- Email: [email protected]
N-vinylpyrrolidone copolymer with pendent thiol groups for ophthalmic applications
Available online at www.sciencedirect.com
Acta Biomaterialia 5 (2009) 1056–1063 www.elsevier.com/locate/actabiomat
Synthesis and characterization of acrylamide/N-vinylpyrrolidone copolymer with pendent thiol groups for ophthalmic applications Guoguang Niu a, Ying Yang c, Hongbin Zhang a, Jun Yang c, Li Song a, Miki Kashima a, Zhou Yang a, Hui Cao a, Yudong Zheng a, Siquan Zhu b,*, Huai Yang a,* a
School of Materials Science and Engineering, University of Science and Technology Beijing 100083, China b Department of Ophthalmology, Tongren Hospital, Capital Medical University, Beijing, China c The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tian Jin 300071, China Received 26 April 2008; received in revised form 17 October 2008; accepted 21 October 2008 Available online 5 November 2008
Abstract Injectable acrylamide/N-vinylpyrrolidinone copolymers with pendent thiol groups were prepared by a radical polymerization and reductive dissolution reaction. The solution of copolymers was re-gelled through oxidation in air or the thiol–disulfide exchange reaction. The re-gelation time could be adjusted from several minutes to several hours by changing the amount of the disulfide exchange reagent. The re-gelled hydrogels possessed high transmittance in the visible region but could block out some of the ultraviolet radiation. Their refractive indexes ranged from 1.34 to 1.35, and their equilibrium water contents were over 95.0%. The morphologies of the hydrogels were analyzed and the porous structure, with pore sizes of 50–300 lm, was noted. The cytotoxicities of the hydrogels were clearly reduced compared with previous results. The experimental results indicated that the injectable copolymers could be used as an artificial vitreous substance or as a scaffold for lens regeneration. Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. Keywords: Disulfide bond; Hydrogel; Vitreous substance; Lens regeneration
1. Introduction Injectable polymers, which perform sol–gel transition in situ, have attracted much attention for their use in biomedical applications, including bioengineering, cell encapsulation and drug delivery [1–3]. Such a manner of forming gels in situ is advantageous in that more complex shapes can be formed across cavities than with the traditional method of shaping before implantation. Meanwhile the trait of minimal invasiveness derived from injectable materials reduces any discomfort and subsequent complications suffered by patients. Solidification of injectable polymers is generally achieved through the methods of *
Corresponding authors. Tel.: +86 10 62333974; fax: +86 10 623333974. E-mail addresses: [email protected] (S. Zhu), yanghuai@mater. ustb.edu.cn (H. Yang).
thermo-gelating, ionic cross-linking and photo-polymerizing [4,5]. The requirements of materials are different depending on the proposed application. For instance, a material should be biocompatible and degradable for drug delivery, while materials used for intraocular lenses (IOLs) need to be optically clear and non-exothermic, with a modulus comparable to natural ones [6]. In the field of ophthalmics, injectable materials have shown potential for use in applications [5]. The research of materials that combine the sol–gel transformation inside the eye with intravitreal drug delivery is a future direction for the vitreous field [7]. The application of injectable IOLs is an effective way to form low modulus lenses which could be accommodated with the movement of a suspensory ligament [5,8]. Meanwhile injectable materials can also be used as bioscaffolds for lens regeneration in capsules for the treatment of cataract [9].
1742-7061/$ - see front matter Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2008.10.015
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Disulfide bonds as common structure elements in many biologically active peptides in the human body [10]. However disulfide bonds can be easily broken down into pendent thiol groups in aqueous solution by special reagents and then reformed through thiol oxidation or the thiol– disulfide exchange reaction [6]. Based on the above properties, biomaterials cross-linked with disulfide bonds have been investigated recently, such as hyaluronan–gelatin hydrogel films for fibroblast growth [11], a chymotrypsinresponsive hydrogel containing peptides [12] and hyaluronan haloacetates [13]. As a novel solidification method of injectable materials, acrylamide (AAm)-based hydrogels containing reversible disulfide have been explored as injectable IOLs and artificial human vitreous [6,14]; however, the high toxicity of AAm has limited their applications in the ophthalmic field [15]. Poly N-vinylpyrrolidone (PVP), which has good biocompatibility, has been used widely in the field of soft contact lenses. To date, there have been no NVP or NVP-based copolymers with thiol groups used as injectable materials. In this paper, NVP-based (AAm-co-NVP) copolymers with pendent thiol groups were synthesized and characterized. Potential applications of these materials in the ophthalmic field are suggested. The AAm-co-NVP copolymers were prepared in two steps: synthesis of poly (AAm-co-NVP) hydrogels cross-linked with disulfide bonds; and dissolution of hydrogels into soluble copolymers by cutting disulfide bonds down into thiol groups. The properties of hydrogels re-gelled from the solution of the above copolymers with thiol groups were analyzed, including re-gelation time, transmittance, refractive index, equilibrium water content and morphology. Meanwhile the cytotoxicities of the hydrogels have also been analyzed.
1057
and reductive dissolution of the hydrogels by DTT to form copolymers with pendent thiol groups. 2.2.1. Synthesis of poly(AAm-co-NVP) hydrogels crosslinked with BAC Groups of hydrogels were synthesized by radical polymerization. APS and TEMED were used as initiators, while BAC was used as cross-linker. A mixture of AAm and NVP at acrylic molar ratios was reacted in 25.0 vol.% ethanol solution. The resulting hydrogel was immersed in deionized water and the water was renewed periodically. The absorbance of the water used in the immersing process was analyzed at 210 nm with a V-570 spectrophotometer (JASCO Corporation, Japan) to ensure the removal of unreacted monomers. The obtained hydrogels were labeled as ANSS. 2.2.2. Reductive dissolution of ANSS hydrogels by DTT The cleavage reaction of disulfide bonds in the ANSS was performed by adding DTT to the crushed hydrogel at room temperature in a proportion of 5.0 mol per mol of BAC. The hydrogel was liquefied into solution completely after magnetic stirring for 4.0 h in nitrogen atmosphere. After adjusted to pH 3.5 with 10.0 vol.% HCl, the solution was poured into plenty of acetone (pH 3.5) and stirred vigorously under a nitrogen atmosphere. The precipitated copolymer (labeled as ANSH) was collected, freeze-dried and stored at 18 °C. The yield of ANSH was calculated based on the equation: Yield % ¼ WANSH =ðWAAm þ WNVP þ WBAC Þ:::::::::::::::::::: ð1Þ where WANSH is the weight of ANSH copolymer and WAAm, WNVP and WBAC are the weights of AAm, NVP and BAC monomers, respectively.
2. Experimental 2.1. Materials and methods NVP was supplied by Jiachen Chemicals Company, (Shanghai, China). N,N-Bis(acryloyl)-cystamine (BAC), N,N,N,N-tetramethylethylenediamine (TEMED), thiazolyl blue (MTT) and 3,3-dithiodipropionic acid (DTDP) were purchased from Aldrich (USA). D,L-Dithiothreitol (DTT), calf serum (CS) and 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) were purchased from Tengyuan Ltd. Company (Beijing, China). AAm and ammonium persulfate (APS) were supplied by Yili Chemical Company (Beijing, China). AAm was re-crystallized twice with water. All the other reagents were analytical grade, without further purification.
