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Journal of Controlled Release 96 (2004) 309–323

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Hydrophobized dextran-spermine conjugate as potential vector for

in vitro gene transfection

Tony Azzama, Hagit Eliyahua, Arik Makovitzkia,Michal Linialb, Abraham J. Domba,*,1

aDepartment of Medicinal Chemistry and Natural Products, Faculty of Medicine, School of Pharmacy, Hebrew University of Jerusalem,

Jerusalem 91120, IsraelbDepartment of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91905, Israel

Received 11 August 2003; accepted 13 January 2004

Abstract

Dextran polysaccharide was grafted by reductive-amination with mixtures of spermine and other natural/synthetic oligoamines

of two to four amine groups. The transfection efficiencies of the polycations thus obtained were assessed in various cell lines, and

found to depend on the spermine contents. Higher spermine ratios of grafted oligoamines resulted in high gene expression,

whereas low to negligible expressions were obtained with lower spermine contents. The effect was explained by spermine residues

which exhibit altered buffering capacity in comparison to other substituted oligoamines. Hydrophobization of dextran-spermine

(D-SPM) was achieved by treating the polymer with N-hydroxysuccinimide derivatives of cholesterol and fatty acids in a mixture

of water/THF. The degree of hydrophobization was in the range of 1–30%mol/mol (hydrophobicmoieties/primary amine) and the

coupling yields were >95% as determined by 1H-NMR. The oleate-modified D-SPM remarkably enhanced the gene expression in

serum rich media, in marked contrast to unmodified D-SPM which resulted with a drastic decrease in the transfection yields.

Modified D-SPM derivatives of other fatty acids and cholesterol showed improved transfection yields in comparison to

unmodified D-SPM, but to a lower extent when compared to oleate modification. The improvement in cell transfection was

attributed to oleate residues which probably play a role in increasing stability and uptake of polycation–DNA complexes.

D 2004 Published by Elsevier B.V.

Keywords: Dextran; Spermine; Reductive-amination; Gene delivery; Hydrophobic

1. Introduction

DNA can be delivered into the cell nucleus using

physical means or using specific carriers that carry the

0168-3659/$ - see front matter D 2004 Published by Elsevier B.V.

doi:10.1016/j.jconrel.2004.01.022

* Corresponding author. Tel.: +972-2-675-7573; fax: +972-2-

675-8959.

E-mail address: [email protected] (A.J. Domb).1 A.J.D. is affiliated with the David R. Bloom Center for

Pharmacy and with the Alex Grass Center for Drug Design and

Synthesis at the Hebrew University of Jerusalem.

genes into the cells for gene expression. Of the

various methods developed for delivering genes, gene

carriers have been extensively investigated as trans-

fecting agents for therapeutic genes in Gene Therapy.

Gene carriers are divided into two main groups: viral

carriers where the DNA to be delivered is inserted into

a virus, and cationic molecular carriers that form

electrostatic interactions with DNA. Successful gene

therapy depends on the efficient delivery of genetic

materials into the cells nucleus and its effective

T. Azzam et al. / Journal of Controlled Release 96 (2004) 309–323310

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expression within these cells [1]. Although at present,

the in vivo expression levels of synthetic molecular

gene vectors are lower than for viral vectors and gene

expression is transient, these vehicles are likely to

present several advantages including safety, low im-

munogenicity, capacity to deliver large genes, and

large-scale production at low cost. The two leading

classes of synthetic gene delivery systems that have

been mostly investigated involve the use of either

cationic lipids or cationic polymers [2].

Cationic polymers, commonly named polycations,

are a leading class of molecular gene-delivery sys-

tems, in part because of their molecular diversity that

can be modified to fine-tune their physicochemical

properties [3,4]. Polyelectrolyte complexes (PEC)

formed between DNA and polycations have been

shown to tightly pack the DNA in the PEC complex,

so that the entrapped DNA is shielded from contact

with DNase [5]. Polycations commonly used in gene

delivery and transfection include polyethylenimine

[6], poly(L-lysine) [7], cationic dendrimers [8], poly-

brene [9], gelatin [10], tetraminofullerene [11],

poly(L-histidine)-graft-poly(L-lysine) [12], and cation-

ic polysaccharides. Although PEC systems have some

advantages over viral vectors, e.g. low immunogenic-

ity and easy manufacture [13,14], several problems

such as toxicity, lack of biodegradability, low bio-

compatibility and, in particular, low transfection effi-

ciency need to be solved prior to practical use [15].

Polycations used in gene delivery are polyamines that

become cationic at physiological conditions. All pol-

ymers contain primary, secondary, tertiary, or quater-

nary amino groups capable of forming electrostatic

complexes with DNA under physiological conditions.

Most polycations are toxic to cells and non-biode-

gradable, while the polymers based on amino acids

such as poly(L-lysines) are immunogenic [16]. More

advanced polymeric gene delivery systems employ

macromolecules with high cationic charge density that

act as endosomal buffering systems, thus suppressing

the endosomal enzymes activity and protecting the

DNA from degradation [6]. Among the various poly-

cations used in gene delivery and transfection, cation-

ic polysaccharides are considered to be the most

attractive candidates. They are natural, non-toxic,

biodegradable, and biocompatible materials and can

be modified easily for improved physicochemical

properties [17,18].

