Cell Penetrating Peptide Conjugated Polymeric Micelles as a High

Oct 1, 2013 - Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, North Da...
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Cell Penetrating Peptide Conjugated Polymeric Micelles as a High Performance Versatile Nonviral Gene Carrier Buddhadev Layek and Jagdish Singh* Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Fargo, North Dakota 58105, United States S Supporting Information *

ABSTRACT: The major goal of this study is to design, synthesize, and evaluate linoleic acid and penetratin dualfunctionalized chitosan (CS-Lin-Pen) as a nonviral gene carrier. The amphiphilic CS-Lin-Pen self-assembles to form cationic micelles in an aqueous environment. These polymeric micelles exhibited excellent hemocompatibility and cell viability, as evaluated by in vitro hemolysis and MTT assay, respectively. When CS-Lin-Pen micelles were added to plasmid DNA (pDNA) solution, the electrostatic interaction between the cationic micelles and anionic pDNA led to the formation of stable CS-Lin-Pen/pDNA polyplexes with ∼100 nm in size. The resultant polyplexes demonstrated ∼5-fold higher cellular uptake as compared to unmodified chitosan. Furthermore, CS-Lin-Pen micelles showed efficient protection of pDNA from DNase I attack and exhibited ∼34−40-fold higher transfection in comparison with unmodified chitosan in HEK 293, CHO, and HeLa cells. These findings illustrate that the CS-Lin-Pen micelles could be exploited as a potential nonviral vector for efficient gene therapy.

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INTRODUCTION Gene therapy is a promising therapeutic strategy for the treatment of a wide range of human diseases by repairing or replacing defective genes, delivering missing genes, or knocking out unwanted gene expression.1,2 However, naked DNA is highly susceptible to nuclease degradation and shows poor cellular uptake and low transfection efficiency.3 Thus, the development of a safe and effective gene delivery system is of paramount importance in the clinical success of gene therapy. Numerous delivery systems have been employed to deliver genes, which are broadly categorized as viral and nonviral vectors. Although recombinant viruses have demonstrated high transfection efficiency and a rapid transcription of the packaged gene, the issues of immunogenicity, carcinogenicity, and limitations in DNA cargo size impose serious concerns on the clinical applications of viral vectors.4−6 These limitations of viral vectors have expedited the hunt for alternative nonviral delivery systems. Among them, cationic polymers including poly ( L-lysine) (PLL), 7 polyethyleneimine (PEI), 8 and polyamidoamine (PAMAM) dendrimers9 have been extensively studied as promising nonviral vectors because of their favorable physicochemical characteristics, strong DNA binding ability, capacity to carry large payload, and flexibility of chemical modifications. Both PEI and PAMAM dendrimers are efficient gene carriers, but both are nonbiodegradable and suffer from strong cytotoxicity, thus, limiting their use for in vivo applications where high polymer concentrations are necessary.10,11 Although biodegradability is among the advantages of PLL, the polyplexes it forms have lower gene transfection efficacy than that of the PEI and the dendrimers. © XXXX American Chemical Society

Chitosan (CS), a natural cationic copolymer of randomly distributed β-(1, 4)-linked D-glucosamine and N-acetyl-Dglucosamine, has been found to be a safer alternative to other nonviral vectors because of its good biocompatibility, biodegradability, and low cytotoxicity while possessing a high cationic charge density.12−14 However, the clinical applications of unmodified CS have been limited by its low gene transfection efficiency. Studies have reported that the low transfection efficiency of CS is primarily attributed to the poor cellular uptake and inefficient release of plasmid DNA (pDNA) from CS/pDNA polyplexes.15−17 To overcome these challenges, numerous CS derivatives have been synthesized by hydrophobic,12,14 hydrophilic,18 pH sensitive,19 and thermoresponsive modification20 using the reactive activities of amino and hydroxyl groups. Among these, hydrophobic modification of CS has demonstrated the most encouraging results. Hydrophobic modifications are expected to enhance the adsorption of the polymer/pDNA polyplexes on the lipophilic cell membrane, confer efficient pDNA protection from nuclease degradation, and assist intracellular pDNA dissociation.21 Moreover, conjugation of hydrophobic segments to CS resulted in amphiphilic cationic polymers which could easily selforganize to form micelles in aqueous milieu. These polymeric micelles can not only facilitate efficient pDNA condensation but also improve the stability of pDNA polyplexes for in vitro and in vivo environments.22 These distinct benefits of Received: August 8, 2013 Revised: September 16, 2013

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Scheme 1. Synthetic Route of Linoleic Acid and Penetratin Dual Functionalized Chitosan

investigated. The efficacy of CS-Lin-Pen/pDNA polyplexes was evaluated by in vitro gene transfection efficiency in human embryonic kidney (HEK 293), Chinese hamster ovary (CHO), and human cervical adenocarcinoma (HeLa) cells.

