Intracellular Uptake and pH-Dependent Release of Doxorubicin from

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Intracellular Uptake and pH-Dependent Release of Doxorubicin from the Self-Assembled Micelles Based on Amphiphilic Polyaspartamide Graft Copolymers Myeongeun Lee,† Jihoon Jeong,‡ and Dukjoon Kim*,† †

School of Chemical Engineering and ‡School of Pharmacy, Sungkyunkwan University, Suwon, Kyunggi 440-746, Republic of Korea ABSTRACT: Biodegradable and pH-sensitive graft copolymers based on polysuccinimide (PSI) were synthesized as intracellular drug carriers. Hydrophobic octadecylamine (C18) and hydrophilic O-(2aminoethyl) polyethylene glycol (PEG, Mw 5000) were grafted on a PSI backbone for amphiphilicity, enabling the formation of a selfassembled micellar structure in aqueous medium. Biotin was conjugated at the end of the PEG segment as the cell penetrating ligand, and hydrazone hydrate was introduced as a cleavable linkage for the release of pH sensitive drug, doxorubicin. The chemical structure of the polymer and degree of substitution of the graft segments were confirmed by Fourier transform infrared (FTIR) and 1H NMR spectroscopy. The size and distribution of the polymer micelles were investigated by dynamic light scattering. The average diameter of the polymer micelles was 290−310 nm with a narrow distribution. Less than 30% of the total DOX loaded in the polymeric micelles was released at pH 7.4, whereas >75% was released at pH 5 in 70 h because of the cleavage of the hydrazone bond in acidic conditions. For the cytotoxicity test, the MCF-7 cell viability in the presence of biotin-conjugated polymer was much lower than that in the presence of a nonconjugated one, as the former had higher probability of cell penetration aided by a biotin ligand. The DOX uptake in MCF-7 cells was analyzed by the confocal laser scanning microscopy. More DOX uptake was observed in acidic conditions because of the cleavage of hydrazone groups in the polymer.

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INTRODUCTION One of the major concerns in the recent drug delivery technology is the minimization of side effect, which may defect the normal cells and tissues.1−4 This side effect is more frequently observed when toxic and strong drugs such as anticancer drugs are used. The site-specific drug delivery technology overcomes such problems in cancer therapy. In the site-specific drug delivery system, the drug is delivered specifically into the target cancer cells to maximize the drug therapy, minimizing side effects.6−8 The typical site-specific drug delivery system is based on the enhanced permeation and retention (EPR) where a few hundred nanometer-scaled particle or micellar carriers containing drugs may penetrate through the relatively loose blood vessels around the tumor cells.5,9,10 While the carriers circulating through the bloodstreams are possibly accumulated in cancer cells by the EPR effect, the cell penetration is an important hurdle to overcome increasing intracellular drug accumulation.11−15 The intracellular delivery is basically driven by endocytosis mechanism; however, it can be enhanced by the cell penetration ligand, interacting with the receptors in cells for feasible cell penetration.16−18 Once the drugs are introduced inside the cells, they are transferred to lysosomes by early endosome at pH 6.5 and late endosome at pH 5. In lysosome, almost all drugs are degraded © XXXX American Chemical Society

by hydrolytic enzymes. The biodegradation process inside cells results in low drug therapic efficiency; therefore, the utilization of the pH-sensitive copolymer carriers is receiving much attention, as it may establish the “proton sponge effect” by which the drugs are escaped from endosomes before degradation.19−23 Doxorubicin (DOX) is a well-known hydrophobic drug for the treatment of several cancers, including breast, lung, prostate, brain, ovarian, cervix, and others. When DOX is chemically conjugated to the drug carrier, it attaches to the carrier by forming a stable covalent bond. In this circumstance drug release kinetics is more controllable and less drug loss is expected. Because the debonding energy of the chemically conjugated drug is relatively high, feasible release of the conjugated drug by a small change of external stimuli such as pH is highly recommended.24−26 For this purpose, one of the effective cleavable linkers reported is hydrazone functional group. When the polymer carrier and DOX are bonded via hydrazone linker, its cleavage in slight acidic condition (slight reduction of pH) easily releases the drug.27−29 Received: August 27, 2014 Revised: November 21, 2014

