Covalent Layer-by-Layer Assembly of Hyperbranched Polyether and

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Covalent Layer-by-Layer Assembly of Hyperbranched Polyether and Polyethyleneimine: Multilayer Films Providing Possibilities for Surface Functionalization and Local Drug Delivery Xiaofen Hu†,‡ and Jian Ji*,† †

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ College of Bioengineering, Zhejiang Chinese Medical University, Hangzhou, 310053, China ABSTRACT: A convenient and simple route to multifunctional surface coatings via the alternating covalent layer-by-layer (LBL) assembly of pnitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO2) and polyethylenimine (PEI) is described. The in situ chemical reaction between HBPO-NO2 and PEI onto aminolyzed substrates was rapid and mild. Results from ellipsometry measurements, contact angle measurements, and ATR−FTIR spectra confirmed the successful LBL assembly of the building blocks, and the surface reactivity of the multilayer films with HBPO-NO 2 as the outmost layer was demonstrated by the immobilization of an amine-functionalized fluorophore. Furthermore, a biomimetic surface was achieved by surface functionalization of the multilayer films with extracellular matrix protein collagen to promote the adhesion and growth of cells. The studies on the drug loading and in vitro release behaviors of the multilayer films demonstrated their application potentials in local delivery of hydrophilic and hydrophobic therapeutic agents.

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polymer networks and therefore are not susceptible to disassembly under varying solution conditions (e.g., strongly acidic, strongly basic, or high ionic strength solutions). Here we report the construction of multilayer films via the alternate covalent reaction of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO2) with polyethylenimine (PEI) on aminolyzed substrates. The great number of active p-nitrophenyloxycarbonyl groups of hyperbranched polyether reacted rapidly and smoothly with the primary amino groups of PEI and provided the possibilities to form cross-linked covalent bonds within the multilayer films.28−31 Furthermore, a significant amount of residual reactive groups in the multilayer films prepared using this sequential covalent strategy could provide unprecedented opportunities for the postfunctionalization and preparing high functionality polymer films.32,33 For example, it is indeed possible to characterize them with a specific biological function (recognition, adhesion, release) through the immobilization of biomolecules on their surface.34−36 We fabricated HBPO-NO2/PEI multilayer films with HBPO-NO2 as the outmost layer that could react readily with the nucleophilic N-terminus of the extracellular matrix (ECM) protein, collagen, to achieve a biomimetic surface and investigated the endothelial cells’ (ECs) adhesion and growth on these biomolecules-functionalized surfaces. We investigated the potential of HBPO-NO2/PEI multilayer films for applications in local drug delivery of multiple

INTRODUCTION Layer-by-layer (LBL) assembly method has been widely used as a versatile technique for the formation of multilayer films with a tailored structure and composition on substrates with different geometry.1−3 Recently, much attention has been paid to the application of LBL films as functional surface coatings of implanted medical devices. They appear as interesting candidates for the local delivery of therapeutic agents,4−7 antibacterial and anticoagulation applications,8−13 control of cell adhesion and growth,14−17 and so on. Hyperbranched polymers are one class of spherical compounds with imperfectly branched structures. The great number of external groups of hyperbranched polymers provides many possibilities to be either functionalized or multifunctionalized for potential applications in biomedicine, pharmacology, and biotechnology.18−20 Furthermore, hyperbranched polymers with the capability of incorporating high loadings of different types of molecules could be used as a functional building block for LBL assembly. We previously reported multifunctional polyelectrolyte multilayer films with anticoagulant and antibacterial activities and an excellent capability of loading and controlled release of hydrophobic drugs via the alternate deposition of heparin-like sulfonated hyperbranched polyether (HBPO− SO3) and chitosan.21 In recent years, the use of covalent bonds to assemble multilayer films has been a more recent area of investigation.22−27 Compared with traditional assembly methods based on multivalent weak interactions (e.g., electrostatic or hydrogen bonding interactions), covalently bound films offer the advantage of higher stability due to the cross-linked © 2011 American Chemical Society

Received: August 16, 2011 Revised: November 2, 2011 Published: November 4, 2011 4264

