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Cross-reactive alginate derivatives for the production of dual ionic-covalent hydrogel microspheres presenting tunable properties for cell microencapsulation Luca Szabó, Carmen Gonelle-Gispert, Elisa Montanari, François Noverraz, Aurélien Bornet, Léo Bühler, and Sandrine Gerber-Lemaire ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00139 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 5, 2019

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ACS Applied Polymer Materials

Cross-reactive alginate derivatives for the production of dual ionic-covalent hydrogel

microspheres

presenting

tunable

properties

for

cell

microencapsulation

Luca Szabó,1 Carmen Gonelle-Gispert,2 Elisa Montanari,2 François Noverraz,1 Aurélien Bornet,3 Léo H. Bühler, Sandrine Gerber-Lemaire1*

1Institute

of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne,

Group for Functionalized Biomaterials, EPFL SB ISIC SCI-SB-SG, Station 6, CH-1015 Lausanne, Switzerland 2University

Hospital of Geneva, Surgical Research Unit, CMU-1, CH-1211 Geneva,

Switzerland 3Institute

of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne,

NMR service, Batochime, CH-1015 Lausanne, Switzerland

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KEYWORDS. Alginate derivatives; Cell microencapsulation; Cross-reactive polymers; Durability; Hybrid hydrogels; In vivo stability; Shape recovery performance.

ABSTRACT The production of hydrogel micropsheres (MS) presenting physical, mechanical and biological properties which can be modulated by their chemical composition is required to enlarge the panel of biomaterials for cell transplantation therapies. Functionalization of sodium alginate (Na-alg) with cross-reactive poly(ethylene glycol) (PEG) derivatives presenting terminal thiol and carbon electrophile functionalities provided novel polymers which upon simple one-step protocol formed hydrogel MS assembled by combination of Ca-alg interactions and sulfur-carbon covalent bonds. Several parameters such as the grafting degree on the alg backbone and the viscosity of the polymer solutions can be adjusted to provide optimal formulation for the capsule formation technology. Compared with pure Ca-alg MS, dual ionic-covalent MS 2 ACS Paragon Plus Environment

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displayed improved mechanical resistance and shape recovery performance. Importantly, under conditions which resulted in complete liquefaction of Ca-alg MS, chemically crosslinked Alg-PEG MS maintained stable spherical morphology. In addition,

these

hydrogels

allowed

excellent

viability

and

functionality

of

microencapsulated Huh7 cells. After transplantation in the peritoneal cavity of immune competent mice, the dual ionic-covalent MS remained free-floating and maintained their integrity over 30 days.

INTRODUCTION Following the pioneering work of Bisceglie on the immobilization of insulin producing cells1 and the concept of "artificial cells" introduced in 1964 by Chang,2 a wide range of biomaterials has been investigated for the immuno-isolation and transplantation of endocrine cells. The microencapsulation of cells into three-dimensional semipermeable hydrogels, which ensure protection from adverse immune response while supporting bi-directional diffusion of nutrients, oxygen, metabolic products and cell wastes, has indeed emerged as a promising strategy for cell-based therapies.3 While many natural and synthetic polymers were investigated for the engineering of 3 ACS Paragon Plus Environment


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hydrogels intended for cell immobilization, the natural polysaccharide sodium alginate (Na-alg)

remains

by

far

the

most

widely

reported

biomaterial

for

cell

microencapsulation due to its favorable gelling properties in contact with divalent cations such as Ca2+ and Ba2+, under mild conditions of pH and temperature.4 Other advantageous properties of hydrogels prepared from Na-alg include high biocompatibility toward many human and xenogeneic cells, promising in vivo performance and the presence of carboxylic and hydroxyl groups amenable to chemical modifications on the polysaccharide backbone.5-9 However, the reversible ionic crosslinking process which makes use of divalent cations to form bridges between adjacent polymer chains is responsible for the weakness of the resulting gels in vitro and in vivo, and the loss of mechanical integrity over time.10 Such hydrogels can be dissolved in case chelators such as phosphate, lactate, citrate and non-gelling cations are present above a certain concentration in the environment. In addition, alg-based microspheres (MS) generally suffer from poor shape recovery performance leading to undesired capsule breakage.11,12 Stabilization of alg hydrogel beads by polyelectrolyte complexation with polycations such as poly(L-lysine), poly(L-ornithine) and poly(Lguanidine) has been implemented,13 even in clinical trials. However, this modification 4 ACS Paragon Plus Environment

