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Antifungal vanillinimino-chitosan biodynameric lmsLuminita Marin, a Iuliana Stoica, a Mihai Mares, b Valentina Dinu, a Bogdan C. Simionescu ac and Mihail Barboiu * ad Vanillinchitosan biodynamers have been prepared and structuremorphology correlations revealed the pathway of progressive incorporation of the aldehyde onto chitosan backbones. Such dynamic biopolymers or biodynamers, generated from reversibly interacting components, oer the possibility to address the dynamic covalent behaviour of the reversible imine-bond formation/hydrolysis equilibria between vanillin and chitosan polymeric backbones. The reaction takes place with very low conversion in acidic aqueous solutions (712%), but the imine bond formation is amazingly improved (80%) when the reaction takes place while solutionsolid state transition and solid state phase-organization events occur. The chitosanvanillin biopolymeric lms described here present interesting Candida albicans antifungal activity compared with other common bacterial strands, which suggests the implementation of these biocompatible materials as thin layer protecting systems for medical devices. Introduction Due to its therapeutic, haemostatic, depressant, immuno-adju- vant and antitumor properties and its non-toxic behaviors 1,2 chitosan (C), the natural polymer obtained from deacetylation of chitin, has become a biomaterial of expanding interest in biomedical, cosmetics, food or agricultural applications. De novo design of synthetic chitosan-based materials as lms, 3 bers, 4 nanoparticles 5 and gels 6 further increases its applicative value. The main challenge in the large scale use of chitosan is its low solubility in water. To overcome this, dierent chitosan modi- cation reactions have been explored. 714 Among them, the reversible reaction of amino groups of chitosan with aldehydes bearing Schibasechitosan received much attention because the functional moieties, reversibly graed onto the chitosan backbone, disrupt the H-bonding network of chitosan, thus improving its solubility. 7 Moreover, it represents a simple synthetic strategy, yielding modied chitosan with improved antimicrobial, antifungal, antitumor activity or for the delivery of insoluble therapeutic agents, etc. 8,9 Ultrasound, 10 microwave, 11 plasma 12 or enzymatic 13 activated synthetic methods have been used to obtain Schibasechitosan derivatives. The yield of imine formation in water is very low, due to imine exchanging or hydrolysis reactions occur very fast in acidic aqueous solu- tions. 14,15 We previously answered this challenge by reporting 14 that the very low conversion degrees (112%) obtained in acidic aqueous solutions can be amazingly improved to 8090% if concentrated hydrogel/solid state irreversible vitrication processes occur or if aldehydes of low solubility react out of water, 15a when the components are in hydrophobic contact. With all these in mind, the further step was to implement these strategies to obtain such biodynamers 16 of high applicative interest. Within this context, our attention was attracted by vanillin (V), a natural product used as a food avoring agent, which presents improved antifungal properties when forming Schibases, 17a or incorporated onto chitosan as a vanillyl unit. 17b Moreover, recent studies demonstrated that vanillin is an e- cient inhibitor of cancer cell migration and metastasis in a mouse model. 17c One should expect that these premises promise the chitosanvanillin imine (CV) biopolymers to be a potentially valuable system for dierent applications. In the present work, chitosanvanillin Schi-base biopolymers were synthesized via imine-bond formation between chitosan and vanillin at dierent molar ratios (C 0.5 V, CV, C 1.5 V, C 2 V, C 3 V, C 4 V, C 5 V), in acidic wateracetone mixtures, in toluene used as a non-solvent for both components (CVT) or by grinding in the absence of solvents (CVM) (Scheme 1). Two aspects were preferentially followed: (1) the chemical investigation of the reaction evolution by spectro- scopic and morphological analyses of resulted materials and (2) the antibacterial and antifungal properties of the synthesized biodynameric lms. We have tested these materials against the most common bacterial agents, while they present interesting activity against Candida albicans fungus strain. a Petru PoniInstitute of Macromolecular Chemistry of Romanian Academy, 41A, Aleea Gr. Ghica Voda, Iasi, Romania b Ion Ionescu de la BradUniversity, Laboratory of Antimicrobial Chemotherapy, 8, AleeaSadoveanu, Iasi, Romania c Gheorghe AsachiTechnical University of Iasi, 73, Bd. Prof. Dimitrie Mangeron, Iasi, Romania d Institut Europ´ een des Membranes ENSCM/UM2/CNRS 5635, IEM/UM2, CC 047, Place Eug` ene Bataillon, F-34095, Montpellier, Cedex 5, France. E-mail: [email protected]; Fax: +33-467-14-91-19 Electronic supplementary information (ESI) available: Experimental details, NMR, IR, AFM, X-ray diraction, antimicrobial activity. See DOI: 10.1039/c3tb20558d Cite this: DOI: 10.1039/c3tb20558d Received 18th April 2013 Accepted 10th May 2013 DOI: 10.1039/c3tb20558d www.rsc.org/MaterialsB This journal is ª The Royal Society of Chemistry 2013 J. Mater. Chem. B Journal of Materials Chemistry B PAPER Published on 10 May 2013. Downloaded by Mount Allison University on 31/05/2013 19:00:09. View Article Online View Journal

