pH-responsive cocktail drug nanocarriers by encapsulating paclitaxel with doxorubicin modified poly(amino acid)

Abstract

We design a pH-responsive cocktail nanocapsule with a suitable size (around 100 nm) via the assembly of doxorubicin (DOX) modified poly(amino acid). Paclitaxel (PTX) is successfully encapsulated in the hydrophobic cavity of the nanocapsules through a non-covalent interaction between PTX and the DOX modified poly(amino acid). Guided by the surface bioconjugated Arg–Gly–Asp (RGD) moieties, a target peptide, nanocapsules are targeted to and uptaken by cancer cells. These nanocapsules are stable under normal physiological pH conditions (pH 7.4) and drug release can be triggered by the relatively low pH (5.0) in cancer cells. Moreover, the drug release can be tracked via the recovered red fluorescence of DOX due to the relief from the self-quenching state as a result of nanocapsule disruption under acidic conditions. The in vitro test results undoubtedly confirm the RGD-mediated synergetic therapeutic efficacy of these cocktail drug nanocapsules. This research paves a new way for the fabrication of smart nanocapsules for cocktail drug delivery.

---

Introduction

Most serious illnesses including AIDS and cancer face the challenge of drug resistance originating from immunity or the self-repairing ability of living beings.^1,2 The combination of multiple antiretroviral agents that act on different stages of the HIV life-cycle, known as cocktail therapy, has proven highly effective in inhibiting HIV replication and AIDS progression.^3,4 Such a strategy has also been employed for combining multiple anticancer agents to act on different sites and different growth stages in clinical cancer chemotherapy.^5–7 However, the therapeutic effect is limited by the poor bioavailability and suboptimal pharmacokinetics due to the hydrophobicity and low molecular weight of the anticancer agents.^8–10 Up to now, drug carriers with a variety of architectures^11–13 including polymer–drug conjugates, micelles, liposomes, nanospheres, nanogels and virus-based nanocarriers^14,15 have been developed to transport drugs in a controlled and targeted fashion.^16–21 But in most of the reported delivery systems, just one anticancer agent was applied because different agents were difficult to be assembled in a single capsule^22–24 due to the different chemical nature of drugs, especially for the combination usage of hydrophobic and hydrophilic drugs.

Hydrophilic doxorubicin (DOX) was commonly absorbed by the negative charged surface^24,25 or embedded in hydrophilic micelle cores⁸ for effective delivery, while hydrophobic paclitaxel (PTX) was often encapsulated in the hydrophobic cores of the self-assembled amphiphilic polymers micelles.²⁶ Recently, some synthesized polymer vesicles were used to load the hydrophobic and hydrophilic anticancer agents in the core and layer, respectively.^27–31 Compared with liposomes and micelles, these exquisitely designed polymers endow the nanocapsules with tunable size and shape, higher drug loading capacity, longer circulation time and controllable drug release.^32–35

Recently, the polypeptide-based polymers have attracted wide attention due to their good biocompatibility and biodegradability.^36–38 Some polypeptide-based drug delivery systems have entered the stage of clinical evaluation.³⁹ However, co-delivery of DOX and PTX still face challenges such as controlled release to target sites. Very recently, we developed the near infrared (NIR) fluorescence tracked Ag₂S/PTX@poly(amino acid) nanocapsules, of which the drug release could be triggered by breaking of pH-responsive hydrogen bonds.⁴⁰

Herein, we design a pH-responsive cocktail drug nanocapsule with suitable size (around 100 nm) via the assembly of amphiphilic poly(amino acid) [polysuccinimide (PSI) functionalized with oleylamine (OAm) and DOX, termed as PSI_OAm-DOX]. PTX is successfully encapsulated in the nanocapsules through hydrophobic interaction and H-bond between PTX and PSI_OAm-DOX. These nanocapsules are pretty stable under physiological conditions (pH 7.4). Guided by the surface bioconjugated Arg–Gly–Asp (RGD) peptide, nanocapsules are targeted to and uptaken by cancer cells and undergo capsule disruption and thus drug release because of hydrogen bond breaking under acidic conditions (pH 5.0, in lysosome) (Scheme 1). Notably, the drug release can be tracked by the recovered red fluorescence of DOX due to the relief from self-quenching state. The in vitro test results undoubtedly confirm the RGD-mediated synergetic therapeutic efficacy of these cocktail drug nanocapsules.

