Network structure studies on γ–irradiated collagen–PVP superabsorbent hydrogels

Author’s Accepted Manuscript Network structure studies on γ–irradiated Collagen–PVP superabsorbent hydrogels Maria Demeter, Marian Virgolici, Catalin Vancea, Anca Scarisoreanu, Madalina Georgiana Albu Kaya, Viorica Meltzer www.elsevier.com/locate/radphyschem

PII: DOI: Reference:

S0969-806X(16)30428-5 http://dx.doi.org/10.1016/j.radphyschem.2016.09.029 RPC7286

To appear in: Radiation Physics and Chemistry Received date: 6 January 2016 Revised date: 19 September 2016 Accepted date: 24 September 2016 Cite this article as: Maria Demeter, Marian Virgolici, Catalin Vancea, Anca Scarisoreanu, Madalina Georgiana Albu Kaya and Viorica Meltzer, Network structure studies on γ–irradiated Collagen–PVP superabsorbent hydrogels, Radiation Physics and Chemistry, http://dx.doi.org/10.1016/j.radphyschem.2016.09.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Network structure studies on γ–irradiated Collagen–PVP superabsorbent hydrogels Maria Demetera,c, Marian Virgolicib, Catalin Vanceaa, Anca Scarisoreanua, Madalina Georgiana Albu Kayad, Viorica Meltzerc* a

National Institute for Lasers Plasma and Radiation Physics, 409 Atomiştilor Street, 077125,

Măgurele, Romania b

Horia Hulubei National Institute of Physics and Nuclear Engineering, 30 Reactorului,

077125, Măgurele, Romania c

University of Bucharest, Faculty of Chemistry, 4-12 Regina Elisabeta Street, 030018,

Bucharest, Romania d

Leather and Footwear Research Institute - INCDTP, Collagen Department, 93 Ion Minulescu

Street, 031215, Bucharest, Romania *

Corresponding author. Tel.: +4/021.314.35.08; fax: +4/021.315.92.49. viomel@gw-

chimie.math.unibuc.ro (V. Meltzer) ABSTRACT Collagen–polyvinylpyrrolidone (PVP) superabsorbent hydrogels were synthesized by γ– irradiation in the absence of oxygen, using high molecular weight PVP and acidic collagen Type I. Sol–gel analysis and swelling experiments were performed in order to determine the gel fraction, network parameters, the yield of cross–linking, respectively scission, as well as to establish the diffusion characteristics of water. Rheological experiments and characterization of the chemical structure before and after irradiation were conducted in order to evaluate the gel character and its stability upon irradiation. The relationship between these parameters and radiation dose was also established. Gel fraction reached up to 90 %, and the p0/q0 ratio (degradation vs. cross–linking ratio) shows a negligible degradation process. The collagen–PVP hydrogels present swelling in the range 1000 – 2000 %, the diffusion exponent (n) was found to be between 0.59 – 0.68. The network parameters as the molecular weights between two successive cross links ( ( ) are ranged between 3.39

), the cross–linking density ( ) and the mesh size

8.08

, 1.24

2.95

respectively 75–134 nm. Keywords: collagen, polyvinylpyrrolidone, radiation cross–linking, hydrogels 1

,

1. Introduction Up to date, few studies regarding the collagen–PVP interaction in solution or in solid state has been carried out (Sionkowska, 2003). The evaluation of surface properties and composition after UV irradiation has been presented by (Sionkowska et al., 2004; Sionkowska et al., 2006). Gamma or electron beam irradiation of collagen–PVP copolymers has been generally related to changes on the physicochemical structure (Leyva-Gomez et al., 2014) or irradiation effects on its bulk structure (Dumitraşcu et al., 2011). Chen et al. evaluated the potential of collagen–PVP hybrid as a hydrogel with anti–inflammatory properties for wound healing (Chen et al., 2013). In terms of radiation chemistry, a hydrogel synthesized by radiation processing techniques, has to present a minimal condition if the cross–linking process predominates – to become an insoluble gel (Chapiro, 1964; Rosiak et al., 1995). To be a superabsorbent hydrogel, the material has to absorb large amounts of water, saline solution or physiological fluids as high as 10 – 1000 times their own weight, owing to the considerable number of hydrophilic groups in their structure (Buchholz, 2002). Because of their unique properties, superabsorbent hydrogels are suitable for use as wound dressings, disposable diapers or as a scaffold for tissue engineering and as a drug delivery release matrix (Buchholz and Peppas, 1994). Collagen based superabsorbent hydrogels have been produced typically by mixing the collagen with other water–soluble polymers and chemical cross–linking agents, in order improve the gel fraction, cross–linking density, swelling capacity and the mechanical stability (Marandi et al., 2011a; Marandi et al., 2011b; Pourjavadi et al., 2006a; Pourjavadi and Kurdtabar, 2007; Pourjavadi et al., 2006b; Pourjavadi et al., 2006c; Sadeghi and Ghasemi, 2012; Sadeghi and Hosseinzadeh, 2010a, b). In previous studies, we reported the obtaining of collagen–PVP gels by electron beam irradiation at high dose rate in presence of oxygen. The main drawback of this approach is the quality of the obtained gel (soft, with low swelling power, as well as with a high yield of degradation). This is probably due to irradiation 2