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h at 70 °C under a nitrogen atmosphere. The product was precipitated in methanol, filtered, and washed several times with water. After drying in a vacuum oven at 70 °C for 24 h, C18-g-PSI (1 g) was dissolved in DMF (10 mL). PEG (1 g) dissolved in DMF (10 mL) was dropwise added to C18-g-PSI/DMF, and the reaction mixture was heated at 70 °C for 48 h under nitrogen gas. The reaction mixture was precipitated in diethyl ether, and the solvent was completely removed using a dialysis membrane for 3 d, followed by freeze-drying. Conjugation of Biotin Ligand to PEG. Biotin, 2 equiv of PEG/ C18-g-PSI, and 1.1 equiv of DCC, along with 3 mol % DMAP, were mixed in DMF (25 mL). The reaction mixture was kept at 0 °C for 5 min and further stirred at room temperature for 3 h. After precipitation in diethyl ether, the solvent was completely removed using a dialysis membrane for 3 d, followed by freeze drying. Next, the pH-sensitive, acid-cleavable linker, hydrazone functional group, was introduced to the PSI backbone. The synthesized biotin-PEG/C18-g-PSI was dissolved in DMF (10 mL). Hydrazone hydrate dissolved in DMF solution was dropwise added to biotin-PEG/C18-g-PSI solution and then stored for 4 h at room temperature for the grafting reaction. After the grafting, the crude product was precipitated in diethyl ether. The precipitate was washed with acetone several times, followed by dialysis for 3 d and freeze-drying. Finally, DOX was grafted on the PSI backbone by adding DOX to a solution of the-synthesized biotinPEG/C18-g-PSI (50 mg) dissolved in DMSO (25 mL), and the reaction mixture was stirred at room temperature for 24 h. To that reaction mixture, TEA was added and then reacted further for 24 h in the dark. After the grafting reaction, the product was purified by dialysis and freeze-drying as mentioned above. The overall grafting reactions are schematically shown in Figure 2. Preparation of Self-Assembled Aggregates from Polyaspartamide Derivatives. The synthesized polymers were dissolved in PBS at pH 7.4. The polymer molecules were self-assembled by hydrophobic interaction to form aggregates comprising inner hydrophobic cores and outer hydrophilic shells. Ultrasonication was applied if necessary. Chemical Identification. Ubbelohde viscometer was used to measure viscosity of the polymer solution in DMF at the concentration of 0.5 g/dL. The temperature was fixed at 25 °C using a thermo controlled bath. The degree of polymerization (n) of polymer was determined from eq 1 provided by Palo Neri et al.

Many polymers based on polypeptides or amino acids are biocompatible and biodegradable without toxicity.30,31 Polysuccinimide (PSI) can be transformed to poly(amino acid) by hydrolysis or ammonolysis reaction.32,33 In this study, a series of biodegradable copolymers based on PSI were synthesized. Hydrophilic O-(2-aminoethyl) polyethylene glycol (PEG, Mw: 5000) and hydrophobic octadecylamine (C18) segments were grafted on PSI to provide amphiphilicity. Hydrazone, a pHsensitive cleavable linker, was introduced between PSI and DOX. Biotin was attached at the end of the PEG segment as a cell penetrating ligand for the interaction with the receptor of the cells. Because of the amphiphilicity of the synthesized graft copolymers, the formation of self-assembled micellar structure in a few hundreds nm diameter is expected as drug carriers. The physical structure and size of the self-assembled micelles were analyzed. The drug release behavior from the polymer micelles triggered by pH change was investigated. The cytotoxic effect and cellular uptake behavior of polymer micelles were tested in human breast cancer cells (MCF-7).

