Materials Science and Engineering C 54 (2015) 239–244
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Insulin particles as building blocks for controlled insulin release multilayer nano-films Xiangde Lin, Daheui Choi, Jinkee Hong ⁎ School of Chemical Engineering & Material Science, Chung-Ang University, 47 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea
a r t i c l e
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Article history: Received 13 February 2015 Received in revised form 6 April 2015 Accepted 15 May 2015 Available online 18 May 2015 Keywords: Diabetic therapy Insulin delivery Insulin nanoparticles Layer-by-layer assembly Long-acting release
a b s t r a c t Insulin nanoparticles (NPs) were prepared by pH-shift precipitation and a newly developed disassembly method at room temperature. Then, an electrostatic interaction-based, layer-by-layer (LbL) multilayer film incorporating insulin NPs was fabricated with poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH), which is described herein as Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n. The positively charged insulin NPs were introduced into the LbL film in the form of biocompatible PAA-insulin NP aggregates at a pH of 4.5 and were released in phosphate-buffered saline (pH 7.4), triggered by changes in the charges of the insulin molecules. In addition, the insulin-incorporated multilayer was swollen because of the different ionic environment, leading also to insulin release. Eighty percent of the insulin was released from the LBL film in the first stage of 3 h, and sustained release could be maintained in the second stage for up to 7 days in vitro, which is very critical for specific diabetic patients. These striking findings could offer novel directions to researchers in establishing insulin delivery systems for diabetic therapy and fabricating other protein nanoparticles applied to various biomedical platforms. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Diabetes therapy incorporating various nanomaterials and approaches to effectively control glycemic stability has received increased attention over the past decades [1–6]. In implementing new methods, different forms of insulin such as supramolecular insulin assemblies (SIA) [7], insulin analogs [8], insulin nanoparticles (NPs) [9], nanonetworks [10], and nanocomplexes [11] have been prepared and widely employed for insulin delivery, in particular via traditional subcutaneous injection. For instance, nano-sized insulin particles could serve as appropriate drug depots at the injection site for long-term release, according to related research by Gupta et al. [7]. A great deal of effort has also been made to establish various insulin delivery systems such as oral administration [12], intranasal therapy [13], gastrointestinal route [14], pulmonary delivery [15], and tablet implantation [16]. However, a number of challenging issues involved in the process of diabetes treatment, including high cost, low compatibility in vivo, common infections, patient compliance, and sudden hypoglycemia, have not yet been overcome in a flawless manner. The layer-by-layer (LbL) technique for multilayer assembly provides a superb route to build up desired films using a range of functional materials, including polymer polyelectrolytes, DNA, proteins, graphene, nanocomplexes, nanoparticles, nanowires, and nanotubes [17–23]. Particular advantages of the LbL method, such as the precise control of film thickness, specific functionality, optional compositions, and ⁎ Corresponding author. E-mail address: [email protected] (J. Hong).
http://dx.doi.org/10.1016/j.msec.2015.05.046 0928-4931/© 2015 Elsevier B.V. All rights reserved.
versatile morphology, have been investigated and demonstrated in the field of multilayered structures ranging from the nano- to microscale [24–26]. Thus, inorganic NPs, also applicable for nearly any type of charged components, were introduced into desired functional multilayers by virtue of electrostatic interactions [22]. Similarly, LbL multilayered films with building blocks of different drugs can be obtained by taking advantage of the molecular interactions among materials, in particular electrostatic interactions or hydrogen bonding [27,28]. Specifically, researchers have been attracted to the design of insulin delivery systems based on LbL thin films. For example, Chen et al. successfully fabricated a glucose-sensitive multilayer film based on a 21-armstar polymer, showing an on–off switch of insulin release in response to in vivo glucose levels [29]. Their group then further developed LbL films constructed from supramolecular insulin assemblies, which were useful for super long-term glycemic control for up to 295 days [30]. Unlike the glucose-sensitive system, insulin release triggered by variations in pH was also observed by Yoshida et al. after exposing a template containing insulin to weakly acidic or neutral solutions [31]. After transcutaneous protein drug delivery was created, we proposed a method for creating insulin-encapsulated nanofilms by LbL, which could be regarded as a nano-container for controlled insulin release [32]. Herein, we aimed to rapidly prepare insulin NPs through pH-shift precipitation and crystal disassembly, which is a novel technique, compared with conventional growth means that can be timeconsuming, limited in high temperature or carried out under denaturation condition. Following preparation, insulin NPs in the form of PAA-insulin NP aggregation were assembled into pH-sensitive
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(PAH/PAA)n multilayered films via the LbL method for sustained insulin release by means of transcutaneous administration [33]. In the first place, considering the toxicity of building blocks of PAH, biocompatible PAA was used as an outside coating on insulin NPs while low molecular weight PAH was assembled inside multilayers, which showed extremely low toxicity [34,35]. In our strategy, in order to improve poor compliance in subcutaneous injections, LbL films were packaged in the form of patches, which could also avoid direct contact between insulin and tissues and prevent hypoglycemia. Positively charged insulin microcrystals were prepared in acetic acid at pH 6.5 because of the isoelectric point (pI) of insulin. Negatively charged PAA-insulin NPs were formed in PAA solution at a pH of 4.5 and were then used as building blocks to construct LbL films by alternating deposition with PAH, which is convenient for multilayer assembly. We demonstrate that the resulting films can be partially deconstructed to release insulin into the surrounding medium in pH 7.4, phosphate-buffered saline (PBS) solution, which is largely due to the release of insulin NPs. It is also observed that insulin release from the prepared films in PBS solution could last for up to 7 days, which is very critical for specific diabetic patients.
2. Materials and methods 2.1. Materials Insulin (from bovine pancreas, Mw ≈ 5,733), poly(acrylic acid) (PAA, Mw ≈ 2,000), poly(allylamine hydrochloride) (PAH, Mw ≈ 15,000), 10 mM phosphate buffered saline (PBS, pH 7.4), and fluorescein isothiocyanate isomer I (FITC, 90%) were purchased from Sigma-Aldrich. All other chemicals and solvents were of analytical grade.
