Targeted Magnetic Hyperthermia Treatment of Cancer Cells

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Article Cite This: ACS Omega 2019, 4, 1931−1940

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Effectiveness and Safety of a Nontargeted Boost for a CXCR4Targeted Magnetic Hyperthermia Treatment of Cancer Cells Van̂ ia Vilas-Boas,*,†,‡,∥ Begoña Espiña,‡ Yury V. Kolen’ko,‡ Manuel Bañobre-Loṕ ez,‡ Marina Brito,‡ Veroń ica Martins,‡,⊥ Jose ́ Alberto Duarte,§ Dmitri Y. Petrovykh,*,‡ Paulo Freitas,‡ and Feĺ ix Carvalho*,† †

ACS Omega 2019.4:1931-1940. Downloaded from pubs.acs.org by 188.68.0.148 on 01/27/19. For personal use only.

UCIBIO-REQUIMTE, Laboratory of Toxicology, Biological Sciences Department, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal ‡ International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal § CIAFEL, Faculty of Sports, University of Porto, Rua Dr. Plácido da Costa 91, 4200-450 Porto, Portugal S Supporting Information *

ABSTRACT: We investigate the effectiveness and safety of a novel magnetic hyperthermia (MHT) protocol, whereby a pretreatment with nontargeted magnetic nanoparticles (MNPs) is used to boost the subsequent iron loading of cancer cells by the targeted immuno-modified MNPs. As a model example, LN229 cancer cells express specific cellsurface receptors (CXCR4) at levels sufficient for diagnostic identification but insufficient for achieving 100% effective monotherapeutic MHT based on CXCR4-targeted MNPs. The nontargeted boost of the iron content overcomes this limitation of the targeted loading and is positively correlated with the maximum temperature reached during MHT treatment of LN229 cells. The effectiveness of the dualpopulation MHT strategy is validated by achieving a 100% lethal outcome for LN229 cancer cells 72 h after the treatment, while its safety is confirmed by the minimal cytotoxicity observed in control experiments with normal HK-2 cells or with an isotypecontrol targeting antibody. Systematic in vitro measurements thus demonstrate that the magnetic loading by targeted MNPs can be significantly increased by the nontargeted boost, even to double the iron concentration, while improving the effectiveness and maintaining the safety of MHT. This validation of the dual-population MHT strategy opens a novel materials-based pathway, unassisted by highly and nonselectively cytotoxic chemotherapeutic agents, to overcome the limited effectiveness of MHT for treating cancer cells that express only moderate levels of cell-surface receptors. functionalized nanoparticles3−7 can enhance the selectivity of MHT for cancer cells, however, typically at the expense of limiting the effectiveness of MHT because of the reduced magnetic loading. Even with highly efficient targeting, whether evaluated by the fraction or concentration of particles associated with individual targeted cells, it is practically difficult to associate with cancer cells an amount of MNPs that is sufficient to induce lethal cytotoxicity via monotherapeutic targeted MHT.3,5,8−11 Our novel strategy proposes to overcome this limitation of the targeted magnetic loading by a boost with a nontargeted MNP population.2 Here, we validate the effectiveness and safety of the dualpopulation MHT strategy. We use as an in vitro model the LN229 cancer cell line that expresses the chemokine cellsurface receptor CXCR412 at diagnostically significant but

1. INTRODUCTION An important promise of magnetic hyperthermia (MHT)1 was to improve the safety of anticancer treatment, by avoiding the side effects of cytotoxic chemotherapeutic agents, while maintaining its effectiveness. Practical implementations of MHT to date, however, often encounter challenges in following through simultaneously on both aspects of that original promise. These challenges provide the primary motivation for us to investigate the effectiveness and safety of our novel monotherapeutic MHT protocol based on two populations of magnetic nanoparticles (MNPs),2 denoted as targeted and nontargeted hereafter. The ultimately desirable MHT treatment should be both effective and safe (selective), producing under the same conditions a temperature-induced 100% lethal cytotoxicity in cancer cells and minimal cytotoxicity in normal cells. Recent MHT studies typically apply an alternating magnetic field (AMF) to a mixture of cells with MNPs, in order to produce a volumetric temperature increase.3−6 Targeting strategies with © 2019 American Chemical Society

Received: August 28, 2018 Accepted: January 11, 2019 Published: January 25, 2019 1931

DOI: 10.1021/acsomega.8b02199 ACS Omega 2019, 4, 1931−1940

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moderate levels, that is, the levels that potentially limit the effectiveness of a targeted MHT strategy,13 even if the targeting itself is highly efficient. The lower CXCR4 expression in LN229, compared to that in the Jurkat (JK) model,2,14,15 enabled us to quantify the contribution of the nontargeted MNPs to the heating produced by MHT, by varying their concentration. This systematic investigation of the effectiveness of the dual-population MHT strategy was complemented by confirming its safety in control experiments with normal HK-2 cells or with targeted MNPs functionalized by an isotype-control (IC) antibody.