2.3. Characterization of ANSH copolymer The synthesis of ANSH was monitored with a microscopic confocal Raman spectrometer (RM2000, Renishaw, UK) with a 632.8 nm wavelength. Dried sample powder was used directly. The amount of free thiol groups (SH) in ANSH was determined by the Ellman method [16]. A gel permeation chromatograph (Shimadzu Corporation, Japan) equipped with a refractive index detector (RID-10A, Shimadzu Corporation, Japan) and a column of G4000PWXL (Tosoh, Tokyo, Japan) was used to measure the molecular weights of ANSH. Deionized water was used as the mobile phase at a rate of 1.0 ml min1. For calibration, polyethylene oxide/glycol references (Agilent Technologies, USA) with molecular weights of 5,9400, 25,910, 4140 and 1480 Da were used.
2.2. Preparation of AAm-co-NVP copolymer with thiol groups
2.4. Re-gelation of ANSH solution
The copolymer was prepared in two steps: synthesis of poly(AAm-co-NVP) hydrogels cross-linked with BAC
ANSH was dissolved in 1.0 mol l1 NaOH solution (nitrogen saturated) under a nitrogen atmosphere, and then
1058
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
the solution was adjusted to pH 7.4 with 10.0 vol.% HCl. Based on the amount of pendent thiol groups in ANSH, DTDP (0.5 M, pH 7.4) was added to the solution (at molar ratios of DTDP in SH of 0, 50.0, 100.0%) in order to achieve the re-gelation of ANSH solution. The formation of hydrogel (labeled as RANSS) was confirmed by the tilting tube method [6]. The synthesis process from AAm-co-NVP hydrogel to RANSS is shown in Scheme 1. 2.5. Equilibrium water content of RANSS RANSS hydrogel, prepared in a tube, was immersed in 0.9% (w/v) NaCl aqueous solution at 37.0 °C for 1 week until swelling equilibrium was achieved. The wet sample was then taken out and weighted, after which the hydrogel was freeze-dried to a constant weight. The equilibrium water content (EWC) of RANSS was calculated according to: EWC ¼ ðWh WP Þ=Wh ::::::::::::::
ð2Þ
where Wh is the weight of the swelled hydrogel and Wp is the weight of the dried hydrogel. 2.6. Transmittance and refractive index of RANSS hydrogel RANSS hydrogel was prepared using a cylindricalshaped mold 10.0 mm in diameter and 4.0 mm deep. Transmittance of RANSS hydrogel was measured from 200 to 800 nm with a V-570 spectrophotometer (JASCO Corporation, Japan). The refractive index (RI) of the hydrogel was determined with an Abbe refractometer (WAY, Shanghai Precision & Scientific Instrument Co. Ltd, China). 2.7. Microscopy analysis of RANSS hydrogel The RANSS hydrogel swelled in 0.9 wt.% NaCl aqueous solution was freeze-dried and coated with gold in a vacuum. The morphology of the sample was observed with a scanning electron microscope (SEM; JSM-5800, JEOL Corporation, Japan).
2.8. Cytotoxic study of RANSS hydrogel The cytotoxic study was performed according to the MTT method described by Hamiton et al. [15]. Chinese hamster ovary (CHO) cells were used at a density of 10,000 celles/16 mm well in 24 well clusters. The media used was 10.0 wt.% CS minimum essential medium (MEM) with antibiotics. The ANSH polymer was dissolved in 10.0 wt.% CS MEM at pH 7.4, and incubated for 48.0 h to allow the SH to form into disulfide bonds. The hydrogel was then removed from the medium by centrifugation and filtration. The resultant medium was added to CHO cells and incubated for 72.0 h for the cytotoxic study. The absorbance of samples was measured at 540.0 nm using an enzyme mark instrument (Labsystems, Fanland). The results obtained were the average data from three measurements. 3. Results and discussion 3.1. Synthesis of poly(AAm-co-NVP) hydrogel with disulfide bonds Groups of hydrogels with different NVP/AAm molar ratios were obtained. The radical polymerization was very fast, with hydrogels being formed within 5.0 min accompanying by the release of plentiful heat. The transformation from liquid solution to solid hydrogel was influenced by the ratio of monomers in the mixture. The lowest monomer concentration (LMC) above which the hydrogel was formed, would rise with increasing NVP content in the monomer mixture. The LMC was about 10.0% with an NVP/AAm molar ratio of 50:50 and increased to about 60.0% as the molar ratio of NVP/AAm changed to 90:10. Increasing the amount of BAC cross-linker in the monomer mixture favored the formation of hydrogels, and thus the LMC decreased with increasing BAC content. The unreacted monomers, initiators and lower molecular weight oligomers in the hydrogels were removed by the immersion method. In the experiments the concentrations of unreactive monomers were reduced to below 10.0 ppm after the hydrogels had been immersed in water for 7.0 days. 3.2. Preparation and characterization of copolymers with pendent thiol groups
Scheme 1. Schematic representation of the preparation and re-gelation of the injectable copolymers. (A) Synthesis of poly(AAm-co-NVP) hydrogel; (B) dissolution of hydrogel into injectable copolymer; (C) re-gelation of copolymer solution into hydrgel)
DTT has the ability to cut the disulfide bonds (-SAS-) selectively into pendant thiol groups (ASH). However, the reactive thiol groups are not stable and form disulfide bonds again through oxidation or the thiol–disulfide exchange reaction. Based on the above reversible properties of disulfide bonds, some kinds of injectable polymers with pendent thiol groups were made. In order to prevent the oxidation of the thiol groups by air, nitrogen protection was necessary during the preparation of the ANSH samples; the influence of pH on the thiol oxidation was also
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 1. Changes in the amount of SH in the ANSH solution with time at different pHs (initial concentration of SH: 3.5 105 mol l1).
analyzed Fig. 1 showed the changes in the amount of thiol with time in pH 3.5 and 7.4 aqueous solution. It can be seen that the amount of thiol changed little at pH 3.5, indicating that the thiol groups were stable in acidic solution. While pH increased to 7.4, the thiol content decreased to about 50.0% within 30.0 min. The fact that thiol groups were easily oxidized in neutral or basic conditions could be attributed to the increase in the concentration of thiol anions in aqueous solution with the rise in pH. Based on the above experimental results, the purification of ANSH was achieved by precipitating ANSH in acidic acetone (pH 3.5) with nitrogen protection. The amount of free thiol groups in ANSH was analyzed by Ellman method. Table 1 and Fig. 2 show the influence of the monomer concentration and the amount of SH in the ANSH copolymers. It was found that the monomer concentration had little influence on the SH content, while the influence of the molar ratio of the monomer mixture on the amount of SH in the ANSH copolymers was obvious. The amount of SH in the ANSH samples increased with increasing NVP content. The results above could be explained by the discrepancy in the monomer’s polarity and its conjugated effect [17]. AAm has a conjugated double bond in its molecular structure, so its polarity and conjugated effect is larger than that of NVP. This discrepancy enables the AAm and NVP to co-polymerize alternately.
Table 1 The influence of monomer composition on the amount of SH in ANSH. NVP:AAm (mol:mol)
BAC (mol.%)
APS (mol.%)
SH content in ANSH (mol.%) Calculated
50:50 50:50 50:50 60:40 70:30 80:20 a b
2.0 1.0 2.0 2.0 2.0 2.0
1.0 1.0 1.5 1.0 1.0 1.0
4.0 2.0 4.0 4.0 4.0 4.0
Calculated based on the monomer composition. Tested with Ellman’s agent.
a
Tested
b
3.20±0.15 1.61±0.06 3.13±0.07 3.54±0.11 4.10±0.07 5.81±0.09
1059
Fig. 2. The influence of monomer concentration on the amount of SH in ANSH (2.0 mol% BAC, 1.0 mol% APS).
