In Vivo Antitumor Activity of Folate-Conjugated Cholic Acid

Article pubs.acs.org/molecularpharmaceutics

In Vivo Antitumor Activity of Folate-Conjugated Cholic AcidPolyethylenimine Micelles for the Codelivery of Doxorubicin and siRNA to Colorectal Adenocarcinomas Muhammad Wahab Amjad,† Mohd Cairul Iqbal Mohd Amin,*,† Haliza Katas,† Adeel Masood Butt,† Prashant Kesharwani,‡ and Arun K. Iyer‡

Mol. Pharmaceutics 2015.12:4247-4258. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/04/18. For personal use only.

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Center for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia ‡ Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory, Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, Michigan 48201, United States ABSTRACT: Multidrug resistance poses a great challenge to cancer treatment. In order to improve the targeting and codelivery of small interfering RNA (siRNA) and doxorubicin, and to overcome multidrug resistance, we conjugated a cholic acid-polyethylenimine polymer with folic acid, forming CA-PEI-FA micelles. CA-PEI-FA exhibited a low critical micelle concentration (80 μM), small average particle size (150 nm), and positive zeta potential (+ 12 mV). They showed high entrapment efficiency for doxorubicin (61.2 ± 1.7%, w/w), forming D-CA-PEI-FA, and for siRNA, forming D-CA-PEI-FA-S. X-ray photoelectron spectroscopic analysis revealed the presence of external FA on D-CA-PEI-FA micelles. About 25% doxorubicin was released within 24 h at pH 7.4, while more than 30% release was observed at pH 5. The presence of FA enhanced micelle antitumor activity. The D-CA-PEI-FA and D-CA-PEI-FA-S micelles inhibited tumor growth in vivo. No significant differences between their in vitro cytotoxic activities or their in vivo antitumor effects were observed, indicating that the siRNA coloading did not significantly increase the antitumor activity. Histological analysis revealed that tumor tissues from mice treated with D-CA-PEI-FA or D-CAPEI-FA-S showed the lowest cancer cell density and the highest levels of apoptosis and necrosis. Similarly, the livers of these mice exhibited the lowest level of dihydropyrimidine dehydrogenase among all treated groups. The lowest serum vascular endothelial growth factor level (VEGF) (24.4 pg/mL) was observed in mice treated with D-CA-PEI-FA-S micelles using siRNA targeting VEGF. These findings indicated that the developed CA-PEI-FA nanoconjugate has the potential to achieve targeted codelivery of drugs and siRNA. KEYWORDS: micelle, nanoparticle, in vivo tumor targeting, electron microscopy, drug delivery, cytotoxicity

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INTRODUCTION

exhibiting stable complexation with siRNA and its exceptional “proton sponge effect” for nanocomplexes.9 Although a huge number of drugs e.g doxorubicin and paclitaxel have shown their anticancer strength, these drugs are facing enormous challenges such as low solubility in water, multidrug resistance, and off-target effects in their clinical trials. To overcome these challenges, a polymeric micellar system composed of amphiphilic block copolymers has been widely studied to enter into clinical trials10,11 or attaining clinical approval (i.e., Genexol in Korea). The development of multifunctional carriers for the codelivery of gene (siRNA) and anticancer drugs has led to the current progress in nanomedicine-derived cancer therapy.12

Numerous studies have exhibited the therapeutic potential of RNAi in the treatment of several diseases including viral infection and cancer.1,2 Apart from this prospective, an effective delivery is vital for the applications of RNAi.3 Presently, nonviral delivery systems such as lipids (e.g., lipofectamine) and cationic polymers, which were also used to carry plasmid DNA (pDNA), are commonly employed as siRNA delivery agents.4 In contrast to cationic lipids, nonviral polymeric vectors possess numerous benefits regarding physiological stability, the ability to easily produce on a large scale, and safety. So far, a range of natural and synthetic polymers have been explored as siRNA or gene delivery vectors such as poly(ethylenimine) (PEI),5 poly(L-lysine),6 chitosan,7 and polyamidoamine dendrimers.8 PEI has been recognized as the most useful carrier among the natural and synthetic polymers due to its properties of © 2015 American Chemical Society

Received: June 4, 2015 Accepted: November 14, 2015 Published: November 14, 2015 4247

DOI: 10.1021/acs.molpharmaceut.5b00827 Mol. Pharmaceutics 2015, 12, 4247−4258

Article

Molecular Pharmaceutics

fetal bovine serum (FBS), and penicillin−streptomycin were purchased from Life Technologies (Carlsbad, California, USA). Six to eight week old female Nu/Nu nude mice were purchased from Charles River laboratories (Taipei, Taiwan). All animal procedures were performed according to an animal care protocol approved by Universiti Kebangsaan Malaysia Animal Ethical Committee. Approximately 1 × 106 DLD-1 cells suspended in 50 μL of saline was subcutaneously inoculated into the right flanks of Nu/Nu mice. The tumor was monitored for length (l) and width (w) by vernier calipers. Synthesis of the CA-PEI-FA Polymer. The last amine group of PEI was conjugated to the side chain carboxyl group at the C-24 position of CA. Briefly, CA was dissolved in THF, followed by the addition of DCC and NHS to activate it for 8 h at at 25 °C. Ice-cold n-hexane was added to the mixture to precipitate the activated CA and dried for 2 h at 40 °C in an oven. Incubation of PEI and activated CA in dichloromethane for 15 h resulted in the formation of a CA-PEI conjugate. A rotary evaporator was used to dry the conjugate. Dilute HCl was used to dissolve the conjugate, which was subsequently precipitated with ice-cold acetone. Afterward, deionized water was used to mix the conjugate, followed by filtration and freezedrying to obtain the CA-PEI conjugate. Three CA/PEI molar ratios (1:1, 1:3, and 3:1) were used to synthesize the conjugates. The carbodiimide reaction facilitated the attachment of FA to amino groups of PEI on the surface of the conjugate.22 Briefly, FA (40 mg) was dispersed into a mixture of triethylamine (TEA, 0.5 mL) and anhydrous DMSO (5 mL), and activated under an anhydrous nitrogen environment by equivalent amounts of NHS and EDC at room temperature for 2 h. CA-PEI (3:1, 100 mg) was added in 25 mL of distilled water, followed by dilution with 25 mL of methanol and stirred until the optically transparent solutions were obtained, subsequent to the addition of activated FA dropwise to CAPEI solution. The mixture was stirred under nitrogen environment for 24 h at room temperature to bind FA onto PEI molecules, and the reaction was terminated by titrating it with 0.1 M sodium hydroxide (NaOH) at pH 9.0. The final mixture was first dialyzed for 3 days against phosphate buffered saline (PBS) at pH 7.4 to eliminate surplus unreacted substrates and later dialyzed for 3 days against distilled water. The polymer was separated by lyophilization. All processes were performed in the dark, and the number of moles of FA was kept constant for all molar ratios (1:1, 1:3 and 3:1) of CA:PEI. Determination of the CMC of CA-PEI-FA. The CMC of surfactants can be determined using various techniques such as conductivity, surface tension, and fluorescence measurements.23−25 Dynamic light scattering (DLS) is a technique well suited for CMC determination. CA-PEI-FA (1 mg/mL in deionized water) was 10-fold serially diluted in separate eppendorf tubes. All dilutions were analyzed on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) using a scattering angle of 90° at 37 °C. The CA-PEI-FA concentration was plotted against the intensity of scattered light (kilo counts/ s). This method was used for all molar ratios of CA-PEI-FA, and all measurements were conducted in triplicate. Preparation of the CA-PEI-FA Polymeric Micelles. CAPEI-FA micelles were prepared by probe sonication for 3 min. The tip of the sonicator was placed directly into the micelle dispersion, which was immersed in an ice bath. Fourier Transform Infrared (FTIR) Spectroscopy. To assess the functional groups, CA-PEI-FA was characterized

