Octopus-like flexible vector for noninvasive intraocular delivery of short

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Octopus-like flexible vector for noninvasive intraocular delivery of short interfering nucleic acids Kuan Jiang, Yang Hu, Xin Gao, Changyou Zhan, Yanyu Zhang, Shengyu Yao, Cao Xie, Gang Wei, and Weiyue Lu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b02596 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 24, 2019

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Octopus-like flexible vector for noninvasive intraocular delivery of short interfering nucleic acids Kuan Jiang†, Yang Hu†, Xin Gao† Changyou Zhan†, ‡, Yanyu Zhang†, Shengyu Yao†, Cao Xie†, Gang Wei*, †, Weiyue Lu† †Key

Laboratory of Smart Drug Delivery, Ministry of Education; Department of Pharmaceutics,

School of Pharmacy, Fudan University, Shanghai 201203, China ‡Department

of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai

200032, China Corresponding author: Gang Wei, Ph.D., Professor Key Laboratory of Smart Drug Delivery, Ministry of Education; Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, P.R.China Tel: +86 21 51980091 Fax: +86 21 51980090 E-mail address: [email protected]

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Abstract Gene therapy is promising for chronic posterior ocular diseases, which are causal factors for severe vision impairment and even blindness worldwide. However, inherent absorption barriers of the eye restrict intraocular delivery of therapeutic nucleic acids via topical instillation. Safe and efficient non-viral vectors for ocular gene therapy are still unmet clinical desires. Herein, an octopus-like flexible multivalent penetratin (MVP) was designed to facilitate therapeutic nucleic acids condensation and delivery using multi-arm polyethylene glycol (PEG) as a core and conjugating penetratin at each end of the PEG arms as outspread tentacles. Among the MVPs, 8-valent penetratin (8VP) stably compacted nucleic acids into positively charged polyplexes smaller than 100 nm, promoting cellular uptake efficiency (approaching 100%) and transfection rate (over 75%). After instilled into the conjunctival sac, 8VP enabled rapid (< 10 min) and prolonged (> 6 h) distribution of nucleic acids in the retina via a non-corneal pathway. In a retinoblastoma-bearing mice model, topical instillation of 8VP/siRNA efficiently inhibited the protein expression of intraocular tumor without toxicity. MVP is advantageous over the commercial transfection reagent in safety and efficiency, therefore provides a promising vector for noninvasive intraocular gene delivery. Key words: posterior ocular diseases, penetratin, gene delivery, topical instillation, non-corneal pathway Chronic posterior ocular diseases account for the most severe vision loss and even irreversible blindness worldwide,1-3 but safe and convenient treatments remain unmet due to various ocular absorption barriers.4 Intraocular injection is the first choice in clinic to generate high local concentration accessible to the retina and choroid, for instance, intravitreal injection of pegaptanib, a pegylated aptamer, for neovascular age-related macular degeneration.5 However, repeated injections are of poor patient compliance, and may lead to severe complications such as ocular hemorrhage and retinal detachment.6 Topical instillation is more desirable, but trace amount of drug was expected for intraocular absorption.4 Cell-penetrating peptides (CPPs), usually short peptides with high permeability in biological membrane, could facilitate intracellular delivery of hydrophilic proteins and nucleic acids.7, 8 We have demonstrated the potential of penetratin, a non-viral derived CPP from antennapedia homeodomain, as an ocular absorption enhancer, which exhibited substantially better biosafety and ocular permeability than some other classic CPPs.9 The eye is an ideal site for gene therapy as it is a relatively small, self-contained and immuneprivileged organ.10, 11 Post-transcriptional gene silencing strategies blazes a trail for treatment of chronic posterior ocular diseases, however, noninvasive gene delivery to the fundus oculi remains challenging, predominantly due to absence of safe and efficient gene carriers.12-14 We previously reported that gene pre-condensation by cationic polymer was indispensable for penetratin mediated plasmid DNA15 or antisense oligonucleotides (ASO)16 delivery from the cornea to the retina. Nevertheless, potential toxicity of the non-biodegradable cationic polymers and uncertain composition of the formed polyplexes hindered further translation. Moreover, the competitive binding between penetratin and cationic polymer to anionic nucleic acids might impair the intrinsic ocular permeability of penetratin. In the present work, we designed an octopus-like flexible multivalent penetratin (MVP) composed of a biocompatible multi-arm polyethylene glycol (PEG) core and several outspread penetratin tentacles. To assemble with anionic nucleic acids, part of penetratin tentacles could spontaneously 2


