Effect of aggregation behavior of gelatin in aqueous solution on the grafting density of gelatin modified with glycidol

Effect of aggregation behavior of gelatin in aqueous solution on the grafting density of gelatin modified with glycidol

Colloids and Surfaces B: Biointerfaces 95 (2012) 201–207 Contents lists available at SciVerse ScienceDirect Colloids and Surfaces B: Biointerfaces j...

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Colloids and Surfaces B: Biointerfaces 95 (2012) 201–207

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Effect of aggregation behavior of gelatin in aqueous solution on the grafting density of gelatin modified with glycidol Jing Xu a,b , Tian-Duo Li a,b,∗ , Xiao-Long Tang b , Cong-De Qiao b , Qing-Wei Jiang b a b

School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China Key Laboratory of Fine Chemicals of Shandong Province, Shandong Polytechnic University, Jinan 250353, PR China

a r t i c l e

i n f o

Article history: Received 22 November 2011 Received in revised form 24 February 2012 Accepted 27 February 2012 Available online 5 March 2012 Keywords: Gelatin Modify Grafting density Glycidol Aggregation behavior Structural transition

a b s t r a c t The effect of aggregation behavior of gelatin in aqueous solution on the grafting density of glycidol grafted gelatin polymers (GGG polymers) was investigated. The grafting density was measured using the Van Slyke method by calculating the conversion rate of free NH2 groups of gelatin. The conversion rate reached peak values at 6% and 14% of the gelatin aqueous solution. SEM micrographs displayed a series of structural transitions (i.e., spherical, spindle, butterfly, irregular and dendritic aggregates) at varying concentrations from 2% to 16% (w/w) at an interval of 2% (w/w). The spindle aggregates reappeared at the concentrations of 6% and 14%. Viscosity measurements indicated that the physicochemical properties of the gelatin solution had changed with increasing concentration. UV and CD analysis indicated that hydrophobic interactions competed with hydrogen bonding, and the random coils partly transformed to ␤-sheet structure by changing the concentration. Zeta potential and pH data confirmed the increasing electrostatic repulsion associated with increasing the hydrophobic region. XPS analysis revealed that the elemental composition of the gelatin particle surface changed with variation in the aggregate structure, determining the monotonic variation of the grafting density with increasing concentration. Results demonstrate that aggregation behavior of gelatin in aqueous solution plays a crucial role in deciding the grafting density of gelatin modified products. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Gelatin, a water-soluble protein produced by the hydrolysis of animal collagen, has been widely used as a biomaterial in pharmaceutical and medical applications because of a range of favorable properties such as biocompatibility, biodegradability and bio-safety [1–3]. Gelatin solutions with concentrations covering the dilute and semidilute solution zones have been studied to explain the self-aggregation of gelatin in aqueous solution [4–8]. It is known that the aggregation process of gelatin is caused by intermolecular interactions and intramolecular folding [9–11]. Self-aggregation of gelatin gives rise to large scale structures above a certain concentration. Such structures have an effective molar mass higher than those of single chain, which leads to an increase of incompatibility with glycidol [12]. Moreover, the chemical reactions abilities in the aggregated structures between free NH2 groups of gelatin and glycidol containing epoxy groups are largely limited and the conversion rate of free NH2 groups in gelatin is incomplete.

∗ Corresponding author at: School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China. E-mail address: [email protected] (T.-D. Li).

Chemical grafting or cross-linking of gelatin is an effective way to introduce stable covalent bonds between protein segments to obtain a modified product which provides greater control of chemical and physical properties compared to natural polymers [13–15]. Grafting density plays a crucial role in deciding the physical and chemical properties of gelatin systems. In literatures, the grafting ratio has been studied by changing the reaction conditions including temperature, pressure, solvent and materials ratio [16–18]. However, the effect of aggregation behavior of gelatin in aqueous solution on the grafting ratio of gelatin has not been thoroughly studied. In the present work, chemical modification of gelatin by glycidol at varying the gelatin concentration in water from 2 to 18% (w/w) was studied. The grafting density (= a decrease in number of NH2 groups per unit mass) of glycidol grafted gelatin polymers (GGG polymers) is investigated using the Van Slyke method by measuring the conversion rate of free NH2 groups of gelatin [19]. The evolution of the aggregate morphology of gelatin with concentration variation is visualized by scanning electron microscopy (SEM) micrographs. The impact of aggregation behavior of gelatin in aqueous solution on the grafting density of GGG polymers is analyzed by ultraviolet (UV) absorption spectra, circular dichroism (CD) analysis, pH values, zeta potential and X-ray photoelectron spectroscopy (XPS) measurements.

