Doxorubicin-conjugated PLA-PEG-Folate based polymeric micelle for tumor-targeted delivery: Synthesis and in vitro evaluation
© Hami et al.; licensee BioMed Central Ltd. 2014
Received: 26 November 2013
Accepted: 25 February 2014
Published: 6 March 2014
Selective delivery of anticancer agents to target areas in the body is desirable to minimize the side effects while maximizing the therapeutic efficacy. Anthracycline antibiotics such as doxorubicin (DOX) are widely used for treatment of a wide variety of solid tumors.
This study evaluated the potential of a polymeric micellar formulation of doxorubicin as a nanocarrier system for targeted therapy of a folate-receptor positive human ovarian cancer cell in line.
DOX-conjugated targeting and non-targeting micelles prepared by the dialysis method were about 188 and 182 nm in diameter, respectively and their critical micelle concentration was 9.55 μg/ml. The DOX-conjugated micelles exhibited a potent cytotoxicity against SKOV3 human ovarian cancer cells. Moreover, the targeting micelles showed higher cytotoxicity than that of non-targeting ones (IC50 = 4.65 μg/ml vs 13.51 μg/ml).
The prepared micelle is expected to increase the efficacy of DOX against cancer cells and reduce its side effects.
Anthracycline antibiotics such as doxorubicin are widely used for treatment of a wide variety of solid tumors and hematological malignancies[1–3], but their clinical use is limited by their low water solubility, severe side effects such as cardiotoxicity and inherent drug resistance[4, 5]. Drug delivery systems such as polymeric nanocarriers[6, 7], liposomes and dendrimers can improve the antitumour efficacy and reduce toxicity of free DOX. Micelles that consist of hydrophilic shell and hydrophobic core are spherical nanoparticulate carriers with unique properties such as high solubility, high stability, appropriate size (20–200 nm) and long circulation in blood[10, 11]. The use of polymeric micelles as carriers of anticancer drugs has been reported in previous studies[12–16].
Selective delivery of anticancer agents to target areas in the body is desirable to minimize the side effects while maximizing the therapeutic efficacy. Non-specific drug delivery often causes adverse effects on normal cells. Folate (FOL) has several advantages over various targeting ligands such as transferrin, peptides and antibodies. FOL has a small size, non-immunogenicity, low molecular weight and stability. Therefore, micellar delivery systems have further been modified with target-specific ligands (FOL) to enhance tumor specificity and improve the tumor uptake by folate receptor-mediated endocytosis. Many approaches have also been described to prepare pH-sensitive micelles that can release the encapsulated drugs in acidic environment of tumors. Bae et al. have reported the pH-sensitive polymeric micelles based on poly (ethylene glycol)-poly(aspartate hydrazone-adriamycin) for DOX delivery that can release the drug in response to acidic pH at endosomes (pH 5.0–6.0) and lysosomes (pH 4.0–5.0). The micelles showed high antitumor activity in C-26 bearing mice. Folate-poly(ethylene glycol)-poly (aspartate hydrazone-adriamycin) micelles For active intracellular drug delivery was also prepared. The micelles showed good antitumor effect in KB cell line. In another study, Liua and co-workers prepared a targeting micelle based on poly(N-isopropylacrylamide-co-N,Ndimethylacrylamide-co-2-aminoethyl methacrylate)-b-poly (10-undecenoic acid) block copolymer and showed that the micelles was able to target the KB cells and release the drug in acidic pH of the tumor. The micelles significantly enhanced KB cell growth inhibition.
Covalent conjugation of anticancer drugs to their nanoparticulate carrier is more advantageous than physical encapsulation of drugs because it helps to stabilize the drug and prevent premature drug release into the blood circulation to assure drug delivery into the cancerous cells. Therefore, in this study, a folate functionalized PLA-PEG block copolymer was synthesized and doxorubicin was conjugated to the block copolymer via a pH-sensitive hydrazone bond. The prepared micelle was characterized for the structure of prepared block copolymer, average size and critical micelle concentration (CMC). The in vitro cytotoxicity of the folate targeting micelle against SKOV3 human ovarian cancer cells was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and compared with the folate-free micelle. Epithelial cancer cell lines such as SKOV3 demonstrate overexpression of folate receptors (FRs). MTT is a tetrazolium salt, which is reduced within the mitochondria in metabolically active viable cells. The resulting formazan crystals are impermeable to the cell membranes and accumulate only in uninjured cells, therefore this assay provides a measure of mitochondrial function following exposure to the test compound.