2.2. Preparation and characterization of PAA-insulin NPs Insulin NPs at 1 mg/mL were prepared according to the modified seed zone method [36]. Briefly, 1 mg/mL bovine insulin was fully dissolved in 0.01 N acetic acid (pH 2.0) under stirring. To the clear solution, 10 N and 1 N NaOH were added in a stepwise manner to increase the pH. Once the pH has reached 10.5 ± 0.5, the aqueous insulin suspension became clear. Subsequently, the pH of the solution was slowly regulated to pH 6.8 by adding 1 N HCl, and the solution became milky. After stirring the solution for 15 min to completely form microcrystals, centrifugation was performed for 10 min at 13,000 rpm. Thereafter, insulin precipitation was obtained, and a 1 mg/mL PAA polyelectrolyte solution at pH 4.5 was added to acquire an insulin suspension. After 15 min of stirring, centrifugation was repeated at 13,000 rpm for 10 min. The supernatant was then separated from the insulin precipitation. Finally, nano-sized insulin NPs in the supernatant were stored at 4 °C in darkness. The morphologies of the insulin NPs and PAA-insulin NPs deposited onto Si/(PAH/PAA)5 and Si/ (PAH/PAA)5PAH substrates were observed by field-emission scanning
electron microscopy (FE-SEM, SIGMA) and transmission electron microscopy (TEM, FEI Tecnai G2), respectively. 2.3. Fabrication and characterization of insulin-incorporated LbL films LbL multilayer films were fabricated on a silicon wafer by handdipping the substrate into various aqueous solutions at room temperature. The silicon substrate was pre-treated for 2 min with oxygen plasma to create a negatively charged surface. For the first step in the LbL process, the substrate was dipped into a positively charged 1 mg/mL PAH aqueous solution (pH 7.5) for 10 min. Then, the substrate was rinsed three times in deionized water for 2 min, 1 min, and 1 min. Next, the substrate was immersed in a negatively charged 1 mg/mL PAA aqueous solution (pH 3.5) for 10 min. Similar to the previous washing step, the substrate was rinsed three times in deionized water. Si/(PAH/PAA)5 was obtained as a basic layer after five alternating deposition cycles. Afterwards, the substrate with the basic layer was dipped into a 1 mg/mL PAH aqueous solution (pH 4.5) and washed three times by 0.01 N acetic acid (pH 4.5) for 2 min, 1 min, and 1 min. Subsequently, the substrate was placed into the as-prepared PAAinsulin NP suspension solution for 10 min. Identical rinsing steps were carried out to remove extra PAA-insulin NPs. This dipping process was repeated until the desired number of bilayers was acquired. The morphologies of the Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n (n = 0, 1, 4, 12, and 20) multilayers were examined via FE-SEM. Film thickness growth was monitored by a profilometer (Dektak 150, Veeco) at three different positions on the film surface. Furthermore, the morphologies of Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n (n = 12 and 20) multilayers were examined using atomic force microscopy (AFM, Park Systems X10) with a scan area of 2 × 2 μm2. 2.4. Film decomposition triggered by pH Film decomposition was characterized through profilometry measurement for a Si/(PAH/PAA)5(PAH/PAA-insulin NPs)20 film. Specifically, the thickness variation of the pristine 2145.3 ± 17.2 nm film, dried by N2 flow, was monitored by profilometry after exposure to a 10 mM PBS solution (37 °C, pH 7.4, 5 mL in a 20-mL vial) for a predetermined period of time. 2.5. Insulin release from LbL film According to the fabrication of insulin-incorporated films, 0.9 mg/mL bovine insulin and 0.1 mg/mL FITC were completely dissolved in 10 mL of 0.01 N acetic acid (pH 2.0) while stirring in darkness. The pristine 2145.3 ± 17.2 nm Si/(PAH/PAA)5(PAH/PAA-insulin NPs-FITC)20 multilayer films (1.5 × 1.5 cm2) were then fabricated by following the assembly process above. After immersing films into 10 mM PBS (pH 7.4), the release of insulin-FITC from multilayers was
Fig. 1. Schematic presentation of the film assembly of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n based on layer-by-layer deposition. (a) Formation of insulin crystals by the seed zone method. (b) Fabrication of insulin nanoparticles. (c) Coating insulin NPs with polyelectrolytes of PAA. (d) Dipping Si/(PAH/PAA)5/PAH substrate in as-prepared PAA-insulin NP solution. (e) Insulin NP deposition process. (f) Layer-by-layer assembly of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n with repeating PAH and PAA-insulin NP deposition.
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Fig. 2. Morphology analysis on insulin crystals, insulin nanoparticles, and PAA-insulin nanoparticles is observed, respectively. (a) FE-SEM image of insulin crystals. (b) FE-SEM image of 26 ± 3.5 nm insulin nanoparticles without PAA coating. (c) TEM image of insulin nanoparticles with PAA.