2. RESULTS AND DISCUSSION This work describes an in vitro validation of a novel dualpopulation MHT strategy2 using a model LN229 cancer cell line. First, the expression levels of CXCR4 are quantified for the LN229 and JK model cancer cells as well as the HK-2 normal cells. Initial incubation with different concentrations of the nontargeted ca. 20 nm superparamagnetic iron oxide nanoparticles (SPIONs) is then tested as a strategy (Scheme 1) to supplement the iron loading produced by the subsequent

Figure 1. CXCR4 expression in LN229 cells. Western blot results for LN229 cells, using JK and HK-2 cells as positive and negative controls, respectively. Results are mean + SD. Differences were estimated using one-way ANOVA (Kruskal−Wallis) followed by Dunn’s multiple comparisons post-test. *p < 0.05 vs HK-2 cells.

Scheme 1. Safe and Effective CXCR4-Targeted MHT Boost with Nontargeted SPIONs

CXCR4 particles (concentration of the particles or cells) do not result in reaching 100% lethal cytotoxicity (Figure S1). Accordingly, in the following, we fix the initial concentration of the targeted MP-CXCR4 and focus on optimizing the parameters of nontargeted SPIONs as a means of increasing the effectiveness of the MHT treatment. Our standard protocol for optimization of the overall particle loading includes four sequential steps: (1) an incubation of adherent cells with a chosen initial concentration of nontargeted SPIONs; (2) removal of the excess free SPIONs; (3) an incubation of adherent cells with 0.264 gFe·L−1 of MP-CXCR4; (4) removal of the excess free MP-CXCR4. This protocol was used to test four initial concentrations of SPIONs (0.176−0.260 gFe·L−1). After the four-step protocol of incubation and rinsing, cells were detached and submitted to a biphasic AMF sequence, which has been previously tested and optimized to kill JK cells.2 The heating profiles, which consist of an initial rise to the maximum temperature (Tmax in Figure 2) followed by an exposure to a constant temperature (Tc in Figure 2) sufficient to induce cytotoxicity,16,17 have been recorded for ca. 6 × 106 cells per sample and different initial concentrations of SPIONs (as indicated in the legend for Figure 2) and fixed initial concentration of antibody-functionalized MPs (0.264 gFe·L−1), including the results for controls with normal HK-2 cells and for targeted MPs functionalized with an IC antibody (MP-IC). During the biphasic AMF exposure in Figure 2, the highest temperatures (in terms of either Tmax or Tc) are clearly reached for the LN229 cells incubated sequentially with SPIONs and MP-CXCR4. Replacing the anti-CXCR4 targeting of MPCXCR4 with an IC antibody (MP-IC) produces consistently lower temperatures, indicating the specificity of the targeted contribution in our strategy. Noncancerous HK-2 cells incubated sequentially with SPIONs and MP-CXCR4 never reached temperatures above the 41 °C threshold, which is considered safe for normal cells.18 The average maximum temperature (Tmax) reached after the first 30 min for each condition provides a convenient index for a quantitative comparison among the experiments (Table 1). Analyzing the slope of the dependence of Tmax on the initial SPION concentration indicates three clearly different regimes

incubation with the constant concentration of the anti-CXCR4 targeted ca. 250 nm magnetic particles (MPs) (MP-CXCR4). The resulting iron loading and the associated interactions between MNPs and cells are then investigated by microscopy and inductively-coupled-plasma−optical emission spectrometry (ICP−OES), followed by the systematic evaluation of the MHT-induced cytotoxicity and the overall safety and effectiveness of the MHT treatment (Scheme 1). 2.1. Assessing CXCR4 Expression Levels. The expression of the CXCR4 receptor in LN229 was assessed by western blot, using JK cancer cells as a positive control and HK-2 normal cells as a negative control. As expected, the expression of CXCR4 is clearly the highest in JK cells, with a moderate expression observed in LN229 cells and the lowest expression in the normal (noncancerous) HK-2 cells (Figure 1). 2.2. Magnetically-Induced Hyperthermia. The hypothetical advantage of using two populations of MNPs for MHT is the ability to overcome an apparent practical limit on improving the effectiveness of MHT via optimization of the single targeted MNP population,2 the limit that should be exacerbated by the moderate expression level of the CXCR4 targets in LN229 cancer cells (Figure 1). Indeed, we find that realistic variations of the basic parameters that could putatively increase the volumetric MHT effect of the targeted MP1932


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Figure 2. Heating profiles of LN229 and HK-2 cells when exposed to different concentrations of SPIONs (0.176−0.260 gFe·L−1) and to CXCR4or IC-functionalized MPs (0.264 gFe·L−1). The dotted line marks 41 °C, the maximum temperature reached by normal (HK-2) cells, to facilitate comparisons between the cell types or particle functionalizations. Tmax refers to the temperature reached at the end of the first heating step; Tc to the average temperature maintained thereafter. Results are mean + SD of at least two independent experiments per condition.

