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REVIEW ARTICLE
Passive Targeting of Cyclophosphamide-Loaded Carbonate Apatite Nanoparticles to Liver Impedes Breast Tumor Growth in a Syngeneic Model S. Tiash and E.H. Chowdhury* Advanced Engineering Platform and Jeffrey Cheah School of Medicine and Health Sciences, MONASH University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, Malaysia
ARTICLE HISTORY Received: November 11, 2016 Accepted: February 10, 2016 DOI: 10.2174/1381612822666160211 141918
Abstract: Despite being widely used for treating cancer, chemotherapy is accompanied by numerous adverse effects as a result of systemic distribution and nonspecific interactions of the drugs with healthy tissues, eventually leading to therapeutic inefficacy and chemoresistance. Cyclophosphamide (Cyp) as one of the chemotherapeutic pro-drugs is activated in liver and used to treat breast cancer in high dose and in combination with other drugs. In an Please provide corresponding author(s) attempt to reduce the off-target effects and enhance the therapeutic efficacy, pH-sensitive photograph carbonate apatite nanoparticles that had predominantly and size-dependently been localized size should be 4" x 4" inches in liver following intravenous administration, were employed to electrostatically immobilize Cyp and purposely deliver it to the liver for activation. Cyp-loaded particles formed by simple 30 min incubation at 37ºC of the DMEM (pH 7.4) medium containing CaCl2 and Cyp, enhanced in vitro cytotoxicity at different degrees depending on the cell types. The size of the particles could be tightly controlled by the amount of CaCl2 required to prepare the particles and thus the bio-distribution pattern inside different organs of the body. Unlike the small particles (~ 200 nm), the large size particles (~ 600 nm) which were more efficiently accumulated in liver, significantly reduced the tumor volume following intravenous injection in 4T1-induced murine breast cancer model at a very low dose (0.17 mg/Kg) of the drug initially added for complex formation, thus shedding light on the potential applications of the Cyp-loaded nano-formulations in the treatment of breast cancer.
Keywords: Cyclophosphamide, breast cancer, carbonate apatite, nanoparticles, bio-distribution, cytotoxicity, tumor regression, immunocompetent mouse. INTRODUCTION Breast cancer is one of the utmost serious threats to women’s health worldwide. Chemotherapy based on the chemical drugs is the most favored strategy to treat cancer and mostly used after surgical removal of tumor to prevent disease recurrence. A diverse number of structurally dissimilar chemical drugs are used to treat cancer with different modes of actions. Finally, all of these drugs induce apoptosis, the programmed cell death in the uncontrolled highly dividing cells of cancerous tissues. However, non-specific interactions of the drugs particularly with healthy dividing cells all over the body cause inadequate drug concentration in the tumor microenvironment diminishing effectiveness of the treatment. As a result, the dose of a drug needs to upturn to achieve the therapeutic activity. On the other hand, toxicity to healthy tissues compromises the doses and frequency of treatment and thus reduces the therapeutic efficacy of the drug with development of chemoresistance. Therefore, it is important to develop a nano-delivery device to carry drugs to the target tumor tissues so as to reduce the distribution of drug to healthy tissues and thus attain more therapeutic action at very low drug doses. Cyclophosphamide (Cyp) is a nitrogen mustard alkylating agent used to treat a board spectrum of cancers including breast cancer and lymphomas and autoimmune disorders [1-7]. As a prodrug it is enzymatically converted in the liver to active metabolite phosphoramide mustard with chemotherapeutic activity. Cyclophosphamide enters into liver cells by unknown transporters and forms active 4-hydroxy cyclophosphamide that rapidly interconverts with *Address correspondence to this author at the Faculty of Medicine, Nursing and Health Sciences, Monash University, Malaysia; Tel: +603-5514-4978; E-mail:
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its tautomer aldophosphamide. Both of these metabolites diffuse out of the liver cells, circulate and passively enter into other cells, for example cancer cells. Aldophosphamide undergoes non-enzymatic elimination reaction to yield the active form phosphoramide mustard. As an alkylating agent, it attaches an alkyl group (CnH2n+1) to the guanine base of DNA at number 7 nitrogen atom of the imidazole ring and thus interferes with DNA replication by forming intrastrand and interstrand DNA crosslinks [8]. The drug is required in high dose to exert its effect [9-11] and used mostly in combined therapy along with other drugs [12-16]. Different immunoregulatory response was observed in animal models treated with different concentration of Cyp (20-200 mg/Kg in mice models) [17-28]. Other adverse drug reactions include chemotherapy-induced nausea and vomiting, bone marrow suppression, stomach ache, hemorrhagic cystitis, diarrhea, darkening of the skin/nails, alopecia (hair loss) or thinning of hair, changes in color and texture of the hair, and lethargy. Therefore, low dose of drug with higher potency is required to reduce the adverse effects while conferring efficacy to regress tumor. Recently developed carbonate apatite nanoparticles (NPs) with higher affinity for nucleic acids [29-35] can also bind to a number of structurally distinct anticancer drugs [36, 37]. These NPs have been used to deliver siRNAs and drugs individually or in combination for cancer therapy both in vitro and in vivo [37-42]. We have previously shown that Cyp could bind to the NPs with stronger affinity and showed more cytotoxic actions than free Cyp [36]. Therefore, we have used this NP-Cyp to treat the breast cancer mouse model developed by subcutaneous injection of 4T1 cells in the mammary pad. As stated earlier, Cyp is converted into its active metabolites in liver; we have therefore aimed at predominantly accumulating intravenously delivered NP-Cyp in liver through passive targeting by controlling the size of NP. We have revealed that © 2016 Bentham Science Publishers
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targeting liver by relatively larger NPs with bound Cyp could significantly reduce tumor volume at even very low concentration (0.17 mg/Kg) of the drug. METHODS AND MATERIALS Materials Dulbecco’s modified eagle medium (DMEM), calcium chloride dehydrate (CaCl2.2H2O), sodium bicarbonate (NaHCO3), thiazolyl blue tetrazolium bromide (MTT), trifluroacetice acid (TFA; CF3COOH), cyclophosphamide (Cyp), dithiotreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich (St Louis, MO, USA). DMEM powder, fetal bovine serum (FBS), tryple express and penicillin-streptomycin were obtained from Gibco BRL (California, USA). Acetonitrile (ACN) was from Fisher Scientific (Loughborough, UK). All the chemicals used for HPLC were HPLC grade. Alexa Fluor (AF) 488-labeled siRNA was purchased from Qiagen. Particle Preparation for In Vitro and In Vivo Study Apatite nanoparticles (NP) and Cyp-loaded nanoparticles (NPCyp) for in vitro study were prepared as described earlier [36]. Briefly, 3 µl or 7 µl of 1 M CaCl2 was mixed with 1 mL of freshly prepared bicarbonate-buffered DMEM (pH adjusted to 7.4), followed by 30 min incubation at 37°C. For fabrication of NP-Cyp complexes, M of Cyp was added together with CaCl2 to DMEM prior to the incubation. For in vivo study, particles were prepared in 100 µL of freshly prepared DMEM media mixed with either 4 µL or 7 µL of 1 M CaCl2 in smaller tubes and incubated for 30 min at 37°C. 0.17 mg/Kg of Cyp was used to prepare the complexes for treating animals. As for bio-distribution study, 4 or 7 µL of 1 M CaCl2 and 2 µM of fluorescent labeled molecule (AF-488 negative siRNA) were used to prepare fluorescent particles. Particle formulations were maintained on ice immediately after preparation till the injection was performed. Size Measurement Size distribution of NP and NP-Cyp complexes formed in 7 mM of CaCl2 for in vitro study was carried out with zeta sizer (Nano ZS, Malvern) after adding 10% FBS to stop particle aggregation and temporarily keeping at 4°C. A refractive index ratio of 1.325 was used for the estimation of particle size. Data were analyzed using Zetasize software 6.20 and all samples were measured in duplicate. For in vivo study, 900 µL of fresh media was added to 100 µL of NP and NP-Cyp complexes formed in either 4 µL or 7 µL of CaCl2, followed by addition of 10% FBS, keeping the samples on ice and measurement of particle diameter. Cell Viability Assay by 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT) Two invasive human ductal carconima cell lines, MDA-MB231 and and one mouse breast cancer cell line, 4T1 were cultured in a 75 cm2 tissue culture flasks (Nunc, Orlando, USA) and maintained in the DMEM media (pH 7.4) supplemented with 10% FBS and penicillin and streptomycin antibiotic, in a 37°C incubator humidified with 5% CO2. One day before treatment, cells from exponentially growth phase were seeded on a 24-well plate (Griener, Frickenhausen, Germany) with each of the wells allowed to have approximately 5104 MCF-7 or 4T1 cells and 1105 MDA-MB231 cells. After 24 hr, the cells were treated with NP, different concentrations of free and NP-Cyp (10% FBS was used to prevent further particle formation during the treatment) for different period of time (44 hr for MCF-7 and 4T1 while 24 hr for MDA-MB231 cells). After 24 hr treatment with MDA-MB231 cells, extracellular NP was washed out briefly with 200 µL of 5 mM EDTA-PBS and re-
Tiash and Chowdhury