Antifungal vanillin–imino-chitosan biodynameric films

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Journal ofMaterials Chemistry B

PAPER

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a“Petru Poni” Institute of Macromolecular

Aleea Gr. Ghica Voda, Iasi, Romaniab“Ion Ionescu de la Brad” University, Labor

AleeaSadoveanu, Iasi, Romaniac“Gheorghe Asachi” Technical University of I

RomaniadInstitut Europeen des Membranes – ENSC

Place Eugene Bataillon, F-34095, Mo

[email protected]; Fa

† Electronic supplementary informationNMR, IR, AFM, X-ray diffraction,10.1039/c3tb20558d

Cite this: DOI: 10.1039/c3tb20558d

Received 18th April 2013Accepted 10th May 2013

DOI: 10.1039/c3tb20558d

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

Antifungal vanillin–imino-chitosan biodynameric films†

Luminita Marin,a Iuliana Stoica,a Mihai Mares,b Valentina Dinu,a

Bogdan C. Simionescuac and Mihail Barboiu*ad

Vanillin–chitosan biodynamers have been prepared and structure–morphology correlations revealed the

pathway of progressive incorporation of the aldehyde onto chitosan backbones. Such dynamic

biopolymers or biodynamers, generated from reversibly interacting components, offer the possibility to

address the dynamic covalent behaviour of the reversible imine-bond formation/hydrolysis equilibria

between vanillin and chitosan polymeric backbones. The reaction takes place with very low conversion

in acidic aqueous solutions (7–12%), but the imine bond formation is amazingly improved (�80%)

when the reaction takes place while solution–solid state transition and solid state phase-organization

events occur. The chitosan–vanillin biopolymeric films described here present interesting Candida

albicans antifungal activity compared with other common bacterial strands, which suggests the

implementation of these biocompatible materials as thin layer protecting systems for medical devices.

Introduction

Due to its therapeutic, haemostatic, depressant, immuno-adju-vant and antitumor properties and its non-toxic behaviors1,2

chitosan (C), the natural polymer obtained from deacetylation ofchitin, has become a biomaterial of expanding interest inbiomedical, cosmetics, food or agricultural applications. De novodesign of synthetic chitosan-based materials as lms,3 bers,4

nanoparticles5 and gels6 further increases its applicative value.The main challenge in the large scale use of chitosan is its lowsolubility in water. To overcome this, different chitosan modi-cation reactions have been explored.7–14 Among them, thereversible reaction of amino groups of chitosan with aldehydesbearing Schiff base–chitosan received much attention becausethe functional moieties, reversibly graed onto the chitosanbackbone, disrupt the H-bonding network of chitosan, thusimproving its solubility.7 Moreover, it represents a simplesynthetic strategy, yielding modied chitosan with improvedantimicrobial, antifungal, antitumor activity or for the delivery ofinsoluble therapeutic agents, etc.8,9 Ultrasound,10 microwave,11

