Hybrid Alginate@TiO2 Porous Microcapsules as a Reservoir of Animal

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Biological and Medical Applications of Materials and Interfaces

Hybrid Alginate@TiO2 Porous Microcapsules as a Reservoir of Animal Cells for Cell Therapy Gregory Leroux, Myriam Neumann, Christophe Meunier, Antoine Fattaccioli, Carine Michiels, Thierry Arnould, Li Wang, and Bao-Lian Su ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15483 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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ACS Applied Materials & Interfaces

Hybrid Alginate@TiO2 Porous Microcapsules as a Reservoir of Animal Cells for Cell Therapy Grégory Leroux,§ Myriam Neumann,§ Christophe F. Meunier,§Antoine Fattaccioli,‡ Carine Michiels,‡ Thierry Arnould,‡ Li Wang§,* and Bao-Lian Su§,#,* §

Laboratory of Inorganic Materials Chemistry (CMI), University of Namur, 61 Rue de

Bruxelles, B-5000 Namur, Belgium. ‡

Laboratory of Biochemistry and Cell Biology (URBC), Namur Research Institute for Life

Sciences (NARILIS) University of Namur, 61 Rue de Bruxelles, B-5000 Namur. #

State Key Laboratory of Advanced Technology for Materials Synthesis and Processing,

Wuhan University of technology, Luoshi Road 122, Wuhan 430070, China. KEYWORDS: hybrid hydrogel, biocompatible synthesis, porous nanostructure, cell encapsulation, enhanced mechanical resistance, long-term cell activity

ABSTRACT: The number of patients suffering from diseases linked with hormone deficiency (e.g. type 1 diabetes mellitus) has significantly increased in recent years. As organ transplantation presents its limits, the design of novel robust devices for cell encapsulation is of great interest. The current study reports the design of a novel hybrid alginate microcapsule reinforced by titania via a biocompatible synthesis from an aqueous stable titania precursor (TiBALDH) and a cationic polyamine (PDDAC) under mild conditions. The biocompatibility 1 ACS Paragon Plus Environment

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of this one-pot synthesis was confirmed by evaluation of the cytotoxicity of the precursor, additive, product and by-product. The morphology, structure and properties of the obtained hybrid microcapsule were characterized in detail. The microcapsule displayed mesoporous, which was a key parameter to allow the diffusion of nutrients and metabolites and to avoid the entry of immune defenders. The hybrid microcapsule also showed enhanced mechanical stability compared to the pure alginate microcapsule, making it an ideal candidate as a cell reservoir. HepG2 model cells encapsulated in the hybrid microcapsules remained intact for 43 days as highlighted by fluorescent viability probes, their oxygen consumption, and their albumin secretion. The study provides a significant progress in the conception of the robust and biocompatible reservoirs of animal cells for cell therapy.

INTRODUCTION Transplantation or injection of xenogeneic cells that produce therapeutic agents, termed as cell therapy,1 can potentially treat multiple human disorders caused by protein or hormone deficiencies, such as type 1 diabetes mellitus,2 Parkinson’s disease,3 Alzheimer’s disease,4 dwarfism,5 anaemia,6 or factor VII or IX deficiency in haemophilia.7 However, injected cells risk the immune rejection from the host, which results in low efficiency of the cell therapy. For example, β-cell transplantation aiming at the treatment of type 1 diabetes mellitus often fails, due to the attack of the patient’s immune system.2 A promising approach to overcome this problem is the immune-isolation of cells using an encapsulation technology.8-11 Cell encapsulation involves the entrapment of cells within a porous system or a semipermeable membrane which enables the preservation of cellular activity and their immuneisolation.12-14 The defined porosity of the membrane not only allows the entrance of essential nutrients and oxygen and the output of the metabolites and molecules of interest (e.g. insulin

2 ACS Paragon Plus Environment

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for treatment of diabetes mellitus), but also prevents host immune responders from destroying the encapsulated cells.12-13 The first work involving immune-isolation was published in 1934 by Bisceglie.15 Since then, various types of materials have been studied for cell encapsulation addressing a wide range of medical applications.16-18 The used materials are mainly polymeric microcapsules or inorganic oxide-based monolithic gels.19 Polymer microcapsules refer to a polymeric matrix with entrapped cells, which often presents as a microsphere with the enclosed cell suspension as a liquid core and a semi-permeable shell as a solid crust.20 Commonly-used biopolymers for cell encapsulation include chitosan,21 polyelectrolytes,22 agarose or collagen,23 and alginate.24 Alginate, a natural copolymer composed of mannuronic acid (M units) and galuronic acid (G units), has attracted a lot of attention in food or medical applications, due to its biocompatibility, low cost, low toxicity, and mild gelation by addition of divalent cations such as Ca2+.25 However, the main problems of alginate capsules is their low mechanical stability and inadequate pore size, which leads to poor cell protection and preservation in cell therapy approaches. The coating of alginate microcapsules with polycationic molecules, such as poly-L-lysine (PLL), poly-D-lysine, poly-L-ornithine (PLO) or chitosan, improves slightly their mechanical resistance,26-28 but not suffiencently to meet the required criteria for cell therapy. In addition, their porosity remains hardly controllable. Inorganic monolithic gels allow the encapsulation of cells in amorphous silica or titania matrices under aqueous conditions. Compared to polymer microcapsules, they present improved mechanical resistance as well as thermal and chemical stability.29 Most importantly, their porosity can be adjusted in synthesis conditions,30-32 which benefits the permeability control of the gels, the protection of the encapsulated cells and the secretion of therapeutic agents. However, toxic by-products of the gelation such as short chain alcohols from their alkoxy precursors or salts



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from their aqueous precursors like Na2SiO3 present harmful influences on the viability and activity of encapsulated cells, thus decreasing the efficiency in cell therapy.33 It appears to be of great interest to combine alginate microcapsules and inorganic gels to obtain a hybrid microcapsule with high biocompatibility, enhanced mechanical stability and adapted porosity for cell therapy on the basis of cell encapsulation. Some methods have been reported to design alginate/silica hybrid microcapsules with an external layer of silica deposited around the alginate sphere.34-35 Alginate/silica hybrid microcapsules present excellent biocompatibility, improved mechanical strength, and well-adapted porosity for the immune-isolation of encapsulated cells.36-42 However, the slow in vivo dissolution of silica highly limits the use of the hybrid microcapsules.43 Titania-based materials usually involve improved pH stability, thermal resistance and mechanical strength. Moreover, they show high biocompatibility towards living bodies, which has been confirmed by implanting a titaniabased drug delivery system into living rats44 or interfacing cells surface for advanced functionalization.45-49 These promising properties indicate the possible use of titania in cell encapsulation approaches. The combination of alginate microcapsules and a crust containing TiO2 which are desired to be more stable than SiO2, could be a solution to improve the longterm use of the hybrid microcapsules for cell therapy with high efficiency. We report here the design and synthesis of a biocompatible hybrid alginate/titania core/shell microcapsules under mild conditions using a fast and facile one-step process (Figure 1A). Titanium(IV) bis(ammonium lactato)dihydroxide (TiBALDH) was chosen as a biocompatible

precursor

for

the

formation

of

the

hybrid

microcapsules.

