Synergistic Nanoparticulate Drug Combination Overcomes Multidrug

Article pubs.acs.org/molecularpharmaceutics

Synergistic Nanoparticulate Drug Combination Overcomes Multidrug Resistance, Increases Efficacy, and Reduces Cardiotoxicity in a Nonimmunocompromised Breast Tumor Model Adam J. Shuhendler,†,∥ Preethy Prasad,†,∥ Rui Xue Zhang,†,∥ Mohammad Ali Amini,† Mei Sun,‡ Peter P. Liu,‡ Robert G. Bristow,§ Andrew M. Rauth,§ and Xiao Yu Wu*,† †

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada Division of Cardiology, Heart and Stroke/Richard Lewis Centre of Excellence, University Health Network, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada § Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5T 2M9, Canada ‡

ABSTRACT: Anthracyclines, commonly employed for cancer chemotherapy, suffer from dose-limiting cardiotoxicity and poor efficacy due to multidrug resistance (MDR). We previously demonstrated that simultaneous delivery of the synergistic drugs doxorubicin (DOX) and mitomycin C (MMC) by polymer−lipid hybrid nanoparticles (PLN) circumvented MDR, increased efficacy, and reduced cardiotoxicity in immuncompromised mice superior to poly(ethylene glycol)-coated (PEGylated) lipososmal DOX (PLD). Herein it is shown that the DOX-MMC combination was also synergistic in MDR EMT6/AR1 murine breast cancer cells and that their nanoparticle formulations were able to overcome the MDR phenotype. In contrast PLD exhibited little or no effect on the MDR cells. For the first time, these differences in in vitro efficacy are shown to be strongly correlated with cellular uptake and intracellular distribution of DOX brought about by DOX formulations (e.g., free solution, PLN vs PLD). To take into consideration the role of an intact immune system and tumor stroma in the response of host and tumor to chemotherapy, use was made of nonimmunocomprised mouse models to study the dose tolerance, cardiotoxicity, and efficacy of DOX-MMC coloaded PLN (DMsPLN) compared to PLD. DMsPLN treatment at 50 mg/m2 DOX and 17 mg/m2 of MMC singly or once every 4 days for 4 cycles were well tolerated by the mice without elevated systemic toxicity blood markers or myocardial damage. In contrast, PLD was limited to a single treatment due to significant total weight loss. The DMsPLN treatment delayed tumor growth up to 312% and 28% in EMT6/WT and EMT6/AR1 models, respectively. This work supports ̈ or anthracycline-resistant tumors. the translational value of DMsPLN for the aggressive management of either naive KEYWORDS: doxorubicin, mitomycin C, drug combination chemotherapy, polymer−lipid hybrid nanoparticles, multidrug resistance, cardiotoxicity

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INTRODUCTION Anthracycline-based adjuvant treatment combinations are the most widely employed therapeutic strategies used against breast cancer.1−3 Anthracycline-based chemotherapy reduces mortality over nonanthracycline-based options and provides generally higher response rates in both locally advanced and metastatic disease.4 In fact, the widespread adoption of anthracycline-based chemotherapy following surgical resection of the tumor is partly credited with the high rates of long-term survival of breast cancer patients.5 However, the high antitumor efficacy of anthracyclines © 2014 American Chemical Society

such as doxorubicin (DOX) is compromised by associated doselimiting systemic toxicity in the form of myelosuppression6 and congestive heart failure.5,7 These serious systemic adverse effects Special Issue: Drug Delivery and Reversal of MDR Received: Revised: Accepted: Published: 2659

January 30, 2014 May 8, 2014 May 15, 2014 May 15, 2014 dx.doi.org/10.1021/mp500093c | Mol. Pharmaceutics 2014, 11, 2659−2674

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observed in cancer cells treated simultaneously with DOX and MMC.26 Furthermore, the higher toxicity of MMC in the hypoxic microenvironment of solid tumors may also contribute to a better therapeutic effect.27 Having learned that optimal synergy of DOX and MMC combination resulted from nanoscale delivery of both drugs to the site of drug action, i.e., DNA of the same cancer cells, we chose to coencapsulate DOX and MMC in the same nanoparticle. By using stealth polymer−lipid hybrid nanoparticles (sPLN) modified from previous work,28−30 it was demonstrated that the nanoscale delivery of the two agents simultaneously overcame multiple efflux pump-mediated MDR phenotypes in human breast cancer cells at much lower doses as compared to free drugs.30,31 The lipid-based nanoparticle system also demonstrated excellent systemic circulation, tumor uptake, and liver-sparing characteristics determined by whole body optical imaging.32 Encouraged by the promising in vitro cell studies and the in vivo biodistribution results, the efficacy and toxicity of DMsPLN was evaluated in sensitive and MDR human mammary tumor xenografts in immunocompromised nude mice. We found up to a 3-fold increase in animal life span, a 10−20% tumor cure rate, undetectable normal tissue toxicity, and decreased tumor angiogenesis, results that were superior to the results of PLD treatment.33 The tolerability of the nude mice to higher DOX doses (i.e., 50 mg/m2) could not be studied due to a DOX caused severe general toxicity in nude mice.33 Owing to the compromised immune system (e.g., lack of T-cells) of nude mice, they could have different patterns of tumor progression and host response to chemotherapeutic agents as compared to nonimmunocompromised mice. Moreover, human cancer cells grown in nude mice could have different stroma−tumor cell interactions as compared to murine cancer cells grown in mice, which could impact on the response of the tumor to chemotherapy.34−36 Therefore, in the present work, use was made of murine EMT6 breast tumors grown in nonimmunocompromised Balb/c mice to investigate the synergy, cellular uptake, and in vivo efficacy of the DOX-MMC combination as compared to PLD. To find out if a true synergy of the DOX-MMC combination, in free and nanoparticle formulations, occurs in wild-type (WT) EMT6/ WT and MDR EMT6/AR1 murine breast cancer cells we conducted median effect analysis and calculated combination indices (CI), whose values indicate synergism (CI < 1), additive (CI = 1), or antagonism (CI > 1). To elucidate whether previously observed superior efficacy of DMsPLN as compared to see if the superior efficacy of DMsPLN is due to differences in cellular uptake, intracellular distribution, and release of DOX, we conducted confocal fluorescence microscopic examination and determined efficacy of DOX in various formulations in WT EMT6/WT and MDR EMT6/AR1 murine breast cancer cells. From the in vitro cytotoxicity studies of various PLN formulations (i.e., DOX or MMC-loaded PLN, DPLN, and MPLN) and their combinations (DPLN + MPLN or DMsPLN), we selected an optimal formulation for in vivo investigations of efficacy, tolerability, and cardiotoxicity.

