Influence of TAT-peptide polymerization on properties …chem1.eng.wayne.edu/~gzmao/Polyplexes Paper...

www.elsevier.com/locate/jconrel NE

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Journal of Controlled Releas

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Influence of TAT-peptide polymerization on properties and

transfection activity of TAT/DNA polyplexes

Devika Soundara Manickama, Harender S. Bishta, Lei Wanb,

Guangzhao Maob, David Oupickya,*

aDepartment of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, USAbDepartment of Chemical Engineering and Materials Science, Wayne State University, Detroit MI 48202, USA

Received 25 August 2004; accepted 22 September 2004

Available online 21 November 2004

Abstract

Use of bioactive cationic peptides as gene carriers is limited by instability of their DNA complexes in vivo and by the loss of

their biological activity due to undesired interactions of their bioactive parts with the DNA. To overcome the two major

limitations, biodegradable high-molecular-weight form of TAT peptide (POLYTAT) sensitive to cellular redox-potential

gradients was synthesized in this study by oxidative polycondensation. Physicochemical and transfection properties of DNA

polyplexes based on POLYTAT were investigated and compared with polyplexes based on TAT polymer prepared by in situ

template-assisted polymerization. Physicochemical properties of TAT-based polyplexes were affected by the molecular weight

and method of polymerization of the TAT peptide. All TAT-based DNA polyplexes exhibited reduced cytotoxicity when

compared with control polyethylenimine (PEI) polyplexes. Polyplexes based on both high-molecular-weight TAT polypeptides

exhibited increased transfection efficiency compared to control TAT peptide but lower than that of PEI polyplexes. The

evidence shows that transfection activity of TAT-based polyplexes is strongly dependent on the presence of chloroquine and

therefore suggests that TAT polyplexes are internalized by an endocytosis. Overall, high-molecular-weight reducible

polycations based on bioactive peptides has the potential as versatile carriers of nucleic acids that display low cytotoxicity and

can prove to be especially beneficial in cases that require surface presentation of membrane-active or cell-specific targeting

peptides.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Polyplex; TAT; Polypeptide; Transfection; Toxicity

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2004.09.018

* Corresponding author. Tel.: +1 313 993 7669; fax: +1 313

577 2033.

E-mail address: [email protected] (D. Oupicky).

1. Introduction

Gene therapy has shown a potential to treat and

prevent a wide variety of genetic and acquired

diseases. In order to fully utilize this potential, safer

and more efficient vectors for delivery of genes are

e 102 (2005) 293–306

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required. Vectors based on polyelectrolyte complexes

of nucleic acids and synthetic cationic polymers

(polyplexes) represent one of the major alternatives

to viral vectors that usually do not raise the safety

issues associated with viruses but unfortunately their

efficiency, despite enormous progress in recent years,

has yet to achieve competitive levels.

Bioactive cationic peptides capable of condensing

nucleic acids have attracted considerable attention as

parts of gene delivery vectors because of a highly

reproducible and scalable production and inherent

specific biological activity. Natural or artificial pep-

tides are often used to supplement other delivery

systems with various biological functions such as cell

specific targeting, membrane destabilizing activity, or

nuclear localization activity [1–3]. When used alone,

small cationic peptides usually exhibit lower cytotox-

icity and are also weaker activators of the complement

system than high-molecular-weight polycations [4,5].

However, the DNA complexes formed with short

cationic peptides are significantly less stable due to

less prominent cooperative effect [6]. Consequently,

such complexes may lack sufficient stability to

survive blood circulation and to protect DNA from

metabolism in vivo [7]. Additional obstacle hindering

efforts to use peptides as gene delivery vectors is that

some of the potentially useful cationic peptides

interact strongly with nucleic acids via their bioactive

parts, which leaves them unavailable for other

interactions and therefore inactive [8]. Published

evidence indicates that such peptides can constitute

effective components of synthetic gene delivery

complexes, as long as sufficient copies are displayed

on the outer surface of the complex [1,9].

A way of overcoming the two major limitations of

the small cationic bioactive peptides, i.e. low extrac-

ellular stability of their complexes with DNA and the

unwanted interactions with DNA resulting in reduc-

tion of their original biological activity, is to use

readily reversible polymeric forms of the peptides.

Such reversible polypeptides can be easily prepared

from peptides with terminal cysteine residues either

by oxidative polycondensation via the terminal

sulfhydryl groups using dimethylsulfoxide (DMSO)-

mediated oxidation [10] or by DNA template poly-

merization in situ [11,12]. In both cases, high-

molecular-weight polypeptides containing disulfide

bonds in the backbone are produced that can undergo

selective intracellular depolymerization mediated by

small redox molecules or redox enzymes [13,14]. An

additional attractive feature of using the disulfide

bonds in the polypeptide structure is their relative

extracellular stability, which reversibly increases the

affinity of the peptide to the nucleic acid during

extracellular delivery [15,16]. High-molecular-weight

reducible polypeptides can also offer better surface

presentation of the peptides on the surface of DNA

complexes. The intracellular depolymerization of

reducible polypeptides favorably affects cytotoxic

properties of their polyplexes and increases rates of

intracellular disassembly, which leads to enhanced

levels of transfection of both therapeutic DNA and

mRNA [13].