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EXPERIMENTAL SECTION

Materials. L-Aspartic acid was purchased from Sigma-Aldrich and used for the synthesis of PSI. Phosphoric acid (85%), triethylamine (TEA), 4-(dimethylamino)pyridine (DMAP), and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Sigma-Aldrich and used as catalysts for the synthesis of polymers. Anhydrous N,N′dimethylformamide (DMF, Sigma-Aldrich), mesitylene, sulfolane (Gibco), dimethyl sulfoxide-d6 (DMSO-d6, Samchun), phosphate buffered saline (PBS, pH 7.6, Samchun), methanol (Samchun), and diethyl ether (Samchun) were used as solvents. Hydrazone hydrate, O(2-aminoethyl)polyethylene glycol (Mw 5000), octadecylamine (C18), and biotin were purchased from Sigma-Aldrich and grafted on the PSI backbone. HABA/Avidin reagent and pyrene were purchased from Sigma-Aldrich and used as the fluorescence probes. RPMI-1640 medium (RPMI), penicillin-streptomycin (100 U/mL, Corning), trypsin-EDTA (0.25%; TE, Gibco)), fetal bovine serum (FBS, Sigma-Aldrich), and thiazolyl blue tetrazolium bromide (MMT, Sigma-Aldrich) were used as chemical agents for the cell viability tests. Doxorubicin hydrochloride purchased from Sigma-Aldrich was used as a drug to be loaded. Synthesis of PSI. PSI was synthesized from L-aspartic acid under acid catalysis by the condensation polymerization as shown in Figure 1. L-Aspartic acid (25 g) was suspended in a mixture of mesitylene (70

1.56 n = 3.52 × ηred

(1)

where, ηred is the reduced viscosity of polymer. FTIR spectra were recorded to identify the characteristic functional groups in the synthesized polymer. The dry polymer powder sample was mixed with KBr in 1:100 wt ratio, and the resulting mixture was compressed to prepare film to measure IR absorption in the wavelength range ∼4000−400 cm−1 using a PerkinElmer FTIR spectrometer (Model SPECTRUM 2000). The chemical structure of the synthesized polyaspartamide derivatives was analyzed by Fourier transform-nuclear magnetic resonance spectroscopy (FT-NMR) using a Uniy Inovq 500 (Varian, U.S.A.) NMR spectrometer. The polymer sample was dissolved in DMSO-d6 before 1H NMR measurement. The presence of biotin in the synthesized polymer was confirmed by Biotin binding assay. HABA/Avidin reagent (100 mg) was dissolved in distilled water (10 mL), and the biotin-conjugated polymer sample was dissolved in PBS solution at pH 7.4 to prepare 5 mg mL−1 concentration. After that, both solutions were mixed together, and UV absorption intensity was measured at 500 nm wavelength using a UV−visible spectrophometer (OPTIZEN 3220UV, Korea). Figure 3 shows the basic theory of this measurement based on the binding between HABA/Avidin and the biotin-conjugated polymer. Physical Structure. The average diameter and distribution of the self-assembled aggregates were measured using an electrophoresis light scattering spectrophotometer (ELS-Z2, OTSUKA, Japan). The polymer sample was dissolved in PBS solution at pH 7.4 at a concentration of 1 mg mL−1 to obtain self-assembled aggregates. Before the measurement, the polymer solution was filtrated through a

Figure 1. Synthetic scheme of PSI. g) and sulfolane (30 g) in the presence of phosphoric acid (15 mmol) as the catalyst. The polymerization reaction was performed at 170 °C for 8 h under nitrogen gas. Water, produced as the byproduct in the polymerization step, was removed using a Dean−Stark trap. The reaction mixture was precipitated in methanol, filtered using a filter paper, and then washed several times with distilled water until neutralization to afford PSI, which was dried in a vacuum oven at 70 °C for 24 h to obtain dry PSI. Synthesis of Polyaspartamide Derivatives. To synthesize C18g-PSI, PSI (10 g) with an appropriated amount of octadecylamine was dissolved in DMF (50 mL), and the reaction mixture was stirred for 24 B

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Figure 2. Synthetic scheme of biotin-PEG/HYD-DOX/C18-g-PASPAM.

Figure 3. Response mechanism of the HABA/Avidin reagent and biotin-conjugated polymeric micelles. concentrations in the range 1 × 10−5 to 1 mg mL−1. Fluorescence spectra were measured using a fluorescence spectrometer (AMINCOBowman Series 2) at 25 °C. The emission wavelength was fixed at 392 nm, and the fluorescence excitation intensity was measured from 300 to 360 nm under the slot width of 2 nm. The CAC value of each polymer sample was determined from the polymer solution concentration at the abrupt increase of the I337/I334 value. In Vitro DOX Release Experiment. Dialysis method was employed to observe the release behavior of the DOX-conjugated nano polymer self-aggregates. The DOX-conjugated polymer sample (10 mg) was kept in a dialysis membrane tube with the cut off molecular weight of 12000 Da. The membrane tube containing

