Cys) Prodrug Nanohydrogels - ACS Publications

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pH/Reduction dual-responsive oxidized alginate-doxorubicin (mPEG-OALDOX/Cys) prodrug nanohydrogels: Effect of complexation with cyclodextrins Tingting Zhou, Jiagen Li, Xu Jia, Xubo Zhao, and Peng Liu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03990 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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pH/Reduction dual-responsive oxidized alginatedoxorubicin (mPEG-OAL-DOX/Cys) prodrug nanohydrogels: Effect of complexation with cyclodextrins Tingting Zhou, Jiagen Li, Xu Jia, Xubo Zhao, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: Novel biocompatible and biodegradable pH/reduction dual-responsive oxidized alginate-doxorubicin (mPEG-OAL-DOX/Cys) prodrug nanohydrogels were designed for tumorspecific intracellular triggered release of anticancer drug DOX, by conjugating DOX via acidlabile Schiff base linkage into the PEGylated oxidized alginate (mPEG-OAL) crosslinked with bioreducible disulfide bond. The effect of the complexation with cyclodextrins (α-CD and β-CD) before or after the crosslinking of the mPEG-OAL on the DOX content and controlled release performance was investigated. It was found that the cyclodextrin inclusion complex prodrug nanohydrogels mPEG(CD)-OAL-DOX/Cys, prepared by crosslinking of the mPEG-OAL after complexation with cyclodextrins, exhibited better pH/reduction dual-responsive controlled release performance than the mPEG-OAL-DOX/Cys(CD) ones prepared by crosslinking of the

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mPEG-OAL before complexation with cyclodextrins, owing to the supramolecular crosslinking of the adjacent pseudopolyrotaxanes. Especially for the cyclodextrin inclusion complex prodrug nanohydrogels mPEG(α-CD)-OAL-DOX/Cys, DOX was released rapidly under lower pH media mimicking the tumor microenvironment and completely released within 48 h, while the premature leakage under the simulated physiological condition was about 40%, whitout burst release in both cases. The cellular toxity and uptake results demonstrated that the mPEG(α-CD)OAL-DOX/Cys prodrug nanohydrogels possessed the similar inhibition against cancer cell growth in comparison with the free DOX and enhanced drug intracellular accumulation.

Keywords: Prodrug nanohydrogels; cyclodextrin inclusion complex; intracellular triggered release; oxidized alginate; DOX

INTRODUCTION Hydrogels, the three-dimensional network structures obtained from a class of synthetic or natural polymers which can absorb and retain a significant amount of water, are one of the most promising classes of polymer-based drug delivery system (DDS) owing to their characteristics, such as easy to prepare, swelling in aqueous medium, controlled release property resulted from their sensitivity towards pH, temperature, reductant or other stimuli,1 especially for those based on the biopolymers, good biocompatibility and biodegradability could also be achieved.2 Thus, many biopolymer-based drug carriers,3 gene carriers,4 and prodrugs5 have been developed as nanohydrogels for the tumor-specific intracellular triggered release, responding to the reductive acidic tumor intracellular media. However, the drug release was determined not only by the isolation of drugs from the drug carriers or prodrugs, but also the diffusion of the drugs out of them,6 unless the matrices of the


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drug carriers or prodrugs could be dissolved in the targeted sites after the isolation of drugs, which has been reported only for the micelles.7 Furthermore, both of them mainly depend on the crosslinking degree and swelling property of the hydrogels.8 Alginate, a kind of natural linear polysaccharide, has been recognized as potential materials for DDSs owing to its non-antigenicity, nontoxicity, satisfactory biocompatibility, and favorable biodegradability together with pH sensitivity.9 However, it could swell better in physiological medium while poorer in the acidic tumor intracellular condition due to its polyanion nature, as well as its derivative, such as oxidized alginate (OAL).10 Such nature would restrict their application in DDS for controlled release of anticancer drugs, for which the release in acidic tumor intracellular condition is desired. For example, not more than 70% of drug could be released from the alginate-based DDSs in a long term release.11 By now, there is no report on the effect of the cyclodextrin-PEG inclusion complex on the controlled release performance of DDSs, although cyclodextrin-based host-guest supramolecular nanosystems with biological stimuli-responsive properties have been widely studied in cancer treatment, especially for delivery of various classes of drugs, therapeutic agents, proteins and genes12,13 and MRI imaging13, in which the cyclodextrin-based host-guest interaction was used for loading drug or chemicals, fabricating the nanosystems by crosslinking or conjugating.14 In the present work, biocompatible and biodegradable pH/reduction dual-responsive oxidized alginate-doxorubicin (mPEG-OAL-DOX/Cys) prodrug nanohydrogels were designed for tumorspecific intracellular triggered release of anticancer drug DOX, by conjugating DOX via acidlabile Schiff base linkage into the PEGylated oxidized alginate (mPEG-OAL) crosslinked with bioreducible disulfide bond. The complexation with cyclodextrins (α-CD and β-CD) was used to tailor the structure of the prodrug nanohydrogels, before or after the crosslinking of the mPEGOAL (Scheme 1). It was found that the α-CD complex prodrug nanohydrogels mPEG(α-CD)-