TAT-peptide, amino acid residues 47–57 of the

transactivating transcriptional activator protein from

HIV-1, is an example of a so-called protein trans-

duction domain that recently attracted considerable

attention. TAT and similar peptides have been ascribed

with the unusual ability to translocate across cell

membranes in a receptor-independent and temper-

ature-independent manner [17,18]. The apparent lack

of a size limit and apparent ability of TAT-peptide to

transduce proteins and larger delivery vehicles

directly into cell cytoplasm seemed an attractive

feature that could have brought significant advantages

to cellular delivery of macromolecular therapeutics

[9,19,20]. The original reports suggested that TAT-

peptide transduction involves direct penetration of the

lipid bilayer caused by the localized positive charge of

the peptide. Recently, however, a number of studies

questioned the validity of the original reports and

suggested that (1) the internalization of TAT is a

temperature- and energy-dependent process, (2) endo-

somal transport is a key component of the mechanism,

and (3) TAT merely increases non-specific binding to

the cell surface [21–24].

The objective of this study is to design more

efficient and less toxic means of delivering nucleic

acids using TAT-derived polypeptides sensitive to

cellular redox-potential gradients. The central

hypothesis is that using high-molecular-weight form

of TAT peptide will permit more effective presenta-

tion of TAT residues on the surface of DNA

polyplexes, which will improve overall efficiency

of gene delivery process due to better interactions of

TAT polyplexes with cell membranes. To test the

D. Soundara Manickam et al. / Journal of Controlled Release 102 (2005) 293–306 295

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hypothesis, biophysical and transfection properties of

DNA polyplexes based on TAT polypeptides pre-

pared either in situ using template-assisted polymer-

ization or pre-formed by oxidative polycondensation

were investigated.

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2. Materials and methods

2.1. Materials

Peptides, GTATG (GGRKKRRQRRRGG, Mr

1568) and CTATC (CGRKKRRQRRRGC, Mr 1660)

were synthesized using a standard Fmoc procedure by

Sigma-GENOSYS and purified to homogeneity by

HPLC using Discovery Bio-Wide-Pore C-18

(250�4.6 mm) column eluted with 0.1% trifluoro-

acetic acid and a gradient of acetonitrile (0�67.5%).

Polyethylenimine (PEI) with average molecular weigh

25,000 was from Aldrich. Plasmid DNA, gWizkHigh-Expression Luciferase (gWIZLuc), containing

luciferase reporter gene was purchased from Aldevron

(Fargo, ND) as 5 mg/mL aqueous solution and used

without further purification.

2.2. Synthesis of POLYTAT polypeptide

The POLYTAT polypeptide was synthesized by

oxidative polycondensation as previously described

[10]. Briefly, 20.8 mg of CTATC was dissolved in 145

AL phosphate buffered saline (PBS) and 70 ALDMSO. The reaction was carried out at room

temperature for 96 h. During the reaction, aliquots

were removed at 12-h time intervals and analyzed by

Size Exclusion Chromatography (SEC). The reaction

was terminated by dilution into 15 mL of 5 mM

HEPES buffer solution (pH 7) after no change in

molecular weight distribution was observed in two

consecutive SEC analyses. The SEC analysis was

performed using CATSEC-300 column eluted with

0.2 M NaCl and 0.1% TFA. Commercially available

poly(l-lysine) (PLL) (Sigma) samples were used to

estimate molecular weight of POLYTAT. Low molec-

ular weight impurities were removed by centrifugal

membrane filters with molecular weight cut-off

10,000. The concentration of POLYTAT was deter-

mined by a colorimetric 2,4,6-trinitrobenzenesulfonic

acid assay using GTATG calibration. Degradability of

POLYTAT was confirmed by SEC following reduc-

tion with 5 mM dithiothreitol.

2.3. Ethidium bromide exclusion assay

The ability of GTATG, CTATC, and POLYTAT to

condense DNAwas confirmed by a standard ethidium

bromide exclusion assay by measuring the changes in

ethidium bromide/DNA fluorescence. DNA (gWI-

ZLuc) solutions at a concentration of 20 Ag/mL were

mixed with ethidium bromide (1 Ag/mL) and fluo-

rescence measured using 360-nm excitation and 520-

nm emission and set to 100%. Background fluores-

cence was set to 0% using ethidium bromide (1 Ag/mL) solution alone. Fluorescence readings were taken

following a stepwise addition of a peptide solution,

and condensation curves for each peptide constructed.

2.4. Formulation of DNA polyplexes

Plasmid DNA (gWIZLuc) solution at a concen-

tration 20 Ag/mL in 10 mM HEPES buffer (pH 7) was

used to prepare all polyplexes in this study. Peptide/

DNA complexes were formed by adding a small

predetermined volume of a peptide (5 mg/mL) to

achieve the desired molar mixing ratio X (X=c (basic

amino acid residues of a peptide or amino groups of

PEI)/c (DNA phosphates)) and mixed by vigorous

vortexing for 10 s. Mass of 325 per one phosphate

group of DNA was assumed in the calculations.

Polyplexes were used in further experiments at least

90 min after formulation to permit efficient template-

assisted polymerization of CTATC in CTATC/DNA

polyplexes.

