Biocompatible Double-Membrane Hydrogels from Cationic Cellulose

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Biocompatible Double-Membrane Hydrogels from Cationic Cellulose Nanocrystals and Anionic Alginate as Complexing Drugs Co-Delivery Ning Lin, Annabelle Gèze, Denis Wouessidjewe, Jin Huang, and Alain Dufresne ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00555 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 2, 2016

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ACS Applied Materials & Interfaces

Biocompatible Double-Membrane Hydrogels from Cationic

Cellulose

Nanocrystals

and

Anionic

Alginate as Complexing Drugs Co-Delivery Ning Lin,† Annabelle Gèze, ‡ Denis Wouessidjewe, ‡ Jin Huang, †∗ and Alain Dufresne§,∥∗ †

School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of

Technology, Wuhan 430070, P. R. China ‡

Univ. Grenoble Alpes, DPM, UMR CNRS 5063, Grenoble, France

§

Univ. Grenoble Alpes, LGP2, F-38000, Grenoble, France

∥

CNRS, LGP2, F-38000 Grenoble, France

KEYWORDS. Double-membrane hydrogels; Cellulose nanocrystals; Surface modification; Alginate; Drug release.

ABSTRACT

A biocompatible hydrogel with a double-membrane structure is developed from cationic cellulose nanocrystals (CNC) and anionic alginate. The architecture of the double-membrane hydrogel involves an external membrane composed of neat alginate, and an internal composite

ACS Paragon Plus Environment

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hydrogel consolidates by electrostatic interactions between cationic CNC and anionic alginate. The thickness of the outer layer can be regulated by the adsorption duration of neat alginate, and the shape of the inner layer can directly determine the morphology and dimensions of the double-membrane hydrogel (microsphere, capsule, and film-like shapes). Two drugs are introduced into the different membranes of the hydrogel, which will ensure the complexing drugs co-delivery and the varied drugs release behaviors from two membranes (rapid drug release of the outer hydrogel, and prolonged drug release of the inner hydrogel). The double-membrane hydrogel containing the chemically-modified cellulose nanocrystals (CCNC) in the inner membrane hydrogel can provide the sustained drug release ascribed to the “nano-obstruction effect” and “nano-locking effect” induced by the presence of CCNC components in the hydrogels. Derived from natural polysaccharides (cellulose and alginate), the novel doublemembrane structure hydrogel material developed in this study is biocompatible and can realize the complexing drugs release with the firstly quick release of one drug and the successively slow release of another drug, which is expected to achieve the synergistic release effects or potentially provide the solution to drug resistance in biomedical application.

INTRODUCTION Since the first report on hydrophilic gels (hydroxyethyl methacrylate) for biological use in 1960,1 hydrogels have been developed in a variety of pharmaceutical and biomedical applications including cell culture, tissue engineering, controlled drug release, vascular prostheses or implants.2,3 Derived from natural or synthetic polymers, hydrogels are three-dimensional and hydrophilic networks, which may absorb from 10-20% up to thousands of times their dry weight of water.4 Attributed to advantages such as high water content, porosity, soft consistency, and similar properties as the natural living tissue, polysaccharide/protein-based hydrogels (e.g.


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alginate, pectin, carrageenan and dextran/collagen, fibrin and polylysine) are biocompatible, biodegradable, and promisingly suitable for biomedical applications.5 In contrast to traditional single-membrane structure hydrogels, multi-membrane hydrogels possess the multilayered structure and tunable physical properties as a new generation of hydrogel materials.6 Since the influential report of Ladet et al. for the design and fabrication of the hydrogels with the multilayered structure from the polysaccharide (alginate or chitosan) in 2008,7 various polyelectrolyte biopolymers were attempted to develop the multi-membrane hydrogel materials during recent five years, including chitosan,8–10 alginate,11,12 sodium hyaluronate13 and carboxymethylcellulose.14 The multi-membrane hydrogels exhibited significant potential for biomedical applications, especially involving cell bioreactors, micropatterning neural cell cultures, tissue engineering and drugs co-delivery.14,15 However, most of the previous studies fabricated the multi-membrane hydrogels with the same component in each membrane (therefore resulting in the unalterable structure and properties), and also limitation for the thickness regulation of each membrane due to the boundary disappearance of two layers with the high treatment duration (therefore commonly limited at 5 to 10 min preparation in the previous reports).7,11 Furthermore, the reported studies on multi-membrane hydrogels mainly focused on the preparation approach, structure and physical properties of the material, but were short of the application investigation of the multi-membrane hydrogels. With the purpose of optimizing the physical and biological properties (such as mechanical support, biological activity etc.), the concept of multicomponent hybrid hydrogel was proposed and attracted the research interest in the biomaterial field. The strategy employs traditional composite approaches in which a filler is either physically entrapped or chemically crosslinked within the hydrogel matrix to produce the composite hydrogel with improved mechanical or


