Materials Science and Engineering C 58 (2016) 622–628

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Stimuli-responsive weak polyelectrolyte multilayer films: A thin film platform for self triggered multi-drug delivery S. Anandhakumar a,⁎, P. Gokul a, A.M. Raichur b a b

SRM Research Institute, SRM University, Kattankulathur, Chennai 603203, India Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India

a r t i c l e

i n f o

Article history: Received 14 May 2015 Received in revised form 12 August 2015 Accepted 22 August 2015 Available online 29 August 2015 Keywords: Stimuli-responsive Multilayer films Dual drug delivery Ciprofloxacin hydrochloride

a b s t r a c t Polyelectrolyte multilayer (PEM) thin film composed of weak polyelectrolytes was designed by layer-by-layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) for multi-drug delivery applications. Environmental stimuli such as pH and ionic strength showed significant influence in changing the film morphology from pore-free smooth structure to porous structure and favored triggered release of loaded molecules. The film was successfully loaded with bovine serum albumin (BSA) and ciprofloxacin hydrochloride (CH) by modulating the porous polymeric network of the film. Release studies showed that the amount of release could be easily controlled by changing the environmental conditions such as pH and ionic strength. Sustained release of loaded molecules was observed up to 8 h. The fabricated films were found to be biocompatible with epithelial cells during in-vitro cell culture studies. PEM film reported here not only has the potential to be used as self-responding thin film platform for transdermal drug delivery, but also has the potential for further development in antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, PEM films prepared by alternate adsorption of oppositely charged polyelectrolytes on to a charged surface have received great attention in the biomedical field [1]. It is mainly due to their ability to be assembled in-situ on a wound bed, be pre-assembled and transferred from a flexible sheeting material to a wound bed or to be directly functionalized on implantable medical devices [2,3]. PEM films owe its popularity to the fact that it can be conformally coated over different surfaces, including biological and synthetic materials [4]. Various PEM film based drug delivery formulations have been developed to deliver therapeutics such as painkillers, anti-inflammatory drugs, antibiotics, proteins and growth factors [5,6]. Among various PEM films reported to date, PEM films made up of weak polyelectrolytes have attracted greater interest because of their distinct and reversible change of properties in response to external stimulus [7,8]. The use of weak polyelectrolytes is especially attractive for drug delivery applications because the risks of inducing toxicity are higher when strong polyelectrolytes are used. The intrinsic property of weak PEM is that the surface properties of the film such as thickness, roughness, composition and permeability could be easily manipulated by controlling the interactions between the polymers and the surface [9–12]. When there is a change in environmental stimulus such as pH, ionic strength and polarity, it ⁎ Corresponding author at: SRM Research Institute, SRM University, Kattankulathur, Chennai 603203, Tamil Nadu, India. E-mail address: [email protected] (S. Anandhakumar).

http://dx.doi.org/10.1016/j.msec.2015.08.039 0928-4931/© 2015 Elsevier B.V. All rights reserved.

induces charge imbalances in the film [8,13,14]. The films can even be destroyed at extreme conditions as the induced imbalance of charges overcompensates for the attractive polymer–polymer and/or polymer– surface interactions [2,15,16]. The softness and ability to swell and contract in aqueous media make these films compatible with biological systems, thus making them potential candidate for different biomedical applications such as cell adhesion, wound healing, drug delivery and antibacterial coatings [17,18]. Drug delivery with polymer films is an emerging field and needs new methodologies and tools to achieve its goals. For instance, there have been strong interests in the specific use of polymeric films for localized drug delivery. Various thin films have been reported for treating various diseases such as periodontal disease, glaucoma, and cancer by using the films directly as a means for drug encapsulation or as a polymer coating on metallic stents, and even as fully biodegradable stents [19–23]. One of the main challenges in fabricating thin film based drug delivery platform is to develop a system which is able to: 1) provide controlled release of loaded molecules, 2) preserve the biological activity of loaded molecules and 3) load more than one drug and/or protein in it. Many attempts have been devoted to achieve these requirements, it includes incorporation of nanoparticles in the film and use of diblock copolymers for the fabrication of polymer films to control and optimize the initial burst release [23,24]. Other factors, such as thickness, stiffness, chemistry, stability, permeability, composition, biofunctionality and dynamics, have also been explored and they can act as a key to modulate the drug release rates and profiles [25–27]. Though several attempts have been made, the success of formulating a single drug