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resulted in poor mechanical strength of the resulting MS as well as low in vivo biocompatibility and long-term durability.14 Alternative approaches consist in the substitution of the ionic interactions by covalent crosslinks which can resist to a larger range of conditions or in the combination of both electrostatic and covalent interactions within the same hydrogel matrix. The development of covalently crosslinked hydrogels for cell microencapsulation requires numerous criteria15 as the process of hydrogel formation should occur in aqueous media, preserve cell viability and functionality, avoid the use of harsh conditions and toxic components and produce gels of defined shape and morphology.16 The general route toward chemically crosslinked or dual ioniccovalent alg hydrogels involves the functionalization of part of the carboxylic groups by esterification or formation of amide linkage to introduce functionalities that are further conjugated to bridge the adjacent alg chains.17 A widely reported method relies on the photo-induced linking of methacrylated alginate derivatives using short exposure to UV18-20 or green light.21 Similarly, complementary cross-reactive functionalities can be introduced on the alg backbone for further conjugation through furan-maleimide22,23 or tetrazine-norbornene24 Diels-Alder cycloadditions, copper catalyzed azide-alkyne [3+2] cycloadditions25 and disulfide bridge formation.26,27 Controlled oxidation of Na5 ACS Paragon Plus Environment


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alg followed by crosslinking with gelatin through imine formation was also reported to produce degradable microcapsules delivering osteoblast-like cells.28 Other strategies were based on the formation of covalently crosslinked coatings to improve the properties of alg-poly(L-lysine)-alg MS.29,30 Condensation of low molecular weight poly(L-lysine) with electrophile-containing polyanion within the core of Ca-alg MS provided spherical cross-linked hydrogel matrices presenting higher stability in the presence of non-gelling ions than pure Ca-alg MS.31 Several of these approaches involve the use of light trigger or chemical crosslinking additives, and reduce the number of carboxylic groups on the alg backbone available for ionic interactions. In order to benefit from the fast gelation property of all carboxylic moieties of alg while improving both the hydrogel stability and elasticity by covalent crosslinking, we recently established a protocol to functionalize the hydroxyl groups of Na-alg with crossreactive poly(ethylene glycol) (PEG) derivatives equipped with end thiol or 1,2dithiolane moieties.32,33 These polymeric materials were suitable for the encapsulation of human cells and mouse insulinoma MIN6 cells in spherical MS, and for transplantation in animal models. Notably, this strategy allowed for the covalent conjugation of ketoprofen within the hydrogel matrix and subsequent controlled release in vivo to reduce the incidence of pericapsular fibrotic overgrowth after transplantation.34 6 ACS Paragon Plus Environment

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The present study proposes to modulate the mechanical properties and durability of ionically cross-linked alg MS by exploring different types of Michael additions (1,4-addition reactions) involving thiol and carbon electrophiles as complementary covalent crosslinking within the hydrogel network. First, we prepared cross-reactive PEG-functionalized alg polymers at different viscosity ranges. Then, these derivatives were combined to produce dual ioniccovalent MS presenting tunable physical properties depending on the covalent linkage which was spontaneously formed in the gelation bath. Finally, the resulting MS were evaluated in vitro for their cell compatibility with Huh 7 cells and in vivo for their stability using male C57BL/6 mice.

EXPERIMENTAL SECTION Materials and methods. Na-alg Kelton HV (lot no. 61650A, [η] = 813 mL g−1 in 0.1 M NaCl, T = 25 °C, G/M = 0.6) was obtained from Kelco (San Diego, USA, CA). Linear PEG starting materials were purchased from Biochempeg (PEG-MAL, MM = 1000 g.mol-1 and PEG-ACR, MM = 2000 g.mol-1) and Sigma (PEG-SH, MM = 2000 g.mol-1). Other reagent-grade solvents (Fluka, Riedel-de-Haën) and chemicals (Aldrich, Acros, Fluka, Sigma, Maybridge, TCI Chemicals, Apollo and Fluorochem) were used without further purification. All reactions were performed in flame-dried glassware under an inert atmosphere of argon. NMR spectra were recorded on Bruker Avance III-400, Bruker Avance-400 or Bruker DRX-400 spectrometers (Bruker, Billerica, MA, USA) at room temperature (rt), unless otherwise stated. 1H frequency is at 400.13 MHz, 13C frequency is at 7 ACS Paragon Plus Environment


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100.62 MHz. Chemical shifts are reported downfield from tetramethylsilane. 1H and 13C signals are reported in ppm and the resonance multiplicity is described as s (singulet), d (doublet), t (triplet), m (multiplet). Coupling constants (J) are given in hertz (Hz). Solvent used for NMR spectroscopy was deuterated water (D2O, Sigma-Aldrich).

