Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene...

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDi rect

Bio organic & Medic inal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene vectors

0968-0896/$ - see front matter � 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.03.048

⇑ Corresponding authors. Tel.: +86 2885415886. E-mail addresses: [email protected] (J. Zhang), [email protected] (W. Zhu),

[email protected] (X.-Q. Yu).� These authors contributed equally to this work.

Please cite this article in press as: Liu, Q. ; et al. Bioorg. Med. Chem. (2013), http://dx .doi.org/10.1 016/j.bmc .2013.03.0 48

Qiang Liu �, Qian-Qian Jiang �, Wen-Jing Yi, Ji Zhang ⇑, Xue-Chao Zhang, Ming-Bo Wu, Yi-Mei Zhang, Wen Zhu ⇑, Xiao-Qi Yu ⇑Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, State Key Laboratory of Oral Diseases, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610064, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Received 16 January 2013 Revised 17 March 2013 Accepted 19 March 2013 Available online xxxx

Keywords:Gene delivery CyclenCationic lipids Tocopherol

A series of novel 1,4,7,10-tetraazacyclododecanes (cyclen)-based cationic lipids bearing his tidine imidazole group 10a–10e were synthesized. These amphiphilic molecules have different hydrophobic tails (longchain, cholesterol or a-tocopherol) and various type of linking groups (ether, carbamate or ester). These molecules were used as non-viral gene delivery vector s, and their structure–activity relation ships were investigated. As expecte d, the imidazole group could largely improve the buffering capabilities comparing to cyclen. The liposomes formed from 10 and dioleoylphosphatidyl ethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles with proper size and zeta-poten tials. Comparing with Lipofectamine 2000, the formed lipoplexes gave lower transfected cells proportion, but higher fluores-cence intens ity, indicating their good intracellular deliverin g abil ity. Furthermore, results indicate that transfection efficiency of the cationic lipids is influenced by not only the hydrophobic tails but also the linking group . The cyclen-based cationic lipid with a-tocopherol hydrophobic tail and an ester linkage could give the highest transfection efficiency in the presence of serum.

� 2013 Elsevier Ltd. All rights reserved.

6
1. Introduction
Gene therapy has gained significant attention over the past two decades as a potential method for survival against many diseases, such as cancer, diabetes, cystic fibrosis, AIDS and cardiovascu lar ail- ments, etc. 1,2 The therapy involves the delivery of a specific gene (DNA) to targeted cells to combat the disease at the level of its ori- gin. Research efforts are currently focused on designing safe and effective gene delivery system that compact and protect oligonucle- otides for gene therapy. 3 Traditional ly, gene delivery systems are broadly based on either viral or non-viral mediated vectors. Viral vectors are significantly more efficient in delivering the gene, however, fundamenta l problems including toxicity, immunog enicity, limitations with respect to targeting of specific cell types, and potential for mutagen esis limited their potential clinic use. 4 Amongphysical and chemical based non-vira l vectors, liposome s have par- ticularly excellent potential for gene delivery applications .3

Lipids are amphiphilic organic molecule s that contain a hydro- philic head and a hydrophobic tail bridged by a linkage. 5 Thehydrophilic head group which may bind to the negatively charged

phosphat e group of nucleic acid generally has one (guanidinium, amine,7–9 pyridiniu m moieties 10 or imidazolium 10,11) or more (polyamines 12–14 or amino acids 15–17) cationic groups. The hydrophobic tail, which may be long hydrocarbo n chain or steroids, represents a non-polar part that could form bilayer aggregates and interact with cellular membran e. 7 The linkage usually contains abiodegra dable chemical bond (ester,12,16,17 amide13,17,18 or carbam- oyl15) or a non-degrad able ether bond. 8,9 The structure of cationic lipids is significant for the transfection efficiency of cationic liposomes.3,5 Since the first report from Felgner and co-worke rs, 19 var-ious systematic modifications have been performed at the hydrophobic parts, linkage regions and the head groups to opti- mize the DNA delivery to various mammalian cell lines. However, the available lipids were still far from the requiremen t of in vivo applicati on because of their potential toxicity and relative low transfecti on efficiency. Consequentl y, the development of novel non-toxi c cationic lipid gene vectors is of great importance.

In our ongoing research of designing efficient novel cationic transfecti on vectors, we recently demonstrat ed the potential of novel 1,4,7,10-tet raazacyclodod ecane (cyclen)-based gene delivery systems.20 The use of cyclen as hydrophi lic head group is based on its unique structural characteri stics. Cyclen has four amine groups with different pKa values (pKa = 10.51, 9.49, 1.6 and 0.8, respectively ).21 The amines with strong basicity (pKa = 10.51 and 9.49) can be highly protonated in neutral pH range, leading to its good cationic property and subsequent binding ability toward

http://dx.doi.org/10.1016/j.bmc.2013.03.048

mailto:[email protected]




http://www.sciencedirect.com/science/journal/09680896

http://www.elsevier.com/locate/bmc


2 Q. Liu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx

DNA. Meanwhile, the cyclic backbone which is hard to self-fold can also retain it high DNA binding affinity.20b,22 However, the other amines on cyclen have too weak basicity to be protonated in the more acidic endosome environment (pH 5.0–6.5). In other words, cyclen lacks the amino groups having the so-called ‘proton sponge effect’, which might benefit the endosome escape. 23 Thus, incorpo- rating pH sensitive group whose pKa value is in the endosom al pH range into the cyclen-ba sed lipid might promote endosome escape, leading to better gene transfection. Considering the appropriate pKa of imidazole (�6),24–26 in this report, we designed and synthesized a series of cyclen–imidazole based cationic lipids which might have good buffering ability, and their structures are shown in Figure 1. Some hydrophobic moieties with different functions were attached to the cyclen–imidazole structure , and their interac- tions with plasmid DNA were studied.

