Article pubs.acs.org/Langmuir
Cisplatin-Mediated Formation of Polyampholytic Chitosan Nanoparticles with Attenuated Viscosity and pH-Sensitive Drug Release Ra-Hye Kang, Ji-Yeong Kwon, Yeojin Kim, and Sang-Min Lee* Department of Chemistry, The Catholic University of Korea, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Korea S Supporting Information *
ABSTRACT: Chitosan is a biocompatible natural polysaccharide, which has been employed as a polymeric scaffold for versatile, systemic delivery platforms and for locally injectable gels with temperature-sensitive viscosity modulation. Despite the extensive investigation on the chemical modification strategies, however, most of the chitosan-based delivery platforms have been focused on the encapsulation of hydrophobic drugs, which can be simply adsorbed on the chitosan scaffolds by hydrophobic interaction via the postparticle-formation drug-loading process. Herein, we present the facile formation of a cisplatin-coordinated chitosan nanoplatform by exploiting the divalent metal (PtII)-mediated conformational changes of chitosan chains, which allows for the simultaneous drug-loading and nanoparticle formation. To this end, the native chitosan has been chemically modified with short polyethylene glycol and malonic acid as a colloidal stabilizer and a bidentate chelating ligand for PtII coordination, respectively. The resulting PtII-modified polyampholytic chitosan (PtII-MPC) has been self-associated in aqueous media by hydrophobic segregation into a compact nanostructure, which exhibited an attenuated viscosity and pH-sensitive release of PtII compounds. Once the cationic drug molecules have been released under mild acidic conditions, the neutralized PtII-free MPC undergoes interchain flocculation near the isoelectric point because of the polyampholytic property, possibly allowing for the facilitated endosomal escape during the cellular endocytosis by the known membrane perturbation property of chitosan.
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INTRODUCTION Chitosan is a biocompatible natural polysaccharide consisting of partially acetylated glucosamine (GlcN), which has been employed as a nontoxic polymeric scaffold for versatile delivery systems. Because of the weakly basic GlcN residues (pKa ≈ 6.5), the aqueous solubility of native chitosan can be easily controlled by the solution pH: it becomes highly soluble in acidic solution, whereas it is significantly aggregated under neutral conditions via increased hydrophobic interactions. Combining this weakly basic GlcN scaffolds of chitosan with reversible cross-linking strategies, versatile chitosan nanoparticles possessing cross-linked gel networks have been developed as a systemic delivery platform.1 For instance, simple mixing of cationic chitosan with polyanionic molecules, such as tripolyphosphate (TPP) and various organic acids, often induced nanoscale aggregation by ionic interactions.2,3 Additionally, the chemical modification with hydrophobic small molecules such as cholic acid and lipids can produce multiple hydrophobic moieties on the chitosan backbone, which leads to the formation of chitosan nanoparticles via hydrophobic interactions.4−6 Although the facile formation of chitosan-based nanoparticles have been successfully achieved through the aforementioned approaches, most of the systems have been exploited to © XXXX American Chemical Society
encapsulate hydrophobic drugs, such as paclitaxel and doxorubicin, which can be loaded inside the cross-linked chitosan networks by nonspecific physical adsorption via the postparticle-formation drug-loading process.5,7 Hence, to encapsulate the nonhydrophobic drugs such as cisplatin, every drug molecule requires a chemical modification for enhanced adsorption on the chitosan scaffolds,6 which is synthetically cumbersome. Additionally, sustained drug leakages have often been observed under physiological conditions because of the spontaneous partitioning of the adsorbed drug molecules to the aqueous phase.4,5 Herein, we demonstrate the facile formation of chemically modified chitosan-based gel-like nanostructures mediated by the coordination of cisplatin pharmacophore, cis[PtII(NH3)2(H2O)2]2+ (referred to here as PtII). To provide colloidal stability and PtII coordination, short polyethyleneglycol (PEG) and malonic acid have been ligated to the GlcN amine moieties of chitosan by peptide coupling. As the endtethered malonic acids can generate a number of anionic malonic amides on chitosan scaffolds, a modified polyamphoReceived: June 15, 2017 Revised: August 3, 2017
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DOI: 10.1021/acs.langmuir.7b02043 Langmuir XXXX, XXX, XXX−XXX
