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. Author manuscript; available in PMC: 2017 Dec 1.
Published in final edited form as: J Inorg Biochem. 2016 Jul 27;165:170–180. doi: 10.1016/j.jinorgbio.2016.07.016

DESIGN AND CELLULAR STUDIES OF A CARBON NANOTUBE-BASED DELIVERY SYSTEM FOR A HYBRID PLATINUM-ACRIDINE ANTICANCER AGENT

Cale D Fahrenholtz a, Song Ding b, Brian W Bernish a, Mariah L Wright b, Ye Zheng b, Mu Yang b, Xiyuan Yao b, George L Donati b, Michael D Gross b, Ulrich Bierbach b,c, Ravi Singh a,c,*
PMCID: PMC5154932  NIHMSID: NIHMS807941  PMID: 27496614

Abstract

A three-component drug-delivery system has been developed consisting of multi-walled carbon nanotubes (MWCNTs) coated with a non-classical platinum chemotherapeutic agent ([PtCl(NH3)2(L)]Cl (P3A1; L = N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG). The optimized P3A1-MWCNTs are colloidally stable in physiological solution and deliver more P3A1 into breast cancer cells than treatment with the free drug. Furthermore, P3A1-MWCNTs are cytotoxic to several cell models of breast cancer and induce S-phase cell cycle arrest and non-apoptotic cell death in breast cancer cells. By contrast, free P3A1 induces apoptosis and allows progression to G2/M phase. Photothermal activation of P3A1-MWCNTs to generate mild hyperthermia potentiates their cytotoxicity. These findings suggest that delivery of P3A1 to cancer cells using MWCNTs as a drug carrier may be beneficial for combination cancer chemotherapy and photothermal therapy.

Keywords: nanoparticle, chemotherapy, triple negative breast cancer, cytotoxicity, photothermal therapy, drug delivery

Graphical abstract synopsis

graphic file with name nihms807941f7.jpg

A platinum-acridine derivative spontaneously forms a complex with multi-walled carbon nanotubes in aqueous buffers. This drug delivery system induces cytotoxic effects distinct from the free drug including severe replication stress and cell death without initiation of apoptosis. The nanotube enables chemo-photothermal cancer therapy with enhanced cell killing following brief laser exposure.

INTRODUCTION

Platinum-based chemotherapeutic drugs such as cisplatin are among the most effective agents available to clinicians for treatment of testicular and ovarian cancer, but have limited ability to prolong survival of breast, colorectal, prostate, or lung cancer patients.[1] This is in large part due to the development of drug resistance and significant toxic side effects, which limit the ability to give patients the drugs at sufficient doses.[2] Second- and third-generation platinum chemotherapeutics such as carboplatin and oxaliplatin show reduced toxic side effects, but intrinsic resistance in many types of cancer and acquired cross-resistance to therapy still limit their efficacy.[1] Resistance to these platinum agents occurs through tolerance of drug-induced DNA damage, insufficient DNA binding, poor drug uptake and increased activity of cellular detoxification pathways.[2] Cisplatin, carboplatin, and oxaliplatin all induce the same type of DNA damage and suffer from cross-resistance.[1, 2]

Recently developed platinum–acridine (PA) hybrid anticancer agents show a potential for cancer cell killing that is far superior to cisplatin.[3, 4] The acridine group functions as a DNA intercalator that when combined with a platinum moiety, leads to rapid mono-adduct formation with nucleotides near the intercalation site resulting in a more severe form of DNA damage than the cross-links induced by cisplatin.[3–9] PAs also form permanent adducts at significantly higher frequency in genomic DNA than cisplatin resulting in effective inhibition of DNA replication and transcription,[6] which results in submicromolar activity against intrinsically platinum-resistant cancers, including breast cancer, in vitro.[3, 10] Second-generation platinum-acridines are less reactive with non-DNA intracellular nucleophiles, which may contribute to their high potency .[11] The non-crosslinking DNA damage differs greatly from current clinical agents and is able to circumvent resistance.[3]

Nanoparticles such as liposomes or polymer-based products are being tested in clinical trials to improve tumor delivery and reduce off-target toxicity of platinum drugs (reviewed in ref [12]). Carbon nanotubes (CNTs), which consist of sheets of sp2 hybridized carbon rolled into single (SWCNT) or multi-walled (MWCNT) tubes, also show promise for the delivery of cisplatin to cancer cells both in vitro and in vivo.[13–19] CNTs have the capacity to cross biological barriers like the cell membrane, [20] improve the blood stability and tumor targeting of encapsulated or conjugated drugs and small molecules,[21–23] and overcome drug resistance. [24] In addition, the unique combination of electrical, thermal, and spectroscopic properties of CNTs offers further opportunities for advances in the detection, monitoring and therapy of cancer that are not available with other drug carriers. [25] CNTs absorb near-infrared radiation (NIR) emitted from lasers, generating intense heat that can be localized to tumors after single[25–27] or multiple rounds of NIR exposure. [28] This type of laser-based heat treatment represents a promising approach for the management of recurrent breast cancer. [29–33] Functionalization of CNTs by acid-oxidation and polymer-coating can render CNTs safe for in vivo use[23, 34, 35] and addition of targeting ligands may increase tumor-specific uptake.[14, 21–23]

Use of CNTs to deliver chemotherapeutic compounds such as doxorubicin or ruthenium-polypyridyl complexes adsorbed onto CNTs by non-covalent π-stacking results in enhanced cancer cell killing in comparison to the free drug.[24, 36] Acridine also strongly adsorbs onto the surface of CNTs and is released allowing intercalation into DNA.[37, 38] CNTs have a large surface area-to-volume ratio which allows high capacity drug loading.[39] Inspired by the well-documented ability of CNTs to act as a strong adsorbent of cationic dyes, [40] we aimed to generate a CNT-based delivery system for a specific platinum-acridine, P3A1,[4, 41] that could combine the high cytotoxic activity of P3A1 with the drug delivery and photothermal therapy capability of CNTs.

EXPERIMENTAL

Cell lines and reagents

The human breast cancer cell lines MDA-MB-231, MDA-MB-468, MDA-MB-436, SUM159, and BT20 were obtained from American Type Culture Collection (ATCC). All cell lines were authenticated by ATCC and tested to be pathogen-free (including Mycoplasma, bacteria, and fungi). All cells were used within 6 months of resuscitation. MDA-MB-231, MDA-MB-468, SUM159, and BT20 were maintained as previously described.[42] MDA-MB-436 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (Sigma Aldrich), 100 IU/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies).

Synthesis

Compound P3A1 ([PtCl(NH3)2(L)]Cl (L = N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide) was synthesized according to a previously reported procedure [41] and isolated as the monochloride salt with an analytical purity of 96% (based on LC-MS; see the Supplementary Figure S1).

Preparation of P3A1-loaded carbon nanotubes

Multi-walled carbon nanotubes (Nanostructured and Amorphous Materials Inc.) were acid-oxidized using a sulfuric acid and nitric acid treatment (3:1 mixture) for 3 hours at 75 °C. The MWCNTs were collected on an Omnipore JV filter (0.1 µM, Millipore) and washed with distilled water. To remove carbonaceous debris formed during the acid-oxidation process, the surface acidized MWCNTs were resuspended in sodium hydroxide (1.0 M), sonicated for 10 minutes, and stirred at room temperature for 30 minutes.[23] MWCNTs were again collected on a filter as above, washed with distilled water and then resuspended in hydrochloric acid (0.1 M) to protonate the carboxyl groups introduced during the oxidation process. After a final water washing and collection step, acid-oxidized MWCNTs were dried overnight at 80 °C. Stocks were prepared by dispersion of MWCNTs in water (4.5 mg/mL) by bath sonication. For drug loading experiments, P3A1-MWCNT[1] was prepared in a single step by adding acid-oxidized MWCNTs (225 µg) to a glass vial containing saline, P3A1 (0–1.7 µmol/mg MWCNT) and DSPE-mPEG (8.55 mg/mL). Similarly, P3A1-MWCNT[2] was prepared in two steps by adding acid-oxidized MWCNTs (225 µg) to a glass vial containing P3A1 (0–1.11 µmol/mg MWCNT) in saline. Finally, DSPE-mPEG was added to the dispersion at a final concentration of 8.55 mg/mL. Each reaction mixture was bath sonicated twice for 15 minutes at 4 °C to prevent overheating, and unbound P3A1 and DSPE-mPEG were separated using spin columns (Vivaspin 100k molecular weight cut-off columns; EMD Millipore). For subsequent experiments, P3A1-MWCNT[1] or P3A1-MWCNT[2] were prepared by mixing the appropriate amounts of stock solutions of MWCNTs (400 µL, 1.8 mg), DSPE-mPEG(5000) (500 µL, 20 mg/mL in water, Nanocs), P3A1 (400 µL, 5 mM in saline), and saline (10x, 124 µL), and mixtures were then sonicated as above. P3A1-containing materialsand platinum-free controls were incubated for 2 hours at 4 °C. Unassociated reagents were removed using a spin column as above. The final product was aliquoted and stored in saline at −80 °C until use.