In recent publications [19,20], we reported on a

new type of biodegradable polycation based on

grafted oligoamine residues on natural polysacchar-

ides, which are effective in delivering plasmids for a

high biological effect. The use of biodegradable

polysaccharide carriers is especially suitable for trans-

fection and biological applications because they are

water soluble, can be readily transported to cells in

vivo by known biological processes, and act as

effective vehicles for transporting agents complexed

with them [21]. More than 300 different polycations

were prepared starting from various polysaccharides

of different molecular weights and oligoamines hav-

ing two to four amine groups. Although most of these

conjugates formed stable complexes with DNA as

determined by ethidium bromide quenching assay

[22], only the dextran-spermine (D-SPM) polycations

of defined molecular weights were found to be active

in transfecting a wide range of cell lines in vitro. The

reason for the transfection of D-SPM conjugate was

attributed to spermine residues, which play a crucial

role in cell transfection.

Success of non-viral gene delivery depends on the

type of carrier materials to bind plasmid DNA and

facilitate the cell uptake of carrier–DNA complex

[23–25]. One of the major obstacles limiting the in

vitro and in vivo gene delivery is the interaction of

carrier–DNA with extracellular fluids. This phenom-

enon is well known and is attributed to proteins

(serum components) adsorption on the surface of the

carrier–DNA complex, which in part could induce the

aggregation/deactivation of the complex and finally to

the reduction in the transfection efficiencies [26–28].

In an attempt to alter the characteristics of the devel-

oped D-SPM, PEGylated derivatives of the polymer

were recently synthesized and tested for their trans-

fection efficiencies in serum rich media. Such modi-

fication resulted in a substantial increase in gene

expression compared to unmodified D-SPM. Also,

in contrast to unmodified D-SPM, PEGylated-D-

SPM complexed with pSV-hGal was shown to induce

gene expression in the liver of mice after i.v. admin-

istration [29]. The use of PEG for this purpose is

attractive since PEG polymers are hydrophilic, safe,

cheap and do not interact with plasma components

[30].

The complexes formed between D-SPM and plas-

mid DNAs is expected to exhibit a hydrophilic surface

T. Azzam et al. / Journal of Controlled Release 96 (2004) 309–323 311

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due to the saccharides building units of the polymer

backbone. Such hydrophilic nature is thought to limit

the cell uptake of the complexes due to the hydro-

phobic nature of the cell membrane. The chemical

modification of D-SPM polycation is expected to

exploit its potential in cell transfection. The objectives

of the present study was to further study the impor-

tance of grafted spermine residues in the success of

transfection, and to test a new series of hydrophobized

D-SPM derivatives in terms of enhancing transfection

and complex stability in serum rich media.

2. Materials and methods

2.1. Materials

All solvents and reagents were of analytical

grade and were used as received. Tetrahydrofuran

(THF) was dried by distillation over sodium/benzo-

phenone. A sage-metering pump model-365 (Orion,

NJ, USA) was used for slow and reproducible

addition of reactants. Dextran with an average

molecular weight of 40 kDa was obtained from

Sigma Chemical Co. (St. Louis, MO, USA). Sper-

mine, spermidine, potassium periodate, sodium bo-

rohydride, cholesteryl chloroformate, octanoyl

chloride, lauroyl chloride, myristoyl chloride,

stearoyl chloride, oleoyl chloride (technical 85%),

and diisopropylethylamine (DIEA) were obtained

from Fluka Chemie (Buchs, Switzerland). N,N-

bis(3-aminopropyl)-1,3-propanediamine [3:3:3],

N,N-bis(3-aminopropyl)-1,2-ethanediamine [3:2:3],

butanediamine, N,N-dimethylpropylenediamine, and

N-hydroxysuccinimide (NHS) were obtained from

Aldrich (Milwaukee, WI, USA). 1H-NMR spectra

were recorded on a Varian 300MHz instrument

using CDCl3, D2O or DMSO-d6 as solvents. Values

were recorded as ppm relative to internal standard

(TMS). Molecular weights of starting polymers and

conjugates were determined on GPC Spectra Phys-

ics instrument (Darmstadt, Germany) containing a

pump, column (Shodex KB-803, Japan) and refrac-

tive index (RI) detector. Molecular weights were

determined according to Pullulan standards (PSS,

Mainz, Germany) with molecular weights between

1700 and 212,000. Eluents used were 0.05 M

NaNO3 for the uncharged polymers and 0.25 M

sodium phosphate buffer (pH 4) for the cationic

polymers [31]. Elemental microanalysis was per-

formed on a Perkin–Elmer 2400/II analyzer.

2.2. Oxidation of dextran

Oxidized dextran was prepared and characterized

as described elsewhere [19,20].