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functionalized by treatment with a solution containing 0.5 mg/mL collagen in acetic acid buffer solution (pH 4, 0.1 M) for 24 h at room temperature. To ensure the complete removal of physically adsorbed collagen, after the immobilization step, we washed these samples extensively with acetic acid buffer solution, rinsed them, and washed them three times with pure water and finally dried in a stream of nitrogen. The blank PET membranes and the multilayer films pretreated with 2-aminoethanol were used as the control. Then, all substrate materials were placed onto 96-well culture plates (NUCLON) and UV-sterilized for 1 h prior to cell seeding. Cell Culture. Human umbilical vein endothelial cell line ECV-304 cells (ATCC; Manassas, VA) were chosen for cell culture studies. Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium at 37 °C and 5% CO2 and supplemented with 10% heatinactivated fetal bovine serum (Sijiqin Biotech, China), penicillin (100 units/mL), and streptomycin (100 μg/mL). Cell Adhesion and Proliferation. Cell adhesion experiments were carried out by seeding the cells on substrates at a density of 5000 cells/cm2 in serum-free medium for 4 h at 37 °C in a 5% CO2 atmosphere; after this time, the substrates were removed from the 96well plates and washed several times with PBS to remove any nonadherent cells. Then, the substrates were placed into new culture plates, basal medium containing 10% fetal calf serum was added, and the substrates were incubated for further cell proliferation experiments. At a certain time point, the cell monolayers on different substrates were stained with fluorescein diacetate (FDA, Sigma), a fluorescent dye that is an indicator of membrane integrity and cytoplasmic esterase activity.38 We added 20 μL of FDA (5 μg/mL in PBS) solution to each culture well and further incubated for 15 min. Then, the samples were washed with PBS and visualized by fluorescence microscope (Bio-Rad Radiance 2100). The 488 nm wavelength of the laser was used to excite the dye. Cells were counted as a function of surface area at 10 different spots on the substrate in triplicate samples. Loading and Release of Ponceau 2R. Ponceau 2R was dissolved in water at 5 mg/mL, and the solution pH was adjusted by adding a proper amount of aqueous NaOH and HCl. Quartz slide deposited with the (HBPO-NO2/PEI)10/HBPO-NO2 films was exposed to the ponceau 2R solution (pH 4, 5, 7, 9, 11, or 12) for 24 h after adequate immersion in water with the same pH, then rinsed three times in water and blown dry with a stream of nitrogen. The amount of ponceau 2R loaded in multilayer films was analyzed by the measurement of the characteristic absorbance at 503 nm. The quartz slide covered with ponceau 2R-loaded multilayer films was immersed in a vial of phosphate-buffered saline (PBS, pH 7.4, 0.1 M) at 37 °C, which was replaced by fresh solution at appropriate time point to ensure constant release conditions. The concentration of ponceau 2R in the PBS solution was analyzed with the UV−vis spectrometer. Loading and Release of Probucal. Quartz slide deposited with the (HBPO-NO2/PEI)10/HBPO-NO2 films was exposed to the probucal solution (1 mg/mL, DMSO) for 24 h after adequate immersion in DMSO to eliminate the 4-nitrophenol yielded in the LBL process, then rinsed three times in water and blown dry with a stream of nitrogen. The amount of Probucal loaded in multilayer films was analyzed by the measurement of the characteristic absorbance at 240 nm. The quartz slide covered with guest molecules-loaded multilayer films was immersed in a vial of PBS (pH 7.4, 0.1 M) at 37 °C. At regular intervals, the quartz slide was removed from the solution, rinsed with pure water, dried under nitrogen flow followed by UV−vis absorption, and then moved to a fresh vial of PBS to maintain sink conditions. The release kinetics of probucal-loaded multilayer films in acetic acid buffer solution (pH 4, 0.1 M) at 37 °C was also investigated. Characterization Techniques. The structure of HBPO-NO2 was confirmed through FTIR and 1H NMR. FTIR spectra were recorded on a Nexus 870 FTIR spectrometer (Thermo Nicolet, Waltham, MA). 1 H NMR spectra were recorded on a 500 MHz Bruker instrument. The surface analysis was carried out with the measurement of ATR−FTIR (E.S.P., MAGNA-IR560, Nicolet Instrument). Contact

therapeutic agents. Because of the distinct properties of the building blocks, the loading behaviors of the multilayer films toward hydrophilic anionic guest molecules could be controlled by the protonation of PEI chains, whereas the hydrophobic hyperbranched polyether chains in multilayers could be regarded as reservoirs for hydrophobic guest molecules. The covalent LBL assembly of HBPO-NO2 and PEI will then provide a facile method to prepare multifunctional multilayer films that exhibit excellent capabilities of control of cell function and local delivery of multiple therapeutic agents.