Cytotoxicity Assays

3–5 × 104 breast cancer cells were plated in 96-well tissue culture plates (BD Falcon) and allowed to attach overnight. Cells were treated at doses indicated in the figures based on loaded P3A1 and MWCNT concentration determined by UV-Vis spectroscopy as described in the supplementary information. An average of 0.84 ± 0.06 and 0.54 ± 0.06 µmol/mg MWCNT was loaded for P3A1-MWCNT[1] and P3A1-MWCNT[2], respectively. Cells were incubated for the specified times and survival was assessed using the CellTiter Glo assay kit (Promega) according to the manufacturers protocol.

Nanoparticle Tracking Analysis

DSPE-mPEG coated MWCNTs, P3A1-MWCNT[1] and P3A1-MWCNT[2] were diluted 1:5000 in degassed Type I water. Hydrodynamic diameters were obtained in quintuplicate using the Nanosight NS500 (Malvern Instruments; software version 3.1, camera level: 16, duration: seconds, threshold: 3).

Dynamic Light Scattering (DLS)

Hydrodynamic diameter and ζ-potential were measured using the Zetasizer Nano ZS90 (Malvern Instruments; software version 6.34) at 25°C with automatic settings. MWCNTs were diluted to ~15 ng/mL for DLS in water or saline for measurements.

Confocal Microscopy

MDA-MB-231 cells (3.5 × 104) were plated in 4-well chamber slides (Nunc), allowed to attach for 48 hours, and then treated with vehicle, MWCNT, P3A1-MWCNT, or P3A1 at 7.5 µM P3A1 or equivalent MWCNT for 6 hours. Cells were washed twice in PBS, fixed in 4% paraformaldehyde, mounted with hardset mounting medium (Vector Labs) and cover slips, and assessed by confocal microscopy (Excitation 372 nm, Emission 449–549 nm; direct interference contrast (DIC)).

Transmission Electron Microscopy

P3A1-MWCNT[1], P3A1-MWCNT[2] or control DSPE-mPEG-coated MWCNT in water were pipetted onto copper-coated formvar grids and then imaged using a Tecnai Spirit transmission electron microscope. MDA-MB-231 cells (3.0 × 106) were plated in 100-mm tissue culture dishes and allowed to attach overnight. Cells were treated with P3A1-MWCNT[1] (20 µg/mL MWCNT) and incubated for the times indicated. Cells were prepared as previously described. [23]

Flow Cytometry

MDA-MB-231 cells (0.75–2 × 106) were plated in 100-mm tissue culture plates (BD Falcon) and allowed to adhere overnight. Cells were then treated with P3A1-MWCNT[1], P3A1, DSPE-mPEG coated MWCNT, P3A1-DSPE-mPEG, DSPE-mPEG, DSPE-mPEG coated MWCNT, P3A1-DSPE-mPEG, or saline at the specified concentrations for 48 and 72 hours. Cells were washed with PBS and co-stained with Annexin V (APC) and propidium iodide (BD Pharmingen) per the manufacturer’s protocol. Briefly, cells were trypsinized, pelleted, washed twice with cold PBS, and then suspended in 1 × Annexin V binding buffer at a concentration of 1 × 106/mL. 1 × 105 cells were then mixed with Annexin V and incubated for 15 minutes at room temperature in the dark then 400 uL of 1 × Binding Buffer with or without propidium iodide (2.0 µg/mL) was added, mixed, and samples were analyzed on the Accuri6 Flow Cytometer (BD Biosciences). Analysis of data was performed using FCS Express version 3 (De Novo Software). For cell cycle analysis, cells were treated as indicated, fixed in 50% ice-cold ethanol, washed once in PBS, and then were treated with FxCycle PI/RNase staining solution (Life Technologies) per the manufacturer’s protocol. Analysis was performed using ModFit software.

Inductively coupled plasma mass spectrometry

MDA-MB-231 (2.0 × 106) cells were plated in 60-mm tissue culture plates and allowed to attach overnight. Cells were then treated with saline, P3A1-MWCNT[1], or P3A1 (1 µM) for 6 hours and then trypsinized, washed twice in PBS, pelleted, and stored at −20 °C. Samples were digested with a mixture of 5 % HCl and 5 % HNO3 using a microwave-assisted digestion system (Ethos UP, Milestone, Sorisole, Italy). The digested samples were then diluted to a final acid concentration of 1 % v/v before Pt determination by inductively coupled plasma mass spectrometry (ICP-MS). Trace metal grade HCl and HNO3 (Fisher, Pittsburgh, PA, USA), and distilled-deionized water (18 MΩ cm, Milli-Q®, Millipore, Bedford, MA, USA) were used to digest the samples and prepare all solutions. Standard reference solutions used for calibration were prepared in 1 % v/v acid (0.5% HCl plus 0.5% HNO3) from a 1000 mg/L Pt stock (SPEX CertPrep, Metuchen, NJ, USA). A tandem ICP-MS (8800 Triple Quadrupole, Agilent, Tokyo, Japan) equipped with a SPS 4 automatic sampler, a Scott-type double pass spray chamber operated at 2 °C, and a Micromist concentric nebulizer was used in all determinations. Helium gas (≥ 99.999% purity, Airgas, Colfax, NC, USA) was used in the ICP-MS’s collision/reaction cell to minimize potential spectral interferences while monitoring the isotope 195Pt. Other relevant instrument operating conditions such as radio frequency applied power, sample depth, carrier gas flow rate, reaction gas flow rate, and the number of sweeps per replicate were 1550 W, 10.0 mm, 1.05 L/min, 4.0 mL/min, and 100, respectively.

Western Blots

MDA-MB-231 cells were seeded (2.5–5.0 × 105) in 60-mm tissue culture dishes (BD Falcon) and allowed to attach overnight. The following day cells were treated for the indicated times. Plates were washed two times with ice cold PBS and lysates harvested in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with protease inhibitors (Thermo Scientific). Protein content was quantified by BCA protein assay (Pierce). Protein (40 µg) was separated using a 12% SDS-PAGE gel (Biorad) and transferred to nitrocellulose membrane (Biorad). Membranes were probed with primary antibodies for cleaved-PARP (Cell Signaling Technologies, D64E10, diluted 1:1000 in 5% milk in tris-buffered saline containing 0.1% Tween-20 (TBS-T) and GAPDH (Cell Signaling Technology, D16H11, diluted 1:10,000 in 5% milk in TBS-Tween). Then membranes were then probed with appropriate horse-radish peroxidase conjugated secondary antibodies (Cell Signaling Technology, diluted 1:1,000–5,000 in 5% milk in TBS-Tween) and developed using an enhanced chemiluminescence detection system (Pierce) according to the instructions provided by the manufacturer.

Carbon nanotube-mediated photothermal therapy

MDA-MB-231 cells were plated in 48-well plates in non-neighboring wells at a cell density of 2.5 × 104 per well and allowed to attach overnight. Medium was aspirated, replaced with phenol-red free RPMI supplemented with 10% FBS, and treated as indicated. Cells were irradiated at a wavelength of 970 nm and an irradiance of 3 W/cm2 using a K-Cube veterinary laser (K-Laser USA). Twenty-four hours later, relative viability was assessed using CellTiter Glo reagent according to the manufacturer’s protocol.