General procedure for the functionalization of TBA-alg with PEG-SH, PEG-ACR and PEG-MAL. For the preparation of lower viscosity Alg-PEG polymers, the procedure was adapted from ref. 33 using TBA-Alg (100 mg), 1,1'-Carbonyldiimidazole (CDI) (39 mg, 1.0 equiv.) and PEG derivatives (PEG-SH, PEG-ACR, PEG-MAL) (0.10 or 0.20 equiv. 0.0239 mmol or 0.0478 mmol). In case of PEG-SH which standed as HCl salt, 1 equiv. of NaHCO3 was added. For the preparation of polymers reaching higher viscosity in aqueous solution, acetic acid (1.0 equiv, 0.239 mmol, 0.014 mg, 0.015 mL) was added prior to the introduction of CDI. Purification of Alg-PEG polymers was performed by dialysis against distilled water for 3 days (dialysis membrane cut-off of 14 kDa) with addition of NaHCO3 (180 mg in 9 L of distilled water) in the dialysis bucket during the last 3 water changes. The pH value was controlled at 7.0. Alg-SH, Alg-ACR or Alg-MAL were obtained as white solids after freeze-drying. 1H-NMR

(400 MHz, D2O) for Alg-SH: δ 4.19 – 3.52 (m, CH-Alg + CH2-CH2-O), 3.23 (t, J =

5.0 Hz, 2H, CH2-SH), 2.97 (t, J = 6.1 Hz 2H, CH2-NH-C(O)-O) (1H NMR spectrum: Supporting Information, Figure S1). 1H-NMR (400 MHz, D2O) for Alg-ACR: δ 6.47 (dd, J = 17.3, 1.0 Hz 1H, CH2=CH-O), 6.24 (dd, J = 17.3, 10.5 Hz 1H, CH2=CH-O), 6.02 (dd, J = 10.7, 0.9 Hz, 1H, 8 ACS Paragon Plus Environment

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CH2=CH-O), 4.37 – 4.36 (m, 2H, CH2-O-C(O)), 4.20-3.33(m, CH-Alg + CH2-CH2-O), 3.23 (t, J = 4.9 Hz, 2H, CH2-NH) (1H NMR spectrum: Supporting Information, Figure S2; DOSY NMR spectrum: Supporting Information, Figure S4). 1H-NMR (400 MHz, D2O) for Alg-MAL: δ 6.88 (s, 2H, 2x =CH), 4.20 – 3.56 (m, CH-Alg + CH2-CH2-O), 3.39– 3.32 (m, 2H, CH2-NH), 2.562.52 (m, 2H, CH2-C(O)-NH) (1H NMR spectrum: Supporting Information, Figure S3).

Determination of the degree of grafting. 1D 1H-NMR of Alg-PEG derivatives (Alg-PEGSH, Alg-PEG-ACR or Alg-PEG-MAL) was run at 800 MHz with a repetition delay of 5s as 1H longitudinal relaxation time constants (T ) of PEG materials (PEG-SH, PEG-ACR or PEG1

MAL) and alginate were measured to be T1 < 1s. Line broadening was set to 0.3 Hz. The baseline was automatically corrected with Bruker Topspin routine, using degree 5 polynomial. The percentage of grafting was estimated by comparison of integration of 1H resonances of PEG fragment (176, respectively 88 protons) and alginate unit (5 protons). Due to overlap, separation between PEG and alginate resonances was performed by deconvolution with Lorentzian functions, using Bruker Topspin curve fitting and line shape analysis tool.

Formation of dual ionic-covalent MS. Cross-reactive Alg-PEG polymers were dissolved in a solution of 100 mM MOPS buffer, pH 7.4, containing 0.4% NaCl, at the concentration indicated in Table 1. The extrusion protocol was adapted from previous work33,34 using a coaxial air-flow droplet generator (Encapsulator B-395 Pro, Büchi Labortechnik AG, Flawil, Switzerland) and CaCl2 (100 mM in MOPS) as ionic crosslinker. For the evaluation of physical properties over time, the MS were stored in the gelation bath. 9 ACS Paragon Plus Environment


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Physical characterization of microspheres. The average diameter (Table 2) of the MS was measured on a batch of thirty MS per polymeric composition using an Olympus AX70 microscope equipped with an Olympus DP70 color digital camera. The mechanical resistance to 90% compression of the initial MS diameter (Table 2) was analyzed using a texture analyzer with a protocol adapted from ref. 33 on ten MS of each polymeric composition.

Permeability measurements. Alg-PEG MS and control glassbeads were incubated for 36 h with fluorescein isothiocyanate (FITC)‐labeled dextran (0.033 mg/mL, MOPS buffer, pH 7.4) of 40, 150, 250 and 500 kDa molecular weights. The MS were then imaged with a confocal microscope (LSM 510 meta ConfoCor3, Zeiss). Permeability of FITC‐dextrans within the MS was quantified using ImageJ software.

Microencapsulation of Huh7 cells. A solution of cross-reactive polymers (1 mL, Alg-SH:AlgACR or Alg-SH:Alg-MAL) containing Huh 7 cells (5.106) previously cultured in Dulbecco Modified Eagle’s Medium (DMEM) complete medium and centrifuged at 250 g for 5 minutes was placed in a 10 mL syringe. The resulting cell suspension was extruded using the protocol described above for the production of empty dual ionic-covalent MS.

