Preparation and Characterization of Methyl Substituted Maleic

Article pubs.acs.org/IECR

Preparation and Characterization of Methyl Substituted Maleic Anhydride: Modified Collagens Destined for Medical Applications Daniela Pamfil,† Manuela Tatiana Nistor,† Lidija Fras Zemljič,‡ Liliana Vereştiuc,§ Maria Cazacu,† and Cornelia Vasile*,† †

Department of Physical Chemistry of Polymers, Romanian Academy, “Petru Poni” Institute of Macromolecular Chemistry, 41 A, Grigore Ghica Vodă Alley, 700487, Iaşi, Romania ‡ Laboratory for Characterization and Processing of Polymers, Institute of Engineering Materials and Design, Faculty of Mechanical Engineering, University of Maribor, 17 Smetanova Ulica, 2000 Maribor, Slovenia § Faculty of Medical Bioengineering, Department of Biological Sciences, Gr. T. Popa University of Medicine and Pharmacy, 9-13 Kogalniceanu Street, 700454, Iaşi, Romania S Supporting Information *

ABSTRACT: Porous modified collagens with vinyl groups were synthesized by reaction with 2,3-dimethylmaleic anhydride or citraconic anhydride. The structure of modified collagen was assessed by analytical determination of the amino groups in collagen and spectroscopic methods (Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (1H NMR)). The amount of protonated amino groups and carboxylic acid moieties of the samples have been determined by potentiometric titration. The dependence of the viscosity of diluted collagen solutions on the concentration and temperature was followed to determine their denaturation transitions. The increased average molecular weight, particle size, and second virial coefficient of modified collagen solutions with respect to the unmodified one proved that modification took place. Coupled thermal analyses (TG/DTG/DTA/FT-IR/MS) also indicated the collagen modification by increased thermal stability and new volatile compounds formed during decomposition. The adhesion tests revealed their potential application in wound dressing and tissue engineering.

1. INTRODUCTION Collagen, mainly soluble, is widely used in the biomedical field and in tissue engineering owing to its low immunogenicity and toxicity and to its unique properties. Besides its great hemostatic and cell-binding properties, collagen exhibits an excellent biocompatibility profile and predictable biodegradability.1 The poor mechanical properties and low stability in various physiological media of the collagen-based polymeric materials can be improved by chemical modification, crosslinking, blending with other polymers, filling, etc.,2 to successfully meet a range of different clinical applications. The purpose of chemical modification of polymers with low molecular weight reagents is to obtain materials with improved and special properties applicable in various fields.3,4 The formation of cross-links involves predominantly the εamino groups of lysine and hydroxylysine, the positions of which in collagen molecules are determined by the primary structure of their molecular chains.5 The blocking of free εamino groups can be achieved by various acylating agents such as imides, anhydrides, and chloroanhydrides.6,4 The anhydrides are highly reactive toward nucleophiles, and they are able to acylate a number of the important functional groups of proteins and other macromolecules.7 Mono-itacon-amide functional groups were incorporated to the native collagen macromolecule8 to increase its reactivity to other monomers like 2hydroxyethyl methacrylate. Collagen-based compositions as adhesives for soft tissues or made into a sealant film for a variety of medical uses such as wound closures and tendon © 2014 American Chemical Society

wraps for preventing adhesion following surgery were described by Kelman and DeVore.9 Prior to polymerization, soluble or partially fibrillar collagen with monomers in solution were chemically modified with an acylating agent, sulfonating agent, or a combination of them. Collagen protein was modified by maleic anhydride to improve its solubility in organic solvents and to increase the reaction rate between isocyanate and collagen protein.10 Other chemically-modified collagen was obtained by reacting native collagen with a di- or tri-carboxylic acid halides, di- or trisulfonyl halides, di- or tri-anhydrides, or di- or tri-reactive active ester coupling agents. Any remaining lysine epsilon amino groups present in the coupled collagen product may be converted to ureido, beta-malicamino carboxyamido, or sulfonamido groups by isocyanate, epoxy succinic acid, acid halide, sulfonyl halide, or active ester amine-modifying agents.11 The resultant soluble product when dissolved in a physiological buffer provides a viscoelastic solution having therapeutic application in a variety of surgical procedures, particularly in ophthalmic surgery. Collagen was also chemically modified with methacrylic anhydride in a manner adapted from a methodology reported by van Den Bulcke et al. for gelatin derivatization.12 Acetylation Received: Revised: Accepted: Published: 3865

October 22, 2013 January 3, 2014 January 22, 2014 February 26, 2014 dx.doi.org/10.1021/ie403563r | Ind. Eng. Chem. Res. 2014, 53, 3865−3879

Industrial & Engineering Chemistry Research

Article

Scheme 1. Colorimetric Reaction between TNBS and the Amino Groups of Proteins

of a 1 wt % NaOH solution. The reactions of chemical modification were carried out in a controlled manner so that the degree of cross-linking was limited and under the same conditions of temperature and duration. The pH of the reaction mixture was preferably maintained at about 8 or 9. In this manner, almost all of the lysine epsilon amino groups present on the collagen molecules are freed from their protonated form and become capable of reaction with either the coupling agent or the modifying agent.11 Several reactions were achieved varying pH and mass ratio between collagen and anhydride. A solution of anhydride (collagen:anhydride, 1:3 or 1:5 g/g) dissolved in DMSO was added dropwise in the reaction flask over collagen solution under continuous stirring at room temperature for 24 h, and the pH was maintained constant by adding a 1 N NaOH solution. Resulting products were then purified by dialysis against deionized water for three days to remove unreacted compounds. The water was changed three times daily. Before performing their characterization, the final products were dried by lyophylization at −50 °C, porous dry materials being obtained. After freeze-drying (48 h), samples were kept in a desiccator over phosphorus pentoxide 98%. 2.3. Investigation Methods. 2.3.1. Characterization by FT-IR and 1H NMR Spectroscopy. The FT-IR spectra were recorded using a Bruker Vertex 70 spectrophotometer, in ATR mode (Golden Gate ATR system equipped with diamond crystal, 45° angle of incidence and one reflection), in the range 600−4000 cm −1 (resolution 2 cm −1, 64 scans), at ambient temperature. The processing of spectra was achieved using OPUS software version 5. 1 H NMR spectra have been acquired on a NMR Jeol UK 400 MHz Eclipse spectrometer in order to confirm the chemical modification of collagen. The measurements were carried out at room temperature and with a frequency of 400 MHz. An amount of 30 mg sample, dried by lyophylization, was dissolved in 2 wt % DCl in D2O at 34 °C for 3 days; then, the spectra were recorded. 2.3.2. Determination of Amino Groups in Collagen and Modified Collagen. Anhydrides can react with the amino groups of collagen. Blocking amines by reaction with anhydrides can be assessed by measuring the number of amino groups before and after blocking using sodium 2,4,6trinitrobenzenesulfonate (TNBS). TNBS solutions, at low concentrations, below 1 wt %, are colorless24 and in UV domain they show an absorbance of 22 000 M−1 cm−1 at 420 nm.25 Therefore, it does not interfere with colored intermediary, calorimetrically measured to determine free amino groups because the conjugates for tested amino groups show an absorbance at 350 nm26 or at lower values depending on the amino group type.

