Zwitterionic Guanidine-Based Oligomers Mimicking Cell-Penetrating

Sep 4, 2012 - For comparison, micelles based on triblock copolymers with a third block with permanently cationic charges, ... View: ACS ActiveView PDF...
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Zwitterionic Guanidine-Based Oligomers Mimicking Cell-Penetrating Peptides as a Nontoxic Alternative to Cationic Polymers to Enhance the Cellular Uptake of Micelles Yoseop Kim, Sandra Binauld, and Martina H. Stenzel* Centre for Advanced Macromolecular Design (CAMD), University of New South Wales, Sydney, NSW 2052, Australia ABSTRACT: The aim of this work is to generate polymer micelles decorated with a synthetic version of cell-penetrating peptides, which are often rich in arginine with its positively charged guanidine group. A methacrylate-based monomer with guanidinium as functional groups was prepared using arginine (M-Arg) as a building block, resulting in a zwitterionic monomer. RAFT (reversible addition−fragmentation chain transfer) polymerization was employed to generate triblock copolymers with poly(methyl methacrylate)-block-poly(polyethylene glycol methyl ether methacrylate) as the first two blocks, which were subsequently chain extended with the guanidine-based monomer to generate micelles with guanidinium functional groups on the surface. To simulate the actual oligoarginine peptide, which only carries cationic charges, the carboxylate group of P(M-Arg) was methylated to convert the zwitterionic polymer into a cationic polymer P(Me-M-Arg). For comparison, micelles based on triblock copolymers with a third block with permanently cationic charges, poly(2-methacryolyloxy ethyl) trimethyl ammonium chloride (PTMA), was prepared. The hydrodynamic diameters of the micelles were approximately 30−40 nm based on DLS and TEM. A direct correlation between surface charge (zeta potential ζ) and cytotoxicity was observed. The micelles based on the zwitterionic P(M-Arg) were nontoxic (ζ = −10 mV at pH = 7), while the methylated version P(MeM-Arg) with a high cationic charge (ζ = +35 mV at pH = 7) were observed to be toxic. The cellular uptake of the block copolymers by OVCAR-3 ovarian cancer cell lines was found to be relatively fast (about 35% in 3 min) reaching an equilibrium after approximately 30 min. Both micelles, with either P(M-Arg) or P(Me-M-Arg) on the surface, showed an enhanced uptake compared to micelles with P(PEGMEMA) as shell only. In fact, the percentage of uptake was similar, with the difference that cells incubated with micelles with P(M-Arg) (zwitterionic) stayed alive, while P(Me-M-Arg) (cationic) led to significant cell death.

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INTRODUCTION Micelles are widely proposed for the controlled delivery of drug due to their core−shell structure and their nanoscopic size.1−7 Depending on the nature of the block copolymer and the environmental conditions, the amphiphilic copolymer can selfassemble into nanoparticles with different morphologies such as micelles and vesicles, where the hydrophobic block forms the core and the hydrophilic block creates the corona of the nanoparticle.1,8−10 The drugs can safely be protected within the hydrophobic core while the hydrophilic shell can alter the pharmacokinetics and biodistribution of the incorporated drug through the interaction with the biological environment.11 The delivery of drug-loaded micelles into the cell has always been a topic of interest7 and the enhancement of cell uptake was found to translate directly to enhanced drug toxicity.12 The ability of several peptides, called cell-penetrating peptides (CPPs) or protein transduction domains (PTDs), to translocate across the cell membrane into the cytoplasm and nucleus in an energy independent13 or receptor-independent manner14 has been described vividly in literature. Examples are the protein antennapedia, which contains penetratin, a short © 2012 American Chemical Society

signal sequence based peptides, and Tat peptides, which have enhanced the transport of a broad spectrum of cargo, ranging from proteins, magnetic nanoparticles and even DNA.15,16 Also, it has been reported that the Tat peptide containing chelates of technetium-99m and rhenium have been efficiently delivered to the cytoplasm and nucleus in human living cells. 17 Furthermore, Tat peptides have successfully delivered 2-Omethyl phosphorothioate antisense oligonucleotides leading to an increase in pharmacological activity without compromising the specificity of the antisense oligonucleotides.18 The improvement of cell uptake of these peptides has been reported to be dependent on the presence of arginine, an amino acid, which contains positively charged guanidinium functional groups, and not on the secondary structure.19,20 For example, Wender et al. noted that the cell penetrating properties of a Darginine oligomer resulted in more than a 100-fold increase in the rate of cellular uptake compared to the Tat peptides.21 Even though the precise mechanism of the cell uptake pathway is still Received: August 28, 2012 Published: September 4, 2012 3418

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Scheme 1. Synthesis of Triblock Copolymers via the RAFT Processa

(1) Chain extension of TMA in DMSO with AIBN at 70 °C. (2) Chain extension of Arg monomer in DMSO with AIBN at 70 °C. (3) Methylation of triblock copolymer with TMS·CH2N2 in MeOH at room temperature for 24 h.

