Dendrimer for Targeted Drug Delivery of HeLa Cells - ACS Publications

Dec 15, 2015 - comparison to G4.5-RGD/DOX is indication that higher drug ... IL6 is a better suited carrier for targeted drug delivery of DOX to cervi...
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IL‑6 Antibody and RGD Peptide Conjugated Poly(amidoamine) Dendrimer for Targeted Drug Delivery of HeLa Cells Shewaye Lakew Mekuria, Tilahun Ayane Debele, Hsiao-Ying Chou, and Hsieh-Chih Tsai* Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei 106, Taiwan ROC S Supporting Information *

ABSTRACT: In this study, PAMAM dendrimer (G4.5) was conjugated with two targeting moieties, IL-6 antibody and RGD peptide (G4.5-IL6 and G4.5-RGD conjugates). Doxorubicin anticancer drug was physically loaded onto G4.5-IL6 and G4.5RGD with the encapsulation efficiency of 51.3 and 30.1% respectively. The cellular internalization and uptake efficiency of G4.5-IL6/DOX and G4.5-RGD/DOX complexes was observed and compared by confocal microscopy and flow cytometry using HeLa cells, respectively. The lower IC50 value of G4.5-IL6/DOX in comparison to G4.5-RGD/DOX is indication that higher drug loading and faster drug release rate corresponded with greater cytotoxicity. The cytotoxic effect was further verified by increment in late apoptotic/necrotic cells due to delivery of drug through receptor-mediated endocytosis. On the basis of these results, G4.5IL6 is a better suited carrier for targeted drug delivery of DOX to cervical cancer cells.

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INTRODUCTION The major concern of most antitumor drugs remains to be the high toxicity which damages normal cells and leads to multiple adverse effects.1 One commonly used method to overcome the setbacks of chemotherapy is to develop suitable drug delivery systems targeted toward carcinogenic cells and leave healthy cells unharmed.2 Doxorubicin (DOX) is an anthracycline antibiotic that is used in the treatment of a wide spectrum of cancers. While the exact mechanism of its antitumor activity remains unclear, DOX is known to intercalate into DNA, block topoisomerase II activity, and thereby prevent DNA replication and cell division.3,4 Researchers have recently used polymer carriers to regulate the amount of DOX released in active form inside cancer cells.5−7 Dendrimer, an extensively branched polymer, has been demonstrated to be a unique platform for the construction of various multifunctional carriers for drug delivery applications.8−13 Poly(aminoadmine) (PAMAM) dendrimers are a family of highly branched and monodispersed synthetic macromolecules with well-defined structure and composition.5,14 These polymers have internal cavities and peripheral functional groups that can be modified to better localize drugs and modulate dendrimer−drug interactions.14−16 Although high generation dendrimers have significantly increased the drug loading efficiency in comparison to low generation dendrimers, their synthesis is complicated and timeconsuming. Therefore, an alternative approach of creating large spaces through dendrimer self-assembly has been developed to enhance the loading capacity of dendrimer-based carriers.17 © XXXX American Chemical Society

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy and is typically accomplished through the introduction of targeting moieties to drug delivery systems.7,18−21 Covalent conjugation of targeting moiety such as folic acid,8,22−24 monoclonal antibodies,25,26 and peptides27−29 to carriers confer receptormedicated active targeting ability toward tumor tissues. On the other hand, targeting efficacy is dependent on the type of ligand and biostability of the ligand on the carrier system.30 The arginine-glycine-aspartate (RGD) tripeptide acts as a specific integrin-ligand that regulates a variety of cell functions to affect cell-adhesion properties and cell migration.31−33 RGD-conjugated carriers can be internalized through integrin alpha (v) beta3 receptors via cell adhesion mediated endocytosis manner.34,35 Several types of cancer cells including cervical cancer cells express interleukin-6 receptor, human estrogen receptor (HER), and glycoprotein 130 (gp130) that mediates the IL-6 signal cascade and allow drug carriers to be internalized through a receptor mediated endocytosis pathway.26,36,37 We previously reported the improved targeting ability of G4.5 PAMAM dendrimer nanoparticles toward cervical carcinoma after IL-6 conjugation.38 In the present work, we designed G4.5 PAMAM-based carriers with either IL-6 or RGD conjugated to serve as the targeting ligand for use to deliver Received: November 12, 2015 Revised: December 15, 2015

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DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B Scheme 1. Synthesis of G4.5-RGD Conjugates through EDC/NHS Chemistry

UV−vis spectroscopy. Loading efficiency was calculated by the equation:

DOX to cervical HeLa cells. Drug loading capacity, pHdependent drug release profile, toxicity, and targeting efficiency of the systems were evaluated and compared.

