www.elsevier.com/locate/jconrel

Journal of Controlled Release 95 (2004) 639–651

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Compositional regulation of poly(lysine-g-(lactide-b-ethylene

glycol))–DNA complexation and stability

Susan Parka,b, Kevin E. Healya,*

aDepartments of Bioengineering and Materials Science and Engineering, University of California at Berkeley,

370 Hearst Memorial Mining Building, Berkeley, CA 94720-1760, USAbDepartment of Biomedical Engineering, McCormick School of Engineering and Applied Science, Northwestern University,

2145 Sheridan Road, Tech E310, Evanston, IL 60208, USA

Received 30 July 2003; accepted 3 December 2003

Abstract

The search for an effective nonviral gene therapy vector has revealed several significant hurdles, such as transient expression

and cytotoxicity, that impede the success of these systems. A terpolymer of poly(lysine-g-(lactide-b-ethylene glycol)) [pK-g-

p(LL-b-EG)] has been developed, which is capable of stably packaging DNAwith significantly less polycation than unmodified

polylysine (pK) systems. A comparison of unmodified pK (DP= 14, Mw = 2930) to pK grafted with p(LL-b-EG) (Mw tot = 7500)

showed that the minimum amine/phosphate ratio (N:Pmin) needed for complete DNA condensation was reduced by 50%.

However, when the molecular weight of pK was reduced (DP= 4, Mw = 838), there was evidence of terpolymer interference

with DNA condensation. Increasing the number of grafted p(LL-b-EG) chains produced a similar result of incomplete DNA

condensation. All terpolymer formulations produced complexes with DNA that had greater resistance to salt-induced

dissociation and short-term exchange with excess DNA. Terpolymer–DNA complexes exhibited approximately zero-order

plasmid release profiles over a period of 6 weeks. The rate of release was dependent on the complex N:P ratio as well as the

molecular weights of pLL and pK. The ability to use terpolymer composition to control complex stability and controlled release

can provide a means for system optimization for sustained expression profiles of exogenous DNA.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Polymers; DNA; Gene therapy; Sustained release

1. Introduction

Gene therapy has been slow to fulfill its clinical

promise due to the complexity of extracellular and

intracellular hurdles associated with each clinical ap-

plication. While clinical trials using viral vectors were

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

doi:10.1016/j.jconrel.2003.12.002

* Corresponding author. Tel.: +1-510-643-3559; fax: +1-510-

643-5792.

E-mail address: kehealy@socrates.berkeley.edu (K.E. Healy).

initially successful, significant downstream side effects

have raised concerns over the legitimacy of the treat-

ment [1,2]. These safety hazards have proven to be a

great setback against widespread application and have

spawned significant research into nonviral alternatives.

Adoption of safer, nonviral option has been limited by

the reduced efficiency in transfecting cells both in vitro

and in vivo.

It has been suggested that one of the hurdles to

effective gene therapy by polycationic systems is the

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intracellular liberation of plasmid from its vector [3–

5]. Schaffer et al. [3] observed that while the largest

polylysine (DP= 180) showed the greatest tendency

to colocalize with plasmids in the nucleus, its

transfection efficiency was the lowest (15.3% using

the reporter pEGFP-C1). Conversely, lower-molecu-

lar-weight pK (DP= 19) did not display any nuclear

colocalization but exhibited a transfection efficiency

of 32.5%. Further investigation revealed that the

largest pK showed minimal dissociation from plas-

mid DNA and the greatest interference with RNA

transcription [3]. Similarly, increasing the degree of

q-amine substitution on high-molecular-weight poly-

lysine (DP= 190) with neutral y-gluconolactone in-

creased the extent of complex dissociation in high

ionic solutions [5,6]. Maximal luciferase plasmid

expression was obtained when 43% of the amines

on pK were substituted. It was hypothesized that the

reduced affinity between polycations and plasmids

was responsible for increased intranuclear dissocia-

tion and subsequent reporter expression.