0.45 μm filter paper to remove any residual impurities. The size and shape of the self-assembled aggregates were investigated using scanning electron microscopy (SEM, Hitachi, S-2400, Japan). Samples were prepared by casting the polymer solution in distilled water (10 mg L−1) onto a slide glass, which was dried in air. The samples were then coated with palladium gold using an ion coater (Eiko IB-3, Japan) before examination. Critical Aggregation Concentration (CAC). For the measurements of CAC, pyrene was introduced as a fluorescent probe. Pyrene dissolved in toluene was added to the PBS solution at pH 7.4. After agitation for 5 h at 60 °C to evaporate all toluene, the polymer sample was dissolved in 6.0 × 10−7 M pyrene solution to prepare varying C

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polymer sample was placed in PBS solution at pH 7.4, and then the external solution (1 mL) was sampled through the membrane at the predetermined time intervals of 1, 2, 3, 4, 5, 24, 48, and 72 h at 37 °C under mild agitation. The UV absorption intensity of the sample was measured using UV−visible spectrophotometer at a wavelength of 482 nm. Free DOX sample was also prepared by the above-mentioned method to measure the UV absorption intensity for a reference. By comparison of the polymer sample and reference sample based on the free drug, the release amount of DOX from polymer was determined. For this release experiment, the pH of the PBS solution was adjusted by adding 0.1 N HCl aqueous solution. In Vitro Cytotoxicity Measurement. To investigate the cytotoxicity effects of the synthesized polymers, the cell viability % of MCF-7 cells was measured in the presence of the polymer sample. A desired amount of cell culture medium, comprising 5% FBS (50 mL) and 0.5% penicillin−streptomycin in 5 mL of RPMI, was placed in the cell culturing flask. After MCF-7 cells were cultured in a 96-well plate at a concentration of 1 × 104 cell/90 μL concentration for 24 h, the DOX-conjugated polymer, DOX-nonconjugated, and free DOX at varying concentrations were added to each well at 10 μL/well. After 24 h, MTT solution (20 μL, 5 mg mL−1) was added, and the cell culture was incubated at 37 °C in a 5% CO2 incubator for 2 h. After the incubation, the culture medium was removed, followed by adding 200 μL of DMSO in each well to dissolve formazan crystals in the cells. After storing in an oven or incubator for 30 min, the UV intensity was measured using a plate reader at 490 nm. Cell viability % was determined from the relative absorption intensity of the polymer sample to that of control, prepared using the pure PBS solution. The PBS solutions with different pH were before prepared using 0.1 N HCl solution. In Vitro Cellular Uptake. CLSM was employed to investigate the cellular uptake behavior of the synthesized biotin-conjugated polyaspartamide derivatives. A desired amount of cell culture medium comprising 5% FBS (50 mL) and 0.5% penicillin−streptomycin in RPMI (5 mL) was placed in a cell culturing flask. After MCF-7 cells were cultured in a 94-well plate for 24 h at a cell concentration of 10− 15 × 104 cell/2 mL, the biotin-conjugated and nonconjugated polymer samples were prepared by dissolving each at pH 6.4 and 7.4, respectively. The pH was adjusted by the same method mentioned above.

Figure 4. 1H NMR spectrum of PSI.