geometry and the high dose rate used to process it (Dumitraşcu et al., 2011). A collagen–PVP system was used as treatment for posmastectomy sequelae in women with breast cancer (Ruiz-Eng et al., 2010). Chimal–Monroy et al. evaluated the effect of γ–irradiated collagen– polyvinylpyrrolidone (PVP) implants on bone regeneration (Chimal–Monroy et al., 1998). None of the above mentioned studies refers to collagen–PVP system properties or characterize it in terms of radiation–induced processes that occur during irradiation cross–linking. Moreover, collagen–PVP superabsorbent hydrogels synthesized by γ–irradiation in absence of oxygen and at moderate radiation dose rate with a high degree of swelling, high gel fraction, low degradation vs. cross–linking ratio and a stable chemical structure have not been reported. In order to optimize the gel production for particular final use, such as the integration of biomacromolecules or drugs in hydrogel, a good understanding of the relationship between the absorbed dose and the final gel properties is required. The aim of this work is to synthesize collagen–PVP superabsorbent hydrogels without adding chemical cross–linkers and to characterize their cross–linked structure in terms of network parameters correlated with the absorbed dose. The yield of cross–linking and scission, swelling, rheological behaviour, water diffusion and chemical structure as a function of the absorbed dose will be investigated, and a reaction mechanism will be proposed as well. 2. Experimental 2.1. Materials and Synthesis of collagen–PVP hydrogels Collagen gel (2.1%) type I was obtained from calf skin by the technology currently used at the Leather and Footwear Research Institute, Bucharest, Romania. The average molecular weight of acidic collagen gel type I was found to be 300 kDa (Trandafir et al., 2007). PVP 5% solution was obtained by dissolving 5 g of PVP 360 (Sigma Aldrich Co., with average molecular weight ̅ = 3.6

), in 95 ml deionised water at 80ºC, under

magnetic stirring until complete dissolution, then cooled down to the room temperature. In

3

order to prepare collagen–PVP solution, equal volumes of 2.1% collagen gel and 5% PVP 360 solutions were mixed and stirred at room temperature until complete homogenization to form the substrate for irradiation. For γ–irradiation the collagen–PVP mixture was placed in 10 ml plastic syringes and before irradiation each sample was carefully degassed and tightly sealed in order to avoid advanced degradation because of oxygen content. Gamma irradiation was performed at the Multipurpose Irradiation Facility-IRASM gamma irradiator (SVST Co–60/B type supplied by Institute of Isotopes co. Ltd. Budapest) from the Horia Hulubei National Institute of Physics & Nuclear Engineering (IFIN-HH). The absorbed dose and dose rate were measured with the ECB/oscillometry dosimetry system. The used dose rate was 1.1 kGy/h and the absorbed doses were in the 5 – 50 kGy range. 2.2. Sol–gel analysis Hydrogel samples of 10 mm diameter were cut into pieces of 4–5 mm thickness. The hydrogels were dried in a vacuum oven to constant weight then immersed in deionised water for 48 h at constant room temperature (25 C). After 48 h, the gels were dried again in vacuum oven at constant temperature to constant weight. The gel fraction was calculated as the average of three determinations. The sol and gel fraction were calculated as: .................................................................................................................................(1) ( )

...................................................................................................................(2)

where the gel fraction (G%) is the ratio of the dry gel weight after washing out the sol content. Wi is the initial weight of dried sample after irradiation and Wd is the weight of the dried insoluble part of sample after extraction with water. .................................................................................................................(3) After reswelling, the swelling degree (SD) has been calculated as a function of the dry (Wd) and swollen (Ws) gel weights, using equation (3). 4

