Poly(acrylic acid)-Grafted Graphene Oxide as an Intracellular Protein

Article pubs.acs.org/Langmuir

Poly(acrylic acid)-Grafted Graphene Oxide as an Intracellular Protein Carrier Thangavelu Kavitha, Inn-Kyu Kang, and Soo-Young Park* Department of Polymer Science and Engineering, Kyungpook National University, Daegu 702-701, Republic of Korea ABSTRACT: A pH-sensitive poly(acrylic acid)-grafted graphene oxide (GOPAA) nanocarrier was synthesized by in situ atom transfer radical polymerization to allow the oral delivery of hydrophilic macromolecular proteins in their active forms to specific cells or organs. The synthesis, morphology, and physiochemical properties of GO-PAA were examined. A model protein, bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) (BSAFITC), was loaded onto GO-PAA through noncovalent interactions and its release was arrested at acidic pH similar to stomach, whereas at pH similar to intestine it was reduced, which paves way for site specific delivery without its degradation in the gastrointestinal tract. Confocal laser microscopy showed that the BSAFITC-loaded GO-PAA was internalized by KB cells by endocytosis and released into cytoplasm. Thus the GO-PAA as a transmembrane transporter is a new class of drug transporters with potential protein delivery applications.

■

many biomedical areas.20 In terms of its therapeutic use, PAA has attracted considerable interest because of its ability to swell reversibly with changes in pH. Its carboxylic acid groups ionize at pH values above the pKa of 4.7.21,22 Therefore, the impregnation of PAA chains onto graphene provides a means of producing pH-responsive materials. This paper reports the synthesis of GO functionalized with PAA (GO-PAA) by in situ atom transfer radical polymerization (ATRP), characterization of GO-PAA, and its potential use as an intracellular protein carrier. Using bovine serum albumin (BSA) as a model protein, the protein loading and release characteristics of the BSA loaded GO-PAA was examined at different pH and their mechanisms were investigated.

INTRODUCTION Proteins, the so-called machines of life, participate in all vital body processes. They perform the essential functions inside cells as enzymes, transduction signals, and gene regulation. They also maintain the balance between cell survival and apoptosis and have enormous potential in biomedical applications, such as cancer therapy,1 vaccination,2 tissue engineering,3 embryonic stem cell regulation,4 regenerative medicine, treating loss-of-function genetic diseases, and imaging.1 Changes in the functions of the intracellular proteins can result in many diseases.5,6 Therefore, the intracellular delivery of active proteins to specific cells and organs is important for maintaining a highly specific set of cell functions as well as avoiding adverse effects and immune responses.6,7 On the other hand, the delivery of hydrophilic macromolecules, such as peptides and proteins, is problematic due to difficulties, such as denaturation in the stomach, degradation by different digestive enzymes, poor cellular uptake, and limited transport into the tissues.8,9 Therefore, a carrier system is needed to overcome these issues. A variety of nanomaterials, such as liposomes,10,11 polymers,12 carbon nanotubes,13 quantum dots, silica, gold, magnetic nanoparticles, and polypeptides,1,14,15 have been tailored for intracellular protein delivery with some success. On the other hand, the carrier loading capacity and the chemical and biological stabilities of the proteins are significant challenges. Recently, graphene and its derivatives have attracted considerable interest because of its biocompatibility, unique conjugated structure, large specific surface area, intrinsic optical properties, low cost, and its noncovalent interactions with small aromatic molecules.16−18 Initially, polyethylene glycol19 was grafted onto graphene oxide (GO) to impart aqueous solubility and stability under physiological conditions. Polyacrylic acid (PAA) is a pH-sensitive, biocompatible polymer that is used in © XXXX American Chemical Society

