Combinatorial library of chalcogen-containing lipidoids for intracellular delivery of genome-editing proteins

Biomaterials xxx (2018) 1e11

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Combinatorial library of chalcogen-containing lipidoids for intracellular delivery of genome-editing proteins Yamin Li a, 1, Tao Yang a, b, 1, Yingjie Yu a, Nicola Shi a, Liu Yang a, Zachary Glass a, Justin Bolinger a, Isaac James Finkel a, Wenhan Li a, Qiaobing Xu a, * a

Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610065, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2018 Received in revised form 7 March 2018 Accepted 8 March 2018 Available online xxx

Protein based therapeutics with high specificities and low off-target effects are used for transient and accurate manipulation of cell functions. However, developing safe and efficient carriers for intracellular delivery of active therapeutic proteins is a long-standing challenge. Here we report a combinatorial library of chalcogen (O, S, Se) containing lipidoid nanoparticles (LNPs) as efficient nanocarriers for intracellular delivery of negatively supercharged Cre recombinase ((-30)GFP-Cre) and anionic Cas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) for genome editing. The structure-activity relationship between the lipidoids and intracellular protein delivery efficiencies was explored and it was demonstrated that the newly developed LNPs are effective for gene recombination in vivo. © 2018 Published by Elsevier Ltd.

Keywords: Lipidoids Protein delivery CRISPR/Cas9 Genome editing

1. Introduction Proteins as the workhorse biomacromolecules play important roles in the cell and life. Alteration, deficiency or malfunction of proteins within the cell often results in serious conditions [1]. Protein-based therapeutics with high tolerances and specificities, as well as low off-target effects have attracted tremendous attention during the last three decades, as either replacement therapy for protein deficiency or to elicit a therapeutic effect [2e4]. One example is the recently developed CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat associated protein 9) platform with high flexibility and specificity for genome editing through the gene deletion, insertion, activation, repression and even epigenetic modification, which could facilitate disease modeling and new treatments for various genetic disorders and infectious diseases [5e8]. However, like many other protein therapeutics, the development of safe and effective intracellular delivery systems for CRISPR/Cas9 and other genome-editing platforms is still a long-standing challenge, due to their relatively

* Corresponding author. E-mail address: [email protected] (Q. Xu). 1 These authors contributed equally.

large hydrodynamic sizes, varying surface physicochemical properties, and vulnerable higher order structures. [9,10]. So far, mechanical and physical delivery methods including microinjection, electroporation and hydrodynamic injection have been employed for CRISPR/Cas9 and other genome editing protein delivery [8,11]. These methods are straightforward and usually highly efficient, but are also invasive and suffer from many practical issues which limit in vivo application [12,13]. Biochemical modification of CRISPR protein with functional targeting ligands like nuclear localization signal (NLS) peptide [14] and supramolecular delivery systems such as lipid and lipid-like (lipidoid) nanoparticles (LNPs) [15e17], polymeric assemblies [18e20], as well as inorganic nanoparticles-based carriers [21e23] have been explored recently. Among these, LNPs are a category of promising nanocarrier candidates which have been demonstrated by us [16], [24e27] and others [28,29] for successful gene and protein delivery applications both in vitro and in vivo. Combinatorial library strategy has been extensively used to synthesize biomaterials for drug delivery applications [24,30]. Libraries of LNPs were synthesized through Michael addition reaction and ring opening reaction of epoxide with amines by combination of different amines head, linker compound and aliphatic chain tail groups [24,28,31e33]. In this study, we report a new library of cationic chalcogen-containing lipidoid nanoparticles

https://doi.org/10.1016/j.biomaterials.2018.03.011 0142-9612/© 2018 Published by Elsevier Ltd.

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for intracellular delivery of genome editing proteins. Due to the structure similarity of those molecules which only differ in one atom, we also elucidated the structure-activity relationship between lipidoids and intracellular protein delivery efficiencies. The chalcogen-containing lipidoids (R-O17X, Fig. 1) were synthesized by reacting lipophilic tails containing O, S and Se ethers (O17O, O17S and O17Se) with commercial available amines (10, 17, 63, etc.). Lipidoid nanoparticles were then fabricated and negatively supercharged Cre recombinase ((-30)GFP-Cre) [15] and anionic Cas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) [16] were loaded through the supramolecular interactions, mainly electrostatic interactions. HeLa-DsRed and GFP-HEK cells were utilized for evaluation of the intracellular protein delivery and genome editing efficiencies. The physicochemical properties of LNPs were examined and the structure-activity relationship was further explored by comparing the apparent pKa and lipid membrane disruption ability. Through the in vivo study using transgenic Cre reporter mice, we showed that these chalcogen-containing lipidoids have the potential for in vivo gene recombination applications.