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biological properties.16 During last decades, few organic or inorganic particles have been developed for composite hydrogels, including nanoclays, silica nanoparticles, metal nanoparticles, hydroxyapatite, carbon nanotubes, graphene oxide etc.17 Recently, because of toxicity issues of inorganic particles, cellulose nanocrystal (CNC) showed promising potential in the development of hydrogel materials derived from its remarkable modulus, biocompatibility and biodegradability, nontoxicity together with its special chemical and morphological tunability.18 Cellulose nanocrystal is a kind of rod-like and rigid nanoparticle extracted from natural biomass resources. As a nanoreinforcing biofiller, CNC has been used to reinforce alginate, poly(N-isopropylacrylamide), poly(vinyl alcohol), agarose, cellulose derivatives, chitosan, cyclodextrin, or dextran matrices, which commonly achieved improved mechanical properties and structural stability for the development of composite hydrogels.17,19,20 Previously, we have reported the development of pH-sensitive microsphere hydrogels from alginate and pristine CNC,21 and in situ supramolecular hydrogels from cyclodextrin and modified CNC 22 as drug carriers. Besides the mechanical and structural stability enhancements, prolonged drug release behavior was also observed for hydrogels, which was attributed to the proposed “nanoobstruction eﬀect” and “nano-locking eﬀect” induced by the presence of rigid CNC in the hydrogel-based drug delivery system. Due to its numerous advantages involving biocompatibility, low toxicity, relatively low cost, and simple gelation with divalent cations, alginate, the biomaterial obtained from natural brown algae, is widely used to prepare physical hydrogels for biomedical applications, especially as carriers for cell encapsulation,23,24 and delivery of proteins 25,26 or drugs.27,28 Regarding alginatebased hydrogels in drug delivery, the strategy is commonly based on the desired drug encapsulation within the 3D network of the hydrogel, which is applied for the steady drug release


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by diffusion and hydrogel disintegration.29 However, because of the uncontrollable degradation (involving loss of divalent ions into the surrounding medium, and subsequent dissolution)30 and the burst drug release, the alginate-based hydrogel must be modified to become a promising carrier as potential controlled drug release material. Despite the fact that cellulose has been used in drug tableting for an extended history, the research on nanocellulose in advanced drug-loaded systems appeared during recent ten years. Even though few studies reported the use of cellulose nanocrystals in the development of drug carriers, only the single-drug delivery systems were investigated as drug carrier without consideration of complexing drugs delivery.31 Inspired from our previous reports,21,22 this study is a prospective research aiming to develop double-membrane hydrogel system from cationic cellulose nanocrystals and anionic alginate as novel drug carrier. The combination of cationic cellulose nanocrystals and alginate was performed using electrostatic interaction and ionic crosslinking, and the biocompatibility and nontoxicity can be preserved for the doublemembrane hydrogel ascribed to both natural polysaccharide components used as the building blocks. The device used (such as the syringe) for the preparation of the inner composite hydrogel (containing both cationic CNC and alginate) can control the shape and size of the doublemembrane hydrogel, while the thickness of the outer hydrogel (neat alginate) can be regulated via the treatment duration (from about 200 to 700 µm ranging from 5 min to 6 h absorption). Combination therapy has shown several potential advantages (e.g. synergistic effects and reversal of drug resistance) and may prove more effective than single-drug therapy.32,33 The controlled and sustained release of growth factor is a hot research topic in the biomedical field,34,35 because growth factor can regulate not only cell growth, differentiation and migration, but also localized vessel formation.30 If the drug delivery system allows a gradual release of the


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active ingredient, it could help ensure therapeutic levels over a prolonged period, overcoming the short half-life of active substances.36 In the present study, two kinds of drugs, antibiotic and growth factor, were introduced into the different layers of the double-membrane hydrogel, involving the ceftazidime hydrate (CH) antibiotics in the external membrane (pure alginate) and the epidermal growth factor human (EGF) in the internal membrane (CCNC and alginate) of the double-membrane hydrogels. It should be pointed out that recent studies reported the maximal stability of epidermal growth factor human (EGF) in buffer solution at pH values ranging from 6.0 to 8.0, which proved the use of this protein as the model drug during a long and stable release period.32,37 Derived from the different components, compositions and structures, two membrane hydrogels will exhibit varied drug release behaviors, which were expected to be the rapid drug release for the antibiotic (the neat alginate hydrogel) and prolonged drug release for the growth factor (the composite hydrogel). The double-membrane hydrogels developed in this study can be used as novel biomedical drug carrier for oral administration or wound dressings. The proposed double-membrane structure for the architecture of drug delivery system can realize the codelivery of two drugs with a synergistic release effect (“cocktails” therapy with antibiotics and growth factor), or potentially provide the solution to drug resistance (“complexing” therapy with two types of antibiotics). MATERIALS AND METHODS Materials. Native cotton fibers were obtained from Whatman filter paper. Sodium alginate (SA), calcium chloride (CaCl2), (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC), branched-polyethylenimine (PEI, Mw = 2.5×104 g.mol-1), sulfuric acid (H2SO4, 98%), hydrochloric acid (HCl, 37%), and HPLC-grade water were purchased from Sigma-Aldrich and used without further treatment. Bovine serum albumin (BSA), epidermal growth factor human


