Efficient gene delivery mediated by helical ... - ACS Publications

Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 256−266

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Efficient Gene Delivery Mediated by a Helical Polypeptide: Controlling the Membrane Activity via Multivalency and LightAssisted Photochemical Internalization (PCI) Xin Xu,†,∥ Yongjuan Li,†,∥ Qiujun Liang,†,∥ Ziyuan Song,‡ Fangfang Li,† Hua He,† Jinhui Wang,† Lipeng Zhu,† Zhifeng Lin,*,§ and Lichen Yin*,†

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†

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, China ‡ Department of Materials Science and Engineering, University of Illinois at UrbanaChampaign, 1304 W Green Street, Urbana, Illinois 61801, United States § Department of Thoracic Surgery, Shanghai General Hospital, Shanghai Jiao Tong University of Medicine, Shanghai 200080, China S Supporting Information *

ABSTRACT: The development of robust and nontoxic membranepenetrating materials is highly demanded for nonviral gene delivery. Herein, a photosensitizer (PS)-embedded, star-shaped helical polypeptide was developed, which combines the advantages of multivalency-enhanced intracellular DNA uptake and light-strengthened endosomal escape to enable highly efficient gene delivery with low toxicity. 5,10,15,20-Tetrakis-(4aminophenyl) porphyrin as a selected PS initiated ring-opening polymerization of N-carboxyanhydride and yielded a star-shaped helical polypeptide after side-chain functionalization with guanidine groups. The star polypeptide afforded a notably higher transfection efficiency and lower cytotoxicity than those of its linear analogue. Light irradiation caused almost complete (∼90%) endosomal release of the DNA cargo via the photochemical internalization (PCI) mechanism and further led to a 6−8-fold increment of the transfection efficiency in HeLa, B16F10, and RAW 264.7 cells, outperforming commercial reagent 25k PEI by up to 3 orders of magnitude. Because the PS and DNA cargoes were compartmentalized distantly in the core and polypeptide layers, respectively, the generated reactive oxygen species caused minimal damage to DNA molecules to preserve their transfection potency. Such multivalency- and PCI-potentiated gene delivery efficiency was also demonstrated in vivo in melanoma-bearing mice. This study thus provides a promising strategy to overcome the multiple membrane barriers against nonviral gene delivery. KEYWORDS: nonviral gene delivery, α-helical polypeptide, multivalency, membrane penetration, photochemical internalization (PCI), endosomal escape

1. INTRODUCTION

penetrating materials represents a promising approach for nonviral gene delivery. Cell penetrating peptides (CPPs), short oligopeptides with sequence-specific membrane activities, are a major category of membrane-permeable materials.22−24 They can efficiently penetrate the biological membranes and facilitate the transmembrane delivery of small molecules, drugs, and nanoparticles.25,26 CPPs normally possess a large number of arginine residues in their primary structure, which contribute essentially to their penetration potency.27 Additionally, a large number of CPPs adopt a helical structure or can transform into helix upon contact with cell membranes, which potentiates the affinity with

Gene therapy holds great potentials toward the treatment of various inherited or acquired diseases.1−5 Nonetheless, the clinical translation of gene therapy has been severely hurdled because of the lack of efficient yet safe delivery methods.6−10 Nonviral gene delivery, especially mediated by cationic polymers (polycations), has attracted much attention because of the inexpensive synthesis, facile purification, scalability, low toxicity, and minimal immunogenicity.11−16 However, the performance of nonviral gene vectors is hampered by various systemic barriers, leading to the relatively low transfection efficiencies.17−20 A major obstacle against polycation-mediated gene delivery is the biological membranes, such as cell membranes, endosomal/lysosomal membranes, and nuclear membranes.17,21 As such, the development of membrane© 2017 American Chemical Society

Received: October 19, 2017 Accepted: December 5, 2017 Published: December 5, 2017 256

DOI: 10.1021/acsami.7b15896 ACS Appl. Mater. Interfaces 2018, 10, 256−266

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ACS Applied Materials & Interfaces

Scheme 1. Star-Shaped Helical Polypeptide Mediates Effective Intracellular DNA Delivery toward Gene Transfection via a Combination of Helix-Mediated Membrane Pore Formation and Light-Triggered, PCI-Assisted Endosomal Rupture