2. Materials and methods 2.1. General All chemicals used for lipidoids synthesis were purchased from Sigma-Aldrich without further purification unless otherwise noted. (
30)GFP-Cre recombinase, S. pyogenes Cas9 (spCas9) and sgRNA were generated according to our previously reported procedures [16]. HeLa-DsRed and GFP-HEK cells were cultured in Dulbecco's modified eagle's medium (DMEM, Sigma-Aldrich) with 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). All 1H NMR spectra were recorded on a Bruker AVIII 500 MHz NMR spectrometer operated in the Fourier transform mode. Hydrodynamic size and polydispersity index of nanoparticles were measured by Zeta-PALS particle size analyzer (Brookhaven Instruments). The apparent pKa values of lipidoids

were determined as previously reported using 2-(p-toluidinynaphthalene-6-sulphonic acid) (TNS, Sigma-Aldrich) as fluorescent probe [34]. TEM measurements were performed on a FEI Technai Transmission Electron Microscope. Fluorescence images of tissue slices were obtained using BZ-X Analyzer fluorescence microscope.

2.2. Synthesis of O17O (Fig. S1) Sodium hydride (0.72 g, 30 mmol) was added to the solution of ethylene glycol (5.6 g, 90 mmol) in anhydrous DMF (30 mL) and stirred for 10 min at 0  C. 1-Bromotetradecane (6.0 g, 20 mmol) and KI (3.3 g, 20 mmol) were then added and the reaction mixture was kept at 95  C for another 4 h. After cooling to room temperature, the mixture was diluted with cold water, extracted with ethyl acetate, and dried over anhydrous sodium sulfate [35]. Compound 1 (3.3 g, yield ~ 65%) was obtained after column chromatography purification on silica gel using n-hexane/ethyl acetate as mobile phase. Then, compound 1 (3.3 g, 12.8 mmol) and triethylamine (TEA, 1.9 g, 19.2 mmol) were dissolved in anhydrous DCM (80 mL). Acryloyl chloride (1.4 g, 15.4 mmol) was added dropwise at 0  C, and the reaction mixture was stirred overnight. After column chromatography purification, O17O was obtained as colorless oil (3.2 g, yield ~ 82%).

2.3. Synthesis of O17S (Fig. S2) To a solution of 2-mercaptoethanol (1.1 g, 14 mmol) in acetonitrile (20 mL) was added 1-bromotetradecane (5.0 g, 18 mmol) and potassium carbonate (3.6 g, 26 mmol). The reaction solution was stirred overnight at 40  C, filtered and concentrated [36]. Compound 2 (1.8 g, yield ~ 48%) was obtained after column chromatography purification on silica gel using n-hexane/ethyl acetate as mobile phase. In a manner similar to that for the preparation of O17O, O17S was synthesized and purified as oil-like liquid (3.5 g, yield ~ 75%).

Fig. 1. (a) Encapsulation of negatively charged GFP-Cre and Cas9:sgRNA into synthetic cationic lipidoid nanoparticles (LNPs) for intracellular protein delivery and genome editing. (b) Synthetic route and lipidoids nomenclature. (c) Chemical structures of amine heads for lipidoids synthesis.

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2.4. Synthesis of O17Se (Fig. S3)

24 h after delivery.

Potassium selenocyanate (1.5 g, 10 mmol) was added in portion to a solution of 2-bromoethanol (1.6 g, 13 mmol) in acetone (50 mL) at room temperature. The solution was heated to reflux for 2 h. After cooling to room temperature, the white precipitate was filtering off and acetone was removed by rotary evaporation under vacuum. Compound 3 was then dissolved in ethanol (25 mL) and sodium borohydride (0.9 g, 24 mmol) was added slowly at 0  C. After the reaction solution turned to colorless, 1-bromotetradecane (4.1 g, 15 mmol) was added through a dropping funnel. The reaction was stopped by adding DI water (10 mL) after 30 min. Then the ethanol was removed under reduced pressure, reaction mixture was diluted with saturated sodium chloride aqueous solution (50 mL), and extracted with DCM (3  50 mL) [37,38]. Compound 5 (1.5 g, yield ~ 46%) was obtained after column chromatography purification on silica gel using n-hexane/ethyl acetate as elute. In a manner similar to that for the preparation of O17O and O17S, O17Se was obtained as oil-like liquid (2.7 g, yield ~ 72%).

2.9. Intracellular delivery of Cas9:sgRNA/LNP

2.5. Synthesis of lipidoids Head amines (Sigma-Aldrich) were mixed with acrylates tails (O17O, O17S, O17Se) at 1/2.4 M ratio in Teflon-lined glass screw-top vials for 48 h at 70  C. The crude products were purified using a Teledyne Isco Chromatography system [16,32].