hydrophobic modification encourage the possibility of developing amphiphilic CS derivatives with enhanced gene transfection efficiency. Potentially, the transfection efficiency of polymeric micelles can be even further enhanced through surface modification with ligands that facilitate cell binding and internalization.23,24 Since the discovery over two decades ago of a protein transcription factor that holds the capability to cross the cell membrane, cell-penetrating peptides (CPPs) have been extensively studied for their ability to transport diverse therapeutic cargoes including pDNA, siRNA, peptide, protein, liposomes, as well as micelles into cells, tissues, and organs.25−27 CPPs are short (5−30 amino acids long) cationic, amphiphilic or hydrophobic peptide sequences capable of cell membrane translocation and nuclear localization of cargo, thereby possessing a great potential as in vitro and in vivo gene delivery vectors.28 Therefore, the chemical conjugation of a CPP to polymeric micelles could offer the merits of increased cellular uptake and efficient intracellular pDNA release for enhanced gene transfection efficiency. The present study explores a unique approach and is aimed at the design, synthesis, and evaluation of a polymeric nanocarrier that provides solutions for the major barriers in gene delivery, such as enzymatic degradation, poor cellular uptake, inefficient pDNA release, and high cytotoxicity. A novel linoleic acid and penetratin dual functionalized CS-based (CSLin-Pen) gene delivery system has been developed to combine the individual merits of cationic micelles and CPP. Linoleic acid was selected as a hydrophobic unit of the system due to its good safety profile and ability to form stable micelles even in highly diluted in vivo conditions.29 Penetratin (RQIKIWFQNRRMKWKKGG), a well-studied CPP derived from the third α-helix of the Drosophila Antennapedia homeodomain protein, has been chosen because of its ability to increase cellular uptake of attached cargos in vitro and in vivo.26,30 The influences of linoleic acid and penetratin conjugation on particle size, surface potential, pDNA complexation and protection, in vitro pDNA release rate, cellular uptake, and in vitro cell viability were

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EXPERIMENTAL SECTION

Materials. Low molecular weight chitosan (MW 50 kDa, 90% deacetylated), fluorescein 5-isothiocyanate (FITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Linoleic acid and N-hydroxysuccinimide (NHS) were procured from Alfa Aesar (Ward Hill, MA, U.S.A.). DNase I enzyme was purchased from Rockland Inc. (Gilbertsville, PA, U.S.A.). 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide HCl (EDC.HCl) was purchased from Creosalus Inc. (Louisville, KY, U.S.A.). Reporter plasmid DNA encoding beta-galactosidase (gWiz-βGal) and green fluorescence protein (gWiz-GFP) were supplied by Aldevron LLC (Fargo, ND, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), HEK 293, CHO, and HeLa cell lines were purchased from American Type Culture Collection (Rockville, MD, U.S.A.). Agarose and β-galactosidase enzyme assay kit were obtained from Promega (Madison, WI, U.S.A.). Fugene was purchased from Roche Diagnostics (Indianapolis, IN, U.S.A.). All other chemicals were of analytical grade and used without further modification. Synthesis of CS-Lin-Pen. Synthesis of CS-Lin-Pen was conducted in two steps as shown in Scheme 1. In the first step, linoleic acid conjugated CS (CS-Lin) was synthesized by coupling the carboxyl group of linoleic acid with the primary amino groups of CS in the presence of EDC/NHS. Briefly, CS (500 mg) was dissolved in 100 mL of deionized water at pH 5. Linoleic acid (0.3 mol/mol of sugar unit of CS) and EDC (5 mol/mol of linoleic acid) were dissolved in 50 mL of ethanol and stirred for 1 h followed by addition of NHS (5 mol/mol of linoleic acid). The mixture of linoleic acid solution containing EDC and NHS was added dropwise into CS solution with constant stirring and allowed to react at 90 °C for 12 h. The resulting product was dialyzed using a dialysis membrane (MWCO: 3.5 kDa, Thermo Scientific, IL, U.S.A.) against deionized water for 48 h in order to remove water-soluble byproducts completely. The dialyzed product was then frozen at −80 °C and lyophilized (temperature −49 °C and vacuum 0.08 mBar) until a stable weight polymer was obtained. In the next step, the lyophilized product was washed three times with ethanol to eliminate unreacted linoleic acid. Finally, the precipitate was filtered B

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through 0.2 μm filter paper and the precipitate was lyophilized to obtain the CS-Lin polymer. In the subsequent step, penetratin was conjugated to CS-Lin using carbodiimide mediated coupling reaction. Briefly, CS-Lin (20 mg) was dissolved in 10 mL of deionized water. Penetratin (5 mg) and EDC (5 mol/mol of penetratin) were dissolved in deionized water and stirred for 1 h followed by addition of NHS (5 mol/mol of penetratin). The activated penetratin solution was added dropwise into CS-Lin solution under constant stirring and the reaction was performed at room temperature for 24 h. Unreacted penetratin and other reagents were eliminated by exhaustive dialysis against deionized water for 48 h followed by lyophilization to get purified CS-Lin-Pen. Polymer Characterization. The polymers were characterized at each step of its derivation through 1H NMR spectroscopy (Mercury Varian 400 MHz NMR) at 25 °C using deuterated water (D2O) as a solvent. Chemical shifts were measured in parts per million (ppm) using the signal of D2O at 4.75 ppm as the internal reference. The degree of linoleic acid substitution of synthesized polymers was determined by 1H NMR spectroscopy. While the amounts of penetratin peptide coupled to CS-Lin-Pen were determined by a micro bicinchoninic acid (Micro BCA) assay using CS-Lin and penetratin as blank control and standard, respectively.31 The critical micelle concentration (CMC) of CS-Lin and CS-LinPen was determined by fluorescence measurement using pyrene as a hydrophobic probe. Pyrene was dissolved in acetone at a concentration of 24 μg/mL. A total of 10 μL of this solution was added into each 10 mL test tube, and the acetone was removed by drying at 50 °C. Two milliliters of CS-Lin or CS-Lin-Pen solution with different concentrations from 1.0 × 10−3 to 1.0 mg/mL were added to the test tube, keeping the final concentration of pyrene to 0.6 μM. The mixture was then sonicated in a bath sonicator for 30 min and the fluorescence emission spectra of pyrene at a wavelength range of 360− 450 nm were recorded on a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon, NJ, U.S.A.) at room temperature. The excitation wavelength was set at 336 nm, the excitation slit width at 2 nm, and the emission slit width at 5 nm. The emission intensity ratio of the first peak (I1, 372 nm) to the third peak (I3, 395 nm) of pyrene was also calculated for the determination of CMC value. Blood Compatibility Study. To study the interaction of cationic CS-Lin-Pen with the negatively charged erythrocytes, we performed in vitro hemolysis assay at increasing polymer concentrations.14 Blood was collected from adult Sprague−Dawley rats into polypropylene tubes containing ethylenediaminetetraacetic acid (EDTA) solution as an anticoagulant. Erythrocytes were harvested by centrifuging the blood at 1500 rpm for 10 min and the pellet was washed three times with PBS at pH 7.4. The erythrocytes were then resuspended in PBS to a final concentration of 5 × 109 cells/mL. The resulting erythrocytes suspension (100 μL) was mixed with 900 μL of polymer solutions at different concentrations and incubated in a reciprocal water bath at 37 °C. After 1 h of incubation, samples were centrifuged at 1500 rpm for 10 min and the absorbance (A) of the supernatant was measured at 540 nm by a spectrophotometer. PBS and Triton X-100 treated erythrocytes served as the controls for 0 and 100% hemolysis, respectively. The percent hemolysis was calculated according to the following equation:

μg/mL) was also prepared in sodium acetate buffer (pH 6.5). The polymer solution was then added to the pDNA solution at various polymer/pDNA weight ratios, gently vortexed for 10 s, and incubated at room temperature for 30 min prior to use. The average hydrodynamic diameter and zeta potential of the micelles (200 μg/ mL) and micelles/pDNA polyplexes were measured using Zetasizer Nano-ZS (Malvern Instruments, Malvern, U.K.) equipped with a nominal 5 mW He−Ne laser operating at 633 nm wavelength and at 90° constant scattering angle. The measurements were performed using the following settings: water was used as dispersant, viscosity for water was 0.8872 cP, refractive index 1.33, temperature 25 °C, equilibration time 120 s, and number of runs 20. The hydrodynamic diameters of micelles or micelles/pDNA polyplexes were calculated by the Stokes−Einstein equation. The Smoluchowski approximation was utilized to obtain zeta potential data from the electrophoretic mobility. Further, the size and morphology of polyplexes were observed using a DI-3100 atomic force microscope (AFM, Veeco, Minnesota, U.S.A.) operating in tapping mode. Samples for AFM were prepared by placing 3 μL of polyplex solution onto freshly cleaved untreated mica plates (grade V-4; 15 × 15 × 0.15 mm3) followed by air drying for 2 h. Consequently, the mica plates were imaged at a scan rate of 1 Hz using a pyramidal cantilever with a spring constant of 2.8 N/m. All measurements were performed in replicates of six. To determine the association efficiency of pDNA, 0.5 mL of the polyplexes were spun down at 30000 × g for 30 min at 4 °C. The supernatant solution was mixed with Hoechst dye 33342 (1 μg/mL) at a ratio of 1:1 (v/v) and analyzed for pDNA content using a spectrofluorophotometer at excitation and emission wavelengths of 350 and 450 nm, respectively. The association efficiency was calculated by the following equation:

association efficiency(%) = (pDNA total − pDNAfree)/pDNA total × 100 Agarose Gel Retardation Assay. To ascertain pDNA binding, polyplexes prepared at different weight ratios were evaluated by gel electrophoresis on a 0.8% (w/v) agarose gel stained with 0.5 μg/mL of ethidium bromide. The electrophoresis was carried out at 80 V for 1 h 20 min in 0.5× Tris-acetate-EDTA buffer (TAE, Bio-Rad, CA, U.S.A.), and the pDNA bands were visualized on a UV transilluminator at 254 nm. Protection against DNase I Degradation. To confirm the ability of CS-Lin and CS-Lin-Pen polyplexes to protect the condensed pDNA from endonucleases, DNase I protection assay was performed as reported earlier.32 CS-Lin/pDNA and CS-Lin-Pen/pDNA polyplexes, containing 2 μg of pDNA in 20 μL of DNase reaction buffer (10 mM Tris- HCl, 2.5 mM MgCl2, 10 mM CaCl2, pH 7.6), were incubated with 1 unit of DNase I at 37 °C for 1 h. Naked pDNA treated with DNase I served as a positive control. The DNase I reaction was terminated by adding 5 μL of 0.1 M EDTA solution. The pDNA was then dissociated from the polyplex by treatment with 10 μL of heparin (5 mg/mL) at 37 °C for 2 h. A qualitative analysis of pDNA degradation was executed by agarose gel electrophoresis, as described above. Fluorescence Labeling of Polymers. FITC-labeled CS, CS-Lin, and CS-Lin-Pen polymers were synthesized by the reaction between the isothiocyanate group of FITC and free primary amino groups of polymers.14 The FITC of 1 mg in 1 mL of dehydrated methanol was added dropwise to 5 mL of 2% w/v solution of polymer with moderate stirring and the reaction was continued in dark at room temperature for overnight. The FITC labeled polymers were precipitated by raising solution pH to 10 with 0.1 M NaOH and washed three times with dehydrated methanol to remove unreacted FITC. The FITC-labeled polymers were dissolved in distilled water and dialyzed against deionized water for 48 h under darkness, followed by lyophilization. Cellular Uptake. To evaluate cellular internalization, FITC-labeled polymers were allowed to form stable polyplexes with pDNA at the weight ratio of 15. HEK 293 cells were seeded onto 6-well plates at 2 × 105 cells/well in 3 mL of DMEM supplemented with 10% FBS 24 h before experimentation. The FITC labeled polyplexes containing 4 μg