Scheme 1 Schematic illustration for fabrication and pH-responsive drug release of cocktail drug nanocapsules.

Experimental

Materials and methods

The dimethyl sulfoxide (DMSO) and oleylamine (OAm) were purchased from Aldrich. All rare-earth nitrates utilized in this work were purchased from Beijing Ouhe Chemical Reagent Co. 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC, Sigma) and N-hydroxysuccinimide (NHS, Acros) were used for bioconjugation. Paraformaldehyde, NaOH, ethanol, Na₂HPO₄, Na₃PO₄, and CHCl₃ were obtained from Beijing Chemical Reagent Co. The poly-succinimide (PSI) was obtained from Shijiazhuang Desai Chemical Company. For cell culture, Dulbecco's modified eagle medium (DMEM), phosphate buffer solution (PBS) and pancreatin were purchased from M&C Gene Technology. All chemicals were analytical grade and used without further purification. Deionized (DI) water was used throughout. Fetal calf serum was obtained from Hangzhou Sijiqing Bioengineer Materials Ltd. Methyl thiazolyltetrazolium (MTT) was obtained from Amresco Inc. The targeting peptide RGD (amino acid sequence: RGDYCYYCYYCC) was obtained from GL Biochem (Shanghai) Ltd.

The size and morphology of the nanocapsules were characterized by a JEM-1200 EX (JEOL) transmission electron microscope (TEM). Fourier-transform infrared (FT-IR) spectra were performed on a JASCO FT/IR-460 PLUS spectrometer (Tokyo). The drug release quantification was performed on a high performance liquid chromatography (HPLC, Angilent Tech.) equipped with G1322A degasser, G1312A bin pump, G1367E 1260 HiP ALS, and G4212B 1260 DAD. The cell imaging was performed on a TCS SP5 two-photon confocal microscopes (Leica). Dynamic light scattering (DLS) particle size analysis was carried out on a Zetasizer Nano-ZS90 (Malvern) zeta and size analyzer. The optical properties of the nanostructures were characterized with a Hitachi F-4600 fluorescence spectrophotometer.

Synthetic procedures

Preparation of amphiphilic comb-like PSI_OAm-DOX. For PSI_OAm-DOX, 1.6 g of polysuccinimide (PSI) was dissolved in 32 mL of N,N-dimethylformamide (DMF) at 90 °C under magnetic stirring followed by the addition of 261 mg of DOX. The mixture was treated at 100 °C for 5 h for DOX bonding onto the PSI backbone by aminolysis reaction. Then, 2.17 mL of OAm were added and treated at 100 °C for another 5 h to allow for OAm bonding onto the backbone. After cooling to room temperature, methanol (80 mL) was added to precipitate the product (PSI_OAm-DOX). Finally, the PSI_OAm-DOX was redispersed into chloroform to get a stock solution with concentration of 200 mg mL⁻¹ after centrifugation and then evaporating the trace amount of residual methanol.

Preparation of the PTX@PSI_OAm-DOX nanocapsules. Into 10 mL of NaOH (5.0 mM) aqueous solution, 1.0 mL of mixture chloroform solution containing PSI_OAm-DOX (40 mg) and PTX (1.0 mg) was added and sonicated (400 W) for 6 min. Subsequently, the chloroform was removed by evaporating at 58 °C for 30 min under magnetic stirring. The nanocapsules were collected and purified by centrifugation at 11 000 rpm for 10 min and redispersed into PBS (1.0 mL). As a control, 40 mg of PSI_OAm-DOX was treated under the identical conditions without PTX. And we quantified the encapsulated PTX using HPLC after breaking up nanocapsules with n-octanol, which was determined to be 0.82 mg mL⁻¹ in the stock solution. In another aspect, the content of DOX could be determined by detecting and calculating the fluorescence intensities of DOX before and after conjugating reaction, we get the DOX concentration that is 0.66 mg mL⁻¹ in the stock solution.

