Article pubs.acs.org/Langmuir
Side-Chain Amino-Acid-Based pH-Responsive Self-Assembled Block Copolymers for Drug Delivery and Gene Transfer Sonu Kumar,† Rituparna Acharya,‡ Urmi Chatterji,‡ and Priyadarsi De*,† †
Polymer Research Centre, Department of Chemical Sciences, Indian Institute of Science Education and Research - Kolkata, BCKV Campus Main Office, Mohanpur 741252, Nadia, West Bengal, India ‡ Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700 019, West Bengal, India S Supporting Information *
ABSTRACT: Developing safe and effective nanocarriers for multitype of delivery system is advantageous for several kinds of successful biomedicinal therapy with the same carrier. In the present study, we have designed amino acid biomolecules derived hybrid block copolymers which can act as a promising vehicle for both drug delivery and gene transfer. Two representative natural chiral amino acid-containing (L-phenylalanine and L-alanine) vinyl monomers were polymerized via reversible addition−fragmentation chain transfer (RAFT) process in the presence of monomethoxy poly(ethylene glycol) based macro-chain transfer agents (mPEGn-CTA) for the synthesis of well-defined side-chain amino-acid-based amphiphilic block copolymers, monomethoxy poly(ethylene glycol)-b-poly(Boc-amino acid methacryloyloxyethyl ester) (mPEGn-b-P(Boc-AA-EMA)). The self-assembled micellar aggregation of these amphiphilic block copolymers were studied by fluorescence spectroscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). Potential applications of these hybrid polymers as drug carrier have been demonstrated in vitro by encapsulation of nile red dye or doxorubicin drug into the core of the micellar nanoaggregates. Deprotection of side-chain Boc- groups in the amphiphilic block copolymers subsequently transformed them into double hydrophilic pH-responsive cationic block copolymers having primary amino groups in the side-chain terminal. The DNA binding ability of these cationic block copolymers were further investigated by using agarose gel retardation assay and AFM. The in vitro cytotoxicity assay demonstrated their biocompatible nature and these polymers can serve as “smart” materials for promising bioapplications.
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INTRODUCTION Molecular self-assembly is a powerful approach for constructing novel supramolecular architectures with unique properties. Particularly, synthetic block copolymers can adopt various supramolecular architectures, such as micelles, vesicles, fibers, nanotubes, and a variety of other morphologies in solution,1,2 depending on many factors including molecular architectures, block compositions and preparation conditions.3,4 Recent advances in the development of multifunctional self-assembled polymer micelles based on functional block copolymers have led the foundation for designing smart nanocarriers that can enhance the efficiency of drug delivery, gene transfer, or delivery of imaging agents.5−8 Inspired by nature, over the past few years tremendous effort has been devoted for the incorporation of biological materials such as proteins to the synthetic block copolymers, which may create a new class of nonbiological macromolecules with biomimetic structures and properties with entirely new synergism.9 Conjugation of naturally occurring amino acid biomolecules, which are the constitutional components of proteins and peptides, into synthetic polymers are of particular interest, because it benefits synthetic polymeric materials in terms of providing much © 2013 American Chemical Society
higher complexity due to their amphoteric nature, chiral recognition, etc.10 Amino acids, peptides and proteins form higher-order secondary structures mediated by intra- and interchain associations via noncovalent interactions such as directed hydrogen bonding, unspecific hydrophobic stacking, electrostatic and dipolar interactions.11−21 Introducing amino acids into the synthetic polymers may also offer several other advantageous characteristics such as viability of further chemical modifications with bioactive molecules, improved biological properties, and interactions with proteins or genes. Amino acid based polymers can be classified into two groups, depending on whether amino acid moieties are borne in the main chain as backbone segments (polypeptide), or in the side chain as pendants.22 In recent years, preparation of side-chain amino-acid-based well-defined polymers in controlled fashion has gained remarkable attention.22,23 In this context, we have described here the synthesis of well-defined diblock copolymers composed of side-chain amino acid block and polyethylene glycol (PEG) segment, via reversible addition−fragmentation Received: June 22, 2013 Published: November 25, 2013 15375