RESULTS

Preparation and physicochemical characterization of P3A1-loaded nanotubes

To test if MWCNTs can be coated with a platinum-acridine (PA) agent, we used the derivative P3A1 (Fig. 1a, i) ([PtCl(NH3)2(L)]Cl (L = N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide). MWCNTs were acid oxidized to generate carboxyl groups (approximately 15–17% as a percentage of overall MWCNT weight (Supplementary Fig. S2)) on their surface, and then base washed to remove adsorbed carbonaceous debris (Fig. 1a, ii). All MWCNTs used in this study were coated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG) (Fig. 1a, iii) to further improve dispersibility and biocompatibility. Thermogravimetric analysis indicated that DSPE-mPEG coated MWCNT contained approximately 75% DSPE-mPEG by weight after purification from excess coating material (Supplementary Fig. S2).

Figure 1. Chemical Structures, quantification of drug loading onto MWCNTs and nanoparticle size distributions.

Figure 1

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(A) Chemical structures of (i) platinum-acridine P3A1, (ii) acid-oxidized carbon nanotubes, and (iii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000] (DSPE-mPEG) are shown. (B) P3A1-MWCNT[1] or (C) P3A1-MWCNT[2] was prepared and unbound P3A1 was separated by size exclusion columns. Data are displayed as input P3A1 compared to MWCNT loaded P3A1 and calculated as µmol P3A1 per mg MWCNT. The hydrodynamic size distributions of (D) DSPE-mPEG coated MWCNT, (E) P3A1-MWCNT[1] and (F) P3A1-MWCNT[2] were assessed by Nanoparticle Tracking Analysis (NTA).

We assessed two methods for preparing P3A1-loaded carbon nanotubes. For the first method, we used a one-step synthesis, in which P3A1, MWCNT, and DSPE-mPEG were mixed simultaneously. The resulting drug-loaded nanoparticles are referred to as P3A1-MWCNT[1]. To determine optimal drug loading conditions, P3A1-MWCNT[1] was prepared with fixed quantities of MWCNT and DSPE-mPEG in the presence of increasing concentrations of P3A1. After adsorption, P3A1 loaded onto MWCNTs was separated from unbound P3A1 and DSPE-mPEG using size-exclusion columns. P3A1 that was not bound to nanoparticles remained in the filtrate, and the unbound drug concentration was determined spectrophotometrically at an acridine-specific wavelength (λmax = 415 nm) to determine the amount of MWCNT-associated P3A1 (Supplementary Fig. S3). The amount of P3A1 loaded onto P3A1-MWCNT[1] relative to the total P3A1 added to the dispersion is shown in Fig. 1b. The highest level of P3A1 was achieved at the highest input of P3A1 (1.7 µmol/mg MWCNT). The ratio of drug input to drug loaded on MWCNTs (P3A1 loaded/P3A1 input) gradually decreased from 100% at a P3A1 input of 0.25 µmol/mg MWCNT to less than 50% at an input greater than 1.5 µmol/mg MWCNT, defining a saturation point.

We used a two-step procedure to prepare a second type of P3A1-coated carbon nanotubes, termed P3A1-MWCNT[2]. P3A1 was first mixed with MWCNTs and then washed using size-exclusion columns to remove unassociated P3A1; in a second step, aqueous DSPE-mPEG was added to the P3A1 and MWCNT mixture. Notably, mixing P3A1 and MWCNTs in the absence of DSPE-mPEG resulted in substantial aggregation and sedimentation of the MWCNTs. However, after coating with DSPE-mPEG in the second step, P3A1-MWCNT[2] readily dispersed in water following sonication. We found a bimodal loading profile for P3A1-MWCNT[2] without apparent saturation at P3A1 to MWCNT ratios of up to 1 µmol/mg (Fig. 1c).

We next examined the size distribution and ζ-potential of uncoated MWCNTs, DSPE-PEG coated MWCNTs, P3A1-MWCNT[1], and P3A1-MWCNT[2] using nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). The results are summarized in Table 1 and representative NTA data are shown in Fig. 1d–f. NTA indicated that P3A1-MWCNT[1] exhibited a monomodal size distribution with uniform light scattering intensity that is nearly identical to DSPE-mPEG coated MWCNT prepared without P3A1. However, P3A1-MWCNT[2] exhibited a broader size distribution with multiple peaks and a more heterogeneous light scattering intensity. DLS also showed that P3A1-MWCNT[2] possessed the largest hydrodynamic diameter in water. Significantly, no increase in the hydrodynamic diameter of P3A1-MWCNT[1] was observed when measured in saline instead of water, indicating that the nanoparticles did not aggregate at this ionic strength. No change in hydrodynamic diameter was seen after storage of P3A1-MWCNT[1] in saline for 10 days at room temperature (Supplementary Fig. S4) indicating that P3A1-MWCNT[1] resisted aggregation under these conditions. In contrast, P3A1-MWCNT[2] tended to aggregate in normal saline as noted by an increase in the hydrodynamic diameter within a few minutes of incubation, though they were more stable in comparison to the uncoated MWCNT (Table 1). The ζ-potentials in water at pH 6 for P3A1-MWCNT[1], P3A1-MWCNT[2], and DSPE-mPEG coated MWCNT were considerably less negative than uncoated MWCNTs due to the expected partial charge shielding effects of DSPE-mPEG and P3A1.

Table 1.

Physicochemical characterization of P3A1-MWCNT constructs.

DLS1
hydrodynamic
diameter in
water (SD2)		 Polydispersity
index in water
(SD)		 DLS
hydrodynamic
diameter in
saline (SD)		 Polydispersity
index in saline
(SD)		 ζ-potential
(SD)		 NTA3
hydrodynamic
diameter in
water (SD)
Uncoated
MWCNT 		152.0 nm
(± 0.6)
 0.262
(± 0.024)		 2826 nm
(± 390)		 0.572
(± 0.038)		 −58.8 mV
(± 4.6)		 171.5 nm
(± 1.8)
DSPE-PEG
coated MWCNT 		135.9 nm
(± 2.9)		 0.228
(± 0.001)		 136.4 nm
(± 0.7)		 0.248
(± 0.008)		 −17.4 mV
(± 0.5)		 132.7 nm
(± 3.4)
P3A1-
MWCNT[1] 		151.3 nm
(± 4.4)		 0.234
(± 0.032)		 138.3 nm
(± 0.9)		 0.235
(± 0.009)		 −12.7 mV
(± 0.9)		 138.9 nm
(± 4.9)
P3A1+
MWCNT[2] 		187.0
(± 1.4)		 0.230
(± 0.003)		 226.0 nm
(± 1.4)		 0.273
(± 0.011)		 −18.0 mV
(± 0.5)		 168.6 nm
(± 2.5)
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1

dynamic light scattering (DLS);

2

standard deviation (SD);

3

nanoparticle tracking analysis (NTA)

We next imaged P3A1-MWCNT[1], P3A1-MWCNT[2], and drug free DSPE-mPEG coated MWCNT by transmission electron microscopy (TEM). Electron-dense regions indicative of the platinum in P3A1 were observed near the MWCNT surface of both P3A1-MWCNT[1] and P3A1-MWCNT[2] but not for DSPE-mPEG-coated MWCNT (Fig. 2a–f). For P3A1-MWCNT[1], platinum was evenly distributed along the nanotube in single or multiple layers, indicating that P3A1 may both stack on the nanotube surface and self-aggregate within the DSPE-mPEG layer. By contrast, for P3A1-MWCNT[2] areas of platinum-related high electron density were found in concentrated pockets. Numerous drug-free and drug-encapsulating micellar vesicles were seen around P3A1-MWCNT[2] but none were found around P3A1-MWCNT[1]. The methods of preparation and structural characteristics of thus obtained nanoparticles are shown schematically in Fig. 2g. Because P3A1-MWCNT[1] exhibited superior colloidal stability and more homogenous drug loading as compared to P3A1-MWCNT[2], we selected this formulation as our lead material for subsequent studies, with other constructs used only as comparative controls. Fourier-Transform Infrared Spectroscopy (FTIR) was performed to further characterize P3A1-MWCNT[1] and determine if association of P1A1 with the MWCNTs involves coordination of platinum to the surface carboxylates. However, peaks corresponding to bond stretching characteristic of both DSPE-mPEG and P3A1 were undetectable when the sample contained MWCNT (Supplementary Fig. S5). Further analysis using inductively-coupled plasma mass spectrometry (ICP-MS) confirmed the presence of platinum in P3A1-MWCNT[1] (Supplementary Fig. S6).

