Communication pubs.acs.org/cm
Bioinspired Design of Stereospecific D‑Protein Nanomimics for HighEfficiency Autophagy Induction Cheng Hu,† Xianghui Xu,*,†,‡ Xiao Zhang,† Yachao Li,† Yunkun Li,† and Zhongwei Gu*,†,‡ †
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, People’s Republic of China College of Materials Science and Engineering, Nanjing Tech University, Nanjing, Jiangsu 210009, People’s Republic of China
‡
S Supporting Information *
I
Herein, we report a de novo design of chirality-specific dendrimeric peptides as abnormal protein nanomimics (PNs) for efficient intracellular accumulation and autophagy induction in various tumor cell lines. Fully taking into account bioinspired macromolecular, topological and stereochemical design, we construct a novel kind of tailor-made D-protein nanomimics ( D-PNs, Scheme 1): well-defined dendrimeric structure to meet
n recent years, autophagy modulation through exogenous materials has aroused intensive attention to develop treatment protocols according to diverse human diseases.1,2 A growing number of laboratorial and clinical data indicate that autophagy activation is beneficial in curing metabolic abnormality, neurodegenerative illness, infectious disease and malignant tumor. Encouragingly, increasing inorganic nanomaterials (such as, carbon nanotubes, gold nanoparticles and lanthanide-based nanocrystals) emerge the great potentials to modulate autophagy,3,4 thereby massive efforts are being devoted to exploit novel nanomaterials as available autophagy inducers.5,6 However, up to now, few autophagy-inducing nanoparticles have met essential requirements for preclinical tests, due to the critical limitations of the inorganic nanomaterials for clinical translation (e.g., uncontrollable preparation and undefined biosafety).7,8 As a result, discovering the feasible nanomaterial-based strategies for efficient autophagy induction remains a challenging and promising task to satisfy advanced biomedical needs. In eukaryotes, intracellular accumulation of abnormal proteins is a leading factor to launch autophagy for degradation or turnover of these macromolecules.9,10 With bioinspirations from this process, mimicking hallmarks of natural abnormal proteins holds huge potential to create desired pharmacological activity for tumor autophagy induction. Fortunately, dendrimeric structures are found to highly resemble nanoscale sizes and three-dimensional (3D) architectures of many endogenous proteins (such as, 3.0 nm insulin and 5.5 nm hemoglobin).11,12 More importantly, recent bioinspired sequence design of peptide dendrimers surprisingly acquires various biological activity, including antibacterial, neurotoxin and our reporting antitumor dendrimers.13−15 On the other hand, specific homochirality as an essential attribute of natural compounds (e.g., L-peptides/proteins and D-carbohydrates) fundamentally determines macromolecular stereochemical structures, and then impacts on biological functions and fates (e.g., recognition and metabolism).16 For this reason, engineering dendrimeric peptides with the opposite chirality to the natural protein is highly expected to obtain unprecedented protein mimics, possibly providing special metabolic behaviors and autophagyinducing activity. However, little attention has been paid to developing artificial chirality-specific dendrimeric peptides, not to mention chirality-dependent autophagy-inducing protein mimics. Motivated by these bioinspirations, we are keen on creating stereospecific protein mimics as autophagy-inducing entities for future clinical applications. © 2017 American Chemical Society
Scheme 1. Schematic Illustrations of Homochiral DDendrimeric Peptides as Chirality-Specific Protein Nanomimics, and the Exact Enantiomers of D- and L-PNs as Autophagy Inducers for Activating Tumor Autophagy