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(EGF) were chosen as the protein drug models, and ceftazidime hydrate (CH) was used as the antibiotics drug model in the release study. EGF is a small mitogenic polypeptide (~6 kDa) from the recombinant expressed in E. coli. CH and BSA were used in the crystalline states. Sodium hydroxide (NaOH), ethanol (95%) and other reagents of laboratory grade were purchased from Carl-Roth. Extraction of cotton CNC via sulfuric acid hydrolysis. Cellulose nanocrystals (CNC) were prepared by H2SO4 hydrolysis of native cotton fiber, according to our previous protocol with a little modification.38 Cotton fiber was milled with a laboratory milling device (LD-T350) to obtain a fine particulate substance, and then extracted in 2 wt% NaOH solution (30.0 g of fibers for 1.2 L solution) overnight at room temperature. Acid hydrolysis was performed at 45 °C with 60 wt% H2SO4 (preheated) for 120 min under mechanical stirring (30.0 g fibers for 600 mL solution). It should be noted that in comparison with the previous report,31 the acid concentration was slightly reduced and increased the duration for the hydrolysis, which was favored by the introduction of more negative sulfate groups on the surface of the prepared CNC. The suspension was diluted with ice cubes to stop the reaction and washed until neutrality by successive centrifugations at 10 000 rpm (revolutions per minute) for 10 min each step and dialyzed against the distilled water for three days. Upon exhaustive dialysis treatment, free acid molecules were removed. The CNC suspension was dispersed by ultrasonic treatment using a Branson Sonifier for three 5 min cycles (with cooling as necessary to prevent overheating). Finally, the released CNC powder was obtained by freeze-drying. Surface cationization of CNC by chemical modification with EPTMAC. Chemical cationization of the H2SO4-hydrolyzed CNC included two steps, which were surface desulfation and then cationization. Firstly, the sulfate ester groups on the surface of CNC were removed


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using alkaline solution according to previous report.39 In a typical procedure, a 9 wt% aqueous suspension of CNC was mixed with a 7 wt% NaOH solution. The desulfation treatment of CNC was performed at 65 °C for 5 h, and the product was collected as desulfated CNC (DCNC). In comparison with pristine CNC, the ζ-potential change of DCNC suspension indicated the absence of surface sulfate ester groups on the nanocrystals. After the desulfation treatment, chemical cationization was performed on the surface of DCNC using (2,3-epoxypropyl) trimethylammonium chloride (EPTMAC) as the cationic agent. Before the cationic modification, DCNC aqueous suspension (2 wt%) was treated with diluted alkaline solution (NaOH, 2 wt%) for 30 min at room temperature. This pre-treatment activates the hydroxyl groups on the surface of DCNC, which ensured the sufficient reaction for subsequent cationic modification. Surface chemical cationization of DCNC started at the dropping of EPTMAC solution into the suspension with the NaOH as the catalyst, and the reaction lasted for 6 hours at 65 °C. The molar ratio of EPTMAC and surface hydroxyl groups of nanocrystals (1.554 mmol/g from our previous study)

40

was controlled as 20:1. After the reaction, the

suspension (approximately 2 wt%) was precipitated in 95% ethanol and the product was collected by centrifugation. The EPTMAC-cationic cellulose nanocrystals (CCNC) were redispersed by dialysis against the distilled water (approximately 2 wt%) for 5 days. Surface cationization of CNC by physical adsorption using branched-PEI. The physical adsorption of polyethylenimine (PEI) on the surface of CNC was achieved by the electrostatic interactions between cationic polymer chains and negative sulfate groups.41 A mixture of 250 mL of redispered CNC aqueous suspension (1 wt%) was stirred continuously with 500 mL of PEI (1 wt%) for 2 h at room temperature. The pH of the mixture was adjusted to 1.5 with HCl (5% v/v


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solution) to enhance the ionic interactions between CNC nanoparticles and PEI chains. After the electrostatic adsorption, the mixture was centrifuged for 10 min and washed with HPLC-grade water to remove the free PEI chains. Finally, the PEI-cationic cellulose nanocrystals (PCNC) were redispersed in the distilled water (approximately 2 wt%) using IKA Ultra-Turrax Disperser. Preparation of the single-membrane hydrogels. In order to investigate the properties of the hydrogels with various components, single-membrane hydrogels were prepared containing sodium alginate (SA) with or without cellulose nanocrystals (CNC, or CCNC, or PCNC). The nanocrystal and alginate concentrations in hydrogels were controlled as 0.15 wt% and 1.5 wt%, respectively, and the crosslinking reaction was performed by pouring the mixture drop by drop with a syringe equipped with a nozzle into a 0.2 M CaCl2 aqueous solution for 1 h. The sol mixture was crosslinked to produce the microsphere-shaped hydrogel through the formation of “egg-box” structure between alginate component and divalent metal cation (Ca2+). The free Ca2+ on the surface of the hydrogel was washed with the distilled water, and the surface water was carefully removed by a filter paper. The mean diameter of microsphere-based hydrogels was determined by the nozzle size, which were 3.00 ± 0.25 mm in the wet state and shrank to ca. 0.90 ± 0.07 mm after freeze-drying. The film-shaped hydrogel can also be prepared by a prefreezing treatment in a Teflon mold and then crosslinking in the CaCl2 aqueous solution. Preparation of the double-membrane hydrogels. The double-membrane microsphere hydrogel was prepared by dropping the single-membrane hydrogel (as the formation of first membrane) into the pure SA solution (1.5 wt%) for various durations (5 min, 20 min, 1.0 h, 3.0 h and 6.0 h). After adsorption of the pure alginate sols, the hydrogel was transferred into the CaCl2 solution (0.2 M) for the crosslinking of the external SA membrane (the formation of second membrane).


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The preparation routine of single-membrane and double-membrane microsphere hydrogels from cationic cellulose nanocrystals and anionic alginate is shown in Figure 1.