Figure 2. Representative TEM images of drug loaded nanotubes.

Figure 2

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Electron micrographs of (A) DSPE-mPEG coated MWCNT, (B–D) P3A1-MWCNT[1], and (E–F) P3A1-MWCNT[2] (scale bar = 100 nm) are shown. WHITE ARROWs indicate some areas of electron dense platinum (P3A1) bound to MWCNTs. BLACK ARROWs indicate aggregates of P3A1 and DSPE-mPEG not bound to MWCNTs. (G) A schematic summarizing the preparation and structures of the P3A1 nanoparticles is shown.

P3A1-loaded nanotubes release drug after being taken up by breast cancer cells

We next quantified the stability of the coating of P3A1 on P3A1-MWCNT[1] over time. When stored in saline at room temperature, 90% of the P3A1 that was initially associated with the MWCNTs (representing 100% of drug input) remained associated with P3A1-MWCNT[1] and only 10% of the amount of drug loaded onto the MWCNTs was released after 10 days (Supplementary Fig. S7). Although the fraction of the drug released from the MWCNTs (relative to the total amount initially bound to the MWCNTs) remained a constant 10% over time, this may be reflective of a dynamic equilibrium between MWCNT-associated and free P3A1 in saline. Both sodium dodecyl sulfate (SDS) or ethanol increase the release of P3A1 from MWCNTs following a brief incubation (Supplementary Fig. S7). For all subsequent experiments, P3A1 constructs were stored frozen at −80 °C in saline to avoid undesired dissociation of the pharmacophore from the carrier. No changes in nanoparticle size or P3A1 content were detected after multiple freeze-thaw cycles (data not shown).

We next evaluated the release of P3A1 from MWCNTs following uptake by cancer cells. MDA-MB-231 breast cancer cells were treated with P3A1, P3A1-MWCNT[1], DSPE-mPEG coated MWCNT or normal saline at equivalent doses of P3A1 or MWCNT. P3A1-MWCNT[1] were taken up by cancer cells and could be detected within cells by electron microscopy following treatment (Fig. 3a). The intrinsic blue fluorescence of the 9-aminoacridine chromophore of P3A1 allowed detection of intracellular P3A1 using confocal fluorescence microscopy.[43] Photomicrographs show blue fluorescence indicative of the presence of P3A1 in both P3A1 only and P3A1-MWCNT[1] treated cells (Fig. 3b). Despite exposure to an equivalent drug dose, P3A1-MWCNT[1] treated cells exhibited a lower fluorescence intensity compared to P3A1-treated cells, which is most likely a consequence of partial quenching of acridine fluorescence in the formulation. By contrast, detection of intracellular platinum by ICP-MS unequivocally confirmed that P3A1-MWCNT[1] had accumulated at a significantly higher level in cells than P3A1 (Fig. 3c).

Figure 3. Evaluation of P3A1-MWCNT[1] uptake and drug release in cancer cells.

Figure 3

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(A) MDA-MB-231 cells were treated with P3A1-MWCNT[1] (20 µg/mL MWCNT) for 16 hours, fixed with glutaraldehyde, and prepared for TEM. Images of P3A1-MWCNT[1] (white arrows) internalized by breast cancer cells are shown. Numbered regions in the lower magnification image (30,000x; scale bar = 500 nm) are shown in higher magnification (90,000x; scale bar =100 nm) in the adjacent images. (B) MDA-MB-231 cells were treated with vehicle, drug-free DSPE-mPEG coated MWCNT, P3A1-MWCNT[1] or P3A1 (7.5 µM P3A1 or 9.8 µg/mL MWCNT) for 6 h, and then assessed for P3A1 fluorescence by confocal microscopy. (C) MDA-MB-231 cells were treated with vehicle, P3A1-MWCNT[1] or P3A1 (1.0 µM) for 6 h, harvested, microwave-assisted digested, analyzed by ICP-MS, and cell-associated platinum content is shown.

P3A1-loaded nanotubes are cytotoxic to breast cancer cells

An initial cytotoxicity assay was performed to assess the effect of dose and incubation time (24, 48, 72 h) on the cytotoxicity of P3A1-MWCNT[1] using MDA-MB-231 cells (Fig. 4a). P3A1-MWCNT[1] was slightly, but significantly (p < 0.05; student’s t-test) less cytotoxic than free P3A1 in the concentration range 5–20 µM after 24 hours and at all concentrations tested at 48 and 72 hours. Both P3A1 and P3A1-MWCNT[1] were significantly (p < 0.05; student’s t-test) more cytotoxic than drug-free, DSPE-mPEG coated MWCNTs at all concentrations and incubation times. Finally, P3A1-MWCNT[1] proved to be significantly more cytotoxic than P3A1-MWCNT[2] (Supplementary Fig. S8).

Figure 4. Quantification of P3A1-MWCNT[1] induced cytotoxicity in breast cancer cells.

Figure 4

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(A) MDA-MB-231 were treated for 24, 48, and 72 h, or (B) MDA-MB-468, BT20, MDA-MB-436 or SUM159 were treated for 48 h with P3A1-MWCNT[1], DSPE-mPEG coated MWCNT (0–26 µg/mL), P3A1 (0–20 µM), or equivalent doses of P3A1-MWCNT[1]. Data are shown as relative viability assessed by CellTiter Glo assay compared to untreated control ± SD.

P3A1-MWCNT[1] consists of multiple components, each of which potentially may contribute to the overall cytotoxicity of the construct. In order to determine which components played a role in the cytotoxicity of our P3A1 drug delivery system, MDA-MB-231 breast cancer cells were treated for 48 hours with P3A1 alone, P3A1-MWCNT[1], P3A1-DSPE-mPEG, or a mixture of P3A1 -DSPE-mPEG and drug-free DSPE-mPEG coated MWCNTs at equivalent P3A1 and MWCNT concentrations (Supplementary Fig. S8). As a control, cells also were treated with dispersions of drug-free DSPE-mPEG or DSPE-mPEG coated MWCNTs prepared using processing conditions identical to the drug loaded constructs. Drug-free DSPE-mPEG did not cause a cytotoxic effect, and DSPE-mPEG coated MWCNTs killed less than 25% of cells at the highest concentration (26 µg/mL of MWCNTs). P3A1-MWCNT[1] was slightly less cytotoxic than respective free P3A1 at all concentrations tested, though substantially more cytotoxic than all other treatments. Importantly, P3A1-DSPE-mPEG was substantially less cytotoxic than P3A1-MWCNT[1], and the addition of drug free, DSPE-mPEG coated MWCNT did not alter the cytotoxicity of P3A1-DSPE-mPEG, indicating that intact P3A1-MWCNT complex, rather than a treatment with a mixture of the individual components, was important for the cytotoxicity of P3A1-MWCNT[1].

To further explore the potential use of P3A1-MWCNT[1] for cancer therapy, we exposed four additional breast cancer cell lines (MDA-MB-436, MDA-MB-468, SUM159 and BT20) to P3A1 alone, P3A1-MWCNT[1], or drug-free DSPE-mPEG coated MWCNT at equivalent doses of P3A1 or MWCNTs for 48 hours and quantified cell viability. P3A1-MWCNT[1] was similarly or slightly less cytotoxic than free P3A1 toward all cell lines tested (Fig. 4b). The cytotoxicity of drug-free DSPE-mPEG coated MWCNT varied across cell lines tested, but was substantially lower than the levels observed for P3A1 and P3A1-MWCNT[1]. The key finding in here is that substantial cytotoxic activity of P3A1 was retained after coating and release from MWCNTs.