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(200 mesh) for 30 s, and the excess solution was blotted with a filter paper. The grid was dried at room temperature for 24 h. Preparation of PEGylated Chitosan (PEG-Chitosan). Oxidation adducts of mPEG550-COOH were prepared using a modified literature procedure.9 To a test tube equipped with a magnetic stir bar was added chitosan (380 mg) and mPEG550-COOH (700 mg, 1.20 mmol) in DI water, whose pH was adjusted to 4−5 with hydrochloric acid. To the resulting solution was added EDC (1.61 g, 8.40 mmol) and stirred overnight at room temperature. The reaction mixture was purified by dialysis (molecular weight cutoff, MWCO = 1000 Da) against DI water (7 × 1000 mL) for 7 days with water change every 24 h. After dialysis, the product was dried by lyophilization and obtained as a colorless product (0.11 g, 29% yield). 1H NMR (300 MHz, D2O): δ 3.69 (m, 48H, −OCH2CH2− of PEG), 3.36 (s, 3H, −OCH3 of PEG), 2.89 (s, 2H, −CH2− of PEGylated amide), and 2.04 (s, 3H, −COCH3 of acetylated chitosan). FTIR: 1758 cm−1 (strong, CO stretch of carboxylic acid). Preparation of MPC. In a test tube equipped with a magnetic stir bar, malonic acid (250 mg, 2.4 mmol) was dissolved in DI water (∼10 mL) with pH adjusted to 4−5 using aqueous NaOH. Next, EDC (3.22 g, 16.8 mmol) was added to activate the carboxyl groups of malonic acid, followed by the addition of PEG-chitosan (50 mg). The peptide coupling reaction was then carried out at the optimal pH range (5.0− 6.0) under vigorous stirring overnight at room temperature. The coupling reaction for a longer period of time did not increase the final degree of malonate conjugation on MPC. The product was purified by dialysis (MWCO = 1000 Da) against DI water (7 × 1000 mL) for 7 days with water change every 24 h. The lyophilized product was obtained as a colorless powder (0.048 g, 96% yield). 1H NMR (300 MHz, D2O): At pH 2: δ 3.66 (m, 48H, −OCH2CH2− of PEG), 3.36 (s, 3H, −OCH3 of PEG), 3.3−3.2 (s, 2H, −CH2− of malonic amide), 3.2−2.9 (broad s, 1H, −CH− of chitosan-free amine group), 2.87 (s, 2H, −CH2− of PEGylated amide), and 2.03−1.99 (s, 3H, −COCH3 of acetylated chitosan). At pH 7: δ 3.66 (m, 48H, −OCH2CH2− of PEG), 3.36 (s, 3H, −OCH3 of PEG), 3.09 (s, 2H, −CH2− of malonic amide), 3.02−2.89 (broad s, 1H, −CH− of chitosan-free amine group), 2.86 (s, 2H, −CH2− of PEGylated amide), and 2.03−1.99 (s, 3H, −COCH3 of acetylated chitosan). FTIR: At pH 4.0: 3400−2400 cm−1 (medium, O−H stretch of carboxylic acid), 3300 cm−1 (weak, N−H stretch of 2′ amide), 1705 cm−1 (medium, CO stretch of carboxylic acid), 1630 cm−1 (strong, CO stretch of amide, amide I), 1520 cm−1 (medium, N−H bending of amide, amide II), 1382 cm−1 (weak, amide III), and 1249 cm−1 (medium, C−O stretch of carboxylic acid). At pH 7.0: 1562 cm−1 (medium, νasym band from COO−) and 1450 cm−1 (strong, νsym band from COO−). Preparation of PtII-Conjugated MPC (PtII-MPC). To a test tube (12 × 75 mm) equipped with a magnetic stir bar was added cisplatin, (cis-[PtII(NH3)2Cl2], 40 mg, 133 μmol) dissolved in warm water (18 mL). To the resulting solution was added silver nitrate (41 mg, 241 μmol, 1.8 equiv) with a trace amount of nitric acid, and the reaction mixture was allowed to stir for 3 days at room temperature in the dark. To completely precipitate out the slowly forming silver chloride, the solution was stored at 4 °C overnight, and the completion of reaction was determined by the addition of a small amount of saturated NaCl solution to an aliquot of the reaction mixture. Silver chloride precipitate was removed by filtration with a centrifugal filter followed by a syringe filter (0.45 μm pore), yielding cis-[PtII(NH3)2(H2O)2]2+ solution (10.7 mM). To a test tube equipped with a magnetic stir bar was added the MPC precursor (20 mg) dissolved in DI water (4 mL), and the solution pH was adjusted to ∼5.0. To the resulting solution was added cis-[PtII(NH3)2(H2O)2] (5.0 equiv to the available malonic amide residues in MPC) followed by stirring at 37 °C for 72 h. As the pH of the reaction mixture slightly decreased due to the generation of proton from the malonic amide ligand coordinated to PtII ions, the reaction progress could be monitored by checking the pH during the reaction. To complete the reaction, pH was adjusted to ∼5.0 by the addition of diluted NaOH solution. The unreacted PtII ions were removed by centrifugal filtration (MWCO = 10 kDa), and the final product was obtained as a yellowish solution. 1H NMR (300 MHz, D2O): δ 3.68
lytic chitosan (MPC) was prepared, which can flocculate at the pH condition near the isoelectric point. The resulting PtIIconjugated MPC (PtII-MPC) exhibited substantially attenuated solution viscosity because of the nanoscale conformational change of PtII-MPC by the PtII-mediated formation of crosslinked gel-like chitosan networks. Under mild acidic conditions, such as those in the endosomal environment, the coordinated PtII ions can be released from the MPC scaffolds, which leads to the reformation of the PtII-free polyampholytic chitosan structure, possibly allowing for the membrane perturbation by the known chitosan property.8
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EXPERIMENTAL SECTION
Materials. Chitosan (low molecular weight, Mν = 125 kDa estimated, 14.5% acetylation), malonic acid (99%), poly(ethylene glycol) methyl ether (mPEG550-COOH, average Mn = 550), N-ethylN′-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), cisplatin (cis-diamminedichloroplatinum(II) >99.9%, trace metal basis), and nitric acid (70%) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and used without further purification. Regenerated cellulose (RC) dialysis membranes were purchased from Spectrum Laboratories (Thermo Scientific, Waltham, MA). Deionized water (DI) was obtained from Human Power I+ ScholarUV (Human Corporation, Seoul, Korea) (18.2 MΩ cm resistivity). Measurements. Instrumental analyses were carried out at the Catholic University of Korea, Center for Research Facilities. Fouriertransformed nuclear magnetic resonance (NMR) spectroscopy was carried out on an AVANCE III 300 MHz spectrometer. Chemical shifts of 1H NMR spectra are reported in ppm against the residual solvent resonance as the