A Small Combinatorial Library of Lipidoids as Nano-vectors for Gene

Jul 13, 2018 - ... Library of Lipidoids as Nano-vectors for Gene Delivery ... Quantitative transfection study with luciferase reporter gene showed tha...
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A Small Combinatorial Library of Lipidoids as Nano-vectors for Gene Delivery Yi-Mei Zhang, Zheng Huang, Xiao-Ru Wu, Ji Zhang, Yan-Hong Liu, and Xiaoqi Yu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00688 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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A Small Combinatorial Library of Lipidoids as Nano-vectors for Gene Delivery Yi-Mei Zhang, Zheng Huang, Xiao-Ru Wu, Ji Zhang*, Yan-Hong Liu and Xiao-Qi Yu* Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, PR China *Corresponding

authors:

[email protected]

(J.

Zhang);

[email protected]

(X.-Q.

Yu);

Fax:

+86-28-85415886

Abstract: A small library of 27 lipidoids was set up by combinatorial approach. These lipidoids were prepared from three polyamines and nine reactants with alkyl tail via epoxide ring-opening

reaction

or

Michael

addition.

The

polyamines

include

TAEA

(tris(2-aminoethyl)amine), TACN (1, 4, 7-triazacyclononane) and Cyclen (1, 4, 7, 10-tetraazacyclododecane), while the structures of nine reactants vary in both reacting groups and chain lengths. The lipidoids were obtained without any solvent and used directly without further purification, and no helper lipid was need for the interaction with DNA. Initial screening by EGFP transfection assay selected six lipidoid nanoparticles with good transfection efficiency for further investigation. Gel electrophoresis, TEM and relative studies reveal that these lipidoids have good DNA condensation capability and the formed DNA complexes have good stability toward serum or nuclease. Quantitative transfection study with luciferase reporter gene showed that TACN-O14 could give 1.8 times higher efficiency than Lipofectamine 2000 in A549 cells, while TAEA-A12 may give 4.2 times higher efficiency in 7402 cells. Moreover, serum would not inhibit their transfection, and even lightly increased efficiencies could be obtained with serum. Mechanism studies including flow cytometry and CLSM revealed that the higher transfection efficiency of the lipidoid might be attributed to the more rapid and efficient endosome/lysosome escape. Keywords: Lipidoids; Gene delivery; Polyamines; Epoxide ring-opening reaction; Michael addition

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INTRODUCTION Gene therapy has great promise for the treatment of many congenital hereditary and acquired diseases. Generally, safe and efficient gene vectors, including viral and non-viral ones,1 are significant for successful gene therapy.2 For decades, to achieve effective delivery of DNA, a number of synthetic materials such as cationic lipids,3-6 polymers,7-8 dendrimers,9-11 inorganic materials,12 and so on13-15 have been developed as potential non-viral vectors. Among these vectors types, cationic lipids have been favored for many advantages, such as low cytotoxicity, good repeatability, easy characterization of structure and high cellular uptake by cell membrane fusion. Since cationic lipid DOTMA was first designed by Felgner et al for gene delivery in 1987.16 Scientists have put a lot of efforts in the development of novel cationic lipids with high efficiency and biocompatibility. In recent years, many new types of cationic lipids such as phospholipids,17-18 gemini lipids,19-20 and bola-type amphiphilic lipids21 have emerged as efficient gene vectors with good biocompatibility. However, many of these lipids have complicated chemical structures, and their preparations usually comprise multiple steps and time-consuming post-process for further separation and purification.3, 22 To overcome such problems, cationic lipidoids were developed and first reported by Akinc et al in 2008.23 Lipidoids are lipid-like materials, but they are different from the conventional cationic lipids in both structure and preparation. First, lipidoids have more than two hydrophobic tails attaching to the polyamine core. Second, lipidoids can be used directly after being dissolved in solution rather than the pre-formation of liposome, which was usually formed by thin film hydration method. Last but not least, numerous lipidoids can be parallel generated by one-step synthesis strategy and the result lipidoids can be used without purification. The synthesis of lipidoids generally based upon several fundamental chemical reactions, such as Michael addition,23-27 ring-opening reaction of epoxide,28-29 click reaction30-32 or alkylation of amine.33-34 Akinc et al carried out Michael addition chemistry to rapidly synthetize over 1,200 lipidoids, in which the lead lipidoid showed excellent ability to carry siRNA or anti-miRNA in both a local and systemic delivery model.23 Love and co-workers also created 126 lipidoids within 3 days by ring-opening reaction of epoxide. Through screening procedure, one lipidoid could exceed previously reported siRNA delivery

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vehicles in nonhuman primates.28 Most of these reported lipidoids achieved good performance by the assistance of several helper lipids (such as DOPE,35 mPEG2000-DMG,23,

36

cholesterol,28-29, 36 DSPC29 etc.). We recently developed several types of cationic lipids and polymers based upon polyamine structures, such as TAEA (triaminoethylamine), TACN (1, 4, 7-triazacyclononane) and cyclen (1, 4, 7, 10-tetraazacyclododecane). These materials could give strong DNA condensation ability, good cellular uptake and higher transfection efficiency than commercial transfection reagents when serving as non-viral gene vectors.19, 37-41 However, their preparation comprised multiple steps and it took times for their synthesis and purification. Herein, to combine our previous studies with the advantage of lipidoids, we prepared a small library of 27 lipidoids through epoxide ring-opening reaction or Michael addition. Three polyamines and nine reactants with alkyl tail were used for the lipidoids construction. These lipidoids were used without further separation and purification, and no helper lipid was applied. Through initial screening by EGFP transfection assay, six lipidoid nanoparticles with good transfection efficiency were selected for further investigation. The lead lipidoids (TACN-O14 and TAEA-A12) exhibited excellent gene delivery properties such as high DNA affinity, good serum tolerance, cellular uptake and endosomal escape capability.