Thus, far, several promising codelivery systems have been developed using polymeric,13 liposomal,14 and silica-based15 cationic nanoparticles. Among many findings, the codelivery of reporter gene and an anticancer drug has been developed successfully in vitro by using copolymers of amphiphilic nature, e.g., PEI and poly(e-caprolactone).16 Inspired by these reports, in this study, a versatile micellar system of PEI and cholic acid (CA) was established for the codelivery of siRNA and doxorubicin. CA, a bile acid synthesized from cholesterol, is an amphiphilic steroid molecule that self-assembles into micelles above critical micelle concentration (CMC). Bile acids, along with the phospholipids alter the permeability of cell membranes. A synthetic vector, PEI, is commonly used in the delivery of genes because of its ability to localize in the nucleus. It is worth mentioning its transfection efficiency, potential to condense nucleic acid, and the capacity to escape from endosomes. These factors encouraged us to synthesize CAPEI polymeric micelles. The ability of CA-PEI micelles to codeliver siRNA targeting the multidrug resistance (MDR) gene and doxorubicin was investigated. MDR is the main obstacle to the successful delivery of chemotherapeutic agents because multidrug resistance can be associated with increased drug efflux, activation of the detoxification system, DNA repair, and blockage of apoptosis.17 Although several attempts have been made to reverse MDR using nanotechnology18,19 and RNAi-based20 approaches, complete prevention of MDR remains challenging. Folic acid (FA) was attached to CA-PEI micelles in order to assist their internalization in cancer cells. The expression of a folate receptor was shown to be elevated in numerous tumor types and that the FA bound to this receptor on the surface of cancer cell with strong affinity;21 this ability makes FA an ideal option to be used for the delivery of micelles to target tumor cells. In this study, we assessed the antitumor potential of CAPEI-FA in cell cultures and in nude mice bearing human colorectal adenocarcinoma (DLD-1), in order to investigate in vivo codelivery efficiency and tumor targeting.

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EXPERIMENTAL SECTION Materials, Cell Culture, and Animals. CA, PEI (average molecular weight [MW] approximately 1300), N-hydroxysuccinimide (NHS), FA, N,N′-dicyclohexylcarbodiimide (DCC), 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), hydrochloric acid (HCl), tetrahydrofuran, triethylamine, chloroform, methanol, dimethyl sulfoxide (DMSO), phosphatebuffered saline (PBS), hematoxylin for microscopy (Hist.), eosin Y, and dichloromethane were purchased from SigmaAldrich (MO, St. Louis, USA). Doxorubicin hydrochloride was obtained from Calbiochem (Darmstadt, Merck KGaA, Germany). Silencer Select Negative Control No. 1 and siRNA targeting the ATP-binding cassette subfamily B member 1 (ABCB1) gene (sense sequence: 5′-GUUUGUCUACAGUUCGUAAtt-3′) were procured from Life Technologies (Carlsbad, California, USA). siRNA targeting the vascular endothelial growth factor (VEGF) gene (sense, 5′-GGAGUACCCUGAUGAGAUCdTdT-3′; antisense, 5′-GAUCUCAUCAGGGUACUCCdTdT-3′) was purchased from First Base (Singapore). The dialysis membrane (Spectra/Por, molecular weight cutoff (MWCO) = 1000 g/mol) was supplied by Spectrum Laboratories (Rancho Dominguez, CA, USA). American Type Culture Collection (Manassas, ATCC, VA) supplied the human colorectal adenocarcinoma (DLD-1) cell line. Roswell Park Memorial Institute (RPMI) 1640 medium, 4248


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PEI-FA polymer was added to PBS to form micelles, with subsequent addition of siRNA. For 15 s, the mixture was vortexed and placed in the dark for 30 min at room temperature to facilitate the complexation among the micelles and siRNA. For D-CA-PEI-FA-S molar ratios 1:1, 3:1, and 1:3, the nitrogen to phosphate ratios were 5, 5, and 15, correspondingly. The siRNA loading efficiency (%) was calculated by determining the concentration of free siRNA in supernatant after centrifugation (35,000g, 15 min). Using a UV−vis spectrophotometer, the concentration of siRNA was determined at a wavelength of 260 nm. The supernatant taken from the D-CA-PEI-FA micelles served as the reference. Determination of siRNA Complexation by the Gel Retardation Assay. The binding efficiency of siRNA to CAPEI-FA and D-CA-PEI-FA was determined by gel electrophoresis. Twenty microliters each of CA-PEI-FA-S and D-CAPEI-FA-S were added into the wells of a precasted agarose gel (4% w/v) plate with SYBR Green. CA-PEI-FA and D-CA-PEIFA micelles were used as negative controls, while free unloaded siRNA was used as a positive control. The duration of electrophoresis was 26 min, as per the supplier’s procedure (2005−2006, Invitrogen, Waltham, MA, USA). After electrophoresis, an UV transilluminator (Invitrogen) was used to visualize the bands of siRNA. Particle Size and Zeta Potential Measurements. The particle size and zeta potential of the micelles were measured by dynamic light scattering technique using a Zetasizer Nano ZS (Malvern Instruments) at 25 °C. Five measurements of particle size and zeta potential each were recorded for all samples, and their means were calculated to document the final results. Surface Chemistry. X-ray photoelectron spectroscopy (XPS; AXIS His-165 Ultra, Kratos Analytical, Shimadzu Corp.) was used to confirm the presence of FA on the surface of D-CA-PEI-FA. In a fixed transmission mode and a pass energy of 80 eV, the spectrum of binding energy was recorded from 0 to 1100 eV. In Vitro Release of Doxorubicin. Freeze-dried samples (15 mg) of D-CA-PEI-FA and D-CA-PEI-FA-S were resuspended in 5 mL of sodium acetate buffer solution (pH 5.0) or PBS (pH 7.4) and placed in a dialysis membrane bag. The dialysis bag was transferred to a beaker containing the same 40 mL of buffer solution and placed on a magnetic stirrer at 37 °C. At predetermined time intervals, the solution around the dialysis bag was removed and analyzed on a UV−vis spectrophotometer at a wavelength of 482.5 nm to determine the concentration of doxorubicin, and was substituted with fresh solution. The cumulative amount of released drug was calculated, and the percentage of drug released from the micelles was plotted against time. In Vitro Cell Viability Analysis Using CA-PEI-FA and DCA-PEI-FA-S Micelles. ATCC supplied the Chinese hamster lung fibroblast (V79) cell lines which were propagated in DMEM supplemented with 1% penicillin−streptomycin and 10% FBS, and maintained in a humidified 5% CO2/95% air atmosphere at 37 °C in an incubator. RPMI-1640 medium supplemented with 1% penicillin−streptomycin and 10% FBS was used to culture DLD-1 cells. The cells were maintained under a humidified 5% CO2/95% air atmosphere at 37 °C. The effect of CA-PEI-FA micelles on cell viability was assessed using V79 cells. The V79 cells were incubated for 24 h at a seeding density of 4 × 104 cells per well in a 96-well culture plate and treated with increasing concentrations of CA-PEI-FA micelles starting from 31.25 to 500 μgmL−1 and incubated for

