Báo cáo khoa học: NovelN,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery – synthesis,in vitro transfection activity, and physicochemical characterization

Novel N,N ¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery – synthesis, in vitro transfection activity, and physicochemical characterization Michael Spelios and Michalakis Savva

Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University, Brooklyn, NY, USA

Keywords cationic lipid; elasticity; FRET; gene delivery; lipoplex

Correspondence M. Savva, Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy & Health Sciences, Long Island University, 75 DeKalb Avenue, Brooklyn, NY 11201, USA Fax: +1 718 780 4586 Tel: +1 718 488 1471 E-mail: msavva@liu.edu

(Received 23 September 2007, revised 5 November 2007, accepted 9 November 2007)

Novel N,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efﬁcacies were induced at + ⁄ ) 4 : 1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at + ⁄ ) 4 : 1 and yielded increased lipofection activity at a lower charge ratio of + ⁄ ) 2 : 1. Ethidium bromide displacement indicated efﬁcient plasmid DNA binding and compaction by the trans- fection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter ‡ 500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir ﬁlm balance studies showed high in-plane elasticity of these derivatives in isola- tion. In agreement with the monolayer experiments, ﬂuorescence polariza- tion studies veriﬁed the ﬂuid nature of the highly transfection-efﬁcient amphiphiles, with gel-to-liquid crystalline phase transitions below physio- logical temperature. The active compounds also interacted with endosome- mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by ﬂuorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain ﬂuidity and minimal cytotoxicity elicit high transfection activity.

The development of highly potent and minimally toxic cationic lipids for nucleic acid delivery depends on generation of meaningful structure–activity relation-

ships. The rational design of efﬁcacious transfection amphiphiles is based on understanding the impact of the lipid structural components on gene each of

doi:10.1111/j.1742-4658.2007.06185.x

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Abbreviations DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, 1,2-dioleoyl-3- trimethylammonium-propane; DPH, 1,6-diphenyl-1,3,5-hexatriene; EGFP, enhanced green ﬂuorescent protein; EtBr, ethidium bromide; FITC, ﬂuoroscein isothiocyanate; FRET, ﬂuorescence resonance energy transfer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBD-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); ONPG, 2-nitrophenyl b-D-galacto pyranoside; PA, 1,2-dipalmitoyl-sn-glycero-3-phosphate; PC, L-a-phosphatidylcholine (egg, chicken); pDNA, plasmid DNA; Rh-PE, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); SFM, serum-free medium; TNS, 2-(p-toluidino)naphthalene-6-sulfonic acid.

H3C

CH3

together. Since the advent of

H3C

seen in numerous

The R group varies with the derivative:

C11H23 for dilauroyl (1,3lb1) C13H27 for dimyristoyl (1,3lb2) C15H31 for dipalmitoyl (1,3lb3) C17H35 for distearoyl (1,3lb4) C17H31 for dioleoyl (1,3lb5)

M. Spelios and M. Savva Novel cytofectins for gene delivery

cationic

chains, promotes highly efﬁcacious in vitro lipofection through efﬁcient binding and compaction of pDNA, increased acyl chain ﬂuidity and high molecular elastic- ity. The current study is a further examination of the 1,3lb cytofectin involving systematic molecular changes; speciﬁcally, determination of the effects of hydrophobic chain length and degree of unsaturation on target gene expression.

Fig. 1. Structure of the 1,3lb derivatives.

Results

Biological analysis

ﬂuorescence

transfer, namely the polar headgroup, the nonpolar tail (either a cholesterol moiety or a pair of aliphatic hydrocarbon chains), and the linker tethering both regions lipofection 20 years ago [1], cationic lipids with new molecular architectures have been developed and analyzed as gene-delivery vectors, as recent publications. Liu et al. [2] synthesized a series of 16 carbamate-linked cationic lipids, differing in their hydrocarbon chain length, quaternary ammonium head and counter-ion species, and examined their bio- logical performance. Spacer modiﬁcations were studied in cholesterol-based and aliphatic gemini cationic lipids to determine their effects on the transfecting abilities of these dimeric surfactants [3–5]. Rajesh et al. [6] were the ﬁrst to report the inﬂuence of linker orientation reversal on the transfection efﬁciencies and physico- chemical properties of two cationic amphiphiles with identical hydrophilic and hydrophobic constituents. Other groups have also recently described the design, syntheses, physicochemical characterization and trans- fection properties of novel cationic amphiphiles [7–11]. In an effort to further delineate the structural prop- erties of these lipofection reagents that confer superior transfection activity, a novel series of N,N¢-diacyl-1,3- lipids diaminopropyl-2-carbamoyl bivalent was synthesized containing a symmetric bis-[2-dimeth- ylamino-ethyl]-amine polar headgroup at the 2-posi- tion and hydrophobic chains at the 1- and 3- positions of the 1,3-diamino-2-propanol backbone (Fig. 1). The series, designated 1,3lb, consists of four saturated lip- ids, ranging in chain length from 12 to 18 carbons, and a single monounsaturated derivative with a double bond between the 9th and 10th carbons of each 18-car- bon chain. Physicochemical characterization of the cat- ionic lipids in lamellar assemblies with and without plasmid DNA and in media of various ionic strengths comprised a variety of studies and techniques, includ- ing pKa determination, isothermal monolayer compres- sion, ethidium bromide anisotropy, displacement, dynamic light scattering, determination of zeta potential, and ﬂuorescence resonance energy transfer (FRET), and is indispensable for elucidating the structural properties of these amphiphiles that induce high transfection activity.

Lipoplexes of 1,3lb cationic lipids with and without helper lipid(s) were examined at various + ⁄ ) charge ratios for transfection activity. The shortest saturated chain derivative 1,3lb1 was completely inefﬁcient at pro- moting lipofection at all charge ratios, both in the absence and presence of neutral colipid(s). Formulations lacking either 1,2-dioleoyl-sn-glycero-3-phosphoetha- nolamine (DOPE) or phospholipid and cholesterol induced the highest levels of reporter gene expression at + ⁄ ) 4 : 1, the exception being 1,3lb4 which exhibited low activity throughout the range of charge ratios tested (Fig. 2A). The dipalmitoyl derivative 1,3lb3 elicited higher in vitro transfection activity than 1,3lb5, contrary to ﬁndings that unsaturated derivatives are typically the

This work is a continuation of a recent study high- lighting the superior gene delivery mediated by the di- myristoyl derivative 1,3lb2 from the aforementioned series as compared to two other cationic lipid vectors, a conformational isomer and a monovalent analog [12]. It was determined that a symmetrical bivalent pH-expandable polar headgroup, in combination with greater intramolecular space between the hydrophobic

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M. Spelios and M. Savva Novel cytofectins for gene delivery

A

1,3lb2

1,3lb3

/

Fig. 3, revealed the same activity trends as quantita- tively determined by the 2-nitrophenyl b-d-galacto pyranoside (ONPG) assay: 1,3lb3 > 1,3lb2 (cid:2) 1,3lb5 > DOTAP > 1,3lb4.

