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Cisplatin Crosslinked Multifunctional Nanodrugplexes for Combination Therapy Weiqi Zhang, and Ching-Hsuan Tung ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16500 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017
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Cisplatin Crosslinked Multifunctional Nanodrugplexes for Combination Therapy Weiqi Zhang and Ching-Hsuan Tung* Molecular Imaging Innovations Institute, Department of Radiology, Weill Cornell Medicine *To whom correspondence should be addressed. 413 East 69th Street, Box 290, New York, NY 10021, USA; E-mail:
[email protected]. Abstract Combination therapy efficiently tackles cancer by hitting multiple action mechanisms. However, drugs administered, simultaneously or sequentially, may not reach the targeted sites with the desired dose and ratio. The outcomes of combination therapy could be improved with a polymeric nanoparticle, which can simultaneously transport an optimal combination of drugs. We have demonstrated a simple one-pot strategy to formulate nanomedicines based on platinum coordination and the non-covalent interactions of the drugs. A naturally occurring polymer, hyaluronan (HA), was chosen as the building scaffold to form a nanodrugplex with cisplatin and aromatic-cationic drugs. The platinum coordination between cisplatin and HA induce the formation of a nano complex. The aromatic-cationic drugs are tightly packed by an electrostatic interaction and π–π stacking. The nanodrugplex bears excellent flexibility in drug combination and size control. It is stable in storage and has favorable release kinetics and targeting capabilities toward CD44, a receptor for HA that is highly expressed on many types of cancer cells.
Keywords: Hyaluronan, drug delivery, combination therapy, self-assembly, noncovalent interactions
Introduction With the rapid development of nanotechnology, nanoparticle-based multidrug delivery has demonstrated to have a maximized therapeutic outcome, with reduced systematic toxicity, in animal models and clinic trials.1-3 The nano-formulation incorporates two or more drugs into one nanoparticle with distinctive solubility and modulates the drug ratio and release behavior. The nanoparticle could be selectively accumulated at the tumor through the enhanced permeation and retention (EPR) effect or tumor-marker mediated delivery by modulating the size and attaching a specific ligand on the nanoparticles.4 Among the various materials used in drug delivery, organic polymers are one of the most promising materials in nanoparticle formation due to its low cost, excellent biocompatibility, low immunogenicity, and availability of various functional groups for chemical modification.5 Drugs can be loaded onto the nanoparticle through a covalent or noncovalent strategy. The covalent strategy will provide better stability; however, the synthesis may require multiple reaction, purification, and verification steps. If catalysts and harsh reaction conditions are used, the drug’s efficacy could be compromised. Furthermore, the stable chemical bond may attenuate the drug’s release from the nanoparticles. In contrast, the non-covalent strategy withholds the drugs less strongly, facilitating the nanoparticle’s formation and drug release.6 Although micelle and liposome encapsulation strategies, based on noncovalent concepts,
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have been widely exploited to load drugs using modified polymers,7 multiblock copolymers8,9,10 or amphiphilic lipids,11 it is still challenging to employ noncovalent interactions to encapsulate multiple drugs with different physicochemical properties into a natural polymer-based drug delivery system with favored properties, including controlled size, drug loading ratio, release behavior, and selective cytotoxicity.
Figure 1 Schematic illustration of the preparation of nanodrugplex using HA, Cis and aromatic-cationic drugs. HA, Cis and aromatic-cationic drug (Gefi et al) were mixed and incubated at 90 °C for different hours and then the mixture was cooled on ice and purified through dialysis. Carboxyl groups of HA were crosslinked by cisplatin through platinum coordination and meanwhile interacted with amines of the aromatic-cationic drug by electrostatic interaction. The π- π stacking of aromatic-cationic drug itself further enhanced the tight packing. The nanodrugplex can be internalized though HA’s receptor, CD44, a protein highly expressed on various cancer cells. Drugs are released in cells with the degradation of HA.
Cisplatin, with low water solubility, is one of the most widely used cancer chemotherapeutics.12 It is able to cross-link DNA through a coordination between platinum and guanine bases on DNA13. Inspired by this inducible coordination process, strategies have been developed to prepare various cisplatin conjugated nanomedicines. For example, complexes of PEG-b-poly(L-glutamic acid) copolymer and cisplatin (NC-6004) is currently under a phase II/III clinical trial for pancreatic cancer.14 A cisplatin bound polylactide nanoparticle has been prepared to co-transport docetaxel.15 In this study, we have developed a one-pot preparation method to build a multi-drug delivery platform that is based on the same crosslinking property,. This one-pot self-assembly method is simple and does not require any chemical modifications to the components. HA, a main component of the extracellular matrix, is composed of a repeat sugar unit of D-glucuronic acid and N-acetyl-D-glucosamine and is already approved by the FDA for cosmetic surgery.16 HA contains a large number of negatively charged carboxyl groups, which allowed it to form coordinate bonds with the platinum on cisplatin.17-19 In the presence of positively charged aromatic drugs, the electrostatic interaction between the drug and HA20 and the hydrophobic π- π stacking of drugs21 further promoted the nanodrugplex’s formation (Figure 1). HA is also a natural ligand for the CD44 receptor that is highly expressed on a panel of
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cancer cells and lymphocytes22. The formed nanodrugplex could also have a targeting potential towards a CD44 overexpressed tumor.