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bind with and condense gene via electrostatic interaction, while others stretch out to implement ocular permeation and intraocular delivery of nucleic acids (Fig. 1A), like an octopus carries cargo and moves forward (Fig. 1B). After being absorbed into the eye or eliminated into the gastrointestinal tract, the peptide tentacles could be readily degraded. Using ASO and siRNA as model genes, MVPs showed advantages both in gene condensation and intraocular delivery in vitro and in vivo, and more tentacles performed better. The biosafety was evaluated and probable absorption route of the MVP-based polyplexes was also discussed.

Figure 1 Construction and characterization of polyplexes. A, multivalent penetratin (MVP) forms polyplexes with nucleic acids (NAs, for example antisense oligonucleotides (ASO) or siRNA) through a simple mixing process. B, in the polyplexes, several cationic penetratin tentacles bind freely with anionic nucleic acids, while others are ready to mediate the noninvasive intraocular delivery, just like an octopus carrying some cargos (nucleic acids) and moving forward. C, morphology of the polyplexes under the transmission electron microscope. Scale bar, 200 nm. D, particle sizes and zeta potentials of the polyplexes. E, time-dependent particle sizes variation of the polyplexes incubated in 10 mM PBS at 34 ± 0.5°C. The data are presented as mean ± standard deviation (SD) (n = 3). F, mean fluorescence intensity of the polyplexes at different concentrations (ASO was labeled with FAM). Ex 490 nm, Em 520 nm. Pe, free penetratin; 4VP, 4-valent penetratin; 8VP, 8-valent penetratin; Lipo, LipofectamineTM 2000. Construction and characterization of polyplexes Although with satisfying ocular permeability, low charge density makes penetratin alone unsuitable for gene condensation. Herein, local density of positive charges was amplified by conjugating several penetratin molecules to a multi-arm PEG core, obtaining 4-valent penetratin (4VP) and 8valent penetratin (8VP), respectively (ASSOCIATED CONTENT: Supporting Information 3