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For CRISPR/Cas9 gene knockout study, GFP-HEK cells were seeded in 48-well plate with a density of 2  104 cell/well. After 24 h of incubation, Cas9:sgRNA/LNP nanoparticles were added to the cells and incubated for 4 h, followed by media changed. After 48 h of incubation, the green emission from GFP was analyzed by flow cytometry. The final Cas9:sgRNA RNP concentration is 25 nM, and lipidoid concentration is 3.3 mg/L. 2.10. In vitro cytotoxicity assay Cell viability was measured by the standard MTT assay. HeLaDsRed or GFP-HEK cells were seeded into 96-well plate with a density of 5  103 cell/well. (
30)GFP-Cre/LNP or Cas9:sgRNA/LNP nanoparticles were added after 24 h of incubation. The final concentration of protein is 25 nM and LNP is 3.3 mg/L. After incubating for 24 h or 48 h, the MTT reagent (5 mg/mL, in 30 mL PBS buffer) was added and the cells were incubated for another 4 h at 37  C. The cell culture media were then carefully removed and 200 mL of DMSO were added. The DMSO solution was transferred into another 96well plate and the absorbance at 570 nm was recorded by microplate reader. All experiments were performed in quadruplicate. 2.11. In vivo protein delivery to Ai14 mouse

2.6. Fabrication of lipidoid nanoparticles and protein/LNP nanocomplexes Lipidoids were fabricated into nanoparticles for all delivery application. Briefly, lipidoids were mix with sodium acetate buffer (25 mM, pH 5.2), sonicated for 30 min in ultrasonic bath and followed by another 30 min of vigorous vortex. The as-prepared LNPs were stored at 4  C. For protein/LNP complexation, lipidoids nanoparticles were mixed with (
30)GFP-Cre or Cas9:sgRNA in PBS buffer (25 mM, pH 7.4) following our previously reported procedures [16] and incubated at room temperature for 30 min.

Formulated LNPs (lipidoid/Cholesterol/DOPE/DSPE-PEG2k ¼ 16/ 4/1/4, weight ratio) were prepared for protein loading and mice injection. Ai14 mice were housed in a temperature and humidity controlled facility with a 12 h light/dark cycle. Two mice in each group were injected with (
30)GFP-Cre/LNPs formulations on day 0 and 5, with 100 mg protein for each injection. Organs including heart, liver, spleen, lung and kidney from all groups were collected 20 days after injections. The tissues were fixed overnight in 4% paraformaldehyde (PFA) before being sectioning into 10 mm slices. The slices were collected and stained with DAPI for fluorescence imaging.

2.7. Phospholipid bilayer membrane disruption ability test 3. Results and discussion Human red blood cells (hRBCs) were washed with PBS buffer three times and collected after centrifugation at 1000 rpm for 5 min. The resulting stock solution (~10% v/v hRBCs) was diluted 3 fold in PBS buffer to give the assay solution. 90 mL of assay solution was mixed with 10 mL of LNPs solutions (final concentration of lipidoids ¼ 3.3 mg/L) and incubated at 37  C for 60 min. Then the samples were centrifuged again at 1000 rpm for 10 min 10 mL of the supernatant was further diluted into 90 mL of PBS buffer, and the absorbance at 405 nm (OD405) was recorded using a microplate reader. The PBS buffer and Triton X-100 (1% v/v) were used as negative and positive controls respectively [39]. 2.8. Intracellular delivery of (
30)GFP-Cre/LNP For the intracellular uptake study, HeLa-DsRed cells were seeded in 48-well plate with a density of 2  104 cell/well. After 24 h of incubation at 37  C, 5% CO2, (
30)GFP-Cre/LNP nanoparticles were added to the cells and incubated for 6 h before fluorescence microscopy and flow cytometry (BD FACS Calibur, BD Science, CA) analysis (green emission from GFP). The final (
30) GFP-Cre protein concentration is 25 nM, and lipidoid concentration is 3.3 mg/L. For the gene recombination functional study, HeLaDsRed cells were treated with same conditions and the red fluorescence emission from DsRed was analyzed by flow cytometry