internal standard (CDCl3 = 7.27 ppm, D2O = 4.8 ppm). The samples were prepared by dissolving 5 ± 1 mg in 0.7 mL of deuterated solvents in a 5 mm NMR tube. Ultraviolet−visible (UV−vis) absorption spectra were obtained using a Lambda 35 spectrophotometer (Perkin Elmer, Waltham, MA). Attenuated total reflectance−Fourier transform infrared (ATR−FTIR) spectroscopy measurements were carried out with a Bruker TENSOR II FTIR spectrometer equipped with a Miracle Single Reflection Horizontal ATR accessory (PIKE Technologies, Inc., Madison, WI). Thermogravimetric analysis (TGA) was carried out with a TGA 4000 analyzer (PerkinElmer, Waltham, MA) under a nitrogen (N2) atmosphere. The samples (10−20 mg) were heated from 40 to 900 °C at a constant heating rate of 10 °C/min. The solution viscosity was measured with an Ubbelohde viscometer (SI Analytics GmbH, Mainz, Germany). For the measurements, a dilute solution of each sample was prepared by dissolving 200 mg of the sample in 20 mM acetatebuffered solution (pH 4.0, 100 mM NaCl), and the measurements were carried out at 25 °C. Dynamic light scattering (DLS) and zeta (ζ) potential measurements were carried out with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK), which uses a 633 nm He−Ne laser with the noninvasive backscatter method (detection at 173° scattering angle). Correlation data were fitted by the method of cumulants to obtain the translational diffusion coefficient (Dt), from which the hydrodynamic diameters (DH) of the nanoparticles were calculated by the Stokes−Einstein equation. The size distribution of the nanoparticles was obtained by the non-negative least-squares (NNLS) analysis. The ζ-potential of each sample was calculated from the electrophoretic mobility values using the Smoluchowski equation. Unless noted otherwise, all samples were dispersed in 10 mM phosphate buffer (pH 7.4) or 10 mM acetate buffer (pH 4.0) for measurements. The data reported represent an average of ten measurements with five scans each. Energy-filtering transmission electron microscopy (EF-TEM) observation was carried out with a LIBRA 120 microscope (Carl Zeiss, Oberkochen, Germany) with a beam voltage of 120 kV and a slow-scan charge-coupled device (CCD) at the National Instrumentation Center for Environmental Management of the Seoul National University. The sample solution (50 μL) was placed on a copper grid B
DOI: 10.1021/acs.langmuir.7b02043 Langmuir XXXX, XXX, XXX−XXX
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Langmuir Scheme 1. Synthetic Scheme of PtII-MPC
Figure 1. (A) 1H NMR spectra of PEG-chitosan at pH 7 (red) and pH 2 (blue) in D2O. Also shown is the 1H NMR spectrum of native chitosan as a comparison (green). The peak “Ac” at 2.1 ppm is due to the N-acetyl moieties attached to the small portion of GlcN residues (∼14%) in the chitosan backbone. (B) 1H NMR spectra of MPC at pH 7 (red) and pH 2 (blue) in D2O. As a comparison, the 1H NMR spectrum of free malonic acid measured in CDCl3 is plotted at the bottom (green). (C) FTIR spectra of MPC prepared at pH 4 (blue) and pH 7 (red). (D) Hydrogen ion titration curves of native chitosan, PEG-chitosan, and MPC with 0.1 M aqueous NaOH. *In native chitosan, the pH change above ∼6.2 cannot be measured because of significant precipitation. (m, 48H, −OCH2CH2− of PEG), 3.36 (s, 3H, −OCH3 of PEG), 3.19 (s, 2H, −CH2− of malonic amide), 2.88 (s, 2H, −CH2− of PEGylated amide), and 2.02 (s, 3H, −COCH3 of acetylated chitosan). FTIR: 1622 cm−1 (medium, νasym band from COO−) and 1325 cm−1 (strong, νsym band from COO−). Determination of PtII Concentration in PtII-MPC. The amount of PtII pharmacophore in PtII-MPC was determined using a modified literature procedure.10 Briefly, an aliquot of PtII-MPC (200 μL) was added to aqueous HCl (2 M, 50 μL) and incubated for ∼3 h to release the PtII ions from PtII-MPC and convert them into cisplatin. Then, the quantity of PtII ions was determined by colorimetric analysis with 10% SnCl2−HCl (2 N) solution with UV−vis spectroscopy, using the predetermined extinction coefficient (ε) of the Sn−Pt complex (19062 M−1 cm−1 at λmax = 402 nm), which was obtained from the PtII standard solution and the calibration curve (Figure S2 in Supporting Information).
PtII Release Studies from PtII-MPC. Cumulative PtII-release amounts were monitored at 37 °C in 10 mM phosphate buffer (pH 7.4) or 10 mM acetate buffer (pH 5.0) containing either 150 mM NaCl or NaNO3 under the isotonic condition. To this end, PtII-MPC solutions (8.25 mL, 8.03 mM in terms of PtII concentration) were added into a semipermeable dialysis tube (MWCO = 10 kDa) and dialyzed against a buffer solution at 37 °C under vigorous stirring. An aliquot of PtII-MPC was collected at a predetermined time, and the platinum contents were measured by the aforementioned SnCl2mediated colorimetric analyses.
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RESULTS AND DISCUSSION Synthesis and Characterization of Chemically MPC. MPC was prepared via two-step modification with methoxyterminated PEG-acid (mPEG-acid, Figure S1 in Supporting C
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Figure 2. (A) 1H NMR spectra of PtII-MPC and PtII-free MPC precursor. (B) FTIR spectra of PtII-MPC, PtII-free MPC precursor, and free cisplatin. (C) TGA thermograms of PtII-MPC, PtII-free MPC precursor, and native chitosan. (Inset) Differential thermogravimetric (DTG) curves of each sample for the estimation of the decomposition temperature (Td).