0927-7765/$ – see front matter Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.041

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50

2.1. Material

45

All materials including gelatin (type A obtained from pigskin, having an approximate MW of 50,000 and isoelectric point at pH = 8 determined by fluorescence measurements) and glycidol, were obtained from China National Medicine Corporation without further purification or treatment. 2.2. Preparation conditions Gelatin was dissolved in distilled water (2–18% w/w) separately, and after 3 h, the gelatin solution was heated to 40 ◦ C to ensure complete dissolution. The pH of each solution was adjusted to 10.0 by NaOH (2.0 mol/L) solution. The glycidol was then slowly added to the gelatin solution at 40 ◦ C with stirring at predetermined glycidol/gelatin ratios (1:1, mol:mol). During reaction, the pH of these solutions was monitored using a pH-meter and held constant by the dropwise addition of NaOH solution (2.0 mol/L). After reacting for 12 h, the solutions obtained were used for viscosity (proRheo R180, Germany), pH (ESC Co., China) and zeta potential measurement (model: ZC-2000, Microtec, Japan) at 40 ◦ C. Then, the solutions were cooled to 5 ◦ C for weight and the content of free NH2 groups was tested at 40 ◦ C by the Van Slyke method. 2.3. SEM observation Taking care to contrast the difference of aggregate structure of the gelatin particles in different solution concentrations, the same quantity was used for the different solutions at 40 ◦ C. The details of the measurement can be found in our previous papers [20,21]. All the dried samples were coated with gold (∼20 nm thickness) using a Sputter Coater SCD-005 (TEC Co., England), and then observed under a Quanta-200 ESEM (FEI Co., Holland). 2.4. XPS analysis XPS spectra were taken using a Thermo Fisher Scientific ESCALAB 250 spectrometer (England) under the Al K␣ line (1486.6 eV). The high-resolution scans were acquired at energy of 20 eV. Compositional analyses (0–1100 eV) and high-resolution scans of the C1s, N1s and O1s regions were carried out on all samples. Data treatment was performed with the Service Physics ESCAVB data reduction software. 2.5. UV analysis UV spectra for the film samples formed from the gelatin solutions with different concentrations were recorded with a UV-2550 PC spectrophotometer (Japan) at room temperature. Corrections for the background were made. Broader features have been observed in the ultraviolet (the strongest at 278 nm). 2.6. CD analysis Measurements were taken using a Jasco J-810 spectropolarimeter (Japan) equipped with a cavity (NESLAB RTE-111) and purged with N2 gas at a flow rate of 35 mL/min. The gelatin solution was placed in 0.1 cm path length cells for detection. Spectra were recorded from 190 to 250 nm with a resolution of 0.2 nm and an accumulation of six scans. The scan speed was 100 nm/min and the response time was 0.25 s.

Conversion of the free -NH 2 groups ( %)

2. Experimental

40 35 30 25 20 15 10 5 0

2

4

6

8

10

12

14

16

18

20

Gelatin concentration (%) Fig. 1. Conversion rate of free NH2 groups with increasing gelatin concentration from 2% to 18% (w/w) at an interval of 2% (w/w). Error limit of conversion rate of free NH2 groups is ±2.1%.