plasma12 or enzymatic13 activated synthetic methods have been

Chemistry of Romanian Academy, 41A,

atory of Antimicrobial Chemotherapy, 8,

asi, 73, Bd. Prof. Dimitrie Mangeron, Iasi,

M/UM2/CNRS 5635, IEM/UM2, CC 047,

ntpellier, Cedex 5, France. E-mail:

x: +33-467-14-91-19

(ESI) available: Experimental details,antimicrobial activity. See DOI:

Chemistry 2013

used to obtain Schiff base–chitosan derivatives. The yield ofimine formation in water is very low, due to imine exchanging orhydrolysis reactions occur very fast in acidic aqueous solu-tions.14,15 We previously answered this challenge by reporting14

that the very low conversion degrees (1–12%) obtained in acidicaqueous solutions can be amazingly improved to 80–90% ifconcentrated hydrogel/solid state irreversible vitricationprocesses occur or if aldehydes of low solubility react “out ofwater”,15a when the components are in hydrophobic contact.With all these in mind, the further step was to implement thesestrategies to obtain such biodynamers16 of high applicativeinterest. Within this context, our attention was attracted byvanillin (V), a natural product used as a food avoring agent,which presents improved antifungal properties when formingSchiff bases,17a or incorporated onto chitosan as a vanillyl unit.17b

Moreover, recent studies demonstrated that vanillin is an effi-cient inhibitor of cancer cell migration and metastasis in amouse model.17c One should expect that these premises promisethe chitosan–vanillin imine (CV) biopolymers to be a potentiallyvaluable system for different applications. In the present work,chitosan–vanillin Schiff-base biopolymers were synthesized viaimine-bond formation between chitosan and vanillin at differentmolar ratios (C0.5V, CV, C1.5V, C2V, C3V, C4V, C5V), in acidicwater–acetonemixtures, in toluene used as a non-solvent for bothcomponents (CVT) or by grinding in the absence of solvents(CVM) (Scheme 1). Two aspects were preferentially followed: (1)the chemical investigation of the reaction evolution by spectro-scopic and morphological analyses of resulted materialsand (2) the antibacterial and antifungal properties of thesynthesized biodynameric lms. We have tested thesematerials against the most common bacterial agents, while theypresent interesting activity against Candida albicans fungusstrain.

J. Mater. Chem. B

Scheme 1 Synthesis of biopolymers CnV (where n ¼ 0.5, 1.5, 1–5) via reversibleimino-covalent bonding of vanillin V.

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Experimental sectionMaterials and methods

All reagents were purchased from Sigma Aldrich and usedwithout further purication. The molecular weight of chitosanwas calculated to be 125 kDa and its degree of acetylation was DA¼ 15%. 1H-NMR spectra were recorded on a BRUKER AvanceDRX 400 MHz spectrometer in D2O using the residual solventpeak as reference. 13C-NMR solid-state spectroscopy was con-ducted by single-contact 50.32 MHz 13C CP-MAS on a Bruker MSLCXP-200 spectrometer tted with a Bruker-z32DR-MAS-DB probe.Solid powder samples in 13C CP-MAS NMR experiments werecontained in a ceramic cylindrical rotor and spun at 4.5 kHz.Contact time for cross-polarization was 2.5 ms and 1400–4000scans accumulated. Spectra were referenced indirectly to a zerovalue for tetramethylsilane (TMS). FTIR measurements wereperformed with a FT-IR Bruker Vertex 70 Spectrometer in thetransmission mode, by using KBr pellets and a standard sampleholder, or dry biopolymer lms with a Gemini sampling

Fig. 1 (a) Pictures of chitosan, C and CnV (n ¼ 1, 2, 3, 4) biodynameric films. AFM