Figure 1. The preparation routine of single-membrane and double-membrane microsphere hydrogels; optical microscope images of (a) the SA/CCNC single-membrane microsphere hydrogel, (b) the SA/CCNC-1h double-membrane microsphere hydrogel. Solid state 13C cross-polarization−magic angle spinning spectroscopy (13C CP-MAS NMR). 13

C CP-MAS NMR characterization was used to check the surface cationization of cellulose

nanocrystals, which was performed on the AVANCE400 solid instrument spectrometer with a MAS rate of 12 kHz. The contact time for CP was 1.5 ms and the decoupling powers were 4 kHz, 18 kHz and 40 kHz for CNC, CCNC and PCNC samples, respectively. The delay time after the acquisition of FID signal was 2 s. Elemental analysis. Derived from the cationic reagents (EPTMAC and PEI), the content of additional nitrogen element (N%) in cationic cellulose nanocrystals (CCNC and PCNC) can be used to calculate the surface cationic substitution. The total content of carbon (C%), oxygen (O%), nitrogen (N%) and sulfur (S%) elements was measured by elemental analysis at Analysis


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Central Service of the Centre National de la Recherche Scientifique (Vernaison, France). Before the analysis, freeze-dried samples were treated under vacuum at room temperature for 8 h. Surface charges. The ζ-potential of cellulose nanocrystals before and after modification was determined using the Zetasizer Nano instrument (Nano ZS, Malvern). The aqueous suspension of four nanocrystals samples (CNC, DCNC, CCNC and PCNC) with a concentration of 0.25 wt% was analyzed by Nano ZS at 25 °C, and all the measurements were performed in triplicate. The standard deviations of the mean ζ-potential values were provided by the instrument software. Atomic force microscopy (AFM). The morphology and dimensions of rod-like nanocrystals was observed by AFM. Suspensions of approximately 0.01 wt% nanocrystals were dispersed in water with ultrasonic dispersion for 30 min and then deposited on a mica substrate. The substrate loaded with nanoparticles was imaged in tapping mode with a Nanoscope IIIa microscope from Veeco Instruments. Silicon cantilevers were used to perform the imaging at a frequency of 264– 339 kHz and a typical radius of curvature of 10-15 nm. Thermal degradation. Thermogravimetric analysis (TGA) can be applied to investigate the change of thermal stability of CNCs upon modification. Furthermore, the content of adsorbed PEI polymers on the surface of PCNC can also be detected with this analysis. The freeze-dried nanocrystal samples were analyzed with a thermal analyzer Perkin-Elmer TGA-6 under nitrogen flow. Samples of ca. 10 mg were heated from 20 to 600 °C at a heating rate of 10 °C min-1. Optical microscopy. The morphology of the single and double-membrane hydrogels was observed by optical microscopy on Zeiss Discovery V20 stereo microscope with a magnification of × 10. The microsphere hydrogel was placed in a cuboid quartz colorimetric utensil, and fully


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immersed in deionized water. The membrane thickness of the hydrogel was measured from the pictures of three replications at different sections. To further determine the double-membrane structure of hydrogels, the freeze-dried hydrogels were observed by optical microscopy with a magnification of × 25. Scanning Electron Microscope (SEM). In order to investigate their integrity and internal morphology, the freeze-dried hydrogels with or without the half-section cutting were further observed by SEM, which was performed on a Quanta 200 FEI device (Everhart-Thornley detector) equipment. Before the observation, all the samples were coated with gold. The SEM observation was performed under an acceleration voltage of 10 kV. In vitro release study (BSA) of the single-membrane hydrogels. Bovine serum albumin (BSA) was used as the model drug to investigate the drug release behavior of the singlemembrane microsphere hydrogels. In the preparation of the drug-loaded microsphere hydrogels, BSA with a final concentration of 0.1% (w/v) was dissolved into the aqueous mixture of alginate and cellulose nanocrystals under continuous stirring. The drug-loaded mixture gel was dropped into the calcium chloride solution (0.2 M) for 1 h to form the crosslinked microsphere. To study the release profiles, the drug-loaded microsphere hydrogels were immersed in buffer solutions with pH values of 2.0 and 7.4 at 37 °C using a dissolution tester (DISTEK-2100A). The selection of pH 2.0 and 7.4 as the drug release conditions was used to simulate the pH conditions of stomach and colon in the body. At predetermined durations, 0.8 mL of the solution was taken out and analyzed by the Bradford method for the concentration of the released BSA at 595 nm using UV-spectrophotometer.42,43 In all the drug release study, another 0.8 mL of fresh buffer solution was added to keep the total solution volume constant after the solution sample was taken. The


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percentage of the cumulative amount of released BSA was determined from the standard calibration curve. All the drug release experiments were repeated twice. In vitro complexing drugs release study of the double-membrane hydrogels. To analyze the different drug release behaviors of double-membrane hydrogels, two model drugs, ceftazidime hydrate (CH) and epidermal growth factor human (EGF), were introduced in the first- and second-membrane, respectively, of hydrogels. Similar as the manipulation of the BSA drug release study, the drug-loaded double-membrane hydrogels were immersed in buffer solutions with a pH value of 7.4 at 37 °C using the dissolution tester to investigate the complexing drugs release. At predetermined durations, 1.0 mL and 0.8 mL of the solution were taken out and analyzed by UV-spectrophotometer for the concentrations of the released CH at 255 nm,44 and the Bradford method for the concentrations of the released EGF at 465 nm.45 All the drug release experiments were repeated for two times. Swelling/erosion behavior. The swelling behavior of the double-membrane hydrogels (with the microsphere shape) was investigated by immersing the hydrogels into pH 7.4 and pH 2.0 buffer solutions. At specific time intervals, the swollen samples were collected and the free water on the surface was carefully removed. The swelling/erosion ratio (SR) was calculated by the dynamic weight change of the hydrogel with respect to the time as Equation (1):

SR =

ሺܹ௦ − ܹௗ ሻ ܹௗ

ሺ1ሻ

where ‫ ܛ܅‬is the weight of the hydrogel in the swollen state and ‫ ܌܅‬represents the initial weight of the hydrogel. All experiments were performed for three times.


