Magnetic Delivery of Paclitaxel by Magnetic Anionic Liposome/Atelocollagen Complexes for Targeted Cancer Therapy

Abstract

Magnetic drug carriers are a valuable tool for site-specific drug delivery, utilizing both passive targeting via the enhanced permeability and retention effect and active targeting through magnetic forces. We previously developed magnetic anionic liposome (Mag-AL)/atelocollagen (ATCOL) complexes and demonstrated their efficient accumulation at the targeted tissue through magnetic attraction and electrostatic interactions. To assess the usefulness of Mag-AL/ATCOL complexes as tumor-targeted drug delivery carriers, we herein prepared paclitaxel (PTX)-loaded Mag-AL/ATCOL complexes and examined their cellular association and cytotoxicity in C26 murine colon adenocarcinoma cells. The biodistribution of the complexes and their antitumor efficacy were also investigated in C26-bearing mice. PTX-Mag-AL/ATCOL complexes exhibited significant binding to C26 cells under an external magnetic field and released PTX in a sustained manner. Consequently, cytotoxic effects against C26 cells were achieved by PTX-Mag-AL/ATCOL complexes with the application of a magnetic field. Moreover, PTX-Mag-AL/ATCOL complexes preferentially distributed in the spleen and liver after their intravenous administration into C26 tumor-bearing mice, while tumor accumulation showed an approximately 2.9-fold augmented by the application of an external magnetic field to the tumor. Due to this magnetic guidance, PTX-Mag-AL/ATCOL complexes significantly inhibited C26 tumor growth. These results indicate that Mag-AL/ATCOL complexes have the potential to improve the therapeutic efficacy of anticancer drugs as magnetic drug carriers.

INTRODUCTION

In cancer chemotherapy, drug delivery technologies that enhance the efficacy of anticancer drugs while reducing adverse effects are highly desired to develop. The most widely used drug delivery system for chemotherapy is nanoparticulate drug carriers, such as liposomes, polymeric micelles, and lipid nanoparticles.^1–3) These drug carriers are able to extend the systemic circulation of the loaded anticancer drugs. Moreover, drug carriers of approximately 100 nm efficiently extravasate from tumor blood vessels and accumulate in tumor tissues via the enhanced permeability and retention (EPR) effect.^1–3) In addition to passive targeting, active targeting techniques may further improve the tumor deposition of nanoparticulate drug carriers. The most extensively studied method for imparting active targeting ability to nanoparticles is the conjugation of specific ligands, such as peptides, aptamers, and antibodies, on the surface of nanoparticles.^4,5) Ligand conjugation facilitates the effective accumulation of nanoparticles in tumor tissues. However, the infiltration of these nanoparticles from blood vessels into tumor tissues relies on the passive EPR effect and, thus, the tumor-infiltrating efficiency of nanoparticles cannot be improved by ligand conjugation.

On the contrary, magnetic drug targeting is a valuable approach for improving the efficacy of not only the tumor accumulation of nanoparticles, but also tumor infiltration.^6,7) The magnetic drug delivery system consists of two steps: 1) the intravenous injection of a magnetic nanoparticle-drug conjugate or magnetic nanoparticles and drug co-incorporated nanoparticulate drug carriers and 2) the application of an external magnetic field to the target site. Magnetic force enables the active guidance of magnetic nanoparticles from the bloodstream toward the targeted tissue. Among various magnetic drug carriers, magnetic nanoparticle-incorporated liposomes (magnetic liposomes) are versatile drug carriers because liposomes have the capacity to load drugs with various physicochemical properties, such as hydrophilic and hydrophobic low-molecular-weight compounds, proteins, and nucleic acids.^8–10) Magnetic cationic liposomes may strongly bind to the target site via electrostatic interactions,^11,12) thereby facilitating more efficient active drug targeting. However, conventional magnetic cationic liposomes exhibit potent cytotoxicity attributed to synthetic cationic lipids, which may lead to adverse effects.¹³⁾

We previously developed magnetic anionic liposome (Mag-AL)/atelocollagen (ATCOL) complexes as a safe and efficient magnetic drug carrier.¹⁴⁾ Mag-AL/ATCOL complexes are composed of nontoxic biocompatible components, anionic phospholipids, cholesterol, ATCOL, and 10-nm superparamagnetic iron oxide nanoparticles. In these complexes, Mag-AL is coated by cationic ATCOL, allowing for efficient binding to the anionic cell surface under an external magnetic field. A previous study demonstrated that intravenously injected Mag-AL/ATCOL complexes accumulated in a specific region of the liver where an external magnetic field was applied.¹⁴⁾ However, the tumor targeting potential of these complexes has yet to be examined.