a

activated.29 The reader is referred to a range of recent excellent review articles on this subject.30−32 Even though the uptake pathways of CPP are still under investigation, these peptides are undoubtedly beneficial for drug delivery purposes. However, there are only a few but highly promising examples of micellar systems conjugated with CPP.33 Although many peptides are easily available, we are aiming at exploring synthetic alternatives, which are probably commercially more viable. A range of studies have shown that polymers with guanidine functionalities can indeed enhance cellular uptake. Examples of guanidine-containing polymers include polymers prepared by Tew and co-workers,34 McCormick and coworkers,35 or Harth and co-workers.36 This approach holds great promise, although the potential toxicity of these cationic groups needs to be considered. In this work, we are aiming at preparing a range of block copolymer micelles using three different types of cationic oligomers as potential cell penetrating moieties. Among them,

under debate, it has been reported that Tat and poly(arginine) oligopeptides might enter the cell via an endocytic independent pathway.19−21 Nori et al. conjugated a Tat peptide with polymers that was subsequently internalized into ovarian mammalian cancer cells through both endocytic and nonendocytic pathways.22,23 Other research groups questioned the spontaneous passage of arginine-rich peptides over the plasma membrane.24,25 In 2003, however, Lundberg et al. showed that the assumption that CPP enter the cells via an energyindependent was based on experimental artifacts.26 An excellent recent summary on the mechanism of uptake has been published by Dowdy and co-workers.27 It has been highlighted that CPPs enter the cells mainly via macropinocytosis.28 However, there is still a small fraction of CPPs that can enter the cells via penetration. This seems particularly the case when a hydrophobic compound was conjugated to the peptide. Interestingly, this does not seem to affect the integrity of the cell membrane and a repair mechanism is immediately 3419

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Table 1. Triblock Copolymer Synthesis Using PMMA-b-P(PEGMEMA) MacroRAFT Agenta polymer 1 2 3 4 5 6

PMMA50-b-P(PEGMEMA)73 PMMA-b-P(PEGMEMA)-b-PTMAx PMMA-b-P(PEGMEMA)-b-PTMAx PMMA-b-P(PEGMEMA)-b-P(M-Arg)x PMMA-b-P(PEGMEMA)-b-P(M-Arg)x PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg)x

[M]/[macroRAFT]

conversion (%)

20:1 50:1 20:1 50:1 obtained by methylation

50 60 55 76

Nx

Mn,SEC (g mol−1)

PDI

10 30 11 38 11

29000 36700 36900 37400 38000 40000

1.16 1.1 1.11 1.23 1.2 1.07

a Repeating units of the third block in the final polymer from NMR and molecular weight and PDI results obtained from SEC; [macroRAFT]/ [AIBN] = 1:0.2 in DMSO at 70 °C for 24 h.

