Polyaspartamide Derivative Nanoparticles with Tunable Surface

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Polyaspartamide Derivative Nanoparticles with Tunable Surface Charge Achieve Highly Efficient Cellular Uptake and Low Cytotoxicity Min Xu,† Yuefang Zhao,† and Min Feng* Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou 510006, People's Republic of China S Supporting Information *

ABSTRACT: Cationic nanocarrier mediated intracellular therapeutic agent delivery acts as a double-edged sword: the carriers promote cellular uptake, but interact nonspecifically and strongly with negatively charged endogenic proteins and cell membranes, which results in aggregates and high cytotoxicity. The present study was aimed at exploring zwitterionic polyaspartamide derivative nanoparticles for efficient intracellular delivery with low cytotoxicity. Poly(aspartic acid) partially grafted tetraethylenepentamine (PASP-pg-TEPA) with different isoelectric points (IEPs) was synthesized. The PASP-pg-TEPA formed zwitterionic nanoparticles with an irregular core and a well-defined shell structure in aqueous medium. Their particle size decreased from about 300 to 80 nm with an increase of the IEP from 7.5 to 9.1. The surface charge of the PASPpg-TEPA nanoparticles could be tuned from positive to negative with a change of the pH of the medium. The nanoparticles with an IEP above 8.5 exhibited good stability under simulated physiological conditions. It was noted that the zwitterionic PASP-pg-TEPA nanoparticles displayed highly efficient cellular uptake in HeLa cells (approximately 99%) in serum-containing medium and did not adversely affect the cell viability at concentrations up to 1 mg/mL. Furthermore, thermodynamic analysis using isothermal titration calorimetry provided direct evidence that these zwitterionic nanoparticles had low binding affinities for serum protein. Therefore, the zwitterionic PASP-pg-TEPA nanoparticles could overcome limitations of cationic nanocarriers and achieve efficient intracellular delivery with low cytotoxicity.

1. INTRODUCTION Cationic nanoparticles as carriers have shown great promise for enhancing cellular delivery of therapeutic agents in the past decade.1,2 Their positively charged surface is known to have a high affinity for cell membranes, which helps the nanoparticles enter cells relatively easily.3 However, the intracellular delivery mediated by cationic nanoparticles acts as a double-edged sword: the carriers promote cellular uptake, but interact nonspecifically and strongly with negatively charged serum proteins and cell membranes, which leads to aggregates, faster clearance, and high cytotoxicity.4,5 The latter has been one of the major limitations to hamper their further application in vivo. Up to now, coating the nanoparticle surface with a hydrophilic, flexible, and nonionic polymer is the major strategy to ameliorate the problems associated with instability, rapid clearance, and cytotoxicity of cationic nanoparticles.6 Poly(ethylene glycol) (PEG) is one of the most commonly used coating materials because PEGylated nanoparticles exhibit reduced nonspecific serum-protein adsorption, prolonged blood circulation times, and low cytotoxicity. This is mostly attributed to the fact that highly mobile PEG chains hide the original surfaces and hydrate with water to provide a steric hindrance that plays an important role in preventing protein adsorption and cell membrane disruption.7 However, the © 2012 American Chemical Society

antifouling PEGylated surfaces also limit the interaction between nanoparticles and target cells, leading to a significant decrease in cellular uptake of nanoparticles.8 Furthermore, a lack of reactive functional groups on PEG limits its potential conjugation of ligands for active targeted delivery. Recently, zwitteration has been developed as an alternative to PEGylation. These zwitterionic molecules were reported to reduce opsonization by serum proteins mainly because of their strong hydration effects under physiological conditions.9 Zwitterionic cysteine conjugated on silica nanoparticles was found to create low-fouling surfaces that successfully repel serum-protein adsorption and achieve long-term stability in human serum solution.10 Zwitterionic phosphorylcholine coated poly(carbonateurethane) nanoparticles showed effective resistance to platelet adhesion and high hemocompatibility.11 Zwitterionic phosphorylcholine even showed a much better ability to stabilize nanoparticles than the neutral PEG.12,13 The zwitteration presents great potential advantages for application in therapeutic and diagnostic agent delivery in vivo. In the present study, we report the novel zwitterionic polyaspartamide derivative poly(aspartic acid) partially grafted Received: January 12, 2012 Revised: July 7, 2012 Published: July 8, 2012 11310