(νasym) and symmetric (νsym) stretchings of the deprotonated carboxylate anions (COO−), respectively. Taken together, both the characteristic amide bands and the pH-dependent change of carboxylate stretching demonstrate the successful end-tethering of malonic amides on MPC, supporting the presence of acid groups for the subsequent coordination of the cisplatin pharmacophore. Given that the malonate conjugation produces a number of anionic charges on the backbone of chitosan scaffolds, the resulting MPC can exhibit a polyampholytic structure, which possesses both acidic and basic groups on a single polymer chain. Hence, the MPC solution was titrated with aqueous NaOH solution, and the pH change was monitored to estimate the ampholytic property of MPC (Figure 1D). As expected, both native chitosan and PEG-chitosan exhibited only a single plateau at pH ∼6, which corresponds to the pKa value of GlcN ammonium groups. However, the unmodified native chitosan started to precipitate at pH ∼6.2, exhibiting limited aqueous solubility (black curve). In stark contrast, MPC showed two distinct inflection points (blue curve) attributed to the presence of both terminal acid groups in malonic amides and GlcN ammonium groups, which clearly demonstrates the well-defined ampholytic property of MPC. Synthesis and Characterization of Cisplatin-Conjugated MPC (PtII-MPC). The chitosan-tethered malonic amides can be employed as a bidentate chelating ligand, which has been used in a similar manner to coordinate the Pt II pharmacophores in several polymer-based delivery systems, such as poly[N-(2-hydroxypropyl)methacrylamide] (poly[HPMA])13 and hyaluronans.14 For the enhanced binding reactivity of the PtII pharmacophore, the labile chloride ions were initially removed from cisplatin by Ag+-mediated precipitation, and the resulting cis-[Pt(NH3)2(H2O)2]2+ ions (PtII) were subjected to bidentate chelation with the malonic amide ligands on the MPC precursor (Scheme 1). The high yield of PtII conjugation was achieved by the simple agitation of MPC solution with a 5-fold excess molar amount of PtII ions at 37 °C for 72 h. Although the N,O-chelation of the PtII compound can be completed in a short time,13 we have employed a longer incubation time to allow for the macromolecular conformational change of PtII-MPC into a compact nanostructure (see the next section). The reaction progress can be monitored by checking the solution pH because of the steady generation of proton ions through the formation of bidentate PtII-malonic amide complexes,15 and the solution pH of the reaction mixture was adjusted to be slightly acidic to
Information) and malonic acid via carbodiimide-mediated peptide coupling (Scheme 1). Because of the limited aqueous solubility of native chitosan above pH ∼5.5, the initial PEGcoupling (PEGylation) reaction was proceeded in a mild acidic solution, whereas the resulting PEG-chitosan allows for the subsequent malonate conjugation carried out at an optimal pH range (pH 5.0−6.0) for carbodiimide-mediated peptide coupling by increased solubility. On the basis of 1H NMR resonance integrations, ∼13% PEGylation and ∼10% malonate conjugation have been achieved for MPC formation (Figure 1A,B). The comparison of the 1H NMR spectra observed under different pH conditions further provides additional chemical information on the molecular structures of the PEG-chitosan derivatives (Figure 1A). By lowering the solution pH, proton peak 3 (originally appeared at 2.8 ppm at pH 7) moved downfield to ∼3.2 ppm because of the significant protonation at GlcN amine groups (blue spectrum). At the same time, several distinct proton peaks newly appeared at 3.59−3.67 ppm under acidic conditions (dashed box in the blue spectrum), suggesting the PEG-mediated hydrogen bond formation between the oxygen atoms of the grafted mPEG chains and the protonated ammonium ions of the GlcN residues, as previously observed for the PEG-modified chitosan under acidic conditions.11 Similarly, the pH-dependent resonance peak shift of the malonate protons has also been observed from MPC. In conjunction with the previously reported 1H NMR results of acid anhydride-modification,7,12 proton peak 8 from malonic amide was initially observed at 3.1 ppm under neutral conditions (red spectrum in Figure 1B), whereas the acidification below the pKa value of the malonic acid (pKa ≈ 2.8) induced the downfield shift of peak 8 to 3.3 ppm (blue spectrum) by significant protonation, suggesting the presence of terminal acid groups on the malonic amide moieties tethered on MPC. The successful ligation of malonates for subsequent PtII coordination was further verified by the comparison of pHdependent FTIR spectra of MPC (Figure 1C). At pH 4.0, the presence of three characteristic amide bands (amide I at 1630 cm−1, amide II at 1520 cm−1, and amide III at 1382 cm−1 in the blue spectrum) clearly indicates the amide linkage of the malonate groups on the GlcN amines of MPC. On the other hand, a distinct ν(CO) stretching band (1705 cm−1) from the acid groups completely disappeared at pH 7.0 (red spectrum), whereas two characteristic bands newly appeared at 1562 and 1350 cm−1, corresponding to the antisymmetric D
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Figure 3. (A) Number-based hydrodynamic diameter (DH) distribution of PEG-chitosan, MPC precursor, and PtII-MPC measured by DLS at pH 4.0 (top) and pH 7.4 (bottom). (B) Zeta potential (ζ) of PEG-chitosan, MPC, and PtII-MPC at pH 4.0, 7.4, and 9.0. (C) Schematic illustration of the morphology changes in PtII-MPC and PtII-free MPC precursor under acidic and neutral conditions. (D−G) TEM images of (D) well-dispersed MPC at pH 2.0, (E) MPC flocculated at pH 7.4, (F) PtII-MPC at pH 7.4, and (G) PtII-MPC after the partial dissociation of PtII ions by dialysis at pH 5.0.
