OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: http://oatao.univ-toulouse.fr/25606 Official URL: https://doi.org/10.1016/j.colsurfb.2018.11.084 To cite this version: Conzatti, Guillaume and Cavalie, Sandrine and Gayet, Florence and Torrisani, Jérôme and Carrere, Nicolas and Tourrette, Audrey Elaboration of a thermosensitive smart biomaterial: From synthesis to the ex vivo bioadhesion evaluation. (2019) Colloids and Surfaces B Biointerfaces, 175. 91-97. ISSN 0927-7765 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Elaboration of a thermosensitive smart biomaterial: From synthesis to the ex vivo bioadhesion evaluation G. Conzatti\ S. Cavaliea, F. Gayet\ J. TorrisaniC, N. Carrèrec,d, A. Tourrettea,* • ClRIMAT, Un1"'1'Slti de Toulouse 3 • PaulSabalier, CNRS, Toulouse, Pranœ b LCC, Univenlli de Toulouse3 • Paul Sabatier, CNRS, Toulouse, Franœ • msERM, Universlti Toulouse3 • PaulSabatier, CentTe deRecherches en Canc.!rologie de Toulouse, Toulouse, Franœ d G<lstrolnttstlnal Swge,y dtpartmmt, Plllpon HospltJJl • CHU de Toulouse, Toulouse, France ART ICLE INFO ABSTR ACT Keywords: Alginate and chitosan are polysaccharides that are widely used in the biomedical field, especially as wound Polysaccltarldes dressings.Controlled bioadhesion is an advanced functionalitythat offersthe potential to reduœ injuries due to PEC the stripping-off of the biomaterial. Herein, we report the efficient grafting of poly•N(isopropylacryamide) PNIPAM (PNIPAM), a thermosensitive polymer that exhibits a lower critical solution temperature(LCST) at 32 •c onthe Smartpolymer alginate/chitosanpolyelectrolyte complex (PEC) surface. bt vitro studies did not exhibit a cytotoxic effect, and Controlledbioadheslcn cells adhered preferentially on the LCST on PNIPAM grafted surfaces, as reported in the literature. Ex vivo investigations revealed that the adhesive behavior of the biomaterials was not the same on the liver and pan· creas.The effect of the temperatureon the bioadhesionto organs was unexpected,as PNIPAM surfacesexhibited higher adhesion at low temperature.The PNIPAM was thereforeable to confer PEC matrix thermosensitivity, but due to the application force, interactions between PNIPAM chains and theirsubstrate could influence bioad· hesion on tissues. 1. Introduction sensitive polymers. [2,3) Poly N (isopropylacrylamide) (PNIPAM) is a widely studied thermosensitive polymer that changes its hydrophilicity Bioadhesion during wound dressing is an important part of bioma around 32 •c, called its lower critical solution temperature (LCST) terial science. Indeed, bioadhesion, defined as the ability of material to [4,5). Therefore, this is an interesting material, as the human body adhere to a biological entity, is required to achieve an effective healing. temperature is close to 37 •c. Once grafted, its ability to change from a However, a highly bioadhesive material could cause injuries and be passive state at room temperature to an active state at body tempera cou nterproductive. Thus, the manipulation of such materials during ture is depaident on chain length and graft density. Nonetheless, the surgery is a critical consideration, as it is sometimes neœssary to adjust difficulty in efficiently characterizing the surface state of polymer the material position precisely when applied to soft organs. scaffolds has oriented the use of PNIPAM for bioadhesion studies to In this context, a controlled bioadhesion would be a great way to ward the surface modification of well controlledsurfaces, such as glass obtain a balance between position holding and easy manipulation. [6) or gold [7). These surfaces are amenable to theoretical models [8). Bioadhesion is a complex phenomenon that involves proteins such as However, the development of biodegradable biomaterials that fibronectin that act as an anchor for the cells. (1) The ability of a sur would allow controlled bioadhesion is of interest when considering face to support cell adhesion is depaident on its ability to support implanted devices.Polysaccharides are a class of such polymers that are protein adsorption. Many factors have to be considered, such as the generally considered biocompatible. Among others, alginate and chit biomaterial stiffness,surface topographyand hydrophilicity.To control osan are relatively inexpensive biopolymers that are well tolerated by bioadhesion, it is necessary to control the interactions between cell the immune system. [9,10) Their combination, in the form of poly adhesion proteins and the biomaterial surface, i.e. the biomaterial electrolyte complexes (PECs), allows the production of biodegradable surface state. absorbent and resistant matrixes that are able to 1imit fluids, such as Numerous studies have been focused on smart biomaterials ob bleedings. They can also be used to disseminate or to release drugs tained by grafting stimuli responsive polymers, such as pH or thermo [11,12). Previously, we reported that calcium reticulated