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tetraethylenepentamine (PASP-pg-TEPA). These PASP-pgTEPA polymers could form zwitterionic polyelectrolyte nanoparticles in aqueous medium which have tunable surface charges at different pH values. It is of interest to explore the utilization of these zwitterionic PASP-pg-TEPA nanoparticles as a potential nanocarrier. The rationale of using the PASP-pgTEPA nanoparticles for an intracellular delivery system is the following: (1) The PASP-pg-TEPA nanoparticles provide both hydrophobic and zwitterionic microenvironments, which could load either hydrophobic or ionic hydrophilic therapeutic agents. (2) Through adjustment of the isoelectric point (IEP), the zwitterionic PASP-pg-TEPA nanoparticles could display appropriately charged surfaces to maintain stability as well as have low cytotoxicity. (3) Compared with PEGylated nanoparticles, the cationic amino groups on zwitterionic nanoparticles could interact with the cell membrane to improve the cellular uptake efficiency. (4) Their rich functional groups (carboxyl groups and amino groups) on the surface also have potential to covalently link a variety of biologically active molecules to alter their in vivo properties and allow targeting. Herein, the surface charge tenability of PASP-pg-TEPA nanoparticles in different pH media and the IEP effects on their stability in simulated physiological media were investigated. The formation of PASP-pg-TEPA nanoparticles in aqueous solution was studied by using pyrene as the fluorescent probe. The cellular uptake and cytotoxicity of the PASP-pgTEPA nanoparticles were specifically evaluated by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.

2. MATERIALS AND METHODS 2.1. Materials. L-Aspartic acid was purchased from Shanghai Baoman Biotechnology. Tetraethylenepentamine was obtained from Sigma-Aldrich. Pyrene with a purity of 99% was purchased from Aladin. Fluorescein isothiocyanate (FITC) was purchased from Sigma. N,N-Dimethylformamide (DMF) was purchased from Sinopharm Chemical Reagent Co. DMF was dried overnight with 4 Å molecular sieves and redistilled before use. The HeLa cell line was obtained from the American Type Culture Collection. RPMI-1640 medium, fetal bovine serum (FBS), trypsin, and penicillin−streptomycin were purchased from Gibco. Dimethyl sulfoxide (DMSO) and MTT were obtained from Sigma-Aldrich. All other reagents were of analytical grade. 2.2. Synthesis of Zwitterionic PASP-pg-TEPA. Polysuccinimide (PSI) was synthesized by thermal polycondensation of L-aspartic acid according to the previously reported method.14 Briefly, 10 g of Laspartic acid was mixed evenly with 3 g of 85% phosphoric acid as the catalyst and the mixture stirred at 40 °C in a vacuum oven for 2 h and then dissolved in 50 mL of anhydrous DMF. The solution was repeatedly precipitated by ethanol to purify PSI. The precipitate PSI was dried at 50 °C overnight in a vacuum. The structure of PSI was confirmed by 1H NMR and FTIR analysis: 1H NMR (CDCl3, ppm) δ 2.45 (t, 2H, −CHCH2NH−), 2.71, 2.86 (s, 3H, DMF, −CH3), 3.29 (d, 1H, −COCHCH2−), 5.23 (m, 1H, −CHCH2NH−). PASP-pg-TEPA was synthesized by ring-opening reactions of PSI with both TEPA and sodium hydroxide (NaOH), as shown in Figure 1. Briefly, 0.5 g of synthesized PSI (5.15 mmol) was dissolved in 10 mL of DMF, and then the resulting solution was added dropwise to a solution of TEPA (0.5−1.0 mole ratio relative to the succinimide unit of PSI) in 10 mL of DMF. The reaction mixture was stirred at room temperature for 2 h. Subsequently, an aqueous solution was added dropwise to the reaction solution to hydrolyze the remaining succinimide unit of PSI. After being stirred for 1 h at 40 °C, the reaction mixture was dialyzed against deionized water for 48 h using a dialysis bag with a 3500 molecular weight cutoff and finally freezedried to yield PASP-pg-TEPA powder. The structure of PASP-pg-

