Drug Loading and Release Behavior Depending on the Induced

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Drug Loading and Release Behavior Depending on the Induced Porosity of Chitosan/Cellulose Multilayer Nano-films Sohyeon Park, Daheui Choi, Hyejoong Jeong, Jiwoong Heo, and Jinkee Hong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00371 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on September 2, 2017

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Molecular Pharmaceutics

Drug Loading and Release Behavior Depending on the Induced Porosity of Chitosan/Cellulose Multilayer Nano-films Sohyeon Park1, Daheui Choi1, Hyejoong Jeong1, Jiwoong Heo1, Jinkee Hong1* 1

School of Chemical Engineering and Material Science, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea *E-mail: [email protected]. Tel: (+82)-02-820-5561

ABSTRACT The ability to control drug loading and release is the most important feature in the development of medical devices. In this research, we prepared a functional nano-coating technology to incorporate a drug-release layer onto a desired substrate. The multilayer films were prepared using chitosan (CHI) and carboxymethyl cellulose (CMC) polysaccharides by the layer-by-layer (LbL) method. By using chemical crosslinking to change the inner structure of the assembled multilayer, we could control the extent of drug loading and release. The crosslinked multilayer film had a porous structure and enhanced water wettability. Interestingly, more of the smallmolecule drug was loaded into and released from the non-crosslinked multilayer film, whereas more of the macromolecular drug was loaded into and released from the crosslinked multilayer film. These results indicate that drug loading and release can be easily controlled according to the molecular weight of the desired drug by changing the structure of the film. KEYWORDS: Polysaccharide, Layer-by-layer, crosslinking, structural change, Drug Loading and Release Behavior 1

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INTRODUCTION With advancements in medical technology, research on multifunctional medical devices is actively being carried out. In particular, many researchers have focused on the development of drug-coated medical devices that can deliver drugs in vivo without additional surgery.1-4 Immunosuppressant-coated stents have been used to inhibit neointimal hyperplasia in atherosclerosis therapy.5, 6 Since drugs with various properties and molecular weights are used in these medical fields, one of the most important aspects of drug-coating technologies is the ability to control loading and release according to the properties and purpose of the drug.7-10 In this regard, the layer-by-layer (LbL) technique, which can form multilayer thin films on substrates, is a practical coating method. The multilayer films are formed by the successive adsorption of various materials, such as polymers, particles, and crystals, via electrostatic interactions, hydrogen bonding, covalent bonding, and bio-specific interactions between the materials.11-13 Furthermore, the thickness of LbL-assembled multilayers can be controlled by the number of times the process repeated.14 The LbL method has no limitations on the materials employed and can be used with diverse substrates regardless of their size and shape.15-18 These properties of the LbL method allow the assembled film to be used as a drug delivery carrier by easily incorporating therapeutics.16, 17, 19, 20 There are many systems that control drug loading and release through differences in the multilayer film constituents, such as molecular weight and ionization, or by introducing cargo such as drug-loaded nanoparticles or micelles in the LbL assembly process.16,

19, 21, 22

For

example, multilayer thin films have been fabricated using the difference in ionization between poly(β-amino ester) (Poly1) and poly(L-lysine) (PLL). Ovalbumin (Ova), which is widely used 2


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as a model protein in immunology research, has a negative charge at pH 6. After preparing Poly1 and PLL multilayers of Ova by LbL, the degree of Ova release was measured. It was observed that the Poly1/Ova multilayer thin film released faster than the PLL/Ova multilayer thin film in the pH 7.4 phosphate-buffered saline solution because of the difference in ionization and the hydrolysis properties of the two polymers.22 In addition, Hammond et al. fabricated LbL multilayer films using polyelectrolyte and amphiphilic block copolymer micelles as carriers for hydrophobic drugs via hydrogen bonding. For example, amphiphilic poly(ethylene oxide)-blockpoly(ϵ-caprolactone) (PEO-b-PCL) copolymers were used to direct hydrophobic drugs toward the hydrophobic PCL in the assembly of drug-containing micelle cores. The multilayer films prepared in this manner were capable of pH-sensitive release.21 Although there are many methods, changing the structure of the multilayer film is the simplest method to overcome the complicated process used in the previous methods, which allows a clear control of drug loading and release.23-25 Here, we first prepared LbL-assembled multilayer films using carboxymethyl cellulose (CMC) and chitosan (CHI). The introduction of crosslink chemistry was performed to easily change the structure of the multilayer films. Both CHI and CMC are linear polysaccharides, hence are biocompatible and biodegradable by enzymatic hydrolysis. Therefore, it can be advantageous to use polysaccharides instead of synthetic polymers as the drug delivery coating materials. Furthermore, the affinity of CMC and CHI is excellent because they have similar chemical structures (Figure 1a).9, 26-28 We hypothesized that structural changes in the polysaccharide-based multilayer film will affect drug delivery. To demonstrate this, we investigated the drug loading and release behavior of the 3