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encapsulation efficiency (%) initial fluorescence intensity = × 100 initial fluorescence intensity − final fluorescence intensity

EXPERIMENTAL SECTION Materials. Ethylenediamine core carboxyl-terminated G4.5 PAMAM dendrimer (G4.5-COOH) was purchased from Dendritech Inc. Doxorubicin hydrochloride (DOX.HCl) was purchased from Cayman Chemical Co., Ltd. 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxylsuccinimide (NHS) were obtained from Acros Organics (Geel, Belgium). Dulbecco’s modified eagle medium (DMEM), penicillin, sodium pyruvate, trypsin, sterilized fetal bovine serum (FBS), and L-glutamine were purchased from Gibco (Carlsbad, CA). Hela cells were obtained from the Bio Resource collection and Research Center (Hsinchu, Taiwan). Monoclonal antihuman interleukin6 (IL-6) antibody was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in phosphate buffered saline (PBS) to a final concentration of 0.5 mg/mL. Arginine−glysine− aspartate (RGD) peptide, 3-[4,5-dimethyl-2-thiazolyl]-2,5diphenyl-2H-tetrazolium bromide (MTT), and D2O were obtained from Sigma-Aldrich as well. Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and propidium iodide (PI) was purchased from Molecular Probes (Eugene, USA). Water used in this study was purified using a Milli-Q Plus 185 system and had a resistivity higher than 18.2 mΩ cm. All other solvents and chemicals were of HPLC grade. Cellulose dialysis membrane with molecular cutoff of 6000−8000 Da was acquired from CelluSept T1 (Braine-l′ Alleud, Belgium). Synthesis of G4.5-RGD and G4.5-IL6 Conjugates. A G4.5 PAMAM solution was prepared by dissolving 10 mg of G4.5 in 2 mL of ultradistilled water and adjusting the pH to 3 with 1N hydrochloric acid. The solution was dried under vacuum and the product desolved in 2 mL of a dimethylformamide (DMF)/water solution (4:1 v/v). To the G4.5 solution, 4 mg of EDC and 2 mg of NHS were added to provide NHS-activated G4.5 after a 14 h reaction. After drying, NHS-activated G4.5 was reacted with RGD in 2 mL of sodium bicarbonate solution (1 M NaHCO3, pH 8.5) for 2 h at a RGD/G4.5 molar ratio of 16:1. The final solution was then extensively dialyzed and lyophilized overnight in a freeze-dryer to obtain the final product. The stepwise synthesis procedure is illustrated in Scheme 1. The synthesis of G4.5-IL6 was described in our previous work and the molar ratio of G4.5 to IL6 was fixed at 2.8 to 1.38 DOX Loading into G4.5-IL6 and G4.5-RGD Complexes. A loading method similar to that described elsewhere was used.39 Briefly, conjugated dendrimer solution (0.5 mg/mL) was added to DOX solution (prepared with DMSO and neutralized with 5 μL triethylamine) at various molar ratios (dendrimer:DOX: 0:1, 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1) and rigorously stirred. Free DOX was removed by dialysis overnight and final products were collected after lyophilization. Dendrimer-drug interaction was probed by 1H NMR and

Characterization Methods. Fourier transform infrared (FTIR) spectra were acquired using a Thermo Nicolet 6700 system, for which samples were prepared on CaF2 pellets and allowed to dry in vacuum. 1H nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance 600 MHz NMR spectrometer with D2O as the solvent. Ultraviolet− visible (UV−vis) spectra were collected on a Jasco V-550 UV− vis spectrophotometer. Fluorescence spectra were recorded at 25 °C using a 1 cm-path quartz cell on a Jasco FP-8300 spectrophotometer equipped with Zenon lamp at the slit width of 10 nm, scan rate of 240 nm/min, and PMT voltage of 400 V. An excitation wavelength of 490 nm was used and fluorescence emission was monitored in the range of 500−800 nm. The ζ-potential of carriers was measured using a Horiba SZ100 nanoparticle analyzer. Cellular internalization was visualized using a Leica TCS-SP2 confocal laser scanning microscope. Finally, apoptotic and necrotic cells were differentiated with commercially obtained kit by flow cytometry. In Vitro Drug Release Kinetics. The drug release of free DOX and DOX encapsulated in modified complexes into phosphate buffer (pH 7.4) and sodium acetate buffer (pH 5.0) was monitored through dialysis under reservoir-sink conditions at a temperature of 37 °C. At each predetermined time point, an aliquot was withdrawn and its concentration determined by UV−vis. The outer phase buffer medium was replenished each time to maintain a constant volume. In order to ensure reasonable comparison between groups, the amount of DOX was kept constant between groups and the study was performed in triplicate. Cumulative release was calculated using the equation: cumulative release (%) =

concentration of drug release × 100 concentration of drug load

Toxicity toward HeLa Cells. Human cervical carcinoma (HeLa) cells were seeded in 96-well plates at a density of 1 × 105/well and grown overnight in DMEM supplemented with 10% FBS. The medium was then replaced with fresh culture medium containing G4.5-COOH, G4.5-RGD, G4.5-IL6, G4.5RGD/DOX, and G4.5-IL6/DOX, or free DOX. The concentration of DOX was identical in all drug-containing wells. After 48 h of incubation, culture medium was replaced with fresh medium containing 20 μL of a MTT (5 mg/mL) solution for 4 h. The medium was replaced with 50 μL of DMSO to dissolve the formazan crystals. Absorbance was read at a test wavelength of 450 nm and reference wavelength of 570 nm. Cell viability was calculated as cell viability (%) = B