are still unresolved, reducing the benefit of clinical breast cancer chemotherapy. To protect the heart from DOX damage, cardioprotective agents (e.g., dexrazoxane, brand name Zinecard) have been used. These agents have shown some protective effect, however, with a lowered antitumor efficacy of DOX, thus limiting their clinical utility.8 The heart and the tumor often share drug sensitivity but require opposite therapeutic outcomes. Therefore, agents protecting the heart may also protect the tumor, leading to poor therapeutic efficacy. As an alternative cardioprotective strategy, anthracycline derivatives (e.g., epirubicin and doxycycline) with less cardiomyocyte damage have been synthesized; however, at equal doses to DOX, 25% to 50% lower efficacy rates relative to DOX were found.9−12 Nanoparticulate DOX formulations, however, have improved the tolerability of DOX chemotherapy in the clinic, attributable to slow DOX release and altered pharmacokinetics and biodistribution.13−15 Poly(ethylene glycol)coated (PEGylated) liposomal DOX (PLD) significantly reduced the risk of cardiotoxicity5,12,16 and myelosuppression13 while maintaining efficacy levels equal to free DOX.8 Nevertheless, new doselimiting effects in the form of palmar plantar erythrodysethesia (PPE) occur in at least 45% of patients.8 This shortcoming of PLD suggested the necessity of developing new nanoparticulate formulations. In addition to dose-limiting and potentially lethal side effects of anticancer drugs, multidrug resistance (MDR) is a major factor contributing to the failure of cancer chemotherapy.17−22 MDR is the intrinsic or acquired cross-resistance of a cancer cell to chemotherapeutic agents of varying and unrelated chemical structures.17−22 While the MDR phenotype may arise from overactive intracellular detoxification processes (e.g., DNA repair and oxidant scavenging), in nearly 40% of breast cancer, MDR is due to the overexpression of membrane drug efflux transport proteins following ineffective therapy.19,20 To avoid the onset of acquired MDR phenotypes, anticancer drugs are often administered as combinations in an effort to enhance antitumor effects while lowering nonoverlapping toxicity associated with each agent.21,23 However, effective drug combinations require rational design with preclinical demonstration of drug combination synergy,22 which may be found through in vitro cytotoxicity studies. Abraham et al. observed that coencapsulation of vincristine with DOX in liposomes produced no better (or lower) therapeutic efficacy in vivo than liposomal vincristine alone. This was shown to be due to a pronounced antagonism of DOX and vincristine when they were added to cells simultaneously.24 The authors emphasized the significance of in vitro drug combination screening for the prediction of whether a coformulated drug combination will act in an antagonistic or synergistic manner. In our previous work, we demonstrated that a DOX and mitomycin C (MMC) combination, a clinically relevant cancer drug combination, generated synergistic cytotoxicity against both sensitive and MDR human breast cancer cells in vitro25 and against murine mammary carcinoma in vivo when delivered intratumorally by microspheres, resulting in 185% delay in tumor growth.26 The significantly enhanced in vivo efficacy was attributed to the optimal delivery schedule and the pharmacological effects of DOX and MMC on subcellular targets, i.e., DNA. Through an extensive in vitro study on the scheduling effect on the cytotoxicity of DOX and MMC combination, it was found that the synergistic cytotoxicity occurred when DOX was added to cancer cells before MMC or simultaneously with MMC.26 Remarkably higher numbers of DNA double strand breaks were

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EXPERIMENTAL SECTION Chemicals and Reagents. All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada) and used without further purification. Nonionic block copolymer Pluronic F-68 (PF68) was a kind gift from BASF Corp. (Florham Park, NJ, USA). Mitomycin C and 2660

dx.doi.org/10.1021/mp500093c | Mol. Pharmaceutics 2014, 11, 2659−2674


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combination of DOX-PLN and MMC-PLN (DPLN + MPLN), DMsPLN, PLD (Caelyx, Janssen Inc., Toronto, ON, Canada), blank PLN, and 0.9% saline (B. Braun Medical Inc., USA). All treatment concentrations were based on the loaded DOX concentration into DMsPLN. After the addition of various formulations, cells were incubated at 37 °C in a 5% CO2 and 95% air humidified incubator over 24 h. Before conducting MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, the treatment medium was removed from each well, and cells were washed once with α-MEM medium (Gibco, Life Technologies). Ten microliters of 12 mM MTT (SigmaAldrich) were added to each well followed by 4 h incubation at 37 °C. Then, 100 μL of 0.1 g/mL sodium lauryl sulfate (Fisher Scientific, New Jersey, USA) dissolved in 0.01 M HCl was added to each well and incubated overnight at 37 °C. The concentration of formazan was analyzed at 570 nm with a spectrophotometer (SPECTRAMAX PLUS384, Molecular Device, CA, USA). Laser Scanning Confocal Microscopy of Intracellular Localization of Doxorubicin. EMT6 (wild-type and resistant) cells were seeded at densities of 30,000 cells in a 35 mm glass bottom culture dish (MatTek Corporation, Ashland, OR, USA) and allowed to grow for 24 h in 3 mL of growth medium at 37 °C in a humidified incubator with 5% CO2 atm. Following this incubation, 2.5 mL of 0.5 μL/mL Hoescht 33342 nuclei acid dye (Molecular Probes, Inc., Eugene, OR, USA) was added into each well and incubated for 20 min at 37 °C. The medium was removed, and each cell type was incubated with the following three formulations: free DOX, DMsPLN, and PLD, at total DOX concentration of 17 μM for 1 h at 37 °C. The solution was then removed and cells were washed three times with prewarmed growth medium. The cells were imaged using Zeiss LSM 510 laser scanning microscopy (Carl Zeiss Canada, Ltd., Toronto, ON, Canada). Cells were imaged at 364 nm (blue channel) and 543 nm (red channel) laser light to visualize nuclei (blue) and DOX (red), respectively. Cellular Uptake of Doxorubicin in Various Formulations. EMT6/WT or EMT6/AR1 cells were plated at the density of 10,000 cells per well in 96-well plates for 24 h. Then 17 μM DOX in free DOX solution, DMsPLN, and PLD was added to each well according to Wong et al.37 After 0.5, 1, 1.5, 2, and 4 h exposures to the treatment, the medium was removed, and the cells were washed twice with ice-cold phosphor buffer saline (PBS), and 200 μL of PBS per well was added. The amount of DOX was measured by fluorescence at excitation 480 nm/ emission 560 nm (SPECTRAMAX GEMINI XS, Molecular Device). Median Effect Analysis. To find out whether DOX-MMC combination and their nanoparticle formulations can actually produce synergistic anticancer effect on the murine MDR cells or not, we performed median effect analysis.30,31,38 The median effect plot of log[( fa)−1 − 1]−1 vs log[D] was generated for the three treatment groups (1) DOX alone, (2) MMC alone, and (3) DOX and MMC together, where D is the drug concentration and fa is the fraction of cells affected. DOX concentration is used for the x-axis of the drug combination plot. From the plot, the slope (m), a measure of the sigmoidicity of the dose effect relationship, and the x-intercept (Dm), the median effect dose, were determined. Mathematical linearization and definition of efficacy parameters then allowed the generation of a combination index as previously described.30,31,38 The dose of the individual drugs and both drugs together that affect a given percent (x%) of the plated