Figure 1. Synthesis of the zwitterionic polyaspartamide derivative composed of PASP-pg-TEPA. TEPA was confirmed by 1H NMR and FTIR analysis: FTIR (KBr, cm−1) 1659 (−COO−), 1538 (−NH3+); 1H NMR (D2O, ppm) δ 2.65−2.79 (−NHCH2CH2NH−), 2.99−3.07 (−CH2COOH). The carbon, hydrogen, and nitrogen contents of the polymer were determined by elemental analysis using a Vario EL (Elementar, Germany) elemental analyzer. Elemental analysis was used to confirm the reaction between TEPA and PSI and calculate the degree of substitution (DS) of the obtained PASP-pg-TEPA. The DS of the TEPA grafted on poly(aspartic acid) was calculated by the following equation:

DS =

[N]m − [N]o × 100 5 × [N]o

where [N]m is the concentration (wt %) of the N element of PASP-pgTEPA with different IEPs, [N]o is the concentration (wt %) of the N element of poly(aspartic acid), and 5 is the number of atoms of the N element in TEPA. The elemental analysis results and DS of PASP-pgTEPA and poly(aspartic acid) are presented in Table S1 (Supporting Information). 2.3. Preparation of Zwitterionic PASP-pg-TEPA Nanoparticles. The zwitterionic PASP-pg-TEPA nanoparticles were prepared by the dialysis method, as shown in Figure 2. In brief, 10 mg of PASPpg-TEPA polymer in 2 mL of DMF was introduced into a dialysis bag (MWCO = 8000−10000) and then dialyzed against 3.0 L of distilled water, which was replaced every 6 h in the course of 24 h. During the dialysis process, DMF was replaced by water, while the zwitterionic PASP-pg-TEPA polymer formed nanoparticles and remained inside the dialysis membrane. 11311

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Figure 2. Schematic diagram of the preparation of zwitterionic PASP-pg-TEPA nanoparticles in aqueous medium. width, 2 nm; high scanning speed, 250 nm min−1. The ratio of intensities at 371 and 380 nm was recorded. 2.8. Cytotoxicity Study. To evaluate the cytotoxicity of zwitterionic PASP-pg-TEPA nanoparticles, MTT colorimetric assays were performed. Briefly, HeLa cells were seeded in 96-well plates at a density of 5000 cells/well. After 24 h the culture medium was replaced with serum-free medium containing PASP-pg-TEPA nanoparticles at serially diluted concentrations from 0.0125 to 1 mg/mL, and then the cells were incubated for another 24 h. Then 20 μL of MTT in 5 mg/ mL phosphate-buffered saline solution was added to each sample. After the samples were incubated for 4 h, the supernatant was aspirated, and the formazan crystals were dissolved in 150 μL of DMSO. Absorption was measured photometrically at 570 nm with a background correction using a Bio-Tek ELX800 enzyme-linked immunosorbent assay (ELISA) reader. Values of eight measurements were normalized to 100% for the control group (exposure to full medium). Cells without addition of MTT were used as a blank for calibration of the spectrophotometer to zero absorbance. The turnover of the substrate relative to control cells was expressed as relative cell viability and was calculated by the Atest/Acontrol × 100 formula. 2.9. Labeling of PASP-pg-TEPA Nanoparticles with FITC. The preparation of FITC-labeled PASP-pg-TEPA nanoparticles was based on the reaction between the isothiocyanate group of FITC and the primary amino groups of PASP-pg-TEPA, as previously described15 with some modifications. A 10 mg portion of PASPA-pg-TEPA powder was dispersed in 10 mL of FITC solution in carbonic acid buffer (0.01 mg/mL, pH 9.0). The dispersion was stirred overnight in the dark at room temperature. The FITC-labeled PASP-pg-TEPA nanoparticles were separated from unreacted FITC by dialysis against 3.0 L of distilled water, which was replaced every 6 h in the course of 2 days under darkness in a dialysis bag (MWCO = 8000−10000). The FITC-labeled PASP-pg-TEPA nanoparticles were then collected by freeze-drying. 2.10. Cellular Uptake and Intracellular Accumulation Level Study. The cellular uptake and intracellular accumulation level of zwitterionic PASP-pg-TEPA nanoparticles were monitored by fluorescence-activated cell sorting (FACS). HeLa cells were seeded 24 h prior to the experiments into 24-well plates at a density of 1.5 × 104 cells/well in 1.0 mL of culture medium. FITC-labeled PASP-pgTEPA nanoparticles with IEPs of 8.5, 8.8, and 9.1 were prepared and then added to the cells. After 4 h of incubation with the FITC-labeled PASP-pg-TEPA nanoparticles in serum-free or serum-containing medium, the cells were rinsed twice with PBS. Subsequently, the cells were treated with trypsin/EDTA for 2 min, collected by centrifugation, suspended in 0.3 mL of PBS, and kept on ice until analysis. The percentage of FITC positive cells and fluorescence intensity were employed to quantify the cellular uptake efficiency and intracellular accumulation levels, respectively, via flow cytometry using the FACSCalibur instrument (Becton-Dickinson) equipped with an

