In situ enzymatic formation of supramolecular nanofibers for efficiently killing cancer cells

Abstract

We report a strategy of in situ forming supramolecular nanofibers of taxol-phosphorylated peptide conjugates catalyzed by phosphatase for efficiently killing cancer cells.

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Supramolecular nanofibers or hydrogels of peptide amphiphiles have been widely studied in last two decades^1,2 and they have shown big promise in drug delivery,³ immune modulation,⁴ cancer cells inhibition,^5,6 sensing,⁷ understanding biological processes,⁸ and regenerative medicine.⁹ Among their applications in drug delivery, nanofibers of drug-peptide amphiphiles have attracted extensive research interests due to several advantages of this novel self-delivery system, including high and designable drug loading, sustained and constant release of drugs, and better biocompatibility to normal tissues or higher selectivity to their targets.^10,11 However, most of these drug delivery systems are formed in vitro and then applied for cell experiments or in vivo study. Recently, Xu group reported a strategy of in situ formation of supramolecular nanofibers catalysed by enzymes for imaging intracellular enzymes or selectively killing cancer cells by supramolecular hydrogelations.^6,12 Liang and co-workers also reported a strategy of intra-cellular formation of nanoparticles of a taxol amphiphile catalysed by an intracellular enzyme of furin to efficiently kill drug resistant cancer cells.¹³ Inspired by these pioneering results, we opted to develop a drug-peptide amphiphile that could respond to an enzyme and form nanofibers in situ for efficiently killing cancer cells.

Taxol is a well-known anti-cancer drug that specifically binds to the tubulin subunit of microtubules and causes the cell death in ovarian, lung and breast cancers. Our recent results demonstrated that taxol-peptide conjugates had excellent self-assembling and gelation ability.^10,14 We therefore designed conjugates of taxol and L-/D-phosphorylated dipeptide of FpY (Scheme 1). Many amphiphiles with dipeptide FF or FY or tripeptide FFY had excellent self-assembling properties.^2,15 We therefore imaged that taxol-FpY or taxol-^DFp^DY might self-assemble into nanostructures after the enzymatic de-phosphorylation by phosphatase. Alkaline phosphatase was generated and secreted highly by cancer cells such as the HeLa and the HepG2 cells, which provided a potential way for the cancer cells to be detected, tracked or treated.¹⁶ Our taxol-phosphorylated peptide amphiphiles might form nanofibers within or around these cancer cells by phosphatase that capable of constantly releasing taxol and therefore efficiently killing cancer cells.

Scheme 1 Chemical structure of the precursors ((taxol-FpY (L-precursor) and taxol-^DFp^DY (D-precursor)) of possible hydrogelators ((taxol-FY (L-gelator) and taxol-^DF^DY (D-gelator)) and schematic hydrogelation process catalysed by alkaline phosphatase (ALP).

The synthesis of designed molecules was described as following (Scheme S-1†). We firstly obtained the phosphorylated dipeptide FpY or ^DFp^DY by standard Fmoc solid phase peptide synthesis (SPPS) and succinated taxol by solution phase synthesis. The N-hydroxylsuccinimide (NHS) activated succinated taxol was then mixed with the phosphorylated dipeptide to afford the title compound. The pure designed compounds were obtained by high performance liquid chromatography (HPLC). After obtaining the compounds (L-precursor and D-precursor), we tested their self-assembling and gelation ability by alkali phosphatase (ALP). The precursors formed viscous and opaque dispersions in phosphate buffer saline (PBS, pH = 7.4, 0.2 wt%) solutions (L-sol and D-sol in Fig. 1A). The dispersions turned into opaque gels after the addition of ALP (L-gel and D-gel, Fig. 1B) within 10 minutes. The minimum gelation concentration of both peptides was about 0.1 wt% (1 mg mL⁻¹).

Fig. 1 Optical image of (A) dispersions of precursors (0.2 wt%) in PBS buffer solution and (B) the formed gels catalyzed by ALP (2 U mL⁻¹), (C) dynamic time sweep of the dispersions after being added of ALP and (D) dynamic frequency sweep of resulting gels at 4 h time point, and TEM image of (E) L-gel and (F) D-gel.

We then used a rheometer to characterize the gelation process and mechanical properties of resulting gels. As shown in Fig. 1C, both elasticity (G′) and viscosity (G′′) values kept increasing after the addition of ALP to the dispersions during the 2 hours' experimental time, suggesting that ALP kept converting the precursors to the gelators. The results obtained by dynamic frequency sweep indicated that the G′ value of L-gel or D-gel was about 10 times bigger than their corresponding G′′ value, suggesting the formation of true gels. The G′ value of D-gel was bigger than that of L-gel, indicating that D-gel was mechanically stronger than L-gel. We also used transmission electron microscopy (TEM) to study the nanostructures in both L-gel and D-gel (Fig. 1E and F). We observed uniform nanofibers in both gels, and the diameter of nanofibers in both gels was about 15–25 nm. These nanofibers entangled with each other to form three dimensional networks for supramolecular hydrogelations. The nanofibers in D-gel was slightly more rigid than those in L-gel, which probably accounted for the stronger mechanical property of D-gel. However, we did not know the exact mechanism for the better mechanical property of D-gel. These observations clearly indicated that our designed compounds could form supramolecular nanofibers and hydrogels by the enzyme ALP (Fig. 2).