doxorubicin HCl were purchased from Polymed Therapeutics Inc. (Houston, TX, USA). Hydrolyzed polymer of epoxidized soybean oil (HPESO) was a gift from Dr. Z. Liu (Food and Drug Administration, Washington, DC, USA). Poly(ethylene glycol)coated (PEGylated) liposomal doxorubicin (Caelyx) was purchased from the pharmacy at the Princess Margaret Hospital (Toronto, ON, Canada). Cell culture medium, α-modified minimal essential medium (α-MEM), and phosphate buffered saline were obtained from the University Health Network Tissue Culture Media Facility (Toronto, ON, Canada). Cell culture media were supplemented with fetal bovine serum (FBS) (Invitrogen, Inc., Burlington, ON, Canada). Formulation and Characterization of Stealth Polymer Lipid Hybrid Nanoparticles. Drug-loaded PLN were formulated as previously described.28,30 Briefly, for making DMsPLN, 50 mg of myristic acid, 4.5 mg of Myrj59, and 8 mg of Myrj52 were heated to 65 °C until fatty acid and block copolymer surfactants were melted. Under stirring, 4 mg of MMC was added to the molten fatty acid, followed by the simultaneous addition of 400 μL of a 12.5 mg/mL solution of DOX in distilled, deionized water and 50 μL of a 50 g/L solution of HPESO in distilled, deionized water. Lastly, 50 μL of 100 mg/mL PF68 and 388 μL of distilled deionized water were added, and the emulsion was stirred at 65 °C for 20 min. The emulsion was sonicated for 5 min using a UP100H probe ultrasonicator (Hielscher USA Inc., Ringwood, NJ, USA), after which time the nanoemulsion was quickly transferred to 2.5 mL of cooled, sterile 0.9% NaCl under stirring. Single agent, DOX or MMC-loaded PLN (DPLN or MPLN) were prepared similarly except that only one drug was added. Particle size and zeta potential were measured for each formulation by dynamic light scattering and electrophoretic mobility, respectively, using a NICOMP 380ZLS apparatus (PSSNICOMP, Santa Barbara, CA, USA). Immediately after formulation, drug loading (weight/weight% [w/w%] drug in fatty acid) and encapsulation efficiency (w/w% loaded drug) were assayed on 200 μL of DMsPLN suspension by recovering the particle-free filtrate through a 0.1 μm filter and assaying MMC and DOX concentrations by ultraviolet spectrophotometry at 364 and 540 nm, respectively. The remaining DMsPLN suspension was incubated at 4 °C overnight with Sephadex SP C-25 anionic dextran microspheres to remove any unencapsulated drugs from the saline phase. The microspheres were then pulled down by centrifugation, and the nanoparticles were decanted into a separate vial, ready for injection. Cell Lines and Maintenance. Both WT and doxorubicinresistant (AR1) mouse mammary sarcoma EMT6 cells were originally obtained from Dr. I. Tannock at the Ontario Cancer Institute (Toronto, ON, Canada) and maintained in our laboratory. The cells were authenticated using short tandem repeat analysis (Toronto Center for Applied Genomics, Toronto, ON, Canada) and were tested for their suitability for use in animals and were found to be pathogen-free (Research Animal Diagnostics Laboratory, Columbia, MO, USA). EMT6/WT cells were grown in α-MEM supplemented with 10% FBS at 37 °C in a humidified incubator with 5% CO2 atmosphere. EMT6/AR1 cells were grown under the same conditions except the growth medium was supplemented with 1.0 μg/mL DOX. In Vitro Cytotoxicity against Cancer Cells. Cells were plated at the density of 10,000 cells per well in a 96-well plate 24 h before being treated with one of the following formulations at various concentrations from 0.01 to 100 μM: free DOX solution, free MMC solution, combination of free DOX and MMC at 1:1.4 molar ratio, DOX-PLN (DPLN), MMC-PLN (MPLN), the 2661



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Figure 1. Response of EMT6/WT and EMT6/AR1 cells in vitro to exposure to various formulations of DOX, MMC, PLN, and PLD. Percent kill (% kill) of EMT6/WT (A,C) or EMT6/AR1 cancer cells (B,D) after 24 h exposure to different formulations was assessed by the MTT cell viability assay. Both EMT6 cell types were treated with free MMC, free DOX, and free DOX + free MMC (A,B) or PLD, MPLN, DPLN, DPLN + MPLN, and DMsPLN (C,D) at various concentrations ranging from 0.01 to 50 μM. Note that the drug concentrations were based on the DOX loading capacity in the PLN. Data points represent the mean ± standard error of the mean (SE) with n = 3 for each formulation.

colonies, Dx1, Dx2, and Dx1,2, respectively, was calculated from eq 1 of refs 30, 31, and 38:

⎡ f ⎤1/ m Dx = Dm⎢ a ⎥ ⎢⎣ 1 − fa ⎥⎦

Ontario Cancer Institute following guidelines set forth by the Canadian Council on Animal Care (CCAC). Tumor Growth and Treatments. One week after arrival and acclimation, the mice were inoculated intramuscularly in the hind leg with 5 × 105 EMT6/WT or EMT6/AR1 cells. A flexible plastic circle template ruler was used to measure the tumor plus leg diameter (TLD) of the mice. Treatments were initiated once TLD reached 7 to 8 mm. The treatment groups comprised mice receiving intravenous injections via the tail vein of saline as a vehicle control (saline, 2 × n = 5), 50 mg/m2 PLD (PLD, n = 5), 50 mg/m2 DMsPLN (DMsPLN, 2 × n = 5), or four treatments of 50 mg/m2 DMsPLN each, with one treatment being administered every 4 days (4 × DMsPLN, 2 × n = 5). This dose level and the multiple dosing schedule were chosen to match the clinical use of PLD. In clinic, PLD is infused intravenously once a week for a minimum of 4 weeks at a total single infusion dose of 50 mg/m2 (DOXIL Monograph). For dose calculations, mouse body surface area was calculated from animal weight according to the following formula:39 (body surface area) = 0.9662 (body weight)0.66, resulting in cumulative DOX doses of 17.2 mg/kg (PLD, DMsPLN) and 68.8 mg/kg (4 × DMsPLN), and mitomycin C doses of 5.8 mg/kg (DMsPLN) and 23.2 mg/kg (4 × DMsPLN). Body weight and TLD of all mice were measured every other day. Mice were euthanized either when there was 20% weight loss or when TLD reached 12 mm, in accordance with CCAC guidelines. Tumor growth delay (TGD) was

(1)

On the basis of the above equation, the combination index (CI) was computed as follows: CI =

D1 D DD + 2 + 1 2 Dx1 Dx2 Dx1Dx2

(2)

CI allows for quantification of synergism or antagonism for the two drugs. A CI 1 indicates synergism, additive effect, and antagonism, respectively. Animal Model. Eight- to ten-week old Balb/C mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Animal trials were performed with group sizes of 5 animals per treatment per trial. For all but the PLD group, the efficacy trial was repeated to result in a total of 10 animals per treatment group. As will be presented in the results section, the severe toxicity of the PLD formulation at the dose level administered prevented a repeat of the initial trial, resulting in a total group size of 5. All animal handling and procedures were conducted under an approved protocol from the Animal Care Committee at the 2662



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Figure 2. Results for median effect analysis of the interaction of DOX and MMC in free form or PLN formulations in EMT6/WT cells. The filled squares, open diamonds, and open squares indicate where the doses studied fall on the combination index curve.

calculated from the mean survival times of each group according to TGD(%) = (Tcontrol − Ttreatment)/Tcontrol × 100.26 Immediately after euthanasia, intact hearts were removed from the animals and flash frozen in liquid nitrogen. Hearts were then fixed from frozen buffered formalin, embedded in paraffin and sectioned to provide a four-chamber view of the heart. Sections were stained with hematoxylin and eosin (H&E) and/or periodic acid Schiff (PAS) stain to analyze cardiomyocyte morphology. Additionally, histological analysis of mice exhibiting complete tumor regression was performed. The tumor-bearing hind legs of the mice were removed, fixed in buffered formalin, embedded in paraffin,

sectioned, and stained with H&E to determine the presence (saline control mice) or absence (4 × DMsPLN mice) of tumor tissue. Blood Collection and Analysis. Starting the day after tumor inoculation, approximately 200 μL of blood was collected into heparin sulfate-coated capillary tubes (Microvette CB 300 LH, Sarstedt Inc., Montreal, QC, Canada) from each animal every 7 days via the saphenous vein. Blood was centrifuged at 1440g for 20 min at 4 °C to isolate plasma, which was immediately flash frozen on liquid nitrogen until processing. Plasma was assayed for blood enzymes as biomarkers for different tissue 2663



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Figure 3. Median effect analysis for the interaction of DOX and MMC in free form or PLN formulations in EMT6/AR1 cells following a 24 h drug exposure. The filled squares, open diamonds, and open squares indicate where the doses studied fall on the combination index curve.

within each cell line, were performed using the Breslow Survival Test.

toxicity using assay kits, namely, lactate dehydrogenase (LDH) (Cayman Chemical Co., Ann Arbor, MI, USA), alanine transaminase (ALT) (Cayman Chemical Co), and creatine phosphokinase (CPK) (BioAssay Systems, Hayward, CA, USA). Statistical Analysis. For in vitro DOX cellular uptake study, statistical significance between two formulations was tested by Student’s t test in MS Excel. Kaplan−Meier survival analysis was carried out in SPSS v.17 (IBM Corp., Somers, NY, USA). Pairwise comparisons between survival times of each treatment group between each trial, as well as each treatment group