2. Results and discussion

2.1. Synthesis of the cyclen–imidazole based amphiphilic lipids

To explore the important relationship between molecular structure and biological function, structure-di versity oriented synthesis has been focused in current chemical biology. 3,5 As shown in Scheme 1, a series of novel cationic lipids with protonated cyclen head group and different hydrophobic tails bridged by a histidine moiety were designed and synthesized . The hydrophobic moieties 1–5 bearing an amino group were firstly prepared through different

NH N

HNNH

NH

HN

N

ON

HN

O

O

NH N

HNNH

NH

HN

O

ON

HN

O

NH N

HNNH

NH

HN

O

ON

HN

O

O

NH N

HNNH

NH

HN

NH

ON

HN

OO

O

5 CF3COOH 10a

10b

10c

10d

NH N

HNNH

NH

HN

O

ON

HN

O

O

10e

O

5 CF3COOH

5 CF3COOH

5 CF3COOH

5 CF3COOH

Figure 1. Structures of the

Please cite this article in press as: Liu, Q. ; et al. Bioorg. Med. Chem. (201

methods . Severel typical hydrophobi c structures including long hydrocarbo n chain, cholesteryl and tocopheryl which may have special propertie s were introduced via amide (1), ether (2, 3),carbamate (4) or ester (5) bonds. On the other hand, cyclen–imidazole moiety 8 was prepared from tri-Boc-cyclen- acetic acid 6 andLhistidine methyl ester 7 in the presence of 1-ethyl-3-(3-dimethyl- aminoprop yl)carbodiimide hydrochlori de (EDC�HCl), N,N-diisopro -pylethyla mine (DIEA) and N-hydroxybenzotr iazole (HOBt).Subseque nt coupling between compound 8 and amines 1–5 gavethe precursor s 9a–9e, respectively. Target lipids 10a–10e were obtained by removing the Boc groups using trifluoroacetic acid in anhydrou s CH 2Cl2. For the five target molecules, 10a–10c (preparedfrom 1 to 3) have different hydrophobic groups, while 10c–10e (pre-pared from 3 to 5) are differed from their linking groups. All new compound s were characterized by NMR and HRMS.

2.2. Buffering capabilit y

As the golden standard for the transfection efficiency of non- viral gene vectors, PEI was known as its good buffering ability and the consequent ‘proton-s ponge effect’. 27 To determine the buffering abilities of the synthesized lipids 10a–10e containing imidazole moiety, acid–base titration studies were conducted over a pH range of 2–10. As shown in Figure 2, comparing with cyclen, the imidazol e-attached molecules showed large improved buffering abilities which were close to PEI. For the different hydrophobic groups, long hydrocarbo n chain (10a) might have the most

O

title lipids 10a–10e.

3), http:// dx.doi.org/10 .1016/j.b mc.2013.03.0 48


NR1

R1

H2NO

H2N OR2 O

R3H2N ONH

OH2N R3 O

H2NO

R3

1 2 3 4 5

R1 = R2 =

OR3 =

cholesteryltocopheryl

N N

NN

NH

OCH3O

N

HN

O

Boc Boc

N N

NN

OH

O

Boc Boc

Boc H2NOCH3

N

HN

OBoc

6 7 8

EDC/HOBt 1) NaOH2) EDC/HOBt/DIEA1-5

2 HCl+DIEA

N N

NN

NH

ON

HN

O

Boc Boc

BocHN

hydrophobic moiety

9a-9e

CF3COOH/CH2Cl2

NH N

HNNH

NH

ON

HN

O

HN

hydrophobic moiety

10a-10e

5CF3COOH

Scheme 1. Synthetic routes of title lipids.

Figure 2. Acid–base titration profiles of 25 kDa PEI, 150 mM NaCl and lipids 10.Lipids, cyclen or PEI (0.05 mmol of amino groups) was first treated with 0.1 N NaOH to adjust pH to 10.0, and then the solution pH was measured after each addition of 20 lL of 0.1 N HCl.

Figure 3. Ethidium bromide displacement assay of plasmid DNA binding abilities for the lipids 10/DOPE liposomes under various N/P ratios in 10 mM Hepes buffer solution. The molar ratio of 10/DOPE was 1:1.

Q. Liu et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx 3

negative effect on the buffering ability, while cholester ol-con- tained 10b gave the best result.

2.3. Interaction s between the liposomes and plasmid DNA

Cationic liposome s are formed from either individual cationic lipid or more frequently from a combinati on of cationic lipid and neutral lipids such as DOPE, which might increase the transfecti on efficiency significantly. Each of cationic lipids was mixed in different molar ratios (1:0, 1:1, 1:2, 1:3 and 1:4) with DOPE to determine the optimized combination. According to the best behavior in the transfection experiment (data not shown), the 10/DOPE ratio of 1:1 was used herein. Ethidium bromide (EB) dye replacemen tand agarose-g el retardant assays were first employed to evaluate their plasmid DNA binding abilities. 15 Figure 3 shows the EB dye replacemen t assay result of relative fluorescence intensity as a


function of N/P charge ratio in 10 mM of HEPES solution (pH7.4). It was shown that the fluorescent intensities were signifi-cantly decreased with the rise of N/P ratio (0–5). At the N/P ratio of 5, only 0–10% of original fluorescent intensities were found. The parameters of CE50 which represent the N/P charge ratios for quenching 50% fluorescence intensity of EB were evaluated to be 2.7, 2.5, 1.6, 1.4 and 1.9 for lipids 10a–10e, respectivel y, indicating that the tocopherol moiety may benefit the DNA affinity (10c–10e). Besides, agarose gel retardation assay was also applied to evaluate the interaction between 10 and pDNA. Results in Figure4 show that completely DNA retardation could be achieved at N/ P of 4 for the liposomes formed from all five lipids. Therefore, both assays demonst rated the strong interactio n of 10 with plasmid DNA.

Average particle size, surface charge and morphology of the lipoplexes may largely influence the apparent cytotoxicity, intracellular uptake and trafficking, and release of the encapsul ated

3), http://dx .doi.org/10.1 016/j.bmc .2013.03.0 48


Figure 4. Electrophoretic gel retardation assays of lipids 10/DOPE/pDNA complexes at different N/P ratios. The molar ratio of 10/DOPE was 1:1.

Figure 6. Cytotoxicity assays of the lipoplexes prepared at various N/P ratio.


gene.28 Figure 5a depicts the N/P ratio depende nce of mean particle sizes of the lipoplexes formed from 10a to 10e . The average diameters were observed in a range of 95–190 nm, which strongly de- pend on the N/P ratio. In a general view, the average diameters gradually dropped with the increase of N/P, and then reach a pla- teau of about 100–140 nm at N/P ratio of 6. This result indicated effective condensation of DNA by the liposomes via polyion com- plexation (PIC).29 EB replacement assay (Fig. 3) had shown that lipids 10a and 10b have relatively lower DNA binding abilities, and as a result, the lipoplexes formed from 10a and 10b showed larger diameters than those from 10c to 10e . On the other hand, surface potentials of the lipoplex nanoparti cles were also measured by DLS, and the results were shown in Figure 5b. Zeta potentials of the five lipoplexes showed a similar trend along with the change of N/P ratio: they increased from ��12 mV to �38 mV when N/P ratio increased from 2 to 8. The negative zeta potentials at N/P of 2 were from the poor DNA binding at this N/P ratio. With the increase of the cationic materials, the zeta potentials increased and turned positive from N/P of 4.