2.5. Molecular weight determination

Apparent weight-average molecular weights (Mw,a)

of DNA complexes were measured using a 7-angle

BiMwA Molecular Weight Analyzer equipped with

30 mW, vertically polarized solid state laser (660 nm)

as a light source (Brookhaven Instruments). The

instrument was calibrated with toluene and the

response of the CCD detectors was normalized with

a nominal 20-nm diameter polystyrene latex in water

(Duke Scientific). The static light scattering data were

analyzed in most cases by linear extrapolation to zero

scattering angle to obtain Mw,a of the DNA com-


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plexes. Calculation of refractive index increments of

the complexes as a function of the molar mixing ratios

was carried out using previously published model of

complex formation [25]. Previously published refrac-

tive index increment value of 0.185 mL/g was used

for DNA. Known refractive index increment of a

polypeptide poly(l-lysine) (0.188 mL/g) was used as

a good approximation for all three TAT formulations.

In all experiments, 1:1 charge stoichiometry and a full

release of counterions were assumed. Extrapolation to

zero concentration was not performed due to very low

concentrations of the complexes. GTATG, CTATC,

and POLYTAT polyplexes at various X were prepared

at DNA concentration of 20 Ag/mL in 10 mM HEPES

(pH 7) 90 min before analysis. Large dust particles

were removed by centrifugation and complexes were

diluted with 10 mM HEPES (pH 7) to the final DNA

concentration of 10 Ag/mL. Standard error of meas-

urement was calculated for each Mw,a determination.

To assess reproducibility of polyplex preparation,

three separate formulations of selected polyplexes

were also analyzed.

2.6. Hydrodynamic radius and zeta potential

determination

The determination of hydrodynamic diameters and

zeta potentials of peptide/DNA complexes was

performed using ZetaPlus Particle Size and Zeta

Potential analyzer (Brookhaven Instruments) equip-

ped with a 35-mW solid state laser (658 nm).

Scattered light was detected at 908 angle and a

temperature of 25 8C. Complexes of gWIZLuc

plasmid with GTATG, CTATC, and POLYTAT were

prepared in 10 mM HEPES (pH 7) as described

above. Mean hydrodynamic diameters were calculated

for size distribution by weight, assuming a lognormal

distribution using the supplied algorithm and the

results are expressed as meanFS.D. of three runs.

Zeta potential values were calculated from measured

velocities using Smoluchowski equation and results

are expressed as meanFS.D. of 10 runs.

2.7. AFM characterization

Ten microliters of DNA or peptide/DNA complex

solution (20 Ag DNA/mL, X=3.0) was placed on

freshly cleaved mica. After 10 min, excess of solution

was removed and surface dried with a gentle stream of

nitrogen. AFM images were acquired using a Multi-

mode Nanoscope III AFM (Digital Instruments) in

tapping mode in air. Height, amplitude, and phase

images were captured, but only height images are

shown here. Silicon probes with a nominal radius of

curvature 10 nm (NSG10, NanoTechnology Instru-

ments, Europe) were used. Scan rate was 1 Hz.

Integral and proportional gains were approximately

0.4 and 0.7, respectively. In some cases, in situ

imaging was conducted by injecting the solution of a

DNA polyplexes into an AFM liquid cell, which was

sealed by an o-ring, and the adsorbed polyplex

structure was obtained in tapping mode in the

solution.

2.8. Cell lines

Murine melanoma cell line B16F10 (CRL-6475)

and human cervical carcinoma cell line HeLa (CCL-2)

were obtained from ATCC. Human endothelial cell

line EA.hy926, derived by a fusion of human

umbilical vein endothelial cells with a human lung

carcinoma A549, was a kind gift from Dr. Edgell

(University of North Carolina). All three cell lines

were maintained in Dulbecco’s Modified Eagle

Medium (DMEM) supplemented with 4 mM l-

alanyl-l-glutamine (GlutamaxR) and 10% fetal

bovine serum (FBS).

2.9. Cytotoxicity

Cytotoxicity of TAT-based DNA polyplexes was

determined by the CellTiter 96R Aqueous Cell

Proliferation Assay (Promega) and compared with

that of PEI/DNA polyplexes. Twenty thousand

EA.hy926 cells were seeded in a 96-well plate. Two

days after reaching confluence, the cells were

incubated in 150 AL of DMEM/FBS with 2.9 AgDNA/mL dose of GTATG/DNA, CTATC/DNA,

POLYTAT/DNA, and PEI/DNA complexes. The

medium was removed after 24 h and replaced with a

mixture of 100 AL fresh DMEM and 20 AL MTS

reagent solution. The cells were incubated for 1 h at

37 8C in CO2 incubator. The absorbance of each well

was then measured at 505 nm to determine cell

viability. The results are expressed as mean% cell

viability relative to untreated cellsFS.D.