using the potassium bromide (KBr) pellet method on a FTIR spectrophotometer (Spectrum 100; PerkinElmer, Waltham, MA, USA). Prior to the experiment, KBr pellets were dried for 1 h at 50 °C in an oven. CA-PEI-FA (1 mg) was mixed with 100 mg of dried KBr and ground in a mortar to homogenize the powder. A thin transparent pellet was then prepared using a stainless steel disc and plunger set (die). Each sample pellet was placed in the FTIR spectrophotometer, and the instrument was run for 16 scans at a resolution of 4 cm−1. The spectra were recorded in the range of 3700−700 cm−1. Nuclear Magnetic Resonance (1H NMR) and Ultraviolet−Visible (UV−Vis) Spectroscopy. Two milligrams of CA-PEI-FA were dissolved in 0.6−0.7 mL of deuterium oxide (D2O) in a small beaker. The solution was subsequently filtered through a Pasteur pipette equipped with a glass wool plug and ejected into an NMR tube to a depth of approximately 4 cm prior to analysis in an NMR spectrophotometer (Bruker Avance III, FT-NMR 600 MHz equipped with a cryoprobe, Billerica, MA, USA). The resultant spectrum was analyzed to confirm the presence of CA-PEI-FA. The degree of substitution was determined by dividing the total integral values of five FA aromatic protons in the region of 6.5−9.0 ppm by the protons of the PEI hydrocarbon backbone at 2.6 ppm. The degree of conjugation of FA was determined by UV−vis spectroscopy. FA solutions of known concentrations were prepared in D2O. Their respective UV absorbance was recorded and plotted against the FA concentration to form a calibration curve. D2O was used to dissolve 10 mg of each sample, and the UV absorbance was recorded at 363 nm to calculate the level of FA from the calibration curve. The degree of conjugation of FA on a weight to weight basis was calculated using the following equation: conjugation of folic acid (wt%) weight of FA in conjugate = × 100 total weight of conjugate

Preparation of Doxorubicin-Loaded CA-PEI-FA (D-CAPEI-FA) Micelles. D-CA-PEI-FA micelles were prepared as follows: doxorubicin hydrochloride (2.5 mg) was dissolved in chloroform and mixed with 2 μL of TEA. CA-PEI-FA was dissolved in methanol. In a glass beaker, the CA-PEI-FA and doxorubicin solutions were mixed and placed in the dark for 1 day. The mixture was added dropwise to deionized water under ultrasonic agitation (using a Sonifier, Branson Ultrasonics Co., Danbury, CT, USA) for 10 min at power level 3. Solvents such as methanol and chloroform were subsequently removed with the help of a rotary evaporator. To eliminate unloaded doxorubicin, D-CA-PEI-FA was dialyzed for 24 h at 20 °C against 1 L of deionized water using a dialysis membrane bag. For the first 12 h, the water was changed after every 2 h and later every 6 h. D-CA-PEI-FA was freeze-dried immediately after dialysis was completed. A calibration curve was constructed using pure doxorubicin. On the basis of the curve, the amount of doxorubicin present in D-CA-PEI-FA micelles was calculated by dissolving them in 4 mL of cosolvent of a methanol and DMSO (1:1) and recording the absorbance at 482.5 nm on a spectrophotometer. Two milligrams of D-CAPEI-FA was dissolved in D2O (0.6−0.7 mL) and ejected into an NMR tube. The samples were run on an NMR spectrophotometer. Preparation of Doxorubicin-Loaded and siRNA Complexed CA-PEI-FA (D-CA-PEI-FA-S) Micelles. The D-CA4249


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Molecular Pharmaceutics Scheme 1. Complete Synthesis Scheme for CA-PEI-FA

an additional 24 h in a 5% CO2/95% air environment at 37 °C. Finally, alamarBlue reagent (20 μL) was introduced to each well of a culture plate containing cells, and the plate was incubated for another 4 h. The absorbance reading from each well was recorded at 570 nm using a microplate reader. The following equation was used to calculate the cell viability (%):

Subsequently, the mice were euthanized, and their tumors were removed. Histology. For metastasis examination, the tumor, lung, spleen, kidney, heart, and liver were removed, followed by fixation in 10% solution of formalin and were later embedded in paraffin for staining with H&E. The micrographs of these organs were taken using a microscope. Enzyme-Linked Immunosorbent Assay (ELISA). ELISA was performed to measure the dihydropyrimidine dehydrogenase (DPD) concentration in the liver, as per the manufacturer’s protocol (Uscn, Wuhan, China). Using the recombinant DPD standard curve, the DPD concentrations in the liver were calculated as ng/mg of protein. An ELISA was also used to detect vascular endothelial growth factor (VEGF) in the mouse sera (Life Technologies), in accordance with the manufacturer’s instructions. For this experiment, two separate groups of mice were treated with micellar formulations containing siRNAtargeting VEGF (D-CA-PEI-SV and D-CA-PEI-FA-SV, where SV denotes VEGF siRNA), in addition to the treatment groups described above. Statistical Analysis. All data are expressed as the mean ± the standard deviation of three readings. GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analyses (one-way analyses of variance, ANOVA, followed by a posthoc Tukey’s test for multiple comparisons) of all data. The significance level was 0.05, and the data were indicated with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