hemolysis(%) = (A polymer − APBS)/(A Triton X − 100 − APBS) × 100 The microscopic examination of erythrocytes, incubated with different polymer concentrations, was performed to further authenticate the results obtained from spectrophotometric analysis. The morphology of the treated erythrocytes, as described above, was observed under an Olympus DP72 light microscope (Melville, NY, U.S.A.). The images were captured and processed using Olympus Cell Sens version 1.5 software. All samples were analyzed in a replicate of four to get statistically relevant results. Particle Size, Zeta Potential, Morphology, and Association Efficiency. The cationic CS-Lin and CS-Lin-Pen polymers were dissolved in 20 mM sodium acetate buffer (pH 6.5) with a concentration of 1 mg/mL and then filtered through cellulose acetate membrane filters (0.2 μm pore size). The pDNA stock solution (200 C

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Figure 1. Polymer characterization using 1H NMR: (A) chitosan (CS) in D2O, (B) linoleic acid (Lin) in DMSO-d6, (C) CS-Lin in D2O, and (D) CS-Lin-Pen in D2O. of pDNA was added to each well and incubated with the cells for 4 h at 37 °C. Likewise, experiments were performed in CHO and HeLa cells. Afterward, the cells were washed three times with cold PBS and trypsinized using 0.25% trypsin-EDTA solution. The trypsinized cells were centrifuged at 1000 rpm to eliminate excess trypsin-EDTA and resuspended in FACS buffer (PBS, 1% bovine serum albumin, and 0.02% sodium azide). The percentages of FITC positive cells were quantified via flow cytometry (Accuri Cytometer Inc., MI, U.S.A.). To visualize cell internalization, the FITC positive cells were further incubated with 2.5 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 20 min to stain the nuclei and observed with a FV300 confocal microscope (Olympus, NY, U.S.A.). To minimize crosstalk between DAPI and FITC, we used barrier filters for both the channels. For DAPI we used BA 430−460 (blocks longer wavelengths above 460 nm and blocks wavelengths shorter than 430 nm) as a barrier filter, while BA 515−550 (blocks longer wavelengths above 550 nm and blocks wavelengths shorter than 515 nm) was used as the barrier filter for FITC channel. In Vitro Release of pDNA. To determine the in vitro release profile of pDNA, PBS with pH 7.4 and pH 5.5 were used as release media. Polyplexes containing 20 μg of pDNA at the weight ratio of 15 were dispersed in 20 mL of release media and incubated at 37 °C under stirring. At predetermined time points, 0.5 mL of polyplex suspension was centrifuged at 30000 × g for 30 min at 4 °C and pDNA content in the supernatant was quantified by staining with Hoechst dye 33342. Cytotoxicity Assay. The cytotoxicity of polymers and polymer/ pDNA polyplexes was tested by MTT colorimetric assay in HEK 293, CHO, and HeLa cells.33 Cells were seeded onto 96-well plates at 5 × 103 cells/well in 150 μL of DMEM containing 10% FBS and cultured for 24 h at 5% CO2 and 37 °C. Following incubation, cells were treated with various concentrations of polymers (100−1000 μg/mL) or polyplexes at different weight ratios (5, 10, 15, and 20). After 48 h, media containing polymers or polyplexes was removed and 100 μL of MTT solution (1 mg/mL in serum-free DMEM) was added to each

well. After 3 h, the unreacted MTT was aspirated carefully and the cells were rinsed with cold PBS. The formazan crystals formed by live cells were dissolved in 150 μL of dimethyl sulfoxide and the absorbance (A) was measured at 570 nm on a microplate reader. The viability of untreated cells was set as 100%; cells without MTT treatment were considered as blank to calibrate the spectrophotometer reading to zero absorbance. The relative cell viability was calculated by the following equation:

cell viability(%) = (A sample /Acontrol ) × 100 In Vitro Gene Transfection. In vitro transfection efficacy of different formulations was evaluated in HEK 293, CHO, and HeLa cells using gWiz-βGal and gWiz-GFP as reporter plasmids. Cells were plated onto 24-well plates at 1 × 105 cells/well with 0.5 mL of 10% FBS containing DMEM media 24 h prior to transfection studies. At the time of the transfection experiment, the culture media was replaced with 0.5 mL of fresh DMEM with 10% FBS. The polyplexes containing 1 μg pDNA at various weight ratios were added to each well and incubated at 37 °C in 5% CO2 for 4 h. The Fugene/pDNA complex and naked pDNA were used as positive and negative control, respectively. Cells were then washed with PBS (pH 7.4) and cultured in fresh 10% FBS containing DMEM for another 44 h. The βgalactosidase expression was quantified using the β-galactosidase enzyme assay kit at 450 nm following the manufacturer’s protocol. The protein content of cell lystate was determined using a Micro BCA protein assay kit (Pierce). The level of β-galactosidase expression was calculated as milliunit (mU) of β-galactosidase/mg of total cell protein. For the GFP transfection, the transfected cells were washed with PBS (pH 7.4) and trypsinized using 0.25% trypsin-EDTA. The percentage of the GFP transfected cells was determined by flow cytometry. The results of flow cytometry analysis were further confirmed by visualizing the GFP positive cells using a confocal laser scanning microscope at 20× magnifications. D