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RESULTS

Properties of Prepared PLN Formulations. The particle size, zeta potential, and morphology of various PLN formulations (DPLN, MPLN, and DMsPLN), encapsulation efficiency, and drug loading of PLN were characterized and reported in previous papers by our group.30,31 All PLN formulations had similar particle size around 130−150 nm in diameter and zeta potential 2664



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Figure 4. (A,B) DOX cellular uptake of free DOX, PLD, or DMsPLN in EMT6/WT or EMT6/AR1 cells. The effect of different formulations on DOX cellular uptake from 0.5 to 4 h in (A) EMT6/WT cells and (B) EMT6/AR1 cells. At predetermined time points, cells were washed with ice-cold PBS buffer, and DOX uptake was measured at λex480 nm/λem560 nm with fluorescence spectrophotometry. Results were normalized with cells treated by 0.9% saline and expressed as mean ± SE (n = 3). The DOX concentration in all treatments was equally set at 17 μM. (C,D) Laser scanning confocal microscopic images (20× objective lens) were acquired to determine DOX intracellular location. Cells were incubated with free DOX, PLD, or DMsPLN for 1 h at a nominal concentration of 17 μM. From left to right: first panel, bright field images of cells; second panel, nuclei (blue); third panel, DOX (red); and fourth panel, DOX/Hoechst overlaid (Merged) images for EMT6/WT cells (C) and for EMT6/AR1 cells (D).

around −20 mV. The polydispersity index of particle size distribution was around 0.3−0.4.31 Encapsulation efficiency of DOX was nearly 90% in both single and dual loaded PLN formulations. MMC encapsulation efficiency in PLN was about 36% in the dual agent formulation and 67−73% in the single agent formulation.30,31 Single or dual drug release kinetics from

PLN was previously determined in 0.05 M Tris HCl buffer to exhibit a biphasic release with initial burst release within 5 h and sustained release over 48 h. In the presence of 0.1 mM CaCl2, a condition closer to in vivo physiological fluid, about 20% of DOX or MMC was released from the PLN, except MMC released (∼10%) from the dual-agent formulation DMsPLN in the first 5 h.30 2665



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Table 1. Effect of Nanoparticle Formulation on Relative Cellular Uptake of Doxorubicin (DOX) in EMT6/WT and EMT6/AR1 Cellsa nanoparticle formulations compared to free DOX EMT6/WT exposure time (h)

DMsPLN

0.5 1 1.5 2 4

45.4% ± 4.0% 27.5% ± 8.5%b 14.5% ± 9.2% 8.4% ± 2.2% 39.1% ± 9.4%b b

DMsPLN compared to PLD

EMT6/AR1 PLD

DMsPLN

PLD

−14.8% ± 6.5% −34.9% ± 2.9%b −35.6% ± 5.8%b −41.3% ± 8.3%b −44.8% ± 6.5%b

35.5% ± 4.4% 49.2% ± 8.8%b 28.2% ± 3.7% 70.1% ± 9.9%b 99.3% ± 17.7%b b

−23.7% ± 8.7% −14.5% ± 5.3% −11.9% ± 6.4% −8.0% ± 8.3% 44.8% ± 19.4%b

b

EMT6/WT

EMT6/AR1

DMsPLN

DMsPLN

70.7% ± 4.7% 95.8% ± 13.0%c 77.9% ± 14.3%c 84.7% ± 3.8%c 151.8% ± 17.1%c c

77.7% ± 5.7%c 74.5% ± 10.3%c 45.5% ± 4.2%c 84.9% ± 10.8%c 37.8% ± 12.2%

a Relative DOX cellular uptake % of DMsPLN was compared to both free DOX and PLD formulations. bSstatistically significant difference between DMsPLN or PLD and free DOX at each time point with p < 0.05. cStatistically significant difference between DMsPLN and PLD in terms of improved DOX cellular uptake at each time point with p < 0.05.

DMsPLN Increase Cytotoxicity in Both Wild-Type and MDR Murine Breast Cancer Cells. The response of EMT6 cells to treatments with DOX and MMC alone and in combination with different formulations was assessed by the MTT cell viability assay. The dose of the drug was based on the measured DOX and MMC loading capacities in the PLN. As a free single anticancer agent, MMC is less effective on a molar basis at killing cancer cells than DOX in both EMT6/WT and EMT6/ AR1 breast cancer cells (Figure 1A,B). In WT EMT6 cells, when DOX-MMC at a 1:1.4 molar ratio was applied concurrently, the combination therapy significantly enhanced the tumor cell kill over individual drug treatment (Figure 1A). However, when the P-gp-overexpressing EMT6/AR1 cells were exposed to free drugs alone or in combination, none of these formulations were effective enough to kill 50% of breast cancer cells at the studied drug concentrations (Figure 1B) compared to its WT counterpart (Figure 1A). The efficacy of various nanparticle formulations, i.e., single agent-loaded DPLN, MPLN, their combinations (DPLN + MPLN), liposomal DOX PLD, and dual agent-loaded PLN (DMsPLN) is compared in Figure 1C ,D. In EMT6/WT cell types, DMsPLN show a 2-fold efficacy as compared to free drug combination with the required DOX dose for 50% cell killing being reduced from D50 = 0.89 μM for free drug combination to D50 = 0.42 μM (Figure 1C). The advantages of PLN formulations are more prominent in EMT6/AR1 cells (Figure 1D). For example, at 50 μM DOX, treatment with PLN formulations increases percent of cell kill by as high as 2-fold compared to that of free drug treatment (Figure 1 B). PLD was the least effective formulation against both WT and MDR EMT6 murine breast cancer cells with no effect on cell kill in the MDR cells (Figure 1C,D). Synergistic Effect of DOX and MMC Observed in Both Wild-Type and MDR Cells. The dose response curves represented in Figure 1 were linearized by plotting log[(fa)−1 − 1]−1 vs. log[D] (Figures 2 and 3), where fa is the fraction of cells killed and D is the drug concentration. Nonparallel curves in the median effect plot suggested nonexclusivity in the mechanism of cytotoxicity of DOX and MMC in the cells, inferring that two anticancer drugs have different subcellular mechanisms of action against breast tumor cells.38 In the median effect plot for both EMT6/WT (Figure 2A,C,E) and EMT/AR1 (Figure 3A,C,E) cell types, R2 > 0.90 indicating the statistical validity of the analysis and conforms to the law of mass action. The median effect dose (Dm) for each formulation required for 50% cell kill was determined by taking the anti-log of the x-intercept. The data was further analyzed to determine the CI, which plots the relationship of the observed efficacy of the combination from the expected efficacy. The derivation of CI for the DOX-MMC 1:1.4

molar ratio was determined using eqs 1 and 2. CI < 1 suggests a synergistic effect of two agents (DOX and MMC); CI = 1 means the combination of two agents is additive; CI > 1 indicates two drugs act antagonistically against each other. Treatment with free DOX + free MMC, DPLN + MPLN, and DMsPLN demonstrated synergistic effects against EMT6/WT murine cancer cells at certain dose levels (Figure 2B,D,F). However, for EMT6/AR1 cells, free DOX + free MMC did not demonstrate synergistic or additive effect. Coencapsulated DOX and MMC within the same PLN (DMsPLN) showed an advantage of synergistic killing of multidrug resistant EMT6 cancer cells, resulting in significant increase in efficacy over single treatment or free single or combination solutions (Figure 3B,D,F). Enhanced DOX Cellular Uptake by Encapsulation in PLN. Cellular uptake of DOX was determined using a fluorescence spectrometer after incubation of the cells with DMsPLN, PLD, or free DOX. Relative cellular uptake of DOX compared to free DOX (RF%freeDOX) was calculated from fluorescence intensity (FI) by RF%freeDOX =

FInanoparticle formulation − F I freeDOX FIfreeDOX

× 100% (3)