absorbance of control cells × 100 absorbance of treated cells DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B In Vitro Cellular Uptake. Intracellular uptake of DOX was qualitatively confirmed using confocal scanning microscopy. HeLa cells were seeded in 24-well plates with coverslips at a density of 1 × 105 cells/well and grown overnight. The medium was then withdrawn and replenished with solutions of G4.5RGD/DOX and G4.5-IL6/DOX. After 24 h incubation, the wells were emptied and washed with PBS three times prior to fixation using 2 mL of a 4% paraformaldehyde (PFA) solution in PBS for 10 min. The coverslips were rinsed with PBS three times and the cells were permeabilized using 2 mL/well of 0.1% Triton X-100 for 10 min. Finally, the coverslips were washed three times with PBS and maintained hydrated with 1.5 mL of PBS during imaging. The coverslips were mounted onto glass microscope slides with one drop of antifade S5 mounting media added to reduce fluorescence bleeding. Flow Cytometry Analysis. For quantitative analysis of cellular binding, flow cytometry was performed using a Becton Dickinson FACS Area III cell sorter with laser excitation set to 370 and 488 nm. The emission filter was set to 450/20 and 530/30 nm band-pass for dendrimer and DOX respectively. HeLa cells were seeded in culture plates at 2 × 106 cells/well for 24 h. The medium was then replaced with control (PBS), free DOX, G4.5-RGD/DOX, and G4.5-IL6/DOX at a DOX concentration of 20 μg/mL respectively. The medium was discarded after 24 h and the cells were washed with PBS, trypsinized, centrifuged (6 min, 1000 rpm), and resuspended in PBS containing 2% FBS for flow cytometry evaluation. Cells treated with PBS were used as the control. A total of 10000 viable cells were evaluated in each experiment, and the FL1fluoresence was analyzed using BD FACS Diva software provided with the system. Cell Apoptosis/Necrosis Analysis. The Annexin V-Alexa Fluor 488 apoptosis detection kit was used to quantify apoptotic and necrotic cells by standard fluorescent activated cell sorting (FACS) assay. Briefly, Hela cells were seeded into 6-well plates at density of 1 × 106 cells/well for 24 h and washed three times with PBS to remove dead cells. Free DOX, G4.5-RGD, G4.5-IL6 (G4.5 concentration of 40 μg/mL), G4.5RGD/DOX, and G4.5-IL6/DOX (DOX concentration of 20 μg/mL) were incubated with cells overnight. The cells were then washed, collected, and resuspended in 500 μL of 1x annexin-binding buffer. Alexa Fluor@ 488 annexin V and PI were added according to the manufacturer’s recommendation. Samples were incubated in the dark for 15 min at room temperature. An additional 400 μL of 1× annexin-binding buffer was added and mixed gently with the samples prior to analysis. Statistical Analysis. All results are representative of at least three sets of independent experiments with samples performed in duplicate or triplicate in each experiment. The data represent the mean ± standard deviation (SD) of measurements.

Scheme 2. Schematic Diagram of G4.5-RGD/DOX and G4.5-IL6/DOX Complexes Interacting with the Cancer Cell Membrane

was investigated both quantitative and qualitatively using fluorescence, UV−vis, and NMR. Fluorescence spectroscopy is a useful tool to study the interaction of DOX with its surrounding molecule.40 Figure 1A and 1B show the fluorescence spectra of free DOX and DOX loaded in G4.5-RGD and G4.5-IL6 complexes. DOX loading was confirmed by initial increase in fluorescence intensity and predominant quenching of DOX fluorescence at the optimum molar ratio. At the dendrimer: DOX ratio of 0.1:1 and 0.2:1, DOX aggregation within the dendrimer complex resulted in a decrease in fluorescence intensity. However, when the dendrimer amount was increased, DOX fluorescence increased as well due to dispersion of drug molecules inside the complex. At the optimum molar ratio of 1:1, fluorescence of the drug/ dendrimer complex decreased due to formation of a stable system. It means that “even we increase the amount of dendrimer, the fluorescence of DOX not change anymore”. Furthermore, a notable decrease in DOX fluorescence was observed in (Figure S1) after dialysis due to noncovalent binding between DOX and the dendrimer which indicates that hydrophobic interactions are mainly responsible for drug loading.41 Drug encapsulation efficiency was calculated to be 30.1 and 51.3% for DOX loaded in G4.5-RGD and G4.5-IL6 complexes, respectively. As shown in Figure 1C, characteristic maximum absorbance of G4.5 is around 290 nm which corresponds to previous reports.38,42,43 The formation of DOX-bounded G4.5-RGD/ DOX and G4.5-IL6/DOX complexes was evidenced by the red color of the solution due to adsorbed DOX. Moreover, a redshift in DOX absorbance from 480 nm to 490 and 495 nm was observed after its incorporation into G4.5-RGD and G4.5-IL6 respectively due to ground-state electron donor−acceptor interactions between drug and dendrimer components.44 The pay load of DOX, as estimated from absorbance intensity, was 9.2 and 9.8 wt % in G4.5-RGD and G4.5-IL6 complexes, respectively. This result suggests that antibody incorporation increased the drug pay load capacity of the carriers and in agreement with the previous report.45 Next, the interaction between DOX and dendrimers was analyzed by multiple Gaussian curves fitting analysis using Origin 8.5 data processing software as shown in Figure S1. The area under each emission band and its correlating factor R2 value are shown in Table S1 and S2. The nature of interaction between DOX and dendrimer complexes can be elucidated by the change in position of the spectra and the ratio of peak A1 to A2 and A3 to A2. The A1/A2 value of DOX loaded in G4.5RGD and G4.5-IL6 was significantly higher than that of free

