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Supramolecular Host-Guest Interaction-Enhanced Adjustable Drug Release Based on #-CyclodextrinFunctionalized Thermoresponsive Porous Polymer Films Yuanwei Su, Jing Dang, Haitao Zhang, Yingyi Zhang, and Wei Tian Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01502 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017
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Supramolecular Host-Guest Interaction-Enhanced Adjustable Drug Release Based on β-CyclodextrinFunctionalized Thermoresponsive Porous Polymer Films Yuanwei Su, Jing Dang, Haitao Zhang, Yingyi Zhang, Wei Tian* MOE Key Laboratory of Material Physics and Chemistry under Extraodinary Conditions and Shanxi Key Laboratory of Macromolecular Science and Technology, School of Science, Northwestern Polytechnical University, Xi’an, 710072, China
ABSTRACT: Drug delivery systems based on stimuli-responsive porous polymer films (PPFs) have been extensively investigated due to their many advantages. However, the ability to adjust drug release from PPFs is not always perfect, and at times, it cannot satisfy real-world requirements. In this paper, supramolecular host-guest interactions were harnessed to overcome the difficulties associated with adjustable release from these systems by incorporating host molecules into the pore walls of thermoresponsive PPFs. β-Cyclodextrin-functionalized porous amphiphilic block copolymer films (β-CD-PBCPFs) with controllable pore parameters, high homogeneity, and large areas were prepared by combining the self-assembly and breath-figure methods. Drug-loaded β-CD-PBCPFs displayed thermoresponsive release behavior, which could be tuned by increasing β-CD content in phosphate-buffered saline. The release was governed by
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the host-guest interactions of the β-CD moieties and drug molecules. The concept of host-guest interaction-enhanced adjustable release could be applied to different drug molecules, such as doxorubicin and metronidazole.
KEYWORDS: Supramolecular Host-Guest Interaction, Adjustable Release, Porous Polymer Film
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1. INTROCUTION Adjustable drug delivery system (DDS) is so significant because it not only can reduce the patient's pain from repeated injections and endless medication but also can reduce the side effects of drugs.1,2 Among the numerous classes of carriers employed for drug delivery purposes, polymer-based carriers have attracted considerable attention due to their excellent biocompatibility and structural versatility.3,4 There are a multitude of polymer-based carriers that have been used to drug release, including nanoparticles,5 microspheres/microcapsules,6 micelles,7 nanofibers,8 hydrogels/nanogels,9 and porous polymer films (PPFs).10-15 Compared to other carriers, PPFs have many advantages. For example, the thicknesses of PPFs can be as much as tens of micrometers. As a result, PPFs have good mechanical strength and dimensional stability.16 Moreover, the release through pore channels is driven by drug diffusion, which provides a safe means of delivery, avoiding the denaturation of drug molecules.17 Additionally, the drugs to be released are not conjugated to or encapsulated by degradable carriers. Thus, the incidence of side effects may be reduced.11 From the viewpoint of practical application, the drugloaded PPFs can be attached onto to the skin or implanted at the site of tumor resection, to prevent local recurrent tumor growth.18 Although PPFs have the above merits when used as a DDS, once the films were prepared, their release behaviors toward drugs are fixed and limited, which means that the PPFs cannot meet the demand for intelligent DDS. As a result, smart PPFs with temperature-,19 pH-,20 ionic strength-,21 light-,22 ultrasound-,23 or magnetic/electric field-24 responsive features have become increasingly attractive. In these cases, the release behaviors from the films can be altered depending on a change in the surrounding environment. However, the controllability of stimuliresponsive PPFs for drug release is not always perfect, and at time they cannot satisfy real-world
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requirements due to their indirect regulation manner. Furthermore, a change in the microenvironment of stimuli-responsive PPF first must be induced by a change in the pore parameters, such as size and morphology, and then the release behaviors of drugs from the PPF can be adjusted. Thus, the regulation mode is limited only to the PPF itself. By establishing interactions between the PPF and drug molecules through the functionalization of the pore walls of PPFs with functional molecules capable of supramolecular interactions, we intend to directly regulate the drug release behavior. On the basis of the above considerations, we propose a supramolecular host-guest interactionenhanced adjustable drug delivery system based on thermoresponsive porous amphiphilic block polymer films. Supramolecular strategies have been considered as a significant approach to designing on-demand drug delivery systems.25-28 In the field of supramolecular chemistry, hostguest interactions are a very important phenomenon that has been extensively investigated.29-31 Cyclodextrins (CDs), including α-, β-, and γ-CD, are common macrocyclic host molecules that exhibit negligible toxicity, good complexation capacities with a variety of hydrophobic drugs, and capabilities to improve bioavailability, all of which render them suitable for functional delivery systems.32,33 Meanwhile, poly(N-isopropylacrylamide) (PNIPAM) is a common temperature-responsive polymers with many potential applications in the biomedical field, including use as controlled delivery vehicles, cell adhesion mediators and protein precipitators.34 In our previous work,35 we constructed a “on-off”-tunable drug delivery system that functioned by photo-triggered self-assembly morphological transitions of supramolecular branched copolymers based on the host-guest interactions of β-CD moieties and azobenzene. Very recently, we successfully prepared a smart single conical nanochannel in a polyimide membrane by functionalizing the pore walls of the nanochannel with CO2-responsive molecules36 or β-
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CD.37 Therefore, we considered constructing the pore walls of the β-CD-functionalized porous amphiphilic block copolymer films (β-CD-PBCPFs) with copolymers of acrylate-modified β-CD and NIPAM monomers as the hydrophilic blocks (Scheme 1a-b). Using this strategy, the release behavior of the β-CD-PBCPFs can be directly adjusted by utilizing the host-guest complexation interactions between the β-CD units on the pore walls and drug molecules (Scheme 1b-e).
