Gemini-Type Tetraphenylethylene Amphiphiles Containing [12]aneN3

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Gemini Type Tetraphenylethylene Amphiphiles Containing [12]aneN3 and Long Hydrocarbon Chains as Nonviral Gene Vectors and Gene Delivery Monitors Ai-Xiang Ding, Zheng-Li Tan, You-Di Shi, Ling Song, Bing Gong, and Zhong-Lin Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01850 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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ACS Applied Materials & Interfaces

Gemini Type Tetraphenylethylene Amphiphiles Containing [12]aneN3 and Long Hydrocarbon Chains as Nonviral Gene Vectors and Gene Delivery Monitors Ai-Xiang Ding,1,2 Zheng-Li Tan,1 You-Di Shi,1 Lin Song,1 Bing Gong,1,3 Zhong-Lin Lu1* 1

Key Laboratory of Radiopharmaceuticals, Ministry of Education; College of Chemistry, Beijing

Normal University, Beijing 100875, China. 2

College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000,

China 3

Department of Chemistry, State University of New York, Buffalo, NY 14260, USA

KEYWORDS: AIE Micelles, Gemini Lipids, Nonviral Gene Vectors, Micelle and Nanoparticle Transition, Gene Delivery Tracing

ABSTRACT: Four gemini amphiphiles decorated with triazole-[12]aneN3 as the hydrophilic moiety and various long-hydrocarbons as hydrophobic moieties, 1-4, were designed to form micelles possessing aggregation-induced emission (AIE) property for gene delivery and tracing. All four amphiphiles give ultralow critical micelle concentrations, are pH/photo-stable and 1

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biocompatible, and completely retard the migration of plasmid DNAs at low concentrations. The DNA-binding abilities of the micelles were fully assessed. The co-aggregated nanoparticles of 1‒ 4 with DNAs could convert back into AIE micelles. In vitro transfections indicated that lipids 1 and 2 and their originated liposomes bearing decent delivering abilities have great potentials as nonviral vectors. Finally, on the basis of the transfection and the transitions between condensates and micelles, lipid 2 was singled out as the first example for real-time tracing of the intracellular deliveries of non-labeled DNA, which provides spatiotemporal messages about the processes of condensates uptake and DNA release.

Introduction To repair and/or substitute a defective gene through trafficking exogenous genetic materials (e.g., DNA, siRNA, mRNA, shRNA) into target cells, the development of gene therapy has attracted wide attention in recent decades as a promising therapeutic strategy for treating various diseases, such as genetic disorder, cancer, AIDS, diabetes etc.1,

2

Since glycoproteins and

glycolipids anchored on cell surface are negatively charged, the uptake of polyanionic naked nucleic acid is rather inefficient. As a result, the development of safe and convenient gene transfer reagents, referred as gene vectors, with highly efficacious delivery abilities is of critical importance.3, 4 As a rule, gene transfer can be accomplished by either using viral or nonviral means. The overwhelming majority of ongoing clinical trials for gene therapy are based on viral vectors that have been demonstrated to possess high transfection efficiency (TE) and effective rapid gene expression.5 However, viral vectors suffer from many inherent shortcomings, such as potential immunogenicity and tumorigenicity, limited DNA compacting ability, complicated production and storage procedures, and other limitations.6,

7

On the contrary, nonviral gene

delivery system can effectively surmount these restrictions. In addition, many other advantages 2


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including easy large-scale preparation, adjustable molecular structure, and target-specific peculiarity have rendered nonviral vectors more adaptive and versatile.8-11 Presently, the main bottleneck on the employ of nonviral systems is their relatively low transfection efficiency (TE) in vivo. To promote TE, a number of compounds and materials including cationic lipids,12,

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polycationic polymers14 or supramolecules,15 inorganic complexes,16 and nanoparticles17 have been prepared and tested as nonviral vectors. Among them, small molecule based cationic lipids, especially those involving gemini surfactants (GS), have attracted considerable attention due to their simple structure, forthright components, high charge-to-mass ratio, superior surfactant properties, enhanced TE, and good biocompatibility.18-21 Lipids with gemini structures are usually characterized as “m-s-m” (m and s represent the numbers of carbon atoms in the long tails and in the linkers, respectively). Many groups have worked on the design of novel GS, in efforts to achieve desired TE by modulating the structures of “m” and/or “s”.22-24 Although some of the reported examples have obtained commendable TE, their delivering efficacies are still far from being comparable to those of viral vectors. Besides, most of currently reported GS-based delivering systems exhibit single functions for gene transfections. These disadvantages have presented an enormous limit on further practical applications. Therefore, developing new GS with non-traditional structures and multifunctional faculties are urgently called. To meet the listed requirements, a series of [12]aneN3-based bifunctional vectors have been reported by us.25-29 The unique unit [12]aneN3 offers multiple nitrogen atoms and an unfolded conformation. By combining a naphthamide moiety and [12]aneN3, we demonstrated both delivering and tracing capacities at the same time. Moreover, to study the configurational impacts on the binding abilities and to trace non-labeled nucleic acids, we also developed several 3