2.3. Diffusion of water The water diffusion characteristics of the superabsorbent gel were evaluated using the data obtained from the swelling experiments. Only the results obtained up to 60 % of maximum swelling (initial stage of swelling) were processed according with the Fick’s law. The dependence of the amount of water absorbed in hydrogels,

, on time , was used for

analysis of water diffusion. Using the kinetics of swelling, plots of , versus

, against

and

were drawn. Diffusion constants ( ), diffusion exponents ( ) and

diffusion coefficients ( ) were calculated from the slops and intercepts of the lines. 2.4. Rheological analysis In order to determine the elastic moduli of the gels ( – elastic modulus and

– viscous

modulus), oscillatory rheological measurements were performed. All measurements were performed at 25oC in the linear viscous elastic region, at the rate of deformation and the frequency range of

, using a Thermo MARS II Rheometer

equipped with a 20 mm diameter plate (plate geometry). The gap size was set 0.139 mm. 2.5. ATR-FTIR Spectroscopy Fourier transform infrared (FTIR) spectra of unirradiated and irradiated samples were taken with a Spectrum 100 instrument (Perkin Elmer, USA). After the sol fraction was removed, the hydrogels samples were dried out until constant weight and used for FTIR investigations. Spectra were acquired in ATR mode and each spectrum consisted of 20 scans / sample, in the 4000 600

wavenumbers range at a resolution of 4

.

3. Results and Discussion 3.1. Mechanism of radiation cross–linking of collagen–PVP hydrogels In dilute aqueous polymer solutions, the ionizing radiation is primarily absorbed by water, leading to the production of hydroxyl radicals ( e.g.

,

and

. The

and

), hydrogen atoms (

) and other species,

radicals are the main reactive species responsible for

5

macroradicals formation, mainly by H abstraction. The main radicals and their chemical structure generated during irradiation of PVP aqueous solution where first proposed by (Davis et al., 1981; Rosiak et al., 1990) and later revisited by (An et al., 2011). A more complex reaction mechanism of PVP structural modification during its irradiation in dilute aqueous solution has been presented by (Sabatino et al., 2013). After the aqueous collagen solution is irradiated, it is assumed that the initiate the formation of collagen–radicals. The

and

radicals

radical extracts a hydrogen atom,

resulting in the formation of a water molecule (Trandafir et al., 2007). The hydrogen atom extracts from the collagen molecule another hydrogen atom, resulting a

molecule, which

appears in the form of gas bubbles during gelation process. When the collagen/PVP/water system is exposed to γ–radiation, a series of macroradicals are formed from collagen and PVP molecules, which give rise to the free–radical recombination reactions. The structures of these radicals and the proposed mechanism pathway of the collagen–PVP cross–linking are shown in Scheme 1. Reaction 1 shows the general mechanism of radiolysis of water. The most stable radicals structures resulted after PVP radiolysis in aqueous solution are shown in Reaction 2. Reaction 3 shows the mechanism for the formation of a collagen–radical. The reactions 4 and 5 show the specific free–radical recombination reactions of collagen and PVP radicals and their cross–linked networks. Based on the reaction mechanisms proposed for cross–linking of collagen (Inoue et al., 2006; Zhang et al., 2012) and collagen–dextran system by irradiation with γ–radiation (Zhang et al., 2015), herein we propose a cross–linking mechanism for the collagen–PVP system (Reaction 6). The reaction takes place by free–radical recombination of one PVP–located radical with one collagen–located radical, resulting in a covalent bond between PVP and collagen macroradicals. These bonds act to stabilize the triple helix structure of collagen that is most susceptible to degradation.