followed by the measurement of fluorescence intensity (λex = 493 nm) via spectrofluorometer (FP-8300, JASCO). 3. Results and discussion 3.1. Analysis on PAA-insulin NPs and assembled multilayers A schematic illustration of the preparation of PAA-insulin NPs and electrostatic interaction-based LbL film assembly is depicted in Fig. 1. Initially, insulin molecules existed in the form of a monomer or dimer in acetic acid (pH 2.0). Along with the increase in pH towards its pI (pH 5.4), the solubility of insulin reached its minimum and insulin was nearly precipitated out. Beyond the pI, insulin precipitation slowly disappeared as the pH increased and the insulin suspension became almost transparent when the pH reached 10.5 ± 0.5. Within this pH range, enough nanoparticles were dispersed in solution, which acted as seeds [36]. After the pH was regulated back to 6.8, microcrystals were formed under supersaturated conditions, as shown in Fig. 1a. Therefore, microcrystals could be clearly observed by FE-SEM (Fig. 2a). When the insulin crystals were immersed in a pH 4.5 environment, nano-sized insulin particles (NPs) on the surface of the crystals were disassembled into the suspension under stirring, which was proved in Fig. 2b. Fig. 1b–c shows the processes of PAA-insulin NP formation in a PAA solution at a pH of 4.5 with 15 min of stirring. Specifically, positively charged insulin NPs rapidly combined with negatively charged PAA
polyelectrolytes in the form of PAA-insulin NP aggregation as shown in Fig. 1c. In order to figure out molecular interactions among building blocks, Zeta potential ξ (Nano Particle Analyzer, SZ-100) of 140 nm insulin NPs and PAA-insulin NPs was measured accordingly. (+) 21.9 mV and (−) 37.4 mV for insulin NPs and PAA-insulin NPs under pH 4.5 condition were obtained, respectively. Positive and negative surface charges of insulin nanoparticles alternated after PAA and PAH coating, which is proved in Supplementary data Fig. A1a. It illustrates that a multilayer assembly among these building blocks could be directed by electrostatic interactions. In addition, novel PAA-insulin NPs also could be observed under TEM (Fig. 2c). After the preparation of PAA-insulin NPs, the Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n thin film coatings were assembled with the aid of functional amine and carboxylic acid groups in PAH and PAA-insulin NPs, respectively. As a result, PAA-insulin NPs with negative charges adhered onto the positively charged PAH surface by means of electrostatic interactions, as illustrated in Fig. 1d and e. At last, LbL assembly film of Si/(PAH/ PAA)5/(PAH/PAA-insulin NPs)n was fabricated using a predetermined number of deposition cycles. Prior to particle deposition, pristine insulin NPs at 26 ± 3.5 nm were prepared and coated without PAA on the outside (size data in Supplementary data Fig. A1b). As shown in Fig. 3a, PAA-insulin NPs with a diameter of 27 ± 3.5 nm were fabricated and deposited onto a substrate with the basic (PAH/PAA)5/PAH layer. In consideration of uniformly distributed charges of the basic layer, PAA-insulin NPs could
Fig. 3. (a) SEM image of PAA-insulin nanoparticles on a basic layer. (b) Thickness growth curve of assembled films of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n (red curve) and Si/(PAH/PAA) 5/(PAH/PAA)n (black curve) measured by profilometry. The mean diameter of PAA-insulin NPs measured by SEM was 27 nm ± 3.5 nm.
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Fig. 4. Top-down FE-SEM images of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayers with different deposition cycles. (a) n = 1; (b) n = 4; (c) n = 12; (d) n = 20. The inset of (a) shows a pristine Si/(PAH/PAA)5 substrate without insulin NPs.
be deposited homogeneously, compared to dropping directly on the sample holder of TEM in Fig. 2c. The growth in the thickness of the Si/ (PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayers was exponential with respect to the number of deposited bilayers, while in the case of Si/(PAH/PAA)5/(PAH/PAA)n, a linear relationship was typically observed, which is demonstrated in Fig. 3b, respectively. This exponential growth regime could be properly explained by the “in” and “out” diffusion of insulin chains during the LbL film build-up process [37]. Taking advantage of the fact that film thickness could readily reach the micro-scale via exponential growth, thick multilayers incorporating proteins can be obtained and easily applied to drug delivery. FE-SEM provides an intuitive way to analyze the morphologies of LbL multilayer films. Fig. 4a–d shows top-down FE-SEM images of (PAH/PAA)5/(PAH/PAA-insulin NPs)n (n = 1, 4, 12, 20) multilayers with different desired numbers of bilayers on silicon wafers. Pristine Si/(PAH/PAA)5 substrates without the deposition of insulin NPs were also analyzed (inset of Fig. 4a), in which the morphology can be distinguished visibly. Initially, insulin NPs were uniformly dispersed onto the basic layer of (PAH/PAA)5/PAH, a process mediated by the
complementary electrostatic phenomenon. As the number of bilayers increased, insulin NPs were stacked and incorporated into multilayers, leading to rapid, exponential growth in thickness. A film consisting of 20 bilayers was fabricated (Fig. 4d), possessing a dense and partially smooth surface, which may have been a result of the adhesion of small insulin molecules in the supersaturated solution and the dominant deposition of the PAH/PAA bilayer in a strong ionic environment (0.01 N, pH 4.5 acetic acid). In addition to FE-SEM characterization, surface morphology analysis of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)12 and Si/(PAH/PAA)5/(PAH/ PAA-insulin NPs)20 was conducted by AFM in tapping mode, as shown in Fig. 5. The dominant forms of insulin NPs with a height of about 20 nm were present in a spherical shape, and some aggregating clusters along with polymeric chains could be observed (Fig. 5a). The surface morphologies of the basic (PAH/PAA)5 layer and (PAH/PAA)5/(PAH/ PAA-insulin NPs)1 were also measured though cross-sectional measurements using AFM, indicating the successful deposition of insulin NPs and the original basic layer with a height of around 10 nm, as shown in Supplementary data Fig. A2. Similar to the FE-SEM observation, the
Fig. 5. AFM images of (a) Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)12 multilayers and (b) Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film in tapping mode.
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Fig. 6. Deconstruction profile of a Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer film in PBS (pH 7.4). (a) Top-down SEM image of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film before immersing in PBS (pH 7.4). (b) Film morphology characterized by FE-SEM after immersing into PBS buffer for 6 h. (c) Film deconstruction profile measured by profilometry in the dried state.
dense surface of the Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film with small insulin molecules was detected from the AFM images, as demonstrated in Fig. 5b. 3.2. Deconstruction of assembled films containing insulin NPs In order to deeply examine the release of insulin from LbL films, an as-prepared Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer was dipped into a 10 mM PBS solution (37 °C, pH 7.4, 5 mL in a 20-mL vial) for a predetermined amount of time. First, the acidity and ionic strength of the dipping solution were transformed from 0.01 M acetic acid (pH 4.5) to 0.01 M PBS (pH 7.4), resulting in the conversion of positively charged insulin NPs into negatively charged ones. As a result, PAA-insulin NPs were deconstructed and insulin molecules were released into the medium, resulting from the same negative surface charges between insulin and PAA. Furthermore, assembled LbL films based on the weak polyelectrolytes of PAH and PAA were swollen in the aqueous environment, which could be demonstrated by the increased thickness of films in a few minutes. Combining all of these factors, insulin molecules could be liberated from the LbL films, which was justified by morphological analysis performed using FE-SEM in Fig. 6a–b. The porous film was formed after 6 h because of insulin particle release, which is demonstrated in Fig. 6a–b. The deconstruction profile of a Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer film in its dried state was recorded at various time points by profilometry (Fig. 6c).
