Molecular nanofibers of paclitaxel form supramolecular hydrogel for preventing tumor growth in vivo

Yue Luo ab, Meng Lib, Yuwei Zhaob, Hao Liuc, Tianxiang Gongb, Ying Hongb, Linfang Du*a and Qian Peng*c
aKey Laboratory of Bio-resources and Eco-environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China. E-mail: dulinfang@scu.edu.cn
bBlood Research Laboratory, Chengdu Blood Center, Chengdu 610041, Sichuan, China
cCancer Center, Hospital of the University of Electronic Science and Technology of China and Sichuan Provincial People's Hospital, Chengdu 610000, Sichuan, China. E-mail: pengqian0522@163.com

Received 8th July 2016 , Accepted 8th August 2016

First published on 10th August 2016


Abstract

A conjugate of tripeptide RGD and paclitaxel can form a nanofibrous hydrogel with a high payload of 66.7%, which can serve as both carrier and cargo for delaying tumor growth in vivo.


Self-assembly, as prevails in nature, has been recognized as a powerful strategy for construction of functional biomaterials.1−3 Inspired by nature, building blocks for such biomaterials have been developed, including DNA,4 polymer,3,5 lipid,6,7 saccharides,8–10 amino acid11–13 and peptides.12,14–19 After two decades in development, peptide-based biomaterials are attracting more attention because of their ease of preparation, good biocompatibility, ready function, and low immunity.2,20,21 Of these biomaterials, hydrogel containing more than 99% water and 1% hydrogelator is the most explored.22,23 Applications of hydrogel include wound healing,24,25 3D cell culture,26–28 enzyme detection29–31 and inhibitor screening,32–34 tissue engineering35,36 immune adjuvant37–40 and drug delivery.41–43 In the past 5 years, most groups have focused on use of hydrophobic drugs as functional groups to connect peptides forming a hydrogel, thus the drugs serve not only as a hydrophobic head for self-assembly, but also as a self-delivery system. For example, the Xu group reported the first example of a self-assembled prodrug incorporating vancomycin, which exhibited improved anti-bacterial activity, even against VRE (vancomycin-resistant enterococci).44 Following this, they introduced a hydrophobic drug (paclitaxel) to an enzyme-modulated peptide self-assembly system, resulting in sustained drug delivery and better water solubility of the hydrophobic drug.45 Inspired by this, several groups including Yang,46 Cui,47,48 and Ulijn49 have explored this area by rationally designing a drug–peptide conjugate. However, most reported work describes only a 24 h drug release profile and low anti-cancer efficacy when compared with the parent drug. There are even fewer papers about in vivo activity of these molecular hydrogels.50,51

In this study, we used a tripeptide RGD, which is a common motif to target integrin of most cancer cells, to connect paclitaxel via an ester bond. The formed precursor, 1, can form a transparent hydrogel via an ester bond break. The formed hydrogel can constantly release the parent drug over more than 1 week. We also demonstrate the activity of this hydrogel both in vitro and in vivo (on a xenografted mouse tumor model).

After the synthesis of 1, we prepared the paclitaxel derivative (compound 3, Scheme S1) with RGE tripeptide, the control group of RGD peptide. We found that for 1, PBS buffer can form transparent hydrogel at a concentration of 0.5 wt% for 1 h. HPLC results (Fig. S5) indicate that the hydrogel formation results from hydrolysis of 1 at the site of the ester bond (Fig. 1). As illustrated in Fig. 1, 1 itself with RGD motif has good solubility in PBS buffer (pH = 7.4), the ester bond between RGD and paclitaxel can hydrolyze to some extent (about 10% at 1 hours, Fig. S6). The hydrolysis process triggers co-assembly of 1 and paclitaxel, the RGD group of 1 which is not hydrolyzed helps stabilize the final hydrogel, resulting in a more stable hydrogel for up to 1 month, rather than paclitaxel precipitate. This hydrolysis process, similar to that reported by the Yang group,46 is an efficient strategy for preparation of nanostructures containing hydrophobic drugs.


image file: c6ra17473f-f1.tif
Fig. 1 Chemical structure of paclitaxel–RGD conjugate and illustration of the self-assembly process.