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RESULTS AND DISCUSSION Loading of Doxorubicin into Modified Dendrimer Carriers. In a previous study, we found that G4.5 PAMAM dendrimer and IL-6 antibody conjugates can assemble into core−shell nanoparticles with potential for use as bioimaging probe for cancer cells.38 In this study, two targeting moieties, RGD and IL-6, were conjugated to G4.5 dendrimer and evaluated for their effect on targeting DOX-loaded complexes toward cervical cancer cells. The cell-targeting mechanism of RGD and IL-6 is illustrated in Scheme 2. Drug encapsulation C

DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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Table 1. ζ-Potential of G4.5-RGD and G4.5-IL6 before and after DOX Loadinga materials G4.5-IL6 G4.5-RGD G4.5-RGD/DOX G4.5-IL6/DOX a

ζ-potential (mV) at 25 °C −6.83 −8.17 −4.4 −4.83

± ± ± ±

2.96 0.32 3.56 1.17

Note: Data present the mean ± standard deviation (n = 3).

Figure 2. Cumulative release of DOX from G4.5-RGD/DOX and G4.5-IL6/DOX complexes at pH 5.0 and pH 7.4 as a function of time.

presence of proton peaks of RGD-tripeptide (methylene peaks between 3.75 and 4.00 ppm) and G4.5 (multiple methylene protons between 2.6 and 3.6 ppm) indicates the successful coupling of G4.5 and RGD in Figure S2B. The peak area is related to the number of protons and thus the area ratio of dendrimer to RGD is approximately 10.98 to 1. Therefore, according to the spectrum, approximately one molecule of dendrimer was linked to 6.89 molecules of RGD. Figure S2C and S2D confirm the incorporation of DOX with dendrimer complexes through the presence of characteristic DOX resonance peaks at δ 1.32 and 8.50 ppm. The degree of drug loading was calculated by area under peak and approximately 10.41 and 10.53 DOX molecules were loaded per G4.5-RGD and G4.5-IL6 respectively. The ζ-potential of complexes has a profound effect on the colloidal stability of the system. Table 1 shows the ζ-potential of carriers before and after DOX loading. The observed increase in potential of the entire system is due to the loading of uncharged DOX into negatively charged dendrimer complexes.46,47 In Vitro DOX Release from Dendrimer Complexes. In vitro drug release was evaluated in PBS and acetate buffer in regard to the acidic microenvironment of tumors. Figure 2 shows the release profile of free DOX and DOX from dendrimer complexes. Nearly 75% of free DOX was rapidly released within 4 h while the release of DOX from complexes followed a biphasic pattern characterized by initial rapid release and long-term sustained manner release.15,48 More specifically, DOX was released relatively slower from G4.5-RGD than G4.5IL6. At 200 h, 51.56% and 69.73% of DOX was released from G4.5-RGD and G4.5-IL6 respectively at pH 5.0 while only 30.96% and 37.33% of DOX was released at pH 7. In pH 5.0, the protonated dendrimer branches (tertiary amine) with

Figure 1. (A) Fluorescence emission intensity of DOX and G4.5RGD/DOX at different molar ratios. (B) Fluorescence emission intensity of DOX and G4.5-IL6/DOX at different molar ratios. (C) Absorbance intensity of DOX (black), G4.5-RGD/DOX (red), and G4.5-IL6/DOX (blue).

DOX which indicate interaction between DOX and both dendrimer complexes. The G4.5-IL6 conjugated was characterized by 1H NMR, which was shown in Figure S2A. On the other hand, the D

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Figure 3. Confocal microscopic images of HeLa cells treated with (A) G4.5-RGD/DOX and (B) G4.5-IL6/DOX complexes for 24 h with the same concentration of DOX.

positive charges under acidic condition are also able to repel the positively charged DOX molecules, thereby speeding up the release of DOX from the interiors, which is in agreement with the previous studies.48,49 As a result, drugs are effectively released under acidic environment in the endosome/lysosome compared to physiological pH conditions. In addition, DOX within G4.5-IL6 was released to a greater extent due to more significant change in morphology of G4.5-IL6 than G4.5-RGD in response to pH change. Cell Uptake of G4.5-RGD/DOX and G4.5-IL6/DOX Conjugates. The lack of selectivity toward tumor cells is the major cause of severe side effects and can be circumvented through targeting of cytotoxic drugs to diseased cells.18 In the confocal scanning micrograph, as shown in Figure 3A, there is weak red fluorescence intensity of G4.5-RGD/DOX compared to G4.5-IL6/DOX (Figure 3 B). One of reason is that the cell uptake of G4.5-RGD/DOX lower than G4.5-IL6/DOX. Another is that DOX was not effectively released from G4.5RGD/DOX. In contrast, the high affinity of HER for IL-6 afforded specific binding and significant internalization of IL-6 modified complexes into HeLa cells through the receptor mediated endocytosis pathway. Furthermore, drug liberation into the nucleus was significantly higher for IL6-conjugated complexes due to the higher multivalency of IL6 at the surface