Fig. 7. Normalized insulin release profile obtained by fluorescence intensity detection of FITC-labeled insulin.
3.3. Insulin release test of LbL films In addition, to further verify that insulin NPs were loaded into multilayer films as building blocks, the release profile of FITC-labeled insulin NPs (insulin NPs-FITC) capable of deconstructing from LbL membranes in PBS solution was accurately monitored by measuring the fluorescence intensity using photoluminescence (PL) spectroscopy. As-prepared LbL films incorporating insulin NPs-FITC were incubated at 37 °C in PBS solution over certain designated periods of time. Normalized insulin release was obtained from the mean intensity peaks from the fluorescence in film-disrupted medium (λex = 493 nm). As shown in Fig. 7, the release rate of insulin-FITC reached up to 48.2% in 5 min, and 79.1% of the insulin-FITC in the LbL film was disassembled into the PBS buffer solution in 3 h. The sustained longterm release after 3 h is of interest and was observed to last for up to 7 days. In conclusion, the insulin release process mainly could divide into 2 stages. The first rapid release stage in 3 h is quite significant for diabetic patients, in terms of high blood glucose level after a meal. Moreover, the second slow release stage could ensure the stable glucose level of patients during sleeping time in particular. Therefore, the result indicates that the assembled multilayers incorporating insulin NPs play a critical role in insulin delivery systems.
4. Conclusions In summary, this work proposed a newly developed method through rapid pH-shift insulin crystallization and disassembly for the fabrication of nano-sized insulin particles by which LbL multilayer films could be uniformly constructed through electrostatic interactions. Obviously, our strategy for synthesizing insulin NPs got rid of the traditional denaturation environment. Above all, in terms of the different release rate of insulin in various forms, we appropriately combined the inherent properties of insulin NPs with the LbL multilayer assembly technique to develop a sustained insulin release for a transcutaneous delivery system. In vitro examination in physiological condition demonstrates that insulin molecules liberated rapidly in the first stage of 3 h (79.1%), and sustained insulin release continued for up to 7 days in the second stage. Hence, effective LbL Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayer films considerably improved the transcutaneous insulin delivery system. In fact, further development of insulin delivery could be carried out based on our research. For example, more variables, such as using different sized insulin particles, incorporating various
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forms of insulin, and coating insulin particles with multilayers could be significantly helpful for controlling insulin release rate. Furthermore, inspired by our mechanism of insulin NP fabrication and LbL assembly, other delivery systems such as those for enzymes and vaccines are likely to be established for biomedical platforms in the near future. Acknowledgements This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2013R1A1A1076126, and 2012M3A9C6050104). Additionally, this work was also supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009986012015) (Rural Development Administration, Republic of Korea), as well as the High Value-added Food Technology Development Program of the Ministry of Agriculture, Food and Rural Affairs (114027-03-1-HD020) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI14C-3266-030014). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.05.046. References [1] R.C. Turner, C.A. Cull, V. Frighi, R.R. Holman, UK Prospective Diabetes Study (UKPDS) Group, Glycemic control with diet, sulfonylurea, metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for multiple therapies (UKPDS 49), JAMA 281 (1999) 2005–2012. [2] A.T. Cheung, B. Dayanandan, J.T. Lewis, G.S. Korbutt, R.V. Rajotte, M. Bryer-Ash, M.O. Boylan, M.M. Wolfe, T.J. Kieffer, Glucose-dependent insulin release from genetically engineered K cells, Science 290 (2000) 1959–1962. [3] A.S. Narang, R.I. Mahato, Biological and biomaterial approaches for improved islet transplantation, Pharmacol. Rev. 58 (2006) 194–243. [4] M.C. Chen, K. Sonaje, K.J. Chen, H.W. Sung, A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery, Biomaterials 32 (2011) 9826–9838. [5] K.M. Bratlie, R.L. York, M.A. Invernale, R. Langer, D.G. Anderson, Materials for diabetes therapeutics, Adv. Healthcare Mater. 1 (2012) 267–284. [6] K. Yoshida, Y. Hasebe, S. Takahashi, K. Sato, J.I. Anzai, Layer-by-layer deposited nanoand micro-assemblies for insulin delivery: a review, Mater. Sci. Eng. C 34 (2014) 384–392. [7] S. Gupta, T. Chattopadhyay, M.P. Singh, A. Surolia, Supramolecular insulin assembly II for a sustained treatment of type 1 diabetes mellitus, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 13246–13251. [8] E. Gershonov, I. Goldwaser, M. Fridkin, Y. Shechter, A novel approach for a watersoluble long-acting insulin prodrug: design, preparation, and analysis of [(2-sulfo)-9fluorenylmethoxycarbonyl] 3-insulin, J. Med. Chem. 43 (2000) 2530–2537. [9] M.M. Bailey, E.M. Gorman, E.J. Munson, C. Berkland, Pure insulin nanoparticle agglomerates for pulmonary delivery, Langmuir 24 (2008) 13614–13620. [10] Z. Gu, A.A. Aimetti, Q. Wang, T.T. Dang, Y. Zhang, O. Veiseh, H. Cheng, R.S. Langer, D.G. Anderson, Injectable nano-network for glucose-mediated insulin delivery, ACS Nano 7 (2013) 4194–4201. [11] Y.F. Fan, Y.N. Wang, Y.G. Fan, J.B. Ma, Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-by-layer adsorption, Int. J. Pharm. 324 (2006) 158–167.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Insulin particles as building blocks for controlled insulin release multilayer nano-films Xiangde Lin, Daheui Choi, Jinkee Hong ⁎ School of Chemical Engineering & Material Science, Chung-Ang University, 47 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea
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Article history: Received 13 February 2015 Received in revised form 6 April 2015 Accepted 15 May 2015 Available online 18 May 2015 Keywords: Diabetic therapy Insulin delivery Insulin nanoparticles Layer-by-layer assembly Long-acting release