cell viability(%) = A570 of treated cells/A570 of control cells × 100

The cytotoxicity of D-CA-PEI-FA-S micelles was investigated using the alamarBlue assay. DLD-1 cells were incubated for 48 h at a seeding density of 2 × 104 cells/well in 96-well culture plates at 5% CO2/95% air environment at 37 °C. The cells were treated with 200 μL of D-CA-PEI-FA-S micelles containing doxorubicin at concentrations of 50, 25, 12.5, 6.25, 1.56, 0.19, and 0.09 μgmL−1. The plates were incubated for 24 h, followed by the introduction of alamarBlue (20 μL) to each well of the plate, and were further incubated for 4 h. The absorbance reading from each well was recorded at 570 nm using a microplate reader. In Vivo Antitumor Effect. The Nu/Nu mice were housed in individually ventilated cages and maintained in an animal facility that fulfilled the guidelines and requirements for biosafety level 2 at the Universiti Kebangsaan, Malaysia. Mice bearing a visible DLD-1 tumor were randomly divided into saline, CA-PEI, doxorubicin, D-CA-PEI, D-CA-PEI-S, D-CAPEI-FA, and D-CA-PEI-FA-S groups, which were administrated the respective treatment intravenously on day 0 and day 7 at a dose of 8 mg/kg body weight. Body weight and tumor volumes were monitored and recorded twice weekly for 20 days. 4250


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RESULTS AND DISCUSSION FA was introduced into CA-PEI to produce CA-PEI-FA polymeric micelles for use as nanocarriers to codeliver doxorubicin and siRNA (Scheme 1). Under anhydrous nitrogen atmosphere, the carboxyl moiety of FA was activated using NHS/EDC chemistry, followed by the amide linkage-coupling with an amine group of PEI to yield CA-PEI-FA. Determination of the CMC of the CA-PEI-FA Polymer. The CMCs of CA-PEI-FA solutions containing various molar ratios of CA to PEI are shown in Figure 1. CA-PEI-FA micelles

hydrophobic steroidal nucleus, an increased level of CA might increase the hydrophobic interactions among the polymer chains in the core of micelle, hence stabilizing the assembly. Furthermore, the CMC of the CA-PEI-FA micelles was lower than that of the CA-PEI micelles. The CMC decreases with the increase in size of the hydrophobic block, that is, micelle formation takes place at lower concentrations of the polymer. This phenomenon is important for the entrapment efficiency (EE) and drug solubilization of micelles.26 CA-PEI micelles with low CMCs would be beneficial, as they are stable against precipitation in blood owing to dilution and also against dissociation. Additionally, the occurrence of embolisms, which can result from high polymer concentrations, could be avoided.27 FTIR Spectroscopy. The presence of fine peaks in the region of 3700−3000 cm−1 represented OH stretching, confirming the presence of CA (Figure 2a). OH stretching vibrations were observed at 3524 and 3324 cm−1, and CH vibrations were observed at 2968, 2935, and 2872 cm−1. A sharp peak at 1715 cm−1 confirmed the presence of CO of the COOH stretching. Moreover, some characteristic CA peaks were observed at 1375−1401, 1329, 1288, 1242, 1121, 1092, 1078, and 1045 cm−1. The peaks at 3323, 3417, and 3545 cm−1 in the FA spectrum corresponded to the secondary NH, NH2, and ring O−H groups. A peak with less intensity appearing at 3110 cm−1 in the FA spectrum indicated O−H stretching of COOH. The most characteristic FA bands (amide I and II) appeared at 1695 and 1572 cm−1, respectively. In the CA-PEI spectrum, the peaks for N−H bending, CO absorbance, and

Figure 1. Critical micelle concentrations of CA-PEI-FA micelles (CA/ PEI = 1:1, 1:3, and 3:1).

possessing a CA to PEI molar ratio of 3:1 had the lowest CMC (80 μM), while the CMC was calculated as 100 μM for both 1:1 and 1:3 molar ratios of CA to PEI. As CA possesses a

Figure 2. FTIR spectra of CA, PEI, FA, CA-PEI, and CA-PEI-FA (a). HNMR spectra of CA-PEI-FA (b) and D-CA-PEI-FA (c). 4251


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Molecular Pharmaceutics C−H and N−H stretching appeared in the CA-PEI spectrum at 1574 cm−1, 1625 cm−1, 2850−2930 cm−1, and 3326 cm−1, respectively. The N−H bending (1574 cm−1) and CO absorbance bands (1625 cm−1) overlapped, appearing as a doublet in the spectrum of CA-PEI. This doublet confirmed the formation of the amide bond between PEI and CA.28 In the CA-PEI-FA spectrum, a small shoulder peak was observed at 1700 cm−1, indicating the presence of FA.29 Moreover, most FA peaks (from 2500 to 3600 cm−1) disappeared from the CA-PEIFA spectrum. Recently, this phenomenon was also observed by other researchers studying FA-targeted micelles.29 1 H NMR and UV−Vis Spectroscopy. In the CA-PEI-FA 1 H NMR spectrum (Figure 2b), proton shifts appeared in the range of 1−2 ppm, corresponding to the characteristic CA peaks. Doublet, triplet, and multiplet peaks appeared between 1 and 2 ppm, corresponding to the characteristic CA peaks. The number of protons in CA was designated by the integration values between 0.8 and 2 ppm. The presence of PEI was indicated by the proton shifts in the region of 2.2−3.52 ppm. The occurrence of weak signals (corresponding to aromatic protons of FA) between 6.6 and 8.7 ppm confirmed the presence of FA in CA-PEI-FA.32 The concentration of FA in the CA-PEI-FA was 26%, whereas the degree of substitution of FA onto CA-PEI was 26.6%, as determined by UV spectroscopy. Moreover, the spectrum of D-CA-PEI-FA (Figure 2c) showed peaks that were characteristic of doxorubicin. These peaks were not present in the CA-PEI-FA spectrum and were in the regions of 4.1−5.5 and 7.2−7.6 ppm. Similar peaks have also been observed by other researchers studying doxorubicinloaded nanoparticles.33 Preparation of D-CA-PEI-FA Micelles. Hydrophobic doxorubicin was loaded in the core (CA) of CA-PEI-FA micelles. Doxorubicin’s affinity for the polymeric micelles can be enhanced by including a suitable hydrophobic block in the copolymer. Yokoyama et al. prepared PEO-poly(L-aspartate) micelles which involved both physical encapsulation and chemical binding of doxorubicin with the polymer.30 Micelles can be fabricated using AB-type copolymers if one fragment of block copolymer provides adequate interchain cohesive interactions in solvent. The drug−core cohesive interaction includes hydrogen bonding, π−π interactions, hydrophobic and electrostatic attraction; however, hydrophobic interaction is the main driving force behind the encapsulation of a majority of anticancer drugs because of their poor water solubility.31 The EE of doxorubicin in the D-CA-PEI-FA micelles was 61.2% (w/ w). Preparation of D-CA-PEI-FA-S Micelles and the Determination of siRNA Complexation by the Gel Retardation Assay. siRNA was complexed with CA-PEI-FA and D-CA-PEI-FA micelles. The zeta potential of CA-PEI-FA and D-CA-PEI-FA micelles decreased after siRNA complexation. Greater siRNA complexation was observed with CA-PEIFA micelles than with D-CA-PEI-FA micelles since their zeta potential was more positive, and siRNA complexation predominantly involves electrostatic attraction. The amount of free siRNA detected was very low, signifying that a majority of siRNA was encapsulated onto micelles. High loading efficiency of siRNA could be attributed to strong electrostatic attraction between PEI and siRNA.34 Agarose gel electrophoresis was used to investigate siRNA complexation with CA-PEI-FA and D-CA-PEI-FA micelles (Figure 3). The mobility of siRNA in an electric field is lost due to the complexation of cationic polymers with siRNA. Thus, a