2.3. Dextran-oligoamine conjugates

Dextran grafted with various oligoamines was

synthesized by the reductive amination method as

described earlier [19,20]. In brief, a solution of

dialdehyde dextran (6.25 mmol of aldehyde groups)

in 100 ml of DDW was slowly added over 5 h (sage

metering pump) to a basic solution containing 1.25

molar equivalents of spermine (or other oligoamine

mixtures) dissolved in 50 ml of borate buffer (0.1 M,

pH 11). The mixture was gently stirred at room

temperature for 24 h, NaBH4 (1 g, 4 equimolar)

was added, and stirring was continued for 48 h under

the same conditions. The reduction was repeated with

an additional portion of NaBH4 (1 g) and with stirring

for 24 h under the same conditions. The resulting

light-yellow solution was poured into a dialysis

membrane (3500 cutoff, Membrane Filtration Prod-

ucts Inc., San Antonio, TX, USA) and dialyzed

against DDW (6� 5 l) at 4 jC for 2 days. The

dialysate was gravimetrically filtered to remove

insolubles and lyophilized to dryness.

Yield: 40% (w/w).

FT-IR (KBr): 1468 (UCH2U, aliphatic), 1653

(UNH2, primary amine), 2935 (CUC, aliphatic),

and 3297 (secondary amine and UOH groups) cm� 1.

2.4. Determination of primary amines by the TNBS

method

The primary amine content was determined accord-

ing to standard protocol [32] with a slight modifica-

tion. In brief, a total of 20 Al of freshly prepared

aqueous TNBS solution (15 mg ml� 1) was separately

added to marked tubes containing up to 0.2 Amol

spermine (or other soluble oligoamine) dissolved in

600 Al of DDW. The mixtures were separately diluted

with 200 Al of sodium bicarbonate buffer (0.8 M, pH

8.5), vortexed for 1 min, and incubated for 2 h at 37


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jC. Then, 600 Al of 1 N HCl aqueous solution was

added to each tube, vortexed for 1 min, and gently

sonicated for 2 min to remove bubbles. Absorbances

of samples were recorded at 410 nm. A sample

containing the same composition (without the oligo-

amine) was used as a reference in the absorbance

measurements. Weighed conjugates (50–500 Ag,depending on the degree of conjugation) were treated

as above, and the primary amine content was calcu-

lated according to the calibration curve.

2.5. Synthesis of cholesterol–NHS carbonate

derivative

To 0.5 g of cholesteryl chloroformate (1.12 mmol)

dissolved in 25 ml of anhydrous THF was added 0.4

ml of anhydrous DIEA (2.5 equimolar) under nitrogen

atmosphere. The mixture was cooled to 0 jC and 200

mg of solid NHS (1.5 equimolar to chloroformate)

was added, and stirring was continued for 2 h at 0 jCand overnight at room temperature. The solvent was

removed under reduced pressure and the crude was

dissolved in 50 ml of diethyl ether and washed with

saturated aqueous NaCl solution (2� 20 ml) and

water (2� 20 ml). The ethereal phase was dried over

anhydrous MgSO4, filtered and evaporated to dryness.

The crude compound was crystallized in dichloro-

methane/methanol (1:5), stored at � 20 jC for 24 h,

filtered and dried in vacuum over P2O5.

Yield: 0.5 g (f 85%); Rf 0.65 (1% methanol in

dichloromethane).1H-NMR (CDCl3): 0.672 (s, 3H), 0.847 (d, 3H),

0.897 (d, 3H), 0.94–2.1 (m, 32H), 2.492 (d, 2H),

2.830 (s, 4H, UCH2CH2U of NHS group), 4.597 (m,

1H), and 5.422 (m, 1H) ppm.

2.6. Synthesis of fatty acid–NHS ester (general

method)

1.33 g of NHS (11.56 mmol) and 1.35 ml of DIEA

(9.5 mmol) were dissolved in 50 ml of anhydrous

THF under inert atmosphere. The mixture was cooled

to 0 jC and 7.8 mmol of the corresponding fatty acid

chloride in 50 ml of anhydrous THF was slowly added

during 1 h. The mixture was stirred at 0 jC for 2 h and

overnight at room temperature. The resulting precip-

itate salt was discarded by filtration and the filtrate

was evaporated under reduced pressure, redissolved in

small amount of dichloromethane and purified over

silica-gel using dichloromethane as eluent. Fractions

containing the product were collected and solvent was

removed under reduced pressure to obtain colorless

oil. The fatty acid–NHS ester was crystallized from

hot ethanol, collected by filtration and vacuum-dried

over P2O5.

The follows summarize the yields, Rf (TLC) and1H-NMR of all NHS-derivatives:

2.6.1. NHS-oleate

Yield: 92%; Rf 0.35 (CHCl3).1H-NMR(CDCl3): 0.874 (t, 3H), 1.38 (m, 20H),

1.74 (m, 2H), 2.004 (m, 4H), 2.594 (t, 2H), 2.83 (s,

4H) and 5.34 (m, 2H) ppm.

2.6.2. NHS-octanoate

Yield: 85%; Rf 0.43 (CHCl3).1H-NMR (CDCl3): 0.874 (t, 3H), 1.210–1.480 (m,

8H), 1.738 (m, 2H), 2.595 (t, 2H) and 2.832 (s, 4H)

ppm.