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2.10. Transfection efficiency in vitro

Transfection activity was analyzed using DNA

(gWIZluc) complexes of GTATG, CTATC, POLY-

TAT, and PEI prepared as described above (DNA

concentration 20 Ag/mL) at the indicated molar

mixing ratio X. All transfection studies were per-

formed in 48-well plates with cells plated 24 h before

transfection at the seeding density of 30,000 cells per

well using a previously described general protocol

[26]. On a day of transfection, the cells were

incubated with the complexes in 150 AL of FBS

supplemented DMEM. Unless stated otherwise, plas-

mid DNA concentration in the incubation media was

2.9 Ag/mL. After a 3-h incubation, cells were washed

with PBS and cultured for additional 24 h in a fresh

DMEM/FBS prior to analysis of reporter gene

expression. The culture medium was discarded and

cell lysates harvested after incubation of cells for 30

min at room temperature in 100 AL of cell lysis

reagent buffer (Promega). To measure the luciferase

content, 100 AL of luciferase assay buffer (20 mM

glycylglycine (pH 8), 1 mM MgCl2, 0.1 mM EDTA,

3.5 mM DTT, 0.5 mM ATP, 0.27 mM coenzyme A)

was automatically injected into 20 AL of cell lysate

and the luminescence was integrated over 10 s using

single tube Sirius luminometer (Zylux). Total cellular

protein in the cell lysate was determined by the BCA

protein assay using calibration curve constructed with

standard bovine serum albumin solutions. The trans-

fection results are expressed as Relative Light Units

(RLU) per mg of cellular protein. At least three

determinations on two separate occasions were

performed for each transfection experiment. Unless

stated otherwise, the results are expressed as mean

RLUFS.D. and where necessary, significant differ-

ences between two groups are determined by Stu-

dent’s t-test and between multiple groups by ANOVA

using Holm test for multiple comparisons. A value of

Pb0.05 was considered statistically significant.

3. Results and discussion

3.1. POLYTAT synthesis

Artificial or naturally derived cationic peptides

have been widely used as part of synthetic gene

delivery systems because of their DNA condensation

ability and possibility to exploit their intrinsic bio-

logical activity for enhancing the gene delivery

process. While DNA condensation is largely unaf-

fected by the amino acid sequence of a cationic

peptide, its biological activity is strongly dependent

on it. Current knowledge suggests that systemic

intravenous delivery of DNA complexes benefits

from using complexes with low disassembly rates.

This requires using high-molecular-weight polyca-

tions and severely limits the use of bioactive cationic

peptides for systemic gene delivery. Synthesis of high-

molecular-weight peptides with a defined sequence

using traditional methods of peptide synthesis or

methods of genetic engineering is complicated and

often not feasible. If a peptide can be engineered to

contain terminal cysteinyl residues (i.e. if the peptide

termini are not required for its biological activity) then

a simple method of oxidative polycondensation can be

used for the synthesis of high-molecular-weight

polypeptides [10]. In this study, a method was used,

which is based on a mild DMSO oxidation of

sulfhydryl groups into disulfides, to synthesize poly-

peptide consisting of the TAT repeating unit (Scheme

1). The reaction reached equilibrium after 4 days,

furnishing high-molecular-weight form of the TAT

peptide (POLYTAT) with molecular weight distribu-

tion shown in Fig. 1 and with an estimated average

molecular weight 9.4�104. The low molecular weight

residues seen in the chromatogram are likely to be

impurities not containing any sulfhydryl groups or

cyclic by-products. The presence of such impurities

was observed also in polypeptides of different amino

acid sequences used in other studies.

3.2. Formulation and physical properties of DNA

polyplexes

One of the objectives of this study was to

determine how a polymerization method influences

properties of polyplexes based on TAT polypeptides.

DNA polyplexes of POLYTAT (POLYTAT/DNA) and

polyplexes prepared by DNA-template-assisted poly-

merization of CTATC peptide (CTATC/DNA) per-

formed as described in Ref. [7] were studied.

Polyplexes based on non-polymerizable GTATG

peptide (GTATG/DNA) served as controls in all

experiments. The ability of all three TAT peptides to

Fig. 1. SEC analysis of POLYTAT. The SEC analysis of POLYTAT

and control PLL and GTATG samples was performed using

CATSEC-300 column eluted with 0.2 M NaCl and 0.1% TFA.

(Molecular weight averages of the PLL standardswere: PLL (14 kDa)

(Mw(viscosity)=14,600, Mw(LALLS)=8300, Mw/Mn not available)

and PLL (240 kDa) (Mw(viscosity)=240,100,Mw(LALLS)=135,000,

Mw/Mn=1.15).

Scheme 1. Schematic representation of the two approaches

leading to DNA polyplexes containing high-molecular-weight

TAT polypeptides.


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condense DNA was tested first using a standard

ethidium bromide exclusion assay (Fig. 2a). The

condensation curves for all three peptides exhibit the

typical transition between molar mixing ratios (X) of

0.8 and 1.0. POLYTAT showed the best condensation

capability with the lowest residual fluorescence. The

exclusion assay was performed by a rapid step-by-step

addition of TAT peptide to DNA solution and is

therefore unlikely to reflect fully polymerized CTATC

Fig. 2. DNA condensation as measured by ethidium bromide

exclusion assay. (a) DNA condensation was measured as a decrease

of fluorescence after addition of GTATG, CTATC, or POLYTAT to

plasmid DNA(20 Ag/mL)/ethidium bromide (1 Ag/mL) solution

using molar mixing ratio X in the range 0–3. A relative fluorescence

of 100 RFU represents the fluorescence of DNA/ethidium bromide

solution, while 0 RFU designates the background fluorescence of

ethidium bromide not intercalated in DNA. (b) DNA polyplexes of

GTATG, CTATC, and POLYTATwere formulated in the presence of

ethidium bromide (1 Ag/mL) at X=3 and mean fluorescence

measured immediately after polyplex formation and after 90 min

in dark at room temperature (meanFS.D., n=3).


