Efficient Gene Delivery Mediated by a Helical Polypeptide: Controlling

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Efficient gene delivery mediated by helical polypeptide: controlling the membrane activity via multivalency and light-assisted photochemical internalization (PCI) Xin Xu, Yongjuan Li, Qiujun Liang, Ziyuan Song, Fangfang Li, Hua He, Jinhui Wang, Lipeng Zhu, Zhifeng Lin, and Lichen Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15896 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017

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Efficient gene delivery mediated by helical polypeptide: controlling the membrane activity via multivalency and light-assisted photochemical internalization (PCI) Xin Xu, a,1 Yongjuan Li, a,1 Qiujun Liang, a,1 Ziyuan Song, b Fangfang Li, a Hua He,a Jinhui Wang, a Lipeng Zhu, a Zhifeng Lin, c,* Lichen Yin a,* a

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 b

Department of Materials Science and Engineering, University of Illinois at

Urbana-Champaign, 1304 W Green St, Urbana, USA c

Department of Thoracic Surgery, Shanghai General Hospital, Shanghai Jiao Tong

University of Medicine

1

These authors contributed equally.

*

Corresponding author:

Email: [email protected] (L. Yin); [email protected] (Z. Lin)

KEYWORDS: non-viral gene delivery, α-helical polypeptide, multivalency, membrane penetration, photochemical internalization (PCI), endosomal escape 1

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ABSTRACT: The development of robust and nontoxic membrane-penetrating materials is highly demanded for non-viral gene delivery. Herein, a photosensitizer (PS)-embedded, star-shaped helical polypeptide was developed, which combines the merit 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-(4-aminophenyl) porphyrin (TAPP) as a selected PS initiated ring-opening polymerization (ROP) of N-carboxyanhydride (NCA), and yielded star-shaped helical polypeptide after side-chain functionalization with guanidine groups. The star polypeptide afforded notably higher transfection efficiency and lower cytotoxicity than 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 three orders of magnitude. Because the PS and DNA cargo were compartmentalized distantly in the core and polypeptide layers, respectively, the generated reactive oxygen species (ROS) 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 non-viral gene delivery.

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1. INTRODUCTION 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 Non-viral 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 non-viral gene vectors is hampered by the 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 nucleus membranes.17, 21 As such, development of membrane-penetrating materials represents a promising approach for non-viral 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 trans-membrane 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 helical structure or can transform into helix upon contact with cell membranes, which potentiates the affinity with 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 3

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As such, we recently developed cationic, guanidinated polypeptides with sufficient backbone length and stable α-helical secondary structure.31-32 However, only part of the polypeptide/DNA polyplexes (~50%) enters the cells via energy-independent permeation, while 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 a well-established principle to strengthen the molecular interactions with cell membranes.37-39 Commonly adopted multivalent arrangement is either based on globular scaffolds or branched dendrimers, and the obtained multivalent CPPs feature 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 impart the materials with potentiated membrane activities, enhanced gene delivery efficiencies, and reduced cytotoxicity. 4

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Photochemical internalization (PCI) is a recently emerged technique in overcoming the endo-lysosomal entrapment.43-44 This modality derives from photodynamic therapy (PDT), a mechanism based on the peroxidation and destruction of endo-lysosomal membranes by reactive oxygen species (ROS) generated from 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 in 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. Based on the above 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 light-assisted “PCI” effect (Scheme 1).49-51 To this end, guanidine-rich, four-armed helical polypeptide was synthesized via ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) initiated by 5,10,15,20-tetrakis-(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. 5

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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 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), 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, USA). The POB-L-Glu-NCA monomer and 6-azidohexylguanidine were prepared as described previously.25 The chemical structures of compound 1~7 were confirmed by 1H NMR (Figure 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, USA). HeLa and RAW 264.7 cells were cultured in DMEM containing 1% non-essential amino acids, 10% fetal bovine serum (FBS), and 1% L-glutamine. B16F10 cells were cultured in RPMI 1640 medium containing 10% FBS, 1% non-essential amino acids, and 1% L-glutamine. Male C57/BL6 mice (6-8 wk) 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. 6