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instrument. Circular dichroism (CD) spectroscopic measurements were carried out in a JASCO J-185 CD spectrometer. For cytotoxicity test, MCF-7 cell line were grown on Eagle’s minimal essential medium (EMEM, HiMedia) containing 10% fetal bovine serum (HiMedia) and 1% penicillin−streptomycin (HiMedia) solution. Formazan crystals produced from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in MTT solubilization solution (In Vitro Toxicology Assay Kit MTT based, Sigma-Aldrich). For microscopic imaging, MCF-7 cells were fixed with 4% paraformaldehyde (Merck) and cells were permeabilized with Triton-X-100 (Sigma). Then, the cells were subsequently stained with 4′,6diamidino-2-phenylindole (DAPI, USB Corporation). Synthesis of Block Copolymers from mPEGn-CTA. Typically, Boc-F-EMA (1.00 g, 2.65 mmol), mPEG2k-CTA (2,400 g/mol, 127.3 mg, 52.99 μmol), AIBN (0.87 mg, 5.30 μmol; 1 mL solution of 8.7 mg AIBN in 10 mL DMF) and DMF (2 mL) were sealed in a 20 mL vial, purged with dry N2 (20 min), and the vial was placed under stirring in a preheated reaction block at 70 °C. After a predetermined time, the vial was cooled in ice−water bath, and a portion of the reaction mixture was analyzed by 1H NMR to know the monomer conversion. Remaining solution was diluted with acetone and the polymer was precipitated into hexanes. The block copolymer, mPEG2k-b-P(Boc-FEMA), was reprecipitated four times from acetone/hexanes and dried under a vacuum at room temperature for 10 h. Micelle Preparation and Characterization. Typically, 10.0 mg of a block copolymer was dissolved in 5 mL of acetone, and the solution was transferred to a dialysis bag (Spectra/por dialysis membrane, molecular weight cutoff (MWCO): 6−8 kDa) and dialyzed against DI water for 48 h (water was replaced after every 2 to 6 h). The prepared micellar solutions were further diluted for AFM and SEM studies. Nile Red/Dox Loaded Micelles Preparation. Nile red and Dox loaded micelles were prepared by a dialysis method. Typically, block copolymer (20 mg) in 2 mL of DMSO was mixed with Dox/nile red (10 mg). The mixture was added dropwise to 10 mL of deionized (DI) water under stirring. The solution was stirred for 2 h at room temperature, and the organic solvent was removed by dialysis (6−8 kDa MWCO) against DI water for 48 h to obtain the drug/dye loaded micelles. The micellar solution was filtered and freeze-dried. Determination of Critical Aggregation Concentration (CAC). A predetermined amount of pyrene in acetone solution was added to volumetric flasks and acetone was then evaporated completely. Different concentrations of polymer micellar solutions were added to pyrene and left to equilibrate with pyrene overnight, the pyrene concentration in the final solution was 6.0 × 10−7 mol/L. The fluorescence intensities of solutions were measured with the excitation wavelength set at 339 nm and the ratios of pyrene probe emission fluorescence intensities at 392 and 373 nm (I392/I373) were plotted as a function of the logarithm of polymer concentrations. The CAC was obtained from the intersection of two tangent plots of intensity ratio I392/I373 versus the logarithm of polymer concentrations (log C).37,38 Deprotection of Boc-Protected Polymers. Deprotection of Boc-groups from block copolymers mPEGn-b-P(Boc-AA-EMA) were carried out by following similar procedure previously reported by our group elsewhere.36 Agarose Gel Retardation Assay. Deprotected block copolymer was dissolved into phosphate buffered saline (PBS) solutions (pH 4.5) at various concentrations and then mixed with 0.1 μg of pDNA (pEGFP-C1 DNA, 4.7 kbp) to a volume of 10 μL. Various complexes were vortexed and incubated for 30 min at room temperature. After adding 1 μL of loading buffer, each complex solution were loaded onto 1% (w/v) agarose gel containing 1x Tris-acetate-EDTA (TAE) buffer solution (pH 8.0). Electrophoresis was carried out at 80 V for 80 min. Naked pDNA diluted with the same buffer without the polymer was used as the control. DNA was illuminated by ethidium bromide, and was visualized with a UV lamp, using a GelDoc system (BIO-RAD). Zeta Potential and Particle Size Measurements. Zeta potential and particle size measurements of the polyplexes of different polymer/ DNA weight ratios in PBS solution were performed by DLS instrument. The polyplexes were preliminarily prepared in 1.0 mL of
chain transfer (RAFT) polymerization technique. The PEG is a Food and Drug Administration (FDA)-approved biocompatible polymer, and PEG containing block copolymers are beneficial for possible future biomedical applications,24,25 because of its biocompatibility, availability, water solubility, and well-known physicochemical properties.26−29 van Hest et al. have reported the synthesis of β-sheet side chain PEG-peptide block copolymer conjugates via atom transfer radical polymerization (ATRP) technique.30,31 Conjugation of PEG in the PEG hybrid block copolymers containing amphiphilic β-strand peptide sequences stabilized the adopted secondary structure of the peptide and also reduced their sensitivity toward pH variations as compared to the native peptide sequence.32 Adams et al. reported the formation of inter- and intrachain β-sheet motif structuring in aqueous solution by the peptidic component of the PEG-b-poly(side-chain peptide) block copolymers prepared by ATRP.33 Herein, we have presented an effective strategy to prepare PEGylated side chain amino-acid-based polymers by RAFT technique. The synthesized amphiphilic block copolymers were able to encapsulate hydrophobic drug/dye molecules, and can easily be transformed to a double hydrophilic biocompatible pH responsive system carrying primary amino functionalities in the side-chain with cationic characteristics. The synthesized biohybrid materials formed polyplexes with plasmid DNA (pDNA), and hence can be applied as gene transfer vehicle.