Table 1. Average Maximum Temperature (Tmax) Reached in MHT Experiments Using Increasing Initial Amounts of Irona initial [Fe] (gFe·L−1) treatment

0.176 + 0.264

0.195 + 0.264

0.217 + 0.264

0.260 + 0.264

slope

r2

LN229 + SPION + MP-CXCR4 LN229 + SPION + MP-IC HK-2 + SPION + MP-CXCR4

42.4 ± 0.5 41.9 ± 1.3 40.7 ± 0.5

42.3 ± 1.8 40.7 ± 1.8 40.9 ± 0.4

45.8 ± 0.6 43.4 ± 2.3 40.0 ± 0.6***

46.9 ± 0.8 43.4 ± 1.9* 41.2 ± 0.2***

10.28 4.32 0.72

0.84 0.49 0.09

Results are mean ± SD of at least two independent experiments. Differences were estimated using regular two-way ANOVA followed by Tukey’s multiple comparisons post-test. *p < 0.05 and ***p < 0.001 vs LN229 + SPION + MP-CXCR4 for the same initial iron amount. The slopes obtained from linear regression fitting are also presented along with the goodness of the fit, r2, which indicates the nearly linear variation between the reached Tmax and the initial iron amount for the LN229 + SPION + MP-CXCR4 samples. a

SPIONs for LN229 cells compared to HK-2 cells (88.27 vs 55.78 pgFe/cell, respectively), in agreement with the previously reported differential uptake of nanoparticles between cancer and normal cells.19−21 Although the generality of this differential uptake cannot be guaranteed, observing it in our in vitro model supports the specific choice of small SPIONs as the nontargeted particle population in our strategy. Incubation with SPIONs is clearly critical for producing the dramatic difference in heating profiles (Figure 2) between LN229 and HK-2 cells: this difference is minimal in the absence of the SPION contribution (Figure S2). Interaction of MP-CXCR4 and MP-IC particles with cells is difficult to visualize by TEM because the large size of the primary particles (ca. 250 nm) limits the possibilities for preparing ultrathin 100 nm sample sections for TEM. Instead, these larger MPs were visualized by optical microscopy using horseradish peroxidase (HRP)-labeled secondary antibody, which targets the primary antibody attached to MPs (Figure 4). The largest concentration of MP-CXCR4 is clearly detected interacting with LN229 cells (Figure 4a). These particles are visualized surrounding the LN229 cell membranes (detail in Figure 4a), almost outlining the shape of the individual cells. In a standard test of the specificity of the anti-CXCR4 targeting, we functionalized MPs with an IC antibody. In contrast to the MP-CXCR4 particles in Figure 4a, the MP-IC particles are retained around the LN229 cells to a much lower extent (Figure 4b), providing an overall estimate for the nonspecific loading due to a combination of nonspecific binding of antibody-functionalized MPs to LN229 cells and any loosely bound or unbound MPs incompletely removed by washing. The predominantly nonspecific nature of this retention is also consistent with the results of the control experiment with HK-

for the three treatments in Table 1: the strongest dependence (r2 = 0.84) was observed for the LN229 + SPION + MPCXCR4 samples, a much weaker dependence (r2 = 0.49) for the LN229 + SPION + MP-IC, and no dependence (r2 = 0.09) for the HK-2 + SPION + MP-CXCR4 samples. For the highest tested iron loading, the Tmax observed for LN229 + SPION + MP-CXCR4 significantly differed from the samples using MP-IC, instead of MP-CXCR4 (Table 1, p < 0.05). The mean Tmax obtained for LN229 cells with MPCXCR4 was higher than the ones obtained for HK-2 cells under the same conditions; nevertheless, the differences only reached statistical significance (p < 0.001) for the two highest tested initial iron amounts (Table 1). 2.3. Interactions between Particles and Cells. The presumptive effect of incubating the cells with nontargeted SPIONs is their uptake by the cells, which can be directly observed by transmission electron microscopy (TEM). While TEM reveals SPIONs internalized in endosomal compartments of both LN229 and HK-2 cells (Figure 3), quantification by ICP−OES showed an increased overall uptake of the

Figure 3. Uptake of SPIONs by LN229 and HK-2 cells. Transmission electron micrographs show that both LN229 (a) and HK-2 (b) cells uptake SPIONs to endosomal compartments (red arrows). Scale bars are 200 nm. 1933