In efforts to encourage intracellular polycation

dissociation from plasmids for improved transfection

and cytotoxicity, several studies have introduced sus-

ceptible moieties into lipid and polymer chemistries

[7–10]. A polylysine analog with hydrolytically labile

ester linkages in the backbone showed significant

plasmid release due to polymer degradation after 24

h [7,8]. The accompanying in vitro studies demon-

strated improved transfection and reduced cytotoxicity

over controls, while in vivo delivery of plasmids

encoding for interleukin-12 reduced murine tumor

size. Polyethyleneimine (pEI) has also been made

hydrolytically degradable by crosslinking low-molec-

ular-weight pEI prepolymers with either succinate

pEG to introduce ester groups or oligo(lactic acid-

co-succinic acid) to produce more slowly degrading

amide bonds [9,10]. Increased reporter gene expres-

sion levels were attributed to vector degradation as a

facilitator of plasmid unpackaging.

Incorporation of disulfide moieties into pEI or

polylysine has enabled polycation degradation by

reducing agents, such as dithiothreitol and glutathi-

one, the latter of which is a natural intracellular

component [11,12]. In addition, strategic placement

of electron-withdrawing carboxyls adjacent to disul-

fide groups in lipids increased susceptibility to reduc-

tion by glutathione and resulted in improved cell

viability and increased transfection levels [13]. Stabi-

lizing disulfide bonds can also be formed after DNA

condensation by oxidation of sulfhydryls on the

condensing agent [14,15].

Because plasmid is not incorporated into the host

genome, the long-term effectiveness of a single appli-

cation of nonviral vectors is hindered by transient

expression. To address this issue, gene therapy sys-

tems that allow sustained delivery of plasmid DNA by

exploiting polymer degradation to control plasmid

release have been developed [16 – 18]. These

approaches have often utilized hydrolytically degrad-

able polyesters that were processed into scaffolds or

microspheres with incorporated plasmid DNA.

Manipulation of component and processing parame-

ters enabled control over plasmid release kinetics that

was correlated to long-term expression over a period

of several weeks [16,18].

In our work, we address the issues of intracellular

unpackaging by minimizing the molecular weight and

amount of polycation in DNA complexes. This strat-

egy should also help minimize cytotoxicity often

incurred by using high-molecular-weight polycations

in vectors [19]. Terpolymers of poly(lysine-g-(lactide-

b-ethylene glycol)) have already been shown to form

submicron complexes with DNA and improve resis-

tance to DNase [20]. Here, we extend the investiga-

tion of these complexes further by determining the

effect of increasing the degree of polylysine substitu-

tion as well as decreasing polylysine length on com-

plex characteristics. In addition, complex stability is

discussed with respect to plasmid exchange with

excess DNA and plasmid release kinetics due to

polylactide degradation.

2. Materials and methods

A heterofunctional polyethylene glycol (NH2-

pEG-OH, Mw = 3400) bearing a terminal amino and

hydroxyl group was purchased from Shearwater

(Huntsville, AL). All other chemicals were pur-

chased from Sigma-Aldrich (St. Louis, MO) and

were used as received, unless otherwise noted. The

plasmid used was a pEGFPLuc vector (6367 bp;

Clontech, Palo Alto, CA) that was amplified in

Escherichia coli and purified according to standard

procedures.

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2.1. Terpolymer synthesis

2.1.1. P(LL-b-EG) synthesis and characterization

A diblock copolymer of poly-L-lactide-b-ethylene

glycol (p(LL-b-EG)) was synthesized and character-

ized as described previously [20]. Briefly, fmoc-

substituted pEG was synthesized by the reaction of

N-(9-fluorenylmethoxycarbonyloxy)succinimide with

NH2-pEG-OH. After purification by precipitation, the

purity and degree of functionalization were assessed

by proton nuclear magnetic resonance spectroscopy

(1H NMR) (Bruker AMX300) in CDCl3.