J. Mater. Chem. B

accessory to collect horizontal attenuated total reectance (ATR)spectra using a ZnSe crystal. Wide Angle X-ray Diffraction(WAXD) was performed on a Bruker D8 Avance diffractometer,using the Ni-ltered Cu-Ka radiation (l ¼ 0.1541 nm). Theworking conditions were 36 kV and 30 mA. All diffractogramswere investigated in the 2O 40� range (2q�) at rt. Films obtainedby casting were used as initial samples for the X-ray measure-ments. Atomic force microscopy (AFM) images were collected insemi-contact mode with a Solver PRO-M, NT-MDT, Russia. As itwas a statistic investigation, the different areas were not“chosen”, the cantilever being simply landed on the surface andregistering data for squares beginning with a scan size of 20 mm(20 � 20 mm2) up to 0.5 mm (0.5 � 0.5 mm2). The arithmeticaverage roughness (Ra) was measured for all explored areas. Foran accurate comparison of the surface characteristics of all lms,the roughness exponent (RE) was calculated as the slope ofroughness versus the scan size, in a double log plot.

Culture media and inoculation

To check the antimicrobial/antifungal activity, the biopolymerlms were dried under vacuum, at 70 �C for 72 h. All microor-ganisms were stored at �80 �C in 10% glycerol. The bacteriawere refreshed in a Mueller–Hinton broth (Merck) at 36 �C, andaerward were inoculated on Plate Count plates (Biokar,France) for purity checking. Fungi were refreshed on Sabourauddextrose agar (SDA) (Biokar, France) and were grown at 36 �C,pH ¼ 7. Using these cultures, microbial suspensions wereprepared in sterile saline solution to obtain turbidity opticallycomparable to that of the 0.5 McFarland standards, yielding a

roughness and topographic profiles of films' surfaces: (b) C, (c) C5V and (d) C2V.

This journal is ª The Royal Society of Chemistry 2013

Table 1 Chitosan conversion into vanillin–imino-chitosana

C0.5V CV C2V C3V C4V CVT CVM

hsolution (%) nd 9 12 11 nd nd ndhsolid (%) 81 78 39 28 nd 34 9hmax (%) 100 100 50 33.3 25 100 100

a CnV, where n ¼ 0.5, 1–5 – synthesised in solution, CVT synthesised intoluene, CVM synthesised by grinding of solids, hsolution (in aqueoussolution) and hsolid (in solid state) calculated from NMR spectra; hmax– the maximum possible yield according to the molar ratio of the

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suspension containing approximately 1 � 108 CFU mL�1 for allmicroorganisms. Volumes of 0.2 mL from each inoculum werespread onto Mueller–Hinton Agar pre-poured in Petri plates.Aer drying, the biopolymer lms were added. To evaluate theantimicrobial properties, the inhibition of growth wasmeasured under standard conditions aer 24 h of incubation at36 �C. All tests were carried out in triplicate to verify the results.The diameter of the inhibition zone around the lms wasmeasured using a calliper.

reagents; nd – not determined.

Synthesis of dynameric mixtures in aqueous solution

To a 2% chitosan C (0.05 g, 0.29 mmol) and 0.7% acetic acidsolution in distilled water, a solution of vanillin V (variousmolar ratios) in 0.2 mL of acetone was slowly dropped for 30minutes. Aer that the reaction vessel was kept at 70 �C for 8 h.The color of the mixture changes to greenish-yellow, indicatingthe imine bond formation. Different –NH2/–CHO molar ratios(1/2; 1/1; 1.5/1; 2/1; 3/1; 4/1 and 5/1) corresponding to the Schiffbases CnV (n¼ 0.5, 1–5) were used. Additional experiments wereperformed in toluene (CVT) at 70 �C for 72 h, or by grinding thereagents with a pestle into an agate mortar (CVM). To perform1H-NMR spectra, deuterated solvents were used. 1H-NMRspectra were registered for all reaction mixtures.