prevent the irreversible formation of μ-hydroxo-diplatinum precipitates.16 After the purification of the unreacted PtII species by centrifugal filtration (MWCO 10 kDa), the PtII loading amounts estimated by SnII-mediated colorimetric quantification10 revealed an ∼12.8 wt % PtII-loading yield in the resulting PtII-MPC. (Figure S2 in Supporting Information). The successful PtII conjugation on PtII-MPC was initially verified by the 1H NMR analysis (Figure 2A). As expected from the coordination of electron-deficient cationic PtII compounds,13 proton peak 8 of the PtII-bound malonic amide ligands shifted downfield to 3.4 ppm from its initial peak position at 3.3 ppm for the PtII-free malonic amide moieties in the MPC precursor. Interestingly, the peak intensity of proton 8 was apparently suppressed because of the increased hydrophobicity of the coordinated PtII compounds,17 indicating the possible formation of a hydrophobically cross-linked gel-like structure of PtII-MPC by PtII conjugation (see the next section).14 Also supporting the PtII conjugation on PtII-MPC is the FTIR analysis, which shows the dramatic changes in the characteristic stretching bands of the malonic amide ligands (Figure 2B). Upon PtII coordination, the νasym band from the carboxylate groups in the malonic amide ligands shifted to a higher wavenumber (1622 cm−1 in the red spectrum), whereas the νsym band shifted to a lower value (1325 cm−1), resulting in an increased separation frequency (Δν) between the νsym and νasym bands compared to that of the PtII-free malonic amide groups in the MPC precursor (blue spectrum). This is consistent with the previous report that demonstrated the increased Δν of carboxylate ligands when a metal complex was formed through a simple monodentate coordination on the carboxylate group, ascribed to the elimination of the chemical
equivalency between the two oxygen atoms in the carboxylate group.18 As such, in conjunction with the previously reported bidentate chelation of PtII ions for the amide-modified malonate ligands, 13 we have postulated that the Pt II pharmacophores were bound to the malonic amide ligands through the bidentate N,O-chelation binding mode (Figure 2B, inset). TGA additionally revealed the successful PtII coordination to the MPC scaffolds, showing a characteristic thermolysis profile of PtII-MPC compared to that of PtII-free chitosan derivatives (Figure 2C). For a clear estimation of inflection points, DTG profiles were also obtained from the first derivatives of TGA thermograms, and they are demonstrated in the inset of Figure 2C. After dehydration at 50−100 °C, both PtII-MPC and the PtII-free MPC precursor exhibited two-step thermal degradation, showing two distinct decomposition temperatures (Td) at 130−160 °C (first Td) and 210−270 °C (second Td). By contrast, the native chitosan exhibited only a single Td at 250 °C, consistent to the previously reported TGA thermogram of chitosan.19 Presumably, the first Td in both MPC and PtII-MPC can be attributed to the decarboxylation at the malonic amide groups tethered on the chitosan backbone,20 which is completely absent in the native chitosan. The TGA thermograms of both MPC and PtII-MPC also revealed an ∼10−12 wt % loss at the first Td, which is a comparable amount of malonate groups on chitosan as measured by 1H NMR in Figure 1B. Notably, PtII-MPC exhibited both first and second Td at lower temperatures compared to those of the PtII-free MPC precursor, which can be attributed to the catalytic activity of platinum compounds in the thermolysis.21 After the complete thermolysis at ∼800 °C, PtII-MPC exhibited ∼10 wt % of mass remaining without further decomposition, which E
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Figure 4. (A) Comparison of reduced viscosity (ηred) measured in 20 mM acetate-buffered solution (pH 4.0, 100 mM NaCl) at 25 °C. (B) pH- and ion-sensitive PtII release profiles for PtII-MPC at 37 °C under neutral (pH 7.4) or acidic (pH 5.0) solution containing either 150 mM NaCl or NaNO3.
corresponds well to the amount of PtII compounds coordinated to the MPC scaffolds. In stark contrast, both MPC and native chitosan exhibited the entire mass loss at 800 °C. Taken together, the TGA and DTG thermograms demonstrated the characteristic thermal property of PtII-MPC, further verifying our postulation on the chemical structure of PtII-MPC. Morphological Changes and Colloidal Properties of PtII-MPC. As the malonate ligation provides polyampholytic properties to the modified chitosan derivatives, we monitored the pH-dependent changes in the colloidal structures of MPC. Initially, DLS measurements exhibited the mean hydrodynamic diameter (DH) of PEG-chitosan being constant over the pH 4.0−7.4 range (DH = 19 ± 4 nm at pH 4.0 and DH = 22 ± 11 nm at pH 7.4, green lines in Figure 3A) possibly due to the enhanced aqueous solubility of chitosan chains by PEGylation. We note that the number-based distribution has been employed in Figure 3A, which agrees well with the size directly visualized by our TEM observation as commonly reported for polymeric nanoparticles22 (vide infra). In stark contrast, the malonate conjugation induced a significant flocculation in MPC at pH 7.4 with substantially increased DH (320 ± 20 nm), whereas the MPC flocculants were dissolved under acidic conditions, showing the mean DH of 28 ± 5 nm at pH 4.0 (blue lines in Figure 3A). Such a pH-dependent solubility change of MPC can be attributed to its polyampholytic structure, which often aggregates at the pH conditions near the isoelectric point because of the reduced electrostatic interpolymer repulsions.23,24 In conjunction with the DLS measurements, the polyampholytic structures of chitosan derivatives can be additionally confirmed by monitoring the zeta potential under various pH conditions. As expected from the cationic GlcN residues in the chitosan scaffolds, a highly positive ζ-potential was observed for PEG-chitosan at both pH 4.0 and 7.4 (71.9 ± 4.4 mV at pH 4.0 and 23.4 ± 1.7 mV at pH 7.4, green line in Figure 3B). However, the subsequent ligation of the anionic malonate groups on MPC substantially neutralized the surface charges; the ζ-potential of MPC exhibited 46.6 ± 5.6 mV at pH 4.0, whereas it further decreased to 7.0 ± 0.4 and −3.8 ± 0.8 mV at pH 7.4 and 9.0, respectively (blue line in Figure 3B), verifying the significant attenuation of electrostatic repulsion between the MPC chains by charge neutralization. The TEM observation also revealed highly flocculated structures of MPC at pH 7.4 (Figure 3E), whereas they were homogeneously dispersed into the particulate morphology at pH 2.0 (Figure 3D). Together with the aforementioned DLS results,