PTX release. 1.5 mL of the stored nanocapsules was diluted to 3.0 mL with PBS and dialyzed against 60 mL of PBS containing sodium salicylate (to help solubilizing PTX in water) at pH 5.0 and 7.4, respectively. 10 mL of eluate outside the dialysis bag (molecule weight cut 14 000) was taken at certain time interval and extracted with 2 mL of octanol at 37 °C for 1 h. Thereafter, 10 mL of PBS (pH 5.0 or pH 7.4) containing sodium salicylate was replenished. Quantification of PTX solution in octanol was performed on HPLC. PTX taken by each time was taken into account and calculated as cumulative release for the final evaluation. Mobile phase of gradient elution was 90/10–0/100 (water–methanol) for 0–60 min and then 0 : 100–90 : 10 (water–methanol) for 60–63 min. The flow rate was 0.5 mL min⁻¹.

Bioconjugation with RGD peptide. 1.0 mL of RGD (1.0 mg in 1.0 mL PBS) and 1.0 mL of PBS (pH 7.4, 0.1 M) containing EDC (0.7 mg) and NHS (0.1 mg) were mixed with 1.0 mL of nanocapsule dispersion, followed by incubating at 25 °C for 4 h. Finally, the nanocapsule-RGD bioconjugates were collected by centrifugation (12 000 rpm, 10 min) and washed with PBS for three times. The obtained RGD conjugated nanocapsules were redispersed in PBS (pH = 7.4, 1.0 mL) and stocked at 4 °C for later use.

In vitro anti-cancer effect evaluation. Briefly, HeLa cells (5 × 10⁴ cells per well) seeded to the bottom of the 96-well cell culture plate were incubated with different amounts (0–200 μg PTX mL⁻¹) of sterilized nanocapsules of PTX@PSI_OAm, PTX@PSI_OAm-RGD, PSI_OAm-DOX-RGD, PTX @PSI_OAm-DOX-RGD at 37 °C for 24 and 48 h, respectively. Thereafter, the cytotoxicity was evaluated via the methyl thiazolyltetrazolium (MTT) assay.

Cell imaging. HeLa cells were seeded on a sterilized glass cover slide and cultured in a 12-well cell culture plate overnight under recommended conditions at 37 °C in 5% CO₂-humidified incubator. Then the PTX@PSI_OAm-DOX-RGD nanocapsules stock solution was added into the cell culture well with a final concentration of 20 μg PTX mL⁻¹. The HeLa cells were incubated with the nanocapsules for 12 h, followed by washing with PBS for 3 times and incubation for another 12 h. As a control, the bare nanocapsules without RGD, were incubated with the HeLa cells under the same conditions for 24 h. Thereafter, the cells on the glass slide were washed with phosphate buffer solution (PBS, pH 7.4, 10 mM) and fixed in 4% paraformaldehyde solution for 15 min. The fluorescence imaging was conducted on a TCS SP5 two-photon confocal microscope (Leica).

Results and discussion

PSI was modified with DOX and OAm to get the hydrophobic comb-like PSI_OAm-DOX,⁴¹ which is notable that DOX should be decorated onto the backbone of PSI first, otherwise the OAm are likely to prevent the conjugation of DOX due to steric hindrance. Chloroform solution containing PSI_OAm-DOX and PTX was emulsified with aqueous solution of NaOH by means of sonication to get PTX@PSI_OAm-DOX nanocapsules. As a control, PSI_OAm-DOX nanocapsules were prepared in the absence of PTX. The effective modification of PSI by DOX and the successful fabrication of PTX@PSI_OAm-DOX nanocapsules were first characterized via the Fourier transform infrared (FTIR) spectroscopy. As shown in Fig. 1, the FTIR spectra of pure PTX, DOX and PSI_OAm were shown as comparison. Compared to the spectrum of PSI_OAm, the main difference is the widening of peak around 3400 cm⁻¹ in the spectra of PSI_OAm-DOX and PTX@PSI_OAm-DOX nanocapsules, which is the preliminary evidence of the drug loading.

Fig. 1 FTIR spectra of the PTX, DOX, PSI_OAm, PSI_OAm-DOX nanocapsules and PTX@PSI_OAm-DOX nanocapsules.