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

delivery system to fulfill all the requirements has only been minimal. Thus it is important to design multilayer films that can provide sophisticated control over the timing and the rate at which the drug is released. In addition, encapsulation and release of two or more drugs from a single system provide many advantages over conventional formulations, such as improved treatment and patient compliance, high drug concentration on site over extended periods and also reduces undesired side effects of the drug. Recently, codelivery of siRNA and cancer drug was reported by the group of Hammond [28] and codelivery of protein and CH by us [29]. In the latter work it was demonstrated that the encapsulated protein and CH were successfully released in a sustained manner when exposed to external triggers such as laser light and ultrasound [29]. Given the ability of multilayer films consisting weak polyelectrolytes to be altered from pore free film to porous film by changing the environmental stimuli such as pH and ionic strength, and to load various drug molecules and to regulate the release of drugs, we are interested in investigating these films for transdermal drug delivery. This method not only offers advantages of needle free drug delivery but also provides an easy option to limit first-pass drug metabolism. We believe that drug and/or protein-loaded multilayer films coated on the surface of the skin adhesive patch or wound dressing could provide several attractive features for transdermal drug delivery: i) multilayers preserve the loaded proteins/biomolecules in native form [30,31]; ii) high drug concentration in the film would provide a strong driving force for the drugs to penetrate into the skin; iii) more than one drug could be loaded in the film and the kinetics of release of individual drug could be easily regulated to optimize the therapeutic response [28,29]; iv) the change in wound pH when compared to normal skin could be successfully utilized to trigger the release of loaded molecules from the film; and v) the versatility of PEM coating on various substrates would allow this concept to be implemented in many different ways, including coatings of simple skin adhesive patches, woven fiber adhesive patches, micro-needle arrays, implants and drug-releasing coatings for stents. In this study, we report a thin film platform for the encapsulation and release of protein and pharmaceutical drug for self triggered (environmental stimuli responsive) drug delivery. Here, protein is only a model molecule, it can be growth factors, antigens and proteins in actual applications. In this work, we extended our research on PEM films composed of weak polyelectrolytes for encapsulation and release of BSA and CH. Since the weak polyelectrolytes have special tendency to realign their polymeric configuration under the influence of pH and ionic strength, it is interesting to study on how these properties can be utilized to release the loaded molecules from the film. This work will not only expand the knowledge on the preparation of PEM films based on weak polyelectrolytes, but also would benefit the application of stimuli-responsive drug delivery. 2. Experiment 2.1. Materials PAH (Mw = 70 kDa), PMA (Mw = 483 kDa) and BSA were all purchased from Sigma Aldrich (India) and used without any further purification. Ciprofloxacin hydrochloride (CH) was a gift by Dr. Reddy's laboratories Ltd., India. Water from a Milli-Q system with a resistivity greater than 18 MΩ cm was used for all experiments. All pH adjustments were done with 0.1 M HCl or 0.1 M NaOH.