General procedure for the synthesis of lms and solid powdersamples

The thin lms were obtained by casting 3 mL biopolymersolution CnV into a DUROPLAN Petri with a 5 cm diameter. Theobtained crude lms were further dried in vacuum, at 70 �C(Fig. 1). Powders of different yellow color intensities were alsoobtained by lm grinding. All resulted biopolymers wereinsoluble in common organic solvents, but soluble in aqueoussolution (pH ¼ 6.2).

Results and discussionSolution and solid state structural characterization of CnVsystems

NMR and FTIR spectroscopies were used to evaluate the prog-ress of imine bond formation in solution and in solid state,respectively. Evidence for imine-bond formation is supportedby the appearance of the vibration band at 1630 to 1640 cm�1

(–CH]N) in the FTIR spectra of lm and powder samples, whilethe intensity of the 1560 cm�1 band (–NH2) decreases and thepeak at 1665 cm�1 related to the aldehyde groups completelydisappears (Fig. 1S†).18a,b The 1H-NMR spectra in solution of thechitosan–vanillin biopolymers as obtained at different molarratios in D2O/(CD3)2CO allow an easy identication of the peakscorresponding to the partial conversion of the chitosan intovanillin–imino-chitosan, an equilibrium being reached aer2 h. Considering the chemical shi of the H2 protons intochitosan (as a measure of amino groups14) and the chemicalshi of the imine protons, the degree of conversion of aminogroups into imine units in solution was calculated using theequation hsol ¼ [ACH]N/(AH2 � 0.85)] � 100, where A representsthe area of the peaks due to the imine proton (ACH]N) and H2

This journal is ª The Royal Society of Chemistry 2013

into chitosan (AH2) and 0.85 is the deacetylation degree of chi-tosan, respectively. As expected, very low conversion degreeswere found in aqueous solution, i.e. 7–12% (Table 1).

Homogeneous solid lms and powders were obtained byfurther evaporation of the solvents from the reaction mixture.The crude products were dried in vacuum and subsequent13C-NMR solid state spectra exhibit major imine peaks at 160ppm19 and minor weak unreacted aldehyde peaks at 200 ppm,14

indicating the conversion of chitosan into imino-substitutedCnV. The degree of conversion of amine groups into iminelinkages was calculated from 13C-NMR spectra as the ratiobetween the imine carbon and C1 chitosan carbon peak areas.The peak at 110 ppm of C1 carbon of chitosan was used becauseits shape was the least superposed with other peaks, a moreaccurate area determination being thus possible. Taking intoaccount that the chitosan used for this study had a deacetyla-tion degree of 85%, the degree of conversion of amino groupsinto imino bonds was calculated with hsolid ¼ [ACH]N/(AC1 �0.85)] � 100, where ACH]N and AC1 represent the areas of theCH]N and C1 integrated peaks and 0.85 is the deacetylationdegree of chitosan, respectively (Table 1). As can be seen inTable 1, the solution–solid state phase change process (vitri-cation) is highly benecial for imine bond formation, the yielddrastically increasing to 80% for C0.5V. This indicates that,under specic conditions – i.e., high reagent concentration andtemperature – the imine formation takes place during the waterremoval process. Moreover, the imine formation takes place at asignicantly lower yield in a non-solvent like toluene (�30%,CVT) or in the absence of any solvent (�9%, CVM). Thus thecondensation reaction is more effective in solution, when thenumber of collisions of the components is higher and thereforeincreasing the number of effective collisions.

Morphological and topographic structure of CnV lms

Imine bond formation on the chitosan backbone implies amodication of the morphology and thus of lm surfacetopography. To monitor these modications, the self-standingCnV exible lms were analyzed by X-ray diffraction and atomicforce microscopy measurements. The morphology of lmsurfaces was investigated by AFM topographic and phasecontrast measurements. The data are presented in Table 2. Thechitosan lm surface displays a granular morphology with20–30 nm diameter grains and a low roughness exponent(Fig. 1b). The lm samples show similar granular morphology

J. Mater. Chem. B

Table 2 Atomic force microscopy dataa

Code RE D/nm Deg/20 mm

Chitosan, C 0.169 20–30 �5 to 39C5V 0.478 90–150 �11 to 19C3V 0.062 80–110 �10 to 26C2V 0.108 20–30 �4 to 2CV 0.32 120–150 �3.5 to 3.5

a RE: roughness exponent; D: diameter size of grains; deg/20 mm: thephase contrast shi for a scan size of 20 mm (20 � 20 mm2).