2.6.3. NHS-laurate


18H), 1.736 (m, 2H), 2.594 (t, 2H) and 2.827 (s, 4H)

ppm.

2.6.4. NHS-myristate

Yield: 83%; Rf 0.32 (CHCl3).1H-NMR (CDCl3): 0.877 (t, 3H), 1.210–1.4930

(m, 20H), 1.765 (m, 2H), 2.597 (t, 2H) and 2.835 (s,

4H) ppm.

2.6.5. NHS-stearate


28H), 1.735 (m, 2H), 2.589 (t, 2H) and 2.828 (s, 4H)

ppm.

2.7. Modification of dextran-spermine with oleic acid

D-SPM conjugate (20 mg, f 26 Amol of primary

amine, TNBS method) was dissolved in 1.5 ml

mixture of DDW:THF (1:2). The solution was vigor-

ously stirred at room temperature using a microstirrer

and 1–30% mol/mol (to primary amine) of stock

NHS-oleate solution in anhydrous THF was added.


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The mixture was stirred at room temperature for 24

h and THF was removed by a flux of nitrogen, diluted

with DDW and dialyzed against DDW to remove

NHS and unbound fatty acids. The remaining mixture

was freeze-dried and the lyophilate was stored under

nitrogen atmosphere.

Average yield: 18 mg (f 90% w/w).1H-NMR (D2O): 0.696 (m, terminal methyl

group of oleate), 1.095 (m, oleate methylene groups),

1.437 (m, Dextran-NHCH2CH2CH2NHCH2CH2CH2

CH2NHCH2CH2CH2CH2NH2), 1.617 (m, Dextran-

NHCH2CH2CH2NHCH2CH2CH2CH2NHCH2CH2

CH2CH2NH2), 2.15–3.26 (m, Dextran-NHCH2CH2

CH2NHCH 2CH2CH2CH2NHCH2CH2 CH2CH2NH2),

3.30–4.45 (m, polysaccharide hydrogens), 4.773 (m,

anomeric hydrogens of glycosidic linkages), and 5.140

(m, double bond of oleate residues) ppm.

D-SPM derivatives modified with 20% mol/mol (to

primary amine) of other fatty acids and cholesterol

were similarly prepared and characterized by 1H-NMR.

2.8. Transfection studies

2.8.1. Preparation of plasmid DNAs

Plasmid DNAs used in the transfection experi-

ments were pCMV-GFP encoding green fluorescence

protein, pSV-hGal encoding h-galactosidase, and

pLNC-luc encoding luciferase. Plasmid DNAs were

separately amplified in a transformant of Escherichia

coli bacteria and isolated from the bacteria by

Qiagen Maxi kit-25 (Qiagen K.K., Tokyo, Japan).

Briefly, the grown bacteria were harvested and lysed

in an aqueous solution of 1 wt.% sodium dodecyl

sulfate (SDS) and 0.1 wt.% RNase A solution in

NaOH (pH 8), and the lysate was neutralized by the

addition of 3 M potassium acetate (pH 5.5). After

separation of the insoluble portion by use of QIA

filter cartridge, the lysate was applied to the Qiagen-

tip (anion-exchange resin), followed by washing with

a buffer containing 1 M NaCl to remove traces of

RNAs and proteins. The plasmid DNA was eluted

with an elution buffer containing 1.25 M NaCl at pH

8.5, de-salted, and precipitated by 2-propanol. The

precipitated plasmid DNA was centrifuged at 14,000

rpm for 10 min at 4 jC and washed twice with 70%-

ethanol aqueous solution to substitute 2-propanol for

ethanol. After centrifugation (14,000 rpm, 2 min, 4

jC), the resulting plasmid DNA was air-dried and

dissolved in a small volume of 10 mM Tris–HCl

and 1 mM EDTA buffer solution. When measured to

assess the purity of plasmid DNA obtained, the ratio

of the absorbance at 260 nm to that at 280 nm

ranged from 1.8 to 2.0.

2.8.2. In vitro transfection

Human embryonal kidney (HEK293), mouse

fibroblasts (NIH3T3), and cervical cancer (HeLa)

cells were used in the transfection experiments. 0.5

Ag of purified plasmid (Qiagen kit) per well of

transfected cells was mixed with D-SPM (or other

cationic derivatives) at a various weight-mixing ratios

ranging from 1 to 20 w/w (polycation/DNA). The

polycation/DNA complex mixtures were diluted to a

final volume of 200 Al with serum-free medium

(SFM) and allowed to stand at room temperature

for 30 min. Twenty-four-well plates, seeded 24 h be-

fore transfection with 1.5� 105 cells per well, were

washed with SFM and the solution of the complexes

was added to the cell wells and cultured at 37 jC in

95% air/5% CO2. 4 h post transfection, cell medium

was replaced with Dulbecco’s modified Eagle’s Me-

dium (DMEM) containing 10% fetal calf serum

(FCS), and the cells were cultured for 48–72 h under

growth conditions. At this time growth medium was

removed, and cell lysates were formed and analyzed

for gene expression. h-Gal kit (Invitrogen Co., USA)

and luciferase assay kit (Promega) were used accord-

ing to manufacturer’s protocols for the evaluation of

h-galactosidase and luciferase activity, respectively.