Quantification of cell viability and albumin secretion capacity of microencapsulated Huh7 cells. Viability and cell death of microencapsulated cells (0.2 106 cells/1 mL/24-well plate) were quantified using the standard protocol previously described.35 Stainers for live and dead cells were fluorescein diacetate (FDA) and propidium iodide (PI), respectively. Quantification of cell viability was performed at day 1, 4 and 7 after microencapsulation using ImageJ and expressed as a percentage.


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Microencapsulated Huh7 cells (0.2 × 106) were seeded in 1 mL complete culture medium in a 24-well Corning Primaria Cell Culture Multiwell Plate (Fisher Scientific, Hampton, NH, USA). Albumin secretion was measured in 24 hour-supernatants at day 4 and 7. Supernatants were frozen until albumin was measured using a 100% specific human albumin ELISA kit (Abcam, Cambridge, UK) applying manufacturer’s instructions.

Transplantation of MS in mice. Transplantation of empty dual ionic-covalent MS was performed 3 days after microbeads preparation. Using a protocol adapted from previous study,34 around 12’700 MS from each polymeric composition (Alg-SH:Alg-ACR or Alg-SH:Alg-MAL) dispersed in Hank’s Balanced Salt Solution (HBSS) were transplanted in the peritoneal cavity of male C57BL/6 mice anesthetized by isoflurane. The experiment was performed in 4 replicates. All animal experiments were approved by the Geneva veterinary authorities (license GE/151/16).

RESULTS AND DISCUSSION Preparation of cross-reactive Alg-PEG derivatives. Having previously established the conjugation of end-functionalized PEG derivatives to the hydroxyl moieties in C3 position of the mannuronic residues and C2 position of the guluronic residues of the alg backbone,33 we envisaged that dual ionic-covalent hydrogels could result from the combination of PEG-grafted alginate polymers presenting complementary thiol and carbon electrophile functionalities. Cross-reactive alginate derivatives were prepared



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by activation of tetrabutyl ammonium (TBA)-alg with 1,1'-carbodiimidazole (CDI), followed by coupling with either PEG-SH (MM = 2000 g.mol-1, 0.2 eq), PEG-ACR (MM = 2000 g.mol-1, 0.1 eq) or PEG-MAL (MM = 1000 g.mol-1, 0.2 eq) (Scheme 1).

Scheme 1. Preparation of Alg-PEG derivatives.

The amount of PEG derivative was adjusted to produce Alg-PEG polymers (Alg-SH, Alg-ACR or Alg-MAL) presenting grafting degrees comprised between 25 and 30% (Table 1). We hypothesized that the in situ formation of imidazole during the activation of alg hydroxyl groups could be responsible for partial degradation of the alginate backbone chain. The addition of 1 eq. of acetic acid to neutralize the generated imidazole during the activation step resulted in Alg-PEG polymers presenting higher viscosities in aqueous solution than the ones produced without addition of acetic acid (Table 1). Chain degradation during the functionalization 12 ACS Paragon Plus Environment

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procedure can thus be tuned by the addition of acetic acid to provide grafted alg derivatives with controlled viscosities in aqueous solution.

Table 1. Characteristics for Alg-PEG Derivatives Product

Concentration

Viscosity Viscosity Degree (mPa.s)a (mPa.s)b of graftingc (%) 72 554 29

Alg-SH

3 wt %

Alg-ACR

3 wt %

57

654

26

Alg-MAL

3 wt %

201

749

28

aReaction

was performed without addition of AcOH; viscosity measured in deionized water at 22°C. bReaction was performed with the addition of 1 equiv. of AcOH; viscosity measured in deionized water at 22°C. cEstablished from 1H-NMR spectra. The expected chemical structures of Alg-PEG polymers were confirmed by NMR spectroscopy. The 1H NMR spectra displayed characteristic signals related to the end-functionalities of the PEG chains: 3.23 (t) ppm for CH2 linked to thiol in Alg-SH (Supporting Information, Figure S1); 6.47 (dd), 6.24 (dd) and 6.02 (dd) ppm for olefinic protons in Alg-ACR (Supporting Information, Figure S2); 6.88 (s) ppm for olefinic protons in Alg-MAL (Supporting Information, Figure S3). Diffusion Ordered Spectroscopy (DOSY) was performed to give evidence for the covalent conjugation of the PEG derivatives to the alginate backbone (Supporting Information, Figure S4). Determination of the grafting degrees was established from 1D 1H-NMR spectra of Alg-PEG polymers run at 800 MHz, by comparing the integration of 1H resonances of PEG fragments and alginate unit. Due to peak overlap, separation between these resonances was performed by deconvolution (Figure 1). Deconvoluted spectra were



Page 14 of 44

obtained by superimposition of the recorded 1H spectra of Alg-PEG derivatives with simulated spectra obtained with a sum of Lorentzian functions (Supporting Information, Figure S5).