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and distinct variable roughened structures for increasingamounts of vanillin substituent. The low substituted C5V lmsample (Fig. 1c) presents larger grains with wide size dispersity,reected in a higher roughness exponent (Table 2). Themediumsubstituted C3V lm samples still show big grains, but with arelatively uniform grain size dispersity and a quite low rough-ness exponent (Table 2). The high substituted C2V lm samplespresent small grains disposed in parallel rows having the inter-rows distance around 140 nm and a very smooth surface(Fig. 1d). Increasing the substitution degree to CV, the grainssize and roughness exponent become larger (see the ESI† fordetails).

Wide angle X-ray diffraction (XRD) characterization of CnV

Further valuable insights on the self-organization of chitosanlms are obtained from the X-ray diffraction (XRD) of the CnV(n ¼ 0, 0.5, 1–5) compound lms (Fig. 2). The different surfacemorphologies observed in AFM micrographs (Fig. 1b) arerelated to a variable long-range order, as detected by XRD(Fig. 2), and reect the degree of functionalization of chitosanbackbones. The XRD pattern of the chitosan C lm shows broadpeaks of low intensity at 2q ¼ 10–15� and 2q ¼ 15–30�, corre-sponding to interplanar distances of 8.0–9.8 A and 4.4–3.2 A,respectively, indicating the presence of hydrated H-bondedchitosan crystalline form II.20 The formation of grains, asobserved by AFM, is due to the exibility of the macromolecularchains which facilitates globular self-assembly. The XRDpatterns of CnV lm samples indicate changes to the degree of

Fig. 2 XRD patterns of C and CnV (n ¼ 1, 2, 3, 5) films at room temperature.Possible structural self-organization of polymeric backbones (see the text fordetails).

J. Mater. Chem. B

crystallinity. The loss of reection intensity in low-angle XRDpatterns of low-functionalized chitosan materials (C5V and C3V)strongly supports the non-periodic vanillin incorporation ontochitosan backbones, which increases the inter-chain distancesand disturbs the H-bonding network of crystalline chitosan inan irregular manner (Fig. 2). These lms present bigger grainsas compared to the chitosan lm, due to a few rigid imine unitsgraed on chitosan backbones. This increases the inter-chaindistances and thus the size of globular macromolecularassemblies. The gain of reection intensity is correlated with alonger range order and thus with a periodic vanillin incorpo-ration on chitosan backbones.

The XRD pattern of the most functionalized biopolymers(CV, C2V) is indicative of the self-organized lamellar nano-phases, such that the main diffraction peaks at 2q ¼ 7� (100),2q ¼ 13.5� (200) and 2q ¼ 20.2� (300) correspond to interplanar,interchain and intermolecular distances of 13.2, 6.6, and 4.4 A,respectively (Fig. 2). The imine bond formation induces anincrease of the inter-chains distance from 9 to 13.5 A, whichcorresponds to interdigitated vanillin layers H-bonded betweenthe chitosan backbones, as simulated by the MM+method. Thisresults in the formation of small grains and thus of lowroughness lm surfaces. Most probably, the formation oflamellar structures is a consequence of the reversibility of theimine bonds that act as a driving force in materialnanostructuring.

Antimicrobial/antifungal activity of CnV lms

To explore the potential antimicrobial/antifungal activity of CnVlms, microbiological measurements were assessed on

Fig. 3 The inhibitory effect of CnV films (n ¼ 1, 1.5, 2) on Candida albicans. Thediameter (mm) (error bars �10%) of the inhibition zone is calculated as thedifference between the diameter of the colony free zone and the diameter ofthe film.