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[12]aneN3-modified tetraphenylethylene (TPE) molecules for nucleic acid sensing and delivery.30 Multi-functionalization can not only provide good nonviral vectors, but also offer a possibility for penetrating the delivering process, which should provide much-needed insights guiding the further optimization of the design of nonviral gene carriers. In recent years, aggregation-induced emission (AIE) has received much attention, which refers to the phenomenon that compounds don’t or faintly emit in dilute solution but become strongly luminescent when the molecules aggregate into nanoparticles or solid films. It is well known that TPE derivatives are a class of compounds that exhibit characteristic AIE, an extremely valuable merit that leads to numerous applications for these compounds in multiple areas.31-36 We also noticed that TPE amphiphiles can self-assemble into micelles in aqueous solution, which inherit the AIE property and thus give the corresponding fluorescence emission.37-46 By taking advantage of this feature, a very few self-assembled AIE micelles as specific dyes have been applied in cell imaging, drug and gene delivery, and preliminarily used for visualization of drug release and gene transfection. Clearly, it is of theoretical and practical significance to develop novel structures with expanded applications. RO

OR

1: R = N

N

N

N

N

N

2: R = 3: R = 4: R =

N H N

N

HN

NH HN 1, 2, 3, 4

Figure 1. Molecular structures of compounds 1‒4. In this research, we have designed four small-molecule TPE amphiphiles (1‒4) with a novel “gemini structure” containing [12]aneN3-triazoles as hydrophilic “heads” and long hydrocarbon chains as hydrophobic “tails” (Figure 1). The AIE, self-assembly, DNA binding abilities, 4


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transfections, and cellular uptakes of these compounds were systematically investigated. We also proved that DNA condensations caused by these reagents were reversible, and these amphiphiles could reassemble into micelles. Particularly, by taking advantage of this unique conversion between the self-assembled micelles and co-assembled nanoparticles, compound 2 was successfully used to track the deliveries of a label-free plasmid DNA (pUC18 DNA). Results and Discussion Synthesis and Characterization: Figure 1, Scheme S1 and S2 show the structures and synthetic routes of compounds 1‒4. Following reported literature procedures, compounds 14,

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15,48 and 1649 were prepared. Through typical McMurry coupling reaction using TiCl4 as the catalyst, the core unit, 5, was obtained and was subsequently treated with 1-bromo-hydrocarbon (three aliphatic hydrocarbons and an olefin) to provide compounds 6‒9. These compounds were further used in reactions with compound 15 in dry THF by using n-BuLi as a strong alkali to give compounds 10‒13. Target compounds 1‒4 were synthesized through highly efficient Cu(I)mediated alkyne‒azide click reactions and subsequent deprotections. Detailed synthetic processes and characterization data can be found in the Supporting Information. Aggregation-Induced Emission: The UV-vis spectra of compounds 1‒4 were recorded to gain a preliminary understanding on their optical behaviors. It was found that all four compounds have a maximum absorption around 345 nm (Figure S1). Due to the amphipathic nature of the four molecules, they manifested no classic AIE properties in the common binary solvent systems, such as THF/water, DMSO/water, ACN/water, etc. In order to observe their AIE phenomena, HCl solution (10 mM) was used as a good solvent to dissolve these molecules to render the formation of the corresponding hydrochlorides, which rendered them insoluble in THF. Thus the nonemissive hydrochlorides in DMSO were accumulated after introducing THF as a poor solvent. 5



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The emission intensities of the formed aggregates were evidently enhanced when the proportion of THF reached 95% (Figure S2). The wavelengths of the emission maxima were also accompanied by a distinct red shift from 400 to 489 nm. Clearly, the aggregation-caused restriction of intramolecular rotation (RIR) was responsible for the observed emission enhancement. These results imply that all of the molecules are AIE-active. Determination of micelle formation: Due to their amphiphilic structures, the four amphiphiles would self-assemble into micelles in aqueous solution as a result of the balance of attractive and repulsive forces between the hydrophilic and hydrophobic components of the amphiphile and the surrounding medium.40 Since TPE derivatives can inherit the characteristic AIE emission upon the formation of nano assemblies, the critical micelle concentrations (CMCs) of the four compounds could be easily determined by recording their fluorescence versus varying concentrations (Figure S3). Figure 2 shows the changes in fluorescence intensities with increasing concentrations, from which the CMCs of the four amphiphiles were determined to be 4.6 × 10-6 M (1), 4.6 × 10-6 M (2), 4.0 × 10-6 M (3), and 4.9 × 10-6 M (4), respectively. Except for 4, the CMCs decreased along with the extensions of the hydrophobic alkyl chain, which is ascribed to the increasing ratios of the hydrophobic and hydrophilic components. The ultra-low CMCs are very close to those of reported TPE amphiphile (TPE-N+) that gave the lowest CMC (2.0 × 10-6 M).40 Moreover, the pH-stabilities and photo-stabilities of the four amphiphiles were also investigated to assess their potentials as fluorescence dyes. In Figures S4 and S5, the largely constant emission intensities over a large range of pH values and long-time UV 365 nm irradiations suggested their outstanding pH/photo-stability. These properties should be of value to practical applications related to biological imaging.