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EXPERIMENTAL SECTION
Materials. Boc-L-phenylalanine (Boc-L-Phe-OH, 99%), Boc-Lalanine (Boc-L-Ala-OH, 99%), and trifluoroacetic acid (TFA, 99.5%) were obtained from Sisco Research Laboratories Pvt. Ltd., India and used as received. The 4-dimethylaminopyridine (DMAP, 99%), 1hydroxybenzotriazole hydrate (HOBt hydrate, 97%), anhydrous N,Ndimethylformamide (DMF, 99.9%), dicyclohexylcarbodiimide (DCC, 99%), 2-hydroxyethyl methacrylate (HEMA, 97%), pyrene (98%), nile red, doxorubicin hydrochloride (Dox.HCl), and monomethoxy poly(ethylene glycol) with molecular weight 2 kDa (mPEG2k) and 5 kDa (mPEG 5k ) were purchased from Sigma. Dox.HCl was deprotonated according to the literature.34 The 2,2′-azobisisobutyronitrile (AIBN, Sigma, 98%) was recrystallized twice from methanol. The NMR solvents such as CDCl3 (99.8% D) and D2O (99.9% D) were obtained from Cambridge Isotope Laboratories, Inc., USA. The solvents such as hexanes, acetone, ethyl acetate, tetrahydrofuran (THF), and dichloromethane (DCM) were purified by standard procedures. The mPEG-based macro chain transfer agents (mPEG2kCTA and mPEG5k-CTA) were prepared according to our previous report.35 The amino-acid based monomers (Boc-AA-EMA) such as Boc-L-alanine methacryloyloxyethyl ester (Boc-A-EMA) and Boc-Lphenylalanine methacryloyloxyethyl ester (Boc-F-EMA) were synthesized as previously reported.36 Instrumentation. The molecular weights and molecular weight distributions (PDI) were determined by gel permeation chromatography (GPC) in THF relative to poly(methyl methacrylate) standards at 35 °C using 1.0 mL min−1 flow rate. The system is equipped with a Waters Model 515 HPLC pump and a Waters Model 2414 refractive index (RI) detector. The 1H NMR measurements were carried out in a Bruker AVANCEIII 500 MHz spectrometer. FT-IR spectra were obtained from a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Fluorescence emission spectra were recorded on a Horiba Jobin Yvon (Fluoromax-3, Xe-150 W, 250−900 nm) fluorescence spectrometer. UV−vis spectroscopic study was performed on Perkin-Elmer Lambda 35 spectrophotometer, with a scan rate of 240 nm/min. Particle size, zeta potential (ξ), and atomic force microscopy (AFM) were carried out as reported elsewhere.35 For field emission scanning electron microscopy (FE-SEM) study, a small amount of the sample solution were drop casted on a glass slide, dried, coated with gold:palladium (20:80) and then micrographs were taken from the Carl Zeiss-Sigma 15376
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PBS solution (pH 4.5) under a predetermined polymer/DNA ratio and 1.86 μg/mL pDNA, and then kept for incubation at room temperature for 30 min before the measurements. In Vitro Cytotoxicity Test. The MTT reduction assay was used for investigating cytotoxicity of prepared polymers. MCF-7 cells were seeded at a density of 4 × 104 cells/well in a 96 well plate and incubated for 24 h to reach ∼80% confluency. Cells were treated with polymer solutions having different concentrations (20 to 200 μg/mL) and incubated for 24 h. A set of untreated control cells were also incubated for the same time period. The untreated control and polymer treatment was performed in triplicates. To each well, 10% culture volume MTT solution was added and was incubated for 4 h. Then, the cell culture medium was disposed off, and MTT solubilizing solution was added equally to the culture media volume. After 10 min of incubation, the absorbance at 570 nm was measured. For fluorescence microscopy, two of the copolymer concentrations with 20 μg/mL and untreated control, MCF-7 cells were seeded at a density of 2 × 104 cells/well in a 96 well plate and incubated for 24 h. After post incubation, these cells were fixed with 4% paraformaldehyde and then stained with DAPI. Finally, it was used for microscopy (BD Pathway855) at 20× magnification. Filter set used for DAPI imaging were excitation filter 380/10 nm, epifluorescence dichroic filter 400DCLP, emission at 430LP with the exposure time of 6 ms. Images were acquired using BD AttoVision software. Cellular Uptake Studies. For confocal microscopy, polymeric micelles (loaded with Dox or nile red) of various concentrations treated and untreated control, MCF-7 cells were seeded at a density of 2 × 104 cells/well in a 96 well plate and incubated for 24 h. After incubation, these cells were fixed with 4% paraformaldehyde, stained with DAPI and images were taken using BD Attovision software. Excitation filter for doxorubicin and nile red were 548/20 and emission collection were by 570LP filters.