P3A1-MWCNTs induce a form of cell death that is distinct from the free drug

Nanoparticle-based drug delivery systems can alter the way drugs are taken up by cells, how drugs are released within cells, and how drugs distribute within cells. This in turn can affect the underlying pathways and mechanisms of cancer cell death induced by the nanoparticle-drug conjugate, which may differ substantially from the free drug.[44] Therefore, we examined the mechanism of cell death induced by P3A1-MWCNT[1] and free P3A1. MDA-MB-231 cells were treated with a fixed dose of P3A1-MWCNT[1] (12.5 µg/mL MWCNT; 10 µM P3A1), P3A1 alone (10 µM), DSPE-mPEG coated MWCNTs (12.5 µg/mL MWCNT), or normal saline for 48 or 72 hours. Propidium iodide (PI) and Annexin V (AnnV) co-staining followed by flow cytometric analysis was performed to allow discrimination between necrosis (PI+; AnnV-), early apoptosis (PI-; AnnV+), and late apoptosis (PI+; AnnV+). The data indicate that free P3A1 and P3A1-MWCNT[1] induced distinctly different mechanisms of cell death (Fig. 5a). P3A1-induced apoptosis was apparent at 48 hours, with increasing cell death (both early and late stage) at 72 hours compared to controls. By contrast, P3A1-MWCNT[1] showed a marked increase in necrosis at 48 and 72 hours without indications of early apoptosis. To confirm this finding we extracted protein from MDA-MB-231 cells treated with P3A1 or P3A1-MWCNT[1] as above for 24, 48 or 72 hours and quantified cleaved poly(ADP-ribose) polymerase 1 (PARP) by immunoblot analysis (Fig. 5b). Cleavage of PARP is a hallmark of apoptosis and can also indicate necrosis.[45] Consistent with the PI/AnnV staining results, cleaved PARP was greatly increased in cells treated with P3A1 alone for 48 hours, but little was detected in cells treated with P3A1-MWCNT[1] for 48 hours. After 72 hours, a high level of cleaved PARP was detected in cells following either treatment.

Figure 5. Analysis of cell death mechanism and cell cycle of breast cancer cells treated with P3A1-MWCNT[1].

Figure 5

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(A) MDA-MB-231 were treated with drug free DSPE-mPEG coated MWCNT (12.5 µg/mL), P3A1 (10 µM), or equivalent dose of P3A1-MWCNT[1] for 48 and 72 hours. Cells were co-stained with propidium iodide (PI) and Annexin V (AnnV), and then staining was assessed by flow cytometry. Data shown are representative of 3 independent experiments. (B) Lysates collected from cells treated as in (A) were probed by Western Blot for cleaved PARP (cl-PARP) following treatment for 24, 48 or 72 hours. GAPDH was used as a loading control. (C) MDA-MB-231 cells were treated with P3A1 (0–80 µM), P3A1-MWCNT[1] (0–20 µM P3A1, 0–25 µg/mL MWCNT), or vehicle (saline) for 48 h. DNA content based upon PI staining was assessed by flow cytometry.

To gain insight into the cell cycle effects, additional flow cytometry experiments were performed. Cells treated with P3A1-MWCNT[1] induced marked S-phase cell cycle arrest (Fig. 5c). Increasing concentrations of P3A1-MWCNT[1] resulted in a loss of replicating cells, with the majority of the cells remaining in G1-phase after 48 hours of treatment. On the other hand, equivalent concentrations of P3A1 did not show a similar S-phase cell cycle arrest. Even when tested at high concentrations (80 µM), P3A1 did not show major alterations in cell cycle distribution. Together, these data suggest that P3A1-MWCNTs caused S-phase arrest and induced cell death by a non-apoptotic mechanism rather than an apoptotic mechanism, both of which are effects that are distinct from those induced by the free drug.

P3A1-loaded nanotubes are effective for multimodal photothermal therapy and drug delivery

Carbon nanotubes produce intense heat when stimulated with tissue-penetrating near-infrared (NIR) radiation [26, 27]. Therefore we examined combined P3A1-MWCNT[1] treatment with photothermal therapy. MDA-MB-231 cells were treated with P3A1-MWCNT[1], control DSPE-mPEG coated MWCNT, or vehicle, then exposed to 980 nm laser irradiation for thermal therapy. We found that P3A1-MWCNT[1] treatment sensitized cells to laser irradiation (3 W/cm2) (Fig. 6a). Control DSPE-mPEG MWCNT treatment did not induce a loss in cell viability after 60 seconds of laser exposure, whereas P3A1-MWCNT[1] treated cells showed a significant reduction in viability under the same laser conditions. Importantly, this enhanced cytotoxicity occurred after only a brief exposure to mild, non-ablative hyperthermia. The maximum temperature reached after 60 seconds of exposure in the presence of P3A1-MWCNT[1] (or DSPE-mPEG coated MWCNTs) was only 44.2 °C (Fig. 6b). Following longer laser irradiation, the heat generated by the nanotubes alone was sufficient to kill most of the treated cells and was not further enhanced by loaded P3A1. By contrast, free P3A1 did not show any increase in cytotoxicity after laser irradiation (Supplementary Fig. S9). To assess whether laser irradiation and/or heat altered the release of P3A1 from P3A1-MWCNT[1], a sample of P3A1-MWCNT[1] was laser irradiated as above (46 °C). Laser irradiation marginally increased the proportion of P3A1 released from P3A1-MWCNT[1] to 11.9±0.2 of the total amount of P3A1 originally loaded onto the tube as compared to11.1±0.4 percent released from a non-irradiated sample of P3A1-MWCNT[1] processed in parallel.

Figure 6. Evaluation of P3A1-MWCNT[1] for multimodal drug delivery and photothermal therapy.

Figure 6

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(A) MDA-MB-231 were treated with DSPE-mPEG coated MWCNT (12.5 µg/mL), P3A1-MWCNT[1] (12.5 µg/mL, 10 µM P3A1) or vehicle in dye free medium, then laser irradiated (970 nm, 3 W/cm2) for the indicated times. Treatment efficacy was assessed by CellTiter Glo assay 24 hours later. Data are expressed as relative viability compared to untreated control ± SD (**p < 0.01, Two-tailed Student’s t-test). (B) Temperature was assessed by thermocouple immediately after laser irradiation in parallel samples treated as in (A).

DISCUSSION

The platinum-acridine derivative P3A1 shows promising results for the treatment of intractable cancers both in vitro and in rodent cancer models. [4] The key to further development of this and similar agents for clinical use will be to improve drug-like properties and reduce dose-limiting toxicities, while retaining anticancer activity. As a step toward the development of a more effective and selective therapeutic agent, we generated a MWCNT-based drug delivery system for P3A1. We showed that P3A1 readily coats MWCNTs in the presence of DSPE-mPEG to form a colloidally stable nanoparticle complex. P3A1-MWCNT[1] delivered more P3A1 to breast cancer cells than treatment with the free drug, were cytotoxic to several models of breast cancer, and induced a form of cytotoxicity that was distinct from the free drug and involved both S-phase cell cycle arrest and non-apoptotic cell death. Owing to the optical-thermal properties of the MWCNT backbone, P3A1-MWCNT[1] also enabled photothermal cancer therapy.