the phospholipid bilayers.28 However, CPPs typically suffer from short length and inadequate cationic charge density for gene transfer and thus can barely mediate effective gene delivery independently.29,30 As such, we recently developed cationic, guanidinated polypeptides with sufficient backbone length and a stable α-helical secondary structure.31,32 However, only a part of the polypeptide/DNA polyplexes (∼50%) enters the cells via energy-independent permeation, whereas the rest is still internalized via endocytosis and will experience endosomal/lysosomal sequestration.32,33 Additionally, the helical polypeptides with excessive membrane activities will induce appreciable cytotoxicity. Therefore, a helical polypeptide with reduced cytotoxicity yet enhanced penetration capabilities toward cellular and endosomal membranes is highly demanded. The geometry in which cationic charges are displayed greatly impacts the cellular uptake efficiency.34−36 Introduction of multivalency into the design of CPPs, such as TAT, oligolysine, HSV-1 VP22, and oligoarginine, has been demonstrated as a well-established principle to strengthen the molecular interactions with cell membranes.37−39 The commonly adopted multivalent arrangement is based on either globular scaffolds or branched dendrimers, and the obtained multivalent CPPs feature the desired cell tolerability with minimal signs of toxicity.40,41 For instance, a tetravalent deca-arginine fused to the p53 protein afforded improved membrane penetration capability with even reduced toxicity.42 Additionally, the multivalent topology with dense molecular architecture and moderate flexibility also alters the DNA condensation capability of polymers to promote gene transfection.39 We thus hypothesized that tailoring the multivalency of guanidine-rich, helical polypeptides would result in potentiated membrane activities, enhanced gene delivery efficiencies, and reduced cytotoxicity of the materials. Photochemical internalization (PCI) is a recently emerged technique for overcoming the endolysosomal entrapment.43,44

This modality is derived from photodynamic therapy (PDT), a mechanism based on the peroxidation and destruction of endolysosomal membranes by reactive oxygen species (ROS) generated from an activated photosensitizer (PS) under light irradiation.45,46 Consequently, it allows the gene cargoes to be released into the cytoplasm, which could subsequently initiate effective transfection. Because the light dose for inducing PCI is much lower than that for PDT, the membrane-destructive effect can be restricted to a small area and the toxicity induced by ROS is limited.47,48 We thus further hypothesized that the gene delivery efficiency of helical polypeptides could be greatly improved by maximizing the endosomal escape level of gene cargoes via the “PCI” effect. On the basis of the above-mentioned understandings, we herein designed a PS-embedded, star-shaped multivalent helical polypeptide, which featured enhanced membrane activities toward intracellular DNA delivery and enabled maximal endosomal release via the light-assisted PCI effect (Scheme 1).49−51 To this end, a guanidine-rich, four-armed helical polypeptide was synthesized via ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) initiated by 5,10,15,20tetrakis-(4-aminophenyl) porphyrin (TAPP) as a selected PS.52 We reasoned that by taking advantage of the structural multivalency and the PCI mechanism the helical polypeptide would afford enhanced transfection efficiency and diminished cytotoxicity. The DNA condensation, cellular uptake, endosomal escape, in vitro and in vivo gene transfection, and cytotoxicity were systemically explored.

2. EXPERIMENTAL SECTION 2.1. Materials, Cells, and Animals. The chemicals were purchased from Energy Chemical (Shanghai, China) and used as received unless otherwise specified. Branched polyethyleneimine with a molecular weight (MW) of 25k (25k PEI), hexamethyldisilazane (HMDS), TAPP, and methyl-β-cyclodextrin (mβCD) were obtained from J&K Scientific (Shanghai, China). Chlorpromazine (CPZ), 257


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ACS Applied Materials & Interfaces Scheme 2. Synthetic Route of the Star Polypeptide

wortmannin (WTM), and genistein (GNT) were purchased from TCI (Shanghai, China). Fluorescein isothiocyanate (FITC), YOYO-1, Hoechst 33258, DAPI, 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA), and Lysotracker-Red were purchased from Invitrogen (Carlsbad, CA). The POB-L-Glu-NCA monomer and 6-azidohexylguanidine were prepared as described previously.25 The chemical structures of compounds 1−7 were confirmed by 1H NMR (Figures S1−S7). HeLa (human cervix adenocarcinoma), RAW 264.7 (mouse monocyte macrophages), and B16F10 (mouse melanoma) were purchased from the American Type Culture Collection (Rockville, MD). HeLa and RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% nonessential amino acids, 10% fetal bovine serum (FBS), and 1% L-glutamine. B16F10 cells were cultured in RPMI 1640 medium containing 10% FBS, 1% nonessential amino acids, and 1% L-glutamine. Male C57/BL6 mice (6−8 weeks) were obtained from Slaccas Experimental Animal Co., Ltd. (Shanghai, China) and were housed in a clean room. The animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee, Soochow University. 2.2. Synthesis of Polypeptides. POB-L-Glu-NCA (100 mg, 0.32 mmol) was dissolved in dry dimethylformamide (DMF, 1.5 mL), into which TAPP (180 μL, 5 mg/mL in DMF, M/I = 60) and 1,5,7triazabicyclo[4.4.0]dec-5-ene (TBD, 64 μL, 0.01 M in DMF) were added in a glovebox. The mixture was stirred at room temperature (RT) for 72 h and was precipitated with cold methanol (30 mL) to obtain TAPP-PPOBLG as a brown solid (Scheme 2). TAPP-PPOBLG (20 mg, 0.072 mmol alkyne groups) was further dissolved in dry DMF (1.0 mL), into which CuBr (1 mg, 0.0072 mmol), N,N,N′,N″,N″pentamethyldiethylenetriamine (4.5 μL, 0.0216 mmol), and 6azidohexylguanidine (144 μL, 0.144 mmol) were added. The reaction mixture was stirred for 2 days at RT, and then, 1 M HCl (1 mL) was added. The final polypeptide containing guanidine side chains (star polypeptide) was dialyzed for 2 days (molecular weight cut-off = 3.5 kDa) and lyophilized. The chemical structure was confirmed by 1H NMR (Figure S9). The linear polypeptide as a control was polymerized from POB-L-Glu-NCA using HMDS as the initiator (M/I = 60), followed by postfunctionalization with 6-azidohexylguanidine, as described above. Its chemical structure was confirmed by 1H NMR (Figure S8). 2.3. Polyplexes Formation and Characterization. DNA and polypeptides were dissolved in deionized (DI) water at 0.1 and 1 mg/ mL, respectively, and they were mixed at determined weight ratios