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non-crosslinked and crosslinked multilayer films using small-molecule and macromolecular model drugs (Figure 1b). We employed fluorescein isothiocyanate (FITC) as the small-molecule model drug; this has a wide range of applications in fluorescence microscopy because of its high absorptivity and good water solubility. Ova, which is widely used as a model protein in immunology research, was employed as the macromolecular model drug.29-32 The key finding was that the effect of structural changes of the film on loading and release differed depending on the molecular weight of the drug.

a)

b)

4


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Figure 1. (a) Chemical structures of chitosan and carboxymethyl cellulose sodium salt. (b) Schematic illustration of the crosslinking-induced structural change of the LbL-assembled CMC/CHI multilayer film, and its effect on the loading of drugs with different molecular weights.

EXPERIMENTAL SECTION Materials. CMC sodium salt (Mw = 250 000); CHI (medium molecular weight, degree of deacetylation = 75~85%); branched polyethyleneimine (bPEI, Mw = 25 000); (3glycidoxypropyl)trimethoxysilane (GPTMS, Mw = 236.34); N-hydroxysulfosuccinimide (NHS); glutaraldehyde 25% solution (Mw = 25 000); and FITC (Mw = 389.38) were obtained from Sigma-Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Mw = 191.71) was purchased from Daejung. Ova was purchased from Bio Basic Inc. Fluoresceinconjugated Ova (Mw = 45 000) and phosphate-buffered saline (PBS, 1X) were obtained from Gibco® Life Technologies. Surface treatment. An epoxy-amine coupling reaction was performed on the substrate so the deposited multilayer thin films would strongly adhere. First, the Si wafer substrate was sonicated in water for 5 min and cleaned by oxygen plasma for 2 min. The oxygen plasma-treated Si wafer was incubated in 2% GPTMS/toluene solution for 12 h, and then rinsed with pure toluene. The dried Si wafer was placed in a 5 mM aqueous solution of bPEI for 6 h and rinsed twice with deionized (DI) water for 4 min.33, 34 Preparation of the polysaccharide multilayer films. We fabricated multilayer films on Si 5



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wafers through LbL assembly using a negatively charged CMC solution and a positively charged CHI solution. The concentrations of both solutions were 1 mg/mL. The epoxy-amine modified Si wafer was first immersed into the CMC solution (pH 4) for 10 min, and subsequently rinsed twice with DI water for 2 min each. Then, the substrate was immersed in the CHI solution (pH 4) for 10 min, followed by rinsing in the same manner. By repeating this cycle n times, a (CMC/CHI)n (n = number of bilayers) film was produced. Crosslink chemistry for the multilayer films. We used EDC/NHS chemistry for the first crosslink step between the multilayers. The Si wafer substrates with the assembled CMC/CHI multilayers were immersed in a 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 0.2 M EDC and 5 mM NHS for 20 min, followed by immersion in a 1X PBS buffer for 10 min. Next, the substrates were immersed in 2.5% glutaraldehyde solution for additional crosslinking for 40 min and rinsed three times with DI water for each 2 min. Characterization of the multilayer films. The thicknesses of the assembled multilayers on the Si wafer substrates were measured using a profilometer (Dektak 150, Veeco). The quantitative analysis of the material deposited onto the substrate during each LbL assembly step was conducted by a quartz crystal microbalance (QCM; QCM200, Stanford Research Systems). The amount of polymer adsorbed, ∆, was obtained by measuring the frequency decrease of the QCM crystal, ∆F, using Sauerbrey’s equation:

∆F(Hz) = −

2

∆

Here, F0 is the fundamental resonance frequency of the crystal (approximately 5.0 MHz), A is the 6


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area of the QCM electrode, and and indicate the quartz shear modulus (2.95 × 1011 g/(cm·s2)) and density (2.65 g/cm3), respectively. By applying these numerical values, the equation can be simplified as follows:35 ∆F(Hz) = −56.6 × ∆ To compare the surface morphology and roughness of the films before and after crosslinking, they were examined by atomic force microscopy (AFM; NX10, Park Systems) and fieldemission scanning electron microscopy (FE-SEM; LIBRA 120, Carl Zeiss). The AFM measurements were analyzed using XEI and Gwyddion software. SEM images were obtained at an acceleration voltage of 5 kV. The static contact angles of the multilayer films were measured three times for each sample using a laboratory-made contact angle goniometer with a chargecoupled device camera (IMT 3, IMT solutions). The volume of the water droplets was 4 µL and Image J software was used for the contact angle analysis. The refractive indices of the multilayer films on Si were measured by ellipsometry (L2W15S830, Gaertner Scientific Corp.) using a 632.8 nm He Ne laser. We performed Fourier transform infrared spectroscopy (FTIR; FT/IR4700, JASCO) and X-ray photoelectron spectroscopy (XPS; Al Kα source, K-Alpha+, Thermo Fisher Scientific) to demonstrate the bonding between the layers during the crosslink reaction. Drug loading and release profiles of the multilayer films. FITC was used as the smallmolecule model and Ova/fluorescein-conjugated Ova (9:1) as the macromolecule model. This was because we adopted a fluorescence-detection method to obtain the drug loading and release profiles. For the loading of the model drugs, non-crosslinked and crosslinked (CMC/CHI)10 filmcoated Si wafers were immersed into a FITC solution (0.5 mg/mL) or Ova/fluorescein7



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conjugated Ova solution (1 mg/mL) for 1 h, and then removed and dried in air. Next, each drugloaded sample was added to 1X PBS buffer (10 mL) and placed in an incubator at 37 °C to stimulate the release of the fluorescence-emitting drug. At each time point, 0.5 mL of the drugcontaining 1X PBS solution was transferred to a microtube, which was then topped up with fresh 1X PBS to make up each sample to the same volume. The amount of drug released was analyzed by measuring the fluorescence of the solution in the microtube using photoluminescence (FP8300, JASCO). To observe the distribution of the drug loaded into and released from the films, we analyzed the confocal laser scanning microscopy (CLSM; LSM700, Carl Zeiss) images of the non-crosslinked and crosslinked CMC/CHI films coated on glass substrates. All samples were irradiated under the same laser conditions.

RESULTS AND DISCUSSION Surface modification for the robust deposition of multilayer films. Oxygen plasma treatment is commonly used to introduce hydroxyl groups onto various substrates. Silanization and epoxyamine coupling is an effective route to form a surface on an inorganic-based substrate on which a multilayer film can be deposited. Hydroxyl groups of the plasma-treated surface form covalent – Si–O–Si– bonds by attacking and displacing the alkoxy groups of the silane.36 In addition, epoxy groups are present on the silanized surface from the epoxy-functionalized silane compound used to achieve the epoxy-amine coupling reaction. The epoxy groups can firmly anchor the amine groups on the surface via a ring opening reaction by nucleophilic addition of the amine.37, 38 Through this surface modification process, the multilayer thin film can be deposited onto a glass or metal oxide surface in addition to organic surfaces.36 8


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a)

b)

Figure 2. (a) Thickness growth curve of the CMC/CHI multilayers. (b) QCM graph showing the frequency change as a function of the number of layers during the assembly of a (CMC/CHI)6 film. Polysaccharide-based multilayer film preparation. We used LbL deposition to prepare multilayer films consisting of CMC and CHI on the Si wafers. CMC and CHI are polysaccharides with different ionization degrees depending on the pH, and were used as polyanions and polycations, respectively. We adjusted the pH of both CMC and CHI solutions to 4, at which point the ionization of CMC and CHI is more than 80%.39, 40 Therefore, the driving force for the formation of the CMC/CHI multilayer films was the strong electrostatic interactions between the –NH2 groups of CHI and the –COOH groups of CMC. We first deposited CMC onto the silane-amine–treated surface, to which it binds by electrostatic interactions and hydrogen bonding. Next, CHI was successively deposited onto the surface that was negatively charged by the CMC, and then CMC was deposited again onto the surface that was positively charged by the CHI. The thickness of the CMC/CHI multilayer film can be controlled by repeating these deposition cycles.40, 41 Profilometry measurements performed on the LbL films constructed on Si 9