On the other hand, the reactive rate of the NVP monomer is higher than that of AAm because NVP has no conjugated double bond in its molecular structure. Therefore more molecules of NVP than of AAm react with the cross-linker BAC during the first stage. These two factors cause the imbalance in the cross-linking density and componential distribution in copolymers: the parts with more NVP have a higher cross-linking density, while the parts with more AAm possess a lower cross-linking density. During the reaction, AAm monomer will be exhausted first if the amount of NVP monomers is far greater than that of AAm. However, the NVP monomer disfavors the transformation from liquid solution to solid hydrogel, and the copolymer molecules without enough cross-linking density would dissolve into water in the immersion step. Thus the soluble parts of the hydrogels increase with increasing NVP content in the monomer mixture. As a result, the crosslinking density of hydrogels was higher than expected, and consequently the amount of thiol groups in the ANSH was higher than calculated (as shown in Table 1). Similarly, most of the NVP and cross-linker (BAC) monomers would be exhausted during the first reaction stage if the amount of NVP was almost equal to that of AAm in the monomer mixture, thus leaving only a small amount of the BAC monomers to react with the AAm monomers. The AAm monomers favored the formation of hydrogels even at a very low monomer concentration and low cross-linking density [6]. During the immersion steps, some copolymer molecules containing a high ratio of NVP and BAC were dissolved, resulting in a decrease in cross-linking density. As a result, the amount of thiol groups was lower than calculated. Fig. 2 shows that the amounts of SH in ANSH tended to be constant with the rise in monomer concentration, because the polarity and conjugated effect in the monomer were constant with the fixed ratio of NVP/AAm monomer in the reactive system. To prove the explanation above, the influence of the monomer ratio on the yield of ANSH was investigated. Fig. 3 shows that the yield of AHSH decreased greatly with increasing NVP content in the monomer mixture, indicating that NVP disfavored the formation of poly(AAm-co-
1060
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 3. The influence of monomer concentration and composition on the yield of ANSH copolymer (2.0 mol.% BAC, 1.0 mol.% APS).
Fig. 5. The influences of monomer concentration and composition on the molecular weight of ANSH copolymer (BAC = 2.0 mol.%, APS = 1.0 mol.%).
3.3. Re-gelation of ANSH solution and characterization of RANSS hydrogels
Fig. 4. Raman spectra of ANSS, ANSH and RANSN (A, B and C, respectively). All the samples were freeze-dried; ANSS obtained with NVP:AAm = 50:50 (mol:mol), BAC = 2.0 mol.%, APS = 1.0 mol.%, monomer concentration = 20.0 wt.%; ANSH was obtained after dissolution of ANSS with DTT; RANSS was obtained after re-gelation with DTDP.
NVP) hydrogels and there were more molecules dissolved into water during the immersion step. A microscopic confocal Raman spectrometer was used to confirm the presence and disappearance of SH groups before and after re-gelation of ANSH, respectively. As shown in Fig. 4, the characteristic absorption of SH stretching vibration (tS-H) in the ANSH sample appeared at 2570 cm1 and disappeared after re-gelation. A peak at 512 cm1, corresponding to the -SAS- stretching vibration (tS-S), was observed in the ANSS sample. The disappearance of tS-S in ANSH indicated the breaking down of disulfide bonds. After the formation of disulfide bonds in the RANSS sample, the tS-S appeared again at 512 cm1. Figs. 5 and 6 show the changes in molecular weights of the ANSH samples obtained under different conditions. Decreasing the NVP content in the monomer mixture and increasing the concentration of the monomer mixture both decreased molecular weight. The fact that samples with more BAC cross-linker possessed a higher molecular weight is consistent with previous reports [6]. The molecular weights of the polymers decreased with increasing APS content.
Re-gelation time is an important parameter to be considered when ANSH is used as an injectable material. The solution of ANSH copolymers with pendent SH groups could form a hydrogel through oxygenation in air or the thiol–disulfide exchange reaction. In this paper DTDP was used asa thiol–disulfide exchange reagent because of its low cell toxicity [18]. Fig. 7 shows pictures of the ANSH solution and the re-gelled hydrogel. Fig. 8 shows the influence of the DTDP content on the re-gelation time of the ANSH solution. ANSH solutions with the correct concentration could also form hydrogels through oxygenation in air; however, the re-gelation time in this case was quite long, about half an hour or even more. Adding DTDP into the ANSH solution would accelerate the regelation rate. When equimolar DTDP and SH was added to the ANSH solution, re-gelation was achieved within 3.0 min. These results were consistent with previous reports [6]. The solutions of ANSH copolymers containing a greater NVP content had a short re-gelation time, resulting from the higher SH content in the ANSH samples. Fig. 9 showed the influences of the ANSH concentration, the amounts of crosslinker and initiator on the re-gelation time of ANSH solution. Increasing the ANSH concentration increases the solution viscosity and decreases the re-gelation time. As shown in group A, the re-gelation time was about 2.0 min when the ANSH concentration was 5.0 wt.%. The re-gelation time was reduced still further, to about 1.0 min, as the concentration was increased to 7.5 wt.%. With the decrease in BAC content or increase in APS content, the formation of hydrogels required a higher ANSH concentration and a longer regelation time, as shown in groups D and E in Fig. 9. The influences of BAC and APS on re-gelation time could be attributed to the molecular weight changes of the ANSH copolymers. Due to the increase in molecular entanglement in aqueous solution, the copolymers with high molecular
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 6. The influences of BAC and APS on the molecular weight of ANSH copolymers (NVP:AAm = 50:50 (molar ratio), monomer concentration = 20.0 wt.%).
1061
Fig. 8. The influence of DTDP amount on the re-gelation time of ANSH. The error bars representing relative variation of data.
Fig. 9. The influence of monomer composition on the re-gelation time of ANSH (NVP:AAm = 50:50 (mol:mol); for groups A, B, C, D and E, the BAC content was 2.0, 2.0 2.0, 2.0 and 1.0 mol.%, respectively; APS was 1.0, 1.0, 1.0, 1.5 and 1.0 mol.%; monomer concentrations were 15.0, 20.0, 25.0, 20.0, 20.0 wt.%; and molecular weights were 10,000, 8900, 8500, 5900, 7000 Da). The error bars represent the relative variation of the data.
Fig. 7. The pictures of ANSH solution and re-gelled hydrogel (a, ANSH solution; b, re-gelled hydrogel).
weight would require a short re-gelation time. For injectable polymers, the correct re-gelation time is crucial for different applications. Adjusting the DTDP content, changing the concentration of the solution and selecting samples with dif-
ferent molecular weights are effective ways to regulate the re-gelation time of an ANSH solution. The transmittances of RANSS hydrogels were investigated using an ultraviolet–visible spectrophotometer. The hydrogels had excellent optical properties in the range of 400–800 nm, as shown in Fig. 10. As reported, the peaks of photopic sensitivity and scotopic sensitivity are at 555 and 506 nm in visible spectrum [19]. The transmittances of all RANSS hydrogels were over 83.0 and 81.2% at 555 and 506 nm, respectively. Meanwhile the transmittance increased gradually with increasing NVP content. The injectable property and the high transmittance in the visible region provide the opportunity for ANSH copolymers to be used as ophthalmic materials, such as artificial human vitreous and scaffold for lens regeneration. However, the retina is sensitive to the light radiation, thus photochemical damage to the retina can easily be caused by excessive ultraviolet radiation [20]. As shown in Fig. 10, RANSS hydrogels can block out some ultraviolet light radiation; in particular, in the range of 200–300 nm the transmittance was below 20.0%.