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Available. [Figure S1]). Owing to the flexible branching spatial structure, MVPs could selfassemble with anionic nucleic acids just by mixing their solution and possess high capability of gene loading, which was significantly superior to free penetratin (ASSOCIATED CONTENT: Supporting Information Available. [Figure S2]). Taking cellular uptake into account, the optimal charge ratio of polyplexes was set at 12:1 (+/-). Among the polyplexes, the particle size of 8VP/ASO was the smallest (95 nm), which was much smaller than the polyplex formed by LipofectamineTM 2000 with ASO (Lipo/ASO, 355 nm), indicating more penetratin tentacles resulted in more compact polyplexes (Fig. 1C, 1D). The zeta potential of 8VP/ASO was comparable to that of Lipo/ASO, both around + 20 mV, which would achieve longer ocular retention, due to interaction between cationic polyplexes and anionic ocular mucous layer.17 Considering that the polyplexes maintained by electrostatic interaction might accompany with poor stability, changes in particle size were monitored to evaluate the storing stability of these polyplexes. After incubated at 34°C for 48 h, the polyplex formed by free penetratin and ASO (Pe/ASO) has aggregated while 4VP/ASO and 8VP/ASO remained relatively stable, as their particle sizes increased about 600 nm, 90 nm and 80 nm, respectively (Fig. 1E). Assembling with MVP induced the fluorescence intensity of FAM-labeled ASO decreased dramatically (Fig. 1F).18 Fluorescence intensity of 8VP-based polyplexes decreased over 6 times compared to naked ASO at a concentration of 400 nM, while about 2 times for 4VP-based polyplexes. Besides, fluorescence intensity of MVP-based polyplexes decreased in a concentrationdependent manner (R2>0.98) suggestive of high stability against dilution. In contrast, serial dilution of Pe/ASO caused sharp change in fluorescence intensity (from 100 nM to 50 nM), indicating dissociation of free penetratin and ASO from the polyplexes. Apart from fluorescence intensity, alteration in fluorescence spectra also revealed improved gene condensation ability of MVPs (ASSOCIATED CONTENT: Supporting Information Available. [Figure S3]), and more penetratin tentacles enabled stable polyplexes. Both compact structure and PEG shielding contributed to the stability of MVP-based polyplexes.19, 20 MVPs enhanced intraocular delivery of ASO both in vitro and in vivo Ocular absorption routes mainly include corneal and non-corneal pathway, where cornea and conjunctiva participate in the major static barrier composition, respectively.21 Herein, human corneal epithelial cells (HCEC) and human conjunctival epithelial cells (NHC) were employed to evaluate cellular uptake of ASO-contained polyplexes. In HCEC cells, mean fluorescence intensities of the cells treated by 4VP/ASO and 8VP/ASO were about 10 and 50 times higher than those treated by naked ASO (ASSOCIATED CONTENT: Supporting Information Available. [Figure S4]), while in NHC cells, the differences became 8 and 120 times higher, respectively (Fig. 2A, 2B), and the polyplexes did not co-localize with lysosomes (ASSOCIATED CONTENT: Supporting Information Available. [Figure S5]). Notably, mean fluorescence intensity of 8VP/ASO treated NHC cells was about 4 times higher than that in HCEC cells, implying 8VP/ASO would be absorbed more by conjunctiva than cornea. Meanwhile, Lipofectamine was chosen as a positive control to compare with MVPs. In NHC cells, mean fluorescence intensity was significantly higher (p < 0.001) in the cells treated by 8VP/ASO than Lipo/ASO. By further comparison, we found that 8VP showed much lower cytotoxicity than Lipofectamine. In HCEC (ASSOCIATED CONTENT: Supporting Information Available. [Figure S4]) and NHC (Fig. 2C) cells , the proportions of single live cells among total events were similar for untreated cells and MVP/ASO treated cells, but the number for Lipo/ASO treated cells was extremely low. Similar proportions of single live cells and cellular 4


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distributions meant MVP/ASO had slight effects on both HCEC and NHC cells, which was also proven by the results of cytotoxicity test (ASSOCIATED CONTENT: Supporting Information Available. [Figure S6]). In contrast, much lower proportion of single live cells after treated by Lipo/ASO revealed the potential cytotoxicity. Compared with Lipofectamine, MVP was composed of PEG and penetratin, both of which were of good biocompatibility.9, 22, 23 Especially the penetratin tentacles could further be degraded by intracellular enzymes. These factors contributed together to the low toxicity of MVP/ASO.

Figure 2 Multivalent penetratin enhanced intracellular delivery and intraocular distribution of ASO. A, B, flow cytometry profiles of the polyplexes and histogram of the mean fluorescence intensity in NHC cells. C, the populations of single live NHC cell were identified by forward scatter/side scatter (FSC/SSC) gating graph. Values in each plot represent proportions of single live NHC cell among total events. D, E, intraocular distribution of ASO in whole eyes, corneas and retinas from the mice treated by different polyplexes. Epi, epithelium; Endo, endothelium; GCL, 5