623

2.3. PEM film preparation Polyelectrolyte solutions of 1 mg/mL were prepared in 0.2 M NaCl solutions. PEM film was assembled at pH 5 which maintained the polyelectrolytes at the fully charged state. Since the quartz substrates are negatively charged, the assembly was started with PAH adsorption. The adsorption step was 15 min, followed by washing three times with water to remove loosely bound molecules from the surface. The adsorption and washing processes were then repeated for PMA. Deposition of one PAH layer and one PMA layer is regarded as one cycle and is termed a single bilayer formation. This process of sequential adsorption and washing was repeated until a desired number of PAH/PMA bilayers were obtained. After assembly, the films were rinsed thoroughly with water, dried in a nitrogen stream and stored in a desiccator for further characterization. 2.4. Loading of BSA and CH in PEM films The procedure for selective deposition of BSA and CH has been described in our previous study [29]. In brief, six bilayers of PAH/PMA PEM films were prepared as described above. Then these samples were dipped in BSA for 30 min at pH 4 and washed three times with pH adjusted water to remove BSA present in supernatant. Here BSA is a model protein, and can be deposited as a layer by multimode interactions such as electrostatic interactions, hydrogen bonding and hydrophobic interactions [18,32]. After BSA adsorption, two bilayers of PAH/ PMA were added to maintain the surface charge negative, which favors the loading of CH by electrostatic interactions [29]. For CH loading, (PAH/PMA)8 films were dipped in CH solution (5 mg/mL) for 30 min at pH 4, which allowed the drug to permeate and be loaded in the film. Rinsing was not performed after CH loading, which provided a means of incorporating excess unbound drug available for immediate diffusive release upon immersion in a physiological environment. It is important to note that the drug excess is advantageous since it could serve as a loading dose to reach therapeutic concentration. The sustained release obtained from loaded drug could be used for maintaining the drug concentration within therapeutic limit for extended periods. The final drug loaded samples were vacuum dried, stored at 4 °C and later used for in-vitro release experiments. Protein/drug loading of the film was quantified as the absorbance difference prior to and after loading protein/drug in the film using a UV–Visible (UV–Vis) spectrophotometer (Nanodrop 2000c, Nanodrop Technologies, U.S.A.). 2.5. Atomic force microscopy (AFM) imaging The surface morphology of PAH/PMA films was investigated by AFM (MFP-3D AFM, Asylum Research, U.S.A). Imaging was done in air by tapping mode using NCR-20 cantilevers having a resonance frequency of 285 kHz and force constant of 42 N/m (Nanoworld, Switzerland). At least 5 images were recorded from different areas of the sample to obtain consistent results. 2.6. Thickness measurements The film thickness was determined using a SE850 spectroscopic ellipsometer (Sentech Instruments, Germany) over a spectral range of 300 to 800 nm at an incidence angle of 70°. At least 5 measurements were made at different spots and averaged.

2.2. Substrate preparation

2.7. In-vitro release experiments

Quartz slides were initially cut into 8 mm × 8 mm pieces and used for LbL assembly of PAH and PMA. Before assembly, quartz pieces were cleaned ultrasonically with isopropanol/water (75 mL/25 mL) mixture for 30 min followed by rinsing three times with water.

To investigate the influence of pH on drug release, the drug loaded films were immersed into 4 mL of pH adjusted water (pH 1.2 or 7.4) in a tightly capped plastic vial maintained at 37 °C in an incubator shaker. The vial was kept sealed during the experiments to minimize the