1,3lb4

) l l e w U m

1,3lb5

DOTAP

( n o i t a r t n e c n o c l a g - β

1100 1000 900 800 700 600 500 400 300 200 100 0

2 +/– charge ratio

B

1,3lb2/D

1,3lb3/D

/

1,3lb4/D

) l l e w U m

1,3lb5/D

DOTAP

( n o i t a r t n e c n o c l a g - β

1100 1000 900 800 700 600 500 400 300 200 100 0

2 +/– charge ratio

C

1,3lb2/D/c

1,3lb3/D/c

/

1,3lb4/D/c

) l l e w U m

1,3lb5/D/c

DOTAP

Fluorescein covalently attached to plasmid allowed visual tracking of cellular uptake. Internalization of exogenous nucleic acid occurred to the greatest extent with the aid of 1,3lb2, as indicated by the higher ﬂuoro- scein isothiocyanate (FITC)-plasmid DNA (pDNA) intensity (green) when compared to the ﬂuorescence yields of labeled plasmid transported via other derivatives (Fig. 4A). Lipoplexes formed with 1,2-di- oleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl (Rh-PE)-labeled dispersions (red) of 1,3lb2 were visualized as distinctly yellow spots, sug- gesting well-associated complexes of nucleic acid and lipid (Fig. 4B); overlaid FITC and rhodamine images of cells transfected using the other transfection-active analogs showed colocalization of plasmid and lipid to a similar degree (not shown). In accordance with these results, the dimyristoyl derivative was the most efﬁcient the transfection-active compounds at condensing of pDNA as monitored by ethidium bromide (EtBr) dis- placement. Images obtained after transfection with 1,3lb1 (results not shown) showed signiﬁcantly fewer cells, and ﬂuorescent patches where no cells were pres- ent, indicating large aggregates with a high afﬁnity for the plate surface that were not internalized and are probably responsible for the elevated cytotoxicity. In fact, except along the edges of the wells where cells were densely packed and multilayered, accounting for the 46% survival (data not shown), no viable cells were detected after exposure to 1,3lb1. Dynamic light- scattering studies revealed 1,3lb1-containing lipoplexes (+ ⁄ ) 4 : 1) of the largest size with a mean diameter around 1 lm.

( n o i t a r t n e c n o c l a g - β

1100 1000 900 800 700 600 500 400 300 200 100 0

2 +/– charge ratio

lipofection efﬁciency of

most transfection-competent [13–15]. The activity of b-galactosidase was approximately two- to threefold greater than with 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP)-mediated gene delivery. Qualitative analysis of transfection efﬁciency by detection of enhance green ﬂuorescent protein (EGFP), as seen in

Use of DOPE and cholesterol to enhance the gene- delivery properties of cationic lipids has been exten- sively documented [16–21]. For 1,3lb2 and 1,3lb3, transfection activity was appreciably reduced at the highest tested charge ratio by the incorporation of DOPE, falling below levels reported for 1,3lb5; a simi- lar result was observed when cholesterol was added (Fig. 2B,C). The 1,3lb5 increased signiﬁcantly at + ⁄ ) 2 : 1, climbing above that of commercially available DOTAP, and rose mod- erately with the inclusion of cholesterol. The distearoyl derivative continued to mediate low levels of transgene expression at all charge ratios, even after the addition of DOPE alone or in combination with cholesterol. Increasing the + ⁄ ) charge ratio beyond 4 : 1 resulted in decreased transfection activity for the most active derivatives in all formulations (data not shown).

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Fig. 2. In vitro transfection activity of cationic lipids in the absence of helper lipid(s) (A) and in the presence of DOPE (B) or DOPE and cholesterol (C), as measured in a murine skin cell line (B16-F0 mela- noma cells) by ONPG assay (n = 3).

M. Spelios and M. Savva Novel cytofectins for gene delivery

A

D

B

E

C

F

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide] reduction analysis revealed a low lipoplexes at all charge ratios and cytotoxicity of

compositions, with cell viability greater than 60%, except for formulations containing 1,3lb1, which were poorly tolerated (data not shown).

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Fig. 3. Fluorescence of EGFP in B16-F0 cells transfected with lipoplexes of (A) 1,3lb1, (B) 1,3lb2, (C) 1,3lb3, (D) 1,3lb4 and (E) 1,3lb5 at ± 4 : 1 in the absence of helper lipid(s), and with (F) DOTAP at ± 2 : 1. Images were acquired at 10 · magniﬁcation.

M. Spelios and M. Savva Novel cytofectins for gene delivery

A

B

1,3lb2

1,3lb3

1,3lb4

1,3lb5

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M. Spelios and M. Savva Novel cytofectins for gene delivery

Physicochemical characterization

pKa studies

well separated from one another, precluding inﬂuences of the hydrophobic anchors of the derivatives with respect to the number of carbon atoms and double bonds in the aliphatic chains. These pKa values deviate from those obtained using vesicles where the deriva- tives are in greater contact with each other, such as the dispersions under investigation, and van der Waals forces between adjacent cationic lipid molecules, as dictated by their hydrophobic chain length and degree of unsaturation, are a major contribution to the extent of the bis-[2-dimethylamino-ethyl]-amine polar head- subsequently, protonation. group hydration, and, Thus, the acid dissociation constants in Table 1 are molecular descriptors of the derivatives and do not necessarily offer insight into the differences in transfec- tion activities.

40 mM Tris, pH7.2

100

Fig. 4. (A) Fluorescence images of B16-F0 cells treated with lipoplexes of FITC–pDNA. (B) FITC (top), rhodamine (center) and overlaid ﬂuo- rescence and brightﬁeld (bottom) images of cells transfected using Rh-PE-labeled (1 mol%) dispersions of 1,3lb2. Lipoplexes were prepared at a charge ratio of ± 4 : 1, and images were captured 4 h after transfection at 20 and 10 · magniﬁcation for (A) and (B), respectively.

) .

x a m

Changes in the membrane surface charge of pH-stable 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ⁄ cho- lesterol vesicles containing 5 mol% 1,3lb amphiphile were monitored by measuring the ﬂuorescence intensity of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) in the lipid bilayers as a function of pH. As expected, the pH titration curves (not shown) of the derivatives overlapped in accordance with their identical polar the experimental data headgroup. Curve ﬁtting of revealed a pKa between 7.1 and 7.4, indicating that only about 30–50% of the tertiary amine groups are charged at physiological pH (Table 1). Higher values were found for the isolated triamine (pKa1 = 8.959, pKa2 = 9.592) [22], and may be attributed to reduced hydration of the cationic lipids compared to the free amine, as well as tight packing of the hydrophobic chains, which promotes charge distribution over adja- cent lipid molecules.

1,3lb1

f o %

1,3lb2

1,3lb3

1,3lb4

1,3lb5

t

( e c n e c s e r o u l f r B E

0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

2.4

+/– charge ratio

The pH titration curves were ﬁtted to a modiﬁed version of the Henderson–Hasselbach equation, which contains an adjustable parameter C that affects the slope of the transition region. In the original equation, this parameter is equal to 1 for a univalent base (and -1 for a monoprotic acid). The pKa values listed in Table 1 for the bivalent lipids are the mean of two acid dissociation constants, one for each of the ioniz- able amino groups, resulting in pH titration curves with slopes of lower steepness (C less than unity).

SFM

100

The TNS assay was used to ascertain the pKa values of the cationic lipids in assemblies where they were

) . x a m

f o

Table 1. Acid dissociation constants of cationic lipids as deter- mined by nonlinear ﬁtting of TNS ﬂuorescence intensity–pH plots.

%

1,3lb1

1,3lb2

1,3lb3

1,3lb4

Lipid C a Coefﬁcient of determinationb % ionization at pH 7.2 pKa

t

( e c n e c s e r o u l f r B E

1,3lb5

0.4

0.8

1.2

2.8

3.2

3.6

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5 7.24 7.09 7.37 7.36 7.42 0.40 0.52 0.40 0.58 0.53 0.991 0.994 0.984 0.996 0.995 41 33 48 48 51

1.6 2.4 2 +/– charge ratio

a C is an adjustable parameter affecting the slope of the transition region of the ﬁtted pH titration curves, as calculated from Eqn (1). b Goodness-of-ﬁt statistics for pKa were assessed within a 95% conﬁdence interval.

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the Fig. 5. Percentage ethidium bromide displacement against charge ratio of lipoplexes in Tris buffer or SFM. Parabolic curve ﬁts of the experimental data (solid points) are shown as dashed lines.

Cationic lipid–pDNA binding studies

activities (Fig. 2A). The mixed-phase state of monolay- ers composed of the poorly transfection-efﬁcient deriv- ative 1,3lb4 is evidenced by an L1-to-L2 transition in the compression isotherm. Speciﬁcally, liquid-expanded behavior was observed up to a surface pressure and mean molecular area of 24 mNÆm)1 and 70 A˚ 2, respec- tively, at which point a transition occurred to a chain- ordered phase. The pressure continued to rise upon the monolayer further surface area reduction until ﬁnally collapsed at 49 mNÆm)1 and 39 A˚ 2. At 37 (cid:2)C, the two-dimensional transition was absent and only a liquid-expanded state was evident. For all derivatives, increased and compression molecular dimensions forces decreased at monolayer collapse in response to elevated temperature. Differences between the mono- layer collapse parameters of the cationic lipids were not as apparent at 37 (cid:2)C; the derivatives possessed similar mean molecular areas at monolayer collapse, and their collapse pressures were nearly identical.

Binding curves of EtBr-intercalated pDNA titrated with aliquots of cationic lipid dispersions are shown in Fig. 5. With respect to the saturated derivatives, there was a reduction in plasmid compaction efﬁciency with increasing hydrophobic chain length. Interestingly, the transfection-inactive lipid 1,3lb1 was the most efﬁcient at condensing pDNA, with complete charge neutraliza- tion of the negative charges of pDNA at about + ⁄ ) 2.3 in low-ionic-strength medium. This ﬁnding is contradictory to previous work suggesting inefﬁcient DNA condensation and dehydration by 12-carbon fatty acid chains [23]. However, 1,3lb1 was also the most toxic to the cells, accounting for its transfection inactivity. The monounsaturated derivative 1,3lb5 dis- played intermediate nucleic acid condensation efﬁcacy, with full EtBr exclusion at approximately + ⁄ ) 3.1. Increasing the ionic strength resulted in complete probe displacement at higher charge ratios for all lip- ids, but there was no effect on the binding trends.

Langmuir monolayer studies

in an all

the distearoyl derivative, exist

Molecular elasticity is correlated with transfection activity, and was assessed by ﬁrst-derivative analysis of the p–A isotherm; the smaller the slope at monolayer collapse (dp ⁄ dAc), the higher the compressibility. The value of dp ⁄ dAc was highest for 1,3lb4 at both 23 (cid:2)C and 37 (cid:2)C, indicative of the lowest in-plane elasticity, and lipoplexes containing the distearoyl analog con- comitantly generated minimum reporter gene expres- sion (Fig. 2A). The monounsaturated 1,3lb5 was found to be the most elastic at 23 (cid:2)C, with a dp ⁄ dAc value of 1 mNÆm)1ÆA˚ )2.

Phase-transition temperature studies

DPH (1,6-diphenyl-1,3,5-hexatriene) was used to probe for cationic lipid bilayer phase changes by monitoring the depolarization of ﬂuorescence of the extrinsic ﬂuorophore in response to temperature. The shortest saturated chain derivative 1,3lb1 and the monounsatu- rated analog 1,3lb5 displayed the most ﬂuid behavior,

The surface properties of the cationic lipids were inves- tigated using the Langmuir ﬁlm balance technique. Monolayers of the cationic lipids, with the exception of liquid- expanded state at 23 (cid:2)C. Monolayer collapse occurred at lower mean molecular areas and higher surface pres- sures as the acyl chain length increased from 1,3lb1 to 1,3lb14 (Table 2). Tighter lipid packing associated with the additional van der Waals forces of longer hydro- interstitial phobic chains more effectively excludes water and reduces surface tension, allowing a greater reduction in the available surface area of the mono- layer prior to its collapse. The dimyristoyl and dioleoyl derivatives 1,3lb2 and 1,3lb5, respectively, shared simi- lar collapse parameters and comparable transfection

M. Spelios and M. Savva Novel cytofectins for gene delivery

Table 2. Monolayer transitiona and collapse parameters of the 1,3lb series. Measurements were performed using Tris buffer (40 mM, pH 7.2) as the subphase. Values reported are the mean of n experiments ± standard deviation.

Mean molecular area (A˚ 2) Phase stateb p (mNÆm)1) dp ⁄ dAc

23 (cid:2)C 23 (cid:2)C 37 (cid:2)C 23 (cid:2)C 37 (cid:2)C 37 (cid:2)C 23 (cid:2)C 37 (cid:2)C

1,3lb1 (n = 4,6) 1,3lb2 (n = 4,3) 1,3lb3 (n = 3,4) 1,3lb4 (n = 11,6) 64.90 ± 3.22 60.61 ± 2.29 55.61 ± 3.41 56.06 ± 3.81 36.59 ± 1.34 36.31 ± 1.37 38.62 ± 0.75 39.89 ± 0.80 1.27 ± 0.07 1.55 ± 0.10 1.32 ± 0.04 2.29 ± 0.51 1.13 ± 0.08 1.04 ± 0.04 1.11 ± 0.03 1.20 ± 0.11 L1 L1 L1 L1

a Phase transition was determined by a discontinuity in the plot of dp ⁄ dA against mean molecular area (not shown). b L1 and L2 indicate the liquid-expanded and liquid-condensed states, respectively.

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59.27 ± 4.89 55.98 ± 2.55 46.91 ± 1.39 38.70 ± 3.03 70.49 ± 3.43a 54.08 ± 3.30 37.75 ± 2.01 41.62 ± 0.94 40.29 ± 0.72 48.68 ± 6.71 23.73 ± 1.09a 39.04 ± 3.81 1,3lb5 (n = 7,7) 57.92 ± 6.41 36.82 ± 2.54 1.00 ± 0.17 1.20 ± 0.14 L1 L1 L1 L2 L1 L1 L1

1,3lb1

1,3lb2

1,3lb3

1,3lb4

1,3lb5

0.4A

0.35

0.3

0.25

0.2

M. Spelios and M. Savva Novel cytofectins for gene delivery

y p o r t o s i n A

0.15

0.1

0.05

T (°C)

B

The behavior of the cationic lipids in two-dimen- sional monolayers and three-dimensional bilayers was compared. As shown in Table 3, a gel-to-liquid crystalline transition temperature below 23 (cid:2)C was found for 1,3lb1, 1,3lb2 and 1,3lb5, coinciding with the ﬂuid state of these lipids as indicated by the p–A isotherm at 23 (cid:2)C and 37 (cid:2)C. The three-dimensional phase transition exhibited by 1,3lb4 at 45 (cid:2)C complies with monolayer compression data indicating the pres- ence of a chain-ordered phase at 23 (cid:2)C. Taking into consideration the onset phase-transition temperature determined from the ﬁrst-derivative proﬁle of the r–T plot (Fig. 6B) instead of the transition midpoint, the all liquid-expanded states at 23 (cid:2)C and 37 (cid:2)C of the dipalmitoyl and distearoyl derivatives, respectively, in monolayers complement the nature of these lipids in bilayer assemblies at these temperatures.

1,3lb1

1,3lb2

T d / r d

Particle size and electrophoretic mobility studies

1,3lb3

1,3lb4

1,3lb5

30 T (°C)

temperatures

existing in a liquid crystalline state within the range of scanned (Fig. 6A). A gel-to-liquid crystalline phase transition was detected for all other derivatives (temperature span 6–7 (cid:2)C), and increased in tandem with acyl chain length (Table 3). Only 1,3lb4 exhibited a three-dimensional phase transition above physiological temperature, signifying an ordered phase during transfection, with tight lipid packing, and induced low reporter gene expression.

Fig. 6. (A) Fluorescence anisotropy of DPH in cationic lipid bilayers as a function of temperature, and (B) ﬁrst-derivative data of r–T plots. Dispersions were prepared with 40 mM Tris, pH 7.2.

In vitro transfection activity was found to be a func- tion of the size of the cationic lipid–pDNA complex. At low ionic strength, lipoplexes of 1,3lb3 had the largest particle size at + ⁄ ) 4 : 1, with a mean diame- ter of approximately 740 nm (Fig. 7A). Cationic lipid- mediated transfection with the dipalmitoyl derivative yielded the highest levels of reporter gene expression at this charge ratio without helper lipid(s), and could be attributed, among other factors, to enhanced sedimen- tation of larger particles onto cells [24,25]. Complexes of plasmid and either 1,3lb2 or 1,3lb5 shared a similar particle size ((cid:2) 0.5 lm), as well as transfection activ- the highest + ⁄ ) charge ratio tested. The ity at poorly transfection-competent lipids 1,3lb1 and 1,3lb4, when complexed to pDNA at + ⁄ ) 4 : 1, generated a particle size of the smallest mean diameter. In serum- free medium, liposome and lipoplex particle sizes for all lipids generally increased, and the overall particle size trends between the derivatives were largely main- tained, the most notable exception being lipoplexes of 1,3lb1 at + ⁄ ) 4 : 1 which exhibited a mean diameter comparable with that of 1,3lb3-containing lipoplexes (Fig. 7B).

phase-transition onset and

Table 3. Midpoint temperatures obtained from curve ﬁts and ﬁrst-derivative analysis, respectively, of the experimental data in Fig. 6.

lipofection at

these charge ratios

Lipid Coefﬁcient of determinationa Tonset ((cid:2)C) Toffset ((cid:2)C) Tm ((cid:2)C) Toffset ) onset

a Best-ﬁt parameters were assessed within a 95% conﬁdence inter- val.

The electrophoretic mobility of all samples was mea- sured and converted to zeta potential. In low-ionic- strength medium (Fig. 7C), the zeta potential remained negative from + ⁄ ) 1 : 1 to 2 : 1, which hindered efﬁ- cient (Fig. 2A). Increasing the + ⁄ ) charge ratio to 4 : 1 resulted in complete neutralization of the negative charges on pDNA by cationic lipid dispersion of all derivatives except 1,3lb4. Lipoplexes of the distearoyl analog con- tinued to display a highly negative zeta potential, an

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1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5 < 6 16.8 31 45.1 < 6 ND 0.998 0.994 0.988 ND – 13 28 42 – – 20 34 48 – – 7 6 6 –

1 400

M. Spelios and M. Savva Novel cytofectins for gene delivery

A

C

1,3lb1

1 200

1,3lb2

)

1,3lb3

1 000

1,3lb1

V m

1,3lb4

)

1,3lb2

800

1,3lb5

1,3lb3

m n (

D

600

1,3lb4

–20

1,3lb5

( l a i t n e t o p a t e Z

400

–40

200

–60

–80

+/– 1:1

+/– 2:1

+/– 4:1

liposomes

+/– 1:1

+/– 2:1

+/– 4:1

liposomes

1 400

B

D

1,3lb1

1,3lb2

1 200

1,3lb3

)

1 000

1,3lb1

V m

1,3lb4

)

1,3lb2

800

1,3lb5

1,3lb3

m n (

D

600

1,3lb4

–20

1,3lb5

( l a i t n e t o p a t e Z

400

–40

200

–60

–80

+/– 1:1

+/– 2:1

+/– 4:1

liposomes

+/– 1:1

+/– 2:1

+/– 4:1

liposomes

phatidylcholine (PC : PA) vesicles [27]. The ﬂuorescent lipid probes 1,2-dioleoyl-sn-glycero-3-phosphoethanol- amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and Rh-PE were incorporated into the lipid bilayer of the anionic vesicle membranes, and the reduction in from nitro- ﬂuorescence resonance energy transfer

respectively. At high ionic

indication that efﬁcient complexation with and conden- sation of pDNA did not occur, as veriﬁed by ethidium bromide displacement (Fig. 5). Interestingly, 1,3lb1- containing lipoplexes showed a positive zeta potential of about 17 mV at + ⁄ ) 4 : 1, higher than and similar to that for lipoplexes of transfection-active 1,3lb3 and 1,3lb5, strength, only negative values of zeta potential were observed for lipoplexes at all charge ratios (Fig. 7D), suggesting a minimal inﬂuence of electrostatic interactions on cell internalization of 1,3lb–pDNA complexes in vitro.

pH 4.0 pH 7.4

Lipid-mixing studies

Fig. 7. Particle size distribution (A,B) and zeta potential (C,D), as determined by dynamic light-scattering and electrophoretic mobility studies, respectively, of cationic lipid dispersions and lipoplexes in 40 mM Tris, pH 7.2 (A,C) and SFM (B,D).

g n i x i m d i p i l

%

1,3lb1

1,3lb2

1,3lb3

1,3lb4

1,3lb5

The membrane fusion activity of the cationic lipids via electrostatic and hydrophobic interactions was mea- sured by ﬂuorescence resonance energy transfer using the NBD-Rh FRET pair [26]. Lipid mixing occurs in various cellular processes, including lipoplex internali- zation and fusion of the internalized cationic lipid– DNA complex with the endosome membrane. To sim- ulate these processes in vitro, membrane fusion studies were conducted at physiological temperature and ionic strength, at pH 7.4 and 4.0, using endosome-mimick- ing 1,2-dipalmitoyl-sn-glycero-3-phosphate : l-a-phos-

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156

Fig. 8. Lipid mixing, as assessed by FRET, of unlabeled 1,3lb dis- persions with endosome-mimicking PC : PA (73 : 25) vesicles lipid probes NBD-PE and Rh-PE labeled with the ﬂuorescent (1 mol% each), after 35 min. Studies were conducted at 37 (cid:2)C and 154 mM ionic strength at physiological and acidic pH.

number of transfection-efﬁcient derivatives to include the dimyristoyl and dipalmitoyl analogs.

studies

binding

(Fig. 5):

benzoxadiazole to rhodamine was monitored upon probe dilution and increased ﬂuorophore separation achieved by fusion with unlabeled cationic liposomes. Figure 8 shows the lipid mixing efﬁciency of 1,3lb dis- persions. The percentage lipid mixing was nearly dou- bled at pH 4.0 compared with pH 7.4 for all cationic lipids except the least active 1,3lb4, which maintained a constant and low membrane fusion efﬁcacy irrespec- tive of the pH. The trends were identical to those of the 1,3lb1 > 1,3lb2 > 1,3lb5 > 1,3lb3 > 1,3lb4. The dimyristoyl derivative exhibited the highest biomembrane fusogenicity of the active analogs, and lipoplexes of this cationic lipid were internalized to the greatest extent (Fig. 4). For 1,3lb2, the percentage lipid mixing was approximately four times greater than for 1,3lb3, despite displaying an approximately 1.4-fold lower transfection activity than the most biologically active compound (Fig. 2A). However, cell viability was compromised to a greater degree with formulations of 1,3lb2 (63% survival com- pared with 84% for 1,3lb3; data not shown). The same holds true for 1,3lb5 in comparison with 1,3lb3.

M. Spelios and M. Savva Novel cytofectins for gene delivery

Discussion

Increased ﬂuidity, or a low gel-to-liquid crystalline phase-transition temperature, of these lamellar assem- blies under physiological conditions is another charac- teristic of the lipid vesicles in isolation that has been identiﬁed as critical for transfection activity [33–35]. An investigation was recently completed regarding the transfection activity and physicochemical properties of a 1,2-diamino-3-propanol series containing an attach- ment of the same bivalent polar headgroup at the 3-position but with linkages of the acyl chains at the the 1,2-diamino-3-propanol 1- and 2- positions of backbone [30]. The 1,3-dialkyl cationic amphiphiles reported herein feature hydrophobic chains of greater interchain distance than their 1,2-dialkyl counterparts, and the impact this has biologically and physicochemi- cally on these vectors is remarkable. Whereas only the dioleoyl derivative of the 1,2lb series generated trans- fection activity and efﬁciently bound and compacted pDNA in the absence of helper lipid(s), increasing the intramolecular space between the acyl chains activated the dimyristoyl and dipalmitoyl derivatives. This struc- tural modiﬁcation also afforded these lipids higher two-plane elasticity and increased ﬂuidity relative to their corresponding 1,2lb analogs, as indicated by the lower compressibility moduli and reduced gel-to-liquid crystalline phase-transition temperatures of the 1,3lb derivatives.

The current project is part of a greater endeavor to understand the structural effects of double-chained amphiphilic molecules and their aggregates, in the pres- ence and absence of pDNA, on cationic lipid-mediated gene delivery. In particular, the + ⁄ ) charge ratio, neu- tral helper lipids, ionic strength, acyl chain length and degree of unsaturation, the number of ionizable amines in the polar headgroup, and the spatial arrangement of the hydrophobic and hydrophilic regions within the lipid molecule have been examined by our laboratory and correlated with transfection efﬁciency in an exploration of structure–function relationships that has spanned several papers [12,28–31]. Generation of such relation- ships is essential to the development of lipofection reagents that are highly potent and minimally toxic.

Remarkably, many of the physicochemical proper- ties of the dilauroyl derivative, which was found to be a completely inefﬁcient delivery system, are character- istic of an ideal cytofectin. Dispersions of this lipid dis- placed EtBr from pDNA and condensed plasmid to the greatest extent, and were fusogenically superior in lipid-mixing studies with endosome-mimicking vesicles. 1,3lb1 liposomes displayed the most ﬂuid behavior out of all the saturated derivatives, as indicated by the absence of a gel-to-liquid crystalline phase transition. In addition, lipoplexes containing 1,3lb1, due to better hydration, possessed the highest zeta potential at the + ⁄ ) charge ratio with the highest reporter gene expression. However, 1,3lb1 solubilizes cell membranes and lyses cells in much the same way as strong micelle- forming surfactants such as Triton X-100, and such high cytotoxicity rendered the dilauroyl derivative totally inactive.

Conclusion

Five cationic lipid derivatives, differing in the length and degree of unsaturation of their hydrophobic chains, were analyzed with reference to their gene-delivery

Ewert et al. [32] identiﬁed the membrane charge den- sity (rM) of cationic lipid vectors that form lamellar complexes with DNA as a key universal parameter governing their transfection efﬁciency. Our previous work with monovalent cationic lipids also shows this dependence on the average charge per unit area of the membrane. Excluding helper lipids, only the dioleoyl derivatives from a series of primary and tertiary 1,3-dialkoylamido monovalent cationic lipids [31], dif- fering in molecular structure from the 1,3lb series by only a single amine in the polar headgroup, elicited transfection activity. Addition of a second amine group and the subsequent increase in rM increased the

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157

Bis-(2-dimethylamino-ethyl)-amine

The synthesis was carried out as previously described [30].

1,3-Dimyristoylamidopropan-2-ol and 1,3-dimyristoyl- amidopropane-2-(p-nitrophenyl) carbonate

for all derivatives

The compounds were synthesized by a procedure similar to those previously described [31].

1,3-Dimyristoylamidopropane-2-[bis-(2-dimethylamino- ethane)] carbamate (1,3lb2)

capabilities. Lipofection mediated by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, resulted in the highest b-galactosi- dase quantities at + ⁄ ) 4 : 1 in the absence of helper lipids. Transfection activities were reduced in the pres- ence of either DOPE alone or in combination with cho- lesterol except 1,3lb5, which maintained its reporter gene expression levels at + ⁄ ) 4 : 1 and yielded increased enzyme activity at a lower charge ratio of + ⁄ ) 2 : 1. EtBr displacement indicated efﬁcient pDNA compaction by the transfec- tion-competent analogs, irrespective of the ion concen- tration in the dispersion medium (Tris buffer or serum-free medium). Dynamic light-scattering and elec- trophoretic mobility studies revealed lipoplexes of active lipids with mean diameters of several hundred nanometers and positive zeta potentials at low ionic strength, or negative values in high-ionic-strength med- ium. Langmuir ﬁlm balance studies showed high hydra- tion and in-plane elasticity of the active derivatives in isolation. In agreement with the monolayer experi- ments, ﬂuorescence polarization studies veriﬁed the ﬂuid nature of the highly transfection-efﬁcient lipids with gel-to-liquid crystalline phase transitions below physiological temperature. FRET experiments revealed a greater degree, compared with the poorly active deri- vative 1,3lb4, of lipid mixing of the active compounds with anionic vesicles.

Lipids of the 1,3lb series were synthesized with the intent of enhancing the transport of exogenous genetic material into cells in vitro, and their design eliminates the need for additional components in the gene-deliv- ery system (i.e. DOPE and cholesterol), providing a simpler yet more potent formulation. The evolution of synthetic cationic lipid carriers in our laboratory and the structure–activity data collected for the various ser- ies contribute to the long-term goals of understanding the mechanism of lipofection, and the rational design of pharmaceutically sound and therapeutically superior nonviral vectors for gene therapy in vivo.

M. Spelios and M. Savva Novel cytofectins for gene delivery

Experimental procedures

To a solution of N,N¢-ditetradecanoyl-1,3-diaminopropane- 2-(p-nitrophenyl)-carbonate (0.0059 mol; 4.32 g) in 30 mL anhydrous CH2Cl2 were added bis-(2-dimethylaminoethyl)- amine (0.0118 mol) and triethylamine (0.00118 mol). The reaction was stirred at room temperature for 3.5 h, after which time the solvent was evaporated under vacuum and the reaction mixture transferred to a separation funnel with 100 mL of water and 100 mL of ethyl acetate. The aqueous layer was discarded, and the organic phase was washed three times with 100 mL saturated potassium bicarbonate solution to remove the p-NO2-phenol. The oily residue that resulted after concentrating the organic layers was dissolved in a minimum amount of chloroform and loaded onto a sil- ica gel column (26 · 2.8 cm). The column was eluted sequentially with 100 mL chloroform, and 1, 3, 5, 7, 8, 9, 10, 12 (300 mL) and 15% methanol ⁄ chloroform. The 10% and 12% fractions were combined and concentrated to give 2.79 g (68%) of N,N¢-ditetradecanoyl-1,3-diaminopropyl-2- carbamoyl-[bis-(2-dimethylaminoethane)] as a waxy mate- rial. The calculated composition for C40H81N5O4 (relative molecular mass 695) was C, 69.06; H, 11.65; N, 10.07. That found was C, 68.54; H, 11.92; N, 9.96. MS (FAB) m ⁄ z 696.4 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 (cid:2)C, TMS) d 6.86–6.83 (t, 2H, HNCO), 4.72–4.70 (m, 1H, CH), 3.45– 3.30 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.35 [m, 4H, (CH2)2N], 2.21–2.20 [d, coherent peak, 12H, N(CH3)2], 2.19–2.11 (t, 4H, CH2CO), 1.58–1.53 (m, 4H, CH2CH2CO), 1.23–1.20 [coherent peak, 40H, 10(CH2)2], 0.84–0.81 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 (cid:2)C, TMS) d 174.82 (NHCO), 156.37 [NC(O)O], 73.47 (CH), 59.28, 58.43, 46.98, 46.79, 40.66, 37.92, 33.10, 30.87, 30.84, 30.72, 30.59, 30.57, 30.55, 27.02, 23.89, 15.36.

Materials

1,3-Dilauroylamidopropane-2-[bis-(2-dimethylamino- ethane)] carbamate (1,3lb1)

Reagents and solvents were purchased from commercial suppliers and were used without further puriﬁcation.

calculated composition for C36H73N5O4

Synthesis

[M+H]+;

N,N¢-diacyl-1,3-diaminopropyl-2-carbamoyl-[bis-(2-dimethyl- aminoethane)] derivatives were synthesized and identiﬁed to purity > 99%, as described below.

The (relative molecular mass 639) was C, 67.61; H, 11.42; N, 10.95. That found was C, 67.62; H, 11.57; N, 10.98. MS (EI) m ⁄ z 1H NMR (400 MHz, CDCl3, 20 (cid:2)C, 641.1. TMS) d 6.79–6.70 (t, 2H, HNCO), 4.73–4.70 (m, 1H, CH), 3.58–3.29 [m, 8H, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.35

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158

(CH2)2N], 2.22–2.21 [d, coherent peak, 12H, [m, 4H, N(CH3)2], 2.19–2.12 (t, 4H, CH2CO), 1.62–1.56 (m, 4H, CH2CH2CO), 1.24–1.21 [coherent peak, 32H, 10(CH2)2], 0.84–0.81 (t, 6H, CH3).

TMS) d 174.81 (NHCO), 156.38 [NC(O)O], 131.40–130.52 (C=C), 73.44 (CH), 65.34, 59.28, 58.46, 46.96, 46.77, 40.58, 37.86, 33.86, 33.77, 33.70, 30.94, 30.90, 30.84, 30.79, 30.71, 30.66, 30.62, 30.53, 30.50, 30.40, 30.38, 30.32, 30.27, 30.12, 28.40, 28.38, 27.65, 23.87, 15.34.

Plasmids

1,3-Dihexanoylamidopropane-2-[bis-(2-dimethylamino- ethane)] carbamate (1,3lb3)

calculated composition for C44H89N5O4

pUC19-b-gal and pEGFP-N1 were propagated in DH5a- competent cells and collected according to standard proto- cols [36]. Plasmid DNA was puriﬁed by gel permeation chromatography using a Sepharose 4B-packed column equilibrated with 2.5 m ammonium acetate. Agarose gel electrophoresis and the spectrophotometrically determined A260 ⁄ A280 ratio veriﬁed that the pDNA was of high quality and purity.

Lipid dispersions and lipoplexes

(relative The molecular mass 751) was C, 70.31; H, 11.85; N, 9.32. That found was C, 68.97; H, 11.60; N, 9.74. MS (FAB) m ⁄ z 752.7 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 (cid:2)C, TMS) d 6.80 (bs, 2H, HNCO), 4.73 (m, 1H, CH), 3.45–3.31 [m, (CH2)2NC(O)O, CH2NHC(O)], 2.41–2.38 [m, 4H, 8H, (CH2)2N], 2.23–2.21 [d, coherent peak, 12H, N(CH3)2], 2.17–2.13 (t, 4H, CH2CO), 1.60–1.57 (m, 4H, CH2CH2CO), 1.25–1.22 [coherent peak, 48H, 12(CH2)2], 0.86–0.83 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 (cid:2)C, TMS) d 174.82 (NHCO), 156.41 [NC(O)O], 73.51 (CH), 59.41, 58.50, 47.11, 46.87, 40.70, 38.00, 33.14, 30.93, 30.89, 30.75, 30.62, 30.59, 27.05, 23.93, 15.39.

1,3-Distearoylamidopropane-2-[bis-(2-dimethylamino- ethane)] carbamate (1,3lb4)

calculated composition for C48H97N5O4

Cationic lipids, DOPE and cholesterol were dissolved sepa- rately in chloroform, and the appropriate volume of each solution was added to 12 · 75 mm borosilicate glass dispos- able culture tubes. The concentration of cationic lipid and the molar ratio of cationic lipid : DOPE : cholesterol in the lipid formulations were maintained at 0.3 mm and ﬁnal 3 : 2 : 1, respectively. Complete evaporation of organic sol- vent, ﬁrst under a stream of nitrogen gas and then by high vacuum desiccation, was followed by hydration of the dry lipid ﬁlms in Tris buffer (40 mm, pH 7.2) at elevated tem- perature with periodic vortexing. Lipoplexes were prepared at + ⁄ ) charge ratios of 1 : 1, 2 : 1 and 4 : 1 by pipetting a constant volume of pDNA solution into an appropriate amount of diluted liposome preparation.

In vitro transfection studies

The (relative molecular mass 807) was C, 71.37; H, 12.02; N, 8.67. That found was C, 70.62; H, 12.18; N, 8.35. MS (FAB) m ⁄ z 808.6 [M+H]+,780.6 [M+-(CH3)2]; 1H NMR (400 MHz, CDCl3, 20 (cid:2)C, TMS) d 6.90–6.88 (bs, 2H, HNCO), 4.72– (CH2)2NC(O)O, 4.70 (m, 1H, CH), 3.47–3.29 [m, 8H, CH2NHC(O)], 2.40–2.36 [m, 4H, (CH2)2N], 2.21–2.20 [d, coherent peak, 12H, N(CH3)2], 2.15–2.11 (t, 4H, CH2CO), 1.58–1.55 (m, 4H, CH2CH2CO), 1.20 [coherent peak, 56H, 14(CH2)2], 0.84–0.81 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 (cid:2)C, TMS) d 174.83 (NHCO), 156.40 [NC(O)O], 73.48 (CH), 59.38 [N(CH3)2], 58.49 [N(CH3)2], 47.07 [(CH2)2N], 46.87 [(CH2)2N], 40.67, 37.96, 33.13, 30.91, 30.87, 30.74, 30.62, 30.59, 27.03, 23.92, 15.33.

1,3-Dioleoylamidopropane-2-[bis-(2-dimethylamino- ethane)] carbamate (1,3lb5)

Aliquots (250 lL) of lipoplexes in serum-free medium con- taining 1 lg pUC19-b-gal plasmid DNA were added to approximately 50 000 B16-F0 cells seeded into each well of a 48-well tissue culture plate at least 12 h before transfec- tion, and maintained in serum medium (Dulbecco’s modi- ﬁed Eagle’s medium supplemented with 10% fetal bovine serum, 50 unitsÆmL)1 penicillin and 50 lgÆmL)1 streptomy- cin). After incubation for 4 h at 37 (cid:2)C in a 5% CO2 in air atmosphere, the lipoplexes were removed and replaced with 0.5 mL fresh serum medium. The cells were lysed after an additional 44 h, and lipofection activity in terms of reporter enzyme levels was quantiﬁed by a microplate colorimetric assay utilizing the b-galactosidase substrate ONPG. Lipo- plex cytotoxicity was assessed by MTT assay.

A similar procedure was followed for cells transfected with pEGFP-N1 pDNA. Fluorescence images of intact cells were acquired 48 h after lipofection using a Zeiss Axiovert 200M inverted microscope (Carl Zeiss, Go¨ ttingen,

The calculated composition for C48H93N5O4 (relative molec- ular mass 803) was C, 71.73; H, 11.58; N, 8.72. That found was C, 70.45; H, 11.53; N, 8.38. MS (FAB) m ⁄ z 804.8 [M+H]+; 1H NMR (400 MHz, CDCl3, 20 (cid:2)C, TMS) d 6.83 (bs, 2H, HNCO), 5.36–5.31 (m, 4H, CH=CH), 4.75–4.73 (m, 1H, CH), 3.56–3.52 [m, 4H, (CH2)2NC(O)], 3.37–3.29 [m, 4H, CH2NHC(O)], 2.44–2.41 [m, 4H, (CH2)2N], 2.26–2.24 [d, coherent peak, 12H, N(CH3)2], 2.19–2.15 (t, 4H, CH2CO), 2.00–1.95 (m, 8H, CH2CH=CHCH2), 1.61 (m, 4H, CH2CH2CO), 1.29–1.25 [coherent peak, 40H, 10(CH2)2], 0.88–0.84 (t, 6H, CH3); 13C NMR (100 MHz, CDCl3, 20 (cid:2)C,

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M. Spelios and M. Savva Novel cytofectins for gene delivery

Langmuir monolayer studies

Germany). For some experiments, a ﬂuorescein label was covalently attached to the pUC19 plasmid, with a labeling efﬁciency of approximately one marker molecule every 20–60 nucleic acid base pairs (Mirus Label ITTM, Mirus Bio Corporation, Madison, WI, USA), and lipoplexes were formed with either unlabeled lipid dispersions or cationic vesicles labeled with 1 mol% Rh-PE. Images were captured after exchange of the lipoplexes for fresh serum medium.

pKa studies

Monomolecular cationic lipid ﬁlms at the air–water inter- face were studied using a computer-controlled KSV Minit- rough (KSV Instruments Ltd., Helsinki, Finland) equipped with a Wilhelmy plate electrobalance (KSV Instruments Ltd.) to measure surface pressure and two symmetrically moving hydrophilic Delrin barriers (Dupont, Wilmington, DE, USA) to reduce the available surface area. Using a gas-tight microliter syringe, 20 lL of cationic lipid solution (0.75 mm in chloroform) were applied to the surface of 140 mL of the subphase (40 mm Tris, pH 7.2) within a thermoregulated Teﬂon trough (364 · 75 mm effective ﬁlm area). After a time lag of 20 min to ensure complete evapo- ration of organic solvent, the monolayer was compressed at a constant rate of 10 mmÆmin)1. Plots of surface pressure (p) against mean molecular area (A) were automatically generated. Molecular compressibility was assessed from ﬁrst-derivative analysis of the p–A isotherm.

Phase-transition temperature studies

Studies were performed at excitation and emission wave- lengths of 321 and 445 nm, respectively. Excitation and emis- sion slit widths were both 5 nm. Dry DOPC ⁄ cholesterol ⁄ cationic lipid ﬁlms (0.95 ⁄ 0.95 ⁄ 0.1 molar ratio) were reconsti- tuted with Tris buffer (40 mm, pH 7.2) to a ﬁnal combined lipid concentration of 2 mm. The aqueous dispersions were dispensed as 100 lL aliquots into 4.5 mL plastic cuvettes with four optical windows, and diluted to 2 mL with buffered solutions (40 mm Tris, 40 mm Mes) of varying pH containing TNS to give a lipid ⁄ probe molar ratio of 100 : 1. Samples were stirred for 30 min before measurement of TNS ﬂuores- cence intensity. Nonlinear ﬁtting of the pH titration curves was performed using psi-plot (version 7.01, Poly Software International, Pearl River, NY, USA) according to a modi- ﬁed version of the Henderson–Hasselbach equation

ð1Þ

F ¼ A þ

B 1 þ 10CðpH (cid:3) DÞ

cationic

where A is the minimum ﬂuorescence, B is the difference between the maximum and minimum emission intensities, C is a parameter affecting the slope of the transition region, and D is equal to the acid dissociation constant (pKa) of the statistics were lipid [12]. Goodness-of-ﬁt assessed within a 95% conﬁdence interval.

Cationic lipid dispersions (0.5 mm) were prepared with 1 mol% DPH, ensuring minimal membrane perturbation by the ﬂuorophore. Studies were conducted at an excitation wavelength of 351 nm (slit width 5 nm) using a Cary (Varian Instruments, Walnut Eclipse spectroﬂuorometer Creek, CA, USA) equipped with motorized polarizers and a temperature-controlled four-window cuvette holder. Anisotropy (r) values of constantly stirred samples at vari- ous temperatures were calculated using the Cary Eclipse advanced reads application, software version 1.1 (Varian Instruments), from ﬂuorescence intensities of emitted light at 430 nm (slit width 5 nm) polarized parallel and perpen- dicular to the illuminating beam. Nonlinear ﬁtting of the r–T proﬁles was performed as described previously [30].

Cationic lipid–pDNA binding studies

Particle size and electrophoretic mobility studies

Measurements were carried out at room temperature using a Malvern Zetasizer Nano ZS system (Malvern Instru- ments, Southboro, MA, USA) validated using polystyrene microspheres (Duke Scientiﬁc Corporation, Palo Alto, CA, USA) of 60 and 220 nm certiﬁed mean diameter. Lipoplex- es were prepared with either Tris buffer (40 mm, pH 7.2), ﬁltered using a 0.2 lm ﬁlter, or with sterile SFM. Lipo- somes were prepared with the same ﬁltered buffer or SFM by diluting cationic lipid dispersions (0.3 mm) to 24 nm. Mean diameters of samples in disposable polymethylmeth- acrylate cuvettes were obtained, using a 633 nm laser, from Gaussian analysis of the intensity-weighted particle size distributions using the instrument software (dispersion technology software 3.00, Malvern Instruments Ltd.). Electrophoretic mobility was measured in a folded capillary

Plasmid DNA (22.5 lg) and EtBr (0.8 lg) were combined in a quartz cuvette and diluted to 22.7 and 0.68 lm, respec- tively, using Tris buffer or serum-free medium (SFM). Cat- ionic lipids (1 mm) were added in 6.8 lL aliquots with continuous stirring. The enhanced ﬂuorescence intensity of intercalated dye was measured at charge ratio increments of + ⁄ ) 0.2 : 1 at an excitation wavelength of 515 nm (slit width 5 nm) and an emission wavelength between 595 and 605 nm (slit width 2.5 nm). The emission readings were trea- ted as described previously [30] to generate plots of percent- age ethidium bromide displaced from cationic lipid-bound pDNA against the lipoplex charge ratio. The data was not light-scattering effects, which caused a corrected for change in the ﬂuorescence signal of less than 2%, as lacking EtBr. The binding determined from samples proﬁles were modeled as parabolic functions of the form y = ax2 ± bx.

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cell (Malvern Instruments) using the laser Doppler veloci- metry technique and converted by the software to zeta potential.

5 Wettig SD, Badea I, Donkuru M, Verrall RE & Fold- vari M (2007) Structural and transfection properties of amine-substituted gemini surfactant-based nanoparti- cles. J Gene Med 9, 649–658.

Lipid-mixing studies

6 Rajesh M, Sen J, Srujan M, Mukherjee K, Sreedhar B & Chaudhuri A (2007) Dramatic inﬂuence of the orien- tation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery. J Am Chem Soc 129, 11408–11420.

7 Antipina MN, Schulze I, Dobner B, Langner A &

Brezesinski G (2007) Physicochemical investigation of a lipid with a new core structure for gene transfection: 2-amino-3-hexadecyloxy-2-(hexadecyloxymethyl)propan- 1-ol. Langmuir 23, 3919–3926.

8 Chen H, Zhang H, McCallum CM, Szoka FC & Guo X (2007) Unsaturated cationic ortho esters for endo- some permeation in gene delivery. J Med Chem 50, 4269–4278.

PC : PA vesicles (73 : 25) containing 1 mol% each of NBD-PE and Rh-PE were prepared in phosphate buffer (pH 7.4) and diluted to 3 mL (25 lm total lipid concentra- tion) in a magnetically stirred quartz ﬂuorescence cell, ther- mostatically controlled at 37 (cid:2)C, with either the same buffer or acetate buffer pH 4.0. The ionic strength of buf- fers was adjusted to 154 mm using NaCl. Negatively charged liposomes were treated with a twofold molar excess of cationic lipids, and emission scans (kex = 475 nm) were recorded at various times over a period of approximately 0.5 h. The extent of fusion between labeled and unlabeled vesicles was calculated as

(cid:3)

(cid:2)

(cid:4) 100

ð2Þ

% lipid mixing ¼

Ft (cid:3) F0 F100 (cid:3) F0

9 Gardner RA, Belting M, Svensson K & Phanstiel O IV (2007) Synthesis and transfection efﬁciencies of new lipophilic polyamines. J Med Chem 50, 308–318.

where Ft, F0 and F100 are the NBD ⁄ Rh ratios of maximum probe ﬂuorescence intensities at a particular time t, before addition of cationic lipids, and in the presence of 0.1% Triton X-100, respectively.

10 Lamarche F, Mevel M, Montier T, Burel-Deschamps L, Giamarchi P, Tripier R, Delepine P, Le Gall T, Cartier D, Lehn P et al. (2007) Lipophosphoramidates as lipidic part of lipospermines for gene delivery. Bioconjug Chem 18, 1575–1582.

11 Takahashi T, Kojima C, Harada A & Kono K (2007)

M. Spelios and M. Savva Novel cytofectins for gene delivery

Acknowledgements

Alkyl chain moieties of polyamidoamine dendron-bear- ing lipids inﬂuence their function as a nonviral gene vector. Bioconjug Chem 18, 1349–1354.

12 Spelios M, Nedd S, Matsunaga N & Savva M (2007)

This work was supported in part by a pre-doctoral fel- lowship in pharmaceutics from the PhRMA Founda- tion (Washington, DC) and by National Institutes of Health Grant EB004863.

Effect of spacer attachment sites and pH-sensitive head- group expansion on cationic lipid-mediated gene deliv- ery of three novel myristoyl derivatives. Biophys Chem 129, 137–147.

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M. Spelios and M. Savva Novel cytofectins for gene delivery