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values are in line with a previous report of 150 pgFe/cell for MHT experiments with folic-acid-conjugated nanoparticles, after a 3 h incubation with HeLa cervical cancer cells.10 Oh et al. also reported similar values for MDA-MB-231 breast cancer cells after a longer, 48 h, incubation with nontargeted, chitosan-coated MPs.22 The presumed causative correlation between the iron content (Table 2) and the heating profiles (Figure 2 and Table 1) is further supported by observing that for HK-2 + SPION + MP-CXCR4 samples neither the iron content nor the heating temperatures show a strong dependence on the initial iron amount from either of the two particle populations. This weak dependence is consistent with the interpretation of the interactions between MNPs and HK-2 cells as predominantly nonspecific. In a quantitative test of the hypothetical correlation between the heating profiles and the iron content, for the LN229 + SPION + MP-CXCR4 samples, there is a positive and significant correlation (Spearman r = 0.74, p = 0.0128) between the Tmax and the iron content retained from both particle populations (Figure 5). The much weaker correlation

Figure 4. Interaction of functionalized MPs with LN229 and HK-2 cells. LN229 cells interact to a larger extent with CXCR4-targeted (a) than with IC-functionalized (b) particles. Furthermore, LN229 cells (a) interact more than do HK-2 cells (c) with CXCR4-targeted nanoparticles. Reconstituted and stained microtome sections (5 μm thickness); scale bars are 20 μm, magnified details of each image shown as insets. (d) Iron quantification by ICP−OES for each of the three conditions. Results are mean + SD of two independent experiments. ***p < 0.001 vs LN229+MP-CXCR4.

2 cells and MP-CXCR4 particles, which shows a similarly low concentration and random distribution of the particles (Figure 4c). The ICP−OES quantification (Figure 4d) of the iron levels in these samples is in agreement with the conclusions from the optical microscopy images in Figure 4. The specific interactions of MP-CXCR4 with LN229 cells resulted in at least doubling the retained amount of magnetic material, compared to the nonspecific retention of either MP-IC by LN229 or MP-CXCR4 by HK-2. Despite the detectable difference in the retained iron content, the differences between the heating profiles of LN229 and HK-2 cells targeted by MPCXCR4 were only minimal (Figure S2), highlighting the importance of the functional evaluation of MHT treatment protocols. Having examined the retention of SPIONs and MP-CXCR4 populations separately, we proceed to evaluate the total iron content per cell retained after the full protocol with both particle populations and four steps of sequential incubation or rinsing (Table 2). In agreement with the observed Tmax results (Table 1), LN229 + SPION + MP-CXCR4 incorporated significantly more iron per cell than did HK-2 samples (Table 2, p < 0.05). For the highest tested initial iron amount, LN229 + SPION + MP-CXCR4 samples accumulated nearly 110 pgFe/cell. These

Figure 5. Iron content per sample and its correlation with Tmax. The iron amount in each sample submitted to MHT was quantified by ICP-OES. Spearman r correlation coefficients and their p values are listed in the legend. A significant positive correlation was found between the iron content and the Tmax reached in LN229 + SPION + MP-CXCR4 samples.

observed for the two control sample groups indicates the importance and effectiveness of choosing the appropriately interacting particle populations for a given cancer cell type. The minimal correlation observed for the HK-2 samples also confirms that other optimized variants of our strategy are likely to remain safe for normal cells.

Table 2. Average Iron Content Retained per Cell (pgFe/cell) with Increasing Initial Iron Amountsa initial [Fe] (gFe·L−1) treatment

0.176 + 0.264

0.195 + 0.264

0.217 + 0.264

0.260 + 0.264

LN229 + SPION + MP-CXCR4 LN229 + SPION + MP-IC HK-2 + SPION + MP-CXCR4

90.0 ± 4.0 80.4 ± 35.8 44.5 ± 16.6*

61.7 ± 6.3 53.5 ± 0.7 53.3 ± 3.6

106.4 ± 16.5 97.7 ± 37.3 41.6 ± 13.3**

108.0 ± 28.7 77.7 ± 4.2 38.0 ± 10.2**

Results are mean ± SD of at least two independent experiments. Differences were estimated using regular two-way ANOVA followed by Tukey’s multiple comparisons post-test. *p < 0.05 and **p < 0.01 vs LN229 + SPION + MP-CXCR4 for the same initial iron concentration.

a

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Figure 6. Analysis of cell death/viability after MHT. (a) Flow cytometry with annexin-V and 7-AAD staining, 2 h after AMF application. Results for each treatment are expressed as the percentage of dying cells (positive for one or both stains). (b) Dot plot of the ★ sample from panel (a) is shown in comparison with untreated cells; + represents the mean for each quadrant. (c) LDH leakage evaluated 24 h after MHT for correlation with Tmax. Spearman r coefficients and their p values for (a) and (c) are listed in the legends. (d) LSCM images with AnV and 7-AAD labeling (color-coded green and red, respectively) for LN229 or HK-2 cells untreated or 20 h after MHT with SPIONs and MP-CXCR4. Scale bars are 20 μm.