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OAL-DOX/Cys, which were prepared by crosslinking of the mPEG-OAL after complexation with α-CD, exhibited better pH/reduction dual-responsive controlled release performance.

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Scheme 1. Schematic illustration of the synthesis of cyclodextrin inclusion complex prodrug nanohydrogels.

EXPERIMENTAL SECTION Materials and Reagents. Sodium alginate (ALG, C.P., Mn = 35,000, Mw/Mn = 6.23) was purchased from Xudong Chem. Co. Ltd., Beijing, China. mPEG-NH2 (Mn = 2000) was available from Beijing Kaizheng Biological Engineering Development Co., Ltd. α-cyclodextrin (BR) and β-cyclodextrin (≥99%) were purchased from Gracia Chemical Technology Co. Ltd. and Tianjin Guangfu Fine Chemical Research Institute, respectively. Sodium periodate was obtained from Haichuan, Co., Ltd. (Zhejiang, China). Cystamine dihydrochloride (Cys) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were provided by Fluorochem Ltd. 1-Hydroxy-2,5-pyrrolidinedione (NHS) was bought from Aladdin Chemistry Co. Ltd. Doxorubicin hydrochloride (DOX) was got from Beijing Huafeng United Technology Co. Ltd. Glutathione was purchased from Tianjin Heowns Biochem.


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Co., Ltd. Other reagents were commercially available and used as received. Double distilled water was used throughout.

Cyclodextrin inclusion complex prodrug nanohydrogels. Oxidized alginate (OAL) and PEGylated oxidized alginate (mPEG-OAL) were synthesized by the concrete oxidation of alginate with sodium periodate and the PEGylation of OAL with mPEG-NH2 with a feeding ratio of 15 mol% to the repeating units of OAL, according to the procedure reported previously.5 The degree of oxidation (DO%, defined as the number of oxidized alginate units per 100 alginate units) of OAL was analyzed to be about 20.0% by traditional iodometry. The PEGylated degree, defined as the percentage of the PEGylated carboxylic acid groups in OAL, was determined to be 7.5 mol% by nitrogen element analysis. Then the α-CD complex prodrug nanohydrogels mPEG(α-CD)-OAL-DOX/Cys were prepared by conjugation of DOX onto the mPEG(α-CD)-OAL/Cys nanohydrogels, which were prepared by crosslinking the α-CD inclusion complex of mPEG-OAL with Cys. Typically, mPEG-OAL (18.0 mg, 0.054 mmol repeating units) and α-CD (50.0 mg, 0.051 mmol) were mixed in 5.0 mL water and stirred overnight at room temperature. Then the pH value of the above solution was adjusted to 6.5 with PBS solution, and EDC (10.4 mg, 0.054 mmol) and NHS (3.3 mg, 0.029 mmol) were added in sequence at 15-minute intervals under stirring to activate the remanent carboxyl groups in OAL. Then, cystamine dihydrochloride (6.0 mg, 0.027 mmol) was added into the mixture solution and the reaction was sustained under stirring for 2 days at room temperature. Finally, the resulting solution was dialyzed against water for 2 days, and the product, the mPEG(α-CD)-OAL/Cys nanohydrogels, was collected by lyophilization.