EXPERIMENTAL SECTION Materials and Methods. Unless otherwise specified, all reagents are purchased commercially and used directly. The nine long chain-contained reactants including E,23, 42 A23 and O series38 (Scheme 1) were obtained according to the literature. The pEGFP-N1 (Clontech, Palo Alto, CA, USA) and pGL-3 (Promega, Madison, WI, USA) were coding for EGFP DNA and luciferase DNA in vitro study, respectively. From Promega (Madison, WI, USA), MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H tetrazolium, inner salt) was obtained. 1640 medium, fetal bovine serum (FBS) and Lipofectamine 2000 (1 µg/µL) were all purchased from Invitrogen Corp. MicroBCA protein assay kit (Pierce, Rockford, IL, USA), luciferase assay kit (Promega, Madison, WI, USA) were purchased commercially and used according to instructions. Human hepatoma carcinoma cell line (7402

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cells), human lung cancer cells (A549 cells) and human lung cells (7702 cells) were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The 1

H NMR spectra and HRMS spectral were measured on a Bruker AM400 NMR spectrometer

and Bruker Daltonics Bio TOF mass spectrometer. FT-IR spectrometer (NEXUS 670FT-IR) was used to characterize structure. ELISA plate reader (model 680, BioRad) and FACS Calibur flow cytometer (BD AccuriTM C6) were used to measure cell survival and cell uptake. Agarose-gel retardation assay for condensation ability and complex stability, ethidium bromide exclusion assay, dynamic light scattering (DLS) for measuring particle sizes and time-dependent particle size change, transmission electron microscopy (TEM) used to observe the morphology of lipidoid solutions and complexes, cell culture, MTS cytotoxicity assay , and flow cytometry were carried out according to previous literature methods.19, 43-44 Preparation of lipidoid nanoparticles and lipidoids/DNA complexes. Polyamine (200 mg) and reactant with long chain (3.3 eq for TAEA and TACN; 4.4 eq for Cyclen) were added to glass vial without solvent. The reaction mixtures were stirred at 90 °C under N2 atmosphere.24, 28, 45

After 7 days, 27 lipidoids were yielded and used without further separation.

A solution with 50% ethanol and 50% sodium acetate buffer (125 mM, pH 5.2) was firstly prepared. Then the lipidoids were added to yield the lipidoid nanoparticle solutions at a concentration of 1.0 mg/mL.28, 36 To prepare the lipidoids/DNA complexes at various mass ratios (w/w), various volume of lipidoids solution (1.0 mg/mL) was added to a constant amount of DNA at room temperature, and mixed for 30 min. Cell culture. A549 cells, 7402 cells and 7702 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin, 10,000 U mL-1). The cell lines were all incubated at 37 ºC in a humidified atmosphere containing 5% CO2. In vitro transfection assay. The transfection of enhanced green fluorescent protein (EGFP) pDNA was carried out in A549, 7402 and 7702 cells with the absence of serum.44 The 96-well plates were used and mass ratios (w/w) were at 2.5 : 1, 5 : 1, 7.5 : 1 and 10 : 1 as the literature.28 Subsequently, according to the obtained result, the six lipidoid nanoparticles (TAEA-A12, TACN-E12, TACN-O12, TAEA-A14, TAEA-O14 and TACN-O14) were ACS Paragon Plus Environment

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chosen for quantitative transfections using pGL-3 DNA. Transfection experiments were conducted in A549, 7402, and 7702 cells with or without serum.44 The 24-well plates were used and mass ratios were at 5 : 1, 7.5 : 1 and 10 : 1. And Lipofectamine 2000 was used as positive control. Confocal Laser Scanning Microscopy (CLSM) Assay. CLSM assay was performed in A549 cells with the presence of serum. 0.8 µg of Cy5-labeled pGL-3 DNA was used in each well. Cells were treated with lipidoid/DNA complexes (optimal w/w ratio of each sample) for different lengths of time (2 h, 4 h, 8 h, 12 h). As literature methods,44 the endosomes/lysosomes and cell nuclear were stained by Lyso Tracker and DAPI. The intracellular distribution of Cy5-labeled DNA transfected was detected using ZEISS LSM 780 at excitation wavelengths of 405 nm for DAPI (blue), 504 nm for Lyso Tracker (green), 633 nm for Cy5 (red), respectively. The co-localization ratios (Pearson's correlation, Rr) were calculated by software (Image Pro Plus 6.0).

