Chemically Conjugating Polyethylenimine with Chondroitin Sulfate

It seems that the pKa values of the 1° and 2° amino groups decreased and that of the 3° amino group increased ... at an N/P ratio of 5 were 195, 32...
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Chemically Conjugating Polyethylenimine with Chondroitin Sulfate to Promote CD44-Mediated Endocytosis for Gene Delivery Yu-Lun Lo,†,§ Kuo-Hsun Sung,†,§ Chien-Chih Chiu,‡ and Li-Fang Wang*,†,‡ †

Department of Medicinal and Applied Chemistry and ‡Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan S Supporting Information *

ABSTRACT: Polyethylenimine (PEI) is one of the most potent synthetic gene delivery vectors because of its high transfection efficiency. Although PEI has been used as a delivery vehicle for a long while, its toxicity is always an issue for clinical applications. In this study, we introduced a low molecular weight PEI of 10 kilodaltons to chondroitin sulfate (CS) via a Michael addition method. By adjusting weight ratios between cationic PEI and anionic CS, the intermolecular or intramolecular, or both, electrostatic interactions of CS-modified PEI (CP) maintained good water solubility but lost some ability to permeate cell membranes. Thus, the cytotoxicity of PEI decreased without sacrificing its gene transfection efficiency. Three CP copolymers with different PEI contents were synthesized and used to prepare polyplexes with plasmid DNA. The pDNA-formed polyplex with a low PEI content (CP(L)) was least cytotoxic and had a transfection efficiency comparable to Lipofectamine/pDNA. The good uptake of CP(L)/pDNA into U87 cells was primarily based on clatherin-dependent and CD44-mediated endocytosis. KEYWORDS: polyethylenimine, chondroitin sulfate, nonviral vector, gene transfection

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INTRODUCTION Polyethyleneimine (PEI) behaves as a gold standard for nonviral gene delivery vectors. Its high transgene efficiency has been widely used to condense plasmid DNA, siRNA, and antisense oligodeoxynucleotide (ODN).1−4 The positive charge of PEI helps to form a complex with gene drugs and facilitates its proton sponge effect.5 However, the positively charged surface characteristics of PEI also result in its inherent cytotoxicity and nonspecific interactions with nontargeted tissue, with blood cells, and with its self-aggregation with serum proteins.6,7 These issues limit its development for in vivo gene therapy. To solve this problem, several approaches have been explored, such as PEGylation of PEI3,8 and cross-linking of a low molecular weight PEI with cleavable cross-linkers.9,10 The incorporated PEG also prevents recognition by the reticuloendothelial system (RES).11,12 The development of gene delivery systems based on polysaccharides has also progressed in parallel to PEG-decorated ones, not only because they prevent recognition by the RES but also because of the presence of derivable groups on sugar chains, which enrich its versatility for chemical reactions with different kinds of molecules for targeting specific cells and tissue.13 In addition, © 2013 American Chemical Society

polysaccharides have the potential to be taken up inside cells via specific receptors. For example, hyaluronic acid (HA) and chondroitin sulfate (CS) have been reported14−16 to be internalized in cells via CD44 receptor-mediated endocytosis. CD44 is a type I transmembrane glycoprotein participating in many cellular functions, such as cell orientation, adhesion, migration, and matrix-cell signaling processes.17 CD44 is overexpressed in many solid tumor cells18 and a common marker for several cancer stem cells, which exhibit highly malignant and chemoresistant properties.19 The bond between HA and the CD44 adhesion molecule may initiate a series of events that begin with modification of adhesion to the matrix and continue with activation of other molecules such as growth factors, degradation of the matrix, angiogenesis, permeation by blood vessels, and extravasation. All of these steps are necessary in the initiation of metastasis.20 Thus, HA has been used as a Received: Revised: Accepted: Published: 664

August 6, 2012 December 7, 2012 January 2, 2013 January 2, 2013 dx.doi.org/10.1021/mp300432s | Mol. Pharmaceutics 2013, 10, 664−676

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amino groups of PEI via ionic bonding interactions. We hypothesized that an optimized CP conjugate protects pDNA, while it circulates in the bloodstream and accelerates pDNA dissociation from the polyplex in the endosomes after being internalized inside cells because of the introduction of the negatively charged CS, which lowers a charge density of PEI. By fine-tuning the composition between PEI and CS, the minimized cytotoxicity of CP and a sufficient release of pDNA were obtainable. In addition to the physicochemical characterizations of the CP copolymers, the cytotoxicity and transfection efficiency of CP/pDNA polyplexes were correlated with their stability, particle size, and intracellular uptake. The mechanism of cellular uptake was studied using a flow cytometer, and the in vitro cell internalization of pDNA into U87 cells was directly visualized using a confocal laser scanning microscope (CLSM).