Material and methods
The IR spectra were recorded on a Nicollet FT-IR Magna 550 spectrometer, Madison, USA. The 1H NMR spectrum was recorded on a Bruker DRX (Avance 500) spectrometer, Rheinstetten, Germany, 500 MHz. A double beam UV-Visible spectrophotometer (model 2100, Shimadzu, Japan) was utilized for spectrophotometric measurements. Dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern Instruments Ltd., UK) was used to determine the dynamic diameter, size distribution and zeta potential of the micelles.
Doxorubicin was purchased from RPG Life Sciences limited (Mumbai, India). L-lactide, poly(ethylene glycol) (PEG) with MW 4000 g/mol, Hydrazine, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimde (DCC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC ), stannous octoate, folate and MTT were obtained from Sigma (St Louis, MO, USA). p-Nitrophenyl chloroformate (p-NPC), acetonitrile, toluene, dichloromethane and acetone (analytical grade) were purchased from Merck (Darmstadt, Germany). RPMI 1640 medium and penicillin/streptomycin solution were obtained from Gibco Invitrogen (Carlsbad, CA, USA). All other chemicals were of analytical grade.
Preparation of folate-conjugated PLA-PEG block copolymer by ring opening polymerization
Preparation of PLA-PEG block copolymer containing terminal carboxylate group was started with the synthesis of monocarboxylated PEG according to the method described by WH Jo et al. with some modifications and followed by ring-opening polymerization of the lactide in the presence of carboxylated PEG. Briefly, vacuum-dried lactide (16 g) and carboxylated PEG (3 g) were allowed to react in anhydrous toluene in the presence of and tin (II) 2-ethylhexanoate (200 mg) as a catalyst at the refluxing temperature of toluene. The PLA-PEG copolymer was extracted by chloroform after evaporation of the reaction solvent. The prepared PLA-PEG–COOH (1 g) was activated by adding EDC (50 mg) and NHS (40 mg) in dimethyl sulfoxide (DMSO, 10 ml) 5 h at room temperature. To prepare the folate-functionalized copolymer, folate-NH2 was synthesized from reaction of folic acid (250 mg) and triethylamine (TEA, 0.5 ml) in the presence of NHS and EDC in methanol. After 2 h, 1 ml of ethylene diamine was added and the reaction continued overnight at room temperature. The prepared folate-NH2 was added to the activated copolymer in DMSO and the reaction continued for additional 48 h. The mixture was then dialyzed against deionized water to remove unreacted folate-NH2. The formation of monocarboxylated PEG and PLA-PEG copolymer was confirmed by infrared (IR) spectroscopy. The conjugation of folate to the copolymer was also confirmed by 1H NMR spectroscopy. The total amount of folate conjugated to copolymer was determined by UV spectroscopy at 365 nm.
Preparation of doxorubicin-conjugated PLA-PEG block copolymer and micelle formation
Conjugation of Hydrazone Derivative of doxorubicin (Hyd-DOX) to the activated PLA-PEG-FOL block copolymer was carried out according to previously described method with some modifications. The terminal PLA in copolymer was activated by adding p-NPC (340 mg) and dry pyridine (230 mg) to PLA-PEG-FOL (3 g) in dry methylene chloride at 0°C, followed by reaction at room temperature under nitrogen atmosphere. The conjugation of DOX-Hyd to freeze-dried activated block copolymer was performed in dry tetrahydrofurane under nitrogen atmosphere for 36 h. In the final step, dialysis method was employed to prepare the micelles. The folate-free micelles were also prepared in a similar way. The doxorubicin content in the copolymer was measured by UV spectroscopy at 490 nm.