In the present study, paclitaxel was loaded into Mag-AL (PTX-Mag-AL), and the cellular association and cytotoxicity of PTX-Mag-AL/ATCOL complexes against cancer cells under an external magnetic field were investigated. Moreover, the biodistribution of the complexes and their antitumor efficacy were evaluated in tumor-bearing mice.

MATERIALS AND METHODS

Cell Culture

C26 murine colon adenocarcinoma cells were gifted from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). C26 cells were maintained at 37°C under a humidified 5% CO₂ atmosphere in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), penicillin G (100 U/mL), and streptomycin (100 μg/mL).

Animals

Six-week-old male BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Animal experiments were performed according to the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals, as approved and promulgated by the U.S. National Institutes of Health (Bethesda, MD, U.S.A.). The study protocol was approved by the Kobe Pharmaceutical University Committee for Animal Care and Use (Approval number: 2024-007). All efforts were made to minimize suffering.

Construction of PTX-Mag-AL/ATCOL Complexes

PTX-Mag-AL composed of 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (NOF Inc., Tokyo, Japan) and cholesterol (Nacalai Tesque, Kyoto, Japan) at a molar ratio of 1 : 1 and 0.1 mg/mL of iron oxide (II, III) (Fe₃O₄) magnetic nanoparticles (Sigma-Aldrich, St. Louis, MO, U.S.A.) was prepared according to our previous study.¹⁴⁾ PTX was added to the lipid mixture at a lipid-to-drug weight ratio of 10 : 1 before evaporation. PTX-Mag-AL was fluorescent labeled with 5 mol% of DiIC18(3) (Wako Pure Chemical Corporation, Osaka, Japan). The unloaded PTX was removed from PTX-Mag-AL by gel filtration using a PD-10 desalting column (GE Healthcare, Buckinghamshire, U.K.). To construct PTX-Mag-AL/ATCOL complexes, PTX-Mag-AL were gently mixed with an equal volume of 20 μg/mL of ATCOL (Koken Co., Ltd. Tokyo, Japan), followed by an incubation at 4°C for 20 min. The particle sizes and ζ-potentials of the complexes were measured using Zetasizer Pro (Malvern Instrument, Worcestershire, U.K.). The encapsulated PTX ratio in Mag-AL was measured by HPLC.

Cellular Association of PTX-Mag-AL/ATCOL Complexes in C26 Cells

C26 cells were seeded on 24-well culture plates at a density of 2 × 10⁴ cells/cm² and cultured for 72 h. PTX solution or PTX-Mag-AL/ATCOL complexes were added to the well at a PTX amount of 5 μg in the presence or absence of 50% FBS, and then incubated at 37°C for 30 min on a magnetic plate (330 mT) (OZ Biosciences, San Diego, CA, U.S.A.). Cells were then washed with phosphate-buffered saline (PBS) and lysed in lysis buffer (0.5% Triton X-100, 2 mM ethylenediaminetetraacetic acid, and 0.1 M Tris, pH 7.8). The obtained lysates were centrifuged at 10000 × g at 4°C for 10 min, and the amount of PTX in the supernatant was measured by HPLC.

PTX Release from PTX-Mag-AL/ATCOL Complexes

PTX-Mag-AL or PTX-Mag-AL/ATCOL complexes were mixed with an equal volume of FBS and loaded into a dialysis bag (Spectra/Por4 Membrane, MWCO: 12000–14000, Repligen, Waltham, MA, U.S.A.). The dialysis bag was placed into 3% bovine serum albumin in PBS and incubated at 37°C for 48 h under shaking conditions at 100 rpm. The concentration of released PTX was determined by HPLC.