The size and shape of the nanocapsules were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS). Indeed, hydrophobic PSI_OAm-DOX is transformed to amphiphilic carboxylated PSI_OAm-DOX after hydrolysation in NaOH aqueous solution, and spontaneously self-assembly into micells with an average size of 200 nm (Fig. 2a and b) driven by the hydrophobic interaction among the comb-teeth like OAm and DOX on the PSI backbone with the help of ultrasonication. In the presence of PTX, the PTX would be encapsulated in the hydrophobic core and the formed PTX@PSI_OAm-DOX nanocapsules shrank to around 100 nm (Fig. 2c and d), compared with that of PSI_OAm-DOX nanocapsules. The size shrinking is supposed to be induced by the H-bond and the hydrophobic–hydrophobic interaction. Moreover, the DLS size of PTX@PSI_OAm-DOX nanocapsules (100 nm) is more suitable for in vivo drug delivery because the nanoparticles with larger hydrodynamic size are more likely retained by the organs of reticuloe endothelin system (RES).

Fig. 2 TEM images (a and c) and DLS analysis (b and d) of PSI_OAm-DOX (a and b) and PTX@PSI_OAm-DOX nanocapsules.

The introduction of the PTX not only endows the nanocapsules with cocktail therapy effects and suitable size for drug delivery (less than 100 nm), but also formulates the controlled drug release mechanism based on the pH-sensitive H-bond.^42,43 As shown in Fig. 3a, the spherical outline of the nanocapsules became dim after incubating under pH 5.0 for 4 h. If extended to 24 h, the nanocapsules were totally disrupted, leaving some tangled polymers in the solution (Fig. 3b). On the other hand, the nanocapsules without PTX maintained the similar spherical morphology under pH 5.0 (Fig. 3c) as shown in Fig. 2a. Furthermore, the polymer PSI_OAm-DOX were replaced by poly(styrene-methyl acrylic acid)(PSMAA) to prepare the PTX@PSMAA nanocapsules. For these nanocapsules, hydrophobic–hydrophobic interaction is the main driving force and they were stable in both pH 7.4 and pH 5.0 PBS (Fig. S1†), which further demonstrate the H-bond based nanocapsule disruption. These results suggest that the acidic environments induced destruction of nanocapsule structures can be attributed to the breaking of pH-responsive H-bond between PTX and PSI_OAm-DOX in nanocapsules. Meanwhile, these results also undoubtedly evidence the successful encapsulation of PTX in the nanospheres.

Fig. 3 TEM images of disrupted PTX@PSI_OAm-Dox nanocapsules in pH 5.0 PBS for 4 h (a) and 24 h (b), and PSI_OAm-DOX nanocapsules in pH 5.0 PBS.

In vitro release studies were performed by incubating the cocktail nanocapsules in pH 7.4 and pH 5.0 PBS at 37 °C, respectively (Fig. 4). The released PTX was quantified via high performance liquid chromatography (HPLC). At pH 7.4, PTX leakage was less than 8% after 48 h. However, if the pH value was decreased to 5.0, the PTX release was distinctly accelerated. After 48 h, over 59% of PTX was released from the nanocapsules. Moreover, no burst release of PTX from the nanocapsules was observed. These results indicate that the pH-responsive release is sustainable, which is highly desirable for achieving prolonged therapeutic action over a longer period of time after administration. Notably, while the PTX release from the destabilized nanocapsules, the comb-like polymer PSI_OAm-DOX would not reassemble into nanostructure as only tangled polymer was observed and in addition, recovery fluorescence of DOX was observed (Fig. 5a), while, the fluorescence of the DOX remained at self-quenching situation after 48 h incubation at pH 7.4 (Fig. 5b). These results indicate the recovered fluorescence of DOX can be used for the fluorescence tracking of drug release.

Fig. 4 pH-responsive release of PTX as a function of time for PTX@PSI_OAm-Dox as detected by HPLC.

Fig. 5 Fluorescence spectra of PTX@PSI_OAm-Dox in PBS at pH 5.0 (a) and pH 7.4 (b) for different incubation time.