Poly(diallyldimethylammonium) chloride (PDDAC) was used to induce the polymerization of TiBALDH to form TiO2 at the surface of alginate capsules. The titania layer not only reinforced the chemical stability and mechanical properties of the microcapsules, but also presented a uniform surface porosity that avoids the entry of host immune defenders. This


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hybrid microcapsule would be of great promise for cell therapy on the basis of cell encapsulation, such as for the encapsulation of insulin-secreting xenogeneic β-cells for the treatment of type 1 diabetes mellitus. In this work, human hepatocellular carcinoma cells (HepG2) have been used as model cells for cell encapsulation because of their similarity with β-cells in terms of size and in shape, their high availability and their continuous secretion of albumin allowing diffusion studies of the hybrid microcapsules.

EXPERIMENTAL SECTION Chemical

materials.

Titanium(IV)

bis(ammonium

lactato)dihydroxide

solution

(TiBALDH, 50 wt.% in H2O), ammonium lactate solution (20 wt.% in H2O), poly(diallyldimethylammonium) chloride solution (PDDAC, 20 wt.% in H2O), 5chlorosalicylic acid (98%), sodium perchlorate (hydrate, 99.99%), sulfuric acid (ACS reagent, 95.0-98.0%), ammonium hydroxide solution (ACS reagent, 28.0-30.0% NH3 basis), alginic acid sodium salt from brown algae (for immobilization of microorganisms), calcium chloride (dihydrate, 99%), ethylene glycol-bis(β-aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA, tetrasodium salt, 97%), hydrochloric acid (Molecular Biology Grade, 36.538.0%), sodium hydroxide (reagent grade, 98%, anhydrous), chloroform (anhydrous, 99%), ethylenediaminetetraacetic acid (EDTA, disodium salt, dihydrate, 99%), fluorescein isothiocyanate Dextran (Dextran-FITC) and carboxylate-modified latex beads (L5155) were purchased from Sigma-Aldrich. Titania nanoparticles (P25) were purchased from Evonik Industries, and ethanol (absolute) were purchased from Fisher. Dulbelcco’s Modified Eagle Medium (DMEM, 31885) and foetal bovine serum (FBS) were provided from Invitrogen (Casbald, USA). Sodium alginate powder was purified as described by de Vos et al.50 Briefly, the commercial powder was dissolved in an aqueous solution of EGTA (1 mM) to remove Mg2+



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and Ca2+ cations. This solution was then passed through 5 to 0.45 µm filters. The alginate fraction was subsequently acidified by HCl (2 M) at 4°C until gelification. This gel was filtered and washed with HCl (0.01 M), chloroform and butanol to eliminate remaining ions and proteins. The raise of the pH to 7.0 with NaOH (0.5 M) allowed the recovery of a clear solution. Finally, absolute ethanol was added to precipitate the purified alginate and a vacuum pump was used to dry the compound. Biological materials. HepG2 cells (Hepatocellular Liver Carcinoma Cell line) were cultivated in Dulbelcco’s Modified Eagle Medium (DMEM, 31885) supplemented with 10 % of foetal bovine serum in 75-cm2 flasks (Costar, Lowell, USA). The cells were incubated at 37 °C (95% air/5 %CO2). After three days of culture, Phosphate Buffer Saline (PBS) was used to wash the cells before their treatment with a trypsin/EDTA mix. The trypsinised cells were centrifuged (1000 rpm for 5 min), collected and then resuspended in fresh culture medium with serum for culture or without serum for encapsulation. Synthesis of hybrid microcapsules. This section describes how the cells are encapsulated within pure alginate microcapsules or hybrid alginate@TiO2 capsules. All the solutions used were previously adjusted at physiological pH (7.4). Cell encapsulation in pure alginate microcapsules. Firstly, cells were collected in fresh buffered culture medium and mixed with purified sodium alginate (3 wt.%). The final cell density was 1.1*106 cells mL-1. The microcapsules were prepared by extruding this mixture into a calcium chloride solution (100 mM) using low-density polyethylene (LDPE) disposable transfer pipets and at a rate of about 1 drop extruded per second. A cross-linking reaction occurred for five minutes between carboxylate functions of polysaccharide chains due to the presence of Ca+2 cations. It resulted in a microcapsule with a liquid core containing the cells and a solid crust. The microcapsules were then harvested and put in the test tubes


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filled with 5.0 mL of fresh culture medium with serum and maintained in cell culture conditions (37 °C, 5 % CO2). Cell encapsulation in hybrid alginate@TiO2 hybrid microcapsules. Hybrid microcapsules were obtained by the procedure presented in Figure 1A. A buffered titanium precursor solution diluted in the culture medium without serum was added to a suspension of cells containing 3.0 wt.% of purified sodium alginate (1:1 vol.). The final cell density was 1.1*106 cells mL-1. This mixture was homogenised by several back and forth aspiration with a LPDE disposable transfer pipet and then directly extruded into a buffered aqueous solution containing calcium chloride and PDDAC (0.4 wt.%) at a rate of about 1 drop per second. The drops were incubated in the solution for five minutes. The drop transformed into a microcapsule with a white solid crust, indicating that a reaction occurred between the titania precursor (TiBALDH) and PDDAC. The hybrid microcapsules were then harvested and put in the test tubes filled with 5.0 mL of fresh culture medium with serum and maintained in cell culture condition (37 °C, 5 % CO2). Cytotoxicity test. The toxicity of the titanium precursor (TiBALDH) used to synthesize the hybrid microcapsules was evaluated by the measurement of the Lactate Dehydrogenase (LDH) released by the cells incubated with tested compounds. LDH is an enzyme located in the cellular cytoplasm that can be detected outside when the plasma membrane becomes permeable, a sign of cell death. LDH was quantified in the samples using a Detection Kit from Roche Applied Science Laboratory (Cytotoxicity Detection KitPLUS). A number of 50000 HepG2 cells was seeded in a 24-well plate for 24 hours. The culture medium was then replaced by the solution of potentially toxic chemicals with different concentrations. After 5 hours of incubation, the supernatant of each well was collected. The remaining cells, attached and alive in the wells, were lysed by Triton X-100 (10%) for the monitoring of intracellular LDH of living cells. The measurement of absorbance at 490 nm allows the determination of