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ganglion cell layer. F, semi-quantitative analysis of mean fluorescence intensity in treated cornea and retina at different time points. The data are presented as mean ± standard deviation (SD) (n = 3 for A and B, n = 5 for F, nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001). Scale bar, 500 µm for D and 100 µm for E. To estimate intraocular distribution and pharmacokinetics of the FAM-labeled polyplexes after topical instillation, fluorescence observation was performed on frozen sections of the whole mice eyes. Topical instillation of naked ASO induced no perceptible green fluorescence of FAM in both anterior (cornea) and posterior (retina) segments (Fig. 2D). For Pe/ASO treated eye, there was only weak green fluorescence in anterior and posterior segments. By contrast, 4VP/ASO or 8VP/ASO treated eye emitted distinct green fluorescence especially in the retina. Topically instilled eye drops would be absorbed via corneal pathway, non-corneal pathway, or both.4, 21 The cornea only allows absorption of amphiphilic small molecules.24 The conjunctiva and sclera constitute major barriers for non-corneal pathway, but more permeable than cornea, which is meaningful for absorption of the hydrophilic macromolecules.25 Topical instillation of MVP-based polyplexes led to little distribution in the cornea but much distribution in the retina, and especially bright green fluorescence could be observed in outer retina (retinal pigment epithelium) than inner retina (ganglion cell layer), which provided a solid evidence that absorption of the polyplexes was from sclera to retina (Fig. 2E). Therefore, we speculated that both corneal and non-corneal pathway existed when the MVP/ASO polyplexes were absorbed, but non-corneal pathway might play a crucial role in their intraocular delivery.26 Based on the ocular distribution, we assumed that after instilled into the conjunctival sac, MVP-based polyplexes would mainly have a journey as following: conjunctiva → sclera → outer retina (retinal pigment epithelium) → inner retina (ganglion cell layer) → vitreous body. Eye sections at different time points revealed the pharmacokinetic behavior of topical instilled polyplexes (ASSOCIATED CONTENT: Supporting Information Available. [Figure S7]). According to semi-quantitative results (Fig. 2F), for 4VP/ASO and 8VP/ASO treated eyes, mean FAM fluorescence intensity peaked within 1 h, following a residence time up to 6 h, and 8VP/ASO performed better. MVPs promoted siRNA across an in vitro blood-retina barrier model As siRNA was one of the most promising RNA interfering therapeutics, a siRNA targeting firefly luciferase gene was also chosen as model nucleic acids to construct polyplexes, whose characterization and cellular evaluation were also implemented (ASSOCIATED CONTENT: Supporting Information Available. [Figure S8-S11]). The MVP/siRNA polyplexes were much smaller than Pe/siRNA and Lipo/siRNA, positive charged, stable in PBS within 48 h, and resistant to dilution. In addition, the 8VP/siRNA polyplexes increased the cellular uptake by 83 and 7 times in human retinal pigment epithelial cells (ARPE-19) and human retinoblastoma cells (WERI-Rb1), and had almost no cytotoxicity, in contrast to the severe cytotoxicity of Lipo/siRNA to these cells. An in vitro blood-retina barrier (BRB) model (Fig. 3A) was established to explore transfer efficiency of the polyplexes across posterior ocular absorption barriers, mainly the retina pigment epithelium layer.27, 28 Fluorescence images of FAM-labeled siRNA distribution in bottom human umbilical vein endothelial cells (HUVEC) layer revealed that green fluorescence could be only observed in 8VP/siRNA treated layer (Fig. 3B). Polyplexes distribution in the whole BRB could be clearly 6


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observed from the 3D fluorescence images of BRB side view (Fig. 3C). Only 8VP/siRNA treated BRB exhibited obvious green fluorescence both in upper ARPE-19 and bottom HUVEC layer, indicating 8VP enabled siRNA efficiently transfer across the BRB. Although there was obvious green fluorescence in the upper ARPE-19 layer, Lipo/siRNA treated BRB exhibited scarcely any green fluorescence in the bottom HUVEC layer.

Figure 3 Transfer behavior of polyplexes across an in vitro blood-retina barrier (BRB) model. A, schematic representation of the BRB model. B, fluorescence images of bottom HUVEC layer in the acceptor side. C, 3D fluorescence images of polyplexes distribution in BRB in the surface mode. The upper side was ARPE-19 cells layer while the bottom side was HUVEC layer in each image. The white middle line was the border of the adjacent front and side profiles. D, fluorescence intensity of the polyplexes in different medium at a series of concentration. The fresh samples refer to the untreated polyplexes. Other samples were mixed with isopyknic 2 M NaOH solution or 0.2 M phosphate buffer (PB, pH7.4) for further measurement. E, fluorescence intensity variation of the polyplexes mixed with isopyknic 2 M NaOH solution. The original samples refer to those extracted directly from the acceptor side. F, penetration profiles of the polyplexes across the in vitro BRB model. G, cumulative amount of the polyplexes permeated across the BRB after incubation for 4 h. The data are presented as mean ± standard deviation (SD) (n = 3, ***p < 0.001, compared to naked siRNA). Scale bar, 100 μm for B and C. 7