6

3.2. Sol-gel analysis For radiation cross–linking of natural and synthetic polymers, the sol–gel analysis allows to estimate parameters such as yield of cross–linking

and scission

, gelation dose (

) as

well as to correlate these parameters with other physico–chemical properties (Gulrez et al., 2011). By irradiation of polymers blends with ionizing radiation, cross–linking and chain scission reactions are usually observed (IAEA-TECDOC-1324, 2003). Fig.1a depicts the evolution of the gel fraction as a function of absorbed dose. The gel fraction is relatively high even at low doses (at 5 kGy it exceeds 60 %) and it increases with absorbed dose. For calculation of the gelation dose

, the Charlesby–Pinner and a modified version of it

(Olejniczak et al., 1991; Rosiak, 1998) equation were used. In order to avoid inaccuracies resulting from the unknown molecular weight distribution of the used polymers, the Charlesby–Rosiak equation (Eq. 4) is used: (

√

)(

)..............................................................................................(4)

where

is sol fraction,

is degradation density,

dose,

is gelation dose and

is cross–linking density,

is radiation

is the virtual dose (the dose necessary to transform the real

sample into a sample of the most probable molecular weight distribution of (Charlesby, 1991). The gelation dose,

= 2)

ratio (degradation vs. cross–linking ratio) and

were calculated according to Eq. 4, using a freely available computer software that has implemented both the Charlesby–Pinner and Charlesby–Rosiak equations (Kadlubowski et al., 2010). The

based on the Charlesby–Pinner equation was found

0.35, while for

the Charlesby–Rosiak equation this value was equal to 0.14 (Table 1). As can be seen in Fig. 1b, a good linear fit in terms of equation 4 is obtained (R2 = 0.99). A higher value of

ratio indicates the presence of a degradation process. In our case, the

was found to be 0.14, which indicates that degradation process is almost negligible.

7

Since cross–linking and scission occurs simultaneously in the polymer during irradiation, the yield of cross–linking,

and degradation

result of irradiation. We calculate the method allows obtaining the

, offer important information about the final

and

from equilibrium swelling experiments. This

value without any information on the polymer molecular

weight. It is also applicable to the more complicated systems, as in our case, where subsequently cross–linking and chain scission occurs when the aqueous polymer solution is irradiated. A cross–linked gel swells to a given extent, depending on the concentration of effective chains ( ), which is related to the average molecular weight between successive crosslinks (Rosiak et al., 1988). The

is the number of crosslink bonds expressed in units of

and is used to measure the radiation–chemical yields. In order to estimate the yield of cross–linking

and degradation

, equations 5 and 6 were used. The

and

values

calculated for collagen–PVP hydrogels are presented in Table 3. (

) (

).....................................................................................(5)

...................................................................................................................(6) Previous studies reported a

value of

obtained for PVP hydrogels in

argon saturated solutions (Benamer et al., 2006). For collagen–PVP system irradiated in aqueous solution no data about the cross–linking yield value could be found. In our case, the maximum value of

was found to be

for 5 kGy and decreases with

the absorbed dose. The degradation yield was more than one order of magnitude lower than the yield of cross–linking, also decreasing with the absorbed dose (Table 3). This is expected, as the cross–linking density ( ) increases with the absorbed dose, leading to a denser hydrogel structure, therefore leaving less opportunities to create new covalent bonds; and, on the same time, less chances for the chain scission reactions to generate degradation. 3.3. Swelling experiments

8

In order to evaluate the absorption capacity, network structure and to calculate the effective crosslink density of hydrogels, the swelling properties were investigated using deionised water. The swelling experiments were carried out until the swelling degree reached a constant value for each sample irradiated at 5–50 kGy. The swelling degree of collagen–PVP hydrogels vs. immersion time in water is presented in Fig. 2. The collagen–PVP hydrogels reached the equilibrium state after 700 minutes, thus suggesting the formation of a dense network structure, as well as formation of a stable hydrogel. The swelling degree decreased with the increase of absorbed dose, which suggests that the cross–linking density increases with the increase of absorbed dose in the investigated hydrogels. 3.4. Diffusion of water The study of water diffusion phenomena in superabsorbent hydrogels is of great interest as it clarifies polymer behaviour in the field of hydrogels applications like biomedicine, pharmaceutical or tissue engineering. This study was conducted in order to support future studies aiming to integrate biomacromolecules or drugs in hydrogel matrix based on collagen and PVP. The diffusion in a cross–linked hydrogels involves the migration of water molecules into pre–existing or dynamically formed spaces in the hydrogel network. Briefly, the swelling process consists in an increase of the distance between hydrogels chains (Karadag et al., 2002). In order to determine the nature of diffusion of water in collagen-PVP hydrogels, the following equation was used: .........................................................................................................................(7) where

and

are the amount of solvent diffused into the hydrogel at the time

equilibrium condition;

and in

is a constant related to the macromolecular network and the exponent

is a number used to characterize the type of diffusion, showing the transport mechanism (Ritger and Peppas, 1987). Usually, for cylindrical shapes, if diffusion is Fickian, while for

is between 0.4 and 0.5,

the diffusion is of a non-Fickian type. Equation 7 9

is applied to the initial step of swelling process, up to 60 % of the maximum (Karadag et al., 1997). In Figs. 3a and 4b are depicted the plots of using the kinetics of swelling. Parameters