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formation off micelles was w further confirmed c b transmisssion electroon microscoppy (TEM) by The fo and dynnamic light sscattering (D DLS) measuurements. The T TEM im mages in Figgure 2 clearlly showed that all of the fourr amphiphilles self-asseembled intoo particles w with controlllable shapees, narrow DLS measuurements (Fiigure S6) inndicated thatt the mean size disttributions annd high disppersivities. D diameteers of the miicelles are 18 nm (1), 21 2 nm (2), 28 nm (3) annd 31 nm (4)), respectiveely, which are conssistent with those measuured by TEM M.

Figure 2. Determinnations of critical micelle concentrrations (CM MCs) and TE EM images (5 μM) of 1 (A), 2 (B), 3 (C), and 4 (D) inn water. λex = 345 nm, 25 °C, scalee bars = 1000 nm.

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Interaactions witth Calf Thyymus DNA (ctDNA): Complexatiions of the ffour compoounds with natural ctDNA werre investigatted by fluorrescence titrrations in T Tris-HCl bufffer (pH 7.44) at room temperaature (Figurre S7 and 3A). Upon aaddition of DNAs, the emission inntensities of o the four compouunds gradually increaseed, and the eenhancemennt rate follow wed the seqquence 1 ˂ 2 ˂ 4 ˂ 3. At 10 µ µg/mL ctDN NA, the fluoorescence inntensity of 11‒4 increased by 1.6, 22.0, 2.4 andd 2.1 fold, respectively. Thereefore, lengthhening the tail, i.e., thhe hydrophhobic chain,, is conduciive to the D Com mpared with 3, the olefinic bond in the hyddrophobic cchain of 4 interactiions with DNA. loweredd its fluoresscence uponn adding ctD DNA. Accoording to ouur previouslly reported work that involvedd [12]aneN N3 modifiedd TPE moleecules as DNA D probes,30 the em missive enhaancements herein sshould be atttributed to tthe transforrmations of the micelles into nanopparticles. Thhe formed nanoparrticles were observed byy scanning electron microscopy (S SEM) measuurements. Inn the SEM images (Figure 3B)), a number of nanoparrticles with nonuniform n m morphologgies and varrying sizes were clearly obseerved, sugggesting thee effective transformaations from m the miceelles into nanoparrticles by coo-aggregatioon behaviorrs between the amphipphilic moleccules and thhe ctDNA biomoleecules.

Figure 3. (A) Plotss of I/I0-1 at 490 nm veersus ctDNA A concentraations in 50 mM Tris-H HCl buffer (pH 7.4)), concentraations = 10 µ µM, λex = 3445 nm, 25 °C; ° (B) SEM M images off 1 (b1), 2 (bb2), 3 (b3),

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and 4 (b4) after additions of 10 µg ctDNA, concentrations = 10 µM, scale bars: 1 µm. Insets of B are the magnifications of a selected region for clarity, scale bars: 500 nm. Gel Electrophoresis Assays: The condensing abilities of the four compounds toward plasmid DNA were evaluated with gel retardation assays (Figure 4). All four compounds were found to effectively retard the migration of pUC18 DNA, and the concentrations for completely prohibiting electrophoretic mobility with compounds 1‒4 were 16 µM, 16 µM, 12 µM, and 30 µM, respectively. Clearly, the positively charged triazole-[12]aneN3 units can effectively neutralize the negatively charged DNA phosphate groups, and the attractive electrostatic interaction is the dominating driving force to retard the mobility.14 As a result, with the same number of protonated moieties, compounds 1‒3 had rather similar concentrations for effective condensation. Moreover, the condensing abilities of these compounds were similar to those of two triazole-[12]aneN3-modified TPE derivatives we reported.30 It is noteworthy that the hydrophobic effects caused by the long alkyl chains also played active roles in the condensing procedures. Longer hydrophobic chains generally contribute more to the retardation. These results agree with the tendencies we previously observed.25,

27

Nevertheless, the condensing

ability of the unique compound 4, which carries a double bond in its long hydrocarbon chain, was obviously weaker than those of the other three compounds, suggesting a major impact on the binding ability caused by the conversion from a saturated hydrocarbon tail to an unsaturated one.