drug carrier or targeting moiety for nanoparticles to treat CD44-overexpressed cancers.21,22 HA is a copolymer composed of D-glucuronic acid and Nacetyl-D-glucosamine. The chemical structure of CS is similar to that of HA, composed of sulfate groups in C4 or C6 of Dglucuronic acid and N-acetyl-D-galactosamine. This characteristic also makes CS a potential candidate for site-specific drug delivery vectors to solid tumors overexpressing the CD44 receptor.14,15 The ternary complex of pDNA/PEI and CS showed transgene efficiency comparable to that of the pDNA/PEI complex, but with minimized cytotoxicity and agglutination of erythrocytes. The intracellular uptake of the ternary complex was hypothesized to have been the result of a CS-specific receptor-mediated energy-dependent process.15 Plasmid-green fluorescent protein (GFP)/PEI coated with 10 kilodalton CS increased transfection efficiency greater than coating it with higher-molecular-weight CS or HA. The transfection efficiency of GFP/PEI/CS in ovarian cancer cells was 6 times higher than that in normal cells. Intraperitoneally injecting PEI, carried with a plasmid harboring the murine granulocyte macrophagecolony stimulating factor (mGM-CSF) gene coated with CS, prolonged mouse survival compared with coating the PEIcarried gene with HA. The mGM-CSF/PEI gene coated with CS, but not with HA, yielded 100% mouse survival.23 HA is also important in tissue engineering and drug delivery systems because of its interaction with HA receptors on cell membranes. A PEI-HA conjugate with 24.2 mol % PEI was a more efficient gene silencer than PEI itself as a target intracellular delivery carrier for siRNA in B16F1 cells, which overexpress HA receptors.16 A decrease in the number of HA carboxyl groups also reduced the number of the recognition sites for the CD44 receptor because of the chemical conjugation between PEI and HA.24 Alternatively, the PEIHA conjugate was synthesized as a pDNA carrier using an imine reaction between periodate-oxidized HA and PEI to avoid consuming the carboxylic groups of HA.25 The branched PEI (bPEI, 25 kDA) was modified by forming nanoconstructs with CS to improve site-specific targeting. The results showed that the nanoconstructs were less cytotoxic and that they increased transfection efficiency more than PEI and several commercial transfection reagents did.14 However, these CS-PEI nanoconstructs were prepared using electrostatic interactions between CS and PEI, which had self-assembled to nanoparticles before they were complexed with pDNA. We synthesized CP via a Michael addition method between amino groups of PEI and methacrylated CS. The merits of this design include the following: (i) we use a low molecular weight PEI of 10 kDas (kDA) to minimize the cytotoxicity of PEI, which has been reported to increase with an increase in molecular weight;26 (ii) we couple CS to PEI in an aqueous solution without using any catalysts and obtain a potentially biodegradable and nontoxic gene delivery carrier; (iii) we save the carboxyl groups of CS to permit the CD44 receptor to recognize them; and (iv) we obtain completely water-soluble CS-modified PEI (CP) by altering the PEI content. Using the Michael addition, three CP copolymers with different PEI contents were synthesized: the amino groups of PEI attacked the introduced double bonds engineered by methacrylation of CS (CSMA). We optimized the PEI content of the CP copolymers, not only to form a stable complex with pDNA but also to facilitate a certain degree of intramolecular interactions between carboxyl and sulfate groups of CS and