,

, vs.

and

vs.

and the diffusion coefficients ( ) were

determined from the slops and intercepts of the lines. For a non–Fickian diffusion mechanism, the cross–linking density is high thus leading to a small amount of bulk water and a decreased diffusion rate (Hill et al., 1999; Krongauz, 2010). It is known that the high water content of hydrogels is the bulk water, which is similar with the bulk water coming from outside of the gel. In a polymeric network of hydrogels, there are, at least three types of water structures, as following: bulk water, primary and secondary bound–water. When a hydrogel is immersed in water, the first type of water that will be present is the primary bound–water, due to the hydration of hydrophilic groups of the polymer. The primary bound–water is very difficult to remove from the gel. As a result the network swells and the hydrophobic groups are exposed leading to the formation of secondary bound–water (Gulrez et al., 2011). The values of diffusion constants ( ), diffusion exponents ( ) and diffusion coefficients ( ) of the collagen–PVP hydrogels are indicated in Table 2. The values of diffusion exponents are in the range 0.59–0.68, therefore the type of water diffusion into collagen–PVP hydrogels was taken as non–Fickian. This means that the water diffusion takes place by stresses and hydrophilic interactions, as opposed to the Fickian type, which is based on a chemical gradient. The non–Fickian type of transport is specific to cross–linked hydrogels (Hill et al., 1999; Krongauz, 2010), therefore the value of diffusion exponent ( ) can be related to the drug transport mechanism in this type of hydrogels. This means that the structure of the hydrogel plays a more important role than the gradient of the drug concentration and the importance of the structure over the concentration gradient increases with the value of diffusion exponent.

10

The diffusion exponents ( ) decreased as function of absorbed dose, while the diffusion coefficient (D) increased up to 10 kGy, followed by a decrease to a lower value than in the case of 5 kGy. This phenomenon has been attributed to the ease with which water molecules can diffuse into hydrogel network. As it can be seen in Table 3, the cross–linking density increases with the absorbed dose, resulting in a denser hydrogel network, which impedes the water diffusion. 3.5. Network structure In order to characterize network structure, the most important parameters are the polymer volume fraction in the swollen state (

), the molecular weight of the polymer chain

between two neighbouring cross–links (

) and the corresponding mesh size ( ). The

polymer volume fraction in the swollen state shows the amount of fluid absorbed and retained by the gel.

, is a measure of the degree of cross–linking of the polymer. The mesh size, or

the distance between two adjacent cross–links describes the space available between the macromolecular chains which allows diffusion and movement of particles (Datta, 2007). The molecular weight between cross–links (

), was calculated through the modified swelling

theory of Flory, usable when the cross–linking takes place in solution (Equation 8). The crosslink density ( ) and the mesh size (ξ) were determined with equations 8 and 9. The crosslink density was calculated as ̅ ̅

̅

[ (

)

(Bray et al.; 1973; Peppas et al., 1976).

]

............................................................................................(8) [(

[

)

(

(

)]

)]

..................................................................................................(9)

̅ is the specific volume of the polymer, calculated as the reciprocal of density. volume of water, was taken 18.0 cm3 mol−1. taken as an average (PVP = 112.88

, the molar

is the monomeric unit of PVP and collagen, and Collagen = 321.32

11

). It is known

that a collagen monomer unit consists mainly of the following amino acid sequence, Gly-X-Y where Gly

=

Glycine, X = Proline and Y = Hydroxyproline

is the Flory characteristic

ratio. This was taken as a weighted average of the characteristic ratios of the polymers, Collagen = 9 (Cao et al., 2013), PVP = 12.3 (Su et al., 2013).

is the carbon–carbon bond

length (0.154 nm). The weight swelling ratio of hydrogels after cross–linking, calculated as:

= hydrogel mass after irradiation/hydrogel dry mass. The polymer volume

fractions in the relaxed state, after cross–linking ( [

was

(

)

), before swelling was calculated as:

] ...............................................................................................(10)

The weight swelling ratio of hydrogels after swelling

was calculated as:

hydrogel

mass after swelling/hydrogel dry mass. The polymer volume fraction of the swollen gel at equilibrium ( [