and succinylation methods of collagen using acetic and succinic anhydrides was done by Istranova et al.4 Physico-chemical changes of collagen under prolonged exposure to concentrated sodium hydroxide solution, formic acid, trifluoroacetic, tetrafluoroethanol, and hexafluoroisopropanol were also studied.13 Potorac et al.14−16 chemically modified collagen with itaconic and maleic anhydrides. In this study, particular attention has been paid to the preparation and characterization of modified collagen with citraconic (CTA) and dimethyl maleic anhydrides (DMA) in order to obtain reactive products with potential use in tissue engineering and/or wound dressing. To our knowledge, until now, these substituted anhydrides have not been used for chemical modification of collagen in order to increase its functionality and widening its applicability. It is known that DMA and CTA are derivatives of maleic anhydride,17 the first is known to yield unstable derivatives at pH near neutrality,18 this property being useful to dissociate the Bovine 6S procarboxypeptidase A by reversible condensation.19 The citraconic anhydride is a versatile reagent used for the synthesis of maleimides, bicyclic pyrrolidines, and co- and terpolymers, as well as for the protection of N-terminal amino acids20 because the reaction is reversible after acylation of amino groups.7 It can supposed that these reactivity characteristics will be imparted also to chemically modified collagen.

2. MATERIALS AND METHODS 2.1. Materials. Acid-soluble collagen, type I + III from bovine skin dermis as 1.21 wt % in H2O2 solution, was supplied by Lohmann & Rauscher GmbH, Germany. This form, produced by extraction, was selected because is not only soluble, but also nondenatured having an intact triple helix conformation which can be much suitable as biomaterial. Soluble collagen can be extracted from collagenous material with neutral salt solutions or weak acids, the second one being more effective.21 Such soluble collagen is easier to chemically characterize22 and has a special and important role in medicine because, being extracted as aqueous solution or gel, can be processed in different forms such as hydrogels, membranes, matrices (spongious), fibers, or tubes.23 The molecular characteristics of collagen used for chemical modification are evaluated below by dynamic/static light scattering comparatively with anhydride-modified collagens. The citraconic anhydride (CTA), 2,3-dimethylmaleic anhydride (DMA), dimethyl sulfoxyde (DMSO) of analytical purity, sodium hydroxide (NaOH), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma-Aldrich, United Kingdom. 2.2. CollagenSubstituted Anhydride Synthesis. The collagen solution (1 wt %) was prepared by dropwise addition 3866

dx.doi.org/10.1021/ie403563r | Ind. Eng. Chem. Res. 2014, 53, 3865−3879


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2.3.4. Total Organic Carbon and Nitrogen (TOC/TN) analysis. Total organic carbon (TOC) and total nitrogen (TN) were evaluated using Multi-N/C 2100 (Analytik Jena, Germany) instrument with selective high-sensitivity detectors for carbon and nitrogen. Solutions of unmodified and modified protein of a concentration of 0.02 wt % in 0.002 M acetic acid were used. The results were expressed as mmol of carbon (or nitrogen) per gram of sample. 2.3.5. Dynamic/Static Light Scattering. Determination of the average molecular weight was done by using static light scattering (SLS) while particle size and zeta potential analysis of the modified and unmodified collagens was achieved by using dynamic light scattering (DLS). The used solvent was acid acetic 0.1 M. For each sample, four solutions with different concentrations were prepared (0.5, 1.5, 2, and 2.5 mg/mL). Solutions were kept in a refrigerator for 5 days, and before determinations, they were ultrasonicated for 1 min. The Malvern Zetasizer Nano ZS instrument (UK) allows the determination of molecular weight and second virial coefficient (A2) of the samples by measuring the intensity of SLS by scattered light of various concentrations of the sample at one angle (173°). The intensity of scattered light produced by a macromolecule is proportional to the product weight average molecular weight (M) and concentration of the macromolecule. The second virial coefficient is a property describing the interaction strength between the particles and the solvent or appropriate disperse medium. The Rayleigh equation34 was applied:

A lower amount of free amines is indicative of a greater extent of modified collagen.27 First, TNBS reacts under relatively mild alkaline conditions at a temperature of 40 °C with free amines forming the unstable Meisenheimer complex, a highly chromogenic orange colored derivative with absorbance at 335 nm (Scheme 1). Afterward, by changing the pH to the acidic values the Meisenheimer complex is rapidly converted in trinitrophenol (TNP)−collagen, a stable yellow product.28 Substitution degree of amino groups was calculated by determining the content of free amino groups of lysine before and after collagen chemical modification. A freeze-dried collagen sample of 3 mg was mixed with 1 mL of 4 wt % NaHCO3 and 1 mL of 0.5 wt % TNBS freshly prepared and heated at 40 °C for 120 min. Then, 2 mL of 6 M HCl were added, and the mixture was autoclaved at 60 °C for 90 min.29 The resulting solution was diluted with 5 mL of distilled water, and the absorbance was measured at 345 nm using a Spectro UV-Vis Dual Beam, 8 Auto Cell, UVS 2700 model, spectrophotometer (Labomed, Inc. Company, U.S.A). The measured absorbance is proportional with the concentration of free amino groups as it was proved by drawing a calibration curve. Because the reaction conditions are very severe, it is possible that a part of collagen to degrade and therefore to the end free-NH2 would increase. To clarify this aspect, the TNBS results have been compared with those obtained by potentiometric titration and total organic carbon and nitrogen (TOC/TN) analysis. As all obtained results are in accordance, it was supposed that determination errors are not significant and a substitution degree was evaluated. Each reported value was the average of at least three experiments. The substitution degree was calculated using the following equation:30 ABS NC − ABSMC (%)SD = × 100 ABS NC

⎛1 ⎞ KC = ⎜ + 2A 2 C ⎟P(θ ) ⎝ ⎠ Rθ M

(2)

where Rθ = Rayleigh ratiothe ratio of scattered light to incident light of the sample, M = sample molecular weight, A2 = second virial coefficient, C = concentration, P(θ) = angular dependence of the sample scattering intensity, and K = optical constant. Dynamic light scattering (DLS) measures Brownian motion and relates this to the size of the particles. Thus, the hydrodynamic diameter (d(H)) can be calculated by the Stokes−Einstein equation:35

(1)

where SD = substitution degree of amino groups, ABSNC = absorbance of the native collagen, and ABSMC = absorbance of the modified collagen. 2.3.3. Potentiometric Titration. Potentiometric titrations were performed at ionic strength of 0.1 M adjusted with KCl. An automatic two-buret instrument (Mettler Toledo T 70), equipped with a combined pH glass electrode (Mettler Toledo DG 117) and filled with aqueous solutions of 0.1 M HCl/0.1 M KCl and 0.1 M KOH/0.1 M KCl, was used.31 Each dried collagen sample (0.045 g) was dissolved in 0.1 M HCl (4.5 mL), and then, all the solutions for investigation were prepared by adding the obtained acid/collagen solution in 25 mL deionized water with very low carbonate content (≤10−6 M).32 The solutions were titrated in forward and backward runs between pH 2.6 and pH 11.5, and the working temperature was 25 °C. The titrant was added at varied intervals of 0.001−0.25 mL. The stability criterion during the analysis after each titrant addition was established at dE/dt = 0.1 mV/10 s, where 10 s was the minimum time for reaching equilibrium conditions between two additions of the titrant. A blank HCl−KOH titration was conducted under the same conditions as above to eliminate any error due to the presence of water contaminants such as dissolved CO2. The titrant volume was normalized to the mass of the titrated collagen samples and expressed as charges per mass (mmol/g) versus pH curve. From the charging isotherm of forward titration, the dissociation constant (pK) was calculated by nonlinear least-squares fitting.32,33