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Compared to FAM-labeled naked siRNA, freshly prepared 8VP/siRNA and Lipo/siRNA showed much weaker fluorescence intensity (Fig. 3D (a)). As a result, it was necessary to dissociate cationic vectors and anionic siRNA for fluorescence quantification of siRNA. Considering these polyplexes were assembled mainly via electrostatic interaction, which would be affected by pH and ionic strength,29 2 M NaOH solution and 0.2 M phosphate buffer (PB) were chosen as dissociation reagents. Mixing with either two reagents caused increase in fluorescence intensity of both 8VP/siRNA and Lipo/siRNA, but still lower than that of naked siRNA at the same concentration (Fig. 3D (b) and 3D (c)). Mixture of the polyplexes and 2 M NaOH had a pH over 14, which was higher than the isoelectric point (pI) of penetratin (pI=12.7),30 consequently 8VP has converted to be negatively charged and would not bind with anionic siRNA by electrostatic interaction. Notably, when mixing 8VP/siRNA with 2 M NaOH, fluorescence intensity of the mixture was still lower than naked siRNA under the same condition, which implied that some other interactions might exist between 8VP and siRNA, probably hydrophilic-hydrophobic interactions.31 After treated with the polyplexes, the samples extracted from the acceptor side also exhibited increase in fluorescence intensity when mixing with NaOH solution (Fig. 3E), about 4 times higher for 8VP/siRNA and 8 times higher for Lipo/siRNA, indicating structural integrity of the post-transferred polyplexes. According to the quantitative results (Fig. 3F), the polyplexes transferring across BRB behaved in a linear manner within 4 h. After incubation for 4 h (Fig. 3G), the amount of 8VP/siRNA traversing the BRB was up to 15%, 3 times more than Lipo/siRNA and 5 times more than naked siRNA. From the in vitro BRB transfer experiment, 8VP/siRNA exhibited more remarkable BRB permeability than Lipo/siRNA, which was merely entrapped in the upper layer probably due to more intensively positive charge (> +40 mV vs +15 mV) and larger particle size (400 nm vs 85 nm).32, 33 Polyplexes inhibited intracellular protein expression in vitro Firefly luciferase (Fluc) is a tracer protein that had no effects on cell proliferation, and inhibition on intracellular Fluc expression has become a reliable method to evaluate efficiency of gene delivery system.34-36 A Fluc and green fluorescent protein (GFP) co-expressed WERI-Rb-1 cell line (Fluc/GFP-Rb-1) was established for evaluation of MVP-based polyplexes (ASSOCIATED CONTENT: Supporting Information Available. [Figure S12]). The polyplexes were incubated with Fluc/GFP-Rb-1 cells in complete medium containing 10% FBS to evaluate inhibition efficiency. As shown in Fig. 4A and 4B, Fluc levels in naked siRNA treated cells were similar to that of untreated cells. For Pe/siRNA, only at high concentration Fluc expression could be downregulated about 20%. However, Fluc levels in MVP/siRNA treated cells significantly decreased (p < 0.001) compared to those treated by naked siRNA. In 4VP/siRNA treated cells, Fluc levels decreased about 43% (200 nM) and 55% (400 nM), while in 8VP/siRNA treated cells, the inhibition efficiency was about 75% at both low and high concentrations. The inhibition efficiency of Lipo/siRNA was 75% at low concentration, similar to 8VP/siRNA, and about 95% at high concentration. As illustrated in the cytotoxicity experiment (Fig. 4C), Lipo/siRNA at both low and high concentrations would induce a toxic effect on the cells. In other words, most of decrease in Fluc levels caused by Lipo/siRNA was due to cytotoxicity of Lipofectamine, instead of gene silence effect induced by siRNA. On the contrary, biocompatible MVP/siRNA could downregulate Fluc levels efficiently, by promoting siRNA function in the cells.