) was calculated as:

(

)

where

] ..............................................................................................(11)

and

were taken as densities of polymer and solvent. The values of

used are presented in Table 3 and the value of

was taken as

. The

densities of dry and swollen hydrogels were determined with a Mettler Toledo Density Kit for analytical balances, in deionised water at constant temperature (25 C). The experimental values of

,

,

, cross–linking density ( ) and mesh size ( ) are summarized in

Table 3. The cross–linking density ( ) is a parameter that describes the characteristics of the gel, generally increasing with the absorbed dose and ranged from . Molecular weights between two successive cross–links ( range absorbed dose. The magnitude of

and 75

) and the mesh size (ξ) are in the

134 nm respectively, and decrease with the

at high radiation dose and a dose rate of 1 kGy/h, can

probably be affected by the partial scission of the collagen–PVP back bone, as previous 12

studies have shown (Leyva-Gomez et al., 2014). The mesh size behaviour with radiation dose can be assigned with forming of low space between the macromolecular chains of the hydrogel, being an important factor for determining mechanical strength, degradability, and diffusivity of water molecules. Previous studies have shown that most hydrogels used in biomedical applications have mesh sizes ranging from 5 to 100 nm, in their swollen state (Datta, 2007). The mesh sizes obtained in this study (75

134 nm) are comparable with the diameter of protein molecules (Tsai,

2007). Given this, diffusion of biomacromolecules into and from collagen–PVP hydrogels is possible, as well as small molecular weight drugs or other pharmaceutical compounds can penetrate through collagen–PVP hydrogels. 3.6. Rheological analysis Oscillatory shear experiments and the resulting plots of

– the elastic modulus and

– the

viscous modulus are currently used to demonstrate the gel character, and to classify it as entanglement network or covalently cross–linked gel. It has been shown that covalently cross–linked gels exhibit an elastic modulus ( ) greater than the viscous modulus ( ) (Dalton et al., 1995). The oscillatory frequency sweep tests were performed in the linear viscoelastic region at frequency of moduli (

and

. The dynamic elastic and viscous

) as function of angular velocity ( ) for collagen–PVP hydrogels are

reported in Fig. 5a and Fig. 5b, respectively. γ–irradiated collagen–PVP hydrogels show a similar and constant elastic modulus curves in the

range. In this range a

predominant elastic behaviour was observed for all irradiated samples, which can suggest the formation of a permanent network upon γ-irradiation. The fact that the value of the elastic moduli ( ) has increased up to 25 kGy (around 100 Pa), and then at 50 kGy has decreased, usually suggests that at higher radiation dose can take place important macromolecular scissions. This is in line with the decreasing of the cross–linking yield with the absorbed dose.

13

All of these can lead to a more rigid structure, which would present lower elastic properties. The same behaviour was observed in the case of viscous moduli (

), where the magnitude

was much lower. 3.7. ATR–FTIR Spectroscopy The structural changes that appeared after γ–irradiation in the collagen–PVP hydrogels were monitored by ATR–FTIR. FTIR spectra of pure collagen and PVP are reported in Figs. 5a and b. Characteristic FTIR spectra of unirradiated and γ–irradiated collagen–PVP hydrogels are shown in Figs. 5c and d. It is well know that collagen and PVP forms miscible blends due to inter–molecular interaction through hydrogen bonding which are visible in FTIR spectra by a frequency shift (Sionkowska, 2003). The main FTIR characteristic absorption bands corresponding to collagen are: amide A at ~ 3300 1600 1700

, amide II at 1500 1550

, amide B at 3070 and amide III at 1200 1300

specific absorption bands corresponding to PVP, are: and

stretch at 2870 2950 and

,

and

stretch at ~ 3470

. The ,

stretching vibration at 1664

;

deformation vibrations of pyrrolic ring at 1370/1420/1459/1490

;

stretching vibrations of amide III band at 1265/1280 3800-2600

, amide I at

. In Fig. 5c the FTIR spectra within

range represents the specific absorption bands of the amide A and amide B

from collagen structure and stretching vibration of functional groups as

,

and

from PVP structure. The specific absorptions bands of unirradiated collagen–PVP hydrogels were found at: 3320, 3075, 2955, 2926 and 2876