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C18 DNA induced by vvarying conncentrationss (labelled oon the top, Figure 4. Retardattions of pUC B), 3 (C), annd 4 (D) in 50 mM Trris-HCl bufffer (pH 7.4)). [pUC18 D DNA] = 9 µM) of 1 (A), 2 (B µg/mL, 37 °C. m es and sizes of the conddensed nanopparticles weere further characterize c d by SEM The morphologie and dynnamic light scattering (DLS) meaasurements (Figure 5). Unlike thee SEM resuults of the compouunds interactting with cttDNA, the rresulting conndensed graanules of pU UC18 DNA As featured narrow size distributions and regular shaapes. The siizes obtaineed from DLS were in a range of 107‒1344 nm. Such size distribuutions weree propitious to cellular eendocytosis and subseqquent gene transfecctions.50, 51 N Noticing thaat the granuular sizes obbserved witth SEM weere somewhat smaller than thoose with DL LS, which was a freqquently obseerved phenoomenon cauused by thee different techniquues of sampple preparatiions. The drried samples in SEM were w prepareed by dehyddrating the condenssed particless into a decrreased apparrent sizes. D DLS provideed the size ddistributionss in situ in which thhe larger coondensates ccontributed m more than thhe smaller oones.52

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mages and D DLS size disstributions of o 1 (A), 2 (B), 3 (C), and 4 (D) ccondensed Figure 5. SEM im pUC18 DNA nanopparticles, scale bars 1.00 μm. [1‒3]: 20 µM, [4]]: 30 µM. [ppDNA]: 9 μgg/mL. NAs were tthen investtigated to provide prreliminary The releases off the comppacted DN whether theese amphiphiles could serve as ppotential canndidates forr nonviral evaluatiions about w gene veectors. Currrently, num merous tactiics, such ass linkage bbreaking, pH H jump, adddition of additivees, etc., havee been deveeloped for trriggering thhe dissociation of DNA A from the coondensing state in vitro. Hereein, a high-cconcentratioon NaCl solution was employed tto release thhe packed As shown inn Figure S88A, after treeating with 600 mM N NaCl, the coondensed DN NAs were DNA. A mostly released, suuggesting that t the conndensation caused by the amphipphilic molecules was reversibble. Althouggh such highly concenttrated NaCll is fatal to the living cell, the exxperiments intendedd to indicatte the potenntial reversibbility of DN NA condenssation. In siitu DNA reelease was 11



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further confirmed by the fluorescence quenching upon addition of highly-concentrated NaCl solutions to the solutions of these amphiphiles in the presence of ctDNAs (Figure S8B). Although the fluorescence was slightly higher than the original intensities, addition of NaCl caused dramatically decreased emission. Added NaCl led to electrostatic competition of sodium ions with the condensing agents, which resulted in effective DNA release and at the same time, switched the co-aggregated nanoparticles back into self-assembled micelles. Moreover, DLS and TEM also verified the reversion from the coaggregated particles to micelles (Figure S9 and S10). In DLS, the particle sizes around 1080 nm assigned to the released ctDNA biomacromolecules and sizes around 18 nm assigned to the reassembled micelles of 2 were both detected after introducing NaCl to the solutions of 2/ctDNA. And the reassembled micelles were directly visualized by TEM (shown by arrows). In vitro stability of DNA condensates: Sustained stability of a nonviral gene delivery system in extracellular solutions is of critical importance. To investigate the stability of the DNAcontaining nanoparticles, a time-dependence particle size analysis by DLS was performed (Figure S11). In physiological conditions, complete DMEM medium and in the presence of serum, the sizes of the particles maintained almost unchanged during an incubation time over 24 h. The satisfactory stability is very conductive to promote the cellular transfection and should be benefit of further applications in vivo. DNA Binding Abilities: The effects of ionic strengthen condensation and the binding capabilities of the four compounds with DNAs were assessed with ethidium bromide (EB) exclusion assays. As depicted in Figure S12, including NaCl in the condensing procedures evidently weakened the binding forces due to partial neutralization of the phosphate backbones,52 which in turn decreased the binding affinities of the four amphiphiles with DNAs. 12