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Finally, DOX was conjugated onto the nanohydrogels via Schiff base linkage.15 8.0 mg of dried nanohydrogels was dispersed in 4 mL mixture of water and ethanol (v/v, 5:3). After 2 drops of glacial acetic acid was added, 1.0 mL of 2.0 mg mL−1 DOX aqueous solution was added and the mixture was partially submerged in ultrasonic wave oscillator for ten minutes 10 min in the dark to ensure adequate mixing of drug and nanohydrogels. Finally, the mixture was heated at 60 °C for 48 h under nitrogen atmosphere in the dark for 2 days. After the conjugation, the prodrug nanoparticles were collected by centrifugation and washed with a mixture of water and ethanol (v/v, 3:1) several times until the supernatant was colorless to ensure the complete removal of the adsorbed DOX via noncovalent bonds such as electrostatic interaction or hydrogen binding. The product was dried under vacuum at room temperature and stored at 4 °C in the dark. The DOX concentration in the supernatant solution was analyzed by UV–vis spectrophotometer (Lambda 35 UV−vis spectrometer) at 480 nm at room temperature, and the DOX content in the nanohydrogels, defined as the mass ratio of DOX and prodrug, was calculated by subtracting the amount of DOX in the supernatant from the initial addition amount of DOX. For comparison, the mPEG(β-CD)-OAL/Cys nanohydrogels were prepared with the similar condition, replacing α-CD with β-CD. The mPEG-OAL/Cys nanohydrogels were prepared with the similar condition without any cyclodextrin, then the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels were prepared by stirring the dispersion of the mPEG-OAL/Cys nanohydrogels with cyclodextrin (α-CD or β-CD) with the same feeding ratio as abovementioned (Scheme 1).

In vitro triggered drug release profiles. The in vitro release behaviors of the mPEG-OALDOX/Cys prodrug nanohydrogels with two different crosslinking degrees were investigated under different pH 7.4 or 5.0, with 10 µM or 10 mM GSH. As typical procedure of drug release,


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the prodrug nanohydrogels were dispersed in 10 mL PBS solution (pH 7.4) or acetate buffer solution (ABS, pH 5.0) with 10 mM GSH and then transferred into dialysis bag (molecular weight cutoff of 14 000). The drug release was considered to start as soon as the dialysis bag was immersed into 130 mL of corresponding PBS or ABS and the temperature was maintained at 37 °C. At certain time intervals, 5 mL dialysate was taken to evaluate the DOX concentration in the dialysate with a UV–vis spectrophotometer. Besides, 5 mL corresponding fresh PBS or ABS solution was added into the drug release system immediately after each sampling to ensure that the total volume of the solution remained constant. The cumulative release rate of DOX from the prodrug nanohydrogels was calculated as mass ratio of released DOX in the total conjugated DOX.

Cellular toxicity assay and uptake. The cytotoxicity of the mPEG(α-CD)-OAL/Cys nanohydrogels and mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels were compared with free DOX by the MTT assay 48 h after incubation with HepG2 cell.5 The cellular uptake of the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels were exhibited though fluorescence microscope (DM 4000B) using HepG2 cells after 24 h incubation to visualize the intercellular distribution of DOX.5

Analysis and characterization. Fourier transform infrared (FTIR) spectra were recorded by using a Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany). The elemental analyses of the samples were assessed by an Elementar Vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). The morphology of the nanohydrogels was examined with a JEM-1200 EX/S transmission electron microscope (TEM) (JEOL, Tokyo, Japan). The samples were dispersed in double


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distilled water with the aid of ultrasonication for 3 h at first, drops of the uniform dispersed solution were deposited on a copper grid covered with a perforated carbon film in sequence, and then the solvent were evaporated off. The mean hydrodynamic diameter and size distribution of the samples were determined by a dynamic light scattering system (BI-200SM, Brookhaven Instruments) using 135 mW intense laser excitation at 514.5 nm at a detection angle of 90° at room temperature.

RESULTS AND DISCUSSION Preparation and characterization of prodrug nanohydrogels. The preparation route and probable structure model of the prodrug nanohydrogels were illustrated in Scheme 1.Alginate, a well-known biocompatible material, has already been researched deeply in various biomedical fields, especially drug delivery systems. This worthwhile material still showed some rough edges, a fatal flaw is their poor biodegradability which arises from the fact that mammal don’t have appropriate degrading enzymes,16 limiting its further applications in the body. Fortunately, there’s always a solution, oxidation with sodium periodate could felicitously solve the resulting problems and benefit the drug loading process through the relative stable Schiff-base bonds rather than electrostatic interaction.8 The grafted PEG brushes could traditionally prolong the remained time of the nanohydrogels in the body during blood circulation,17 and make the nanohydrogels more biocompatible. Cystamine was also adopted in our drug carriers systems to stabilize the formation of these nanohydrogels, also due to its famous disulfide bonds and resulting biodegradability.18 However, it is difficult to control the particle size of the biopolymers by covalent crosslinking them in aqueous solution, although it could be improved to some extent by the PEGylation.15