placed with serum-containing DMEM media for another day. 3 mM of CaCl2 was used to prepare NP and NP-Cyp for cell treatment. After completing cell incubation for a total period of 44 hr, 50 µL of MTT (5 mg/mL in PBS) was added to each well for formation of formazan crystals by metabolically active cells. After 4 hr, the media was discarded and 300 µL of dimethyl sulfoxide (DMSO) was added to each well and plates were agitated on built-in plate shaker for 20 sec to dissolve the dark blue crystals of formazan. For control, cells were kept untreated in the media containing no exogenous CaCl2. Formazan quantification in the form of optical density (OD) was performed at 595 nm wavelength with 630 nm of reference wavelength using plate reader (microplate spectrophotometer, Biorad). The cell viability for free and NP-Cyp was calculated using the following equation:
Each experiment was done in triplicate and expressed in graphs as mean± SD of % of cell viability. Moreover % of cytotoxicity for free and NP-Cyp was calculated in terms of media (untreated) and NP treated cells respectively and enhancement in cytotoxic effect of the drugs due to the complexation with NPs was calculated as follows:
Where CYNP, CY free drug and CYNP drug represent the % of cytotoxicity due to NP, free drugs and NP-drugs, respectively. Enhancement in toxicity was calculated for all different concentrations and expressed as mean± SD. Bio-Distribution of NP in 4T1 Induced Tumor Mouse Model Female Balb/c mice (6-8 weeks old) of 15-20 gm of body weights were purchased from School of Medicine and Health Science animal facility, Monash University. The mice were maintained in 12: 12 light: dark condition and provided with ab libitum and water. All the experiments were done in accordance with the regulations imposed by Monash University Animal Welfare Committee. Approximately 1×105 4T1 cells (in 100 µL PBS) were injected subcutaneously on the mammary pad of mice (considered as day 1) and the mice were checked regularly for the outgrowth of tumor by touching the area of injection by index finger. For bio-distribution study, mice were administered with fluorescent AF-488 labeled neg. siRNA either in free or NP-bound form (groupings was done as stated in Table 1) through tail vein injection when the tumor volume reached to 13.20±2.51 mm3. Mice were sacrificed humanly by cervical dislocation after 4 or 24 hr of administration as specified under group names in Table 1. In addition, untreated mice were also sacrificed when the tumor volume reached to 13.20±2.51 mm 3 for comparison as control. After sacrifice, liver, kidney, spleen, lung, brain and tumor were collected and washed in chilled PBS for two times followed by addition of 1mL lysis buffer (0.072 gm DTT, 1% NP-40 in 50 mL of 1X PBS) per 500 mg of tissue mass. Tissues were lysed using the mechanical homogenizer (eppendrof) with four strokes intermittently while maintaining the samples on ice till a complete homogenized solution was made. The solutions of tissue lysates were centrifuged for 30 min at 4°C and 13,000 rpm. 100 µL of supernatant was added on 96-well opti-plate (Nunc) for measuring fluorescence intensity of AF-488 labeled siRNA with 2030 multilabel reader vitorTM X5 (Perkin Elmer) attached with Perkin Elmer 2030 manager software using λex = 490 nm and λem = 535 nm. Data were represented as mean± SD of fluorescence inten-
Passive Targeting of Cyclophosphamide-Loaded Carbonate Apatite
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Table 1. Treatment groupings for tumor regression analysis used in this experiment.
sity/500 mg of tissue mass after the values were blank-corrected using untreated group of mice for each tissue. Treatment of NP-Cyp in 4T1 Induced Breast Cancer Murine Model When the volume of the outgrowth tumor reached to an average 13.20±2.51 mm3, mice were grouped in different assemblies (6 mice per group) randomly and treated intravenously (tail-vein) at the right or left caudal vein as specified in Table 2. The second dose was administered after 3 days of the 1st dose. The gross body weights of mice were monitored and the lengths and widths of the outgrowth tumor were measured using the vernier caliper in mm scale for 30 days and the mice were monitored for their activities and then humanly sacrificed by cervical dislocation. The volume of the tumor was calculated using the following formula
The data have been presented here as mean± SD of tumor volume from each group. Statistical Analysis Statistical analysis was done using the SPSS (version 17 for windows) for in vivo (tumor regression and bio-distribution study) data. LSD post-hoc test for one way ANOVA was used to analyse and compare the significant difference between different treated groups. For bio-distribution study, data were compared among treated and untreated groups for each respective organ. Data were considered statistically significant when *p