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showed a linear multilayer growth with increasing deposition cycles. From six bilayers onward, the thickness increased by approximately 350 nm per three bilayers, and the final thickness of 21 bilayers was 2.2 µm (Figure 2a). We also analyzed the adsorption behavior of CMC and CHI in the multilayer films using QCM. Figure 2b shows the increase in the amount of CMC/CHI deposited as a function of the number of layers. Specifically, the QCM frequency decreased with an increasing number of layers, indicating the successful assembly of the CHI and CMC film. We performed QCM measurements up to 12 layers (six bilayers), and an exponential mass increase was observed from six layers (three bilayers). This is because of the “in and out diffusion” of the polymer chains due to the Coulombic interactions and entropy increase.42

Figure 3. XPS C 1s spectra of the (CMC/CHI)10 films (a) and crosslinked (CMC/CHI)10 films 10


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(b). XPS N 1s spectra of the (CMC/CHI)10 films (c) and crosslinked (CMC/CHI)10 films (d). Crosslinking the prepared multilayer films. We employed EDC/NHS chemistry for crosslinking the CMC/CHI multilayers. NHS is used to prepare amine-reactive esters of the carboxyl (–COOH) groups for crosslinking. Carboxylates can react with NHS in the presence of a carbodiimide such as EDC, resulting in semi-stable NHS esters. These may then be reacted with the amines (–NH2) of CHI to form amide crosslinks. Although NHS is not required for carbodiimide reactions, its use greatly enhances the coupling efficiency.43-45 Therefore, amide bonds between the carboxyl groups of CMC and amines of CHI were formed by immersing the prepared (CMC/CHI)10 films in an EDC/NHS solution. Next, the crosslinked (CMC/CHI)10 films were immersed in a glutaraldehyde solution to further crosslink the multilayers.46, 47 Glutaraldehyde is a powerful reagent for crosslinking amine groups, and it is a possible candidate to replace EDC/NHS because of its ability to form amide bonds. Glutaraldehyde treatment involves the formation of covalent bonds between two polysaccharides using bifunctional reagents containing reactive end groups that react with functional groups such as primary amines.48 XPS analysis was conducted to confirm the successful crosslinking of the (CMC/CHI)10 films. As seen in Figure 3, the C 1s spectrum of the (CMC/CHI)10 film was deconvoluted into only three peaks: C–C, C–O, and O=C–O. That of the crosslinked (CMC/CHI)10 film includes an additional C=N peak, resulting from the crosslink with glutaraldehyde, and a C=O peak, corresponding to the amide bonds. Furthermore, the N 1s spectrum of the (CMC/CHI)10 film is composed of only two peaks, attributed to N–C and NH2 (or NH3+). Similarly, the crosslinked (CMC/CHI)10 film gave two additional peaks, attributed to N=C and –N–(C=O)–, with a decrease in the NH2 (or NH3+) peak 11



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intensity.49-51 As shown in the FTIR spectra in Figure 4, the O–H peaks of the non-crosslinked and crosslinked (CMC/CHI)10 films were observed at lower wavenumbers than the O–H peaks in the CMC and CHI spectra. This means that not only electrostatic interactions but also hydrogen bonds were formed between CMC and CHI during the LbL deposition, which decrease the O–H bond strength. In the (CMC/CHI)10 film spectrum, there are two noticeable peaks around 3400 cm-1, attributed to the overlap of the O–H and N–H signals, and a peak at 1640 cm-1, attributed to C=O–O. The spectrum of the crosslinked (CMC/CHI)10 film shows the same overlapping peaks around 3400 cm-1, with peaks at 1680 cm-1 (O=C) and 1645 cm-1 (N–H) corresponding to amide bonds. These results demonstrate that the (CMC/CHI)10 multilayer films were successfully crosslinked.52-54