The relative cellular uptake of DOX in DMsPLN as compared to PLD (RF%PLD) was calculated by RF%PLD =

FIDMsPLN − FIPLD × 100% FIPLD

(4)

Among the three formulations, DMsPLN treatment resulted in the highest cellular uptake of DOX in both EMT6/WT and EMT6/AR1 cells, followed by free DOX and PLD as measured by relative DOX fluorescence from 0.5 to 4 h (Figure 4A,B). At 4 h, PLD and free DOX did not show significant difference in terms of DOX intracellular accumulation. Table 1 shows the effect of nanoparticle formulations on relative DOX cellular uptake in DMsPLN and PLD as compared to free DOX in EMT6 WT and AR1 cells (first four columns). It is seen that DMsPLN significantly enhanced DOX uptake up to 99%, especially by EMT6/AR1 cells (p < 0.05 at most time points). In contrast, PLD had a negative effect on DOX uptake with reductions up to 45% except at 4 h by EMT6/AR1 cells, although this adverse effect became smaller in the MDR cells. Thus, the difference between DMsPLN and PLD uptake became up to 151% and 85% in EMT6/WT and EMT6/AR1 cells, respectively. DOX concentration in cells continuously increased with time in the DMsPLN treated cells, whereas free DOX and PLD appeared to reach a plateau within 2 h in EMT6/WT cells. It is noted that 2666



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cellular uptake of all DOX formulations by EMT6/AR1 cells is consistently lower than EMT6/WT cells (Figure 4A,B). This result was similar to the findings by Wong et al., which showed lower cellular levels of DOX delivered by DPLN in EMT6/AR1 cells than in EMT6/WT cells.28 Intracellular distribution of DOX after 1 h treatment with various formulations was examined using confocal fluorescence microscopy. Free DOX and DMsPLN resulted in DOX localization in the nuclei of EMT6/WT cells, while DOX from PLD was confined to the cytoplasm (Figure 4C). In EMT6/AR1 cells, nuclear localization of DOX was only observed in the DMsPLN treated cells (Figure 4D). DOX fluorescence was barely detectable in the EMT6/AR1 cells following PLD treatment (Figure 4D). These results explain in part why PLD was the least effective in EMT6/WT cells and had no effect on EMT6/AR1 cells (Figure 1D). DMsPLN Are More Effective than PLD in Both WildType and Multidrug Resistant Tumors in Mice. In order to evaluate the efficacy of DMsPLN in the immune system, intact mouse model EMT6/WT or EMT6/AR1 leg tumors in Balb/c mice were used. Figure 5A,B show the tumor growth in saline control mice. PLD, the current clinically employed nanoparticulate formulation of DOX, was used as the comparator group. The toxicity of PLD at the dosing interval and dose level (50 mg/m2 DOX) in this study was too severe to warrant repeated administrations of that formulation (see below). Results of the single PLD experiments for EMT6 and EMT6/ AR1 mice are shown in Figure 5C,D, respectively. Little effect of PLD was seen on other tumor types. In the treatment of EMT6/ WT tumors, there was a significant difference (p < 0.05) in the efficacy of PLD relative to DMsPLN (Figure 5C,E). A clear enhancement of tumor growth delay with a single equivalent dose of DMsPLN over PLD was evident. With 4 × DMsPLN treatment, there was an even greater tumor growth delay with 80% of the treated mice in this groups showing significant tumor regression before regrowth and 20% (2/10) showing complete tumor regression (Figure 5G,I [same data as in G on an expanded time scale]). The trend for efficacy against EMT6/AR1 tumors having an MDR phenotype were qualitatively the same as that for the WT tumors, with DMsPLN being more effective than PLD (Figure 5D,F). In addition, some mice in the 4 × DMsPLN group (Figure 5H) responded with longer tumor growth delay relative to a single administration of DMsPLN (Figure 5F). However, quantitatively, the effectiveness of DMsPLN on EMT6/AR1 growth delay was much less than for EMT6/WT tumors. Average tumor growth curves for various treatment groups up to 40 days for mice bearing EMT6/WT and up to 19 days for mice bearing EMT6/AR1 tumor were compared in Figures 5K,L. Note that the average data were only presented when the number of survived mice was three or more in order to calculate standard deviations. The therapeutic benefit of DMsPLN was obviously seen from Figures 5K,L. These trends of enhanced survivorship were recapitulated by Kaplan−Meier survival analysis for EMT6/WT (Figure 6A) and EMT6/AR1 (Figure 6B) tumors. The individual trials (n = 5 per trial) for all but the PLD treatment groups were assessed for homogeneity of survival times. The two trials were not found to be significantly different statistically in terms of survival times, after pooling the two trials for the saline, DMsPLN, and 4 × DMsPLN groups (data not shown). For the treatment of EMT6/ WT tumors, the PLD treatment was significantly more effective than the saline control (p < 0.05) but was less effective than either of the DMsPLN treatment arms (p < 0.05), with mean survival

Figure 5. Tumor growth curves for breast tumor-bearing mice treated with saline (control), liposomal doxorubicin (PLD), doxorubicin and mitomycin C coloaded polymer−lipid hybrid nanoparticle single injection (DMsPLN), or four injections (4 × DMsPLN), 4 days apart. (A−J) Tumor-plus-leg diameter vs time for individual mice (each curve represents one animal); note that the x-axis scale has been expanded in panels I and J relative to all other panels to show the entire duration of the survival study. Two trials were performed for saline, DMsPLN, and 4 × DMsPLN with n = 5 each trial, where solid lines represent animals in the first trial and dotted lines represent animals in the second trial. (K,L) Average tumor-plus-leg diameter vs time for (K) EMT6/WT and (L) EMT6/AR1 breast tumor-bearing mice treated with various formulations. The data are presented as average ± standard deviation. Note that the average data were not presented when the number of survived mice was smaller than three.

times of 15.0, 27.2, and 56.1 days for PLD, DMsPLN, and 4 × DMsPLN groups, respectively, as compared with 13.6 days for saline control (Figure 6C). These survival values translated into TGD of 10% for PLD treatment, as compared to 100% for DMsPLN and 312% for 4 × DMsPLN treatments (Figure 6C). 2667



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Figure 7. Gross anatomical photographs of the right hind leg of EMT6/ WT tumor-bearing Balb/C mice treated with (A) saline or treated with (B) 4 × DMsPLN. Histological analysis of formalin fixed, paraffin embedded, H&E stained tissue sections from the right hind leg of the same EMT6/WT tumor-bearing Balb/C mice in panels A and B, treated with (C) saline or (D) 4 × DMsPLN, respectively. Light microscope images were obtained at 20× objective. FIS, fibroskeletal invasive sarcoma; SM, skeletal muscle; FE, fibrovascular epithelium.