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Cyclodextrins (α-CD or β-CD) are able to thread onto chains of polymers to form polypseudo-rotaxanes with the following major driving forces of the complexation: hydrophobic and van der Waals interactions between the inner surface of the CD ring and the hydrophobic sites of the guest polymers.19 The inner cavity diameter of β-CD is about 0.60 nm, which is slightly larger than that of α-CD (about 0.47 nm). The cavity size of α-CD and the cross-sectional area of the PEG chain match with each other perfectly. β-CD can also form a poly-pseudo-rotaxane with PEG, although the cavity size of β-CD is a little larger than the cross-sectional area of the PEG chain. The complexation of PEG chains with cyclodextrin would change their chain rigidity and solubility, thus making the crosslinked nanohydrogels to be uniform.20 Furthermore, the inner structure of the crosslinked nanohydrogels would change and the drug release behavior from the crosslinked nanohydrogels (especially the diffusion of drug molecules) might be affected.

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Figure 1. FTIR spectra of ALG, OAL, mPEG-OAL and mPEG(α-CD)-OAL/Cys nanohydrogels.


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It has been reported that more aldehyde groups in OAL would lead to the worse biocompatibility, and the cytocompatibility of OAL with lower degree of oxidation (DO% < 30%) was similar to normal biocompatible alginate.21 The DO% was controlled to be 20.0% in the present work. After the oxidation, the absorbance band at 1740 cm−1, which corresponded to stretching vibration of C=O of aldehyde groups,15 appeared in the FTIR spectrum of the product (Figure 1), confirming the successful synthesis of OAL. Then the OAL was PEGylated by amidation between the carboxyl groups in OAL and the end amino group of mPEG-NH2. The graft density of PEG in mPEG-OAL was calculated to be 7.5% by N element analysis. The remarkable absorbance at 1110 cm−1 of the C−O−C stretching vibration in the FT-IR analysis also confirmed the successful introduction of PEG chains.5 Furthermore, the infrared characteristic peak at 1665 cm−1 of the stretching vibration of carbonyl of the amide bond (amide I band) and the absorbance at 1544 cm−1 of the in-plane bending vibration of N–H (amide II band) suggested the formation of amide groups, by which the PEG chains were grafted onto the OAL. And the absorbance peaks of the asymmetrical and symmetrical stretching vibrations of – CH2– at 2945 cm−1 and 2885 cm−1 increased after the PEGylation. The mPEG(α-CD)-OAL/Cys nanohydrogels were prepared by crosslinking the mPEG-OAL with slight excess of Cys in order to ensure the complete consumption of the carboxyl groups in OAL, after complexation with α-CD. It would avoid the drug loading via electrostatic interaction, which would lead to obvious burst release. The relative peak intensity of the amide groups increased, indicating the successful crosslinking reaction with Cys. Although the characteristic absorbance of asymmetric vibration of the C-O-C glycosidic bridges at 1155 cm-1, coupled symmetric stretching vibration of C-C and C-O at 1079 and 1029 cm-1 of α-CD (Figure S1) were overlapped with those of PEG, the O–H stretching absorbance shifted from 3408 cm-1 to 3402


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cm−1 and became sharper in comparison with that of mPEG-OAL due to the strong absorbance of hydroxyl groups in α-CD, revealing the successful complexation with α-CD.22 TEM technique was used to compare the particle size and morphology of the α-CD inclusion complex nanohydrogels mPEG-OAL/Cys(α-CD) and (b) mPEG(α-CD)-OAL/Cys. As shown in Figure 2, the mPEG-OAL/Cys(α-CD) nanohydrogels exhibited bigger aggregates, while the mPEG(α-CD)-OAL/Cys nanohydrogels were approximately uniform with a small size below 50 nm, although their morphology was not regular spheres. It demonstrated that the crosslinking after inclusion complexation with α-CD was beneficial to the nanohydrogel formation.

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Figure 2. TEM images of the (a) mPEG-OAL/Cys(α-CD) and (b) mPEG(α-CD)-OAL/Cys nanohydrogels.