a b s t r a c t Insulin nanoparticles (NPs) were prepared by pH-shift precipitation and a newly developed disassembly method at room temperature. Then, an electrostatic interaction-based, layer-by-layer (LbL) multilayer film incorporating insulin NPs was fabricated with poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH), which is described herein as Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n. The positively charged insulin NPs were introduced into the LbL film in the form of biocompatible PAA-insulin NP aggregates at a pH of 4.5 and were released in phosphate-buffered saline (pH 7.4), triggered by changes in the charges of the insulin molecules. In addition, the insulin-incorporated multilayer was swollen because of the different ionic environment, leading also to insulin release. Eighty percent of the insulin was released from the LBL film in the first stage of 3 h, and sustained release could be maintained in the second stage for up to 7 days in vitro, which is very critical for specific diabetic patients. These striking findings could offer novel directions to researchers in establishing insulin delivery systems for diabetic therapy and fabricating other protein nanoparticles applied to various biomedical platforms. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Diabetes therapy incorporating various nanomaterials and approaches to effectively control glycemic stability has received increased attention over the past decades [1–6]. In implementing new methods, different forms of insulin such as supramolecular insulin assemblies (SIA) [7], insulin analogs [8], insulin nanoparticles (NPs) [9], nanonetworks [10], and nanocomplexes [11] have been prepared and widely employed for insulin delivery, in particular via traditional subcutaneous injection. For instance, nano-sized insulin particles could serve as appropriate drug depots at the injection site for long-term release, according to related research by Gupta et al. [7]. A great deal of effort has also been made to establish various insulin delivery systems such as oral administration [12], intranasal therapy [13], gastrointestinal route [14], pulmonary delivery [15], and tablet implantation [16]. However, a number of challenging issues involved in the process of diabetes treatment, including high cost, low compatibility in vivo, common infections, patient compliance, and sudden hypoglycemia, have not yet been overcome in a flawless manner. The layer-by-layer (LbL) technique for multilayer assembly provides a superb route to build up desired films using a range of functional materials, including polymer polyelectrolytes, DNA, proteins, graphene, nanocomplexes, nanoparticles, nanowires, and nanotubes [17–23]. Particular advantages of the LbL method, such as the precise control of film thickness, specific functionality, optional compositions, and ⁎ Corresponding author. E-mail address: [email protected] (J. Hong).
http://dx.doi.org/10.1016/j.msec.2015.05.046 0928-4931/© 2015 Elsevier B.V. All rights reserved.
versatile morphology, have been investigated and demonstrated in the field of multilayered structures ranging from the nano- to microscale [24–26]. Thus, inorganic NPs, also applicable for nearly any type of charged components, were introduced into desired functional multilayers by virtue of electrostatic interactions [22]. Similarly, LbL multilayered films with building blocks of different drugs can be obtained by taking advantage of the molecular interactions among materials, in particular electrostatic interactions or hydrogen bonding [27,28]. Specifically, researchers have been attracted to the design of insulin delivery systems based on LbL thin films. For example, Chen et al. successfully fabricated a glucose-sensitive multilayer film based on a 21-armstar polymer, showing an on–off switch of insulin release in response to in vivo glucose levels [29]. Their group then further developed LbL films constructed from supramolecular insulin assemblies, which were useful for super long-term glycemic control for up to 295 days [30]. Unlike the glucose-sensitive system, insulin release triggered by variations in pH was also observed by Yoshida et al. after exposing a template containing insulin to weakly acidic or neutral solutions [31]. After transcutaneous protein drug delivery was created, we proposed a method for creating insulin-encapsulated nanofilms by LbL, which could be regarded as a nano-container for controlled insulin release [32]. Herein, we aimed to rapidly prepare insulin NPs through pH-shift precipitation and crystal disassembly, which is a novel technique, compared with conventional growth means that can be timeconsuming, limited in high temperature or carried out under denaturation condition. Following preparation, insulin NPs in the form of PAA-insulin NP aggregation were assembled into pH-sensitive
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(PAH/PAA)n multilayered films via the LbL method for sustained insulin release by means of transcutaneous administration [33]. In the first place, considering the toxicity of building blocks of PAH, biocompatible PAA was used as an outside coating on insulin NPs while low molecular weight PAH was assembled inside multilayers, which showed extremely low toxicity [34,35]. In our strategy, in order to improve poor compliance in subcutaneous injections, LbL films were packaged in the form of patches, which could also avoid direct contact between insulin and tissues and prevent hypoglycemia. Positively charged insulin microcrystals were prepared in acetic acid at pH 6.5 because of the isoelectric point (pI) of insulin. Negatively charged PAA-insulin NPs were formed in PAA solution at a pH of 4.5 and were then used as building blocks to construct LbL films by alternating deposition with PAH, which is convenient for multilayer assembly. We demonstrate that the resulting films can be partially deconstructed to release insulin into the surrounding medium in pH 7.4, phosphate-buffered saline (PBS) solution, which is largely due to the release of insulin NPs. It is also observed that insulin release from the prepared films in PBS solution could last for up to 7 days, which is very critical for specific diabetic patients.
2. Materials and methods 2.1. Materials Insulin (from bovine pancreas, Mw ≈ 5,733), poly(acrylic acid) (PAA, Mw ≈ 2,000), poly(allylamine hydrochloride) (PAH, Mw ≈ 15,000), 10 mM phosphate buffered saline (PBS, pH 7.4), and fluorescein isothiocyanate isomer I (FITC, 90%) were purchased from Sigma-Aldrich. All other chemicals and solvents were of analytical grade.
