a aplicação do CMA (100kHz e 225,72Oe) com NPM recoberta com aminosilana foi mais eficiente na diminuição da taxa de proliferação celular do que quando revestidas com dextrana. Este estudo apresentou valores de SAR adequada, na faixa de 10 a 100W/g – valores considerados típicos de SAR para este tipo de aplicação.⁽³⁰⁾ Em nosso estudo, as NPM de óxido de ferro (magnetita) também foram recobertas com aminosilana e o valor médio de SAR destas NPM foi de 115,4±6,0W/g. Estudos clínicos reportados na literatura⁽³¹^–³³⁾ também têm usado este tipo de NPM recoberto com aminosilana, e os parâmetros de aplicação de MHT são semelhantes aos usados em nosso estudo de GBM on-a-chip. Estas características similares, como também a utilização de células tumorais C6 que mimetizam o GBM humano, auxiliam na transposição dos dados para o modelo humano, permitindo melhor avaliação de abordagens terapêuticas alternativas, como o MHT, combinadas ou não com outras técnicas em doenças com alta severidade e baixa resposta aos tratamentos convencionais como são as GBM.

Atualmente, o modelo organ on-a-chip de GBM tem sido utilizado para avaliar a capacidade de modelar a progressão de regiões hipercelulares do GBM, observada em pacientes, imitando a obstrução de vasos sanguíneos e modelando a entrega de nutrientes e gradientes de oxigênio durante a evolução do GBM;⁽³⁴⁾ triagem de drogas de alto rendimento e liberação prolongada de drogas;⁽³⁵^–³⁷⁾ avaliar o compartimento vascular que apresenta uma rede de vasos em comunicação com tumores sólidos 3D imitando microambiente do tumor, incluindo o conhecido como efeito Enhanced Permeability and Retention (EPR),⁽³⁸⁾ entre outros. Portanto, a optimização da terapia da MHT em um dispositivo microfluídico que mimetize as características do GBM se apresenta com potencial aplicação para o translacional em humanos.

O modelo de GBM on-a-chip do presente trabalho proporcionou bases para a implementação da metodologia da técnica de MHT, visando avaliar seu potencial terapêutico em GBM temporalmente, embora nosso modelo tenha apresentado um fator limitante, que foi a falta da rede vascular associada ao tecido tumoral, mas que deve ser implementada em trabalhos futuros.

CONCLUSÃO

O presente estudo mostrou eficiência da terapia de magneto-hipertermia no tratamento de tumor de glioblastoma on-a-chip, com lise de todas as células tumorais após 30 minutos de terapia de magneto-hipertermia, usando nanopartículas de óxido de ferro recobertas com aminosilana (usadas em ensaios clínicos). Também, o valor de taxa de absorção específica foi o tipicamente usado em ensaios de terapia de magneto-hipertermia em tumores de glioblastoma humano.

AGRADECIMENTOS

Esta pesquisa foi financiada pelo Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (400856/2016-6) e Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2016/21470-3; 2014/50983-3).

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PERMALINK

Magnetic hyperthermia therapy in glioblastoma tumor on-a-Chip model

Javier Bustamante Mamani

Bruna Souto Marinho

Gabriel Nery de Albuquerque Rego

Mariana Penteado Nucci

Fernando Alvieri

Ricardo Silva dos Santos

João Victor Matias Ferreira

Fernando Anselmo de Oliveira

Lionel Fernel Gamarra

ABSTRACT

Objective:

Methods:

Results:

Conclusion:

INTRODUCTION

OBJECTIVE

METHODS

Magnetic nanoparticles

Description of magnetic hyperthermia equipment

Determination of specific absorption rate of magnetic nanoparticles of iron oxide

Cell pre-culture of C6 lineage

3D cell culture of C6 cells on-a-chip

Assay of magnetic hyperthermia process in glioblastoma on-a-chip

Evaluation of efficiency of magnetic hyperthermia process in glioblastoma on-a-chip

RESULTS

Assessment of specific absortion rate of iron oxide magnetic nanoparticles

Figure 3. Heating curve of magnetic nanoparticles submitted to alternating magnetic field of 300 Gauss and frequency of oscillation of 305kHz. The inset shows the distribution of values of rate of specific absorption of magnetic nanoparticles.

3D cell culture of C6 cells on-a-chip

Figure 4. Microscopy images of clear field of 3D cell culture of C6 cells on-a-chip. Images of cells adhere to central cavity of chip after (A) 4 hours and (B) 48 hours of culture (4X); (C) C6 cell colony and their morphology (20X) and (D) Details of cell proliferation (4X).

Assessment of efficiency of magnetic hyperthermia therapy in glioblastoma on-a-chip

DISCUSSION

CONCLUSION

ACKNOWLEDGEMENTS

REFERENCES

Terapia de magneto-hipertermia no modelo de tumor de glioblastoma on-a-Chip

Javier Bustamante Mamani

Bruna Souto Marinho

Gabriel Nery de Albuquerque Rego

Mariana Penteado Nucci

Fernando Alvieri

Ricardo Silva dos Santos

João Victor Matias Ferreira

Fernando Anselmo de Oliveira

Lionel Fernel Gamarra

RESUMO

Objetivo:

Métodos:

Resultados:

Conclusão:

INTRODUÇÃO

OBJETIVO

MÉTODOS

Nanopartículas magnéticas

Descrição do equipamento de magneto-hipertermia

Determinação da taxa de absorção específica das nanopartículas magnéticas de óxido de ferro

Pré-cultura celular da linhagem C6

Cultura celular 3D das células C6 on-a-chip

Ensaio do processo de magneto-hipertermia no glioblastoma on-a-chip

Avaliação da eficiência do processo de magneto-hipertermia no glioblastoma on-a-chip

RESULTADOS

Avaliação da taxa de absorção específica das nanopartículas magnéticas de óxido de ferro

Figura 3. Curva de aquecimento das nanopartículas magnéticas submetidas a um campo magnético alternado de 300 Gauss e frequência de oscilação de 305kHz. O inset mostra a distribuição dos valores da taxa de absorção específica das nanopartículas magnéticas.

Cultura celular 3D das células C6 on-a-chip

Figura 4. Imagens de microscopia de campo claro da cultura celular 3D de células C6 on-a-chip. Imagem de células aderidas na cavidade central do chip após (A) 4 horas e (B) 48 horas de cultivo (4X); (C) Colônia de células C6 e sua morfologia (20X) e (D) Detalhes da proliferação celular (4X).

Avaliação da eficiência da terapia da magneto-hipertermia no tumor de glioblastoma on-a-chip

DISCUSSÃO

CONCLUSÃO

AGRADECIMENTOS

ACTIONS

PERMALINK

RESOURCES

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