2.4. Transmission Electron Microscopy (TEM). The zwitterionic PASP-pg-TEPA nanoparticles were prepared according to the aforementioned formation protocol. The freeze-dried powders were dispersed in ddH2O (dd = double-distilled) with a final concentration of 1 mg/mL. Drops (10 μL) of freshly prepared solution were placed on a 300-mesh Formvar carbon coated copper grid and allowed to equilibrate for 1 min. The solution was wiped off with filter paper, and the grids were then stained with 2.5% phosphotungstic acid for 1 min and allowed to air-dry. Images were taken using a JEM100CX transmission electron microscope set to an accelerating voltage of 80 kV. 2.5. Measurement of the ζ Potential and Transmittance of the Zwitterionic PASP-pg-TEPA Nanoparticle Dispersion. To determine the IEP of the PASP-pg-TEPA nanoparticles, the ζ potential and transmittance of the PASP-pg-TEPA nanoparticle dispersion at different pH values were measured. The PASP-pg-TEPA nanoparticle dispersion was prepared at a concentration of 10 mg/mL in 10 mM NaCl. The ζ potential of the nanoparticle dispersion was monitored with a Zetasizer NS 90 with a He−Ne laser beam (Malvern Instruments, Malvern, U.K.). All measurements were done at a wavelength of 633 nm at 25 °C with a fixed scattering angle of 90°, and the pH values ranged from 4 to 11. The transmittance of the nanoparticle dispersion at different pH values was measured with a UV−vis spectrophotometer at 500 nm. Adjustment of the pH of the solution was by careful addition of 0.1 N HCl with stirring 2.6. Stability in Simulated Physiological Medium. The stability of PASP-pg-TEPA nanoparticles under simulated physiological conditions was monitored by size measurements. The hydrodynamic particle diameter was measured by using photon correlation spectroscopy (PCS) on a Malvern Zetasizer NS90 (Malvern Instruments). The instrument was equipped with a 10 mW helium neon laser producing light at a wavelength of 633 nm. PASP-pg-TEPA nanoparticles were dispersed in 10 mM NaCl solution, physiological saline (150 mM NaCl) solution, physiological phosphate-buffered saline (PBS; 8 g of NaCl, 0.2 g of KCl, 2.9 g of Na2HPO4·12H2O, and 0.2 g of KH2PO4 in 1000 mL of Milli-Q water), and DMEM (Dulbecco's modified Eagle's medium) cell culture medium with 10% FBS, respectively. Each data point is comprised of three independent experiments. 2.7. Pyrene Fluorescence Probe. PASP-pg-TEPA nanoparticle formation was examined by fluorescence experiments with pyrene as the probe. Fluorescence spectra were recorded on an SAFAS flx spectrofluorometer. A serial dilution of PASP-pg-TEPA was prepared with a pyrene concentration of 6 × 10−7 M. Both the excitation and emission spectra were measured. After repeated testing and debugging, the optimal conditions for determining pyrene fluorescence were established as follows: excitation wavelength, 333 nm; emission wavelengths, 371 and 380 nm; excitation slit width, 2 nm; emission slit 11312