Diblock copolymers of p(LL-b-EG) were synthe-

sized with pLL of three different degrees of polymer-

ization by reacting freshly recrystallized L-lactide and

fmoc pEG at monomer/hydroxyl (M/OH) ratios of 40

and 75. To produce the diblock copolymer of lowest

molecular weight, a M/OH ratio of 75 was used and

the reaction was stopped after 2 h. Stannous octoate

was used at a constant lactide monomer/initiator ratio

(M/I) of 300 to initiate the reaction. All reactants were

added to anhydrous toluene in a sealed reaction vessel

under dry nitrogen and refluxed at 100 jC under a

gentle flow of dry nitrogen. The copolymer (p(LL-b-

EG)-fmoc) was purified by precipitation and its total

molecular weight was assessed using an Agilent 1100

HPLC system with two Polymer Laboratories mixed E

columns in series and methylene chloride as the mobile

phase. The detectors used included a multiangle light

scattering detector, refractive interferometer (Wyatt

Technology, Santa Barbara, CA), and a photodiode

array detector (Agilent Technologies, Mountain View,

CA). The dn/dc values for each diblock copolymer in

methylene chloride were determined experimentally

using a refractive interferometer (Wyatt Technology).

For comparison, 1H NMR was also used to calculate

molecular weight by using the known molecular

weight of pEG as an internal standard.

The hydroxyl on the polylactide end of the diblock

copolymer was then functionalized to be amine-reac-

tive (su-p(LL-b-EG)-fmoc) with disuccinimidyl car-

bonate (DSC) (0.5 M) and 4-dimethyl aminopyridine

(DMAP). The product was purified by dissolution and

precipitation into cold ether until the aromatic DMAP

protons (in CDCl3) at 6.45 and 8.20 ppm disappeared.

Percent substitution was calculated using the ratio of

the reappeared succinimidyl peak area at 2.82 ppm to

that of pEG at 3.63 ppm (in CDCl3).

S. Park, K.E. Healy / Journal of C

2.1.2. pK grafting

The final terpolymer of poly(lysine-g-(lactide-b-

ethylene glycol)) was synthesized by coupling su-

p(LL-b-EG)-fmoc to the q-amines on poly-L-lysine

(pK) in dimethyl sulfoxide (DMSO) at a 1:1 molar

ratio. Three different molecular weights of pK were

reacted: 8800 (DP= 42), 2930 (DP= 14), or 838

(DP= 4). The nomenclature for the resulting terpol-

ymers is pKY-Xk, where Y denotes the degree of pK

polymerization (4, 14, or 42) that has been grafted to a

p(LL-b-EG) diblock copolymer with a combined

molecular weight of approximately X000 (5k, 7k, or

11k). Terpolymers reacted at a 1:3 or 1:5 molar ratio

of pK42:p(LL-b-EG) (5k) are denoted as pK42-5k(3)

and pK42-5k(5), respectively.

The amount of pK present in each terpolymer was

determined by 1H NMR in deuterated dimethyl sulf-

oxide (DMSO-d6). For comparison, a binding assay

using trinitrobenzyl sulfonic acid (TNBS) was per-

formed using a modified standard protocol [21]. The

reaction was performed in a solution of 75% DMSO

and 25% sodium bicarbonate buffer (pH = 8.5) for 1

h at 37 jC. Hydrochloric acid was immediately added

to prevent hydrolysis of the product. The solution

absorbance was read at 436 nm and the amount of q-amine per milligram of polymer was calculated using

a calibration curve made with known amounts of

pK42.

2.2. Particle formation and characterization

2.2.1. Complex formation

Complexes were formed by adding pK or pK-g-

p(LL-b-EG) in DMSO (1 or 2 mg/ml) to DNA in

ultrapure water (UPW; 18 MV/cm) in Teflonk vials.