3.1. Lipidoids synthesis O, S, Se ethers containing lipophilic acrylates tails, O17O, O17S and O17Se were synthesized at first and their structures were characterized by 1H NMR spectra (see Experimental Section and Figs. S1-S3). Then commercial available amine heads (10, 17, 63, etc., Fig. 1) were mixed with acrylates stoichiometrically and reacted at 70  C for 48 h [16,32]. Lipidoids were purified by Teledyne Isco Chromatography system, characterized by 1H NMR and ESI-MS (Fig. S4), and named as amine number (R) and O17X (R-O17X, X ¼ O, S and Se) as shown in Fig. 1. The typical 1H NMR and ESI-MS spectra of 76-O17O, 76-O-17S and 76-O17Se are shown in Fig. 2a and b. 3.2. Lipidoid nanoparticles fabrication and characterization The lipidoids nanoparticles were fabricated through simple ultrasonication and vortex procedures (see Experimental Section) in sodium acetate buffer (25 mM, pH 5.2). Hydrodynamic sizes and polydispersity index (PDI) of LNPs were measured by dynamic laser scattering (DLS) analysis (Figs. S5-S6). As shown in Fig. 2c, most of the O, S and Se ethers containing LNPs have the averaged hydrodynamic diameter () between 100 and 300 nm, and the PDI in

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Fig. 2. (a) 1H NMR and (b) ESI-MS spectra of 76-O17O, 76-O17S and 76-O17Se. (c) Statistical analysis of averaged hydrodynamic diameter () distribution of LNPs. (d) Typical hydrodynamic diameter distributions of 76-O17O, 76-O17S and 76-O17Se LNPs.

the range 0.1e0.3 (Fig. S6), which are comparable with our previously reported synthetic lipidoid nanoparticles sizes and expected to be suitable for intracellular protein delivery application [16,32]. After further analysis (Fig. 2c; the values shown above the bars represent the percentages of LNPs with O17O, O17S and O17Se tails located in certain range of diameter out of all 51 LNPs), it was found that ~53% of LNPs with O17O tails, ~82% of O17S LNPs and ~65% of O17Se LNPs located in the smaller size region (<200 nm), which is probably resulted from the effect of incorporated chalcogen atoms on the supramolecular self-assembly behaviors in aqueous solutions. Typical size distribution profiles of 76-O17O ( ¼ 170.1 nm, m2/G [2] ¼ 0.37), 76-O17S ( ¼ 114.3 nm, m2/ G [2] ¼ 0.24) and 76-O17Se ( ¼ 129.4 nm, m2/G [2] ¼ 0.18) LNPs are shown in Fig. 2d. The morphologies of LNPs were further studied by the transmission electron microscopy (TEM). As shown in Fig. 3a, spherical particles were observed in the images of 76-O17O, 76-O17S and 76O17Se LNPs, and the measured number-averaged sizes (145 nm, 94 nm, 133 nm for 76-O17O, 76-O17S and 76-O17Se, respectively) are comparable with the hydrodynamic diameters as determined by DLS (Fig. 2d and S5). The morphologies of other LNPs including 80-O17O, 80-O17S and 80-O17Se, were also examined and the TEM images are shown in Fig. S7, in which spherical particles were observed as well. Then the stability of as-prepared LNPs was examined by DLS and fluorescence measurements. As shown in Fig. 3b, the time-dependent DLS measurements revealed that no evident aggregation of the 76-O17O, 76-O17S and 76-O17Se LNPs

occurred during five days of storage under room temperature, with the relative size change < ±15%. Fluorescence resonance energy transfer (FRET) pair, DiO and DiI, loaded 76-O17Se LNPs also showed negligible FRET ratio (I575/(I575þI505)) variations in five days of storage (Fig. 3c), which indicated the structure integrity and long-term storage stability of the LNPs [40,41]. 3.3. In vitro screening of LNPs for protein delivery A Cre recombinase protein fused to a negatively supercharged GFP variant ((-30)GFP-Cre), which has been shown to complex with cationic LNPs through electrostatic attraction and other types supramolecular interactions, was used as a model cargo protein. The cellular uptake of LNPs could be determined by direct analysis of intracellular GFP fluorescent intensity [16]. HeLa-DsRed cells were used in this study, which can also express red fluorescent DsRed upon Cre-mediated recombination to facilitate the functional study of delivered proteins in the following section. The (
30)GFP-Cre protein loaded LNPs (GFP-Cre/LNPs) were prepared at first by simply mixing precalculated amount of aqueous solution of LNPs and protein at ambient conditions. The GFP-Cre protein loading efficiencies of 76-O17O, 76-O17S and 76-O17Se were determined as 85.6%, 95.1% and 97.0%, respectively (Fig. S8). The hydrodynamic diameters, size distribution, zeta-potential of GFP-Cre/LNPs are shown in Figs. S9 and S10. GFP-Cre/76-O17Se showed increased hydrodynamic size (~253.6 nm) and reduced zeta-potential value (~-19.4 mV) comparing to blank 76-O17Se

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Fig. 3. (a) Typical TEM images and (b) relative size variations of 76-O17O, 76-O17S and 76-O17Se LNPs. Scale bar ¼ 100 nm. (c) Fluorescent emission intensities and FRET ratios of DiO/DiI loaded 76-O17Se LNPs during storage.