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EB is a frequently used dye that stains double-stranded DNAs. Upon intercalation into adjacent DNA base pairs and groove binding with the secondary structure of DNA, EB emits red fluorescence. Therefore, quenching of the fluorescence by replacing EB molecules in the DNAs is a valid approach to evaluate the DNA-binding ability of extraneous molecules. In Figure S13, upon addition of the four compounds, the emission intensities of EB-containing ctDNA solutions gradually diminished. Deduced from the plots of 1-I/I0 (Figure 6), the apparent binding constants (Kapp) of the four amphiphiles 1-4 were determined to be 4.51 × 106, 4.35 × 106, 6.21 × 106, and 4.97 × 106 M-1, respectively. These results implied that the longer the alkyl chain, the stronger the binding affinity. Changing the saturated hydrocarbon chain to an unsaturated one weakened the binding strength, which is consistent with the results obtained above. However, compared with our reported TPE molecules without the long hydrophobic chains,30 the binding affinities of the four TPE derivatives herein were much weaker, indicating that incorporating long alkylchains can help reducing the binding strength between condensing agents and DNAs, which should favor DNA disaggregation in the cellular metabolism process. Combination of AGE assays and EB displacement, we conclude that the mutual electrostatic interactions between the amphiphiles and DNA molecules is the major driving force for the formation of condensed nanoparticles, while some other supramolecular interactions such as hydrophilic and hydrophobic interactions, π-π stacking and hydrogen-bond interactions also play active roles.

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Figure 6. Plots of 1 − I/I0 vs concentrationns of the fouur amphiphhiles, λex= 5337 nm, [EB]] = 20 µM, 2 °C, 5mM M Tris-HCl, 50 mM NaC Cl, pH 7.4. [DNA] = 100 μM, 25 Studiies of Cellu ular Uptakee: To examiine whetherr these miceelles and thee condensatees of nonlabeled pUC18 DN NA induced by the fouur compoundds can be innternalized into cells bby cellular H cells were incuubated with the four am mphiphiles aand the correesponding transporrt systems, HepG2 condenssates for 4.00 h and thenn observed bby using coonfocal laser scanning m microscopyy (CLSM). In Figurres 7, S14 aand S15, CLSM C imagees suggested that thesee micelles aand their coondensates can be ttaken up byy the cells. For the miccelles (Figuure 7A and S14), the blue b fluoresccence was visible tthroughout the t cytoplassm, but not in the nucleeus, indicatinng that the micelles m didd not enter into thee nucleus. W With the aiid of fluoreescence, thee cellular sttructures were w visuallyy imaged. Consequuently, thesse micelles hhave a greaat potential as dyes of self-assembbled nanopaarticles for stainingg the cells. H However, thhe subcellullar investigaations of theese blue conndensates prresented a rather distinct d form m (Figure 7B B and S15). The condeensates centrralized as blue b fluoresccence dots in a certtain area off the cells (yyellow arrow ws). These dots were m mainly locaated in the ccytoplasm, but quitte a few dotts also entered into thee nucleus. T The discrimiinating pattterns and loocations of the distrributions deemonstratedd that the coondensates themselves successfullly penetrateed into the 14


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cells. Thhe cellular uptake abillities of HeppG2 cells oon the four compoundss and their fabricated condenssates of pU UC18 DNAss were quanntitatively eevaluated byy flow cytoometric assaay (Figure S16). Thhe percentaages of the innternalized cells were nnearly 100% % under theese two circuumstances due to aall of the internalized ceells had an oobvious red shift in the fluorescencce intensitiees. Despite the factt that the em mission intensities of the four coompounds ccould be evvidently enhhanced by adding D DNAs, all oof these com mpounds annd condensaates showedd similar fluuorescence intensities. We specculate that iit was causeed mainly byy the intake of lower am mounts of condensates compared to that of free m micelles, leaading to sim milar fluoreescence inttensities dissplayed by the flow cytomettry. Underlyying the aboove results, the easy ceellular uptakkes confirmeed the possiibilities of using thhese gemini amphiphilees as effectivve nonviral gene vectorrs.

Figure 7. CLSM im mages (20× ×) of HepG22 cells incubbated with tthe four am mphiphiles (A A, 20 μM, a1: 1, aa2: 2, a3: 33, and a4: 44) and the condensatees of pUC118 DNA coondensed byy the four amphiphhiles (B, [1‒‒3]: 20 µM, [4]: 30 µM M, b1: 1, b22: 2, b3: 3, aand b4: 4) foor 4.0 h. Im mages of A were obbtained from m the blue channels, im mages of B were w the meerged imagees of bright fields and blue chaannels. [pDN NA]: 9 μg/m mL, scale baars: 20 μm. 15



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Cytottoxicity: Considering their potenntial applicaation in the area of gene therappeutics as nonvirall gene vecctors and trracers, the cellular toxxicities of the deliverring agentss must be understoood. The w widely used MTT assayy of assessiing eukaryootic cell viaabilities wass adopt to assess tooxicity. As presented iin Figure 8,, the resultss for the fouur different types of caancer cells (HepG22, A549, HE EK293T andd HeLa) indiicated excelllent biocom mpatibilities possessed bby all four compouunds. Despitte that increeased cytotooxicity was observed w with increassing feedingg dosages, all of thhe cells mainntained ˃800% metaboliic viabilitiess at concenttrations up to 30 μM. Inn addition, the perccentages of viable cellss in the pressence of thee four comppounds weree no less thaan 60% in most caases at conceentrations aas high as 50 μM. As a result, the inherent biological hyppotoxicity suggestss that the foour amphiphhiles are suittable as nonnviral gene ccarriers.