624

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

evaporative loss. 3 mL of sample was extracted from the vial at regular intervals to estimate the released amount, which is then replaced with 3 mL of pre-warmed pH adjusted water. To investigate the influence of ionic strength on drug release, the release experiments were performed in NaCl solutions by varying the salt concentration from 0 to 0.4 M. In all cases, the release amount was directly estimated by measuring the absorbance of supernatant at predetermined time interval. 2.8. Antibacterial studies Antibacterial studies were performed with the supernatant solutions collected at different time periods during release by disc diffusion method. The overnight grown Escherichia coli (E. coli) culture in Luria–Bertani broth containing about 3 × 106 colony forming units (CFU) per mL was spread using a sterilized cotton swab on nutrient agar plates. Sterile discs of 6 mm were loaded with released drug solutions, air dried and placed on the surface of the agar plates. After 18 h of incubation at 37 °C, the zone of inhibition (ZOI) around the discs was measured. Experiments were performed in triplicates and the mean results were recorded. 2.9. Cell viability experiments Kidney epithelial cells (VERO cell line) with a cell density of 5 × 103 cells/mL were cultured on tissue culture polystyrene (TCPS) plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The pH of the medium was maintained at 7.4. After separating the cells from the growth surface using trypsin, the cells were resuspended in fresh medium containing 0.4% trypan blue (Sigma) and counted in a hemocytometer. Trypan blue exclusion was performed to determine viable cells prior to seeding. Before cell adhesion studies, the multilayer films were sterilized by spraying with 70% ethanol. After washing with phosphate buffered saline (PBS), the PEM films were seeded with epithelial cells (cell density of 5 × 103 cells/mL) in a 24-well plate containing fresh medium. Cells seeded on TCPS plates served as control samples. The cell proliferation on PEM films was determined over a period of 7 days by MTT assay [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide]. In living cells, thiazolyl blue tetrazolium bromide dye is reduced to purple formazan by the presence of cellular oxidoreductase enzymes. 10 μL of 12 mM MTT was added for cell incubation at 37 °C for 4 h in the darkness. The media were then separated and washed with PBS. The formazan salt produced was dissolved with dimethylsulfoxide, and the absorbance of the solution was measured at 560 nm to estimate the formazan concentration. The cell viability was estimated by comparing the absorbance of cells contained on control well to the cells cultured on PEM films. An Olympus inverted phase contrast microscope was used for all experiments to capture the images of the cell morphology, density, and spreading on the multilayer surfaces over 7 days. The medium was changed every other day and the cells were photographed daily. 2.10. Statistical analysis All quantitative data are expressed as the mean ± standard deviation (SD). Statistical analysis was determined using single factor analysis of variance. p-Value less than or equal to 0.05 was considered to be statistically significant. 3. Results and discussion An ideal thin film drug delivery system should be easy to assemble from readily available layer components, exhibits good responsiveness and provides controlled release of loaded molecules. PAH and PMA are well known weak polyelectrolytes, where cumulative residual charge − arising from the ionization of NH+ groups significantly 2 and COO

influences the configuration as well as the performance of the assembled films. Stimuli-responsiveness of the film due to ionization is an additional benefit for drug delivery as it does not only control the rate of release but also favor in reducing cytotoxicity related issues. In this study, we investigate the fabrication of PAH/PMA thin films and demonstrate its applicability as a stimuli-responsive drug delivery system. Fig. 1 shows the schematic representation of PAH/PMA PEM film preparation and stimuli-responsive release of loaded protein and drug. Taking the advantage of electrostatic interaction between PAH and PMA [31], LbL assembly of PAH and PMA was performed on glass substrate to fabricate (PAH/PMA) PEM films. Fig. 2 shows AFM images of asprepared films and films treated at various pH ranges to investigate the pH responsiveness of assembled films. Notably, the freshly prepared film was uniform and appeared smooth without any pore and other discontinuity (Fig. 2c). The average roughness and thickness of the film were about 5.17 ± 0.5 and 21.4 ± 1.5 nm, respectively. When the film was treated with different pH water, the film morphology changed from continuous pore free to porous structure. Notably, the film morphology turns into porous one with the formation of new pores. The formation of new pores in the film increases the roughness of film as estimated from the AFM measurements (Supplementary information). It is noteworthy that the influence of pH on PEM film is significant at acidic and basic pH ranges when compared to neutral pH. The transformation is attributed to repulsion between the same functional groups when the degree of ionization of other polymer decreases as a function of pH [33]. The pore formation occurs due to repulsion between unbal− anced NH+ groups induce 2 groups at acidic pH range whereas COO rupture at basic pH range [33–35]. Carboxylate groups of PMA transform from dissociated to associated form when pH is lowered less than pKa value (pKa value of PMA is 3.8) [35]. This results in the formation of unbalanced amine groups in the acidic pH range. The unbalanced amine groups induce repulsion with the neighbors and results in the formation of pores on the film. On the other hand, PMA is fully charged at pH 7.4 and has more number of ionized groups to counter ionized amine groups of PAH [35], hence the film is smooth and intermixed without any discontinuity. When the pH is increased to 9, it favors the formation of free (unbalanced) carboxylate groups and induces charge imbalances in the film as explained above. Similarly, it results in the formation of porous structure with a kind of patterned peaks and valleys at alkaline pH range as shown in Fig. 2e&f. It is noteworthy that the color of the AFM images changes from gold to black when pH is increased or decreased from the assembly pH. This indirectly implies that there is a decrease in the thickness of the film. It is believed that pH change induces charge imbalances in the film, hence some of the loosely bound polymers detach from the surface. Thus significant decrease in thickness is observed when the pH is increased or decreased from the assembly pH, which corroborates well with thickness measurements (Supporting information). It can be concluded that the films are smooth and intermixed only in the neutral range and form porous structure when pH is changed. The influence of ionic strength on film characteristics was studied by varying the salt concentration from 0 to 0.4 M NaCl at pH 5. The AFM images at different ionic strengths show that pore formation occurs as a function of ionic strength (Fig. 3). The morphology of the film treated with pH 5 water (0 M NaCl) was found to be smooth and featureless and almost resembled freshly prepared film (data not shown). On the contrary, nanopores in the range of 50–100 nm were observed on film surface when the films were treated with 0.1 M NaCl. When the concentration was increased, these pores grew and widened as a function of salt concentration as shown in Fig. 3b&c. The presence of salt screens the interaction between charged segments and the film loosens and forms nanopores [36,37]. Though pore formation and growth were observed with increase in ionic strength, it did not contribute significantly to the roughness value until 0.2 M NaCl (Supporting information). When the concentration was increased to 0.4 M NaCl, the roughness increased from 4.9 ± 0.5 to 6.8 ± 0.5 nm. The presence of salt reduces the