Figure 4. FT-IR spectra of CMC (black), CHI (brown), the non-crosslinked (CMC/CHI)10 film (green), and the crosslinked (CMC/CHI)10 film (red). Morphology analysis of the multilayer films. We hypothesized that the dense structure of the multilayer film would change to a porous structure because of the additional bond formation and 12


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reassembly between the crosslinked polymer chains.40, 55-57 To investigate this hypothesis, we analyzed the morphology of the films by SEM and AFM. The AFM images in Figure 5 show that the roughness of the standard (CMC/CHI)10 film was higher than that of the crosslinked (CMC/CHI)10 film; the root mean square (RMS) roughness values were 8.073 nm and 13.535 nm, respectively (Figure 5). The SEM images in Figure 5 also clearly show the morphology change that arises because of the porous structure of the crosslinked film. To obtain the porosity of the multilayer films, we measured the refractive indices of the films by ellipsometry and calculated their porosities using the Lorentz-Lorenz equation: − 1 1

= (1 − )

− 1

1

Figure 5. AFM images and cross-section SEM image of the non-crosslinked (CMC/CHI)10 film (a~c) and the crosslinked (CMC/CHI)10 film (d~f). A table exhibiting the properties of multilayer 13



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films: thickness measured using a profilometer, roughness measured using AFM, and nanoporosity calculated using the Lorentz-Lorenz equation (g). Here, Nf, N0, and P indicate the refractive index of the multilayer film, the refractive index of CMC (or CHI), and porosity, respectively. In this case, N0 was calculated to be approximately 1.52, equal to the refractive indices of both CMC and CHI. As a result, the porosities of the noncrosslinked and crosslinked (CMC/CHI)10 films are 2% and 63%, respectively (Figure 5).58, 59 The increase of porosity by crosslinking was also observed in the Brunauer-Emmett-Teller (BET) analysis (Figure S2). We also found that the thickness of the multilayer film increased by about 48 nm after crosslinking, which was due to the swelling that generally occurs in the liquid-phase crosslinking process.60, 61

Figure 6. Static water contact angles of the non-crosslinked and crosslinked (CMC/CHI)10 films. Meanwhile, the increase in roughness also affects the wetting behavior of the film surface. Since CMC and CHI have hydrophilic functional groups, –COOH and –NH2, the multilayer films are also hydrophilic. A hydrophilic surface composed of a hydrophilic material increases its surface roughness and surface energy upon crosslinking, resulting in a super-hydrophilic surface. This 14


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well-known principle is based on the Wenzel and Cassie-Baxter equation.62 Figure 6 shows the static water contact angles of the film surfaces. As expected, the contact angle values of the (CMC/CHI)10 film and crosslinked (CMC/CHI)10 film were approximately 36.5° and 5°, respectively.

Figure 7. Confocal laser scanning microscopy (CLSM) of fluorescein isothiocyanate (FITC) loaded into and released from (a, b) a standard (CMC/CHI)10 film and (c, d) a crosslinked (CMC/CHI)10 film. CLSM of ovalbumin loaded into and released from (e, f) a standard (CMC/CHI)10 film and (g, h) a crosslinked (CMC/CHI)10 film. Also shown are the accumulated 15



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release curves of (i) FITC and (j) ovalbumin (n = 3).

Drug loading and release behaviors of the multilayer films. We used a diffusion loading method to investigate the effect of film surface area on drug loading and release. Figure 7a–d exhibits the CLSM images of the FITC loaded into and released from the non-crosslinked and crosslinked (CMC/CHI)10 films. Despite the increased surface area caused by crosslinking, the amount of drug loading was greater in the non-crosslinked film. This can be explained by two reasons. The first factor is that FITC is a small molecule, so can be loaded into a dense film regardless of the porosity. The second factor is the remained functional group activity of the noncrosslinked multilayer films. For small molecules, such as FITC, the interaction between the functionalized film and the drug has a greater influence than that of the film surface area on loading and release. We also measured the amount of drug released using photoluminescence. As shown in Figure 7i, the amounts of FITC released from the non-crosslinked and crosslinked (CMC/CHI)10 films were 106.95 µg/mL and 32.82 µg/mL, respectively. In other words, the noncrosslinked film released a larger amount of the drug. The release behavior can be explained in relation to the stability of the films. The graph in Figure S3 is the result of the 10BL film stability in 1X PBS where the drug release experiment. The release of the drug from the film is mainly influenced by diffusion and film degradation. In the graph, the crosslinked (CMC/CHI)10 film shows a slight degradation of the film for 48 hours, so the release in this film would have been caused by a simple diffusion of the drug model molecule (FITC and Ova). Also, the crosslinked (CMC/CHI)10 film not only has a porous structure due to crosslinking but also has weak activity of a functional group capable of binding with a drug molecule, so that diffusion of a drug 16