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EXPERIMENTAL SECTION

Materials. HBPO (average Mw ca. 4354, Mw/Mn = 1.74) was provided kindly by Professor Deyue Yan of Shanghai Jiao Tong University (China). 4-Nitrophenyl chloroformate was purchased from Shanghai D&R Finechem and used as received. PEI (average Mw ca. 25 000 (LS)) was purchased from Aldrich Chemical. Ponceau 2R was purchased from Shanghai Hufeng Biotechnology. The aminolyzed quartz slides (1 cm × 2 cm) and silicon wafers were obtained by the reaction of 3-(triethoxysilyl)propan-1-amine (KH550) and hydroxyl groups on the surface.37 The aminolyzed PET films were prepared by the aminolysis in 1, 6-hexanediamine solution (propanol, 0.06 g/mL) for 6 h at 60 °C.9 Synthesis of HBPO-NO2. HBPO (1.00 g, 8.6 mmol hydroxyl groups) was dissolved in dried THF (100 mL), and an excess of triethylamine (1.5 mL) was added. 4-Nitrophenyl chloroformate (2.00 g, 9.9 mmol) in THF solution (20 mL) was added dropwise to the mixture and then allowed to react overnight with stirring. The resulting solution was filtered and concentrated through rotary evaporation. The brown product was purified by precipitation from diethyl ether three times to remove the unreacted 4-nitrophenyl chloroformate and dried in vacuo (yield = 1.79 g, 74%). 1H NMR (DMSO-d6, ppm): 0.79 (−CH2CH3), 1.34 (−CH2CH3), 3.03−3.33 (−CH 2 − OCH 2 −, −CH 2 OH), 4.13 (−(CCH 2 OCH 2 )), 7.44 (−COO−ArH), 8.22 (NO2−ArH). FTIR peak ν cm−1: 3434, 2966, 2931, 2876, 1764, 1529, 1348, 1216, 1116, 1046. Preparation of Multilayer Films. Solutions of HBPO-NO2 or PEI were prepared in chloroform solution (2 mg/mL). The aminolyzed substrates were alternately immersed into solutions of HBPO-NO2 and PEI for 10 min each. Between each deposition step, the substrates were rinsed with pure chloroform and blown dry with a stream of nitrogen. This cycle was repeated until the desired number of HBPO-NO2/PEI layers was reached. Multilayer films obtained by dipping the substrate n + 1 times in the solution of HBPO-NO2 and n times in the solution of PEI, named (HBPO-NO2/PEI)n/HBPO-NO2, were investigated in the following. 4-Nitrophenol, the byproduct of the reaction between HBPO-NO2 and PEI, could be eliminated from the multilayer films simply by immersion in DMSO or water. This was a necessary step before further investigation of the multilayer films. Immobilization of Amine-Functionalized Molecules. Immobilization of Fluorescent Dye. To demonstrate the possibility of surface modification of the multilayer films with amine-functionalized molecules, an amine-functionalized fluorophore (tetramethylrhodamine cadaverine) was employed as reported.29 In brief, 10 μL of a concentrated solution of tetramethylrhodamine cadaverine (40 mg/ mL, in DMSO) was dropped onto the (HBPO-NO2/PEI)10/HBPONO2 film-coated quartz slide. These solutions were allowed to sit for 20 h, after which time the films were rinsed generously using DMSO and soaked in DMSO at 37 °C. At predetermined time, films were removed, dried, and characterized by fluorescence microscopy and UV−vis absorption. The (HBPO-NO2/PEI)10/HBPO-NO2 films pretreated in a DMSO solution of ethane-1,2-diamine (50%, v/v) for 24 h were used as the control. Immobilization of Collagen. First, PET membranes deposited with the (HBPO-NO2/PEI)5/HBPO-NO2 films were immersed in pure water overnight to eliminate the 4-nitrophenol yielded in the LBL process, then rinsed three times in pure water, blown dry with a stream of nitrogen, and dried in vacuo. The multilayer films were collagen4265