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RESULTS AND DISCUSSION

Synthesis and Characterization of CS-Lin-Pen. Herein we describe the design, synthesis, and evaluation of linoleic acid and penetratin conjugated CS (CS-Lin-Pen) cationic micelles as a safe and efficient gene carrier. The dual functionalized CSLin-Pen serves a number of distinct purposes: (1) CS offers cationic primary amine for electrostatic interactions with anionic pDNA, (2) linoleic acid confers enhance membrane adsorption of polymer/pDNA polyplexes and promote intracellular pDNA release, while (3) penetratin facilitates cellular uptake and nuclear localization of the polyplexes. Synthesis of CS-Lin-Pen was conducted in two steps. In the first step, CSLin was successfully synthesized by amidification reaction between the carboxyl group of linoleic acid and the amine groups of CS using EDC/NHS as a zero-length cross-linker.34 The second step involved synthesis of CS-Lin-Pen by reacting the free carboxyl group of penetratin with one of the primary amines of the CS-Lin in presence of EDC/NHS. 1 H NMR spectrum was utilized to confirm the conjugation of linoleic acid and penetratin to CS backbone. The 1H NMR spectra of CS, linoleic acid, CS-Lin, and CS-Lin-Pen are shown in Figure 1. The proton peak of −COOH of linoleic acid (δ = 12 ppm) was observed in the 1H NMR spectrum of linoleic acid, but disappeared in the 1H NMR spectrum of CS-Lin or CS-Lin-Pen. Furthermore, the −CH3 protons peak of linoleic acid (δ = 0.8 ppm) appeared in the 1H NMR spectrum of CSLin and CS-Lin-Pen. The graft ratio of linoleic acid, which can be defined as the number of linoleic acid groups per 100 sugar units of CS, was calculated by the peak areas of −CH3 protons of linoleic acid (δ = 0.8 ppm) and −CH3 protons of CS (δ = 1.9 ppm) in association with the deacetylation degree of CS. The obtained graft ratio was found to be ∼15%. The multiple weak peaks at δ7−7.5 ppm in the 1H NMR spectrum of CSLin-Pen belong to the aromatic protons of phenylalanine and tryptophan in the penetratin sequence. The degree of penetratin substitution was determined by Micro BCA, which indicates that every 52 sugar units of CS conjugated one penetratin peptide. The synthesized CS-Lin and CS-Lin-Pen polymers could easily self-aggregate to form micelles in an aqueous environment. The CMC is an important characteristic of an amphiphilic material, representing micelle formation ability. The aggregation behavior of these polymers was examined by fluorometry using pyrene as a molecular probe.35 Figure 2 demonstrates the changes of the fluorescence intensity ratio of the first peak to the third peak (I1/I3) of pyrene against the logarithm of polymer concentration. At the low polymer concentration, the I1/I3 ratio remains nearly constant. With further increase in polymer concentration, the intensity ratio begins to decrease sharply, indicating the onset of micelle formation. At or above the CMC, inclusion of pyrene into the micelles led to amplification of the overall fluorescence intensity. However, the I3 of pyrene increased considerably faster than that of the I1, resulting in a sharp decrease in the I1/ I3 ratio. The CMC of CS-Lin was found to be ∼15 μg/mL. Moreover, it was found that the conjugation of penetratin to CS-Lin did not alter the CMC value. Blood Compatibility Study. The nonspecific interactions of cationic formulations with the anionic membrane of erythrocytes can cause severe hemolysis. In addition, these interactions may adversely affect the half-life, target ability, and reproducibility of the medication.36 The blood compatibility of

Figure 2. Plots of pyrene I1/I3 fluorescence intensity ratio vs logarithm of polymer concentration in water at room temperature. Arrow indicates the CMC values of polymers.

the cationic micelles was evaluated by spectrophotometric measurement of hemoglobin release from erythrocytes after 1 h incubation with various concentrations of polymers at 37 °C.37 As shown in Figure 3A, cationic micelles did not induce significant hemolysis in our experiment, suggesting that the micelles did not affect the integrity of the erythrocyte membrane. The visual observation of the hemolytic activity was in complete agreement with the spectrophotometric analysis (Figure 3B). The morphological changes of the treated erythrocytes were observed under light microscope at 40× magnification. As revealed in microscopic examination (Figure 4), the erythrocytes treated with cationic micelles did not exhibit any visible damage to their membranes and were comparable with those in PBS (pH 7.4). Conversely, erythrocytes incubated with Triton X-100 (1% v/v) were severely damaged, showing release of hemoglobin, and a large extent of erythrocyte debris. The results of spectrophotometric analysis together with microscopic observation confirm the good hemocompatibility of cationic micelles investigated and support their prospect for in vivo applications. Particle Characteristics. The particle size and surface charge of polyplexes are two important factors in determining the efficacy of cellular uptake and downstream gene expression. It has been reported that particles less than 200 nm in size are efficiently internalized by most cell types and a positive surface charge may induce greater interaction with the anionic proteoglycans of the cell membrane, triggering endocytosis.38 The average hydrodynamic diameters and zeta potentials of polyplexes were determined by dynamic light scattering (DLS) method. At the weight ratio of 1, CS-Lin-Pen/pDNA polyplexes had negative surface charge, signifying the amount of polymer was insufficient to condense pDNA completely. As the polymer/pDNA weight ratio increased, the zeta potential increased and the diameter of polyplexes was found to be decreased (Table 1), which perhaps is due to the better condensation of pDNA in the presence of a higher density of available amines surrounding the polyplexes. Sizes of the polyplexes were distributed within a fairly narrow range as indicated by low polydispersity index (PDI; 0.13−0.27). It is worth mentioning that the hydrodynamic diameters of the CSLin-Pen/pDNA polyplexes were significantly lower than CS/ pDNA polyplexes, indicating formation of compact nanoparticles. As is known, CS/pDNA polyplexes are formed due to the electrostatic interaction between cationic CS and the E