2.4. Cytotoxicity of the lipoplexes

Low cytotoxicity of a synthetic gene delivery carrier was another key factor for clinic gene therapy, and the cytotoxicities of the prepared lipoplexes were examined in A549 cell line by MTT assay. The results shown in Figure 6 revealed that the variation of both hydrophobic tails and linkages would influence the cytotoxicity. Under all tested N/P ratios, lipoplex formed from 10bshowed higher toxicity than those from 10c. Considering the sole structural difference between 10b and 10c, we speculate that a-tocopherol moiety may be more biocompatib le than cholesterol moiety in this kind of cationic lipids. For the lipoplexes form from a-tocopherol-co ntained molecule s (10c–10e), the one from 10dshowed higher toxicity, especiall y in higher N/P ratios, suggestin gthat carbamate linkage might not be good choice. 20 On the contrary, the long chain tail may benefit the biocompatibili ty, and the lipopolex from 10a showed highest cell viability. All of these lipoplexes showed lower cytotoxicity than lipofectamine 2000, indicating their potential as gene delivery vectors.

Figure 5. Mean particle sizes (a) and zeta-potentials (b) of the lipoplexes form


2.5. In vitro gene transfection

The report gene of enhanced green fluorescent protein (eGFP)was used in the in vitro gene delivery to directly visualize the transfected cells. Lipids 10 mediated eGFP expression in A549 cells was observed by an inverted fluorescent microscope, and lipofectamine 2000 was used as control. Lipoplexe s were prepared at three N/P ratios (4, 6 and 8), and the transfection images are shown in Figure 7. The transfection mediated by five lipoplexes all showed the strongest fluorescent emission at N/P of 6, while tocopherol -containe d lipids 10c–10e (Fig. 7B, E and H) gave much better results than 10a (Fig. 7J) and 10b (Fig. 7K) bearing other hydrophobic groups. The eGFP images showed similar high transfection effi-ciency of 10c–10e comparing to lipofectamine 2000 (Fig. 7L). To further measure the levels of eGFP expression, subsequent flowcytometr y assay was conducte d, taking both % eGFP positive cells and RFI (relative fluorescence intensity) into account. The RFI is de- fined as the relative fluorescence intensity in eGFP positive cells,

ed from 10a to 10e under various N/P ratios (DLS at room temperature).



Figure 7. Fluorescent microscope images of A549 cells transfected by 10c (A–C), 10d (D–F), 10e (G–I), 10a (J), 10b (K) and lipofectamine 2000 (L). Lipid/DOPE ratio was 1:1, N/P ratios were 4 (A, D, G), 6 (B, E, H, J, K) and 8 (C, F, I). The cells were observed by fluorescence microscopy after 24 h transfection.


and lipofectamine 2000 was used as standard. Considering the weak transfection efficiency of 10a and 10b, only 10c–10e was applied to flow cytometry assay. As shown in Figure 8a, the best transfection efficiency of 10c–10e was obtained at the N/P ratio of 6, and the proportio ns of eGFP positive cells were about 30% of that involving lipofectamine 2000. 10d showed slightly lower efficiency than 10c and 10e, which might be ascribed to its higher cytotoxicity (Fig. 6). However, to our delight, the RFI of 10c–10ewere about 1.4 times higher than the control (Fig. 8b). A higher RFI value correlates positivel y with a higher eGFP expression. 30

From these results it was suggested that the lipoplexes formed from cyclen-tocop herol materials might face weaker endocytosis than those containing lipofecta mine 2000, but these particles exhi- bit much better intracellular behavior, leading to higher eGFP expression.

The transfection efficiencies of tocopherol-conta ined lipids 10c–10e were further investigated in the presence of serum. The serum incompa tibility of cationic lipids during gene transfecti on was believed to be caused by the adsorption of negatively charged serum proteins onto the positivel y charged lipoplex surface, therefore preventing their efficient interaction with cell membrane and subsequent endocytosis. The eGFP assays were conducted with the

Figure 8. Transfection efficiencies of 10c–10e at various N/P ratios in comparison to lipowas 1.0 lg/well. Data were expressed as ratio of eGFP transfected cells (a) and RFI (b) o


medium containing 10% fetal bovine serum (FBS). Similarly , the fluorescent microscope images showed the strongest fluorescenceat N/P of 6. Figure 9 shows the relative flow cytometry assay results, and the proportio n of eGFP transfected cells within the experime nts using 10c–10e were still less than that observed in experime nt using lipofectamine 2000. The transfection efficienciesof 10c and 10d were dramatically decreased comparing to the experime nts without serum. But on the contrary, 10e gave even better result with serum, especially at higher N/P ratios (6 and 8). These results demonstrate that 10e has higher serum-re sistance capability , which might be caused by the ester bond in the molecular structure . It is reported recently that a-tocopherol tail and ester linkage-con tained lipid showed high serum compatibilit y and increased transfection efficiency with the increase of the serum concentr ation. 17 Like cholesterol, tocopherol can also change the configuration of the liposome s to resist protein-induced disrup- tion, but in a more pronounced manner, thus making tocopherol- containe d liposome s as efficient delivery vectors in vivo. 17,31

Results herein further demonstrat e that not only the hydrophobic moiety plays important role in the transfecti on process with serum, the linking group also have significant effect on the gene delivery results.

fectamine 2000. The molar ratio of lipid/DOPE was 1:1, and concentrations of DNA btained from flow cytometry analysis.



Figure 9. Transfection efficiencies of 10c–e at various N/P ratios in the presence of 10% serum in comparison to lipofectamine 2000. The molar ratio of lipid/DOPE was 1:1, and concentrations of DNA was 1.0 lg/well. Data were expressed as ratio of eGFP transfected cells (a) and RFI (b) obtained from flow cytometry analysis.


3. Conclusions

Cyclen was combined with imidazole moiety to improve its buffering ability. Several cyclen–imidazole-conta ined amphiphilic molecules bearing various hydrophobi c tails and linkages were designed and synthesized. These molecules were used as non-viral gene delivery vectors, and their structure–activity relationships were investigated . The target lipids showed good buffering capability, high DNA binding ability, and can condense DNA into stable lipoplex nanoparticl e with proper size and zeta-potent ial. Although the proportio n of transfected cells was lower than that using commercially available lipfectamine 2000, these lipids showed dis- tinctly higher RFI, indicating their good intracellular delivering ability. Tocopherol was found to be the best choice of hydrophobic group. Furthermore, besides the hydrophobi c moiety, the structure of the linkage also has significant effect on the transfection results, especially in the experiments with serum. The cyclen-ba sed cationic lipid with a-tocopher ol hydrophobic tail and an ester linkage could give even higher transfection efficiency in the presence of serum. These results may help us to develop novel non-viral gene vectors with higher efficiency and lower cytotoxic ity.