Table 1. RAFT Block Copolymerization of Boc-AA-EMA Monomers in the Presence of mPEGn-CTA at 70 °C in DMF block copolymera mPEG2k-bP(Boc-FEMA) mPEG5k-bP(Boc-FEMA) mPEG2k-bP(Boc-AEMA) mPEG5k-bP(Boc-AEMA)
time (min)
convb (%)
Mn,GPCc (g/mol)
PDIc
Mn,theod (g/mol)
Mn,NMRe (g/mol)
240
70
14 100
1.25
15 610
13 760
270
55
11 000
1.18
15 780
14 040
240
78
12 800
1.21
14 150
16 570
270
60
10 600
1.16
14 430
11 630
a
Molar ratio of [monomer]/[mPEGn-CTA]/[AIBN] = 50/1/0.1. Determined by 1H NMR spectroscopy in CDCl3. cMeasured by GPC. d The theoretical molecular weight (Mn,theo) = ([Boc-AA-EMA]0/ [mPEGn-CTA]0 × molecular weight (MW) of Boc-AA-EMA × conversion) + (MW of mPEGn-CTA). eDetermined by 1H NMR study. b
copolymers in CDCl3, the typical resonance signals corresponding to both the block are clearly visible. Comparison of the integration areas from the terminal −CH2−CH2- protons (from the HOOC−CH2−CH2-C(CN)(CH3)- chain end) at 2.3−2.7 ppm and the repeating unit protons at 3.8−4.6 ppm (for −OCH2-CH2-O- and chiral proton) allowed calculation of the number-average molecular weight (Mn,NMR) from NMR spectroscopy. The Mn,NMR values must typically be considered an upper bound since in the final polymer there may be some unreacted macro-CTA. Reasonable agreement between Mn,GPC, Mn,theo, and Mn,NMR values (Table 1) indicate that mPEGnmacro CTA can be successfully employed for the preparation of side-chain amino-acid-based block copolymers with a relatively narrow PDI. Deprotection of Boc-Groups from Block Copolymers. To obtain terminal primary amino groups in the side chain of these amino-acid-based block copolymers, we removed Bocprotecting groups from the amino acids by using TFA/DCM (1:1, v/v) at room temperature (Scheme 1). Successful deprotection of Boc- groups from the block copolymers were confirmed by the disappearance of Boc-proton signals at about 1.4 ppm in the 1H NMR spectrum shown in Figure 2C for mPEG2k-b-P(H2N−F-EMA) (also see Figure S1B in the Supporting Information for mPEG2k-b-P(H2N-A-EMA)). In addition, Boc-deprotection was further confirmed by FT-IR study. As an example, the absorption peak at 1517 cm−1 attributed to the N−H (amide II band) in mPEG2k-b-P(BocF-EMA) and mPEG2k-b-P(Boc-A-EMA) disappeared after deprotection reactions (see Figure S2 in the Supporting Information) because of the conversion of Boc-group into the -NH2 functionality. After Boc-deprotection, amphiphilic block copolymers mPEGn-b-P(Boc-AA-EMA) are converted to double hydrophilic block copolymers mPEGn-b-P(H2N-AAEMA), hence they are readily soluble in aqueous medium. pH Responsiveness of Deprotected Block Copolymers. In recent years, a variety of chiral and responsive polymers poly[(meth)acrylamide]s with free carboxylic acid group containing amino acid moiety in the side-chain have been developed that can show pH responsiveness due to the protonation/deprotonation of carboxyl groups.22 Herein, the terminal primary amino groups in the side-chain of as synthesized polymers mPEGn-b-P(H2N-AA-EMA) can be
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RESULTS AND DISCUSSION mPEG-CTA-Mediated RAFT Polymerization of AminoAcid-Based Monomers. To design PEGylated block copolymers consisting of side-chain amino acid pendants, we performed RAFT polymerization of methacrylate derivatives of amino acid in the presence of mPEG based macro-CTA using [Boc-AA-EMA]/[mPEGn-CTA]/[AIBN] = 50/1/0.1 in DMF at 70 °C. Herein, we have adopted two representative aminoacid-based vinyl monomers Boc-F-EMA and Boc-A-EMA because phenylalanine is the most hydrophobic amino acid and alanine is the least hydrophobic chiral amino acid according to the hydrophobicity values at pH 7.39 Also, we have employed two different chain lengths of mPEG-based macro-CTA, mPEG2k, and mPEG5k for providing different extent of hyrdrophilicity in the block copolymers. The block copolymers, mPEGn-b-P(Boc-AA-EMA), were obtained with good yields and their number average molecular weights (Mn,GPC) were determined from GPC (Table 1). Figure 1 shows that after block copolymerization GPC chromatograms have shifted toward higher molecular weight region compared to mPEGn-CTA, suggesting effective chain extension, although traces of unreacted macro-CTA remained present in the block copolymer samples. Similar observations were already reported using mPEG macro-CTA for the polymerization of isoprene.40 In the GPC traces of the block copolymers, the shoulder at the low-molecular-weight region corresponds mPEGn-CTA and the Mn,GPC obtained for the block copolymers (Table 1) are little lower than the theoretical number average molecular weight (Mn,theo) determined by 1H NMR monomer conversion. The structure of the block copolymers were confirmed by 1H NMR spectroscopy (Figure 2A for the mPEG2k-b-P(Boc-F-EMA) and Figure S1A in the Supporting Information for the mPEG2k-bP(Boc-A-EMA)). In the 1H NMR spectra of the block 15377
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Figure 1. GPC traces of mPEGn-CTA’s and corresponding block copolymers: (A) polymers from mPEG2k-CTA, and (B) polymers from mPEG5kCTA.