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Figure 3. (A) Graphical representation of percent hemolysis in rat erythrocytes upon incubation with micelles at incremental concentrations. PBS, pH 7.4 and 1% (v/v) Triton X-100 were used as negative control and positive control, respectively. Data represent the mean ± SD (n = 4). (B) Photo images of test tubes containing supernatant from erythrocytes exposed to different concentrations of micelles.

into cationic micelles; consequently both the electrostatic and hydrophobic interaction promotes pDNA condensation to form compact nanoparticles with improved stability. Moreover, there was no change in particle diameter and zeta potential following the conjugation of peptide ligand to CS-Lin micelles. At the polymer to pDNA weight ratio of 15, the average size of the polyplexes was ∼100 nm (Figure 5A,B) with a net positive surface charge, which would be favorable for cellular uptake and gene expression both in vitro and in vivo. The morphology of the polyplexes prepared at the polymer to pDNA weight ratio of 15 was observed using AFM. Under the microscope, polyplexes were spherical in shape with a mean diameter of ∼70 nm (Figure 5C,D). The difference in particle size measured by two different techniques could be explained by the fact that AFM measures the size of dry particles, while DLS determines the hydrodynamic diameter of the polyplexes in buffer solution. The pDNA association efficiency of CS/pDNA, CS-Lin/pDNA, and CS-Lin-Pen/pDNA polyplexes at the weight ratio of 15 was 98.2, 96.5, and 95.7%, respectively.

Figure 4. Microscopic images of erythrocytes exposed to different polymers at 500 μg/mL concentration, PBS, and 1% (v/v) Triton X100. Images were taken at 40× magnification using Olympus DP72 microscope.

anionic pDNA, which usually lead to uncontrollable large particles. However, linoleic acid conjugation imparted amphiphilicity to the CS molecule which could easily self-assemble

Table 1. Particle Size and Zeta Potential of Micelles and Their Corresponding pDNA Polyplexes in 20 mM Sodium Acetate Buffer at pH 6.5a avg particle size (nm) ± SD (PDI)b samples

micellesc

CS

a

CS-Lin

70.5 ± 3.7

CS-Lin-Pen

72.7 ± 4.2

zeta potential (mV) ± SD

DNA loaded micelles

micellesc

DNA loaded micelles

polymer/DNA weight ratio

320.1 ± 9.2 (0.35) 269.5 ± 7.3 (0.26) 237.4 ± 8.4 (0.27) 210.9 ± 6.7 (0.25) 195.2 ± 8.6 (0.21) 190.1 ± 6.8 (0.27) 159.8 ± 4.3 (0.18) 127.8 ± 5.7 (0.15) 102.9 ± 4.1 (0.15) 98.7 ± 5.2 (0.13) 190.2 ± 6.2 (0.25) 162.0 ± 3.5 (0.19) 130.3 ± 4.8 (0.16) 104.2 ± 5.6 (0.15) 100.3 ± 5.7 (0.14)

45.4 ± 2.5

9.4 ± 0.8 22.3 ± 1.1 25.2 ± 0.9 26.5 ± 1.3 28.6 ± 1.1 −8.3 ± 1.8 14.3 ± 0.4 17.1 ± 0.5 18.1 ± 0.6 18.9 ± 0.4 −6.6 ± 1.7 8.8 ± 0.3 16.0 ± 0.6 18.4 ± 0.4 19.4 ± 0.6

1 5 10 15 20 1 5 10 15 20 1 5 10 15 20

33.6 ± 1.8

35.4 ± 1.6

Data are mean ± SD (n = 6). bPDI: polydispersity index. cMicelles were prepared at a concentration of 200 μg/mL. F

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Figure 5. Size distribution of (A) CS-Lin/pDNA (w/w = 15) and (B) CS-Lin-Pen/pDNA (w/w = 15) polyplexes obtained by DLS method. Atomic force microscopy images of (C) CS-Lin/pDNA (w/w = 15) and (D) CS-Lin-Pen/pDNA (w/w = 15) polyplexes. A total of 3 μL of each formulation was placed on a freshly cleaved untreated mica plate, and images were recorded in tapping contact mode.

Figure 6. (A) Gel retardation assay of CS-Lin/pDNA and CS-Lin-Pen/pDNA polyplexes at various weight ratios. (B) DNase I protection and release assay to evaluate the pDNA protective ability of CS-Lin/pDNA and CS-Lin-Pen/pDNA polyplexes against endonucleases degradation at different weight ratios. Naked pDNA (ND) was used as a control.

Gel Retardation Assay. For efficient gene delivery, it is important that pDNA is condensed into stable particles to protect the pDNA from extracellular enzymatic degradation and to allow for adequate cell uptake. The pDNA binding efficiency of cationic micelles was monitored by agarose gel retardation assay using naked pDNA as a control. Polyplexes, formulated at different weight ratios from 0.2 to 20, were electrophoresed to determine the optimal weight ratio required for complete condensation of the pDNA. Figure 6A showed

that the electrophoretic mobility of pDNA was completely retarded when the polymer/pDNA weight ratio attained ≥5, indicating that both CS-Lin and CS-Lin-Pen micelles bound pDNA strongly. Results obtained from the gel retardation assay are in complete harmony with the zeta potential measurements. DNase Protection Assay. DNase I is a nonspecific endonuclease that catalyzes the hydrolytic cleavage of DNA backbone. Thus, the integrity of pDNA in the presence of DNase I is a prerequisite to ensure proper function of pDNA in G