biotin (PEG/C18-g-PSI), and polymer with biotin (biotin-PEG/ HYD/C18-g-PASPAM) are shown in Figure 6 for comparison. As shown in Figure 6, pure HABA/Avidin reagent showed a strong absorption band at 500 nm. The spectrum of PEG/C18g-PSI also showed a similar absorption band at 500 nm because of the presence of undestroyed HABA/avidine complex. Compared to the UV−visible spectrum of PEG/C18-g-PSI, the UV−visible spectrum of biotin-PEG/HYD/C18-g-PASPAM spectrum, however, showed a significant absorption loss at 500 nm because of the bond formation between avidin and biotin, destroying the HABA/Avidin complex. CAC of Biotin-PEG/HYD/C18-g-PASPAM. The synthesized biotin-PEG/HYD/C18-g-PASPAM was dissolved in PBS solution at pH 7.4 containing pyrene, and the excitation spectra of the fluorescence were measured at different polymer concentrations. At very low polymer concentration, the band shift was not observed at 334 nm in the excitation spectra of pyrene. With increasing polymer concentration, however, the band shift at 337 nm was noticeable, along with the increase in the intensity, owing to the formation of hydrophobic cores where the pyrene coexists with other hydrophobic molecules by hydrophobic interaction. Therefore, the CAC value was determined for the polymer concentration at the abrupt increase of the I337/I334 value, as shown in Figure 7. The low CAC value indicated the stability of polymer micelles in dilute aqueous media. Characterization of Biotin-PEG/DOX-HYD/C18-g-PASPAM Self-Assembled Micelles Size and Size Distribution. The size and distribution of self-assembled micelles formed from the PASPAM derivatives were analyzed at pH 7.4 buffer solution according the type of substituent functional groups. Also, the size and structure of the micelles were photographed using SEM in dry state. Figure 8a shows the size distribution of micelles formed by the two polymer systems, PEG/C18-g-PSI and biotin-PEG/DOX-HYD/C18-g-PASPAM. The average size of PEG/C18-g-PSI micelles before the conjugation to DOX was 240 nm, which was smaller than that of Biotin-PEG/DOXHYD/C18-g-PASPAM (310 nm) with DOX conjugation. This average diameter increase was caused by the additional aggregation of hydrophobic DOX and biotin molecules in the core structure of micelles. The additional hydrophobic molecules in the core structure did not have significant effect

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RESULTS AND DISCUSSION Synthesis of Polyaspartamide Derivatives. PSI was synthesized from L-aspartic acid in acidic conditions by the condensation polymerization. The reduced viscosity of the synthesized polymer was 27, corresponding to a molecular weight of 60000 g/mol from eq 1. 1H NMR spectrum of the synthesized PSI is shown in Figure 4, and the peaks in the range 5.1−5.3 ppm arise from the methine protons, and the other two peaks in the range 3.2−3.5 and 2.4−2.5 ppm are from methylene protons in PSI. For the formation of self-assembled micelles in aqueous media, hydrophobic octadecylamine (C18) and hydrophilic PEG groups were grafted on the PSI backbone. The biotin and hydrazone bond were introduced as a cellular uptake ligand and pH-sensitive cleavable bond for DOX release, respectively. All characteristic protons in the graft polymer were well assigned, as shown in Figure 5a−c. The degree of substitutions of C18 and PEG graft groups was calculated from the ratio of peak area of the characteristic proton in each group to that in the PSI main chain. When C18 and PEG were fed at the ratio of 20 and 10 mol % to PSI repeating unit, the actual degree of substitution was 16 and 8 mol %, respectively. The degree of substitution of biotin and DOX was 0.7 and 0.3 mol %, respectively, when those were fed at 1 and 0.5 mol %. The presence of biotin in the synthesized biotin-PEG/HYD/ C18-g-PASPAM was confirmed by the HABA/Avidin assay. The UV−visible spectra of HABA/Avidin complex, polymer without D

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Figure 6. UV−vis spectra of HABA/Avidin complex after addition of no biotin conjugated and biotin conjugated micelles.

Figure 7. I337/I334 ratios for the biotin-PEG/HYD/C18-g-PASPAM system at different concentrations at pH 7.4.

Figure 9 shows the pH effect on the average micelle size of PEG/DOX-HYD/C18-g-PSI and biotin-PEG/DOX-HYD/C18g-PASPAM systems. The polymeric micelles of a few hundreds micrometer diameter were formed at pH < 7.4, and their structure was stable without noticeable size variation in acidic condition. The presence of biotin groups in the polymer increased with increasing average micelle size because of the increase in the hydrophobic core diameter associated with hydrophobicity. The release of DOX was triggered at pH < 6.1 by the cleavage of hydrazone bond; however, it did not reduce the micelles size significantly, as shown in Figure 9, indicating that the absence of small amount of DOX induced noticeable conformational rearrangement of molecules in the hydrophobic core. The micelle diameter of the PEG/DOX-HYD/C18-g-PSI system without biotin was slightly smaller than that of biotinPEG/DOX-HYD/C18-g-PASPAM. In Vitro DOX Release Behavior. The DOX release behavior from biotin-PEG/HYD-DOX/C18-g-PASPAM micelles was monitored using a UV−visible spectrophotometer. Biotin-PEG/HYD-DOX/C18-g-PASPAM (10 mg) was dispersed in PBS solution at pH 7.4 to form micelles, and the DOX release content was measured at 37 °C and pH 7.4. The calibration curve relating the UV absorption intensity at 482