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Scheme 1. Synthesis Route for the Formation of HBPO-NO2

angle measurements were taken using a KRUSS DSA100 contact angle system at ambient temperature employing the sessile drop. The measurement of the sessile method was performed at the 15th second after the probe (water droplet) contacted the polymer. For each sample, at least 10 measurements were used to calculate the average contact angle. AFM images were performed in the tapping mode under ambient conditions using a commercial scanning probe microscope, Seiko SPI3800N, equipped with a silicon cantilever, Nanosensors, typical spring constant 40 N·m −1. Spectroscopic ellipsometry was carried out using an M-2000 (J. A. Wollam) to measure the film thickness on silicon wafers. UV−vis spectra were obtained on a Shimadzu model UV-2550 spectrometer.

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RESULTS AND DISCUSSION

Synthesis of HBPO-NO 2 . p-Nitrophenyloxycarbonyl group-terminated hyperbranched polyether HBPO-NO2 was synthesized as shown in Scheme 1. The terminal hydroxyl groups of HBPO were reacted with 4-nitrophenyl chloroformate in THF. Figure 1 shows the 1H NMR spectra of HBPO

Figure 2. FTIR spectra of HBPO and HBPO-NO2.

absorption peaks were not observed on that of HBPO, which further confirmed the formation of p-nitrophenyloxycarbonyl group-terminated hyperbranched polyether. Fabrication and Characterization of HBPO-NO2/PEI Multilayer Films. Hyperbranched polyether decorated with active p-nitrophenyloxycarbonyl groups could react readily with primary amines under mild conditions (Scheme 2). The crossScheme 2. Reaction between HBPO-NO2 and Primary Amines

linked multilayer films were formed through an alternate deposition of HBPO-NO2 and PEI on aminolyzed substrates based on in situ chemical reaction between the functional groups. Ellipsometry measurement was employed to confirm the successful LBL assembly, and a linear film growth was observed (Figure 3). On average, the bilayer thickness was ∼4.5 nm. The surface wettability of the multilayer films was investigated. The contact angle results in Figure 4 showed the evolution of the alternate deposition of HBPO-NO2 and PEI layers. As indicated in Figure 4, the contact angle of the multilayer film’s surface displayed an oscillating variation with increasing number of layers. It increased after each deposition of a HBPO-NO2 layer on top of the multilayer films (fractional number) except for the primary deposition on the KH550modified substrates and then dropped following the deposition of a PEI layer on top (integral number). Because of the

Figure 1. 1H NMR spectra of HBPO and HBPO-NO2 in d-DMSO.

and HBPO-NO2, respectively. Compared with HBPO, two new proton peaks (peaks a and b) assigned to the protons of phenyl ring from the attached p -nitrophenyloxycarbonyl group at δ 7.44 and 8.22 appeared in the 1H NMR spectrum of HBPONO2. These results indicated that the reactive ester groups have been grafted onto the surface of HBPO successfully, and the degree of the terminal hydroxyl groups changed into the pnitrophenyloxycarbonyl groups was estimated to be ∼60% by measuring the peak areas at 7.44 (or 8.22) and 0.79 ppm. Figure 2 shows the FTIR spectra of HBPO and HBPO-NO 2, respectively. The absorption peaks at 1764 and 1216 cm −1 were obvious in the FTIR spectrum of HBPO-NO2 because of the absorption of terminal ester groups. The absorption peaks at 1529 and 1348 cm−1 corresponded to the nitro groups. These 4266

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number of amino groups reacted during the process of PEI layer formed on the 4.5- and 9.5-bilayered films was measured at about 0.18 and 0.49 nmol/cm2, respectively. Repeated in situ chemical reaction of HBPO-NO2 with PEI on the aminolyzed PET surface resulted in the change of chemical structures on the substrates. Figure 6 showed the

Figure 3. Thickness of assembled films at various number of bilayers.

Figure 6. ATR-FTIR spectra of aminolyzed PET membranes before and after (HBPO-NO2/PEI)n multilayer films formed.