Figure 8. Cytotoxiccity of A549 (A), HEK K293T (B), H HeLa (C) annd HepG2 (D D) cells culttured with o compounnds 1‒4. differennt concentrattions (μM) of In Viitro Gene T Transfection: A qualitative evaluaation of the transfectionn abilities oof the four lipids aand the lipoosomes (coomposed byy lipids 1‒4 with DO OPE in a 11:1 molar rratio) was conductted by transffecting pEG GFP-N1 repoorter gene in HepG2 ceell lines. Exxperiments uusing cells 16


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FP and DOP PE/pEGFP, and blank cells c as conttrols were performed. T The results culturedd with pEGF shown iin Figure 9 indicates thhat the four ccompounds are able to deliver the GFP geness into cells and leadd to expresssion of GFP. Lipids 1 annd 2 showed higher traansfection effficiencies ((TEs) than lipids 3 and 4. In adddition, the densities of the cells trransfected bby the liposomes of 1/D DOPE and 2/DOPE E were significantly ennhanced andd were compparable to thhe outcomee of the com mmercially availablle lipofectam mine 2000 (Lipo20000). The incoorporation oof auxiliaryy DOPE, a naturally occurrinng lipid, thuus can effecctively enhaance the trannsfection effficiencies oof the geminni lipids 1 and 2. F For 3 and 44, however,, complexattions with DOPEs D resuulted in insiignificant inncrease of green fluuorescence spots.

Figure 9. Expressioons of gene encoding fo for GFP trannsferred intoo HepG2 celll lines by (aa) 1, (b) 2, PE, (i) Lip22000, (j) pE EGFP, (k) (c) 3, (dd) 4, (e) 1//DOPE, (f) 2/DOPE, (gg) 3/DOPE, (h) 4/DOP DOPE/ppEGFP, (l) bblank. Conccentrations = 30 μM, [ppEGFP-N1] = 9 μg/mL. Next, the expresssion of lucifferase was ddetermined to quantitattively assesss the TEs oof the four lipids annd liposomees by transfeecting pGL--3 plasmid iin HepG2 ceell lines. Ass it is well kknown, the concenttrations and ratios of D DOPE play critical c rolees in the trannsfection prrocesses, whhich exert 17



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large influence on the TEs. Accordingly, transfection at different concentrations and molar ratios of lipids with DOPEs were measured in detail. TEs were reported as RLU (relative light unit) per mg of total protein content. As shown in Figure S17 and 10A, lipid concentrations have a major impact on TEs. The optimal transfection concentrations for lipids1‒4 are all at 30‒40 μM. The TEs of lipids 1 and 2 are much higher than those of lipids 3 and 4. Compared with Lipo2000, the best TEs of lipids 1‒4 are 55.4%, 59.6%, 1.4% and 4.9%, respectively. Although showing less favorable capacities to deliver pGL-3 plasmid in comparison with Lipo2000, lipids 1 and 2, as small molecules for transferring genes without DOPE, may serve as nonviral gene carriers due to their low cytotoxicity, precise structures and straightforward components. The cell transfections in HepG2 cell lines by using the liposomes of 1/DOPE ‒ 4/DOPE are discussed below (Figure S18‒S21 and 10B). Based on the obtained results, two conclusions can be made: first, liposomes 1/DOPE and 2/DOPE greatly boosted the TEs. For 3 and 4, however, the corresponding liposomes did not promote the TEs. In fact, their TEs were still much lower than those of liposomes 1/DOPE and 2/DOPE and Lipo2000. Second, the concentrations and molar ratios of DOPE with the lipids greatly affected the TEs. Under optimal conditions, i.e., with the molar ratios of lipids 1 and 2 with DOPE being 1/0.5 and 1/1 at a concentration of 30 µM, the best TE of 1/DOPE was comparable to Lipo2000 (98%), and that of 2/DOPE was even better than Lipo2000 (175%). Results from the transfection assays in HepG2 cells clearly demonstrate that the two gemini lipids hold great promise as nonviral gene carriers. Under optimal conditions, transfection assays mediated by these lipids and liposomes in other cells, including A549, HeLa and HEK293T were also carried out to give a comprehensive inspection on the TEs. As shown in Figure S22 and 10C, among the four types of cancer cells tested, the best TEs are observed with the HEK293T cell lines, then the HeLa cell lines, followed 18