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Storage stability. The storage stability of the double-membrane hydrogel was evaluated by the drug leakage rate in water. About 600 mg double-membrane hydrogel (20 microspheres) with CH- and EGF-loading were put in the distilled water at room temperature. At predetermined durations, the solution was taken out and analyzed by UV-spectrophotometer for the leakage of CH and EGF drugs. To keep constant the solution volume, fresh buffer solution with the same volume as the taken sample was added. RESULTS AND DISCUSSION Surface cationization of cellulose nanocrystals was realized by two approaches, involving the conversion of surface hydroxyl groups to cationic quatemary ammonium groups from “chemical grafting”, and the surface coverage by cationic polymer chains from “physical adsorption”. These two synthesis routines and chemical structures of cationic cellulose nanocrystals (CCNC and PCNC) are shown in Figure 2.

Figure 2. Synthesis pathways of cationic cellulose nanocrystals with the chemical grafting and physical adsorption strategies.


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C (CP/MAS) solid-state NMR analysis was used to investigate the surface modification on

cellulose nanocrystals, which is shown in Figure S1. The chemical shifts of carbon atoms from the glucopyranose units of cellulose were assigned to C1 (105 ppm), C4 crystalline (89 ppm), C2,3,5 (71-75 ppm), and C6 crystalline (65 ppm) on the spectra of pristine CNC, cationic CCNC and PCNC. The weak signals of chemical shifts located at 84 ppm and 63 ppm were attributed to the C4 and C6 of cellulose amorphous regions, which reflected the highly crystalline nature of cellulose nanocrystals.46 In comparison with pristine CNC, two extra weak peaks appeared at 45 ppm and 37 ppm on the spectrum of PCNC, which were assigned to the –CH2–CH2– groupsfrom adsorbed PEI chains on the surface of the nanocrystals. On the spectrum of CCNC, the additional chemical shift located at 55 ppm indicated the appearance of –CH3 groups on the structure of modified nanocrystals. In contrast to the three nanocrystals’ spectra, the broadening of peaks for CCNC and invariability of peaks for PCNC were observed in contrast to that of pristine CNC, which confirmed the surface modification on CCNC and PCNC. The degree of cationic modification (CCNC and PCNC) was determined from the results of elemental analysis (as shown in Table S1 in Supporting Information). According to the calculation of the nitrogen element content (N%) in modified nanocrystals, 27.6% of the surface hydroxyl

groups

of

cellulose

nanocrystals

were

replaced

by

cationic

hydroxypropyltrimethylammonium groups, which indicates that most of C6-OH was converted to cationic groups during the EPTMAC-chemical modification. The degree of adsorbed PEI chains on the surface of sulfated cellulose nanocrystals was 10.2% by weight, which shows the importance of electrostatic interaction between high molecular weight PEI chains and sulfated cellulose nanocrystals during the physical modification.


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Resulting from the presence of sulfate groups, H2SO4-hydrolyzed CNC exhibit a negatively charged surface. Through the physical or chemical modification, the negative charged surface of CNC can be transformed to positive charged surface for CCNC and PCNC. In the colloidal system of charged rod-like nanoparticles, the dispersion medium and the stationary layer of fluid attached to the dispersed nanoparticles provide the potential difference, which is named zeta potential (ζ-potential).40 The average ζ-potential values and distributions for the four nanocrystal types (pristine CNC, desulfated DCNC, and cationic PCNC, CCNC) are shown in Figure 3. Derived from the esterification of H2SO4 hydrolysis, negatively-charged sulfate groups were introduced on the surface of pristine CNC (-51.0 ± 7.7 mV). The broad distribution of CNC ζpotential may be attributed to the incomplete hydrolysis and somewhat self-aggregation in aqueous suspension. After the surface cationization, the surface of the nanocrystals was significantly converted to positively-charged, as indicated by the ζ-potential values of +37.3 ± 5.4 mV and +38.9 ± 3.6 mV for PCNC and CCNC, respectively. Furthermore, it is worth noting that in comparison with pristine CNC, the ζ-potential distribution for PCNC and CCNC is narrowed after the surface modification.


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Figure 3. Zeta potential distribution of pristine nanocrystals (CNC), cationic nanocrystals (CCNC and PCNC), as well as desulfated nanocrystals (DCNC). The morphology and dimensions of cellulose nanocrystals were observed and measured by AFM at different observation scales. As shown in Figure 4, the three types of cellulose nanocrystals, viz. negatively-charged CNC, and positively-charged CCNC and PCNC, exhibited the typical rod-like or needle-like morphology and homogeneous dispersion in water, with a length of about 100-300 nm and a width of about 15-30 nm. In fact, the presence of surface charges on nanocrystals (regardless of the sign of the charge) can provide enhanced electrostatic repulsion among nanoparticles, and therefore promote the dispersion of the nanocrystals in aqueous suspension.47 With the removal of surface charges, such as the desulfated nanocrystals (DCNC), the nanoparticles commonly self-aggregated or flocculated at the bottom of the aqueous suspension due to the lack of the electrostatic repulsion (not shown).