■

MATERIALS AND METHODS

Materials. Graphite with a mean particle size of 25 μm was obtained from Samjung C & G Inc. (S. Korea). Tert-butyl acrylate (tBA, Aldrich, 98%) was purified by passing it over alumina and stored at 4 °C. Copper(I) bromide (CuBr, Sigma Aldrich, 98%) was purified by mixing with glacial acetic acid, washing with methanol, and drying in a vacuum oven. N,N,N,N,N-pentamethyldiethylenetriamine (PMDETA, Sigma Aldrich, 99%) was dried using a 4 Å molecular sieve. Trifluoro acetic acid, 1,3-diaminopropane, N-hydroxysuccinimide (NHS), and N-(3-(dimethylamino) propyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl), 2-bromo-2-methylpropionyl bromide (98%), fluorescein isothiocyanate-labeled bovine serum albumin (BSAFITC), high glucose Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS), (3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%), trypsin, and all other reagents mentioned in this article were purchased from Sigma Aldrich and used as received. Received: November 9, 2013 Revised: December 17, 2013

A

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 1. Synthesis of GO-PAA. Synthesis of GO. GO was synthesized using a modification of Hummer’s method.23 Briefly, dried graphite powder (4 g) was mixed with NaNO3 (2 g) and H2SO4 (92 mL) at 400 °C of GO-PAA and GO-I suggests that the amount of PAA grafted on GO was approximately 35 wt % of GO-PAA. Figure 5a shows the Raman spectra of the GO, GO-I, and GO-PAA powder samples at the laser excitation wavelength of 532 nm. In all the samples, two prominent D and G bands that reflect the amount of disorder and the relative degree of

Figure 6. GO-PAA particle size and surface charge as a function of pH.

hydrodynamic size of GO-PAA increased continuously with increasing pH because of the increased charge carried by PAA, and the zeta potential of GO-PAA decreased with increasing pH and became negative when the pH was higher than the pKa of PAA (∼4.7).30 These results suggest that at low pH, the protonation (positive charge) of carboxyl groups of PAA caused chain shrinkage and a decrease in particle size, whereas at high pH, deprotonation (ionization, negative charge) led to chain stretching due to electrostatic repulsive interactions between the chains.31 AFM images and height profiles of GO and GO-PAA (Figure 7) showed that GO existed as platelets with a lateral dimension of micrometer range and thickness of ∼1−2 nm. The thickness of GO-PAA (Figure 7d) increased to ∼7 nm and the lateral dimension decreased to ∼200 nm. The increased thickness of GO-PAA indicated the successful grafting of PAA onto the surface. The decrease in lateral dimensions of GO-PAA platelets might have been caused by sonication during their

Figure 5. (a) Raman and (b) XPS survey spectra of GO, GO-I, and GO-PAA. D

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 7. (a,b) Tapping mode AFM images and (c,d) height profiles of (a,c) GO and (b,d) GO-PAA, respectively.

between BSA and GO.32 This fluorescence quenching revealed a strong interaction between BSAFITC and GO-PAA. The loading ratio of BSAFITC onto GO-PAA was determined by UV−vis spectroscopy. Figure 7c shows the BSAFITC loading on GO-PAA as a function of the BSAFITC concentration (C0). The amounts of BSAFITC loaded increased with increasing C0, reaching saturation at 95% for C0 values > 1500 μg/mL. Interestingly, this saturation level was much higher than the 64−75% of other nanocarrier, such as liposomes.11 The driving force for the high payload of proteins is the stronger hydrophobic and electrostatic interactions between the GO surfaces and proteins. To examine the feasibility of GO-PAA as a hydrophilic drug carrier, the release of BSAFITC from GO-PAA-BSAFITC was examined at 37 °C in different buffer solutions. Figure 9 shows the protein retained on GO-PAA at a pH of 3.0, 7.4, and 8.0. At pH 3, which is similar to pH in a fasting stomach, no release of protein was observed even after 3 days, whereas at pH 7.4, which is similar to the pH of the body fluids in intercellular spaces, ∼ 20% of the loaded protein was released in 3 days. On the other hand, at pH 8 (intestinal pH), ∼79% of the loaded proteins was released within 3 days. The mechanism of this release at molecular level was the change of the PAA chain conformation. PAA is a weak anionic polyelectrolyte whose charge is dependent on pH. At lower pH, the PAA chains are protonated to be shrunk and at high pH, the PAA chains are