100 as a positive control (92.3 ± 9.1% cell death for HK-2 cells and 99.3 ± 6.2% cell death for LN229 cells). In good agreement with the percentage of dying cells measured by cytometry (Figure 6a), cell death levels were equal to or greater than 25% for all the LN229 + SPION + MP-CXCR4 samples, and significantly and positively correlated with the observed Tmax (Figure 6c, Spearman r = 0.75, p = 0.0174). On the other hand, cell death was always kept below 20% for all the HK-2 + SPION + MP-CXCR4 samples (Figure 6c). The strongest indication of the effectiveness of the MHT treatment is provided by the cell viability measurements using a metabolic rate assay (Figure 7). Exposure to the AMF alone (Figure S3) or to particles alone (white and gray bars, Figure 7) did not produce significant toxicity in any of the cell lines, confirming that the cytotoxicity measured for LN229 + SPION + MP-CXCR4 samples after AMF application is MHTinduced. Compared to the similarly treated HK-2 samples, LN229 + SPION + MP-CXCR4 samples exposed to the highest initial concentration of SPIONs (corresponding to ca. 110 pgFe/cell loading, Table 2) have significantly fewer viable cells: less than 10% viable cells 24 h after MHT and practically no viable cells 72 h after MHT (purple bars, Figure 7). This highly effective MHT outcome at both 24 and 72 h end points for LN229 + SPION + MP-CXCR4 samples is nearly identical to that previously achieved for analogously treated JK cells,2 demonstrating that both moderate (LN229) and high (JK) “baseline” loading by the targeted MP-CXCR4 particles can be boosted by the nontargeted SPIONs to produce effective MHT. The nearly identical outcomes achieved in vitro for LN229 and JK cells also indicate that the two-population

2.4. Cytotoxic Outcome. The cytotoxic effects of MHT on LN229 and HK-2 cells were analyzed at different time points after AMF application for evidence of a correlation with the maximum temperature reached during the MHT treatment. Two hours after MHT, samples were incubated with phycoerythrin-labeled annexin-V (AnV-PE) and 7-aminoactinomycin D (7-AAD) and analyzed by flow cytometry. The number of cells showing positivity for one or both labels was counted and presented as “percentage of dying cells” (Figure 6a), which strongly correlated with the Tmax for the LN229 + SPION + MP-CXCR4 samples (Spearman r = 0.96, p = 0.0028). For most of these samples, more than 25% (dashed horizontal line, Figure 6a) of the cells stained positive for both labels, whereas for the control conditions (LN229 + SPION + MP-IC and HK-2 + SPION + MP-CXCR4), the percentage of dying cells remained below this level. A representative dot plot of the event distribution of a LN229 + SPION + MP-CXCR4 sample is provided in Figure 6b, in comparison with untreated control cells. In agreement with the flow cytometry results, laser scanning confocal microscopy (LSCM) images taken 20 h after MHT showed increased double positive, round and detached cells for LN229 + SPION + MP-CXCR4 compared to HK-2 + SPION + MP-CXCR4 samples (Figure 6d). The leakage of lactate dehydrogenase (LDH) to the extracellular medium is a sign of membrane disruption and, consequently, effective cell death. To complement the cytometry data in Figure 6a, LDH leakage levels were measured 24 h after MHT (Figure 6c), using 0.5% Triton X1935