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Figure 4 Inhibition efficiency of polyplexes on firefly luciferase (Fluc) expression in vitro. A, B, qualitative and semi-quantitative results of inhibition on Fluc expression of Fluc/GFP-Rb-1 cells by siRNA-contained polyplexes. Cells were treated with complete medium containing different polyplexes at a concentration of 200 nM or 400 nM (siRNA) for 4 h, following another 20 h incubation in complete medium at 37°C. C, cytotoxicity of the polyplexes to WERI-Rb-1 cells. Results were expressed as percent of the values obtained from untreated cells via a CCK-8 assay. The data are presented as mean ± standard deviation (SD) (n = 3 for A and B, n = 5 for C, nsp > 0.05, *p < 0.05, ***p < 0.001, compared to naked siRNA at the same concentration). Topical instilled polyplexes inhibited protein expression of intraocular tumor Retinoblastoma is the most common primary intraocular malignancy in children and needs timely management once confirmed.3 With progression of the disease, tumor cells would invade the whole eye, especially the retina and vitreous body. Therefore, it is a suitable intraocular disease model to evaluate the efficiency of posterior ocular gene delivery system. Fluc/GFP-Rb-1 cells were seeded in the subretinal position of Balb/c nude mice to establish an intraocular orthotopic tumor model as previously reported (Fig. 5A).37 Tumor-bearing mice were randomly grouped and received treatment by instilling of various formulations in the conjunctival sac according to schedule in Fig. 5B. Bioluminescence from the tumor was detected to reflect siRNA silence effects. Considering individual differences, the bioluminescence intensity of each subject on the day before treatment (D0) was assigned as the origin and the quantitative changes in bioluminescence intensity during subsequent days (self-compared to D0) were evaluated. According to the bioluminescence signals and growth curves (Fig. 5C, 5D), the eyes treated by 8VP/siRNA showed a much slower increase in tumor bioluminescence intensity than those treated by other formulations during treatment. On the D13, inhibition effects of 8VP/siRNA began to show significant advantages over Lipo/siRNA, while on the D15, bioluminescence intensity increase of 8VP/siRNA (7.5 folds, compared to D0) treated eyes was very significantly lower than those treated by Lipo/siRNA (25 folds, compared to D0). Moreover, ocular irritation of these polyplexes was also evaluated. There was no histological alteration in cornea after receiving treatment for 15 d (ASSOCIATED CONTENT: Supporting Information Available. [Figure S13]), indicating the polyplexes were safe to the cornea under the therapeutic regimen. Although with similar in vitro cellular uptake and gene silencing efficiency, 8VP/siRNA performed better in vivo than Lipo/siRNA, probably due to the advantages enabled by octopus-like 8VP with improved gene condensation and ocular permeability, which resulted in smaller particle size, proper positive charge, satisfying stability, and eventually better intraocular delivery efficiency.

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Figure 5 Inhibition efficiency of siRNA-contained polyplexes on firefly luciferase (Fluc) expression in orthotopic intraocular tumor. A, establishment and treatment of the mice model, and ocular appearance of tumor-bearing mice. B, time schedule of establishment, treatment and evaluation of tumor-bearing mice. C, time-dependent in vivo bioluminescence images of the eyes transplanted with orthotopic tumor. D, semi-quantitative inhibition efficiency of the polyplexes on Fluc expression of the tumor. Each mouse in various groups received the polyplexes equal to 1 μg siRNA every day during the treatment. The data are presented as mean ± standard deviation (SD) (n ≥ 3, nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, compared to Lipo/siRNA) Based on the systemic in vitro and in vivo evaluation, 8VP was proven as a promising vector for noninvasive intraocular delivery of both ASO and siRNA, better than the commercial gene transfection reagent both in safety and efficiency. Besides ASO (Mw 6 kD) and siRNA (Mw 14 kD), it seems reasonable to speculate that 8VP also has the potential to alter clinical administration route of the ophthalmological gene medicines with similar physicochemical property, for example aptamer pegaptanib whose Mw of nucleic acid part is about 10 kD, to a noninvasive mode. Moreover, 8VP could also be used for delivery of miRNA and plasmid DNA, which would further expand its application fields as a “universal” gene vector. Notably, the safety feature of 8VP makes it potentially useful in clinic. Therefore, this work provided a facile and efficient strategy for 10


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noninvasive intraocular gene therapy, and proposed that non-corneal pathway predominated the intraocular absorption of these polyplexes. Furthermore, some penetratin derivatives were also reported elsewhere, which exhibited ameliorative ocular permeability without changing the electropositivity of wild-type penetratin.23 To construct multivalent penetratin derivatives might afford more efficient vectors for noninvasive intraocular gene delivery.