4. Experimental section

4.1. Materials and methods

All chemical s and reagents were obtained commerc ially and were used as received. Anhydrous chloroform and dichlorometha ne were dried and purified under nitrogen by using standard methods and were distilled immediately before use. [4,7,10-tri s(tert-butoxy-carbonyl)-1,4,7,10-tetraaza- cyclododecan- 1-yl] acetic acid (com-pound 6, Scheme 1),32 ether containing a-tocopherol amine (com-pound 1)9 and long chain-containe d amine (compound 3)6 weresynthesized according to the literature. High molecular weight PEI (branched, average molecular weight 25 kDa), and MTT (3-(4,5-dimethylthi azol-2-yl)-2,5-diphenyltetrazo lium bromide) were pur- chased from Sigma–Aldrich (St. Louis, MO, USA). The plasmids used in the study were pEGFP-N1 (Clontech, Palo Alto, CA, USA). The 1640 Medium and fetal bovine serum were purchase d from Invitrogen Corp. A549 (human lung adenocarci noma epithelial cells) was pur- chased from the American Type Culture Collection (ATCC).

4.2. Preparation of cyclen-based cationic lipids

4.2.1. Synthesis of conjugate 8A solution of triBoc-cyclen- acetic acid 6 (2.12 g, 4 mmol) and

histidine methyl ester dihydrochlo ride (968 mg, 4 mmol) in CH 2Cl2


(50 mL) was cooled to 0 �C, then N-hydroxybenzotr iazole (HOBt,648 mg, 4.8 mmol), EDC �HCl (920 mg, 1.2 mmol) and N,N-diisopro -pylethyla mine (DIEA, 1.55 g, 12 mmol) were added and the reac- tion mixture was stirred for 1 h at 0 �C and at room temperat ure overnight. The mixture was then washed with saturated aqueous NaHCO3 solution (2 � 20 mL) and saturated brine (20 mL). The organic phase was dried over Na 2SO4, filtered, and concentr ated to afford a white solid, which was further purified by column chromatography on silica gel (CH2Cl2/MeOH = 20:1, v/v) to yield 8 (2.0 g, 2.93 mmol, 73.3%). 1H NMR (CDCl3, 400 MHz): d = 7.55 (s, 1H, imidazole), 6.81 (s, 1H, imidazol e), 4.80 (t, 1H, J = 9 Hz, NH–CH), 3.71 (s,3H, O–CH3), 3.56–2.95 (m, 16H, cyclen-H , cyclen-CH 2, imidazol e- CH2), 2.79–2.64 (m, 4H, cyclen-H), 1.44 (s, 27H, Boc-H). HR-MS (ESI): C32H56N7O9 [M+H]+, 682.4134, found 682.4140.

4.2.2. Preparation of compou nd 2Phthalim ide potassium (2.2 mmol) and cholester ol-5-en-3 b-

oxyethane bromide (2 mmol) were added into DMF (20 mL), and the solution was heated at 100 �C for 12 h. Solvent was evaporated in vacuum. The crude product was dissolved in ethanol (20 mL),and hydrazine hydrate (2 mL) was then added to the solution. After refluxed at 80 �C for 2 h, the solvent was evaporated in vacuum. The residue was dissolved in ethyl acetate (40 mL) and the organic layer was washed with brine for three times (3 � 20 mL). Organic solvent was evaporated to get an oil. The product was isolated as a pure material upon column chromatograp hy on silica gel (CH2Cl2/MeOH/NH3�H2O = 20:1, v/v) to yield compound 2 as white solid. Yield 30%: 1H NMR (CDCl3, 400 MHz): d = 5.34 (s, 1H, C@C–H), 3.50 (t, 2H, J = 5.2 Hz, O–CH2), 2.84 (t, 2H, J = 5.2 Hz, O–CH2),3.15 (m, 1H, cholester ol-H), 2.37–0.84 (m, 40H, cholesterol-H),0.66 (s, 3H, cholester ol-H). HR-MS (ESI): C29H52NO [M+H] +,430.4043 , found 430.4038.

4.2.3. Preparation of compou nd 4a-Tocopherylch loroformate (2 mmol) in 20 mL CH 2Cl2 was

added drop-wise to a solution of ethylenedia mine (20 mmol, 10 equiv) in 30 mL CH 2Cl2 and stirred at room temperature for 24 h. The mixture was then washed with water (30 mL) and saturated brine (30 mL). The organic phase was dried over Na 2SO4, fil-tered, and concentr ated to afford a oil, which was further purifiedby column chromatography on silica gel (CH2Cl2/MeOH = 20:1, v/v)to yield compound 4 as an oil. Yield 95%: 1H NMR (CDCl3,400 MHz): d = 3.34 (t, 2H, J = 5.6 Hz, NH–CH2), 2.90 (t, 2H, J = 5.6 Hz NH 2–CH2), 2.57 (t, 2H, Ph-CH 2), 2.07 (s, 3H, Ph-CH 3),2.05 (s, 3H, Ph-CH 3), 2.00 (s, 3H, Ph-CH 3), 1.78 (m, 2H, C–CH2,J = 6.8 Hz), 1.55–1.03 (m, 24H, –(CH2)9, –(CH)3, C–CH3), 0.87–0.83(m, 12H, –CH3). HR-MS (ESI): C32H57N2O3 [M+H]+, 517.4364, found 517.4357 .




4.2.4. Preparat ion of compound 5N,N0-Dicyclohexylcar bodie (2.2 mmol) in 10 mL CH 2Cl2 was

added drop-wise to a solution of a-tocopherol (2 mmol), N-(tert-butoxycarbony l)glycine (Boc-glycine, 2 mmol) and DMAP (0.02 mmol) in 30 mL CH 2Cl2. After stirred at room temperature for 12 h, the insoluble precipitate was filtered off, and the solvent was removed to afford an oil, which was further purified by column chromatography on silica gel (CH2Cl2/MeOH = 20:1, v/v) to yield Boc-protected intermediate as an oil. Yield 50%: 1H NMR (CDCl3, 400 MHz): d = 4.21 (s, 2H, NH–CH2), 2.58 (t, 2H, Ph-CH 2,J = 6.4 Hz), 2.08 (s, 3H, Ph-CH 3), 2.00 (s, 3H, Ph-CH 3), 1.97 (s, 3H, Ph-CH3), 1.82 (m, 2H, C–CH2), 1.55–1.05 (m, 33H, Boc-H, –(CH2)9,–(CH)3, C–CH3), 0.86 (m, 12H, –CH3). HR-MS (ESI): C36H62NO5

[M+H]+, 588.4623 , found 588.4620. Compound 5 can be obtained by removing the N-(tert-butoxycarb onyl)-protected group of the intermediate with CF 3COOH, and used without any purification.