copolymer -CH2−CH2 peak (δ = 0.8−1.2 ppm). The composition of the block copolymer was calculated to be PMMA 50 -b-P(PEGMEMA)73 (Mn,theo = 27000 g mol−1). Synthesis of Methacrylate-Based Monomers with Guanidine in the Functional Group. M-Arg (Arginine Methacrylate). Arginine methacrylate monomer was prepared according a procedure used for the acetylation of arginine, although with some modifications.37 LArginine hydrochloride (21.0 g, 0.0997 mol) and sodium bicarbonate (16.8 g, 0.2 mol) was added to 100 mL of water. The solution was cooled to 5 °C, 12.5 mL of methacrylic anhydride (0.11 mol) was added dropwise over a period of 10 min, and the mixture was stirred for 20 min. A few drops of concentrated ammonia−water were added to adjust the pH to 8. The solution was filtered and washed three times with dichloromethane. The sample was freeze-dried and stored at −7 °C 1 H NMR (300.17 MHz, CDCl3, 25 °C): δ (ppm) = 9.22 (t, 1H, CH2COOH), 7.73 (t, 1H, CH2NH2CNH), 5.57 (s, 1H, CCH), 5.00 (s, 1H, CCH), 3.90 (m, 1H, NHCH), 1.87 (s, 3H, CH3), 1.32 (m, 4H, NHCH2CH2). Synthesis of Triblock Copolymers via the RAFT Process. PMMA-bP(PEGMEMA)-b-P(M-Arg). The block copolymer synthesized previously has been used as macroRAFT agent and chain extended with the arginine methacrylate M-Arg monomer. Monomer (1.9 × 10−2 g, 1.39 × 10−4 mol or 4.8 × 10−2 g, 3.485 × 10−4 mol) was mixed with the block copolymer (macroRAFT agent; 1.0 × 10−1 g, 6.97 × 10−6 mol) and AIBN (1.2 × 10−4 g, 1.4 × 10−6 mol) in 5 mL of DMSO. The solution was degassed with nitrogen for 30 min and the polymerization was carried out at 70 °C for 24 h. The polymer was purified by dialysis against distilled water with 14000 MWCO membranes over 2 days. Polymers were freeze-dried and stored at −7 °C. The results are summarized in Table 1. PMMA-b-P(PEGMEMA)-b-PTMA. (2-Methacryolyloxy ethyl) trimethyl ammonium chloride (TMA; 1.7 × 10−2 g, 6.68 × 10−5 mol and 3.5 × 10−2 g, 1.67 × 10−4 mol) were mixed with the block copolymer (macroRAFT agent; 1.0 g, 3.342 × 10−6 mol) and AIBN (1.1 × 10−4 g, 6.68 × 10−7 mol) in 5 mL of DMSO. The samples were degassed for 30 min with nitrogen and the polymerization was carried out at 70 °C for 24 h. Polymers were purified by dialysis using 14000 MWCO membranes over 2 days followed by freeze-drying and stored at −7 °C. The results are summarized in Table 1. Methylated PMMA-b-P(PEGMEMA)-b-P(M-Arg). To a solution of the PMMA-b-P(PEGMEMA)-b-P(M-Arg) triblock copolymer (500 mg) in 2 mL of dichloromethane was added an excess of trimethylsilyldiazomethane (10 equiv/arginine unit) and the mixture was stirred for 24 h. The polymer was purified by precipitation in diethyl ether (×2) and dried under vacuum. Self-Assembly of Triblock Copolymers into Micelles. A total of 30 mg of the copolymers synthesized previously was dissolved in 8 mL of DMF. The samples were dialyzed against distilled water for 48 h using membrane (MWCO 3500) dialysis. The water was replaced every 6 h. Cell Culture. Human ovarian cancer cell line, OVCAR-3, was grown in RPMI-1640 [2 × 10−3 M L-glutamine, 1.5 g L−1 sodium bicarbonate, 0.010 M 2- hydroxyethylpiperazinesulfonic acid (HEPES), 4.5 g L−1 glucose, 10−3 M sodium pyruvate] medium supplemented with 10% fetal bovine serum (FBS). The cells were grown in 5% CO2 at 37 °C. In Vitro Cell Proliferation Assay. Sulforhodamine B assay (SRB assay) has been used to study the cell proliferation. In brief, OVCAR-3

two of these oligomers are based on the guanidine carrying amino acid arginine. A triblock copolymer will be prepared using reversible addition/fragmentation chain transfer (RAFT) polymerization based on poly(methyl methacrylate) as the hydrophobic block and poly(oligo ethylene glycol methyl methacrylate) as well as a cationic polymer as the two hydrophilic blocks (Scheme 1). Particularly, the effect of the third block structure on the overall charge of the micelles and on the cellular uptake was the focal point of this study.

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EXPERIMENTAL SECTION

Materials. Toluene (Ajax, 99.4%), N,N-dimethyl acetamide (DMAc; Aldrich, 99.9%), dimethyl sulfoxide (DMSO; Ajax, 98.9%), N,N-dimethyl formamide (DMF; Aldrich), diethyl ether (Univar), petroleum spirit (Aldrich), sodium hydrogen carbonate (NaHCO3; Univar), L-arginine hydrochloride (Aldrich), and trimethylsilyldiazomethane (Aldrich) were used without any further purification. Monomers were destabilized by passing them over a column of basic alumina and stored at −7 °C. 2,2-Azobisisobutyronitrile (AIBN) and 4,4-Azobis(4-cyanovaleric acid) (ACVA) were purified by recrystallization from methanol. The RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB) was prepared according to the procedure described elsewhere. Methods. Synthesis of Poly(methyl methacrylates) PMMA via the RAFT Process. MMA (1.2 g, 4.0 × 10−3 mol), CPADB RAFT agent (3.3 × 10−2 g, 4.0 × 10−5 mol), and initiator AIBN (3.9 × 10−3 g, 8.0 × 10−6 mol) were mixed in toluene (20 mL) in a round-bottom flask. The flask containing the mixture was sealed and degassed with nitrogen gas in an ice bath for 1 h. The flask was immersed in an oil bath at 70 °C. The polymerization was terminated after 24 h by placing the flask in an ice bath for 5 min and introducing air. The polymer was purified three times by precipitating in petroleum spirit and centrifuged at 6000 rpm for 5 min. The solvent was decanted and the polymer was dried under reduced pressure overnight. The conversion was determined by 1H NMR (CDCl3) from the monomer methyl vinylic peak (-CCH2, δ = 5.7−6.0 ppm) and the −O−CH3 peak (δ = 3.8−4.2 ppm). A conversion of 50% has been obtained which it is equivalent to 50 MMA repeating units. Synthesis of Poly(poly(ethylene glycol) methyl ether methacrylates)-block-Poly(methyl methacrylates) PMMA -b- P(PEGMEMA) via the RAFT Process. PMMA with 50 repeating units (Mn,SEC = 5000 g mol−1) was used as a macroRAFT agent for chain extension with PEGMEMA and fluorescein-O-methacrylate. PEGMEMA (2.28 g, 7.6 × 10−3 mol) and fluorescein-O-methacrylate (1.6 × 10−1 g, 4.0 × 10−4 mol) were mixed with PMMA macroRAFT agent (0.40 g, 8.0 × 10−5 mol) and AIBN (2.6 × 10−3 g, 1.6 × 10−5 mol) in 10 mL of toluene. The mixtures were purged with nitrogen for 30 min in an ice bath to avoid the evaporation of solvent and monomers. The polymerization was carried out in an oil bath at 70 °C for 24 h. The reaction was terminated by reducing the temperature in an ice bath for 5 min and by introducing air. Precipitation in anhydrous diethyl ether was performed to remove the unreacted monomer. After decanting the solvent, the polymers were dried under reduced pressure overnight. The conversion was determined by 1H NMR (CDCl3) from the PEGMEMA monomer vinylic peak (-CCH2, δ = 5.7−6.0 ppm), P(PEGMEMA) -CH2−CH2 peak (δ = 3.8−4.2 ppm), and block 3420