After 20 min, an additional volume of water was

added and the solutions were lyophilized. The final

DNA concentration before lyophilization was 5 Ag/ml

and the amount of polymer added varied on the

desired amine/phosphate charge ratio (N:P). The total

volume of DMSO used was kept constant and below

10% vol/vol to minimize solvent effects on conden-

sation. Complexes were resuspended in UPW for

further characterization, unless otherwise noted.

2.2.2. Degree of condensation

Ethidium bromide (EB) was used as an indicator of

condensation since it becomes intensely fluorescent

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Table 1

1j Amine content of terpolymers

Amol NH2/mg

TNBS

Polymer

NMR

Percent amines

substituted

pK42-5k 3.55F 0.20 3.20 1.8

pK42-5k(3) 1.79F 0.24 1.17 11.1

pK42-5k(5) 1.31F 0.11 0.96 14.3

pK42-7k 2.00F 0.15 1.81 4.3

pK14-7k 0.70F 0.06 0.50 24.4

pK4-7 0.12F 0.01 0.55 21.9

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upon intercalation with free DNA but cannot bind to

condensed DNA. Resuspended particles were made

and resuspended as described above, mixed with 1

mM EB, and the relative fluorescence unit (RFU) was

measured in a black 96-well plate with a microplate

fluorimeter (Molecular Devices, Sunnyvale, CA) (ex:

339 nm, em: 610 nm, cutoff: 515 nm). The autofluor-

escence of free EB was subtracted from each sample

value and then normalized to the maximum fluores-

cence obtained for DNA with no polymer added

(percent maximum RFU). DNA condensation was

considered to be complete when EB fluorescence

reached background levels. Measurements were per-

formed on triplicate samples.

2.2.3. Assessing complex size

Complexes were made with pK4, pK14, pK4-7k,

and pK14-7k at their respective N:Pmin and resus-

pended in ultrapure water. A drop of each suspension

was dried on a cleaned silicon wafer and coated with

f 10 nm carbon. Each sample was visualized using a

cold field emission scanning electron microscope

(Hitachi S-5000) using an accelerating voltage of 5 kV.

Resuspended complexes were also analyzed for

apparent Mw and rg using static light scattering, as

described in detail previously [20]. Briefly, sample

dilutions were carefully measured by weight, and the

final concentration was considered to be the total

amount of DNA plus polymer used. Samples were

manually injected into a multiangle light scattering

detector using a flow rate of 0.2 ml/min and filtered

ultrapure water was the mobile phase. The data used

for analysis were limited to that from seven low-angle

detectors (36j–90j), since these low angles are the

most critical to obtain the apparent molecular weight

and rg* of complexes. The lowest measurable angles at

14j and 26j were excluded since low-angle detectors

were the most sensitive to contaminating particles and

were easily saturated. Samples were run in triplicate

and each run produced for at least 20 data points.

Only those data with a Debye fit of less than 10%

error were used for further analysis.

2.2.4. Plasmid exchange with excess DNA

Complexes were made at a constant pK amine/

DNA phosphate charge ratio (N:P) of 1.5 using pK4-

7k, pK14-7k, and pK42-7k, or their unmodified pK

counterparts pK4, pK14, and pK42. Particles were

resuspended in incomplete Dulbecco’s phosphate-

buffered saline (PBS; pH= 7.2; Gibco-BRL). Aliquots

containing approximately 1.8 Ag of DNA were dis-

tributed into microcentrifuge tubes containing a 3 M

excess of calf thymus DNA bound to cellulose beads.

Samples were incubated at 37 jC for various lengths

of time, after which they were centrifuged and the

supernatant was frozen until analysis. The supernatant

was concentrated using YM-100 Microcon filters

(Millipore, Bedford, MA) until the final volume was

approximately 10 Al. In each well, 7 Al of the

concentration was used for electrophoresis on a 1%

agarose gel (4.7 V/cm, 1 h) with Tris–acetate–EDTA

(pH = 8.0) running buffer. Gels were stained with 0.5

Ag/ml ethidium bromide and visualized using a stan-

dard transilluminator.