before incubation at RT for 30 min. The resultant polyplexes were assessed for DNA condensation by agarose (1%) electrophoresis. The DNA condensation level was also quantitatively monitored using the ethidium bromide (EB) exclusion assay.53,54 The ζ potential and particle size of the polyplexes were recorded by dynamic laser scattering (DLS, Malvern Zetasizer). Release of DNA from polyplexes was explored using a heparin replacement assay.31 Heparin, at various final concentrations, was mixed with polyplexes, and the mixture was incubated at 37 °C for 1 h. The DNA condensation level was assessed using the EB exclusion assay.54 2.4. In Vitro Gene Transfection. Cells were seeded on 96-well plates and cultured at 37 °C until reaching 70% confluence. The polyplexes (0.1 μg DNA per well) were incubated with cells in optiMEM for 4 h. Cells were then irradiated by 661 nm light (Maestro, In vivo Imaging System) at the power density of 5 mW/cm2 for 4 min after the removal of polyplexes. Nonirradiated cells served as a control. Cells were further incubated in DMEM supplemented with 10% FBS for another 20 h before quantification of the luciferase expression level using a Bright-Glo luciferase assay kit (Promega). A bicinchoninic acid assay (BCA) kit (Pierce) was used to measure the cellular protein level. Results were represented as relative luminescence unit per 1 mg protein (RLU/mg protein). Polyplexes formed by 25k PEI and DNA (optimal w/w = 1) were used as a control in the above-mentioned studies. To monitor the transfection efficiency of polyplexes in the presence of serum, cells were treated with polyplexes in DMEM supplemented with 10% FBS for 24 h at different DNA doses. 2.5. In Vitro Cell Uptake and Internalization Mechanism. DNA was labeled with YOYO-1, and polypeptide/YOYO-1-DNA polyplexes were prepared similarly as mentioned above. HeLa cells were seeded on 96-well plates and cultured at 37 °C until reaching 70% confluence. The polyplexes were incubated with cells (0.1 μg YOYO-1-DNA/well) in opti-MEM for 4 h. The cells were then washed with heparin-containing PBS (20 U/mL) three times and lysed using the RIPA lysis buffer (100 μL/well). The cellular uptake level of YOYO-1-DNA was determined by a microplate reader (λex = 480 nm, λem = 530 nm), and the protein level was determined using the BCA kit. The cellular uptake level was represented as ng YOYO-1-DNA associated with 1 mg of cellular protein. The cell uptake level of polypeptide/DNA polyplexes was also measured at 4 °C or in the presence of various endocytic inhibitors to explore the internalization mechanism. Briefly, HeLa cells were treated with polyplexes (polymer/DNA = 10, w/w) at 4 °C for 4 h. 258


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Figure 1. (A) CD spectra of polypeptides (0.01 mg/mL) in the aqueous solution. (B) Helical stability of the star polypeptide (0.01 mg/mL) at various pH values or NaCl concentrations, as indicated by the molar ellipticity at 222 nm. rpm, 4 °C). The supernatant was measured for the luciferase expression level as described for in vitro transfection. 2.9. Statistical Analysis. Statistical analysis was performed using Student’s t-test, and the differences between the test and control groups were considered to be significant at *p < 0.05 and very significant at **p < 0.01 and ***p < 0.001.