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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 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD, 64 µL, 0.01 M in DMF) were added in a glovebox. The mixture was stirred at RT for 72 h, and was precipitated with cold methanol (30 mL) to obtain TAPP-PPOBLG as 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 (PMDETA, 4.5 µL, 0.0216 mmol), and 6-azidohexylguanidine (144 µL, 0.144 mmol) were added. The reaction mixture was stirred for two days at RT and then 1 M HCl (1 mL) was added. The final polypeptide containing guanidine side chains (star polypeptide) was dialyzed for two days (MWCO = 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 post-functionalization with 6-azidohexylguanidine as described above. Its chemical structure was confirmed by 1H NMR (Figure S8). 2.3. Polyplexes formation and characterization. DNA and polypeptide were dissolved in 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. Thee DNA condensation level was also quantitatively monitored by using the ethidium bromide (EB) exclusion assay.53,54 Zeta potential and particle size of the polyplexes were 7

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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 were cultured at 37 °C until reaching 70% confluence. The polyplexes (0.1 µg DNA per well) were incubated with cells in opti-MEM 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 removal of polyplexes. Non-irradiated cells served as a control. Cells were further incubated in DMEM supplemented with 10% FBS before quantification of the luciferase expression level using a Bright-Glo luciferase assay kit (Promega, USA). A bicinchoninic acid assay (BCA) kit (Pierce, USA) 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 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 8

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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) for 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. 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. Otherwise, cells were pre-treated 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 denoted as 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 on 96-well plate (70% confluence) were incubated with polypeptide (2 µg/well) and FITC-Tris (1 µg/well) in opti-MEM at 37 °C for 2 h. Then, cells were washed by heparin-containing PBS for 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 9

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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 confocal laser scanning microscope (CLSM). HeLa cells on 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 for 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. Non-irradiated cells served as a control. The colocalization ratio between YOYO-1-DNA and Lysotracker-Red-stained endolysosomes was quantified according to the previously reported method.54 2.7. Cytotoxicity. Cells were seeded on 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 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 percentage viability of control cells which 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 10

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five groups (four mice per group) which received intratumoral injection of either polyplexes (20 µg DNA/mouse) or PBS (∼50 µL/injection). Group 1 and 2 received star polypeptide/DNA polyplexes, while mice in group 1 were irradiated (661 nm, 5 mW/cm2) for 10 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 for 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 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 differences between test and control groups were judged to be significant at *p < 0.05 and very significant at **p < 0.01 and ***p < 0.001.

3. RESULTS AND DISCUSSION 3.1.

Synthesis

and characterization of polypeptides.

Prior

to

the

TAPP-initiated polymerization, NCA polymerization was first performed by using aniline (AN) to validate whether it can efficiently initiate the ROP of NCA. According to the MALDI-TOF analysis, the MW of the obtained AN-PPOBLG (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 relatively low polydispersity index (PDI= 1.23) was obtained via ROP initiated by TAPP, a PS that bears 11

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four-armed aniline.56 The degree of polymerization (DP) of TAPP-PPOBLG determined by GPC analysis was 60 (Mn = 17188 g/mol), which was in agreement with the expected value. As a control, PPOBLG with similar MW (DP = 66, Mn = 18096 g/mol) was synthesized with HMDS as the initiator (Figure S11 and Table S1). In order 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 similar absorption curves to free TAPP, which also demonstrated that the 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 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 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 endo/lysosomes. 3.2. Polyplexes formation and characterization. The gel retardation assay was first conducted to assess the DNA condensation capability of the linear and star polypeptides, which showed that they could effectively condense DNA at the 12

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polymer/DNA weight ratios ≥ 2 (Figure 2A). In consistence, the EB exclusion assay also quantitatively revealed that over 90% of the DNA could be condensed by the star and 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. Due to their potent DNA condensation capabilities, both the star and linear polypeptides 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 higher zeta potential (~40 mV) than linear 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 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, while more DNA was released at further increased heparin concentrations. At the same heparin concentration, higher DNA release amount was noted for star polypeptide than its linear analogue. Similar findings were observed from the gel retardation assay (Figure S18). Such result thus suggested that the star polypeptide, although featuring comparable DNA condensation level to its linear analogue, could promote DNA release in the presence of polyanions, which would be favorable toward intracellular 13

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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 the linear polypeptide at the polymer/DNA weight ratios of 15 and 20, indicating that the star-shaped topology greatly contributed to the effective gene delivery. The remarkably higher transfection efficiencies of the star polypeptide were also noted in B16F10 and RAW 264.7 cells (Figure 4B and 4C), which outperformed commercial transfection reagent 25k PEI by two orders of magnitude. Remarkably compromised transfection efficiency in serum is a critical drawback of polycations. We thus further explored the transfection efficiencies of star polypeptide/DNA polyplexes in the presence of 10% FBS. As shown in Figure 4D, the transfection efficiencies of star polypeptide were greatly compromised in the presence of 10% FBS at the DNA amount of 0.1 µg/well. However, at the increased DNA amount 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 mediate endosomal escape via localized generation of ROS to disrupt the endosomal membranes, thus potentiating the transfection efficiencies.59 As such, we thereafter 14