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

625

Fig. 1. Schematic showing our research methodology for the fabrication of PEM films for stimuli-responsive drug and protein delivery. A, glass substrate; A–B, LbL deposition (PAH/PMA)6 layers; B–C, BSA loading; C–D, additional (PAH/PMA)2 layer deposition; D–E, CH loading; E–F, stimuli-responsive release.

interaction between charged segments and the polymer structure becomes more compacted and folded [8,38]. Significant decrease in thickness proves that the polyelectrolytes probably start detaching from the surface. These results corroborate well with previous investigations [8,37,39] that higher salt concentration reduces polymer–polymer and polymer–surface interactions thus causing less interpenetrated and unstable films, which leads to detachment of polymer and formation of porous films. 3.1. Protein and drug loading studies For multi-drug delivery, we have investigated the encapsulation of CH and BSA in multilayer film and its subsequent release. It is reported in our previous study that porous and supramolecular structure of the polyelectrolyte multilayer film could be effectively used to load CH and BSA within the polymeric network of the film [29]. In brief, small water soluble drugs like CH could be easily loaded in the porous network of the film whereas large molecules like BSA (proteins) can be

loaded in the film as a layer. The loading process was investigated using UV–Vis spectroscopy by measuring the absorbance difference of supernatant prior to and after loading. BSA can be directly loaded into the PEM film using multi-mode interactions such as electrostatic interactions, hydrogen bonding and hydrophobic interactions [18]. Here, the loading was performed at pH 4, where the film was porous due to ionization of PMA. In addition, it lies well below the isoelectric point of BSA (4.8), hence facilitates higher loading [40,29]. For the case of CH encapsulation, two mechanisms contributed to the amount of CH loaded into the film: (a) electrostatic interaction between protonated CH and negatively charged PMA on the surface of the film; and (b) normal diffusion through the pores in the polymeric network due to the concentration gradient. The estimated amount of loading was about 600–700 μg/cm2 for BSA and 400–600 μg/cm2 for CH. It is noteworthy that this film showed higher loading under same conditions when compared to their counterparts, e.g., film made up of strong polyelectrolytes [29]. The loading is almost 30–40% higher than that of (PAH/dextran sulfate) films, where there is no significant change in

Fig. 2. AFM image of LbL assembled (PAH/PMA)8 PEM film to show the influence of pH on morphology of the film. (a) pH 1.5, (b) pH 3, (c) pH 5, (d) pH 7, (e) pH 9 and (f) pH 11. Scale bar =5 μm.