RESULTS AND DISCUSSION Synthesis and characterization of lipidoids. As illustrated in Scheme 1, the 27 target lipidoids were prepared from 3 polyamines and 9 reactants with alkyl long chain. The polyamines included TAEA (tris(2-aminoethyl) amine), TACN (1, 4, 7-triazacyclononane) and Cyclen (1, 4, 7, 10-tetraazacyclododecane), which contain three or four 1º/2º amino groups in each molecule and commonly used in the construction of cationic lipids or polymers.44, 46-47 The nine reactants with alkyl tails, which were simply prepared from long chain alcohol/amine and acryloyl chloride or epichlorohydrin, varied in both the reaction sites (acrylate, acrylamide or epoxide) and the length of tail (C12, C14 and C16), resulting in lipidoids with different linking groups (esters, amides or hydroxyls, described as E, A, O in Scheme 1, respectively). By combinatorial approach, 27 lipidoids were prepared simultaneously after stirring the mixtures of polyamine/long chain reactant at 90 °C for 7 days with the mole ratio of 1 : 3.3 (for TAEA and TACN) or 1 : 4.4 (for Cyclen). Since polyamines with 3~4 amino groups were used, to obtained higher conversion, a relatively long reaction time was needed. All the resulted lipidoids were mixtures with different numbers of alkyl chains and directly used without

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further purification. 1H NMR and Mass spectra of 6 representative lipidoids (Figure S1 and S2) revealed that most of the long chain substrates reacted and attached to polyamine, and the resulted lipidoids were mixtures with different numbers of alkyl chains. FT-IR spectra of TACN-O14 also proved the existence of hydroxyl group in the lipidoid, demonstrating the occurrence of epoxide ring-opening reaction (Figure 1A).

Scheme 1. A library of 27 lipidoids prepared from polyamines and reactants with alkyl tails.

The length of alkyl chain would affect the solubility of the lipidoids, and those with longer (C16) chains could not well dissolve in water. For the soluble ones, the lipidoids were added to a solution containing 50% ethanol and 50% sodium acetate buffer (125 mM, pH 5.2), resulting in lipidoid nanoparticle solution at a concentration of 1.0 mg/mL. Transmission electron microscopy (TEM, Figure 1B) and dynamic light scattering (DLS, Figure 1C) were used to observe the morphology and particle size distribution of TAEA-O14 and TACN-O14. The TEM image shows that the nanoparticles of TAEA-O14 and TACN-O14 were uniformly dispersed with spherical appearance. In aqueous solution (1.0 mg/mL), their particle size was ~500 nm and with small dispersion.

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Figure 1. (A) FT-IR spectra of TACN-O14; (B) Nanoparticles of TAEA-O14 (1 mg mL−1) and TACN-O14 (1 mg mL−1) observed by TEM, with scale bar of 1µm; (C) Size distribution of TAEA-O14 and TACN-O14 lipidoid nanoparticles.

Interaction between lipidoids and plasmid DNA. The DNA condensation ability of the materials were roughly estimated by using 9 lipidoids (with C14 tailes). Agarose gel retardation results revealed that all lipidoids gave similar DNA retardation ability, and full retardation could be achieved at w/w of 5~7.5 (Figure S3). Then, six typical lipidoids with different headgroups, chain length and linking moieties were chosen for further studies. These lipidoids were also chosen for their better performance in preliminary transfection experiment. As shown in Figure 2, TAEA-derived showed slightly stronger DNA retardation ability than TACN-derived ones, and full retardation could be observed at mass ratio of 5-7.5. This might be attributed to the higher charge density of TAEA, which contains 4 amino groups in the molecule. Further, ethidium bromide (EB) dye replacement assay was also carried out to study the binding strength between lipidoids and DNA.48 The EB that intercalated with DNA base pairs would be replaced by the lipidoids, resulting in the fluorescence quenching. Results in Figure S4 confirmed the EB replacement ability of the lipidoids. Similar to the gel retardation results, TAEA-derived lipidoids exhibited stronger fluorescence quenching ability. TAEA-A12 showed the strongest DNA binding ability, and the w/w ratio for 50% quenching was 1.20.

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Figure 2. Agarose gel retardation of lipidoids/pDNA complexes at various w/w ratios. 0.125 µg of pUC-19 DNA was used for each sample.

Nucleases are widely distributed in blood and tissues, DNase is an endonuclease that may catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone.49 It is necessary to confirm that the lipidoids may protect DNA from digestion by these biomolecules. The stability of lipidoid/DNA complexes was also investigated by gel electrophoresis assay. Results in Figure 3 show that naked DNA was completely degraded after 2 h incubation with nuclease (Lane 2 for DNA). Whereas all of the six lipidoids showed the ability to efficiently protect DNA from degradation, and the protected DNA could be released by heparin. Similarly, the stability of the lipidoids/DNA complexes against serum was also studied. No released DNA was found on the gel even after 2 h incubation with 10% or 50% of serum (Figure S5A). After treatment with heparin, DNA could be released in most case except TACN-E12 (with 50% serum, Figure S5B), which has the weakest DNA binding ability (Figure 2 and S4). The complex stability assays against DNase and serum both demonstrate the good DNA protection ability of these lipidoid nanoparticles.

Figure 3. DNA protection by lipidoids against DNase. For DNA control, Lane 1: naked DNA; Lane 2: with DNase for 2 h. For each lipidoids group, Lane 1: vector/DNA complex (w/w = 10); Lane 2: with DNase for 2

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h; Lane 3: with heparin (no DNase); Lane 4: with DNase for 2 h followed by heat-inactivation of DNase and then treated with heparin. The complexes were all at the w/w ratio of 10. 0.125 µg of pUC-19 DNA was used for each sample.