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MATERIALS AND METHODS Preparing and Characterizing PEI-Conjugated CS (CP). An oral grade CS was used and its number average MW was 58 kDA in equivalent dextran standards, as determined using Gel Permeation Chromatography (GPC). CSMA was synthesized as previously described.27 The degree of methacrylation on the CS was controlled at 70%. PEI (MW = 10 kDA) was grafted onto the CSMA using three different molar ratios of 0.1, 0.3, and 0.5 via Michael addition. After PEI had completely dissolved in double deionized (DD) water, the aqueous solution of CSMA, at a concentration of 1 mg/mL, was added dropwise to the PEI solution (1 mg/mL) and stirred at room temperature (RT) for 48 h. The resulting crude product was purified by dialysis using a dialysis membrane against DD water (MWCO 25 000 DA) for 48 h. The final product was yielded using lyophilization. 1 H nuclear magnetic resonance (NMR) experiments were recorded using a Varian Mercury plus-200 (200 MHz) NMR spectrometer in D2O at a concentration of 10 mg/mL. Fouriertransform infrared (FT-IR) spectra were acquired using a System 2000 spectrophotometer. The dried samples were ground with KBr powder and pressed into pellets for FT-IR measurements. Sixty-four scans were signal-averaged (range, 4000−400 cm−1) at a resolution of 4 cm−1. The molecular weight and molecular weight distribution of polymers were measured by GPC using an Aglient 1100 series equipped with PL aquagel−OH 40 and PL aquagel−OH 60 columns. A phosphate buffer solution at pH 7.4 was used as an eluent at a flow rate of 1 mL/min. Six monodisperse dextran standards were used to generate a calibration curve. The PEI content of CP was determined by measuring the cuprammonium complex formed between PEI and copper(II) at 630 nm using a UV−visible spectrophotometer.28 The PEI content of CP was calculated based on a standard calibration curve of PEI(10K). The buffer capacity was analyzed by acid/ base titration. Each 20 mg sample was added to 20 mL of 150 mM NaCl, and the pH was adjusted to 9.5 using 0.1 N NaOH. The titration was done at 25.0 ± 0.1 °C by adding 0.1 N HCl under an atmosphere of CO2-free N2. The solubility of CP was estimated from transmittance measurements and compared with a physical mixing using the same amount of CSMA and PEI(10K) as CPs at pH 7. The wavelength was set at 630 nm using a UV−visible spectrophotometer. Preparing and Characterizing CP/pDNA Polyplexes. pEGFP-C1 plasmid driven by a cytomegalovirus (CMV) 665

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promoter, pGL3-control plasmid with a Hind III/Xba I firefly luciferase cDNA fragment, and wild-type p53 cDNA cloned into the pCDNA vector were introduced into the E. coli strain DH5α and purified using a kit. CP was dissolved in DD water to a final concentration of 1 mg/mL. The pDNA concentration was fixed at 3 μg/100 μL in DD water to measure DNA binding and at 4 μg/500 μL for other measurements. Equal volumes of CP and pDNA solutions were then mixed and immediately vortexed at high speed for 60 s. The DNA binding ability of polyplexes was evaluated using agarose gel electrophoresis. The polyplexes were prepared at different N/P ratios. The stability of the polyplexes with and without 10% FBS was analyzed using gel electrophoresis with 0.8% agarose in Tris-acetate-EDTA (TAE) with ethidium bromide (EtBr) (1 μg/mL). A current of 100 V was applied to the gels for 40 min, and DNA retention was visualized under UV illumination at 365 nm. The hydrodynamic diameter and zeta potential of polyplexes were measured. Light scattering measurements were done with a laser at 633 nm and a 90° scattering angle. Polystyrene nanospheres (220 ± 6 nm and −50 mV) were used to verify the performance of the instrument. The particle size and zeta potential of each polyplex was measured three times. The size and morphology of polyplexes were also observed using a transmission electron microscope (TEM). A carbon-coated 200-mesh copper specimen grid was glow-discharged for 1.5 min. Ten microliters of the polyplexes were deposited on a TEM grid and allowed to dry for three days at RT before examining the samples with the TEM. Cell Experiments. U87 cells (a human glioblastoma cell line) and 3T3 cells (a mouse fibroblast cell line) were cultivated at 37 °C under humidified 5% CO2 in MEM, supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin. U87 cells were seeded in 96-well tissue culture plates at a density of 5 × 103/well in MEM containing 10% FBS. The cytotoxicity of the polymers was evaluated by determining cell viability after 48 h of incubation with various concentrations of CPs (1−200 μg of polymer/mL). The cytotoxicities of the polyplexes were examined at various N/P ratios of CPs/pDNA for 48 h after they had been incubated at 37 °C for 6 h. The number of viable cells was determined by estimating their mitochondrial reductase activity using the tetrazolium-based colorimetric method (MTT conversion test).29 In vitro transgene expression was done in U87 cells at a density of 1 × 105/well in 12-well plates and incubated in MEM containing 10% FBS for 24 h before transfection. Polyplexes with N/P ratios from 1 to 7 were prepared using different amounts of polymers and a fixed pDNA amount of 4 μg to a final volume of 500 μL. After 6 h of incubation, the medium was replaced with 1 mL of fresh complete-medium, and the cells were incubated for 48 h post-transfection. The GFP expression was directly visualized using a fluorescence microscope. For the luciferase assay, the procedures stated above were repeated to determine the transfection efficiency in U87 cells or 3T3 cells. To quantify the luciferase expression, transfected cells were twice rinsed gently with 1 mL of 0.1 M PBS, added to a 200 μL/well of lysis buffer, and allowed to stand overnight at −20 °C. The luciferase activity was monitored using a microplate scintillation and luminescence counter after mixing the contents of a 50 μL/well of supernatant with the contents of 50 μL/well of luciferase assay reagent. The total protein