626

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

Fig. 3. AFM investigation to show the influence of ionic strength on film morphology. (a) 0.1 M, (b) 0.2 M and (c) 0.4 M.

the film morphology as a function of pH and ionic strength. Thus, it confirms that this film is not only capable of loading two or more drugs in its polymeric network without any modification but also achieves higher loading efficiency. 3.2. In-vitro release studies The drug release from the loaded films was investigated by UV–Vis spectroscopy. To investigate the influence of pH and ionic strength on drug release, films loaded with BSA and CH were subjected to different release conditions. Two mechanisms contributed to the amount of drug or protein released from the films: (a) normal diffusion through pores in the polymeric network due to the concentration gradient; and (b) release due to increased permeability when pH or ionic strength changes. The environmental stimuli such as ionic strength and pH affect the interaction between the drug and films and favor drug release by weakening the drug/protein–film interactions. For concentration gradient induced drug release, the drug release occurs through diffusion from high concentration (film surface) to lower concentration (bulk). The amount of drug released was directly estimated by measuring the absorbance of supernatant solution. The investigations show that the changes in pH and ionic strength significantly influence the drug release (Fig. 4). In all the cases, the release was found to be biphasic with the initial burst release at first 0.5–1 h, followed by sustained release up to 8 h. The initial burst release is due to excess drug or protein present on the surface of the film. After burst release, the drug or protein concentration in the film decreases, which in turn reduces concentration gradient and hence release. It can be seen that the CH release is higher at pH 1.2 than that of pH 7.4 (Fig. 4a). Decrease in pH significantly increases the film permeability, hence reducing the film resistance to drug diffusion and favors higher release. For instance, the total amount of CH release observed at pH 7.4 was about 69% which increased to 86% when pH was

decreased to 1.2. The amount of BSA released was found to be 65% at pH 7.4. Since BSA is denatured at pH 1.5 [31], BSA release experiments were not performed at pH 1.5. To investigate the influence of ionic strength on drug release the experiments were performed at different NaCl concentrations varying from 0 to 0.4 M NaCl. It can be seen that the amount of drug release was increased as a function of ionic strength as shown in Fig. 4b. When ionic strength increased to 0.4 M NaCl, the amount released was about 77% for BSA and 85% for CH. The enhanced release is mainly due to two factors: (a) salt screens the interaction between the drug and film; and (b) increased permeability of the film as a function of concentration. The release of CH is higher in all the conditions than that of BSA, which is due to higher mobility of CH. The difference in amount released between BSA and CH confirms that the factors such as mobility, hydrodynamic diameter and film resistance play a significant role in the release process. 3.3. Cell culture and antibacterial studies In perspective of using this film as thin film drug delivery platform for transdermal applications, cell viability studies on multilayer films were investigated using kidney epithelial cells. Fig. 5 shows the cell viability of control and PEM thin films cultured over a period of one week. It can be seen that the number of cells increased as a function of time. The percentage absorbance of cells seeded on multilayer film was about 94% after 1 day, which was increased approximately to 225% after 7 days. This increase in absorbance indicates better attachment and growth of cells. The interpretation is corroborated well with cell adhesion studies, as shown in Fig. 6. For cell adhesion experiments, cells were directly seeded on sterilized multilayer samples and cellular activity like cell adsorption and proliferation was investigated by studying the morphology of the cells. Notably, the cell growth was increased as a function of time. The cells were spherical and less spread on the surface during the initial

Fig. 4. Drug release as a function of (a) pH and (b) ionic strength.