a prerequisite of controllable and biosafety nanotherapeutics, 3D highly branched architecture to mimic nanoscale contours of endogenous autophagy-inducing proteins, chiral repeat units based on D-amino acids for building abnormal stereochemical structure and generating robust autophagy-inducing activity, and abundant lysine residues to facilitate tumor accumulation of artificial protein nanomimics. We anticipate that the intracellular sustained accumulation of chirality-specific D-PNs would activate efficient autophagy mimicking acute turnover of endogenous redundant proteins. Moreover, we disclose the macromolecular and biological mechanisms of D-PNs in continuous autophagy induction, as compared with the enantiomers (L-PNs). To the best of our knowledge, this is the first demonstration of chirality-specific protein nanomimics as highly efficient organic autophagy-inducing entities for tumor autophagy activation in vitro and in vivo. Received: May 14, 2017 Revised: July 23, 2017 Published: July 26, 2017 7658
DOI: 10.1021/acs.chemmater.7b01982 Chem. Mater. 2017, 29, 7658−7662
Communication
Chemistry of Materials We first constructed homochiral D-dendrimeric peptides with chiral D-lysine as branching units and tris(2-aminoethyl)amine as a C3-symmetric core via divergent approach.17−20 To avoid racemization in dendrimeric constructions, the C3-symmetric core was reacted with excessive N-Boc-protected D-lysine under the special conditions, including specific coupling catalytic system (HBTU/HOBT/DIPEA).21 After careful purification and deprotection, the first generation of D-PNs was obtained. To mimic the proper nanoscale and architecture of natural proteins, we repeated these processes to stepwise produce a family of homochiral D-PNs (Gn D-PNs, n = 1, 2, 3 or 4). Analogously, we constructed the enantiomeric L-PNs based on chiral L-lysine. The detailed synthetic procedures and characterizations can be found in the Supporting Information. It is essential to note that precise macromolecular structure of our PNs is the most prominent advantage to satisfy the fundamental requirements of clinical translation. The matrixassisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF MS) provided direct evidence for accurate macromolecular structures of L- and D-PNs. As shown in Figure 1A, molecular weight of protonated G4 D-PNs (5911.6)
found that optical activity of L- and D-PNs revealed nonlinear relationship with respect to the number of chiral amino acids, because 3D steric hindrance of dendrimeric architecture and secondary structures within PNs forced chiral monomer units to adopt conformation.22−24 Hence, circular dichroism (CD) was carried out to expound the secondary structures of protein nanomimics (Figure 1D). Remarkably, the CD spectra for Dand L-PNs (from G1 to G4) demonstrated perfect mirror-image symmetry in an aqueous solution at 25 °C, implying the protein mimics were quite exact enantiomers. Entirely opposite signals on optical activity and circular dichroism confirmed that the Land D-PNs kept accurate stereochemical architectures undergoing the step-by-step macromolecular constructions. Taken together, these PNs closely imitated precise structures and highly order architectures of naturally occurring proteins. If chirality-specific D-PNs are capable of arousing autophagy, they will become a pioneering example of peptide-based autophagyinducing nanomimics. Once the dendrimeric structure, nanostructure and stereochemical architectures were well confirmed, the fourth generation of dendrimeric peptides were used as representative L- and D-PNs for the following investigation. We employed an autophagy probe of acridine orange (AO) to investigate the possibility and degree of PN-inducing autophagy in a human hepatocellular carcinoma HepG2 cell line. Obviously, the maximum red autolysosomes (observed by confocal laser scanning microscopy, CLSM, Figure 2A) and the highest red/ green ratio (calculated by fluorescence-activated cell sorting, FACS, Figure 2B) were found in D-PN-treated tumor cells, reflecting the highest autophagy-inducing activity. The
Figure 1. Macromolecular, nanostructural and stereochemical characterizations of homochiral protein nanomimics. (A) MALDITOF MS results, (B) TEM images, (C) ORD curves and (D) CD spectra of L- and D-PNs.