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Figure 3. Typical hydrodynamic diameter (Dh) and distribution of (a) mPEG-OAL/Cys, (b) mPEG-OAL/Cys(α-CD), (c) mPEG-OAL/Cys(β-CD), (d) mPEG(α-CD)-OAL/Cys and (e) mPEG(β-CD)-OAL/Cys.


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Considering the particle aggregation might caused by the shrinkage of dispersion droplets during the drying process in TEM sampling, it is difficult to present the dispersibility of the nanohydrogels. Additionally, the nanohydrogels will be used in their swollen state, but not dried state. So the DLS technique was used for the hydrodynamic diameter (Dh) and distribution analysis. All the nanohydrogels showed single-peak distribution (Figure 3), indicating they could be dispersed well in water. The mPEG-OAL/Cys nanohydrogels without cyclodextrin showed a Dh of 194 nm (Figure 3a). After inclusion complexation with α-CD or β-CD, the data increased to 214 nm or 250 nm respectively, and the distribution became wider (Figure 3b and 3c). The results indicated that the CDs had entered the mPEG-OAL/Cys nanohydrogels, and both the Dh and distribution of the mPEG-OAL/Cys(β-CD) nanohydrogels were bigger than those of the mPEG-OAL/Cys(α-CD) nanohydrogels due to the bigger size of β-CD. As for the mPEG(CD)-OAL/Cys nanohydrogels prepared by crosslinking after the inclusion complexation of PEG with cyclodextrin, the Dh of 289 nm and 370 nm were obtained with α-CD and β-CD, respectively (Figure 3d and 3e). The data were much bigger than those of the nanohydrogels without cyclodextrin, or complexation with cyclodextrin after crosslinking. In the two cases, cyclodextrin is easy to form inclusion complexation of PEG in the solution, and the inclusion complexes could be wrapped in the formed nanohydrogels during the crosslinking. While in the cases of the mPEG-OAL/Cys(α-CD) and mPEG-OAL/Cys(β-CD) nanohydrogels prepared by crosslinking before complexation with cyclodextrin, less amount of CDs could enter into the nanohydrogels due to their crosslinked structure. As for the mPEG(CD)-OAL/Cys nanohydrogels, before the covalent crosslinking with Cys, pseudopolyrotaxanes were assembled with PEG block and cyclodextrin, and the adjacent pseudopolyrotaxanes would form supramolecular crosslinking structure,23 which increased the chain rigidity of the mPEG-OAL


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and therefore led to lower crosslinking degree with Cys (Scheme 1). Thus, their Dh were much bigger than those of the nanohydrogels without cyclodextrin. Interestingly, the mPEG(α-CD)OAL/Cys presented a very narrow distribution, within 283-297 nm. In the present work, DOX was conjugated onto the nanohydrogels in acidic water/ethanol mixture, in order to avoid the DOX-loading via electrostatic interaction. After conjugating DOX onto the nanohydrogels via acid-labile Schiff base linkage, the DOX contents of 36.8%±0.4%, 35.3%±0.7%, 40.6±0.9%, and 39.3%±1.2% were obtained for the mPEG(α-CD)-OALDOX/Cys, mPEG(β-CD)-OAL-DOX/Cys, mPEG-OAL-DOX/Cys(α-CD), and mPEG-OALDOX/Cys(β-CD) prodrug nanohydrogels, respectively. It was found that the cyclodextrin species showed negligible influence on the DOX contents in the prodrug nanohydrogels, demonstrating the drug conjugation was not affected by the inclusion complexation with CDs, which just changed the crosslinking structure of the nanohydrogels in fact. However, the adding modes of CDs exhibited obvious effect on the DOX contents in the prodrug nanohydrogels. The former two ones, in which the covalent crosslinking degrees were lower due to the supramolecular crosslinking of the adjacent pseudopolyrotaxanes, possessed the lower drug contents, maybe due to the volume steric hindrance of the CDs in the mPEG(CD)-OAL/Cys nanohydrogels.