Cytotoxicity of PTX-Mag-AL/ATCOL Complexes against C26 Cells

PTX solution or PTX-Mag-AL/ATCOL complexes at a PTX concentration of 0.05–5 μg/mL were added to C26 cells seeded in 48-well culture plates and incubated at 37°C for 30 min on a magnetic plate. Cells were washed twice with PBS and further incubated at 37°C for 24 h. Cell viability was then measured using Cell Counting Reagent SF (Nacalai Tesque) and a Synergy HTX multimode microplate reader (Agilent Technologies Japan, Ltd., Tokyo, Japan).

In Vivo Tissue Distribution of PTX-Mag-AL/ATCOL Complexes

PTX-Mag-AL was radio-labeled with Indium-111 (¹¹¹In) using the remote loading method according to our previous study.¹⁵⁾ Tumor-bearing mice were prepared by subcutaneous injection of C26 cells (1 × 10⁶ cells/100 μL) into the back of BALB/c mice. When the tumor volume reached 500 mm³, 200 μg of ¹¹¹In-labeled PTX-Mag-AL or PTX-Mag-AL/ATCOL complexes was intravenously administered to tumor-bearing mice. Immediately after administration, the tumor tissue was located on a neodymium-iron-boron permanent magnet disk (24 × 20 mm, 500 mT, Magna Co., Ltd., Tokyo, Japan) for 1 h. Mice were then sacrificed, and the heart, lung, kidney, spleen, liver, and tumor were collected. The amount of PTX-Mag-AL in each organ was quantified by measuring radioactivity using a PerkinElmer 2480 WIZARD2 automatic gamma counter (PerkinElmer Japan Co., Ltd., Kanagawa, Japan).

Antitumor Efficacy of PTX-Mag-AL/ATCOL Complexes in C26 Tumor-Bearing Mice

When the tumor volume of C26 tumor-bearing mice reached 100 mm³, saline, PTX solution, Mag-AL/ATCOL complexes, or PTX-Mag-AL/ATCOL complexes were intravenously injected into mice at a dose of 2 mg PTX/kg four times (days 0, 2, 4, and 6). Immediately after administration, the tumor tissue was located on a permanent magnet for 1 h. Tumor volume and body weight were recorded every other day.

Analytical Method

Acetonitrile was mixed with each sample at a 10 : 1 volume ratio. The mixture was centrifuged at 10000 × g at 4°C for 10 min, and the supernatant was collected and evaporated. The residue was re-dissolved in the HPLC mobile phase (0.1% phosphoric acid : acetonitrile = 1 : 1). The mobile phase was delivered at 1.1 mL/min. The concentration of PTX was measured using the HPLC system composed of an LC-20AD pump, SIL-20A autosampler, and SPD-20A UV/vis detector (Shimadzu, Kyoto, Japan) set at 227 nm, and the system was equipped with a CMB-20A system controller and ODS column (5C₁₈, 150 × 4.6 mm i.d., Nacalai Tesque).

Statistical Analysis

Results are presented as the mean ± or + standard deviation (S.D.) of three or four experiments. An ANOVA was used to test the significance of differences between groups. Two-group comparisons were performed using the Student’s t-test. Multiple comparisons between all groups were performed using the Tukey–Kramer test.

RESULTS AND DISCUSSION

Cellular Association and PTX Release Property of PTX-Mag-AL/ATCOL Complexes

The cellular association of PTX-Mag-AL/ATCOL complexes in C26 cells was evaluated. The particle size and ζ-potential of PTX-Mag-AL were 113.3 ± 8.7 nm and −48.2 ± 1.9 mV, respectively. The PTX encapsulation efficiency of Mag-AL was 50.6 ± 7.9%. When PTX-Mag-AL formed complexes with ATCOL, the particle size was not significantly affected (117.1 ± 5.3 nm), whereas the ζ-potential increased (−22.9 ± 3.2 mV). As shown in Fig. 1A, the amount of PTX in C26 cells incubated with PTX-Mag-AL/ATCOL complexes was approximately 16.7 times higher than that in cells treated with PTX solution in the presence of a magnetic field. Regarding the effects of FBS, the cellular-associated amount of PTX by PTX-Mag-AL/ATCOL complexes significantly decreased in the presence of FBS under conditions without magnetic field application. Fleischer et al.¹⁶⁾ previously reported that the cellular binding of anionic Au nanoparticles was significantly inhibited by the adsorption of serum proteins onto the surface of Au nanoparticles. The present result is in accordance with this finding. On the contrary, PTX-Mag-AL/ATCOL complexes efficiently bound to C26 cells following the application of a magnetic field even in the presence of FBS. This result indicates that magnetic force overcomes the inhibitory effects of serum proteins on the cellular association of PTX-Mag-AL/ATCOL complexes.