Otherwise, cells were pretreated with inhibitors including CPZ (10 μg/mL), mβCD (5 mM), GNT (100 μg/mL), and WTM (10 μg/mL) for 30 min at 37 °C, followed by addition of the polyplexes and incubation at 37 °C for 4 h. Results were represented as the percentage uptake level of control cells that were treated with polyplexes at 37 °C without inhibitors. The “pore formation” capability of polypeptides was evaluated by assessing the uptake level of fluorescein-tris(hydroxymethyl)methanethiourea (FITC-Tris), a membrane-impermeable dye, in the presence of polypeptides.55 Cells in a 96-well plate (70% confluence) were incubated with polypeptides (2 μg/well) and FITC-Tris (1 μg/ well) in opti-MEM at 37 °C for 2 h. Then, the cells were washed with heparin-containing PBS three times and lysed. The FITC-Tris concentration in the lysate was measured by the microplate reader (λex = 480 nm, λem = 530 nm), and the protein concentration was measured by the BCA kit. Results were expressed as μg FITC/mg protein. Free FITC-Tris in the absence of polypeptides was used as a negative control. 2.6. Endosomal Escape. The endosomal escape of polyplexes was observed by a confocal laser scanning microscope (CLSM). HeLa cells in 24-well plates (30% confluence) were incubated with polyplexes (polymer/YOYO-1-DNA = 10, w/w) at 0.5 μg YOYO-1-DNA/well in opti-MEM for 4 h. Cells were washed with heparin-containing PBS four times, irradiated (661 nm, 5 mW/cm2) for 4 min, and stained with Hoechst 33258 (5 μg/mL) as well as Lysotracker-Red (200 nM) before CLSM observation. Nonirradiated cells served as a control. The colocalization ratio between YOYO-1-DNA and Lysotracker-Redstained endolysosomes was quantified according to the previously reported method.54 2.7. Cytotoxicity. Cells were seeded in 96-well plates and cultured at 37 °C until reaching 70% confluence. Polyplexes were added (0.1 μg DNA/well) and incubated with cells in opti-MEM for 4 h, and the cells were further incubated in fresh DMEM containing 10% FBS for another 20 h. The cell viability was thereafter monitored using the MTT assay.32 Results were expressed as the percentage viability of control cells that did not receive polyplexes treatment. 2.8. In Vivo Transfection. Polypeptides and DNA were dissolved in sterile DI water at 20 and 1 mg/mL, respectively, and were mixed at the weight ratio of 40. B16F10 cells (1 × 107 cells) were subcutaneously injected to male C57/BL6 mice (18−20 g) at the left flank. When the tumor reached 100 mm3, mice were divided into five groups (four mice per group), which received an intratumoral injection of either polyplexes (20 μg DNA/mouse) or PBS (∼50 μL/ injection). Groups 1 and 2 received star polypeptide/DNA polyplexes, whereas mice in group 1 were irradiated (661 nm, 5 mW/cm2) for 20 min at 6 h post injection. Group 3 received linear polypeptide/DNA polyplexes, group 4 received 25k PEI/DNA polyplexes, and group 5 received PBS. Mice were sacrificed after 48 h, and tumors were collected, washed with PBS three times, and homogenized with protease inhibitor-containing lysis buffer. The lysate was frozen and thawed for three cycles, followed by centrifugation for 20 min (12 000

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Polypeptides. Prior to the TAPP-initiated polymerization, NCA polymerization was first performed using aniline (AN) to validate whether it can efficiently initiate the ROP of NCA. According to MALDI-TOF analysis, the MW of the obtained ANPPOBLG (Mn = 5578.52 g/mol) at M/I = 20 agreed well with the expected MW (Mn = 5578.05 g/mol), suggesting that NCA polymerizations could be effectively initiated by AN (Figure S10). Then, well-defined TAPP-PPOBLG with a relatively low polydispersity index (=1.23) was obtained via ROP initiated by TAPP, a PS that bears four-armed aniline.56 The degree of polymerization (DP) of TAPP-PPOBLG determined by gel permeation chromatography analysis was 60 (Mn = 17 188 g/ mol), which was in agreement with the expected value. As a control, PPOBLG with a similar MW (DP = 66, Mn = 18 096 g/mol) was synthesized with HMDS as the initiator (Figure S11 and Table S1). To further verify the content of TAPP in the star polypeptide, the UV−vis spectrum was recorded. As shown in Figure S12, TAPP-PPOBLG had absorption curves similar to those of free TAPP, which also demonstrated that TAPP could effectively trigger NCA polymerization. The mass fraction of TAPP in TAPP-PPOBLG was calculated to be 2.6%, which was appropriate for achieving the PCI effect. The side chains of both TAPP-PPOBLG and PPOBLG were functionalized with guanidine groups via the “click” chemistry,57 yielding star and linear polypeptides with desired aqueous solubility. The helical structure of the polypeptides was evaluated by circular dichroism (CD).32 In the CD spectra, double minima at 208 and 222 nm were observed, indicating the α-helical structures of star and linear polypeptides (Figure 1A). The calculated helicities of linear and star polypeptides were 57 and 66%, respectively. Moreover, the helical conformation of the star polypeptide was stable within a wide range of pH (1−9) and ionic length (0−0.3 M, Figure 1B), which allowed it to mediate helicity-dependent membrane penetration in the neutral extracellular compartment or in the acidic endosomes/lysosomes. 3.2. Polyplexes Formation and Characterization. The gel retardation assay was first conducted to assess the DNA 259


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Figure 2. DNA condensation by polypeptides at various polymer/DNA weight ratios, as evaluated by the gel retardation assay (A) and the EB exclusion assay (B) (n = 3). N represents naked DNA. (C) DNA release from polyplexes in the presence of heparin at various concentrations (polymer/DNA = 15:1, w/w) (n = 3).