The particle size of vector/DNA complex would partially affect the transfection performance. Dynamic light scattering (DLS) assay was applied to measure the particle size, and the results are shown in Figure 4. Particle size was largely dependent on the mass ratio. Generally, the size reduced with the increase of lipidoid dosage and finally reached 150-300 nm at the w/w of 10. What’s more, the morphology of selected complex was further investigated by transmission electron microscopy (TEM). It was shown that TAEA-A12/DNA (w/w of 10) and TACN-O14/DNA (w/w of 7.5) complexes were almost spherical particles with the diameters in the range of 100-200 nm. After incubation with 10% FBS, the exterior of the complex was retained and became smaller and more regular (Figure S6), suggesting its good serum tolerance. The stability of TACN-O14/DNA complexes in PBS or serum solutions was also studied by DLS, and the results show that the complexes could well retain their particle size within 24 h (Figure S7). The smaller size in the presence of serum was consistent with the results in TEM (Figure S6).

Figure 4. Mean particle sizes of the lipidoid/DNA complexes at various w/w ratio (5, 7.5 and 10) measured by DLS at 25 °C. The complexes with 1 µg of pUC-19 DNA was dissolved in 1 mL of dd-H2O. Data represent mean ± SD (n = 3).

In vitro transfection. The transfection performance of the lipidoids was first investigated by using pEGFP-N1 plasmid DNA as reporter gene in three cell lines, and Lipofectamine 2000 was used as positive control. The lipidoid/DNA complexes were incubated with cells for 24 h

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with mass ratios (w/w) of 2.5 : 1, 5 : 1, 7.5 : 1 and 10 : 1. The lipidoids with longer hydrophobic tails (C16) gave poor transfection in all of the three cell lines. This might be due to their lower water solubility caused by the longer aliphatic chain. Besides, several complexes also induced low EGFP expression. Figure 5 gives the fluorescent microscope images from the transfection mediated by the six lipidoid nanoparticles with relatively higher efficiencies. The hydrophobic chains of C12 or C14 seemed to be suitable for gene delivery, and the best transfection efficiencies in three cell lines were all close to Lipofectamine 2000. The hydrophilic headgroup and linking moiety also have distinct effect on the transfection. Lipidoids derived from Cyclen may not induce good gene transfection, and TAEA or TACN was better choice for such strategy. For TAEA-C12 lipidoids group, only amide-contained lipidoids exhibited good transfection (column #1, Figure 5), while ester or hydroxyl-contained ones gave lower efficiency. However, the hydroxyl-contained linking moiety is suitable in other series of lipidoids (column #3, #5 and #6), indicating that the structure-activity relationship of lipidoids is complicate and needs further in-depth studies for better clarification. For the different cell types, TAEA-A12 gave relatively higher transfection efficiency in A549 and 7402 cells, while TAEA-O14 induced the best transfection in 7702 cells. For the better performance in the EGFP expression assay, such six lipidoids were chosen for further study.

Figure 5. Fluorescent microscope images of pEGFP-transfected cells. Each image was obtained under optimized w/w ratio. And the scale car means 200 µm. 0.2 µg of pEGFP DNA was used for each well, and Lipofectamine 2000 (0.5 µL) was used as positive control.

Quantitative transfections were subsequently performed by using pGL-3 as reporter gene, and Lipofectamine 2000 was used for comparison. The transfection efficiencies were

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investigated in two types of cancer cell lines (A549 and 7402 cells) and one normal cell line (7702 cells). The mass ratio of 5, 7.5 and 10 were chosen for the transfection according to the gel electrophoresis and cytotoxicity results. TACN-O14 gave the best efficiency in A549 cells, and up to 1.8 times higher efficiency than Lipofectamine 2000 could be achieved (Figure 6A). Meanwhile, TAEA-A12 had much better performance in 7402 cells (4.2 times higher than Lipofectamine 2000, Figure 6B). It’s worth noting that in the EGFP assay (Figure 5), the efficiency of the lipidoids was not higher than Lipofectamine 2000. This could be due to the different type of DNA cargo and different measurement method for transfection efficiency. Further, the transfection was seldom affected serum, even increased efficiency was obtained with the presence of FBS. Cell selectivity could be preliminarily observed for the lipidoids, especially TACN-O14, which gave much higher transfection efficiency in A549 cells than in other cell lines. In addition, the transfection efficiency of these lipidoid nanoparticles were much lower in normal cells (relative to Lipofectamine 2000, Figure 6C), suggesting that such materials might have smaller effect on normal tissue in the treatment of tumor.

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Figure 6. Luciferase gene expression transfected in (A) A549, (B) 7402 and (C) 7702 with or without the presence of serum. Each material was tested at various w/w ratio (5, 7.5 and 10) by in comparison with Lipofectamine 2000. Data represent mean ± SD (n = 3). 0.8 µg of pGL-3 DNA was used for each well, and 2 µL of Lipofectamine 2000 was used at the optimal mass ratio of 2.5.

The cytotoxicity of the vector/DNA complexes were estimated by MTS-based cell viability assays in two cells lines (A549 and 7702). As shown in Figure 7, the increase of mass ratio generally led to more cell death, and the final cell viability (w/w = 15) was comparable to Lipofectamine 2000. Under the same mass ratio (2.5), the toxicity of the lipidoids was much lower than Lipofectamine 2000. The chemical structure seemed to have little effect on the toxicity, and the six lipidoids gave similar results in this assay. Since the transfection efficiency of the non-viral vectors is not directly related to its cytotoxicity, and the DNA used in MTT assay is not therapeutic DNA which may lead to cell death, no obvious cancer cell selectivity was observed in this assay, and the cytotoxicity was mainly affected by the material concentration.

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Figure 7. Relative cell viabilities (to negative control as 1) caused by the six lipidoids toward A549 (A) and 7702 (B) cells. Each material was tested at w/w ratio of 2.5, 5, 7.5, 10 and 15. Lipofectamine 2000 was used at the optimal mass ratio of 2.5. Data represent mean ± SD (n = 3). 0.2 µg of pGL-3 DNA was used for each well, and 0.5 µL of Lipofectamine 2000 was used at the optimal mass ratio of 2.5.

