English

• 1.   Introduction

  Chirality is ubiquitous in nature including ribose and amino acids. In the organisms and biological processes, chirality greatly affects the biological activities and functions for optically active small molecules and macromolecules [1 -4]. In particular, chirality can regulate the secondary structures in polypeptide blends and has effect on the physiochemical properties [5]. For example, the factors affecting the physiochemical properties of polypeptides including secondary structures [6], molecular weights [7], charge density [8] and the nanoparticle size [9, 10] have been investigated.

  In our previous work [11], we synthesized a series of poly(S-(o-nitrobenzyl)-_L-cysteine) (_L-PNBC) based polypeptide copolymers, which exclusively presented β-sheet conformation and photoresponsive self-assembly behaviors for on-demand drug release. However, how the chirality of PNBC influences its secondary structures and cellular internalization process is unknown. In this study, we prepared three kinds of polypeptides including poly(S-(o-nitrobenzyl)-_L-cysteine) (_L-PNBC), poly(S-(onitrobenzyl)-_D-cysteine) (_D-PNBC) and poly(S-(o-nitrobenzyl)-_{L, D}-cysteine) (_{L, D}-PNBC) with different degrees of polymerization (DP) and chirality. We found that the chirality not only had profound effect on the secondary conformation, but also affected their cell uptake behaviors.

  2.   Results and discussion

  2.1   Secondary conformations of _{L, D}-PNBC polypeptides

  In this study, we prepared three kinds of polypeptides including poly(S-(o-nitrobenzyl)-_L-cysteine) (_L-PNBC), poly(S-(o-nitrobenzyl)-_D-cysteine) (_D-PNBC) and poly(S-(o-nitrobenzyl)-L, D-cysteine) (_{L, D}-PNBC) with different DPs ranging in 10, 40, 80, 120, 160 and moderate molecular weight distributions (M_W/M_n = 1.4-1.7). There are several methods to measure and analyze the secondary structure of polypeptides. First, by Fourier transform infrared (FT-IR) spectra, some researches [12, 13] indicate that when the volume fraction of the PBLG blocks is increased, the a-helix conformation is increased while β-sheets and random coil contents are systematically decreased. Second, circular dichroism (CD) signatures of fibrils derived from L and _D-Ac-(FKFE)₂-NH₂ peptides are mirror images corresponding to β-sheet secondary structure and random coil [14]. So, we investigated the secondary structure of these polypeptides by FT-IR (Fig. 1) and CD (Fig. 2).From Fig. 1A FT-IR spectra, as the content of _L-NBC-NCA increased, β-sheet conformation was decreased and then increased. On the contrary, the random coil was increased and then reduced.Meanwhile, whatever the _L-NBA-NCA content was, the a-helix conformation did not exist, as shown in Fig. 1B. When the content of _L-NBA-NCA is 100%, the intermolecular interactions are strengthened because of the π-π effects of neighboring phenyls in chains, resulting in forming regular structure [15]. Similar conclusion can be obtained by analyzing CD profiles in Fig. 2A.

  图 1

  

  图 1  (A) FT-IR spectra of copolymers; (B) relationship between chirality and conformation; (C) relationship between DP and conformation; (D) comparison of β-sheet conformation before and after photocleavage.

  Figure 1.  (A) FT-IR spectra of copolymers; (B) relationship between chirality and conformation; (C) relationship between DP and conformation; (D) comparison of β-sheet conformation before and after photocleavage.

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  图 2

  

  图 2  Circular dichroism (CD) spectra (A) relationship between chirality and conformation; (B) comparison of β-sheet conformation before and after photocleavage.

  Figure 2.  Circular dichroism (CD) spectra (A) relationship between chirality and conformation; (B) comparison of β-sheet conformation before and after photocleavage.

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  As shown in Fig. 1C, when the ratio of _L-NBC-NCA was 50%, as DP increased, the β-sheet continuously increased but the random coil constantly decreased. This was due to the fact that the increasing DP resulted in the stronger intermolecular interactions to induce the regular secondary structure. After the NB groups were completely photocleaved by 365 nm UV irradiation, the b-sheet all decreased independent of the content of _L-NBC-NCA (Fig. 1D and Fig. 2B). This was due to the facts that NB groups have steric effect on hindering chains to freely rotate while the chains can freely rotate to become random coil after photocleavage.

  2.2   Drug loading and release properties of _{L, D}-PNBC polypeptides

  The CPT loading particles were prepared by nanoprecipitation method and it was found the particle size of the three CPT loading particle were bigger than the corresponding blank particle (Table S1 in Supporting information). This was probably because with CPT-loaded, the intermolecular forces of polymers became weaker, resulting in increasing the particle size. The particle size was about 117-144 nm, as determined by DLS in Fig. 3A -C.Compared with the TEM images of three kinds of nanoparticles in Fig. 3D -F, the CPT loading particle formed nearly spherical particle with the sizes (100-120 nm) slightly smaller than those by DLS.