Figure 3. Particle size (A) and ζ potential (B) of polyplexes in DI water at various polymer/DNA weight ratios, as determined by the DLS measurement.

Figure 4. In vitro transfection efficiencies of polyplexes at various polymer/DNA weight ratios in HeLa (A), B16F10 (B), and RAW 264.7 (C) cells in the serum-free medium (n = 3). (D) Transfection efficiencies of the different polyplexes in HeLa cells at various polymer/DNA weight ratios and different DNA contents (per well) in the presence of 10% serum (n = 3).

linear polypeptides at polymer/DNA weight ratios ≥ 2 (Figure 2B). These results substantiated that the star and linear polypeptides with different architectures displayed comparable DNA binding affinities. Owing to their potent DNA condensation capabilities, both the star and linear polypeptides

condensation capability of the linear and star polypeptides, which showed that they could effectively condense DNA at the polymer/DNA weight ratios ≥ 2 (Figure 2A). In consistence with this, the EB exclusion assay also quantitatively revealed that over 90% of the DNA could be condensed by the star and 260


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Figure 5. In vitro transfection efficiencies of polyplexes at various polymer/DNA weight ratios in HeLa (A), B16F10 (B), and RAW 264.7 (C) cells with or without light irradiation (661 nm, 5 mW/cm2, 4 min) (n = 3).

Figure 6. Intracellular kinetics of polyplexes and light-promoted endosomal escape via the PCI effect. (A) Cellular uptake levels of polymer/YOYO1-DNA polyplexes in HeLa cells (n = 3). (B) Cellular uptake of polyplexes in HeLa cells at 4 °C or in the presence of various endocytic inhibitors (n = 3). (C) Cellular uptake level of FITC-Tris in HeLa cells in the presence of polypeptides (n = 3). (D) CLSM images of HeLa cells treated with star polypeptide/YOYO-1-DNA polyplexes (w/w = 10) with or without light irradiation (661 nm, 5 mW/cm2, 4 min) (bar = 10 μm). (E) Colocalization ratios between YOYO-1-DNA and Lysotracker-Red following the same treatment in (D) (n = 50).

could complex DNA to form ∼150 nm polyplexes with positive surface charges at polymer/DNA weight ratios ≥ 2 (Figure 3).

Additionally, the star polypeptide/DNA polyplexes revealed a higher ζ potential (∼40 mV) than that revealed by the linear 261


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was remarkably higher than the fluorescence intensity of cells without light irradiation (5 au). Because the generated ROS would cause oxidization and damage of DNA molecules to impede gene transfection, we thereafter explored the DNA integrity upon light irradiation. As shown in Figure S15, after light irradiation (661 nm, 5 mW/cm2) of the star polypeptide/ DNA polyplexes for up to 20 min, the fluorescence intensity of the DNA band after electrophoresis was negligibly reduced. In comparison, ∼50% of the DNA molecules were degraded after light irradiation of the physical mixture of linear polypeptide/ DNA polyplexes and TAPP for 20 min. Such a huge discrepancy could be attributed to the distant separation of TAPP and DNA molecules that were localized in the core and the polypeptide layer and thus the generated ROS with high concentrations would not directly interact with the DNA molecules. 3.4. Intracellular Kinetics. The gene delivery efficiencies of polyplexes are largely related to their intracellular kinetics.60 Therefore, we first investigated the cellular uptake level of polypeptide/YOYO-1-DNA polyplexes in HeLa cells. As shown in Figure 6A, the linear and star polypeptides displayed a dramatically higher cellular uptake than that by 25k PEI, wherein the star polypeptide outperformed its linear analogue by ∼2-fold. We also performed flow cytometry analysis to demonstrate the cellular uptake percentages of different polyplexes in HeLa cells (Figure S17).61 In direct comparison, commercial transfection reagent 25k PEI showed the uptake level of ∼30%, whereas both star and linear polypeptides demonstrated a higher uptake level (∼90 and ∼60%, respectively). More importantly, star polypeptides showed a notably higher uptake level than that of its linear analogue, which was consistent with the above-mentioned results from spectrofluorimetry analysis. These results substantiated that the helical polypeptides could mediate effective intracellular delivery of DNA cargoes because of their potent membrane activities and the multivalent star topology was more favorable for triggering the cellular uptake of polyplexes, mainly attributed to the relatively higher charge density of star polypeptides that benefits the interactions with cell membranes. The internalization mechanism of polyplexes was also probed using various endocytic inhibitors. Clathrin-mediated endocytosis (CME) is inhibited by CPZ, caveolae-mediated endocytosis is inhibited by mβCD and GNT, and macropinocytosis is inhibited by WTM.62 As depicted in Figure 6B, the uptake level was decreased by 65% at 4 °C, suggesting that energy-dependent endocytosis mainly contributed to the cell uptake of polyplexes. Moreover, the uptake level was dramatically reduced by CPZ and WTM (∼40%) but not GNT or mβCD (Figure 6B), which suggested that the star polypeptide/DNA polyplexes were mainly endocytosed via CME and macropinocytosis rather than caveolae. It has been reported that the helical polypeptides can puncture pores on cell membrane to mediate potent membrane penetration as well as efficient diffusion of nucleic acids into cells.32 We thus investigated the pore formation capability of the linear and star polypeptides. A membrane-impermeable fluorescent dye, FITC-Tris, was utilized as a biomarker, and its cellular uptake level after coincubation with polypeptides was monitored to reflect the pore formation extent.55 As shown in Figure 6C, free FITC-Tris negligibly entered HeLa cells, whereas after coincubation with helical polypeptides, its uptake level was dramatically enhanced. Additionally, the star polypeptide mediated a significantly higher uptake level of