d(H) =

KT 3πηD

(3)

where d(H) = hydrodynamic diameter, k = Boltzmann’s constant, η = viscosity, T = absolute temperature (K), and D = diffusion coefficient. The cumulants analysis gives a good description of the size that is comparable with other methods of analysis for spherical, reasonably narrow monomodal samples, i.e. with polydispersity below a value of 0.1. For samples with a slightly increased width, the Z-average size and polydispersity will give values that can be used for comparative purposes. For broader distributions, where the polydispersity is over 0.5, it is unwise to rely on the Z-average mean, and a distribution analysis should be used to determine the peak positions. The intercept varied between 0.85 and 0.95; therefore the measurements have been performed in good conditions. The cumulants analysis of the DLS gives two values: Z-average size, an intensity mean value for the size, and a width parameter known as the polydispersity, or the polydispersity index (PDI). A PDI of 1 indicates large variations in particle size; a reported value of 0 means that there is no variation in size.36 It is a dimensionless 3867



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Scheme 2. (a) Reaction between Collagen and CTA; (b) Reaction between Collagen and DMA

number extrapolated from the autocorrelation function.37 Zeta potential measurements were determined by means of the same instrument a Zetasizer Model Nano ZS provided by Malvern Instruments, UK. Each zeta potential value represents an average of three recordings at a constant temperature of 25 ± 0.02 °C. 2.3.6. Viscosity Measurements of Diluted Collagen Solutions. The dependence of the viscosity of diluted collagen solutions on the concentration and temperature was followed. The measurements of viscosity depending on the concentration were performed using an Ubbelohde viscometer with dilution and suspended level with a capillary diameter of 0.5 mm at 25 ± 0.2 °C. The efflux time was determined both for the solvent (to) (0.1 M acid acetic solution) and for the collagen solutions (t) in the same solvent with protein concentrations within 0.035−0.075 wt %. From these data, the reduced (ηsp/c) and inherent viscosity (ln ηrel/c) values were determined. Before these measurements, all the solution samples were kept in a refrigerator (7 °C) for 5 days and filtered through a filter with pore diameters of 0.45 μm. The effect of temperature on the viscosity behavior was studied using an AVS 450 automatic Ubbelohde viscometer, manufactured by Schott. A 15-mL solution (0.2 wt %) was incubated for 10 min at the given temperature from 28 to 45 °C, and then, the efflux time (t) was recorded. The efflux time (to) of 0.1 M acetic acid solution (collagen-based samples solvent) was also determined under the same conditions. The relative viscosity was plotted against the temperature (ηrel = f(t)). Fractional viscosities were calculated for each temperature as follows (eq 4):38 F (T ) =

where F(T) = fractional viscosity at the given temperature and ηsp(T) = measured specific viscosity at the given temperature. These fractional viscosities were plotted against the temperatures and the denaturation temperature (Td) was taken to be the temperature where fractional viscosity was 0.5. This is the temperature at which the triple helix structure of collagen in solution is disintegrated into random coils.39 Another method to determinate the Td was the fitting of the data obtained which was conducted following the sigmoidal model described in Boltzmann equation40 by Origin Lab program version 8. The intrinsic viscosity [η] provides information about the hydrodynamic volume of a macromolecule in a solvent. Several mathematical equations are available in the literature for determining the intrinsic viscosity [η] of a polymer solution, by graphical extrapolation. The most commonly employed equations are those of Huggins and Kraemer:41 ηsp c

ln ηrel c

= [η]B − KB × [η]B 2 × c

(5)

(6)

where [η]A, [η]B = Huggins and Kraemer intrinsic viscosity and KA, KB = Huggins and Kraemer coefficients which will be adequate to evaluate the solvent quality used in collagen dilution. 2.3.7. Thermal Analysis by TG/DTG/DTA/FT-IR/MS. The thermal stability study of the collagen-based materials in an inert atmosphere was performed using a simultaneous thermogravimetric/differential thermal analysis system: model STA 449F1 Jupiter (Netzsch-Germany). Samples with a mass of 8.5−8.7 mg were heated with 10 °C/min, in open Al2O3 crucibles, from 30 to 600 °C. The volatile compounds have been washing away under a nitrogen flow of 50 mL/min, and

ηsp(T ) − ηsp(45 °C) ηsp(28 °C) − ηsp(45 °C)

= [η]A + KA × [η]A 2 × c

(4) 3868



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Figure 1. 1H NMR spectra of unmodified (a) and anhydride-modified collagen with CTA (b) and DMA (c).

for the protection of the thermo-balance, a purge flow of 20 mL/min was used. The analyses of volatile compounds were performed by coupling the thermo-balance with a FT-IR spectrophotometer, Vertex-70 model (Bruke-Germany), and a mass spectrometer, QMS 403C Aëolos model (NetzschGermany). Volatile compounds transfer from thermo-balance to analysis instruments were conducted through a polytetrafluorethylene line to FT-IR spectrophotometer and quartz capillary to mass spectrometer; thus, the analysis was being made in real time during the heating program and thermal decomposition. The TG collected data were processed with Proteus and OPUS 6.5 software which was used to analyze the FT-IR data. The ionized fragments collected, under electron impact with ionization energy of 70 eV, in mass spectrometer chamber were analyzed with Aeolos 7.0 software in spectrum scanning mode and in the range of m/z 1−200. 2.3.8. Adhesion Test. The bioadhesive tests for the collagens modified by reaction with anhydrides were evaluated by using a TA.XTPlus Texture Analyzer (Stable Micro Systems, UK) equipped with a 5 kg load cell. The lyophilized samples cut into circular shape with diameter of 6 mm were attached with a double-sided adhesive tape to the mobile support. The mobile support of the adhesion machine has similar dimensions with the sample and executes a transverse movement. The sample took contact with the dialysis membrane which was positioned on the fixed support of the adhesion machine. The boiled dialysis film surface was applied, 0.75 mL buffer solution with a pH 7.4 and a temperature of 37 °C (the same as body temperature). For membranes of dialysis, Medicell International Ltd. dialysis tubing with a 25.4 mm internal diameter and molecular weight cutoff of 12−14 kDa was used. About the parameters used for sample characterization concerning the adhesion test, it can be mentioned that the sample was lowered with a speed of 0.5 mm/s to contact the dialysis film with a force of 50 g and kept in contact time of 10 s. It was then

withdrawn at a constant speed of 10 mm/s to a distance of 10 mm. Using the Texture Exponent software package of the TA.XTPlus, the values of work of adhesion (mm mN) were calculated which is the product of distance and the maximum detachment force needed to separate the sample from the test surface. In the literature are mentioned studies about the advantages of this available computerized texture analyzer which gave good precision results on gelatin materials when relatively simple guidelines are followed.42 The reported values are the average of at least three determinations.