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by the A549 and HepG2 cell lines. The highest TEs (% of Lipo2000) are summarized in Table S1. Despite that better transfection results (absolute transfection values) were obtained in the subsequent three cell lines in comparison with HepG2 cells, the TEs were slightly or even significantly lower than that of Lipo2000. Moreover, the results also indicated that the best TE of liposome 1/DOPE were superior to those of 2/DOPE in Hela and HEK293T cell lines, which made a great divergence to the transfections in HepG2 cell lines. Due to distinct transfection pathways possessed by the different cell types, the different TEs of the four lipids and liposomes between various cell lines might be acceptable.53,

54

Above all, the four gemini amphiphiles

exhibit great potential for expanding novel structures with rigid TPE frameworks as generally applicable nonviral gene vehicles.

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Figure 10. (A) Reesult histoggrams preseenting the rrelative trannsfection effficiencies oof varying l (B) L Luciferase concenttrations of lipids 1‒4 with Lipo22000 as control in HeepG2 cell lines; 20


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expressions transfected by liposomes (b1) 1/DOPE, (b2) 2/DOPE, (b3) 3/DOPE, (b4) 4/DOPE with molar ratios of 1/0.5‒1/3 at varying concentrations. (C) Result histogram of luciferase expressions mediated by lipids 1‒4 and liposomes 1/DOPE‒4/DOPE under the respective optimal conditions. Concentrations for lipids 1, 2, liposomes 1 and 2 were 30 μM, and for lipids 3 and 4, liposomes 3 and 4 were 40 μM, the ratios of 1/DOPE, 2/DOPE, 3/DOPE and 4/DOPE were 2/1, 1/1, 1/2 and 1/2. [pGL-3] = 9 μg/mL. Based on the transfection data, several points can be made. First, among the four gemini lipids, pGL-3 plasmid could be favourably transfected in all of the selected cell types by lipids 1 and 2 without the help of natural lipid DOPE, while lipids 3 and 4 exhibited rather low TEs. In addition, incorporations with helper lipid DOPE seemed beneficial to increase the TEs of 1 and 2, whereas it had no effect on improving the transfection abilities of 3 and 4. Second, the short-length lipids 1 and 2 and their liposomes exhibited fairly good TEs, which were comparable to those of Lipo2000 in many cases. Even more impressive is that liposome 2/DOPE generated 1.75-fold higher TE than Lipo2000 in HepG2 cell lines. Furthermore, lipids and liposomes (1 and 2, 1/DOPE and 2/DOPE) with shorter-length chains showed much higher TEs than their longerchain counterparts (3 and 4, 3/DOPE and 4/DOPE). Nevertheless, the cellular uptake studies showed all four lipids could effectively deliver their condensed DNAs into the cells. Two possible explanations are presented as the most likely based on what was reported: on one hand, shorter hydrocarbon chains generally accelerate the intermembrane transfer rate of the lipid monomers and lipid membrane mixing, which leads to increased fluidity of the bilayers of liposomes, gives rise to potentially disrupted endosomes and ensues endosomal escape of DNAs.25,

55

On the other hand, an elongated tail generally strengthens the stability of the

compacted supramolecules, which hampers DNA detachment, thus reduces the corresponding 21



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TEs.56 Third, compared with lipid 3 and its liposome 3/DOPE, 4 and 4/DOPE showed relatively higher TEs, indicating that replacing a saturated aliphatic chain with an unsaturated one could improve, to certain degree, the transfection ability. The underlying reason may be associated with the potential influence on membrane fluidity,57-60 which suggests that introducing unsaturated alkyl chains can bring out a more susceptible change of the supramolecular condensates.61 Briefly, gemini amphiphiles 1 and 2 showed great potential in gene delivery and are worthy of being extended to the design of other TPE-based nonrival gene vectors. The obtained results should furnish valuable guidance for the development of new synthetic gemini lipids. Tracing the delivery of non-labeled and FAM-labeled DNAs (pUC18 and FAM-DNA): Based on the observed transfections, the differentiated morphology features, the distribution characteristics of the self-assembled AIE micelles, co-assembled nano-aggregates in the living cells, and the self-transition from the condensates to micelles in solutions, compound 2 was selected as representative for tracking the delivering and releasing processes of pUC18 DNA (no fluorescence) and FAM-labelled DNA (green fluorescence). As shown in Figure 11, at 0.5 h, only a small fraction of blue-emission condensates entered into the cells, and the condensates mainly gathered together around the cytomembrane. Therefore, the resultant cellular fluorescence was extremely weak. However, more condensates transferred into the cells after 2.0 h, and the majority of them were found to locate in the cytoplasm. A close survey revealed that some condensates had translocated into the nucleus. As the incubation lasted, the number of the condensates internalized into the cells continued to increase (4.0 h). It can be seen that a large portion of condensates had covered the nucleus. These results demonstrate that this unique gene vector can indeed enter into the nucleus together with DNAs, something that is very rare in 22