Figure 2. 1H NMR spectra of block copolymer (A) mPEG2k-b-P(Boc-F-EMA) in CDCl3, (B) mPEG2k-b-P(Boc-F-EMA) in D2O, and (C) corresponding deprotected polymer mPEG2k-b-P(H2N−F-EMA) in D2O (* denotes the solvent resonances).
molecular weight (chain length) of mPEG block increases overall hydrophilicity of the block copolymer. Self-Assembly of Block Copolymers. Although the 1H NMR spectrum of mPEG2k-b-P(Boc-F-EMA) in CDCl3 (Figure 2A) shows all the proton resonance peaks from both the block segments, corresponding 1H NMR spectrum in D2O displayed peaks only from hydrophilic mPEG block and there was a complete disappearance of proton signals from Boc-F-EMA repeating units. This could be due to the possible suppressed molecular motion of the aggregated hydrophobic P(Boc-FEMA) segments surrounded by the hydrophilic mPEG block (Figure 2B), strongly suggesting the formation of a stable higher-order morphology in aqueous medium.42,43 Then, critical aggregation concentration (CAC) value was determined for the aqueous solutions of these amphiphilic block copolymers by fluorescence spectroscopy technique using pyrene as a hydrophobic fluorescent probe because of its high sensitivity to the local polarity of the medium.35 A red shift from 373 to 392 nm of pyrene fluorescence spectrum is observed, with the increment of polymer concentration, indicating that pyrene molecules are partitioned into a less polar environment. The ratios of pyrene probe fluorescence
reversibly protonated and deprotonated by adjusting the pH of their aqueous solutions.41 To determine pH responsiveness of the double hydrophilic block copolymers, we analyzed their aqueous solutions (0.2 wt %) as a function of pH at 27 °C using UV−vis spectroscopy, by determining the transmittance at 500 nm (Figure 3A for mPEG2k-b-P(H2N−F-EMA) and Figure S3A in the Supporting Information for mPEG2k-bP(H2N-A-EMA)). At lower pH, the primary amine moiety is completely protonated and the polymer solution was transparent. With the increasing pH of the solution, it transformed from transparent to opaque and gradually precipitation occurs, demonstrating the obvious pH-responsive property of these polymers. The reduction of 50%T of the deprotected block copolymer solutions were observed at pH 5.6, 6.4, 5.8, and 6.9 for the mPEG2k-b-P(H2N−F-EMA), mPEG2k-b-P(H2N-AEMA), mPEG5k-b-P(H2N−F-EMA), and mPEG5k-b-P(H2NA-EMA), respectively. These pH-responsive phase transitions were reversible and lower transition pH for phenylalanine containing block copolymers are due to the higher hydrophobicity of phenylalanine than alanine. For the same series, responsive pH for block copolymers with mPEG2k are lower than the mPEG5k containing polymers because increasing 15378
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Scheme 1. Synthesis of mPEGn-CTA-Derived Amino-Acid-Based Block Copolymers by RAFT Polymerization, and Their Drug/ Dye Encapsulation and DNA Complexation
Figure 3. (A) Effect of pH on the transmittance of aqueous solutions of deprotected mPEG2k-b-P(H2N−F-EMA) block copolymer at 500 nm (0.2 wt % solution), and (B) CAC determination of mPEG2k-b-P(Boc-F-EMA), where inset in B shows variation in fluorescence emission spectra of encapsulated pyrene dye.