content of the cell lysate was examined using a BCA protein assay kit and done according to the manufacturer’s instructions. For p53 expression, U87 cells were seeded in a 6-well plate at density 2 × 105/well in MEM containing 10% FBS for 24 h. Before the transfection assay, the culture medium was replaced with 2 mL of MEM without 10% FBS, and CP(L)/p53 polyplexes were added for 6 h of incubation. PEI(25K) and Lipofectamine were used as positive controls. After 48 h of transfection, the transfected cells were lysed with a 50 μL/well of RIPA lysis buffer. The collected cell lysates were frozen at −20 °C overnight for complete lysis, and the protein concentration was determined using a BCA protein assay kit. An equal amount of protein (30 μg) was separated using 10% SDS-PAGE gel and transferred to a nitrocellulose (NC) membrane. The NC membranes were blocked in blocking buffer (5% nonfat milk powder dissolved in Tris-buffered saline buffer containing 0.1% Tween20 [TBST]) at room temperature. After 1 h of incubation, the blocked NC membranes were probed with a 1:3000 dilution of p53 antibody in 1% nonfat milk-TBST buffer at 4 °C overnight. The next day, the NC membrane was washed for 10 min with TBST 3 times and then incubated with a 1:4000 dilution of peroxidase-conjugated secondary antibody in TBST for 1 h at RT. The NC membrane was washed three times with TBST and developed using an enhanced chemiluminescence (ECL) detection system. Confocal Laser Scanning Microscope (CLSM). The intracellular delivery of pDNA was observed using a CLSM. U87 cells were seeded at a density of 1.5 × 105/well in 12-well plates containing one glass coverslip/well in MEM supplemented with 10% FBS, and then incubated for 24 h. The polyplex was prepared at an N/P ratio of 7 of CP(L) and Cy5labeled pGL3-control plasmid. The pGL3-control plasmid was labeled with Cy5 using a nucleic acid labeling kit. The cells were exposed to the polyplexes for various time periods at 37 °C. The coverslips were twice washed gently with 1 mL of 0.1 M PBS and then treated with a 100 nM green fluorescent dye (Lysotracker: DND-26) for 2 h at 37 °C. The coverslips were removed, washed, placed in a new empty well, and treated with 1 mL of 3.7% paraformaldehyde in 0.1 M PBS for 15 min to fix the cells. The cells were treated with 1 mL/ well of 0.1% Triton X-100 and incubated for 10 min. After DAPI staining, the cells on the coverslips were mounted with fluorescent mounting medium on glass slides. A CLSM was used for cell imaging. CD44 Receptor Expression in U87 Cells and 3T3 Cells. An FITC conjugate anti-CD44 antibody against CD44 receptor assay was used to measure the expression of CD44 receptor in U87 and 3T3 cells. The cells were trypsinized and then washed in PBS buffer. The cells (3 × 105) were then resuspended in PBS buffer and incubated with antihuman/mouse CD44 FITC antibody for 30 min on ice. The CD44 antibody concentration was based on the manufacturer’s instructions. After the incubation, the cells were washed three times with PBS, resuspended in 1 mL of PBS buffer, and then analyzed using a flow cytometer. Uptake in the Presence of Endocytosis Inhibitors. To understand the endocytosis pathway of CPs/pDNA polyplexes, four inhibitors were tested.30,31 FITC-conjugated CP was synthesized using a slightly modified method.32 U87 cells were seeded at a density of 2 × 105/well in 6-well plates in MEM supplemented with 10% FBS and incubated for 24 h. The cells were preincubated with 10 μg/mL chlorpromazine, 200 nM genistein, 50 nM wortmannin, or 1 mg/mL CS, respectively. 666