Besides the effective pH-controlled PTX release, it is necessary to evaluate the potential toxicity, delivery safety and therapy effect of the cocktail drug nanocapsules for drug delivery applications. The cytotoxicity of the drug nanocapsules to HeLa cell line was evaluated by the MTT method. Fig. 6 shows the cell viability after 48 h of incubation with nanocapsules at different concentrations. When the concentration of PTX reaches 200 μg mL⁻¹, the viability of HeLa cells still keeps over 92%. The IC₅₀ of PTX@PSI_OAm-DOX and PSI_OAm-DOX-RGD are far more than 200 μg mL⁻¹, respectively. That is to say, PTX@PSI_OAm-DOX nanocapsules have negligible cytotoxicity to HeLa cells under normal physiological conditions (pH 7.4, 37 °C) since the DOX was located inside the nanocapsules by hydrophobic–hydrophobic interaction. On the other hand, if bioconjugated with cancer cell targeting peptide, RGD, the nanocapsules could specifically bind to the integrin α_vβ₃ on the HeLa cell surface, which would help to penetrate the cell membranes mediated by the RGD and undergo the lysosome digestion (pH 5.0). Both of the PTX and PSI_OAm-DOX act as anticancer drugs to kill the cell effectively, in which only 10% cells are still alive. The IC₅₀ of PTX@PSI_OAm-RGD and PTX of PTX@PSI_OAm-DOX-RGD are 100 μg mL⁻¹ and 75 μg mL⁻¹, respectively. These results indicate that the PTX@PSI_OAm-DOX nanocapsules are safe until they are uptaken by the cancer cells. Meanwhile, as control, PTX@PSI_OAm-RGD nanocapsules without DOX conjugating and PSI_OAm-DOX-RGD nanocapsules without PTX show significant cytotoxicity to HeLa cells, but the therapy effect is much lower than the PTX@PSI_OAm-DOX-RGD nanocapsules, indicating the cocktail drug nanocapsules have synergetic anticancer effects.

Fig. 6 HeLa cell viability following 48 h exposure to different nanocapsules at different concentrations in cell culture media (pH 7.4), respectively.

It is more interesting to study the pH responsiveness of PTX@PSI_OAm-DOX nanocapsules in living cells. The cellular uptake of PTX@PSI_OAm-DOX nanocapsules is evaluated to determine whether the pH-responsive nanocapsules are effective in the intracellular environments. As shown in Fig. 7a, without the guidance of targeting peptide RGD, PTX@PSI_OAm-DOX does not penetrate the cell membrane even that the cells are exposed to the nanocapsules for 24 h. As to the PTX@PSI_OAm-DOX-RGD nanocapsules, they are first incubated with cells for 12 h and then removed from the culture media, followed by further 12 h culture. As shown in Fig. 7b, the cells exposed to PTX@PSI_OAm-DOX-RGD nanocapsules display the relatively weak red fluorescence of DOX in cytoplasm. By prolonging the incubation time to 24 h, the cells display much stronger red fluorescence due to everlasting lysosome digestion (pH 5.0). It should be noted that the DOX or DOX–polymer can act as anticancer drug in cytoplasm as well as in nucleus, even without entering the cell.⁴⁴ These results further indicate the acidic environments of the lysosome play a key role in the nanocapsule degradation, drug release and fluorescence recovery.

Fig. 7 Fluorescence imaging of HeLa cells incubated with (a) PTX@PSI_OAm-Dox without RGD for 24 h, (b) PTX@PSI_OAm-DOX-RGD nanocapsules for 12 h, and (c) PTX@PSI_OAm-DOX-RGD nanocapsules for 12 h with another 12 h incubation.

Conclusions

We present a novel poly(amino acid) nanocapsule for simultaneous loading of doxorubicin and paclitaxel for cocktail drug delivery with enhanced synergetic chemo-therapy efficacy. DOX and OAm was covalently linked to PSI to get the hydrophobic polymer–drug conjugate PSI_OAm-DOX. PTX is then encapsulated in hydrophobic cavity of the PSI_OAm-DOX nanocapsules through hydrophobic interaction and H-bond. After bioconjugation with cancer cell targeting peptide (RGD), PTX@PSI_OAm-DOX-RGD nanocapsules are specifically uptaken by HeLa cells and undergo the lysosome digestion at low pH (5.0), which results in the nanocapsule disruption and drug release. This PTX@PSI_OAm-DOX-RGD nanocapsule is a promising co-delivery system of PTX and DOX for chemotherapeutics-involved cocktails for cancer therapy. This research paves a new way for the fabrication of smart nanocapsules for cocktail drug delivery and will draw wide attention in the fields of nanomaterials, biotechnology and nanomedicine.