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Scheme 2. Synthesis of Methacrylate-Based Monomer from Arginine M-Arg and the Protonation of the Guanidine to the Guanidinium Group at Physiological pH Value

Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a JEOL 1400 transmission electron microscope. The instrument operates at an accelerating voltage of 100 kV. Samples were negative stained with phosphotungstic acid (2% w/w). A Formvarcoated grid was cast by putting it onto the surface of a polymer aqueous solution for 1 min. Excess solution was removed using filter paper. In the staining process, the cast grid was gently put onto the surface of a drop of phosphotungstic acid for 30 s. The stained grid was dried under air. Zeta Potential. The zeta potential of particles was measured by phase analysis light scattering (PALS, Brookhaven BI-90 PALS). The pH of the solutions was adjusted by adding a few drops of hydrochloric acid and sodium hydroxide using a pH meter. Samples were filtered before analysis, and each sample was run five times at 25 °C.

cells were seeded in 96-well plates (3000 cells per well) with culture medium 10% FBS RPMI at 37 °C in 5% CO2 environment for 24 h. The medium was refreshed with 0.2 mL of a solution consisting of 0.1 mL medium and 0.1 mL of micelle solution resulting in final micelle concentrations of 10, 25, 50, 125, and 250 μg mL−1. The cells were incubated at 37 °C for 72 h. Subsequently, the medium was removed and the cells were fixed in 10% (w/v) TCA for 30 min at 4 °C and washed five times with tap water, followed by staining with 0.4% (w/v) SRB dissolved in 1% acetic acid for 20 min, and washed again five times with 1% acetic acid. After drying overnight, 100 μL of 0.010 M Tris (pH = 10.5) solution was added to solubilize the dye. Absorbance was measured at 570 nm using a S960 plate reader (Metertech, Taiwan). Nontreated (no micelle, only media) cells were used as controls. The optical density (OD) was used to calculate cell viability.

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cell viability(%) = (OD570,sample − OD570,blank )

RESULTS AND DISCUSSION Synthesis of Triblock Copolymers via the RAFT Process. RAFT polymerization has been demonstrated to be a suitable method for the synthesis of block copolymers.39−42 It is a robust technique that can be applied to a large range of monomers and allows synthesis of polymers with targeted molecular weight and narrow molecular weight distributions.43 Polymers synthesized via the RAFT process usually carry a thiocarbonylthio end group (Scheme 1). This end group can be cleaved via aminolysis or other means creating a reactive end group for the conjugation with biomolecules such as proteins44 or for other reactive avenues such as thiol−ene reactions.45 Concerns about the potential toxicity of the RAFT end group have been raised, but several studies demonstrated that the toxicity can generally be neglected.46−48 When designing a block copolymer by RAFT, the order of preparation of the blocks determines the final position of the living RAFT end group. In this work, the PMMA macroRAFT agent was prepared first, followed by chain extension with the hydrophilic monomers. In theory, the reverse order would be possible because all building blocks are based on methacrylates, but the low solubility of the cationic polymers can be challenging, especially when attempting to add the hydrophobic PMMA block. Therefore, MMA homopolymer synthesis was performed first using CPADB RAFT agent at 70 °C in toluene. A homopolymer with a theoretical molecular weight of 5000 g mol−1 and a very narrow molecular weight distribution with a polydispersity index (PDI) of 1.1 was obtained. Purification was carried out via precipitation of the polymer in anhydrous diethyl ether. The polymer was subsequently employed as macroRAFT agent for the chain extension with PEGMEMA in toluene at 70 °C. To avoid PEGMEMA homopolymerization, the concentration of the initiator AIBN was maintained below the macroRAFT agent concentrations. The theoretical molecular weight based on the conversion was 26900 g mol−1 and the experimental molecular weight based on SEC was 29000 g mol−1. Deviations between the molecular weights can be assigned to the polystyrene SEC calibrations. However, a very narrow molecular weight distribution with a PDI of 1.16