Fig. 2 IC₅₀ values of precursors and gelators determined by the MTT assay to inhibit (A) HeLa and (B) HepG2 cells (**: p < 0.01; ***: p < 0.001).

We tested the anti-cancer ability of the precursors and the resulting gelators. We treated the HeLa and HepG2 cells by both precursors, resulting gels, and taxol, respectively for 48 h, and then determined the cell viability by the MTT assay. The results in Fig. 3 and Table S-1† indicated that the IC₅₀ value of L-precursor was lower than that of L-gelator, which was 122 and 202 nM for HeLa cells, respectively. We observed a similar trend for D-precursor and D-gelator (96 and 297 nM for HeLa cells, respectively). We also obtained similar results in HepG2 cells, and the precursors could inhibit HepG2 cells more efficiently than the gelators (Table S-1†). The better inhibition capacity of the precursor than that of the gelator to cancer cells was probably due to the enhanced uptake of the precursor by cells than that of the gelator. For instance, the intracellular taxol concentration in HeLa cells treated with D-precursor was about 4 times higher than that treated with D-gelator (Fig. S-2 and Table S-3†). For normal cells such as NIH 3T3 cells, the precursors were also more toxic to them (Table S-2†). However, the D-precursor showed a 3-fold lower IC₅₀ value to HeLa cells than D-gelator, while D-precursor was 1.7 times more toxic to 3T3 cells. Cancer cells had higher phosphatase expression levels, which might account for the bigger differences in inhibition capacity between precursors and gelators. Cui and co-workers demonstrated that nanofibers showed a much lower cellular uptake compared with nanospheres.¹⁸ In our study, the enhanced uptake of the precursor than the gelator was probably due to the membrane permeability differences between them. The precursor could freely diffuse into the cells, while the nanofibers showed a lower uptake due to the larger size. These results were also consistent with reported ones that precursors of gelators could kill cancer cells more efficiently than the gelators probably due to the in situ formation of nanofibers within or around cells.^13,17 Compared the IC₅₀ value of D-precursor and L-precursor, the one of D-precursor (122 and 65 nM for HeLa and HepG2 cells, respectively) was lower than that of L-precursor (962 and 131 nM for HeLa and HepG2 cells, respectively). D-peptides had slightly better stability in biological environments than L-peptides,¹⁹ and recent studies have showed that D-peptides could self-assemble around cells by enzymatic reactions to inhibit cell growth. These hypothesises might account for the lower IC₅₀ value of D-precursor.^13,20

Fig. 3 The immune-fluorescence images obtained by anti-beta tubulinantibody coupling with Alexa Fluor® 488 Goat Anti-Mouse IgG (H + L) and DAPI of the HeLa cells treated with (A) D-precursor and (B) D-gelator at the concentration of 1 μM at different time points.

To gain insights into the inhibition capacity difference between the precursor and the gelator, we used the immunofluorescence antibody to label and study the dynamics of tubulin alignments. As showed in Fig. 3, the tubulins (green) in HeLa cells changed from flexible to rigid ones after the treatment with D-precursor (1 μM, Fig. 3A). The regular morphology of tubulin started to disrupt at 12 h time point, and we observed cell shrinkage and hardly saw regular morphology of tubulin alignments at 24 h time point. These observations suggested that our D-precursor could induce HeLa cells apoptosis at during the 12–24 h time period. For HeLa cells treated with the same molar concentration of D-gelator (Fig. 3B), the tubulin alignments also turned from flexible into rigid at 6 h time point. However, the cell shrinkage and tubulin alignments disruption were delayed to about 48 h time point. These observations confirmed the better inhibition capacity of the precursor than the gelator.

In summary, we reported another example of results that precursors of gelators of drug amphiphiles could inhibit cancer cells more efficiently than their corresponding gelators probably due to the in situ formation of nanofibers and gels within or around cells. Our study, in combine with other pioneering works,^6,13,17 suggested that the strategy of in situ formation of nanomedicines around cancer cells or tumor tissues was a very useful and powerful one for cancer therapy. The enzyme of phosphatase was over-expressed in several cancers including ovarin cancers and breast cancers, the strategy of using phosphatase to catalyse the formation of nanomedicines in tumor sites might be developed into a useful one for the treatment of these cancers.

Acknowledgements

This work is supported by Science and Technology Guiding Project of Guangdong Province (2013B091500071), Tianjin MSTC (15JCZDJC38100), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13023).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Details of synthesis and characterization of the compounds, details of experimental procedure, rheology, and congress curve of cell inhibition assay. See DOI: 10.1039/c6ra01676f

‡ The authors contribute equally to this work.

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