1062
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 10. Transmittances of RANSS hydrogels with 4.0 mm thickness (the concentration of RANSS with increasing NVP was 7.5, 7.5, 5.0 and 5.0 wt.%, respectively).
Table 2 The refractive indexes of re-gelled hydrogels. RANSS concentration (wt.%)
5.0 7.5 10.0
NVP:AAM (mol:mol) 50:50
60:40
70:30
80:20
1.3409 1.3451 1.3491
1.3412 1.3454 –
1.3415 – –
1.3418 – –
Table 2 shows the changes in the refractive index (RI) with the concentration and composition of samples. Due to the low concentration of RANSS, the RIs of hydrogels ranged from 1.34 to 1.35, which is very close
to the RI of the human vitreous body (RI 1.33) [7]. Increasing the RANSS concentration would lead to a rise in the RI; however, sharply increasing the viscosity of the solution would result in difficulties of operation. The correct sample concentration is in the range of 5.0–10.0 wt.%. The equilibrium water contents (EWCs) of RANSS hydrogels were analyzed. The results showed that EWC values of all samples were over 95.0%, which is very close to the value of human vitreous body (a transparent jelly-like substance containing 97.0–99.0% water) [7]. The high EWC of RANSS could be attributed to the low cross-linking density in the hydrogel and the high hydrophilicity of the macromolecules. Highly hydrophilic hydrogels, such as RANSS hydrogels, are more suitable for use as artificial vitreous substance because the hydrogels show improved interfacial energetics and improved conformity with the retinal surface [7]. Moreover, an ideal vitreous substance must allow the transfer of necessary metabolites and proteins inside the vitreous body, and to or from the neighboring tissues [7]. Based on the above consideration, the morphologies of RANSS hydrogels were observed. Fig. 11 shows SEM photographs of the RANSS samples. RANSS hydrogels had the porous structure, with a pore size of 50–300 lm, due to its high EWC. The void space within the hydrogel network is big enough for the diffusion of nutrient materials and the release of metabolites from the cells. Transparent RANSS hydrogels with a porous structure are also suitable for use as a scaffold for lens regeneration. After a cataract extraction, ANSH copolymer solution
Fig. 11. The SEM photographs of RANSS hydrogels. (a1, a2) NVP:AAm = 60:40 (molar ratio); (b1, b2) NVP:AAm = 70:30 (mol:mol).
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
1063
Acknowledgement This research is supported by the grant from Chinese National Science and Technology Supporting Item with No. 2006BAI03A09. References
Fig. 12. Plots of RANSS concentration vs. the value of MTT staining.
containing lens epithelial cells is injected into capsular bags and solidified into a porous hydrogel. Lens epithelial cells have the capacity to divide in the porous structure, and breed non-dividing enucleated lens fiber cells which, together with the porous material, form the internal lens substance [21]. The cytotoxicities of the RANSS hydrogels were analyzed based on the MTT method. Fig. 12 showed the changes in the degree of MTT staining with the RANSS hydrogel concentration. It can be seen that the MTT staining decreased gradually with increasing RANSS concentration. However, the 50.0% point, or IC50 value, was not obtained because the cytotoxicities of the RANSS samples were low under the experimental conditions. Compared with the previous results [15], it could be seen that the cytotoxicities of RANSS hydrogels were obviously lower than those of the reported materials. 4. Conclusion Injectable acrylamide/N-vinylpyrrolidinone copolymers with pendent thiol groups were prepared. The SH content of the copolymers was influenced by the monomer composition and the concentration of the monomer mixture. By adjusting the DTDP content, the concentration and composition of reactive copolymers could regulate the re-gelation time of ANSH solution. The re-gelled hydrogels had high transmittance in the visible region, and their RIs and EWCs were about 1.34–1.35 and over 95.0%, respectively. The hydrogels possessed a porous structure, with pore size ranging from 50 to 300 lm; their cytotoxicities were lower than those of the previous materials reported. The experimental results indicate that injectable ANSH copolymers could be used in the ophthalmic field, e.g. as artificial vitreous substance and scaffold for lens regeneration.
[1] Liu H, Li H, Cheng W, Yang Y, Zhu M, Zhou C. Novel injectable calcium phosphate/chitosan composites for bone substitute materials. Acta Biomater 2006;2:557–65. [2] Burdick JA, Anseth KS. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials 2002;23(22):4315–23. [3] Kang GD, Cheon SH, Khang G, Song SC. Thermosensitive poly(organophosphazene) hydrogels for a controlled drug delivery. Eur J Pharm Biopharm 2006;63:340–6. [4] Kretlow JD, Klouda L, Mikos AG. Injectable matrices and scaffolds for drug delivery in tissue engineering. Adv Drug Deliv Rev 2007;5(4–5):263–73. [5] de Groot JH, van Beijma FJ, Haitjema HJ, Dillingham KA, Hodd KA, Koopmans SA, et al. Injectable intraocular lens materials based upon hydrogels. Biomacromolecules 2001;2:628–34. [6] Aliyar HA, Hamilton PD, Ravi N. Refilling of ocular lens capsule with copolymeric hydrogel containing reversible disulfide. Biomacromolecules 2005;6:204–11. [7] Soman N, Banerjee R. Artificial vitreous replacements. Bio-Med Mater Eng 2003;13:59–74. [8] Nishi O. Refilling the lens of the rabbit eye after endocapsular cataract surgery. Folia Ophthalmol 1987;38:1615–8. [9] Gwon A. Lens regeneration in mammals: a review. Surv Ophthamol 2006;51:51–62. [10] Rabenstein DL, Weaver KH. Kinetics and equilibria of the thiol/ disulfide exchange reactions of somatostatin with glutathione. J Org Chem 1996;61:7391–7. [11] Shu XZ, Liu Y, Luo Y, Roberts MC, Prestwich GD. Disulfide crosslinked hyaluronan hydrogels. Biomacromolecules 2002;3:1304–11. [12] Plunkett KN, Berkowski KL, Moore JS. Chymotrypsin responsive hydrogel: application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides. Biomacromolecules 2005;6:632–7. [13] Serban MA, Prestwich GD. Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels. Biomacromolecules 2007;8(9):2821–8. [14] Aliyar HA, Foster WJ, Hamilton PD, Ravi N. Towards the development of an artificial human vitreous. Polym Preprints 2004;45(2):469–70. [15] Hamilton PD, Aliyar HA, Ravi N. Biocompatibility of thiolcontaining polyacrylamide polymers suitable for ophthalmic applications. Polym Preprints 2004;45(2):495–6. [16] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. [17] Pan ZR. Polymer Chemistry. Beijing: Chemical Industry Press; 2002, pp. 90–98. [18] Hisano N, Morikawa N, Iwata H, Ikada Y. Entrapment of islets into reversible disulfide hydrogels. J Biomed Mater Res 1998;40:115–23. [19] Wyszecki G, Stiles WS. Color Science. New York: John Wiley & Sons; 1967. [20] Noell WK, Walker VS, Kang BS. Retinal damage by light in rats. Invest Ophthalmol 1966;5:450–73. [21] Lipman RD, Taylor A. The in vitro replicative potential and cellular morphology of human lens epithelial cells derived from different aged donors. Curr Eye Res 1987;6:1453–7.
