A Novel pH-Responsive Nanogel for the Controlled Uptake and

ARTICLE pubs.acs.org/JPCC

A Novel pH-Responsive Nanogel for the Controlled Uptake and Release of Hydrophobic and Cationic Solutes Simona Argentiere,*,† Laura Blasi,† Giovanni Morello,† and Giuseppe Gigli‡ † ‡

Nanoscience Institute of CNR, Via Arnesano, 73100, Lecce, Italy National Nanotechnology Laboratory (NNL) of CNR-Nanoscience Institute, Universita del Salento, Via Arnesano, 73100 Lecce, Italy

bS Supporting Information ABSTRACT: The study of the mechanisms underlying the uptake and release of bioactive molecules from stimuli-responsive nanoparticles is the key to their application in biomedical fields. Here, we present the pH-controlled uptake and release of two chemically different solutes from newly synthesized nanogels, showing their potential as pH-controlled dual drug delivery systems. Nanogels were synthesized by emulsion copolymerization of methacrylic acid (MAA) and methyl acrylate (MA), and then characterized by FT-IR, 1H NMR, and dynamic light scattering (DLS). To study the encapsulation and delivery properties of PMA-PMAA nanogels, a hydrophobic thiophene fluorophore (TF) and a cationic drug, doxorubicin (DX), were chosen. The uptake and release experiments were run at different pH values and monitored by absorption and both steady-state and time-resolved photoluminescence (PL) spectroscopy. Interestingly, the release of TF and DX was obtained under basic and acidic conditions, respectively, thus demonstrating a pH-controlled dual drug release by PMA-PMAA nanogels depending on the chemical nature of the tested solutes.

’ INTRODUCTION Nanogels represent a promising class of soft materials for drug delivery and controlled release of bioactive molecules. They exhibit several attractive features over other particulate delivery systems, namely, stability, ease of synthesis, and good control over particle size.1,2 To date, nanogels responsive to external stimuli, such as temperature and light, have been synthesized for the controlled drug delivery. However, pH responsive nanogels exploit the pH changes within the body for the selective response in specific tissues or cellular compartments.3,4 Further, they are even more versatile because of the numerous pH gradients that exist in both normal and disease states. For example, some kinds of cancers and inflamed tissues have a pH lower than the physiological value of 7.4. Moreover, in cellular compartments the pH drops from 6.2 (within the early endosome) to 4.5 (in the lysosomes), giving a large change in proton concentration.5,6 Recently, nanogels based on poly(methacrylic acid) (PMAA) have been explored for the targeted delivery of drugs7,8 and tissue engineering.9 PMAA nanogels exhibit a sharp volume phase change upon increasing pH, thanks to the carboxylic groups attached on their polymer chains. In particular, under acidic conditions, the carboxylic groups are not ionized, and the PMAA nanogels are in a collapsed state, whereas at higher pH values, the Coulombic repulsions between the deprotonated carboxylic groups induce the swelling of the nanogels.10,11 For example, this behavior has been exploited to protect sensitive drugs from r 2011 American Chemical Society

the acidic environment of the stomach and in cases where a specific release in the gastrointestinal tract is required.12 Monodispersed nanogels over a wide range of sizes have been synthesized by a number of different methods, and copolymerization of methacrylic acid has been mostly carried out by soapfree seeded emulsion polymerization13,14 and seed feed emulsion polymerization.9,15 However, the soap-free seeded technique gave large particles, which could limit their use in biomedical applications, whereas the latter is usually characterized by complex multistep procedures. In our group, the synthesis of pH-responsive nanogels by a two-step procedure involving emulsion polymerization of methyl acrylate and subsequent acidic hydrolysis has been widely investigated.16,17 However, a one-pot procedure should be faster and easier to run than a two-step procedure. The absence of the acidic hydrolysis should also allow us to obtain the pH-responsive nanogels under very mild conditions. Accordingly, a novel synthesis of pH-responsive nanogels has been developed by a one-step emulsion copolymerization of methacrylic acid (MAA) and methyl acrylate (MA) in aqueous solution. The obtained nanoparticles were characterized by FT-IR and 1H NMR, whereas their morphology was studied both in solution by

Received: May 27, 2011 Revised: July 20, 2011 Published: July 22, 2011 16347

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Scheme 1. Preparation of PMA-PMAA Nanogel

Figure 1. Molecular structures of doxorubicin hydrochloride (DX) and oligothiophene (TF) fluorophore.

dynamic light scattering (DLS) and in solid state by tapping mode AFM (see Supporting Information). To determine the suitability of PMA-PMAA nanogels as pHsensitive carriers for biomedical applications, two chemically different solutes were chosen: one a hydrophobic thiophene (TF) fluorophore and the other a cationic drug, doxorubicin (DX). Thiophenes are fluorescent probes characterized by high optical stability, bright fluorescence, and large Stokes shifts.18 Conversely, DX is one of the most potent and extensively used chemotherapeutic agents in modern cancer treatment.19,20 The uptake and release of both solutes from PMA-PMAA nanogels were monitored as a function of pH by UV
vis absorption and steady-state photoluminescence (PL) spectroscopy, whereas the quenching phenomena were studied by timeresolved PL measurements. Results indicated that uptake and release were controlled by changing the environmental pH. In particular, the uptake of both DX and TF were greater at basic pH values; thus, the physical entrapment was the driving mechanism in the uptake process. The release of TF and DX was instead obtained under basic and acidic conditions, respectively; therefore, a pH-controlled dual drug release was observed depending on the chemical nature of the solutes. Accordingly, PMA-PMAA nanogels could provide great potential to realize systems for the release of two or more bioactive molecules at different release profiles.

’ EXPERIMENTAL METHODS Materials. Methyl acrylate (MA), methyl methacrylate (MMA), methacrylic acid (MAA), sodium dodecyl sulfate (SDS), ammonium persulphate (APS), sodium thiosulphate (STS), and doxorubicin hydrochloride (DX) were obtained from Sigma Aldrich and used without further purification. Deionized water with a minimal resistivity of 18.0 MΩ cm was employed in the synthetic procedure. 500 -Methylsulfanyl-[2,20 ;50 ,200 ]terthiophene-5-carboxylic acid 2,5-dioxopyrrolidin-1-yl ester (TF)21 was provided by Mediteknology srl. The chemical structures of TF and DX are reported in Figure 1. Synthesis of PMA-PMAA Nanogels. The polymeric nanogels were prepared using micelles as nanoscaled reactors. In brief, small unilamellar vesicles were prepared by mixing SDS (250 mg, 0.867 mmol) and water (50 mL) at room temperature (RT) and sonicating them for 15 min. This dispersion was heated to ca. 60 °C in nitrogen atmosphere. The initiating agents APS (30 mg, 0.13 mmol) and STS (20 mg, 0.13 mmol) were added to the SDS vesicle dispersion. Finally, methyl acrylate (MA) and methacrylic acid (MAA) monomers were added to the mixture in a molar ratio of 10:1. The overall procedure is summarized in Scheme 1. The solution turned turbid within 10 min, indicating the successful beginning of radical polymerization. The reaction

was carried out for 2 h and was stopped by cooling it down in an ice bath. The reaction mixture was then ultracentrifugated (30000 rpm, 1 h). After ultracentrifugation, a gel layer was found to be formed between a white precipitate on the bottom of the tube (which likely consisted of exceeding surfactant and unreacted monomers) and the solution on the top of the tube. Afterward, the obtained nanogels were further purified by gel filtration on a Sephadex G-25 column (NAP-25, GE-Healthcare) and dried under nitrogen flow to obtain a white powder (64% yield). Chemical Characterization of PMA-PMAA Nanogels: FT-IR and 1H NMR. The PMA-PMAA nanogels were freeze-dried using a LIO-5P apparatus (Cinquepascal s.r.l., MI, Italy). The FT-IR spectrum was acquired (ATR) by means of a Jasco FT/IR-6300. The main absorption bands were the following (cm
1): 3550 (m), 3252 (m), 2954 (s), 2850 (m), 1730 (s), 1442 (s), 1165 (s), 1057(m), 972 (m), 828 (m), and 762 (m). The lyophilized PMA-PMAA nanogel was suspended in CDCl3, and the 1H NMR spectrum was obtained on a Bruker spectrometer operating at a frequency of 400 MHz. The chemical shifts are reported in parts per million downfield (δ) from TMS: 3.67 (s, 3H, CH3 of MA), 1.4
2.5 (m, 5H, CH2CH of MA and CHCH of MAA), and 1.25 (s, 3H, CH3 of MAA). Dynamic Light Scattering and Zeta Potential Measurements. The size and zeta potential of PMA-PMAA nanogels were measured using a Zetasizer Nano ZS90 (Malvern Instrument). Nanogel size was measured by dynamic light scattering (DLS) at a fixed angle of 90°. Zeta potential was determined via electrophoretic light scattering to evaluate the stability of the nanogel particles. For both measurements, the nanogel was diluted in a 1.0 mM MES buffer, and the pH was adjusted at different values by adding submicroliter amounts of 0.5 M NaOH and 0.5 M HCl. The suspension was filtered through a Millipore membrane filter (0.22 μm pore size) to remove dust particles. All of the measurements were carried out at RT and every sample was measured five times. Polystyrene nanospheres (60 ( 5 nm; Duke Scientific Corporation) were employed to calibrate the instrument. Titration. A 15.0 mg sample of the tested nanogel was suspended in 10 mM NaOH to fix the initial Na+ concentration and pH of the titration. Then the bulk titration was run with 100 mM HCl. The back-titration was finally performed with 16348

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100 mM NaOH. Both the titration and back-titration were run slowly for 3 h to allow for proper equilibration. Scanning Probe Microscopy. The size and shape of PMAPMAA nanogels were examined using an ambient AFM operated in true noncontact mode (XE-100, PSIA, Korea) using NCHR silicon nitride cantilevers (Nanosensors, St. Louis, MO; spring constant 42 N/m; tip radius pKa of the nanogel; see also Figure 5). When the uptake was run 16351

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Figure 9. Absorbance (A,C) and fluorescence (B,D) spectra of DX (dashed line) and the PMA-PMAA nanogel (dotted line) at the same concentrations of the experiments (2  10
4 M and 1.0 g/L, respectively), and of the nanogel after the uptake of DX (solid line) at pH 6.8 (A,B) and 9.0 (C,D). λex= 480 nm.

Figure 10. Plot of the average lifetime versus pH of DX-loaded nanogels and comparison to the time constant of the control solution.

at pH 9.0, DX was electrically neutral, whereas the nanogel was even more negatively charged. According to the absorbance spectra, the uptake of DX was 0.083 and 0.115 (mg DX/mg nanogel) at pH 6.8 and 9.0, respectively. The absorbance spectra in Figure 9 showed that the uptake was almost quantitative at pH 9.0 and decreased with decreasing pH. However, the discrepancy between absorbance and fluorescence data leads us to determine the quenching of DX at the two encapsulation rates by time-resolved characterization (Figure 10). These data were finally compared with the average lifetime of the DX control solution. While the control solution decayed in about 1.04 ns (ascribable to its intrinsic lifetime), the PMA-PMAA nanogels loaded with DX at increasing pH levels underwent faster and faster decay, thus demonstrating a gradual increasing of both the quenching phenomenon and the encapsulation rate. Additionally, DLS and zeta potential measurements have been made both before and after the DX uptake, to evaluate the influence of the encapsulated DX molecules on both the volume and surface charge of the PMA-PMAA nanogels. The zeta

Figure 11. Absorbance spectra of the PMA-PMAA nanogel after the uptake of DX at pH 6.8 (dashed line) and 9.0 (solid line), and the release at pH 5.5 (dotted line). λex= 480 nm.

potential of the nanogel before and after the uptake of DX at pH 6.8 was
43.1 mV and
36.6 mV, respectively. This reduction can be ascribed to the increase of the positive DX molecules onto the nanogel surface. It was also observed that the DX uptake was associated with an increment of the hydrodynamic diameter from 106 to 175 nm. Indeed, the protonated DX molecules are highly hydrated, and their encapsulation within the nanogel caused a significant swelling. Finally, the release of DX was enhanced when nanogels loaded by doxorubicin were suspended in acidic solutions at pH 5.5. The release of DX molecules has been ascribed to the pH-induced shrinking of the PMA-PMAA nanogels.32 Absorbance spectra in Figure 11 indicate the complete release of DX from the nanogel network.

’ CONCLUSIONS The uptake and release of two different solutes from newly synthesized PMA-PMAA nanogels are described, highlighting their potential for pH-activated drug delivery. 16352

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The Journal of Physical Chemistry C Nanogels were synthesized by polymerizing methacrylic acid (MAA) with methyl acrylate (MA), then they were chemically (FT-IR, 1H NMR) and morphologically (AFM, DLS) characterized. The drug loading and release capacities of the PMA-PMAA nanogels were tested using one hydrophobic oligothiophene (TF) fluorophore and one hydrophilic and charged solute, doxorubicin (DX). The PMA-PMAA nanogels were demonstrated to be suitable systems for encapsulating both the solutes and releasing them in a dual, pH-dependent way. In particular, high loading capacities were achieved at basic pH values for both of the solutes, whereas the release of TF and DX was obtained under basic and acidic conditions, respectively. Since the release process was mainly controlled by the nature of the encapsulated solutes, bioactive molecules and drugs could be targeted to both basic or acidic compartments within the body by properly choosing their chemical structures. According to these findings, the PMA-PMAA nanogels represent versatile carriers for dual, pH-activated drug release.

’ ASSOCIATED CONTENT

bS

Supporting Information. Influence of ionic strength and monomer type on the nanogel swelling, titration of pH-insensitive nanogels and AFM characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +39 0832 298373. Fax: +39 0832 298386. E-mail: simona. [email protected].