polypeptide/DNA polyplexes (∼20 mV), probably owing to the unique four-armed structure of the star polypeptide with higher cationic charge densities. The particle size of star polypeptide/DNA polyplexes was negligibly enhanced in 10% FBS within 2 h (Figure S13), indicating the desired stability of the polyplexes in serum. Heparin, at different concentrations, was then utilized to mediate DNA release from polyplexes. As shown in Figure 2C, at the heparin concentration of 0.05 mg/mL, DNA started to be released from star polypeptide/DNA polyplexes, whereas more DNA was released at further increased heparin concentrations. At the same heparin concentration, a higher amount of DNA release was noted for the star polypeptide than that for its linear analogue. Similar findings were observed from the gel retardation assay (Figure S18). Such a result thus suggested that the star polypeptide, although featuring a DNA condensation level comparable to that of its linear analogue, could promote DNA release in the presence of polyanions, which would be favorable toward intracellular DNA release and effective gene transfection. 3.3. In Vitro Transfection. The gene transfection capabilities of the linear and star polypeptides were first assessed in HeLa cells in the absence of serum. As shown in Figure 4A, the star polypeptide displayed 14-fold higher transfection efficiencies than those of the linear polypeptide at the polymer/DNA weight ratios of 15 and 20, indicating that the star-shaped topology greatly contributed to effective gene delivery. The remarkably higher transfection efficiencies of the star polypeptide were also noted in B16F10 and RAW 264.7 cells (Figure 4B,C), which outperformed commercial transfection reagent 25k PEI by 2 orders of magnitude. Remarkably compromised transfection efficiency in serum is a critical drawback of polycations. We thus further explored the transfection efficiencies of the star polypeptide/DNA polyplexes in the presence of 10% FBS. As shown in Figure 4D, the transfection efficiencies of the star polypeptide were greatly compromised in the presence of 10% FBS when the amount of DNA was 0.1 μg/well. However, with the increased amount of DNA of 0.3 μg/well, the transfection efficiencies could be markedly recovered, which were only several fold lower than those under serum-free condition. Such observations may be attributed to the excessive amount of free polypeptides that could counteract the binding of serum proteins to enhance the stability of polyplexes.58 The PCI effect induced by the light irradiation of PS is an efficient strategy to mediating endosomal escape via localized generation of ROS to disrupt the endosomal membranes, thus potentiating the transfection efficiencies.59 As such, we thereafter monitored the transfection efficiencies of the star polypeptide/DNA polyplexes upon light irradiation. As shown in Figure 5, the transfection efficiencies of polyplexes were enhanced by 6−8 folds in all three test cell lines (HeLa, B16F10, and RAW 264.7) upon light irradiation, leading to superiority over commercial transfection reagent 25k PEI by several hundred folds. It was therefore demonstrated that the TAPP-incorporated star polypeptide could enable lightenhanced gene transfection by taking advantage of the PCI effect. The ROS generation by the photoactivated photosensitizer was further demonstrated. As shown in Figure S14, after light irradiation (661 nm, 5 mW/cm2) for 4 min, green fluorescence of the ROS probe, DCFH-DA, could be clearly observed with the quantified fluorescence intensity density of 36 au, which 262


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Figure 7. Cytotoxicity of polyplexes in HeLa (A), B16F10 (B), and RAW 264.7 (C) cells as determined by the MTT assay (n = 3).

irradiation (661 nm, 5 mW/cm2, 4 min, Figure S16). These results thus substantiated that the star polypeptide, although affording higher membrane activity than that of the linear polypeptide, possessed lower cytotoxicity, presumably because fewer helical motifs would have direct contact with the cell membranes. 3.6. In Vivo Transfection. To further verify whether the light-enhanced, star polypeptide-mediated gene delivery could be applied in vivo, the transfection efficiencies of polyplexes were assessed in xenograft melanoma via intratumoral injection. In consistence with the in vitro data, the star polypeptide displayed remarkably higher transfection efficiencies than those of the linear polypeptide and commercial reagent 25k PEI (Figure 8). Additionally, light irradiation (661 nm, 5 mW/cm2,