Acta Biomaterialia 5 (2009) 1056–1063 www.elsevier.com/locate/actabiomat
Synthesis and characterization of acrylamide/N-vinylpyrrolidone copolymer with pendent thiol groups for ophthalmic applications Guoguang Niu a, Ying Yang c, Hongbin Zhang a, Jun Yang c, Li Song a, Miki Kashima a, Zhou Yang a, Hui Cao a, Yudong Zheng a, Siquan Zhu b,*, Huai Yang a,* a
School of Materials Science and Engineering, University of Science and Technology Beijing 100083, China b Department of Ophthalmology, Tongren Hospital, Capital Medical University, Beijing, China c The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tian Jin 300071, China Received 26 April 2008; received in revised form 17 October 2008; accepted 21 October 2008 Available online 5 November 2008
Abstract Injectable acrylamide/N-vinylpyrrolidinone copolymers with pendent thiol groups were prepared by a radical polymerization and reductive dissolution reaction. The solution of copolymers was re-gelled through oxidation in air or the thiol–disulfide exchange reaction. The re-gelation time could be adjusted from several minutes to several hours by changing the amount of the disulfide exchange reagent. The re-gelled hydrogels possessed high transmittance in the visible region but could block out some of the ultraviolet radiation. Their refractive indexes ranged from 1.34 to 1.35, and their equilibrium water contents were over 95.0%. The morphologies of the hydrogels were analyzed and the porous structure, with pore sizes of 50–300 lm, was noted. The cytotoxicities of the hydrogels were clearly reduced compared with previous results. The experimental results indicated that the injectable copolymers could be used as an artificial vitreous substance or as a scaffold for lens regeneration. Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. Keywords: Disulfide bond; Hydrogel; Vitreous substance; Lens regeneration
1. Introduction Injectable polymers, which perform sol–gel transition in situ, have attracted much attention for their use in biomedical applications, including bioengineering, cell encapsulation and drug delivery [1–3]. Such a manner of forming gels in situ is advantageous in that more complex shapes can be formed across cavities than with the traditional method of shaping before implantation. Meanwhile the trait of minimal invasiveness derived from injectable materials reduces any discomfort and subsequent complications suffered by patients. Solidification of injectable polymers is generally achieved through the methods of *
Corresponding authors. Tel.: +86 10 62333974; fax: +86 10 623333974. E-mail addresses: [email protected] (S. Zhu), yanghuai@mater. ustb.edu.cn (H. Yang).
thermo-gelating, ionic cross-linking and photo-polymerizing [4,5]. The requirements of materials are different depending on the proposed application. For instance, a material should be biocompatible and degradable for drug delivery, while materials used for intraocular lenses (IOLs) need to be optically clear and non-exothermic, with a modulus comparable to natural ones [6]. In the field of ophthalmics, injectable materials have shown potential for use in applications [5]. The research of materials that combine the sol–gel transformation inside the eye with intravitreal drug delivery is a future direction for the vitreous field [7]. The application of injectable IOLs is an effective way to form low modulus lenses which could be accommodated with the movement of a suspensory ligament [5,8]. Meanwhile injectable materials can also be used as bioscaffolds for lens regeneration in capsules for the treatment of cataract [9].
1742-7061/$ - see front matter Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2008.10.015
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Disulfide bonds as common structure elements in many biologically active peptides in the human body [10]. However disulfide bonds can be easily broken down into pendent thiol groups in aqueous solution by special reagents and then reformed through thiol oxidation or the thiol– disulfide exchange reaction [6]. Based on the above properties, biomaterials cross-linked with disulfide bonds have been investigated recently, such as hyaluronan–gelatin hydrogel films for fibroblast growth [11], a chymotrypsinresponsive hydrogel containing peptides [12] and hyaluronan haloacetates [13]. As a novel solidification method of injectable materials, acrylamide (AAm)-based hydrogels containing reversible disulfide have been explored as injectable IOLs and artificial human vitreous [6,14]; however, the high toxicity of AAm has limited their applications in the ophthalmic field [15]. Poly N-vinylpyrrolidone (PVP), which has good biocompatibility, has been used widely in the field of soft contact lenses. To date, there have been no NVP or NVP-based copolymers with thiol groups used as injectable materials. In this paper, NVP-based (AAm-co-NVP) copolymers with pendent thiol groups were synthesized and characterized. Potential applications of these materials in the ophthalmic field are suggested. The AAm-co-NVP copolymers were prepared in two steps: synthesis of poly (AAm-co-NVP) hydrogels cross-linked with disulfide bonds; and dissolution of hydrogels into soluble copolymers by cutting disulfide bonds down into thiol groups. The properties of hydrogels re-gelled from the solution of the above copolymers with thiol groups were analyzed, including re-gelation time, transmittance, refractive index, equilibrium water content and morphology. Meanwhile the cytotoxicities of the hydrogels have also been analyzed.
1057
and reductive dissolution of the hydrogels by DTT to form copolymers with pendent thiol groups. 2.2.1. Synthesis of poly(AAm-co-NVP) hydrogels crosslinked with BAC Groups of hydrogels were synthesized by radical polymerization. APS and TEMED were used as initiators, while BAC was used as cross-linker. A mixture of AAm and NVP at acrylic molar ratios was reacted in 25.0 vol.% ethanol solution. The resulting hydrogel was immersed in deionized water and the water was renewed periodically. The absorbance of the water used in the immersing process was analyzed at 210 nm with a V-570 spectrophotometer (JASCO Corporation, Japan) to ensure the removal of unreacted monomers. The obtained hydrogels were labeled as ANSS. 2.2.2. Reductive dissolution of ANSS hydrogels by DTT The cleavage reaction of disulfide bonds in the ANSS was performed by adding DTT to the crushed hydrogel at room temperature in a proportion of 5.0 mol per mol of BAC. The hydrogel was liquefied into solution completely after magnetic stirring for 4.0 h in nitrogen atmosphere. After adjusted to pH 3.5 with 10.0 vol.% HCl, the solution was poured into plenty of acetone (pH 3.5) and stirred vigorously under a nitrogen atmosphere. The precipitated copolymer (labeled as ANSH) was collected, freeze-dried and stored at 18 °C. The yield of ANSH was calculated based on the equation: Yield % ¼ WANSH =ðWAAm þ WNVP þ WBAC Þ:::::::::::::::::::: ð1Þ where WANSH is the weight of ANSH copolymer and WAAm, WNVP and WBAC are the weights of AAm, NVP and BAC monomers, respectively.
2. Experimental 2.1. Materials and methods NVP was supplied by Jiachen Chemicals Company, (Shanghai, China). N,N-Bis(acryloyl)-cystamine (BAC), N,N,N,N-tetramethylethylenediamine (TEMED), thiazolyl blue (MTT) and 3,3-dithiodipropionic acid (DTDP) were purchased from Aldrich (USA). D,L-Dithiothreitol (DTT), calf serum (CS) and 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) were purchased from Tengyuan Ltd. Company (Beijing, China). AAm and ammonium persulfate (APS) were supplied by Yili Chemical Company (Beijing, China). AAm was re-crystallized twice with water. All the other reagents were analytical grade, without further purification.