ATR-FTIR spectra of aminolyzed PET substrate and 15- and 30-bilayer HBPO-NO2/PEI multilayer films on the substrates, respectively. The wide absorption ranging from 3500 to 2400 cm−1 corresponded to the overlap of the peaks of amino groups and alkyl chains introduced by the LBL process. The absorption peak near 1120 cm−1 became wider with an increase in the number of bilayers deposited because of the adsorption of polyether chains in HBPO-NO2. Further inspection of the image of 30-bilayer HBPO-NO2/PEI multilayer films revealed the presence of an absorption peak at 1638 cm−1. This peak corresponded to the amide carbonyl group formed upon reaction of HBPO-NO2 with PEI. Although there was no obvious spectroscopic absorption of amide carbonyl group in 15-bilayer multilayer films, the successful formation of HBPONO2/PEI multilayer films based on covalent bonds was visually observed from the color change of PEI solution to bright yellow as the concentration of byproduct 4-nitrophenol increased during LBL fabrication (Scheme 2). Furthermore, we performed subsequent experiments to determine the possibility of immobilization of amine-functionalized molecules onto HBPO-NO2/PEI multilayer films. The multilayer films with HBPO-NO2 as the outmost layer contained large amounts of active p-nitrophenyloxycarbonyl groups on the surface, which could react readily with primary amines. The images of (HBPO-NO 2/PEI)10/HBPO-NO2 multilayer films treated with amine-functionalized fluorescent dye, tetramethylrhodamine cadaverine, clearly displayed the presence of areas with red fluorescence after soaking in DMSO for 1 (Figure 7a) or 6 h (Figure 7b). Fluorescent dyes weakly adsorbed on the multilayer films could be removed via fully rinse. The images in Figure 7c,d correspond to the identical experiments conducted using (HBPO-NO2/PEI)10/HBPONO2 multilayer films that were first pretreated by reacting the active ester groups with ethane-1,2-diamine. In here, the fluorescence on the multilayer films almost disappeared after fully rinsing in DMSO. These images suggested that the fluorescence observed in Figure 7b arose from the presence of fluorescent dyes that have reacted with HBPO-NO2 and were

Figure 4. Contact angle of HBPO-NO2/PEI multilayer films assembled on aminolyzed quartz slides. Fractional numbered films were with HBPO-NO2 as the outermost layer, whereas integral numbered films were with PEI as the outermost layer.

multiple polar terminal nitro groups in HBPO-NO2, the chemical immobilization of HBPO-NO2 monolayer increased the surface wettability of the aminolyzed substrates, but compared with the polyelectrolyte PEI, HBPO-NO2 with a hyperbranched polyether skeleton exhibited hydrophobicity. Further inspection of Figure 4 revealed that multilayer films with comparative stable surface wettability were constructed and fully covered the substrates after five circles of in situ chemical reaction of HBPO-NO2 with PEI onto the surface. The surface morphologies of the multilayer films were analyzed using tapping mode AFM as shown in Figure 5. Once full

Figure 5. Topographical AFM images of (a) (HBPO-NO 2/ PEI)5HBPO-NO2 films and (b) (HBPO-NO2/PEI)10 films.

coverage was achieved, the growth of multilayer films caused a rougher topography. It was probably related to the remarkable increasement of the reactive capability of the multilayers according to the results from the determination of the byproduct concentration in the assembly solutions. The 4267

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Figure 7. Fluorescence micrographs of (a,b) (HBPO-NO2/PEI)10/HBPO-NO2 multilayer films and (c,d) the multilayer films pretreated by reaction with ethane-1,2-diamine, treated with drops of a concentrated solution of tetramethylrhodamine cadaverine and subsequently imaged after soaking in DMSO for (a,c) 1 h and (b,d) 6 h, respectively. (e) UV−vis spectra of tetramethylrhodamine cadaverine-immobilized multilayer films after soaking in DMSO for 6 h.

bound covalently to the multilayer films. The characterization of fluorophore-treated films by UV−vis spectrometer also demonstrated the successful covalent immobilization of tetramethylrhodamine cadaverine on (HBPO-NO 2/PEI)10/ HBPO-NO2 multilayer films and further illuminated that the active p-nitrophenyloxycarbonyl groups in HBPO-NO2 played an important role in surface modification of the multilayer films via mild in situ chemical reaction with amine-functionalized molecules (Figure 7e). Cell Adhesion and Proliferation on HBPO-NO2/PEI Multilayer Films. According to the aforementioned results, the surface properties of HBPO-NO2/PEI multilayer films with HBPO-NO2 as the outmost layer could be tailored by mild reaction with amine-functionalized molecules. Collagen-functionalized (HBPO-NO2/PEI)5/HBPO-NO2 multilayer films (11 L-collagen) were fabricated on PET substrates, and their ability to induce adhesion of ECs was evaluated. At the same time, blank PET membranes and 2-aminoethanol pretreated multilayer films (11 L) were used as the control. ECs viability may be directly related to the adhesion and proliferation of cells on substrates. Figure 8 compared the cell densities on the