/(OD570,control − OD570,blank ) × 100 To further study the effect of toxicity and cell uptake of methylated PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) micelles, the triblock copolymer were mixed with the diblock copolymer PMMA-bP(PEGMEMA) at molar ratios of 50:50, 25:75, and 10:90. The mixed polymers were micellized in a similar method described above and were incubated with OVCAR-3 cells at concentration of 100 μg mL−1 at 37 °C for 24 h. Cell viability was measure using SRB method. Cell Uptake via Immunofluorescence and Fluorescence Reader. OVCAR-3 cells were incubated in a 96-well plate (10000 cells well−1) with culture medium 10% FBS RPMI-1640 at 37 °C in 5% CO2 environment for 24 h. The medium was refreshed and 0.2 mL of solution was added to each well, which was prepared from 0.1 mL of 10% FBS RPMI-1640 and 0.1 mL of distilled water fluorescence micelles, at a micellar concentration of 100 μg mL−1. The solutions were subsequently incubated at 37 °C/5% CO2 for different time intervals (3 min up to 3 h). OVCAR-3 cells were washed three times with PBS solution. Cell uptake of the fluorescent micelles was confirmed using confocal fluorescence microscopy set at λex 535 nm and λem 590 nm. Fluorescence intensity of the medium was measured using the fluorescence reader at λex 535 nm and λem 590 nm by comparing the fluorescent intensity of the solution at t = 0 with the fluorescent intensity at different time points.38 Analysis. Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR spectra were recorded using a Bruker 300 MHz spectrometer; samples were analyzed in CDCl3 and DMSO at 25 °C. Size Exclusion Chromatography (SEC). Molecular weight distributions of the block copolymers were determined by size exclusion chromatography (SEC) using a Shimadzu modular system, comprising an autoinjector, a Phenomenex 5.0 μm bead-size guard column (50 × 7.5 mm), followed by three linear 300 × 7.8 mm Phenogel columns (50A, 100A, and 500A, 5 μm particle size) and a differential refractive index detector. The eluent was N,N-dimethylacetamide (DMAc; 0.05% w/v LiBr, 0.05% BHT) at 50 °C with a flow rate of 1 mL min−1. The system was calibrated using narrow polystyrene standards ranging from 500 to 106 g mol−1. The samples were prepared followed by filtration using a filter with a pore size of 0.22 μm, which eliminates higher aggregates Dynamic Light Scattering (DLS). Hydrodynamic diameters were obtained using a Malvern Zetasizer Nano ZS (He−Ne laser 633 nm, max 5mW). Samples were filtered before analyzing, and measured at least three times at 25 °C. 3421

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Self-Assembly of Triblock Copolymers into Micelles. The triblock copolymers were dissolved in DMF and dialyzed against distilled water resulting in the formation of guanidinium- or ammonium-decorated micelles. The micellar systems were investigated using dynamic light scattering (DLS) and transmission electron microscope (TEM; Figure 2) to

suggested that the polymerization had proceeded in a living manner (Table 1). The unique properties of cell-penetrating peptides including guanidinium-rich transporters (GRT) that might improve cellular uptake are well-known.35,49 The aim of this paper is to synthesize polymeric micelles rich in guanidinium cations to mimic these groups. Therefore, a methacrylate-based monomer containing a guanidinium functional group has been synthesized by modification of the amino acid arginine (Scheme 2).37 The modification lead to a monomer with zwitterionic structure in physiological conditions: the guanidine group is protonated, while the carboxylic group is deprotonated (M-arg). To investigate the influence of the overall charge of the micelles on their cytotoxicity and interaction with the cells, the monomer based on arginine (M-Arg) as well as a commercial cationic monomer as control experiment, (2-methacryolyloxy ethyl) trimethylammonium chloride (TMA), were used to chain-extend the block copolymer (PMMA 5 0 -b-P(PEGMEMA)73; Scheme 1). Chain extension using M-Arg and TMA were performed using the same conditions and two different monomer/macroRAFT agent (diblock copolymer) ratios. The polymerizations led to the generation of four triblock copolymers with 10 or 30 repeating units of TMA (PMMA-b-P(PEGMEMA)-b-PTMA) and 11 and 38 blocks of M-arg (PMMA-b-P(PEGMEMA)-b-P(M-Arg)), respectively. All the copolymers showed narrow polydispersity based on the SEC results (Figure 1), which suggests that the polymerization had proceeded in a living manner (Table 1).

