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Supramolecular Hydrogels from Cisplatin-Loaded Block Copolymer Nanoparticles and r-Cyclodextrins with a Stepwise Delivery Property Wen Zhu,† Yanli Li,‡ Lixin Liu,*,‡ Yongming Chen,*,† Chun Wang,§ and Fu Xi† Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100190, China, College of Life Science, Graduate University of the Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China, and Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States Received August 1, 2010; Revised Manuscript Received September 15, 2010
A stepwise anticancer drug delivery system based on an injectable supramolecular hydrogel was presented. In this system, poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAA) block copolymer nanoparticles containing cisplatin were released by erosion of the hydrogels and then the cisplatin was released from the nanoparticles by exchanging with chloride ions. By mixing R-cyclodextrins (R-CDs) and the PEG-b-PAA micelles with their PAA cores loaded with the cisplatin in water, the novel supramolecular hydrogels were generated by threading R-CDs onto the PEG segments and forming physical cross-links of molecular necklaces. The gelation properties could be tuned by changing concentrations of the polymers and cisplatin, their feeds, and by adding PEG homopolymers or Pluronic copolymers as additives. Structures and properties of the supramolecular hydrogels containing cisplatin were studied by wide-angle X-ray diffraction (XRD) and rheology measurements, respectively. The thixotropic effect of the hydrogels and their reversible sol-gel transition were confirmed. In vitro hydrogel erosion experiments were conducted and cisplatin release in saline and pure water was quantified. Hydrogel erosion produced discrete nanoparticles from which cisplatin was released completely in saline. In contrast, the hydrogels were eroded into nanoparticles in pure water, but no cisplatin could be released. In vitro cytotoxicity studies showed that the cisplatinloaded hydrogels inhibited the growth of human bladder carcinoma EJ cells with a similar potency as that of the free cisplatin, whereas the hydrogels without cisplatin showed no cytotoxicity. These results suggested that the cisplatin-coordinated PEG-b-PAA/R-CD supramolecular hydrogels hold great potential as an injectable system for sustained delivery of cisplatin in cancer therapy.
Introduction Supramolecular hydrogels are polymer networks formed through physical cross-links of polymer segments that hold a large amount of water. As one of the most promising types of biomaterials, they have been widely explored to serve as drug delivery systems and to encapsulate cells for tissue engineering.1-3 Owing to the weakly noncovalent nature of the cross-links, such hydrogels can be broken by shear force and, therefore, show gel-sol reversible transformation. Such a thixotropic effect endows the physical hydrogels with injectability that is wellsuited for localized, minimally invasive delivery of drugs. The injected sols form gels in situ in the body and may dissolve gradually in vivo in body fluids and, as a result, provide sustained release of drugs. Most of these injectable hydrogels have been used for delivering hydrophilic therapeutics, such as small hydrophilic drug molecules, proteins, peptides, and oligonucleotides.4 A few injectable hydrogel systems have been developed for controlled release of anticancer drugs, including 5-fluorouracil,5 doxorubicin,6 chlorambucil,7 and platinum metal-based drugs.8 Cyclodextrins are a series of cyclic oligosaccharides composed of six, seven, or eight D(+)-glucose units linked by R-1,4* To whom correspondence should be addressed. Tel.: +86 10 6265 9906. Fax: +86 10 6255 9373. E-mail:
[email protected] (Y.C.);
[email protected] (L.L.). † Institute of Chemistry, The Chinese Academy of Sciences. ‡ Graduate University of the Chinese Academy of Sciences. § University of Minnesota.