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(14) Hsu, S.-C.; Lee, C.-F.; Chiu, W.-Y. J. Appl. Polym. Sci. 1999, 71, 47–57. (15) Lally, S.; Mackenzie, P.; LeMaitre, C. L.; Freemont, T. J.; Saunders, B. R. J. Colloid Interface Sci. 2007, 316, 367–375. (16) Argentiere, S.; Blasi, L.; Ciccarella, G.; Barbarella, G.; Cingolani, R.; Gigli, G. Macromol. Symp. 2009, 281, 69–76. (17) Argentiere, S.; Blasi, L.; Ciccarella, G.; Barbarella, G.; Cingolani, R.; Gigli, G. J. Appl. Polym. Sci. 2010, 116, 2808–2815. (18) Barbarella, G.; Zambianchi, M.; Pudova, O.; Paladini, V.; Ventola, A.; Cipriani, F.; Gigli, G.; Cingolani, R.; Citro, G. J. Am. Chem. Soc. 2001, 123, 11600–11607. (19) Hortobagyi, G. N. Drugs 1997, 54, 1–7. (20) Bertin, P. A.; Smith, D.; Nguyen, S. T. Chem. Commun. 2005, 3793–3795. (21) Barbarella, G.; Zambianchi, M.; Ventola, A.; Fabiano, E.; Della Sala, F.; Gigli, G.; Anni, M.; Bolognesi, A.; Polito, L.; Naldi, M.; Capobianco, M. Bioconjugate Chem. 2006, 17, 58–67. (22) Huang, C.-F.; Chang, F.-C. Polymer 2003, 44, 2965–2974. (23) Sukumaran, S. K.; Beaucage, G. Europhys. Lett. 2002, 59, 714–720. (24) Saunders, B. R.; Crowther, H. M.; Vincent, B. Macromolecules 1997, 30, 482–487. (25) Kang, K.; Kan, C.; Du, Y.; Liu, D. Eur. Polym. J. 1998, 34, 997–1006. (26) Helfferich, F. Ion Exchange; McGraw-Hill: New York, 1962. (27) Eichenbaum, G. M.; Kiser, P. F.; Simon, S. A.; Needham, D. Macromolecules 1998, 31, 5084–5093. (28) Alvarez-Puebla, R. A.; Arceo, E.; Goulet, P. J. G.; Garrido, J. J.; Aroca, R. F. J. Phys. Chem. B 2005, 109, 3787–3792. (29) Hoare, T. R.; Kohane, D. S. Polymer 2008, 49, 1993–2007. (30) Kiser, P. F.; Wilson, G.; Needham, D. Nature 1998, 394, 459–462. (31) Lynch, I.; de Gregorio, P.; Dawson, K. A. J. Phys. Chem. B 2005, 109, 6257–6261. (32) Ogawa, K.; Wang, B.; Kokufuta, E. Langmuir 2001, 17, 4704–4707.

’ ACKNOWLEDGMENT We are grateful to Mediteknology srl for providing us the thiophene compound used in this study. Dr Alessandra Quarta, Benedetta Antonazzo, Raffaella Tunno and Mariangela Mortato are kindly acknowledged for their technical support. ’ REFERENCES (1) Raemdonck, K.; Demeester, J.; De Smedt, S. Soft Matter 2009, 5, 707–715. (2) Kabanov, A. V.; Vinogradov, S. V. Angew. Chem., Int. Ed. 2009, 48, 5418–5429. (3) Lyon, L. A.; Meng, Z.; Singh, N.; Sorrell, C. D.; St. John, A. Chem. Soc. Rev. 2009, 38, 865–874. (4) Qiu, Y.; Park, K. Adv. Drug Delivery Rev. 2001, 53, 321–339. (5) Gerweck, L. E.; Seetharaman, K. Cancer Res. 1996, 56, 1194– 1198. (6) Schmaljohann, D. Adv. Drug Delivery Rev. 2006, 58, 1655–1670. (7) Kim, J. O.; Sahay, G.; Kabanov, A. V.; Bronich, T. K. Biomacromolecules 2010, 11, 919–926. (8) Xu, X.; Fu, S.; Wang, K.; Jia, W.; Guo, G.; Zheng, X.; Dong, P.; Guo, Q.; Qian, Z. J. Microencapsulation 2009, 26, 642–648. (9) Saunders, J. M.; Tong, T.; Le Maitre, C. L.; Freemont, T. J.; Saunders, B. R. Soft Matter 2007, 3, 486–494. (10) Zhou, S.; Chu, B. J. Phys. Chem. B 1998, 102, 1364–1371. (11) Blasi, L.; Argentiere, S.; Morello, G.; Barbarella, G.; Cingolani, R.; Gigli, G. Acta Biomater. 2010, 6, 2148–56. (12) Wang, K.; Xu, X.; Wang, Y.; Yan, X.; Guo, G.; Huang, M.; Luo, F.; Zhao, X.; Wei, Y.; Qian, Z. Int. J. Pharm. 2010, 389, 130–138. (13) Kang, K.; Kan, C.; Du, Y.; Liu, D. Eur. Polym. J. 2005, 41, 439–445. 16353

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