Cells transfected with pCMV-GFP were analyzed for

gene expression using fluorescence microscope in-

strument (model Axiovert 35, Zeiss, Jena, Germany).

The yield of transfection (% transfection) was calcu-

lated by counting the fluorescent cells in a field of a

particular well, and dividing the number of fluores-

cent cells by the number of total cells in the same

field. In some cases, the degree of gene expression

was normalized to total protein content using the

standard BCA assay kit (Pierce, USA). For the

transfection experiments in serum-containing medi-

um, SFM was replaced with 10% FCS at the com-

plexation and uptake stages, followed by the same

procedure described above.

Cell transfection with Calcium phosphate reagent

(Sigma) was performed according to a well docu-

mented protocol [33,34]. Transfection applying


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DOTAP/Chol 1/1 (Avanti Polar lipids Inc., Alabama,

USA) and FuGen 6 (Roche Diagnostics, Indianapolis,

IN) based lipid formulations were performed accord-

ing to manufacturer’s protocols.

3. Results and discussion

3.1. Chemistry

3.1.1. Dextran-oligoamine based conjugates: synthe-

sis and characterization

Grafting of the representative oligoamine or

oligoamines mixtures to oxidized dextran was per-

formed by means of reductive amination method as

described earlier [19,20]. The oligoamines used for

conjugation were the naturally occurring spermine

and spermidine, and other oligoamines of two to

four amine groups. Also, dextran-oligoamine con-

jugates substituted with two different oligoamines at

various mole ratios were prepared and characterized

(Scheme 1).

Scheme 1. Reductive amination of typical dextran gra

The polycations were characterized by nitrogen

elemental analysis (%N), primary amine content

(TNBS) and average molecular weights (GPC) as

shown in Table 1. Group A represents seven dextran

derivatives conjugated to spermine (#1), spermidine

(#7), and spermine/spermidine mixed oligoamines

(#2–6) at varying mole ratios ranging from 9/1 to

1/9, respectively. The average nitrogen (%N) and

primary amine contents found for this group was

11.0F 0.30 and 1.25F 0.08 (Amol mg� 1), respec-

tively. The remarkable decrease in the average

molecular weights of conjugates in comparison to

starting dextran (40 kDa) is explained by the

random and extensive aminolysis of glycoside link-

ages during conjugation [35].

Group B of Table 1 shows a series of cationic

dextran derivatives conjugated to various commercial

oligoamines and their mixtures with spermine. The

derivative conjugated to N,N-dimethyl-1,3-propanedi-

amine (#7, Group B) was shown to exhibit a low and

negligible TNBS value as expected due to the absence

of primary amine groups. On the other hand, when

fted to mixed spermine/spermidine oligoamines.

Table 1

Chemical characterization of dextran derivatives grafted with various oligoamine mixturesa

# Substituted oligoamine(s)b % SPMc %Nd Amol mg� 1 (TNBS)e Mw (kD) Mn (kD) P

Group A

1 SPM 100 11.28 1.37F 0.05 10.07 4.50 2.23

2 SPM/SPD 90 10.89 1.29F 0.04 11.74 4.77 2.46

3 SPM/SPD 70 11.35 1.20F 0.12 9.43 4.10 2.29

4 SPM/SPD 50 10.59 1.10F 0.10 11.08 4.35 2.54

5 SPM/SPD 30 11.34 1.26F 0.05 8.87 3.92 2.26

6 SPM/SPD 10 10.68 1.36F 0.03 11.50 4.68 2.45

7 SPD 0 10.96 1.22F 0.02 10.80 4.47 2.42

Group B

1 [3:3:3] 0 9.58 1.07F 0.08 6.14 4.19 1.46

2 SPM/[3:3:3] 70 10.51 1.37F 0.02 7.42 3.78 1.96

3 [3:2:3] 0 9.67 1.28F 0.05 6.95 5.19 1.34

4 SPM/[3:2:3] 70 11.29 1.41F 0.03 10.48 4.34 2.41

5 [4] 0 9.05 2.43F 0.12 5.70 4.68 1.22

6 SPM/[4] 70 9.24 1.17F 0.04 8.89 4.02 2.21

7 N-[3]-N(CH3)2 0 9.68 0.05F 0.02 13.72 11.03 1.24

8 SPM/N-[3]-N(CH3)2 70 10.76 1.09F 0.04 11.05 4.60 2.40

a Reaction conditions: oxidized dextran (f 50% dialdehyde) and the appropriate oligoamine or oligoamine mixtures (1/1.25, aldehyde/(total

oligoamine)) were allowed to react under similar conditions as described in the Experimental section.b Abbreviations: SPM (spermine); SPD (spermidine); [3:3:3] (N,N-bis(3-aminopropyl)-1,3-propanediamine); [3:2:3] (N,N-bis(3-amino-

propyl)-1,2-ethanediamine); [4] (1,4-butanediamine); N-[3]-N(CH3)2 (N,N-dimethyl-1,3-propanediamine).c Percent of spermine content in dextran– (mixed oligoamine) conjugate.d Found nitrogen content (elemental analysis).e Amount of primary amine (Amol mg� 1) determined by the TNBS method (meanF S.D.). Mw, Mn, and polydispersity ( P=Mw/Mn) were

determined by GPC as described in the Experimental section for the polycationic polymers.