Fig. 1. Cellular Association and PTX Release Property of PTX-Mag-AL/ATCOL Complexes

(A) C26 cells were incubated with PTX solution or PTX-Mag-AL/ATCOL complexes at a concentration of 5 μg PTX/mL at 37°C for 30 min in the presence or absence of a magnetic field. Each value represents the mean + S.D. (n = 4). ^**p < 0.01, significantly different from the corresponding group without the magnet. ^††p < 0.01, significantly different from the corresponding group without FBS. (B) PTX-Mag-AL or PTX-Mag-AL/ATCOL complexes were mixed with an equal volume of FBS in a dialysis bag, and were then incubated at 37°C for 48 h under shaking conditions. The released PTX amount was measured by HPLC. Each value represents the mean ± S.D. (n = 4). PTX-Mag-AL/ATCOL: paclitaxel magnetic anionic liposome/atelocollagen.

The PTX release property of PTX-Mag-AL/ATCOL complexes was also assessed. Sustained PTX release from Mag-AL was observed in the presence of FBS, and the formation of complexes with ATCOL did not affect the PTX release property of Mag-AL (Fig. 1B). This PTX release profile of PTX-Mag-AL is similar to that of PTX-loaded polyethylene glycol (PEG)-modified liposomes.^17,18) Therefore, Mag-AL is capable of stably encapsulating PTX under conditions containing serum, while Mag-AL is more likely to interact with serum proteins as described above.

In Vitro Cytotoxic Effects of PTX-Mag-AL/ATCOL Complexes against C26 Cells

We investigated the cytotoxicity of PTX-Mag-AL/ATCOL complexes against C26 cells. A dose-dependent growth inhibitory effect against C26 cells was observed with PTX solution (IC₅₀: 0.53 μg PTX/mL), and more potent cytotoxicity was obtained with PTX-Mag-AL/ATCOL complexes (IC₅₀: 0.13 μg PTX/mL) (Fig. 2). Moreover, the cytotoxicity of the complexes was augmented under magnetic field application (IC₅₀: 0.05 μg PTX/mL). This IC₅₀ value was significantly lower than that of PTX-loaded PEG-modified liposomes (IC₅₀: 1.8 μM = 1.54 μg PTX/mL),¹⁹⁾ strongly suggesting that PTX-Mag-AL/ATCOL complexes efficiently delivered PTX into C26 cells with the application of a magnetic field. We previously demonstrated that the majority of Mag-AL/ATCOL complexes remained on the cell surface and were not internalized into cells after cellular binding.^15,20) Based on this finding, we consider PTX-Mag-AL/ATCOL complexes to continuously release PTX on the cell surface, leading to prolonged exposure to PTX and the enhanced growth inhibition of C26 cells.

Fig. 2. In Vitro Cytotoxicity of PTX-Mag-AL/ATCOL Complexes against C26 Cells

C26 cells were incubated with PTX solution, Mag-AL/ATCOL complexes, or PTX-Mag-AL/ATCOL complexes at 37°C for 30 min in the presence or absence of a magnetic field. Cells were then washed and incubated for 24 h. Cell viability was measured using the WST-8 assay. PTX-Mag-AL/ATCOL: paclitaxel magnetic anionic liposome/atelocollagen.

Figure 2 also shows that high concentrations of empty (PTX-free) Mag-AL/ATCOL complexes resulted in the significant loss of cell viability under magnetic field application. Since Mag-AL/ATCOL complexes were hardly internalized into cells,^15,20) the cytotoxicity induced by the cellular internalization of the complexes is considered to be negligible. Therefore, the reduction in C26 cell viability caused by empty Mag-AL/ATCOL complexes may be attributable to the physical impact of their magnetically guided collision with C26 cells. However, empty Mag-AL/ATCOL complexes exhibited no cytotoxicity without magnetic field application. Based on these findings, we assume that Mag-AL/ATCOL complexes themselves do not damage normal tissues where a magnetic field is not applied.