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vitro or in vivo.39 To authenticate that the synthesized micelles provide efficient protection of pDNA from endonucleases, DNase I protection assay was performed and analyzed by agarose gel electrophoresis. The results of the DNase I protection experiment are demonstrated in Figure 6B. The naked pDNA was completely degraded by DNase I, while pDNA showed no evidence of degradation when complexed with cationic micelles. These results clearly demonstrate that under physiological conditions where the presence of different endonucleases significantly influences the integrity of naked pDNA, cationic micelles provide efficient protection of pDNA and are suitable for in vivo conditions. Cellular Uptake. The ability of the polyplexes to traverse the cell membranes is essential for efficient gene expression. Figure 7 illustrates the distribution of different polyplexes in

To replicate the existing pH environment of cytoplasm and nucleus, PBS at pH 7.4 was employed as the release media. Figure 8 depicts the in vitro cumulative release rate of pDNA

Figure 8. In vitro pDNA release profiles of CS-Lin/pDNA, CS-LinPen/pDNA, and CS/pDNA polyplexes prepared at a weight ratio of 15 in pH 7.4 PBS at 37 °C. Data represent the mean ± SD (n = 4).

from the polyplexes prepared at the weight ratio of 15. After 48 h of incubation, compared to CS/pDNA polyplexes from which about 8.9% of the complexed pDNA was released, the cumulative release percentages from CS-Lin/pDNA and CSLin-Pen/pDNA were increased to 31.5 and 30.6%, respectively. These results indicate that the hydrophobic linoleic acid conjugation accelerated the pDNA release rate, which could be attributable to the hydrophobicity-induced weakening of the electrostatic interaction between pDNA and cationic micelles. Therefore, the improved pDNA release profiles along with excellent pDNA protection ability of cationic micelles render them suitable as an efficient gene carrier. To simulate the environment in the lysosomes, a release study was also performed at pH 5.5. A similar trend was noticed but the overall cumulative pDNA release of polyplexes was ∼32−37% lower than that at pH 7.4 media (Figure S2). This could be explained by the higher amount of protonated amine groups in the polymer at pH 5.5 compared to pH 7.4, which resulted in tighter binding between polymer and pDNA. Cytotoxicity Assay. Polycations are known to possess some inherent cytotoxicity due to their strong electrostatic interactions with the negatively charged cell membrane.42 Thus, in addition to the hemocompatibility study, in vitro cytotoxicity assay was performed to exemplify that the synthesized micelles were safe and did not damage the cells during the uptake process. In this study, the cytotoxicity of cationic micelles was examined at different concentrations in HEK 293, CHO, and HeLa cells by MTT assay. As shown in Figures 9A, S3A, and S3B, the cationic micelles did not alter the cell viability to a concentration of 1 mg/mL, which was ∼50-fold higher than the optimal polymer concentration used in the transfection assay. Moreover, the conjugation of peptide ligand did not exert any negative impact on the cell viability of the micelles. The cytotoxicity of the micelles/pDNA polyplexes at various weight ratios ranging from 5 to 20 was further established in different cell lines. As demonstrated in Figures 9B, S3C, and S3D, micelles/pDNA polyplexes did not show any change in cell

Figure 7. Live cell confocal microscopic images of HEK 293 cells treated with (A) CS/pDNA, (B) CS-Lin/pDNA, and (C) CS-LinPen/pDNA polyplexes at the weight ratio of 15. Cells are specified by the bright field (BF) image. The nuclei of the cells were stained with DAPI (blue) and polymers were labeled with FITC (green). The merged image shows an overlap of the polyplexes and nuclei within the cells. Images were taken at 20× magnification after 4 h of incubation with different formulations.

HEK 293 cells after 4 h of uptake. The CS/pDNA polyplexes exhibited the lowest number of fluorescence (green) positive cells while the dual-functionalized CS-Lin-Pen/pDNA polyplexes showed maximum internalization. The confocal images also indicate that CS/pDNA and CS-Lin/pDNA polyplexes were mainly accumulated around the perinuclear section of the cytoplasm, whereas an intense overlap of polyplexes and nuclei was observed in the case of CS-Lin-Pen/pDNA. Nuclei were stained with DAPI, showing blue fluorescence. The quantitative estimation of cellular uptake further authenticated the efficiency of CS-Lin-Pen/pDNA polyplexes over CS/pDNA and CS-Lin/ pDNA polyplexes (Figure S1). For CS/pDNA polyplexes, we observed only 14.7−20.1% FITC positive cells, while cellular uptake was enhanced by 2.7−3.1-fold for CS-Lin/pDNA polyplexes in different cell lines. The uptake percentage of CS-Lin-Pen/pDNA polyplexes in HEK 293, CHO, and HeLa cells was 95.4, 80.8, and 74.3%, respectively. These data emphasized the importance of penetratin conjugation for transportation of the polyplexes into multiple cell lines. In Vitro Release of pDNA. After the polyplexes penetrate the cytoplasm, a very tight binding between pDNA and its carriers imposes difficulty in unpacking of pDNA cargoes and thereby hinders gene expression.40 Integration of hydrophobic segments to the polymeric backbone has emerged as an effective strategy to diminish polymer/pDNA binding force.41 H

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Figure 9. Cytotoxicity of (A) CS-Lin and CS-Lin-Pen micelles at different concentrations and (B) CS-Lin/pDNA and CS-Lin-Pen/pDNA polyplexes at various weight ratios was evaluated in HEK 293 cells using a MTT assay. Data represent the mean ± SD (n = 4).

Figure 10. In vitro transfection efficiency of CS/pDNA, CS-Lin/pDNA, and CS-Lin-Pen/pDNA polyplexes prepared at the optimal weight ratio of 15. (A) Level of β-galactosidase expression in HEK 293, CHO, and HeLa cells. (B) Flow cytometric determination of GFP transfected HEK 293, CHO, and HeLa cells. Fugene (w/w = 3) was used as a positive control. Data represent the mean ± SD (n = 4). “*” Indicates significantly (p < 0.01) higher than Fugene.