these pH-dependent zeta potential measurements further suggest that the colloidal morphology of MPC can indeed be modulated by the ampholytic charge conversion of MPC scaffolds under various pH conditions. Given that the PtII conjugation was carried out in a slightly acidic solution to prevent the μ-hydroxo-diplatinum complex formation,16 a well-dispersed colloidal structure of MPC was expected during the PtII conjugation reaction under mild acidic conditions, where the dispersed colloidal structure of the parent MPC can be preserved to the subsequent PtII-MPC (Figure 3C). Indeed, the mean DH of PtII-MPC (DH = 31 ± 8 nm, red lines in Figure 3A) measured by DLS under acidic conditions exhibited a size similar to that of the PtII-free MPC precursor (DH = 28 ± 5 nm, blue lines in Figure 3A). Interestingly, the PtII coordination inhibited the flocculation of PtII-MPC under neutral conditions, showing a mean DH of 32 ± 19 nm in contrast to the PtII-free MPC precursor, which flocculated significantly under the same pH conditions. As hypothesized previously, this well-dispersed colloidal structure of PtII-MPC under neutral conditions can be attributed to the PtII-mediated formation of MPC gel-like networks by hydrophobic crosslinking, which induces the segregation of PtII-MPC chains into a collapsed globular structure in aqueous solution. Additional DLS measurements under isotonic conditions equivalent to the physiological media (150 mM NaCl) also revealed a similar size of PtII-MPC but a narrow distribution (DH = 28 ± 9 nm) because of the ion-mediated charge screening effect25 (Figure S3 in Supporting Information). Consistently, previous reports demonstrated that the coordination of divalent metal ions to the carboxylate-containing polymers induced a globular segregation of the resulting metal-coordinated polymer complexes.17,26 Hence, the coordinated PtII pharmacophores in our PtII-MPC should become more hydrophobic, eventually leading to the formation of globular gel structures by hydrophobic cross-linking. Indeed, TEM observation clearly supported the PtII-mediated nanoscale particle formation of PtII-MPC under neutral conditions (Figure 3F) in contrast to the highly flocculated MPC precursor under the same pH conditions. As the PtII coordination induced significant morphology changes, we compared the pH-dependent ζ-potential of PtIIMPC against those of other chitosan derivatives. Not surprisingly, the highly positive ζ-potential of PtII-MPC (47.3 ± 3.7 mV, red line in Figure 3B) under acidic conditions was essentially unchanged from that of the parent MPC (46.6 ± 5.6 mV), which can be ascribed to the modification of the anionic F
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Langmuir Scheme 2. pH-Sensitive Release of Cisplatin Pharmacophore, cis-[Pt(NH3)2(H2O)2]2+ from PtII-MPC
malonate groups on both MPC and PtII-MPC by protonation and PtII coordination, respectively. However, unlike the MPC precursor that exhibited neutralized charges under high pH conditions, PtII-MPC still retained the positive ζ-potentials, showing 20.8 ± 1.6 and 19.3 ± 1.5 mV at pH 7.4 and 9.0, respectively. This aspect suggests the chemical basis for the enhanced colloidal stability of PtII-MPC by electrostatic repulsion in the current stage, whereas the positive surface charge should be passivated for further biological applications because of the potential toxicity of cationic materials. Viscosity Changes and PtII Release Profiles of PtIIMPC. The PtII-mediated segregation of chitosan scaffolds induced dramatic changes in the viscosity of PtII-MPC. Generally, the solvent-induced conformational changes into a collapsed globular structure often decrease the viscosity of the polymer solution because of the reduced intermolecular interactions among the segregated particles.27 Hence, such morphology-dependent viscosity changes have been commonly observed for a wide range of polymer systems,28,29 and we have also compared the reduced viscosity (ηred) of PtII-MPC against those of other chitosan derivatives in diluted solution at 25 °C (Figure 4A). As expected from the known semiflexible conformation of chitosan30 and its PEGylated derivative31 under mild acidic conditions, the native chitosan exhibited a sharp increase in ηred as a function of the concentration, whereas relatively low ηred was observed for PEG-chitosan over the same concentration range. By contrast, MPC exhibited mostly increased ηred compared to that of PEG-chitosan, possibly due to the ampholytic structures of MPC, which can enhance the interchain interactions.32 Notably, PtII-MPC exhibited much attenuated ηred with relatively small changes as a function of the polymer concentration above ∼0.0014 g/mL. As hypothesized previously, this low ηred of PtII-MPC can be attributed to the conformational segregation of chitosan chains into a compact globular structure via PtII coordination. Interestingly, ηred of PtII-MPC increased inversely as the concentration decreased below ∼0.0014 g/mL, approaching that of the PtII-free MPC precursor. We ascribed such a different aspect of ηred at a low concentration of PtII-MPC possibly to the reversible dissociation of PtII ions from the malonic amide ligands on MPC scaffolds by the significant dilution of PtII-MPC under mild acidic conditions. (Figure S4 in Supporting Information). The reversible dissociation of PtII pharmacophores in a pHdependent manner has been commonly observed with versatile chelating ligands such as amido malonate13 and N-acetyl lysine,15 which possess a bidentate N,O-chelating binding mode similar to that of our malonic amide ligand. Hence, the temporal release of PtII from PtII-MPC was monitored under various pH and ionic conditions to verify the reversible dissociation of PtII ions. To this end, PtII-MPC was dialyzed against pH 5.0 or 7.4 buffered solution, each of which contains either 150 mM chlorides or equimolar nitrates for the isotonic condition. The PtII release rate in the chloride-free solution was
additionally monitored as the control because the labile chloride ion has been known to enhance the dissociation of PtII-coordination bonds33 in contrast to the nitrate, which is relatively inert for PtII compounds. Indeed, PtII-MPC was highly stable under chloride-free neutral conditions, showing only 8.2% release of PtII ions after 2 days (blue dashed line in Figure 4B). In stark contrast, the PtII release rate was significantly increased under acidic conditions: ∼42% PtII was released during the initial 36 h and ∼57% release was achieved after 96 h (green line). This acid-sensitive release of PtII can be attributed to the acid-labile malonic amide ligand, which can promote the dissociation of the PtII complex under acidic conditions (Scheme 2). After the 48 h-incubation of PtII-MPC at pH 5.0, TEM observation revealed the intriguing morphology change of PtII-MPC into irregular shapes (Figure 3G) by the release of PtII ions, which leads to the slightly flocculated MPC structure. The presence of chloride anions in the acidic medium further increased the PtII release rate by the Cl−-mediated dechelation process of PtII ions, showing an ∼63% release of PtII after the initial 24 h and more than 70% release at 48 h (red line).