Figure 3. Gel retardation assay. Well 1, DNA ladder; 2, free siRNA; 3, CA-PEI-FA; 4, CA-PEI-FA-S; and 5, D-CA-PEI-FA-S.

polymer’s ability to complex with siRNA is reflected by the retardation of siRNA’s mobility in electrophoresis. The migration of siRNA was completely retarded with both CAPEI-FA-S and D-CA-PEI-FA-S, indicating complete neutralization of the siRNA’s negative charge.35 Prior to siRNA complexation, CA-PEI-FA micelles exhibited an average hydrodynamic size of 125 nm and a zeta potential of +7.5 mV. A reduction in the zeta potential (decreased by 3.5 mV to +4.02 mV) of CA-PEI-FA was observed after complexation with siRNA, reflecting the neutralization of cationic CA-PEI-FA by anionic siRNA. Similarly, D-CA-PEI-FA micelles exhibited an average particle size of 130 nm and a zeta potential of +5.5 mV before siRNA complexation. A reduction in the zeta potential (decreased by 3.36 mV to +2.14 mV) of D-CA-PEIFA was observed after complexation with siRNA. The decrease in micelle size after siRNA complexation can be attributed to electrostatic attraction among PEI (highly cationic) and siRNA (highly anionic). A small positive charge and size are important for electrostatic assembly and cell internalization. Particle Size and Zeta Potential Analysis. Zeta potential,36 particle size,37 and receptor-mediated38 internalization are three crucial determinants of cellular micelle uptake. Generally, a positive zeta potential enables the linking of micelles to cells through electrostatic interactions, although a high density of cations in direct contact with the cell membrane can result in cytotoxicity. The endocytosis of the nanoparticles is facilitated by small particle size. The cell internalization and delivery specificity of nanoparticles can be enhanced by attaching a targeting ligand to nanoparticles. The mean particle diameter of freshly prepared CA-PEI-FA micelles in this study was determined by DLS (Table 1). The self-aggregation behavior of CA-PEI-FA in an aqueous medium was influenced by numerous forces such as hydrogen bonding between hydrophilic segments, hydrogen bonding of water molecules and hydrophilic segments, and the hydrophobic−hydrophobic associations between CA segments. Apart from governing selfaggregation of the amphiphilic CA-PEI-FA polymer, these interactions also determine the aggregate size. The micellar CAPEI-FA solution was obtained by sonication, and the mean particle size obtained at all molar CA/PEI ratios was less than 200 nm, with a uniform distribution. The nanoparticle size between 10 and 100 nm is considered to be ideal for passive tumor targeting.39 4252


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although a high positive charge may increase nanoparticle interactions with negatively charged proteins. Surface Chemistry. The elements on the surface of micelles were recognized by measuring specific binding energy (eV) using XPS. The occurrence of FA on the surface of micelles can be investigated using the N 1s region of the XPS spectrum. Figure 4 exhibits the characteristic FA peak at 400 eV in the wide scan of CA-PEI-FA and D-CA-PEI-FA. The characteristic N peak intensity was greater in CA-PEI-FA than in D-CA-PEI-FA. This might be due to the interaction of some FA with doxorubicin. In general, the characteristic N peaks increased in sharpness and intensity as the degree of FA substitution increased. The N-containing aromatic polymer, PEI, exhibited weak π−π satellite features shifted several electronvolts from the main N peak. In Vitro Release of Doxorubicin. Doxorubicin release behaviors from D-CA-PEI-FA and D-CA-PEI-FA-S were compared at 37 °C in aqueous solutions of pH 5.0 and pH 7.4. pH 5.0 was selected to simulate the acidic lysosomal atmosphere. As per Figure 5, a characteristic release pattern containing two stages was witnessed in all four tests, i.e., an early fast release followed by a slow sustained release for up to several days. Furthermore, the release of doxorubicin from both micelles was quicker at pH 5.0. The quicker doxorubicin release under acidic conditions can be attributed to accelerated degradation of the CA core and reprotonation of doxorubicin’s amino group. A similar pH-reliant release of doxorubicin was reported in other doxorubicin-loaded micelles,43 and this was supposed to improve intracellular release of druge following endocytic entry into tumor cells and subsequent entrapment within endosomes/lysosomes. Additionally, both FA conjugation and siRNA complexation were reported to delay the release of doxorubicin, as compared to that observed previously using CA-PEI micelles.44 In Vitro Cell Viability in the Presence of CA-PEI-FA Micelles. Biocompatibility is a major concern for biomaterials, and we therefore performed a preliminary evaluation of micellar cytotoxicity using the alamarBlue assay. A cell viability assay (Figure 6a) indicated that the tested concentrations of CA-PEIFA micelles were nontoxic to V79 cells. No marked reduction in cell viability was observed, even at a high concentration of

Table 1. Zeta Potential and Average Particle Size of CA-PEI Micelles at Different Molar Ratios and of CA-PEI-FA, CAPEI-FA-S, D-CA-PEI-FA, and D-CA-PEI-FA-S Micellesa

a

micelle formulations

zeta potential (mV)

particle size (nm)

CA-PEI 1:1 CA-PEI 1:3 CA-PEI 3:1 CA-PEI-FA CA-PEI-FA-S D-CA-PEI-FA D-CA-PEI-FA-S

+12.2 ± 1.2 +18.7 ± 1.5 +9.2 ± 1.1 +7.5 ± 1.0 +4.0 ± 1.5 +5.5 ± 1.1 +2.1 ± 1.0

172 148 164 125 120 130 118

± ± ± ± ± ± ±

5.2 6.7 8.6 7.1 6.7 8.6 7.1

polydispersity index 0.326 0.287 0.296 0.316 0.226 0.357 0.334

± ± ± ± ± ± ±

0.04 0.04 0.02 0.03 0.02 0.04 0.03

CA-PEI 3:1 was used to synthesize CA-PEI-FA.