4.2.5. Preparat ion of precursors 9Compound 8 (341 mg, 0.5 mmol) was dissolved in methanol

(20 mL). 2 N NaOH (10 mL) was added. After stirring for 2 h at room temperature , the solvent was removed under reduced pressure. The solution was acidified to pH 3 by 2 N HCl, and then the solvent was removed under reduced pressure to obtain a white product which was directly coupled with amine 1–5 (0.5 mmol)in the presence of HOBt (81 mg, 0.6 mmol), EDC �HCl (115 mg, 0.6 mmol) and DIEA (259 mg, 2 mmol) in 50 mL CH 2Cl2 at 0 �Cfor 1 h and at room temperature overnight. The mixture was then washed with saturated aqueous NaHCO 3 solution (2 � 20 mL) and saturated brine (20 mL). The organic phase was dried over Na 2SO4,filtered, and concentrated to afford an oil solid, which was further purified by column chromatography on silica gel (CH2Cl2/MeOH = 20:1, v/v) to yield the synthetic precursor.

Compound 9a (375 mg, 0.32 mmol, yield: 64%): 1H NMR (CDCl3,400 MHz): d = 11.76 (s, 1H, imidazol e), 7.49 (s, 1H, imidazole), 6.83 (s, 1H, imidazole), 4.55 (s, 1H, NHC–H), 3.90 (s, 2H, NH–CH2), 3.34–3.20 (m, 20H, cyclen-H, CON–CH2, cyclen-CH 2, imidazole-C H2),1.51–1.36 (m, 31H, Boc-H, CONCH 2–CH2), 1.51–1.23 (m, 52H, (CH2)13), 0.85 (t, 6 H, J = 6.4 Hz, –CH3). HR-MS (ESI): C65H122N9O9

[M+H]+, 1172.936 0, found 1172.9364. Compound 9b (380 mg, yield: 70%):1H NMR (CDCl3, 400 MHz):

d = 11.79 (s, 1H, imidazole), 7.54 (s, 1H, imidazole), 6.75 (s, 1H, imidazole), 5.31 (s, 1H, C@C–H), 4.51 (t, 1H, NHC–H, J = 6.8 Hz),3.37–3.08 (m, 20H, cyclen-H, NH–CH2–CH2–O, cyclen-CH 2, imidazole-CH2), 2.96–2.87 (m, 1H, cholesterol-H), 2.75–2.62 (m, 4H, cyclen-H), 2.09–1.78 (m, 6H, cholesterol-H), 0.94–0.83 (m, 62H, Boc-H, cholesterol-H), 0.64 (s, 3H, cholesterol-H). HR-MS (ESI):C59H100N8O9 [M+H]+, 1079.7843, found 1079.7842.

Compound 9c (337 mg, 0.3 mmol, yield: 60%):1H NMR (CDCl3,400 MHz): d = 11.76 (s, 1H, imidazole), 7.51 (s, 1H, imidazole),6.78 (s, 1H, imidazole), 4.55 (t, 1H, J = 6.8 Hz, NHC–H), 3.90 (s,2H, NH–CH2), 3.34–3.20 (m, 20H, cyclen-H , CON–CH2, cyclen- CH2, imidazole-C H2), 1.51–1.36 (m, 31H, Boc-H, CONCH 2–CH2),1.23 (m, 52H, CH 2), 0.85 (t, 6H, J = 6.4 Hz, –CH3). HR-MS (ESI):C62H107N8O10 [M+H]+, 1123.8105, found 1123.8113.

Compound 9d (385 mg, 0.325 mmol, yield: 65%): 1H NMR (CDCl3, 400 MHz): d = 11.82 (s, 1H, imidazole), 7.78 (s, 1H, imidazole), 6.82 (s, 1H, imidazole), 4.48 (t, 1H, NHC–H, J = 6.0 Hz), 3.33–3.09 (m, 22H, cyclen-H , cyclen-CH 2, imidazol e-CH 2, NH–CH2–CH2–NH, Ph-CH 2), 2.94–2.73 (m, 4H, cyclen-H), 1.99 (s, 3H, Ph- CH3), 1.94 (s, 3H, Ph-CH 3), 1.92 (s, 3H, Ph-CH 3), 1.74 (t, 2H, J = 6.8 Hz, C–CH2), 1.51–1.05 (m, 51H, Boc-H, –(CH2)9, –(CH)3, C–CH3), 0.83 (t, 12H, J = 6.8 Hz, –CH3). HR-MS (ESI): C65H122N9O9

[M+H]+, 1166.816 3, found 1166.8157. Compound 9e (337 mg, 0.3 mmol, yield: 58.7%): 1H NMR

(CDCl3, 400 MHz): d = 11.78 (s, 1H, imidazole), 7.50 (s, 1H, imidazole), 6.80 (s, 1H, imidazole), 4.62 (t, 1H, NHC–H, J = 6.0 Hz), 4.15 (s,


2H, NH–CH2), 3.33–3.15 (m, 18H, cyclen-H , cyclen-CH 2, imidazole- CH2, Ph-CH 2), 2.96–2.71 (m, 4H, cyclen-H), 1.99 (s, 3H, Ph-CH 3),1.92 (s, 3H, Ph-CH 3), 1.90 (s, 3H, Ph-CH 3), 1.74 (t, 2H, C–CH2,J = 6.8 Hz), 1.51–1.05 (m, 51H, Boc-H, –(CH2)9, –(CH)3, C–CH3),0.83 (t, 12H, J = 6.8 Hz, –CH3). HR-MS (ESI): C62H105N8O11 [M+H]+,1137.789 7, found 1137.7898.

4.2.6. Synthesis of title lipids Compound 9 (2 mmol) was suspended in anhydrous dichloro-

methane (5 mL), and then, a solution of trifluoroacetic acid (5 mL) in anhydrou s dichlorometha ne (5 mL) was added dropwise under ice bath and N2 atmosphere. And then, the obtained mixture was stirred at room temperature for 6 h. After the solvent and trifluoroacetic acid were removed, lipid 10a was directly obtained as oil. Lipids 10b–10e were obtained as white solid by treating the residues with anhydrous ethyl ether twice.

