Doxorubicin-loaded cholic acid-polyethyleneimine micelles for targeted delivery of antitumor drugs: synthesis, characterization, and evaluation of their in vitro cytotoxicity
© Amjad et al.; licensee Springer. 2012
Received: 30 October 2012
Accepted: 16 December 2012
Published: 28 December 2012
Doxorubicin-loaded micelles were prepared from a copolymer comprising cholic acid (CA) and polyethyleneimine (PEI) for the delivery of antitumor drugs. The CA-PEI copolymer was synthesized via pairing mediated by N,N’-dicyclohexylcarbodiimide and N-hydroxysuccinimide using dichloromethane as a solvent. Fourier transform infrared and nuclear magnetic resonance analyses were performed to verify the formation of an amide linkage between CA and PEI and doxorubicin localization into the copolymer. Dynamic light scattering and transmission electron microscopy studies revealed that the copolymer could self-assemble into micelles with a spherical morphology and an average diameter of <200 nm. The CA-PEI copolymer was also characterized by X-ray diffraction and differential scanning calorimetry. Doxorubicin-loaded micelles were prepared by dialysis method. A drug release study showed reduced drug release with escalating drug content. In a cytotoxicity assay using human colorectal adenocarcinoma (DLD-1) cells, the doxorubicin-loaded CA-PEI micelles exhibited better antitumor activity than that shown by doxorubicin. This is the first study on CA-PEI micelles as doxorubicin carriers, and this study demonstrated that they are promising candidates as carriers for sustained targeted antitumor drug delivery system.
KeywordsMicelles Nanoparticles Cholic acid Polyethyleneimine Doxorubicin
Several therapeutic anticancer drugs, although pharmacologically effective in cancer treatment, are restricted in their clinical applications because of their severe toxicity. The severe toxicity is usually due to the lipid solubility of most of the anticancer drugs (>70%) and the therapeutic doses that are often very high. Doxorubicin is one of the most successful drugs for targeting a broad range of cancers. Nevertheless, its clinical use is hindered by its side effects, which include cardiotoxicity and acquired drug resistance. To overcome these complications, researchers have placed an emphasis on developing nanoscale anticancer drug carriers for improving therapeutic efficacy in addition to reducing unwanted side effects.
Polymeric micelles self-assembled from amphiphilic copolymers have gained much interest for use in targeted anticancer drug delivery since they have a number of physico- and bio-chemical advantages over other types of nanocarriers. Polymeric micelles are virus-sized with a core-shell structure having a hydrophobic core and a hydrophilic shell and, more significantly, inherent stealth. Polymeric micelles seem ideal for the targeted and controlled delivery of hydrophobic anticancer drugs, including paclitaxel and doxorubicin, in that they significantly increase their water solubility, extend their circulation time, passively target tumor tissues, increase their bioavailability, have tremendous biocompatibility, and are degradable in vivo into nontoxic products. Several types of polymer blocks can be used to form micelles, of which the most studied include poly(α-hydroxy esters) (such as polylactide, polyglycolide, and poly(ε-caprolactone)), polyether, hydrotrophic polymers, and poly(amino acids). Several attempts have been made to formulate stable polymeric micelles with new surfactant combinations to achieve ideal drug delivery in vitro as well as in vivo.
Cholic acid (CA), a bile acid, is an amphiphilic steroid molecule naturally synthesized from cholesterol, which organizes into micelles above the critical micelle concentration (CMC). Bile acids, together with the phospholipids, vary the permeability of cell membranes. Some bile acids form hydrogen-bonded aggregates with some drugs, which may lead to alterations in drug bioavailability. Polyethyleneimine (PEI) is a cationic synthetic vector mainly used for gene delivery owing to its high nucleic acid condensing potential, ability to escape endosomes, nuclear localization capability, and promising transfection efficacy both in vitro and in vivo.
We synthesized doxorubicin-loaded cholic acid-polyethyleneimine (CA-PEI) micelles as an antitumor drug delivery system. The antitumor activity of the doxorubicin-loaded CA-PEI micelles was then tested using human colorectal adenocarcinoma (DLD-1) cells.
CA, PEI (average molecular weight (MW) approximately 1,300), N,N’-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), hydrochloric acid (HCl), triethylamine, tetrahydrofuran, and dichloromethane were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). The Spectra/Por™ dialysis membrane (MW cutoff (MWCO) = 1,000 g/mol) was purchased from Spectrum Labs (Rancho Dominguez, CA, USA).
Synthesis of the CA-PEI copolymer
The resulting conjugates were dried using a rotary evaporator and dissolved in dilute HCl followed by precipitation with cold acetone. Finally, they were dissolved in deionized water, filtered, and freeze-dried.