3. RESULTS AND DISCUSSION 3.1. Chemical Structure of Modified Collagen-Spectroscopical Methods Results. At alkaline pH (pH 8−9), the anhydride reacts with amines to form amide linkages with an extending terminal carboxylate group by opening the anhydride ring and effectively transforming the amine function into a carboxylate. The process of collagen modification at the amino group is described by Scheme 2. Bonding between carbon atoms from the carbonyl group (CO) of anhydride with the amino group (NH2) of collagen occurred by a nucleophilic addition−elimination reaction. The result was getting a modified collagen (desired acylated product) which has a vinyl group and a carboxyl in its chemical structure. Reaction between collagen and CTA can lead at two different linkages with amino groups of collagen depending on the substituent (methyl) positions with respect to the carbonyl group (Scheme 2a). The major side reaction was hydrolysis of the anhydride. In aqueous solutions, the anhydrides may break down by the addition of one molecule of water to yield two carboxylate groups.7 Hydrolysis and aminolysis (reaction with protein) are competing reactions involving nucleophilic substitution. The presence of an excess of the anhydride in the reaction medium 3869



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usually is enough to minimize the effects of competing hydrolysis. However, the anhydride excess can also lead to other side reactions as cross-linking of the collagen chains. A nucleophile (such as OH− group or an amino group of a collagen molecule) attacks the carbon atom using a pair of its own electrons. Since both hydrolysis and aminolysis yield the release of carboxylic acid functionalities, the medium becomes acidic during reaction. The pH of the reaction solution has an important effect on the rate of the nucleophilic substitution reaction. Basic solutions tend to drive the nucleophilic substitution reaction, resulting in faster rates.43 This requires either the presence of a strongly buffered environment to maintain the pH or periodic monitoring and adjustment of the pH with a base as the reaction progresses. Both collagen and obtained modified collagens are white porous materials. The yield of reaction was about 90 wt % with respect to the total amount of reactants. The structural characterization of the collagens was performed by FT-IR and 1H NMR spectroscopy. FT-IR data (not shown) indicated conformational changes in collagen materials structure. Compared to the unmodified collagen, some differences were observed in the FT-IR spectra. In the DMA-modified collagen spectra was identified an intensification of the peak 2912 cm−1, corresponding to the aliphatic group CH, and of the peak 1060 cm−1, corresponding to the COH stretching. Also, a new band at 1685 cm−1 was indicative for ester group CO. New bands have been identified at 2869 and 2810 cm−1 in the spectra of the CTAmodified collagen which correspond to the aliphatic groups and also an intensification of the band at 698 cm−1 indicated the formation of the new groups of CHCH (vinyl group) which exists in the CTA structure. In the 1H NMR spectrum (Figure 1) of the collagen modified with CTA (collagen: CTA, 1:3 g/g) three new signals were observed compared with the unmodified collagen spectrum. Around 6 ppm appeared a signal assigned to the vinyl group (CH) and at 8.7 ppm appeared a signal attributed to secondary amide (NH). The methyl group (CH3) was evidenced by at least one new signal at 2.1 ppm. In the spectrum of the collagen modified with DMA (collagen: DMA, 1:3 g/g) two signals at 8.7 and 2.1 ppm associated with the amide group and two methyl groups were presented. 3.2. TNBS Assay Results. The reaction of TNBS with primary amino groups of collagen was used to determine the free amine groups remained in the protein mass44 and the evaluation of the substitution degree according to procedure described in the experimental section. In Table 1 are presented the results related to the substitution degrees of modified collagen samples. Compared to CTA, an increased reactivity to collagen was accomplished by DMA with a substitution degree of 10.6 because of more susceptibility to hydrolyze of CTA compared to DMA. However, DMA and CTA can be at least as promising as the itaconic anhydride and maleic anhydride recently mentioned by Potorac at al.16 which had registered a substitution degree of 10.3 and 5.1 respectively. 3.3. Potentiometric Titration and TOC/TN Results. In order to study the protonation−deprotonation of amino and carboxyl groups, a potentiometric titration of the collagen based materials in aqueous solution were performed. The charging isotherms (the reference sample is presented as an example in Figure 2) normalized to the mass of sample indicates

Table 1. Anhydride Modified Collagen Samples and Their Substitution Degrees sample name* DMA 7.2 DMA 8.2 DMA 10.6 CTA 5.6 CTA 8.4 CTA 9.6

type of anhydride used for collagen modification

mass ratio of collagen:anhydride (g/g)

pH

substitution degree (mol %)

dimethylmaleic anhydride dimethylmaleic anhydride dimethylmaleic anhydride citraconic anhydride

1:5

9

7.21

1:5

8

8.18

1:3

8

10.6

1:5

8

5.6

citraconic anhydride

1:3

8

8.37

citraconic anhydride

1:5

9

9.6

*

Symbol of sample indicates type of anhydride used and substitution degree.

Figure 2. Charging isotherm obtained for the pure collagen determined by potentiometric titration.

amphoteric character of collagen. It can be observed that the collagen molecules are strongly positively charged at low pH owing to the protonation of all terminal amino groups. If collagen contains a considerable proportion of lysine and hydroxylysine, it is normal to obtain a high base-binding capacity. At higher pH, collagen is negatively charged due deprotonated carboxyl groups. It was posible to clearly show in the case of unmodified collagen the acid and base-binding maxima from positive and negative plateau of curves and accounts of 0.75 and 0.18 mmol/g, respectively (Figure 2). The isoelectric point (zero charge value) of the curve was around 7.3. At this point, the equilibrium between the adsorption and release of protons prevails (there is no excess of negative or positive charges). Earlier titration curves of collagen show isoelectric points of 8.2645 or 6.9546 and acid-binding capacity and base-binding capacity of 0.85−0.90 and 0.38−0.45 mmol/ g.47−50 Our results were somehow similar to the literature ones. The forward and backward titration curves were almost coincident proving that collagen samples were stable, no precipitations were observed, and backward titration was also reversible. The average pKa values of the terminal carboxylic acid and amine of unmodified collagen were 4.3 and 10.1, respectively (Table 2). The obtained values were in accordance with theory which proved our appropriate determination51 and reliability of model. At the pH value of 4.3, half of the carboxylic acids of the collagen function are deprotonated, and all of the amines are protonated. Also, it can be noted that around the pH value of 3870



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collagen type I.45 All the values (average molecular weight of 398 KDa, denaturation temperature of 31 °C, isoelectric point of 7.2) obtained for unmodified collagen were close to those find in literature, and they changed after reaction with both anhydrides (Table 4).