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known systems. Furthermore, this delivery behavior could effectively prevent the DNase in cytoplasm from degrading the released DNA, which might be responsible for the decent transfections shown by the lipids. After incubating for 4 h with the condensates, followed by carefully washing with PBS (0.5 mL) buffer 10 times, and then culturing in fresh mediums for an additional 20 h, CLSM images at 24 h were captured. Interestingly, similar to the cellular uptakes with free micelles, the fluorescence were well dispersed throughout the cells instead of locating at a certain position in the form of condensates. The conversion of the fluorescence distributions may be caused by the detachment of the DNAs from the condensates and reassembled AIE micelles, which in turn affirmed the in situ transition from the condensates to micelles in living cells. Further evidence suggesting such a transition was gained by monitoring the delivering processes of FAM-DNA (Figure S23 and 12). The uptakes during the initial four hours offered similar results as above. Besides, meticulous examination on the results at 6.0 and 24 h revealed the separation of FAM-DNA from the condensates and the reassembled blue-emission AIE micelles (yellow arrows). These observations clearly illustrate the advantages of reversible, supramolecular packing by using AIE micelles as self-indicating gene delivery systems. With the aid of spatiotemporal transition of the varied aggregation morphologies and fluorescence colors (micelles in “blue” color, FAM-DAN in “green” color, and condensates in “cyan” color), the delivery of DNAs, including label-free and labeled DNAs, were monitored. This concept has provided a new approach for tracking the cellular uptake and release of biomacromolecules. It is anticipated that this strategy could be adopted for applications in other related domains.

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mages (40×)) of HepG2 cells inccubated witth lipid 2 Figure 11. Time dependent CLSM im DNA. [2] = 20 μM, [DN NA] = 9 μgg/mL, scale bars: b 20 μm m. condenssed pUC18 D

Figure 12. CLSM images (40×) of HepG G2 cells incuubated with lipid 2 conddensed FAM M-DNA at 6.0 and 24 h. [2] = 20 μM, [DN NA] = 9 μg//mL, scale bbars: 20 μm.. 24


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Conclusions In the present work, four new TPE based gemini amphiphiles, referring to 1‒4, consisting of two hydrophilic units (triazole-[12]aneN3) and two different-length hydrophobic chains were successfully fabricated for simultaneous DNA condensation, delivery, and tracking of non-labled gene transfer process. The four small-molecule amphiphiles self-assembled into well-defined micelles inheriting intrinsic AIE natures, which showed exceedingly low CMCs (4.02‒4.94 × 106

M), strong fluorescence, and good pH and photo stabilities. The four amphiphiles could also

co-aggregate with ctDNAs into nanoparticles that change assembled morphology and enhance fluorescence. Compounds 1‒4 possessed excellent DNA condensation abilities and nice biocompatibilities. GFP and luciferase expressions in vitro proved that amphiphilic lipids bearing saturated, short and/or unsaturated, long hydrophobic chains are more adaptive as gene vectors. Cellular uptake studies revealed the desired permeability possessed by the four micelles and their DNA complexes. The different morphologies and distribution features, along with the interconversion of the nanocondensates and micelles of compound 2 in living cells successfully proved it to be a good candidate in building nonviral gene conveyers that exhibit AIE characteristics for tracking the intracellular gene delivering process. This work has opened a new avenue to design novel gemini amphiphiles with AIE natures. It has also shed additional insights into traceable gene delivery systems. The system described here can be anticipated to offer valuable information for the fabrication of other more efficient traceable TPE based amphiphiles. Further work in these aspects are in progress. ASSOCIATED CONTENT

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Supporting Information. Detailed information on the experimental section (Materials and instrumentations, general procedures for synthesis and measurements) and additional data (spectra and characterizations) are included in Supporting Information. AUTHOR INFORMATION Corresponding Author * Corresponding authors, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This article was supported by the financial assistance from the Nature Science Foundation of China (21372032 and 91227109), the Fundamental Research Funds for the Central Universities, Beijing Municipal Commission of Education, the Program for Changjiang Scholars and Innovative Research Team in University. REFERENCES (1)

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84x44mm (300 x 300 DPI)


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Gemini-Type Tetraphenylethylene Amphiphiles Containing [12]aneN3

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