Letter pubs.acs.org/journal/abseba
Inkjet Printing-Based Patchable Multilayered BiomoleculeContaining Nanofilms for Biomedical Applications Moonhyun Choi, Jiwoong Heo, Miso Yang, and Jinkee Hong* School of Chemical Engineering and Material Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea S Supporting Information *
ABSTRACT: Thin films including biocompatible polymers and biological materials as building blocks can be produced with a variety of critical film characteristics, including various materials, thicknesses, roughnesses, amounts of compound released, and release rates for biomedical purposes. We developed a multilayer fabrication system via high-throughput layer-by-layer (LbL) assembly of a nanofilm with inkjet printing to facilitate practical biomedical applications. Our system was used to generate biomolecule (ovalbumin and basic fibroblast growth factor)-containing printed LbL films. This is the first demonstration of the clinical benefits of nanofilm-type nanobiomaterials based on molecular organization, suggesting that novel therapeutic human skin patches could be realized without the need for conventional surgical practices. KEYWORDS: free-standing skin patch, inkjet printing, layer-by-layer (LbL) assembled multilayer, biomolecules
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INTRODUCTION Recently, multilayer structures have attracted significant attention as intelligent coatings for biomedical applications. The generation of multilayered thin films on a variety of surfaces to provide both high drug loading and precise control over the release of active biomolecules, such as proteins,1−3 DNA,4 growth factors,5,6 and enzymes,7 has significant potential to expand the development of new delivery coatings for biomedical technologies. In particular, efforts to prepare nanothin films incorporating bioactive materials have focused on practical applications for humans and the programmed release of therapeutics from multilayer films.8,9 Layer-by-layer (LbL) assembly is an excellent method for depositing multilayered nanofilms with bioactive materials onto any surface through molecular interactions.10,11 Based on physical interactions, one of the biggest advantages of LbL is minimal damage or denaturation of bioactive materials; thus, it can be used to build thin films with a variety of functional molecules such as vaccines,12−14 nucleic acids,15 proteins,16−19 growth factors,20−23 cancer drugs,24−26 and micronanoparticles.27−29 LbL nanofilm studies have shown the enormous potential of this method using a variety of materials and various driving forces. Vertically multilayered structures can be organized by tuning the interactions during LbL assembly. Compared with bulk films, versatile and well-organized multilayer nanofilms with bioactive materials have unique physical properties, such as high transparency, noncovalent adhesion, and high flexibility. Many studies have focused on developing techniques for transferring nanofilms for practical biomedical applications. Free-standing or patchable nanofilms have been applied for surgical repair, drug delivery, and wound © XXXX American Chemical Society
healing on the skin surface or surgical site. Sheet-shaped nanofilms have several advantages over spherical carriers; these include a larger surface area for targeting, the potential application of multilayered structures leading to heterosurface modification, and unique dynamics as a result of increased flexibility. With respect to the fabrication of these LbL nanofilms, it is important to improve the yield rate and production speed. In particular, bioactive materials are highly sensitive and have a short half-life; therefore, it is important to minimize the reaction time. Because substrates are immersed in aqueous solutions during LbL assembly, the bioactive materials can be easily damaged or denatured through direct contact between the substrate and solution. Inkjet printing is of considerable interest for thin film application to alleviate these problems by reducing contact. The inkjet technique can be widely applied in a variety of industries, is a noncontact process, and is more economical and productive than conventional methods.30,31 In addition, the inkjet printing technique is very useful for applying expensive and sensitive biomolecules, such as proteins, DNA, growth factors, etc., because the ink drop placement can be precisely controlled using small droplets with accurate volumes. In this study, we report the potential biomedical application of inkjet-printed multilayer films. More concretely, we report the development of versatile methods of fabricating multilayered nanofilms containing bioactive materials using inkjet Received: March 5, 2017 Accepted: April 24, 2017 Published: April 24, 2017 A
DOI: 10.1021/acsbiomaterials.7b00138 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 1. (a) UV−vis absorption spectra and (b) thickness growth curves of (CHI/HA+OVA−Texas red)n (where n = the number of bilayers) and (BPEI +bFGF/HEP)n printed LbL films. AFM images of (c) (CHI/HA+OVA−Texas red)n and (d) (BPEI+bFGF/HEP)n printed LbL films.