Table 2. pKa Values for Carboxylic and Amino Groups of Collagen Samples sample name

average pKa for COOH (pK1)

amount of protonated −COOH groups [mmol/kg]

average pKa for NH2 (pK2)

amount of protonated −NH3+ groups [mmol/kg]

4.3 4.7 4.8 4.7 5.1

33 53 70 46 81

10.1 10 10 9.9 9.9

13 9 2 7 4

collagen CTA 8.4 CTA 9.6 DMA 7.2 DMA 10.6

Table 4. M̅ w, A2, Z-average, PDI, and Zeta Potential Value Results after Analysis of the Collagens Solutions sample collagen DMA 7.2 DMA 8.2 DMA 10.3 CTA 5.6 CTA 8.4 CTA 9.6

10, half of −NH3+ content is still dissociated. Therefore, in the collagen structure (at high alkaline pH) will be present significant deprotonated terminal amino groups which are very suitable to react with anhydrides; this again explaining the optimum alkaline pH for modification reaction that is presented in our paper. The same procedure (as mentioned in section 2.3.3) of calculation of pK values of amino and carboxylic groups was established for modified collagens. The determined values are presented in Table 2. From the results in the Table 2 it can be observed that functional groups with higher pKa decreased (amino groups) after collagen modification and those with pKa around 4.5−5.1 that belong to carboxyl groups increased. This proved the success of collagen modification. After titration, the modified collagen had a less amount of NH2 groups showing that the reaction between collagen and anhydrides succeeded by blocking the free amino groups of collagen. Regarding the increase of COOH groups (in COO− form) and simultaneous decrease of amino groups, the most successful modification can be attributed to the sample CTA 9.6 and DMA 10.6. By using the TOC/TN method, no significant changes were observed for the nitrogen content (Table 3), but some changes

collagen

CTA 8.4

CTA 9.6

DMA 7.2

DMA 10.6

average TOC (mmol/g) average TN (mmol/g)

33.21

39.44

39.77

35.49

40.66

6.54

6.81

6.89

5.92

6.82

A2 (mL mol/g2) 10 4

Z-average (nm)

PDI

zeta potential (mV)

398 876 1395 819

1.8 10.44 24.85 14.12

410 1170 2100 2700

0.94 0.54 0.17 0.41

22.9 25.3 28.0 24.1

558 525 598

22.45 25.97 17.56

2880 1840 2290

0.13 0.56 0.42

28.4 26.3 26.0

The average molecular weight (M̅ w) and second virial coefficient (A2) increased after chemical modification and with the increasing substitution degree which means formation of strong inter- and intramolecular interactions in accordance with the variation of KA and KB values (see below). The PDI of collagen was high indicating heterogeneity by size, as also it appears from the corresponding curve where two peaks were presented (Figure 3). PDI decreased after modification, the samples becoming much homogeneous. Particle size increased by at least 5 times, and the particle size distribution was changed after modification, which is indicative of collagen triple helices modification by the anhydrides and also the formation of some large collagen agglomerates. Zeta potential values were within the limits of the solution with good stability for all pH values studied. The values of the zeta potential of collagen solutions were included in a tight range which denotes that after modification the collagen solution remained inside the range of stability. 3.5. Effect of Concentration and Temperature on the Rheological Behavior of Collagen and AnhydrideModified Collagen Solutions. Collagen solutions are highly structured systems which, even at low concentrations, are characterized by a high viscosity. The graphs of reduced viscosity versus concentration (Figure S1 in the Supporting Information) for all studied samples are straight lines in a wide range of concentrations. It was observed that the viscosities of CTA-modified collagen differ rather insignificantly from the viscosities of DMA-modified collagen. However, a major difference can be marked between the viscosities of the unmodified collagen solution and the modified collagen solutions, which indicates that the shape and dimension of collagen macromolecules are changed by modification. Therefore, the presence of the acylated groups in the chemical structure of the collagen leads to a considerable increase of the macromolecule dimensions which gave a high viscosity to solution. Intrinsic and inherent viscosity had significantly increased after modification with both anhydrides proving that modification took place with changes in conformation of macromolecules (Table 5) and strong interactions are present, both intermacromolecular and with solvent, as high values of the KA and KB (which increases 20 or 8 times, respectively, for modified collagen with respect to the value of unmodified

Table 3. Content of Total Organic Carbon (TOC) and Total Nitrogen (TN) in Collagen-Based Samples amounts

M̅ w (KDa)

were evident in the content of total carbon atoms. Modified collagens shown a higher number of carbon atoms in respect with unmodified collagen which was derived from the anhydrides structure. After titration, the modified collagen has a less amount of NH2 groups showing that the reaction between collagen and anhydrides succeeded by blocking the free amino groups of collagen. It can be concluded that the results obtained by TOC/TN and titration methods are in a good concordance with the results obtained by TNBS assay described above; in all cases a decrease in the content of amino groups after collagen functionalization reaction was observed. 3.4. DLS/SLS Results. From the literature data it is wellknown that collagen exhibits many distinct properties such as high average molecular weight (about 200 and 100 kDa for β and β chains, respectively) and narrow molecular weight distribution, basic isoelectric point of 8.26, triple helix to coil translation in the process of denaturation at around 37.5 °C for 3871



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The heat transformation of collagen to gelatin involves disintegration of the collagen triple helical structure in random coils leading to the change in physical properties like viscosity and light scattering.53 The denaturation temperature (Td) depends on the type of collagen and the treatments experienced during the technological process of collagen extraction.54 Td varies with collagen provenance, values of 28−35 °C being found for rat-tail-tendon collagen.55 The Td of mammalian collagens is constant of 39−40 °C but varies from 5 to 30 °C for fish54 and 31−35 °C depending on static or dynamic conditions of determination, shearing could induce a decrease of collagen Td.56 Figure 4 shows the pattern of changes in the viscosity of modified collagen solutions on heating. As can be seen, all solutions of the modified collagen exhibited a sharply decrease of the ηrel versus temperature from 28 to 38 °C and then the ηrel was almost steady at high temperatures. The change in the ηrel of the unmodified collagen solution is presented in the insert of Figure 4, and it also presents a decrease in the same temperature range; two regions of viscosity decrease (transitions) can also be detected. The fractional viscosity also decreased with an increase of temperature and the values of the transition temperatures obtained are found to be similar. The transition from a triple helix to a globular coil structure of collagen involved the breakage of hydrogen bonds between the adjacent polypeptide chains of collagen molecules and the changes of intact trimers into individual chains or dimers, causing the sharp decrease of ηrel at the same time.56 The minimal difference between denaturation temperatures of collagens is indicative of minimal differences in the extents of stable structure.57 Td was determined with a good accuracy by the graphical method. The reduced χ2, for all fitted curves, was χ2 ≤ 0.1; therefore, the use of Boltzmann equation was recommended. Also, another way to establish if such an approach is proper is the correlation factor R2, which has to take values close to 1. For the studied systems, values around 0.9 were found, which indicates that the approach was suitable. The unmodified collagen after heating revealed two inflection points (31 and 42 °C) probably because the collagen sample used contains two types of structures (I + III) or there are two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates has better thermal resistance than the smaller one. Modified collagen samples have only low temperature denaturation, the conformation being mainly uniform. It is known that collagen is practically insoluble in water having a significant strength. When insoluble collagen is converted to acid-soluble collagen by lowering the pH of solution, then this strength is lost and solubility will increase. But Td will decrease with acidification of the solution, indicating lower thermal stability of the protein at low pH.58 The CTA 5.6 and DMA 7.2 anhydride modified-collagen samples, with the lowest degree of substitution, were more stable and had a higher Td (32−33 °C) compared with those which have a bigger degree of substitution which showed a lower Td (31 °C; Table S1 in the Supporting Information). It can be concluded that collagen samples with a bigger SD are less thermally resistant in acidic medium of pH ≤ 3.2. As the denaturation temperature has not substantially changed after anhydrides modification, it can be considered that the structure of collagen as a triple helix form was not substantially changed. The change of the temperature could be an effective treatment for adjusting

Figure 3. Plot of size distribution by intensity for unmodified collagen and DMA (a) modified collagen solutions (0.75 wt %); unmodified collagen and CTA (b) modified collagen solutions (0.75 wt %).