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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, 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 light-enhanced gene transfection by taking advantage of the “PCI” effect. The ROS generation by photo-activated 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 a.u., which was remarkably higher than the fluorescence intensity of cells without light irradiation (5 a.u.). Because generated ROS would cause oxidization and damage of DNA molecules to impede the 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 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. 15

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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 dramatically higher cellular uptake than 25k PEI, wherein the star polypeptide outperformed its linear analogue by ~2 fold. We also performed the flow cytometry analysis to demonstrate the cellular uptake percentages of different polyplexes in HeLa cells.61 In direct comparison, commercial transfection reagent 25k PEI showed the uptake level of ∼30%, while both star and linear polypeptides demonstrated higher uptake level (∼90% and ∼60%, respectively). More importantly, star polypeptide showed notably higher uptake level than its linear analogue, which was consistent with the above results from spectrofluorimetry analysis. These results substantiated that the helical polypeptides could mediate effective intracellular delivery of DNA cargoes due to 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 by using various endocytic inhibitors. The clathrin-mediated endocytosis (CME) is inhibited by CPZ; the caveolae-mediated endocytosis is inhibited by mβCD and GNT, while macropinocytosis is inhibited 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 16

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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 co-incubation with polypeptides was monitored to reflect the pore formation extent.55 As shown in Figure 6C, free FITC-Tris negligibly entered HeLa cells, while after co-incubation with helical polypeptides, its uptake level was dramatically enhanced. Additionally, the star polypeptide mediated significantly higher uptake level of FITC-Tris than the linear polypeptide, which substantiated that the star polypeptide with multi-arm 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 incubation, extensive distribution of green fluorescence (YOYO-1-DNA) was observed inside the cells, but green fluorescence largely overlapped with the red fluorescence (Lysotracker Red), leading to a quantified colocalization ratio of ~65% (Figure 6E). Such result accorded well with the cellular uptake study, in that more than half of the polyplexes entered cells via endocytosis, and thus they were entrapped in the endo/lysosomes to impede 17

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effective transfection. For the rest of YOYO-1-DNA that was internalized via non-endocytosis, it 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 with 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 similar case also works for helical polypeptides. Particularly, polypeptides with potent membrane activities will contribute to high transfection efficiencies, while at 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 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 to the linear polypeptide and 25k PEI, the 18

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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 irradiation (661 nm, 5 mW/cm2, 4 min, Figure S16). These results thus substantiated that the star polypeptide, although affording higher membrane activity than 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 polypeptides displayed remarkably higher transfection efficiencies than the linear polypeptide and commercial reagent 25k PEI (Figure 8). Additionally, light irradiation (661 nm, 5 mW/cm2, 10 min) further elevated the transfection efficiency of star polypeptide by ~3 folds. 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 polypeptide which enabled

multivalency-enhanced

intracellular

DNA

delivery

efficiency

and

light-strengthened endosomal escape. The star polypeptide thus showed notably 19

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higher transfection efficiency and lower cytotoxicity than 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 PEI by 2~3 orders of magnitude. Such multivalency-enhanced 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 non-viral gene delivery. The PS-embedded, star polypeptide would serve as a favorable addition to existing transfection materials, and may find potential utilities toward anti-cancer gene therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed description of instrumentation, synthetic routes, 1H NMR spectra, and additional experimental results (PDF). AUTHOR INFORMATION Corresponding Author *Email:[email protected] (L. Yin); [email protected] (Z. Lin) Author Contributions 1

X. X., Y. L., and Q. L. contributed equally to this work. 20

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Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS 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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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.

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Scheme 2. Synthetic route of the star polypeptide.

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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.

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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 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).

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

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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 content (per well) in the presence of 10% serum (n = 3).

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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).

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Figure 6. Intracellular kinetics of polyplexes and light-promoted endosomal escape via the “PCI” effect. (A) Cellular uptake levels of polymer/YOYO-1-DNA polyplexes in HeLa cells (n = 3). (B) Cellular uptake of polyplexes in HeLa cells at 4 ºC 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).

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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).

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

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