In vitro controlled release performance. The pH-triggered drug release performance of the two kinds of prodrug nanohydrogels was compared in vitro in different media at 37 °C: pH 7.4 PBS + 10 mM GSH mimicking the physiological media and pH 5.0 ABS + 10 µM GSH mimicking the tumor intracellular media. There was no burst release in all the in vitro release profiles, demonstrating that DOX was conjugated onto these nanohydrogels via covalent bond, Schiff base bonds here, but not the weak interaction such as electrostatic interaction. It is because that


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slight excess of Cys was used for the crosslinking in order to ensure the complete consumption of the carboxyl groups in OAL in the present work. Besides, the acidic media was used for the conjugation of DOX to avoid the DOX-loading via electrostatic interaction. And the possible loaded DOX via noncovalent bonds had been washed off with the mixture of water and ethanol (v/v, 3:1) several times. For the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels (Figure 4A), only about 42% of DOX were released in the media of pH 5.0 ABS + 10 µM GSH within more than 50 h, without distinct difference between the two ones containing α-CD or β-CD, similar effect as on their DOX contents. At pH 7.4 PBS + 10 mM GSH media, the cumulative release was only 30% or 19%, respectively. The controlled release performance was far less than ideal, although the release profiles showed the pH/reduction dual-responsive characteristic. Furthermore, the cumulative release ratios from the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels in the simulated tumor intracellular media within 50 h were much lower than that from the mPEGOAL-DOX/Cys prodrug nanohydrogels without CD of 59%. It indicated that the inclusion complexation with CDs after crosslinking with Cys could hinder the DOX release. Upon dilution in these media without CDs in the in vitro release profiles, the CD molecules might fall off from their supermolecular complexes with PEG due to the reaction equilibrium, dissolving into the media or adsorbed on the nanohydrogels via hydrogen bond. The CD molecules adsorbed on the nanohydrogels would form supermolecular complexation with the cleaved DOX molecules, thus hindering the DOX release. As for the mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels (Figure 4B), all DOX was released from the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels and more than 92% was release from the mPEG(β-CD)-OAL-DOX/Cys prodrug nanohydrogels in the simulated tumor


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intracellular media within 48 h, due to the cleavage of the disulfide crosslinking linkage with GSH, although the inclusion complex is more hydrophobic than PEG and CD,24 which is bad to the swelling of the prodrug nanohydrogels, as well as the diffusion of the small species such as DOX, GSH and H+. And only about 40% of DOX were released in the simulated physiological media within 48 h, without distinct difference between the two ones containing α-CD or β-CD. Interestingly, the DOX release from the mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels was faster than that from the mPEG-OAL-DOX/Cys ones in the simulated tumor intracellular media, due to the supramolecular crosslinking of the adjacent pseudopolyrotaxanes. For the same reason, all the cumulative release ratios of the mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels in both the simulated physiological and tumor intracellular media were higher than those from the mPEG-OAL-DOX/Cys(CD) ones.

90

120

α-CD, pH 7.4 + 10 µM GSH α-CD, pH 5.0 + 10 mM GSH β-CD, pH 7.4 + 10 µM GSH β-CD, pH 5.0 + 10 mM GSH

(A)

80 70

(B)

100

No CD, pH 5.0 + 10 mM GSH

Cumulative release (%)

Cumulative release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 50 40 30 20 10 0

80 60 40 α-CD, pH 7.4 + 10 µM GSH α-CD, pH 5.0 + 10 mM GSH β-CD, pH 7.4 + 10 µM GSH β-CD, pH 5.0 + 10 mM GSH

20 0

0

10

20

30

40

50

60

0

10

20

30

40

50

Time (h)

Time (h)

Figure 4. In vitro cumulative release profiles of the mPEG-OAL-DOX/Cys(CD) (A) and mPEG(CD)-OAL-DOX/Cys (B) prodrug nanohydrogels.


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The drug release mechanism was determined by fitting the in vitro release profiles in the various release kinetic models (Figures S2-S4) and the results are summarized in Tables 1-3. All the values of the regression coefficient (R2) in the zero-order and first-order models were lower than the other two models, indicating the two models were not applicable to such microgels in the present work.25 Comparing the correlation coefficient values, the mPEG-OALDOX/Cys(CD) prodrug nanohydrogels gave a good fit to Higuchi model with R2 > 0.97 (Figure S3), thus drug release mechanism was assumed to be diffusion controlled. However, this model fails to explain the influence of swelling of the matrix upon hydration and gradual erosion of the matrix. In the Korsmeyer-Peppas model, all the release exponents were in the range of 0.43 0.85 indicating their release mechanism of anomalous (non-Fickian) transport, except for the mPEG-OAL-DOX/Cys(α-CD) prodrug nanohydrogels in the simulated physiological media, following a Fickian diffusion mechanism in which drug release mechanism through diffusion, i.e., diffusion controlled release.26