  图 3

  

  图 3  The size distribution of _L-PNBC/CPT (A), _D-PNBC/CPT (B) and _L-D-1-1/CPT (C) particle; the TEM images of _L-PNBC/CPT (D), _D-PNBC/CPT (E) and _L-D-1-1/CPT (F) particle.

  Figure 3.  The size distribution of _L-PNBC/CPT (A), _D-PNBC/CPT (B) and _L-D-1-1/CPT (C) particle; the TEM images of _L-PNBC/CPT (D), _D-PNBC/CPT (E) and _L-D-1-1/CPT (F) particle.

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  Ding et al. [15] reported that the DOX-loaded micelle with poly (_L-Leu) core exhibited a quick DOX release because of the loose micellar core induced by the rigid polypeptide block. The cumulative CPT releases of three kinds of CPT loading particles were tested at 37 ℃ (Fig. 4A). Similarly biphasic drug release profiles were given for these nanoparticles [11], which also indicated that the drug release properties of the CPT-loading particles were not related to the chirality.

  图 4

  

  图 4  (A) The cumulative CPT release of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (B) the cell viability of _L-PNBC, _D-PNBC and _L-D-1-1 particle; (C) the cell viability of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (D) the intensity of free CPT, _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle in different incubation time (^*p < 0.05).

  Figure 4.  (A) The cumulative CPT release of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (B) the cell viability of _L-PNBC, _D-PNBC and _L-D-1-1 particle; (C) the cell viability of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (D) the intensity of free CPT, _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle in different incubation time (^*p < 0.05).

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  2.3   Biological properties of _{L, D}-PNBC polypeptides

  2.3.1   Cell viability

  According to the research of Liao et al. [16], the half-maximal inhibitory concentration (IC₅₀) of CPT was 2-6 μg/mL. The test of cytotoxicity was measured by MTT method. These particles have less toxicity and the cell viability was above 80% in Fig. 4B. As for CPT loading particles in Fig. 4C, as the concentration of particle was increased, the cytotoxicity was increased. When it was reached to 100 μg/mL, the cell viability was only 10%. When the concentration of CPT was 2 μg/mL, the cell viability was 50%. The IC₅₀ order of different CPT loading particles and free CPT was _L-PNBC/CPT < _L-D-1-1/CPT < _D-PNBC/CPT < free CPT (Table S2 in Supporting information). These data suggested that _L-PNBC/CPT was the quickest to be taken by HeLa cells and _D-PNBC/CPT was the slowest one to enter cells. That is to say, the chirality of these polypeptides had effect on the related particles to enter HeLa cells. This conclusion was further clarified by the following cellular uptake experiments.

  2.3.2   Cellular uptake

  The cellular uptake was measured by flow cytometry and fluorescence microscope. Compared to free CPT, the fluorescence intensity of CPT-loaded particles became stronger (Fig. 4D). This was probably because that CPT loading nanoparticle entered cells by an endocytosis process while free CPT only diffused into cells.Moreover, the fluorescence intensity of the cells incubated with _L-PNBC/CPT was the strongest, and the _D-PNBC/CPT one was the weakest. This suggested that the chirality of polypeptides affected CPT loading particles to enter cells. Most amino acids found in natural proteins occur in the L configuration and cell membranes also contain a number of natural proteins. Thus, the polypeptides with higher L configuration show favorable interactions with cell membranes [17]. Similarly, when the cell culture time was 6 h, there was only little pink region overlapped between blue (CPT color) and red (the nucleus was stained as red by propidium iodide) from the image of free CPT-incubated cells (Fig. 5A). This means only little CPT was internalized by cells even for 6 h. Conversely, there was stronger pink intensity from the cells cultured with other three CPT loading particles (Fig. 5B -D), indicating more CPT was carried into cells. Moreover, _L-PNBC/CPT incubated cells showed the strongest pink fluorescence while _D-PNBC/CPT was the least one. These data are basically consistent with the abovementioned flow cytometry and cytotoxicity.

  图 5

  

  图 5  Fluorescence microscope images of free CPT (A), _L-PNBC/CPT (B), _D-PNBC/CPT (C) and _{L, D}-1-1/CPT (D) particle after incubation 6 h.

  Figure 5.  Fluorescence microscope images of free CPT (A), _L-PNBC/CPT (B), _D-PNBC/CPT (C) and _{L, D}-1-1/CPT (D) particle after incubation 6 h.