linkages, which are named as R-, β-, or γ-CD, respectively. CDs may be selectively threaded onto some linear polymer chains, and the resulting supramolecular complex tends to aggregate to form packed columns that are the physical crosslinks of hydrogels. It has been reported that PEGs of high molecular weight,9 grafted PEGs,10-12 star-PEGs,13 and PEG block copolymers14 formed hydrogels by inclusion interaction with R-CD rings. This kind of supramolecular hydrogel was found to be thixotropic and reversible, making it an excellent injectable material. In addition, both CDs and PEG are known to be biocompatible and bioabsorbable.9 The CD-based hydrogels can be first incorporated with bioactive agents, such as drugs, proteins, vaccines, or nucleotides, then injected into the tissue and served as a depot for controlled release.15 So far, the CD-based hydrogels have been applied for controlled release of hydrophilic molecules such as dextran-fluorescein isothiocyanate14,16 and doxorubicin hydrochloride.17,18 Cisplatin is a well-known hydrophobic anticancer drug exhibiting high antitumor activity and has been widely applied in clinical application for cancer chemotherapy.19 It is known that cisplatin can be loaded in and release from hydrogels. Leung et al. have reported an injectable cisplatin-epinephrine loaded bovine collagen gel for clinical study of unresectable hepatocellular carcinoma.20 Casolaro et al. have prepared the cisplatin complexed hydrogels containing R-amino acid residues for cancer therapy.21 Konishi et al. have investigated the in vivo controlled release of cisplatin from a gelatin hydrogel.22 Gelpart (commercially available in Japan),23 chitosan hydrogels,24 and
10.1021/bm100889j 2010 American Chemical Society Published on Web 10/19/2010
Hydrogels from Nanoparticles and R-Cyclodextrins Scheme 1. Illustration of Formation and Structure of the Supramolecular Hydrogel Containing PEG-b-P(AA-cisplatin) Micelles and R-CDs
other hydrogels25-28 were reported for cisplatin loading. However, most of these studies used chemically cross-linked hydrogels, which could have difficulty in body clearance, and the cross-linking agents could be potentially toxic. Moreover, free cisplatin displays significant toxic side effects such as chronic neurotoxicity, acute nephrotoxicity, and very short plasma circulation time, which limit its clinical use. Incorporating cisplatin in nanoparticles has led to a prolonged plasma circulation and accumulation in solid tumors due to the enhanced permeability and retention (EPR) effect.29 Kataoka et al. have investigated cisplatin complex micelles of poly(ethylene glycol)b-poly(amino acid) via interaction of platinum(II) atoms and carboxylic groups, which enables a sustained cisplatin release in blood circulation.30,31 Also, cisplatin can be loaded into amphiphilic polymer micelles by hydrophobic interaction and delivered by diffusion.32,33 The cisplatin loaded polymeric micelles have a great advantage for drug targeting to solid tumors, which improves the bioavailability of drugs and survivals of patients. Above examples on delivering free anticancer drugs using polymeric hydrogels have shown a promising application. However, the released free drugs from hydrogels would still face the same problems that the parent drugs have, like the short plasma circulation and strong side effects due to no passive targeting. To combine both drug release advantages of physical cross-linked injectable hydrogels and nanoparticles, here we present a new type of supramolecular hydrogels composed of cisplatin-coordinated polymeric micelles and R-CDs. As shown in Scheme 1, cisplatin induces poly(ethylene glycol)-b-poly(acrylic acid) (PEG-b-PAA) self-assembly into PEG-b-P(AAcisplatin) micelles by coordination between platinum(II) atoms and carboxylic groups of PAA blocks in neutral aqueous solution. R-CDs are then added, and the inclusion complexes between CDs and PEG corona of PEG-b-P(AA-cisplatin) form the cross-links of hydrogels. To deliver drugs, the PEG-b-P(AAcisplatin) micelles are released from the dissolved hydrogels and cisplatin is released from the PEG-b-P(AA-cisplatin) micelles in saline. The addition of PEG homopolymers or Pluronic copolymers makes the hydrogels more robust, showing that these gels can be tuned easily and, thus, may provide an ideal cisplatin delivery system. Compared with other hydrogel/ micelle and hydrogel/microsphere systems reported previously,34-37 the hydrogelated micelles discussed in this work provide indispensable cross-links, the micellar cores, as an integral part of the network structure, which represents a unique delivery system using supramolecular hydrogels.