Figure 5. 1H NMR spectra of (a) PEG/HYD/C18-g-PASPAM, (b) biotin-PEG/HYD/C18-g-PASPAM, and (c) biotin-PEG/HYD-DOX/ C18-g-PASPAM.

on the size distribution. The SEM image of the PEG/C18-g-PSI sample is shown in Figure 8b. The self-assembled particles were spherical, and their size was around 100 nm which was a little smaller than that analyzed from DLS (average diameter 240 nm) because this SEM image was taken in the dry state. E

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Figure 10. In vitro DOX release behaviors from the biotin-PEG/ HYD/C18-g-PASPAM sample (a) at the two different pHs of 5 and 7.4 and (b) at pH 7.4, followed by pH 5.0. At least three samples were examined at the same condition, and the average value was taken for the data.

Figure 8. (a) Particle size distribution and (b) SEM image of PEG/ DOX-HYD/C18-g-PASPAM and biotin-PEG/DOX-HYD/C18-g-PASPAM.

release at pH 5 was attributed to the cleavage of hydrazone bond in acidic condition. In both the cases, about 90% of the total release content during 70 h occurred in 10 h. The DOX released from the polymer at pH 7.4 was presumably physically dispersed drugs rather than the chemically conjugated ones. This pH-dependence of DOX releasing behavior from polymer micelles is supplemented in Figure 10b. While only a small amount of DOX (∼20% of the total loading content) was released to reach equilibrium during 10 h at pH 7.4, the abrupt increase in DOX release (up to 80%) was observed when pH was reduced to 5. The release of the chemically conjugated drug was triggered by the cleavage of hydrazone linker in acidic condition. In Vitro Cell Viability. The cell viability of breast cancer cells, MCF-7, in the presence of PEG/HYD/C18-g-PASPAM, PEG/HYD-DOX/C18-g-PASPAM, biotin-PEG/HYD/C18-gPASPAM, and biotin-PEG/HYD-DOX/C18-g-PASPAM was measured using the MTT method, and the results are shown in Figure 11. In Figure 11b,d, the cell toxicity of DOXconjugated polymer samples were compared with that of the free drug. As the concentration of free drug in this case was the same as that of the polymer, its actual content was larger than that conjugated in the polymer sample. For PEG/HYD/C18-gPASPAM and biotin-PEG/HYD/C18-g-PASPAM with no DOX conjugation, the cell viability was similar. The cell viability

Figure 9. Average particle size of PEG/DOX-HYD/C18-g-PASPAM and biotin-PEG/DOX-HYD/C18-g-PASPAM systems according to pH.

nm wavelength to the free DOX concentration was obtained prior to this measurement. Less than 30% of the total DOX loaded in the Biotin-PEG/HYD-DOX/C18-g-PASPAM was released at pH 7.4, but >75% was released at pH 5 in the same amount of time (70 h), as shown in Figure 10a. This fast F

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Figure 11. Concentration effect on the in vitro cytotoxicity of (a) PEG/HYD/C18-g-PASPAM, (b) PEG/HYD-DOX/C18-g-PASPAM, (c) biotinPEG/HYD/C18-g-PASPAM, and (d) biotin-PEG/HYD-DOX/C18-g-PASPAM systems at pH 6.4 and 7.4. Statistically significant differences in cytotoxicity were analyzed using Student’s t test (*P < 0.05). In (b) and (d), the concentration of free DOX drug is the same as that of the DOXconjugated polymer sample.

In Vitro Cellular Uptake. The penetration behavior of the synthesized polymer micelles into MCF-7 cells was investigated by CLSM. The confocal laser intensity was measured for biotin nonconjugated and biotin-conjugated polymers after each sample was maintained for 5, 15, and 30 min at different pHs, and the results are shown in Figure 12a,b. Both figures show that the intensity increases with increasing duration time, because more polymer molecules penetrate into the cells by endocytosis. The biotin-conjugated polymer sample, biotinPEG/HYD-DOX/C18-g-PASPAM, however, illustrates much higher uptake in cells than the biotin nonconjugated one, PEG/ HYD-DOX/C18-g-PASPAM, because of the cell penetrating activity of biotin. The biotin-PEG/HYD-DOX/C18-g-PASPAM sample shows the highest intensity after exposure for 30 min at pH 6.4. More DOX uptake was observed in acidic conditions owing to the cleavage of hydrazone groups in the polymer molecules.