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from that of nascent PEI (the peak between δ 2.4 and 2.7 ppm attributed to the CH2 protons of PEI shown in Figure 1B). A similar observation has been reported in the PEI-grafted HA system.25 In FT-IR measurements, CP(L) showed a combination of peaks from CSMA and PEI(10K), where the PEI stretching appeared at 1647 cm−1 (1°N−H), 1577 cm−1 (2°N−H), 1471 cm−1 (CH2), and 1317 cm−1 (C−N), and the CSMA stretching bands appeared at 1646 cm−1 (amide I) and 1350 cm−1 (S O) (Figure 1D). All of the NMR and FT-IR spectra are clear evidence of a successful reaction between CSMA and PEI. The left shift of elution time in the GPC diagram of CP(H) to that of CSMA was also a good evidence (Supporting Information Figure S2). Acid/base titration profiles were created to determine the proton buffering capacity of CPs. Solutions were adjusted to pH ∼9.5 using 0.1 N NaOH and titrated using 0.1 N HCl. CSMA did not show any buffering capacity, while CPs showed a buffer capacity similar to that of PEI(10K) (Supporting Information Figure S3). Several apparent pKa values of 8.595, 7.378, 6.485, 5.685, and 4.765 for PEI(10K) and those of 8.368, 7.079, 6.469, 5.975, and 5.207 for CP(H) were obtained. However, the apparent pKa values were only seen at 8.315, 7.064, and 5.917 for CP(M) and at 8.407, 7.022, and 5.718 for CP(L). The values were attributable to the protonation of primary (1°), secondary (2°), and tertiary (3°) amino groups, respectively. It seems that the pKa values of the 1° and 2° amino groups decreased and that of the 3° amino group increased after the introduction of CS to PEI. PEI is a high charge density polymer and its protonation state of a site will electrostatically affect the protonation of nearby sites. In addition, CS is a polyelectrolyte bearing negative charges. Its apparent pKa value was estimated to be 4.4−4.5 by pH titration.33 At a pH condition close to the pKa of the 1° and 2° amino groups, the high deprotonation degree of CS enhances the interactions between PEI and CS. The cumulative build-up of multiple charge−charge interactions leads to deviation in the protonation behavior and results in a lower pKa value of CPs. On contrast, at a pH condition close to the pKa of the 3° amino group, the protonated CS can form hydrogen bonding interactions itself and reduces the interactions with PEI. Accounting for such H-bonding interactions may lead to an increase in the pKa value of the 3° amino group after the introduction of CS to PEI. Nevertheless, the apparent pKa values (5.975, 5.917, and 5.718, respectively, to CP(H), CP(M), and CP(L)) decreasing with decreasing the amount of PEI in CP was not clearly understood yet. All these values are close to a reported value of PEI, approximately 5.5.34 To test the solubility of CPs with different pH values, we prepared CPs at a concentration of 1 mg/mL. Transmittances of CPs were close to that of CSMA (larger than 99.4%) at a pH ranging between 1.2 and 11.5. Comparing the solubility of CPs with a physical mixing of CSMA and PEI using the same weight ratio as chemically synthesized CPs at pH 7.0, the physical complexes of CP(M) and CP(L) formed precipitates, but CP(H) showed a turbidity with 42.4% transmittance. On the contrary, solutions of the chemically synthesized CP(L), CP(M), and CP(H) remained clear (Supporting Information Figure S4). Characterizing CPs/pDNA Polyplexes. The hydrodynamic diameters of polyplex particles ranged between 69 and 182 nm and decreased with an increase in the N/P ratio for all CP/pDNA polyplexes (Table 1). For example, the hydro-

3T3 cells were seeded at the same density as U87 cells and pretreated with 1 mg/mL CS only. After 30 min of incubation, the medium containing the inhibitors was changed to fresh MEM, and the CP(L)/pDNA polyplex at an N/P ratio of 7 were treated and incubated for another 2 h. Next, the cells were trypsinized, centrifuged, and resuspended in 1 mL of cold PBS and then analyzed using the flow cytometer. Statistical Methods. Means, SD, and SE of the data were calculated. Differences between the experimental groups and the control groups were tested using Student’s-Newman-Keuls’ test, and P < 0.05 was considered significant.

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RESULTS Preparing and Characterizing CP. Various amounts of PEI were grafted onto CS via a Michael addition method, where the amino groups of PEI attacked the double bonds of CSMA (Scheme 1) in DD water without using any catalysts. Scheme 1. Chemical Reaction of CSMA and PEI(10K) To Form PEI-g-CS (CPs) and the Complex Formation with pDNA

The synthesis of CSMA was as described elsewhere.27 The degree of methacrylation on CS was controlled at ∼70%. The concentration of PEI in CPs was determined by measuring the cuprammonium complex formed between PEI and copper(II) at 630 nm using a UV−vis spectrophotometer (Supporting Information Figure S1). The PEI weight percentages of CPs using the PEI molar ratio of 0.5, 0.3, and 0.1 in feed were 83.7, 68.0, and 57.5 wt %, respectively. Three different PEI contents, CP(H), CP(M), and CP(L), corresponded to high, medium, and low PEI content. Figure 1 shows the 1H NMR spectra of CSMA, PEI(10K), and CP(L) in D2O. The peak shown at δ 1.89 ppm (C in Figure 1A) was ascribed to methyl groups adjacent to the double bonds, and that at δ 1.99 ppm (D in Figure 1A) corresponded to the methyl groups on CSMA, respectively. In the NMR spectrum of CP(L) (Figure 1C), two distinctive proton peaks of the CSMA double bonds shown at δ 5.65 and 6.10 ppm (A and B in Figure 1A) disappeared, and a new peak appeared at δ 1.1 ppm (E in Figure 1C). The proton peaks appeared at δ 2.3−3.1 ppm in Figure 1C, which was different 667

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Figure 1. NMR spectra of (A) methacrylated chondroitin sulfate (CSMA), (B) polyethylenimine (PEI(10K)), and (C) PEI-grafted CSMA copolymer (CP(L)) in deuterium water, and (D) their FT-IR spectra.