Flow cytometry was applied to further study the internalization of complexes in A549 and 7402 cells. All the six lipidoids and Lipofectamine 2000 were complexed with Cy5-labeled DNA at the optimal mass ratio. After 4 h incubation, the percentages of Cy5-positive cells and the relative fluorescence intensity (RFI) were calculated (Figure 8). Similar to the luciferase transfection results, both the uptake cell percentage and RFI induced by the complexes gave slight increase after the participation of serum. That might be attributed to the smaller particle size of complexes (Figure S6 and S7) which would favor cellular uptake.50-51 Results in Figure 8 basically coincide with those obtained in luciferase assay, TACN-O14 induced much higher cellular uptake, especially RFI, in A549 cells, while TAEA-A12 gave the best performance in 7402 cells. The cellular uptake level of the two lipidoid nanoparticles in the respective cells was comparable to that of Lipofectamine 2000. Thus their higher transfection efficiency (Figure 6) may come from their better intracellular behavior.

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Figure 8. Cellular uptake (histogram) and RFI (dot plot) of vector/DNA complexes at optimal transfection w/w ratio in A549 (A) and 7402 (B) cells. Data shows the representative mean ±SD (n = 3). 0.8 µg of Cy5-labeled pGL-3 DNA was used in each well, and 2 µL of Lipofectamine 2000 was used at the optimal mass ratio of 2.5.

The intracellular distribution of DNA transfected by the vetors was subsequently observed by using confocal laser scanning microscopy (CLSM). The DNA and cell nuclei were stained by Cy5 (red) and DAPI (blue), respectively. The lipidoid/DNA complexes were prepared at the optimal mass ratio, and the experiments were performed in A549 cells with the presence of serum. As shown in Figure 9A, all the vectors could efficiently carry Cy5-labeled DNA into cells. On the other hand, most of the lipidoids and Lipofectamine 2000 could deliver DNA to perinuclear region, and the red fluorescence signals were mainly found around the nuclei. The red-blue co-localization Pearson’s correlation of CLSM images was calculated and shown in Figure 9B. TACN-O14 had the highest co-localization ratio, suggesting that more DNA carried by TACN-O14 could enter the nucleus within 4 h than those delivered by other lipidoids. This may contribute much to its higher transfection efficiency (Figure 6A).

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Figure 9. (A) CLSM images of A549 cells transfected with complex (at optimal w/w ratio) after 4 h incubation. (B)The co-localization ratios calculated by software. 0.8 µg of Cy5-labeled pGL-3 DNA was used in each well, and 2 µL of Lipofectamine 2000 was used at the optimal mass ratio of 2.5.

CLSM was further applied to investigate the endosome/lysosome escape capability of the complexes by taking the images at different incubation times (2, 4, 8 and 12 h). TACN-O14 and Lipofectamine 2000 were selected for this assay, and the endosome/lysosomes were stained with Lyso Tracker Green (Figure 10). The images show the difference between the endosomal escape behavior of the complexes formed from TACN-O14 and Lipofectamine 2000. After 2 or 4 h incubation, more yellow fluorescent signals were observed in the merged images from the transfection mediated by TACN-O14, suggesting that more amount of

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internalized DNA located in late endosome than the case involving Lipofectamine 2000. With the extension of incubation time to 12 h, more and more red signals reappeared, indicating the escape of DNA (or lipidoid/DNA complexes). However, on the contrary, the red signals decreased with the increase of yellow signals in the 12 h transfection process by Lipofectamine 2000. The calculated data (Figure 10B) also show that for the transfection mediated by TACN-O14, the co-localization ratio began to drop from 4 h incubation. Meanwhile, the co-localization ratio increased continuously in the transfection with Lipofectamine 2000. Such results reflect that TACN-O14 might induce endosome escape more rapidly. Since the DNA internalization mediated by TACN-O14 is not better than that by Lipofectamine 2000, the higher transfection efficiency of the lipidoid might come from the better endosomal escape.

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Figure 10. (A) Intracellular distribution of Cy5-labeled pDNA (red) complexed with Lipofectamine 2000 and TACN-O14 at optimal w/w ratio in A549 cells after different incubation time. Acidic late endosomes and lysosomes were stained with LysoTracker Green (green). (B) Time-dependent changes in co-localization ratios of the complexes containing Cy5-labeled pDNA (red signals) with late endosomes and lysosomes (green signals). 0.8 µg of Cy5-labeled pGL-3 DNA was used in each well, and 2 µL of Lipofectamine 2000 was used at the optimal mass ratio of 2.5.

CONCLUSIONS In summary, a small library of 27 lipidoids was obtained by combinatorial approach from three commonly used small molecule polyamines and nine reactants with alkyl tails. Initial screening by EGFP transfection assay selected six lipidoid nanoparticles with good transfection efficiency for further studies. These lipidoids have good DNA condensation capability and the formed DNA complexes have good stability toward serum or nuclease. Quantitative transfection study with luciferase reporter gene showed that TACN-O14 could give 1.8 times higher efficiency than Lipofectamine 2000 in A549 cells, while TAEA-A12 may give 4.2 times higher efficiency in 7402 cells. Moreover, their transfection efficiencies were not inhibited by serum, but even lightly increased. Mechanism studies including flow cytometry and CLSM revealed that the higher transfection efficiency of the lipidoid might be attributed to the more rapid and efficient endosome/lysosome escape. These lipidoids represent a potential type of non-viral gene vectors with high transfection efficiency, good serum tolerance and convenience in preparation and use.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

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H NMR and MS spectra of lipidoids, preliminary electrophoretic gel retardation results,

ethidium bromide dye replacement assay, complex stability studies against serum via electrophoretic gel retardation assay, TEM images of lipidoid/DNA complexes without or with the presence of serum, the size change of the complexes over incubation time.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21472131, 21672155). We also thank the Comprehensive Training Platform of Specialized Laboratory, College of Chemistry, and the Analytical & Testing Center of Sichuan University for sample analysis.