Figure 6. Kaplan−Meier survival curves for (A) EMT6/WT and (B) EMT6/AR1 tumor-bearing mice treated with saline, 50 mg/m2 PLD, 50 mg/m2 DMsPLN, or 4× 50 mg/m2 DMsPLN. (C) Quantitative comparison of the mean survival time in days and the tumor growth delay of each treatment group for each tumor type. Values are the mean of the pooled group survival values and standard error of the mean (SE). † , Significantly different mean survival time relative to saline group (p < 0.05); §, significantly different mean survival time relative to PLD group (p < 0.05); *, significantly different mean survival time relative to DMsPLN group (p < 0.05).

treatment arm was performed. This dose-limiting toxicity also precluded a multiple dosing schedule of the PLD formulation. In contrast, even at 4-fold dose levels of DMsPLN (200 mg/m2), dose-limiting toxicity was not observed (Figure 9G,H). Blood enzyme levels [CPK (Figure 9A,B), LDH (Figure 9C,D), and ALT (Figure 9E,F)] were measured serially once a week over the 7- and 3-week course of treatment for EMT6/WT and EMT6/ AR1 tumor-bearing animals, respectively (Figure 9). Although the differences in blood enzyme activities between the treatment groups were not statistically significant (p > 0.05), the trends in blood enzyme levels consistently suggested lower toxicity of DMsPLN and 4 × DMsPLN treatment relative to PLD therapy in both tumor models. PLD treatment resulted in an increase in CPK levels (Figure 9A,B) and LDH levels (Figure 9C,D) between weeks 1 and 2 of treatment. In the EMT6/WT group, LDH levels were elevated at weeks 4 and 5 of the DMsPLN and 4 × DMsPLN treatments, respectively (Figure 9C), and ALT levels were elevated for both groups from week 4 onward (Figure 9E). Considering the total body weight measurements, although not conclusive statistically, these elevations observed in DMsPLN and 4 × DMsPLN groups could be indicative of tumor cell death and not necessarily nonspecific tissue damage.37 Histological examination of the heart allowed the cardioprotective potential of DMsPLN as a drug delivery method to be compared to PLD (Figure 10). In PLD-treated mice, vertical sections of the heart showed an enlarged size with cardiac chamber dilatation, particularly enlarged ventricles (Figure 10D) compared with saline (Figure 10A), DMsPLN (Figure 10B), and 4 × DMsPLN (Figure 10C) treatment groups. The histology of heart tissues from PLD-treated mice showed loss of myocardial fibers, atrophic change (Figure 10H,L), and degenerative alterations delineated by PAS staining (Figure 10P,T). These changes were observed in multiple, distinct patches throughout heart tissue from animals treated with 50 mg/m2 PLD but were not seen in heart tissue from any other group. However, a dosedependent anomaly was noticed in the myocardium of DMsPLN-treated mice that was not apparent in saline-treated animals: vacuole-like structures were noted within the myocardial cells, most prominently in the cross-sectional view of

In treating EMT6/AR1 tumors, only the DMsPLN and 4 × DMsPLN treatments showed significant enhancement of therapeutic efficacy over the saline control (p < 0.05). These treatment arms were also significantly better than the PLD group (p < 0.05). To examine whether at the end of treatment the animal with TLD smaller than the initial size at the beginning of the treatment was in fact due to real regression of tumor, we conducted histological examination of the leg inoculated with the EMT6/WT tumor. Compared to saline control with significant swelling of the upper leg noted at a TLD of 12 mm (Figure 7A), 4 × DMsPLN-treated mice showed sustained eradication of the tumor from the leg muscle both at a gross anatomical (Figure 7B) level and histological (Figure 7D) level. The tumor had completely disappeared with 4 × DMsPLN treatment, resulting in a TLD of 5.5 mm (Figure 7B). Histologically, focal intraskeletal carcinoma (FIC) can clearly be seen in saline treated tumor, surrounded by a thin capsule-like layer of skeletal muscle (SM), and finally by overlying fibrovascular epithelium (FE) (Figure 7C). However, after treatment with 4 × DMsPLN and tumor regression, the FIC characteristic of the tumor is undetectable (Figure 7D). DMsPLNs Improved the Dose-Limiting Toxicity Profile as Compared with PLD. Longitudinal recording of animal body weight over the survival time of the animal was used as a surrogate marker of whole animal health, with losses of 20% of total initial body weight serving as a toxic end point requiring euthanasia of the animal. For both EMT6/WT and EMT6/AR1 models, none of the saline control or DMsPLN group members demonstrated weight loss nearing 20% (Figure 8A,B,E,F). However, for mice receiving a single administration of PLD, severe total body weight loss, nearing if not equal to 20%, was observed in all animals (Figure 8C,D). Because of this severe toxicity of the administered dose of 50 mg/m2, only a single trial of this 2668



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Figure 8. Total body weight of individual mice bearing EMT6/WT (A,C,E,G) or EMT6/AR1 tumors (B,D,F,H) were generated by serial weighing over the length of the efficacy trial. Each curve represents one animal. The dashed black line on each plot represents the toxic cut off of 20% body weight loss. Please note the 4-fold difference in the “days post-incubation” scale for WT versus AR1 tumor bearing mice.

myocardium of DMsPLN (Figure 10J,R) and 4 × DMsPLN (Figure 10K,S). The vacuoles were more numerous and of greater size in the 4 × DMsPLN groups, suggesting a dose-dependent effect of the DMsPLN treatment. At this high dose, some vacuolelike structures were apparent in the longitudinal view of the myocardium (Figure 10G,O) in both the cytoplasm and the nuclei. Although the nature of these vacuoles was not known, the myocardium did not display any other morphological change indicative of a toxic response.

localization of DOX, and cytotoxicity in WT and MDR EMT6 murine breast cancer cells. DMsPLN enhanced DOX uptake by up to 99%, whereas PLD reduced DOX uptake by up to 45% compared to free DOX in vitro (Table 1). After 1 h incubation, the DMsPLN taken up by the cells were transported to the nuclear region even in the EMT6/AR1 cells (Figure 4C,D). However, PLD taken by EMT6/WT cells were confined in the cytoplasm with little DOX detected in the nuclei (Figure 4C). Because the primary mechanism of DOX is its intercalation between two adjacent DNA bases and inhibition of the action of topoisomerase II,40,41 adequate intracellular concentration of DOX, particularly in the cell nuclei, are required to achieve an anticancer effect. Our results showed that cellular uptake and nuclear DOX transport of PLD were the lowest among the three DOX formulations studied, which is in agreement with the trend of in vitro efficacy (Figure 1). Although cellular uptake of PLD was moderate in EMT6/WT cells, unavailability of DOX in the nuclei damped DOX efficacy. It is known that PLD release DOX

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DISCUSSION DOX Formulation Plays an Important Role in Cellular Uptake, Nuclear Localization, and Cytotoxicity of DOX. Having investigated three DOX formulations, i.e., free DOX solution, PEGylated liposomal DOX (PLD), and polymer−lipid nanoparticle (PLN) encapsulated DOX at cellular and intracellular levels, we found for the first time a strong correlation between DOX formulation, cellular uptake, intracellular 2669



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Figure 9. Blood enzyme levels indicate toxicity of the PLD but not the DMsPLN treatment. Serial blood collection and analysis of plasma enzyme levels were performed for EMT6/WT (A,C,E) and EMT6/AR1 (B,D,F) tumor-bearing animals. CPK (A,B), LDH (C,D), and ALT (E,F) were assayed in the saline (blue), PLD (green), DMsPLN (purple), and 4 × DMsPLN (red) groups. Data represents mean and standard deviation of three plasma samples.

very slowly with a release half-life t1/2 = 118 h and that >90% DOX is retained inside the liposomes over 24 h as determined both in vitro and in vivo.3−5 In addition, the liposomes do not seem to enter the nuclei even after entry to the EMT6/WT cells as observed in this work (Figure 4C). Inadequate DOX release and low level entry to nuclei may contribute to the reduced cytotoxicity of PLD as compared to free DOX, DPLN, and DMsPLN. One might suspect that surfactant PF68 used in the PLN formulations might influence the cellular uptake and cytotoxicity of the nanoparticles, as Pluronic block copolymers have been reported to modulate P-gp-mediated drug transport.42−44 In a series of studies conducted by Kabonov and co-workers, P-gp modulating effect of Pluronic P85 and L61 on cell uptake of rhodamine 123 and DOX cytotoxicity in cancer cells was found, however, not so for PF68 due to its low hydrophobicity.42,43 In more recent work, Huang et al. reported that PF68 at 0.03% to 0.3% was found to affect P-gp-mediated transport of celiprolol in Caco-2 cells.44 In our nanoparticle formulation, 50 μL of 100 mg/mL PF68 were added in 400 + 388 μL of distilled deionized water containing other solid components, then the nanoemulsion was transferred to 2.5 mL of 0.9% NaCl solution (saline). For the in vitro cytotoxicity tests, the DOX concentration ranging from 0.01 to 100 μM was prepared by adding DMsPLN suspensions to culture medium. The resultant maximum