2.3. Characterization of ANSH copolymer The synthesis of ANSH was monitored with a microscopic confocal Raman spectrometer (RM2000, Renishaw, UK) with a 632.8 nm wavelength. Dried sample powder was used directly. The amount of free thiol groups (SH) in ANSH was determined by the Ellman method [16]. A gel permeation chromatograph (Shimadzu Corporation, Japan) equipped with a refractive index detector (RID-10A, Shimadzu Corporation, Japan) and a column of G4000PWXL (Tosoh, Tokyo, Japan) was used to measure the molecular weights of ANSH. Deionized water was used as the mobile phase at a rate of 1.0 ml min1. For calibration, polyethylene oxide/glycol references (Agilent Technologies, USA) with molecular weights of 5,9400, 25,910, 4140 and 1480 Da were used.
2.2. Preparation of AAm-co-NVP copolymer with thiol groups
2.4. Re-gelation of ANSH solution
The copolymer was prepared in two steps: synthesis of poly(AAm-co-NVP) hydrogels cross-linked with BAC
ANSH was dissolved in 1.0 mol l1 NaOH solution (nitrogen saturated) under a nitrogen atmosphere, and then
1058
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
the solution was adjusted to pH 7.4 with 10.0 vol.% HCl. Based on the amount of pendent thiol groups in ANSH, DTDP (0.5 M, pH 7.4) was added to the solution (at molar ratios of DTDP in SH of 0, 50.0, 100.0%) in order to achieve the re-gelation of ANSH solution. The formation of hydrogel (labeled as RANSS) was confirmed by the tilting tube method [6]. The synthesis process from AAm-co-NVP hydrogel to RANSS is shown in Scheme 1. 2.5. Equilibrium water content of RANSS RANSS hydrogel, prepared in a tube, was immersed in 0.9% (w/v) NaCl aqueous solution at 37.0 °C for 1 week until swelling equilibrium was achieved. The wet sample was then taken out and weighted, after which the hydrogel was freeze-dried to a constant weight. The equilibrium water content (EWC) of RANSS was calculated according to: EWC ¼ ðWh WP Þ=Wh ::::::::::::::
ð2Þ
where Wh is the weight of the swelled hydrogel and Wp is the weight of the dried hydrogel. 2.6. Transmittance and refractive index of RANSS hydrogel RANSS hydrogel was prepared using a cylindricalshaped mold 10.0 mm in diameter and 4.0 mm deep. Transmittance of RANSS hydrogel was measured from 200 to 800 nm with a V-570 spectrophotometer (JASCO Corporation, Japan). The refractive index (RI) of the hydrogel was determined with an Abbe refractometer (WAY, Shanghai Precision & Scientific Instrument Co. Ltd, China). 2.7. Microscopy analysis of RANSS hydrogel The RANSS hydrogel swelled in 0.9 wt.% NaCl aqueous solution was freeze-dried and coated with gold in a vacuum. The morphology of the sample was observed with a scanning electron microscope (SEM; JSM-5800, JEOL Corporation, Japan).
2.8. Cytotoxic study of RANSS hydrogel The cytotoxic study was performed according to the MTT method described by Hamiton et al. [15]. Chinese hamster ovary (CHO) cells were used at a density of 10,000 celles/16 mm well in 24 well clusters. The media used was 10.0 wt.% CS minimum essential medium (MEM) with antibiotics. The ANSH polymer was dissolved in 10.0 wt.% CS MEM at pH 7.4, and incubated for 48.0 h to allow the SH to form into disulfide bonds. The hydrogel was then removed from the medium by centrifugation and filtration. The resultant medium was added to CHO cells and incubated for 72.0 h for the cytotoxic study. The absorbance of samples was measured at 540.0 nm using an enzyme mark instrument (Labsystems, Fanland). The results obtained were the average data from three measurements. 3. Results and discussion 3.1. Synthesis of poly(AAm-co-NVP) hydrogel with disulfide bonds Groups of hydrogels with different NVP/AAm molar ratios were obtained. The radical polymerization was very fast, with hydrogels being formed within 5.0 min accompanying by the release of plentiful heat. The transformation from liquid solution to solid hydrogel was influenced by the ratio of monomers in the mixture. The lowest monomer concentration (LMC) above which the hydrogel was formed, would rise with increasing NVP content in the monomer mixture. The LMC was about 10.0% with an NVP/AAm molar ratio of 50:50 and increased to about 60.0% as the molar ratio of NVP/AAm changed to 90:10. Increasing the amount of BAC cross-linker in the monomer mixture favored the formation of hydrogels, and thus the LMC decreased with increasing BAC content. The unreacted monomers, initiators and lower molecular weight oligomers in the hydrogels were removed by the immersion method. In the experiments the concentrations of unreactive monomers were reduced to below 10.0 ppm after the hydrogels had been immersed in water for 7.0 days. 3.2. Preparation and characterization of copolymers with pendent thiol groups
Scheme 1. Schematic representation of the preparation and re-gelation of the injectable copolymers. (A) Synthesis of poly(AAm-co-NVP) hydrogel; (B) dissolution of hydrogel into injectable copolymer; (C) re-gelation of copolymer solution into hydrgel)
DTT has the ability to cut the disulfide bonds (-SAS-) selectively into pendant thiol groups (ASH). However, the reactive thiol groups are not stable and form disulfide bonds again through oxidation or the thiol–disulfide exchange reaction. Based on the above reversible properties of disulfide bonds, some kinds of injectable polymers with pendent thiol groups were made. In order to prevent the oxidation of the thiol groups by air, nitrogen protection was necessary during the preparation of the ANSH samples; the influence of pH on the thiol oxidation was also
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 1. Changes in the amount of SH in the ANSH solution with time at different pHs (initial concentration of SH: 3.5 105 mol l1).
analyzed Fig. 1 showed the changes in the amount of thiol with time in pH 3.5 and 7.4 aqueous solution. It can be seen that the amount of thiol changed little at pH 3.5, indicating that the thiol groups were stable in acidic solution. While pH increased to 7.4, the thiol content decreased to about 50.0% within 30.0 min. The fact that thiol groups were easily oxidized in neutral or basic conditions could be attributed to the increase in the concentration of thiol anions in aqueous solution with the rise in pH. Based on the above experimental results, the purification of ANSH was achieved by precipitating ANSH in acidic acetone (pH 3.5) with nitrogen protection. The amount of free thiol groups in ANSH was analyzed by Ellman method. Table 1 and Fig. 2 show the influence of the monomer concentration and the amount of SH in the ANSH copolymers. It was found that the monomer concentration had little influence on the SH content, while the influence of the molar ratio of the monomer mixture on the amount of SH in the ANSH copolymers was obvious. The amount of SH in the ANSH samples increased with increasing NVP content. The results above could be explained by the discrepancy in the monomer’s polarity and its conjugated effect [17]. AAm has a conjugated double bond in its molecular structure, so its polarity and conjugated effect is larger than that of NVP. This discrepancy enables the AAm and NVP to co-polymerize alternately.
Table 1 The influence of monomer composition on the amount of SH in ANSH. NVP:AAm (mol:mol)
BAC (mol.%)
APS (mol.%)
SH content in ANSH (mol.%) Calculated
50:50 50:50 50:50 60:40 70:30 80:20 a b
2.0 1.0 2.0 2.0 2.0 2.0
1.0 1.0 1.5 1.0 1.0 1.0
4.0 2.0 4.0 4.0 4.0 4.0
Calculated based on the monomer composition. Tested with Ellman’s agent.
a
Tested
b
3.20±0.15 1.61±0.06 3.13±0.07 3.54±0.11 4.10±0.07 5.81±0.09
1059
Fig. 2. The influence of monomer concentration on the amount of SH in ANSH (2.0 mol% BAC, 1.0 mol% APS).