Compound 10a (yield 99%):1H NMR (CDCl3, 400 MHz): d = 14.43 (s, 1, imidazole), 8.29 (s, 1H, imidazol e), 6.82 (s, 1H, imidazole),4.59 (t, 1H, NHC–H, J = 6.6 Hz), 3.94 (s, 2H, NH–CH2), 3.30–2.77(m, 24H, cyclen-H, CON–CH2, cyclen-CH 2, imidazol e-CH 2), 1.46–1.38 (m, 4H, CONCH 2–CH2), 1.17–1.02 (m, 52H, CH 2), 0.79 (t, 6H, J = 6.2 Hz, –CH3). 13C NMR (CDCl3, 100 MHz): d = 173.5, 171.0, 168.9, 133.3, 129.4, 117.9, 55.8, 53.5, 50.5, 50.2, 47.8, 47.1, 44.9, 43.4, 42.8, 41.0, 32.0, 31.9, 31.1, 29.8, 29.7, 29.5, 29.4, 28.6, 27.6, 27.1, 27.0, 26.8, 22.8, 14.2. HR-MS (ESI): C50H98N9O3 [M+H]+,872.7787 , found 872.7784.

Compound 10b (yield 97%): 1H NMR (CDCl3, 400 MHz):d = 14.52 (s, 1H, imidazol e), 8.18 (s, 1H, imidazole), 7.35 (s, 1H, imidazol e), 5.31 (s, 1H, C@C–H), 4.60 (t, 1H, NHC–H, J = 6.2 Hz),3.37–3.08 (m, 24H, cyclen-H , NH–CH2–CH2–O, cyclen-CH 2, imidazole-CH2, cholester ol-H), 2.31–1.79 (m, 6H, cholesterol-H), 1.50–0.84 (m, 35H, Boc-H, cholesterol-H), 0.64 (s, 3H, cholesterol-H).13C NMR (CDCl3, 100 MHz): d = 173.4, 171.0, 140.5, 122.1, 118.3, 115.4, 79.6, 56.9, 59.4, 50.3, 42.4, 39.7, 39.2, 36.9, 36.0, 32.0, 31.3, 28.1, 24.1, 23.0, 22.7, 21.2, 19.4, 18.9, 18.5, 12.0. HR-MS (ESI): C45H79N8O3 [M+H]+, 779.6270, found 779.6274.

Compound 10c (yield 95%): 1H NMR (CDCl3, 400 MHz):d = 14.50 (s, 1 H, imidazole), 8.44 (s, 1H, imidazole), 7.35 (s, 1H, imidazol e), 4.63 (t, 1H, NHC–H, J = 6.8 Hz), 3.58–3.36 (m, 8H, cyclen-H, cyclen-CH 2, imidazole-C H2, NH–CH2–CH2–O), 3.16–2.81(m, 18H, cyclen-H, Ph-CH 2) 2.05 (s, 6H, Ph-CH 3), 1.98 (s, 3H, Ph- CH3), 1.72 (t, 2H, C–CH2, J = 6.8 Hz), 1.49–1.05 (m, 24H, –(CH2)9,–(CH)3, C–CH3), 0.83 (t, 12H, J = 7.4 Hz, –CH3). 13C NMR (CDCl3,100 MHz): d = 173.3, 170.8, 148.4, 147.1, 133.1, 129.6, 127.5, 125.7, 123.4, 120.3, 118.1, 117.7, 114.5, 75.1, 44.6, 43.4, 42.6, 40.6, 40.1, 39.5, 37.7, 37.4, 33.0, 31.2, 31.1, 28.1, 27.1, 24.9, 24.6, 23.5, 22.8, 21.2, 19.9, 19.7, 12.5, 11.8, 11.6. HR-MS (ESI):C47H83N8O4 [M+H]+, 823.6532, found 823.6540.

Compound 10d (yield 95%): 1H NMR (CDCl3, 400 MHz):d = 14.52 (s, 1H, imidazol e), 8.96 (s, 1H, imidazole), 7.76 (s, 1H, imidazol e), 4.58 (t, 1H, NHC–H, J = 6.0 Hz), 3.66–2.81 (m, 26H, cyclen-H, cyclen-CH 2, imidazole-CH 2, NH–CH2–CH2–O, Ph-CH 2), 2.48 (s, 3H, Ph-CH 3), 2.45(s, 3H, Ph-CH 3), 2.41 (s, 3H, Ph-CH 3), 1.74 (t,2H, C–CH2, J = 6.8 Hz), 1.52–1.06 (m, 24H, –(CH2)9, –(CH)3, C–CH3), 0.83 (t, 12H, J = 6.8 Hz, –CH3). 13C NMR (CDCl3, 100 MHz):d = 173.3, 170.8, 156.1, 149.4, 140.6, 130.2, 128.0, 126.3, 122.9, 118.4, 117.6, 115.4, 75.4, 44.5, 42.8, 39.5, 37.6, 37.4, 32.9, 31.3, 28.1, 24.9, 24.6, 22.9, 22.8, 21.2, 19.9, 19.7, 12.8, 11.9. HR-MS (ESI): C48H84N9O5 [M+H]+, 866.6590, found 866.6586.

Compound 10e (yield 93%): 1H NMR (CDCl3, 400 MHz):d = 14.48 (s, 1H, imidazol e), 8.96 (s, 1H, imidazole), 7.35 (s, 1H, imidazol e), 4.72 (t, 1H, NHC–H, J = 6.0 Hz), 4.22 (s, 2H, NH–CH2),3.37–2.80 (m, 22H, cyclen-H, cyclen-CH 2, imidazole-C H2, Ph- CH2), 1.99 (s, 3H, Ph-CH 3), 1.93 (s, 3H, Ph-CH 3), 1.90 (s, 3H, Ph- CH3), 1.74 (t, 2H, C–CH2, J = 6.8 Hz), 1.52–1.04 (m, 24H, –(CH2)9,–(CH)3, C–CH3), 0.83 (t, 12H, J = 6.8 Hz, –CH3). 13C NMR (CDCl3,




100 MHz): d = 173.2, 171.4, 149.8, 140.4, 133.1, 129.6, 126.7, 125.2, 123.2, 117.9, 75.5, 43.3, 39.5, 37.6, 37.4, 32.9, 32.8, 31.2, 28.1, 24.9, 24.6, 22.8, 22.7, 21.1, 19.9, 19.7, 12.8, 11.8. HR-MS (ESI):C47H81N8O5Na [M+Na] +, 859.6144, found 859.6125.

4.3. Acid–base titration

Lipid 10 (0.25 mmol of amino groups) was dissolved in 5 mL of 150 mM NaCl aqueous solution, and 0.1 M NaOH was added to adjust the pH to 10.0. Aliquots (20 lL for each) of 0.1 M HCl were added, and the solution pH was measured with a pH meter (pHS-25) after each addition. For comparison, PEI (25 kDa), cyclen and a blank were used under same experimental condition s.