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Figure 4. AFM images of CNC (a, a′), CCNC (b, b′), and PCNC (c, c′) at different observation scales (images a, b, c: 3.3 × 3.3 µm; images a′, b′, c′: 1.1 × 1.1 µm). Another important physical property that is expected to be affected by different surface groups of cellulose nanocrystals is thermal stability. As reported previously, the presence of surface sulfate groups induces a sharp decrease of thermal degradation temperature (Td) for cellulose nanocrystals.48 In this study, as shown in Figure S2, the Td values for DCNC and CCNC (with the removal of surface sulfated groups) increased by 60 °C in comparison with that of pristine CNC. Similar as the DCNC, PCNC also exhibited an improvement of thermal stability despite the preservation of sulfate groups on the surface and physical adsorption of cationic PEI chains. It has been reported that the combination of poly(ethylene oxide) (PEO) chains and sulfated CNC can improve the thermal stability of CNC through the protection and shielding effects of polymeric chains to sulfate groups.49 Similar phenomenon may explain the improvement of thermal stability for PEI-adsorbed nanocrystals (PCNC). The optical microscopy observation of the microsphere hydrogels with single or doublemembrane structures are shown in Figure 5. With the control of the absorption duration for pure alginate, the thickness of the second membrane (the external layer) can be regulated from about 200 µm (5 min) to 700 µm (6 h). Interestingly, different from the structure of hydrogels treated for 5 min, 20 min and 1 h, the hydrogels with the absorption durations of 3 h and 6 h exhibited even three-layer structure, which may be attributed to the high content of absorbed alginate but insufficient Ca2+ crosslinking treatment (the Ca2+ crosslinking for all the double-membrane hydrogels was controlled as 1 h). The thickness variation of the pure alginate membrane was measured using optical microscopy observation and results are summarized in Table S2. Besides the preparation of double-membrane microsphere hydrogels, double-membrane hydrogels with a


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capsule-like and film-like shape can also be developed through the control of the shape of the first membrane hydrogel (as shown in Figure S3). The integral and half-section appearance of the double-membrane microsphere hydrogels after freeze-drying treatment was observed by optical microscopy. As shown in Figure S4, the double-membrane structure of the hydrogel can be observed from the thin and transparent layer (pure alginate membrane) as the “shell” together with the compact and spherical layer (alginate/nanoparticles membrane) as the “core”.

Figure 5. Optical micrographs of the microsphere hydrogels (the y axis represents the adsorption duration of pure alginate for the formation of the external hydrogel membrane). The integral and internal morphology of the hydrogel after freeze-drying was revealed by SEM, as shown in Figure 6. From the panoramic images of the single-membrane hydrogels (images a


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and b), it can be observed that the SA/CCNC hydrogel (Figure 6b) exhibit a more regular and entire spherical morphology in comparison with that of the collapsed surface morphology for the SA hydrogel (Figure 6a). Similar morphologies were also observed for the SA/CNC and SA/PCNC hydrogels, as shown in Figure S5. This is an indication that the presence of rigid cellulose nanoparticles can prevent the structural collapse and enhance the structural stability of the alginate-based hydrogels. The double-membrane structure of the freeze-dried SA/CCNC-1h and SA/CCNC-3h hydrogels can be observed from the cross-sectional SEM images (Figure 6c′ and 6d′), which presented a thin membrane covering the surface of the internal hydrogel. Similar double-membrane structures were also observed for the freeze-dried SA/CCNC-6h and SA/CCNC-20min hydrogels, but difficult to be distinguished on the images of the SA/CCNC5min hydrogel due to the thinness of its external membrane (not shown).

Figure 6. The panoramic and cross-sectional SEM images of (a and a′) SA, (b and b′) SA/CCNC single-membrane hydrogel; and (c and c′) SA/CCNC-1h, (d and d′) SA/CCNC-3h doublemembrane hydrogels after freeze-drying treatment. Before the use of expensive protein drug in the drug release study, the typical and cheap protein BSA was chosen as model to investigate the possible drug release behavior of the hydrogels. The


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drug release behavior (BSA) of the single-membrane hydrogels was pH-sensitive, as shown in Figure 7 representing the cumulative drug release curves of four single-membrane hydrogels in the pH 7.4 and pH 2.0 buffer solutions. Under the pH 7.4 condition, the four single-membrane hydrogels, SA, SA/CCNC, SA/CNC and SA/PCNC exhibited the BSA release behaviors with the different sustained effects. As shown in Figure 7a, in comparison with the rapid BSA release of the pure alginate hydrogel SA (3 days), the SA/CNC and SA/PCNC hydrogels provided a prolonged BSA release of 6 days, and the SA/CCNC hydrogel even exhibited a controlled BSA release of 8 days. On the basis of the formation of a stable crosslinked structure, the effect of prolonged BSA release for the SA/CCNC hydrogel can be attributed to the “nano-obstruction effect” and “nano-locking effect” induced by the rigid CCNC nanoparticles and possible electrostatic interactions between the cationic nanocrystals and alginate in the hydrogel.22 Nanoobstruction effect derived from the presence of nanoparticles in the drug carrier will prolong the diffusion route of the drug molecules. If there exists strong interactions between the nanoparticles and matrix (such as electrostatic interaction between CCNC and alginate in this study), the drug delivery system can further provide the diffusion delay of the drug release, which can be named as “nano-locking” effect. However, the SA/CNC hydrogel can only provide the “nano-obstruction effect” to sustain the BSA release derived from the obstruction of the nanoparticles, which showed the BSA release period of 6 days in the pH 7.4 condition. The SA/PCNC hydrogel containing the branched PEI polymer exhibited a prolonged BSA release effect similar to SA/CNC, which may result from the crosslinking competition between cationic PEI/alginate and Ca2+/alginate in the structure of the hydrogel. Under the pH 2.0 condition, the amount of the BSA release for all the hydrogels obviously decreased in comparison with those at pH 7.4, because the alginate will become more compact in the acidic condition and thus less