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extracellular and intracellular LDH activity and the evaluation of the toxicity of compounds. In addition, the cytotoxicity of the titanium precursor, the additive, the product and the byproduct of the encapsulation process were further evaluated using LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific). Again, 50000 HepG2 cells were seeded in a 24-well plate for 24 hours. The culture medium was then replaced by the solution of potentially toxic chemicals with different concentrations. After 5 hours of incubation, the culture medium was removed. 200 µL of the staining solution was added directly to cells. The mixture was incubated for 30 min at 22 °C and subsequently examined using a Multizoom AZ-100 microscope (Nikon). Material properties. The hydrolysis and condensation of TiBALDH in the presence of PDDAC has been highlighted by micrographs collected in brightfield mode using a Multizoom AZ-100 microscope (Nikon). Micrographs of hybrid alginate@TiO2 hybrid microcapsules were taken over time during incubation (1, 5, 20, 30 minutes). The same experiment was also performed with microcapsules synthesized without TiBALDH, and without PDDAC. Microcapsules of different compositions were dehydrated with ethanol and dried through a process of critical point drying with liquid carbon dioxide in order to avoid the structural collapse of the microcapsules. The porosity of the samples was determined via the adsorption and desorption of nitrogen at -196 °C using a volumetric adsorption analyzer (Micromeritics Tristar 3000). Prior to the measurement, the samples were degassed overnight. The morphology and structure of the samples were observed using field emission scanning electron microscopy (FE-SEM) (JEOL JSM-7500F, JEOL). The chemical composition of the samples was evaluated using an energy-dispersive X-ray analysis system (EDX) with an acceleration voltage of 15.00 kV and a working distance of 8 mm.


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The mechanical stability of microcapsules with different compositions was evaluated using two different methods. Firstly, 100 microcapsules were placed in a buffered 50 mM EDTA solution (pH 7.4) under stirring (60 rpm) and the number of intact microcapsules was counted in function of time. This analysis protocol was chosen to mimic the degradation mechanism of alginate microcapsules due to the progressive exchange of Ca2+ ions by Na+ cations. The 5-chlorosalicylic acid assay was performed to quantify the TiO2 in the hybrid microcapsules. Briefly, a microcapsule was dissolved in 1.0 mL of concentrated sulfuric acid. 2.5 mL of 5-chlorosalicylic acid (2.5% in absolute ethanol), 2.5 mL of sodium perchlorate (1 M), 7.5 mL of absolute ethanol and 10.0 mL of deionized water were added to the dissolved microcapsule. The pH of the solution was adjusted to 4 using concentrated NH4OH. The solution was filled up with deionized water to 50 mL. The absorbance of the solution was monitored at 355 nm using a Lambda 35 UV/visible spectrophotometer (Perkin Elmer) and the quantity of TiO2 in the microcapsule determined using a standard curve elaborated from the TiBALDH solution with different concentrations. The mass diffusion of the alginate@TiO2 microcapsules was evaluated using different fluorescent probes with various hydrodynamic diameters. Briefly, 60 microcapsules were synthesized and then mixed with 6.0 mL PBS (pH 7.4) containing 0.4 mg L-1 of DextranFITC with various molecular weight cut-off (MWCO: 10, 70 and 250 kDa) or 200 µL of green fluorescent carboxylate-modified latex beads (2.5% solids). The concentration evolution of each substance were determined at room temperature for several days. The fluorescence intensity of the different molecules was recorded using a LS45 Luminescence Spectrometer (Perkin Elmer). The excitation/emission wavelengths were respectively 494/518 nm for Dextran-FITC and 470/515 nm for latex beads. The fluorescence intensity of the probes without microcapsules was taken as the reference.



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Cell viability and metabolic activity of encapsulated cells. The viability of encapsulated cells was assessed by measuring the oxygen consumption using a Clark’s electrode (HansaTech Instruments). For each type of microcapsules, 3 of them were added in the vessel of the device, containing 1.0 mL of culture medium. The respiration activity of entrapped HepG2 cells was determined just after the encapsulation and kept as the reference value (100 %) over the whole experiment. The cell viability was corroborated by the use of a vital dye probe (LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells, Thermo Fisher Scientific). Entrapped cells were incubated within the staining solution for 30 minutes at 22°C. Micrographs were taken using confocal laser scanning microscopy (CLSM). The secretion of the hybrid microcapsules with encapsulated cells was monitored over time by the concentration of human albumin in the medium. The cell-laden microcapsules were collected and replaced by fresh culture medium every three or four days. The concentration of human albumin was measured using a ELISA kit commercialized by AssayPro (Human Albumin ELISA Kit). Statistical analysis. The percentages of LDH released by HepG2 cells incubated with different concentrations of TiBALDH were compared to the basal death of the cells (control). Results were analysed by unpaired Student’s t-test, using XLSTAT 2013 software (Addinsoft) and with p < 0.05 being considered as significant.

RESULTS AND DISCUSSION Design of the hybrid microcapsules. Titania (TiO2) can be synthesized by various methods such as chemical vapor deposition, electrodeposition, ion beam-assisted process, sol-gel process, or hydrothermal methods. However, the methods other than the mentioned sol-gel process are hardly applicable for cell encapsulation because of the harsh conditions of


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synthesis, involving high temperature and pressure. It is well known that the sol-gel process, could allow the cell encapsulation by mixing cells with a sol before the condensation reaction which leads to the formation of a monolithic gel containing the cells under mild conditions.51 These amorphous hydrogels, such as silica gels, can be obtained via the hydrolysis and condensation of alkoxy precursors. Unlike silica sol-gel synthesis, the hydrolysis and condensation rates of titania alkoxy precursors are very fast and difficult to be controlled, because titanium, as a transition metal, possesses an unsaturation of coordinance that makes its alkoxy precursors very electrophilic and very reactive towards water. As a consequence, instead of gels, precipitates are obtained. More importantly, the by-products of this preparation method which generally refers to short-chain alcohols presents huge negative effects on the phospholipid bilayers of cell membranes,52 which results in the limitation of this method on cell encapsulation. It can be concluded that sol-gel processes using alkoxytitanium are not adapted for cell encapsulation. TiBALDH, a water stable molecule, can serve as a precursor in the synthesis of titania. With the assistance of long chain polyamines, such as poly-L-lysine (PLL), Poly(diallyldimethylammonium) chloride (PDDAC) or amine-terminated dendrimers, TiBALDH hydrolyzes and condenses into titania (Figure 1B), which has been reported as a promising material-coating approach.45-46, 48-49, 53-54 The formed titania indicates an anatase phase according to the X-ray diffraction (XRD) pattern. (Figure S1) Compared with the sol-gel processes of alkoxytitanium, the titania synthesis using TiBALDH avoids the formation of cytotoxic short-chain alcohols, which makes TiBALDH a potential biocompatible titania precursor in the formation of hybrid microcapsules for cell encapsulation.