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as suggested by similar condensation profiles for

GTATG and CTATC. Previous reports suggested that

the template-assisted polymerization of similar cati-

onic peptides proceeds rapidly and requires less than

30 min for completion [11]. Fig. 2b shows the residual

fluorescence intensity immediately after complex

formation and after a 90-min incubation. Significant

decrease of the fluorescence intensity observed for

CTATC/DNA suggests tightening of the polyplex

structure due to underwent polymerization. No such

decrease was observed for GTATG/DNA polyplexes.

Addition of a polycation to DNA solution results in

the DNA condensation and a formation of primary

complexes, which can then undergo a secondary

association until a colloidal equilibrium is reached.

Low molecular weight polycations usually form

polyplexes with a very low colloidal stability leading

to a formation of large particles. It can be assumed

that CTATC and GTATG peptides form primary

polyplexes of similar properties. In case of GTATG/

DNA, these polyplexes undergo rapid aggregation;

resulting in large hydrodynamic diameters (Fig. 3).

Size of the CTATC/DNA polyplexes, on the other

hand, will be determined by the balance between the

rate of aggregation and the rate of stabilizing template

polymerization. The stabilizing effect of the template

polymerization on the hydrodynamic diameter of

CTATC/DNA polyplexes is clearly demonstrated in

Fig. 3. Influence of template polymerization of CTATC on

aggregation of primary DNA polyplexes. Immediately after

preparation of CTATC and GTATG complexes with plasmid DNA

at X=3, hydrodynamic diameters were measured in 30-s intervals by

dynamic light scattering.

Fig. 3. Although the instrumentation available in the

authors’ laboratory does not permit capturing the early

phases of aggregation of primary complexes immedi-

ately after polycation addition, it nevertheless allows

to clearly demonstrate the stabilizing effect of

template polymerization of CTATC.

Biological activity of DNA polyplexes is affected

by their molecular parameters [27]. Parameters such

as size, surface charge, and molecular weight are

known to be controlled by the molecular weight of the

used polycations. Fig. 4 shows hydrodynamic diam-

eter, zeta potential, and apparent molecular weight for

all three TAT-based polyplexes prepared at various

molar mixing ratios, X. Fig. 4a confirms that increas-

ing X leads to a formation of smaller polyplexes. As

expected, POLYTAT forms polyplexes with the

smallest size of the three formulations due to its high

molecular weight that permits effective colloidal

stabilization via formation of stabilizing shell of

non-interacting parts of POLYTAT molecules on the

surface of the polyplexes [28]. This was further

confirmed by the observed highest positive zeta

potential of POLYTAT/DNA (Fig. 4b). Fig. 4c

confirms that GTATG forms a highly aggregated

polyplexes with the highest apparent molecular

weight of the three TAT-peptide forms tested. Appa-

rent molecular weight of the POLYTAT/DNA poly-

plexes decreases with increasing X, confirming the

effect of POLYTAT excess on stabilization of the

polyplexes against aggregation as observed for the

dependence of hydrodynamic diameters on X. In

contrast, apparent molecular weight of CTATC/DNA

increases with increasing X. The increase of molecular

weight, combined with decrease of the size of CTATC

polyplexes, would indicate increase of structural

density of CTATC polyplexes with increasing excess

of CTATC peptide. Typically, however, the structural

density of polyplexes based on high-molecular-weight

polycations increases at XN1 with increasing excess of

the polycation because of the intra-complex repulsive

action of excessive polycation [29–31]. Further

physical studies of CTATC/DNA polyplexes are

required to fully confirm and explain the molecular

weight behavior of CTATC/DNA polyplexes.

To determine possible influence of the polymer-

ization method on morphology of the polyplexes,

POLYTAT/DNA and CTATC/DNA were visualized

by AFM (Fig. 5). Both CTATC and POLYTAT

Fig. 5. Morphology of the polyplexes as determined by AFM. The

morphology was evaluated for (a) plasmid DNA (image size 1�1Am2 and z-range=1.5 nm), (b) CTATC/DNA (image size 4�4 Am2

and z-range=15 nm), and (c,d) POLYTAT/DNA formed in water

using X=3. POLYTAT/DNA polyplexes were visualized (c) in air

(image size 2�2 Am2 and z-range=15 nm) or (d) in a solution using

the liquid cell (image size 2�2 Am2 and z-range=50 nm).

Fig. 4. Molecular parameters of TAT-based DNA polyplexes. The

polyplexes were formulated at X=1.2, 2, and 3 using GTATG,

CTATC, and POLYTAT and (a) hydrodynamic diameter, (b) zeta

potential, and (c) apparent molecular weight of the polyplexes were

determined at 25 8C.


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changed the DNA morphology from wormlike chains

to compact particles. Although a distinct subpopula-

tion of rods among the typical spherical particles was

observed in CTATC/DNA polyplexes (Fig. 5b), no

clear difference in the morphology was observed

between CTATC and POLYTAT polyplexes (Fig. 5b

vs. c). The sizes of the rod-like particles were about 4

nm in height, 80 nm in length, and 40 nm in width.

The images in Fig. 5a–c were acquired in a dry state.

To determine the influence of drying on the polyplex

morphology, POLYTAT polyplexes were imaged

directly in the solution using a liquid cell (Fig. 5d).