Figure 4. Flow cytometry analysis of HeLa cells treated with (A) PBS, (B) free DOX, (C) G4.5-RGD/DOX, and (D) G.5-IL6/DOX for 24 h.

of dendrimers which strengthened ligand−receptor binding and increased targeting ability.9 In contrary, internalization of RGDconjugated systems occurs through cell adhesion mediated endocytosis via integrins receptors and the intracellular accumulation of these targeted systems can offer prolonged drug delivery.35,50 Flow cytometry was used to quantify the cellular uptake of free DOX and DOX loaded in modified G4.5 complexes after 24 h of treatment. Negative control of untreated cells (Figure 4A) revealed complete lack of fluorescence while cells treated E

DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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ization of complexes into HeLa cells, the binding efficiency of G4.5-RGD/DOX and G4.5-IL6/DOX was 58.5% and 56.3% respectively. These results confirm that cellular uptake was significantly improved by both RGD and IL-6 receptor mediated pathway mechanisms. In Vitro Cytotoxicity. Cytotoxicity is one of the most critical considerations for biomaterials and it is known that the in vitro cytotoxicity of PAMAM dendrimers depends on the concentration, peripheral functional group, and type of cell line used.51,52 The MTT assay was adopted to access the cytotoxicity of modified G4.5 complexes and the results reveal that the nanoparticles themselves are not toxic toward HeLa cells (Figure S3). Doxorubicin loaded in modified complexes exhibited significantly lower cytotoxicity than free DOX due to the slow release of DOX from nanoparticles (Figure 5). However, when compared to negative control, both G4.5RGD/DOX and G4.5-IL6/DOX resulted in significant decrease in cell viability. HeLa cells incubated with DOX at 20 μg/mL in the form of free DOX, G4.5-RGD/DOX, and G4.5-IL6/DOX were viable at 30.67 ± 10.01%, 53.33 ± 6.00% and 49.05 ± 8.02% respectively. IC50 values for free DOX, G4.5-RGD/ DOX, and G4.5-IL6/DOX were calculated to be 12.3, 21.8, and 18.1 μg/mL, respectively. The higher IC50 values of G4.5/DOX complexes in comparison to free DOX may be the result of

Figure 5. MTT assay of HeLa cell viability after treatment with free DOX dissolved in PBS, G4.5-RGD/DOX, and G4.5-IL6/DOX complexes at different DOX concentration. The data are expressed as mean ± SD (n = 3).

with DOX exhibited fluorescence due to diffusion of DOX across the membrane (Figure 4B). As calculated from Figure 4C and 4D where high fluorescence indicates the internal-

Figure 6. Apoptotic/necrotic cell populations as determined by flow cytometric analysis with Annexin V-Alexa fluor 488 and propidium iodide (PI) staining after incubating HeLa cells with (A) PBS as control, (B) free DOX, (C) G4.5-RGD/DOX, and (D) G4.5-IL6/DOX. The lower-left and upper-left quadrants in each section indicate the populations of normal cells and early necrotic cells, respectively, whereas the lower-right and upperright quadrants in each panel indicate the populations of early and late apoptotic cells, respectively. F

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continuous drug release from the drug delivery vehicle up to 48 h. On the other hand, the IC50 value of G4.5-IL6/DOX was lower than that of G4.5-RGD/DOX due to the latter has a slower drug release rate. Cell death mechanism was assessed by flow cytometry and cells were double stained for viability (negative for PI) and apoptosis (positive for Alexa Fluor 488 Annexin V). As shown in Figure 6A, majority of the cells were localized in Q3 with 99.1% of viable cells in the control group. After treatment with DOX (Figure 6B), G4.5-RGD/DOX (Figure 6C), and G4.5IL6/DOX (Figure 6D), complete elimination of viable cells was observed. The percentage of early apoptotic cells after free DOX, G4.5-RGD/DOX, and G4.5-IL6/DOX treatment was 26.7, 62.5, and 29.7%, respectively, while the percentage of late apoptotic cells was 73.3, 37.5, and 70.3% respectively. The enhanced apoptosis induced by G4.5-IL6/DOX in comparison to G4.5-RGD/DOX is likely the result of enhanced cellular uptake associated with antibody mediated endocytosis.

CONCLUSIONS The current study demonstrates that the conjugation of cancer cell targeting moieties RGD and IL-6 to G4.5 dendrimers and the subsequent formation of a drug delivery system that is capable of delivering the anticancer drug Doxorubicin. The DOX-loaded G4.5-RGD and G4.5-IL6 conjugates were responsive to environmental pH and released significantly higher amount of DOX under acidic conditions due to the expulsion of positively charged DOX molecules from protonated hydrophobic dendrimer cores. In comparison to the RGD-conjugated system, IL6-conjugated system exhibited relatively higher cellular internalization, higher drug release, more toxicity and resulted in greater percentage of cells in late apoptotic stage after treatment. These differences may be attributed to higher multivalent ligand density at the surface of the dendrimer in IL6 and thus more efficiency delivery through receptor-mediated endocytosis. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b11125. Fluorescence emission spectra of free DOX, G4.5-RGD/ DOX, and G4.5-IL6/DOX before and after dialysis, Gaussian profile of G4.5-RGD/DOX and G4.5-IL6/ DOX after dialysis, 1H NMR of G4.5-RGD, G4.5-RGD/ DOX, and G4.5-IL6/DOX, and the cytotoxicity of G4.5 PAMAM, G4.5-RGD, and G4.5-IL6 against HeLa cells (PDF)