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spermine was added in excess to N,N-dimethyl-1,3-

propanediamine oligoamine (#8, Group B), a relative-

ly high primary amine content was obtained (f 1.1

Amol mg� 1).

3.1.2. Hydrophobization of dextran-spermine with

fatty acids and cholesterol

Preliminary hydrophobization attempts of D-SPM

conjugate with fatty acids chlorides resulted in a low

degree of modification, probably due to rapid hydro-

lysis of the acyl chloride groups in aqueous media

(data not shown). Therefore, NHS-activated fatty

acids were prepared and applied for the modification

of D-SPM. Such derivatives were previously applied

and with high coupling yields in the modification of

monoclonal antibodies [36] and more recently to

poly(L-lysine)s [37,38]. Saturated fatty acid chlorides

of C8–C18 and oleyl chloride were treated with

NHS under anhydrous conditions and in the presence

of base to obtain the corresponding NHS-ester deriv-

atives in relatively high yields. These derivatives

were purified by silica-gel column chromatography

and crystallized from hot ethanol. The purity of the

compounds was >99% as determined by TLC and1H-NMR. Cholesterol activated with NHS-carbonate

group was similarly prepared from NHS and choles-

teryl chloroformate.

D-SPM conjugate was hydrophobized with in-

creasing amounts of fatty acid (1–30% mol/mol, fatty

acid/primary amine) by adding the corresponding

fatty-acid NHS ester to a concentrated solution of

D-SPM in a mixture of water/THF (Scheme 2).

Dialysis was proved efficient to purify the modified

polycations from liberated NHS and traces of un-

bound fatty acids as evaluated by TLC. More than

95% binding (relative to starting feed) was obtained

as determined by 1H-NMR. At low degrees of

fatty acid/cholesterol modification (1 to about 20%

mol/mol), water-soluble derivatives were obtained.

Higher degrees of modification (20% and 30% mol/

mol) resulted in slightly turbid solutions in aqueous

media.

Scheme 2. Hydrophobization of D-SPM conjugate with NHS-oleate.


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3.2. Transfection studies

3.2.1. Role of grafted spermine residues in cell

transfection

Various cationic polysaccharide derivatives hav-

ing multiple amine functionalities of various

grafted oligoamines were previously developed

and tested for their transfection efficiencies in a

wide range of cell lines [19]. Although most of

these conjugates formed stable complexes with

DNAs as determined by the ethidium bromide

quenching assay [22], only the D-SPM based

polycation was found to be active in cell transfec-

tion. The reason for the transfection efficiency of

certain polycations (i.e. D-SPM) is probably related

to the unique complexation properties and the

buffering capacity of grafted spermine residues

[39]. To emphasize the importance of grafted

spermine moieties in cell transfection, a series of

polycations were synthesized by grafting dextran

with mixtures of spermine and other oligoamines

as illustrated in Table 1.

In the first experiment, dextran polycations

grafted with a mixture of spermine/spermidine at

varying mole ratios (Group A, Table 1) were tested

for their transfection efficiencies. The mole ratios of

grafted spermine/spermidine were varied from 9/1 to

1/9, respectively. In addition, two control polycations

were grafted with either spermine or spermidine. The

transfection efficiencies of these polymers were eval-

uated in NIH3T3/pLNC-luc system as a function of

spermine percentages to total grafted oligoamine, and

the results were recorded at the optimal weight-

mixing ratio (polymer/DNA) as shown in Fig. 1a.

When spermine (without spermidine) was grafted to

dextran polysaccharide, high luciferase activity

(f 30,000 RLU/mg protein) was obtained similar

to a DOTAP/Chol 1/1 cationic lipid control (data not

shown). Incorporation of spermidine at 1/9 mole

ratio (i.e. 90% spermine) resulted in a 20% decrease

in luciferase expression. Higher spermidine substitu-

tions resulted in sharp loss of the luciferase expres-

sion (Fig. 1a). In a similar transfection experiment,

conjugates grafted with mixtures of spermine and

other oligoamines (Group B, Table 1) were tested for

their transfection efficiencies in a HEK293/pSV-hGalsystem (Fig. 1b). Dextran conjugated to N,N-bis(3-

aminopropyl)-1,3-propanediamine ([3:3:3], #1), N,N-

Fig. 1. (a) NIH3T3 cells transfected with pLNC-luc applying cationic dextran derivatives grafted with spermine/spermidine at various mole

ratios (2 to #7 of Group A, Table 1). Transfection efficiency was recorded relatively to D-SPM based conjugate (#1 of Group A, Table 1). (b)

HEK293 cells transfected with pSV-hGal applying dextran-(mixed oligoamine) based conjugates (Group B, 1 to #8; Table 1). The transfection

was recorded as OD (Experimental section) and was not corrected to total protein contents in cell wells.