nanoparticles ( 170.1 nm, zeta-potential 23.4 mV), which are consistent with our previous study. Furthermore, the stability of GFP-Cre/LNPs complexes was determined by time-dependent DLS measurements and results are shown in Fig. S10. All the samples tested (GFP-Cre/76-O17O, GFP-Cre/76-O17S and GFP-Cre/76O17Se) showed <±20% of relative size changes in 5 days incubation which indicate their good stability under storage. For the intracellular delivery, after incubation with GFP-Cre/LNPs nanoparticles for 6 h, the GFP positive cells were observed using fluorescence microscopy, harvested and counted by flow cytometry. As shown in Fig. 4a, comparing with the control group, which is untreated HeLa-DsRed cell, bright green fluorescence emission could be detected from the GFP-Cre/Lpf2k (Lpf2k, commercial transfection agent, Lipofectamine 2000), GFP-Cre/76-O17O, GFP-Cre/76O17S and GFP-Cre/76-O17Se treated cells. The naked protein, (
30) GFP-Cre, treated cells, however, showed negligible fluorescence emission comparing to lipid-facilitated delivery systems, which indicated that the naked (
30)GFP-Cre protein cannot efficiently enter into the HeLa-DsRed cells. The intracellular (
30)GFP-Cre protein delivery efficiencies were further quantified by flow cytometry. As shown in Fig. 4b and S11, both of the naked (
30) GFP-Cre protein and control group showed low portions of GFP positive cells, which is consistent with the results of fluorescence microscopy observation (Fig. 4a) and our previously reported results [16]. However, in the presence of LNPs, the proportions of GFP positive cells are increased, located in the range of 4e42% and most of them are around 12e18%. Some of the delivery efficiencies of LNPs were even comparable with that of Lpf2k (~31% of GFP positive cells). For instance, the proportions of GFP positive cells treated with (
30)GFP-Cre protein loaded 400-O17Se, 80-O17Se and 77O17Se LNPs were 42%, 39% and 37%, respectively (Fig. S11). Furthermore, the internalization mechanisms of these protein/LNPs complexes were studied through pretreatment of HeLa-DsRed cells

with endocytosis inhibitors, including sucrose (a clathrin-mediated endocytosis inhibitor), methyl-b-cyclodextrin (M-b-CD, a cholesterol-depleting agent), dynasore (dynamin II inhibitor) and nystatin (a caveolin-mediated endocytosis inhibitor). As shown in Fig. S12, the internalization efficiencies of all three types of protein/ LNPs (GFP-Cre/76-O17O, GFP-Cre/76-O17S, GFP-Cre/76-O17Se) are significantly suppressed by sucrose and M-b-CD, and treating cells with dynasore could also decrease the intracellular delivery efficacy of GFP-Cre/76-O17Se nanoparticles. Overall, these results indicate that clathrin, plasma cholesterol and dynamin play important roles in the cellular uptake of these GFP-Cre protein complexed chalcogen-containing LNPs.

3.4. Investigation of structure-activity relationship Although the library of O, S and Se ether-containing lipidoids is relatively small with 51 lipidoids studied, we investigated the structure-activity relationship between LNPs and intracellular protein delivery efficacies. Lipidoids with >20% GFP positive cells treated with (
30)GFP-Cre protein/LNP nanoparticles were defined as efficacious LNPs (red data points in Fig. 4b) comparing with the bulk LNPs (black data points) at first. The lipidoids library was then categorized into three groups according to their hydrophobic tail structures (O17O, O17S and O17Se); each tail made up 33.3% of the library. In the efficacious LNPs group, 21.4%, 28.6% and 50% of lipidoids are with O17O, O17S and O17Se tails respectively. Therefore, the relative hit rates of LNPs with O17O, O17S and O17Se tails are
11.9%,
4.7% and 16.7%, respectively, relative to the initial library (Fig. 4c). In other words, lipidoids with O17O and O17S tails were significantly underrepresented among LNPs with delivery efficacy >20%, while lipidoids with O17Se tail was overrepresented, suggesting that O17Se tails are associated with efficacious LNPs.