Experimental Section Materials. Methoxy poly(ethylene glycol) (PEG45-OH, Mn 2000, Alfa) was dried by azeodistillation with benzene and freeze-dried. tertButyl acrylate (tBA, 99%, Aldrich) was dried over CaH2. Poly(ethylene
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glycol) (PEG227, Mn 10000, Alfa), poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127, PEG125-bPPG40-b-PEG125, Mn 13400, Mw/Mn 1.02, Aldrich), R-CD (AP grade, Wacker Chemie AG), trifluoroacetic acid (TFA, 99%, Merck-Schuchardt), 2-bromoisobutyryl bromide (98%, Aldrich), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Duchefa), cisplatin (>99%, Boyuan Ltd. China), and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%, Aldrich) were obtained and used without further purification. Dialysis bags with molecular weight cutoff (MWCO) of 3500 Da were obtained from Viskase. All other chemicals and solvents were of analytical grade and were used as received. Characterization Methods. 1H NMR spectra were obtained on a Bruker DMX400 spectrometer. CDCl3 and DMSO-d6 were used as solvents and TMS as internal standard. Apparent molecular weights, Mn, and molecular weight distributions, Mw/Mn, of the polymers were determined by size exclusion chromatography (SEC) equipped with a Waters 515 pump, a Waters 2414 refractive index detector, and a combination of column Styragel HT-2, HT-3, and HT4 with the effective molecular weight ranges were 100-10000, 500-30000, and 5000-600000, respectively. Linear polystyrene standards were applied for calibration. The eluent was THF at a flow rate of 1 mL/min at 35 °C. Particle size measurement was carried out with a Brookhaven Zetaplus analyzer (Brookhaven Instruments). The stock solutions were filtered through filters of 0.45 µm pore size to remove large particles before detection. UV spectra were obtained using a UV-1601 UV-visible spectrophotometer (Shimadzu). Fourier transform infrared (FT-IR) spectra of the samples were collected on a Nicolet Avatar 330 FT-IR spectrometer at frequencies ranging from 500 to 3800 cm-1. XRD patterns of the samples were recorded on a Rigaku D/max 2500 X-ray powder diffractometer with Cu KR (1.54 Å) radiation (40 kV, 40 mA). The proportional counter detector collected data at a rate of 2θ ) 2° min-1 over the range of 2θ ) 5-35°. The morphology of the freezedried hydrogel samples was obtained by scanning electron microscopy (SEM, JSM6700F). The samples were coated with Pt before observation. Synthesis of PEG-b-PAA Block Copolymers. PEG-b-PAA copolymers were synthesized by a combination of atom transfer radical polymerization (ATRP) of tBA initiated by PEG45-Br and hydrolysis of PtBA according to the literature.38 Mw/Mn of PEG-b-PtBA block copolymers and the numbers of tBA repeating units (n) were determined by SEC and 1H NMR analysis, respectively. The tBA units transformed completely into AA units after hydrolysis as confirmed by the 1H NMR and FT-IR analysis. 1H NMR (400 MHz, DMSO-d6) δ 1.51 (s, -CH2CH(COOH)-), 2.21 (s, -CH2CH(COOH)-), 3.51 (s, -OCH2CH2O-), 11.10-12.90 (broad, -COOH). Preparation of the Supramolecular Hydrogels. A general protocol for formation of supramolecular hydrogel was supplied with G1 as an example. In a 1.5 mL tube, 50 mg of PEG45-b-PAA50 was dissolved in 250 mg of water and the pH was adjusted to 7 by adding dilute NaOH solution. The final mass of the solution was 440 mg by adding water. Then 10 mg of cisplatin was added to the polymer solution and the mixture was shaken at 37 °C for 72 h. A total of 550 mg of R-CD solution (14.5 wt %) was then added, and the mixture was ultrasonicated for 20 min. The drug loaded hydrogel was then formed at still. The hydrogel formation by different recipes was listed in Table 2. For the G7-10, PEG227, or Pluronic F127 solution was added to the micelle solution as additives. Rheological Studies. The viscosity of the hydrogels was measured at 37 °C using a TA AR2000 stress-controlled rheometer with 25 mm parallel plates. The chosen gap was ∼800 µm for all measurements. A preshear rate of 30 s-1 was applied if necessary. In Vitro Cisplatin Release. In vitro release of cisplatin from the supramolecular hydrogels was studied in 0.01 M phosphate-buffered saline (PBS, pH 7.4) with 0.16 M NaCl and in distilled water, respectively. The cisplatin-coordinated supramolecular hydrogel (500 mg) was put into a 1.5 mL tube filled with buffer solution before being sealed with a dialysis membrane (MWCO ) 3500 Da). The tube was then submerged fully into a beaker with 400 mL of buffer solution.