3. Results and discussion 3.1. Chemical modification of gelatin by glycidol The chemical modification of gelatin through chemical reactions between free NH2 groups in gelatin and glycidol containing epoxy groups was performed in a water medium (Scheme 1). The concentration of the gelatin solutions ranged from 2% (w/w) to 18% (w/w) at an interval of 2% (w/w). The result of calculating conversion rate of free NH2 groups suggests that the grafting density is significantly affected by the gelatin concentration. Fig. 1 shows that after the conversion rate of free NH2 groups reaches a peak value at 6%, and then reaches another peak value at 14%. The results show that the variation of the conversion rate with increasing concentration is not monotonic, which cannot be explained by the standard viewpoint [22]. 3.2. Aggregation morphology evolution To visualize the evolution of the system morphology, SEM micrographs were taken at varying concentrations from 2% (w/w) to 16% (w/w) at an interval of 2% (w/w) of the gelatin aqueous solutions. The micrographs are shown in Fig. 2, and serve to illustrate how the microstructure develops with increasing concentration. Micrograph 2a shows some irregular spherical aggregates at the concentration of 2% (w/w) and a size distribution of gelatin particles ranging from 270 nm to 330 nm. However, after increasing the concentration to 4% (w/w), several spindle aggregates (about 1.5 ␮m) have appeared as indicated by the arrow shown in micrograph 2b. It can be seen in micrograph 2c that the spindle aggregates diameter has increased (about 2 ␮m), and the shape has more distinct at 6% (w/w) compared to micrograph 2b. A series of change in aggregation morphology of the gelatin particles was observed by SEM, including irregular aggregates (about 400–500 nm), network aggregates, butterfly aggregates and irregular aggregates in which the surface structure was more like a sepak takraw (about 1.0 ␮m), as shown in micrographs d–f. However, after increasing the concentration to 14% (w/w) the spindle aggregates (about 3–4 ␮m) reappeared (Fig. 2g). Although the shape of the spindle aggregates at 14% is in quite similar to the morphology at 6%, the size of the aggregates increased, which is an unfavorable factor for chemical reaction because of the larger scale structure. Finally, micrograph 2h displays the microstructure at 16% (w/w) (dendritic aggregates, about

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203

Scheme 1. GGG polymer synthesis.

Fig. 2. Structural transition of gelatin particles investigated by SEM at varying concentration. (a) 2% (w/w); (b) 4% (w/w); (c) 6% (w/w); (d) 8% (w/w); (e) 10% (w/w); (f) 12% (w/w); (g) 14% (w/w); (h) 16% (w/w).

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1.6 1.4

a

1.2 100 1.0

ce~12.2% Intensity

Dynamic viscosity (mPa/S)

3.6

2 10

0.8 0.6 0.4 0.2

c*~3.7%

0.7

0.0 -0.2

1

10 -0.4

Gelatin concentration (%)

0

3.3. Viscosity characterization Polyelectrolyte solution properties have been extensively studied by means of viscosity measurement over the past 60 years [3,23]. Viscosity plays a key role in the analysis of gelatin chain aggregation in aqueous solution [10]. In this study, the viscosity was measured at 40 ◦ C in a salt-free solution as a function of concentration. The three usual scaling regimes (dilute, semidilute unentangled, and entangled) for polymer solutions in the coiled state were clearly evident. The scaling theory is appropriate for gelatin solutions. Specific viscosities vs. concentration profiles were obtained for gelatin solutions that cover the overlap concentration and the semidilute unentangled regimes, as shown in Fig. 3. Below the overlap concentration c* ≈ 3.7% (w/w), hydration generates a repulsive force between pairs of particles [9,24–26]. However, in this region, the exponent (0.7) deviated somewhat from the literature reported which might be caused by the discrepancy in the distribution of molecular weight [3]. Above the entanglement concentration ce ≈ 12.2% (w/w), a power law  ∼ c3.5 was observed. It was consistent with expectations for entangled polyelectrolyte solutions. In the intermediate concentration range of ∼3.7–12.2% (w/w), the solution viscosity scales with concentration with an exponent of 2. This value is expected for semidilute, unentangled solutions in a -solvent [3]. In this regime, the hydrophobic and electrostatic interactions form among gelatin residues.