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REFERENCES

(1) Singh, Y.; Palombo, M.; Sinko, P. J. Recent Trends in Targeted Anticancer Prodrug and Conjugate Design. Curr. Med. Chem. 2008, 15 (18), 1802−1826. (2) Marks, P. A.; Breslow, R. Dimethyl Sulfoxide to Vorinostat: Development of This Histone Deacetylase Inhibitor as an Anticancer Drug. Nat. Biotechnol. 2007, 25 (1), 84−90. (3) Chen, N.-T.; Wu, C.-Y.; Chung, C.-Y.; Hwu, Y.; Cheng, S.-H.; Mou, C.-Y.; Lo, L.-W. Probing the Dynamics of Doxorubicin-DNA Intercalation during the Initial Activation of Apoptosis by Fluorescence Lifetime Imaging Microscopy (FLIM). PLoS One 2012, 7 (9), e44947. (4) Bassi, L.; Palitti, F. Anti-topoisomerase Drugs as Potent Inducers of Chromosomal Aberrations. Genet. Mol. Biol. 2000, 23, 1065−1069. (5) Gillies, E.; Frechet, J. Dendrimers and Dendritic Polymers in Drug Delivery. Drug Discovery Today 2005, 10 (1), 35−43. (6) Soliman, G. M.; Sharma, A.; Maysinger, D.; Kakkar, A. Dendrimers and Miktoarm Polymers Based Multivalent Nanocarriers for Efficient and Targeted Drug Delivery. Chem. Commun. 2011, 47 (34), 9572−87. (7) Zhang, J.; Lan, C. Q.; Post, M.; Simard, B.; Deslandes, Y.; Hsieh, T. H. Design of Nanoparticles as Drug Carriers for Cancer Therapy. Cancer Genomics Proteomics 2006, 3 (3−4), 147−157. (8) Zhu, J.; Shi, X. Dendrimer-Based Nanodevices for Targeted Drug Delivery Applications. J. Mater. Chem. B 2013, 1 (34), 4199. (9) Lee, C. C.; MacKay, J. A.; Frechet, J. M. J.; Szoka, F. C. Designing Dendrimers for Biological Applications. Nat. Biotechnol. 2005, 23 (12), 1517−1526. (10) Kesharwani, P.; Jain, K.; Jain, N. K. Dendrimer as Nanocarrier for Drug Delivery. Prog. Polym. Sci. 2014, 39 (2), 268−307. (11) Madaan, K.; Kumar, S.; Poonia, N.; Lather, V.; Pandita, D. Dendrimers in Drug Delivery and Targeting: Drug-Dendrimer Interactions and Toxicity Issues. J. Pharm. BioAllied Sci. 2014, 6 (3), 139−150. (12) He, H.; Wang, Y.; Wen, H.; Jia, X. Dendrimer-Based Multilayer Nanocarrier for Potential Synergistic Paclitaxel-Doxorubicin Combination Drug Delivery. RSC Adv. 2014, 4 (7), 3643−3652. (13) Shi, C.; Guo, D.; Xiao, K.; Wang, X.; Wang, L.; Luo, J. A DrugSpecific Nanocarrier Design for Efficient Anticancer Therapy. Nat. Commun. 2015, 6, 7449. (14) Esfand, R.; Tomalia, D. A. Poly(amidoamine) (PAMAM) Dendrimers: from Biomimicry to Drug Delivery and Biomedical Applications. Drug Discovery Today 2001, 6 (8), 427−436. (15) Zhang, M.; Guo, R.; Kéri, M.; Bányai, I.; Zheng, Y.; Cao, M.; Cao, X.; Shi, X. Impact of Dendrimer Surface Functional Groups on the Release of Doxorubicin from Dendrimer Carriers. J. Phys. Chem. B 2014, 118 (6), 1696−1706. (16) Lo, S.-T.; Kumar, A.; Hsieh, J.-T.; Sun, X. Dendrimer Nanoscaffolds for Potential Theranostics of Prostate Cancer with a Focus on Radiochemistry. Mol. Pharmaceutics 2013, 10 (3), 793−812. (17) Wei, T.; Chen, C.; Liu, J.; Liu, C.; Posocco, P.; Liu, X.; Cheng, Q.; Huo, S.; Liang, Z.; Fermeglia, M.; et al. Anticancer Drug Nanomicelles formed by Self-Assembling Amphiphilic Dendrimer to Combat Cancer Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2015, 112 (10), 2978−2983. (18) Muvaffak, A.; Gurhan, I.; Gunduz, U.; Hasirci, N. Preparation and Characterization of a Biodegradable Drug Targeting System for Anticancer Drug Delivery: Microsphere-Antibody Conjugate. J. Drug Target 2005, 13 (3), 151−159. (19) Brannon-Peppas, L.; Blanchette, J. O. Nanoparticle and Targeted Systems for Cancer Therapy. Adv. Drug Delivery Rev. 2012, 64, 206−212. (20) Menjoge, A. R.; Kannan, R. M.; Tomalia, D. A. DendrimerBased Drug and Imaging Conjugates: Design Considerations for Nanomedical Applications. Drug Discovery Today 2010, 15 (5−6), 171−85. (21) Dosio, F.; Brusa, P.; Cattel, L. Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components. Toxins 2011, 3 (7), 848−883.