Conclusion In the present work, safe and efficient intraocular delivery of ASO and siRNA was enabled by an octopus-like flexible multivalent penetratin, for example 8VP. Branched spatial structure and sufficient flexible cationic penetratin tentacles of 8VP significantly improved the capability of gene condensation and stability of formed polyplexes. Besides remarkable cellular uptake, 8VP-based polyplexes could efficiently permeate an in vitro BRB model with intact structure. We also demonstrated that these polyplexes could be absorbed into the eyes mainly through a non-corneal pathway, and topical instillation of siRNA-loaded polyplexes was efficient to inhibit protein expression in an intraocular retinoblastoma model. More importantly, 8VP exhibited obvious advantages over a commercial transfection reagent both in safety and efficiency. It could reasonably be expected that non-viral vectors based on the strategy of biocompatible multivalent peptides would promote development and translation of noninvasive gene delivery and intraocular gene therapy.

Supporting Information Experimental sections, additional results, and expanded discussions.

Acknowledgments We are grateful for the financial support from the National Natural Science Foundation of China (81573358 to G. W., 81673361 to C. Z. and 81690263 to W. L.).

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Reference 1. Ting, D. S. W.; Cheung, G. C. M.; Wong, T. Y. Clin. Exp. Ophthalmol. 2016, 44, 260-277. 2. Mitchell, P.; Liew, G.; Gopinath, B.; Wong, T. Y. Lancet 2018, 392, 1147-1159. 3. Dimaras, H.; Kimani, K.; Dimba, E. A. O.; Gronsdahl, P.; White, A.; Chan, H. S. L.; Gallie, B. L. Lancet 2012, 379, 1436-1446. 4. Weng, Y.; Liu, J.; Jin, S.; Guo, W.; Liang, X.; Hu, Z. Acta Pharm. Sin. B 2017, 7, 281-291. 5. Kourlas, H.; Schiller, D. S. Clin. Ther. 2006, 28, 36-44. 6. Sampat, K. M.; Garg, S. J. Curr. Opin. Ophthalmol. 2010, 21, 178-183. 7. Copolovici, D. M.; Langel, K.; Eriste, E.; Langel, Ü. ACS Nano 2014, 8, 1972-1994. 8. Pescina, S.; Ostacolo, C.; Gomez-Monterrey, I. M.; Sala, M.; Bertamino, A.; Sonvico, F.; Padula, C.; Santi, P.; Bianchera, A.; Nicoli, S. J. Controlled Release 2018, 284, 84-102. 9. Liu, C.; Tai, L.; Zhang, W.; Wei, G.; Pan, W.; Lu, W. Mol. Pharmaceutics 2014, 11, 12181227. 10. Solinís, M. Á.; Del Pozo-Rodríguez, A.; Apaolaza, P. S.; Rodríguez-Gascón, A. Eur. J. Pharm. Biopharm. 2015, 95, 331-342. 11. Garoon, R. B.; Stout, J. T. Curr. Opin. Ophthalmol. 2016, 27, 268-273. 12. Thakur, A.; Fitzpatrick, S.; Zaman, A.; Kugathasan, K.; Muirhead, B.; Hortelano, G.; Sheardown, H. J. Biol. Eng. 2012, 6, 7. 13. Cideciyan, A. V.; Jacobson, S. G.; Drack, A. V.; Ho, A. C.; Charng, J.; Garafalo, A. V.; Roman, A. J.; Sumaroka, A.; Han, I. C.; Hochstedler, M. D.; Pfeifer, W. L.; Sohn, E. H.; Taiel, M.; Schwartz, M. R.; Biasutto, P.; Wit, W. D.; Cheetham, M. E.; Adamson, P.; Rodman, D. M.; Platenburg, G.; Tome, M. D.; Balikova, I.; Nerinckx, F.; Zaeytijd, J. D.; Van Cauwenbergh, C.; Leroy, B. P.; Russell, S. R. Nat. Med. 2019, 25, 225-228. 14. Thomas, C. E.; Ehrhardt, A.; Kay, M. A. Nat. Rev. Genet. 2003, 4, 346-358. 15. Liu, C.; Jiang, K.; Tai, L.; Liu, Y.; Wei, G.; Lu, W.; Pan, W. ACS Appl. Mater. Interfaces 2016, 8, 19256-19267. 16. Tai, L.; Liu, C.; Jiang, K.; Chen, X.; Wei, G.; Lu, W.; Pan, W. Nanomedicine: Nanotechnol., Biol. Med. 2017, 13, 2091-2100. 17. Ruponen, M.; Urtti, A. Eur. J. Pharm. Biopharm. 2015, 96, 442-446. 18. Biswas, S.; Deshpande, P. P.; Navarro, G.; Dodwadkar, N. S.; Torchilin, V. P. Biomaterials 2013, 34, 1289-1301. 19. Islam, M. A.; Xu, Y.; Tao, W.; Ubellacker, J. M.; Lim, M.; Aum, D.; Lee, G. Y.; Zhou, K.; Zope, H.; Yu, M.; Cao, W.; Oswald, J. T.; Dinarvand, M.; Mahmoudi, M.; Langer, R.; Kantoff, P. W.; Farokhzad, O. C.; Zetter, B. R.; Shi, J. Nat. Biomed. Eng. 2018, 2, 850-864. 20. Xu, X.; Wu, J.; Liu, S.; Saw, P. E.; Tao, W.; Li, Y.; Krygsman, L.; Yegnasubramanian, S.; De Marzo, A. M.; Shi, J.; Bieberich, C. J.; Farokhzad, O. C. Small 2018, 14, 1802565. 21. Agrahari, V.; Mandal, A.; Agrahari, V.; Trinh, H. M.; Joseph, M.; Ray, A.; Hadji, H.; Mitra, R.; Pal, D.; Mitra, A. K. Drug Delivery Transl. Res. 2016, 6, 735-754. 22. Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. Adv. Drug Delivery Rev. 2016, 99, 2851. 23. Jiang, K.; Gao, X.; Shen, Q.; Zhan, C.; Zhang, Y.; Xie, C.; Wei, G.; Lu, W. Acta Biomater. 2017, 63, 123-134. 24. Ruberti, J. W.; Sinha Roy, A.; Roberts, C. J. Annu. Rev. Biomed. Eng. 2011, 13, 269-295. 25. Bisht, R.; Mandal, A.; Jaiswal, J. K.; Rupenthal, I. D. Wiley Interdiscip. Rev.: Nanomed. 12