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argon laser with an excitation wavelength of 488 nm. The filter setting for emission was 530/30 nm bandpass. Data were acquired in linear mode and visualized in linear mode. 2.11. Isothermal Titration Calorimetry (ITC) Assay. The binding affinities of serum proteins for PASP-pg-TEPA nanoparticles were assayed by ITC using the VP-ITC calorimeter (MicroCal, GE Healthcare) at 25 °C. A 1430 μL volume of three PASP-pg-TEPA nanoparticle samples, namely, with IEPs of 8.5, 8.8, and 9.1, was added into the sample cell. To determine the binding affinity, 300 μL of FBS stock solution was added stepwise. Typically, 20 injections of 10 μL volume were made with intervals of 300 s between each addition. The titration cell was stirred continuously at 1000 rpm. The heat of the FBS dilution in the deionized water alone was subtracted from the titration data (both normalized to 0) for each experiment. Titration data were integrated and analyzed using the Origin software provided by MicroCal. Figure 3. Normalized pyrene fluorescence intensities (I371/I380) for PASP-pg-TEPA nanoparticles with serially diluted concentrations (blue line, spectrum of pyrene with PASP-pg-TEPA nanoparticles, 10 g/L; green line, spectrum of pyrene with PASP-pg-TEPA nanoparticles, 1 g/L; orange line, spectrum of pyrene with PASP-pg-TEPA nanoparticles, 0.1 g/L; red line, spectrum of pyrene in pure water (negative control).

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Zwitterionic PASP-pg-TEPA. The zwitterionic polyaspartamide derivatives based on PASP-pg-TEPA were synthesized by ring-opening reactions of PSI with both TEPA and sodium hydroxide, as shown in Figure 1. The total ring-opening of the imide structure of PSI was through a two-step reaction: The first was TEPA partial opening of succinimidyl ring moieties, resulting in TEPA grafted onto the main chains. Subsequently, the opening of the rest of the succinimidyl ring moieties was through hydrolysis by sodium hydroxide to obtain carboxyl groups. The obtained zwitterionic polyaspartamide derivative, PASP-pgTEPA, was composed of poly(aspartic acid) as the main chain with both pendent carboxyl groups and amine groups arranged on the main chain randomly. The DS of PASP-pg-TEPA calculated by elemental analysis was based on the concentration (wt %) of the N element, as shown in Table S1 (Supporting Information). The nitrogen content of PASP-pg-TEPA increased from 8.85% to 13.86−15.22% after reaction with TEPA, owing to the extra nitrogen atoms introduced from TEPA, indicating the introduction of amine groups. The DS of the obtained PASP-pg-TEPA calculated by the equation was 11.32%, 13.33%, and 14.40%, corresponding to IEPs of 8.5, 8.8 and 9.1, respectively (Table S1). The reaction conditions were controlled at relatively low concentrations and room temperature to successfully induce the ring-opening reaction but reduce the side reaction of cross-linking of PSI. 3.2. Formation of Zwitterionic PASP-pg-TEPA Nanoparticles with Different Isoelectric Points. This zwitterionic polymer, PASP-pg-TEPA, containing both positively charged amine groups and negatively charged carboxyl groups could form polyelectric zwitterionic nanoparticles (80−300 nm in diameter depending on the IEP) in aqueous medium, as shown in Figure 2. The process of forming zwitterionic nanoparticles was monitored by pyrene-based fluorescence probes. The ratio of vibronic bands in the pyrene fluorescence spectra (I371/I380) calculated from the intensities at 371 and 380 nm was plotted at serially diluted PASP-pg-TEPA nanoparticle concentrations, as shown in Figure 3. The I371/I380 value was 1.22 when the concentration of PASP-pg-TEPA nanoparticles was 10 mg/mL, which confirmed the formation of supermolecular objects with a hydrophobic environment.16 With further dilution of the nanoparticle dispersion, the I371/I380 value gradually increased to 1.80, which indicated the shrinkage of the hydrophobic area with the opening of zwitterionic nanoparticles. When the concentration was below 0.1 mg/mL, the I371/I380 value remained constant at about 1.80, which is