Table 1. Hydrodynamic Diameters and Zeta Potentials of CP/pDNA Polyplexes in DD Watera CP(H) Cp/pDNA N/P N/P N/P N/P a

= = = =

1 3 5 7

CP(M)

CP(L)

size (Dh) (nm)

zeta (mV)

size (Dh) (nm)

zeta (mV)

size (Dh) (nm)

zeta (mV)

182.50 ± 10.6 167.90 ± 1.13 119.50 ± 4.38 76.25 ± 5.31

−24.10 ± 0.99 9.66 ± 0.33 17.90 ± 2.69 20.00 ± 2.40

142.20 ± 14.2 128.40 ± 16.2 133.90 ± 0.21 87.23 ± 9.27

−23.30 ± 1.13 2.16 ± 2.81 15.90 ± 1.48 15.60 ± 1.84

127.80 ± 18.0 120.90 ± 7.78 85.99 ± 0.11 68.83 ± 11.8

−18.60 ± 0.21 19.20 ± 0.14 16.20 ± 0.42 22.80 ± 3.25

The concentration of samples was 0.1 mg/mL, and the temperature was 25°C. The data are means ± SD of an experiment done in triplicate.

Figure 2. Transmission electron microscope (TEM) images of CP(H)/pDNA polyplexes. The numeral at the top of each image indicates an N/P ratio of CP(H) and pDNA.

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Table 2. Hydrodynamic Diameters and Zeta Potentials of CPs/pDNA Polyplexes at Various N/P Ratios in DD Water Containing 10% FBS at Different Time Pointsa zeta ± SD (mV) (Dh ± SD (nm)) CP(H)

time

N/P = 1

N/P = 3

N/P = 5

N/P = 7

0h

−11.2 ± 2.3 (112.0 ± 2.1) −12.1 ± 2.2 (129.9 ± 6.0) −16.6 ± 0.1 (105.8 ± 6.5) −10.2 ± 1.6 (62.8 ± 8.2) −9.1 ± 0.4 (68.6 ± 11.6) −11.2 ± 0.8 (58.6 ± 5.6) −12.7 ± 0.0 (143.4 ± 16.3) −17.1 ± 0.4 (123.8 ± 16.9) −13.4 ± 0.5 (105.1 ± 23.9)

−15.9 ± 2.1 (119.1 ± 7.1) −18.3 ± 0.4 (142.0 ± 8.1) −15.1 ± 0.6 (150.6 ± 4.5) −14.9 ± 0.4 (129.9 ± 6.4) −17.7 ± 0.3 (162.5 ± 1.6) −15.4 ± 1.6 (152.0 ± 1.4) −16.3 ± 0.5 (157.1 ± 2.1 −17.2 ± 0.8 (173.8 ± 6.2) −16.8 ± 3.0 (156.4 ± 12.9)

−18.0 ± 0.4 (194.8 ± 36.2) −19.7 ± 0.4 (327.8 ± 17.7) −19.3 ± 1.6 (506.0 ± 55.4) −17.7 ± 0.8 (188.7 ± 1.2) −19.0 ± 1.6 (259.7 ± 11.6) −19.1 ± 0.1 (296.7 ± 17.0) −13.9 ± 0.1 (115.8 ± 8.8) −16.3 ± 0.4 (155.0 ± 1.4) −16.3 ± 1.0 (142.9 ± 6.0)

−13.6 ± 2.1 (94.9 ± 1.6) −15.0 ± 0.3 (91.3 ± 1.1) −16.5 ± 0.6 (106.2 ± 7.6) −10.8 ± 0.6 (60.6 ± 1.8) −13.6 ± 0.8 (56.4 ± 0.5) −13.9 ± 0.4 (58.3 ± 0.3) −13.1 ± 0.5 (40.0 ± 2.2) −14.5 ± 0.8 (44.7 ± 6.5) −15.4 ± 1.4 (61.5 ± 2.9)

2h 4h CP(M)

0h 2h 4h

CP(L)

0h 2h 4h

a

The concentration of samples was 0.1 mg/mL, and the temperature was 25 °C. The data are means ± SD of an experiment done in triplicate.