The results show that sizes and shapes of the spherical

POLYTAT/DNA particles corresponded to those seen

in Fig. 5c (~45 nm in height, ~150 nm in diameter; the

actual diameter is smaller due to tip convolution in

AFM analysis).

3.3. Cytotoxicity of DNA polyplexes

A successful gene delivery carrier should be able

to deliver transcriptionally active gene to the cell

nucleus without negatively affecting normal func-


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tions of the host cell. Cytotoxicity of the TAT-based

polyplexes was evaluated in human endothelial cell

line EA.hy926 and compared with cytotoxicity of

PEI/DNA (Fig. 6). The selected human endothelial

cell line represents an important study object for

toxicity of blood-borne polyplexes [32,33]. The

EA.hy926 cell line was derived by a fusion of

human umbilical vein endothelial cells with a

human lung carcinoma A549 and maintains the

differentiated properties of endothelium including

expression of von Willebrand factor (vWF), Weibel-

Palade bodies and even angiogenesis. Because the

expression levels of some differentiated properties,

including vWF, do not reach maximal levels until

several days after the culture has reached its final

cell density, the experiments were performed with

confluent cells. The results show that all TAT-based

polyplexes are less toxic than PEI polyplexes and

thus confirm previous findings of low cytotoxicity

of similar reducible polypeptides when compared

with non-biodegradable polycations [13]. The tox-

icity of polycations such as PEI is a well-known

phenomenon and has been reported by a number of

investigators in both in vitro and in vivo experi-

ments [34,35]. Based on the published evidence,

three mechanisms can be postulated by which

polycations exert their cytotoxic activity [33,35–

37]: (1) direct destabilization of plasma membranes,

(2) destabilization of intracellular membranes (e.g.,

lysosomal, nuclear), and (3) interference of poly-

Fig. 6. Cytotoxicity evaluation. The MTS assay was used to assess

the viability of human endothelial EA.hy926 cells following

transfection for 24 h with GTATG, CTATC, POLYTAT, and PEI

polyplexes. Results are shown as a mean and standard deviation

from triplicate samples.

cations with vital cellular processes by interacting

with proteins and nucleic acids. Structural features

most affecting the way polycations interact with cell

membranes include charge density, molecular

weight, type of charged center, and molecular

flexibility. High cationic charge densities and highly

flexible polymers, such as PEI, are usually expected

to cause greater cytotoxic effects than those with

low cationic charge density and more rigid chains. It

is hypothesized that low cytotoxicity of the disul-

fide-containing polypeptides, such as POLYTAT, is

a direct consequence of the reduced binding affinity

for cell membranes and vital protein and nucleic

acid molecules following rapid intracellular decrease

of molecular weight of these polycations.

3.4. Transfection activity

Polyplex-mediated gene delivery is a multi-step

process that can be potentially highly influenced by

the subcellular degradation of the reducible polyca-

tions used in this study. In addition to the reduction of

cytotoxicity discussed previously, molecular weight of

a polycation controls the affinity to DNA, which can

be directly linked to disassembly rates of the poly-

plexes and can affect transcriptional availability of the

delivered DNA [13]. It was hypothesized that

increased rates of disassembly of POLYTAT/DNA

and CTATC/DNA polyplexes promote better tran-

scription of the delivered DNA, which could result in

enhanced transfection activity. Transfection activity of

POLYTAT/DNA and CTATC/DNA was therefore

investigated in three cell lines and the results were

compared with those obtained for control PEI/DNA

polyplexes. GTATG/DNA polyplexes served as addi-

tional control used to determine the influence of TAT

molecular weight on transfection activity. It was

further hypothesized that using the polymeric form

of TAT peptide (POLYTAT) would result in a better

surface presentation of the TAT residues on the

surface of DNA complexes, which would be benefi-

cial for interactions with cell membranes and lead to a

higher transfection efficiency.

The initial transfection studies were performed in

B16F10 mouse melanoma cells, HeLa human cer-

vical carcinoma cells, and EA.hy926 human endo-

thelial cells using a DNA dose of 0.5 Ag per well

(2.9 Ag/mL) (Fig. 7). To estimate the influence of

Fig. 7. Transfection efficiency of TAT- and PEI-based polyplexes in

a panel of cell lines. GTATG, CTATC, and POLYTAT polyplexes

were formulated at X=3, while control PEI polyplexes at X=8. A

single DNA dose (0.5 Ag/well; 2.9 Ag/mL) was used in all three cell

lines. The luciferase reporter gene expression was measured 24 h

after 4-h incubation of the polyplexes with the cells either in the

absence or presence of 100 AM chloroquine. The luciferase

expression in relative light units (RLU) is normalized to mg of

cellular protein and reported as the mean and standard deviation

obtained from triplicate transfections.


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polyplex capability to avoid or to escape endo/

lysosomal pathway on the overall expression of

luciferase reporter gene, all experiments were con-

ducted in the presence and absence of chloroquine.

Polyplex-mediated transfection of fast growing

B16F10 cells resulted in the expected high levels

of luciferase gene expression (Fig. 7a). Almost a

100-fold difference between the transfection activity

of GTATG/DNA and high-molecular-weight forms of

TAT/DNA in the absence of chloroquine confirms

the importance of molecular weight of polycations

for high transgene expression levels. The difference

between low and high-molecular-weight forms of

TAT peptide further increased in transfection experi-

ments conducted in the presence of chloroquine.