Figure 2. Hydrodynamic diameter via DLS of copolymer PMMA-bP(PEGMEMA)-b-P(M-Arg) (top) and PMMA-b-P(PEGMEMA)-bPTMA (bottom) including TEM images (negative staining).

determine shape and size. DLS measurements for all systems showed the formation of micelles with approximate sizes between 20−35 nm (Figure 2). The polydispersity index (PDI) was found to be less than 0.2, which suggests a reasonably narrow micelle size distribution. Factors affecting the size and morphology of self-assembled aggregates include the relative hydrophobic block length, content of water within the solvent mixture, the nature and presence of ions, homopolymers surfactants and different polydispersities.50 Although the molecular weight of the triblock copolymers are approximately the same according to the SEC analysis in DMAc, which is a good solvent for the polymer and therefore leads to individual chain, the resulting micelles in water vary slightly in size (Table 2). This is not surprising because the different cationic charges lead to chain repulsion resulting in chain stretching, thus, an increase in the micelle size. Table 2. Hydrodynamic Diameter Dh and PDI of Different Polymeric Micelles Measured by DLS (Water)

Figure 1. Molecular weight distribution obtained by SEC of diblock copolymer 1 (BI) and triblock copolymers 2−6 (Table 1).

1 2 3 4 5 6

The triblock copolymers based on M-Arg has a zwitterionic structure under physiological conditions. This contrasts the cationic structure of peptides based on arginine where the carboxylate group is involved in the formation of the amide bond. To simulate oligoarginine as control substance, post functionalization was performed to generate a cationic polymer. Methylation of the carboxylic group of the PMMA-bP(PEGMEMA)-b-P(M-Arg) copolymer using trimethylsilyldiazomethane is an established procedure for methylation that allows complete modification within a short period of time (Scheme 1). The modification step was complete within a day without any visible side reactions leading to PMMA-bP(PEGMEMA)-b-P(Me-M-Arg) (Table 1).

polymer

Dh (nm)

PDI

PMMA50-b-P(PEGMEMA)73 PMMA50-b-P(PEGMEMA)73-b-PTMA10 PMMA50-b-P(PEGMEMA)73-b-PTMA30 PMMA50-b-P(PEGMEMA)73-b-P(M-Arg)11 PMMA50-b-P(PEGMEMA)73-b-P(M-Arg)38 PMMA50-b-P(PEGMEMA)73-b-P(Me-M-Arg)11

22 23 25 32 34 35

0.17 0.19 0.23 0.15 0.14 0.17

Zeta Potential Characterization. The cationic nature of molecules or particles are associated with cytotoxic effects.51 For example, cationic lipids containing tertiary or quaternary amines result in high cytotoxicity caused by the interference with critical enzymes such as PKC.52 The quaternary ammonium functionality is more toxic than the tertiary one. The high toxicity can be partly prevailed by delocalizing the positive charge of the cationic group throughout a heterocyclic 3422

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ring. Pyridinium or guanidinium, as the substitution for the ammonium functionality, was found to be significantly less toxic.51 Yingyongnarongkul et al.,53 synthesized guanidinium containing transfection agents among a library of different cationic lipids and demonstrated that these compounds were safer. However, the MTT assay revealed that at high concentrations even these compounds show high cytotoxicity. To evaluate the influence of the third block on the overall charge of the micelles, zeta potential measurements were performed at different pH values (Figure 3). Micelles based on

Figure 4. Cell viability of OVCAR-3 in percent after incubation with different copolymer micelles for 72 h; Bl stands for PMMA-bP(PEGMEMA) block copolymer.

noticeable toxic effect on OVCAR-3 cell lines, especially at higher concentrations. The toxicity of the PTMA based polymers has even been exceeded by the guanidinium based polymer PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg). The high toxicity of these two types of polymers is not surprising considering the cationic charge. It has been repeatedly highlighted that materials containing guanidine are toxic to the point that polymers with guanidine functionalities have been proposed as anticancer agents.54 Aminoguanidine can act as an antioxidant, a nitric oxide synthase inhibitor (NOS) and as an inhibitor of advanced glycosylation end products (AGEs).54 Considering the high toxicity of guanidine, it was surprising to see the zwitterionic triblock copolymer, PMMA-bP(PEGMEMA)-b-P(M-Arg) was found to be nontoxic for the tested concentration range and even slightly enhanced cell growth (Figure 4). It is interesting to note that while PMMA-b-P(PEGMEMA)b-P(M-Arg) is nontoxic, the methylated version PMMA-bP(PEGMEMA)-b-P(Me-M-Arg) displays the highest toxicity. The order of toxicity was found to be in sharp agreement with the measured zeta potential. A higher (more positive) zeta potential translated directly to a lower cell viability (Figures 3 and 4). The high toxicity of PMMA-b-P(PEGMEMA)-b-P(Me-MArg) permitted further cell uptake studies. The triblock copolymer was therefore mixed with PMMA-b-P(PEGMEMA) block copolymers to generate micelles with weight ratios of 10, 25, and 50% of PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) to be able to obtain a relationship between polymer composition, toxicity and later cell uptake. After 24 h of incubation with the OVCAR-3 cell lines the cell viability was significantly reduced although the fraction of surviving cells was sufficient to allow cell uptake studies (Figure 5). Cell Uptake of Micelles. Arginine, in particular its guanidinium functionality, is well-known to enhance the uptake of polymers,35,36 but usually at a price of higher polymer carrier toxicity. The zwitterionic structure was shown to be nontoxic within the frame of the study, but the central question is if this may affect the cellular uptake. To investigate the interaction between the OVCAR-3 cells and the micelles synthesized in this work, micelles were labeled with fluorescein. The chosen polymer concentration was set to 100 μg mL−1, which is well above the critical micelle concentration (CMC) of the polymer because earlier studies