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bis(3-aminopropyl)-1,2-ethanediamine ([3:2:3], #3),

1,4-butanediamine ([4], #5), and N,N-dimethyl-1,3-

propanediamine (N-[3]-N(CH3)2, #7), resulted in low

transfection yields in comparison to D-SPM and

calcium phosphate (CaPi). On the contrary, when

70% of such grafted oligoamines were replaced with


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spermine (polymers 2, 4, 6 and #8; Fig. 1b), high

luciferase expression was obtained similar to D-SPM

and calcium phosphate. These results strongly indi-

cate the effect of the oligoamine structure on the

transfection yield, with spermine being the most

effective.

3.2.2. Hydrophobized dextran-spermine derivatives

According to the results of Fig. 1, it was decided to

focus on the most active polycation form (i.e. D-SPM)

Fig. 2. (a) HEK293 cells transfected with pSV-hGal applying D-SPM polyc

transfection was performed in 10% FCS medium and the h-Gal expres(Experimental section). (b) Inverted fluorescent microscopy of pCMV-GF

and modified D-SPM-20% oleate (II).

for further chemical modifications. One of the major

obstacles in the in vitro and in vivo transfection is the

presence of serum. Serum components are believed to

interact with the DNA–polycation complexes and to

induce aggregation/deactivation of the complex and

finally to a marked reduction in the transfection

efficiencies [27,28]. To emphasize this effect, five

identical D-SPM based conjugates were prepared,

characterized and tested for their transfection efficien-

cies in serum-containing medium (10% FCS) using

ation chemically modified with increasing amount of oleic acid. The

sion was compared to the commercial transfecting reagent Fugen

P transfected HEK293 cells in 10% FCS of unmodified D-SPM (I)


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the NIH3T3/pLNC-luc system. The presence of 10%

FCS remarkably reduced the luc expression by about

80–85% compared with the expression in SFM (data

not shown).

In order to improve the transfection in the pres-

ence of serum, fatty side groups were attached to the

polycation to protect the polymer–DNA complex

from serum proteins, and facilitate cellular uptake

of hydrophobic complexes through the cell mem-

brane [40,41]. The majority of cationic lipids com-

monly used in cell transfection are composed of di-

oleate hydrophobic tails connected via various

spacers to different cationic head groups [42–45].

In analogy to these constructs, the leading polycation

(i.e. D-SPM) was modified with increasing amounts

of oleic acid to evaluate the transfection efficiencies

of these modified derivatives as functions of oleate

and serum contents.

Fig. 2a summarizes the effect of oleate content of

D-SPM on the transfection efficiencies. The transfec-

tion was performed in 10% FCS medium applying the

HEK293/pSV-hGal system. Unmodified D-SPM (i.e.

0% oleate) resulted in low expression yields (OD

0.032) as expected. The highest hGal activities whereobtained with 10–30% oleate contents where nearly

equal expression to the FuGen 6 (Roche Diagnostics)

control was obtained. Fig. 2b shows a typical fluo-

rescent imaging of pCMV-GFP transfected HEK293

cells using D-SPM conjugate (I), and D-SPM-20%

oleate (II) in 10% FCS. It can be clearly concluded

Fig. 3. Effect of oleate content of D-SPM on the transfection efficiencies in

the activity obtained with unmodified D-SPM in SFM. The data was reco

from both images that oleate modification significant-

ly increased the transfection yields. Similar substantial

increase in the transfection efficiency of D-SPM-20%

oleate was obtained with the HeLa cell line (data not

shown).

The effect of oleate modification of D-SPM on

the transfection efficiencies in higher serum-contain-

ing medium (i.e. 20% FCS) was evaluated in

NIH3T3 cells applying pLNC-luc as the marker

gene. Fig. 3 shows the transfection efficiencies of a

series of three D-SPM conjugates modified with

increasing amounts of oleate (10–30% mol/mol).

The transfection yields were recorded relative to

luciferase readings obtained with unmodified D-

SPM in parallel transfection experiment conducted

with the same cell line and in SFM. The transfection

yields in this experiment were recorded as

meanF S.D. (n = 5). At 10% and 20% oleate mod-

ifications, nearly 25% and 45%, respectively, of the

gene expression was retained relative to the gene

expression obtained with unmodified D-SPM in

SFM. Modification of the polymer with 30% oleate

resulted in nearly 75% preservation of the gene

expression in comparison to D-SPM in SFM.

The substantial increase in the transfection effi-

ciencies of the hydrophobized D-SPM in serum-rich

media was attributed, as stated earlier, to the chemi-

cally bound oleate moieties which act as shielding

agents and keep the complexes intact from interaction

with serum components. To emphasize the importance

20% FCS medium. The luciferase activity was recorded relatively to

rded as meanF S.D. (n= 5).