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

627

Fig. 5. MTT cytotoxicity test on control and PEM films after 1, 3, 5 and 7 days of cell culture.

days. After acclimatizing to the new conditions, the cells had adhered and well spread on the sample surfaces, with a needle like morphology. It is noteworthy that the spaces between the cells reduced uniformly in all directions as a function of time, which confirms cellular growth on the multilayer surfaces. Fig. 7 shows the ZOI of released CH from the films at different time intervals. The ZOI was observed for all the samples, indicating significant microbial susceptibility against bacterial pathogen, E. coli. It can be seen that ZOI decreases as a function of time due to decrease in concentration of CH. The antibacterial property of the film continued up to 8 h, beyond that there was no ZOI. These results confirm that stimuli-responsive films reported here have sufficient biocompatibility and antibacterial properties, thus have significant potential in fields such as transdermal drug delivery, antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents.

Fig. 7. Antibacterial activity of released CH against the pathogen E. coli. Photograph shows the ZOI of drug as a function of release time.

4. Conclusion We have reported a thin film platform made up of weak polyelectrolytes for dual drug delivery and demonstrated successful release of BSA and CH. The changes in the environmental stimuli such as pH and ionic strength significantly influence the film morphology and induce rupture. The loaded drug concentration in the film was about 600–700 μg/cm2 for BSA and 400–600 μg/cm2 for CH. The pore formation due to charge imbalances in the film could be effectively exploited for the release of loaded BSA and CH. When the film has more ionized

Fig. 6. Microscopic images to show the cell adhesion and growth on the multilayer samples. (a–c) Multilayer samples; (d) control sample. Cell growth after (a) 2, (b) 4 and (c, d) 7 days. Scale bar =100 μm.

628

S. Anandhakumar et al. / Materials Science and Engineering C 58 (2016) 622–628

groups in its polymeric network, the release is faster. The drug-loaded films showed initial burst release, followed by sustained release up to 8 h. The fabricated films showed excellent biocompatibility with kidney epithelial cells during in-vitro cell culture experiments. Thus PEM films with performance metrics like the one presented here have the potential to be effective in transdermal drug delivery, antimicrobial or antiinflammatory coatings on implants and drug-releasing coatings for stents. Acknowledgements The authors thank the Institute Nanoscience Initiative, Indian Institute of Science for microscopy facilities. Funding was provided from the institute funding of SRM University. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.08.039. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

G. Decher, Science 277 (1997) 1232–1237. P.T. Hammond, Curr. Opin. Colloid Interface Sci. 4 (1999) 430–442. T. Boudou, T. Thomas Crouzier, K. Ren, G. Blin, C. Picart, Adv. Mater. 21 (2009) 1–27. C. Picart, Curr. Med. Chem. 15 (2008) 685–697. C.S. Peyratout, L. Dahne, Angew. Chem. Int. Ed. 43 (2004) 3762–3783. A. Volodkin, A. Skirtach, H. Möhwald, Adv. Polym. Sci. 240 (2011) 135–161. S.S. Shiratori, M.F. Rubner, Macromolecules 33 (2000) 4213–4219. S.T. Dubas, J.B. Schlenoff, Macromolecules 34 (2001) 3736–3740. H.G.M. Van de Steeg, M.A. Cohen Stuart, A. De Keizer, B.H. Bijsterbosch, Langmuir 8 (1992) 2538–2546. J.B. Schlenoff, H. Ly, M. Li, J. Am. Chem. Soc. 120 (1998) 7626–7634. D. Yoo, S.S. Shiratori, M.F. Rubner, Macromolecules 31 (1998) 4309–4318. R.R. Netz, J.F. Joanny, Macromolecules 32 (1999) 9013–9025. G.B. Sukhorukov, A.A. Antipode, A. Voigt, E. Donath, H. Möhwald, Macromol. Rapid Commun. 22 (2001) 44–46.