2.2. Preparation and characterization of PAA-insulin NPs Insulin NPs at 1 mg/mL were prepared according to the modified seed zone method [36]. Briefly, 1 mg/mL bovine insulin was fully dissolved in 0.01 N acetic acid (pH 2.0) under stirring. To the clear solution, 10 N and 1 N NaOH were added in a stepwise manner to increase the pH. Once the pH has reached 10.5 ± 0.5, the aqueous insulin suspension became clear. Subsequently, the pH of the solution was slowly regulated to pH 6.8 by adding 1 N HCl, and the solution became milky. After stirring the solution for 15 min to completely form microcrystals, centrifugation was performed for 10 min at 13,000 rpm. Thereafter, insulin precipitation was obtained, and a 1 mg/mL PAA polyelectrolyte solution at pH 4.5 was added to acquire an insulin suspension. After 15 min of stirring, centrifugation was repeated at 13,000 rpm for 10 min. The supernatant was then separated from the insulin precipitation. Finally, nano-sized insulin NPs in the supernatant were stored at 4 °C in darkness. The morphologies of the insulin NPs and PAA-insulin NPs deposited onto Si/(PAH/PAA)5 and Si/ (PAH/PAA)5PAH substrates were observed by field-emission scanning
electron microscopy (FE-SEM, SIGMA) and transmission electron microscopy (TEM, FEI Tecnai G2), respectively. 2.3. Fabrication and characterization of insulin-incorporated LbL films LbL multilayer films were fabricated on a silicon wafer by handdipping the substrate into various aqueous solutions at room temperature. The silicon substrate was pre-treated for 2 min with oxygen plasma to create a negatively charged surface. For the first step in the LbL process, the substrate was dipped into a positively charged 1 mg/mL PAH aqueous solution (pH 7.5) for 10 min. Then, the substrate was rinsed three times in deionized water for 2 min, 1 min, and 1 min. Next, the substrate was immersed in a negatively charged 1 mg/mL PAA aqueous solution (pH 3.5) for 10 min. Similar to the previous washing step, the substrate was rinsed three times in deionized water. Si/(PAH/PAA)5 was obtained as a basic layer after five alternating deposition cycles. Afterwards, the substrate with the basic layer was dipped into a 1 mg/mL PAH aqueous solution (pH 4.5) and washed three times by 0.01 N acetic acid (pH 4.5) for 2 min, 1 min, and 1 min. Subsequently, the substrate was placed into the as-prepared PAAinsulin NP suspension solution for 10 min. Identical rinsing steps were carried out to remove extra PAA-insulin NPs. This dipping process was repeated until the desired number of bilayers was acquired. The morphologies of the Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n (n = 0, 1, 4, 12, and 20) multilayers were examined via FE-SEM. Film thickness growth was monitored by a profilometer (Dektak 150, Veeco) at three different positions on the film surface. Furthermore, the morphologies of Si/(PAH/PAA)5(PAH/PAA-insulin NPs)n (n = 12 and 20) multilayers were examined using atomic force microscopy (AFM, Park Systems X10) with a scan area of 2 × 2 μm2. 2.4. Film decomposition triggered by pH Film decomposition was characterized through profilometry measurement for a Si/(PAH/PAA)5(PAH/PAA-insulin NPs)20 film. Specifically, the thickness variation of the pristine 2145.3 ± 17.2 nm film, dried by N2 flow, was monitored by profilometry after exposure to a 10 mM PBS solution (37 °C, pH 7.4, 5 mL in a 20-mL vial) for a predetermined period of time. 2.5. Insulin release from LbL film According to the fabrication of insulin-incorporated films, 0.9 mg/mL bovine insulin and 0.1 mg/mL FITC were completely dissolved in 10 mL of 0.01 N acetic acid (pH 2.0) while stirring in darkness. The pristine 2145.3 ± 17.2 nm Si/(PAH/PAA)5(PAH/PAA-insulin NPs-FITC)20 multilayer films (1.5 × 1.5 cm2) were then fabricated by following the assembly process above. After immersing films into 10 mM PBS (pH 7.4), the release of insulin-FITC from multilayers was
Fig. 1. Schematic presentation of the film assembly of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n based on layer-by-layer deposition. (a) Formation of insulin crystals by the seed zone method. (b) Fabrication of insulin nanoparticles. (c) Coating insulin NPs with polyelectrolytes of PAA. (d) Dipping Si/(PAH/PAA)5/PAH substrate in as-prepared PAA-insulin NP solution. (e) Insulin NP deposition process. (f) Layer-by-layer assembly of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n with repeating PAH and PAA-insulin NP deposition.
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Fig. 2. Morphology analysis on insulin crystals, insulin nanoparticles, and PAA-insulin nanoparticles is observed, respectively. (a) FE-SEM image of insulin crystals. (b) FE-SEM image of 26 ± 3.5 nm insulin nanoparticles without PAA coating. (c) TEM image of insulin nanoparticles with PAA.