Table 2

NIH3T3 transfected pLNC-luc applying D-SPM conjugate modified

in two forms with increasing amounts of oleic acid

% Oleate contenta Luciferase (RLU per mg protein)b

Chemical

modificationcPhysical

formulationd

5 1493 Low ( < 25)

10 1650 Low ( < 25)

20 2817 Low ( < 25)

30 10,240 Low ( < 25)

DOTAP/Chol (1/1)e 9980

The transfection applying all derivatives and formulations were

performed in 20% FCS medium unless sited otherwise.a Degree of modification in mol/mol (oleic acid/primary amine).b Relative Light Units (RLU) of luciferase normalized to 1 mg

protein (Experimental section).c Chemically bound oleic acid via amide linkage.d Physically bound oleic acid via ionic interactions.e DOTAP/Chol (1/1) lipid control was evaluated in 10% FCS

medium.


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of the chemically bound oleate moieties in increasing

the stability against serum components, D-SPM for-

mulations with increasing amounts of unbound oleic

acid were prepared and tested for their transfection

activities in serum-rich media. Such formulations

were prepared by simply mixing a water solution of

the D-SPM with an appropriate amount of oleic acid

in THF. The mixtures were allowed to stir at room

Fig. 4. HEK293 cells transfected with pCMV-GFP applying D-SPM conju

various saturated fatty acids (C8–C18), and cholesterol. Transfection w

qualitatively by fluorescent microscopy (Experimental section). D-SPM-2

temperature for at least 1 h, followed by evaporation

of THF by a flux of nitrogen gas and lyophilization to

dryness. The resulting formulations exhibit oleate

moieties connected to D-SPM by weak acid–base

salt interactions (UNH3+ �OOCU), which could be

easily replaced upon interaction with DNA.

Table 2 shows the transfection efficiencies in 20%

FCS medium (NIH3T3/pLNC-luc) obtained with D-

SPM polycations covalently-modified or ionically-

bound with increasing amounts of oleic acid (5–30%

mol/mol). Chemical modification with oleate resulted

with an increase in the transfection efficacy, whereas

when the ionically-bound oleate formulations were

applied, negligible luciferase readings were obtained

with all oleate contents. The results strongly empha-

size that chemically bound oleate moieties act as

shielding agents that increase the stability of poly-

mer–DNA complex against serum proteins. DOTAP/

Chol (1:1) in 10% FCS medium showed similar

luciferase readings compared to D-SPM-20% oleate

in 20% FCS medium system.

To further evaluate the importance of oleate

moieties to the altered transfection characteristics,

D-SPM was chemically modified with other types of

hydrophobic moieties and the transfection efficien-

cies of these derivatives were qualitatively evaluated

in HEK293/pCMV-GFP system (Fig. 4). The hydro-

gate chemically modified with 20% mol/mol (to primary amine) of

as performed in 10% FCS and the%transfection was determined

0% oleate and Fugen were used as positive controls.


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phobic group content in all derivatives was chosen

to be 20% (mol/mol) based on the result described

above. The hydrophobic moieties used in the mod-

ification were saturated fatty acids of C8–C18, and

cholesterol. D-SPM-20% oleate and FuGen 6 were

used as positive controls. Although these derivatives

showed improved transfection characteristics in com-

parison to unmodified D-SPM, no substantial in-

crease in the transfection efficiencies was observed.

These results suggest that oleate moieties enhance

cellular uptake in marked extent compared to other

fatty acids and cholesterol. Current studies focus on

understanding the mechanism of action of D-SPM-

oleate by means of cellular uptake and trafficking.

4. Conclusions

A series of cationic polysaccharides were synthe-

sized by grafting dextran with spermine in combination

with other oligoamines of two to four amino groups.

The transfection efficiencies of these polycations were

assessed in various cell lines and marker genes, and

found to depend on the content of grafted spermine.

Polycations grafted with higher spermine content

resulted in high gene expression, whereas lower sper-

mine content resulted in low transfection yields. The

results emphasize the importance of spermine moieties

in cell transfection, probably due to the altered buffer-

ing capacity and complexation of spermine residues

compared with other grafted oligoamines.

The leading D-SPM polycation was further modi-

fied with increasing amounts of hydrophobic moieties

including C8–C18 saturated fatty acids, oleic acid and

cholesterol. Although hydrophobized polycations

showed altered transfection characteristics compared

to the unmodified D-SPM, only the oleate-modified

polycations showed significant increase in the trans-

fection efficiencies. The oleate-modified D-SPM fa-

cilitated transfection in serum containing medium (up

to 20% FCS), in marked contrast to unmodified D-

SPM which resulted in low to negligible transfection

yields in 10% and 20% FCS. The altered transfection

yields obtained with oleate-modified D-SPM were

explained by the unique hydrophobic nature of oleate

residues which enhances on the one hand cellular

uptake, and on the other hand reduces the interaction

with serum components.

Acknowledgements

Tony Azzam is grateful to the Ministry of Science,

Israel, for the financial assistance. This work was

supported in part by the AFIRST, French–Israeli

Cooperation on Gene Therapy, and by the US–Israel

Binational Fund (BSF).

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