detected by MALDI-TOF MS agreed well with theoretical value (5911.4). In the meantime, the purity of D-PNs reached to 99.9% measured by the reversed-phase high-performance liquid chromatography (RP-HPLC, Figure S26). And transmission electron microscopy (TEM) images confirmed both G4 L- and D-PNs had well-defined and unique nanostructures about 8 nm (Figure 1B), which were indeed analogous to endogenous protein. Next, we investigated the stereochemical properties of homochiral PNs using optical rotation studies. As shown in optical rotatory dispersion (ORD) curves in an aqueous solution at 20 °C, each generation of D-PNs showed a sustained growth in the value of the specific rotation [α] with the increase of wavelength, suggesting Cotton effects of D-PNs (Figure 1C). Interestingly, L-PNs had diametrically opposite sign and magnitude rotations to the corresponding D-PNs in the ORD curves, presenting excellent X axial symmetry. In addition, we
Figure 2. Stereospecific protein nanomimics for activating tumor autophagy. (A) CLSM images and (B) FACS analysis for AO-stained HepG2 cells after incubation with L-PNs, D-PNs (100 μg mL−1) and Rapa (50 nM) for 24 h (n = 3, *p < 0.005). (C) TEM images for the ultrastructural analysis of HepG2 cells treated with L- and D-PNs for 24 h, respectively. Autophagosomes (◆) and autolysosomes (★) were noted in the TEM images. (D) Western blot assay for autophagyrelated LC3 protein expression in HepG2 cells after treatment with 100, 200 and 300 μg mL−1 L- and D-PNs for 24 h, respectively. (E) Fluorescence images for cryosections of HepG2 tumor after intratumoral injection with normal saline, L- and D-PNs, including Alexa Fluor 488 stained LC3 channel (green) and Hoechst 33342stained nucleus channel (blue). 7659
DOI: 10.1021/acs.chemmater.7b01982 Chem. Mater. 2017, 29, 7658−7662
Communication
Chemistry of Materials autophagy-inducing efficiency of D-PNs indicated that chiralityspecific design succeeded in providing novel protein nanomimics for efficient autophagy activation, even exceeding utility of clinical autophagy inducer (rapamycin, Rapa, *p < 0.005). To affirm the existence of tumor autophagy, cell ultrastructures were analyzed by TEM after treatment with PNs (Figure 2C). By comparison with the untreated HepG2 cells (Figure S31), the cytoplasm of D-PN-treated tumor cells was strikingly filled with autophagosomes (incipient and multilamellar vacuoles, ◆) and autolysosomes (electron-dense vacuoles, ★), supporting ultrastrong autophagy-inducing effects of D-protein mimics. However, only a small number of autophagic vacuoles can be found in the L-PN-treated cells. Following gold-standard ultrastructural analysis, we checked the molecular biology evidence on the conversion of microtubule-associated protein light chain 3 (LC3) from LC3-I (18 kDa) to LC3-II (16 kDa) after their location at autophagosomal membranes.25,26 Western blot results showed that much larger amount of LC3-II protein was expressed after incubation with D-PNs (100 μg mL−1) as compared to L-PNs treatment (Figure 2D), which was consistent with the CLSM and TEM results. Additionally, increasing PN concentration markedly increased the LC3-II protein level, suggesting the dosedependent pharmacological effects of PNs. Next, we assessed whether the homochiral PNs could serve as efficient autophagyinducing nanotherapeutics for in vivo application using nude mice bearing HepG2 xenograft tumors. After intratumoral injection of L-PNs or D-PNs every 24 h for three times (5 mg per kg body weight), cryosections of tumor tissue were analyzed by immunohistochemical staining with LC3 antibody (Figure 2E). Distinguishing from the untreated and L-PNs group, the largest number of green dots in the D-PNadministrated group represented the high LC3-II generation and autophagy initiation, confirming strong autophagy-inducing activity of D-PNs in vivo (green fluorescence). And Western blot result confirmed that D-PNs effectively upregulated the LC3-II protein level in vivo (Figure S34). In conclusion, these results demonstrated that the stereospecific D-PNs exhibited extremely high autophagy-inducing activity both in vitro and in vivo. Afterward, we turned to disclose the chirality-dependent mechanism of the homochiral PNs on highly efficient autophagy-inducing activity. It is worth mentioning that numerous lysine residues of amino groups on PNs tremendously improved their water solubility, which even reached up to about 65 g per 100 g H2O for L- and D-PNs (Figure S35). More importantly, both L- and D-lysine-rich PNs achieved rapid and effective