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Figure 1. The formation of the hybrid alginate@TiO2 microcapsule. (A) The fabrication procedure of hybrid alginate@TiO2 microcapsules. The mixture of sodium alginate and TiBALDH is dropped into a solution of calcium chloride and PDDAC to form hybrid alginate@TiO2 microcapsules through the ionic crossing-linking of alginate and the hydrolysis and condensation of TiBALDH. (B) The hydrolysis and condensation processes of TiBALDH in the formation of hybrid alginate@TiO2 microcapsules. The fabrication process of the hybrid alginate@TiO2 microcapsule is depicted in Figure 1A. When the mixture of sodium alginate and TiBALDH was dropped into a solution of calcium chloride and PDDAC, a series of reaction took place in the outmost layer of the drops towards the formation of hybrid alginate@TiO2 microcapsules. A calcium alginate shell was formed around the drop through the ionic cross-linking between the alginate and Ca2+ ions. Meanwhile, the PDDAC molecules in the solution were adsorbed on the surface of the 12 ACS Paragon Plus Environment

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alginate drop by electrostatic interaction. The adsorbed PDDAC molecules induced the hydrolysis and condensation of the TiBALDH molecules inside the drops to form a titania coating. (Figure 1B) The core of the formed microcapsules was a liquid mixture of sodium alginate and TiBALDH. With the incubation of the microcapsules in the solution of calcium chloride and PDDAC, the hybrid calcium alginate-TiO2 shells became thicker through the continuous processes of the cross-linking, hydrolysis and condensation. In consequence, hybrid alginate@TiO2 microcapsules were obtained. Cytotoxicity of the formation process of the hybrid microcapsules. In order to confirm the biocompatibility of the hybrid microcapsules for the encapsulation of HepG2 cells, the cytotoxicity of the titania precursor, the additive, the product and the by-product of the formation process were studied. This study can help evaluate the concentration upper limit of these compounds which can be tolerated by HepG2 cells after 5 or 24 hours of incubation. The results of the cytotoxicity test of the precursor TiBALDH on HepG2 cells are presented in Figure 2. Live/dead viability probes, consisting of green-fluorescent calcein-AM and red-fluorescent ethidium homodimer-1, were used to test the viability of HepG2 cells after the incubation in the TiBALDH solution with different concentrations. When a cell culture was incubated in the medium containing these probes, the metabolically active cells were stained by green-fluorescent calcein-AM as a result of the active intracellular esterase, indicative of living cells, whereas the cells with broken/permeabilised plasma membranes were stained with red-fluorescent ethidium homodimer-1, indicative of dead cells. Fluorescent microscopy on suspensions of the probe-stained HepG2 cells (Figure 2A) indicates the percentage of living cells in all experimental groups is close to 100%, even though the high concentration of TiBALDH (> 100 mM) influenced on the attachment of HepG2 cells. The long-term incubation in the TiBALDH solution with a high concentration resulted the loss of cell activity. After 24-hour incubation, the viability of the cells incubated



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in a TiBALDH solution with a high concentration (500 mM) decreased to ~ 30 %, nevertheless, the cells incubated in the TiBALDH solution with lower concentration (≤ 250 mM) still presented the high viability of > 70 %. (Figure S2) The cytotoxicity of the precursor TiBALDH on HepG2 cells was also evaluated by the measurement of the LDH released by the cells. LDH is an enzyme located in the cytoplasm that can be detected outside due to the permeable plasma membrane of the dead cells. The percentages of LDH released by the cells of the different experiment groups are presented in Figure 2B. The percentage of LDH released by the cells of basal control is 14.9(±2.9) %, while the ones released by the cells incubated in the TiBALDH solution with the concentrations from 1 mM to 500 mM remain between 10 % to 23 %, which supports that TiBALDH did not have negative effects on the permeability of the plasma membrane of the cells, indicative of non-cytotoxicity. Two evaluations of the cytotoxicity of the precursor TiBALDH towards HepG2 cells confirms the innocuity of TiBALDH (≤ 250 mM) when mixed directly with HepG2 cells for a long period of time and its potential to serve as a TiO2 precursor employed in the fabrication of hybrid microcapsules for animal cell encapsulation.


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Figure 2. Cytotoxicity of TiBALDH on HepG2 cells after 5 hours of incubation. (A) Fluorescent micrographs of the cytotoxicity kit stained HepG2 cells after incubated in the TiBALDH solution with different concentrations. “Control” represents the blank experimental group in which cells are incubated without the TiBALDH. Green fluorescent cells are alive, while red fluorescent ones are dead. (B) The release of the intracytoplasmic enzyme LDH of the HepG2 cells after incubated in TiBALDH solution with different concentrations. Results are expressed as means ± standard deviations for three independent measurements (n=3). “Control” represents the blank experiment group in which cells are incubated without the TiBALDH.



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The cytotoxicity of the reaction additive PDDAC on HepG2 cells has also been investigated. The result (Figure S3A) indicates the high cytotoxicity of PDDAC. Even a low concentration of PDDAC solution (0.1 wt.%) could lead to the cell broken and cell deactivation. This is due to the high positive charge density of this long-chain polymer. PDDAC can strongly bond on the cell membrane, thus inducing the formation of holes in the cell membrane, and finally resulting in the cell apoptosis. In our encapsulation, PDDAC, serving as an additive to induce the formation of titania shells around alginate microcapsules, is mostly located on the surface of the microcapsules, thus the direct contact with the encapsulated cells and the toxic influences on the encapsulated cells are avoided. According to the chemical equation of the titania synthesis (Figure 1B), the product and the by-products are titania and ammonium lactate, respectively. In order to confirm the biocompatibility of the encapsulation procedure, the cytotoxicity of these two compounds on HepG2 cells were tested. Titania presented high biocompatibility at all test concentrations, even though the high concentration of titania suspension (>250 mM) affected the attaching behaviours of the cells. (Figure S3B) Ammonium lactate showed non-toxicity on HepG2 cells at the low concentration (< 100 mM), but high toxicity at the high concentration (> 250 mM). (Figure S3C) The chemical kinetics of the synthesis of titania in the formation of hybrid microcapsules was investigated to quantify the generated product and by-product. The amount of titania formed around an alginate microcapsule was determined by the 5-chlorosalicylic acid assay allowing the titanium (IV) quantitation. The incubation time in the PDDAC-containing CaCl2 solution was maintained constant while the initial concentration of the titania precursor was varied. 1 mL mixture of alginate and titania precursor can generate 20 microcapsules. The quantity of the generated titania was lower than the initial quantity of the titania precursor. (Figure S4) It indicates that only part of precursor had taken part in the reaction, which was