Materials and methods Materials Hyaluronan (HA) (MW=1000 kDa) was purchased from LifeCore (Chaska, MN). Gefitinib (Gefi) was from LC Laboratories (Boston, MA). Methotrexate (MTX), Rhodamine B (Rb), Chloroquine (CQ), Camptothecin (CPT), Paclitaxel (PTX), cis-Diammineplatinum(II) dichloride (cisplatin, Cis), o-Phenylenediamine (OPDA) and Dimethylformamide (DMF) were purchased from Sigma (St. Louis, MO). Nanodrugplex synthesis HA was dissolved in ddH2O at the concentration of 5 mg/ml. Cis (15 mg/ml) was dissolved in ddH2O with heating at 90 °C. The concentration of Rb in ddH2O, and Gefi and MTX in DMF was set at 10 mg/ml. Briefly, the Cis solution was first mixed with Rb, Gefi, or MTX of different weight ratios in an eppendorf tube at 90 °C. 800 µl of HA was then added and the final volume of the mixture was fixed at 1.2 ml by adding water. The completed mixture was then incubated at 90 °C for pre-determined hours. After incubation, the acquired solution was cooled on ice for 15 min, transferred to a dialysis tube (MWCO=3.5K, Thermo Fisher) and dialyzed in 1L of ddH2O at room temperature. The dialysis was conducted at room temperature for 1 day. The dialysis water was collected and changed for 4 times. The free drug in the collected water was quantified based on their absorbance, according to a corresponding standard curve. Absorbance at 330 nm, 303 nm, and 544 nm was used to quantify Gefi, MTX and Rb, respectively. The incorporated drug was calculated by subtracting the free drug from the drug input during synthesis. The Cis content was determined using the o-phenylenediamine (OPDA) method described in the supplementary information. The incorporation and loading efficiency of the drug were expressed as follow: =
× 100% ℎ
× 100% ℎ To probe the effects of the input drug on the nanodrugplex, the HA and Cis to drug (Gefi, MTX or Rb) ratios were set as 16:9:1, 16:9:2, 16:9:4, 16:9:6 and 16:9:8. The 2 hour incubation time was fixed at 90 °C. The final products were designated as HA/Cis/Gefi, HA/Cis/MTX and HA/Cis/Rb (16:9:1), (16:9:2), (16:9:4), (16:9:6) and (16:9:8), respectively. To confirm the influence of the Cis input on the nanodrugplex, the HA, and Cis to Gefi or Rb ratio was varied as 16:1:4, 16:3:4, 16:6:4, 16:9:4 and 16:15:4 and the HA, Cis to MTX ratios were fixed at 16:1:2, 16:3:2, 16:6:2, 16:9:2 and 16:15:2 while the 90 °C incubation time was set for 2 hours. The resulting nanodrugplex was named as HA/Cis/Gefi and HA/Cis/Rb (16:1:4), (16:3:4), (16:6:4), (16:9:4) and (16:15:4) or HA/Cis/MTX (16:1:2), (16:3:2), (16:6:2), (16:9:2) and (16:15:2). To probe the effects of the total incubation time at 90 °C on the nanodrugplex, the HA, Cis, to Gefi, MTX or Rb ratios were fixed at 16:9:4, 16:9:2 and 16:9:4, respectively. The incubation times at =
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90 °C were 1, 2, 4, 8 and 12h. The acquired nanodrugplexes were named HA/Cis/Gefi, HA/Cis/MTX and HA/Cis/Rb (1h), (2h), (4h), (8h) and (12h). To prepare HA/Cis/CQ/Rb, Cis, CQ and Rb were first mixed as described above, and then 800 µl of 5 mg/ml HA was added. The weight ratio of HA to Cis, CQ and Rb was set at 8:6:2:1. After a 6-hour incubation at 90 °C, the mixture was cooled on ice and subjected to dialysis against water. The final CQ and Rb contents were quantified based on their specific absorbance (CQ: 343 nm and Rb: 544 nm), as mentioned above, because their absorption peak had minimal overlap. A HA/Cis/Rb particle with the same reaction condition was set as the control with a weight ratio fixed at 8:6:1 during the synthesis. UV-Vis-NIR absorption spectroscopy The absorption spectrum of the formed nanodrugplex was conducted on a Cary 60 UV-Vis Spectrophotometer. Briefly, the HA/Cis/Gefi, HA/Cis/MTX and HA/Cis/Rb were diluted to 0.1 mg/ml in an HA concentration using water. Approximately 500 µl of the above solution was scanned from 200 nm to 800 nm using a spectrophotometer. The wavelength was set at 1 nm. Dynamic light scattering (DLS) analysis The hydrodynamic size of the nanodrugplex was measured using a Zetasizer Nano-S (Malvern Instruments, UK). Around 50 µl of the acquired nanodrugplex was diluted by 950 µl H2O and subjected to DLS analysis. The zeta potential of the nanodrugplex was recorded with a Zeta PALS analyzer (Brookhaven Instruments, Holtsville, NY). Before the