almost the same as the value when pyrene is dissolved in water. The shift of I371/I380 indicated the slow opening of the selfassembled supermolecular objects into free polymer chains and reduction in the hydrophobic environment within the nanoparticles.17 The formation of PASP-pg-TEPA nanoparticles showed the concentration-dependent self-assembly behavior. The formation should involve both electrostatic and hydrophobic interactions based on adsorption of oppositely charged groups in PASP-pg-TEPA intramolecules and intermolecules. TEM images showed that the morphology of PASP-pg-TEPA nanoparticles was of spherical type (Figure 4). By close observation, the most interesting finding was that the PASP-pgTEPA nanoparticles showed a special core−shell-type structure. The core was irregular in shape, and the shell had a well-defined spherical shape. The bright white part should be assigned to the irregular hydrophobic core composed of the deionized and charge-neutralized PASP-pg-TEPA segments. The dark gray part corresponds to the hydrophilic shell of the ionized PASPpg-TEPA segments. The pyrene probe experiment also proved the PASP-pg-TEPA nanoparticles contained a hydrophobic core, which was consistent with the core−shell image of TEM. These PASP-pg-TEPA nanoparticles had zwitterionic properties as a native protein. We evaluated the IEP of PASP-pgTEPA nanoparticles by measuring light transmittance of zwitterionic nanoparticle samples at 500 nm at different pH values (4−11), as shown in Figure S3 (Supporting Information). The PASP-pg-TEPA nanoparticles were clear with high light transmittance at pH 4 and then turned milky with an increase in the pH of the dispersion, even precipitated with light transmittance sharply decreasing near the IEP; subsequently, the precipitate dissociated, and the nanoparticle dispersion was clear again. The pH value corresponding to the lowest transmittance of the samples was regarded as the IEP of the PASP-pg-TEPA nanoparticles. Four PASP-pg-TEPA nanoparticle samples having IEPs of 7.5, 8.5, 8.8, and 9.1 were prepared. The IEP of these zwitterionic PASP-pg-TEPA nanoparticles depended on the DS of TEPA. The higher the degree of substitution, the higher the IEP. The DS of TEPA in a zwitterionic PASP-pg-TEPA molecule could be controlled by the feed ratio of TEPA to PSI. 11313

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Figure 4. TEM images and size distribution of zwitterionic PASP-pg-TEPA nanoparticles having IEPs of (A) 9.1 and (B) 8.5.

3.3. Tunable Surface-Charge Property of Zwitterionic PASP-pg-TEPA Nanoparticles. Nanoparticles with cationic surface charge are known to efficiently enter cells in vitro because of adsorptive interactions with negatively charged mucopolysaccharide on the cell membrane.3 A cationic nanoparticle-mediated drug and gene delivery system such as chitosan, polylysine, and poly(ethylenimine) has been widely employed to enhance cellular delivery of drugs and genes.18,19 However, these cationic nanoparticle delivery systems show extremely low delivery efficiency in vivo because their highly positive surface charges induce cell membrane damage and aggregates due to nonspecific interaction with serum proteins. These deficiencies of cationic nanoparticles lead to high cytotoxicity as well as low stability under physiological conditions.5 Here, the surface charges of PASP-pg-TEPA nanoparticles were investigated by monitoring the ζ potential at different pH values, as shown in Figure 5A. The ζ potential changed from positive to negative and passed through zero in the pH range of 4−11. The value at which the surface of PASPpg-TEPA nanoparticles exhibited a neutral net electrical charge correlated with their IEPs. This result demonstrated that these zwitterionic PASP-pg-TEPA nanoparticles had unique reversible surface charge properties as native proteins (Figure 5B) which carry net positive charges below their IEP and net negative charges above it. The surface charge of these zwitterionic PASP-pg-TEPA nanoparticles may be tuned by changing the pH of the medium to provide a low cationic charged surface. The lower surface charge density of the nanoparticles may weaken the interactions with oppositely

charged blood components and meanwhile maintain their efficient cellular uptake. Furthermore, in comparison to the neutral physiological condition (pH 7.4), the mildly acidic conditions existing in tumor tissues (pH 6.8) and in endosomes (pH 5−6) would lead to higher ionization degrees of these zwitterionic PASP-pg-TEPA nanoparticles and may promote carried therapeutic and diagnostic agent release at the tumor and intracellular sights by taking advantage of the lower pH of these microenvironments. 3.4. Stability of Zwitterionic PASP-pg-TEPA Nanoparticles under Simulated Physiological Conditions. Very stable nanocarriers under physiological conditions are an important prerequisite for successful systemic drug and gene delivery in vivo.20 The physical stability of zwitterionic PASPpg-TEPA nanoparticles under serum-containing, physiological pH, and salt conditions was studied by dynamic light scattering. Compared with the size measurement in a low salt medium (10 mM NaCl), the PASP-pg-TEPA nanoparticles with an IEP above 8.5 did not show significant differences in size in 10% serum-containing medium, PBS (pH 7.4), and physiological saline (0.9% NaCl) (Figure 6). Furthermore, after incubation with the three aforementioned types of simulated physiological medium for 10 and 24 h at 37 °C, these zwitterionic nanoparticles with an IEP above 8.5 also showed no obvious changes in size. However, the PASP-pg-TEPA nanoparticles having a near neutral IEP of 7.5 exhibited a much larger particle size above 1 μm in all media, very likely due to their isoelectric point precipitation. Thus, no further testing for the unstable sample with an IEP of 7.5 was performed. The reason the 11314