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probably because the reaction could only occur on the surface of microcapsules with the assistance of adsorbed PDDAC while the titania precursor in the core of the alginate drop had not reacted. The quantity of synthesized titania increased linearly until it reached a limit of about 6000 nmol per microcapsule. (Figure S4) It is worth to noting that when the concentration of TiBALDH is below 250 mM, the concentration of generated titania and ammonium lactate is below 100 mM (5000 nm per microcapsule * 20). The titania and ammonium lactate with this concentration is biocompatible to HepG2 cells (Figure S3B, S3C), which evidences that using TiBALDH with a concentration equal to or below 250 mM is appropriate to synthesize hybrid microcapsules for cell encapsulation. Formation of the hybrid microcapsules. The formation of the hybrid microcapsule was recorded using optical microscope. The microcapsule was incubated in the PDDACcontaining CaCl2 solution. (Figure 3A) The microcapsules were synthesised by dropping a mixture of alginate and TiBALDH into a CaCl2 solution without PDDAC (Figure 3B) and that prepared by extruding alginate without TiBALDH in a PDDAC-containing CaCl2 solution (Figure 3C) were set as control groups. The brightfield mode micrographs were collected after the incubation of 1, 5, 20, 30 min, respectively. (Figure 3A-C) A white layer around the microcapsule formed with both the titanium precursor and the polycation could be observed by means of the naked eyes. In optical microscope, the optical transparency of the microcapsule became opaque. (Figure 3A) The optical transparency of the microcapsule was conserved when only the titanium precursor (Figure 3B) or the polycation (Figure 3C) was involved in the microcapsule synthesis. These results indicate that a dense layer around the alginate microcapsule could be formed through the hydrolysis and condensation of the titanium precursor with the assistance of the polycationic macromolecule PDDAC. The final microcapsule is presented in the form of a robust white-coloured bead with the diameter of about 5 mm.



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Figure 3. The evolution of the transparency and size of the microcapsules in function of the reaction time. (A-C) Microscope images illustrating the reaction between PDDAC and TiBALDH to form an external layer of TiO2 around the alginate microcapsule. Brightfield pictures taken over time of (A) an alginate/TiBALDH/PDDAC microcapsule, (B) an alginate/TiBALDH micropauses, and (C) alginate/PDDAC microcapsules. (D) The diameter of the three kind microcapsules with as a function of the reaction time. Results are expressed as means ± standard deviations for three independent measurements (n=3).


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The diameter of these microcapsules is presented in Figure 3D as a function of the reaction time. The interaction between the polycationic macromolecule PDDAC and negatively charged alginate led to the condensation of the microcapsule, resulting in the diameter decrease. When the titania precursor was used alone, the diameter of the microcapsule was similar to that of the pure alginate microcapsule, due to the non-interaction between the titania precursor and alginate. The diameter of the microcapsule prepared with PDDAC and TiBALDH is between that of the microcapsule prepared with PDDAC and that of the microcapsule prepared with TiBALDH, indicating that TiBALDH reacted to form a condensed inorganic titania layer which led to the decrease of the diameter and the formed condensed layer was strong enough to prevent the shrinkage of the microcapsule by the interaction between PDDAC and alginate.

Structure and chemical composition of the hybrid microcapsules. The morphology, structure and chemical composition of the alginate@TiO2 microcapsules were characterised. The pure alginate microcapsules, the microcapsules synthesized by dropping a mixture of alginate and TiBALDH into a CaCl2 solution without PDDAC, and the microcapsules prepared by extruding alginate without TiBALDH in a PDDAC-containing CaCl2 solution were set as the control groups. In order to assess the structure and chemical composition, microcapsules were dried under supercritical conditions with liquid carbon dioxide to avoid the structural collapse. The dried microcapsules were characterized using scanning electron microscope (SEM) equipped with an energy-dispersive X-ray analysis system (EDX).



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Figure 4. Structure and chemical composition of the hybrid microcapsules. (A) SEM micrographs of a purified alginate microcapsule (A1), the microcapsule surface (A2), and the microcapsule core (A3), together with an EDX spectrum of the microcapsule surface (A4). (B) SEM micrographs of an alginate/PDDAC microcapsule (B1), the microcapsule surface (B2), and the microcapsule core (B3), together with an EDX spectrum of the microcapsule surface (B4). (C) SEM micrographs of an alginate/TiBALDH microcapsule (C1), the microcapsule surface (C2), and the microcapsule core (C3), together with an EDX spectrum of the microcapsule surface (C4). (D) SEM micrographs of an alginate/TiBALDH/PDDAC microcapsule (D1), the microcapsule surface (D2), and the microcapsule core (D3), together with an EDX spectrum of the microcapsule surface (D4). The microcapsules were supercritically dried with CO2 prior to analyses. All dried microcapsules maintained their original spherical morphology, even though their diameters decreased dramatically due to the supercritical drying processes (Figure 4A1, 4B1, 20 ACS Paragon Plus Environment

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4C1, 4D1). The surface of the pure alginate microcapsule (Figure 4A2) and the alginate/PDDAC microcapsule (Figure 4B2) present wrinkles in micrometer-scale due to the dehydration of the polymeric spheres. However, because of the addition of titania precursor, the

surface

of

the

alginate/TiBALDH

microcapsule

(Figure

4C2)

and

the

alginate/TiBALDH/PDDAC microcapsule (Figure 4D2) are smoother in micrometer-scale and seem to be harder. It is noted that some cracks were generated on the surface of the alginate/TiBALDH/PDDAC microcapsule (Figure 4D1) during the drying processes, probably because of the component separation of the heterogeneous shells composed of calcium alginate and titania. EDX analyses on the surface of microcapsules with different compositions were performed to highlight the formation of titania layer in the hybrid microcapsules (Figure 4A4, 4B4, 4C4, 4D4). The presence of calcium, carbon and oxygen were detected on the surface of the pure alginate microcapsule (Figure 4A4) and the surface of the alginate/PDDAC microcapsule (Figure 4B4), indicating the presence of a layer of calcium alginate at their outmost surface. Titanium EDX signal could be detected in the alginate/TiBALDH/PDDAC microcapsule (Figure 4D4), which evidenced the formation of a titania layer around the alginate core by the hydrolysis and condensation of the precursor. The absence of titanium peak in the EDX spectrum of the surface of the alginate/TiBALDH microcapsule confirms that a titania coating cannot be formed without the assistance of PDDAC (Figure 4D3). The structure of the core of the microcapsules was characterized using SEM. The cross section of the pure alginate microcapsule, the alginate/PDDAC microcapsule, the alginate/TiBALDH microcapsule and the alginate/TiBALDH/PDDAC microcapsule were shown in Figure 4A3, 4B3, 4C3, and 4D3, respectively. A spongy, porous structure was observed

in

all

kinds

of

microcapsules.