synthesis or ATRP conditions.19 The size of GO studied by DLS was reduced from 1.5 ± 0.7 (μm) to 153 ± 49 (nm) by sonication for 24 h which confirms the sonication effect. Whatever the reason, small GO platelets might be better as a drug carrier. In addition, the blunt edge of the GO-PAA compared to the sharp edges of the GO sheets suggested polymer wrapping and folding on the GO sheets.32 UV−vis and PL spectroscopy of GO-PAA-BSAFITC was performed to confirm the loading, as shown in Figure 8. The UV−vis spectrum of BSAFITC (Figure 8a, green) before loading revealed two absorption peaks at ∼276 and ∼490 nm, which are characteristic of BSA and FITC, respectively, and the UV− vis spectrum of GO-PAA showed a continuous decrease in intensity with increasing wavelength without particular peaks. The spectrum of GO-PAA-BSAFITC (Figure 8a, blue) exhibited the same peaks as BSAFITC but at a reduced intensity on the superimposed spectrum of GO-PAA (Figure 8a, red). Furthermore, the FITC peak at 486.5 nm of BSAFITC was red shifted slightly to 491.2 nm in GO-PAA-BSAFITC, indicating successful loading of the dye-tagged protein onto the GO-PAA nanocarrier. Figure 8b shows the PL spectra of BSAFITC before and after loading onto GO-PAA. The quenching of the FITC fluorescence of GO-PAA-BSAFITC compared to free BSAFITC was attributed to efficient fluorescence resonance energy transfer between GO and FITC through π−π between FITC of BSA and GO and hydrophobic and electrostatic interactions E

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Figure 8. (a) UV−vis and (b) PL spectra of BSAFITC before and after loading on GO-PAA, and (c) loading of BSAFITC onto GO-PAA, as a function of the BSAFITC concentration.

prevent protein release. At the intestines pH (pH 8), negative repulsive forces due to deprotonated COO− groups causes chain stretching which paves the way for protein to come out of the carrier. Therefore, the devised GO-PAA might act as a nanocarrier for hydrophilic drugs, prevent denaturation in the upper gastrointestinal tract, and allow release in the intestine. The stability of the proteins against enzyme degradation is an important factor of successful protein-based therapy. The stability of BSA loaded on GO-PAA compared to that of native BSA at the same concentration was examined by SDS-PAGE (Figure 9). SDS-PAGE revealed a band of the undigested BSA and the intensity of the band exhibited its amount. As depicted in the Figure 10, a distinct band of the BSA band for GO-PAABSAFITC as that of the control was observed. On the other hand, free BSAFITC was digested almost completely by trypsin even after 1 h, and digestion by trypsin was complete after 6 h, as evidenced by the complete disappearance of the BSA band in the BSAFITC samples. The detailed mechanism of the protection afforded by GO-PAA is unclear,33 even though steric hindrance by grafted PAA is a possibility. In addition, zeta potential measurements of BSAFITC and GO-PAA-BSAFITC at pH 3.0, 7.4, and 8.0 (Table 1) revealed BSAFITC to have similar zeta potentials to those of GO-PAA-BSAFITC at all measured pH, suggesting that the characteristics of the loaded protein were

Figure 9. Release of BSAFITC from GO-PAA at pH 3, 7.4, and 8 at 37 °C.

deprotonated to be swelled by electrostatic repulsions between them. For example, the −COOH groups of PAA chains are protonated at pH 3 (similar pH in the gastrointestinal tract) resulting in chain contraction and thus acting as a gate to F