concentration in the 96-well plate was thus only 0.006% PF68, which was 2−20% of those reported by Huang et al.44 Thus, we do not expect a noticeable positive effect of PF68 on the cellular uptake and cytotoxicity of the PLN formulations. Nevertheless, it would be interesting to examine whether adding PF68 to PLD would enhance its effect on MDR cells. Synergy of Free and Nanoparticulate DOX-MMC Combination. Combination chemotherapy has been widely investigated clinically and preclinically.22−24,30,31 To assess if a drug combination produces a synergistic effect, median effect analysis, a mathematical treatment of drug interactions based on the law of mass action, is the method of choice.38 In the present study, we employed this method to analyze the effect of DOXMMC combinations in different formulations in vitro in EMT6/ WT and MDR EMT6/AR1 cells. CI was used to determine whether each formulation of DOX and MMC combination was synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1). On the basis of these in vitro results a promising drug formulation was selected for in vivo study. In the in vitro cytotoxicity study, combinations of free DOX and free MMC demonstrated superior reductions in cell viability over monochemotherapy (Figure 1A). This result is consistent with our previous work, which has demonstrated this synergism in EMT6/WT cells using a clonogenic assay after coadministering free DOX and MMC at a variety of concentrations.6 However, the free DOX and MMC 2670



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In fact, the mean survival times of previously reported antitumor efficacy of DOX-based micro-26,51 and nanoparticulate29 formulations and free chemotherapeutic agents against EMT6/WT mouse models did not exceed more than 10 days growth delay over the saline control. In the only report where an anthracyclinebased chemotherapeutic regimen was assessed against an EMT6/AR1 mouse model, there was no survival advantage over the saline control.49 However, in this difficult to treat breast cancer model, the DMsPLN showed significantly greater therapeutic efficacy over any previous anthracycline-based interventions, as well as over the clinically used PLD in both sensitive and MDR tumor models (Figures 5 and 6). This enhancement over PLD may result from the synergistic effect of DOX and MMC as evidenced in our in vitro studies.25,26 DMsPLN treatment has achieved statistically significant (p < 0.05) tumor growth delay for the first time in vivo against the highly resistant EMT6/AR1 tumor type. However, the efficacy in the MDR tumor is not as high as in the WT tumor (Figures 5 and 6). The inability of DMsPLN to completely overcome the MDR phenotype of EMT6/AR1 tumors as they did in vitro28,30 may be due to faster growth of the MDR tumor in vivo compared to the WT tumor as well as other factors. In addition, increased membrane rigidity of resistant cells compared to the WT cells could have impaired the endocytic process of DMsPLN.52 Drug resistant tumors induced a more robust and more rapid response to chemotherapeutic stress over WT tumors in vivo.53 MDR tumorbearing mice have altered metabolism relative to WT tumorbearing animals with negative alterations of pharmacokinetic parameters, such as a reduced area under the curve.53 Since nanoparticle penetration into tumor tissue is an important determinant of the success of drug delivery and therapeutic efficacy,54,55 the collagen-rich extracellular matrix of MDR tumors may limit the penetration of nanoparticles into tumor tissue, preventing effective chemotherapy. Furthermore, other factors in tumor matrix may contribute to tumor associated physiological parameters such as perfusion and lymphatic drainage, thus influencing nanoparticle retention.56 More investigations on the dose and treatment schedule as well as extracellular matrix modulation are needed for further improving the response of MDR tumor to nanomedicine. Previously we have demonstrated a significantly higher TGD with DMsPLN treatment in immunocompromised mice bearing human MDA-MB 435/MDR1 breast tumor33 compared to the TGD observed in the nonimmunocomprised Balb/c mice with EMT6/AR1 tumor in the present study. We speculate that this significant difference can be ascribed to the aggressiveness of the EMT6/AR1 cells resulting in much faster tumor growth than MDA-MB 435/LCC6/MDR1 cells as seen previously. The host−cancer cell interactions in the tumor microenvironment could also play a role in the current study when murine EMT6 breast cancer cells were used to establish tumor in mice.34−36 Another difference between EMT6/AR1 and MDA-MB 435/ LCC6/MDR1 cell lines is that the latter was obtained by MDR1 gene transfection, while the former cell line was selected by adding a low dose of DOX, similar to the clinical scenario of acquired MDR following chemotherapy. Thus, the MDR mechanism of EMT6/AR1 cells could be more complex than the MDR1 gene overexpressing cells, a possibility that deserves further investigation. Possible Mechanisms of Low Systemic and Cardiotoxicity of DMsPLN as Compared to PLD. Equal in clinical importance to the achieved level of antitumor efficacy, the DMsPLN formulation, even at 4× total cumulative doses, was

Figure 10. DMsPLN formulation shows no evidence of treatmentrelated cardiotoxicity, while PLD formulation exhibits treatment-related cardiomyocyte degeneration. Hearts were analyzed at 1.2× magnification for whole organ morphology (A−D) and at 40× magnification for longitudinal (E−H,M−P) and cross-sectional view (I−L,Q−T). White arrowheads represent regions of myocardial degeneration in H&E stained sections, and black arrowheads indicate regions of myocardial damage indicated by PAS staining.

combination failed to exert a synergistic effect against MDR EMT6/AR1 cells (Figure 3A). A synergistic effect of the DOXMMC combination was only observed when they were encapsulated in PLN and administered together either by DPLN + MPLN or DMsPLN (Figures 1 and 2). These results indicate that the nanoparticle carrier, PLN, in fact enabled the synergistic effect of DOX and MMC in the MDR cells. High in Vivo Efficacy of DMsPLN Likely Resulted from Nanoscale Delivery of DOX and MMC. The investigation of the in vivo efficacy of DOX-MMC nanoparticle formulation was pursued in a murine breast tumor model that responded poorly to free DOX treatment. Although in vitro cytotoxicity data suggested combination treatment using single agent-loaded nanoparticles, DPLN plus MPLN, may be better than DMsPLN in the EMT6/AR1 cells (Figure 1D), no significant difference between these two treatment regimens was found in the EMT6/WT cells. Moreover, it is well documented that DOX and MMC have completely different pharmacokinetic profiles with elimination half-life (t1/2) being 7−20 h and 7−90 min, respectively.45−49 If DPLN and MPLN are administered systemically, the chance for DOX and MMC to enter the same cancer cell is slim, especially after 1 h or so, which is unlike that in a cell culture dish. Therefore, we selected DMsPLN for in vivo studies. We chose PLD, a clinically employed liposomal formulation of DOX, as the comparator to evaluate the efficacy and toxicity profile of DMsPLN at clinically relevant doses. PLD-based treatment reduces the acute cardiotoxicity of DOX;12 however, it increases the incidence of PPE8,50 and has not conclusively demonstrated higher therapeutic efficacy.12 The murine EMT6 tumor, while not human, provided an animal model of aggressive breast cancer, demonstrating a rapid growth rate with short mean survival times in untreated animals. 2671



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PLD, poly(ethylene glycol)-coated (PEGylated) lipososmal DOX; TGD, tumor growth delay; TLD, tumor plus leg diameter

significantly less toxic than a single dose of PLD to the animal as a whole, as indicated by normal body weight values (Figure 8), and to the heart, as indicated by both plasma CPK levels (Figure 9) and myocardial histological examination (Figure 10). The severe anorexia observed in this work with PLD has been noted elsewhere as a side effect of this treatment in mice.15 The mechanism of DOX cardiotoxicity is not entirely clear, but doxorubicinol (DOXol), the major phase I metabolite of DOX, has been implicated.57,58 DOXol is the product of C13 carbonyl reduction of DOX, mediated predominantly by aldo−keto reductase isoforms 1C3 and 7A2 and by carbonylreductase isoform 1 and, to a lesser extent, isoform 3.57,58 The tissue expression levels of these metabolizing enzymes and the biodistribution of the nanoparticle drug carriers may help explain the reduced cardiotoxicity of the DMsPLN formulation relative to PLD. For all of these enzymes, their expression in the liver significantly exceeds their expression in the heart.57,58 The stealth PLN formulation has been shown to effectively avoid hepatic uptake,32 in contrast to the large hepatic retention of stealth liposomal formulations.59 If in fact the liver is responsible for the majority of DOXol production and DMsPLN can evade liver capture, the metabolism of DOX to DOXol may be reduced by using PLN formulations. By limiting DOXol production, cardiotoxicity may be avoided. Further studies investigating this hypothesis are currently under way. Regardless of mechanism, by reduction of the cardiotoxicity that severely limits the total dose of DOX, DMsPLN could be administered at more frequent and numerous intervals, with the potential for more aggressive and effective breast cancer chemotherapy.