Figure 3. Zeta potential of copolymer micelles in water at different pH values. Bl stands for PMMA-b-P(PGEMEMA) block copolymer. Polymer concentration = 250 μg mL−1.

PMMA-b-P(PEGMEMA) diblock copolymer showed a slight negative charge within the measured pH values. As mentioned previously, PMMA-b-P(PEGMEMA)-b-PTMA triblock copolymer micelles have been synthesized as a control sample to evaluate the toxic effect of a permanently localized positive charge. Within the pH range of interest for drug delivery carriers, such as the pH range of the cell culture medium and the cytoplasm of the cells, the particles are positively charged. As expected, the micelle with the longer PTMA block had a larger positive zeta potential due to the higher number of cationic charges in the polymer. The unusual shape of the curve of PMMA-b-P(PEGMEMA)-b-PTMA30 was reproducible and may be the result of some changes in the polymer chain conformation when the chloride counterion is slowly replaced by the increasing amount of hydroxyl ions. The zeta potential of the PMMA-b-P(PEGMEMA)-b-P(M-Arg) triblock copolymer micelles have been determined using the same procedure. The measured zeta potential was negative indicating that the negatively charged carboxylate more than counterbalanced the positively charged guanidinium group. Moreover, the methylated equivalent, the PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) triblock copolymer micelle, was positively charged throughout the pH range of interest (Figure 3). In Vitro Cell Proliferation Assay. OVCAR-3 cell lines were chosen for the biological evaluation. Initially, the cytotoxicity of the block copolymer and triblock copolymer micelles was tested using sulforhodamine B assay (SRB assay). Briefly, 3000 cells were incubated with polymer concentrations ranging from 250, 125, 50, 25 to 10 μg mL−1 for 72 h at 37 °C. Figure 4 shows the percentage viability of OVCAR-3 cells relative to the control sample. The PMMA-b-P(PEGMEMA) micelles are not cytotoxic within the chosen concentration window similar to earlier studies.38,47,48 In contrast, PMMA-bP(PEGMEMA)-b-PTMA triblock copolymer micelles have a 3423

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Figure 5. Cell viability of OCVAR-3 in percent after incubation of various copolymer micelles at 37 °C for 72 h composed of a mixture of PMMA-b-P(PEGMEMA) and PMMA-b-P(PEGMEMA)-b-P(Me-MArg) blocks with varying ratios between both polymers.

Figure 7. Cellular uptake (in wt%) by OVCAR-3 cells of PMMA-bP(PEGMEMA) and PMMA-b-P(PEGMEMA)-b-P(M-Arg) copolymer micelles monitored over 2 h. Bl stands for PMMA-bP(PEGMEMA) block copolymer.

have shown that disassembly can hamper the cellular uptake of polymers.12,38 Figure 6 displays the fluorescence micrographs of

uptake of micelles.48,55 The uptake percentage of PMMA-bP(PEGMEMA) block copolymers reached a saturation point after an incubation time of 30 min at approximately 43 wt % uptake. Further incubation for one (Figure 8) or two days did

Figure 6. Life OVCAR-3 cells under the light microscope (left) and the confocal fluorescence microscope showing the distribution of green fluorescence PMMA-b-P(PEGMEMA) micelles within the cells (polymer concentration = 100 μg mL−1).

OVCAR-3 cells incubated with green fluorescent micelles. The cell lines were washed three times with PBS buffer solution after different time intervals. The green nanoparticles were clearly visible inside the cells (Figure 6). Quantitative results of the cell uptake were achieved by measuring the fluorescence intensity of the media before and after incubation with the cells (Figure 7). This method is only suitable when the uptake is relatively high compared to the initial amount of micelles and is beyond the error of the method. This approach has been successfully applied in earlier studies and is therefore employed here as well.38,47 However, this method is only suitable when the particles do not adhere to the outer membrane layer and are clearly engulfed into the cell. Complementary confocal fluorescence microscopy analysis is therefore essential to confirm the location of the micelles. Uptake studies of PMMA-b-P(PEGMEMA)-b-PTMA and the pure methylated PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) triblock copolymer micelles were omitted due to their cytotoxic effects. Cell uptake studies of PMMA-b-P(PEGMEMA) micelles and PMMA-b-P(PEGMEMA)-b-P(M-Arg) micelles were performed over a short period of time (0−180 min) and up to 2 days. The initial cell uptake of block copolymers was very fast, reaching about 35% in 3 min, which is typical for the