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  3.   Conclusions

  We synthesized a series of polypeptides _{L, D}-PNBC with different DPs and chirality. Both FT-IR and CD analyses demonstrated that the chirality of polypeptides had profound effect on tuning the second conformation of β-sheet. The particle sizes and drug release properties were not related to the chirality. More _L-PNBC/CPT nanoparticles entered HeLa cells than _D-PNBC/CPT and _{L, D}-1-1/CPT ones, giving a lowest IC₅₀.

  4.   Experimental

  4.1   Preparation of camptothecin (CPT) loaded nanoparticles in aqueous solution

  According our previous work [11], _{L, D}-PNBC polypeptides were synthesized by the ring-opening polymerization of S-(o-nitrobenzyl)-_L-cysteine N-carboxyanhydride (_L-NBC-NCA) and/or _D-NBC-NCA using benzylamine as initiator in DMF solution at room temperature. These chiral copolymers denoted as _L-PNBC, _L-D-1-1, _L-D-3-1, _L-D-1-3 and _D-PNBC have DP = 10, and _L-D-1-1, _L-D-3-1, _L-D-1-3 denote the molar ratios of _L-NBC-NCA to _D-NBC-NCA of 1:1, 3:1, and 1:3, respectively. Using a dialysis method [18, 19], a typical procedure for the fabrication of CPT-loaded particles in aqueous solution is as follows. Both _{L, D}-PNBC (10 mg) and CPT (5 mg, 8.6 mmol) were dissolved in 2 mL of DMF. The solution was added to distilled water (20 mL) gradually at a speed of 30 μL/min using a microsyringe until the formation of nanoparticles. The resulting nanoparticles solution was then put into a dialysis bag (MWCO = 3500) and subjected to dialysis against 4 × 1 L of distilled water for 24 h. The CPT loading nanoparticles were denoted as _L-PNBC/CPT, _D-PNBC/CPT, and _L-D-1-1/CPT.

  4.2   In vitro CPT release

  The CPT-loaded nanoparticles (150 mg) with or without CPT were suspended in 2 mL PBS (pH 7.4) and incubated at 37 ℃ on a shaker. At regular intervals (0.5, 1, 2, 4, 6, 12, 24, 36 and 48 h) samples were removed and centrifuged for 10 min at 20, 000 RCF.The nanoparticle pellet was discarded and 970 mL of the supernatant was removed and added to 30 mL of quantification fluid (DMSO:1 mol/L HCl:10% SDS) [20, 21]. Control curves were constructed by spiking blank particle samples with known quantities of CPT at 365 nm by UV-vis. Three samples were measured for each time point.

  4.3   In vitro cell viability and cellular uptake

  Cell viability was measured by the MTT assay [22 -24]. After incubating HeLa cancer cell line with _L-PNBC/CPT, _D-PNBC/CPT, _L-D-1-1/CPT, _L-PNBC, _D-PNBC and _L-D-1-1 particles with respective concentrations in 96 well-plates for 48 h, the solutions were removed and washed with PBS three times. Fresh medium containing 20 mL Cell Titer-Blue reagent was added to each well, followed by 4 h incubation at 37 ℃. The cell viability results were quantified using fluorescent plate-reader.

  To explore the variations of the cytotoxicity between the _L-PNBC/CPT, _D-PNBC/CPT, _L-D-1-1/CPT and free CPT, the cellular uptake of these NPs by HeLa cells was examined by flow cytometry and fluorescence microscope.

  Acknowledgment

  The authors are grateful for the financial support of National Natural Science Foundation of China (No. 21474061).

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.11.006

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  Figure 1  (A) FT-IR spectra of copolymers; (B) relationship between chirality and conformation; (C) relationship between DP and conformation; (D) comparison of β-sheet conformation before and after photocleavage.

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  Figure 2  Circular dichroism (CD) spectra (A) relationship between chirality and conformation; (B) comparison of β-sheet conformation before and after photocleavage.

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  Figure 3  The size distribution of _L-PNBC/CPT (A), _D-PNBC/CPT (B) and _L-D-1-1/CPT (C) particle; the TEM images of _L-PNBC/CPT (D), _D-PNBC/CPT (E) and _L-D-1-1/CPT (F) particle.

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  Figure 4  (A) The cumulative CPT release of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (B) the cell viability of _L-PNBC, _D-PNBC and _L-D-1-1 particle; (C) the cell viability of _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle; (D) the intensity of free CPT, _L-PNBC/CPT, _D-PNBC/CPT and _L-D-1-1/CPT particle in different incubation time (^*p < 0.05).

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  Figure 5  Fluorescence microscope images of free CPT (A), _L-PNBC/CPT (B), _D-PNBC/CPT (C) and _{L, D}-1-1/CPT (D) particle after incubation 6 h.

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