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Table 1. Characteristics of Block Copolymers block copolymera PEG45-b-PtBA50 PEG45-b-PtBA30
Mn (NMR)a
Mn (SEC)b
Mw/Mnb
8400 5800
17200 9900
1.07 1.11
a Composition and number average molecular weights Mn of the block copolymers were estimated from 1H NMR spectra. b Mn and Mw/Mn (where Mw is the weight average molecular weight) of the block copolymers were determined by SEC.
The beaker was maintained in a water bath of 37 °C with constant 100 rpm stirring. At regular time intervals, 0.5 mL of the medium was removed for UV analysis for its drug content and fresh buffer solution (0.5 mL) was added to keep the volume constant. Quantitative determination of cisplatin was performed as follows: the sample solution was mixed with an equal volume of o-phenylenediamine (OPDA) solution (1.2 mg/mL in DMF). The mixture was placed in a 100 °C water bath for 10 min, and the absorbance of the solution was measured at 703 nm. The amount of cisplatin in the sample was calculated in reference to standard solutions of free cisplatin.33 A profile showing the cumulative amount of drug release as a function of time was plotted for each release condition. To measure the nanoparticles released from the hydrogels, we detected the solution inside the tube by DLS during or after the release process. A control experiment of cisplatin loaded directly in the PEG227/RCD hydrogel (PEG227 13 wt %, R-CD 8 wt %, cisplatin 0.5 wt %) without PEG-b-PAA polymer was carried out in 0.01 M PBS (pH 7.4) with 0.16 M NaCl and in distilled water using the same method above. In Vitro Cytotoxicity Assay. Human bladder carcinoma EJ cell line was used to evaluate cytotoxicity of the supramolecular hydrogels. All growth media was prepared by supplementing RPMI 1640 with 5% penicillin-streptomycin, 10% fetal bovine serum, and sterilized with 0.2 µm filter prior to use. Cells were counted by a hemocytometer and seeded into 96-well plates at a density of 3000 cells per well. The plates were incubated in a humidified 37 °C environment with 5% CO2 for 24 h. Free cisplatin, PEG-b-P(AA-cisplatin) micelles and cisplatinloaded hydrogels were added to the cells with a final cisplatin concentration of 12.5 mg/L. The cells were then incubated for 24, 48, and 72 h before adding 20 µL of MTT solution into the medium of each well. After incubation for another 4 h, 0.15 mL of DMSO was added to each well to dissolve the MTT crystals formed. The plates were vigorously shaken before measuring the relative color intensity using a microplate reader (MULTISCAN MK-III, Thermo-Electron Co.). A test wavelength of 570 nm and a reference wavelength of 630 nm were used. The cytotoxicity of gels without cisplatin was also determined at a concentration of 1.75 g/L using the same method. Each experiment was repeated four times at each sample concentration.