It

is

worth

to

note

that

the

alginate/TiBALDH/PDDAC microcapsule presented a uniform porous structure and



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possessed smaller-sized pores in comparison with other microcapsules (Figure 4D3), which could benefit the control on the mass penetration of the microcapsule when applied in the encapsulation of therapeutic agents-releasing cells for cell therapy. Porosity of the hybrid microcapsules. Porosity plays an important role in the cell therapy with cell-laden microcapsules. A microcapsule with appropriate porosity cannot only protect encapsulated cells from the attack of cytotoxic agents by the size limitation of the pores, but also facilitate the release of therapeutic agents for cell therapy with high efficiency. In order to design hybrid microcapsule with optimal porosity, the synthesis parameters such as the reaction additives, the concentration of the titania precursor, and reaction time, were investigated in detail. The microcapsules were dried under supercritical conditions, then their porosity was characterized by nitrogen adsorption and desorption measurement. The isotherms and pore size distribution are presented in Figure 5.


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Figure 5. Porosity of the hybrid microcapsules. (A) Nitrogen adsorption-desorption isotherms and (B) pore size distribution of the microcapsules formed by different reaction additives. (C) Nitrogen adsorption-desorption isotherms and (D) pore size distribution of the hybrid alginate@TiO2 microcapsules formed with different concentrations of titania precursor. (E) Nitrogen adsorption-desorption isotherms and (F) pore size distribution of the hybrid alginate@TiO2 microcapsules formed with different reaction time. According to the IUPAC, the isotherms of the pure alginate microcapsules, the microcapsules formed with PDDAC, and the microcapsules formed with TiBALDH belong to type II isotherms, while the isotherm of microcapsules formed with PDDAC and TiBALDH belongs to type IV isotherm (Figure 5A). The corresponding pore size distribution indicates that our hybrid alginate@TiO2 microcapsules have mesopores with a uniform pore size of ~18 nm. However, other microcapsules show ambiguous pore size distributions. These results demonstrate that the hybridization of titania and alginate could induce the formation of uniform mesopores instead of the large and irregular pores of the pure alginate microcapsules. In addition, the concentration of the titania precursor had a great influence on the porosity of the formed hybrid alginate@TiO2 microcapsules (Figure 5C). The pore size of the hybrid microcapsules decreased with the increase of the titania precursor concentration (Figure 5D). A high TiBALDH concentration (500 mM) led to the formation of dense titania layers, resulting in the low porosity. Furthermore, the reaction time also presented a close relationship with the porosity of the hybrid microcapsules (Figure 5E, 5F). It can be pointed out that 5 min have been confirmed as a suitable reaction time for the synthesis of the porous hybrid microcapsules. In a short reaction time (1 min), porous titania layers could not be formed. However, a long-time reaction could decrease the porosity of the microcapsules due to the formation of the dense titania layers. Consequently, the hybrid alginate@TiO2 microcapsules synthesized in an appropriate condition possessed optimal porosity and 23 ACS Paragon Plus Environment


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uniform mesopores, which could contribute to the well-control on the mass diffusion between the external and internal of the microcapsules, thus potentially improving their efficiency in cell therapy.

Figure 6. Mass diffusion of the hybrid microcapsules. Study of the diffusion of fluorescent probes into hybrid alginate@TiO2 microcapsules (TiBALDH, 100 mM). 100% corresponds to the fluorescence intensity of the solution without microcapsules. The hydrodynamic radius of molecules allows the estimation of the MWCO: 4, 12 and 22 nm for the dextran-FITC, and 30 nm for the latex carboxylated-modified beads. The mass diffusion of hybrid alginate@TiO2 microcapsules were determined by the measurement of the quantity of fluorescent probes with different hydrodynamic diameter that enabled to diffuse into the internal of the microcapsules. These molecules simulate the immune defenders that may enter into the microcapsule to attack the entrapped cells. Two types of probes were used: the fluorescein isothiocyanate-dextran (Dextran-FITC) with various diameters (10, 70 and 250 kDa) and latex beads with a diameter of 30 nm. According to the Stoke equation, the different Dextran-FITC probes have a diameter of 4, 12 and 22 nm,


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respectively. Figure 6 shows the evolution of the fluorescence intensity of solutions in which the microcapsules are added. The experiment was performed over a period of 7 days. A rapid diffusion (48 h) of more than 15 % of the smallest molecules before reaching equilibrium was observed. The other molecules seemed difficult to diffuse as 91 %, 93 % and 95 % of them stayed outside of the microcapsules, respectively. The pore size of the microcapsule shell can be hypothesized to be between 4 and 12 nm, which coincides with the pore size distribution of the microcapsules analysed by isotherm (Figure 5D). It is worth to note that the hybrid alginate@TiO2 microcapsules process pores with two different sizes (Figure 5D). The pores in the microcapsule shell mainly composed of titania and calcium alginate present a diameter of ~12 nm, while the pores in the microcapsule core mainly composed of alginate have a diameter of ~50 nm. The molecular weight of the smallest immune defender (IgG, immunoglobulin G) is evaluated around 150 kDa.55 According to the relationship between molecular weight and Stokes radius, it matches with a size of about 20 nm.56-58 Therefore, the small pores in the shell enable to serve as a filter for immune-isolation and a channel for the diffusion of the nutrients and metabolites, while the large pores in the core can provide space for the metabolism of encapsulated cells. Moreover, in order to investigate the effect of the porosity on the mass diffusion behaviour, a comparison between those of microcapsules with different compositions has been made. The fluorescein isothiocyanate-dextran (Dextran-FITC) with 250 kDa has been used in this study, due to the similarity between its diameter (22 nm) and the size of IgG. The results are shown in Figure S5. They indicate that the probes diffused very quickly into the pure alginate and alginate/TiBALDH microcapsules, only 50 %-70 % of the probe molecules retained in the solution after 60 minutes. In contrast, the alginate/PDDAC and hybrid alginate@TiO2 microcapsules retained more than 80% of probes within the solution. It is worth noting that the diffused probe quantity in the hybrid alginate@TiO2 microcapsules was less than those in