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Fig. 4. (a) Typical images of (
30)GFP-Cre protein and (
30)GFP-Cre loaded 76-O17O, 76-O17S and 76-O17Se LNPs treated HeLa-DsRed cells. Scale bar ¼ 200 mm. (b) Percentage of GFP positive cells shown for 51 LNPs tested. Data points marked in red for LNPs induced high level of transfection. (c) The tails (O17O, O17S and O17Se) influenced (
30)GFP-Cre protein transfection activity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

According to previous studies, the delivery efficiencies of LNPs are also related to the chemical structures of amine heads, hydrophobic tails, substitution numbers and apparent pKa values, etc. [29,33,42,43] In this study, for further elucidate the structureactivity relationship of O, S, Se ethers containing lipidoids, effects of apparent pKa value and phospholipids bilayer membrane disruption ability of the LNPs were further analyzed. Apparent pKa values were measured following the previously reported

procedures using 2-(p-toluidinyl)naphthalene-6-sulphonic acid (TNS) as fluorescent probe (Fig. S13) [29,34]. The phospholipids bilayer membrane disruption ability of LNPs was evaluated using human red blood cells (hRBCs) as model and hemoglobin as the chromophore reporter agent [39,44,45]. Absorbance at 405 nm (OD405) was recorded to assess the amount of released hemoglobin, using PBS buffer and Triton X-100 (1% v/v) as negative and positive controls, respectively (Fig. S14), in which higher OD405

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Fig. 5. (a) Apparent pKa values and (b) phospholipid bilayer membrane disruption ability influenced (
30)GFP-Cre protein delivery efficiency. Relative hit rates of efficacious LNPs of LNPs with (c) two, one or none properties possessed and (d) O17O, O17S, O17Se tails.

values indicate stronger membrane disruption capabilities. As shown in Fig. 5a and b, the apparent pKa and OD405 values of LNPs were plotted against the percentages of GFP positive cells for each LNP, and it was found that (gated with blue dash lines in Fig. 5a and b) most of the efficacious nanoparticles (with GFP positive cells > 20%) were located in the regions of pKa >5.1 (85.7% of total amount of efficacious nanoparticles) and OD405 > 0.2 (78.6% of total amount of efficacious nanoparticles). After further examination, it was found that these two properties have striking effects on (
30)GFP-Cre protein transfection efficiencies in HeLa-DsRed cells. As shown in Fig. 5c, when LNPs possess both of properties (i.e., pKa >5.1 and OD405 > 0.2), the relative hit rate to be able to mediate high transfection efficiency was 77%. When one or two of the properties was removed from the LNPs, the likelihood of achieving high transfection efficiency of (
30)GFP-Cre protein into HeLaDsRed cells dropped significantly to 8e33%. Furthermore, as to the structure-activity relationship, it was found that, for LNPs with O17O, O17S and O17Se tails, the relative hit rates of above mentioned efficacy criteria were (pKa >5.1/OD405 > 0.2)
1.9%/14.6%, 0.99%/4.2% and 0.99%/10.5%, respectively (Fig. 5d). It was clear that both of the two properties were underrepresented in the group of LNPs with O17O tails, which is consistent with the results shown in Fig. 4c, in which O17O tail was underrepresented in the efficacious lipidoid. While both properties of high pKa and OD405 values were overrepresented in the group of LNPs with O17S and O17Se tails. It was presumed that the surface charge property determined by apparent pKa values as well as membrane

disruption ability of LNPs, which is considered to be related to the facilitated endosomal escape process are critical factors for achieving highly efficient intracellular delivery of bioactive macromolecules, which has been demonstrated by our group and others [16,29]. Furthermore, according to the results shown in Fig. 5c and d, comparing with the apparent pKa values, the membrane disruption ability of these LNPs appears to be the more influential factor in determining in vitro (
30)GFP-Cre protein delivery efficiency into HeLa-DsRed cells. By analyzing LNPs with O17Se tails, we found that amine head molecules containing 1e2 nitrogen atoms, and at least one tertiary amine group, with less than 3 tail groups are most likely to be efficacious for LNPs for GFPCre delivery (Fig. S15). 3.5. (-30)GFP-Cre protein delivery for gene recombination and cytotoxicity assay The top 12 of LNPs identified through intracellular delivery screening experiments (Fig. S11) were further tested for gene recombination using HeLa-DsRed model cells. The expression of DsRed from Cre protein mediated gene recombination was analyzed after 24 h of co-incubation with free (
30)GFP-Cre protein and protein loaded LNPs. As shown in Fig. 6a, naked (
30)GFPCre protein could not induce DsRed expression, due to its low internalization ability, which is consistent with the results of fluorescence microscopy observation and flow cytometry analysis (Fig. 4a and S11) [16]. Most of the tested LNPs, on the other hand,

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Fig. 6. (a) DsRed expression and (b) cell viability of HeLa-DsRed cells treated with (
30)GFP-Cre and (
30)GFP-Cre loaded LNPs.

could efficiently deliver (
30)GFP-Cre protein and induce gene recombination, with 14e46% of the cells positive for DsRed. Specifically, some of the top LNPs (76-O17S (40.8%), 76-O17Se (36.1%), 77-O17S (38.6%), 77-O17Se (31.0%), 78-O17Se (37.8%) and 80-O17S (45.6%)) were identified with comparable or even higher transfection efficiencies when compared with Lpf2k (33.5%). Then, the MTT assay against HeLa-DsRed cells was conducted. As shown in Fig. 6b and 76-O17S, 76-O17Se, 77-O17S, and 77-O17Se LNPs showed low cytotoxicity, as >80% cells were alive, which indicated good biocompatibility as delivery platforms for bioactive molecules [46e50], when compared to Lpf2k, 400-O17Se 78-O17Se, 80-O17S and 80-O17Se, of which the cell viability was 67e77%. Above all, 76O17S, 76-O17Se, 77-O17S, and 77-O17Se were identified with high intracellular protein delivery and Cre-mediated genome recombination efficacies, while showed lower cytotoxicity than Lpf2k.