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CONCLUSIONS In summary, we have demonstrated the facile formation of nanoscale gel-like chitosan networks via PtII-mediated hydrophobic cross-linking of chemically modified chitosan derivatives, where the short mPEG and malonic acids have been ligated as a colloidal stabilizer and a chelating ligand for PtII pharmacophores, respectively. The malonic amide ligands on the chitosan scaffolds can coordinate the PtII ions in the N,Ochelating bidentate binding mode, which then leads to the conformational change of PtII-MPC into nanoparticulate aggregation. As a result, PtII-MPC exhibited a substantially attenuated solution viscosity, which is highly beneficial for the facile injection and filtration processes, whereas the positive surface charges of PtII-MPC arising from the cationic PtII pharmacophores can be regulated by the surface passivation with anionic biocompatible polymers such as alginic acids for further biological applications.34 Once the PtII pharmacophores are released in a pH- and ion-sensitive manner, the remaining PtII-free MPC scaffolds can flocculate under mild acidic conditions such as those in cellular endosomes, possibly allowing for the facilitated endosomal escape of drug molecules by the known membrane perturbation property of chitosan.8
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b02043. 1 H NMR spectra of mPEG and oxidized mPEG, UV−vis absorption spectra and calibration curve for the SnIImediated colorimetric quantification of PtII compounds, pH- and ion-dependent size changes of PtII-MPC, and G
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Langmuir
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time-dependent coordination stability analysis of PtIIMPC (PDF)
(12) Xu, P.; Bajaj, G.; Shugg, T.; Van Alstine, W. G.; Yeo, Y. Zwitterionic Chitosan Derivatives for pH-Sensitive Stealth Coating. Biomacromolecules 2010, 11, 2352−2358. (13) Sood, P.; Thurmond, K. B.; Jacob, J. E.; Waller, L. K.; Silva, G. O.; Stewart, D. R.; Nowotnik, D. P. Synthesis and Characterization of AP5346, a Novel Polymer-Linked Diaminocyclohexyl Platinum Chemotherapeutic Agent. Bioconjugate Chem. 2006, 17, 1270−1279. (14) Ohta, S.; Hiramoto, S.; Amano, Y.; Sato, M.; Suzuki, Y.; Shinohara, M.; Emoto, S.; Yamaguchi, H.; Ishigami, H.; Sakai, Y.; Kitayama, J.; Ito, T. Production of Cisplatin-Incorporating Hyaluronan Nanogels via Chelating Ligand−Metal Coordination. Bioconjugate Chem. 2016, 27, 504−508. (15) Lee, S.-M.; O’Halloran, T. V.; Nguyen, S. T. Polymer-Caged Nanobins for Synergistic Cisplatin−Doxorubicin Combination Chemotherapy. J. Am. Chem. Soc. 2010, 132, 17130−17138. (16) Appleton, T. G.; Hall, J. R.; Neale, D. W.; Thompson, C. S. M. Reactions of the cis-diamminediaquaplatinum(II) cation with 2aminomalonic acid and its homologs, aspartic and glutamic acids. Rearrangements of metastable complexes with carboxylate-bound ligands to N,O-chelates and formation of di- and trinuclear complexes. Inorg. Chem. 1990, 29, 3985−3990. (17) Nishiyama, N.; Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Preparation and Characterization of Self-Assembled Polymer−Metal Complex Micelle from cisDichlorodiammineplatinum(II) and Poly(ethylene glycol)−Poly(α,βaspartic acid) Block Copolymer in an Aqueous Medium. Langmuir 1999, 15, 377−383. (18) Deacon, G. B.; Phillips, R. J. Relationships between the carbonoxygen stretching frequencies of carboxylato complexes and the type of carboxylate coordination. Coord. Chem. Rev. 1980, 33, 227−250. (19) Kaya, M.; Akata, I.; Baran, T.; Menteş, A. Physicochemical Properties of Chitin and Chitosan Produced from Medicinal Fungus (Fomitopsis pinicola). Food Biophys. 2015, 10, 162−168. (20) Smiglak, M.; Holbrey, J. D.; Griffin, S. T.; Reichert, W. M.; Swatloski, R. P.; Katritzky, A. R.; Yang, H.; Zhang, D.; Kirichenko, K.; Rogers, R. D. Ionic liquids via reaction of the zwitterionic 1,3dimethylimidazolium-2-carboxylate with protic acids. Overcoming synthetic limitations and establishing new halide free protocols for the formation of ILs. Green Chem. 2007, 9, 90−98. (21) Patel, H. A.; Bocchini, S.; Frache, A.; Camino, G. Platinum nanoparticle intercalated montmorillonite to enhance the char formation of polyamide 6 nanocomposites. J. Mater. Chem. 2010, 20, 9550−9558. (22) Shah, P. N.; Lin, L. Y.; Smolen, J. A.; Tagaev, J. A.; Gunsten, S. P.; Han, D. S.; Heo, G. S.; Li, Y.; Zhang, F.; Zhang, S.; Wright, B. D.; Panzner, M. J.; Youngs, W. J.; Brody, S. L.; Wooley, K. L.; Cannon, C. L. Synthesis, Characterization, and In Vivo Efficacy of Shell CrossLinked Nanoparticle Formulations Carrying Silver Antimicrobials as Aerosolized Therapeutics. ACS Nano 2013, 7, 4977−4987. (23) Sfika, V.; Tsitsilianis, C. Association Phenomena of Poly(acrylic acid)-b-poly(2-vinylpyridine)- b-poly(acrylic acid) Triblock Polyampholyte in Aqueous Solutions: From Transient Network to Compact Micelles. Macromolecules 2003, 36, 4983−4988. (24) Casettari, L.; Vllasaliu, D.; Mantovani, G.; Howdle, S. M.; Stolnik, S.; Illum, L. Effect of PEGylation on the Toxicity and Permeability Enhancement of Chitosan. Biomacromolecules 2010, 11, 2854−2865. (25) Huang, Y.; Lapitsky, Y. Monovalent Salt Enhances Colloidal Stability during the Formation of Chitosan/Tripolyphosphate Microgels. Langmuir 2011, 27, 10392−10399. (26) Ikeda, Y.; Beer, M.; Schmidt, M.; Huber, K. Ca2+ and Cu2+ Induced Conformational Changes of Sodium Polymethacrylate in Dilute Aqueous Solution. Macromolecules 1998, 31, 728−733. (27) Seo, M.; Beck, B. J.; Paulusse, J. M. J.; Hawker, C. J.; Kim, S. Y. Polymeric Nanoparticles via Noncovalent Cross-Linking of Linear Chains. Macromolecules 2008, 41, 6413−6418. (28) Yamauchi, K.; Lizotte, J. R.; Long, T. E. Thermoreversible Poly(alkyl acrylates) Consisting of Self-Complementary Multiple Hydrogen Bonding. Macromolecules 2003, 36, 1083−1088.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Sang-Min Lee: 0000-0003-0430-387X Author Contributions
R.-H.K. and J.-Y.K. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2016R1D1A1B03934733) and the 2014 Research Fund of the Catholic University of Korea.