protein collagen, 11 L-collagen multilayer films were more adapted to initial cell attachment, resulting in cell density of 168 ± 41 cells/mm2, which corresponds to a ∼13 fold increase compared with that found on the control multilayer films (p < 0.01). The increased number of cells on the different substrates at the end of the culture period exhibited that proliferation occurred on all substrate surfaces. For 11 L-collagen multilayer films, cell density of 283 ± 37 cells/mm2 was observed after 48 h of culture. Compared with the two control samples, cell density of ECs cultured on 11 L-collagen multilayer films was significantly higher (p < 0.01). As illustrated by the fluorescence micrographs in Figure 9, a large number of cells attached on the surface of 11 L-collagen multilayer films and spread well; moreover, the formation of a confluent layer of ECs after 48 h of incubation time was confirmed. The cell density results, in combination with the fluorescence micrographs study, showed that the surface modification of collagen on multilayer films induced a significant improvement in cell adhesion and spreading. HBPO-NO2/PEI Multilayer Films as Local Drug Delivery Systems. The protonation of PEI in multilayer films could provide efficient loading of hydrophilic anionic guest molecules. To evaluate the feasibility of this approach, we conducted experiments using anionic dye ponceau 2R as a model drug and a postdiffusion procedure to incorporate the dye into preassembled multilayer films. The protonation of PEI depended on the pH value, which could be utilized for pHcontrolled loading.39 First, protonated HBPO-NO2/PEI multilayer films with different concentration ratios of H+ to the repeating unit of PEI were successfully fabricated by pretreatment of the multilayer films in aqueous solutions at different pH values. Subsequently, the capability of the protonated multilayer films in loading was investigated. The UV−vis absorption spectra of ponceau 2R-loaded (HBPO-NO 2/ PEI)10/HBPO-NO2 multilayer films were presented in Figure 10. The multilayer films showed the capability of loading in the pH range from 5 to 11. The maximal absorbance was measured at pH 7, and further pH adjustment resulted in the decrease in the amount of ponceau 2R loaded in the multilayer films. It was well known that PEI was deprotonated in basic environment; therefore, the ability of PEI to trap the anionic dye became weak with the increase in pH value, and there was almost no ponceau 2R within the multilayer films at pH 12 (data not shown). In the case of acid solution, the protonation of

Figure 8. ECs attachment and growth on blank PET membranes, 2aminoethanol pretreated (HBPO-NO2/PEI)5/HBPO-NO2 films (11 L), and collagen-functionalized (HBPO-NO2/PEI)5/HBPO-NO2 films (11 L-collagen).

different substrates in 4 h adhesion experiments and further proliferation experiments. After 4 h of adhesion, low cell densities of 24 ± 10 and 13 ± 8 cells/mm2 were observed on the control samples, blank PET membranes, and 11 L multilayer films, respectively. With the introduction of ECM 4268

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Figure 9. Fluorescence microscopy images of ECs attachment and growth on blank PET membranes, 2-aminoethanol pretreated (HBPO-NO 2/ PEI)5/HBPO-NO2 films (11 L), and collagen-functionalized (HBPO-NO2/PEI)5/HBPO-NO2 films (11 L-collagen).

Figure 10. UV−vis spectra of ponceau 2R-loaded (HBPO-NO2/ PEI)10/HBPO-NO2 multilayer films. The influence of the pH value on loading behaviors of the multilayer films was investigated.