Similar effect of molecular weight of TAT peptides

on transfection activity was observed in HeLa cells

(Fig. 7b). Polyplex-mediated transfection of the

endothelial cells EA.hy926 is very inefficient and

the levels of luciferase expression provided by all

three TAT formulations were near-background levels

in the absence of chloroquine (Fig. 7c). The presence

of chloroquine in the transfection media resulted in

more than 10-fold increase of luciferase expression

mediated by CTATC and POLYTAT polyplexes but

no increase was observed for GTATG/DNA. Control

PEI polyplexes proved to be superior to the TAT-

based polyplexes when transfections were conducted

in the absence of chloroquine in all three cell lines.

The data in Fig. 7 clearly demonstrate the benefits of

increasing the molecular weight of TAT peptide for

transfection activity of its DNA polyplexes. As

shown in the previous sections, the polymerization

method of the TAT peptides affects physicochemical

properties of their DNA polyplexes. The transfection

results obtained for CTATC/DNA and POLYTAT/

DNA were, therefore, compared to elucidate if the

polymerization method affects also the transfection

activity. In all three cell lines tested, no significant

differences in luciferase expression levels were

observed for CTATC and POLYTAT polyplexes.

The only statistically significant difference was

observed in transfections of HeLa cells conducted

in the presence of chloroquine, where CTATC/DNA

polyplexes unexpectedly displayed about 150-fold

increased activity compared to POLYTAT/DNA. In

repeated experiments, the increased activity of

CTATC/DNA was confirmed to exhibit an average

16-fold (n=4) higher luciferase expression than

POLYTAT/DNA. Finally, analysis of the influence

of the presence of chloroquine during transfection

Fig. 8. Influence of DNA dose on transfection efficiency. GTATG,

CTATC, and POLYTAT polyplexes were formulated at X=3 and

control PEI polyplexes at X=8. Transfections were performed in

B16F10 mouse melanoma cells (a) in the absence of chloroquine

and (b) in the presence of 100 AM chloroquine. Results are shown

as mean relative light units (RLU) expressed per mg of cellular

proteinFS.D. of three replicate assays.


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showed that levels of luciferase expression mediated

by both CTATC and POLYTAT polyplexes are

significantly enhanced by chloroquine. On the other

hand, PEI/DNA polyplexes demonstrated only low

levels of potentiation by chloroquine due to their

inherent capability to escape endo/lysosomal traffick-

ing pathway. These results clearly suggest that the

TAT-based polyplexes are most likely internalized by

an endosomal cell uptake and that they do not have

any significant inherent membrane active properties

that would facilitate their direct translocation across

plasma membrane.

The efficiency of polyplex-mediated transfection

is greatly influenced by the DNA dose used. The

transfection studies showed in Fig. 7 were conducted

using a relatively high DNA dose (2.9 Ag/mL) that

could mask potential differences in the activity of

CTATC and POLYTAT polyplexes. Dependence of

luciferase reporter gene expression in B16F10 cells

on the DNA dose for CTATC, POLYTAT, and

control PEI polyplexes was therefore evaluated

(Fig. 8). In the absence of chloroquine (Fig. 8a),

the transfection activity of POLYTAT/DNA poly-

plexes decreases more slowly than that of CTATC/

DNA polyplexes in the DNA concentration range

above 0.5 Ag/mL, but is consistently lower than the

transfection activity of PEI/DNA polyplexes. At the

lowest DNA concentrations tested, the luciferase

expression of all three tested vectors drops signifi-

cantly to almost background levels. Transfecting the

cells in the presence of chloroquine results in high

levels of luciferase expression for all three tested

vectors. Unlike PEI polyplexes, the TAT-based

polyplexes appear to preserve their activity without

a significant decrease of luciferase expression to a

DNA dose of about 0.5 Ag/mL. As a result,

chloroquine potentiation of the TAT-mediated trans-

fection increases with decreasing DNA dose. In case

of CTATC/DNA polyplexes, for example, luciferase

expression increased 980-fold in the presence of

chloroquine at DNA dose 2.9 Ag/mL and 12,400-fold

at the lower DNA dose of 0.3 Ag/mL.

Molar mixing ratio used in polyplex formulation

not only affects the physicochemical properties as

demonstrated in Fig. 4, but more importantly for the

transfection activity, it determines the amount of free

polycation present in the formulations. For polyplexes

such as PEI/DNA that rely on buffering capability of

polycations for their activity, the presence of free

polycation is crucial for their transfection activity both

in vitro and in vivo [38,39]. To permit direct

comparison of the influence of CTATC and POLYTAT

polyplex composition on transfection activity with

that of PEI polyplexes, charge ratio Z was used

instead of molar mixing ratio X (Fig. 9). For both

TAT-based polypeptides tested, the Z and X ratios are

almost equal but they vary significantly for PEI

because of the variation in charge density in the PEI

chain. The compositions of the polyplexes are, there-

fore, expressed in terms of the charge ratio Z to

account for the variation in the content of charged

Fig. 9. Influence of charge ratio on transfection activity. GTATG,

CTATC, POLYTAT, and PEI polyplexes were formulated at various

charge ratios, Z, and transfections were performed in B16F10

mouse melanoma cells (a) in the absence of chloroquine and (b) in

the presence of 100 AM chloroquine using a single DNA dose (0.2

Ag/well; 1.25 Ag/mL). Results are shown as mean relative light units

(RLU) expressed per mg of cellular proteinFS.D. of three replicate

assays.