internalization in tumor cells (Figure 3A), overcoming the low membrane permeability of widely used peptide entities (especially linear D-peptides).27 Excellent internalization was a critical first step in achieving the intracellular accumulation of PNs. Because it is well-known that bioactivity and metabolism of endogenous and artificial proteins are completely regulated by stereospecific enzyme catalysis during autophagy, we next investigated the biological behaviors of chirality-specific PNs under the biomimetic enzyme condition. As shown in the Figure 3B, we confirmed that both L- and D-PNs maintained nice molecular stability in the presence of bovine serum albumin (BSA), which had no proteolytic activity. Next, it was evident that L-PNs having the same homochirality as natural proteins were gradually degraded by a common protease of parenzyme, showing above 50% decomposition within 24 h. On the contrary, stereospecific D-
Figure 3. Chirality-dependent autophagy mechanisms based on biological behaviors of L- and D-PNs. (A) Internalization efficiency of L- and D-PNs to HepG2 cells within 24 h. (B) Biological stability of L- and D-PNs in the biomimetic solutions containing BSA or parenzyme at 37 °C for 48 h (n = 3, samples were determined by HPLC). (C) FRET quenching design for visualizing biodegradation of protein mimics (top left). Fluorescence spectra for L-FITC-PNs-BHQ (dash line) and D-FITC-PNs-BHQ (solid line) after incubation with parenzyme at the preset time (right), and the corresponding fluorescence imaging in 96-well microplate (bottom left). (D) Schematic illustrations for autophagy processes and mechanisms of L-PNs (involving biodegradation and recycle) and D-PNs (involving continuous autophagy-inducing activity).
PNs were very stable even incubation with parenzyme for 48 h, indicating that the D-homochirality definitively affected their interactions with biological active substances. To visually illustrate their chirality-dependent biological fates, we endowed L- and D-PNs with image-guide function based on a fluorescence resonance energy transfer (FRET) quenching principle as follows (Figure 3C): PNs were labeled with fluorescein isothiocyanate (FITC) to introduce distinct fluorescence signals, and then FITC-PNs were decorated with black hole quencher (BHQ) to extinguish FITC fluorescence (FITC-PNs-BHQ, Figure S40).28 Then, incubation of L-FITCPNs-BHQ with parenzyme solution triggered the cleavage of Lprotein mimics and the separation of FITC and BHQ, resulting in a remarkable increase of FITC fluorescence intensity within 48 h (13.0-fold). In contrast, D-FITC-PNs-BHQ were always at quenching state in the same conditions, supporting ineffective enzymolysis to homochiral D -PNs. Simultaneously, the alterations were captured by a fluorescence imaging system at different enzyme exposure time points in a 96-well microplate (bottom left). The FITC signals of L-FITC-PNs-BHQ were gradually activated with enzyme catalysis, whereas D-FITCPNs-BHQ always had no obvious fluorescence signals. Altogether, the stereospecific D-protein nanomimics as autophagy-inducing nanomaterials activated highly efficient autophagy in tumor cells, thanks to the resistance to enzymatic degradation, high intracellular accumulation and continuous autophagy induction (Figure 3D). 7660
DOI: 10.1021/acs.chemmater.7b01982 Chem. Mater. 2017, 29, 7658−7662
Communication
Chemistry of Materials
reversal. As the first exploration on stereospecific protein nanomimics for autophagy induction, this work not only substantiates our assumption on creating stereospecific dendrimeric peptides as bioinspired autophagy-inducing nanomimics but also opens up a new direction in discovering unprecedented protein nanomimics for autophagy-associated tumor treatment.
Finally, we investigated the broad spectrum of our proteinmimicking nanotheraputics to several tumors using a specific autophagic reporter of monodansylcadaverine (MDC). After 24 h treatment with L- and D-PNs, MDC-positive structures confirmed that our protein nanomimics indeed activated the autophagy of mouse breast 4T1 cancer cells, mouse melanoma B16 cells, human lung adenocarcinoma A549 cells, human cervical HeLa cancer cells (Figure S44) and D-PNs always offered much higher autophagy-inducing efficiency than L-PNs in the tumor cell lines. Meanwhile, quantitative fluorescence intensity for MDC-positive autophagic vacuoles manifested that chirality-specific D-PNs could serve as robust inducers for autophagy-defective human breast MCF-7 cancer cells and homologous drug-resistant MCF-7R cells (Figure 4, *p
