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 docx

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 o

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

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 each of the lipid structural components on gene

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)

doi:10.1111/j.1742-4658.2007.06185.x

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 transfecpolariza-tion-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

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.

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 together Since the advent of lipofection

20 years ago [1], cationic lipids with new molecular

architectures have been developed and analyzed as

gene-delivery vectors, as seen in numerous 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-diaminopropyl-2-carbamoyl bivalent cationic lipids

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- posi2-posi-tions

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 pKadetermination, isothermal monolayer

compres-sion, fluorescence anisotropy, ethidium bromide

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

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

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

Results Biological analysis 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

N

N

H3C

O

O

R

O

O

R

H3C

H3C

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)

Fig 1 Structure of the 1,3lb derivatives.

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

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 1,3lb5 > DOTAP > 1,3lb4

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

of the transfection-active compounds at condensing 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

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 lipofection efficiency of 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)

0

100

200

300

400

500

600

700

800

900

1000

1100

A

B

C

1,3lb2 1,3lb3 1,3lb4 1,3lb5 DOTAP

0

100

200

300

400

500

600

700

800

900

1000

1100

+/– charge ratio

+/– charge ratio

+/– charge ratio

1,3lb2/D 1,3lb3/D 1,3lb4/D 1,3lb5/D DOTAP

0

100

200

300

400

500

600

700

800

900

1000

1100

1,3lb2/D/c 1,3lb3/D/c 1,3lb4/D/c 1,3lb5/D/c DOTAP

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).

MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide] reduction analysis revealed a low

cytotoxicity of lipoplexes at all charge ratios and

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

A

B

C

D

E

F

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.

A 1,3lb2

1,3lb3

1,3lb4

1,3lb5

B

Physicochemical characterization

pKastudies

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

headgroup Curve fitting of the experimental data

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

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)

The TNS assay was used to ascertain the pKa values

of the cationic lipids in assemblies where they were

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 pKavalues 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-group hydration, and, subsequently, protonation 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

Table 1 Acid dissociation constants of cationic lipids as

deter-mined by nonlinear fitting of TNS fluorescence intensity–pH plots.

Coefficient of determination b

% ionization

at pH 7.2

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 pK a were assessed within a 95%

confidence interval.

0 20 40 60 80 100

+/– charge ratio

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

SFM

0 20 40 60 80 100

+/– charge ratio

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

Fig 5 Percentage ethidium bromide displacement against the charge ratio of lipoplexes in Tris buffer or SFM Parabolic curve fits

of the experimental data (solid points) are shown as dashed lines.

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.

Cationic lipid–pDNA binding studies

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

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 the distearoyl derivative, exist in an all

liquid-expanded state at 23C 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-phobic chains more effectively excludes interstitial

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

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)1and 70 A˚2, respec-tively, at which point a transition occurred to a chain-ordered phase The pressure continued to rise upon further surface area reduction until the monolayer finally collapsed at 49 mNÆm)1 and 39 A˚2 At 37C, the two-dimensional transition was absent and only a liquid-expanded state was evident For all derivatives, molecular dimensions increased and compression 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 C; the derivatives possessed similar mean molecular areas at monolayer collapse, and their collapse pressures were nearly identical 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 23C and 37 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 23C, with a dp ⁄ dAcvalue 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, Table 2 Monolayer transition a and collapse parameters of the 1,3lb series Measurements were performed using Tris buffer (40 m M ,

pH 7.2) as the subphase Values reported are the mean of n experiments ± standard deviation.

1,3lb1 (n = 4,6) 59.27 ± 4.89 64.90 ± 3.22 37.75 ± 2.01 36.59 ± 1.34 1.27 ± 0.07 1.13 ± 0.08 L 1 L 1

1,3lb2 (n = 4,3) 55.98 ± 2.55 60.61 ± 2.29 41.62 ± 0.94 36.31 ± 1.37 1.55 ± 0.10 1.04 ± 0.04 L1 L1 1,3lb3 (n = 3,4) 46.91 ± 1.39 55.61 ± 3.41 40.29 ± 0.72 38.62 ± 0.75 1.32 ± 0.04 1.11 ± 0.03 L1 L1 1,3lb4 (n = 11,6) 38.70 ± 3.03

70.49 ± 3.43 a

56.06 ± 3.81 48.68 ± 6.71

23.73 ± 1.09 a

39.89 ± 0.80 2.29 ± 0.51 1.20 ± 0.11 L 2

L1

L 1

1,3lb5 (n = 7,7) 54.08 ± 3.30 57.92 ± 6.41 39.04 ± 3.81 36.82 ± 2.54 1.00 ± 0.17 1.20 ± 0.14 L1 L1

a Phase transition was determined by a discontinuity in the plot of dp ⁄ dA against mean molecular area (not shown) b L1and L2indicate the liquid-expanded and liquid-condensed states, respectively.

existing in a liquid crystalline state within the range

of temperatures scanned (Fig 6A) A gel-to-liquid

crystalline phase transition was detected for all other

derivatives (temperature span 6–7C), 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

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 23C 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 23C and 37 C The three-dimensional phase transition exhibited by 1,3lb4 at 45C complies with monolayer compression data indicating the pres-ence of a chain-ordered phase at 23C 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 23C and 37 C of the dipalmitoyl and distearoyl derivatives, respectively, in monolayers complement the nature of these lipids in bilayer assemblies at these temperatures

Particle size and electrophoretic mobility studies

In vitro transfection activity was found to be a func-tion of the size of the cafunc-tionic 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 ( 0.5 lm), as well as transfection activ-ity at the highest +⁄) charge ratio tested The 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)

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 lipofection at these charge ratios (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

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

A

B

0 10 20 30 40 50 60

0 10 20 30 40 50 60

T (°C)

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

T (°C)

1,3lb2 1,3lb3 1,3lb4 1,3lb5

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 m M Tris, pH 7.2.

Table 3 Midpoint and onset phase-transition temperatures

obtained from curve fits and first-derivative analysis, respectively,

of the experimental data in Fig 6.

Lipid

T m

(C)

Coefficient of

determination a

T onset

(C)

T offset

(C) Toffset) onset

a

Best-fit parameters were assessed within a 95% confidence

inter-val.

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, respectively At high ionic 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

Lipid-mixing studies

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-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 fluorescence resonance energy transfer from

nitro-0

200

400

600

800

A

B

C

D

1 000

1 200

1 400

1 000

1 200

1 400

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

0

200

400

600

800

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

–80 –60 –40 –20

–80 –60 –40 –20

0 20 40 60 80

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

0 20 40 60 80

1,3lb1 1,3lb2 1,3lb3 1,3lb4 1,3lb5

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 m M Tris, pH 7.2 (A,C) and SFM (B,D).

0 10 20 30 40 50 60

pH 4.0

pH 7.4

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

154 m M ionic strength at physiological and acidic pH.

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 binding studies (Fig 5): 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

Discussion

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 abspres-ence 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

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 rMincreased the

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

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 1- and 2- positions of the 1,2-diamino-3-propanol 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

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