This journal is ª The Royal Society of Chemistry 2013

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Escherichia coli (ATCC 25922 Gram-negative) or Staphylococcusepidermidis (ATCC 1228 Gram-positive) bacteria and Candidaalbicans (ATCC 10231) fungus, using an adapted diffusionmethod.21 As illustrated in Fig. 3, an important growth inhibi-tory effect was observed for all chitosan–vanillin lms in thecase of the Candida albicans fungus strain, while in the case ofEscherichia coli and Staphylococcus epidermidis bacterial strainsthe microorganism growth was inhibited only on small areasthat were in direct contact with the active sites of the lms,particularly for the lms with high imine linkage content(Fig. 5S†). The fungus growth was progressively inhibited underthe lm surfaces and on a large surrounding area, clearlyindicating an increasing inhibitory effect – vanillin contentrelationship. This behavior suggests that the inhibitory effect ofCnV lms is most probably the result of the reversible vanillinrelease as long as it is consumed in microorganism inhibition.

Conclusions

Schiff base vanillin–chitosan biopolymers were obtained bycondensation of various molar ratios of vanillin and chitosan inhomogeneous aqueous and heterogenous non-solvent systemsand or by grinding the solid state reagents. Themorphology andantifungal activity of the biopolymeric lms were characterized.The present results reveal that the imine bond formation onchitosan polymeric backbones takes place with very lowconversion degrees (around 10%) in acidic aqueous solutions,but is substantially increased (conversion �80%) when solu-tion–solid state transition and solid state phase-organizationevents occur.14,22 The vanillin, of very low water solubility, mightreact “out of water” with the chitosan backbones, but the yieldremains very low (see CVT and CVM examples – Table 1).23 Theprogressive incorporation of vanillin onto hydrophilic chitosanbackbones produces the depletion of the very strong H-bondedpolymeric chains and of the globular self-organized chitosanparticles inducing, in the rst step, the increase of their sizes.The hydrophobic swelling due to vanillin residues at the inter-faces reaches a critical step when lamellar self-organizedmorphologies are created. The hydrophilic chitosan–hydro-phobic vanillin alternative layers are probably responsible forthe more compact organization of self-organized small parti-cles. The hydrophobic region dened by “vanillin interfaces”along the chitosan backbone might have a protecting environ-mental effect against the hydrolysis and the stabilization of theimino-bonds.23

This work sheds light on the experimental conditions of thevanillin imine-bond formation on chitosan polymeric back-bones. Very low conversion degrees were obtained in acidicaqueous solutions, contrary to previous literature reportsstating the formation of stable imino-chitosan derivatives insuch experimental conditions. These reactions take place whenconcentrated solid state vitrication occurs. Moreover, thevanillin–chitosan conjugates may have interesting antibacterialproperties. Among the various fungal pathogens, attackingimmune-compromised patients, Candida albicans accounts forthe majority of systemic infections with mortality rates rangingfrom 50 to 100%.24 Hospital-acquired infections by Candida

This journal is ª The Royal Society of Chemistry 2013

albicans formed on the surface of implantable medical deviceshave become a major health concern. Until now, good anti-fungal action against Candida albicans has been reported forsolutions of low molecular weight chitosan (32, 38 kDa).25 Inthis regard, the CnV biodynameric materials with promisingCandida albicans antifungal activity described here open thedoor to implement these biocompatible dynameric lms as thesolid thin layer protecting systems for medical devices.

Acknowledgements

This work was nancially supported by ANR 2010 BLAN 717 2and DYNANO, PITN-GA-2011-289033 projects (see http://www.dynano.eu), by the Romanian National Authority forScientic Research, CNCS – UEFISCDI grant, project numberPN-II-ID-PCCE-2011-2-0028, and by the European Social Fund –

“Cristofor I. Simionescu” Postdoctoral Fellowship Programme(ID POSDRU/89/1.5/S/55216), Sectoral Operational ProgrammeHuman Resources Development 2007–2013.

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