Scheme 1 Schematic illustration of host-guest interaction-enhanced adjustable release based on β-CDfunctionalized thermoresponsive porous amphiphilic block copolymer films (β-CD-PBCPFs). (a) Structure of amphiphilic block copolymer; (b) Preparation of β-CD-PBCPFs through breath figure method; (c) DOX loading into the pores of β-CD-PBCPFs, and then molecular recognition between β-CD and DOX; and (d) Adjustable release based on the host-guest interactions between β-CD and DOX.
Herein, a series of β-CD-PBCPFs with host-guest binding sites located on the pore walls were prepared for adjustable drug delivery. We first synthesized a series of polystyrene-b-poly(N-
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isopropylacrylamide-co-acrylate-β-cyclodextrin] block copolymers with different contents of βCD via successive reversible addition-fragmentation chain transfer (RAFT) polymerizations. These copolymer precursors were then used to prepare β-CD-PBCPFs by combining of the selfassembly and breath-figure (BF) method. Generally, the BF method can facilitate efficient fabrication of ordered two-dimensional micro- and submicroporous polymer films.38,39 The pore parameters and surface wettability of β-CD-PBCPFs were facilely modulated by changing the βCD content in the hydrophilic segments of the block copolymers. The release of doxorubicin (DOX) from DOX-loaded β-CD-PBCPFs was investigated in phosphate-buffered saline.
2. EXPERIMENTAL SECTION 2.1. Materials. Styrene (St) was commercially obtained from Guangdong Guang Hua Science and Technology Co., Ltd. Before usage, St was carefully distilled under reduced pressure. Nisopropylacrylamide (NIPAM) was purchased from Tokyo Chemical Industry Co., Ltd. Doxorubicin hydrochloride (DOX·HCl, 98%) was obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. 2,2’-Azobis(isobutyronitrile) (AIBN; Fluka, 99%) was recrystallized before use. 1,4-Dioxane and N,N-dimethylformamide (DMF) were dried before use. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and antibiotics were purchased from Sigma Chemical Co., USA. The RAFT agent, 3-(benzylthiocarbonothioylthio) propanoic acid,40 and acrylate-β-cyclodextrin (GMA-EDA-β-CD) were synthesized using previously described methods.41 Poly(ethylene terephthalate) (PET) film was purchased from LASPEF Plastic Co. Ltd. and cleaned with acetone three times before use. Deionized water was used in all experiments after being ultrafiltrated to 18.0 MΩ. CR was obtained from Shanghai Chemical Technology Co., Ltd.
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2.2. Synthesis of PS-b-P(GMA-EDA-β-CD-co-NIPAM) PS-macroCTA was synthesized by RAFT
polymerization.
