β-Cyclodextrin polyrotaxane monoaldehyde: a novel bio-crosslinker with high biocompatibility

Abstract

In this paper, we prepared a multi-functional bio-crosslinker: β-cyclodextrins (β-CD) polyrotaxane monoaldehyde (β-CD–PR-4). Compared to conditional crosslinkers, such as GA and EDC, this multi-functional bio-crosslinker has better crosslinking efficiency and lower cytotoxicity.

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Collagen is the most abundant protein and a primary component of extracellular matrices in mammalian connective tissue.¹ It has better biodegradability and biocompatibility, weak antigenicity and low toxicity² with broad applications.³ However, the weak mechanical properties, low thermal stability and high biodegradation rate of native collagen are not sufficient for its application in vivo or in vitro.⁴ The cross-linking of collagen is a way to improve the defects mentioned above. However physical methods for crosslinking does not result in an adequate degrees of crosslinking although they avoid the potentially cytotoxicity.⁵ For chemical crosslink, the most widely used crosslinkers contains glutaraldehyde (GA), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or genipin.^6–9 But these conditional crosslinkers always exhibited evident defect, such as cytotoxicity, insufficient crosslinking effect or some chromogenic reaction. Therefore, it is necessary to develop an alternative crosslinker which showed excellent crosslinking efficiency as well as cytocompatibility.

Modified cyclodextrins (CD)-based polyrotaxane supramolecular were proved to be biodegradable and toxicologically acceptable.^10,11 Many researchers^12–16 have prepared CD-based polyrotaxane by threading many CD units onto a polymer chain to acquire the polypseudorotaxane, and subsequently using anthryl, dinitrophenyl, naphthyl, trinitrophenyl trityl groups and various organic molecules as stoppers. However, using these stoppers brings some defects, such as cytotoxicity, indissolvable and the polyrotaxane also lacking of reactive functional groups, which limits its applications. So, we prepared tens of CD-based polyrotaxane by threading many β-CD units onto a poly(propylene glycol)bis(2-aminopropylether) (PPG–NH₂, MW ≈ 2000) chain and then capped the resultant polypseudorotaxane using β-CD monoaldehydes as stoppers, and then reduction with NaBH₄, and the then, it was directly oxidized in high yield to the corresponding monoaldehyde by cyclized 2-iodoxybenzoic acid (IBX) in dimethylsulfoxide (DMSO). And the last we evaluate its cytotoxicity and use it to crosslink collagen.

As shown in Fig. 1, β-CD-based polypseudorotaxane (β-CD–PR-1) and β-CD-based polyrotaxanes (β-CD–PR-2) was synthesized by the known methods.¹⁷ The reduction β-CD-based polyrotaxanes (β-CD–PR-3) was synthesized after the upper reaction. Namely, 50 μl acetic acid dropped in the reaction solution and continued to reflux for 2 h. After that, a sufficient amount of NaBH₄ was added to this reaction mixture, and allowed to react for 24 h at room temperature. Then, it dropped into 200 mL acetone, stirred for 1 h and stood for 3 h. The precipitated products were collected by centrifugation, then washed twice with ethanol and dried under vacuum at 35 °C to get the β-CD–PR-3 complexes. β-CD-based polyrotaxane monoaldehydes (β-CD–PR-4) was synthesized according to literature procedure.²⁰ Briefly, 0.58 g β-CD–PR-3 and 0.36 g IBX were dissolved in 6 mL DMSO. Then magnetic stirred for 24 hour at 25 °C and the reaction mixture was dropped into 200 mL acetone, stirred for 1 h and then stood for 3 h. After centrifugation, the precipitate dried under vacuum at 30 °C. After that it was dissolved in water, stirred for 4 h, the precipitate was removed by filtration. At last, lyophilization of the filtrate afforded the desired product.

Fig. 1 Schematic of synthesis of β-cyclodextrins polyrotaxane monoaldehydes.

We have done the XRD measurements of β-CD and β-CD–PR-1. The three strong peaks of pure β-CD appear at 10.6°, 12.5°, and 19.5°. It indicates a cage-type crystal structure.¹⁸ The diffractogram of β-CD–PR-1 showed a diffraction pattern different from that of β-CD. The three strong peaks appear at 5.7°, 11.5°, and 17.8°. This constitutes primary evidence that a different crystal type was formed.