On the other hand, the reactive rate of the NVP monomer is higher than that of AAm because NVP has no conjugated double bond in its molecular structure. Therefore more molecules of NVP than of AAm react with the cross-linker BAC during the first stage. These two factors cause the imbalance in the cross-linking density and componential distribution in copolymers: the parts with more NVP have a higher cross-linking density, while the parts with more AAm possess a lower cross-linking density. During the reaction, AAm monomer will be exhausted first if the amount of NVP monomers is far greater than that of AAm. However, the NVP monomer disfavors the transformation from liquid solution to solid hydrogel, and the copolymer molecules without enough cross-linking density would dissolve into water in the immersion step. Thus the soluble parts of the hydrogels increase with increasing NVP content in the monomer mixture. As a result, the crosslinking density of hydrogels was higher than expected, and consequently the amount of thiol groups in the ANSH was higher than calculated (as shown in Table 1). Similarly, most of the NVP and cross-linker (BAC) monomers would be exhausted during the first reaction stage if the amount of NVP was almost equal to that of AAm in the monomer mixture, thus leaving only a small amount of the BAC monomers to react with the AAm monomers. The AAm monomers favored the formation of hydrogels even at a very low monomer concentration and low cross-linking density [6]. During the immersion steps, some copolymer molecules containing a high ratio of NVP and BAC were dissolved, resulting in a decrease in cross-linking density. As a result, the amount of thiol groups was lower than calculated. Fig. 2 shows that the amounts of SH in ANSH tended to be constant with the rise in monomer concentration, because the polarity and conjugated effect in the monomer were constant with the fixed ratio of NVP/AAm monomer in the reactive system. To prove the explanation above, the influence of the monomer ratio on the yield of ANSH was investigated. Fig. 3 shows that the yield of AHSH decreased greatly with increasing NVP content in the monomer mixture, indicating that NVP disfavored the formation of poly(AAm-co-
1060
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 3. The influence of monomer concentration and composition on the yield of ANSH copolymer (2.0 mol.% BAC, 1.0 mol.% APS).
Fig. 5. The influences of monomer concentration and composition on the molecular weight of ANSH copolymer (BAC = 2.0 mol.%, APS = 1.0 mol.%).
3.3. Re-gelation of ANSH solution and characterization of RANSS hydrogels
Fig. 4. Raman spectra of ANSS, ANSH and RANSN (A, B and C, respectively). All the samples were freeze-dried; ANSS obtained with NVP:AAm = 50:50 (mol:mol), BAC = 2.0 mol.%, APS = 1.0 mol.%, monomer concentration = 20.0 wt.%; ANSH was obtained after dissolution of ANSS with DTT; RANSS was obtained after re-gelation with DTDP.
NVP) hydrogels and there were more molecules dissolved into water during the immersion step. A microscopic confocal Raman spectrometer was used to confirm the presence and disappearance of SH groups before and after re-gelation of ANSH, respectively. As shown in Fig. 4, the characteristic absorption of SH stretching vibration (tS-H) in the ANSH sample appeared at 2570 cm1 and disappeared after re-gelation. A peak at 512 cm1, corresponding to the -SAS- stretching vibration (tS-S), was observed in the ANSS sample. The disappearance of tS-S in ANSH indicated the breaking down of disulfide bonds. After the formation of disulfide bonds in the RANSS sample, the tS-S appeared again at 512 cm1. Figs. 5 and 6 show the changes in molecular weights of the ANSH samples obtained under different conditions. Decreasing the NVP content in the monomer mixture and increasing the concentration of the monomer mixture both decreased molecular weight. The fact that samples with more BAC cross-linker possessed a higher molecular weight is consistent with previous reports [6]. The molecular weights of the polymers decreased with increasing APS content.
Re-gelation time is an important parameter to be considered when ANSH is used as an injectable material. The solution of ANSH copolymers with pendent SH groups could form a hydrogel through oxygenation in air or the thiol–disulfide exchange reaction. In this paper DTDP was used asa thiol–disulfide exchange reagent because of its low cell toxicity [18]. Fig. 7 shows pictures of the ANSH solution and the re-gelled hydrogel. Fig. 8 shows the influence of the DTDP content on the re-gelation time of the ANSH solution. ANSH solutions with the correct concentration could also form hydrogels through oxygenation in air; however, the re-gelation time in this case was quite long, about half an hour or even more. Adding DTDP into the ANSH solution would accelerate the regelation rate. When equimolar DTDP and SH was added to the ANSH solution, re-gelation was achieved within 3.0 min. These results were consistent with previous reports [6]. The solutions of ANSH copolymers containing a greater NVP content had a short re-gelation time, resulting from the higher SH content in the ANSH samples. Fig. 9 showed the influences of the ANSH concentration, the amounts of crosslinker and initiator on the re-gelation time of ANSH solution. Increasing the ANSH concentration increases the solution viscosity and decreases the re-gelation time. As shown in group A, the re-gelation time was about 2.0 min when the ANSH concentration was 5.0 wt.%. The re-gelation time was reduced still further, to about 1.0 min, as the concentration was increased to 7.5 wt.%. With the decrease in BAC content or increase in APS content, the formation of hydrogels required a higher ANSH concentration and a longer regelation time, as shown in groups D and E in Fig. 9. The influences of BAC and APS on re-gelation time could be attributed to the molecular weight changes of the ANSH copolymers. Due to the increase in molecular entanglement in aqueous solution, the copolymers with high molecular
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 6. The influences of BAC and APS on the molecular weight of ANSH copolymers (NVP:AAm = 50:50 (molar ratio), monomer concentration = 20.0 wt.%).
1061
Fig. 8. The influence of DTDP amount on the re-gelation time of ANSH. The error bars representing relative variation of data.
Fig. 9. The influence of monomer composition on the re-gelation time of ANSH (NVP:AAm = 50:50 (mol:mol); for groups A, B, C, D and E, the BAC content was 2.0, 2.0 2.0, 2.0 and 1.0 mol.%, respectively; APS was 1.0, 1.0, 1.0, 1.5 and 1.0 mol.%; monomer concentrations were 15.0, 20.0, 25.0, 20.0, 20.0 wt.%; and molecular weights were 10,000, 8900, 8500, 5900, 7000 Da). The error bars represent the relative variation of the data.
Fig. 7. The pictures of ANSH solution and re-gelled hydrogel (a, ANSH solution; b, re-gelled hydrogel).
weight would require a short re-gelation time. For injectable polymers, the correct re-gelation time is crucial for different applications. Adjusting the DTDP content, changing the concentration of the solution and selecting samples with dif-
ferent molecular weights are effective ways to regulate the re-gelation time of an ANSH solution. The transmittances of RANSS hydrogels were investigated using an ultraviolet–visible spectrophotometer. The hydrogels had excellent optical properties in the range of 400–800 nm, as shown in Fig. 10. As reported, the peaks of photopic sensitivity and scotopic sensitivity are at 555 and 506 nm in visible spectrum [19]. The transmittances of all RANSS hydrogels were over 83.0 and 81.2% at 555 and 506 nm, respectively. Meanwhile the transmittance increased gradually with increasing NVP content. The injectable property and the high transmittance in the visible region provide the opportunity for ANSH copolymers to be used as ophthalmic materials, such as artificial human vitreous and scaffold for lens regeneration. However, the retina is sensitive to the light radiation, thus photochemical damage to the retina can easily be caused by excessive ultraviolet radiation [20]. As shown in Fig. 10, RANSS hydrogels can block out some ultraviolet light radiation; in particular, in the range of 200–300 nm the transmittance was below 20.0%.