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permeable, but highly swell at the higher pH condition. As shown in Figure 7b, derived from the expected electrostatic interactions between cationic cellulose nanocrystals and anionic alginate, the total cumulative BSA release for the SA/CCNC and SA/PCNC hydrogels was lower than 3% in comparison with SA and SA/CNC (without interactions) hydrogels (about 6%) after 10 days.

Figure 7. Drug release study (BSA) of single-membrane hydrogels in (a) pH 7.4 and (b) pH 2.0 buffer solutions. Before the complexing drugs release study, the standard curves and equations of the concentrations for both drugs with UV-absorbance were constructed, as shown in Figure S6. The chemical structure of the drug ceftazidime hydrate (CH) is shown in Figure S7. The complexing


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drugs co-delivery of the double-membrane hydrogels containing the antibiotics drug (CH) and growth factor (EGF) are shown in Figure 8a. Regarding the release of CH, the three kinds of double-membrane hydrogels including CCNC, CNC and PCNC displayed similar behaviors, with rapid release of the drug molecules within three days under the pH 7.4 condition. Derived from the same component and structure of the three hydrogels, the rapid CH release can be attributed to the swelling and disintegration of the external pure alginate-based membrane hydrogel. During the first three-days of CH release, no signal of the EGF release can be detected, which indicated the separated and ordered drugs release from the double-membrane hydrogels. Concerning the SA/CNC-1h and SA/PCNC-1h double-membrane hydrogels, the EGF release started after four days, and rapidly released from the sixth to eighth day. However, the SA/CCNC-1h double-membrane hydrogel exhibited a different EGF drug release behavior, which showed a prolonged drug release lasting for 7-8 days (starting release from the fourth day and reaching an equilibrium at the eleventh or twelfth day). It seems that the cationic cellulose nanocrystals resulting from the surface chemical modification (CCNC) can provide a remarkable effect to sustain the drug release in the hydrogel, while a weak effect of the controlled drug release was observed for the pristine and cationic cellulose nanocrystal resulting from the physical modification (CNC and PCNC) in the hydrogels. The UV spectrum of CH and EGF release further indicates the change of the drugs absorbency at the critical times for the SA/CCNC-1h double-membrane hydrogel (associated with the drugs concentration and release rate in the buffer solution), as shown in Figures 8b and c.


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Figure 8. Complexing drugs release study involving ceftazidime hydrate (CH, the open symbols) and epidermal growth factor human (EGF, the solid symbols) of the double-membrane hydrogels in the pH 7.4 buffer solution (a) drugs release profiles, (b) UV spectra of CH release for the SA/CCNC-1h double-membrane hydrogel at the critical times, (c) UV spectra of EGF release for the SA/CCNC-1h double-membrane hydrogel with the Bradford method at the critical times. In order to clearly observe the structural changes and make a comparison with the complexing drugs release behavior, the SA/CCNC-1h double-membrane hydrogel was used for the swelling/erosion experiment and optical microscopy at the critical durations under both pH 7.4 and pH 2.0 conditions. As shown in Figure 9a, the SA/CCNC-1h hydrogel presented two phases during the swelling-erosion experiment resulting from the special structure of two-layered


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hydrogel. The outer membrane hydrogel (with neat alginate) underwent swelling and absorbed water during the first day, which can be observed through the thickness and transparency increases of the external membrane hydrogel (from the optical microscopy images). The swelling ratio of the hydrogel gradually reduced from the first to third day, which can be attributed to the erosion of the external membrane hydrogel. After three days swelling/erosion, the SA/CCNC-1h hydrogel exhibited a single-membrane hydrogel structure composed of CCNC and alginate (as shown in the optical microscopy image). The swelling process of the composite hydrogel for this membrane lasted for about five days, and reached the high swelling ratio of about 200% attributed to the mechanical enhancement and structure stabilization from the architecture of cationic CCNC and anionic alginate. After eight days, the double-membrane hydrogel went through the second erosion of the internal composite hydrogel, which can be observed through the crack from the optical microscopy image. Regarding the swelling/erosion behavior of the double-membrane hydrogel under the pH 2.0 condition (Figure 9b), the SA/CCNC-1h hydrogel showed a low swelling/erosion ratio of about -4% after 10 days, indicating the small compacting behavior and structural stability of the alginate-based hydrogel under the acidic condition. The optical microscopy observation at different durations further proved the preservation of the double-membrane structural integrity for the hydrogel under the pH 2.0 condition. The swelling/erosion results reflected the structural change of the SA/CCNC-1h double-membrane hydrogel under the different pH conditions, which was generally consistent with the complexing drugs release behavior.