Figure 3. Agarose gel electrophoresis to test pDNA retention in polyplexes prepared at various N/P ratios (A) without 10% FBS, (B) after standing for 10 min with 10% FBS, and (C) after standing for 2 h with 10% FBS. Naked pDNA was used as a reference, and the numerals of each graph indicate the N/P ratios of CPs and pDNA.

dynamic diameter of CP(H)/pDNA was 182 nm at an N/P of 1 and 76 nm at an N/P of 7. Surface morphologies of the polyplexes were visualized using a TEM (Figure 2). The TEM micrographs show that the CP(H)/pDNA polyplexes had an irregular spherical shape and also decreased the particle size

with an increase in the N/P ratio. The zeta potentials of CPs/ pDNA were negative at an N/P of 1 and turned positive at an N/P ≥ 3 (Table 1). The N/P ratio of CPs/pDNA ≥ 5 showed a stable zeta potential with a value ≥15 eV. 669

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Molecular Pharmaceutics

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The hydrodynamic diameters and zeta potentials of the polyplexes were also measured at three time points in the presence of 10% FBS to simulate a physiological condition. The CPs/pDNA polyplexes had negative zeta potentials without any correlation to the N/P ratio and the duration period in FBS solution (Table 2). In the presence of FBS, the hydrodynamic diameters of CP(H)/pDNA and CP(M)/pDNA polyplexes were stable at the N/P ratios of 1 and 7, but increased dramatically with time at the N/P ratios of 3 and 5. The hydrodynamic diameters of CP(H)/pDNA at an N/P ratio of 5 were 195, 328, and 506 nm, respectively, left standing in 10% FBS for 0, 2, and 4 h. The interference of FBS on particle size was minimized in CP(L)/pDNA polyplexes. The polyplex of CP(L) and pDNA at an N/P ratio of 7 was used to study the effect of different pH values on particle diameters and zeta-potentials (Supporting Information Figure S5). The particle diameters of CP(L)/pDNA were stable at pHs of 5 and 7, increased at pH 8, and deformed at pH 9. The hydrodynamic diameter increased 5.4-fold from 72 nm at pH 5 to 386 nm at pH 8. The zeta potentials were 18.3, 11.6, −4.3, and −12.7 mV, respectively, to the pH values of 5, 7, 8, and 9. The surface morphologies of polyplexes in different pH values were also visualized by TEM, which showed the same pH dependence as we observed in DLS results. As previously mentioned in the pH titration, the pKa values of CP(L) were 8.407, 7.022, and 5.718 . At pH 9, the deprotonation of CP(L) resulted in remarkably reducing the electrostatic interaction forces with the negatively charged pDNA. The binding ability of CPs and pDNA was studied using an agarose gel electrophoresis retardation assay. To verify the protection of pDNA against serum digestion, the electrophoretic mobility analysis of CPs/pDNA polyplexes was tested after it had been left standing in 10% FBS at RT for 10 min or 2 h. Without FBS, the pDNA was well-complexed at all ratios of CP(H)/pDNA, but only at N/P ratios ≥3 of CP(M)/pDNA and CP(L)/pDNA (Figure 3A). With 10% FBS, the pDNA was exposed at N/P = 1 of CP(H)/pDNA (Figure 3B). The pDNA was degraded after 2 h of incubation with 10% FBS (Figure 3C). With this test result, we ensured the stability of pDNA complexed with CPs if we prepared CPs/pDNA at an N/P ratio ≥3. Cytotoxicity. Cell viability decreased dramatically with PEI(25K) at a concentration ≥10 μg/mL (Figure 4A), but with CP(M) and CP(L), cell viability still remained ≥80% at a concentration of 20 μg/mL. At a concentration ≥50 μg/mL, cytotoxicity with CP(H) and CP(M) was similar to that with PEI(25K) and PEI(10K), but with CP(L), cell viability was significantly higher (P < 0.01). The CPs/pDNA with N/P ratios ranging between 1 and 7 showed minimal cytotoxicity compared with PEI(25K)/pDNA at an N/P ratio of 10 (Figure 4B). The lower cytotoxicity of the polyplexes may be because of the lower PEI content in the CPs/pDNA preparation as well as because of the incorporation of naturally nontoxic CS. In Vitro Gene Transfection. Three plasmid DNAs, pEGFP-C1, pGL3-control, and p53, were used to test the transfection efficiency of CPs as a nonviral gene vector. The formation of similar particle diameters was confirmed by DLS when CP(L) was complexed with three different plasmid DNAs at an N/P ratio of 7. The hydrodynamic diameters of polyplexes were 88 ± 11, 82 ± 4, and 87 ± 3 nm, when using pEGFP-C1, pGL3-control, and p53 as a plasmid, respectively (Supporting Information Figure S6).