2.2.5. Plasmid release from complexes

Complexes were formed as described earlier using

the following formulations: pK14 (N:P= 1.2), pK14-

7k (N:P= 0.62 and 1.2), pK42 (N:P= 0.8), pK42-7k

(N:P= 0.62), and pK42-11k (N:P= 0.42). Three sepa-

rate runs of triplicate samples (n= 9) were performed

for each sample. Complexes were resuspended in 1 ml

of incomplete Dulbecco’s PBS for a final concentra-

tion of 5 Ag DNA/ml and incubated at 37 jC. Atcertain time points, 50 Al of the sample dispersion was

removed and added to iDPBS in black 96-well plates

for a total volume of 100 Al. For samples with very

high DNA concentrations, the amount of sample used

for measurement was reduced to 25 or 15 Al and

compensated for accordingly in calculations. Because

the degrading complexes could not be separated from

released DNA, the tested samples contained both

complexed and free DNA. PicoGreen (Molecular

Probes, Eugene, OR) was used according to the

Fig. 1. The effect of increasing the number of p(LL-b-EG) chains grafted to pK. Polylysine with a degree of polymerization of 42 was reacted

with p(LL-b-EG) (Mw = 5800) at a molar ratio of (.) 1:1, (D) 1:3, or (n) 1:5.

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manufacturer’s protocol (ex: 485 nm, Em: 535 nm,

cutoff: 515 nm) and quantitated only the amount of

free DNA present. Because removal of aliquots also

depleted the sample dispersion of degrading com-

plexes, the volume was not replenished with buffer

in order to avoid diluting the remaining amount of

complexes. However, the extremely dilute concentra-

tion of complexes eliminated the need to add buffer to

maintain perfect sink conditions.

Fig. 2. The effect of decreasing pK length. Polylysine with a degree o

(Mw = 7500) at a 1:1 molar ratio.

For electrophoretic analysis, aliquots of 15 Al werealso removed from samples and frozen until later use.

Saved samples were thawed and concentrated using

Microcon YM-100 centrifuge filters. Approximately

150 ng of the combined samples was run per well on a

1% agarose gel (7.5 V/cm, 30 min) in 1� Tris–

acetate–EDTA running buffer. Concentrated samples

from day 42 were also linearized by HindIII digestion

(10 U, 37 jC, 1 h) and run for 1 h on a 1% agarose gel.

f polymerization of either 4 or 14 was grafted with p(LL-b-EG)

Fig. 3. Static light scattering analysis of complexes at their respective N:Pmin for apparent molecular weight (Mw*) and apparent radius of

gyration (rg*). Bars marked with * (for rg*) or # (for Mw*) are significantly different from all other groups (ANOVA, p< 0.01, Tukey HSD).

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A molecular weight ladder (Invitrogen, Carlsbad, CA)

and pEGFPLuc plasmid stock were run in conjunction

with samples for comparison. Gels were stained with

SYBR Gold (Molecular Probes) according to the

manufacturer’s protocol and visualized using a stan-

dard transilluminator and digital camera with a UV-2

and yellow 12 lens filter.

2.2.6. Polylactide degradation in pK14-7k

The bulk hydrolysis of the pLL segment was

assessed by incubating individual samples (1.5–2.0

mg each) of pK14-7k in distilled water at 37 jC. Atcertain time points, one sample was removed, lyoph-

ilized, and redissolved in deuterated DMSO. 1H NMR

spectroscopy was used to calculate the molecular

weight of pLL by using the methine peak at 5.19

ppm and the pEG ether peak (3.5 ppm) as an internal

standard.

Fig. 4. Cold field emission scanning electron micrographs of

complexesmade at their respective N:Pmin. The bar in each represents

200 nm.