Figure 1. Deconvolution spectra of Alg-PEG derivatives. Deconvolution spectra decomposed in regions interpreted as part of alginate signal contribution (blue), aliphatic peak of PEG including 13C satellites (black) and other functional groups belonging to the different components (green) that have to be subtracted from the alginate contribution. Microsphere formation and characterization. Cross-reactive Alg-PEG derivatives (1:1 AlgSH:Alg-ACR and 1:1 Alg-SH:Alg-MAL) were dissolved in aqueous 3-(N-morpholino) 14 ACS Paragon Plus Environment

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propanesulfonic acid (MOPS) (100 mM, pH = 7.4) containing 0.4% NaCl, at an initial polymer concentration of 3 wt%. The resulting solutions (2 mL) were extruded into a gelation bath containing CaCl2 as ionic crosslinker to produce MS by concomitant formation of ionic interactions and covalent sulfur-carbon bonds resulting from Michael addition of thiol to acrylate or maleimide moieties (Figure 2). The MS were assessed for their size, mechanical resistance to compression (Table 2), elasticity/shape recovery and chemical stability in sodium citrate solutions, at different time points over one week. For comparison, MS were formed from pure Na-alg (3 wt % in MOPS) under the same conditions. Other molar ratios (1:2 and 2:1) of starting Alg-PEG derivatives were attempted but led to large size distribution of the resulting MS and deviations from spherical morphology (data not shown).

Figure 2. Bright-field microscopy images of Ca-alg MS (A), Alg-SH:Alg-ACR MS (B) and Alg-SH:Alg-MAL MS (C). MS were produced from polymer solutions at 3 wt % concentration. Images were retrieved on the day of MS production (scale bar 1.0 mm). As presented in Table 2, the diameter of dual ionic-covalent MS is regularly decreasing over time while their mechanical resistance upon compression to 90% of their initial diameter is increasing (from 1.29 at day 0 to 1.74 N/mm3 at day 7 for Alg-SH:Alg-ACR MS; from 1.40 at day 0 to 2.68 N/mm3 at day 7 for Alg-SH:Alg-MAL MS). These observations are consistent with the formation of the covalent network which occurs at a slower rate than the initial and fast ionic gelation process. With an increasing number of sulfur-carbon bonds the MS network 15 ACS Paragon Plus Environment


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becomes more dense and rigid, resulting in higher mechanical resistance. The nature of the chemical bond influences the mechanical properties of the dual ionic-covalent network as AlgSH:Alg-MAL MS show the highest resistance to uniaxial compression. Upon storage in the gelation bath, pure Ca-alg MS do not show significant change of their diameter over one week. After an initial increase over the first 3 days, Ca-alg MS undergo a decrease of their mechanical resistance upon longer storage time. This observation is consistent with previous report on the properties of Ca-alg MS produced from a variety of commercially available alginate polymers and incubated in different storage media.11 Variations observed on Ca-alg MS during the first days of storage can be attributed to reordering of the gelled network before stabilization of the system in the incubation medium. Interestingly, after prolonged storage (14 days), the size and mechanical resistance of dual ionic-covalent MS remain stable (data comparable to the ones measured after 7 days), while Ca-alg MS present decreased mechanical resistance (1.25 N/mm3).

Table 2. Size and Mechanical Resistance to Uniaxial Compression of MS

Day 0

Day 3

Day 7

1006 ± 114

Alg-SH: Alg-ACR 1158 ± 125

Alg-SH: Alg-MAL 1174 ± 149

Resistanceb (N/mm3)

1.51 ± 0.36

1.29 ± 0.10

1.40 ± 0.23

d3a (m)

968 ± 59

1130 ± 71

1080 ± 115

Resistanceb (N/mm3)

2.00 ± 0.56

1.60 ± 0.11

1.91 ± 0.25

d7a (m)

976 ± 69

1068 ± 89

1050 ± 125

Resistanceb (N/mm3)

1.55 ± 0.40

1.74 ± 0.15

2.68 ± 0.12

Characteristics

Ca-alg

d0a (m)

aMeasured

with an Olympus AX microscope equipped with Olympus DP70 color digital camera. Results are the means ± SD of independent measurements on 30 MS. bMechanical resistance to uniaxial compression to 90% of the initial MS diameter, volume-normalized, measured on a texture analyzer. Results are the means ± SD of independent measurements on 10 MS. 16 ACS Paragon Plus Environment

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The shape recovery performance of Alg-SH:Alg-ACR, Alg-SH:Alg-MAL and Ca-alg MS was evaluated by applying 10 successive compressions to 90% of the initial MS diameters, at day 0, day 3 and day 7 after the production of the MS (Figure 3).