4

6

8

10

12

14

16

18

20

0 -5

b

-10

θ ( 10-3deg cm2/dmol)

20 ␮m). Although the sample undergoes a process of dehydration under the vacuum SEM measurement conditions, few changes in morphology were observed during the measurements. This observation possibly occurs because the higher hydrophobicity caused tighter packing of the hydrophobic region in aqueous solution. Moreover, the aggregate structure of gelatin was confirmed by freeze-drying techniques to avoid complicates sample handling. Fig. 2 reveals that the morphology of the gelatin aggregate displayed a series of changes with the concentration variation. The variation of morphology and size of the gelatin aggregates can be caused by microenvironmental changes, such as different solutions concentrations. We deduced that the elemental composition of the gelatin particles surface would change with the transition to aggregate structure. To achieve better understanding, sample characterizations, including viscosity, UV, CD, zeta potential, pH and XPS experiments, were carried out.

2

Gelatin concentration (%)

Fig. 3. Specific viscosity of gelatin at 40 ◦ C in salt-free solution as a function of concentration (2%-20%, w/w) measured with a proRheo R180 rotary viscosimeter. Error limit of specific viscosity is ±0.5%.

-15 -20 -25

d) c) b)

-30 -35 190

a) 200

210

220

230

240

Wavelength (nm) Fig. 4. (a) Intensity variation of UV–vis spectra at  (278 nm) recorded with gelatin concentration ranging from 2% to 20% (w/w) at an interval of 1% (w/w). Error limit of intensity at  (278 nm) is ±1.5%. (b) The CD spectra were recorded from freshly diluted gelatin solutions from the original solution of (a) 2% (w/w), (b) 6% (w/w), (c) 10% (w/w), (d) 14% (w/w).

3.4. UV and CD spectroscopy analysis Among the twenty naturally occurring amino acids, only tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) have aromatic UV chromophores, and thus are the subject of most laser spectroscopic studies on amino acids so far [27]. Specific UV spectra show that the spectra are similar to each other but with different relative intensities at max (278 nm), as shown in Fig. 4a. UV intensity is affected by many factors, such as free and bound chromophores or chromophore aggregation. The intensity of the band at 278 nm is relatively weak at 2–3% compared to that of the band at higher concentrations, but a fluctuation over a small range in the intensity was observed upon further increasing the concentration: UV intensities were reduced at 6% and at 14% (w/w), but enhanced at other concentrations. Moreover, intensity at 10% and 12% (w/w) was not recorded on curve because max moved to 279 nm at the two concentrations. The results indicate that the intensity of the band was disturbed by some weak interactions. The hydrogen bonding is mainly controlled by hydration at 2%. With a further increase in concentration, hydration is partly destroyed and the hydrophobic interactions of macromolecules chains become stronger [24–26]. The possibility of intermolecular or intramolecular hydrogen between gelatin chains is improved. Therefore, we

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Fig. 5. Aggregation morphology of gelatin at 6% (w/w) obtained by SEM micrographs. (a) Adding urea to inhibit hydrogen bonding; and (b) adding SDS to test hydrophobic interactions.

suggest that hydrophobic interactions and hydrogen bonding are competitive with each other as concentration varies from 4% to 20%. CD relies on the interaction of circularly polarized light with optically active (chiral) compounds. The far-UV region of the spectrum (240–180 nm) includes the different forms of regular secondary structure for proteins. For any given protein, the CD spectrum in this region can be analyzed in terms of the content of ␣-helix, ␤-sheet, ␤-turn, etc. The CD spectrum of gelatin which was recorded from freshly diluted each specimen in distilled water showed the occurrence of a negative Cotton effect around 198 nm (Fig. 4b, curves a–d), indicating that the solution was composed predominantly of a random coil component [28–30]. The negative

cotton effect rapidly decreased, while a negative peak at 223 nm characteristic of ␤-sheets weakly increased along with the gelatin concentration. The observed conversion suggests that the conformation of the gelatin partly transforms from a random coil to a ␤-sheet or other secondary structure with increasing concentration. The UV results indicate that hydrophobic interactions increase and compete with hydrogen bonding with increasing concentration. Hydrophobic interactions, as nonspecific interactions, are the major driving force for protein folding and possibly cause chain aggregation. The strength of the electrostatic repulsion between charged residues is enhanced in the hydrophobic region

Fig. 6. Aggregation morphology of gelatin investigated by SEM micrographs. (a) Varying pH value using 1 M NaOH to pH = 11 at 10% (w/w); (b) adding 0.01 M NaCl to improve electrostatic repulsion at 6% (w/w) and (c) 10% (w/w).