Results and Discussion Preparation and Characterization of Injectable Hydrogels. The PEG-b-PAA used for loading cisplatin was prepared first through ATRP of tBA initiated with the bromoisobutyryl PEG followed by hydrolysis. Two block copolymer samples, PEG45-b-PAA50 and PEG45-b-PAA30, were obtained and their properties were shown in Table 1. Formation of the cisplatin-loaded polymer nanoparticles was confirmed by characterization of the aqueous mixed solution of PEG-b-PAA and cisplatin. The average diameters of PEG45-b-P(AA50cisplatin) (Cp 0.01 wt %, CcisPt 0.003 wt %, [COOH]/[Pt] ) 11:1) and PEG45-b-P(AA30-cisplatin) (Cp 0.01 wt %, CcisPt 0.003 wt %, [COOH]/[Pt] ) 9:1) micelles were 35 and 29 nm, respectively, as determined by DLS shown in Figure 1. Cp and CcisPt stands for the concentrations of PEG-b-PAA polymers and cisplatin, respectively. Similar to the literature, the cisplatin should be complexed into the core of particles by exchanging the chloride with carboxylic unit.31
Figure 1. Size distributions of (a) PEG45-b-P(AA50-cisplatin) and (b) PEG45-b-P(AA30-cisplatin) micelles.
To form the hydrogels, R-CD solutions were mixed with the aqueous solutions of cisplatin loaded polymer nanoparticles and the detailed conditions with the feed and concentration were listed in Table 2. For the hydrogel sample G1 from PEG45-bPAA50 in Table 2, the molar ratios of [COOH]/[Pt] and [R-CD]/ [EG] were 14:1 and 1:5, respectively. As shown in photos in Figure 2b, the mixture solution formed hydrogels in about 2 h and an opaque appearance was characteristic of the CD threading induced gelation. The light yellow color was due to the presence of cisplatin being incorporated. When the CcisPt was decreased to 0.5 wt %, while the Cp and CCD (CCD stands for the concentration of R-CD) remained unchanged, that is, the sample G2 from the same block copolymers and the ratio of [COOH]/ [Pt] was changed to 27:1, whereas the molar ratio of [R-CD]/ [EG] remained unchanged, hydrogels were obtained but the gelation time became longer (ca. 5 h). For the sample G3 from the copolymer with shorter PAA segments, the molar ratios of [COOH]/[Pt] and [R-CD]/[EG] were comparable to that of the G1 and the drug nanoparticles loaded hydrogels were still obtained. However, for the sample G4 with lower CcisPt, no hydrogel but an emulsion was obtained. When the Cp was decreased to 2 wt % and CcisPt was 0.5 wt % (samples G5 and G6), no gelation was observed. The results above indicated that PEG-b-P(AA-cisplatin) micelles formed supramolecular hydrogels in the presence of R-CD. However, gelation, which took hours, was relatively slow. These gels were mechanically weak and did not even form at lower cisplatin or polymer concentrations. It is known that PEGs of high molar mass and Pluronic copolymers may also form the supramolecular hydrogels by forming inclusion complex with R-CDs.9,14b For the former case, the partial threading complex tends to aggregate to form crystalline structure whereas those unthreaded PEG segments swell and capture water. For the latter case, addition of CDs will induce gelation at lower polymer concentration. Therefore, we added 3 wt % of PEG227
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Table 2. Composition and Gelation Results of Cisplatin-Loaded Samples samplea
polymerb (Cp)
CcisPt
COOH/Ptc
R-CD/EGc
gelation timed
result
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10
I (5 wt %) I (5 wt %) II (5 wt %) II (5 wt %) I (2 wt %) II (2 wt %) I (2 wt %) + III (3 wt %) II (2 wt %) + III (3 wt %) I (2 wt %) + IV (3 wt %) II (2 wt %) + IV (3 wt %)
1 wt % 0.5 wt % 1 wt % 0.5 wt % 0.5 wt % 0.5 wt % 0.5 wt % 0.5 wt % 0.5 wt % 0.5 wt %
14:1 27:1 11:1 22:1 11:1 9:1 11:1 9:1 11:1 9:1
1:5 1:5 1:7 1:7 1:2 1:3 1:10 1:11 1:9 1:10
ca. 2 h ca. 5 h ca. 2 h
gel gel gel sol sol sol gel gel gel gel