Acknowledgements

The authors gratefully acknowledge for the financial support by the National Natural Science Foundation of China (21475007 and 21275015), the State Key Project of Fundamental Research of China (2011CB932403), and the Fundamental Research Funds for the Central Universities (YS1406). We also thank the support from the "Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology".

Notes and references

1. A. Calmy, F. Pascual and N. Ford, N. Engl. J. Med., 2004, 350, 2720–2721.
2. P. Bouwman and J. Jonkers, Nat. Rev. Cancer, 2012, 12, 587–598.
3. J. Cohen, Science, 2005, 309, 1002–1005.
4. P. N. Nehete, B. P. Nehete, P. Manuri, L. Hill, J. L. Palmer and K. J. Sastry, Vaccine, 2005, 23, 2154–2159.
5. C. Bock and T. Lengauer, Nat. Rev. Cancer, 2012, 12, 494–501.
6. M. Martin, M. A. Segui, A. Anton, A. Ruiz, M. Ramos, E. Adrover, I. Aranda, A. Rodriguez-Lescure, R. Grosse, L. Calvo, A. Barnadas, D. Isla, P. M. del Prado, M. Borrego, J. Zaluski, A. Arcusa, M. Munoz, J. M. Vega, J. R. Mel, B. Munarriz, C. Llorca, C. Jara, E. Alba, J. Florian, J. F. Li, J. A. Garcia-Asenjo, A. Saez, M. Rios, S. Almenar, G. Peiro, A. Lluch and G. Investigators, N. Engl. J. Med., 2010, 363, 2200–2210.
7. J. A. Sparano, M. L. Wang, S. Martino, V. Jones, E. A. Perez, T. Saphner, A. C. Wolff, G. W. Sledge, W. C. Wood and N. E. Davidson, N. Engl. J. Med., 2008, 358, 1663–1671.
8. A. Gabizon, H. Shmeeda and Y. Barenholz, Clin. Pharmacokinet., 2003, 42, 419–436.
9. N. K. Ibrahim, N. Desai, S. Legha, P. Soon-Shiong, R. L. Theriault, E. Rivera, B. Esmaeli, S. E. Ring, A. Bedikian, G. N. Hortobagyi and J. A. Ellerhorst, Clin. Cancer Res., 2002, 8, 1038–1044.
10. P. J. O'Dwyer, J. P. Stevenson and S. W. Johnson, Drugs, 2000, 59(suppl 4), 19–27.
11. Q. B. Xiao, Y. T. Ji, Z. H. Xiao, Y. Zhang, H. Z. Lin and Q. B. Wang, Chem. Commun., 2013, 49, 1527–1529.
12. S. Y. Han, Y. X. Liu, X. Nie, Q. Xu, F. Jiao, W. Li, Y. L. Zhao, Y. Wu and C. Y. Chen, Small, 2012, 8, 1596–1606.
13. J. F. Hui, X. Y. Zhang, Z. C. Zhang, S. Q. Wang, L. Tao, Y. Wei and X. Wang, Nanoscale, 2012, 4, 6967–6970.
14. M. Verwegen and J. Cornelissen, Macromol. Biosci., 2015, 15, 98–110.
15. Y. J. Ma, R. J. M. Nolte and J. Cornelissen, Adv. Drug Delivery Rev., 2012, 64, 811–825.
16. S. S. Wu, X. Huang and X. Z. Du, J. Mater. Chem. B, 2015, 3, 1426–1432.
17. Z. W. Chen, Z. H. Li, Y. H. Lin, M. L. Yin, J. S. Ren and X. G. Qu, Biomaterials, 2013, 34, 1364–1371.
18. Z. W. Chen, Z. H. Li, Y. H. Lin, M. L. Yin, J. S. Ren and X. G. Qu, Chem.–Eur. J., 2013, 19, 1778–1783.
19. K. Sugano, M. Kansy, P. Artursson, A. Avdeef, S. Bendels, L. Di, G. F. Ecker, B. Faller, H. Fischer, G. Gerebtzoff, H. Lennernaes and F. Senner, Nat. Rev. Drug Discovery, 2010, 9, 597–614.