REFERENCES 1. Liu, F.; Huang, L. Development of Non-Viral Vectors for Systemic Gene Delivery. J. Controlled Release 2002, 78 , 259-266. 2. Guo, X.; Huang, L. Recent Advances in Nonviral Vectors for Gene Delivery. Acc. Chem. Res. 2011, 45, 971-979. 3. Shirazi, R. S.; Ewert, K. K.; Leal, C.; Majzoub, R. N.; Bouxsein, N. F.; Safinya, C. R. Synthesis and Characterization of Degradable Multivalent Cationic Lipids with Disulfide-Bond Spacers for Gene Delivery. Biochim. Biophys. Acta 2011, 1808, 2156-2166. 4. Koynova, R.; Tenchov, B.; Wang, L.; MacDonald, R. C. Hydrophobic Moiety of Cationic Lipids Strongly Modulates Their Transfection Activity. Mol. Pharm. 2009, 6, 951-958. 5. Zhi, D.; Zhang, S.; Cui, S.; Zhao, Y.; Wang, Y.; Zhao, D. The Headgroup Evolution of Cationic Lipids for Gene Delivery. Bioconjugate Chem. 2013, 24, 487-519. 6. Bhattacharya, S.; Bajaj, A. Advances in Gene Delivery through Molecular Design of Cationic Lipids. Chem. Commun. 2009, 4632-4656. 7. Yue, Y.; Wu, C. Progress and Perspectives in Developing Polymeric Vectors for In Vitro Gene Delivery. Biomater. Sci. 2013, 1, 152-170. 8. Kaur, S.; Prasad, C.; Balakrishnan, B.; Banerjee, R. Trigger Responsive Polymeric Nanocarriers for Cancer Therapy. Biomater. Sci. 2015, 3, 955-987. 9. Luo, K.; Li, C.; Li, L.; She, W.; Wang, G.; Gu, Z. Arginine Functionalized Peptide Dendrimers as Potential Gene Delivery Vehicles. Biomaterials 2012, 33, 4917-4927. 10. Sheikhi Mehrabadi, F.; Fischer, W.; Haag, R. Dendritic and Lipid-Based Carriers for Gene/Sirna Delivery (A Review). Curr. Opin. Solid State Mater. Sci. 2012, 16, 310-322. 11. Liu, H.; Wang, H.; Yang, W.; Cheng, Y. Disulfide Cross-Linked Low Generation Dendrimers with High Gene Transfection Efficacy, Low Cytotoxicity, and Low Cost. J. Am. Chem. Soc. 2012, 134, 17680-17687. 12. Loh, X. J.; Lee, T.-C.; Dou, Q.; Deen, G. R. Utilising Inorganic Nanocarriers for Gene