Figure 2. Total cumulative release profiles of (a) OVA from (CHI/HA+OVA−Texas red)20,30,40 printed LbL films and (b) bFGF from (BPEI +bFGF/HEP)20,30,40 printed LbL films in PBS buffer. (c) Release ratio (%) profiles of OVA from (CHI/HA+OVA−Texas red)20,30,40 printed LbL films and (d) bFGF from (BPEI+bFGF/HEP)20,30,40 printed LbL films. B
DOI: 10.1021/acsbiomaterials.7b00138 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 3. (a) Schematic representation of the inkjet-printed nanofilms for skin delivery. (b) Photographs taken in the light and dark of printed (CHI/HA+OVA−Texas red)20 LbL films supported by double-sided adhesive tape. (c) Schematic illustration of the fabrication of free-standing nanofilms floating in solution. (d) Spin-coated photoresist layer. (e) Printed (GO+/GO−)100 LbL films on the photoresist layer. (f) Printed (GO +/GO−)100 LbL films detached from the substrate and floating in acetone. (g) Printed (GO+/GO−)100 LbL films on human skin.
nanofilms printed onto silicon wafers using AFM. The AFM images of the dried films (Figure 1c, d) showed that each printed LbL nanofilm exhibited a distinct surface morphology. The printed LbL nanofilms containing OVA were generally rougher than the bFGF-loaded printed nanofilms. We confirmed that the roughness of the nanofilms was dependent on the thickness of the films. As shown in Figure 2, the release profiles of the films display two distinct steps with an initial burst release of between 34% (OVA) and 67% (bFGF) during the first 6 h, followed by slow release between 7 and 148 h. This phenomenon is caused by the swelling and disassembly of the printed LbL films driven by noninterdiffusion. Most of the OVA or bFGF is released initially as the printed LbL films degrade; then, the OVA or bFGF that is close to the surface is released slowly because of strong interactions (mainly electrostatic interaction and partially hydrogen bonding, hydrophobic interaction, van der Waals interaction) between the compound and surface.36−38 When incubated in the PBS solution, the printed LbL films composed of (CHI/HA+OVA−Texas Red)20,30,40 and (BPEI +bFGF/HEP)20,30,40 rapidly released OVA and bFGF. As shown in Figures 2a, c, the differences in the release behaviors of the (CHI/HA+OVA−Texas Red)n nanofilms reflects the differences in the incorporation rates depending on the increased loading with increasing number of layers. The release kinetics of the (BPEI+bFGF/HEP)n nanofilms are shown in Figures 2b, d; because there is a burst-like release profile, the time scale of the release is independent of the amount released. Most of the bFGF was released initially as the printed LbL films were disassembled; subsequently, the bFGF that was proximal to the surface was slowly released because of strong interactions. The release patterns from the different bilayers were similar; however, the total amount of released protein can be controlled through the number of layers. In the case of (bFGF/HEP)20,30,40, the total amounts released by the films
printing. To investigate the practical applications of printed nanofilms, we focus on the convenient manipulation of biomolecule-containing printed nanofilms involving a simple patchable method and transfer from a solid surface to human skin using a water-soluble sacrificial layer. In summary, we applied inkjet printing to construct a new biomedical material, i.e., a patchable and free-standing printed nanofilm containing biomolecules, and demonstrated the release of the inkjetprinted nanofilm onto human skin.
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RESULTS AND DISCUSSION We first demonstrated LbL assembly of blended charged materials using inkjet printing. The blend approach for polymer deposition to form LbL thin films has been studied extensively. Caruso and co-workers reported that the properties of multilayered nanofilms constructed from blended chargedmaterial solutions can be controlled.32−34 We used two kinds of biomolecules: OVA as a model protein and bFGF as a bioactive material. bFGF has been shown in preliminary animal research to protect hearts from injuries related to heart attacks by reducing tissue damage and promoting improved function.35 UV−vis spectroscopy was used to monitor the sequential adsorption of the layers. Figure 1a shows the UV−vis spectra of printed films composed of (CHI/HA+OVA−Texas Red)n, where n is the number of bilayers, and (BPEI+bFGF/HEP)n. The UV−vis absorption spectra of OVA−Texas Red and HEP have absorption maxima at 596 and 195 nm, respectively. The appearance of both absorption maxima indicates the continuous accumulation of layers. The linear relationship between the intensity of the adsorption and number of bilayers is evidence that uniform LbL assembly was achieved. As shown in Figure 1b, printed LbL films of (CHI/HA+OVA−Texas Red)n and (BPEI+bFGF/HEP)n exhibited linear growth and were thin. The morphologies of the biomolecule-containing printed nanofilm surfaces were observed by analyzing (CHI/HA +OVA−Texas Red)20,30,40 and (BPEI+bFGF/HEP)20,30,40 LbL C