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formed a hydrated layer around the PASP-pg-TEPA nanoparticles, acting as a steric hindrance.21 The repulsive forces and steric hindrance between zwitterionic nanoparticles prevented their aggregation. The high stability of the zwitterionic PASPpg-TEPA nanoparticles under simulated physiological conditions would be desired for biomedical applications in vivo. 3.5. Low Cytotoxicity of Zwitterionic PASP-pg-TEPA Nanoparticles. The cationic nanocarriers cause well-known adverse side effects, especially high cytotoxicity, which hampered the development for in vivo application.22 The cytotoxicity of these zwitterionic PASP-pg-TEPA nanoparticles was evaluated to conclude their safety profile for possible further applications. The colorimetric MTT assay was carried out, and the effect of PASP-pg-TEPA nanoparticles on HeLa cell viability was determined as a function of the concentration, as shown in Figure 7. Figure 7 indicates that the PASP-pg-

Figure 5. (A) ζ potential of zwitterionic PASP-pg-TEPA nanoparticles at different pH values. (B) Schematic diagram of tunable surfacecharge properties of zwitterionic PASP-pg-TEPA nanoparticles.

Figure 7. Cell viability of zwitterionic PASP-pg-TEPA nanoparticles having different IEPs.

TEPA nanoparticle samples with IEPs of 8.5 and 8.8 retained high cell viability (>90%) at all tested concentrations up to 1000 μg/mL. The PASP-pg-TEPA nanoparticles with an IEP of 9.1 showed a slight decrease of cell viability (>80% at a concentration of 800 μg/mL). Compared with branched poly(ethylenimine) (25 kDa) as a typical polycation, all the zwitterionic nanoparticles were much less toxic to HeLa cells and exhibited no cytotoxicity under 200 μg/mL. This was possibly because of their relatively lower net positive surface charges, which were not high enough to irreversibly disrupt the structural balance of the oppositely charged cell membrane. 3.6. Low Affinities of Zwitterionic PASP-pg-TEPA Nanoparticles for Serum Proteins. Serum-containing media usually showed inhibitory effects on the cellular uptake efficiency of cationic nanocarriers due to the fact that cationic nanocarriers interact nonspecifically and strongly with negatively charged serum proteins.4 ITC was used to investigate the binding affinities of PASP-pg-TEPA nanoparticles to proteins in serum. PEG6000, a nonfouling material, was used as a negative control to compare to PASP-pg-TEPA nanoparticles in terms of exothermic quantity. The ITC responses were recorded during titration of FBS with PASP-pg-TEPA nanoparticles, as shown in Figure 8. The top curves in Figure 8 show the raw data obtained during each injection. Each peak

Figure 6. Stability of zwitterionic PASP-pg-TEPA nanoparticles under simulated physiological conditions (blue bars, incubation period of 4 h; red bars, incubation period of 10 h; green bars, incubation period of 24 h).

PASP-pg-TEPA nanoparticles with an IEP above 8.5 were stable under physiological conditions might be that when the environmental pH was below their IEP, these zwitterionic nanoparticles were predominantly positively surface charged and exhibited repulsive forces. Also, the water molecules 11315

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Figure 8. Typical ITC data recorded for zwitterionic PASP-pg-TEPA nanoparticles and FBS interactions. The area underneath each injection peak (top panels) was equal to the total heat released for that injection. When this integrated heat was plotted against the volume of FBS added to PASPpg-TEPA nanoparticles in the cell, a complete binding isotherm for the interaction was obtained (bottom panels).

represents a related heat variation with the injection of a small aliquot of FBS into the ITC reaction cell containing the PASPpg-TEPA nanoparticle dispersion or PEG 6000. The bottom curves correspond to the integrated calorimetric response of PASP-pg-TEPA nanoparticles, subtracting that of deionized

water, plotted against the total volume of titrate added. The interaction strengths between proteins and the samples were evaluated on the basis of the exothermicity during the initial stages of interaction.23 In the titration profiles the initial exothermic quantities of all PASP-pg-TEPA nanoparticles and 11316