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Nanobiotechnol. 2018, 10, e1473. Hughes, P.; Olejnik, O.; Changlin, J.; Wilson, C. Adv. Drug Delivery Rev. 2005, 57, 20102032. Hornof, M.; Toropainen, E.; Urtti, A. Eur. J. Pharm. Biopharm. 2005, 60, 207-225. Hamilton, R. D.; Leach, L. Methods Mol. Biol. 2011, 686, 401. Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Nat. Rev. Genet. 2014, 15, 541-555. Holm, T.; Bruchmann, J.; Scheynius, A.; Langel, U. Lett. Appl. Microbiol. 2012, 54, 39-44. Singh, T.; Murthy, A. S. N.; Yang, H.; Im, J. Drug Delivery 2018, 25, 1996-2006. Li, Y.; Tang, C.; Zhang, E.; Yang, L. Int. J. Pharm. 2016, 515, 20-29. Blanco, E.; Shen, H.; Ferrari, M. Nat. Biotechnol. 2015, 33, 941-951. Prescher, J. A.; Contag, C. H. Curr. Opin. Chem. Biol. 2010, 14, 80-89. Ball, R. L.; Hajj, K. A.; Vizelman, J.; Bajaj, P.; Whitehead, K. A. Nano Lett. 2018, 18, 38143822. Xu, X.; Wu, J.; Liu, Y.; Yu, M.; Zhao, L.; Zhu, X.; Bhasin, S.; Li, Q.; Ha, E.; Shi, J.; Farokhzad, O. C. Angew. Chem., Int. Ed. 2016, 55, 7091-7094. Lemaitre, S.; Poyer, F.; Marco, S.; Freneaux, P.; Doz, F.; Aerts, I.; Desjardins, L.; Cassoux, N.; Thomas, C. D. Invest. Ophthalmol. Visual Sci. 2017, 58, 3055-3064.

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Octopus-like flexible vector for noninvasive intraocular delivery of short

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