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

showed little fluorescence. GO-PAA initially transported BSA to the endosomes and then to the cytoplasm, and the transported BSA was finally diffused through the cells. The internalization pathway of GO-PAA-BSAFITC is energy-dependent endocytosis, which is a well-known mechanism.13 To confirm the endocytosis mechanism for the internalization GOPAA-BSAFITC the KB cells were incubated with GO-PAABSAFITC for 24 h at 4 °C at which the cell activity was reduced sufficiently to prevent endocytosis. Figure 11a,c shows CFM images of the uptake of GO-PAA-BSAFITC by the KB cells after incubation for 24 h at 37 or 4 °C. The cells incubated at 4 °C exhibited lower GO-PAA-BSAFITC uptake, showing that endocytosis is the main uptake mechanism. Biocompatibility is the most important requisite of any new material intended for introduction into a living system. The biocompatibility of GO-PAA was assessed using a MTT assay in which fibroblast cells were treated with GO-PAA. Figure 11d shows the relative cell viabilities with and without GO-PAA as a function of the incubation time. The relative cell viability with and without GO-PAA were almost similar over a 5 day period even at a high concentration of 100 mg/L. These results suggest that fibroblast proliferation and viability is a little affected by the internalization of GO-PAA.

Figure 10. SDS-PAGE analysis of BSAFITC with or without (control) trypsin and of GO-PAA-BSAFITC treated with trypsin for 1, 3, or 6 h.

Table 1. Zeta Potentials of Free BSAFITC and GO-PAABSAFITC at Various pH pH

BSAFITC (mV)

GO-PAA-BSAFITC (mV)

3.0 5.0 7.4 8.0

+13.5 −10.3 −17 −31.3

+15.7 −12.5 −21.3 −27.6

similar to that of the native protein. These results also confirmed that the GO-PAA protected the adsorbed proteins from enzymolysis and retained the biological activity of the protein delivered. The prominence of GO-PAA as a transmembrane nanovector dwells in competent cellular internalization, which forms the basis of drug delivery. The uptake of GO-PAA-BSAFITC and BSAFITC by the KB cells after incubation for 24 h at 37 °C was examined by CFM (Figure 11). The cells incubated with GOPAA-BSAFITC for 24 h at 37 °C (Figure 11a) exhibited green fluorescence, whereas the cells treated with free BSAFITC

■

CONCLUSIONS The GO-PAA synthesized by in situ ATRP was biocompatible with the cells, hosted protein drugs, protected the proteins from the harsh gastric environment, and released the drugs under physiological conditions. The proteins loaded on GO-PAA were protected from enzymatic hydrolysis. The intracellular uptake of the GO-PAA-BSAFITC occurred via an energydependent endocytosis pathway. Therefore, this pH-responsive

Figure 11. CFM images of the uptakes of (a,c) GO-PAA-BSAFITC and (b) BSAFITC by KB cells after incubation for 24 h at (a,b) 37 and (c) 4 °C, and (d) relative cell viabilities in the absence (control) and presence of 10 and 100 mg/L GO-PAA in PBS buffer as determined by MTT. G

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Robust Free-Standing Paper Composed of a TWEEN/Graphene Composite. Adv. Mater. 2010, 22 (15), 1736−1740. (17) Wang, Y.; Li, Z. H.; Hu, D. H.; Lin, C. T.; Li, J. H.; Lin, Y. H. Aptamer/Graphene Oxide Nanocomplex for in Situ Molecular Probing in Living Cells. J. Am. Chem. Soc. 2010, 132 (27), 9274−9276. (18) Zhang, Y. B.; Ali, S. F.; Dervishi, E.; Xu, Y.; Li, Z. R.; Casciano, D.; Biris, A. S. Cytotoxicity Effects of Graphene and Single-Wall Carbon Nanotubes in Neural Phaeochromocytoma-Derived PC12 Cells. ACS Nano 2010, 4 (6), 3181−3186. (19) Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J. PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. J. Am. Chem. Soc. 2008, 130 (33), 10876−10877. (20) Zhao, Y.; Kang, J.; Tan, T. W. Salt-, pH- and temperatureresponsive semi-interpenetrating polymer network hydrogel based on poly(aspartic acid) and poly(acrylic acid). Polymer 2006, 47 (22), 7702−7710. (21) Lee, J. W.; Kim, S. Y.; Kim, S. S.; Lee, Y. M.; Lee, K. H.; Kim, S. J. Synthesis and characteristics of interpenetrating polymer network hydrogel composed of chitosan and poly(acrylic acid). J. Appl. Polym. Sci. 1999, 73 (1), 113−120. (22) Alarcon, C. D. H.; Pennadam, S.; Alexander, C. Stimuli responsive polymers for biomedical applications. Chem. Soc. Rev. 2005, 34 (3), 276−285. (23) Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339−1339. (24) Yang, Y. F.; Wang, J.; Zhang, J.; Liu, J. C.; Yang, X. L.; Zhao, H. Y. Exfoliated Graphite Oxide Decorated by PDMAEMA Chains and Polymer Particles. Langmuir 2009, 25 (19), 11808−11814. (25) Bao, H. Q.; Pan, Y. Z.; Ping, Y.; Sahoo, N. G.; Wu, T. F.; Li, L.; Li, J.; Gan, L. H. Chitosan-Functionalized Graphene Oxide as a Nanocarrier for Drug and Gene Delivery. Small 2011, 7 (11), 1569− 1578. (26) Kavitha, T.; Abdi, S. I. H.; Park, S.-Y. pH-Sensitive nanocargo based on smart polymer functionalized graphene oxide for site-specific drug delivery. Phys. Chem. Chem. Phys. 2013, 15 (14), 5176−5185. (27) Jung, I.; Dikin, D.; Park, S.; Cai, W.; Mielke, S. L.; Ruoff, R. S. Effect of Water Vapor on Electrical Properties of Individual Reduced Graphene Oxide Sheets. J. Phys. Chem. C 2008, 112 (51), 20264− 20268. (28) Stankovich, S.; Dikin, D. A.; Piner, R. D.; Kohlhaas, K. A.; Kleinhammes, A.; Jia, Y.; Wu, Y.; Nguyen, S. T.; Ruoff, R. S. Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carbon 2007, 45 (7), 1558−1565. (29) Liu, J. Q.; Tao, L.; Yang, W. R.; Li, D.; Boyer, C.; Wuhrer, R.; Braet, F.; Davis, T. P. Synthesis, Characterization, and Multilayer Assembly of pH Sensitive Graphene-Polymer Nanocomposites. Langmuir 2010, 26 (12), 10068−10075. (30) Hu, Y.; Chen, Y.; Chen, Q.; Zhang, L. Y.; Jiang, X. Q.; Yang, C. Z. Synthesis and stimuli-responsive properties of chitosan/poly (acrylic acid) hollow nanospheres. Polymer 2005, 46 (26), 12703− 12710. (31) Hu, Y.; Jiang, X. Q.; Ding, Y.; Ge, H. X.; Yuan, Y. Y.; Yang, C. Z. Synthesis and characterization of chitosan-poly(acrylic acid) nanoparticles. Biomaterials 2002, 23 (15), 3193−3201. (32) Selvin, P. R. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 2000, 7 (9), 730−734. (33) Shen, H.; Liu, M.; He, H. X.; Zhang, L. M.; Huang, J.; Chong, Y.; Dai, J. W.; Zhang, Z. J. PEGylated Graphene Oxide-Mediated Protein Delivery for Cell Function Regulation. ACS Appl. Mater. Interfaces 2012, 4 (11), 6317−6323.

nanocargo is a suitable carrier for protein delivery. Moreover, this advanced establishment highlights its potential for many treatments as well as applications to metabolic manipulation.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Tel: +82-53-950-5630. Fax: +82-53950-6623. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF-2011-0020264). REFERENCES

(1) Gu, Z.; Biswas, A.; Zhao, M. X.; Tang, Y. Tailoring nanocarriers for intracellular protein delivery. Chem. Soc. Rev. 2011, 40 (7), 3638− 3655. (2) Shi, L.; Sings, H. L.; Bryan, J. T.; Wang, B.; Wang, Y.; Mach, H.; Kosinski, M.; Washabaugh, M. W.; Sitrin, R.; Barr, E. GARDASIL (R): Prophylactic human papillomavirus vaccine development - From bench top to bed-side. Clin. Pharmacol. Ther. 2007, 81 (2), 259−264. (3) Baldwin, S. P.; Saltzman, W. M. Materials for protein delivery in tissue engineering. Adv. Drug Delivery Rev. 1998, 33 (1−2), 71−86. (4) Kim, D.; Kim, C. H.; Moon, J. I.; Chung, Y. G.; Chang, M. Y.; Han, B. S.; Ko, S.; Yang, E.; Cha, K. Y.; Lanza, R.; Kim, K. S. Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins. Cell Stem Cell 2009, 4 (6), 472− 476. (5) Walsh, G. Biopharmaceutical benchmarks 2010. Nat. Biotechnol. 2010, 28 (9), 917−924. (6) Leader, B.; Baca, Q. J.; Golan, D. E. Protein therapeutics: A summary and pharmacological classification. Nat. Rev. Drug Discovery 2008, 7 (1), 21−39. (7) Andrady, C.; Sharma, S. K.; Chester, K. A. Antibody-enzyme fusion proteins for cancer therapy. Immunotherapy 2011, 3 (2), 193− 211. (8) Khafagy, E. S.; Morishita, M.; Onuki, Y.; Takayama, K. Current challenges in non-invasive insulin delivery systems: A comparative review. Adv. Drug Delivery Rev. 2007, 59 (15), 1521−1546. (9) Sonaje, K.; Lin, K. J.; Wang, J. J.; Mi, F. L.; Chen, C. T.; Juang, J. H.; Sung, H. W. Self-Assembled pH-Sensitive Nanoparticles: A Platform for Oral Delivery of Protein Drugs. Adv. Funct. Mater. 2010, 20 (21), 3695−3700. (10) Park, S. J.; Choi, S. G.; Davaa, E.; Park, J. S. Encapsulation enhancement and stabilization of insulin in cationic liposomes. Int. J. Pharm. 2011, 415 (1−2), 267−272. (11) Kim, S. K.; Foote, M. B.; Huang, L. The targeted intracellular delivery of cytochrome C protein to tumors using lipid-apolipoprotein nanoparticles. Biomaterials 2012, 33 (15), 3959−3966. (12) Verheyen, E.; van der Wal, S.; Deschout, H.; Braeckmans, K.; de Smedt, S.; Barendregt, A.; Hennink, W. E.; van Nostrum, C. F. Protein macromonomers containing reduction-sensitive linkers for covalent immobilization and glutathione triggered release from dextran hydrogels. J. Controlled Release 2011, 156 (3), 329−336. (13) Kam, N. W. S.; Dai, H. J. Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc. 2005, 127 (16), 6021−6026. (14) Bale, S. S.; Kwon, S. J.; Shah, D. A.; Banerjee, A.; Dordick, J. S.; Kane, R. S. Nanoparticle-Mediated Cytoplasmic Delivery of Proteins To Target Cellular Machinery. ACS Nano 2010, 4 (3), 1493−1500. (15) Slowing, I. I.; Trewyn, B. G.; Lin, V. S. Y. Mesoporous Silica Nanoparticles for Intracellular Delivery of Membrane-Impermeable Proteins. J. Am. Chem. Soc. 2007, 129 (28), 8845−8849. (16) Park, S.; Mohanty, N.; Suk, J. W.; Nagaraja, A.; An, J. H.; Piner, R. D.; Cai, W. W.; Dreyer, D. R.; Berry, V.; Ruoff, R. S. Biocompatible, H

dx.doi.org/10.1021/la404337d | Langmuir XXXX, XXX, XXX−XXX