analysis. As shown in Figure 4, uniform micellar aggregates were observed from the self-assembly of mPEG2k-b-P(Boc-FEMA) in aqueous medium, having an average diameter of about 420 nm with the height of 100 nm (see Figure S4 in the Supporting Information). However, in the case of mPEG2k-bP(Boc-A-EMA), two kinds of well-defined micellar morphology was observed. As illustrated in Figure 5, frame 1 shows small preformed primary micelles with an average diameter of 230 nm with the calculated height of the micelle of about 50 nm and the height profile plot indicates smooth micellar surface (see Figure S5 in the Supporting Information), whereas frame 2 depicts the formation of uniform large multimolecular composite structures, formed by the secondary aggregation of those primary micelles in aqueous solution, with an average diameter of about 390 nm having calculated height of 80 nm with smooth micellar surface (see Figure S6 in the Supporting Information). Although nonuniform spherical morphology was observed for mPEG5k-b-P(Boc-F-EMA) (see Figure S7 in the Supporting Information), the self-assembled mPEG5k-b-P(BocA-EMA) produced well-defined micellar aggregates with an
intensities at 392 and 373 nm (I392/I373) were plotted against the log C of the block copolymer. As shown in Figure 3B and Figure S3B in the Supporting Information, at low concentrations of block copolymers, the intensity ratio I392/I373 remained almost unchanged and once the copolymer concentration reached a particular value (CAC) it increased abruptly, indicating the formation of aggregate. The CAC values were obtained by the intersection of the lines drawn through the flat region points at low concentrations and the abruptly increasing regions at high concentrations. The achieved CAC values of mPEG2k-b-P(Boc-F-EMA) and mPEG2k-b-P(Boc-A-EMA) were 0.98 and 0.75 μg/mL, respectively. The obtained CAC values indicated the high tendency of the amphiphilic block copolymers to self-assemble in aqueous solution.44 Further, the solution of mPEGn-b-P(Boc-AA-EMA) in acetone was dialyzed against DI water and the resulting aqueous solution was employed to investigate the selfassociation of these amphiphilic diblock copolymers toward higher-order structure formation by both AFM and SEM 15379
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Figure 4. AFM images ( (A)height, (B) amplitude, (C) three-dimensional), and (D) SEM image of mPEG2k-b-P(Boc-F-EMA) (samples were prepared from 0.1 mg mL−1 aqueous solution).
Figure 5. AFM height image of mPEG2k-b-P(Boc-A-EMA) block copolymer (samples were prepared from 0.1 mg mL−1 aqueous solution).
Figure 6. (A) AFM amplitude, (B) phase, and (C) SEM images of mPEG5k-b-P(H2N-A-EMA) block copolymer (prepared from 0.1 mg mL−1 aqueous solution). Inset: enlarged view.
aggregation in our case appeared because of the presence of amino acid moieties in the hydrophobic block, which induced secondary interactions. The self-association of Boc-deprotected double hydrophilic mPEGn-b-P(H2N-AA-EMA) block copolymers in aqueous
average diameter of 340 nm (see Figure S8 in the Supporting Information). Formation of such large composite structures by the secondary aggregation mechanism of initially formed small micelles in aqueous solution are reported in the literature.45−49 We believe that these type of possible secondary micellar 15380
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Figure 7. CD spectra of block copolymers in methanol; (A) mPEG2k-b-P(Boc-A-EMA) and mPEG2k-b-P(H2N-A-EMA), and (B) mPEG2k-b-P(BocF-EMA), and mPEG2k-b-P(H2N−F-EMA).
Figure 8. (A) Electrophoretic mobility of pDNA in the polyplexes formed by mPEG2k-b-P(H2N-A-EMA) polymer at different polymer/DNA weight ratios, pDNA without polymer was used as control, and (B) AFM image of polyplex at polymer/DNA weight ratio of 40.