The zeta potential of CA-PEI micelles of all molar ratios was positive. The molar ratio (1:3) of CA-PEI micelles containing maximum PEI content exhibited the highest (positive) zeta potential (+18.7 ± 1.5 mV), whereas a 3:1 molar ratio produced the lowest zeta potential. FA conjugation decreased the zeta potential. Zeta potential is a vital determinant of the cellular uptake mechanism and efficiency, as well as the in vivo fate of nanoparticles,40,41 though the optimal zeta potential (e.g., negative, neutral or positive) and charge densities were documented to differ between nanoparticles, as determined by their abilities to avert unspecific accumulation in undesired sites, lessen nonspecific clearance of nanoparticles, and extend blood circulation time. However, PEG−PDLLA micelles of negative and neutral charge showed no significant difference in the kinetics of blood clearance. Still, micelles with negative charge remarkably decreased the nonspecific uptake by the spleen and liver in comparison with micelles of neutral charge, which was due to the presence of electrostatic repulsion between micelles (negatively charged) and cell surface.42 On the basis of the aforementioned information, we believed that the optimal positive surface charge and smaller size of the CA-PEI-FA-S and D-CA-PEI-FA-S micelles (Table 1) may be ideal for adequate ligand-targeted codelivery and cellular uptake of doxorubicin and siRNA. In addition, a slight positive zeta potential might improve the in vivo stability of the nanoparticle,

Figure 4. XPS wide scan spectra of CA-PEI-FA and D-CA-PEI-FA on the left. The spectra on the right show the relative nitrogen signals at high resolution of both formulations. 4253


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PEI-FA and D-CA-PEI-FA-S caused the death of cells in a concentration-dependent manner, and there was no significant difference between their effects. This indicated that FA conjugation enhanced the activity of D-CA-PEI micelles, while siRNA loading did not produce additional cytotoxicity. The in vivo cytotoxic effectiveness of doxorubicin loaded onto micelles is expected to be further enhanced, owing to the EPR effect of micelles. So, the adverse effects and toxicity of doxorubicin can be reduced by the selective micellar uptake by cancer cells. In Vivo Antitumor Effect. Tumor growth in the saline group was rapid, with the mean tumor volume expanding from 30 mm3 to 1977 mm3 within 20 days. The CA-PEI micelletreated mice showed a similar tumor growth profile, reaching a tumor volume of 1937 mm3, which indicated that these micelles had little physiological activity (Figure 7a). Doxorubicin and DCA-PEI produced considerable tumor inhibition. D-CA-PEI-S also inhibited tumor growth, suggesting that reducing expression of the MDR gene suppressed tumor growth in vivo. D-CA-PEI-FA and D-CA-PEI-FA-S micelles enhanced these tumor inhibitory effects because of FA-mediated tumor targeting. Consistent with the in vitro cytotoxicity data, no significant variation among the effects of D-CA-PEI-FA and DCA-PEI-FA-S was found. This comprehensive in vivo study revealed that FA conjugation enhanced the antitumor activity of CA-PEI micelles but showed that siRNA coloading did not significantly enhance this effect. Since body weight change is an indicator of systemic toxicity, this was measured in parallel with the antitumor effect (Figure 7b). Mouse body weight in the CA-PEI-treated groups was not significantly different from that of the saline-treated mice, indicating that these nanoparticles did not cause significant systemic toxicity. The D-CA-PEI-FA- and D-CA-PEI-FA-Streated groups did not display obvious body weight fluctuations. However, their tumor weights were much lower than those of the other groups, indicating that D-CA-PEI-FA and D-CA-PEI-FA-S micelles did not negatively influence the health of the mice; this contrasted with free doxorubicin-treated animals, which had larger tumors but showed decreased body weight. This suggested that D-CA-PEI-FA-S, which possesses efficient antitumor activity together with low systemic toxicity,

Figure 5. Cumulative release of doxorubicin (%) from D-CA-PEI-FA and D-CA-PEI-FA-S micelles at pH 5 and 7.4. The release of doxorubicin from each formulation at pH 5 and 7.4 was statistically analyzed.

500 μg/mL CA-PEI-FA. These results suggest that CA-PEI-FA micelles are nontoxic to normal cells. In Vitro Cytotoxicity of D-CA-PEI-FA-S Micelles. As per Figure 6b, the inhibition (%) of cancer cells was higher in the presence of D-CA-PEI-FA-S micelles than in the presence of DCA-PEI-FA micelles. The incorporation of doxorubicin into the D-CA-PEI-FA and D-CA-PEI-FA-S micelles improved their cytotoxic potential against cancer cells. The IC50 values for DCA-PEI-FA (4.88 μg/mL) and D-CA-PEI-FA-S (3.88 μg/mL) micelles were lower than those determined previously for DCA-PEI (5.85 μg/mL) or free doxorubicin (10.58 μg/mL). The high IC50 of doxorubicin and low inhibition (%) of cells, in contrast to the corresponding values for D-CA-PEI-FA and DCA-PEI-FA-S micelles, may reflect the removal of doxorubicin by drug efflux pumps from the tumor interstitium45 and high retention and permeability of micelles in the tumor. Additionally, the improved permeation of D-CA-PEI-FA-S micelles increases drug delivery to the target site and hence increases the retention time of doxorubicin at the site of action. Both D-CA-

Figure 6. In vitro cell viability assays after the exposure of V79 cells to CA-PEI-FA micelles (a). In vitro cytotoxicity of D-CA-PEI-FA and D-CA-PEIFA-S micelles against DLD-1 cells after 24 h (b). In panel b, at each doxorubicin concentration, inhibition (%) of D-CA-PEI, D-CA-PEI-FA, and DCA-PEI-FA-S was compared to doxorubin for statistical analysis. 4254


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Figure 7. In vivo antitumor effect (a,b): the tumor inhibiting rate at day 21 (a) and body weight of tumor-bearing mice (b). In panel a, all groups were compared with doxorubicin for statistical analysis. In weight gain (%) of b, all groups were compared with saline for statistical analysis.