1062
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
Fig. 10. Transmittances of RANSS hydrogels with 4.0 mm thickness (the concentration of RANSS with increasing NVP was 7.5, 7.5, 5.0 and 5.0 wt.%, respectively).
Table 2 The refractive indexes of re-gelled hydrogels. RANSS concentration (wt.%)
5.0 7.5 10.0
NVP:AAM (mol:mol) 50:50
60:40
70:30
80:20
1.3409 1.3451 1.3491
1.3412 1.3454 –
1.3415 – –
1.3418 – –
Table 2 shows the changes in the refractive index (RI) with the concentration and composition of samples. Due to the low concentration of RANSS, the RIs of hydrogels ranged from 1.34 to 1.35, which is very close
to the RI of the human vitreous body (RI 1.33) [7]. Increasing the RANSS concentration would lead to a rise in the RI; however, sharply increasing the viscosity of the solution would result in difficulties of operation. The correct sample concentration is in the range of 5.0–10.0 wt.%. The equilibrium water contents (EWCs) of RANSS hydrogels were analyzed. The results showed that EWC values of all samples were over 95.0%, which is very close to the value of human vitreous body (a transparent jelly-like substance containing 97.0–99.0% water) [7]. The high EWC of RANSS could be attributed to the low cross-linking density in the hydrogel and the high hydrophilicity of the macromolecules. Highly hydrophilic hydrogels, such as RANSS hydrogels, are more suitable for use as artificial vitreous substance because the hydrogels show improved interfacial energetics and improved conformity with the retinal surface [7]. Moreover, an ideal vitreous substance must allow the transfer of necessary metabolites and proteins inside the vitreous body, and to or from the neighboring tissues [7]. Based on the above consideration, the morphologies of RANSS hydrogels were observed. Fig. 11 shows SEM photographs of the RANSS samples. RANSS hydrogels had the porous structure, with a pore size of 50–300 lm, due to its high EWC. The void space within the hydrogel network is big enough for the diffusion of nutrient materials and the release of metabolites from the cells. Transparent RANSS hydrogels with a porous structure are also suitable for use as a scaffold for lens regeneration. After a cataract extraction, ANSH copolymer solution
Fig. 11. The SEM photographs of RANSS hydrogels. (a1, a2) NVP:AAm = 60:40 (molar ratio); (b1, b2) NVP:AAm = 70:30 (mol:mol).
G. Niu et al. / Acta Biomaterialia 5 (2009) 1056–1063
1063
Acknowledgement This research is supported by the grant from Chinese National Science and Technology Supporting Item with No. 2006BAI03A09. References
Fig. 12. Plots of RANSS concentration vs. the value of MTT staining.
containing lens epithelial cells is injected into capsular bags and solidified into a porous hydrogel. Lens epithelial cells have the capacity to divide in the porous structure, and breed non-dividing enucleated lens fiber cells which, together with the porous material, form the internal lens substance [21]. The cytotoxicities of the RANSS hydrogels were analyzed based on the MTT method. Fig. 12 showed the changes in the degree of MTT staining with the RANSS hydrogel concentration. It can be seen that the MTT staining decreased gradually with increasing RANSS concentration. However, the 50.0% point, or IC50 value, was not obtained because the cytotoxicities of the RANSS samples were low under the experimental conditions. Compared with the previous results [15], it could be seen that the cytotoxicities of RANSS hydrogels were obviously lower than those of the reported materials. 4. Conclusion Injectable acrylamide/N-vinylpyrrolidinone copolymers with pendent thiol groups were prepared. The SH content of the copolymers was influenced by the monomer composition and the concentration of the monomer mixture. By adjusting the DTDP content, the concentration and composition of reactive copolymers could regulate the re-gelation time of ANSH solution. The re-gelled hydrogels had high transmittance in the visible region, and their RIs and EWCs were about 1.34–1.35 and over 95.0%, respectively. The hydrogels possessed a porous structure, with pore size ranging from 50 to 300 lm; their cytotoxicities were lower than those of the previous materials reported. The experimental results indicate that injectable ANSH copolymers could be used in the ophthalmic field, e.g. as artificial vitreous substance and scaffold for lens regeneration.
[1] Liu H, Li H, Cheng W, Yang Y, Zhu M, Zhou C. Novel injectable calcium phosphate/chitosan composites for bone substitute materials. Acta Biomater 2006;2:557–65. [2] Burdick JA, Anseth KS. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials 2002;23(22):4315–23. [3] Kang GD, Cheon SH, Khang G, Song SC. Thermosensitive poly(organophosphazene) hydrogels for a controlled drug delivery. Eur J Pharm Biopharm 2006;63:340–6. [4] Kretlow JD, Klouda L, Mikos AG. Injectable matrices and scaffolds for drug delivery in tissue engineering. Adv Drug Deliv Rev 2007;5(4–5):263–73. [5] de Groot JH, van Beijma FJ, Haitjema HJ, Dillingham KA, Hodd KA, Koopmans SA, et al. Injectable intraocular lens materials based upon hydrogels. Biomacromolecules 2001;2:628–34. [6] Aliyar HA, Hamilton PD, Ravi N. Refilling of ocular lens capsule with copolymeric hydrogel containing reversible disulfide. Biomacromolecules 2005;6:204–11. [7] Soman N, Banerjee R. Artificial vitreous replacements. Bio-Med Mater Eng 2003;13:59–74. [8] Nishi O. Refilling the lens of the rabbit eye after endocapsular cataract surgery. Folia Ophthalmol 1987;38:1615–8. [9] Gwon A. Lens regeneration in mammals: a review. Surv Ophthamol 2006;51:51–62. [10] Rabenstein DL, Weaver KH. Kinetics and equilibria of the thiol/ disulfide exchange reactions of somatostatin with glutathione. J Org Chem 1996;61:7391–7. [11] Shu XZ, Liu Y, Luo Y, Roberts MC, Prestwich GD. Disulfide crosslinked hyaluronan hydrogels. Biomacromolecules 2002;3:1304–11. [12] Plunkett KN, Berkowski KL, Moore JS. Chymotrypsin responsive hydrogel: application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides. Biomacromolecules 2005;6:632–7. [13] Serban MA, Prestwich GD. Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels. Biomacromolecules 2007;8(9):2821–8. [14] Aliyar HA, Foster WJ, Hamilton PD, Ravi N. Towards the development of an artificial human vitreous. Polym Preprints 2004;45(2):469–70. [15] Hamilton PD, Aliyar HA, Ravi N. Biocompatibility of thiolcontaining polyacrylamide polymers suitable for ophthalmic applications. Polym Preprints 2004;45(2):495–6. [16] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. [17] Pan ZR. Polymer Chemistry. Beijing: Chemical Industry Press; 2002, pp. 90–98. [18] Hisano N, Morikawa N, Iwata H, Ikada Y. Entrapment of islets into reversible disulfide hydrogels. J Biomed Mater Res 1998;40:115–23. [19] Wyszecki G, Stiles WS. Color Science. New York: John Wiley & Sons; 1967. [20] Noell WK, Walker VS, Kang BS. Retinal damage by light in rats. Invest Ophthalmol 1966;5:450–73. [21] Lipman RD, Taylor A. The in vitro replicative potential and cellular morphology of human lens epithelial cells derived from different aged donors. Curr Eye Res 1987;6:1453–7.