In Vivo Tissue Distribution of PTX-Mag-AL/ATCOL Complexes in C26 Tumor-Bearing Mice

The in vivo distribution of PTX-Mag-AL/ATCOL complexes in normal and tumor tissues was evaluated after their intravenous injection into C26 tumor-bearing mice. Both PTX-Mag-AL and PTX-Mag-AL/ATCOL complexes mainly distributed in the spleen and liver. On the contrary, the lung accumulation of PTX-Mag-AL was increased by the formation of the complexes with ATCOL (Fig. 3). This may be attributed to the positive charge of ATCOL because previous studies demonstrated that cationic nanoparticles preferentially accumulated in the lung.^21,22)

Fig. 3. In Vivo Tissue Distribution of PTX-Mag-AL/ATCOL Complexes in C26 Tumor-Bearing Mice

¹¹¹In-labeled PTX-Mag-AL or ¹¹¹In-labeled PTX-Mag-AL/ATCOL complexes were intravenously injected into C26 tumor-bearing mice. An external magnetic field was applied to the tumor tissue for 1 h, and the accumulated amounts of PTX-Mag-AL in the heart, lung, kidney, spleen, liver, and tumor were then measured. Each value represents the mean + S.D. (n = 5). ^**p < 0.01, significantly different from the corresponding group without the magnet. ^†p < 0.05; ^††p < 0.01, significantly different from the corresponding group without complex formation with ATCOL. PTX-Mag-AL/ATCOL: paclitaxel magnetic anionic liposome/atelocollagen.

In previous studies on the tumor-targeted delivery of magnetic liposomes, the duration of external magnetic field application to the tumor was set to within 1 h.^23,24) Based on these reports, we preliminarily examined the effect of the duration of magnetic field exposure on the tumor accumulation of PTX-Mag-AL/ATCOL complexes. Supplementary Figure S1 showed that tumor accumulation of the complexes was higher after 1 h of magnetic field exposure than after 30 min. However, no significant difference was observed between 1 and 1.5 h of exposure. This would be because PTX-Mag-AL/ATCOL complexes exhibited rapid clearance from the blood, with a plasma concentration of approximately 1.4% of dose/mL at 1 h after intravenous administration. Therefore, in the present study, a magnetic field was applied to the tumor for 1 h immediately after the intravenous injection of PTX-Mag-AL/ATCOL complexes. When an external magnetic field was applied to the tumor tissue for 1 h, the accumulated amount of PTX-Mag-AL/ATCOL complexes in the tumor showed an approximately 2.9-fold increase (Fig. 3). On the contrary, the accumulation of the complexes in normal tissues (heart, lung, kidney, spleen, and liver) did not significantly differ with or without the application of a magnetic field. The increase in tumor accumulation of PTX-Mag-AL/ATCOL complexes under magnetic field application is greater than that of magnetic cationic liposomes composed of cationic lipid previously reported (approximately a 1.5-fold increase).¹²⁾ This may be due to the differences in their surface charges. Previous magnetic cationic liposomes exhibited a much higher ζ-potential (approximately 28 mV) than PTX-Mag-AL/ATCOL complexes. Therefore, those liposomes were likely to interact nonspecifically with vascular endothelial cells, whereas PTX-Mag-AL/ATCOL complexes were presumed to selectively bind to cells within tumor tissues with the help of magnetic field application.