Multiple groups have reported the use of CNTs and other fullerenes,[13–19] as well as liposomal and polymeric nanoparticles,[12, 46] for delivery of conventional platinum chemotherapeutics. In general, these previous studies focused on encapsulation of cisplatin or cisplatin derivatives within the lumen of CNTs as a strategy to prevent premature drug activation and for slow drug release following cell uptake.[13, 15–17] Alternatively, other CNT-based platinum chemotherapy delivery systems rely on covalent attachment to tether the functional platinum group to the CNT surface via a cleavable linker. [14, 18, 19] While these studies clearly demonstrate the capacity of using CNT for targeted drug delivery and controlled release, further clinical development may be hampered due to reliance on intracellular cleavage and modification of the platinum complex for activation, complicated linker synthesis, or inherent problems common to classical platinum pharmacophores. [1] Our system does not rely on activation of a prodrug and requires no modification of the drug for loading onto CNTs. In addition to π-stacking interactions, [37, 38, 47] electrostatic force between the negatively charged MWCNTs and positively charged P3A1, which exist as 2+ charged cations at neutral pH, [4] likely plays a role in the interaction of P3A1 with CNTs. Furthermore, the carboxylate groups on the MWCNTs may help further stabilize the platinum moieties via direct coordination. [48]

P3A1-MWCNT[1] offers several potential advantages over previously described platinum therapeutic agents. First, we showed that P3A1-MWCNT[1] was effective for the treatment of five triple-negative breast cancer (TNBC) cell lines (Fig. 5) at a dose that is less than a tenth of the reported dose of cisplatin required for a similar effect on MDA-MB-231 cells.[3] TNBC patients show the highest levels of tumor recurrence, the lowest five-year survival rates of all breast cancer subtypes,[49] and do not benefit from current molecularly targeted therapies.[50] The high activity we establish for P3A1 and P3A1-MWCNT[1] treatment of TNBC cells offers the possibility of expanding platinum therapy to TNBC patients.

We assessed two methods for preparing P3A1 loaded carbon nanotubes (P3A1-MWCNT[1] and P3A1-MWCNT[1]) and noted a difference in P3A1 loading when assessed by electron microscopy. It appeared that the presence of DSPE-mPEG during loading of P3A1 onto P3A1-MWCNT[1] acted to stabilize the particle suspension against aggregation and allowed for a more even coating of P3A1. However, addition of P3A1 in the absence of DSPE-mPEG induced aggregation of P3A1-MWCNT[2] and the high drug concentration present on the surface of the nanotubes may have caused partitioning of P3A1 into DSPE-mPEG vesicles during the subsequent coating process. The micellar structures that appear on the surface of P3A1-MWCNT[2] visualized by TEM may explain the larger hydrodynamic diameter and more heterogeneous size distribution of P3A1-MWCNT[2] as compared to P3A1-MWCNT[1].

We found that P3A1-MWCNT[1] induced severe replication stress which prevented cells from passing through S-phase and progressing through the cell cycle, leading to cell death without initiation of apoptosis; whereas free P3A1 induced apoptosis without a significant effect on progression through the cell cycle. P3A1-MWCNT[1] may be beneficial compared to free P3A1 as it immediately prevents breast cancer cells from dividing. Mutations or loss of the tumor suppressor p53, which is common in many cancers including TNBC,[51] can prevent induction of apoptosis.[52] We show that P3A1-MWCNT[1] induces non-apoptotic cell death in a p53 mutant TNBC cell line, MDA-MB-231, and P3A1-MWCNT[1] was cytotoxic to three additional TNBC cell lines that harbor p53 mutations (BT20, MDA-MB-436, MDA-MB-468). These data suggest that P3A1-MWCNT[1] may be useful for treatment of cancers deficient in p53 or apoptotic machinery.

Serum or other components of the culture media could play a role in the extracellular release of P3A1, but the increased platinum delivery by P3A1-MWCNT and the different modes of cell death and cell cycle effects induced by free P3A1 and P3A1-MWCNT[1] suggest that the nanoparticle-drug conjugate is delivered intact to cells. Confocal fluorescence microscopy showed that cell-associated P3A1 fluorescence intensity was less for P3A1-MWCNT[1] treated cells compared to cells treated with free P3A1 at equivalent doses (Fig. 3b); however, cell-associated platinum assessed by ICP-MS was higher in P3A1-MWCNT[1]-treated cells compared to P3A1-treated cells (Fig. 3c). Because acridine fluorescence is quenched when associated with CNTs[38] and P3A1-MWCNT[1] delivers more platinum to cells in comparison to treatment of cells treated with an equivalent amount of free P3A1 (as shown by ICP-MS), it is reasonable to suggest that at least a portion of the P3A1 is still CNT-associated following cell uptake. CNTs may serve as a P3A1 “reservoir” within in the cells. Therefore, the difference between the activity of P3A1 and P3A1-MWCNT[1] possibly lies within the delivery of the chemotherapeutic with regards to drug release, intracellular trafficking and localization, maximal achieved dose, as well as dosing rate.

Thermal therapy shows promise for the treatment of many types of cancer.[29] Due to the MWCNT backbone, our drug delivery system has the inherent capability for combination NIR stimulated photothermal therapy and P3A1 treatment. We show that photothermal activation of P3A1-MWCNT[1] is more effective than photothermal treatment of drug-free MWCNTs or P3A1-MWCNT[1] alone (Fig. 6). Importantly, this effect occurs under mild hyperthermia conditions (44 °C) and a brief exposure (60 seconds) to laser irradiation and therefore would be suitable as part of a treatment regimen designed to spare healthy breast tissue in a clinical setting.[30] We previously found that similar NIR exposure does not affect the cytotoxicity of MWCNT,[27, 28]. The small increase in the amount of P3A1 released following laser irradiation of P3A1-MWCNT[1] compared to drug release from non-irradiated P3A1-MWCNT[1] is likely insufficient to account for the increased cytotoxicity. Therefore this increase possibly is caused by chemo-sensitization due to hyperthermia rather than greater drug release.[25]

The long-term rationale behind using nanoparticles, including CNTs, as drug delivery vehicles is the capacity for nanoparticles to accumulate in tumors at a higher rate than small molecules. This is due to the leaky and tortuous nature of tumor blood vessel architecture, a phenomenon known as the enhanced permeability and retention (EPR) effect.[53] Phospholipid-polyethylene glycol coated CNT, like those used in this report, can greatly increase blood residence and tumor selective delivery of small molecules and chemotherapeutics adsorbed onto their surface.[21–23] Acid-oxidation and polymer-coating can mitigate acute CNT toxicity in vivo,[23, 34, 35] and growing evidence suggests in vivo biodegradation mechanisms that could dramatically reduce any potential long-term risks of CNT exposure[54, 55]. CNTs are degraded within a few days by peroxidase enzymes from both plants and animals[56] and inside the cells by myeloperoxidase,[57] eosinophile peroxidase[58] or other reactive oxygen species mediated pathways.[55, 59] Moreover, enzymatically degraded CNTs appear to be less toxic as compared to their pristine counterparts.[57] As more understanding is developed regarding the degradation of CNTs in biological environments, it may be possible to design CNTs that leave no long-term biological footprint.[54]

CONCLUSION

Here we show the development of a new anti-neoplastic agent based upon a versatile MWCNT drug delivery system loaded with the chemotherapeutic agent P3A1, a platinum-based antineoplastic agent that is an order of magnitude more effective than current analogs.[3] The P3A1-MWCNT complex is easily formed in aqueous solution minimizing processing and purification steps, and enables combined chemo- and photothermal therapy for multimodal cancer treatment regimens. The unique combination of tumor cell replicative stasis and non-apoptotic cell death induced by P3A1-MWCNT[1] may lead to improvements in cancer therapy using P3A1 or similar drugs. Our system offers further possibilities for improved tumor accumulation, selective targeting, cancer imaging enabled by the MWCNT backbone.[25] We therefore believe that this agent warrants further preclinical investigation for the treatment of breast and other cancers.

Supplementary Material

Highlights.

  • Efficient coating of a platinum-acridine agent on multi-walled carbon nanotubes

  • Drug release from the nanotube complex following cell uptake

  • Promising therapeutic effect in triple negative breast cancer

  • Nanoparticle-specific induction of S-phase arrest and non-apoptotic cell death

  • Combined photothermal and chemotherapy on a single delivery platform

Acknowledgments

This work was supported in part by grant NCI R00CA154006 (R.S.), by grant NCI R01CA101880 (U.B.) and by start-up funds from the Wake Forest School of Medicine Department of Cancer Biology. C.D.F was supported by NCI T32CA079448. We acknowledge support from the National Science Foundation’s Major Research Instrumentation Program (NSF-MRI, grant CHE-1531698) and from Wake Forest University Comprehensive Cancer Center (WFUCCC) Cellular Imaging Shared Resource and the Cell Viral Vector Core Laboratory supported in part by NCI CCSG P30CA012197. We also thank the National Institutes of Health and National Cancer Institute for grant 1R13CA200223-01A1 (Conference Organization support, 1st International Symposium on Clinical and Experimental Metallodrugs in Medicine: Cancer Chemotherapy, CEMM), from which interesting discussions contributed to this work. We thank Jessica Swanner, Jerod Sears, Brittany Eldridge, Ken Grant, Paula Graham, Yelena Karpova and Dr. David Ornelles of Wake Forest School of Medicine of Wake Forest University for assistance in data collection and analysis.