4.4. Amplification and purification of plasmid DNA

pEGFP-N1 plasmids were used as the enhanced green fluores-cent protein reporter gene, which was transformed in Escherichiacoli DH5 a. Plasmid was amplified in E. coli grown in LB media at 37 �C and 220 rpm overnight. The plasmids were purified by an EndoFree TiangenTM Plasmid Kit. Then, the purified plasmids were dissolved in TE buffer solution and stored at �20 �C. The integrity of plasmids was confirmed by agarose gel electrophor esis. The pur- ity and concentratio n of plasmids were determined by the ratio of ultraviolet (UV) absorbances at 260 nm to 280 nm.

4.5. Preparation of cationic liposome s and liposome–DNAcomplexes (lipoplexes)

Individual cationic lipid (0.0025 mmol) or its mixture with DOPE in the desired mole ratio was dissolved in anhydrous chloroform (2.5 mL) in autoclave d glass vials. Thin films were made by slowly rotary-evap orating the solvent at room temperature. Last trace of organic solvent was removed by keeping these films under vacuum above 8 h. The dried films and PBS buffer (10 mM, pH 7.4)were preheated to 70 �C, and then the buffer was added to the films to the final lipid concentration of 1 mM. The mixtures were vortexed vigorously until the films were complete ly resuspended. Sonication of these suspensions for 20 min in a bath sonicator at 60 �C afforded the correspondi ng cationic liposomes that were stored at 4 �C.

To prepare the lipid/DOPE/pD NA complexes (lipoplexes), various amounts of cationic lipids were mixed with a constant amount of DNA by pipetting thoroughly at various N/P ratio, and the mixture was incubate d for 30 min at room temperature . The theoreti- cal N/P ratio represents the charge ratio of cationic lipid to nucleotide base (in molar) and was calculated by considering the average nucleotide mass of 350.

4.6. Ethidium bromide replacemen t assay

The ability of lipids 10 to condense DNA was studied using ethidium bromide (EB) exclusion assays. Fluorescence spectra were measured at room temperature in air by a Horiba JobinYvon Flu- oromax-4 spectrofluorometer and corrected for the system re- sponse. EB (5 lL, 1.0 mg mL �1) was put into quartz cuvette containing 2.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazinee- thanesulfon ic acid (HEPES) solution (pH 7.4). After shaking, the fluorescence intensity of EB was measured. Then CT DNA (10 lL,1.0 mg mL �1) was added to the solution and mixed symmetrically ,and the measured fluorescence intensity is the result of the interaction between DNA and EB. Subsequentl y, the solutions of lipid 10(1.0 mmol L�1, 3.2 lL for each addition) were added to the above solution for further measurement. All the samples were excited at 520 nm and the emission was measured at 600 nm.


4.7. Agarose-ge l retardation assay

Lipid 10/DOPE/pDNA complexes at different N/P ratios (theamino groups of lipids to phosphate groups of DNA) ranging from 1 to 8 were prepared by adding an appropriate volume of lipids (inPBS solution) to 0.125 lg pUC-19 DNA. The complexes were incubated at 37 �C for 30 min. After that the complexes were electro- phoresed on the 1.0% (W/V) agarose gel containing gelred and with Tris–acetate (TAE) running buffer at 110 V for 30 min. DNA was visualized with a UV lamp using a BioRad Universal Hood II.

4.8. Lipoplex particle sizes and zeta potential s

Sizes and zeta potential s of the lipid/pDNAlipop lex at various N/ P ratios were analyzed at room temperature on a dynamic light scatterin g system (Zetasizer Nano ZS, Malvern instruments Led)at 25 �C. The lipoplex particle solution was first prepared by mixing 10/DOPE liposomes and pDNA (2 lg/mL) under diverse N/P charge ratios in 1 mL deionized water.

4.9. Cell culture

Human non-small-c ell lung carcinom a A549 cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic s (penicillin–streptomycin, 10,000 U mL �1) at 37 �C in ahumidified atmosphere containing 5% CO 2.

4.10. MTT cytotoxic ity assay

Toxicity of lipoplexes toward A549 cells was determined by using MTT (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) reduction assay following literature procedures. 33 About5000 cells/well were seeded into 96-well plates. After 24 h, optimized lipid/DOP E formulat ions were complexed with 0.5 lg of DNA at various N/P ratios and incubate d for 30 min at rt; 100 lLof lipoplexes were added to the cells in the absence of serum. After 4 h of incubation, lipoplex solutions were removed, and 200 lL of medium with 10% FBS was added. After 24 h, 20 lL of MTT solution was added after 20 lL medium was moved out of the plate, and the cells were incubate d for another 4 h. Blue formazan crystals were seen at well when checked under microscope. Media were removed, 150 lL of DMSO was added per well, and then, plates were incubate d on a shaker for 10 min at room temperat ure to dissolve blue crystal. The absorbance was measured using a microtiter plate reader. The % viability was then calculated as [[A490(treated cells)-background ]/[A490(Untreated cells)-background]] � 100. Lipo- plexes prepared from Lipofectamine 2000 were used as control.

4.11. In vitro transfection

In order to obtain about 80% confluent cultures at the time of transfecti on, 24-well plates were seeded with 80,000 cell/well in 500 lL antibiotic-free media 24 h before transfection. For the preparation of lipoplexes applied to cells, various amounts of liposome sand DNA were serially diluted separately in both serum- and antibiotic-free 1640 culture medium; then, the DNA solutions were added into liposome solutions drop by drop and vortexed gently, after which the mixtures were incubated at room temperat ure for about 30 min to obtain lipoplexes of desired N/P ratios, the finallipoplexes volume was 200 lL, and the DNA was used at a concentration of 1 lg/well unless otherwise noted. After 30 min of com- plexation, old cell culture medium was removed from the wells, cells were washed once with serum-fr ee 1640, and the above 200 lL lipoplexes was added to each well. The plates were then incubate d for 4 h at 37 �C in a humidified atmosphere containing 5% CO 2. At the end of incubation period, medium was removed,




and 500 lL of fresh 1640 medium containing 10% FBS was added to each well. Plates were further incubated for a period of 24 h before checking the reporter gene expression.

For fluorescent microscopy assays, cells were transfected by complexes containing pEGFP-N1. After 24 h incubation, the microscopy images were obtained at the magnification of 40 �and recorded using Viewfinder Lite (1.0) software. Control transfection was performed in each case using a commercially available transfection reagent Lipofectami ne 2000™ based on the standard conditions specified by the manufac ture.

For FACS analysis, cells were transfected by complexes containing pEGFP-N1. After 24 h incubation, cells were rinsed twice with PBS, and followed by trypsinization and centrifugation at 1400 rpm for 3 min to harvest the cells. Then, the harvested cells were resuspended in PBS, and flow cytometer was employed to measure the fluorescent intensity.