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Page 18 of 22

Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Delivery. Biomater. Sci. 2016, 4, 70-86. 13. Yang, X.-Z.; Dou, S.; Wang, Y.-C.; Long, H.-Y.; Xiong, M.-H.; Mao, C.-Q.; Yao, Y.-D.; Wang, J. Single-Step Assembly of Cationic Lipid–Polymer Hybrid Nanoparticles for Systemic Delivery of siRNA. ACS Nano 2012, 6, 4955-4965. 14. Harmon, A. M.; Lash, M. H.; Sparks, S. M.; Uhrich, K. E. Preferential Cellular Uptake of Amphiphilic Macromolecule–Lipid Complexes with Enhanced Stability and Biocompatibility. J. Controlled Release 2011, 153, 233-239. 15. Hadinoto, K.; Sundaresan, A.; Cheow, W. S. Lipid–Polymer Hybrid Nanoparticles as A New Generation Therapeutic Delivery Platform: A Review. Eur. J. Pharm. Biopharm. 2013, 85, 427-443. 16. P.L. Felgner, T. R. G., M. Holm, R. Roman, H.W. Chan, M. Wenz, J.P. Northrop, G.M. Ringold, M. Danielsen. Lipofection: A Highly Efficient, Lipid-Mediated DNA-Transfection Procedure. Proc. Nati. Acad. Sci. USA 1987, 1987, 7413. 17. Pierrat, P.; Creusat, G.; Laverny, G.; Pons, F.; Zuber, G.; Lebeau, L. A Cationic Phospholipid–Detergent Conjugate as a New Efficient Carrier for siRNA Delivery. Chem. – Eur. J. 2012, 18, 3835-3839. 18. McKinlay, C. J.; Waymouth, R. M.; Wender, P. A. Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery. J. Am. Chem. Soc. 2016, 138, 3510-3517. 19. Zhang, Y.-M.; Liu, Y.-H.; Zhang, J.; Liu, Q.; Huang, Z.; Yu, X.-Q. Cationic Gemini Lipids with Cyclen Headgroups: Interaction with DNA and Gene Delivery Abilities. RSC Adv. 2014, 4, 44261-44268. 20. Kumar, M.; Jinturkar, K.; Yadav, M. R.; Misra, A. Gemini Amphiphiles: A Novel Class of Nonviral Gene Delivery Vectors. 2010, 27, 237-278. 21. Khan, M.; Ang, C. Y.; Wiradharma, N.; Yong, L.-K.; Liu, S.; Liu, L.; Gao, S.; Yang, Y.-Y. Diaminododecane-Based Cationic Bolaamphiphile as A Non-Viral Gene Delivery Carrier. Biomaterials 2012, 33, 4673-4680. 22. Behr, J. P.; Demeneix, B.; Loeffler, J. P.; Perez-Mutul, J. Efficient Gene Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine-Coated DNA. P. Natil. Acad. Sci. 1989, 86, 6982-6986. 23. Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, V.; Hossain, N.; Bacallado, S. A.; Nguyen, D. N.; Fuller, J.; Alvarez, R.; Borodovsky, A.; Borland, T.; Constien, R.; de Fougerolles, A.; Dorkin, J. R.; Narayanannair Jayaprakash, K.; Jayaraman, M.; John, M.; Koteliansky, V.; Manoharan, M.; Nechev, L.; Qin, J.; Racie, T.; Raitcheva, D.; Rajeev, K. G.; Sah, D. W. Y.; Soutschek, J.; Toudjarska, I.; Vornlocher, H.-P.; Zimmermann, T. S.; Langer, R.; Anderson, D. G. A Combinatorial Library of Lipid-Like Materials for Delivery of Rnai Therapeutics. Nat Biotech 2008, 26, 561-569. 24. Wang, M.; Alberti, K.; Varone, A.; Pouli, D.; Georgakoudi, I.; Xu, Q. Enhanced Intracellular siRNA Delivery using Bioreducible Lipid-Like Nanoparticles. Advanced Healthcare Materials 2014, 3, 1398-1403. 25. Cho, S.-W.; Goldberg, M.; Son, S. M.; Xu, Q.; Yang, F.; Mei, Y.; Bogatyrev, S.; Langer, R.; Anderson, D. G. Lipid-Like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells. Adv. Funct. Mater. 2009, 19, 3112-3118. 26. Sun, S.; Wang, M.; Knupp, S. A.; Soto-Feliciano, Y.; Hu, X.; Kaplan, D. L.; Langer, R.;

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Anderson, D. G.; Xu, Q. Combinatorial Library of Lipidoids for In Vitro DNA Delivery. Bioconjugate Chem. 2012, 23, 135-140. 27. Wang, M.; Sun, S.; Alberti, K. A.; Xu, Q. A Combinatorial Library of Unsaturated Lipidoids for Efficient Intracellular Gene Delivery. ACS Synth. Biol. 2012, 1, 403-407. 28. Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.; Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T.; Frank-Kamenetsky, M.; Yip, K. N.; Alvarez, R.; Sah, D. W. Y.; de Fougerolles, A.; Fitzgerald, K.; Koteliansky, V.; Akinc, A.; Langer, R.; Anderson, D. G. Lipid-Like Materials for Low-Dose, in Vivo Gene Silencing. P. Natil. Acad. Sci. 2010, 107, 1864-1869. 29. Dong, Y.; Eltoukhy, A. A.; Alabi, C. A.; Khan, O. F.; Veiseh, O.; Dorkin, J. R.; Sirirungruang, S.; Yin, H.; Tang, B. C.; Pelet, J. M.; Chen, D.; Gu, Z.; Xue, Y.; Langer, R.; Anderson, D. G. Lipid-Like Nanomaterials for Simultaneous Gene Expression and Silencing in Vivo. Advanced Healthcare Materials 2014, 3, 1392-1397. 30. Sheng, R.; Luo, T.; Li, H.; Sun, J.; Wang, Z.; Cao, A. ‘Click’ Synthesized Sterol-Based Cationic Lipids as Gene Carriers, and The Effect of Skeletons and Headgroups on Gene Delivery. Biorg. Med. Chem. 2013, 21, 6366-6377. 31. Alabi, C. A.; Love, K. T.; Sahay, G.; Yin, H.; Luly, K. M.; Langer, R.; Anderson, D. G. Multiparametric Approach for The Evaluation of Lipid Nanoparticles for siRNA Delivery. P. Natil. Acad. Sci. 2013, 110, 12881-12886. 32. Li, L.; Zahner, D.; Su, Y.; Gruen, C.; Davidson, G.; Levkin, P. A. A Biomimetic Lipid Library for Gene Delivery through Thiol-yne Click Chemistry. Biomaterials 2012, 33, 8160-8166. 33. Li, L.; Wang, F.; Wu, Y.; Davidson, G.; Levkin, P. A. Combinatorial Synthesis and High-Throughput Screening of Alkyl Amines for Nonviral Gene Delivery. Bioconjugate Chem. 2013, 24, 1543-1551. 34. Salvatore, R. N.; Nagle, A. S.; Jung, K. W. Cesium Effect:  High Chemoselectivity in Direct N-Alkylation of Amines. J. Org. Chem. 2002, 67, 674-683. 35. Fenton, O. S.; Kauffman, K. J.; McClellan, R. L.; Appel, E. A.; Dorkin, J. R.; Tibbitt, M. W.; Heartlein, M. W.; DeRosa, F.; Langer, R.; Anderson, D. G. Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery. Adv. Mater. 2016, 28, 2939-2943. 36. Whitehead, K. A.; Dorkin, J. R.; Vegas, A. J.; Chang, P. H.; Veiseh, O.; Matthews, J.; Fenton, O. S.; Zhang, Y.; Olejnik, K. T.; Yesilyurt, V.; Chen, D.; Barros, S.; Klebanov, B.; Novobrantseva, T.; Langer, R.; Anderson, D. G. Degradable Lipid Nanoparticles with Predictable in Vivo Sirna Delivery Activity. Nature Commun. 2014, 5, 4277. 37. Yi, W.-J.; Yu, X.-C.; Wang, B.; Zhang, J.; Yu, Q.-Y.; Zhou, X.-D.; Yu, X.-Q. TACN-Based Oligomers with Aromatic Backbones for Efficient Nucleic Acid Delivery. Chem. Commun. 2014, 50 , 6454-6457. 38. Yi, W.-J.; Zhang, Q.-F.; Zhang, J.; Liu, Q.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-Q. Cyclen-Based Lipidic Oligomers as Potential Gene Delivery Vehicles. Acta Biomater. 2014, 10, 1412-1422. 39. Zhang, Q.-F.; Wang, B.; Yin, D.-X.; Zhang, J.; Wu, W.-X.; Yu, Q.-Y.; Yu, X.-Q. Linear TACN-Based Cationic Polymers as Non-Viral Gene Vectors. RSC Adv. 2014, 4, 59164-59174. 40. Wang, B.; Yi, W.-J.; Zhang, J.; Zhang, Q.-F.; Xun, M.-M.; Yu, X.-Q. TACN-Based