decreased slightly with polymer concentration but was still very high (>90%), up to 0.2 mg mL−1 and (>80%) up to 0.4 mg mL−1. No significant pH-dependence was observed in this experiment. For PEG/HYD-DOX/C18-g-PASPAM and biotinPEG/HYD/C18-g-PASPAM with DOX conjugation, the cell viability was much lower than that of the former samples, as the toxicity was mostly governed by the presence of DOX and not the pure polymer molecules. The toxicity effect of DOX increased significantly with increasing concentration. The presence of biotin in the polymer has some effect on the toxicity of DOX conjugated samples. The biotin-conjugated polymer sample resulted in a lower cell viability than the nonconjugated one, as biotin aids the penetration of the DOXconjugated polymer micelles into the cells leading to the accumulation of more DOX molecules in the cells. The cell viability in the presence of the biotin-conjugated polymer sample was even lower than that of the free drug-loaded system, because biotin delivers the drugs more efficiently into the cells, owing to the effective cell penetration associated with an interaction between the biotin ligand and cell receptors.

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CONCLUSION Biodegradable pH-sensitive polyaspartamides were synthesized for application in intracellular drug delivery carriers. To form a G

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at pH 6.4 showed the highest intensity. The results from these in vitro experiments suggest that the present biotin-PEG/HYDDOX/C18-g-PASPAM system can be a potential candidate for systemic anticancer drug delivery. The coupling of passive and ligand-mediated active targeting capabilities of biotin-PEG/ HYD-DOX/C18-g-PASPAM may result in a significantly enhanced accumulation of DOX in a tumor region, which helps to achieve desired cytotoxicity of the anticancer drug in tumor cells at a reduced dose. This biotin-PEG/HYD-DOX/ C18-g-PASPAM micelle system has much better stability than other polymer micelles due to covalent bonding of the drug, and thus, it may have a potential impact on translation to human application.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82-31-290-7250. Fax: 031-290-7270. E-mail djkim@ skku.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) Grant funded by the Korean government (MEST; 2010-0027955 and 2012R1A2A1A05026313).

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REFERENCES

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Figure 12. Confocal laser scanning microscope images of (a) PEG/ HYD-DOX/C18-g-PASPAM and (b) biotin-PEG/HYD-DOX/C18-gPASPAM after incubation for 5, 10, and 30 min at different pHs.

self-assembled micellar structure, PEG and C18 groups were grafted on the PSI backbone as hydrophilic and hydrophobic segments, respectively. The hydrazone bond, an acid-cleavable linker, was introduced to the PSI backbone for the release of drug (DOX) conjugated in acidic conditions. Biotin was attached at the end of the PEG segment as a cell penetrating ligand. Two polymeric systems, biotin conjugated and nonconjugated, were synthesized for comparison of micelle structure, cell viability, and cell penetration behaviors. The average diameter of the biotin-conjugated PSI (310 nm) was slightly larger than that of nonconjugated one (290 nm). Less than 30% of the total DOX loaded in the biotin-PEG/HYD/ C18-g-PASPAM was released at pH 7.4, whereas >75% was released at pH 5 in the same time (70 h). The fast release at pH 5 was attributed to the cleavage of hydrazone bond in acidic condition. In cytotoxicity tests, the MCF-7 cell viability in the presence of a biotin-conjugated polymer was much lower than that in the presence of nonconjugated one, as the former had higher probability than the latter for the penetration into the cells via interaction between the ligand and the receptor. The cell viability in the bioconjugated system was even lower than that of the free DOX drug because of more efficient intracellular drug delivery associated with the biotin ligand. The cell penetration behavior of the synthesized polymer micelles into MCF-7 was investigated by CLSM. At low pH, higher intensity was detected resulting from the release of DOX via cleavage of hydrazone bond in polymer molecules. The biotin-conjugated polymer sample after an exposure for 30 min H

dx.doi.org/10.1021/bm501272c | Biomacromolecules XXXX, XXX, XXX−XXX

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dx.doi.org/10.1021/bm501272c | Biomacromolecules XXXX, XXX, XXX−XXX