Acknowled gments

This work was financially supported by the National Program on Key Basic Research Project of China (973 Program, 2012CB720603 ,2013CB32890 0), the National Science Foundation of China (No.21232005) and State Key Lab of Oral Diseases, Sichuan University, China (SKLODSCUKF2012-02). J.Z. thanks the Program for New Cen- tury Excellent Talents in University (NCET-11-0354). We also thank Analytical & Testing Center of Sichuan University for structural analysis of the compounds .

References and notes

1. Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259. 2. Guo, X.; Huang, L. Acc. Chem. Res. 2012, 45, 971. 3. Bhattacharya, S.; Bajaj, A. Chem. Commun. 2009, 4632. 4. Check, E. Nature 2003, 423, 573. 5. Zhi, D.; Zhang, S.; Wang, B.; Zhao, Y.; Yang, B.; Yu, S. Bioconjugate Chem. 2010,

21, 563. 6. Mevel, M.; Kamaly, N.; Carmona, S.; Oliver, M. H.; Jorgensen, M. R.; Crowther,

C.; Salazar, F. H.; Marion, P. L.; Fujino, M.; Natori, Y.; Thanou, M.; Arbuthnot, P.; Yaouanc, J. J.; Jaffres, P. A.; Miller, A. D. J. Controlled Release 2010, 143, 222.

7. Radchatawedchakoon, W.; Krajarng, A.; Niyomtham, N.; Watanapokasin, R.; Yingyongnarongkul, B. Chem. Eur. J. 2011, 17, 3287.

8. Bhavani, K.; Srilakshmi, P. V. Bioconjugate Chem. 2011, 22, 2581.


9. Bhavani, K.; Srilakshmi, P. V. J. Med. Chem. 2011, 54, 548. 10. Zhu, L.; Lu, Y.; Miller, D. D.; Mahato, R. I. Bioconjugate Chem. 2008, 19, 2499. 11. Medvedeva, D. A.; Maslov, M. A.; Serikov, R. N.; Morozova, N. G.; Serebrenikova,

G. A.; Sheglov, D. V.; Latyshev, A. V.; Vlassov, V. V.; Zenkova, M. A. J. Med. Chem. 2009, 52, 6558.

12. Zhang, Q. F.; Yang, W. H.; Yi, W. J.; Zhang, J.; Ren, J.; Luo, T. Y.; Zhu, W.; Yu, X. Q. Bioorg. Med. Chem. Lett. 2011, 21, 7045.

13. Khan, M.; Ang, C. Y.; Wiradharma, N.; Yong, L. K.; Liu, S. Q.; Liu, L. H.; Gao, S. J.; Yang, Y. Y. Biomaterials 2012, 33, 4673.

14. Maslov, M. A.; Kabilova, T. O.; Petukhov, I. A.; Morozova, N. G.; Serebrennikova, G. A.; Vlassov, V. V.; Zenkova, M. A. J. Controlled Release 2012, 160, 182.

15. Sheng, R. L.; Luo, T.; Zhu, Y. D.; Li, H.; Sun, J. J.; Chen, S. D.; Sun, W. Y.; Cao, A. M. Biomaterials 2011, 32, 3507.

16. Li, L.; Song, H. M.; Luo, K.; He, B.; Nie, Y.; Yang, Y.; Wu, Y.; Gu, Z. W. Int. J. Pharm. 2011, 408, 183.

17. Bhavani, K.; Srilakshmi, P. V. Mol. Pharm. 2012, 9, 1146. 18. Srujan, M.; Chandrashekhar, V.; Reddy, R. C.; Prabhakar, R.; Sreedhar, B.;

Chaudhuri, A. Biomaterials 2011, 32, 5231. 19. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.;

Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84,7413.

20. (a) Huang, Q. D.; Zhong, G. X.; Zhang, Y.; Ren, J.; Zhang, J.; Fu, Y.; Zhu, W.; Yu, X. Q. PLoS One 2011, 6, e23134; (b) Huang, Q. D.; Ou, W. J.; Chen, H.; Feng, Z. H.; Wang, J. Y.; Zhang, J.; Zhu, W.; Yu, X. Q. Eur. J. Pharm. Biopharm. 2011, 78, 326; (c) Liu, J. L.; Ma, Q. P.; Huang, Q. D.; Yang, W. H.; Zhang, J.; Wang, J. Y.; Zhu, W.; Yu, X. Q. Eur. J. Med. Chem. 2011, 46, 4133; (d) Huang, Q. D.; Ren, J.; Chen, H.; Ou, W. J.; Zhang, J.; Fu, Y.; Zhu, W.; Yu, X. Q. ChemPlusChem 2012, 584; (e)Huang, Q. D.; Ren, J.; Ou, W. J.; Fu, Y.; Cai, M. Q.; Zhang, J.; Zhu, W.; Yu, X. Q. Chem. Biol. Drug. Des. 2012, 79, 879.

21. Izalt, R. M.; Pawlak, K.; Bradahaw, J. S. Chem. Rev. 1991, 91, 1721. 22. Fujiwara, T.; Hasegawa, S.; Hirashima, N.; Nakanishi, M.; Ohwada, T. BBA–

Biomembranes 2000, 1468, 396. 23. Varkouhi, A. K.; Scholte, M.; Storm, G.; Haisma, H. J. J. Controlled Release 2011,

151, 220. 24. Emilie Bertrand, E.; Goncalves, C.; Billiet, L.; Gomez, J. P.; Pichon, C.;

Cheradame, H.; Midoux, P.; Guegan, P. Chem. Commun. 2011, 47, 12547. 25. Midoux, P.; Pichon, C.; Yaouanc, J. J.; Jaffres, P. A. Br. J. Pharmacol. 2009, 157,

166.26. Shrestha, R.; Elsabahy, M.; Florez-Malaver, S.; Samarajeewa, S.; Wooley, K. L.

Biomaterials 2012, 33, 8557. 27. Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Controlled Release 1999, 60, 149. 28. Gratton, S.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.;

Desimone, J. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11613. 29. Wiradharma, N.; Tong, Y. W.; Yang, Y. Y. Biomaterials 2009, 30, 3100. 30. Biswas, J.; Mishra, S. K.; Kondaiah, P.; Bhattacharya, S. Org. Biomol. Chem. 2011,

9, 4600. 31. Halks-Miller, M.; Guo, L. S.; Hamilton, R. L., Jr. Lipids 1985, 20, 195. 32. Jeon, J. W.; Son, S. J.; Yoo, C. E.; Hong, I. S.; Song, J. B.; Suh, J. Org. Lett. 2002, 4,

4155.33. Deck, L. M.; Baca, M. L.; Salas, S. L.; Hunsaker, L. A.; Vander Jagt, D. L. J. Med.

Chem. 1999, 42, 4250.



Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene...

Documents

Transcript of Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene...