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Langmuir 2002, 18, 4944-4952
Dually Responsive Microgels from Polyether-Modified Poly(acrylic acid): Swelling and Drug Loading Lev Bromberg, Marina Temchenko, and T. Alan Hatton* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received December 28, 2001. In Final Form: April 4, 2002 Gel microparticles composed of lightly cross-linked poly(acrylic acid) networks, onto which polyether chains (Pluronic F127) are grafted, are introduced. The hydrophobic poly(propylene oxide) chains aggregate within the microgel structure, and the resulting aggregates are capable of solubilizing hydrophobic drugs, such as taxol. At temperatures where the Pluronic chains are not aggregated, the microgels behave like networks without spatial heterogeneity. Upon formation of aggregates within hydrogels, their equilibrium swelling diminishes, and the swelling behavior indicates non-Gaussian chain distribution. The kinetics of gel swelling shows unusual temperature dependence of the effective diffusion coefficient, indicative of chain rearrangement within a certain temperature range. The microgels exhibit high ion-exchange capacity for cationic hydrophilic drugs. The potential for the newly obtained microgels to be used as drug carriers is discussed.
Introduction The delivery of potent drugs in common use in cancer treatment is currently performed mostly through intravenous drug infusions.1 The infusions, or injections, lead to an initial rapid increase and subsequent decay, below therapeutic levels, of the drug concentration in blood. Thus, the infusions need to be performed frequently, exacerbating the potential for side effects. Therefore, the therapeutic potential for protracted infusions is still confined to a subset of patients. Toxicity side effects have led to numerous attempts at development of new formulations for drug delivery such as long-circulating liposomes and micelles,2,3 water-soluble polymer-drug conjugates,4 biodegradable gels and microparticles,5,6 and drug formulations more suitable for oral administration. Oral treatment with cytotoxic anticancer drugs is preferred as this delivery route is convenient to patients, reduces administration costs, and facilitates the use of more chronic treatment regimens.7 Low oral bioavailability has usually limited the development of cancer treatment by the oral route. In the present study, we set out to test the hypothesis whether temperature- and pH-sensitive gel microparticles are feasible vehicles for oral delivery of anticancer drugs. We built on our previous work on copolymers of poly(acrylic acid) (PAA) and poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) (PEO-PPO-PEO copolymers, trademark Pluronic).8-37 Such Pluronic-PAA copolymers typically consist of about 50 wt % of PAA chains (1) DelaFlor-Weiss, E.; Uziely, B.; Muggia, F. M. Ann. Oncol. 1993, 4, 723-733. (2) Sparano, J. A.; Winer, E. P. Semin. Oncol. 2001, 28 (4 Suppl 12), 32-40. (3) Batrakova, E. V.; Dorodnych, T. Y.; Klinskii, E. Y.; Kliushnenkova, E. N.; Shemchukova, O. B.; Goncharova, O. N.; Arjakov, S. A.; Alakhov, V. Y.; Kabanov, A. V. Br. J. Cancer 1996, 74, 1545-1552. (4) Duncan, R.; Gac-Breton, S.; Keane, R.; Musila, R.; Sat, Y. N.; Satchi, R.; Searle, F. J. Controlled Release 2001, 74, 135-146. (5) Mallery, S. R.; Pei, P.; Kang, J.; Ness, G. M.; Ortiz, R.; Touhalisky, J. E.; Schwendeman, S. P. Anticancer Res. 2000, 20, 2817-2825. (6) Mitra, S.; Gaur, U.; Ghosh, P. C.; Maitra, A. N. J. Controlled Release 2001, 74, 317-323. (7) Malingre, M. M.; Beijnen, J. H.; Schellens, J. H. Invest. New Drugs 2001, 19, 155-162. (8) Bromberg, L. E.; Lupton, E. C.; Schiller, M. E.; Timm, M. J.; McKinney, G. U.S. Patent 5,939,485, 1999.
bonded to the polyether backbone of the Pluronic copolymer, resulting in graft-comb structures of high molecular weight (above 105 g/mol).22 The synthetic scheme for the Pluronic-PAA copolymers involves free-radical polymerization of acrylic acid with chain transfer to the Pluronic copolymer resulting in C-C bonding between PAA chains and Pluronic:16,21,22
where R• is the free radical, XmH is Pluronic, and AA is the acrylic acid monomer. Introduction of a divinyl crosslinker (XL) results in the appearance of PAA networks with grafted Pluronics: (9) Bromberg, L. In Handbook of Surfaces and Interfaces of Materials; Nalwa, H. S., Ed.; Academic Press: San Diego, 2001; Vol. 4, Chapter 7. (10) Bromberg, L. In Handbook of Polyelectrolytes and Their Applications; Tripathy, S. H., Nalwa, S., Eds.; American Scientific Publishers: Stevenson Ranch, CA, 2002, in press. (11) Bromberg, L. E.; Mendum, T. H. E.; Orkisz, M.; Ron, E. S.; Lupton, E. C. Polym. Mater. Sci. Eng. 1997, 76, 273-275. (12) Orkisz, M. J.; Bromberg, L.; Pike, R.; Lupton, E. C.; Ron, E. S. Polym. Mater. Sci. Eng. 1997, 76, 276-277. (13) Bromberg, L.; Orkisz, M.; Roos, E.; Ron, E. S.; Schiller, M. J. Controlled Release 1997, 48, 305-308. (14) Bromberg, L. E.; Mendum, T. H. E.; Orkisz, M. J.; Lupton, E. C.; Ron, E. S. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1997, 38, 602-603. (15) Bromberg, L. E.; Orkisz, M. J.; Ron, E. S. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1997, 38, 626-627. (16) Bromberg, L. J. Phys. Chem. B 1998, 102, 1956-1963. (17) Bromberg, L. E.; Ron, E. S. Adv. Drug Delivery Rev. 1998, 31, 197-221. (18) Bromberg, L. E.; Goldfeld, M. G. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1998, 39, 681-682. (19) Bromberg, L. Macromolecules 1998, 31, 6148-6156. (20) Bromberg, L. Langmuir 1998, 14, 5806-5812. (21) Bromberg, L. Ind. Eng. Chem. Res. 1998, 37, 4267-4274. (22) Bromberg, L. J. Phys. Chem. B 1998, 102, 10736-10744.
10.1021/la011868l CCC: $22.00 © 2002 American Chemical Society Published on Web 05/17/2002
Microgels from Modified Poly(acrylic acid)
One Pluronic-PAA molecule contains numerous PAA and Pluronic segments. Semidilute aqueous solutions of Pluronic-PAA copolymers exhibit intrachain aggregation with a signature of hydrophobic domains detectable by fluorescentandelectronparamagneticresonanceprobes,25,26,37 as well as interchain aggregation (micellization) above certain critical temperatures.20,26 The Pluronic-PAA conformation is strongly pH-dependent.22,25 In the present work, we modified the Pluronic-PAA copolymers by introducing a covalent cross-linker (ethylene glycol dimethacrylate) into their structure, which resulted in an interesting new species of gels. Intramolecularly crosslinked macromolecules with a globular structure are termed microgels.38 These microgels swell greatly in water, depending on pH and temperature. The pH sensitivity of our hydrogels loaded with drugs can be exploited in oral drug delivery, since at low pH, such as in the stomach, these hydrogels remain collapsed, while the drastic pH change in the upper small intestine leads to dramatic swelling and thus the drug can be released. Judging by the superior bioadhesion properties of the parent un-crosslinked Pluronic-PAA copolymers,10,15,17,31 our new microgels should adhere strongly to the mucosa, a property allowing for a longer residence time. The carboxylic pendent groups, which can act as calcium binders leading to epithelial cell junction opening,39,40 may enhance the absorption of drugs through the epithelial cell monolayer of the upper small intestine. Since the dangling Pluronic chains attached to the PAA network aggregate, the novel microgels can solubilize hydrophobic drugs such as taxol. Hence, the microgels can be potential carriers of both hydrophobic and ionic anticancer drugs. Experimental Section Materials. Nonionic copolymer Pluronic F127 NF was obtained from BASF Corp. and used without further treatment. It has the following characteristics: formula, EO100PO65EO100; (23) Bromberg, L.; Salvati, L. Bioconjugate Chem. 1999, 10, 678686. (24) Bromberg, L. E. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1999, 40, 616-617. (25) Bromberg, L. E.; Barr, D. P. Macromolecules 1999, 32, 36493657. (26) Huibers, P. D. T.; Bromberg, L. E.; Robinson, B. H.; Hatton, T. A. Macromolecules 1999, 32, 4889-4894. (27) Bromberg, L.; Magner, E. Langmuir 1999, 15, 6792-6798. (28) Bromberg, L.; Temchenko, M. Langmuir 1999, 15, 8627-8632. (29) Bromberg, L. E.; Temchenko, M.; Colby, R. H. Langmuir 2000, 16, 2609-2614. (30) Plucktaveesak, N.; Bromberg, L. E.; Colby, R. H. Proceedings of the XIIIth International Congress on Rheology, Cambridge, U.K., 2000; Vol. 3, pp 307-309. (31) Bromberg, L. J. Pharm. Pharmacol. 2001, 53, 109-114. (32) Bromberg, L. J. Pharm. Pharmacol. 2001, 53, 541-547. (33) Ho, A. K.; Bromberg, L. E.; O’Connor, A. J.; Perera, J. M.; Stevens, G. W.; Hatton, T. A. Langmuir 2001, 17, 3538-3544. (34) Colby, R. H.; Plucktaveesak, N.; Bromberg, L. Langmuir 2001, 17, 2937-2941. (35) Bromberg, L. Ind. Eng. Chem. Res. 2001, 40, 2437-2444. (36) Olivieri, L.; Seiller, M.; Bromberg, L.; Ron, E.; Couvreur, P.; Grossiord, J.-L. Pharm. Res. 2001, 18, 689-693. (37) Ho, A. K.; Bromberg, L. E.; Huibers, P. D. T.; O’Connor, A. J.; Perera, J. M.; Stevens, G. W.; Hatton, T. A. Langmuir 2002, 18, 30053013. (38) Funke, W.; Okay, O.; Joos-Mu¨ller, B. Adv. Polym. Sci. 1998, 136, 139-234. (39) Lehr, C. M. Eur. J. Drug Metab. Pharmacokinet. 1996, 21, 139148. (40) Torres-Lugo, M.; Peppas, N. A. Macromolecules 1999, 32, 66466651.
Langmuir, Vol. 18, No. 12, 2002 4945 nominal molecular weight, 12 600; molecular weight of PPO segment, 3780; 70 wt % of EO; and cloud point above 100 °C. Acrylic acid (99%, vinyl monomer), ethylene glycol dimethacrylate (98%, divinyl cross-linker), dodecane (99+%, solvent), and 4,4′azobis(4-cyanovaleric acid) (75+%, azo initiator) were purchased from Aldrich Chemical Co. and used as received. Lauroyl peroxide (97%, redox initiator) was obtained from Fluka Chemie AG (Switzerland). Poly(vinylpyrrolidinone-co-1-hexadecene) (Ganex V-216) (dispersion stabilizer) was obtained from International Specialty Products (Wayne, NJ). Doxorubicin hydrochloride and taxol (paclitaxel), both of 99% purity, were obtained from Hande Tech USA (Houston, TX), a subsidiary of Yunnan Hande Technological Development Co. (Kunming, P. R. China). Mitomycin C and mitoxantrone dihydrochloride (97%) were obtained from Sigma-Aldrich Co. and used as received. All other chemicals, gases, and organic solvents of the highest purity available were obtained from commercial sources. Microgel Synthesis. Synthesis was carried out on a laboratory scale in an adiabatic mode. Acrylic acid (40 mL) was partially neutralized by addition of 5 M NaOH aqueous solution (0.5 mL). Pluronic (24 g) was dissolved in the resulting solution under nitrogen, and a desired amount of ethylene glycol dimethacrylate (EGDMA) was added. Amounts of EGDMA ranged from 1.1 µL to 1.1 mL, and the molar ratio of the EGDMA to acrylic acid set in the reaction mixture designates the degree of cross-linking of the microgels [XL, mol % ) 100 × (number of moles of EGDMA/ number of moles of acrylic acid)] in what follows. Lauroyl peroxide (100 mg) and 4,4′-azobis(4-cyanovaleric acid) (100 mg) were dissolved in 2 mL of acrylic acid and added to the solution of Pluronic in acrylic acid. The resulting solution was deaerated by nitrogen bubbling for 0.5 h and added to a three-necked 0.5-mL flask containing a 1 wt % solution of Ganex V-216 in dodecane (200 mL). The flask was vigorously stirred by a mechanical stirrer and deaerated by constant nitrogen purge from the bottom. Then the flask was heated to 70 °C using an oil bath and kept at that temperature under stirring and nitrogen purge. After about 1 h, the formation of white particles was observed on the flask walls. The reaction was continued at 70 °C for another 3 h. Then the reactor was disassembled, and the contents of the reactor were filtered using Whatman filter paper (retention size, 10 µm). The microgel particles were extensively washed by hexane and dried under vacuum. The level of the monomer in the wash-outs in such a procedure is typically only 1-2% of the initial acrylic acid loading, due to the extremely high efficiency of monomer incorporation into the copolymers.21,35 Spherical particles were observed under a microscope. Particle sizing was performed in hexane using a ZetaPlus zeta potential analyzer (Brookhaven Instruments Co.). A typical batch of particles with XL ) 1 mol % was measured to have an effective median diameter of 13 mm and a polydispersity of 1.4. To ascertain the grafting of Pluronic segments onto crosslinked poly(acrylic acid) networks, a procedure described elsewhere35 was applied. In brief, a particulate sample was suspended in 1 M NaOH for 3 days and lyophilized. The sample was then placed into a Soxhlet extractor charged with dichloromethane and kept under reflux for 2 days. The wash-outs were collected, evaporated under vacuum, and weighed. Preliminary experiments demonstrated negligible solubility of poly(sodium acrylate) and total solubility of the Pluronic, respectively, in dichloromethane. The fraction of the Pluronic washed from the particles was negligible, within experimental error ((5% of the initial Pluronic content). In a separate series of experiments, the effective content of unbound species in the gel phase was measured by separating the gelled phase from unbound species using centrifugation.35 For separation, 5 w/v% particle suspensions were equilibrated at 4 °C in deionized water and the pH was adjusted to 7 by 5 M NaOH as needed. The suspensions were then centrifuged at 10 000g for 0.5 h, and the supernatant was carefully removed. The concentrated suspension was then additionally dialyzed against an aqueous solution (pH 7) at 4 °C (membrane molecular weight cutoff, 2 000 000; Spectrum Laboratories). The separated polymer solutions and gel suspensions were snapfrozen, lyophilized, and weighed. The weight fraction of the unbound phase in all measurements was less than 10 wt % of the total weight of the particles. Hence, for all practical purposes, the overall composition of the microgels in the present study
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corresponded to that set in the reactor. That is, the weight ratio of Pluronic to poly(acrylic acid) in the particles was 43:57. Microgel Swelling Experiments. The ability of microgels to absorb water was studied using a volumetric method. Single microgel particles were placed into glass capillary tubes (internal diameter, 1-1.2 mm) using suction pressures applied by an Ultramicro Accropet filler/dispenser (Carolina Biological Supply Co., Burlington, NC) via a rubber connector. The tubes were placed into a homemade glass thermostated cuvette and observed under a Nikon TMS inverted microscope (Nikon Corp., Tokyo, Japan) equipped with a model IV-550 video microscaler (For-A Co., Tokyo, Japan) and a Hitachi color video monitor. An analogous experimental setup was described by Eichenbaum et al.41 The boundaries of the spherical particles were fitted with the microscaler, and the particle diameter was measured with an accuracy of (0.5 µm or better. Initially, the diameter of a dry particle or a particle swollen under the initial pH and temperature (d0) was measured. Then, either the capillary tube was filled gently with water with an appropriate pH or the particle was removed from the initial tube and placed into another tube, which itself was then placed into a different solution and/or subjected to a different temperature. The diameter of the swollen particle (ds) was measured over time. In equilibrium swelling experiments, the particles were allowed to swell for 24 h, after which no changes in the particle size were observed at any temperature. The equilibrium volume ratio S ) V/V0 was defined as S ) (ds/ d0)3. In kinetic experiments, the volume changes of the particles were videotaped and the diameter changes were measured at low-speed replays. Measurements at a given pH and temperature were conducted with 3-5 different particles in different capillary tubes. Average S values are reported throughout. Bulk Microgel Titration. To obtain apparent pKa and ionexchange capacity of the microgels, bulk titration was performed at 20 °C using a 736 GP Titrino potentiometric titration system (Metrohm Ltd, Herisau, Switzerland). Titration of microgels in their acidic form was carried out with a solution of 0.1 M NaOH and 75 mM NaCl at a ratio of 150 mL of solution per 1 g of dry gel. The apparent pKa was calculated using the HendersonHasselbach equation:42
pKa ) pHi + log[Na+] - log[X/2] where [Na+] is the bulk sodium concentration, [X/2] is the concentration of carboxyl groups when half of the acrylic acid mers are protonated, and pHi is the pH at the inflection point on the titration curve. For microgels with XL ) 1 mol %, the following parameters were found: [X] ) 0.021 M, water content of 1/2 converted microgel ) 65.6 wt %, maximum COOH capacity ) 6.12 mequiv/g dry, pKa ) 4.79. These results correspond well with the known pKa values of poly(acrylic acid) gels,43 un-crosslinked Pluronic-PAA copolymers,22 and some other anionic microgels.44 Microscopy. The dried samples were mounted onto a scanning electron microscopy (SEM) stub with nonconductive glue and sputter-coated with gold (200-300 Å). Images of the particles were taken at various magnifications using a JEOL 6320 FE6SEM microscope. Solute Loading. Water-Soluble Solutes. The maximum loading level of doxorubicin, mitoxantrone, and mitomycin C into microgels was measured using a Millipore Ultrafree-MC centrifugal filter device (Millipore Co.). A microgel was suspended in Tris buffer (5 mM, pH 7.0), and 50 µL of the suspension (2 mg gel/mL buffer) was equilibrated with a 3.0 mM stock solution of a drug (450 µL) for 16 h while shaking.44,45 Shaking was performed using a KS10 orbital shaker (BEA-Enprotech Corp., Hyde Park, MA) in an environmental chamber at 37 °C. In the case of doxorubicin, the pH of the microgel suspensions equilibrated with the stock drug solution was varied by the addition of small (41) Eichenbaum, G. M.; Kiser, P. F.; Simon, S. A.; Needham, D. Macromolecules 1998, 31, 5084-5093. (42) Helfferich, F. Ion Exchange; Dover Publications: New York, 1995. (43) Katchalsky, A.; Michaeli, I. J. Polym. Sci. 1955, 15, 69-86. (44) Eichenbaum, G. M.; Kiser, P. F.; Dobrynin, A. V.; Simon, S. A.; Needham, D. Macromolecules 1999, 32, 4867-4878. (45) Kiser, P. F.; Wilson, G.; Needham, D. J. Controlled Release 2000, 68, 9-22.
Bromberg et al. amounts of 5 M NaOH or HCl solutions, and temperature was varied from 15 to 45 °C. After equilibration, the microgel particles were filtered off by centrifugation (10 000g, 0.5 h) and the supernatant was assayed for drug concentration. A Shimadzu model 1600 PC spectrophotometer with a temperature-controlled quartz cuvette (path length, 1 cm) was used for electronic absorption measurements. The extinction coefficients of doxorubicin (λ ) 482 nm) and mitoxantrone (λ ) 614 nm) were determined at pH 7.0 to be 12 200 and 22 100 M-1 cm-1, respectively. The concentration of mitomycin C was assayed by high-performance liquid chromatography (HPLC) using a Capcell Pak MF Ph-1 (100 × 4.6 mm i.d.; particle size, 5 µm) column (Phenomenex, Torrance, CA). The HPLC was a Hewlett-Packard 1090 system with an autosampler and a variable wavelength UV detector controlled by the HPLC Chemstation software (Hewlett-Packard). Deionized water was used as the mobile phase (flow rate, 1 mL/min; injection volume, 25 µL), and detection was carried out at 365 nm.47 The typical retention time of mitomycin C was 4.88 min. The drug uptake was expressed as
U (mmol drug/g gel) ) [(Ac - Ar)/Ac]VCs/Mgel where Ac and Ar are the absorbance or HPLC readings in the appropriately diluted stock solution and in the system equilibrated with microgel, respectively, V ) 0.5 mL is the total volume of the system, Cs ) 3 µmol/mL is the concentration of the stock solution, and Mgel ) 0.1 mg is the microgel mass. The U values were measured in triplicate for each drug and for each temperature, pH, and gel, respectively. In a control series of experiments, equilibration of 6 µmol/mL doxorubicin with microgels for 1 week yielded U values close (within experimental error) to the ones obtained with 3 µmol/mL solutions under otherwise identical conditions (see above). This ensured equilibrium U values. Hydrophobic Solutes. The loading of taxol into microgels was measured by equilibrating taxol adsorbed onto steel beads with the 1 wt % suspension of microgels (pH 7.0). Stainless steel beads (1-3 µm diameter) were soaked in a 10 mM solution of taxol in acetonitrile, following by stripping off the solvent in a rotary evaporator. The beads were used in order to enhance the area of contact between the microgel suspension and taxol. The beads were separated into several fractions. One fraction was added to a polypropylene vial containing the microgel suspension (0.5 mL), and the vial was gently shaken in a horizontal position in an environmental chamber at 20 or 37 °C. Then the beads were recovered from the suspension by using a magnet. The beads were dried under vacuum and placed into acetonitrile (0.5 mL), where taxol was extracted after shaking overnight. The solvent fraction was assayed for taxol concentration using HPLC. The control fraction of loaded beads was subjected to the extraction without equilibration with the microgel suspension. The solubility of taxol in water was measured at 37 °C by sonicating 5 mg of the drug suspension in 0.5 mL of water placed in a polypropylene vial for 15 s followed by centrifugation at 10 000g for 3 min.47 The supernatant was then removed and evaporated under vacuum, and the taxol traces were dissolved in acetonitrile and assayed by HPLC. Taxol concentrations were measured in triplicate using the HPLC system described above. The chromatography assay comprised the use of a Capcell Pak C18 UG 120 (150 × 4.6 mm i.d.; particle size, 3 mm) column (Phenomenex), acetonitrile-0.1% phosphoric acid in deionized (DI) water (55: 45 v/v, 1.3 mL/min) as a mobile phase, and UV detection at 227 nm.48 The typical retention time of the taxol peak was 3.46 min. Pyrene loading was studied in order to assess the temperaturedependent aggregation within microgels. 37 A stock solution of 1 mM pyrene in absolute methanol was prepared, from which 1-3 µL was added to 3.0 mL of an aerated aqueous sample. The sample was then allowed to equilibrate for 24 h at a given temperature, and excitation and emission (λex ) 335 nm) spectra were recorded. 25 Fluorescence spectra were recorded using a 10 (46) Song, D.; Au, J. L. J. Chromatogr., B 1996, 676, 165-168. (47) Niethammer, A.; Gaedicke, G.; Lode, H. N.; Wrasidlo, W. Bioconjugate Chem. 2001, 12, 414-420. (48) Lee, S.-H.; Yoo, S. D.; Lee, K.-H. J. Chromatogr., B 1999, 724, 357-363.
Microgels from Modified Poly(acrylic acid)
Figure 1. SEM image of the Pluronic-PAA-EGDMA particles with effective cross-linking degree XL ) 1 mol %. The particles were removed from the reactor, washed with hexane, and dried.
Figure 2. SEM image of the particles as in Figure 1 that were suspended in deionized water at 2 wt % and were allowed to equilibrium swell for 2 days at ambient temperature. Then the particles were snap-frozen and lyophilized. The particles were freeze-fractured in liquid nitrogen. mm path length quartz cell in a thermostated cuvette holder using a Shimadzu model RF-5301 PC spectrofluorophotometer under controlled temperature conditions (slit widths, 1.5 nm). The ratio of the intensities of the first (373 nm) to the third (384 nm) vibronic peak (I1/I3) in the emission spectra of the monomer pyrene was used to estimate the polarity of the pyrene microenvironment. In the control experiments, the I1/I3 values were measured in 1 wt % Pluronic F127 and Pluronic-PAA (without cross-linking) aqueous solutions, as described elsewhere.37
Results and Discussion SEM Particle Characterization. SEM images were taken to visualize the shape and structure of as-prepared and swollen and lyophilized microgels. Particles resulting from the synthesis described in the Experimental Section were spherical (Figure 1) and were generally in the range of 4-60 µm in size, with the majority of the particles being smaller than 10 µm in diameter. When equilibrium swollen in water and lyophilized, the particles greatly expanded in size, exhibiting round structures with very smooth gel surfaces, devoid of any visible pores of more than ca. 0.5-1 µm size (Figure 2). As we will see later, the absence of the macropores in the gel particles leads to a collective diffusion coefficient of the polymer that is much lower than that for the porous media. When lyophilized from suspensions of over 1 wt % concentration, the equilibriumswollen particles were fused together, showing interpenetration of the polymer along the outer edges (Figure 3). Characterization of Microgels by the Pyrene Solubilization Technique. Pyrene has been widely used as a hydrophobic fluorescent probe in polymeric solutions, including solutions of Pluronic-PAA,25,37 because the ratio I1/I3 of the intensities of the first (λ1 ≈ 373 nm) and the third (λ3 ≈ 384 nm) vibronic bands in its emission spectrum
Langmuir, Vol. 18, No. 12, 2002 4947
Figure 3. Particles as in Figure 2 at higher magnification.
Figure 4. Temperature dependency of the I1/I3 intensity ratio of pyrene in 1 w/w% aqueous solutions (pH 7.0) of Pluronic F127, un-cross-linked Pluronic-PAA, and a suspension of Pluronic-PAA cross-linked by ethylene glycol dimethacrylate (XL ) 1 mol %). The pH is 7.0 throughout. The data points for 1% F127, 1% Pluronic-PAA, and the 1% microgel suspension are given by open circles, open triangles, and filled squares, respectively.
is a sensitive measure of the polarity of the solvating environment for the pyrene, decreasing with increasing hydrophobicity. Pyrene partitions preferentially into the most hydrophobic regions such as aggregates, hydrophobic cores of the micelles, and so forth and thus provides a measure for both the presence and the hydrophobicity of such regions.37 Figure 4 shows the effect of temperature on the I1/I3 ratio in aqueous solutions of Pluronic F127, PluronicPAA, and a suspension of microgels of Pluronic-PAA cross-linked by EGDMA. In Pluronic F127 solutions, the I1/I3 ratio is roughly equal to that in water at temperatures below the critical micellization temperature (CMT), indicating a strongly polar environment. At temperatures above the CMT, the I1/I3 decreases sharply as pyrene partitions to a more hydrophobic environment as micellar aggregates begin to form. When micellization begins, the pyrene reports to hydrophobic PPO-rich micelle cores, at which point the environment sensed by the probe is independent of the F127 concentration.37 In Pluronic-PAA solutions, where PAA is covalently bound to the Pluronic F127, the I1/I3 is below 1.3 throughout the temperature range, indicating a polarity lower than that in the equivalent PAA solution at the same pH.37 This is evidence of the formation of sterically constrained, hydrophobic domains of Pluronic onto which several PAA segments are attached.16,22 Such domains, where pyrene must be located, can be invoked to explain the overall low I1/I3 values in the Pluronic-PAA solutions. Moreover, the I1/I3 versus T plots reveal opposite trends in Pluronic-PAA and Pluronic solutions. Namely, while
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Figure 5. Equilibrium swelling of microgel particles in deionized water at pH 7.0 as a function of temperature. The numbers stand for the degree of cross-linking (XL) in molar percent.
in Pluronic solutions the I1/I3 is close to that in water below the CMT and sharply decreases when the pyrene gets solubilized into the hydrophobic PPO cores of the Pluronic micelles above CMT, the I1/I3 in Pluronic-PAA is low at T < CMT and increases above the CMT (for CMT values in Pluronic-PAA solutions, see refs 16 and 22). This trend is even more striking in the cross-linked Pluronic-PAA microgels (Figure 4). Namely, the I1/I3 is below 0.85, indicating a strongly nonpolar environment, but increases at T > 20 °C up to 1.45 at 25 °C, at which point (corresponding to the formation of a massive number of micelles, compare with the CMT of Pluronic and Pluronic-PAA) the I1/I3 values start decreasing. The observed trends can be explained by redistribution of pyrene between hydrophobic PPO domains, “frozen” by PAA grafting and cross-linking, and micellar aggregates that form at elevated temperatures due to the rearrangement of polymer segments. Overall, the trends observed with cross-linked microgels reinforce our previous observation of domains more hydrophobic than micelles in uncross-linked Pluronic-PAA37 and provide evidence of temperature-dependent sensitivity of solubilization by the microgels. Moreover, formation of interchain micelles may enhance cross-linking of the microgels22 and thus influence their swelling. Experiments described below did confirm this hypothesis. Equilibrium Swelling of Microgels. Effect of pH and Temperature. In a study of equilibrium swelling, the microgel particles were allowed to equilibrium swell at a certain initial pH and temperature to yield d0 (see Experimental Section). Then the microgel particle was immersed into a solution of different pH and temperature to yield the equilibrium microgel diameter ds. The effects of temperature and pH on equilibrium swelling of microgels characterized by several cross-linking ratios are shown in Figures 5 and 6. The temperature dependency of the equilibrium swelling (Figure 5) at temperatures above 25 °C, corresponding to the formation of the micellelike aggregates (compare with Figure 4), confirms the appearance of additional physical cross-links due to the aggregation of dangling Pluronic segments within the cross-linked PAA. The relative collapse of the lightly crosslinked microgels is about 2-fold that of the microgels that have been cross-linked more densely by EGDMA, which is due to the intuitively expected differences in contributions of the physical cross-links. Similarly, the effect of the PAA chain ionization at pH > 5 (pKa of microgels is at 4.79, see Experimental Section) is less pronounced for
Figure 6. Equilibrium swelling of microgel particles in deionized water at 15 and 37 °C as a function of pH. The numbers stand for the degree of cross-linking (XL) in molar percent.
more densely cross-linked microgels at 37 °C than at 15 °C, where physical cross-links have not been formed (Figure 6). Figure 5 shows that the maximum microgel swelling at pH > 5 decreased with the degree of cross-linking XL. That is, the greater the extent of cross-linking, the less the gels swelled from their most condensed state when placed in the high-pH solutions. This result is analogous to the one recently described for poly(methacrylic acidco-acrylic acid) microgels.44 Eichenbaum et al.44 observed a linear relationship between the equilibrium swelling ratio of ionized gels and the number of monomers between cross-links. They invoked the Flory-Huggins theory,49 modified to account for the ion binding, the change in the Flory interaction parameter with swelling,50 and the nonGaussian elasticity of the microgel matrix, to predict the pH dependency of the equilibrium swelling of the microgels quantitatively. In our case, however, the relatively low degrees of permanent cross-linking (XL up to only 1 mol %) and the generally unknown (and also pH- and temperature-dependent) structure of the microgels make it difficult to estimate the Flory interaction parameter (χ), without making liberal assumptions. Therefore, we correlate the equilibrium swelling measured with fixed pH and T for microgels of varying cross-linking ratios. Effect of the Length of the Subchain. Eichenbaum et al.44 estimated the length of the subchains, that is, the number of monomers between cross-links, from experimental measurements of gel pore sizes. The linear dimensions for the mer and cross-linker were estimated from an analysis of the known bond lengths and angles of their respective molecular structures. The presence of the Pluronic dangling chains randomly bonded within the (49) Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953. (50) Hasa, J.; Ilavsky, M. J. Polym. Sci., Polym. Phys. Ed. 1975, 13, 263-274.
Microgels from Modified Poly(acrylic acid)
Figure 7. Equilibrium swelling of microgel particles in deionized water at pH 7.0 as a function of the effective length of the subchain, N. The solid lines show linear fits with the slopes of 0.3 and 0.6 for the data obtained at 15 and 37 °C, respectively (R2 g 0.98).
PAA network makes an analogous calculation impossible. Therefore, in the present study we relate the XL ratio to an effective subchain length N, which accounts only for the number of acrylic acid monomers between EGDMA cross-links. The expression for N as a function of XL is derived from the “random-walk” polymerization model:51
N ) [a6cxl(cxl + cm)]-1 where a ) 10vxlvm, vxl ) 0.2 M is the molar volume of the cross-linker (EGDMA), and vm ) 0.063 M-1 is the molar volume of the monomer (acrylic acid); cxl and cm are the molar concentrations of the cross-linker and monomer, respectively. The results of equilibrium swelling experiments with microgels cross-linked by EGDMA are shown in Figure 7. The dry particles of diameter d0 were allowed to equilibrium swell in deionized water (pH 7.0) at temperatures above and below the CMT, ≈20 °C, of the Pluronic-PAA,22 yielding ds. The results shown in Figure 7 indicate that at 15 °C, where the microgels are devoid of interchain aggregates, the swelling ratio S scales as S ∝ N0.3, which according to the Flory-Huggins theory is indicative of the Gaussian chain statistics, typical for covalently cross-linked gels without spatial heterogeneity.49,51 However, at 37 °C, the elasticity of the microgel becomes higher due to the appearance of additional physical cross-links (Pluronic chains with hydrophobic PPO segments). The swelling ratio scales as S ∝ Nx, with x e 0.6, consistent with nonGaussian chain statistics.51 Overall, the results in Figure 7 show high absorbency of the microgels at the salt-free limit (swelling ratio up to 300 and higher) and useful temperature sensitivity of the water uptake. Moreover, the observed trends allow for the prediction of the equilibrium swelling capacity of the microgels as a function of the initial monomer/cross-linker ratio set in the reactor. Kinetics of Swelling. The kinetics of swelling is one of the foremost characteristics of microgels intended for drug delivery. The time-dependent radius of a spherical gel particle r(t) is given by Tanaka and Fillmore:52
Langmuir, Vol. 18, No. 12, 2002 4949
Figure 8. Kinetics of swelling of microgel microspheres in water (15 °C, pH 7.4). The numbers designate the effective cross-linking ratio, XL, in mol %.
where r0 is the initial radius of the gel particle, D is the diffusion coefficient, τn ) r∞2/(n2π2D) is the nth mode relaxation time,53 and ∆r0 ≡ r∞ - r0. In most practical cases, the relaxation is dominated by the longest relaxation time τ1, and thus the effective diffusion coefficient is given by
D ) De ≡ (r∞/π)2/τ1 and when t g τ1, the relative diameter change becomes
d∞ - d(t) 6 = 2 exp[-t/τ1] d∞ - d0 π
-1
∆r(t) ) ∆r0(6/π2)
∑n-2 exp(-t/τn)
(51) Bromberg, L.; Grosberg, A. Y.; Matsuo, E. S.; Suzuki, Y.; Tanaka, T. J. Chem. Phys. 1997, 106, 2906-2910. (52) Tanaka, T.; Fillmore, D. J. Chem. Phys. 1979, 70, 1214-1218.
or simply
ln
(
)
dt - d∞ ) const - t/τ1 d0 - d∞
(1)
Here, d0, dt, and d∞ are the initial particle diameter, the diameter at time t, and the diameter at equilibrium swelling, respectively. Figure 8 shows the kinetics of swelling of PluronicPAA-EGDMA microgels in water (pH adjusted to 7.4) as a function of the cross-linking ratio (XL) in terms of eq 1. As is seen, kinetics can be described by straight lines (R2 > 0.98 in all cases), the slopes of which yield the effective diffusion coefficient (De). The effect of the crosslinking density on the diffusion coefficient is evident when the XL is expressed in terms of the effective length of the subchain (Figure 9). The effective diffusion coefficient decreases as the elastic chains between cross-links become shorter and thus relax more rapidly. Analogous dependencies are commonly observed with poly(sodium acrylate)based superabsorbent materials that lack macropores.54 However, an opposite trend, that is, an increase in the De values, is observed with an increase in the cross-linking density if the gels have macropores,55 because the volume fraction of the macropores (which have rapid relaxation modes) increases as the subchains become shorter. These results are consistent with the observation that the particle surfaces are smooth and nonporous (Figures 1 and 2). (53) Shibayama, M. In Gels Handbook; Osada, Y., Kajiwara, K., Fushimi, T., Hirasa, O., Hirokawa, Y., Matsunaga, T., Shimomura, T., Wang, L., Eds.; Academic Press: New York, 2001; Vol. 1, pp 82-97. (54) Buchholz, F. L. In Modern Superabsorbent Polymer Technology; Buchholz, F. L., Graham, A. T., Eds.; Wiley-VCH: New York, 1998; Chapter 5, pp 167-227. (55) Ikkai, F.; Shibayama, M. J. Polym. Sci., Part B: Polym. Phys. 1996, 34, 1637-1645.
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Bromberg et al.
Figure 9. Effective diffusion coefficient (De) of the microgels as a function of the effective length of the subchain (N). Conditions are as in Figure 8. The straight line is shown to guide the eye only.
Figure 10. Temperature dependency of the effective diffusion coefficient of the microgels. Effective cross-link ratio XL ) 0.01 mol %, pH 7.4. The dashed line illustrates the I1/I3 trend (compare with Figure 4).
Since our gels exhibit significant temperature dependencies of their equilibrium swelling (Figure 5), it was of interest to examine the effect of temperature on the gel diffusion (Figure 10). The diffusion coefficient showed marked temperature dependency, with De values having a minimum in the range 25-31 °C, which corresponds to the maximum I1/I3 values. The following scenario can be offered to explain this observation. As we discussed in the preceding paragraphs, at T > 20 °C the hydrophobic, constrained microdomains formed within the particle in the process of its synthesis begin to dissociate into the copolymer segments that can rearrange themselves more freely. Such a rearrangement of the PPO segments leads to formation of intermolecular aggregates acting as crosslinks. The appearance of the physical cross-links in addition to the permanent EGDMA cross-links is responsible for the longer relaxation times, and thus a lower De in a frozen state of the gel. At temperatures above 25 °C, aggregation of the PPO segments leads to formation of larger, micellelike aggregates,37 capable of solubilizing more pyrene and thus leading to the decrease in the observed I1/I3 value. As the temperature rises above 31 °C, both the fraction of the PPO segments involved in the formation of the “intraparticle” micelles and the fraction of water-rich regions of the particle become larger, resembling an intraparticle phase separation. The relaxation of the polymer-rich phases is very slow,53 but the overall relaxation of the particle as observed under the microscope might be determined by the relaxation of the water-rich, loose structures. As the fraction of the loose regions of the particles becomes larger, the overall rate
Figure 11. Molecular structures of the drugs used in the loading studies.
of diffusion grows. Analogous temperature dependencies have been observed by Tanaka et al.56 for poly(Nisopropylacrylamide) gels near their lower critical solution temperature (LCST). As the gel collapsed near the LCST, forming aggregates, it reached a frozen state characterized by a sharply lower De, which increased above the LCST as polymer-rich and water-rich regions formed in the gel. Drug Loading. Figure 11 shows the molecular structures of the three cationic drugs and one uncharged drug that were loaded into the microgels. All of these compounds are currently in clinical use as anticancer drugs.57 Doxorubicin, mitoxantrone, and mitomycin C are mono-, di-, and trivalent cationic weak bases, respectively. (56) Tanaka, T.; Sato, E.; Hirokawa, Y.; Hirotsu, S.; Peetermans, J. Phys. Rev. Lett. 1985, 55, 2455. (57) Baker, A. F.; Dorr, R. T. Cancer Treat. Rev. 2001, 27, 221-233. Messori, A.; Trippoli, S. Anticancer Drugs 1998, 9, 909-916. Roy, J. A.; Piccart, M. J. Curr. Opin. Oncol. 1995, 7, 517-522. Trudeau, M.; Pagani, O. Semin. Oncol. 2001, 28 (4 Suppl 12), 41-50. Sculier, J. P.; Ghisdal, L.; Berghmans, T.; Branle, F.; Lafitte, J. J.; Vallot, F.; Meert, A. P.; Lemaitre, F.; Steels, E.; Burniat, A.; Mascaux, C. Br. J. Cancer 2001, 84, 1150-1155. Drakos, P.; Bar-Ziv, J.; Catane, R. Am. J. Clin. Oncol. 1994, 17, 502-505. Arcamone, F. Doxorubicin: Anticancer Antibiotics; Academic Press: New York, 1981.
Microgels from Modified Poly(acrylic acid)
Langmuir, Vol. 18, No. 12, 2002 4951
Table 1. Properties of Anticancer Drugs and Their Equilibrium Uptake by the Pluronic-PAA Microgels (Cross-Linking Ratio, XL ) 1 mol %; Ion-Exchange Capacity, 6.12 mmol/g) at pH 7.0 drug
MWa
Log P
uptake ( SD, mmol/g
mitomycin C mitoxantrone doxorubicin
334.1 444.2 543.5
-0.4b 0.24c 1.85b
Taxol
853.3
4d
5.31 ( 1.86 (37 °C) 3.70 ( 0.56 (37 °C) 2.97 ( 0.33 (20 °C) 2.26 ( 0.37 (37 °C) (2.27 ( 0.90) × 10-3 (20 °C) (6.97 ( 0.87) × 10-3 (37 °C)
a Molecular weights are given for free bases and not hydrochloride salts. b Reference 58. c Calculated using ClogP Program (BioByte Corp.) d Reference 47.