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monotherapeutic context.4,24 As an example, Thomas and coworkers incubated cancer (CD44+) versus normal (CD44−) cells from mouse, during 1 h, with hyaluronic-acid-conjugated particles (to target CD44 antigen) to perform in vitro MHT.24 Even though the observed temperature was not specified, the study reported cell viability levels of ca. 30% for the cancer cells, without damaging normal cells. In another study, galactosamine-conjugated nanoparticles were used to perform targeted in vitro MHT (780 kHz, 19 kA·m−1, 20 min), reaching for liver cancer cells low viability levels comparable to the ones observed in our work;4 the application of this approach may be limited by the wide expression of asyaloglycoprotein receptors, to which galactosamine binds, in normal liver cells.25 Most of the reports on targeted MHT in the literature, however, find MHT alone to be insufficient to kill cancer cells; accordingly, MHT is mainly being used in combination with chemotherapy agents.3,10,23 For example, Mi and co-workers developed Herceptin-conjugated nanoparticles and tested their MHT potential in breast cancer cells. The MHT efficiency of these particles, when submitted to AMF, was low (a 40% reduction in cell viability), and a multimodal nanoparticle system, integrating nanoparticles and docetaxel in a copolymer matrix conjugated with Herceptin, was then proposed with very promising results:23 the synergistic effects of chemotherapy and targeted MHT lead to cell viability levels below 20%. Using AMF parameters very similar to the ones used in our study, Kruse et al. described the use of CREKA-functionalized nanoparticles for targeted MHT in a lung cancer cell line. Considerably high initial magnetite concentration (3 gFe3O4·L−1 ≈ 2.17 gFe·L−1) was necessary to reach temperatures from 41 to 45 °C during 20 min, which reduced cell viability to 50%. The MHT strategy was, in the end, improved by an additive effect of cisplatin.3 Another work using folate-conjugated nanoparticles for in vitro targeted MHT reported a 35% decrease in cell viability 24 h after the application of a 265 kHz and 27 kA·m−1 AMF during 10 min. This targeted MHT strategy was chosen to sensitize cancer cells for chemotherapy with doxorubicin, further decreasing cell viability levels to ca. 10%.10 Comparison to the above literature examples clearly demonstrates the practical benefits of our two-population strategy for MHT, as we have been able to achieve comparable or better effectiveness in monotherapeutic MHT context, in a single MHT session and without the use of highly and nonselectively cytotoxic chemotherapeutic agents.

Figure 7. Cell viability after MHT with the highest tested amount of iron. Cells in LN229 + SPION + MP-CXCR4 samples showed significantly lower cell viability than did the similarly treated HK-2 cells at both 24 and 72 h after MHT. In LN229 + SPION + MPCXCR4 samples, no viable cells were observed 72 h after MHT. The particles alone did not significantly affect LN229 and HK-2 cell viability (gray and white bars, respectively). Results are mean + SD. Differences were estimated using regular two-way ANOVA followed by Tukey’s multiple comparisons post-test. ****p < 0.0001 vs untreated cells (dashed line); **p < 0.01 and ***p < 0.001 between the identified samples, for the same end point.

MHT strategy can be effective against both adherent and suspension cell lines, respectively. 2.5. Overall Safety and Effectiveness of MHT Protocol. Our original proposal and demonstration of the two-population approach to MHT2 left open the question of the extent to which the nontargeted population of SPIONs can be used to compensate the insufficient effectiveness of MHT produced by a single targeted population. Using nontargeted SPIONs to provide a relatively high fraction of the total iron loading also raised a concern about possible cytotoxic side effects for the nonspecifically loaded normal cells. In this work, the LN229 model cancer cell line provides a platform for investigating both of the above concerns. Although suspension cell lines, such as JK, may be intrinsically well suited for experiments with volumetric hyperthermia,2 practical considerations, including a more direct comparison to the existing literature,3−6,10,23 make an adherent LN229 cell line appropriate for this investigation. The moderate expression of the CXCR4 cell-surface receptor (Figure 1) in LN229 cells intrinsically limits the iron loading that can be achieved by the targeted MP-CXCR4 population alone. Accordingly, the boost required from the nontargeted SPIONs to induce sufficiently high temperatures under MHT conditions (Figure 2) is comparable to the iron loading produced by the targeted MP-CXCR4 population (Tables 1 and 2). Successfully achieving 100% lethal cytotoxicity (Figure 7) thus confirms that our strategy is not limited to small fractional increases of the iron loading by the nontargeted SPIONs. At the same time, minimal cytotoxicity observed in all the control experiments (Figures 6 and 7) strongly indicates that the nonspecific loading of normal cells under our protocol does not result in undesirable side effects of the MHT treatment. In other words, the tests in this work indicate that generalized variants of our strategy are likely to be both selective and effective in terms of MHT outcome. The controls carried out in the present work (Figures 2, 4−7; S2−S4) also clearly demonstrate that each of the elements of our strategy (a targeted MP-CXCR4 population, a nontargeted SPION population, and AMF-induced MHT heating) is critically important for its overall success. 2.6. Comparative MHT Outcome. There are only a few reports in the literature on in vitro targeted MHT in a

3. CONCLUSIONS This work validates the effectiveness and safety of a novel dualpopulation MHT strategy, whereby a population of nontargeted SPIONs is used to boost the loading of cancer cells by targeted MNPs. The model LN229 cancer cell line that expresses the CXCR4 cell-surface receptor at diagnostically significant but moderate levels provides the means of testing the effectiveness of the dual-population MHT strategy under realistic in vitro conditions and of quantifying the contribution of the nontargeted SPIONs, at the level of both MHT heating profiles and MHT outcomes. The strong positive correlation between the SPION concentration and MHT outcomes for the LN229 cancer cells confirms that the nontargeted SPIONs can be used in a wide range of concentrations to boost the 1936