3.6. In vivo GFP-Cre delivery for gene recombination in Ai14 mouse Delivering genome editing proteins in vivo has the therapeutic potential for a wide range of genetic diseases. Driven by the in vitro screening results, the potency of using newly developed O, S, and Se ethers containing LNPs to deliver (
30)GFP-Cre protein in vivo for Cre-mediated gene recombination was further explored. Ai14 mouse model was used for this purpose, which has a genetically

integrated loxP-flanked STOP cassette that prevents the transcription of red fluorescent protein, tdTomato. Upon Cre mediated gene recombination, the STOP cassette is removed, resulting in tdTomato expression [51]. Considering the different performances of cargo loaded LNPs in vitro and in vivo, three LNPs with same amine heads and different tails (76-O17O, 76-O17S and 76-O17Se) were tested in this study. For in vivo study, formulated LNPs (lipidoid/Cholesterol/ DOPE/DSPE-PEG2k ¼ 16/4/1/4, weight ratio) were prepared. The hydrodynamic size, polydispersity and zeta-potential of formulated 76-O17O, 76-O17S and 76-O17Se were determined at first and the results are shown in Fig. S16. Further study revealed that the formulated nanoparticles with comparable intracellular delivery efficiencies (Fig. S17) and neglectable hemolysis capabilities (Fig. S18) are much more stable than their unformulated counterparts in the presence of serum proteins (Fig. S19). Furthermore, unformulated 76-O17S and 76-O17Se LNPs showed much higher stability against serum proteins comparing to 76-O17O LNPs, which may also related to their intracellular delivery performances. Then, Ai14 mice were injected (tail vein injection) with (
30) GFP-Cre loaded formulated LNPs (GFP-Cre/76-O17O, GFP-Cre/76O17S and GFP-Cre/76-O17Se) at Day 0 and Day 5 (100 mg of protein for each injection). Organs including heart, liver, spleen, lung and kidney were collected at Day 20 and analyzed for the tdTomato expression. As shown in Fig. 7 and Fig. S20, under the same preparation and imaging conditions, strong tdTomato signals were observed in the sections of lung from GFP-Cre/76-O17S and GFPCre/76-O17Se injected mice. Fluorescence images with lower magnification and larger field of view are shown in Fig. S21, from which it is clear that comparing with control and GFP/76-O17O injected group, the GFP/76-O17S and GFP/76-O17Se injection could induce Cre-mediated genome recombination efficiently in the lung. The mechanism for lung targeted delivery remains unclear and is currently under investigation. Furthermore, from the perspective of chemistry, both the in vitro screening results and the in vivo tests showed that lipidoids with same amine heads and different hydrophobic tails could possess very different physicochemical properties, intracellular delivery efficacies, and genome recombination profiles, which further emphasized the important role that the chemistry played in the designing of effective and safe delivery vehicles for therapeutic reagents. Additionally, from a therapeutic perspective, the in vivo delivery of CRISPR/Cas9 system would be more relevant than Crelox system. Therefore, the possibility of using these LNPs to deliver CRISPR/Cas9 RNP for genome editing was further explored. 3.7. Cas9:sgRNA RNP delivery for genome modification The Cas9:sgRNA RNP targeting genomic GFP reporter gene and GFP-HEK cells were used for this purpose. The zeta-potential (Cas9:sgRNA/76-O17Se,
13.2 mV), hydrodynamic diameter (Cas9:sgRNA/76-O17Se, 314.7 nm), size distribution and stability of Cas9:sgRNA loaded LNPs (Cas9:sgRNA/76-O17O, Cas9:sgRNA/76O17S and Cas9:sgRNA/76-O17Se) were determined by DLS and the results are shown in Figs. S9 and S10. The sizes of 76-O17O, 76O17S and 76-O17Se LNPs increased to 274e325 nm after complexing with Cas9:sgRNA and the Cas9:sgRNA/LNPs showed relative good stability during 5 days storage with <±20% relative size variations observed. The morphologies of Cas9:sgRNA loaded LNPs were examined by TEM, and typical image of Cas9:sgRNA loaded 76-O17Se LNP (Cas9:sgRNA/76-O17Se) was shown in Fig. S22. For the intracellular delivery, GFP-HEK cells were treated with Cas9:sgRNA/LNPs nanocomplexes for 48 h, harvested and the GFP gene knockout efficacy was further analyzed by flow cytometry. As shown in Fig. 8a, naked Cas9:sgRNA RNP could not induce GFP gene knockout, while the knockout efficiency of Cas9:sgRNA/Lpf2k is