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the alginate/PDDAC microcapsules. A direct relationship is established between the pore size distribution and the diffusion behaviour. Associated with the analysis of the pore size distribution of four kinds of microcapsules (Figure 5B), it can be pointed out that probes with a diameter of 22 nm can easily diffuse into pure alginate and alginate/TiBALDH microcapsules due to the presence of macropores. The hybrid alginate@TiO2 microcapsules with the pore size < 22 nm and the alginate/PDDAC with pore size ~ 30 nm prevented the probe diffusion by the size-selectivity of mesopores. The hybrid alginate@TiO2 microcapsules with smaller pore size show stronger influences on the probe diffusion. In consequence, our hybrid microcapsules with uniform mesopores are able to prevent the diffusion of molecules in which size is larger than 22 nm, demonstrating its potential ability for immune-isolation and applicability for in vivo cell therapy. Mechanical strength of the hybrid microcapsules. The mechanical strength of the microcapsules is crucial for the sustainability of the material in cell therapy. This property was firstly evaluated by mixing the microcapsules with various compositions into a PBS buffer supplemented with EDTA. This solution is supposed to reproduce conditions where alginate-based materials are progressively degraded because of the exchange of calcium ions by sodium ions. The alginate@TiO2 microcapsules synthesized with the TiBALDH concentration of 100 mM and 250 mM were compared to pure alginate microcapsules used as a positive control. The intact microcapsules were counted at several time intervals. The results are depicted in Figure 7. A rapid dissolution of pure alginate microcapsules was observed due to the dissociation of calcium alginate by sodium ions. After 1h incubation, almost no pure alginate microcapsules could be observed in the buffer, while 100 % of the hybrid alginate@TiO2 microcapsules still remained intact. After 48 h incubation, 52 % hybrid alginate@TiO2 (100 mM) microcapsules and 65 % hybrid alginate@TiO2 (250 mM) microcapsules remained intact, demonstrating that a denser titania layer formed by the


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precursor with a higher concentration showed an improved stability and mechanical strength. Furthermore, 44 % hybrid alginate@TiO2 (250 mM) microcapsules still presented a totallypreserved spherical morphology after 4 months (data not shown).

Figure 7. Mechanical strength of the hybrid microcapsules. Evaluation of the mechanical resistance of pure alginate microcapsules and hybrid alginate@TiO2 microcapsules synthesized with the TiBALDH concentration of 100 mM or 250 mM. The microcapsules were incubated in a 50 mM EDTA PBS solution and the number of intact microcapsules was counted over time. Results are expressed as means ± standard deviations for three independent measurements (n=3). The mechanical strength of microcapsules with different compositions was characterized by the measurement of Young’s modulus that gives information about the stiffness of an elastic material (Table 1). The results show that the deposition of a layer of PDDAC around purified alginate capsule improved the tightness of the material (+19 %) while it was not the case by simply mixing TiBALDH with alginate. When TiO2 layers were formed around alginate microcapsules via the reaction between TiBALDH and PDDAC, the rigidity of the



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microcapsules was significantly enhanced as an increase of 51% in the Young’s modulus, which evidenced that our hybrid alginate@TiO2 microcapsules possessed an enhanced stability and mechanical strength. Table 1. Determination of mechanical strength of microcapsules with different compositions. Composition of microcapsules

E (kPa)

Pure alginate

78

Alginate/TiBALDH

70

Alginate/PDDAC

93

Alginate@TiO2 (100 mM)

118

Alginate@TiO2 (250 mM)

118

Cell encapsulation of the hybrid microcapsules. Owing to the similarity in size and in shape with β-cells and the continuous secretion of albumin, HepG2 cell have been used as a model cell to be encapsulated in the hybrid alginate@TiO2 microcapsules towards the study on drug delivery or release of the hybrid microcapsules.


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Figure 8. Structure of the hybrid microcapsules with encapsulated HepG2 cells. (A) SEM micrograph of a cross section of an alginate@TiO2 microcapsule with encapsulated HepG2 cells. (B) SEM micrograph of the shell of an alginate@TiO2 microcapsule with encapsulated HepG2 cells. (C) SEM micrograph of the core of an alginate@TiO2 microcapsule with encapsulated HepG2 cells. (D) SEM micrograph of the encapsulated HepG2 cells. The microcapsule was supercritically dried with CO2 prior analyses. Figure 8A shows a cross section of a hybrid alginate@TiO2 capsule with encapsulated HepG2 cells, presenting the difference of structures between the shell and the core of the microcapsule, indicative of a core@shell structure. The structure of the shell (Figure 8B) and the core (Figure 8C) of the microcapsule with encapsulated cells is as similar as that of the microcapsules without cells (Figure 4D2, 4D3), which demonstrates that the cell encapsulation procedure did not influence the morphology and structure of the hybrid microcapsules and confirms the properties of the hybrid microcapsules analysed above could be applicative in the cell-laden case. Importantly, HepG2 cells encapsulated in the core of the microcapsule can be observed to have a well-preserved morphology (Figure 8D), evidencing the applicability of the microcapsules and the cell encapsulation procedure. Viability of the encapsulated HepG2 cells. The viability of the HepG2 cells encapsulated in the alginate or alginate@TiO2 microcapsules was investigated by Live/Dead viability probes and the consumption of oxygen. A fluorescent probe, consisting of calcein-AM and ethidium homodimer-1, was used to stain the encapsulated HepG2 cells. The metabolically active cells were stained with greenfluorescent calcein-AM by the active intracellular esterase, indicative of living cells, whereas the cells with damaged plasma membranes were stained with red-fluorescent ethidium homodimer-1, indicative of dead cells. The stained cells encapsulated within microcapsules were observed 2 hours after the encapsulation using CLSM. The cells entrapped within all 29 ACS Paragon Plus Environment


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microcapsules presented a high viability of more than 80 % (Figure 9A). The viability of the cells entrapped within the pure alginate microcapsules decreased to about 30 % after 1 week cultured in the medium, probably because the low porosity led to the insufficiency of the oxygen supplement, thus negatively influencing the entrapped cells. However, the viability of the cells encapsulated in our hybrid alginate@TiO2 microcapsules maintained the high viability of > 60 %, even though the cell death increased slightly (Figure 9B). Some nucleic acids released from the damaged cells were bound on the shell of microcapsules by the positive charged PDDAC and reacted with ethidium homodimer-1 to generate that red fluorescent shown as a red line in the shell of the hybrid microcapsules (Figure 9B). All these results evidence the high biocompatibility of our hybrid alginate@TiO2 microcapsules and its high applicability in cell encapsulation.