Figure 8. H&E-stained sections of organs collected from mice after treatment with the different micelle formulations.

can act as a promising nanoplatform to codeliver chemotherapeutic drugs and nucleic acids into tumor interstitia, resulting in more effective cancer treatment. Histology. The lungs, heart, liver, kidneys, spleen, and tumor of the animals were analyzed in all treatment groups (Figure 8). The tumors from the saline- and CA-PEI-treated groups appeared more hypercellular and exhibited a higher level of nuclear polymorphism in the H&E-stained tissue sections, as compared with those collected from the other therapeutic groups. The tumor cells were polygonal, containing scanty to moderate cytoplasm and pleomorphic vesicular nuclei, with a prominent nucleolus. The tumor cells were arranged in groups and islands, with occasional glandular formation. The tumor could be classified as a poorly differentiated adenocarcinoma. The lung sections showed intra-alveolar hemorrhage, with no tumor deposits. The myocardium and endocardium sections appeared normal, and no tumor was observed. The central vein of the liver was normal. Moreover, the hepatocytes and portal triad were also normal, with no signs of DLD-1 tumor. The

spleen section showed extramedullary hematopoiesis without metastasis. The kidney sections also had a normal morphology without metastasis. The doxorubicin-treated group showed a tumor composed of polygonal cells containing scanty cytoplasm and pleomorphic vesicular nuclei, with a prominent nucleolus. The tumor cells were arranged in groups and islands with a glandular formation, and there were areas of apoptosis and necrosis. Increased mitosis was also observed. The tumor could be referred to as a poorly differentiated adenocarcinoma. The lung section showed areas of congestion and septae with edema and neutrophilic infiltration. No tumor deposit was present. The myocardium, endocardium, and liver sections appeared normal, with no evidence of a tumor. The spleen section showed extramedullary hematopoiesis without metastasis. The kidney had normal glomeruli, tubules, and blood vessels. Apart from the tumor size, similar histology was seen in the other groups, including those treated with the D-CA-PEI, D-CA-PEI-S, D-CA-PEI-FA, and D-CA-PEI-FA-S micelles. D-CA-PEI-FA and D-CA-PEI4255


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Figure 9. Dihydropyrimidine dehydrogenase (DPD) expression in the liver of mice treated with saline, CA-PEI, doxorubicin, D-CA-PEI, D-CA-PEIS, D-CA-PEI-FA, or D-CA-PEI-FA-S (a). The effect of the different micellar formulations on the expression of VEGF in serum (b). All of the groups were compared with saline for statistical analysis.

discovered to be a mitogen and survival factor for endothelial cells.52 The survival role is facilitated via upregulation of the inhibitors of apoptosis family members, upregulation of Bcl-2, and activation of the phosphatidylinositol 3-kinase-Akt signaling pathway.53,54 The expression of VEGF is controlled in a complex manner at the transcriptional and translational levels via several tumor and oncogenic suppressor pathways, by the estradiol receptor and by hypoxia.55 Tumor growth and metastasis are highly reliant on angiogenesis, as observed from the preclinical and clinical studies conducted on colon cancer. Moreover, VEGF was found to be the main angiogenic factor.56 VEGF levels were analyzed in sera from the sacrificed mice in each study group (Figure 9b). Two additional treatment groups (D-CA-PEI-SV and D-CA-PEI-FA-SV) were included in this study, specifically for VEGF analysis. These groups were treated in the same way as the other groups, except that siRNA targeting VEGF was used, instead of siRNA targeting ABCB1. The serum VEGF level was significantly elevated by more than 7-fold in tumor-bearing mice treated with saline (53.6 pg/mL), as compared with healthy mice (7.4 pg/mL). This showed that VEGF is an important cancer biomarker. The effect of the CAPEI micelles on VEGF (53.6 pg/mL) was comparable to that of saline. The level of VEGF in serum was decreased in doxorubicin-treated mice. Although doxorubicin has no reported effect on VEGF, this slight decrease in VEGF most likely reflected the cytotoxicity of doxorubicin. The D-CA-PEI micelles produced similar results as those obtained with doxorubicin alone. The D-CA-PEI-SV micelles considerably reduced the VEGF serum level. The most apparent reason for this is the SV-mediated silencing of VEGF. The D-CA-PEI-Streated group had a VEGF level that was similar to that of the doxorubicin- or D-CA-PEI-treated mice. This may be because ABCB1, and not VEGF, siRNA was present in these micelles. The combined effects of doxorubicin and ABCB1 siRNA were predominantly cytotoxic, causing a slight decrease in the VEGF level. FA targeting greatly enhanced the activity of the D-CAPEI-FA micelles, producing a serum VEGF level of 37.8 pg/mL in these mice. The highest anti-VEGF activity was observed following the administration of the D-CA-PEI-FA-SV micelles

FA-S micelles showed the highest antitumor activities, and tumor tissues from these groups had the lowest cancer cell density and the highest level of cell apoptosis and necrosis. This phenomenon was previously observed by researchers investigating the codelivery of anticancer drugs and siRNA with antitumor activity.46 This indicated that the optimal size and zeta potential of these nanoparticles, FA-mediated tumor targeting, and enhanced doxorubicin release in the acidic environment optimized their in vivo cytotoxicity. Liver DPD Level. An increase in the intratumoral DPD level is reported to be one of the causes behind drug resistance.47 DPD is extensively dispersed in various organs, particularly in the liver.48 In the current study, DPD protein expression was investigated in the liver tissues of the treated mice (Figure 9a). After the induction of DLD-1 xenografts, an elevated level of DPD protein was observed in the liver tissues. The level of liver DPD was decreased in doxorubicin-treated mice, in contrast to saline- or CA-PEI-treated animals. This may indicate the cytotoxic potential of doxorubicin, which reduced the tumor severity, and hence, the DPD level decreased. However, the administration of hydrophobic doxorubicin without a carrier only produced a moderate effect. D-CA-PEI showed an enhanced effect of doxorubicin on the DPD level. D-CA-PEI-S had an additional synergistic effect on the DPD level, although this effect was not significantly better than that of D-CA-PEI. The liver DPD levels in mice treated with D-CA-PEI-FA and D-CA-PEI-FA-S were 1224.8 and 1174.6 ng/mg, respectively. As compared to saline, a significant reduction in the level of DPD was seen in mice treated with DCA-PEI-S and D-CA-PEI-FA-S (Figure 9a). This could be attributed to multiple factors, such as doxorubicin, folate targeting, particle size, and zeta potential. Tumor progression and metastasis are greatly reliant on neoangiogenesis, which may arise either from bone marrowderived endothelial precursor cells, circulating endothelial cells, or from preexisting blood vessels.49 VEGF is among one of the chief regulators of physiological and pathological angiogenesis.50 Its expression is upregulated in several tumors, such as primary and metastatic colorectal adenocarcinoma.51 VEGF via interaction with VEGFR-1 and VEGFR-2 was 4256