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REFERENCES
(1) Li, Y.; Maciel, D.; Rodrigues, J.; Shi, X.; Tomás, H. Biodegradable Polymer Nanogels for Drug/Nucleic Acid Delivery. Chem. Rev. 2015, 115, 8564−8608. (2) Bodnar, M.; Hartmann, J. F.; Borbely, J. Preparation and Characterization of Chitosan-Based Nanoparticles. Biomacromolecules 2005, 6, 2521−2527. (3) Aktaş, Y.; Yemisci, M.; Andrieux, K.; Gürsoy, R. N.; Alonso, M. J.; Fernandez-Megia, E.; Novoa-Carballal, R.; Quiñoá, E.; Riguera, R.; Sargon, M. F.; Ç elik, H. H.; Demir, A. S.; Hıncal, A. A.; Dalkara, T.; Ç apan, Y.; Couvreur, P. Development and Brain Delivery of Chitosan−PEG Nanoparticles Functionalized with the Monoclonal Antibody OX26. Bioconjugate Chem. 2005, 16, 1503−1511. (4) Kim, J.-H.; Kim, Y.-S.; Park, K.; Lee, S.; Nam, H. Y.; Min, K. H.; Jo, H. G.; Park, J. H.; Choi, K.; Jeong, S. Y.; Park, R.-W.; Kim, I.-S.; Kim, K.; Kwon, I. C. Antitumor efficacy of cisplatin-loaded glycol chitosan nanoparticles in tumor-bearing mice. J. Controlled Release 2008, 127, 41−49. (5) Zhao, Z.; He, M.; Yin, L.; Bao, J.; Shi, L.; Wang, B.; Tang, C.; Yin, C. Biodegradable Nanoparticles Based on Linoleic Acid and Poly(βmalic acid) Double Grafted Chitosan Derivatives as Carriers of Anticancer Drugs. Biomacromolecules 2009, 10, 565−572. (6) Nascimento, A. V.; Singh, A.; Bousbaa, H.; Ferreira, D.; Sarmento, B.; Amiji, M. M. Combinatorial-Designed Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles for Encapsulation and Delivery of Lipid-Modified Platinum Derivatives in Wild-Type and Resistant Non-Small-Cell Lung Cancer Cells. Mol. Pharmaceutics 2015, 12, 4466−4477. (7) Chen, C.-K.; Wang, Q.; Jones, C. H.; Yu, Y.; Zhang, H.; Law, W.C.; Lai, C. K.; Zeng, Q.; Prasad, P. N.; Pfeifer, B. A.; Cheng, C. Synthesis of pH-Responsive Chitosan Nanocapsules for the Controlled Delivery of Doxorubicin. Langmuir 2014, 30, 4111−4119. (8) Fang, N.; Chan, V. Interaction of Liposome with Immobilized Chitosan during Main Phase Transition. Biomacromolecules 2003, 4, 581−588. (9) Fishman, A.; Acton, A.; Lee-Ruff, E. A Simple Preparation of PEG-Carboxylates by Direct Oxidation. Synth. Commun. 2004, 34, 2309−2312. (10) Milner, O. I.; Shipman, G. F. Colorimetric Determination of Platinum with Stannous Chloride. Anal. Chem. 1955, 27, 1476−1478. (11) Jang, J. H.; Choi, Y. M.; Choi, Y. Y.; Joo, M. K.; Park, M. H.; Choi, B. G.; Kang, E. Y.; Jeong, B. pH/temperature sensitive chitosang-(PA-PEG) aqueous solutions as new thermogelling systems. J. Mater. Chem. 2011, 21, 5484−5491. H
DOI: 10.1021/acs.langmuir.7b02043 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir (29) Park, T.; Zimmerman, S. C. Interplay of Fidelity, Binding Strength, and Structure in Supramolecular Polymers. J. Am. Chem. Soc. 2006, 128, 14236−14237. (30) Morris, G. A.; Castile, J.; Smith, A.; Adams, G. G.; Harding, S. E. Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach. Carbohydr. Polym. 2009, 76, 616−621. (31) Bhattarai, N.; Ramay, H. R.; Gunn, J.; Matsen, F. A.; Zhang, M. PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release. J. Controlled Release 2005, 103, 609−624. (32) Dobrynin, A. V.; Colby, R. H.; Rubinstein, M. Polyampholytes. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 3513−3538. (33) Li, S.-D.; Howell, S. B. CD44-Targeted Microparticles for Delivery of Cisplatin to Peritoneal Metastases. Mol. Pharmaceutics 2010, 7, 280−290. (34) Schütz, C. A.; Juillerat-Jeanneret, L.; Käuper, P.; Wandrey, C. Cell Response to the Exposure to Chitosan−TPP//Alginate Nanogels. Biomacromolecules 2011, 12, 4153−4161.
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