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Figure 9. Swelling/erosion behavior of the SA/CCNC-1h double-membrane hydrogel under (a) pH 7.4 and (b) pH 2.0 conditions. Based on the results of in vitro drug release and swelling behavior of single- and doublemembrane hydrogels, a possible complexing drugs release model for the double-membrane hydrogel prepared from cationic CCNC and anionic SA was proposed. As shown in Figure 10, the external membrane (1-layer) of neat alginate went through the swelling, partial and complete disintegration to rapidly release the antibiotics drugs under the pH 7.4 condition. Regarding the inner membrane (2-layer), there was the formation of the enhanced skeleton from the


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electrostatic interaction between rigid nanoparticles (cationic CCNC) and alginate (anionic), which facilitated the structural stability of the internal hydrogel. Therefore, longer swelling and disintegration procedure was expected for the internal membrane than for the external membrane under the pH 7.4 condition. Derived from structural construction of CCNC in the hydrogel, the presence of nanoparticles can provide a “nano-obstruction effect” to prolong the drug release for the partial disintegration, and meanwhile play a “nano-locking effect” to further prevent the burst release of the drugs during the gradual disintegration of the hydrogel. It is worth noting that the double-membrane hydrogel as the drug delivery system realized the complexing drugs release with the firstly quick release of one drug and the successively slow release of another drug.

Figure 10. Proposed complexing drugs release model for the double-membrane hydrogel with the formation of cationic CCNC and anionic alginate under the pH 7.4 condition. The storage stability of the double-membrane hydrogel can be defined as the stability of the drugs entrapment, which can be evaluated by the drug leakage rate in water. As shown in Figure S8, the SA/CCNC-1h hydrogel can preserve its structural stability in water for more than two weeks, which presented the low leakage rates of the CH and EGF drugs. Moreover, the stored hydrogel maintained the spherical morphology and double-membrane architecture after eight and


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fourteen days in comparison with the original hydrogel. The good storage stability of the SA/CCNC-1h double-membrane hydrogel is important for its potential practical application as a biomedical material. CONCLUSIONS Alginate-based hydrogels possessing a double-membrane structure were prepared as novel complexing drugs carriers. The presence of cationic cellulose nanocrystals (CCNC) in the inner membrane can enhance the structural stability of the hydrogel through electrostatic interactions between cationic nanoparticles and anionic alginate. The different compositions and properties of the external and internal hydrogels provided the varied effects of the drugs release, which achieved the complexing drugs co-delivery with the rapid release of antibiotic and then sustained release of growth factor. The biocompatible hydrogels investigated in this study exhibited a special double-membrane structure and alternative drugs release behaviors, which showed their potential in the oral administration and wound dressing in biomedical application. Besides the rigid cellulose nanocrystals, cationic cellulose nanofibrils as flexible nanofilaments could also be applied to the alginate-based double-membrane hydrogel system, which may induce crosslinking between nanofibrils and alginate through chains entanglement, avoiding the step of Ca2+ crosslinking. In addition, despite of the reported nontoxicity for the natural components (cellulose nanocrystals and alginate), the cytotoxicity and in vivo study of the double-membrane hydrogels are also significant for the practical and clinic application of the hydrogels. All these studies are in progress and will be present in future work. ASSOCIATED CONTENT


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Supporting Information: Elemental analysis; thickness of the external membrane hydrogel for the double-membrane hydrogels;

13

C (CP/MAS) solid-state NMR spectra for CNC, ECNC, and

PCNC; TGA thermograms for CNC, DCNC, ECNC, and PCNC; photos of double-membrane SA/ECNC hydrogels with capsule-like shape and film-like shape; optical micrographs of the microsphere hydrogels after freeze-drying; SEM images of single-membrane hydrogels after freeze-drying; standard curves and equations of the BSA concentration and UV-absorbance from the Bradford method; ceftazidime hydrate concentration and UV-absorbance; epidermal Growth Factor human concentration and UV-absorbance from the Bradford method; storage stability of the double-membrane hydrogel in water. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author ∗ Alain Dufresne, Email: [email protected]. Address: The International School of Paper, Print Media and Biomaterials (Pagora), CS10065, 38402 Saint Martin d'Hères Cedex, France. Tel.: +33 476826995; fax: +33 476826933. ∗ Jin Huang, Email: [email protected]. Address: 122 Luoshi Road, Wuhan University of Technology, Wuhan 430070, P. R. China. Tel: +86 27 87749300; fax: +86 27 87749300. ACKNOWLEDGMENT LGP2 is part of the LabEx Tec 21 (Investissements d’Avenir - grant agreement n°ANR-11LABX-0030) and of the PolyNat Carnot Institut (Investissements d’Avenir - grant agreement n°ANR-11-CARN-030-01). This study was supported by Fundamental Research Funds for the Central Universities (Self-Determined and Innovative Research Funds of WUT: 2016IVA084).


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Table of Content

Biocompatible Double-Membrane Hydrogels from Cationic

Cellulose

Nanocrystals

and

Anionic

Alginate as Complexing Drugs Co-Delivery Ning Lin,† Annabelle Gèze, ‡ Denis Wouessidjewe, ‡ Jin Huang, †∗ and Alain Dufresne§,∥∗ †

School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of

technology, Wuhan 430070, P.R. China, ‡

Univ. Grenoble Alpes, LGP2, F-38000, Grenoble, France

§

Univ. Grenoble Alpes, DPM, UMR CNRS 5063, Grenoble, France

∥

CNRS, LGP2, F-38000, Grenoble, France


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Biocompatible Double-Membrane Hydrogels from Cationic Cellulose

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