. In this range of the FTIR spectra, we observed an increase

of absorption bands intensity with increasing of absorbed dose, except for those samples irradiated at 5 kGy. The most important shifting towards lower wavenumbers was found for the sample irradiated at 50 kGy, from 3320 position of band located at 3075

to 3308

. For all irradiated samples, the

has remained unchanged. The frequency of amide A

14

and amide B bands depends of collagen conformational structure. In some cases, when structural order of a protein is affected, the frequency of these bands decreases as well. When a shifting of these absorption bands towards lower wavenumbers is observed, the conformational structure of collagen is often affected by degradation of hydrogen bonds (Rabotyagova et al., 2008). In Fig. 5d is shown a detailed FTIR spectrum in the 3000–2800 being specific to

and

range, this interval

bonds stretching vibration. In this range, for 0 kGy sample,

three main characteristic peaks were detected and noted with 1, 2 and 3, as follows: 2956 (1), 2928

(2) and 2878

(3). With increasing of absorbed dose from 5 kGy

up to 50 kGy, a slightly shifting towards higher wavenumbers was a observed, from 2949 to 2954

attributed to the peak 1. Peak 2 had a decreasing tendency, from 2928

to 2923

, instead at the higher absorbed dose an obviously increasing in bands

intensity was observed. In the case of peak 3, for those samples irradiated at 5 kGy only one shifting was observed, from 2878

to 2851

(indicated in Fig. 5d as peak 4).

Likewise, in this range was observed an increase of band intensity with increasing of absorbed dose. The integrity of collagen molecule after –irradiation was checked by calculating of the peak ratios intensity of amide III (1240 1450

) to the peak intensity of absorption band located at

(Figueiró et al., 2004). The ratio of these band intensities offer the possibility to

verify if the collagen triple–helical structure was damaged. In this study the ratio of 1240 /1450

absorbnace for pure collagen, was found to be 1.09, while for unirradiated

collagen–PVP was 0.88. When the collagen–PVP was irradiated, the ratio decreased with absorbed dose, having a maximum value of 0.96 at 5 kGy and the lowest value was equal to 0.79 at 50 kGy. This result indicates that a certain amount of collagens triple–helical structure

15

was damaged, after irradiation with higher absorbed dose than 25 kGy, respectively 50 kGy. For lower doses, 5 kGy and 10 kGy, the cross–linking reactions predominate. 4. Conclusions A collagen–PVP hydrogel was obtained by γ–irradiation in the absence of oxygen and without chemical cross–linking agents. The obtained hydrogel presents superabsorbent capacity, with a swelling value up to 2000 % and high gel fraction, above 90 %. The sol–gel analysis shows that the cross–linking process predominates over the degradation process, regardless of the absorbed dose. The molecular weight between cross–linking

and the corresponding mesh size are

decreasing with the absorbed dose. However, even at the highest dose, the mesh size is large enough to allow the use of this type of hydrogel for some drugs delivery applications. The decrease in the mesh size is a direct consequence of the increasing cross–linking density with the absorbed dose. The yield of cross–linking and scission decrease with the absorbed dose, but the cross–linking phenomena predominates over the degradation. A similar trend is observed for the swelling degree, which decreases due to the increase of the cross–linking density. The fact that the elastic modulus is higher than the viscous modulus came to confirm the collagen–PVP system is being cross–linked by irradiation and not degraded. The decreasing of the elastic modulus at higher doses is associated with the macromolecular scission; therefore large doses can be detrimental to the elastic properties of the hydrogels. The decreases of diffusion exponent with the absorbed dose are directly correlated with the increase of the cross–linking density, which leaves less available space in the hydrogel matrix. As a consequence, less bulk water will be accommodated in a highly irradiated hydrogel. The FTIR analysis shows that the triple helix of collagen is preserved even at high absorbed dose. A reaction mechanism for the cross–linking of collagen and PVP was proposed. It