3. Results

3.1. Terpolymer characterization

Molecular weight analysis of p(LL-b-EG) diblock

using GPC-MALLS and 1H NMR agreed well, as

reported previously [20]. The characteristic 1H NMR

peaks of the final pK-g-p(LL-b-EG) terpolymer were

as follows: (DMSO-d6) 1–1.56 (s, 4H) and 1.33 (s,

2H); 2–2.76 (s, 2H); 3–8.05 (d, 1H); and 4–4.25 (s,

1H). Table 1 summarizes the results from the primary

amine determination. The data generated by NMR

agree best with TNBS data for pK42-5k. As greater

amounts of p(LL-b-EG) were grafted onto pK or as the

degree of polymerization for pK decreased, increas-

ingly disparate results were produced.

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3.2. Assessing terpolymer interaction with DNA

Fig. 1 shows the condensation profiles of terpol-

ymers with different amounts of p(LL-b-EG) grafted

to pK as a function of amine/phosphate charge ratio

(N:P). The curves for both pK42-5k(3) and pK42-

5k(5) were shifted to the left, indicating more ad-

vanced stages of DNA condensation at low N:P ratios

when compared to that of pK42-5k. However, the

reduction in fluorescence for both terpolymers dis-

played an uncharacteristic plateau above background

levels.

The effect of pK length on plasmid condensation is

illustrated in Fig. 2. The N:Pmin was 1.3 and 1.2 for

pK4 and pK14, respectively. Grafting p(LL-b-EG) to

pK shifted each of the condensation profiles to the

left, suggesting more advanced condensation at lower

N:P. Complexes made with pK14-7k were able to

reduce the N:Pmin by approximately 50%. This obser-

vation is consistent with results comparing pK42 to

pK42-7k, as previously reported [20]. However, pK4-

7k condensation of plasmid resulted in an obvious

S. Park, K.E. Healy / Journal of C

Fig. 5. Electrophoretic analysis of plasmid released from complexes in the

pK or corresponding terpolymers at a N:P ratio of 1.5 were incubated wi

Complexes made with unmodified polylysines (pK4, pK14, and pK42) d

formulations (pK14-7k and pK42-7k) formed complexes with complete sta

was due to sheared DNA from the DNA cellulose beads. A DNA mark

comparison.

plateau at approximately 10% maximum RFU. It

appeared that the fluorescence levels began to ap-

proach zero as N:P increased past 1.1 but did not do

so completely in the N:P range investigated.

3.3. Assessing complex size

The results of static light scattering analysis of

complexes for apparent molecular weight (Mw*) or

apparent radius of gyration (rg*) are shown in Fig. 3.

Complexes made with terpolymer had a Mw* of ap-

proximately one order of magnitude lower than their

respective unmodified polylysine counterparts. How-

ever, rg* values for terpolymer complexes were larger

(ANOVA, p < 0.01, Tukey HSD) than their unmodi-

fied pK complements. In addition, complexes made

with pK14 or pK14-7k had significantly smaller rg*

values (ANOVA, p < 0.01, Tukey HSD) than those

made with their pK4-based (pK4 or pK4-7k, respec-

tively) analogs. Scanning electron microscopy (SEM)

confirmed that nanometer-scale particles were formed

(Fig. 4).

presence of excess DNA. Complexes made with either unmodified

th cellulose-bound DNA in incomplete Dulbecco’s PBS (pH= 7.2).

isplayed immediate destabilization by salt (t = 0). Two terpolymer

bility for the duration of the experiment. The smearing in each lane

er ladder (M) as well as pEGFPLuc stock (S) were also run for

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3.4. Plasmid exchange with excess DNA