Table 1. Drug release data of the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels fitted in zero-order and first-order models. Zero-order

First-order

Samples R2

K0

R2

K

α-CD pH=7.4+10 µM GSH

0.8934

0.4922

0.5531

0.0168

α-CD pH=5.0+10 mM GSH

0.9063

0.7426

0.5346

0.0212

β-CD pH=7.4+10 µM GSH

0.9311

0.2688

0.7115

0.0120

β-CD pH=5.0+10 mM GSH

0.9352

0.6622

0.6490

0.0173

No CD pH=5.0+10 mM GSH

0.9106

1.0457

0.6307

0.0205


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Table 2. Drug release data of mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels fitted in zeroorder and first-order models. Zero-order

First-order

Samples R2

K0

R2

K

α-CD pH=7.4+10 µM GSH

0.9376

2.7748

0.5911

0.0873

α-CD pH=5.0+ 10 mM GSH

0.9694

9.9231

0.7794

0.1212

β-CD pH=7.4+10 µM GSH

0.9541

3.5550

0.8076

0.0852

β-CD pH=5.0+10 mM GSH

0.9588

7.1569

0.8104

0.1189

Table 3. Fitting results with Higuchi and Korsmeyer-Peppas models. Higuchi Prodrug nanohydrogels

Korsmeyer-Peppas

Releasing media R2

k

R2

n

mPEG-OAL-

pH 7.4 +10 µM GSH

0.9776

0.4621

0.9578

0.3282

DOX/Cys(α-CD)

pH 5.0 +10 mM GSH

0.9888

0.7975

0.9193

0.7374

mPEG-OAL-

pH 7.4 +10 µM GSH

0.9816

0.3035

0.9799

0.4358

DOX/Cys(β-CD)

pH 5.0 +10 mM GSH

0.9907

0.7446

0.9719

0.6458

mPEG(α-CD)-OAL-

pH 7.4 +10 µM GSH

0.9727

1.7169

0.8958

0.8137

DOX/Cys

pH 5.0 +10 mM GSH

0.9918

5.0728

0.9848

0.9769

mPEG(β-CD)-OAL-

pH 7.4 +10 µM GSH

0.9901

1.4573

0.9408

0.5496

DOX/Cys

pH 5.0 +10 mM GSH

0.9879

3.6240

0.9866

0.9616


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The cumulative release of the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels were no more than 43% in both simulated media, indicating that the hydrolysis of Schiff base bonds was a reversible process,27 and some parts of the cleaved DOX could not be diffused out but was attracted to the nanohydrogels through the formation of new Schiff base bonds.28 In the simulated tumor intracellular media, the bioreducible disulfide crosslinkage was cleaved off and the crosslinking degree decreased for both mPEG-OAL-DOX/Cys(α-CD) and mPEG-OALDOX/Cys(β-CD), favoring the diffusion of the cleaved DOX molecules. However, as in the simulated physiological media, most of the disulfide crosslinkage was remained, and the β-CD in the prodrug nanohydrogels would form the supermolecular structure with the cleaved DOX molecules,29 thus hindering the formation of the new Schiff base bonds. The mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels showed a relative better fit to Korsmeyer-Peppas model in the simulated physiological media (Figure S4), although both regression coefficients were not so ideal, with the release exponents were in the range of 0.43 0.85. Compared with the mPEG-OAL-DOX/Cys(CD) prodrug nanohydrogels, the difference should be caused by the supramolecular crosslinking of the adjacent pseudopolyrotaxanes in the mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels. As in the simulated tumor intracellular media, both the covalent disulfide crosslinkage and the acid-labile Schiff base linkage were cleaved off, and the cleaved DOX molecules were protonated. So high cumulative release were achived of > 60%. The relative lower cumulative release from the mPEG(β-CD)-OAL-DOX/Cys prodrug nanohydrogels than the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels also resulted from the supermolecular complexation structure of β-CD with the cleaved DOX molecules.