Figure 3. Resistance to 10 successive compressions of Ca-alg MS, Alg-SH:Alg-ACR MS and Alg-SH:Alg-MAL MS, measured at day 0 (A), day 3 (B) and day 7(C). Error bars represent the standard deviation from n = 10 measurements. 17 ACS Paragon Plus Environment


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As previously reported,11 Ca-alg MS show moderate elasticity (20% shape recovery after 10 compressions), with no significant evolution over one week of storage in MOPS solution. Interestingly, the recovery performance of the dual ionic-covalent Alg-SH:AlgACR and Alg-SH:Alg-MAL MS improves over time and reaches 40% shape recovery after one week. This evolution is attributed to the formation of the covalent crosslinkings which occur at a slower rate than the ionic interactions. After 7 days, no significant evolution of these properties was observed (data not shown at day 14), indicating that formation of the dual network of electrostatic and covalent crosslinkings reached completion. Exchange of calcium ions with monovalent non-gelling ions such as sodium and potassium affects the integrity of Ca-alg MS, which can eventually lead to complete dissolution of the capsules.36 Ca-alg MS and dual ionic-covalent MS were exposed to increasing concentrations of sodium citrate to assess the ability of the covalent network to maintain stable and spherical shape in the presence of non-gelling ions (Table 3).

Table 3. Stability of MS upon exposure to sodium citrate. 18 ACS Paragon Plus Environment

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sodium citrate 5 mM

10 mM

20 mM

30 mM

40 mM

Ca-alg MS

Stable

Stable

Stable

Alg-SH:Alg-ACR MS

Stable

Stable

Stable

Stable

Stable

Alg-SH:Alg-MAL MS

Stable

Stable

Stable

Stable

Stable

concentrationa

aAfter

Dissolved Dissolved

storage in the gelation bath for 7 days, MS were recovered by filtration (70 m)

and immersed in aqueous solution of sodium citrate at the indicated concentrations for 24 hours. Stability was evaluated by observation of the MS under an Olympus AX70 microscope.

As observed in previous studies,37 when exposed to monovalent non-gelling ions, the limited stability of Ca-alg MS became visible as dissolution of the caspules occurred for concentrations above 20 mM. In contrast, Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS remained homogeneous and spherical

at all concentrations tested. The

liquefaction of the Ca-alg ionic interactions did not affect the diameter of Alg-SH:AlgACR MS, which remained constant from 5 to 40 mM (Supporting Information, Figure S6). The average diameter of Alg-SH:Alg-MAL MS increased by 45% upon exposure to increasing concentration of sodium citrate from 5 to 20 mM. However, from 30 mM, the size of the MS remained stable. It was already observed that molecular weight and 19 ACS Paragon Plus Environment


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structure of PEG components can largely influence the swelling behavior of pure PEG hydrogels.38 In our study, the different molecular weights of PEG-MAL (MM = 1000 g.mol-1) and PEG-ACR (MM = 2000 g.mol-1) components as well as the chemical composition of the cross-linked polymer in the vicinity of the thiol-carbon covalent bond, seemed to be responsible for the different behavior of the dual ionic-covalent network in the presence of Na+ ions. Noteworthy, for both polymeric compositions, the spherical shape was maintained, highlighting the contribution of the covalent crosslinkings to reinforce the integrity of the MS toward exposure to non-gelling ions which are present under physiological conditions (i.e. in vivo).

Permeability. The permeability profiles of Alg-SH:Alg-ACR, Alg-SH:Alg-MAL and Caalg MS were compared to verify that the covalent crosslinkings did not affect the diffusion properties of the hydrogels. The MS were incubated with fluorescein isothiocyanate (FITC)-dextran standards (40 to 500 kDa molecular weights), in MOPS solution (100 mM, pH = 7.4). After 36 hours under gentle stirring, permeability of FITCdextrans within the MS was quantified on cross-sectional confocal images of the beads (triplicates) using pixel quantification with ImageJ (Figure 4).


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Figure 4. Relative permeability of FITC-dextrans within Alg-SH:Alg-ACR, Alg-SH:Alg-MAL and Ca-alg MS as a function of FITC-dextrans molecular weight after 36 hours of incubation under gentle stirring. Error bars represent the standard deviation from n = 3 measurements. Quantification was performed using ImageJ. The three systems presented permeability profiles similar to previously reported data for alginate-based MS.39 The higher infiltration capacity of Alg-SH:Alg-MAL MS suggests that this system could provide better diffusion of nutrients, oxygen and metabolic products than Alg-SH:Alg-ACR MS, in the context of cell microencapsulation.

Cell microencapsulation. The feasibility of cell microencapsulation in Alg-SH:Alg-ACR, and Alg-SH:Alg-MAL MS was confirmed using human hepatocellular carcinoma cells (Huh7). These cells were previously used by us and others to evaluate the cell compatibility26 and antiviral protective effect40 of alginate-based encapsulation 21 ACS Paragon Plus Environment


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hydrogels. Encapsulation of Huh7 cells, performed under physiological conditions by extrusion in a gelation bath containing CaCl2, resulted in homogeneous cell distribution within both MS types (Figure 5A). Cell viability, assessed by FDA/PI staining at days 1, 4 and 7 after encapsulation (Supporting Information, Figure S7), averaged more than 90% for both polymeric compositions (Figure 5B) and remained constant over extended time (staining at day 15 showed similar viabilities, data not shown).