PECs showed •Corresponding author. E-maila ddress: Audrey.tourrette@univ•t1se3.fr (A. Tourrette). https://doi.org/10.1016/j.colsurfb.2018.11.084 an interesting resistance toward enzymes, along with a good balance 12,000 g/mol and dried under vacuum. between degradation and resistance in saline environment [12]. How The theoretical molecular weight, Mnth, of the polymer was calcu ever, only a few studies report the development of biodegradable lated with Eq. 1: wound dressings with thermo controlled bioadhesion by the grafting of []mono 0 PNIPAM on polysaccharide substrates [13]. To date, no studies are Mnth =××MMmono CTA []CTA 0 (1) devoted to the exploration of a thermosensitive PEC. Herein, the grafting of a functional PNIPAM COOH polymer in a where Mmono represents the molecular weight of the monomer, [mono]0 fi brush structure on alginate/chitosan PEC lm surfaces through NHS/ its initial concentration, %conv is the percentage of conversion of the EDC coupling was studied. To control the chain length of the grafted monomer (considered as 100%) and [CTA]0 the initial concentration of PNIPAM, it was synthesized by reversible addition fragmentation chain the CTA. transfer (RAFT) polymerization, a controlled radical polymerization, [4] and compared to a commercial polymer. The grafting strategy was 2.4. Characterization of the PNIPAM COOH also set to modulate the graft density of the PNIPAM brush. The re sulting films were characterized by X ray photoelectron spectroscopy The molar mass of the PNIPAM was measured by size exclusion (XPS) prior to characterization of cytotoxicity and thermo controlled chromatography (SEC) using DMF/LiBr (10 mM, refractive index of bioadhesion with human cells (in vitro) and organs (ex vivo). 1.431) as solvent. Before analysis, the samples were filtered through a 10 μm nylon filter. Detection was performed by the measurement of the 2. Materials and methods refractive index variation (Optilab rEX detector) combined to a light scattering analyzer (miniDawn, 690 nm). Mass determination was per 2.1. Materials formed using the ASTRA software with a dn/dc of PNIPAM in DMF equal to 0.0731 mL/g. [18] Sodium alginate, chitosan, calcium chloride, acetic acid, N The functionality of PNIPAM was determined by COOH pH metric Isopropylacrylamide (NIPAM), poly (N Isopropylacrylamide) (PNIPAM, titration of the polymer (0.5 g in 20 mL H2O) with NaOH (0.02 M). The − Mw = 5000 g.mol 1), N Hydroxysuccinimide (NHS), N (3 pH was measured using a 766 Calimatic pH meter (KCl electrode). Dimethylaminopropyl) N’ ethylcarbodiimide hydrochloride (EDC), ethylene diamine (EDA), Tetrahydrofuran (THF), dimethylsulfoxide 2.5. Surface functionalization (DMSO), Methyl 2 (dodecylthiocarbonothioylthio) 2 methylpropio nate (CTA) and 4.4′ Azobis(4 cyanovalericacid) (radical initiator, The grafts on the surface of the PECs were achieved through an ACVA), as well as all solvents, were provided by Sigma Aldrich. NHS/EDC coupling. To increase the reactivity of the PECs (PEC and The alginate and chitosan were characterized as previously re PEC EDA), an acidic treatment was performed by immersing the PECs ported. [12] The G/M unit ratio of alginate was estimated using a water in water (HCl, pH 2.5) for 1 h prior to any reaction. suppressive NMR method to M/G = 2.7 [14]. Viscosimetry determined its molecular weight to be 340 kDa, and α and κ parameters to be 1.13 2.5.1. Grafting of EDA on alginate/chitosan PECs and 6.9 × 10 − 4 mL.g 1, respectively. [15] Using the Shigemasa To increase the grafting density on the films, EDA was previously method, the degree of acetylation (DA) of the chitosan was estimated to grafted on the desired samples (PEC EDA). Briefly, PECs (30 g) were be 23% [16] with Mw = 1 300 kDa (α = 0.93, κ = 1.81 × 10 − 1 ml. immersed in DMSO (0.6 mL) containing NHS (100 mmol/L) and EDC g 1). [17] NIPAM was purified by hexane recrystallization prior to use. (200 mmol/L) for 3 h before EDA addition (0.6 mL, final concentration Other products were used in the as received state. of 7.2 mol/L). The mixture was left to react for 12 h. The mixture was slightly agitated on a gyratory rocker. Films were thoroughly rinsed 2.2. Alginate/chitosan PECs elaboration with EtOH and DMSO to remove any unreacted species. The procedure for the PEC elaboration was based on our previous 2.5.2. Grafting of PNIPAM COOH on alginate/chitosan PECs fi studies with minor modi cations. [11,12] The sodium alginate was The grafting of the PNIPAM COOH was performed both on EDA dissolved in water at a concentration of 1.5% w/v and mechanically grafted PECs (PEC EDA) and on native PECs (PEC). The PNIPAM stirred overnight (500 rpm). In parallel, the chitosan was dissolved in COOH (2 mmol/L) was activated for 3 h with NHS (25 mmol/L) and acidic water (acetic acid, 0.34% v/v) at a concentration of 1.5% w/v EDC (50 mmol/L) in DMSO (1 mL). PEC of PEC EDA (30 g) were added and mechanically stirred overnight (500 rpm).