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Figure 9. (A) Cellular uptake efficiency and (B) intracellular levels of zwitterionic PASP-pg-TEPA nanoparticles in HeLa cells. (C) Representative flow cytometry histogram showing the intracellular levels of zwitterionic PASP-pg-TEPA nanoparticles in HeLa cells in 4 h.

interact strongly with negatively charged serum proteins. The results were consistent with those of our ITC measurements. The protein adsorption resistance of zwitteration was recently confirmed by Schlenoff’s research group.9

PEG6000 were approximately 4.0 and 5.0 kcal/mol, respectively. These results demonstrated that the zwitterionic PASPpg-TEPA nanoparticles had the advantage of serum resistance as well as PEG6000. The results are consistent with those previously reported.9 3.7. Efficient Cellular Uptake by Cancer Cells. It is wellknown that the passivation effect of the PEG molecule chain on the surface of cationic nanoparticles reduces cytotoxicity but depresses cellular uptake as well, which turned out to be a dilemma for the PEG application in the modification of cationic polymers. Herein, the cellular uptake and intracellular accumulation levels of these zwitterionic FITC-labeled PASPpg-TEPA nanoparticles with different IEPs were evaluated. The percentages of FITC-positive cells and FITC-fluorescence mean values were measured by flow cytometry, which represent the cellular uptake efficiency and intracellular accumulation levels of PASP-pg-TEPA nanoparticles, respectively. When the FITC-labeled PASP-pg-TEPA nanoparticles with different IEPs were incubated with HeLa cells for 4 h in both 10% serumcontaining and serum-free conditions, the cellular uptake of these zwitterionic FITC-labeled PASP-pg-TEPA nanoparticle samples with different IEPs showed high uptake efficiency (approximately 99%), as shown in Figure 9A. However, the intracellular accumulation levels of PASP-pg-TEPA nanoparticles were dependent on their IEP (Figure 9B). The zwitterionic nanoparticles having an IEP of 9.1 achieved 1.8and 2.2-fold higher intracellular levels than those nanoparticles having an IEP of 8.5 in both serum-free and serum-containing media. Moreover, it is well-known that the presence of serum could reduce the cellular uptake efficiency of cationic nanocarriers.24 In this study, serum in culture media did not show any significant inhibitory effects on both cellular uptake and intracellular accumulation levels. These results also implied that these zwitterionic PASP-pg-TEPA nanoparticles did not

4. CONCLUSION In the present study, the zwitterionic polyaspartamide derivative PASP-pg-TEPA having different IEPs formed zwitterionic PASP-pg-TEPA nanoparticles in aqueous medium. These zwitterionic nanoparticles contained an irregular core and a well-defined shell. Their particle size decreased from 300 to 80 nm with an increase in IEP from 7.5 to 9.1. The reversible surface charged properties of PASP-pg-TEPA nanoparticles could be tuned from positive charges to negative charges below and above the IEP. PASP-pg-TEPA nanoparticles with an IEP above 8.5 exhibited good stability under simulated physiological conditions. Cellular uptake and cytotoxicity studies demonstrated that the tested zwitterionic PASP-pg-TEPA nanoparticles showed highly efficient cellular uptake with low cytotoxicity. Furthermore, serum in culture media did not show any significant inhibitory effects on either cellular uptake or intracellular levels. The ITC thermodynamic analysis revealed that these zwitterionic nanoparticles had low binding affinities for serum proteins. Therefore, the zwitterionic PASP-pg-TEPA nanoparticles could overcome the limitation of cationic nanoparticles and achieve efficient intracellular delivery with low cytotoxicity.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed structural characterization of PASP-pg-TEPA, including NMR, FT-IR, elemental analysis, and light transmittance measurements and effects of salt concentration and pH on the 11317

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formation of PASP-pg-TEPA nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +8620 39943119. Fax: +8620 39943119. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the Fundamental Research Funds for the Central Universities (Grant 09ykpy67) and the Undergraduates’ Innovation Project of Guangdong Province Funds (Grant 201002256) for their financial support of this research.

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REFERENCES

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dx.doi.org/10.1021/la3025028 | Langmuir 2012, 28, 11310−11318