Analysis of the conjugates
To assess their functional groups, drug-loaded and blank conjugates were characterized using a Fourier trans-form infrared (FTIR) spectrophotometer (Spectrum 100, PerkinElmer, Waltham, MA, USA) using the potassium bromide (KBr) disc method. For each sample, 16 scans were obtained at a resolution of 4 cm−1 in the range of 4,000 to 700 cm−1. Further characterization of the conjugates was also performed using nuclear magnetic resonance (NMR) spectroscopy (Bruker Avance III, FT-NMR 600 MHz with cryoprobe, Germany). The CMCs of the micelles were determined using the dynamic light scattering method (Zetasizer Nano ZS, Malvern Instruments, Malvern, Worcestershire, UK) at 37°C with a scattering angle of 90°. The alterations in light intensity were recorded, and a graph was plotted for the molar concentrations of the samples versus the mean intensity. A sharp increase in the intensity signified the formation of micelles. Samples for morphological investigations were prepared by air-drying a drop of the micellar suspension on a carbon-coated formvar film on a 400-mesh copper grid. The morphology of the micelles was then visualized by transmission electron microscopy (TEM; Tecnai™ Spirit, FEI, Eindhoven, The Netherlands) at 220 kV and under various magnifications. The conjugates were observed under a light microscope (FluoView FV1000, Olympus, Tokyo, Japan). The X-ray diffraction (XRD) patterns of the CA-PEI conjugates were analyzed with an X-ray diffractometer (D8 ADVANCE, Cu Kα = 1.54184 Å, Bruker, WI, USA). The thermal behavior of the conjugates was investigated by differential scanning calo-rimetry (DSC) (Diamond DSC, PerkinElmer, Waltham, MA, USA).
Preparation of the doxorubicin-loaded CA-PEI micelles
Doxorubicin hydrochloride (2.5 mg) was dissolved in 2 mL chloroform and mixed with 2 μL of triethylamine. CA-PEI copolymers of different molar ratios (1:1, 1:2, 1:4, 3:1, and 4:1) were dissolved in 2 mL methanol. The doxorubicin and CA-PEI copolymer solutions were mixed in a glass vial and kept in the dark for 24 h. The solution was then poured drop by drop into deionized water (20 mL) under ultrasonic agitation using a sonifier (Branson Ultrasonics Co., Danbury, CT, USA) at a power level of 3 for 10 min. The organic solvents namely chloroform and methanol were then completely removed by vacuum distillation using a rotary evaporator. The doxorubicin-loaded micelle solution was then dialyzed against 1 L of deionized water for 24 h at 20°C using a cellulose membrane bag (MWCO = 1,000) to remove unloaded doxorubicin. The deionized water was substituted every 2 h for the first 12 h and then every 6 h. Immediately after this, the product was freeze-dried. The extent of doxorubicin loaded into the micelles was determined from a calibration curve of pure doxorubicin. Freeze-dried doxorubicin-loaded micelles were dissolved in 4 mL of a DMSO and methanol mixture (1:1), and the absorbance was measured at 480 nm using a UV-1601 spectrophotometer (Shimadzu Corp., Kyoto, Japan).
In vitro drug release study
The drug release experiment was carried out in vitro. A doxorubicin-loaded micelle solution previously prepared by dialysis was used for release analysis. This solution was introduced into the dialysis membrane. Subsequently, the dialysis membrane was placed in a 200-mL beaker with 100 mL of phosphate-buffered saline (PBS). This beaker was placed on a magnetic stirrer with a stirring speed of 100 rpm at 37°C. At suitable intervals, 3 mL samples were taken from the release medium and an equivalent volume of fresh medium was added. The concentration of doxorubicin in each sample was measured by ultraviolet–visible spectrophotometry at 480 nm.
Human colorectal adenocarcinoma (DLD-1) and Chinese hamster lung fibroblast (V79) cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). DLD-1 cells were cultured and maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium, whereas V79 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Both cell lines were supplemented with 10% FBS and 1% penicillin-streptomycin and maintained at 37°C in a humidified 5% CO2/95% air atmosphere.
Results and discussion
Formation and characterization of the CA-PEI micelles
The facially amphipathic CA was introduced into PEI to prepare stable CA-PEI micelles as carriers for the delivery of doxorubicin. The CA terminal carboxyl group that was principally activated using DCC/NHS chemistry was conjugated to the PEI amine group via an amide linkage to obtain the CA-PEI conjugate (Figure1).
The freeze-drying process produced white crystalline CA-PEI conjugates where their morphology was observed under the light microscope as shown in Figure2b. The synthesized conjugates appeared as slender, needle-shaped small units. Each unit could be distinguished separately, and the length of the units varied slightly.
High CMCs are a key problem linked to micelle formulations given intravenously or diluted in blood. Low CMCs of CA-PEI micelles would thus offer some benefits, such as stability against dissociation and precipitation in blood due to dilution. In addition, embolism caused by the elevated amount of polymers used for the micelle formation could be avoided.
DLC and EE of doxorubicin-loaded micelles
DLC (% w/w)
EE (% w/w)
In vitro cell cytotoxicity
Here, we report the synthesis of doxorubicin-loaded novel CA-PEI micelles for the first time. The conjugates readily formed micelles, which exhibited a uniform spherical morphology as observed by TEM. XRD analysis revealed that the conjugates had a crystalline structure. Increasing the quantity of incorporated doxorubicin decreased the release rate of the drug. Doxorubicin-loaded CA-PEI micelles had an enhanced antitumor activity against tumor cells in vitro compared with that of doxorubicin itself. In contrast, when blank micelles were exposed to normal (V79) cells, they did not exhibit considerable toxicity. Together, these results indicate the potential of doxorubicin-loaded CA-PEI micelles as carriers for targeted antitumor drug delivery system.
Critical micelle concentration
Drug loading content
Dulbecco’s modified Eagle’s medium
Differential scanning calorimetry
Fourier transform infrared
Hydrogen nuclear magnetic resonance
Molecular weight cutoff
Transmission electron microscopy
This project was funded by a Research University Grant (UKM-GUP-SK-07-23-045) from Universiti Kebangsaan Malaysia (UKM) and Science Fund (02-01-02-SF0738) from the Ministry of Science, Technology and Innovation, Malaysia.
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