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AUTHOR INFORMATION

Corresponding Author

*(H.-C.T.) E-mail: [email protected]. Telephone: +886-2-27303625. Fax: +886-2-27303733. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Ministry of Science and Technology, Taiwan (MOST 103-2221-E-011-035) and the National Taiwan University of Science and Technology (102H451201) for providing financial support. G

DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B (22) Thomas, T. P.; Huang, B.; Choi, S. K.; Silpe, J. E.; Kotlyar, A.; Desai, A. M.; Zong, H.; Gam, J.; Joice, M.; Baker, J. R. Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic. Mol. Pharmaceutics 2012, 9 (9), 2669−2676. (23) Singh, P.; Gupta, U.; Asthana, A.; Jain, N. K. Folate and Folate− PEG−PAMAM Dendrimers: Synthesis, Characterization, and Targeted Anticancer Drug Delivery Potential in Tumor Bearing Mice. Bioconjugate Chem. 2008, 19 (11), 2239−2252. (24) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. PAMAM Dendrimer-Based Multifunctional Conjugate for Cancer Therapy: Synthesis, Characterization, and Functionality. Biomacromolecules 2006, 7 (2), 572−579. (25) Thomas, T. P.; Patri, A. K.; Myc, A.; Myaing, M. T.; Ye, J. Y.; Norris, T. B.; Baker, J. R. In Vitro Targeting of Synthesized AntibodyConjugated Dendrimer Nanoparticles. Biomacromolecules 2004, 5 (6), 2269−2274. (26) Shukla, R.; Thomas, T. P.; Peters, J. L.; Desai, A. M.; KukowskaLatallo, J.; Patri, A. K.; Kotlyar, A.; Baker, J. R. HER2 Specific Tumor Targeting with Dendrimer Conjugated Anti-HER2 mAb. Bioconjugate Chem. 2006, 17 (5), 1109−1115. (27) Liu, J.; Gray, W. D.; Davis, M. E.; Luo, Y. Peptide and Saccharide-Conjugated Dendrimers for Targeted Drug Delivery: A Concise Review. Interface Focus 2012, 2 (3), 307−324. (28) Shukla, R.; Thomas, T. P.; Peters, J.; Kotlyar, A.; Myc, A.; Baker, J. R., Jr. Tumor Angiogenic Vasculature Targeting with PAMAM Dendrimer-RGD Conjugates. Chem. Commun. 2005, 46, 5739−5741. (29) Li, Z.; Huang, P.; Zhang, X.; Lin, J.; Yang, S.; Liu, B.; Gao, F.; Xi, P.; Ren, Q.; Cui, D. RGD-Conjugated Dendrimer-Modified Gold Nanorods for in Vivo Tumor Targeting and Photothermal Therapy. Mol. Pharmaceutics 2010, 7 (1), 94−104. (30) Yin, H.-Q.; Mai, D.-S.; Gan, F.; Chen, X.-J. One-Step Synthesis of Linear and Cyclic RGD Conjugated Gold Nanoparticles for Tumour Targeting and Imaging. RSC Adv. 2014, 4 (18), 9078. (31) Tan, T.-W.; Yang, W.-H.; Lin, Y.-T.; Hsu, S.-F.; Li, T.-M.; Kao, S.-T.; Chen, W.-C.; Fong, Y.-C.; Tang, C.-H. Cyr61 Increases Migration and MMP-13 Expression Via αvβ3 Integrin, FAK, ERK and AP-1-Dependent Pathway in Human Chondrosarcoma Cells. Carcinogenesis 2009, 30 (2), 258−268. (32) Caswell, P.; Norman, J. Endocytic Transport of Integrins During Cell Migration and Invasion. Trends Cell Biol. 2008, 18 (6), 257−263. (33) Aguzzi, M. S.; Fortugno, P.; Giampietri, C.; Ragone, G.; Capogrossi, M. C.; Facchiano, A. Intracellular Targets of RGDS Peptide in Melanoma Cells. Mol. Cancer 2010, 9, 84−84. (34) Lucie, S.; Elisabeth, G.; Stéphanie, F.; Guy, S.; Amandine, H.; Corinne, A.-R.; Didier, B.; Catherine, S.; Alexeï, G.; Pascal, D.; et al. Clustering and Internalization of Integrin α(v)β(3) With a Tetrameric RGD-synthetic Peptide. Mol. Ther. 2009, 17 (5), 837−843. (35) Bareford, L. M.; Swaan, P. W. Endocytic Mechanisms for Targeted Drug Delivery. Adv. Drug Delivery Rev. 2007, 59 (8), 748− 758. (36) Galien, R.; Garcia, T. Estrogen Receptor Impairs Interleukin-6 Expression by Preventing Protein Binding on the NF-kappaB Site. Nucleic Acids Res. 1997, 25 (12), 2424−2429. (37) Narazaki, M.; Yasukawa, K.; Saito, T.; Ohsugi, Y.; Fukui, H.; Koishihara, Y.; Yancopoulos, G.; Taga, T.; Kishimoto, T. Soluble forms of the Interleukin-6 Signal-Transducing Receptor Component gp130 in Human Serum Possessing a Potential to Inhibit Signals through Membrane-Anchored gp130. Blood 1993, 82 (4), 1120−1126. (38) Mekuria, S. L.; Tsai, H.-C. Preparation of Self-Assembled CoreShell Nano Structure of Conjugated Generation 4.5 Poly (amidoamine) Dendrimer and Monoclonal Anti-IL-6 Antibody as Bioimaging Probe. Colloids Surf., B 2015, 135, 253−260. (39) Li, J.-M.; Wang, Y.-Y.; Zhao, M.-X.; Tan, C.-P.; Li, Y.-Q.; Le, X.Y.; Ji, L.-N.; Mao, Z.-W. Multifunctional QD-Based Co-Delivery of siRNA and Doxorubicin to HeLa Cells for Reversal of Multidrug Resistance and Real-Time Tracking. Biomaterials 2012, 33 (9), 2780− 2790. (40) Pietrzak, M.; Wieczorek, Z.; Stachelska, A.; Darzynkiewicz, Z. Interactions of Chlorophyllin with Acridine Orange, Quinacrine