Figure 11. Release profile of ponceau 2R-loaded multilayer films prepared at different pH values.

ponceau 2R at the azo moiety provided a Coulombic repulsion to limit the incorporation of the dye into HBPO-NO2/PEI multilayer films. Consequently, less loading was obtained at pH 5, and a dramatic decrease in the capability of incorporating ponceau 2R occurred at pH 4. The data in Figure 10 demonstrated that a significant amount of anionic guest molecules could be successfully introduced into HBPO-NO 2/ PEI multilayer films via a simple postdiffusion process in mild neutral solution. Next, the release behavior of ponceau 2R-loaded HBPONO2/PEI multilayer films was investigated in PBS solutions at 37 °C. All dye-loaded multilayer films prepared at different pH values (pH 5, 7, 9) showed gradually release (Figure 11). The amount of dye released from the LBL films was strongly dependent on the pH value used for the loading, with only ∼32 μg/cm2 of dye released after 48 h at pH 9. Upon adjustment of the pH value, the amount of dye released was considerably higher, with ∼41 μg/cm2 of dye released at pH 5 and ∼44 μg/ cm2 at pH 7.

We previously have proven that hyperbranched polyether with a hydrophobic core used as a building block for LBL assembly introduced hydrophobic nanodomains into multilayer films, which could be regarded as nanoreservoirs for hydrophobic guest molecules.21 In the present work, hydrophobic drug probucal was chosen and incorporated into (HBPO-NO2/ PEI)10/HBPO-NO2 multilayer films via postdiffusion successfully. The release behavior of probucal-loaded multilayer films was first investigated in PBS solution (pH 7.4, 0.1 M). A burst release of 23% within the first 1.5 h was present in Figure 12; subsequently, the release of the drug was dramatically slowed, then no significant release was noticed in PBS even after a dipping time of several days due to the strong affinity between the guest molecules and hydrophobic nanodomains in the films. Approximately only 33% of the drug was released in the selected experimental time window. Past reports demonstrated that the variation of external pH could accelerate the release of guest molecules from weak polyelectrolyte multilayers.40−42 As previously mentioned, the 4269

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ACKNOWLEDGMENTS This work was financially supported from the National Science Fund for Distinguished Young Scholars(51025312), the Natural Science Foundation of China (50830106), The National Basic Research Program of China (2011CB606203), and Open Project of State Key Laboratory of Supramolecular Structure and Materials (SKLSSM201103). We thank Prof. Deyue Yan and Yongfeng Zhou at Shanghai Jiao Tong University for beneficial discussions and the offer of hyperbranched poly(3-ethyl-3-oxetanemethanol) (HBPO).

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Figure 12. Release profile of probucal incorporated in (HBPO-NO2/ PEI)10/HBPO-NO2 multilayer films. The influence of the pH value of buffer solutions on release behaviors was investigated.

protonation of PEI chains in HBPO-NO2/PEI multilayer films could be controlled by the variation of external solution conditions. The increase in the number of effective charges on PEI chains in acid environment improved the wettability of the multilayer films and consequently facilitated the concentration gradient-induced diffusion of guest molecules from the multilayer films. To assay the influence of the external pH on the release behavior, the release profile of probucal-loaded HBPO-NO2/PEI multilayer films immersed in acetic acid buffer solution (pH 4, 0.1 M) was obtained. As shown in Figure 12, the percentage of release in solution of pH 4 obviously increased, with ∼73% of the drug released within 3 days. Then, a sustainable slow release occurred, reaching a cumulative release of ∼78% after 9 days. However, a significant part of probucal was still trapped within the multilayer films and was essentially not affected by the pH variation. This result revealed that the guest molecules were successfully incorporated into the hydrophobic nanodomains resulting from the hydrophobic hyperbranched polyether chains; moreover, the covalently cross-linked HBPO-NO2/PEI multilayer films exhibited good stability in an acid environment.

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CONCLUSIONS p-Nitrophenyloxycarbonyl group-terminated hyperbranched polyether (HBPO-NO2) was synthesized and employed as a building block to fabricate cross-linked multilayer films via the alternating in situ chemical reaction with PEI onto aminolyzed substrates. Results from the surface characterization experiments during the LBL process verified the progressive growth of films. Furthermore, collagen-functionalized (HBPO-NO2/ PEI)5/HBPO-NO2 multilayer films were constructed to promote endothelialization on the substrates via the postmodification of HBPO-NO2-terminated multilayer films. In addition, results from the loading and release experiments verified the potential of the multilayer films as local delivery carriers of hydrophilic anionic or hydrophobic therapeutic agents. The facile method reported here to prepare multifunctional multilayer films may have good potential for surface modification of implanted medical devices.

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