Figure 5. Microencapsulation of Huh7 cells. (A) Representative images of Huh7 cells microencapsulated in Alg-SH:Alg-ACR, and Alg-SH:Alg-MAL MS at day 0; scale bar: 200 m. (B) Quantification of viable cells with fluorescein diacetate (FDA) and nonviable cells with propidium iodide (PI) at days 1, 4 and 7 after microencapsulation and culture. Values are expressed as % of the total cell area (n = 6). Quantification was performed using ImageJ on photographs of stained microencapsulated cells (representative photographs are given in supporting information, Figure S7).


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The functionality of microencapsulated Huh7 cells was assessed by quantification of albumin released in the culture medium at days 4 and 7 after encapsulation (Figure 6). Both polymeric composition allowed sustained secretion and diffusion of albumin. The amount of albumin secreted from Huh7 cells microencapsulated in Alg-SH:Alg-MAL MS was higher, which could be correlated with the higher permeability observed for this polymeric composition. These data suggest that Alg-SH:Alg-ACR and Alg-SH:AlgMAL hydrogels support both the viability and functionality of encapsulated Huh7 cells, the later composition providing a more favorable environment as illustrated by the higher secretion of albumin.

Figure 6. Quantification of the production albumin by Huh7 cells encapsulated within Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS at days 4 and 7. Values correspond to albumin released during a 24h-incubation by 0.2 106 encapsulated Huh7 cells placed in 1 ml.



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Transplantation of dual ionic-covalent MS. The in vivo compatibility of Alg-SH:Alg-ACR and Alg-SH:Alg-MAL hydrogels was assessed by transplantation of MS formed from both polymeric compositions in the peritoneal cavity of immune competent mice, with follow-up periods of 15 and 30 days. At both time points, the abdominal cavity of mice contained free-floating MS (Supporting Information, Figure S8), without any sign of inflammation or connective tissue formation. A representative amount of transplanted MS was retrieved (500 L) for examination by light microscopy (Figure 7). The integrity and sphericity of Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS was maintained over time, which confirmed the high stability of the hydrogel matrices composed of ionic interactions and carbon-sulfur covalent bonds between cross-reactive PEG chains.


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Figure 7. Representative images of Alg-SH:Alg-ACR, and Alg-SH:Alg-MAL MS retrieved from the peritoneal cavity of immune competent mice at days 15 and 30 post transplantation. Scale bar: 200 m.

CONCLUSION Despite great promise, the use of microencapsulated allo- and xenogeneic cells in clinical applications remains a challenge which is partly due to the lack of materials presenting optimal properties to support long-term cell functionality and in vivo durability. A strategy was developed to synthesize cross-reactive PEGylated alginate derivatives presenting controlled grafting degrees and tunable physical properties. In particular, the degree of degradation of the alginate backbone chain during the coupling reaction was modulated by the addition of 1 equiv. of AcOH to produce polymer solutions of various viscosities, which is a key parameter for the encapsulator settings. In comparison to pure Ca-alg MS, the MS obtained from the combination of Alg-SH with either Alg-ACR or Alg-MAL presented higher mechanical resistance to uniaxial compression and improved shape recovery performance upon multiple compressions. Using a simple one-step extrusion protocol, MS are formed by fast ionic Ca-alg interactions and slower covalent Michael addition (1,4-addition) of thiol to acrylate or thiol to maleimide, without the need for additional chemical crosslinker. The evolution 25 ACS Paragon Plus Environment


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of size, mechanical resistance and elasticity over time suggests that completion of the dual ionic-covalent network is achieved within 7 days. While Ca-alg MS suffer from rapid dissolution upon exposure to non-gelling ions above a certain concentration, both Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS maintained their spherical morphology in sodium citrate solutions (up to 40 mM concentration). The suitability of these systems for cell microencapsulation was demonstrated using Huh7 cells which showed excellent viability and albumin secretion capacity over two weeks. Interestingly, Huh7 cells encapsulated in Alg-SH:Alg-MAL MS produced higher amounts of albumin, which can be correlated with the higher permeability of this polymeric composition. After transplantation in immune competent mice, Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS remained free-floating in the peritoneal cavity, up to 30 days. Both MS types were easily retrieved, highlighting their good stability under physiological conditions. The production of spherical hydrogels which maintain the favorable properties of pure Caalg MS (cell biocompatibility, simple formation protocol) and improve their shortcomings, in particular their limited durability under physiological conditions and their lack of elasticity, provides a new basis for future therapies based on cell transplantation. Alg-SH:Alg-ACR and Alg-SH:Alg-MAL MS, which are easily formed by 26 ACS Paragon Plus Environment

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ionic interactions and spontaneous covalent crosslinkings, offer tunable physical properties, favorable cell encapsulation environment and in vivo stability.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publication website. 1H

NMR and DOSY NMR spectra (Figures S1-S4), superimposition of deconvoluted spectra

and raw 1H NMR data (Figure S5), stability in sodium citrate solutions (Figure S6), quantification of microencapsulated Huh7 cells (Figure S7), macroscopic inspection of AlgSH:Alg-ACR and Alg-SH:Alg-MAL MS after transplantation in immune competent mice (Figure S8).