A
25
mL
Schlenk
flask
was
charged
with
3-
(benzylthiocarbonothioylthio)propanoic acid (13.6 mg, 0.05 mmol), St (3165 mg, 30.4 mmol), AIBN (0.82 mg, 0.005 mmol) and 6 mL of 1,4-dioxane. After being deoxygenated through three freeze-pump-thaw cycles, the above mixture was heated to 110 °C in an oil bath with constant stirring. After 22.5 h, the mixture was then exposed to air and cooled to room temperature to terminate the reaction. The viscous mixture was then dissolved in tetrahydrofuran (THF), precipitated in excess methanol, and filtered. The above process was repeated three times. The resulting product was dried at room temperature under vacuum for 48 h. Yield: ~81%. FT-IR (KBr): 1064 cm−1 (ν, −C=S); 1705 cm−1 (ν, C=O); 3025 cm−1 (ν,–C–H in benzene). 1H NMR (CDCl3, δ, ppm): 6.41–7.32 (5H, Ph), 2.17 (2H, –CH2–COOH), 1.42 (–CH2–), 1.83 (–CH–). A series of PS-b-P(GMA-EDA-β-CD-co-NIPAM)s were obtained using similar RAFT polymerization conditions. PS-macroCTA (492 mg, 0.015 mmol), GMA-EDA-β-CD (98.85 mg, 0.075 mmol), NIPAM (16.97 mg, 0. 15 mmol) and AIBN (0.246 mg, 0.0015 mmol) were dissolved in DMF (3 mL). The resulting solution was sealed in a Schlenk flask and deoxygenated by three freeze-pump-thaw cycles, and the flask was heated to 90 °C in an oil bath with constant stirring. After 48 h, the mixture was exposed to air and cooled to ambient temperature to stop the polymerization and then dialyzed (molecular weight cut off: 3500) in DMF for 7 d. Subsequently, the excess DMF was removed under reduced pressure. The viscous mixture was dissolved in DMF, precipitated in excess deionized water, and filtered. The final product was dried under vacuum for 48 h. Yield: ~86.13%. The other samples were synthesized using the similar procedure. FT-IR (KBr): 1643 cm−1 (ν, −C=O); 1450 cm−1 (ν, –N–H); 3025 cm−1 (ν, –C–
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H in benzene); 1368 cm−1 (ν, –C–H in β-CD). 1H NMR (DMSO-d6, δ, ppm): 5.7 (2,3–OH of βCD), 4.83 (1–H of β-CD), 6.35–7.32 (5H, Ph-); 4. 2 (2H, –O–CH2–), 0.85 (6H, –(CH2–CH3)2−). 2.3. Preparation of β-CD-PBCPFs The films were prepared by the BF method according to the reference.42 A series of PS-b-P(GMA-EDA-β-CD-co-NIPAM)s were dissolved in CS2 to obtain 4 mg/mL homogeneous solutions. The polymer solution was then dropped onto the surface of PET film and placed under a humid airflow (25 °C and > 80% RH). A turbid solution was soon formed, accompanied by the evaporation of CS2. After solidification, the films were dried at room temperature. 2.4. Drug loading and release The obtained β-CD-PBCPFs were wet by immersing in 10 mL phosphate buffered saline (PBS, pH 7.4) in a 25 mL Schlenk flask for 30 min. Then, the medium was discarded and a solution of DOX·HCl (6 mL, 9 mg mL-1, pH = 7.4) was added to the flask. The mixture was degassed by pump for 30 sec. The flask was heated at 60 °C in an oil bath for 0.5 h. To remove the HCl from DOX·HCl, triethylamine (2 equiv. DOX·HCl) was added to the flask, which was then sealed. The mixture was degassed using a pump for another 30 sec. The flask was heated at 60 °C in an oil bath for 3.5 h. Afterward, the films were dried at room temperature. The loading capacity of β-CD-PBCPFs was calculated from the sum of the release drug in experiment and the residual drug in the porous films, which can be calculated by dissolving these films in DMF. Next, the release experiments were conducted at 37 °C and 25 °C. DOX-loaded PBCPFs were individually immersed in 30 mL phosphate buffered saline (PBS, pH 7.4), and then placed in a water-bath shaker with 120 rpm at either 37 °C or 25 °C. At certain time intervals, samples (4 mL) were taken out from the release solutions, and fresh PBS (4 mL) was added. The
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accumulated release amount of DOX was determined using UV-Vis spectroscopy by measuring absorption at 485 nm. 2.5. Cell culture The HeLa cell line used as the in vitro model was provided by the College of Life Sciences, Nankai University. DMEM supplemented with 10% FBS and antibiotics was used to culture the cells. Next, the cells were cultured in a humidified atmospher (37 °C, 5% CO2). HeLa cells were detached by trypsin-EDTA solution and rinsed after the cells had reached approximately 90% confluence. These cells were used for the following experiments. 