FITC-Tris than that of the linear polypeptide, which substantiated that the star polypeptide with a multiarm structure and higher cationic charge density afforded stronger pore formation capabilities. CLSM was adopted to investigate whether the star polypeptide/YOYO-1-DNA polyplexes could effectively escape from endo/lysosomes, a vital step for effective gene transfection.47 As shown in Figure 6D, after 4 h of incubation, extensive distribution of green fluorescence (YOYO-1-DNA) was observed inside the cells, but green fluorescence largely overlapped with red fluorescence (Lysotracker-Red), leading to a quantified colocalization ratio of ∼65% (Figure 6E). Such a result accorded well with the cellular uptake study, in that more than half of the polyplexes entered the cells via endocytosis and thus they were entrapped in the endo/lysosomes to impede effective transfection. For the rest of YOYO-1-DNA polyplexes were internalized via nonendocytosis, they would not experience endosomal entrapment and thus could be subsequently transported into the nuclei. When the cells were subjected to light irradiation (661 nm, 5 mW/cm2) for 4 min after incubation with the star polypeptide/YOYO-1-DNA polyplexes for 4 h, green fluorescence markedly separated from red fluorescence (Figure 6D) and the colocalization ratio decreased from 65 to 15% (Figure 6E). These results substantiated that light irradiation of the PS (TAPP) facilitated the endosomal escape of polyplexes by generating ROS that destabilized the endosomal membrane, the so-called PCI effect. By combining the helical polypeptide-mediated pore formation on cell membranes and PCI-assisted endosome rupture, effective intracellular delivery of DNA cargoes could be achieved, followed by almost complete escape from endo/ lysosomes. 3.5. Cytotoxicity. Besides transfection efficiency, cytotoxicity is another important factor that needs to be considered during the design of gene vectors. Cytotoxicity and transfection efficiency are often contradictory, and a similar case also works for helical polypeptides. Particularly, polypeptides with potent membrane activities will contribute to high transfection efficiencies, while in the meantime, the excessive membrane activity will also induce appreciable cytotoxicity. Therefore, it is of great necessity to balance the transfection efficiency and cytotoxicity and thus polymers with high transfection efficiency and diminished cytotoxicity are ideal.54 Herein, the cytotoxicities of polyplexes were assessed in HeLa, B16F10, and RAW 264.7 cells by the MTT assay. Compared with the linear polypeptide and 25k PEI, the star polypeptide displayed notably higher cell viability, especially at high polymer/DNA weight ratios of 15 and 20 (Figure 7). In addition, no significant change of cell viability was observed before and after light

Figure 8. In vivo transfection efficiencies of polyplexes following intratumoral injection in B16F10 xenograft tumor-bearing mice at 20 μg DNA/mouse (n = 4).

20 min) further elevated the transfection efficiency of star polypeptide by ∼3-fold. These results again confirmed the advantages of the star polypeptide for gene delivery and also demonstrated the light-enhanced gene transfection in vivo as a result of facilitated endosomal escape due to the PCI effect.

4. CONCLUSIONS In conclusion, we developed PS-embedded, star-shaped helical polypeptides, which enabled multivalency-enhanced intracellular DNA delivery efficiency and light-strengthened endosomal escape. The star polypeptide thus showed a notably higher transfection efficiency and lower cytotoxicity than those of its linear analogue. Light irradiation caused almost complete (∼90%) endosomal release of the DNA cargo via the PCI mechanism and further led to a 6−8-fold increment of the transfection efficiency, outperforming commercial reagent 25k 263