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groups in PEI. In the calculation of Z, it was assumed

that 45% of total amino groups of PEI is charged at

pH 7 [40]. In the absence of chloroquine, the

transfection activity of PEI/DNA decreased abruptly

at Zb3 while both CTATC/DNA and POLYTAT/DNA

did not show a significant decrease until Zb2 (Fig.

9a). POLYTAT/DNA polyplexes showed higher luci-

ferase expression than CTATC/DNA at all charge ratio

tested. The low DNA dose used in this transfection

experiment (0.2 Ag/well; 1.25 Ag/mL) disadvantaged

PEI polyplexes that require a critical mass of PEI for

efficient buffering function. Addition of chloroquine

to the transfection resulted in the expected increase of

luciferase expression for all three vectors tested (Fig.

9b). Similar to transfection conducted in the absence

of chloroquine, activity of PEI polyplexes exhibited a

strong dependence on the presence of free PEI and

decreased significantly as the charge ratio decreased.

In contrast, only a limited decrease in luciferase

expression was observed for POLYTAT and CTATC

polyplexes. Indeed, a 90-fold higher transfection

activity was measured for POLYTAT polyplexes at

the lowest Z tested compared with PEI polyplexes.

Large chloroquine potentiation effect was observed

for TAT polyplexes (7,400-fold increase in case of

CTATC polyplexes), again suggesting inefficient

cytoplasmic delivery of these vectors. The less

pronounced dependence of transfection activity of

TAT-based polyplexes on charge ratio used suggests

less significant reliance, if any at all, on the free

polycation than in case of PEI polyplexes. Higher

luciferase reporter gene expression observed for the

TAT-based polyplexes compared with PEI polyplexes

(Fig. 9b) is likely to be at least partly a result of

efficient intracellular degradation of TAT polypep-

tides and subsequent improved transcriptional avail-

ability of DNA. Better performance of POLYTAT

polyplexes is likely due to better surface presentation

of the TAT residues than in case of CTATC

polyplexes and therefore more efficient interactions

with cell membranes.

The central hypothesis of this study was that

using the polymeric form of the TAT peptide

(POLYTAT) will permit better surface presentation

of the TAT residues and that this results in a more

efficient cellular delivery of DNA due to TAT

translocating capability. In addition to the protein

and particle transduction properties of TAT peptide, a

nuclear localization function of the TAT peptides was

recently reported that can further enhance the trans-

fection activity of their DNA complexes [41].

Cellular uptake of TAT and other so-called protein

transduction domains have been ascribed in the

literature to an energy- and receptor-independent

mechanism that does not involve endocytosis and is

capable to deliver even large cargo [18,19]. These

studies were recently revisited and the mechanism of

cellular uptake reevaluated. The current evidence

therefore shows that the cell internalization of TAT

peptides and other protein transduction domains is an

energy-dependent process involving classical adsorp-


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tive endocytosis [21–23]. The data presented in this

study fully confirm the current mechanism of TAT

internalization as documented by the strong depend-

ence of transfection activity of TAT-based polyplexes

on the presence of chloroquine, which suggests

endocytic cell uptake of these vectors. As suggested

by the very low luciferase expression in slowly

dividing endothelial cells, the data also do not seem

to confirm previous findings that the TAT nuclear

localization sequence is involved in enhancing gene

transfer [41].

4. Conclusion

Biodegradable, high-molecular-weight polycation

based on TAT peptide has been successfully synthe-

sized and its physicochemical properties, cytotoxicity,

and transfection activity evaluated. Physicochemical

properties of TAT-based polyplexes were affected by

the molecular weight and method of polymerization

of the TAT peptide. All TAT-based DNA polyplexes

exhibited reduced cytotoxicity when compared with

control PEI polyplexes. Evaluation of transfection

activity confirmed the importance of high molecular

weight of polycations for efficient transgene expres-

sion and suggested a contribution of the polymer-

ization method to the overall efficiency of the cellular

gene delivery process mediated by the TAT polypep-

tides. Higher luciferase reporter gene expression

observed for the TAT-based polyplexes compared

with the PEI polyplexes under selected experimental

conditions is likely to be, at least partly, a result of

efficient intracellular degradation of TAT polypep-

tides and subsequently improved transcriptional avail-

ability of DNA. TAT-based polyplexes appear to be

internalized by adsorptive endocytosis and their

transfection activity is strongly dependent on the

presence of chloroquine. Overall, it is expected that

reducible polycations based on bioactive peptides will

evolve into versatile carriers of nucleic acids that

display low cytotoxicity and will prove especially

beneficial in cases that require surface presentation of

membrane-active or cell-specific targeting peptides or

in development of non-toxic alternatives to PEI that

require a presence of large quantities of free high-

molecular-weight polycations to effectively buffer

endosomal pH.

Acknowledgment

This work was supported by AACP New Inves-

tigators Program from The American Foundation for

Pharmaceutical Education, by National Science Foun-

dation (CTS-0221586), and by Wayne State Univer-

sity Research Grant.

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