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

Author contributions The manuscript was written through contributions of all authors. LS designed and performed the synthesis of alginate derivatives, prepared and characterized the crosslinked microspheres, contributed to the cell compatibility assessment. EM and 27 ACS Paragon Plus Environment


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CGG performed the cell encapsulation and transplantation experiments, and evaluated cell viability. FN contributed to the development of the grafting procedure on Na-alg. AB developed the deconvolution method to determine degrees of grafting by NMR spectroscopy. CGG, LHB and SG supervised the project, designed the experiments and prepared the manuscript. All authors have given approval to the final version of the manuscript.

Funding source The authors acknowledge financial support from the Swiss National Science Foundation (SNSF grants CR23I2_152974 and 310030E-164250), the Foundation Insuleman and the Enable program.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Dr Christine Wandrey for scientific discussions, Emilie Baudat and Anto Barisic for their technical support with NMR experiments, Dr Ruud Hovius for support with 28 ACS Paragon Plus Environment

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confocal microscopy as well as Joël Pimenta and Nadja Perriraz for excellent technical assistance.

ABBREVIATIONS ACR, acrylate; Alg, alginate; CDI, carbodiimidazole; DMEM, Dulbecco Modified Eagle’s Medium; DOSY, Diffusion Ordered Spectroscopy; FDA, fluorescein diacetate; HBSS, Hank’s Balanced Salt Solution; MAL, maleimide; MS, microsphere; MOPS, 3-(N-morpholino) propanesulfonic acid; Na-alg, sodium alginate; PEG, poly(ethylene glycol); PI, propidium iodide; ppm, parts per million; TBA, tetrabutyl ammonium; wt, weight.



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FOR TABLE OF CONTENTS ONLY



Preparation of Alg-PEG derivatives. 79x119mm (300 x 300 DPI)

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Deconvolution spectra of Alg-PEG derivatives. Deconvolution spectra decomposed in regions interpreted as part of alginate signal contribution (blue), aliphatic peak of PEG including 13C satellites (black) and other functional groups belonging to the different components (green) that have to be subtracted from the alginate contribution. 84x185mm (96 x 96 DPI)



Bright-field microscopy images of Ca-alg MS (A), Alg-SH:Alg-ACR MS (B) and Alg-SH:Alg-MAL MS (C). MS were produced from polymer solutions at 3 wt % concentration. Images were retrieved on the day of MS production (scale bar 1.0 mm). 94x72mm (96 x 96 DPI)


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Resistance to 10 successive compressions of Ca-alg MS, Alg-SH:Alg-ACR MS and Alg-SH:Alg-MAL MS, measured at day 0 (A), day 3 (B) and day 7(C). Error bars represent the standard deviation from n = 10 measurements. 106x215mm (96 x 96 DPI)



Relative permeability of FITC-dextrans within Alg-SH:Alg-ACR, Alg-SH:Alg-MAL and Ca-alg MS as a function of FITC-dextrans molecular weight after 36 hours of incubation under gentle stirring. Error bars represent the standard deviation from n = 3 measurements. Quantification was performed using ImageJ. 93x85mm (96 x 96 DPI)


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Microencapsulation of Huh7 cells. (A) Representative images of Huh7 cells microencapsulated in Alg-SH:AlgACR, and Alg-SH:Alg-MAL MS at day 0; scale bar: 200 m. (B) Quantification of viable cells with fluorescein diacetate (FDA) and nonviable cells with propidium iodide (PI) at days 1, 4 and 7 after microencapsulation and culture. Values are expressed as % of the total cell area (n = 6). Quantification was performed using ImageJ on photographs of stained microencapsulated cells (representative photographs are given in supporting information, Figure S7). 95x78mm (96 x 96 DPI)



Quantification of the production albumin by Huh7 cells encapsulated within Alg-SH:Alg-ACR and Alg-SH:AlgMAL MS at days 4 and 7. Values correspond to albumin released during a 24h-incubation by 0.2 106 encapsulated Huh7 cells placed in 1 ml. 103x79mm (96 x 96 DPI)


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Representative images of Alg-SH:Alg-ACR, and Alg-SH:Alg-MAL MS retrieved from the peritoneal cavity of immune competent mice at days 15 and 30 post transplantation. Scale bar: 200 μm. 133x103mm (96 x 96 DPI)



TOC graphic 111x64mm (96 x 96 DPI)


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Cross-reactive alginate derivatives for the ... - ACS Publications

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