DOI: 10.1021/acsbiomaterials.7b00138 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering with 20, 30, and 40 bilayers were 1.19, 1.25, and 1.55 ng/cm2, respectively. Although they normally have short durations, release bursts result in the highest release rate in the initial stages and may be the optimal delivery method for various bioapplications. Rapid release or high initial rates of delivery may be desirable for wound treatment, targeted delivery, pulsatile release, etc. For example, during initial wound treatment, an early release burst provides instantaneous relief for the patient followed by sustainable release to promote deliberate healing. In general, one of the current challenges with a burst release pattern is that it is unpredictable; thus, even when a burst release profile is desirable, the amount released cannot be easily controlled. The most important aspects in developing drug delivery platforms are accurate prediction of the timing of the burst release and quantification of the drugs or proteins released from smart platforms. Therefore, by utilizing the inkjet-based printing technique, we can fabricate customized burst-release nanofilms with desired drug loadings, suitable release profiles, and compatible sizes. For successful practical application of printed nanofilms, we developed two different application methods. First, to stick printed nanofilms to the skin, we generated simple adhesive patches using double-sided adhesive tape (Figure 3a). We fabricated printed (CHI/HA+OVA−Texas red)20 LbL films in a model setting comprising protein multilayer thin films coated onto model skin patch substrates (Figure 3b). Using the inkjet printing technique, LbL-assembled films composed of several materials with various functionalities (i.e., shapes and mixtures) were fabricated on silicon wafers, PDMS, and flexible substrates (Figure S1). Furthermore, Figure S2 shows the high potential throughput of the inkjet printing technique for mass production of LbL-assembled films. Patches of (PAH-FITC/PAA)20 LbL nanofilms (3 × 3 cm) were printed onto A4-sized polypropylene substrate. In addition, we prepared printed LbL films patches incorporating OVA for transcutaneous delivery and placed the resultant (CHI/HA+OVA−Texas red)20 multilayer patches on skin. Second, we fabricated a free-standing printed LbL nanofilm to generate a substrate-independent patch (Figure 3c). The synthesis involved covering a silicon wafer with an acetonesoluble photoresist (SU-8 2150) sacrificial layer (Figure 3d) followed by dipping the wafer in BPEI solution (pH 5.5) for 20 min to generate a positive charge on the photoresist sacrificial layer. We then printed GO+/GO- LbL films onto the photoresist sacrificial layer (Figure 3e). Upon fabrication of the GO nanofilm, the sacrificial layer was dissolved in acetone, and the GO nanofilms on the substrate gradually detached from the edges of the substrate. After 30 min, the polysaccharide nanosheet was fully detached without any shape or size (0.5 cm2) distortion (Figure 3f). To transfer the GO nanofilms from the substrate onto human skin, we prepared a poly(vinyl alcohol) (PVA) layer as a hydrophilic sacrificial layer. We chose PVA as a suitable material for the sacrificial layer because it is a water-soluble polymer that does not adversely affect skin. The PVA layer was prepared by spin-coating a 20 wt % PVA aqueous solution onto a polypropylene (PP) substrate. The PVA layer was then spontaneously released from the acetone-insoluble PP substrate within a few seconds by immersion in acetone. To assess the practical applications of the printed GO+/GOLbL nanofilms, we attached a patch of the nanofilm onto the left arm skin of a human subject. The printed GO LbL
nanofilms were then released from the PVA-coated PP substrate within a few seconds via dissolution of the PVA layer with a drop of deionized water. The printed GO nanofilms on the skin were barely visible from the top under visible light (Figure 3g). The intrinsic color of the modified printed GO nanofilms confirmed that the shape and size of the printed GO nanofilms were preserved on the skin. Furthermore, black regions were barely detected, which demonstrates the successful transfer of the GO nanofilms onto skin.
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CONCLUSION In summary, our results suggest that an inkjet-based highthroughput assembly system for fabricating multilayered nanofilms can be effectively designed to generate structures of desired shapes on various substrates with control of the release pattern of functional molecules for practical biomedical applications. Using bioactive molecules, we succeeded in constructing patchable and freestanding multilayered nanofilms using an inkjet-based LbL method. We demonstrated transfer of nanofilms containing various materials from solid substrates onto human skin. These inkjet-printed nanofilms will have applications in the field of skincare.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00138. Experimental details on materials and methods sections, and photograph images of printed LbL films on the flexible vinyl substrate and PET film (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +82-2-820-5561. ORCID
Jinkee Hong: 0000-0003-3243-8536 Author Contributions
M.C. performed the experiments, analyzed the data, and wrote the manuscript. M.C. and J.W.H. participated in data interpretation and made graphene materials. M.Y. participated in release experiments. J.H. conceived and designed the study. Funding
This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning of Korea government 2012M3A9C6050104 and 2016M3A9C6917405. Additionally, this research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C-3266, HI15C1653). Notes
The authors declare no competing financial interest.
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DOI: 10.1021/acsbiomaterials.7b00138 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsbiomaterials.7b00138 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