16

shows the formation of the main macroradicals, formed by irradiation of the collagen–PVP and their cross–linked network formed upon free–radical recombination reactions. Acknowledgments This work has been financed by the National Authority for Research and Innovation in the frame of Nucleus programme – contract 4N/2016. The authors gratefully acknowledge to the Prof. Dr. Murat Şen and PhD student Hande Hayrabolulu from Haceteppe University (Turkey) who support us with the rheological experiments. References An, J.-C., Weaver, A., Kim, B., Barkatt, A., Poster, D., Vreeland, W.N., Silverman, J., AlSheikhly, M., 2011. Radiation-induced synthesis of poly(vinylpyrrolidone) nanogel. Polymer 52, 5746-5755. Benamer, S., Mahlous, M., Boukrif, A., Mansouri, B., Youcef, S.L., 2006. Synthesis and characterisation of hydrogels based on poly(vinyl pyrrolidone). Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 248, 284-290. Buchholz, F.L., 2002. Superabsorbent Polymers, Encyclopedia of Polymer Science and Technology. John Wiley & Sons, Inc. Buchholz, F.L., Peppas, N.A., 1994. Superabsorbent Polymers: Science and Technology. American Chemical Society, Wasington D.C. Bueno, V.B., Bentini, R., Catalani, L.H., Petri, D.F.S., 2013. Synthesis and swelling behavior of xanthan-based hydrogels. Carbohyd Polym 92, 1091-1099. Cao, A., Tang, Y.L., Liu, Y., Yuan, H.X., Liu, L.B., 2013. A strategy for antimicrobial regulation based on fluorescent conjugated oligomer-DNA hybrid hydrogels. Chem Commun 49, 5574-5576. Chapiro, A., 1964. Radiation Chemistry of Polymers. Radiation Research Supplement 4, 179191. Charlesby, A., 1991. Past and Future-Trends in Polymer Irradiation. Radiat Phys Chem 37, 510. Chen, J.H., Chen, J.X., Song, J.Y., 2013. Collagen-PVP hybrid based anti-inflammatory hydrogel for wound repairing. J Control Release 172, E129-E130. Chimal-Monroy, J., Bravo-Ruiz, T., Furuzawa-Carballeda, G.J., Lira, J.M., de la Cruz, J.C., Almazan, A., Krotzsch-Gomez, F.E., Arrellin, G., Diaz de Leon, L., 1998. Collagen-PVP accelerates new bone formation of experimentally induced bone defects in rat skull and promotes the expression of osteopontin and SPARC during bone repair of rat femora fractures. Ann N Y Acad Sci 857, 232-236.

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Scheme 1. The basic reaction for the γ–radiation cross–linking of the collagen–PVP hydrogel and the proposed reaction mechanism. 20

Figure 1. a) Gel fraction of collagen–PVP hydrogels as function of radiation dose; b) Sol–gel data plotted in co-ordinates corresponding to Eq. (4). Figure 2. The swelling degree of collagen–PVP hydrogels vs. immersion time in water Figure 3. a) Swelling kinetic curves of collagen–PVP hydrogels; b) Plot of

, vs.

of collagen–PVP hydrogels Figure 4. a) Elastic ( ) modulus and b) viscous modulus (

) as function of angular

frequency of collagen–PVP hydrogels Figure 5. a) FTIR spectra of pure collagen; b) FTIR spectra of pure PVP; c) FT-IR spectra of collagen–PVP hydrogels in the range 3800–2600 hydrogels in the range 3000–2800

; d) FTIR spectra of collagen–PVP

.

Table 1. Gelation dose and degradation vs. crosslinking degree according Charlesby-Pinner and Charlesby-Rosiak equation Sol-gel analysis (kGy) Correlation (R2) Table 2. Parameters ,

Charlesby-Pinner Eq.

Charlesby-Rosiak Eq.

1.57 0.08 0.35 0.14 2.19 0.97 0.99 and diffusion coefficients (D) for collagen-PVP hydrogels

Dose,(kGy) D 5 -2.56 0.68 0.08 10 -2.28 0.66 0.11 25 -2.31 0.58 0.09 50 -2.38 0.59 0.07 Table 3. Network parameters of γ–irradiated collagen–PVP

(

) ( 5 10 25 50

) 1.26 1.12 1.19 1.22

( 0.11 0.12 0.12 0.13

0.04 0.04 0.05 0.06

) (

) ( 1.24 1.95 2.00 2.95

8.09 5.12 5.00 3.39

21

) 134 99 95 75

(

) 3.65 3.25 1.25 0.89

(

) 10.2 9.11 3.50 2.51

Highlights  

Collagen–Polyvinylpyrrolidone γ–radiation cross–linking is described ,

and ξ were calculated by for the case when the cross–links are introduced in

solution 

Relation between absorbed dose and swelling degree, network parameters or rheology parameters was established



Radiation–induced mechanism of collagen–PVP cross–linking in aqueous solution is proposed

22

23

24

Scheme 1

25