20. S. Ganta, H. Devalapally, A. Shahiwala and M. Amiji, J. Controlled Release, 2008, 126, 187–204.
21. S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991–1003.
22. J. Hureaux, F. Lagarce, F. Gagnadoux, M. C. Rousselet, V. Moal, T. Urban and J. P. Benoit, Pharm. Res., 2010, 27, 421–430.
23. J. Y. Lee, K. H. Bae, J. S. Kim, Y. S. Nam and T. G. Park, Biomaterials, 2011, 32, 8635–8644.
24. Z. J. Zhang, J. Wang, X. Nie, T. Wen, Y. L. Ji, X. C. Wu, Y. L. Zhao and C. Y. Chen, J. Am. Chem. Soc., 2014, 136, 7317–7326.
25. W. H. Chiang, W. C. Huang, Y. J. Chang, M. Y. Shen, H. H. Chen, C. S. Chern and H. C. Chiu, Macromol. Chem. Phys., 2014, 215, 1332–1341.
26. X. X. Chuan, Q. Song, J. L. Lin, X. H. Chen, H. Zhang, W. B. Dai, B. He, X. Q. Wang and Q. Zhang, Mol. Pharm., 2014, 11, 3656–3670.
27. W. C. Ma, A. R. Xu, J. Y. Ying, B. Li and Y. Jin, J. Biomed. Nanotechnol., 2015, 11, 1193–1200.
28. P. Tanner, P. Baumann, R. Enea, O. Onaca, C. Palivan and W. Meier, Acc. Chem. Res., 2011, 44, 1039–1049.
29. W. Tian, J. Y. Liu, Y. Guo, Y. Y. Shen, D. J. Zhou and S. R. Guo, J. Mat. Chem. B, 2015, 3, 1204–1207.
30. Q. Xu, Y. X. Liu, S. S. Su, W. Li, C. Y. Chen and Y. Wu, Biomaterials, 2012, 33, 1627–1639.
31. S. H. Hu, S. Y. Chen and X. H. Gao, ACS Nano, 2012, 6, 2558–2565.
32. F. Liu, J. W. Hu, G. J. Liu, C. M. Hou, S. D. Lin, H. L. Zou, G. W. Zhang, J. P. Sun, H. S. Luo and Y. Y. Tu, Macromolecules, 2013, 46, 2646–2657.
33. F. H. Meng and Z. Y. Zhong, J. Phys. Chem. Lett., 2011, 2, 1533–1539.
34. A. Rosler, G. W. M. Vandermeulen and H. A. Klok, Adv. Drug Delivery Rev., 2012, 64, 270–279.
35. Q. Yang, K. Wang, J. J. Nie, B. Y. Du and G. P. Tang, Biomacromolecules, 2014, 15, 2285–2293.
36. Y. F. Huang, S. C. Lu, Y. C. Huang and J. S. Jan, Small, 2014, 10, 1939–1944.
37. J. A. MacKay, M. N. Chen, J. R. McDaniel, W. G. Liu, A. J. Simnick and A. Chilkoti, Nat. Mater., 2009, 8, 993–999.
38. C. C. Zhou, M. Z. Wang, K. D. Zou, J. Chen, Y. Q. Zhu and J. Z. Du, ACS Macro Lett., 2013, 2, 1021–1025.
39. D. Peer, J. M. Karp, S. Hong, O. C. FaroKhzad, R. Margalit and R. Langer, Nat. Nanotechnol., 2007, 2, 751–760.
40. S. Huang, S. Peng, Y. B. Li, J. B. Cui, H. L. Chen and L. Y. Wang, Nano Res., 2015 DOI:10.1007/s12274-12015-10702-12275.
41. S. Huang, M. Bai and L. Y. Wang, Sci. Rep., 2013, 3, 2023–2027.
42. B. G. Xing, C. W. Yu, K. H. Chow, P. L. Ho, D. G. Fu and B. Xu, J. Am. Chem. Soc., 2002, 124, 14846–14847.
43. B. S. Kim, S. W. Park and P. T. Hammond, ACS Nano, 2008, 2, 386–392.
44. T. R. Triton and G. Yee, Science, 1982, 217, 248–250.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra05939a

‡ These two authors contributed equally to this work.

---