ABBREVIATIONS

AnnV

annexin V

CNT

carbon nanotube

DLS

dynamic light scattering

DMEM

Dulbecco’s Modified Eagle’s Medium

DNA

deoxyribonucleic acid

DSPE-mPEG

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-5000]

EPR

enhanced permeability and retention

FBS

fetal bovine serum

FTIR

Fourier-transform infrared spectroscopy

GAPDH

glyceraldehyde 3-phosphatase dehydrogenase

ICP-MS

inductively coupled plasma mass spectrometry

MWCNT

multiwalled carbon nanotube

NIR

near-infrared radiation

NTA

nanoparticle tracking analysis

P3A1

([PtCl(NH3)2(N-(2-(acridin-9-ylamino)ethyl)-N-methylproprionimidamide)

PA

platinum-acridine

PARP

poly ADP Ribose

PBS

phosphate buffered saline

PI

propidium iodide

RPMI

Roswell Park Memorial Institute medium

SWCNT

single walled carbon nanotube

TBS

tris-buffered saline

TEM

transmission electron microscopy

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

C.D.F., U.B. and R.S. conceived the research. C.D.F., S.D., M.Y., X.Y., B.W.B, M.L.W., Y.Z., M.D.G., G.L.D., U.B. and R.S. developed the methodology, carried out the experiments, and performed data analysis. C.D.F, U.B. and R.S. wrote the main manuscript text. All authors reviewed and edited the manuscript.

Contributor Information

Cale D. Fahrenholtz, Email: cfahrenh@wakehealth.edu.

Song Ding, Email: dings0@wfu.edu.

Brian W. Bernish, Email: bbernish@wakehealth.edu.

Mariah L. Wright, Email: mariah.l.wright@alumni.wfu.edu.

Ye Zheng, Email: zheny9@wfu.edu.

Mu Yang, Email: yangm11@wfu.edu.

Xiyuan Yao, Email: yaox15@wfu.edu.

George L. Donati, Email: donatigl@wfu.edu.

Michael D. Gross, Email: grossmd@wfu.edu.

Ulrich Bierbach, Email: bierbau@wfu.edu.