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quantified using Coomassie Plus (Bradford) Protein kit (product 23236, Thermo Fisher Scientific, Inc., Rockford, IL USA), and 25 μg of protein were diluted 1:1 in 2× Laemmli buffer with 5% β-mercaptoethanol and heated at 95 °C, for 5 min. Samples were kept at −20 °C until analysis. Samples were loaded in a 4−15% Mini-PROTEAN TGX Precast Protein Gels (catalog number 4561085) (Bio-Rad Laboratories, Hercules, CA, USA), and electrophoretically separated at a constant voltage of 75 V using a Tris-glycine running buffer [25 mM Tris base, 192 mM glycine, and 0.1% SDS (w/v), pH 8.3]. Proteins were then transferred to a nitrocellulose membrane using a transfer buffer [20% methanol (v/v) in 25 mM Tris base and 192 mM glycine, pH 8.3] at a constant voltage of 100 V, for 3 h. A Mini-PROTEAN Tetra cell system (Bio-Rad Laboratories, Hercules, CA, USA) was used for the electrophoresis and transfer procedures. Membranes were rinsed in Tris-buffered saline solution [TBS: 20 mM Tris base, 300 mM NaCl, pH 8.0], blocked for 2 h, at room temperature (RT), in blocking buffer [5% nonfat powdered skim milk (w/v) in TBS solution with 0.05% Tween 20 (v/v) (TBS-T)] and then incubated overnight, at 4 °C, with a mouse monoclonal anti-human-CXCR4 antibody (clone 44717, catalog number MAB173, R&D Systems, Abingdon, United Kingdom) or an anti-α-tubulin antibody (catalog number T6074, Sigma-Aldrich Inc.), in a 1:2000 dilution, in blocking buffer. After incubation with the primary antibody, membranes were washed three times (10 min each) with TBS-T and incubated with the secondary antibody [antimouse IgG-HRP polyclonal antibody (1:5000 in blocking buffer)], for 1 h, at RT. Following two more washes in TBS-T (10 min each), bands were revealed using ECL prime chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), according to the supplier’s instructions, and visualized using a G:BOX Chemi XT4 (Syngene, Cambridge, UK). Quantification of band intensity was performed using ImageJ analysis software (NIH, USA). Final results, collected from three independent experiments, represent the ratio between CXCR4 and αtubulin band intensities. 4.4. Incubation of Cells with MNPs. A previously developed protocol2 was used with some modifications. In the first incubation step with a nontargeted particle population, 3 × 106 cells per T25 culture flask were incubated for 150 min in cell culture medium at 37 °C with increasing concentrations of poly(acrylic acid)-coated SPIONs. The ca. 20 nm SPIONs synthesized in-house had been chosen as the nontargeted population because they have properties that are known to be beneficial for MHT applications.1,26 Specifically, the synthesis of these SPIONs is well established27 and results in nearly monodisperse27,28 MNPs with extensively characterized physicochemical27,29,30 and magnetic27,28 properties, efficient heating demonstrated under MHT conditions,2,27 and successful experience using them as unmodified MNPs in previous MNP−cell interaction studies.2,29 The total initial iron concentration of those SPIONs was 0.176, 0.195, 0.217, or 0.260 gFe·L−1. Excess of free SPIONs was then removed, and the cellular monolayer was washed with Hank’s balanced salt solution (HBSS) with calcium and magnesium (+/+). Concurrently, ca. 250 nm composite dextran iron oxide particles (catalog number 09-20-252, NanoMag-D, micromod Partikeltechnologie GmbH, Rostock, Germany), with proteinA at the surface, were functionalized (incubation for 30 min, at RT, with agitation) with anti-CXCR4 antibody or an IC

effectiveness of MHT and to overcome the limitations imposed by the moderate expression of the targeted receptor. The concomitant observations of minimal cytotoxicity in all the control experiments, in turn, confirm the safety and selectivity of our MHT strategy. On the basis of this in vitro validation, we expect that the translational potential of our strategy can be further explored in the future to provide safe and effective MHT treatments for different types of cancer cells that express diagnostically significant levels of cell-surface biomarkers.