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and it favors the formation of a ␤-sheet structure, which causes the extension of molecular chains. Moreover, CD analysis also demonstrates that the ␤-sheet structure increases with hydrophobic increase.

22000

a

N1s

d:14%

18000

b) c)

3.6. Effect of different factors For the purpose of confirming the contribution of different interactions, a series of experiments were carried out. After adding urea to inhibit hydrogen bonding, the original spindle aggregates were partly destroyed at 6% (w/w) (Fig. 5a). The result indicates that inhibiting hydrogen bonding is adverse to forming spindle aggregates, which illustrates that hydrogen bonding plays a key role in causing the collapse of molecular chains. Then, SDS was added to test the hydrophobic interactions. The aggregation morphology exhibited no obvious change compared to the initial morphology at the same concentration, but the size of the aggregates increased (Fig. 5b). The result indicates that hydrophobic interactions play a crucial role in aggregation, and positively correlate with the aggregate diameter. Fig. 6a exhibits the aggregation morphology of gelatin particles with varying pH to pH = 11, the shape of the aggregates transformed to spindle aggregates at 10% (w/w). Upon increasing or decreasing the value of the pH, positive or negative net charges form, and the electrostatic repulsion among the charged residues are increased. The increase in electrostatic repulsion makes the spherical aggregates transform to spindle aggregates. The transition reflects the delicate balance of the charge interaction. The conclusion is further confirmed by the evolution of the aggregation morphology of the gelatin particles after adding NaCl. The spindle morphology partly transformed to ribbon morphology at 6% (w/w) (Fig. 6b). Fig. 6c shows that the spherical aggregates changed completely to spindle aggregates at 10% (w/w). This result implies that the molecular

14000

c:10%

d)

a)

a:2%

12000 10000 8000 398

6000

399

400

401

402

Bonding Energy(eV)

4000 2000 394

396

398 400

402

404

406

408

410

412

414

Bonding energy (eV) 60000

b

O1S a)

50000

531 531.6 532.2

d:14%

b)

40000

Intensity(a.u.)

To further investigate the effect of these interactions, the zeta potential and pH were measured [6,31,32]. The pH of the system is controlled by the solvent when the gelatin concentration is lower than 3%. With a further increase in gelatin concentration, a different pH was observed. The pH increased to 6.93 at 5–6% (w/w) but sharply deceased to 6.82 at 7% (w/w), and dropped continuously to 6.75 at 12% (w/w). However, the pH rose slowly to 6.80 at 15% (w/w). The increase in the pH at some concentrations can be attributed to ionization of the NH2 groups. Combining the UV and CD data, it is concluded that the ionization of the NH2 groups is enhanced because the NH2 groups are isolated from their surroundings or aggregates due to hydrophobic interaction. The number of charged chains would be increased with increasing ionization of the NH2 groups. Great care has been taken to confirm the pH analyses. The zeta potential displays a gradual decrease with increasing concentration. But, it exhibits a weak increase at 6%, 10% and 14% (w/w), which reflects the increase in the charge density at these concentrations. From these results, it is deduced that electrostatic repulsion varies with variation in the number of charged chains, such as lysine/arginine side chains. These results show that the evolution of aggregate structure in gelatin in aqueous solution is driven by weak noncovalent interactions, including hydrogen bonding, van der Waals forces, electrostatic interactions and hydrophobicity effects. Although these interactions might be relatively insignificant in isolation, when combined they are sufficient to generate well-ordered aggregate structures. However, it is necessary to further evaluate the contribution of the interactions to the aggregate structure.

Intensity(a.u.)