Table 1 lists molecular weights, n-octanol-to-water partition coefficients (P), and equilibrium uptake of the drugs by the microgels characterized by XL ) 1 mol % and maximum ion-exchange capacity of 6.12 mmol/g (measured by bulk titration as described in the Experimental Section). All of the uptake values in Table 1 were less than or equal to the maximum microgel capacity for protons. A pronounced dependence was observed with the weak bases: the smaller, more hydrophilic, and more charged the solute, the higher its loading into the microgels. The uptake of the hydrophobic, neutral taxol was generally 1000-fold lower than that of the basic drugs, indicating a different mechanism of uptake. While the bases that are counterions to the microgel are loaded via Donnan ion-exchange equilibria,41,44 taxol is solubilized by the hydrophobic PPO domains in the microgels. The latter notion is supported by the fact that the equilibrium solubility of taxol in water at 20 °C was measured to be 1.4 ( 0.8 µM, which is over 16-fold and 50-fold less than the equilibrium solubility in 1 wt % microgel suspension at 20 and 37 °C, respectively. The characteristic increase in taxol loading capacity at temperatures above CMT provides additional evidence to this suggested mechanism of taxol solubilization into micellelike aggregates within the microgels. The micelles in Pluronic-PAA solutions typically have a solubilizing capacity higher than that of the small hydrophobic domains existing below the CMT.37 The solubilizing capacity of the microgels for taxol is at least equal to that of Pluronic-PAA micelles for other hydrophobic solutes such as steroid hormones.14,28 The ability of microgels to effectively load and hold taxol, combined with their mucoadhesive properties,10,15,17,31 is a feature important for localized delivery. General trends important for drug loading via the ionexchange mechanism were studied using the potent chemotherapeutic drug doxorubicin (Figures 12-14). As the degree of carboxyl group ionization increases with pH, the ion-exchange capacity of the microgels increases, reaching about half of the maximum capacity found by titration, indicating that the loading of doxorubicin can be limited by the available free volume of the network (Figure 12). Notably, the pH dependencies of the equilibrium swelling (Figure 6) and doxorubicin loading coincide. Similar results were observed with poly(methacrylic acid) microgels.45 The effects of steric “crowding” of the drug within the microgel network and the availability of the carboxyls for the ion exchange are highlighted by the effects of temperature (Figure 13) and cross-linking density (Figure 14). The collapse of the microgels at elevated temperature (Figure 5) due to the appearance of physical cross-links leads to less volume within the microgel network available for hosting the drug, and thus lower equilibrium loading at higher tempera-
Figure 12. Equilibrium uptake of doxorubicin by microgels (XL ) 1 mol %) as a function of pH at 37 °C.
Figure 13. Effect of temperature on equilibrium uptake of doxorubicin by microgels (XL ) 1 mol %) at pH 7.0.
Figure 14. Equilibrium uptake of doxorubicin by microgels (XL ) 1 mol %) as a function of the effective length of the subchain (N) at pH 7.0 and 37 °C.
tures. Analogously, the longer subchain allowing for a looser network and higher swelling (compare with Figure 7) leads to a slightly higher equilibrium loading of doxorubicin. It is believed that the observed trends, along with the very high overall capacity of our microgels for doxorubicin, will allow for the proper design of the chemotherapeutic drug delivery. One might envision microgels loaded with both taxol and doxorubicin, which would be a feature unique for the microgels described in the present study. Concluding Remarks The study dealt with microgels that consist of loosely cross-linked poly(acrylic acid) onto which Pluronic F127 segments are grafted. Temperature dependencies of the equilibrium swelling and I1/I3 values originating from the
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Figure 15. A scheme representing thermoreversible aggregation of Pluronic dangling chains (shown by solid lines) into aggregates within gel microparticles.
pyrene spectra indicate that at certain temperatures below 25 °C the microgel particles contain chains of Pluronic, possibly linked with PAA segments by multiple covalent bonds (thus creating hydrophobic microdomains, frozen into the microgel structure). Interestingly, the dependency of the equilibrium swelling of the microgels on the effective subchain length in salt-free water at low temperatures suggests Gaussian chain statistics. This can be explained by relative homogeneity of the microgel structure, despite the presence of hydrophobic domains. At elevated temperatures, the dangling Pluronic chains rearrange, creating aggregates that act as physical cross-links, lowering the equilibrium swelling of the microgels (Figure 15). Such microgels cross-linked by both covalent and physical crosslinks possess non-Gaussian chain statistics. Importantly, the “intragel” aggregates are capable of solubilizing
Bromberg et al.
hydrophobic drugs such as taxol, while the presence of carboxyls allows for the loading of positively charged drugs. One may hypothesize that microgel particles doubly loaded with, for instance, both taxol and doxorubicin may be very potent anticancer drug delivery vehicles. Permanently cross-linked copolymeric micelles have generated much interest primarily because of their potential applications as drug carriers.10,59 The micelles, cross-linked either within the hydrophilic shell or the hydrophobic core, close the gap between macromolecules (Stokes radii below 10 nm) and microparticles (size 100 nm and higher). The lesser size of the cross-linked micelles is important for certain drug administration routes such as percutaneous lymphatic delivery or extravasation into solid tumors.10 The present work introduces an unusual hybrid between cross-linked micelles and microparticles, that is, a microparticle within which numerous micelles can be formed. The size of our gel microparticles (hundreds of microns, when swollen) would not allow for their intravenous administration. However, their high capacity for both hydrophilic and hydrophobic drugs, combined with their mucoadhesive nature, would certainly be advantageous for oral and topical drug delivery. LA011868L (58) Hansch, C.; Leo, A. Exploring QSAR; American Chemical Society: Washington, DC, 1995; Vols. 1 and 2. (59) Thurmond, K. B.; Kowalewski, T.; Wooley, K. L. J. Am. Chem. Soc. 1996, 118, 7239-7240. Thurmond, K. B.; Remsen, E. E.; Kowalewski, T.; Wooley, K. L. Nucleic Acids Res. 1999, 27, 2966-2971. Ro¨sler, A.; Vandermeulen, G. W. M.; Klok, H.-A. Adv. Drug Delivery Rev. 2001, 53, 95-108.