measurement was conducted, 250 µl of the nanodrugplex was mixed with 1 ml H2O. For all DLS analysis, each sample was measured at least twice. Transmission electron microscopy (TEM) About 5 µl of the nanodrugplex (0.8 mg/ml in HA) was dropped and left on a copper grid for 2 minutes. The extra solution was dried using filter paper. One drop of Uranyl acetate (1.5% in H2O) was then added for negative stains. The grid was stained for 1 min at room temperature; the staining solution was wicked away using filter paper. The grid was observed using a JEOL 1400 Transmission Electron Microscope with various magnifications. To analyze the diameter distribution of the nanodrugplex from TEM observations, the size of at least 200 particles was manually counted. Stability of nanodrugplex HA/Cis/Gefi, HA/Cis/MTX and HA/Cis/Rb (1h), (2h), (4h), (8h) and (12h) nanodrugplexes were stored in water at 4 °C. After the time points of 1, 2, 3, 5, 7, 11 and 15 weeks, the size and absorption spectra of the nanodrugplex was monitored by DLS and spectrophotometry analysis. The fluorescent spectra of HA/Cis/Rb were also recorded with an Rb concentration set at 10 µg/ml. All of the measurements were conducted at least in duplicate. Drug release of nanodrugplex in PBS One milliliter of the nanodrugplex solution was sealed in a dialysis tube (MWCO=3.5kDa) and submerged in 80 ml PBS in a beaker. The drug release was conducted at 37 °C in an incubator with a rotation speed at 100 rpm. At the time points of 0.5, 1, 2, 4, 8, 12, 24, 48, 72 and 96 hours, 1.6 ml PBS was collected and an equal volume of fresh PBS was added back. The Gefi, MTX, Rb or CQ content in the collected PBS was quantified based on their corresponding absorbance.
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Meanwhile, 250 µl of collected PBS was subjected to OPDA method mentioned above to quantify Cis. Cis was calculated according to the standard curve of Cis dissolved in PBS. Cell culture Human breast cancer cell lines MDA-MB-231 and MCF-7 were acquired from the American Type Culture Collection (ATCC, Manassas,VA). Cells were cultured in a DMEM medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin and maintained at 37 °C in an incubator with humidified air containing 5% CO2. Fluorescent microscopy MDA-MB-231 and MCF-7 cells (4000/well) was seeded in a 96-well plate (black, clear bottom) and left overnight for cell attachment. The cells were incubated with Rb, HA/Cis/Rb (2h) containing 6 µM Rb, and the same concentration of HA/Cis/Rb but with 10 folds of extra HA in the medium for 30 minutes. Post incubation, the cells were washed twice using PBS and then re-incubated in fresh DMEM medium. After 0, 3 and 15-hours of further incubation, the cells were imaged using EVOS fluorescent microscope (Life Technologies). To probe the cellular localization of HA/Cis/Rb and HA/Cis/CQ/Rb, 15000 MDA-MB-231 cells were seeded in a chamber slide and cultured overnight to let cells adhere. Cells were incubated with 12 µM free CQ, 6 µM free Rb, HA/Cis/Rb containing 6 µM Rb, and HA/Cis/CQ/Rb containing 12 µM CQ and 6 µM Rb for 23 hours. Then the cells were washed twice and stained with 2 µM LysoTracker blue together with 50 nM MitoTracker green (Life Technologies) for 1h at 37 °C. After staining, the cells were rinsed twice using PBS before fluorescent imaging. The DAPI, GFP, and RFP channels were used to image LysoTracker blue, MitoTracker green, and Rb, respectively. When cell imaging was performed, the culture medium was switched to a phenol red free DMEM medium. MTS assay About 4000 MDA-MB-231 or MCF-7 cells were seeded in a 96-well plate and incubated overnight to allow cell adherence. In order to compare the cytotoxicities between free drug, free drug combination, and the multidrug nanodrugplex (HA/Cis/Gefi (2h) and HA/Cis/MTX (2h)), cells were treated by 12 µM Cis, 15 µM Gefi, 5 µM MTX, 12 µM Cis + 15 µM Gefi, 12 µM Cis + 5 µM MTX, HA/Cis/Gefi containing 12 µM Cis and 15 µM Gefi, and HA/Cis/MTX containing 12 µM Cis and 5 µM MTX. Each treatment was conducted in triplicate and cells without treatments were set as the control. After 2 and 3-days incubation, the cells were rinsed with PBS and the cell viability was evaluated by MTS assay kit (Promega). Statistic analysis The statistic difference was determined by two-sided student’s t-test. *P