2.6. In vitro cytotoxicity estimation The cytotoxicity of PBCPFs prepared from PS318-bPNIPAM13, PS318-b-P(GMA-EDA-β-CD6-co-NIPAM8), and PS318-b-P(GMA-EDA-β-CD9-coNIPAM5) before and after DOX loading (0.20 cm × 0.20 cm) were evaluated by the MTT cell proliferation assay according to previous report.43 A pristine silicon wafer (0.20 cm × 0.20 cm） was used as the control. First, HeLa cells were split into 48-well cell-culture plates with the final cell concentration of 1 × 104 cells/well and incubated as above. After 24 h incubation, PBCPFs and β-CD-PBCPFs were separately placed into each well of 48-well plate with 200 µL DMEM supplemented with 10% FBS and antibiotics. After 48 h incubation, MTT solution (40 µL, 5.0 mg/mL) was added to each well and incubated for a further 4 h, and then the medium contained MTT was discarded, and 150 µL DMSO was added. After shaking for 20 min, the absorbance was measured immediately with an Elx-800 microplate reader (BioTek, USA) at 570 nm. 2.7. In vitro release DOX-loaded β-CD-PBCPFs prepared from PS318-b-PNIPAM13 (0.20 cm × 0.20 cm), PS318-b-P(GMA-EDA-β-CD6-co-NIPAM8) (0.15 cm × 0.15 cm), and PS318-bP(GMA-EDA-β-CD9-co-NIPAM5) (0.13 cm × 0.13 cm) were used to determine the in vitro cellular uptake of DOX. First, HeLa cells were split into the 12-well plates to give a final cell concentration of 1 ×104 cells/well, and the resulting solutions were incubated in a humidified
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atmospher (37 °C, 5% CO2) for 24 h. Then, the medium was removed, and the DOX-loaded βCD-PBCPFs were separately placed into 12-well cell-culture plates with 200 µL DMEM (10% FBS and antibiotics). After incubating for 4 h at 37 °C and 25 °C, DOX-loaded β-CD-PBCPFs and medium were discarded, and the cells were washed thrice with PBS (pH 7.4). Finally, CLSM with an excitation wavelength of 488 nm was employed for fluorescence imaging after the nuclei were stained with Hoechst 33242 and immobilized with GA solution (2%). 2.8. Statistics Statistical analysis was conducted using the Student’s t-test with values of p＜ 0.05 considered to be statistically significant.44 2.9. Characterizations Characterization of polymer structure, porous film parameters, and drug release behavior as well as cellular cytotoxicity are described in detail in Supporting Information.
3. RESULTS AND DOSCUSSION 3.1. Synthesis of PS-b-P(GMA-EDA-β-CD-co-NIPAM) A series of PS-b-P(GMA-EDA-βCD-co-NIPAM) amphiphilic block copolymers with various β-CD contents were synthesized via two-step RAFT polymerizations to be used in preparing β-CD-PBCPFs (Scheme S1). The PS macromolecular chain transfer agent (PS-macroCTA) was prepared first by homogeneous RAFT polymerization of styrene in 1,4-dioxane using 3-(benzylthiocarbonothioylthio)propanoic acid as the RAFT agent and AIBN as the initiator. Subsequently, the PS-b-P(GMA-EDA-β-CD-coNIPAM)s were synthesized in the second step: the well-controlled RAFT polymerization of the NIPAM and GMA-EDA-β-CD monomers using the PS-macroCTA described above and AIBN as the initiator.
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The macromolecular structure of PS-macroCTA was confirmed by 1H NMR (Figure S1) and FTIR spectra (Figure S2). The final structure of PS-b-P(GMA-EDA-β-CD-co-NIPAM) was characterized by size exclusion chromatography/multiangle laser light scattering (SEC-MALLS) (Figure 1), 1H NMR (Figure S3) and FTIR (Figure S4). The weight-average molecular weights (Mw,SEC) and distributions (MWD) of the resulting polymers were determined by SEC/MALLS. As shown in Figure S5, a symmetric and monomodal elution peak in the high molecular weight region was clearly observed on the SEC/MALLS curves of a series of block copolymers. As seen in Table S1, the Mw,SEC values of block copolymers were close, which was also confirmed by the peak area integrals in the 1H NMR spectra. Furthermore, the degree of polymerization ratios of the hydrophobic PS segment to the hydrophilic P(NIPAM-co-GMA-EDA-β-CD) segment of these block copolymers were calculated by 1H NMR (Table S1). This result indicated that the chain length of the hydrophilic segment of these block copolymers was close, which was beneficial for adjusting the content of β-CD in the hydrophilic segment. The content of β-CD was important for regulating the pore parameters and release behavior of the final β-CDPBCPFs.45 Additionally, the MWD of all block copolymers were narrow (MWD