Incubation of pK–DNA complexes with excess

DNA suggested that complexes were relatively un-

stable and readily exchanged (Fig. 5). Merely adding

a physiologically relevant buffer solution to unmod-

S. Park, K.E. Healy / Journal of646

Fig. 6. (A) Plasmid release profiles from complexes incubated at 37 jC(N:P= 0.62), (.) pK14-7k (N:P= 1.2), (o) pK42 (N:P= 0.8), (D) pK42-7

released was quantitated using PicoGreen (n= 9). (B) Curve fitting of

terpolymer–DNA complexes: (n) pK14-7k (N:P= 0.62) [ y= 1.87x, R2 = 0

(N:P= 0.62) [ y = 0.55x, R2 = 0.92].

ified pK complexes readily destabilized them, as

indicated by samples taken at t = 0 h. Complex

resistance to salt dissociation and exchange with

excess DNA appeared to increase with increasing

pK length. Particles made with pK4-7k showed

improved stability over those made with unmodified

for various time points for (x) pK14 (N:P= 1.2), (n) pK14-7k

k (N:P= 0.62), and (*) pK42-11k (N:P= 0.42). The amount of DNA

selected data revealing zero-order plasmid release kinetics from

.92]; (.) pK14-7k (N:P= 1.2) [ y = 1.28x, R2 = 0.86]; (D) pK42-7k

Table 2

Bulk degradation of pLL in pK14-7k

Incubation time (days)

0 7 14 28 35 42

DPpLL 18.0 17.2 16.9 16.4 16.0 14.8

Fig. 7. Plasmid released from complexes incubated at 37 jC for

various time points and run on a 1% agarose gel (1� TAE,

pH= 8.0). The parenthesis indicate the N:P ratio used to make

complexes. The pooled samples were taken at the same time as

those used for the PicoGreen assay and run against a DNA marker

ladder (M) as well as pEGFPLuc stock (S).

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pK; however, there was still some detectable levels

of release plasmid. Complexes made with pK14-7k

and pK42-7k terpolymers were able to completely

stabilize complexes against salt dissociation and

prevent exchange with excess DNA for the duration

of the study. The background smearing in each lane

is attributed to shearing of the DNA bound to

cellulose, as evidenced by the last lane. Similar

results were seen when using an incubation buffer

of pH of 5.5 (data not shown).

3.5. Plasmid release from complexes

Particles made with unmodified pK14 at its

N:Pmin immediately showed high levels of fluores-

cence due to destabilization incurred by buffer salts

(Fig. 6A). Complexes made with pK14-7k terpol-

ymers demonstrated good initial stability against salt

dissociation but slowly released increasing amounts

of plasmid over a 6-week period. Table 2 shows the

progressive decrease in molecular weight of the

pLL segment of pK14-7k as a function of time.

Over a 6-week time period, the degree of polymer-

ization of pLL was reduced by approximately 18%

due to hydrolysis. Comparison of release profiles of

pK14 and pK14-7k (N:P= 1.2) shows that even

though an equivalent amount of polylysine was

used for each sample set, only terpolymer–DNA

complexes demonstrated controlled release of DNA.

Complexes made with pK42 showed significantly

better stability in buffer than pK14 and little plas-

mid was released after 6 weeks. Complexes made

with pK42-7k also showed controlled release of

plasmid over time but displayed less release than

pK14-based terpolymers. Fig. 6B illustrates linear

regression fits for the data of three of the terpoly-

mer formulations shown in Fig. 6A. Analysis of

quantitative plasmid release shows that terpolymer–

DNA complexes exhibit approximately zero-order

release kinetics.

Electrophoretic analysis of plasmid release from

complexes supported trends observed by the Pico-

Green assay, in addition to revealing the integrity of

the DNA (Fig. 7). Low-molecular-weight DNA

components appeared at long time points, particular-

ly in samples from terpolymer complexes. In addi-

tion, the prevalence of an extra band increased with

time, and was confirmed to be linearized plasmid by

Fig. 8. Comparison of plasmid released from complexes after 42 days in incubation (� ) before and (+) after HindII digestion.

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comparison to HindIII-digested pGFPLuc plasmid

(Fig. 8).