4. EXPERIMENTAL SECTION 4.1. Reagents. All reagents used in this study were of analytical grade or of the highest grade available. Fetal bovine serum (FBS) was purchased from HyClone UK, Ltd., Northumberland, England, United Kingdom. Penicillin (10 000 U·mL−1) and streptomycin (10 000 μg·mL−1), herein referred to as Pen−Strep; Dulbecco’s modified Eagle’s medium (DMEM) high glucose (product D5648); sodium pyruvate; nicotinamide adenine dinucleotide reduced form; Triton X100; 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); ethylenediaminetetraacetic acid (EDTA); sodium dodecyl sulfate (SDS); KH2PO4; K2HPO4·3H2O; CaCl2; NaCl; glycerol; paraformaldehyde; and bovine serum albumin (BSA) were purchased from Sigma Inc. (St. Louis, MO, USA). DMEM with nutrient mixture F-12 (DMEM/F-12) and GlutaMAX (catalog number 31331), trypsin 0.25%-EDTA, fungizone (250 μg·mL−1), and human transferrin (4 mg·mL−1) were purchased from Gibco Laboratories (Lenexa, KS, USA). Anti-mouse IgG-HRP polyclonal antibody (catalog number NA931) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). BD Pharmingen AnVPE and 7-AAD were purchased from BD Biosciences, USA. Anti-human CXCR4, clone 12G5, low-endotoxin, and azidefree monoclonal antibody produced in mouse (catalog number 306512), and its matched IC, mouse anti-human monoclonal IgG2a (catalog number 402202), were purchased from BioLegend Inc. (San Diego, CA, USA). 4.2. Cell Culture. Human cell lines from glioblastoma (LN229, ATCC CRL-2611) and normal kidney (HK-2, ATCC CRL-2190) were purchased from the American Type Culture Collection (Manassas, VA, USA). LN229 cells were grown in DMEM (high glucose) medium supplemented with 10% FBS and 1% Pen−Strep, and HK-2 cells were grown in DMEM/F12 GlutaMAX, supplemented with 10% FBS, 1% Pen−Strep, 2.5 μg·mL−1 fungizone, and 5 μg·mL−1 human transferrin. JK cells (ATCC TIB-152) were kept in Roswell Park Memorial Institute (RPMI-1640, product R6504 Sigma-Aldrich Inc.) cell culture medium supplemented with 10% FBS and 1% Pen− Strep. All the cell lines were maintained at 37 °C, in a controlled atmosphere of 5% CO2. 4.3. CXCR4 Expression in LN229 and HK-2 Cells Assessed by Western Blot. Approximately 1 × 106 cells per well were seeded in six-well plates and grown for 24 h before being scraped (2 wells per condition) in 1 mL ice-cold PBS, collected to microcentrifuge tubes, and centrifuged at 850g, for 5 min, at 4 °C. Cells were then lysed using ice-cold radioimmunoprecipitation assay buffer (50 mM Tris−HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA) with 1% (v/v) protease (complete, Mini, EDTA-free protease inhibitor cocktail, Roche) and phosphatase (1 mM Na3VO4, 1 mM NaF) inhibitors for 30 min at 4 °C, and centrifuged at 16 000g, 10 min, at 4 °C. Protein content in the supernatant was 1937


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Mayer’s haematoxylin. Optical images were collected using an inverted wide-field microscope Eclipse Ti-E, Nikon. 4.7. ICP−OES for Iron Quantification. To measure the amount of iron in each sample, after MHT, an aliquot of each sample was digested in 1 mL HCl 37%, at RT, for 72 h, and then diluted to a final volume of 50 mL with ultrapure water (Milli-Q, Merck Millipore). Samples were then analyzed by ICP−OES (three repeated measurements per sample; in all cases, the coefficient of variation was 0.6 and p < 0.05. All statistical analysis was carried out using GraphPad Prism software v6 (San Diego, CA); p values under 0.05 were considered statistically significant.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02199.

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Parameters affecting the heating profile and MHT outcome for LN229 cells; the heating profile of LN229 and HK-2 cells with MP-CXCR4; the effect of the AMF on cells; the outcome differences between cancer (LN229) and normal (HK-2) cells; sample preparation for TEM; annexin-V and 7-AAD labeling for flow cytometry; LDH leakage assay (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (V.V.-B.). *E-mail: [email protected] (D.Y.P.). *E-mail: felixdc@ff.up.pt (F.C.). ORCID

Vânia Vilas-Boas: 0000-0002-4798-2158 Begoña Espiña: 0000-0002-7645-2834 Yury V. Kolen’ko: 0000-0001-7493-1762 Manuel Bañobre-López: 0000-0003-4319-2631 Marina Brito: 0000-0002-8973-104X Dmitri Y. Petrovykh: 0000-0001-9089-4076 Present Addresses ∥

In Vitro Toxicology and Dermato-Cosmetology Department, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. ⊥ INESC−Microsistemas e Nanotecnologias, Rua Alves Redol 9, Lisbon 1000-029, Portugal. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V.V.-B. acknowledges FCT (Portugal) for the PhD fellowship (grant SFRH/BD/82556/2011). This work received financial support from project no. NORTE-01-0145-FEDER-000024, supported by the Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). B.E. acknowledges the project Nanotechnology Based Functional Solutions (NORTE-010145-FEDER-000019), supported by Norte Portugal Regional Operational Programme (NORTE2020) under the PORTUGAL 2020 Partnership Agreement, through the ERDF.

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REFERENCES

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Targeted Magnetic Hyperthermia Treatment of Cancer Cells

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