REFERENCES

  • 1.Kelland L. Nat Rev Cancer. 2007;7:573–584. doi: 10.1038/nrc2167. [DOI] [PubMed] [Google Scholar]
  • 2.Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo M, Kroemer G. Oncogene. 2012;31:1869–1883. doi: 10.1038/onc.2011.384. [DOI] [PubMed] [Google Scholar]
  • 3.Graham LA, Suryadi J, West TK, Kucera GL, Bierbach U. Journal of medicinal chemistry. 2012;55:7817–7827. doi: 10.1021/jm300879k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ma Z, Choudhury JR, Wright MW, Day CS, Saluta G, Kucera GL, Bierbach U. Journal of medicinal chemistry. 2008;51:7574–7580. doi: 10.1021/jm800900g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Dutta S, Snyder MJ, Rosile D, Binz KL, Roll EH, Suryadi J, Bierbach U, Guthold M. Cell biochemistry and biophysics. 2013;67:1103–1113. doi: 10.1007/s12013-013-9614-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Qiao X, Zeitany AE, Wright MW, Essader AS, Levine KE, Kucera GL, Bierbach U. Metallomics : integrated biometal science. 2012;4:645–652. doi: 10.1039/c2mt20031g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Smyre CL, Saluta G, Kute TE, Kucera GL, Bierbach U. ACS medicinal chemistry letters. 2011;2:870–874. doi: 10.1021/ml2001888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Graham LA, Wilson GM, West TK, Day CS, Kucera GL, Bierbach U. ACS medicinal chemistry letters. 2011;2:687–691. doi: 10.1021/ml200104h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Choudhury JR, Rao L, Bierbach U. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 2011;16:373–380. doi: 10.1007/s00775-010-0733-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ding S, Qiao X, Kucera GL, Bierbach U. Chemical communications. 2013;49:2415–2417. doi: 10.1039/c3cc38957j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ma Z, Rao L, Bierbach U. Journal of medicinal chemistry. 2009;52:3424–3427. doi: 10.1021/jm900451y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Oberoi HS, Nukolova NV, Kabanov AV, Bronich TK. Advanced drug delivery reviews. 2013;65:1667–1685. doi: 10.1016/j.addr.2013.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ajima K, Murakami T, Mizoguchi Y, Tsuchida K, Ichihashi T, Iijima S, Yudasaka M. ACS nano. 2008;2:2057–2064. doi: 10.1021/nn800395t. [DOI] [PubMed] [Google Scholar]
  • 14.Dhar S, Liu Z, Thomale J, Dai H, Lippard SJ. Journal of the American Chemical Society. 2008;130:11467–11476. doi: 10.1021/ja803036e. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Li J, Pant A, Chin CF, Ang WH, Menard-Moyon C, Nayak TR, Gibson D, Ramaprabhu S, Panczyk T, Bianco A, Pastorin G. Nanomedicine : nanotechnology, biology, and medicine. 2014;10:1465–1475. doi: 10.1016/j.nano.2014.01.004. [DOI] [PubMed] [Google Scholar]
  • 16.Muzi L, Menard-Moyon C, Russier J, Li J, Chin CF, Ang WH, Pastorin G, Risuleo G, Bianco A. Nanoscale. 2015;7:5383–5394. doi: 10.1039/c5nr00220f. [DOI] [PubMed] [Google Scholar]
  • 17.Yoong SL, Wong BS, Zhou QL, Chin CF, Li J, Venkatesan T, Ho HK, Yu V, Ang WH, Pastorin G. Biomaterials. 2014;35:748–759. doi: 10.1016/j.biomaterials.2013.09.036. [DOI] [PubMed] [Google Scholar]
  • 18.Bhirde AA, Patel V, Gavard J, Zhang G, Sousa AA, Masedunskas A, Leapman RD, Weigert R, Gutkind JS, Rusling JF. ACS nano. 2009;3:307–316. doi: 10.1021/nn800551s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Feazell RP, Nakayama-Ratchford N, Dai H, Lippard SJ. Journal of the American Chemical Society. 2007;129 doi: 10.1021/ja073231f. 8438-+ [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lacerda L, Russier J, Pastorin G, Herrero MA, Venturelli E, Dumortier H, Al-Jamal KT, Prato M, Kostarelos K, Bianco A. Biomaterials. 2012;33:3334–3343. doi: 10.1016/j.biomaterials.2012.01.024. [DOI] [PubMed] [Google Scholar]
  • 21.Liu Z, Chen K, Davis C, Sherlock S, Cao Q, Chen X, Dai H. Cancer Res. 2008;68:6652–6660. doi: 10.1158/0008-5472.CAN-08-1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Shao W, Paul A, Zhao B, Lee C, Rodes L, Prakash S. Biomaterials. 2013;34:10109–10119. doi: 10.1016/j.biomaterials.2013.09.007. [DOI] [PubMed] [Google Scholar]
  • 23.Fahrenholtz CD, Hadimani M, King SB, Torti SV, Singh R. Nanomedicine (Lond) 2015;10:2481–2497. doi: 10.2217/NNM.15.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wang N, Feng Y, Zeng L, Zhao Z, Chen T. ACS Appl Mater Interfaces. 2015;7:14933–14945. doi: 10.1021/acsami.5b03739. [DOI] [PubMed] [Google Scholar]
  • 25.Singh R, Torti SV. Advanced drug delivery reviews. 2013;65:2045–2060. doi: 10.1016/j.addr.2013.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Torti SV, Byrne F, Whelan O, Levi N, Ucer B, Schmid M, Torti FM, Akman S, Liu J, Ajayan PM, Nalamasu O, Carroll DL. Int J Nanomedicine. 2007;2:707–714. [PMC free article] [PubMed] [Google Scholar]
  • 27.Eldridge BN, Bernish BW, Fahrenholtz CD, Singh R. ACS Biomaterials Science & Engineering, American Chemical Society. 2016 doi: 10.1021/acsbiomaterials.6b00052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Ding X, Singh R, Burke A, Hatcher H, Olson J, Kraft RA, Schmid M, Carroll D, Bourland JD, Akman S, Torti FM, Torti SV. Nanomedicine (Lond) 2011;6:1341–1352. doi: 10.2217/nnm.11.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zhao Z, Wu F. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology. 2010;36:1149–1155. doi: 10.1016/j.ejso.2010.09.012. [DOI] [PubMed] [Google Scholar]
  • 30.Dowlatshahi K, Francescatti DS, Bloom KJ. American journal of surgery. 2002;184:359–363. doi: 10.1016/s0002-9610(02)00942-x. [DOI] [PubMed] [Google Scholar]
  • 31.Roti JLR. Int J Hyperther. 2008;24:3–15. [Google Scholar]
  • 32.Emami B, Song CW. Int J Radiat Oncol Biol Phys. 1984;10:289–295. doi: 10.1016/0360-3016(84)90015-4. [DOI] [PubMed] [Google Scholar]
  • 33.Burke AR, Singh RN, Carroll DL, Wood JC, D’Agostino RB, Jr, Ajayan PM, Torti FM, Torti SV. Biomaterials. 2012;33:2961–2970. doi: 10.1016/j.biomaterials.2011.12.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Singh R, Pantarotto D, Lacerda L, Pastorin G, Klumpp C, Prato M, Bianco A, Kostarelos K. Proc Natl Acad Sci U S A. 2006;103:3357–3362. doi: 10.1073/pnas.0509009103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Burke AR, Singh RN, Carroll DL, Owen JD, Kock ND, D’Agostino R, Jr, Torti FM, Torti SV. Biomaterials. 2011;32:5970–5978. doi: 10.1016/j.biomaterials.2011.04.059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ali-Boucetta H, Al-Jamal KT, McCarthy D, Prato M, Bianco A, Kostarelos K. Chemical communications. 2008:459–461. doi: 10.1039/b712350g. [DOI] [PubMed] [Google Scholar]
  • 37.Meng Y, Liu FY, Han JF, Sun SG, Fan JL, Song FL, Peng XJ. Mater Sci Eng B-Adv. 2012;177:887–891. [Google Scholar]
  • 38.Mackiewicz N, Delaire JA, Rutherford AW, Doris E, Mioskowski C. Chemistry. 2009;15:3882–3888. doi: 10.1002/chem.200801863. [DOI] [PubMed] [Google Scholar]
  • 39.Prato M, Kostarelos K, Bianco A. Acc Chem Res. 2008;41:60–68. doi: 10.1021/ar700089b. [DOI] [PubMed] [Google Scholar]
  • 40.Gong JL, Wang B, Zeng GM, Yang CP, Niu CG, Niu QY, Zhou WJ, Liang Y. J Hazard Mater. 2009;164:1517–1522. doi: 10.1016/j.jhazmat.2008.09.072. [DOI] [PubMed] [Google Scholar]
  • 41.Ding S, Qiao X, Kucera GL, Bierbach U. Journal of medicinal chemistry. 2012;55:10198–10203. doi: 10.1021/jm301278c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Swanner J, Mims J, Carroll D, Akman S, Furdui C, Torti S, Singh R. International Journal of Nanomedicine. 2015;10:3937–3953. doi: 10.2147/IJN.S80349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Pickard AJ, Liu F, Bartenstein TF, Haines LG, Levine KE, Kucera GL, Bierbach U. Chemistry. 2014;20:16174–16187. doi: 10.1002/chem.201404845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Li X, He Q, Shi J. ACS nano. 2014;8:1309–1320. doi: 10.1021/nn4046985. [DOI] [PubMed] [Google Scholar]
  • 45.Gobeil S, Boucher CC, Nadeau D, Poirier GG. Cell Death Differ. 2001;8:588–594. doi: 10.1038/sj.cdd.4400851. [DOI] [PubMed] [Google Scholar]
  • 46.Dhar S, Gu FX, Langer R, Farokhzad OC, Lippard SJ. Proc Natl Acad Sci U S A. 2008;105:17356–17361. doi: 10.1073/pnas.0809154105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Roquelet C, Lauret JS, Alain-Rizzo V, Voisin C, Fleurier R, Delarue M, Garrot D, Loiseau A, Roussignol P, Delaire JA, Deleporte E. Chemphyschem. 2010;11:1667–1672. doi: 10.1002/cphc.201000067. [DOI] [PubMed] [Google Scholar]
  • 48.Lee HY, Mohammed KA, Goldberg EP, Kaye F, Nasreen N. Am J Cancer Res. 2015;5:603–615. [PMC free article] [PubMed] [Google Scholar]
  • 49.Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA. Clin Cancer Res. 2007;13:4429–4434. doi: 10.1158/1078-0432.CCR-06-3045. [DOI] [PubMed] [Google Scholar]
  • 50.Hudis CA, Gianni L. Oncologist. 2011;(16 Suppl 1):1–11. doi: 10.1634/theoncologist.2011-S1-01. [DOI] [PubMed] [Google Scholar]
  • 51.Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Lonning PE, Borresen-Dale AL. Proc Natl Acad Sci U S A. 2001;98:10869–10874. doi: 10.1073/pnas.191367098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Fridman JS, Lowe SW. Oncogene. 2003;22:9030–9040. doi: 10.1038/sj.onc.1207116. [DOI] [PubMed] [Google Scholar]
  • 53.Fang J, Nakamura H, Maeda H. Advanced drug delivery reviews. 2011;63:136–151. doi: 10.1016/j.addr.2010.04.009. [DOI] [PubMed] [Google Scholar]
  • 54.Kotchey GP, Zhao Y, Kagan VE, Star A. Advanced drug delivery reviews. 2013;65:1921–1932. doi: 10.1016/j.addr.2013.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Elgrabli D, Dachraoui W, Menard-Moyon C, Liu XJ, Begin D, Begin-Colin S, Bianco A, Gazeau F, Alloyeau D. ACS nano. 2015;9:10113–10124. doi: 10.1021/acsnano.5b03708. [DOI] [PubMed] [Google Scholar]
  • 56.Allen BL, Kichambare PD, Gou P, Vlasova II, Kapralov AA, Konduru N, Kagan VE, Star A. Nano Lett. 2008;8:3899–3903. doi: 10.1021/nl802315h. [DOI] [PubMed] [Google Scholar]
  • 57.Kagan VE, Konduru NV, Feng WH, Allen BL, Conroy J, Volkov Y, Vlasova II, Belikova NA, Yanamala N, Kapralov A, Tyurina YY, Shi JW, Kisin ER, Murray AR, Franks J, Stolz D, Gou PP, Klein-Seetharaman J, Fadeel B, Star A, Shvedova AA. Nat Nanotechnol. 2010;5:354–359. doi: 10.1038/nnano.2010.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Andon FT, Kapralov AA, Yanamala N, Feng WH, Baygan A, Chambers BJ, Hultenby K, Ye F, Toprak MS, Brandner BD, Fornara A, Klein-Seetharaman J, Kotchey GP, Star A, Shvedova AA, Fadeel B, Kagan VE. Small. 2013;9:2721–2729. doi: 10.1002/smll.201202508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Kagan VE, Kapralov AA, St Croix CM, Watkins SC, Kisin ER, Kotchey GP, Balasubramanian K, Vlasova II, Yu J, Kim K, Seo W, MallampaIli RK, Star A, Shvedova AA. ACS nano. 2014;8:5610–5621. doi: 10.1021/nn406484b. [DOI] [PMC free article] [PubMed] [Google Scholar]

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