16000

3.5. Zeta and pH analysis

b:6%

20000

c)

531.2

c:10%

d)

30000

b:6%

20000

a: 2% 530 531 532 533 Bonding Energy (eV)

10000 0 525

530

535

540

545

Bonding energy (eV) Fig. 7. (a) XPS spectrum of the N1s core level recorded with gelatin particles from gelatin aqueous solutions at (a) 2% (w/w), (b) 6% (w/w), (c) 10% (w/w), (d) 14% (w/w) using deposition method. (b) XPS spectrum of the O1s core level recorded from with gelatin particles from gelatin aqueous solutions at (a) 2% (w/w), (b) 6% (w/w), (c) 10% (w/w), (d) 14% (w/w) using deposition method.

chains possibly arrange themselves in a stretched state under electrostatic repulsion. 3.7. XPS analysis XPS analysis was carried out for these samples at 2%, 6%, 10%, and 14% (w/w) prepared from the gelatin solutions using the deposition method. The O1s, N1s and C1s core-level spectra were recorded. Fig. 7a shows that the N1s core-level spectrum exhibits a single component at 399.7 eV assigned to the NH bond [33,34]. The spectrum shows that the N1s bonding energy has a slight peak shift and the strength of the N1s bonding energy has a small variation with increasing concentration, which illustrates the effect of hydrogen bonding. A series of O1s XPS spectra for the CO peak at varying concentrations were observed. The peak shifts from 531 eV to 532.2 eV, which is assigned to the COO− bonds or the COOH bonds (Fig. 7b). The peak shifted 1 eV and 0.4 eV toward higher bonding energy at 2% and 10% (w/w) respectively compared to that at 6% (w/w). The result reflects the impact of hydrogen bonding on the O1s bonding energy [35]. Combined with other analyses, it is concluded that hydration forms at 2% (w/w), and intermolecular or intramolecular hydrogen bonds replace the hydration and become an important interaction with increasing concentration. However, the OH cluster plays a key role in affecting the O1s bonding energy at 10%

J. Xu et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 201–207 Table 1 N/O composition ratio recorded from gelatin particles from gelatin aqueous solutions at different gelatin concentrations using deposition method. Gelatin concentration (w/w)

2%

6%

10%

14%

N/O

0.2134

0.7516

0.3782

0.8126

207

Acknowledgments This work is partly supported by the National Natural Science Funds of China (No. 21176147) and the Shandong Province in the Young Scientist Award Fund of China (No. 02BS109). Appendix A. Supplementary data

(w/w). The O1s bonding energy is mainly affected by hydrophobic interactions at 6% (w/w). The variation in the elemental composition of the surface of the gelatin particles was measured by XPS at 2%, 6%, 10% and 14% (w/w). As shown in Table 1, N/O reached peak values at 6% (w/w) and 14% (w/w), values that were higher than at 2% (w/w) and 10% (w/w). The results indicate that the elemental composition of the surface of the gelatin particles is altered by structural transitions, which is essential for understanding the variation of grafting intensity with increasing concentration. 4. Conclusions The aggregation behavior of gelatin in aqueous solution was studied by SEM, UV, CD, zeta potential, pH and XPS. The aggregate structure of gelatin formed in aqueous solution at different concentrations largely affected the grafting density of the GGG polymer. Whereas, spherical aggregates observed in 2% (w/w) were indicative of the action of hydration, the structural transitions at other concentrations were strongly influenced by hydrogen bonding, hydrophobic interactions and electrostatic repulsion. An increase in the hydrophobic interaction with increasing concentration imposes a direct influence on the electrostatic repulsion between the charged amino acid head groups, and the increase in the surface curvature of the aggregates is attributed to the increase in the electrostatic repulsion. The formation of ␤-sheet hydrogen bonding among the hydrophobic residues plays a crucial role in the axial growth of aggregates. The collapse or extension of the gelatin chains was caused by a secondary structural change, leading to structural transitions and a change in the elemental composition of the gelatin particles surfaces, which affected the variation of the grafting intensity as the concentration increased. The work is essential for providing a theoretical basis to link aggregate structure with grafting density, leading to better control of the structure/performance relationship in gelatin-modified materials.

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