Figure 4. MTT assay of the cytotoxicity of CPs and their CPs/pDNA polyplexes to U87 cells as a function of (A) CPs concentrations (n = 8), and (B) polyplexes at different N/P ratios (n = 6, *p < 0.01). The N/P ratio of PEI(25K)/pDNA was 10.

The relative green fluorescence expression from pEGFP-C1 was traced using a fluorescence microscope (Figure 5A,B). The green fluorescence expression was clearly observed when the N/P ratio of CPs/pEGFP increased to 5. At an N/P ratio of 7, the green fluorescence was comparable to that of Lipofectamine. Because the pEGFP-C1expression increased with an increase in the CPs/pDNA ratio in the fluorescence study, the N/P ratio was adjusted to >7 for luciferase gene expression studies using pGL3-control plasmid. The quantitative transfection ability of polyplexes was measured and compared with that of PEI(25K)/pGL3 and Lipofectamine/pGL3 in the presence and absence of 10% FBS. In the absence of FBS, the transfection efficiencies of CPs/pGL3 increased as the N/P ratio increased, and they were higher than those of PEI and Lipofectamine at the N/P ratio ≥5 (the left panel of Figure 5C). The transfection efficiencies of CPs/pGL3 decreased in the presence of 10% FBS, but they were still comparable to those of PEI and Lipofectamine (the right panel of Figure 5C). We tested p53 protein expression transfected by CP(L)/p53 using a Western blotting assay. The p53 protein expression was clearly seen at the N/P ratio of PEI(25K)/p53 = 10 and of CP(L)/p53 = 15, using GAPDH as an internal control (Figure 5D). The degree of p53 protein expression of both systems was about 2.5-fold superior to that of Lipofectamine (Figure 5E). 670

dx.doi.org/10.1021/mp300432s | Mol. Pharmaceutics 2013, 10, 664−676

Molecular Pharmaceutics

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Figure 5. Gene expression in U87 cells: (A) fluorescent images of pEGFP-C1 expression using a CPs/pDNA polyplex at a N/P ratio of 7, (B) relative fluorescence intensity of pEGFP-C1 expression of CPs/pDNA polyplexes with various N/P ratios, (C) luciferase activity of pGL3-Control expression normalized with protein amounts (n = 3) in the presence (right panel) and absence (left panel) of 10% FBS, (D) p53 expression of CP(L) using Western blotting, and (E) the p53 expression fold relative to Lipofectamine (n = 3, *p < 0.01). The N/P ratio of PEI(25K)/pDNA was 10.

uptake of pDNA in red fluorescence increased with incubation time (Figure 6). At 4 h, a portion of internalized Cy5-labeled pDNA overlapped the endolysosomes (see white arrows), while most of the pDNA molecules reached the perinuclear region in the U87 cells (see the white square). Several mechanisms of endocytosis have been reported to be involved in the cellular uptake of polyplexes.35 Three major mechanisms were tested using their corresponding chemical

Cellular Internalization. Because the CP(L)/pDNA polyplex at an N/P ratio of 7 had a stable hydrodynamic diameter with time, we selected this polyplex for a detailed study of cellular internalization. To directly visualize pDNA internalization into U87 cells, we labeled pGL3-control plasmid with fluorescent Cy5 dye and traced the Cy5-labeled pGL3 using a CLSM. The nuclei were stained with DAPI in blue and the endolysosomes with Lysotracker in green. Intracellular 671

dx.doi.org/10.1021/mp300432s | Mol. Pharmaceutics 2013, 10, 664−676

Molecular Pharmaceutics

Article

Figure 6. CLSM images of U87 cells exposed to CP(L)/Cy5-labeled pGL3 (1 μg/well) at an N/P ratio of 7 at different time points. Blue, nuclei (DAPI); green, Lysotracker; red, Cy5-labeled pGL3 plasmid DNA.

ternary complexes except PEI/pDNA/CS showed little uptake and gene expression in B16−F10 cells. Pathak et al.14 physically fabricated CS-PEI nanoconstructs with various contents of CS as a pDNA carrier. Although they demonstrated that the nanoconstructs were significantly less toxic and more efficient for transfection than branched PEI(25K) and some commercial transfection reagents, the hydrodynamic diameters of the CSPEI nanoconstructs increased by 92−207 nm when 1−5 wt % of CS was added. After they had been complexed with pDNA, their size increased by 186−352 nm in the presence of 1−5 wt % of CS. Because particle size is a factor for deciding the extravasation rate of nanoparticles from the bloodstream, as well as recognition by the RES, in general, nanoparticle sizes of 10−200 nm are preferred for in vivo delivery37 (particles >10 nm to escape renal clearance and