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Figure 9. Viability of the encapsulated HepG2 cells. (A) CLSM micrographs (brightfield, calcein-AM (green), ethidium homodimer-1 (red), emerged) of the viability kit stained HepG2 cells cultured 2 hours after the encapsulation. Green fluorescent cells are alive, while



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red fluorescent ones are dead. Scale bars, 500 µm. (B) CLSM micrographs (brightfield, calcein-AM (green), ethidium homodimer-1 (red), emerged) of the viability kit stained HepG2 cells cultured 1 week after the encapsulation. Green fluorescent cells are alive, while red fluorescent ones are dead. Scale bars, 500 µm. (C) Oxygen consumption of the HepG2 cells encapsulated in pure alginate microcapsules and alginate@TiO2 microcapsules synthesized with the TiBALDH concentration of 100 mM and 250 mM as a function of time. 100 % corresponds to the oxygen consumption of the cells entrapped within the microcapsule after the encapsulation (2.3*102 µmol of O2 hour-1 mirocapsule-1). Results are expressed as means ± standard deviations for three independent measurements (n=3). The consumption of oxygen by the encapsulated HepG2 cells, refleting directly their metabolic acitivity, was measured by a Clark’s electrode. Figure 9C shows that the metabolic acitivity of the cells encapsulated within our hybrid microcapsules can be maintained over 43 days as the relative respiration increased above 100 %. The oxygen consumption by the cells encapsulated in our hybrid microcapsules was higher than the consumption by the cells encapsulated in the pure alginate microcapsules, which indicates the high biocompatibility of our hybrid microcapsules. The results are in accordance with the viability obtained using CLSM (Figure 9B). A rapid increase in the value was observed in the microcapsules probably due to cell proliferation. These results also indicate the high biocompatiblity of our hybrid microcapsules for the long-term activity of the encapsulated HepG2 cells, making the hybrid microcapsules a promising cell reservoir for cell therapy. Secretion of the hybrid microcapsules with encapsulated HepG2 cells. In order to confirm the possibility of the therapeutic agent release towards cell therapy, the secretion of the human albumin from the microcapsules with encapsulated HepG2 cells has been studied. Every three or four days, the medium was collected and replaced by fresh medium so that the results can be expressed as a daily concentration release, presented in Figure 10. The 32 ACS Paragon Plus Environment

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molecule weight of the albumin is around 65 kDa. The albumin was detected in the medium, confirming not only the viability of the encapsulated HepG2 cell for the generation of albumin but also the porosity of the hybrid microcapsules for the albumin release from the internal of the microcapsules into the medium. The daily secretion of the albumin increased with the incubation time, which indicated an increasing in activity of the encapsulated cells. A similar phenomenon has been stated by the oxygen consumption of HepG2 cells (Figure 9C). It is worth to note that the albumin quantity released from the hybrid microcapsules (250 mM) was more than that released from the hybrid microcapsules (100 mM), which could attribute to their different pore size. The pore size of the hybrid microcapsules (250 mM) was larger than the one of the hybrid microcapsules (100 mM) (Figure 5D), which contributed to an easier mass diffusion. In consequence, the results demonstrate that molecules as large as 65 kDa can easily diffuse outside of the hybrid microcapsules due to their adapted porosity. The hybrid microcapsules are thus adapted for the encapsulation of therapeutic cells. In future, they could become promising “artificial organs” which enable the recovery of severe diseases by the release of therapeutic agents.

Figure 10. Secretion of the hybrid microcapsules with encapsulated HepG2 cells. Amount of human albumin secreted by HepG2 cells encapsulated in hybrid alginate@TiO2 33 ACS Paragon Plus Environment


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microcapsules incubated in the medium. Concentrations show the daily albumin release from the microcapsules. Results are expressed as means ± standard deviations for three independent measurements (n=3). CONCLUSIONS The study presents the design of a novel hybrid microcapsule with improved properties for cell encapsulation approaches. It consists in the hybridization of biocompatible purified alginate microcapsules with a titania coating synthesised by the hydrolysis and condensation of an aqueous stable precursor (TiBALDH) induced by a long chain quaternary ammonium polymer (PDDAC). This synthesis is performed by a facile and rapid one-step process under mild and biocompatible condition. The hybrid alginate@TiO2 microcapsules demonstrated a high porosity, allowing the diffusion of nutrients and metabolites and protecting the encapsulated cells from the attack of the host immune system by their uniform mesoporous pores. In addition, the hybrid microcapsules also revealed significantly improved mechanical properties compared to pure alginate microcapsule, which contributed to their high stability for long-term applications. Most importantly, the synthesis procedure and the formed hybrid microcapsules showed high biocompatibility towards human model HepG2 cells. The HepG2 cells encapsulated in the hybrid alginate@TiO2 microcapsules revealed long-term viability, high activity and preserved functionality such as diffusion of molecules of interest like albumin outside of the microcapsules, which make it an ideal cell reservoir to encapsulate therapeutic cells to treat several human diseases or disorders such as type 1 diabetes mellitus and to substitute the radical and delicate organ transplantation. The present work shows that our alginate@TiO2 microcapsules give a pioneering inspiration of hybrid hydrogels for its application in cell encapsulation towards cell therapy.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. XRD pattern of the TiO2, Figure S1; cytotoxicity of TiBALDH on HepG2 cells after 24 hours of incubation, Figure S2; cytotoxicity of PDDAC, TiO2 nanoparticles and ammonium lactate on HepG2 cells after 5 hours of incubation, Figure S3; chemical kinetics of the synthesis of titania in the formation of hybrid microcapsules, Figure S4; Mass diffusion of the microcapsules with different compositions, Figure S5. (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (L. Wang) *E-mail: [email protected] (B. L. Su) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R52) and “Algae Factory” (1610187) European H2020 program financed by FEDER and Wallonia Region of Belgium. B. L. Su acknowledges the Chinese Central Government for an “Expert of the State” position in the Program of the “Thousand Talents”, and the Chinese Ministry of Education for a “Changjiang Chair Professor” position. G. Leroux thanks the University of Namur for his assistant position to realize his PhD research. We are grateful to Dr. Marie-Ève Duprez of the University of Mons for the



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measurements of the Young’s modulus of the microcapsules. We thank Dr. Jun Jin and Prof. Yu Li from the State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of technology, Wuhan, China for their help. REFERENCES 1.

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Table of Contents Graphic


Hybrid Alginate@TiO2 Porous Microcapsules as a Reservoir of Animal

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