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REFERENCES

(1) Buzdar, A. U. Topoisomerase IIα Gene Amplification and Response to Anthracycline-Containing Adjuvant Chemotherapy in Breast Cancer. J. Clin. Oncol. 2006, 24, 2409−2411. (2) Moore, M. J.; Ehrlichman, C. Pharmacology of Anticancer Drugs. In The Basic Science of Oncology, 3rd ed.; Tannock, I. F., Hill, R. P., Eds.; McGraw-Hill: Toronto, 1998; pp 370−391. (3) Pacini, P.; Rinaldini, M.; Algeri, R.; Guarneri, A.; Tucci, E.; Barsanti, G.; Neri, B.; Bastiani, P.; Marzano, S.; Fallai, C. FEC (5-Fluorouracil, Epidoxorubicin and Cyclophosphamide) versus EM (Epidoxorubicin and Mitomycin-C) with or without Lonidamine as First-Line Treatment for Advanced Breast Cancer. A Multicentric Randomised Study. Final Results. Eur. J. Cancer. 2000, 36, 966−975. (4) Bergh, J.; Jonsson, P. E.; Glimelius, B.; Nygren, P. A Systematic Overview of Chemotherapy Effects in Breast Cancer. Acta Oncol. 2001, 40, 253−281. (5) Smith, L. A.; Cornelius, V. R.; Plummer, C. J.; Levitt, G.; Verrill, M.; Canney, P.; Jones, A. Cardiotoxicity of Anthracycline Agents for the Treatment of Cancer: Systematic Review and Meta-Analysis of Randomised Controlled Trials. BMC Cancer 2010, 10, 337. (6) Gabizon, A.; Shmeeda, H.; Barenholz, Y. Pharmacokinetics of Pegylated Liposomal Doxorubicin: Review of Animal and Human Studies. Clin. Pharmacokinet. 2003, 42, 419−436. (7) Shi, Y.; Moon, M.; Dawood, S.; McManus, B.; Liu, P. P. Mechanisms and Management of Doxorubicin Cardiotoxicity. Herz 2011, 36, 296−305. (8) Leonard, R. C.; Williams, S.; Tulpule, A.; Levine, A. M.; Oliveros, S. Improving the Therapeutic Index of Anthracycline Chemotherapy: Focus on Liposomal Doxorubicin (Myocet). Breast 2009, 18, 218−224. (9) Lai, H. C.; Yeh, Y. C.; Ting, C. T.; Lee, W. L.; Lee, H. W.; Wang, L. C.; Wang, K. Y.; Lai, H. C.; Wu, A.; Liu, T. J. Doxycycline Suppresses Doxorubicin-Induced Oxidative Stress and Cellular Apoptosis in Mouse Hearts. Eur. J. Pharmacol. 2010, 644, 176−187. (10) Hryniuk, W.; Frei, E.; Wright, F. A. A Single Scale for Comparing Dose-Intensity of All Chemotherapy Regimens in Breast Cancer: Summation Dose-Intensity. J. Clin. Oncol. 1998, 16, 3137−3147. (11) Mouridsen, H. T.; Alfthan, C.; Bastholt, L.; Bergh, J.; Dalmark, M.; Eksborg, S.; Hellsten, S.; Kjaer, M.; Peterson, C.; Skovsgard, T.; et al. Current Status of Epirubicin (Farmorubicin) in the Treatment of Solid Tumours. Acta Oncol. 1990, 29, 257−285. (12) van Dalen, E. C.; Michiels, E. M.; Caron, H. N.; Kremer, L. C. Different Anthracycline Derivates for Reducing Cardiotoxicity in Cancer Patients. Cochrane Database Syst. Rev. 2010, CD005006. (13) Charrois, G. J.; Allen, T. M. Drug Release Rate Influences the Pharmacokinetics, Biodistribution, Therapeutic Activity, and Toxicity of Pegylated Liposomal Doxorubicin Formulations in Murine Breast Cancer. Biochim. Biophys. Acta 2004, 1663, 167−177. (14) Cui, J.; Li, C.; Guo, W.; Li, Y.; Wang, C.; Zhang, L.; Zhang, L.; Hao, Y.; Wang, Y. Direct Comparison of Two Pegylated Liposomal Doxorubicin Formulations: Is AUC Predictive for Toxicity and Efficacy? J. Controlled Release 2007, 118, 204−215. (15) Mamidi, R. N.; Weng, S.; Stellar, S.; Wang, C.; Yu, N.; Huang, T.; Tonelli, A. P.; Kelley, M. F.; Angiuoli, A.; Fung, M. C. Pharmacokinetics, Efficacy and Toxicity of Different Pegylated Liposomal Doxorubicin Formulations in Preclinical Models: Is a Conventional Bioequivalence Approach Sufficient to Ensure Therapeutic Equivalence of Pegylated Liposomal Doxorubicin Products? Cancer Chemother. Pharmacol. 2010, 66, 1173−1184. (16) Safra, T.; Muggia, F.; Jeffers, S.; Tsao-Wei, D. D.; Groshen, S.; Lyass, O.; Henderson, R.; Berry, G.; Gabizon, A. Pegylated Liposomal Doxorubicin (Doxil): Reduced Clinical Cardiotoxicity in Patients Reaching or Exceeding Cumulative Doses of 500 mg/m2. Ann. Oncol. 2000, 11, 1029−1033. (17) Longley, D. B.; Johnston, P. G. Molecular Mechanisms of Drug Resistance. J. Pathol. 2005, 205, 275−292.

AUTHOR INFORMATION

Corresponding Author

*(X.Y.W.) Tel: (416) 978-5272. E-mail: [email protected]. ca. Author Contributions ∥

These authors (A.J.S., P.P., and R.X.Z.) contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors sincerely acknowledge the Canadian Breast Cancer Foundation−Ontario Region for funding the in vivo studies of this project, Canadian Institutes of Health Research for an equipment grant and funding the in vitro studies, Natural Sciences and Engineering Research Council of Canada (NSERC) for equipment grants, NSERC CGS scholarship and Ben Cohen top up fund to AJS, University Open Scholarship to PP and RZ, Dr. Z. Liu (National Center for Agricultural Utilization Research, US Department of Agriculture) for providing HPESO sample, and Jean Flanagan and Debbie Squires (Ontario Cancer Institute Animal Colony) for technical assistance with the animal model and blood collection procedures.

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ABBREVIATIONS USED ALT, alanine transaminase; CI, combination index; CPK, creatine phosphokinase; DMsPLN, doxorubicin and mitomycin C coloaded stealth polymer−lipid hybrid nanoparticles; DOX, doxorubicin; DOXol, doxorubicinol; DPLN, doxorubicin loaded polymer−lipid hybrid nanoparticles; MDR, multidrug resistance; MPLN, mitomycin C loaded polymer−lipid hybrid nanoparticles; PF68, Pluronic F-68 nonionic block copolymer; 2672



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