Table 5. Viscometric Data for Unmodified and AnhydrideModified Collagen: [η]A, [η]B, and R2 (Correlation Factor) reduced viscosity

inherent viscosity 2

sample name

[η]A (dL/g)

R

collagen DMA 7.2 DMA 8.2 DMA10.3 CTA 5.6 CTA 8.4 CTA 9.6

0.091 7.39 6.78 7.03 5.27 6.84 9.42

0.986 0.998 0.999 0.995 0.999 0.999 0.997

[η]B (dL/g)

R2

0.102 8.97 8.34 8.52 6.62 8.67 11.35

0.985 0.993 0.987 0.979 0.998 0.995 0.962

collagen) indicate. The increase in collagen dimensions in the solution according to SLS, DLS, and intrinsic viscosity (Tables 4 and 5) is very high. Such drastic changes may be the result of both modification of terminal amine groups and also partial cross-linking of a few molecules after modification with anhydrides. At the concentration used for molecular weight and particle size determination, the partial cross-linking does not affect the solubility; all solutions are clear and transparent. Vasilev et al. confirmed that solutions are prone to gelation and at a temperature of 35−40 °C collagen is irreversibly destroyed as a result of the denaturation of the dissolved protein.52 Generally, the increase of temperature induces structural transition in the collagen network which results in a shrinkage of the protein structure. 3872



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Figure 4. Plots of relative viscosity (ηrel) versus temperature for various modified collagen solutions and for unmodified collagen.

collagen viscosity for casting and filling according to the results of other authors.59 3.6. Thermal Analysis Results. The thermal analyses results contribute to a more efficient characterization of primary and secondary structure of collagen containing materials. The relevance of thermal stability evaluation and decomposition products results from the increased interest of using collagen materials in various applications and the possibility to distinguish between various kind of collagen by using thermal characteristics, the TG/DTG/DTA curves being considered as materials “fingerprints”.60,61 Coupling these techniques with FT-IR/MS spectra to analyze volatile products brings new insights on structure modification and material characteristics, as their high sensitivity to the changes in collagen structures is known. Collagen is characterized by a polypeptide sequence of glycine-proline-X and glycine-X-hydroxyproline, where the X component is a different amino acid, other than the three amino acids mentioned and by high hydroxyproline content. The proline aminoacid is pyrrolidine-2-carboxylic acid. The collagen proteins are most composed from α-amino acids in L configuration of which thermal stability differs from acid to acid. Generally, the thermal decomposition of collagen starts around 250 °C, but amounts of undecomposed aminoacids can been detected at high temperatures.62 The thermogravimetric curves for collagen and anhydridemodified materials (Figure S2 in the Supporting Information) indicated the presence of two thermal processes of mass loss. The thermal behavior of materials is primarily dictated by the behavior of collagen. It is know from the literature that the thermal stability of proteins, including collagen, is governed by interchange of hydrophobic and electrostatic interactions between the polymer chains. The first step of mass loss corresponds to two overlapped processes, including the denaturation of collagen and elimination of the free and bonded water existing in the material. On the basis of the initial decomposition temperature summarized in Table 6, it can be concluded that the chemical modification of collagen with CTA or DMA increased the

Table 6. Thermal Characteristics of Collagen-Based Samples second process

sample collagen CTA 9.6 DMA 10.6

first process Tmax, °C

Tonset, °C

Tmax, °C

Tendset, °C

mass loss, % (30−570 °C)

71 82 77

262 273 270

326 318 326

375 373 371

72 69 72

thermal stability of the materials in the solid state. Also, the collagen modified with anhydrides registered an improved thermal stability than itaconic anhydride (70 °C) and maleic anhydride (80 °C).14,16 The second process of thermal degradation included sequential decomposition reactions superposed in multiple mass loss steps on a extend temperature range. Making a comparison between the onset temperatures (Tonset), higher values of 273 and 270 °C was obtained for modified products in the second process (compared to the onset for unmodified collagen of 262 °C) signifying a higher heat resistance. All the samples presented similar residual mass with a slight increase of the residue for DMA 10.6 due to the presence of dimethyl groups in the structures, which usually increase the thermal stability of the materials which containing them. Also, the CTA 9.6 sample was individualized by the existence of three peaks on the TG curve with peak temperature at 316, 324, and 336 °C which indicate either that the thermal decomposition of the CTA 9.6 occurs in three really quick steps with formation in the end of the first stage of derived compounds weakly in terms of thermal stability which will degrade with increasing the temperature or that the sample could contain isomers as it was shown in the reaction scheme (Scheme 2a). 3.6.1. Analysis of Volatile Thermal Decomposition Compounds. The evolution of FT-IR absorption bands arising from volatile compounds involved during the thermal analysis was collected as a 3D data cube. The identification of the volatile compounds has performed using two databases for bands assessment in FT-IR and MS spectra,63,64 and they reflect 3873



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Figure 5. In situ FT-IR spectra of volatile thermal decomposition products resulted at various temperatures from collagen (a) and anhydridemodified collagen: CTA 9.6 (b) and DMA 10.6 (c).

An increase of volatile compounds containing −OH bond emission with increasing temperature was characteristic for all samples. The thermal degradation starts with the denaturation of triple helix of collagen, process without mass loss but with loss of free water molecules continues with the release of bonded water. This phase is observed on FT-IR spectra by the appearance of absorption bands characteristic for −OH bond vibration (Figure 5) and MS spectra by increasing the m/z 18 signal and the ionized fragment at m/z 17 (Figure 6). New absorption bands on collagen FT-IR spectra recorded at 260 °C are attributed to amide and amine ionized fragments, also the new double bonds with absorption in infrared range at 929 cm−1 and 718 cm−1 are attributed to a trans νCH of (CH CH)2 group and ionized fragments on MS spectra with m/z 52 ÷ 54. Absorption band at 964 cm−1 attributed to the vibrational band of NH from ammonia and at 670 cm−1 shifted to 718 cm−1 with temperature was responsible to the CH symmetric from acetylene. The thermal decomposition of collagen starts with the cleavage of the weak bond of polypeptide sequences, followed by a series of chain reaction which will cause uncontrolled cleavage of polypeptide chains and formation of new compounds as a result of recombination reaction of these fragments. Polypeptide molecules decomposed by several pathways through dehydration, decarboxylation, decarbonylation, deamination, and desulfurization of molecules, accompanied by emission of H2, H2O, CO2, NH3, and H2S. The amount of volatile compounds and the rate of decomposition depend on α or β aminoacid type in the

different pathways of degradation for anhydride-modified materials compared with unmodified collagen. Following a primary analysis of the FT-IR spectra (Figure S3 in the Supporting Information), it can be remarked that the emission of gaseous phase took place on a well-defined temperature range for the collagen-modified materials starting after 7 min for water release and 23 min for thermal decomposition of CTA 9.6 sample and after 26 min for DMA 10.6 sample. The volatile compounds emission from collagen decomposition took place even at the beginning of heating in a wider temperature range which could be explained by the existence of different sequences of amino acids that form the polypeptide chains which decompose independently. It can conclude that anhydride-modified collagen samples are more thermally stable than native collagen. Because collagen has a non-homogeneous distribution of the aminoacids and the fragments with methyl groups and aromatic nucleus manifest a raised stability, the temperature caused cleavage of the protein in unequal fragments of polypeptide and derived structures. Selective spectra as 2D spectra were extracted from 3D spectra at 40, 60, and 260 °C and at maximum temperature of decomposition specific for each sample (Figure 5). A high amount of volatile compounds with −OH bonds were released during the thermal decomposition at low temperature, identified as belonging to the water from the collagen. The weak vibrational bands between 3800 and 3900 cm −1 corresponded to −OH stretching bond, and the board band from 3531 to 3060 cm−1 included overlapping symmetric and asymmetric stretching vibration of the −OH group. 3874