The ¹H NMR spectra of the β-CD–PR-1, β-CD–PR-2, β-CD–PR-3 and β-CD–PR-4 samples are shown in Fig. 2. In the ¹H NMR spectrum, the peaks were identified and were found to belong to both β-CD and PPG–NH₂ molecules. Such as δ (ppm): 1.03–1.05 (CH₃ for PPG), 3.56 (C₂–H, C₄–H), 3.64 (C₃–H, C₅–H, C₆–H), 4.43 (C₆–OH), 4.83 (C₁–H), 5.68 (C₂–OH, C₃–OH). There are also a shift in some of the CD-protons.¹⁹ The results suggest that the β-CD–PR-1 may be the inclusion complex. Furthermore, a ¹H NMR spectrum of β-CD–PR-1 shows that the integral area ratio between methyl protons of PPG–NH₂ (a molecule of PPG–NH₂ 2000 contains ca. 102 methyl protons, δ = 1.03–1.05 ppm) and C₁–H of β-CD protons (a molecule of β-CD contains 7 C₁–H protons, δ = 4.83 ppm) is 7.9 : 7.0. In the case of β-CD–PR-2, this ratio decreases to 6.8 : 7.0. According to these ¹H NMR data, we can calculate that there are ca. 13 β-CD units in β-CD–PR-1 and ca. 15 β-CD units in β-CD–PR-2. Moreover, the signals assigned to the protons (–CH=N–, δ = 7.95 ppm) is observed in the ¹H NMR spectra of β-CD–PR-2. These results clearly indicate that two β-CD cavities are successfully introduced at the amino terminals of β-CD–PR-1. Compared with the ¹H NMR spectra of the β-CD–PR-2, in the ¹H NMR spectra of the β-CD–PR-3, the signals assigned to the protons (–CH=N–, δ = 7.95 ppm) has disappeared, and a new single is observed at δ 9.70 in the spectra of β-CD–PR-4. It is the characteristic single signal of the formyl proton, so it proved to generate aldehyde groups. Simultaneously, a new signal of an anomeric proton at δ 4.93 appears at the expense of the signal at δ 4.93. And the ratio of these two signals is 1 : 6, which is the indicative of the monooxidation, and these results are consistent with Jing Hu²⁰ and Cornwell.²¹ The molecular weight of β-CD–PR-4 is about 18 950.

Fig. 2 The ¹H NMR spectra of the β-CD–PR-1, β-CD–PR-2, β-CD–PR-3 and β-CD–PR-4.

Fig. 3 shows the results of cytotoxicity test by the addition of β-CD–PR-4 to L929 fibroblasts. While the conditional cross-linker EDC and GA were as control. The cytotoxicity test was according to literature procedure.^22,23 The cytotoxicities of β-CD–PR-4 were not even observed at the concentration of 0.1 mg mL⁻¹. However, the crosslinker EDC and GA began to show cytotoxicity at the concentration of 10⁻³ mg mL⁻¹, and they could kill about 79.3% and 89.7% cells compared to that of β-CD–PR-4 at the concentration of 0.1 mg mL⁻¹, respectively. The results show that the β-CD–PR-4 has better compatibility than the conditional crosslinkers.

Fig. 3 Viability of L929 fibroblasts exposed to various crosslinker at different concentrations: β-CD–PR-4 (), EDC () and GA ().

The ninhydrin²⁴ assay was used to estimate the crosslinking efficiency of β-CD–PR-4 to collagen, and the results were shown in Fig. 4. The crosslinking index of collagen increased with the increase of the crosslinkers concentrating. For β-CD–PR-4, the maximum crosslinking degree is around 87.1% at the reactive functional groups concentration of 0.012 mM mL⁻¹, which was higher than that of EDC and GA, whose crosslinking degree is 69.3% and 79.0%, respectively, at the same concentration. The high crosslinking ability of β-CD–PR-4 to collagen should be attributed to strong binding, as β-CD–PR-4 had both covalent and non-covalent interactions with collagen. Hence, aldehydic functionality in β-CD–PR-4 covalently crosslinked with amino groups of collagen and the hydroxyl groups could involve in hydrogen bonding interaction that brings significant increase in thermal and enzymatic stability.²⁵

Fig. 4 Crosslinking efficiency of different crosslinker concentrations of β-CD–PR-4 (), EDC () and GA ().

After cross-link, we tested the denaturation temperature of the collagen with DSC, and the results were shown in Fig. 5. The thermal stability of the collagen increased with increasing in β-CD–PR-4 concentration. The denaturation temperature of un-crosslinked collagen was about 58.0 °C, while that of β-CD–PR-4 treated collagen would increase with the increase of β-CD–PR-4 concentration. After cross-linked with 0.012 mM mL⁻¹ β-CD–PR-4, the denaturation temperature of collagen would increase by 23.6 °C. The increased in thermal stability could be related to the increasing in the number of crosslinking, as they decreased the entropy of transition²⁶ which would lead the collagen gel to be stiffer. In addition, the special and rigid structure of β-CD–PR-4 could also improve the thermal stability of the collagen effectively.

Fig. 5 DSC thermographs of pure collagen and different concentrations (mM mL⁻¹) of β-CD–PR-4 crosslinked collagen.

In summary, we had successfully prepared a multi-functional bio-crosslinker: β-cyclodextrins (β-CD) polyrotaxane monoaldehydes (β-CD–PR-4) in a convenient and efficient method. On the basis of this method, we could synthesis CD-capped polyrotaxanes monoaldehydes or multi-aldehydes with appropriate molecular weights by changing the reaction conditions or selecting various polymer chains. It exhibited low cytotoxicity to L929 cells and could cross-link the collagen, and also had no chromogenic reaction. The maximum crosslinking degree is around 87.1%, which was much higher than that of EDC and GA at the same concentration. In addition, it could improve the thermal stability of the collagen effectively. Our results revealed the application of β-CD–PR-4 as a multi-functional bio-crosslinker. It also has the potential to be applied in the field of molecular machines, tissue engineering scaffolds, human biological sensors, especially used as drug controlled release carrier materials.²⁷

Acknowledgements

This work was supported by National Natural Science Foundation of China (no. 51232002, 51273072) and the Natural Science Foundation of Guangdong Province (2012A080800015, 2012A080203010), which are gratefully acknowledged.

Notes and references

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