CS-Lin-Pen micelles gave the maximum gene expression, which was ∼37- and 34-fold higher, respectively, as compared to CS. Transfections performed with Fugene were used as a positive control. The results of β-galactosidase expression in three different cell lines revealed that the transfection potencies of CS-Lin-Pen were significantly (p < 0.01) higher than Fugene. It was also noticed that the transfection efficiencies of CS-Lin, CS-Lin-Pen, and Fugene were cell line dependent, as stated by previous reports,43,44 and the sequential order was HEK 293 > CHO > HeLa. Gene transfection efficiency of CS-Lin and CS-Lin-Pen polymers was also evaluated using green fluorescence protein encoding plasmid DNA (pGFP). The transfection efficiency was estimated by quantifying the percentage of GFP positive cells, as measured by flow cytometry analysis (Figure 10B). In HEK 293 cells, transfection efficiency of CS-Lin-Pen micelles was found to be improved ∼13.2-fold compared to unmodified CS, which was in agreement with the results of pβ-gal

viability compared to the control. Unlike PEI and other cationic polymers, CS-Lin-Pen did not show any visible cytotoxicity, as evidenced by MTT assay. This finding suggests the great promise of the synthesized CS-Lin-Pen micelles as a suitable gene delivery vector for repetitive administration of large quantities of genes of interest in clinical applications. In Vitro Gene Transfection. In vitro gene transfection ability of CS-Lin and CS-Lin-Pen polymers was first evaluated in HEK 293, CHO, and HeLa cells using β-galactosidase (pβgal) as a reporter gene. To decide the optimal weight ratio of polymer/pDNA polyplexes for transfection, weight ratios ranging from 5 to 20 were investigated for initial screening (Figure S4), and the optimized weight ratio of 15 was used for all transfection studies. As depicted in Figure 10A, the naked pβ-gal showed very little to no β-galactosidase expression. In the case of HEK 293 cells, the CS-Lin-Pen/pβ-gal polyplexes induced the maximum β-galactosidase expression, which was ∼40- and 2-fold higher as compared to CS/pβ-gal and CS-Lin/ pβ-gal polyplexes, respectively. In CHO and HeLa cells also, I

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multiple cell lines. More importantly, the dual-functionalized micelles showed excellent hemocompatibility and no cytotoxicity when used as transfection reagents. These findings cumulatively reinforce the expectation that CS-Lin-Pen micelles could symbolize a potential nonviral alternative to gene therapy.

expression data. A similar trend was observed in CHO and HeLa cells The transfection potential of different polyplexes was further scrutinized by confocal laser scanning microscopy. As revealed in Figure 11, no GFP positive cells were detected when the

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ASSOCIATED CONTENT

S Supporting Information *

Quantitative analysis of cellular uptake by flow cytometry, in vitro pDNA release at pH 5.5, cytotoxicity of micelles and micelles/pDNA polyplexes in CHO and HeLa cells, and effect of polymer/pDNA weight ratio on gene transfection efficiency of micelles. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

Figure 11. Confocal microscopic images of HEK 293 cells transfected with (A) naked pGFP, (B) CS/pGFP (w/w = 15), (C) CS-Lin/pGFP (w/w = 15), (D) CS-Lin-Pen/pGFP (w/w = 15) polyplexes, and (E) Fugene/pGFP (w/w = 3). HEK 293 cells were incubated with different formulations for 4 h, and the GFP expression was evaluated after 48 h of transfection. Images were taken at 20× magnification.

*Telephone: +1-701-231-7943. Fax: +1-701-231-8333. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to greatly acknowledge the financial support provided by NSF EPSCoR Grant No. EPS-0814442.

transfection was carried out by naked pGFP. Only a few GFP positive cells with weak fluorescence intensities were recorded when CS was utilized as a gene carrier. Conversely, the CS-LinPen/pGFP resulted in highly intense fluorescence in most of the exposed HEK 293 cells. Hence, the results of confocal microscopic observation reinforce our conclusions drawn from the quantitative assessments of pβ-gal and pGFP expression data. These results indicate that CS-Lin-Pen micelles could efficiently transfer both the pβ-gal and pGFP reporter plasmids into multiple cell lines. The higher transfection efficacy of CSLin to CS might be attributable to the hydrophobic linoleic acid modification, which is recognized as a favorable option to enhance the stability of polyplexes in biological milieu, to improve cellular uptake, and to increase unpacking of complexed pDNA. The superiority of the CS-Lin-Pen/pDNA over CS-Lin/pDNA polyplexes emphasized the significance of penetratin mediated enhanced cellular uptake and nuclear trafficking of polyplexes into target cells. Together with the excellent cell viability and in vitro gene expression experiments, CS-Lin-Pen was established as a safe, simple, and efficient gene delivery vector which not only facilitates the cellular uptake of the pDNA, but also effectively transports pDNA to the nucleus for transcription.

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CONCLUSIONS Literature archives numerous gene delivery vectors. However, only a few of them exhibited promising results in clinical trials. The primary reasons for failure in the clinical setting include poor gene transfection efficiency and high toxicity. In this report, we have designed and synthesized chitosan-based cationic micelles as potential nonviral gene delivery systems. The synthesized micelles exhibited efficient pDNA binding ability to form stable polyplexes with ∼100 nm diameters and provide effective protection of the condensed pDNA from DNase I degradation. The linoleic acid and penetratin dualfunctionalized chitosan CS-Lin-Pen was the most potent vector, ∼34−40 times more effective than unmodified chitosan in J

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