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carriers for Redox-Responsive Delivery of Doxorubicin. Biomater. Sci. 2016, 4, 412−417. (9) Islam, M. A.; Reesor, E. K. G.; Xu, Y.; Zope, H. R.; Zetter, B. R.; Shi, J. Biomaterials for mRNA Delivery. Biomater. Sci. 2015, 3, 1519− 1533. (10) Wu, J. P.; Cheng, B.; Roffler, S. R.; Lundy, D. J.; Yen, C. Y.; Chen, P.; Lai, J. J.; Pun, S. H.; Stayton, P. S.; Hsieh, P. C. Reloadable Multidrug Capturing Delivery System for Targeted Ischemic Disease Treatment. Sci. Transl. Med. 2016, 8, No. 365ra160. (11) Srinivasachari, S.; Liu, Y.; Zhang, G.; Prevette, L.; Reineke, T. M. Trehalose Click Polymers Inhibit Nanoparticle Aggregation and Promote pDNA Delivery in Serum. J. Am. Chem. Soc. 2006, 128, 8176−8184. (12) Li, Y.; Humphries, B.; Wang, Z.; Lang, S.; Huang, X.; Xiao, H.; Jiang, Y.; Yang, C. Complex Coacervation-Integrated Hybrid Nanoparticles Increasing Plasmid DNA Delivery Efficiency In Vivo. ACS Appl. Mater. Interfaces 2016, 8, 30735−30746. (13) Thambi, T.; Park, J. H.; Lee, D. S. Stimuli-Responsive Polymersomes for Cancer Therapy. Biomater. Sci. 2016, 4, 55−69. (14) Wang, M.; Alberti, K.; Varone, A.; Pouli, D.; Georgakoudi, I.; Xu, Q. B. Enhanced Intracellular siRNA Delivery using Bioreducible Lipid-Like Nanoparticles. Adv. Healthcare Mater. 2014, 3, 1398−1403. (15) Cheng, Q.; Du, L. L.; Meng, L. W.; Han, S. C.; Wei, T.; Wang, X. X.; Wu, Y. D.; Song, X. Y.; Zhou, J. H.; Zheng, S. Q.; Huang, Y. Y.; Liang, X. J.; Cao, H. Q.; Dong, A. J.; Liang, Z. C. The Promising Nanocarrier for Doxorubicin and siRNA Co-delivery by PDMAEMABased Amphiphilic Nanomicelles. ACS Appl. Mater. Interfaces 2016, 8, 4347−4356. (16) Chen, C. K.; Law, W. C.; Aalinkeel, R.; Nair, B.; Kopwitthaya, A.; Mahajan, S. D.; Reynolds, J. L.; Zou, J.; Schwartz, S. A.; Prasad, P. N.; et al. Well-Defined Degradable Cationic Polylactide as Nanocarrier for the Delivery of siRNA to Silence Angiogenesis in Prostate Cancer. Adv. Healthcare Mater. 2012, 1, 751−761. (17) Liu, Y.; Du, J.; Choi, J. S.; Chen, K. J.; Hou, S.; Yan, M.; Lin, W. Y.; Chen, K. S.; Ro, T.; Lipshutz, G. S.; et al. A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors. Angew. Chem., Int. Ed. 2016, 55, 169−173. (18) Liu, B.; Li, C.; Cheng, Z.; Hou, Z.; Huang, S.; Lin, J. Functional Nanomaterials for Near-Infrared-Triggered Cancer Therapy. Biomater. Sci. 2016, 4, 890−909. (19) Yang, Q.; Cai, J.; Sun, S.; Kang, X.; Guo, J.; Zhu, Y.; Yan, L.; Jing, X.; Wang, Z. Polymer Nanoparticle Delivery of Dichloroacetate and DACH-Pt to Enhance Antitumor Efficacy and Lower Systemic Toxicity. Biomater. Sci. 2016, 4, 661−669. (20) Cutlar, L.; Gao, Y.; Aied, A.; Greiser, U.; Murauer, E. M.; Zhou, D.; Wang, W. A Knot Polymer Mediated Non-Viral Gene Transfection for Skin Cells. Biomater. Sci. 2016, 4, 92−95. (21) Ping, Y.; Liu, C. D.; Tang, G. P.; Li, J. S.; Li, J.; Yang, W. T.; Xu, F. J. Correction: Functionalization of Chitosan via Atom Transfer Radical Polymerization for Gene Delivery. Adv. Funct. Mater. 2011, 20, 3106−3116. (22) Yang, L.; Li, W.; Kirberger, M.; Liao, W.; Ren, J. Design of Nanomaterial Based Systems for Novel Vaccine Development. Biomater. Sci. 2016, 4, 785−802. (23) Johnson, R. N.; Chu, D. S.; Shi, J.; Schellinger, J. G.; Carlson, P. M.; Pun, S. H. HPMA-Oligolysine Copolymers for Gene Delivery: Optimization of Peptide Length and Polymer Molecular Weight. J. Controlled Release 2011, 155, 303−311. (24) Patra, S.; Roy, E.; Madhuri, R.; Sharma, P. K. The Next Generation Cell-Penetrating Peptide and Carbon Dot Conjugated Nano-Liposome for Transdermal Delivery of Curcumin. Biomater. Sci. 2016, 4, 418−429. (25) Milletti, F. Cell-Penetrating Peptides: Classes, Origin, and Current Landscape. Drug Discovery Today 2012, 17, 850−860. (26) Snyder, E. L.; Dowdy, S. F. Cell Penetrating Peptides in Drug Delivery. Pharm. Res. 2004, 21, 389−393. (27) Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. The Design of Guanidinium-Rich Transporters and Their

PEI by 2−3 orders of magnitude. Such a multivalencyenhanced and PCI-potentiated gene delivery efficiency was also noted in vivo following intratumoral injection. This study therefore provides a promising, light-assisted strategy to overcome the multiple membrane barriers against nonviral gene delivery. The PS-embedded, star polypeptide would serve as a favorable addition to existing transfection materials and may find potential utilities toward anticancer gene therapy.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b15896. Detailed description of instrumentation, synthetic routes, 1 H NMR spectra, and additional experimental results (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.L.). *E-mail: [email protected] (L.Y.). ORCID

Lichen Yin: 0000-0002-4573-0555 Author Contributions ∥

X.X., Y.L., and Q.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51403145, 51573123, and 51722305), the Ministry of Science and Technology of China (2016YFA0201200), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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