After demonstration of hydrogel formation, TEM was used to examine the nanostructure in the hydrogel. Bundles of nanofibers with diameter of 20–30 nm were observed, which entangle with each other and gelation water to form a 3D-network, resulting in a jelly-like structure. Replacement of RGD with RGE shows no significant difference in gelation ability and morphology, with nanofibers 20–30 nm in diameter and 30–50 μm in length. To examine the self-assembling ability of the hydrogel, a rheometer was used to test its mechanical property. As shown in Fig. 2C, the storage modulus (G′) of gel I (formed by hydrolysis of compound 1) and gel II (formed by hydrolysis of compound 3) is bigger than its corresponding loss modulus (G′′) at a concentration of 0.5 wt%, indicating the presence of an elastic and viscous three-dimensional network in the hydrogel. The storage modulus of both gels is about 40 Pa, suggesting a weak gel, which is common for a supramolecular hydrogel based on peptide.


image file: c6ra17473f-f2.tif
Fig. 2 (A) and (B) TEM images of gel I (formed by hydrolysis of 1) and gel II (formed by hydrolysis of 3) at a concentration of 0.5 wt% in PBS solution (pH = 7.4) for 1 h (inset: optical images of the gels). Scale bar is 500 nm. (C) Dynamic frequency sweep of gel I and gel II after 1 h of gel formation at the strain of 1.0%.

The release profile of the (active) parent drug from a hydrogel is critical to its function. An experiment was performed to determine the paclitaxel release from the hydrogel. As shown in Fig. 3, under physiological temperature conditions (37 °C), both hydrogels exhibit two stages in their release profiles: at 8 hours and 24 hours. There was a burst release of gel I at 8 hours, then release of about 0.5 μg per mL per hour after 24 hours. For gel II, paclitaxel was released at rate of about 0.6 μg per mL per hour and 0.4 μg per mL per hour at the two stages, respectively. Both gels can release paclitaxel for as long as 192 h at the constant release rate. These results suggest the potential of these hydrogels for sustained and constant delivery of anti-cancer drugs.


image file: c6ra17473f-f3.tif
Fig. 3 Accumulative release profile of paclitaxel from gel I and II at the concentration of 0.5 wt% (pH = 7.4) at 37 °C for (A) the first 24 h and (B) all the test time of 192 h. Each experiment was performed by 3 times.

Next, the cytotoxicity of 1 was tested. Compared with paclitaxel, 1 has similar cytotoxicity against 4T1 cell lines (Fig. S7), indicating that the conjugate of RGD and paclitaxel does not compromise the activity of paclitaxel. However, 3 showed much less toxicity than paclitaxel. To further demonstrate the efficacy of hydrogel I, the in vivo anti-tumor efficacy of hydrogel I and II in breast tumor was evaluated. When the size of tumor was about 300 mm3, hydrogels were injected intratumor at dosages of 40 mg kg−1. As shown in Fig. 4, just one dose of hydrogel I can delay tumor growth during the tested period. The final volume of tumors was about 1000 mm3 and 338% bigger than the original volume (∼300 mm3). However, the final relative tumor volumes were 940 mm3 and 348% in groups given 10 mg kg−1 of paclitaxel (intratumor injected 10 mg kg−1 every other day), or intratumor injected with PBS solution as control, respectively. Compared with the control group, the body weight of mice did not decrease in the groups administered the hydrogel. These results suggest that RGD decorated paclitaxel hydrogel can efficiently delay growth of breast cancer tumors.


image file: c6ra17473f-f4.tif
Fig. 4 (A) Hydrogel-inhibited 4T1 tumor growth and (B) body weight of mice during the experiment period. Female Balb/c mice were s.c. injected with 5 × 105 4T1 cells on the right flank. Bars shown are mean ± SE, and differences among groups are determined using one-way ANOVA analysis. The asterisks indicate that difference between PBS group and other group. *p < 0.05.

Conclusions

In summary, a tripeptide decorated paclitaxel was developed with drug loading of 66.7%, which is higher than in most previously reported studies. The conjugate of RGD and paclitaxel can form a supramolecular hydrogel with long sustained drug release capacity. Both in vitro and in vivo experiments suggest that this hydrogel does not compromise the activity of paclitaxel. Although the activity of the present hydrogel did not significantly enhance the activity of paclitaxel in vivo, which is common for most hydrogels, the hydrogel can be considered as a new formation of paclitaxel, not only enhancing the water solubility of paclitaxel by about 6000-fold, but it can also be combined with other anti-cancer drugs for combination therapy.52,53 Compared with other hydrogels, which show cytotoxicity and short time drug release, the present hydrogel exhibits anti-cancer efficacy both on cells and on a xenografted mouse tumor model, suggesting practical applications for this system in future.

Acknowledgements

This work was supported by the Hospital Doctor Foundation of Sichuan Provincial People's Hospital (Grant No. 30305030834). All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals, and protocols were approved by the Institutional Animal Care and Use Committee in College of Life Sciences, Sichuan University.

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Footnotes

Electronic supplementary information (ESI) available: Details experimental procedure and characterization, and MTT result. See DOI: 10.1039/c6ra17473f
These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2016
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