Figure 8. Cellular uptake (in wt%) of PMMA-b-P(PEGMEMA)-bP(M-Arg) micelles and micelles prepared from a mixture of PMMA-bP(PEGMEMA) and PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) after 1 day. Bl stands for PMMA-b-P(PEGMEMA) block copolymer.

not increase the amount. The uptake of the zwitterionic PMMA-b-P(PEGMEMA)-b-P(M-Arg) triblock copolymer micelles was not significantly different from the diblock copolymer up to an incubation time of 30 min. However, the particles decorated with guanidinium groups showed an increase of uptake until 180 min of incubation without reaching the saturation point. An extended period of time did not increase the uptake significantly and the percentage of uptake settled between 60 and 70%. It should be pointed out that the uptake of the micelles with its PEG chains on the surface is not surprising and commonly observed.4 In fact, the discussed type 3424

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of micelles here, PMMA-b-P(PEGMEMA), is efficiently been taken up by endocytosis.56 CPPs are therefore not responsible for uptake, but they can potentially enhance the amount that can be accumulated in the cell. The zwitterionic shell obviously enhanced the uptake of micelles compared to the micelle with a PEG shell only. This result needs to be seen in the context that the zeta potential measurements (Figure 3) exposed an overall negative charge of the micelles with the zwitterionic structure. It has always been argued that it is the cationic charge that enhances interaction with the cell membrane. To put the uptake results of the zwitterionic PMMA-b-P(PEGMEMA)-b-P(M-Arg) micelles into the right context, the cellular uptake of methylated PMMA-b-P(PEGMEMA)-b-(Me-M-PArg) micelles were studied because their overall positive charge (Figure 3) should enhance this process. Due to the high toxicity of this polymer, it had to be mixed with P(PEGMEMA)-b-PMMA at mole ratios of 10, 25, and 50% to lower the overall toxicity (Figure 5). After 24 h of incubation with 100 μg mL−1 of the mixed micelles, the uptake of the micelles was improved compared to the diblock copolymer micelles alone (Figure 8). Increasing amount of PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) in the mixed micelle led to higher uptake. Extrapolation of these values to 100% PMMA-b-P(PEGMEMA)-b-P(Me-M-Arg) would result in a value of around 60−70%, which is in the same order of magnitude to the nontoxic, zwitterionic PMMA-b-P(PEGMEMA)-b-P(M-Arg) micelle (Figure 8). This may indicate that it is not the charge of the polymer that influences the uptake, but the presence of the guanidinium functionality alone.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors like to thank Yoon Ji Kim for help with the experiments. M.S. thanks the ARC (Australian Research Council) for funding in form of a Future Fellowship (FT0991273). M.S. thanks the ARC for financial support for this project (DP110102409). The authors would like to thank the Centre for Advanced Macromolecular Design (CAMD) and UNSW Mark Wainwright Analytical Centre for support.

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REFERENCES

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CONCLUSIONS The aim of this work was to design guanidine-decorated micelles for drug delivery to might improve the cell uptake. Guanidine-base monomers have been successfully obtained by modification of the amino acid arginine leading to a monomer with zwitterionic structure with an negatively charged carboxylic acid group and a positive charged guanidinium functionality. Chain-extension using RAFT polymerization of a diblock copolymer PMMA-b-P(PEGMEMA) led to triblock copolymers PMMA-b-P(PEGMEMA)-b-P(M-Arg) with the third block carrying guanidinium groups. The zwitterionic structure was subsequently converted to a cationic structure by esterification of the carboxylate resulting in PMMA-bP(PEGMEMA)-b-P(Me-M-Arg). The zeta potential of the micelle with zwitterionic structure was slightly negative. In contrast, micelles with only guanidinium carried a positive overall charge. Cell viability studies found that the zeta potential was in direct correlation with toxicity. While the zwitterionic structure was nontoxic, the cationic version after methylation was highly cytotoxic against OVCAR-3 ovarian cancer cell lines. The initial cellular uptake of micelles was found to be fast (∼35% in 3 min). The long-term cell uptake for the block copolymer reached an equilibrium state at around 45%, whereas the uptake of the guanidinium-rich zwitterionic micelles kept increasing to 60−70%. Comparison of the uptake of the zwitterionic micelle with the methylated micelle PMMAb-P(PEGMEMA)-b-P(Me-M-Arg) shows no significant differences. It seems, therefore, that not the overall charge of the micelle is responsible for the uptake but the presence of guanidinium groups alone. Using the zwitterionic structure led to a nontoxic drug carrier system whose performance is comparable to the cationic, but toxic, drug carriers. 3425

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