[14] Y. Lvov, A.A. Antipode, A. Mamedov, H. Möhwald, G.B. Sukhorukov, Nano Lett. 1 (2001) 125–128. [15] W. Tong, C. Gao, H. Möhwald, Macromolecules 39 (2006) 335–340. [16] T. Mauser, C. Déjugnat, G.B. Sukhorukov, J. Phys. Chem. B. 110 (2006) 20246–20253. [17] B. Thierry, F.M. Winnik, Y. Merhi, M. Tabrizian, J. Am. Chem. Soc. 125 (2003) 7494–7495. [18] Z.Y. Tang, Y. Wang, P. Podsiadlo, N.A. Kotov, Adv. Mater. 18 (2006) 3203–3224. [19] R.K. Agarwal, D.H. Robinson, G.I. Maze, R.A. Reinhardt, J. Control. Release 23 (1993) 137–146. [20] S.F. Huang, J.L. Chen, M.K. Yeh, C.H. Chiang, J. Ocul. Pharmacol. Ther. 21 (2005) 445–453. [21] Y. Dong, Z. Zhang, S.S. Feng, Int. J. Pharm. 350 (2008) 166–171. [22] U. Westedt, M. Wittmar, M. Hellwig, P. Hanefeld, A. Greiner, A.K. Schaper, T. Kissel, J. Control. Release 111 (2006) 235–246. [23] J.K. Jackson, J. Smith, K. Letchford, K.A. Babiuk, M. Lindsay, P. Signore, W.L. Hunter, K.Y. Wang, H.M. Burt, Int. J. Pharm. 283 (2004) 97–109. [24] H.J. Lim, H.Y. Nam, B.H. Lee, D.J. Kim, J.Y. Ko, J.S. Park, Biotechnol. Prog. 23 (2007) 693–697. [25] A. Kumar, A. Srivastava, I.Y. Galaev, B. Mattiasson, Prog. Polym. Sci. 32 (2007) 1205–1237. [26] A.K. Bajpai, S.K. Shukla, S. Bhanu, S. Kankakee, Prog. Polym. Sci. 33 (2008) 1088–1118. [27] M. Catauro, F. Bollino, F. Papale, S. Pacifico, J. Drug Deliv, Sci. Technol. 26 (2015) 10–16. [28] Z.J. Deng, S.W. Morton, E. Ben-Akiva, E.C. Dreaden, K.E. Shopsowitz, P.T. Hammond, ACS Nano 7 (2013) 9571–9584. [29] S. Anandhakumar, A.M. Raichur, Acta Biomater. 9 (2013) 8864–8874. [30] A. Yu, Y. Wang, E. Barlow, F. Caruso, Adv. Mater. 17 (2005) 1737–1741. [31] S. Anandhakumar, V. Nagaraja, A.M. Raichur, Colloids Surf. B 78 (2010) 266–274. [32] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102. [33] E. Kharlampieva, S.A. Sukhishvili, Langmuir 19 (2003) 1235–1243. [34] T. Mauser, C. Dejugnat, H. Mohwald, G.B. Sukhorukov, Langmuir 22 (2006) 5888–5893. [35] T. Mauser, C. Dejugnat, G.B. Sukhorukov, Macromol. Rapid Commun. 25 (2004) 1781–1785. [36] Z. Sui, D. Salloum, J.B. Schlenoff, Langmuir 19 (2003) 2491–2495. [37] N.G. Hoogeveen, M.A. Cohen Stuart, G.J. Fleer, J. Colloid Interface Sci. 182 (1996) 146–157. [38] R.A. McAloney, M. Sinyor, V. Dudnik, M.C. Goh, Langmuir 17 (2001) 6655–6663. [39] S.T. Dubas, J.B. Schlenoff, Langmuir 17 (2001) 7725–7727. [40] S. Anandhakumar, M. Debapriya, V. Nagaraja, A.M. Raichur, Mater. Sci. Eng. C 31 (2011) 342–349.

Stimuli-responsive weak polyelectrolyte multilayer films: A thin film platform for self triggered multi-drug delivery.

Polyelectrolyte multilayer (PEM) thin film composed of weak polyelectrolytes was designed by layer-by-layer (LbL) assembly of poly(allylamine hydrochl...
2MB Sizes 1 Downloads 9 Views