Particle size and zeta potential measurements by dynamic light scattering (DLS)
Particle size, polydispersity index (PDI) and zeta potential of blank and DOX-conjugated polymeric micelles were measured using a Zetasizer (Nano-ZS, Malvern Instruments Ltd., UK). All measurements were performed in triplicate.
Determination of the critical micelle concentration (CMC)
The CMC of prepared micelles was estimated by fluorescence spectroscopy using pyrene as a fluorescent probe. Briefly, 10 ml of DOX-conjugated copolymer aqueous solutions with different concentrations (0.05 μg/ml to 500 μg/ml) were added to the volumetric flasks containing solvent-dried pyrene (10-7 M). The solutions were sonicated at 40°C for 30 minutes, followed by stirring for 24 h at room temperature. The excitation wavelength was set at 334 nm and the intensity ratios of I383 to I372 were plotted as a function of concentration of the block copolymer solutions. The CMC of DOX-conjugated micelle was taken from the copolymer concentration at which the relative fluorescence intensity ratio began to increase.
In vitro cytotoxicity studies
SKOV3 human ovarian cancer cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. The cytotoxic activity of free DOX and chemically conjugated DOX in targeting (DOX-Hyd-PLA-PEG-FOL) and non-targeting micelles against SKOV3 cells was measured using MTT assay. The cells were seeded in 96-well plates at 10,000 cells per well and incubated for 24 h before test. Free DOX and DOX-conjugated targeting and non-targeting PLA-PEG micelles were dissolved and diluted in the growth medium to give different concentrations of DOX (equivalent DOX concentrations; 10-2 - 105 ng/ml). The blank micelle concentrations (0.01-100 μg/ml) in RPMI 1640 were also prepared. The old media in the plates were replaced with 100 μl of the media containing the test compounds. After 72 h incubation, 20 μl of MTT solution (5 mg/ml) was added to each well and the plates were then maintained in incubator for an additional 4 h. The media containing MTT were then removed and the purple formazan crystals were dissolved in 100 μl of DMSO. The absorbance of the formazan crystals was read with a Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT) at 570 nm. The IC50 values (The concentration of the test compounds at which 50% cell growth is inhibited) were calculated by using GraphPad Prism Software (Version 5.04, GraphPad Software, San Diego California, USA).
All experiments were carried out three times and results were expressed as mean ± SD. All data analyses were performed using GraphPad Prism version 5.04. The significance level was set at p < 0.05.
Results and discussion
Preparation of doxorubicin-conjugated PLA-PEG-Folate micelle
Spectral data of the prepared compounds
IR (KBr) Vmax in cm-1
1H-NMR (CDCl3) δ in ppm
1755 (C=O in PLA), 1091(C-O-C in PEG), 1635 (C=O in PEG)
3.6 (-OCH2CH2- in PEG), 1.6 (CH3 in PLA), 5.2 (CH in PLA)
7.1, 8.1, 1(FOL),
7.5, 8.4 (p-NPC)
Particle size distribution
Particle size, polydispersity index and zeta potential of blank and DOX-conjugated micelles prepared by the dialysis method
Zeta potential (mV)
Particle size (nm)
-12.47 ± 2.92
0.25 ± 0.06
176.71 ± 12.61
-10.24 ± 1.57
0.28 ± 0.04
188.43 ± 8.96
-7.12 ± 1.23
0.16 ± 0.05
182.19 ± 6.38
The CMC of DOX-conjugated PLA-PEG block copolymer
In vitro cytotoxicity studies
Doxorubicin-conjugated PLA-PEG-folate micelles with the hydrazone linkage were prepared with active targeting capability. This formulation showed a superior cytotoxicity compared to non-targeting ones against a folate-receptor positive cell line. The prepared DOX-conjugated micelles with folate ligand, appropriate size and low CMC value have a great potential for in vivo applications in cancer therapy.
This work was financially supported by grant from Research Council of Tehran University of Medical Sciences grant No.90-02-87-11816.
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