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(4) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E.; Busini, V.; Hossain, N. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 2008, 26, 561−569. (5) Merdan, T.; Kopecek, J.; Kissel, T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv. Drug Delivery Rev. 2002, 54, 715−758. (6) Miyata, K.; Kakizawa, Y.; Nishiyama, N.; Yamasaki, Y.; Watanabe, T.; Kohara, M. Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver. J. Controlled Release 2005, 109, 15−23. (7) Maclaughlin, F.; Mumper, R.; Wang, J.; Tagliaferri, J.; Gill, I.; Hichicliffe, M. Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J. Controlled Release 1998, 56, 259−272. (8) Wood, K.; Little, S.; Langer, R.; Hammond, P. A family of hierarchically selfassembling linear-dendritic hybrid polymers for highly efficient targeted gene delivery. Angew. Chem., Int. Ed. 2005, 44, 6704−6708. (9) Boussif, O.; Lezoualc’h, F.; Zanta, M.; Mergny, M.; Scherman, D.; Demeneix, B. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (10) Yap, T.; Carden, C.; Kaye, S. Beyond chemotherapy: targeted therapies in ovarian cancer. Nat. Rev. Cancer 2009, 9, 167−181. (11) Moebus, V.; Jackisch, C.; Lueck, H.; Bois, A.; Thomssen, C.; Kurbacher, C. Intense dose-dense sequential chemotherapy with epirubicin, paclitaxel, and cyclophosphamide compared with conventionally scheduled chemotherapy in high-risk primary breast cancer: mature results of an AGO phase III study. J. Clin. Oncol. 2010, 28, 2874−2880. (12) Wang, Y.; Gao, S.; Ye, W.; Yoon, H.; Yang, Y. Co-delivery of drugs and DNA from cationic core-shell nanoparticles self-assembled from a biodegradable copolymer. Nat. Mater. 2006, 5, 791−796. (13) Zhu, C.; Jung, S.; Luo, S.; Meng, F.; Zhu, X.; Park, T. Codelivery of siRNA and paclitaxel into cancer cells by biodegradable cationic micelles based on PDMAEMA-PCL-PDMAEMA triblock copolymers. Biomaterials 2010, 31, 2408−2416. (14) Xu, Z.; Zhang, Z.; Chen, Y.; Chen, L.; Lin, L.; Li, Y. The characteristics and performance of a multifunctional nanoassembly system for the co-delivery of docetaxel and iSur-pDNA in a mouse hepatocellular carcinoma model. Biomaterials 2010, 31, 916−922. (15) Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. Engineered design of mesoporous silica nanoparticles to deliver doxorubicin and P-glycoprotein siRNA to overcome drug resistance in a cancer cell line. ACS Nano 2010, 4, 4539−4550. (16) Qiu, L. Y.; Bae, Y. H. Self-assembled polyethylenimine-graftpoly(e-caprolactone) micelles as potential dual carriers of genes and anticancer drugs. Biomaterials 2007, 28, 4132−4142. (17) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discovery 2006, 5, 219−234. (18) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2007, 2, 751−760. (19) Nie, S. M.; Xing, Y.; Kim, G. J.; Simons, J. W. Nanotechnology applications in cancer. Annu. Rev. Biomed. Eng. 2007, 9, 257−288. (20) Izquierdo, M. Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther. 2005, 12, 217−227. (21) Low, P.; Henne, W.; Doorneweerd, D. Discovery and development of folic-acid based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc. Chem. Res. 2008, 41, 120−129. (22) Li, J. M.; Wang, Y. Y.; Zhang, W.; Su, H.; Ji, L. N.; Mao, Z. W. Low-weight polyethylenimine cross linked 2-hydroxypopyl-β-cyclodextrin and folic acid as an efficient and nontoxic siRNA carrier for gene silencing and tumor inhibition by VEGF siRNA. Int. J. Nanomed. 2013, 8, 2101−2117. (23) Birdi, K.S. Handbook of Surface and Colloid Chemistry; CRC Press, Boca Raton, FL, 1997.

(VEGF: 24.4 pg/mL). FA-mediated tumor targeting of these micelles and entrapment in the acidic tumor endosomal/ lysosomal compartments caused rapid release of doxorubicin and VEGF siRNA, which directly inhibited VEGF expression and thus lowered serum VEGF levels. The cytotoxic effect of doxorubicin also decreased VEGF levels. Lastly, the D-CA-PEIFA-S micelles produced a VEGF level (36.4 pg/mL) similar to that of the D-CA-PEI-FA micelles.

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CONCLUSIONS D-CA-PEI-FA micelles were successfully prepared and characterized, and subsequently siRNA was complexed with these micelles to produce D-CA-PEI-FA-S with a high siRNA loading efficiency. The zeta potential of D-CA-PEI-FA (+5.5 mV) micelles decreased after siRNA complexation (+2.14 mV), while XPS analysis revealed the presence of FA outside D-CAPEI-FA micelles. Doxorubicin showed a two-phase release from D-CA-PEI-FA-S micelles, with an immediate release followed by a sustained-release profile. Furthermore, doxorubicin was released much faster at pH 5.0 as compared to that at pH 7.4. The D-CA-PEI-FA and D-CA-PEI-FA-S micelles were found to inhibit in vivo tumor growth; however, no significant difference in their in vitro cytotoxic activity or in vivo antitumor effect was observed. It can be concluded that the FA presence enhanced micellar activity, whereas the presence of siRNA did not significantly increase their antitumor activity. The body weight of the mice in the D-CA-PEI-FA-S-treated group did not obviously fluctuate, indicating that the micelles did not exhibit significant systemic toxicity. Histological analysis revealed that tumor tissue from mice treated with D-CA-PEI-FA or D-CAPEI-FA-S had the lowest cancer cell densities and the highest level of apoptosis and necrosis. Moreover, other vital organs were not affected by these treatments. The liver DPD levels were the lowest in mice treated with D-CA-PEI-FA or D-CAPEI-FA-S, as shown by ELISA. A group of mice treated with micelles containing siRNA-targeting VEGF instead of ABCB1 (D-CA-PEI-FA-SV) showed the lowest serum VEGF levels. These results indicated that the developed nanoconjugate, CAPEI-FA, has the potential to achieve targeted codelivery of doxorubicin and siRNA.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +603-9289 7690. Fax: +603-2698 3271. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by an Exploratory Research Grant Scheme (ERGS/1/2013/skk02/UKM/02/4) from Ministry of Higher Education Malaysia.

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REFERENCES

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In Vivo Antitumor Activity of Folate-Conjugated Cholic Acid

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