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Figure 6. continued

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Figure 6. MS spectra of the volatile compounds evolved during the thermal decomposition of collagen-based samples.

collagen structure.65 From this point of view, serine has a better stability compared with glycine or alanine due to the presence of hydroxymethyl groups. Due to the composition of collagen in aminoacids with different thermal stability, the decomposition reactions followed several pathways which may lead to fragmentation of whole molecule or scission of functional groups. The thermal decomposition reactions were responsible for producing significant quantities of carbon dioxide and ammonia. However, the volatile compounds detected during the pyrolysis (each compound was assigned considering the most probable ionized fragments according to literature data) of collagen material were carbon dioxide (only the most important m/z values are given in the following) (m/z 44), ammonia (m/z 15), H2 (m/z 2), CH4 (m/z 16), C2H4 (m/z 28), C2H6 (m/z 30), C3H6 (m/z 42), CH2CCH2 (m/z 40), CH3CCH (m/z 40), CH2 CHCH2CH3 (m/z 56), HCN (m/z 27), CH3CN (m/z 41), CH5CN (m/z 43), CH2CHCN (m/z 53), acetone, acids, alcohols, amides, nitriles, and small amounts of pyrrolidine (m/ z 71). Differences in thermal decomposition products evolution from unmodified collagen and anhydride-modified collagen appeared in the evolution curves of ionized fragment with m/z below 53 (Figure 6a−j); at higher m/z values, the evolution curves overlapped.

Under temperature action, the maleic anhydrides decompose by homogeneous unimolecular reactions in acetylene, carbon monoxide, and carbon dioxide, in equimolar amounts (Table S3 in the Supporting Information). CTA thermally decomposed over 450 °C giving carbon dioxide, carbon monoxide, and propyne (on MS spectra the m/ z 40 and its ionized fragments and on FT-IR spectra at 2254 cm−1 for CC and CH3 at 1456 cm−1) which underwent some polymerization to trimethylbenzenes.66 It have been noticed that speeding up the thermal decomposition lead to emission of large quantities of carbon dioxide and compounds with functionalities that appeared at lower wavenumber then 1750 cm−1. The band stretching at 2360 cm−1, the asymmetric stretching vibration at 2350 cm−1, as well the deformation vibration at 667 cm−1 confirmed the elimination of CO2. The main volatile compounds identified during the thermal decomposition of the anhydride-modified collagen samples are: acetaldehyde, ammonia, acetic acid, formic acid, methylamine, acetone, alcohols (ethanol, methanol, isopropyl alcohol) and several amides as formamide, acetamide, N-methyl acetamide, propion amide and nitriles. At 260 °C the anhydride-modified materials presented in MS spectra a high emission of CO2 and additional volatile compounds with m/z 26 assigned to C2H2 fragment (DMA 10.6) and m/z 27 for C2H3, m/z 28 for CO, 3876



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lead to the formation of fluid filled pockets which are ideal for bacterial proliferation. A uniform adherence will not only reduce infection but can also reduce pain and promote wound healing.67 Understanding the interaction between collagen based material surfaces and tissue is critical, and control of wound dressing−tissue surface interactions continues to be an important factor for consideration in the design of biocompatible surfaces. The adhesion tests evidenced the highest values of the work of adhesion for DMA 10.6 and CTA 9.2 materials to which were registered a maximum ∼500 mm mN with respect to 234 mm mN for unmodified collagen (Figure 7).

C2H4, and m/z 29 for C2H5, CHO fragments (CTA 9.6). The evolutions of the ionized fragments from polypeptide groups in MS spectra versus time are represented in Figure 6a−j. At the final temperature of decomposition on mass spectra were recorded volatile compounds with m/z 91 for C7H7 (tropolium ion) produced from cleavage of benzene (m/z 78) or pyrrolidine (m/z 71) ring and also the ionized fragment at m/z 92 (C7H8). Particularities in evolution curve patterns of volatile compounds were observed for m/z 16 (Figure 6b) CTA 9.6 sample. The emission of the m/z 16 ionized fragment took place in a very low amount up to 30 min, and then, the evolution was intensified to high temperature, while the emission of the ionized fragment from volatile compounds with m/z 17 occurred in two distinct steps with peaks at 33 and 50 min, respectively. The m/z 17 versus time curves (Figure 6c) attributed to ionized fragments from water, ammonia, and formamide presented different emission profiles during the decomposition of samples. Thus high amounts of free and bound water were emitted after 9.3 min for collagen and DMA 10.6 and after 33.6 min due to the decomposition reactions with release of water molecules, ammonia, and formamide.The curve of CTA 9.6 shown a second peak at 50 min of evolution time. Two steps of evolution of the ionized fragments with m/z 18 (Figure 6d) assigned mainly to water loss were found with peaks at 9.5 and 31 min for collagen and DMA 10.6 and with a little retarded evolution for CTA 9.6, the peaks at 10.5 and 34 min, respectively. A retarded evolution with 1 or 2 min of the ionized fragments with m/z 22, 38, 39, and 44 (Figure 6e−g, Figure 6i) was also characteristic to CTA 9.6. The m/z 42 could be assigned to ionized fragments from acetone and acetamide, and it has a particular evolution for each sample. A curve with two peaks at 34 and 45 min was obtained for evolution of these compounds from collagen, while a curve with a single peak at 45 min was characteristic for anhydride-modified collagen. The CTA 9.6 modified collagen sample had also a supplementary peak at 26 min in the evolution curves of the fragments with m/ z 46 (Figure 6j). The evolution of other ionized fragments presented a maximum of evolution at 35−43 min, and it was mentioned that no differences appeared between samples. It is worth mentioning that from all these evolution curves the collagen sample modified with citraconic anhydride showed a particular behavior, very complex, and this can be due to the possibility to obtain according to the reaction scheme of two isomers (Scheme 2). Analysis of the volatile products evolved from substituted modified collagen indicates both retardation in time of evolution start of various products because of increased thermal stability evolution curves show particular characteristics for each kind of anhydride used and also new products (m/z 22, 38, 39, and 46 for CTA 9.6) are formed as a consequence of chemical modification of collagen. 3.7. Adhesion Test Results. Adherence has been considered an important requirement for an ideal skin substitute. Adherence more than any other factors shows the importance of this requirement and a wound covering which does not readily adhere to the wound surface may even greatly diminish other properties. A temporary skin substitute should adhere rapidly to the dry and wet wound surface with sufficient strength to resist lifting and slipping. Adherence must be intimate and uniform because small areas of nonadherence will

Figure 7. Work of adhesion for anhydride-modified collagen samples.