Mustard and Doxorubicin Analyzed by Light Absorption and Fluorescence Spectroscopy. Biophys. Chem. 2003, 104 (1), 305−313. (41) Liu, Z.; Sun, X.; Nakayama-Ratchford, N.; Dai, H. Supramolecular Chemistry on Water-Soluble Carbon Nanotubes for Drug Loading and Delivery. ACS Nano 2007, 1 (1), 50−56. (42) Triulzi, R. C.; Micic, M.; Orbulescu, J.; Giordani, S.; Mueller, B.; Leblanc, R. M. Antibody-Gold Quantum Dot-PAMAM Dendrimer Complex as an Immunoglobulin Immunoassay. Analyst 2008, 133 (5), 667−672. (43) Pande, S.; Crooks, R. M. Analysis of Poly(amidoamine) Dendrimer Structure by UV-Vis Spectroscopy. Langmuir 2011, 27 (15), 9609−9613. (44) de la Escosura, A.; Martínez-Díaz, M. V.; Guldi, D. M.; Torres, T. Stabilization of Charge-Separated States in PhthalocyanineFullerene Ensembles through Supramolecular Donor-Acceptor Interactions. J. Am. Chem. Soc. 2006, 128 (12), 4112−4118. (45) King, H. D.; Dubowchik, G. M.; Mastalerz, H.; Willner, D.; Hofstead, S. J.; Firestone, R. A.; Lasch, S. J.; Trail, P. A. Monoclonal Antibody Conjugates of Doxorubicin Prepared with Branched Peptide Linkers: Inhibition of Aggregation by Methoxytriethyleneglycol Chains. J. Med. Chem. 2002, 45 (19), 4336−4343. (46) Chandra, S.; Dietrich, S.; Lang, H.; Bahadur, D. Dendrimer− Doxorubicin conjugate for enhanced therapeutic effects for cancer. J. Mater. Chem. 2011, 21 (15), 5729. (47) Chandra, S.; Noronha, G.; Dietrich, S.; Lang, H.; Bahadur, D. Dendrimer-magnetic nanoparticles as multiple stimuli responsive and enzymatic drug delivery vehicle. J. Magn. Magn. Mater. 2015, 380, 7− 12. (48) Fu, F.; Wu, Y.; Zhu, J.; Wen, S.; Shen, M.; Shi, X. Multifunctional Lactobionic Acid-Modified Dendrimers for Targeted Drug Delivery to Liver Cancer Cells: Investigating the Role Played by PEG Spacer. ACS Appl. Mater. Interfaces 2014, 6 (18), 16416−25. (49) Wang, Y.; Cao, X.; Guo, R.; Shen, M.; Zhang, M.; Zhu, M.; Shi, X. Targeted Delivery of Doxorubicin into Cancer Cells using a Folic Acid-Dendrimer Conjugate. Polym. Chem. 2011, 2 (8), 1754−1760. (50) Muro, S.; Gajewski, C.; Koval, M.; Muzykantov, V. R. ICAM-1 Recycling in Endothelial Cells: a Novel Pathway for Sustained Intracellular Delivery and Prolonged Effects of Drugs. Blood 2005, 105 (2), 650−658. (51) Jones, D. E.; Ghandehari, H.; Facelli, J. C. Predicting Cytotoxicity of PAMAM Dendrimers using Molecular Descriptors. Beilstein J. Nanotechnol. 2015, 6, 1886−1896. (52) Jevprasesphant, R.; Penny, J.; Jalal, R.; Attwood, D.; McKeown, N. B.; D’Emanuele, A. The Influence of Surface Modification on the Cytotoxicity of PAMAM Dendrimers. Int. J. Pharm. 2003, 252 (1−2), 263−266.

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DOI: 10.1021/acs.jpcb.5b11125 J. Phys. Chem. B XXXX, XXX, XXX−XXX