In Vivo Antitumor Efficacy of PTX-Mag-AL/ATCOL Complexes

The antitumor efficacy of PTX-Mag-AL/ATCOL complexes was evaluated in C26 tumor-bearing mice. PTX-Mag-AL/ATCOL complexes exhibited higher antitumor efficacy than PTX solution (Fig. 4A). The greatest suppression of tumor growth was achieved when PTX-Mag-AL/ATCOL complexes were combined with the application of an external magnetic field to the tumor tissues. These results are in accordance with the in vitro cytotoxic activity (Fig. 2). In addition, a significant body weight loss was not observed in any treatment group (Fig. 4B), suggesting that PTX-Mag-AL/ATCOL complexes did not exert adverse effects. Giordano et al.²⁵⁾ reported that the intratumoral PTX concentrations at 4 h after intravenous administration of PTX solution (60 mg/kg) in several tumor-bearing mice were approximately 9–21 μg/g, corresponding to 0.6–1.4% of dose/g tumor. Thus, the tumor accumulation of PTX-Mag-AL/ATCOL complexes under magnetic field application (0.9% of dose/g tumor in Fig. 3) was not significantly greater than that of PTX solution. Nevertheless, PTX-Mag-AL/ATCOL complexes showed a more potent antitumor efficacy than PTX solution. This may be due to the higher retention of the complexes in the tumor compared with PTX solution. It has been reported that a large proportion of PTX in the tumor is eliminated within 12 h after intravenous administration of PTX solution,²⁶⁾ whereas liposomal PTX remains in the tumor even at 48 h post-administration.¹⁹⁾

Fig. 4. In Vivo Antitumor Efficacy of PTX-Mag-AL/ATCOL Complexes in C26 Tumor-Bearing Mice

PBS, PTX solution, Mag-AL/ATCOL complexes, or PTX-Mag-AL/ATCOL complexes were intravenously administered to C26 tumor-bearing mice at a dose of 2 mg/kg as PTX four times every other day (days 0, 2, 4, and 6). An external magnetic field was applied to the tumor tissue for 1 h immediately after administration. The tumor volume (A) and body weight (B) of each mouse were measured every other day. Each value represents the mean ± S.D. (n = 6). ^**p < 0.01, significantly different from PBS. ^††p < 0.01, significantly different from PTX-Mag-AL/ATCOL complexes without the magnet. PBS: phosphate-buffered saline; PTX-Mag-AL/ATCOL: paclitaxel magnetic anionic liposome/atelocollagen.

Although PTX-Mag-AL/ATCOL complexes exhibited a significant antitumor efficacy following four-time administrations and application of a magnetic field, our previous report demonstrates that a comparable antitumor efficacy was obtained with a single administration of PTX-loaded polyethylene glycol (PEG)-modified liposomes (PTX-PEG-Lip) at the same PTX dose.¹⁹⁾ This would be because PTX-PEG-Lip exhibits significantly higher retention in blood circulation than PTX-Mag-AL/ATCOL complexes, resulting in greater tumor accumulation via the EPR effect (approximately 12% of dose/g tumor). Even if PTX-Mag-AL/ATCOL complexes can be magnetically guided, applying a magnetic field to the tumor is not expected to further enhance their tumor accumulation unless the complexes remain in the bloodstream. Therefore, it is considered that prolonging the blood circulation time of PTX-Mag-AL/ATCOL complexes could improve their tumor accumulation under magnetic field application and antitumor efficacy. We previously attempted surface modification of Mag-AL/ATCOL complexes with polyethylene glycol (PEG), which is commonly used to improve the blood circulation time of liposomes.^19,27) However, PEG modification of Mag-AL interfered with the formation of complexes between Mag-AL and ATCOL. As an alternative approach to improve the blood circulation time of liposomes without PEG modification, it has been reported that replacing cholesterol in liposomes with asiatic acid or ginsenoside Rg3 prolonged the blood circulation time of liposomes.^28,29) Based on these findings, we are currently developing new PTX-Mag-AL/ATCOL complexes with prolonged blood circulation by incorporating these materials into Mag-AL. In addition, application of an external magnetic field can only accumulate PTX-Mag-AL/ATCOL complexes to superficially located tumors. Therefore, future studies will explore the implantation of magnets to target tumors located in deeper tissues in orthotopic mouse tumor models.

In conclusion, we herein demonstrated that Mag-AL/ATCOL complexes efficiently loaded PTX and enabled its sustained release. The PTX-Mag-AL/ATCOL complexes prepared showed a significant cellular association with the application of a magnetic field even under conditions with serum. Therefore, PTX-Mag-AL/ATCOL complexes efficiently accumulated in solid tumors to which an external magnetic field was applied and exhibited potent anti-tumor efficacy. These results indicate the potential of the combination of Mag-AL/ATCOL complexes with an external magnetic field as a novel cancer therapy based on an active targeting strategy.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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