followed by the measurement of fluorescence intensity (λex = 493 nm) via spectrofluorometer (FP-8300, JASCO). 3. Results and discussion 3.1. Analysis on PAA-insulin NPs and assembled multilayers A schematic illustration of the preparation of PAA-insulin NPs and electrostatic interaction-based LbL film assembly is depicted in Fig. 1. Initially, insulin molecules existed in the form of a monomer or dimer in acetic acid (pH 2.0). Along with the increase in pH towards its pI (pH 5.4), the solubility of insulin reached its minimum and insulin was nearly precipitated out. Beyond the pI, insulin precipitation slowly disappeared as the pH increased and the insulin suspension became almost transparent when the pH reached 10.5 ± 0.5. Within this pH range, enough nanoparticles were dispersed in solution, which acted as seeds [36]. After the pH was regulated back to 6.8, microcrystals were formed under supersaturated conditions, as shown in Fig. 1a. Therefore, microcrystals could be clearly observed by FE-SEM (Fig. 2a). When the insulin crystals were immersed in a pH 4.5 environment, nano-sized insulin particles (NPs) on the surface of the crystals were disassembled into the suspension under stirring, which was proved in Fig. 2b. Fig. 1b–c shows the processes of PAA-insulin NP formation in a PAA solution at a pH of 4.5 with 15 min of stirring. Specifically, positively charged insulin NPs rapidly combined with negatively charged PAA
polyelectrolytes in the form of PAA-insulin NP aggregation as shown in Fig. 1c. In order to figure out molecular interactions among building blocks, Zeta potential ξ (Nano Particle Analyzer, SZ-100) of 140 nm insulin NPs and PAA-insulin NPs was measured accordingly. (+) 21.9 mV and (−) 37.4 mV for insulin NPs and PAA-insulin NPs under pH 4.5 condition were obtained, respectively. Positive and negative surface charges of insulin nanoparticles alternated after PAA and PAH coating, which is proved in Supplementary data Fig. A1a. It illustrates that a multilayer assembly among these building blocks could be directed by electrostatic interactions. In addition, novel PAA-insulin NPs also could be observed under TEM (Fig. 2c). After the preparation of PAA-insulin NPs, the Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n thin film coatings were assembled with the aid of functional amine and carboxylic acid groups in PAH and PAA-insulin NPs, respectively. As a result, PAA-insulin NPs with negative charges adhered onto the positively charged PAH surface by means of electrostatic interactions, as illustrated in Fig. 1d and e. At last, LbL assembly film of Si/(PAH/ PAA)5/(PAH/PAA-insulin NPs)n was fabricated using a predetermined number of deposition cycles. Prior to particle deposition, pristine insulin NPs at 26 ± 3.5 nm were prepared and coated without PAA on the outside (size data in Supplementary data Fig. A1b). As shown in Fig. 3a, PAA-insulin NPs with a diameter of 27 ± 3.5 nm were fabricated and deposited onto a substrate with the basic (PAH/PAA)5/PAH layer. In consideration of uniformly distributed charges of the basic layer, PAA-insulin NPs could
Fig. 3. (a) SEM image of PAA-insulin nanoparticles on a basic layer. (b) Thickness growth curve of assembled films of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n (red curve) and Si/(PAH/PAA) 5/(PAH/PAA)n (black curve) measured by profilometry. The mean diameter of PAA-insulin NPs measured by SEM was 27 nm ± 3.5 nm.
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Fig. 4. Top-down FE-SEM images of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayers with different deposition cycles. (a) n = 1; (b) n = 4; (c) n = 12; (d) n = 20. The inset of (a) shows a pristine Si/(PAH/PAA)5 substrate without insulin NPs.
be deposited homogeneously, compared to dropping directly on the sample holder of TEM in Fig. 2c. The growth in the thickness of the Si/ (PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayers was exponential with respect to the number of deposited bilayers, while in the case of Si/(PAH/PAA)5/(PAH/PAA)n, a linear relationship was typically observed, which is demonstrated in Fig. 3b, respectively. This exponential growth regime could be properly explained by the “in” and “out” diffusion of insulin chains during the LbL film build-up process [37]. Taking advantage of the fact that film thickness could readily reach the micro-scale via exponential growth, thick multilayers incorporating proteins can be obtained and easily applied to drug delivery. FE-SEM provides an intuitive way to analyze the morphologies of LbL multilayer films. Fig. 4a–d shows top-down FE-SEM images of (PAH/PAA)5/(PAH/PAA-insulin NPs)n (n = 1, 4, 12, 20) multilayers with different desired numbers of bilayers on silicon wafers. Pristine Si/(PAH/PAA)5 substrates without the deposition of insulin NPs were also analyzed (inset of Fig. 4a), in which the morphology can be distinguished visibly. Initially, insulin NPs were uniformly dispersed onto the basic layer of (PAH/PAA)5/PAH, a process mediated by the
complementary electrostatic phenomenon. As the number of bilayers increased, insulin NPs were stacked and incorporated into multilayers, leading to rapid, exponential growth in thickness. A film consisting of 20 bilayers was fabricated (Fig. 4d), possessing a dense and partially smooth surface, which may have been a result of the adhesion of small insulin molecules in the supersaturated solution and the dominant deposition of the PAH/PAA bilayer in a strong ionic environment (0.01 N, pH 4.5 acetic acid). In addition to FE-SEM characterization, surface morphology analysis of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)12 and Si/(PAH/PAA)5/(PAH/ PAA-insulin NPs)20 was conducted by AFM in tapping mode, as shown in Fig. 5. The dominant forms of insulin NPs with a height of about 20 nm were present in a spherical shape, and some aggregating clusters along with polymeric chains could be observed (Fig. 5a). The surface morphologies of the basic (PAH/PAA)5 layer and (PAH/PAA)5/(PAH/ PAA-insulin NPs)1 were also measured though cross-sectional measurements using AFM, indicating the successful deposition of insulin NPs and the original basic layer with a height of around 10 nm, as shown in Supplementary data Fig. A2. Similar to the FE-SEM observation, the
Fig. 5. AFM images of (a) Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)12 multilayers and (b) Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film in tapping mode.
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Fig. 6. Deconstruction profile of a Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer film in PBS (pH 7.4). (a) Top-down SEM image of Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film before immersing in PBS (pH 7.4). (b) Film morphology characterized by FE-SEM after immersing into PBS buffer for 6 h. (c) Film deconstruction profile measured by profilometry in the dried state.
dense surface of the Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 film with small insulin molecules was detected from the AFM images, as demonstrated in Fig. 5b. 3.2. Deconstruction of assembled films containing insulin NPs In order to deeply examine the release of insulin from LbL films, an as-prepared Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer was dipped into a 10 mM PBS solution (37 °C, pH 7.4, 5 mL in a 20-mL vial) for a predetermined amount of time. First, the acidity and ionic strength of the dipping solution were transformed from 0.01 M acetic acid (pH 4.5) to 0.01 M PBS (pH 7.4), resulting in the conversion of positively charged insulin NPs into negatively charged ones. As a result, PAA-insulin NPs were deconstructed and insulin molecules were released into the medium, resulting from the same negative surface charges between insulin and PAA. Furthermore, assembled LbL films based on the weak polyelectrolytes of PAH and PAA were swollen in the aqueous environment, which could be demonstrated by the increased thickness of films in a few minutes. Combining all of these factors, insulin molecules could be liberated from the LbL films, which was justified by morphological analysis performed using FE-SEM in Fig. 6a–b. The porous film was formed after 6 h because of insulin particle release, which is demonstrated in Fig. 6a–b. The deconstruction profile of a Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)20 multilayer film in its dried state was recorded at various time points by profilometry (Fig. 6c).