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Cellular toxicity assay and uptake. The cytotoxicity of the mPEG(α-CD)-OAL/Cys nanohydrogels and mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels were compared with free DOX by the MTT assay 48 h after incubation with HepG2 cell. As shown in Figure 5, the cell viability was higher than 86% after incubation in presence of 20 µg/mL mPEG(α-CD)OAL/Cys nanohydrogels, revealing the excellent cytocompatibility. As for the mPEG(α-CD)OAL-DOX/Cys prodrug nanohydrogels, the cell viability decreased with increasing the concentration of the prodrug nanohydrogels. However, the cell viabilities were higher than those with free DOX with the same concentration. Taking into account of the DOX content of 36.8%, the actual DOX concentration was 7.36 µg/mL after 48 h incubation with 20 µg/mL prodrug nanohydrogels. The cell viability in such case was in middle of the values after 48 h incubation with 5 and 10 µg/mL DOX, demonstrating that the prodrug nanohydrogels exhibited a similar inhibition against cancer cell growth in comparison with the free DOX although the DOX was released in a sustained mode from the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels.

mPEG(a-CD)-OAL/Cys mPEG(a-CD)-OAL-DOX/Cys DOX

100 80 Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60 40 20 0 0

2.5 5 10 Concentration (µg/mL)


20

20

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Figure5. Cell viability of the mPEG(α-CD)-OAL/Cys nanohydrogels, mPEG(α-CD)-OALDOX/Cys prodrug nanohydrogels, and free DOX determined by the MTT assay 48 h after with HepG2 cell.

To reveal the successful cellular uptake of the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels by the HepG2 cells, they were incubated for 24 h and the intercellular distribution of DOX was exhibited though fluorescence microscope (DM 4000B). The cell nuclei showed blue fluorescence after stained with Hoechst 33258, while DOX exhibited strong red fluorescence (Figure 6). Such fluorescence positions were completely overlapped in the merged image, revealing that the released DOX was mainly accumulated in the cell nuclei. Also due to the effective cellular uptake and internalization, the mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels possessed the enhanced drug intracellular accumulation, and promosing inhibition against cancer cell growth as a result.

Figure 6. Cellular uptake of HepG2 cells stained by Hoechst, mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels micelles after 24 h via the fluorescence microscope. Each images, from left to right field, represented cell nuclei in bright flied, stained by the Hoechst, DOX fluorescence, the lapped image, respectively.


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CONCLUSIONS In summary, the effect of the cyclodextrins (species and adding models) on the drug contents and controlled release performance of novel pH/reduction dual-responsive oxidized alginatedoxorubicin (mPEG-OAL-DOX/Cys) prodrug nanohydrogels were investigated for the first time in the present work. The results showed that the mPEG(CD)-OAL-DOX/Cys prodrug nanohydrogels, prepared by crosslinking of the mPEG-OAL after complexation with cyclodextrins, exhibited better pH/reduction dual-responsive controlled release performance, owing to the supramolecular crosslinking of the adjacent pseudopolyrotaxanes, regardless of the cyclodextrin species. However, the complexation of the cleaved DOX with β-CD would postpone the DOX release, so the cyclodextrin inclusion complex with β-CD was revealed to a promising approach to tailor the structure and subsequently controlled release performance of the final prodrug nanohydrogels. The mPEG(α-CD)-OAL-DOX/Cys prodrug nanohydrogels could release DOX completely in the simulated tumor intracellular media within 48 h, while the premature leakage under the simulated physiological condition was about 40%. The cellular toxity results demonstrated the excellent bicompatibility of the mPEG(α-CD)-OAL/Cys nanohydrogels, as well as the similar inhibition against cancer cell growth in comparison with the free DOX. And the fluorescence microscope analysis revealed that the released DOX was mainly accumulated in the cell nuclei. Such excellent pH/reduction dual-responsive controlled release performance make them promising DDS for DOX delivery.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website


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Langmuir

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

ACKNOWLEDGMENTS This project was granted financial support from the Program for New Century Excellent Talents in University of the Ministry of Education of China (Grant No. NCET-09-0441) and the "Chunhui Project" of the Ministry of Education of China (Grant no. Z2012116).

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For Table of Contents Use Only

pH/Reduction dual-responsive oxidized alginate-doxorubicin (mPEGOAL-DOX/Cys) prodrug nanohydrogels: Effect of complexation with cyclodextrins Tingting Zhou, Jiagen Li, Xu Jia, Xubo Zhao, and Peng Liu*

DOX Cys

mPEG-OAL-DOX/Cys(CD)

Cys

DOX

OAL

PEG

Cystamine

CD

DOX

mPEG(CD)-OAL-DOX/Cys


27

Cys) Prodrug Nanohydrogels - ACS Publications

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