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molecule can proceed smoothly. On the other hand, non-crosslinked (CMC/CHI)10 films exhibited significant decrease in thickness by film degradation. And, the non-crosslinked film has a functional group activity for interaction with drug model molecules. Especially, isothiocyanate groups of FITC form the bond with primary amine groups of CHI by a nucleophile-electrophile interaction. In practice, FITC has been used for detection of chitosan nanoparticles or evaluation of chitosan multilayer film degradation. is often conjugated to chitosan for chitosan detection.63,

64

The non-crosslinked films have amine groups that can

interact with the isothiocyanate groups, whereas the crosslinked films already form tight bonds between their functional groups by EDC and glutaraldehyde, thus it is expected that there are few amine groups capable of bonding with isothiocyanate groups. Thus, it is concluded that in the case of non-crosslinked films, release of chitosan and FITC would be released in a conjugated form by decomposition of the film or diffusion of FITC. In contrast, in the case of Ova, the drug loading and release amounts were larger in the case of the crosslinked film. Since Ova is a macromolecule, its loading to a significant depth in the dense (CMC/CHI)10 film is difficult, whereas the higher surface area of the porous crosslinked (CMC/CHI)10 film allows increased drug loading into the inner structure.23,

24, 65, 66

The CLSM image in Figure 7e shows the

crosslinked (CMC/CHI)10 film generates a much stronger fluorescence compared to the noncrosslinked film. We also confirmed the Ova release behavior of the crosslinked (CMC/CHI)10 film, which releases more than the standard (CMC/CHI)10 film, corresponded to the amount loaded (Figure 7j). This means that for macromolecules such as Ova, the surface area has a significantly greater effect on drug loading and release than the interaction between the drug and the multilayer film. 17



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CONCLUSIONS We studied the effect of structural changes of LbL-assembled multilayer films on drug loading and release. We first fabricated biocompatible CMC/CHI multilayer films using LbL assembly via the electrostatic interactions between CMC and CHI, and then observed the film growth behaviors using a profilometer and QCM. In addition, we introduced crosslink chemistry in the assembled CMC/CHI multilayer film to change its structure, and confirmed successful crosslinking by XPS and FTIR. We also investigated the effect of crosslinking on the film properties, including thickness, morphology, and wettability, using SEM, AFM, and contact angle measurements. The porosity and roughness of the crosslinked CMC/CHI films were greater because crosslinking induced additional bond formation and reassembly between the polymer chains. The interesting finding was that the effect of the structural changes on loading and release differed depending on the molecular weight of the drug. We conclude that these results are due to the increased surface area and decreased activity of the polymer functional groups upon crosslinking. This approach presents a method for preparing coatings that can control drug loading and release for use in various medical devices, by a technique that is easier and more effective than conventional methods.

ASSOCIATED CONTENT 18


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Supporting Information Available: The following files are available free of charge. Method and Result for cell viability testing of the films. Method and Result for BET analysis.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected].

ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP; Grants 2012M3A9C6050104); a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI); and the Ministry of Health & Welfare, Republic of Korea (Grants HI14C3266). This research was also supported by the Chung-Ang University Excellent Student Scholarship in 2016.

ABBREVIATIONS LbL, layer-by-layer assembly CMC, carboxymethyl cellulose 19



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CHI, chitosan Ova, ovalbumin QCM, quartz crystal microbalance FE-SEM, field-emission scanning electron microscope AFM, atomic force microscopy CLSM, confocal laser scanning microscopy

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