Fig. 7. Normalized insulin release profile obtained by fluorescence intensity detection of FITC-labeled insulin.
3.3. Insulin release test of LbL films In addition, to further verify that insulin NPs were loaded into multilayer films as building blocks, the release profile of FITC-labeled insulin NPs (insulin NPs-FITC) capable of deconstructing from LbL membranes in PBS solution was accurately monitored by measuring the fluorescence intensity using photoluminescence (PL) spectroscopy. As-prepared LbL films incorporating insulin NPs-FITC were incubated at 37 °C in PBS solution over certain designated periods of time. Normalized insulin release was obtained from the mean intensity peaks from the fluorescence in film-disrupted medium (λex = 493 nm). As shown in Fig. 7, the release rate of insulin-FITC reached up to 48.2% in 5 min, and 79.1% of the insulin-FITC in the LbL film was disassembled into the PBS buffer solution in 3 h. The sustained longterm release after 3 h is of interest and was observed to last for up to 7 days. In conclusion, the insulin release process mainly could divide into 2 stages. The first rapid release stage in 3 h is quite significant for diabetic patients, in terms of high blood glucose level after a meal. Moreover, the second slow release stage could ensure the stable glucose level of patients during sleeping time in particular. Therefore, the result indicates that the assembled multilayers incorporating insulin NPs play a critical role in insulin delivery systems.
4. Conclusions In summary, this work proposed a newly developed method through rapid pH-shift insulin crystallization and disassembly for the fabrication of nano-sized insulin particles by which LbL multilayer films could be uniformly constructed through electrostatic interactions. Obviously, our strategy for synthesizing insulin NPs got rid of the traditional denaturation environment. Above all, in terms of the different release rate of insulin in various forms, we appropriately combined the inherent properties of insulin NPs with the LbL multilayer assembly technique to develop a sustained insulin release for a transcutaneous delivery system. In vitro examination in physiological condition demonstrates that insulin molecules liberated rapidly in the first stage of 3 h (79.1%), and sustained insulin release continued for up to 7 days in the second stage. Hence, effective LbL Si/(PAH/PAA)5/(PAH/PAA-insulin NPs)n multilayer films considerably improved the transcutaneous insulin delivery system. In fact, further development of insulin delivery could be carried out based on our research. For example, more variables, such as using different sized insulin particles, incorporating various
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forms of insulin, and coating insulin particles with multilayers could be significantly helpful for controlling insulin release rate. Furthermore, inspired by our mechanism of insulin NP fabrication and LbL assembly, other delivery systems such as those for enzymes and vaccines are likely to be established for biomedical platforms in the near future. Acknowledgements This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2013R1A1A1076126, and 2012M3A9C6050104). Additionally, this work was also supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009986012015) (Rural Development Administration, Republic of Korea), as well as the High Value-added Food Technology Development Program of the Ministry of Agriculture, Food and Rural Affairs (114027-03-1-HD020) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HI14C-3266-030014). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.05.046. References [1] R.C. Turner, C.A. Cull, V. Frighi, R.R. Holman, UK Prospective Diabetes Study (UKPDS) Group, Glycemic control with diet, sulfonylurea, metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for multiple therapies (UKPDS 49), JAMA 281 (1999) 2005–2012. [2] A.T. Cheung, B. Dayanandan, J.T. Lewis, G.S. Korbutt, R.V. Rajotte, M. Bryer-Ash, M.O. Boylan, M.M. Wolfe, T.J. Kieffer, Glucose-dependent insulin release from genetically engineered K cells, Science 290 (2000) 1959–1962. [3] A.S. Narang, R.I. Mahato, Biological and biomaterial approaches for improved islet transplantation, Pharmacol. Rev. 58 (2006) 194–243. [4] M.C. Chen, K. Sonaje, K.J. Chen, H.W. Sung, A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery, Biomaterials 32 (2011) 9826–9838. [5] K.M. Bratlie, R.L. York, M.A. Invernale, R. Langer, D.G. Anderson, Materials for diabetes therapeutics, Adv. Healthcare Mater. 1 (2012) 267–284. [6] K. Yoshida, Y. Hasebe, S. Takahashi, K. Sato, J.I. Anzai, Layer-by-layer deposited nanoand micro-assemblies for insulin delivery: a review, Mater. Sci. Eng. C 34 (2014) 384–392. [7] S. Gupta, T. Chattopadhyay, M.P. Singh, A. Surolia, Supramolecular insulin assembly II for a sustained treatment of type 1 diabetes mellitus, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 13246–13251. [8] E. Gershonov, I. Goldwaser, M. Fridkin, Y. Shechter, A novel approach for a watersoluble long-acting insulin prodrug: design, preparation, and analysis of [(2-sulfo)-9fluorenylmethoxycarbonyl] 3-insulin, J. Med. Chem. 43 (2000) 2530–2537. [9] M.M. Bailey, E.M. Gorman, E.J. Munson, C. Berkland, Pure insulin nanoparticle agglomerates for pulmonary delivery, Langmuir 24 (2008) 13614–13620. [10] Z. Gu, A.A. Aimetti, Q. Wang, T.T. Dang, Y. Zhang, O. Veiseh, H. Cheng, R.S. Langer, D.G. Anderson, Injectable nano-network for glucose-mediated insulin delivery, ACS Nano 7 (2013) 4194–4201. [11] Y.F. Fan, Y.N. Wang, Y.G. Fan, J.B. Ma, Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-by-layer adsorption, Int. J. Pharm. 324 (2006) 158–167.
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