(related to its conformation in solution) was investigated by CD spectroscopic measurements. Figure 7A depicts the CD spectrum of mPEG2k-b-P(Boc-A-EMA) in methanol, which displays a strong positive signal at 209 nm and a weak negative signal at 237 nm. Herein, the positive signal at around 209 nm is probably due to the n→π* transition of the carboxyl chromophore and the negative peak at 237 nm can generally be attributed to the π1→π* transition of the amide chromophore.50,51 However, corresponding Boc-deprotected mPEG2kb-P(H2N-A-EMA) block copolymer exhibited only a positive intense CD signal at 210 nm. As illustrated by Figure 7B, a weak positive peak at 202 nm along with a strong positive signal at 220 nm is apparent in the CD spectrum of mPEG2k-b-P(BocF-EMA), whereas corresponding Boc-deprotected polymer mPEG2k-b-P(H2N−F-EMA) shows only a strong positive signal at 219 nm. Typically, the peak at 220 and 202 nm can be attributed to the n→π* and π→π* transitions, respectively, of the carboxyl chromophore.50,52,53 These results suggest the existence of a specific conformation of these block copolymers in methanol solution.51 Characterization of Polymer/DNA Complexes. The surface charges (ξ) of various Boc-deprotected block copolymers in DI water (1.0 mg mL−1) at pH 5.0 were measured by DLS. The ξ values of mPEG2k-b-P(H2N−FEMA), mPEG2k-b-P(H2N-A-EMA), mPEG5k-b-P(H2N−FEMA) and mPEG5k-b-P(H2N-A-EMA) were found to be +9.7, +9.6, +11.8 and +8.5 mV, respectively. The positive ξ values are due to the presence of ammonium (−NH3+) pendants at acidic pH, indicating cationic nature of the deprotected block copolymers. The electrophoretic mobility study of pDNA, in the polyplexes formed by mPEG2k-b-
medium were also investigated by AFM and SEM. Their aqueous solutions (0.1 mg/mL) were sonicated for 1.0 h and drop-casted over microscopic glass coverslip. In the case of small PEG block-containing copolymers, for example, in phenylalanine-based mPEG2k-b-P(H2N-F-EMA) block copolymer, micellar aggregates was observed (see Figure S9 in the Supporting Information), and in alanine-based block copolymer mPEG2k-b-P(H2N-A-EMA), defined micellar morphology with an average diameter of 620 nm was observed (see Figure S10 in the Supporting Information). In these cases, the generation of micellar morphology was probably exhibited because of the domination of hydrophobicity by methacrylate backbone over the hydrophilicity by terminal primary amino groups and short PEG segment, whereas in the former case, the presence of phenyl groups that imparts hydrophobicity and also π−π interactions can induce aggregation. Furthermore, in the case of the longer PEGylated block-containing copolymers, for example, in mPEG5k-b-P(H2N-F-EMA), micelle to twisted ribbon-like nonuniform aggregated morphology was observed (see Figure S11 in the Supporting Information), and in mPEG5k-b-P(H2N-A-EMA), spherical to rodlike morphology of varying dimensions appeared (Figure 6, also see Figures S12 and S13 in the Supporting Information). Interestingly, in these cases, the increased hydrophilicity by larger PEG segment and terminal primary amino group dominates over the other hydrophobic as discussed parameters, and creates the macrosupramolecular self-associated morphologies induced by possible secondary interactions among amino acid block segments. Chiroptical Property of Block Copolymers. The chiroptical behavior of the amino-acid-based block copolymers 15381
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Figure 9. Cytotoxicity of block copolymers on MCF-7 cells, (A) mPEG2k-b-P(H2N−F-EMA) (blue color), mPEG5k-b-P(H2N−F-EMA) (pink color), (B) mPEG2k-b-P(H2N-A-EMA) (blue color), mPEG5k-b-P(H2N-A-EMA) (pink color), and (C) CLSM images of MCF-7 cells treated with block copolymers having concentration at 20 μg/mL.
0.1 μg of pDNA to a volume of 10 μL to achieve polymer/ DNA polyplex at a mass ratio of 40:1. Then, 2 μL aliquots of mPEG2k-b-P(H2N-A-EMA)/pDNA complex was adsorbed on silicon wafers and after drying AFM image was recorded. Figure 8B shows that the plasmid DNA was complexed with mPEG2kb-P(H2N-A-EMA) and formed large globular particles.56 The AFM profiles of this complex demonstrated the average diameter and height of 450 and 50 nm, respectively (see Figure S15 in the Supporting Information). The obtained larger size renders the aggregation of preformed polyplex to larger agglomerates due to lack of repulsion, because the surface charges of polymers get reduced in buffer solutions due to the higher ionic strength.57 And this phenomenon was further supported by the size distribution profiles of the polyplexes at different weight ratio of polymer/DNA as depicted by Figure S14B in the Supporting Information. The mPEG2k-b-P(H2N-AEMA) polymer tended to compact pDNA into less stable particles with lower hydrodynamic diameter sizes at initial stage (130 nm at weight ratio 5), and then their polyplexes particle sizes gradually increased to approach 330 and 510 nm at weight ratio 10 and 50, respectively, exhibited formation of large agglomerates,58 possibly because of the tendency of aggregation of the polymer itself (without DNA) in PBS solution of particle size 230 nm (see Figure S14B in the Supporting Information). These results indicates positively charged amino-acid-based block copolymers can be used for polyplex formation for gene delivery.29 Cytotoxicity of Amino-Acid-Based Block Copolymers. Because cytotoxicity assessment is crucial before biomedical applications of polymers, we have evaluated in vitro cytotoxicity of as synthesized amino-acid-based block copolymers mPEGn-