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Cationic Lipids with Amino Acid Backbone and Double Tails: Materials for Non-Viral Gene Delivery. Bioorg. Med. Chem. Lett. 2014, 24, 1771-1775. 41. Huang, Z.; Zhang, Y.-M.; Cheng, Q.; Zhang, J.; Liu, Y.-H.; Wang, B.; Yu, X.-Q. Structure-Activity Relationship Studies of Symmetrical Cationic Bolasomes as Non-viral Gene Vectors. J. Mater. Chem. B 2016, 4, 5575-5584. 42. Xun, M.-M.; Xiao, Y.-P.; Zhang, J.; Liu, Y.-H.; Peng, Q.; Guo, Q.; Wu, W.-X.; Xu, Y.; Yu, X.-Q. Low Molecular Weight PEI-Based Polycationic Gene Vectors via Michael Addition Polymerization with Improved Serum-Tolerance. Polymer 2015, 65, 45-54. 43. Huang, Z.; Liu, Y.-H.; Zhang, Y.-M.; Zhang, J.; Liu, Q.; Yu, X.-Q. Cyclen-Based Cationic Lipids Containing A Ph-Sensitive Moiety as Gene Delivery Vectors. Org. Biomol. Chem. 2015, 13, 620-630. 44. Zhang, Q.-F.; Yi, W.-J.; Wang, B.; Zhang, J.; Ren, L.; Chen, Q.-M.; Guo, L.; Yu, X.-Q. Linear Polycations by Ring-Opening Polymerization as Non-Viral Gene Delivery Vectors. Biomaterials 2013, 34, 5391-5401. 45. Sun, S.; Wang, M.; Alberti, K. A.; Choy, A.; Xu, Q. DOPE Facilitates Quaternized Lipidoids (Qlds) for In Vitro DNA Delivery. Nanomed. Nanotechnol. Biol. Med. 2013, 9, 849-854. 46. Zhang, Q.-F.; Yu, Q.-Y.; Geng, Y.; Zhang, J.; Wu, W.-X.; Wang, G.; Gu, Z.; Yu, X.-Q. Ring-Opening Polymerization for Hyperbranched Polycationic Gene Delivery Vectors with Excellent Serum Tolerance. ACS Appl. Mater. Interfaces 2014, 6, 15733-15742. 47. Wang, H.-J.; Liu, Y.-H.; Zhang, J.; Zhang, Y.; Xia, Y.; Yu, X.-Q. Cyclen-Based Cationic Lipids with Double Hydrophobic Tails for Efficient Gene Delivery. Biomater. Sci. 2014, 2, 1460-1470. 48. Lleres, D.; Dauty, E.; Behr, J.-P.; Mély, Y.; Duportail, G. DNA Condensation by An Oxidizable Cationic Detergent. Interactions with Lipid Vesicles. Chem. Phys. Lipids 2001, 111, 59-71. 49. Wang, J.; Dou, B.; Bao, Y. Efficient Targeted Pdna/Sirna Delivery with Folate–Low-Molecular-Weight Polyethyleneimine–Modified Pullulan as Non-Viral Carrier. Materials Science and Engineering: C 2014, 34, 98-109. 50. Spagnou, S.; Miller, A. D.; Keller, M. Lipidic Carriers of siRNA:  Differences in the Formulation, Cellular Uptake, and Delivery with Plasmid DNA. Biochemistry 2004, 43, 13348-13356. 51. Xiang, S.; Tong, H.; Shi, Q.; Fernandes, J. C.; Jin, T.; Dai, K.; Zhang, X. Uptake Mechanisms of Non-Viral Gene Delivery. J. Controlled Release 2012, 158, 371-378.

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Table of contents graphic A Small Combinatorial Library of Lipidoids as Nano-vectors for Gene Delivery Yi-Mei Zhang, Zheng Huang, Xiao-Ru Wu, Ji Zhang*, Yan-Hong Liu and Xiao-Qi Yu*

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