Therefore, the adhesion was improved for the anhydride modified collagens with a higher reactivity. Nishad Fathima et al.68 showed that work of adhesion can be used as indicator for the stabilization of collagen and an increase in the rate of work of adhesion would promote adsorption while a decrease leads to hindered adsorption. Their findings are in accordance with our results

4. CONCLUSIONS Two substituted anhydrides (2,3-dimethylmaleic anhydride (DMA), citraconic anhydride (CTA)) have been used to bring new modifications in the soluble collagen structure. The changes in the structure of modified collagen were assessed by free amino groups determination, potentiometric titration and TOC/TN analyses, and FT-IR and 1H NMR spectroscopy. An increased reactivity to collagen was registered for DMA compared to CTA. Modified collagen have an increased viscosity and molecular weight in solution compared to unmodified collagen, indicating that shape and dimensions of collagen macromolecules were changed after modification. Second virial coefficient, particle size analysis, and zeta potential of the modified collagen show increased values with respect to unmodified collagen because of modification and also some aggregates formation by stronger interactions involving carboxylic groups after anhydride moiety incorporation. Denaturation temperature in acidic medium was decreased with increasing substitution degree. The chemically modified collagen with anhydrides presented improved thermal stability in the solid state with a particular pathway of decomposition for each individual sample. The main compounds resulting from the thermal decomposition were identified in FT-IR and MS spectra as amide, amine, 3877



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(11) Scholz, M. T. Modified collagen compound and method of preparation. U.S. Patent 4,883,864, Nov 28, 1989. (12) Bulcke, A. V. D.; Bogdanov, B.; Rooze, De N.; Schacht, E.; Cornelissen, M.; Berghams, H. Structural and Rheological Properties of Methacrylamide Modified Gelatin Hydrogels. Biomacromolecules 2000, 1 (1), 31−38. (13) Doillon, C. J.; Drouin, R.; Cote, M. F.; Dallaire, N.; Pageau, J. F.; Laroche, G. Chemical inactivators as sterilization agents for bovine collagen materials. J. Biomed. Mater. Res. 1997, 37 (2), 212−221. (14) Potorac, S.; Popa, M.; Maier, V.; Lisa, G.; Verestiuc, L. New hydrogels based on maleilated collagen with potential applications in tissue engineering. Mat. Sci. Eng. CBio. S. 2012, 32, 236−243. (15) Potorac, S.; Popa, M.; Verestiuc, L.; Cerf, D. Le. New semi-IPN scaffolds based on HEMA and collagen modified with itaconic anhydride. Mater. Lett. 2012, 67, 95−98. (16) Potorac, S.; Popa, M.; Picton, L.; Dulong, V.; Verestiuc, L.; Cerf, D. Le. Collagen functionalized with unsaturated cyclic anhydridesinteractions in solution and solid state. Biopolymers 2014, 101, 228− 236, DOI: 10.1002/bip.22319. (17) Trubetskoy, V. S.; Hagstrom, J. E.; Budker, V. G.; Wolff, J. A.; Rozema, D. B.; Monahan, S. D. Compositions and methods for drug delivery using pH sensitive molecules. U.S. Patent 6,919,091 B2, Jul. 19, 2005. (18) Dixon, H. B. F.; Perham, R. N. Reversible blocking of amino groups with citraconic anhydride. Biochem. J. 1968, 109 (2), 312−314. (19) Puigserver, A.; Desnuelle, P. Dissociation of Bovine 6S Procarboxypeptidase A by Reversible Condensation with 2, 3Dimethyl Maleic Anhydride: Application to the Partial Characterization of Subunit III. Proc. Natl. Acad. Sci. U.S.A. 1975, 72 (6), 2442− 2445. (20) Fischer, A.; Schwarz, A.; Wandrey, C.; Bommarius, A. S.; Knaup, G.; Drauz, K. Preparative-scale enzyme-catalyzed peptide synthesis using solubilizing N-terminal protecting groups. Biomed. Biochim. Acta 1991, 50 (10−11), 169−174. (21) Davidson, R. J.; Cooper, D. R. Intermolecular Relationship between Neutral-salt-soluble and Acid-soluble Collagen. Nature 1968, 217, 168−169. (22) Davison, P. F. Solvents for collagen. U.S. Patent 4,983,721, Jan 8, 1991. (23) Albu, M. G.; Titorencu, I.; Ghica, M. V. Biomaterials Applications for Nanomedicine; Pignatello, R., Ed.; InTech: Europe, 2011; Chapter 17, pp 333−358. (24) TNBS (2,4,6-trinitrobenzene sulfonic acid); 1% Solution Safety Data Sheet. Fed. Regist. 2012, 77, 58. (25) Sugimura, H.; Moriguchi, T.; Kanda, M.; Sonobayashi, Y.; Nishimura, H. M.; Ichii. T.; Murase, K.; Kazama, S. Molecular Densities of Aminosilane Self-Assembled Monolayers as Estimated by Chemical Labeling. http://www.mtl.kyoto-u.ac.jp/groups/sugimura-g/PDF/ Sugimura-LB13P100.pdf (accessed Dec 22, 2013). (26) Gao, A.; Yang, X.; Zhang, C.; Long, G.; Pu, J.; Yuan, Y.; Liu, H.; Li, Y.; Liao, F. Facile spectrophotometric assay of molar equivalents of N-hydroxysuccinimide esters of monomethoxyl poly-(ethylene glycol) derivatives. Chem. Central J. 2012, 6, 142. (27) Chang, R. C.; Olsen, D. R.; Polarek, J. W.; Williams, K. E. Method for producing double-crosslinked collagen. U.S. Patent 0207,671, Aug 25, 2011. (28) Hinterwirth, H.; Strobl, M.; Al-Dubai, H. Analytical methods for detection of small amounts of amino groups on solid surfaces: a survey. Monatsh. Chem. 2010, 141, 291−299. (29) Ofner, C. M.; Budnis, W. A. Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices. Pharm. Res. 1996, 13 (12), 1821−1827. (30) Zhang, M.; Li, G. Partial characterization of fish skin collagen cross-linked by N-hydroxysuccinimide activated adipic acid. J. Aquat. Food Prod. Tech. 2013, DOI: 10.1080/10498850.2012.693578. (31) Pinto Zarth, C. S.; Fras Zemljič, L.; Č akara, D.; Bračič, M.; Pfeifer, A.; Kleinschek, K. S.; Heinze, T. Charging Behavior and Stability of the Novel Amino Group Containing Cellulose Ester

nitriles, ammonia, aldehyde, acids, alcohols, and other ionized fragments of these compounds. Such modified collagens have enhanced solubility in acidic media and high reactivity and could have potential medical applications as such or as hydrogels because of improved adhesion to the tissues used for wound dressing.

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ASSOCIATED CONTENT

S Supporting Information *

(Figure S1) Concentration dependence of reduced viscosity on collagen solutions. (Table s1) Values obtained through fractional viscosity and the Boltzmann equation. Figures S2 and S3 and Tables S2 and S3 show some data concerning the thermal behavior of the collagen-based materials. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +40 232 217454. Fax: +40 232 211299. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial supported by Romanian ANCS UEFISCDI through bilateral collaboration Romania− Slovenia, “Functionalization of synthetic polymers for development of new antimicrobial packaging” 525/2012.

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REFERENCES

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