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Fig. 7. Typical fluorescence images of sections of lungs obtained from PBS and GFP-Cre loaded formulated LNPs injected Ai14 mice. Blue channel: DAPI; red channel: tdTomato. Scale bar ¼ 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

relatively high, with 63% of GFP negative cells, which are consistent with previously reported results [16]. When using O, S, and Se ether-containing LNPs as the delivery vehicles, the GFP-HEK cells showed 14%e58% GFP expression loss. In particular, 50.2%, 57.7%, 54.7% and 57.4% of GFP knockout were observed when cells were treated with Cas9:sgRNA loaded 76-O17Se, 80-O17Se, 81-O17Se and 400-O17Se LNPs. Based on the results of gene recombination of Cre protein in HeLa-DsRed cells and GFP gene knockout of Cas9:sgRNA RNP delivery in GFP-HEK cells, it was safe to conclude that our lipidoids could efficiently deliver genome editing proteins into different mammalian cell lines in vitro, with some of the LNPs' efficacies comparable with commercial available transfection reagent Lpf2k. Other than efficiency, cytotoxicity and biocompatibility issues should also be considered [52e55]. Therefore, the in vitro cytotoxicity of Cas9:sgRNA/LNPs against GFP-HEK cells was also evaluated by the MTT assay. As shown in Fig. 8b, the cell viabilities were determined as 67%e119% after incubation with Cas9:sgRNA/LNPs at 37  C for 48 h, revealing that the some of the LNPs are almost non-

cytotoxic to GFP-HEK cells, while some of them are as toxic as Lpf2k (cell viability ~66%) under the same experimental conditions. After further analysis, it was found that, two of the top LNPs with high Cas9:sgRNA delivery efficiencies, i.e. 80-O17Se and 400-O17Se, possessed comparable cytotoxicity with Lpf2k (68.2% and 66.7% of cell viability, respectively), which is also consistent with cytotoxicity results against HeLa-DsRed cells as shown in Fig. 6b. However, 76-O17Se and 81-O17Se LNPs showed both high Cas9:sgRNA transfection efficiency (50.2% and 54.7%) and low cytotoxicity (76.3% and 97.7% of cell viability after 48 h of incubation) profiles, which were preferred as effective and safe candidates for in vivo tests. Considering the different physicochemical properties of GFP-Cre and CRISPR/Cas9 RNP loaded LNPs, both of the biodistribution and genome editing efficiencies at specific organs and tissues would not be necessarily identical. Therefore, fabrication and optimization of CRISPR/Cas9 RNP loaded LNP formulations tailored for in vivo genome editing at specific organs are currently undergoing in our group for further applications.

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Y. Li et al. / Biomaterials xxx (2018) 1e11

Conflicts of interest The authors declare no competing financial interest. Acknowledgments This work was supported by National Science Foundation, China Grant DMR 1452122. We thank Drs David Kaplan and Lorenzo Tozzi for providing materials. T. Yang is sponsored by the China Scholarship Council (CSC). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.biomaterials.2018.03.011. References

Fig. 8. (a) GFP knockout and (d) cell viability of GFP-HEK cells treated with Cas9:sgRNA and Cas9:sgRNA/LNPs.

4. Conclusion In summary, we report here the synthesis of a O, S, and Se ethercontaining lipidoids library and the evaluation of these lipidoids for in vitro (
30)GFP-Cre and Cas9:sgRNA genome editing protein delivery. The initial intracellular delivery screening using GFP reporter indicated that lipidoids with O17Se tails are more likely to form efficacious LNPs. Furthermore, the physicochemical properties of LNPs including hydrodynamic size, morphology, apparent pKa values and phospholipids bilayer membrane disruption abilities were studied and it was concluded that the membrane disruption ability appears to be the influential factor in determining (
30)GFP-Cre protein delivery efficiency into HeLa-DsRed cells. Cre protein mediated gene recombination and Cas9:sgRNA induced gene knockout with proteins loaded LNPs were examined and lipidoids candidates with high delivery efficiency and low cytotoxicity were identified. Further in vivo study indicated the potential of newly synthesized LNPs for in vivo gene recombination. More studies regarding the fabrication, characterization and optimization of CRISPR/Cas9 RNP loaded LNP formulations are undergoing in our group for further in vivo application.

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