P(H2N-A-EMA) at different polymer/DNA weight ratios ranging from 0.1 to 10, was conducted to further investigate the ability of amino-acid-based block copolymers to bind DNA and to characterize their ionic interaction and electrolytic stabilities (Figure 8A).54 In the gel, larger migration of pDNA indicates the less stability of the ionic polyplex, which also denotes weaker interaction between polymer and pDNA. It was observed that the polyplex at weight ratio from 0.1 to 1 showed migration of pDNA across the gel similar to that observed for the free pDNA. With further increment in weight ratio, the amount of migrated pDNA decreased and all the bands of the polyplex were found at different positions from free pDNA, indicating stable polyplex formation at higher ratios of polymer/DNA and all pDNA molecules were involved in the formation of complexes with the amino acid block of the polymer. The achieved ratio for complete complexation of pDNA by PEGylated amino-acid-based block copolymer (in this case polymer/DNA = 2) depends on the amino acid block length and the mPEG block would influence and tend to coat the bundling of pDNA with amino acid side chains.55 Herein, large amount of cationic polymer was required to attain complete DNA complexation due to the steric hindrance of the PEG chain,56 and this can be further lowered by increasing amino acid block length, i.e., extent of cationic charge. The poymer/DNA weight ratio dependence of zeta-potentials of the polyplexes illustrated the increment in zeta potential from −12.8 to +7.0 mV with the gradual increase of the weight ratio up to 100 (see Figure S14A in the Supporting Information). DNA-binding ability was further confirmed by AFM and DLS study. The mPEG2k-b-P(H2N-A-EMA) polymer was dissolved into PBS solution (pH 4.5) and then mixed with 15382
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Figure 10. CLSM images for the uptake of mPEG2k-b-P(Boc-A-EMA)-nile red and mPEG2k-b-P(Boc-F-EMA)-Dox in MCF-7 cells treated at different concentrations for 24 h.
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CONCLUSIONS The mPEG-based macro-CTA can be used for the successful RAFT polymerization of amino acid side-chain containing methacrylate monomers to prepare well-defined amphiphilic block copolymers, mPEGn-b-P(Boc-AA-EMA). The block copolymers self-assemble in aqueous medium and produced micellar aggregates, where encapsulation of nile red based dye or doxorubicin based anticancer drug can be achieved. The CLSM studies revealed intracellular accumulation of dye/drug, strongly suggests their capability of being reservoir for cancer detection or cancer treatment application. Boc-group deprotection provided double hydrophilic block copolymers with terminal primary amine functionalities in the amino acid pendants, hence resulting cationic surface charge and pH responsive hydrophilic polymers, which were forming unique morphologies in aqueous medium due to their macrosupramolecular self-assembly. The hydrophilic block copolymers are nontoxic to cancer cells and are effective for binding with pDNA. These well-defined side-chain amino-acid-based block copolymers with cationic, biocompatible, and pHresponsive properties may provide new opportunities and fundamental guidelines to design promising single “smart” carrier with excellent performance for an efficient dual drug and gene delivery system in biomedicine.
b-P(H2N-AA-EMA), in MCF-7 (human breast cancer) cells using MTT assay. Figures 9A and B, shows noncytotoxic characteristics of these block copolymers at concentrations up to 200 μg/mL following long-term incubation (24 h). Moreover, in both the set of block copolymers with varying mPEG block length (mPEG2k- and mPEG5k-), the cellular cytotocicity was in well analogous to each other. Confocal microscopic study of Boc-deprotected block copolymers treated MCF-7 cells revealed no obvious change in nuclear structure with retained morphological integrity of the cells as compared to the control (Figure 9C). The cellular uptake of dye- (nile red) or drug (Dox)-loaded PEGylated block copolymer based micelles were further determined by confocal laser scanning microscopy (CLSM) images. Figure 10A shows dose dependent cellular uptake of hydrophobic nile red dye loaded mPEG2k-b-P(Boc-A-EMA) in MCF-7 cells after 24 h post treatment. Diffused accumulation of dye loaded mPEG2k-b-P(Boc-A-EMA) micelles in the intracellular matrix as well as in the nucleus demonstrated their potential application as a biomarker for pharmaceutical applications.59 Furthermore, the fluorescence microscopy images of MCF-7 cells treated with doxorubicin-based anticancer drug loaded mPEG2k-b-P(Boc-F-EMA) micelles further confirmed their dose-dependent cellular internalization, which further exhibited their promising application for cancer treatment (Figure 10B). These results demonstrate potential application of the studied PEGylated amino-acid-based block copolymers as a promising drug delivery system.
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ASSOCIATED CONTENT
S Supporting Information *
1
H NMR and FT-IR spectra of block copolymers, AFM and SEM images of block copolymers, CAC and pH response curves of polymer, zeta potential, size, and AFM image of 15383
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polymer/pDNA complexes. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (P. De). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The financial support of this work by the Department of Science and Technology (DST), New Delhi, India [Project SR/ S1/OC-51/2010] is gratefully acknowledged. We thank Dr. Sankar Maiti (Department of Biological Sciences, Indian Institute of Science Education and Research-Kolkata) for the electrophoresis study.
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