4. Discussion

The development of nonviral transfection vehicles

requires addressing the myriad hurdles that impede

efficient and sustained cellular expression. Primary

consideration should be given to clinical feasibility

and extracellular events, including submicron plasmid

packaging, tissue specificity, and cellular uptake.

Once internalized, complexes must be able to escape

endosomal degradation as well as deliver the genetic

information into the nucleus in a competent form for

transcription. As with any current vectors, polylysine

has limitations that must be considered and improved

upon if it is to be a viable clinical therapy. This work

extends our investigation of a terpolymer system that

addresses the issues of plasmid unpackaging by uti-

lizing hydrolytically labile bonds and low-molecular-

weight polymer components.

Terpolymer characterization for primary amine

content by 1H NMR and a TNBS binding assay

showed that there was good agreement between the

two techniques for pK42 with a low degree of

substitution (Table 1). The results between 1H NMR

and TNBS analysis became more disparate when the

degree of pK substitution increased or pK length

decreased. Since the TNBS technique is a binding

assay that requires a calibration curve to compute

amounts, the choice of standard molecule can have a

significant effect on results. For previous assays,

pK42 was used and the effect of minor modification

proved to be minimal in its interference in TNBS

binding [20]. However, when the percent pK substi-

tution increased [either by increasing the amount of

p(LL-b-EG) grafted or decreasing pK length], the

steric hindrance exerted by grafted p(LL-b-EG) be-

came more significant and unmodified pK42 becomes

less appropriate as a standard. Consequently, all

subsequent calculations requiring primary amine con-

tent utilized data acquired by 1H NMR.

Previous work has demonstrated that p(LL-b-EG)

grafted onto pK significantly affected DNA binding

dynamics as well as protection of DNA from nuclease

degradation [20]. While increasing the pLL segment

length can reduce the amount of pK needed for

complete condensation, increasing the number of

grafted p(LL-b-EG) chains can have a different effect

on plasmid condensation (Fig. 1). Both terpolymers,

pK42-5k(3) and pK42-5k(5), have fluorescence pro-

files that are shifted to the left of pK42-5k, suggesting

that DNA condensation is more advanced than with

pK42-5k at low N:P ratios. However, neither terpoly-

mer was able to completely reduce ethidium bromide

fluorescence to background levels at the N:P ratios

investigated. A similar result was seen in Fig. 2 when

pK4 was substituted with p(LL-b-EG) (7k). These

results, supported by previously published data, sug-

gested that there could be two competing effects

generated by the greater number of p(LL-b-EG)

chains with respect to pK length [20]. One effect

could be attributed to increased terpolymer hydropho-

bicity (via pLL), which has been implicated as a

secondary force that facilitates DNA condensation

[22–24]. This effect was observed previously when

increasing the molecular weight of the pLL segment

grafted to pK42 decreased the N:Pmin [20]. Secondly,

the greater number of hydrophilic pEG chains can

interfere with polylysine binding to DNA due to its

large radius of gyration. [25,26]. The end group was

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not considered a significant influence on complex

formation since replacement of the fmoc group with

biotin did not change the performance of pK14-7k in

its interaction with DNA. While many studies have

successfully condensed plasmid DNA with pK–pEG

block copolymers, a survey of the literature suggests

that that pEG interference becomes significant when

its Mw is near or greater than that of the pK backbone

[25,26]. However, when considering pK-g-p(LL-b-

EG) terpolymers, the pLL segment can counteract

this effect by imparting enough hydrophobicity and

distance between pK and pEG, as evidenced by the

performance of pK14-7k. From a clinical standpoint,

this could result in nanoparticles with higher surface

densities of pEG needed for ‘‘stealth’’ behavior or

presentation of active molecules (i.e., peptides) on a

‘‘stealth’’ background.

Binding dynamics of polycations to DNA can also

be elucidated by static light scattering analysis of

complexes [20,27]. Complexes made with unmodified

pK14 showed greater Mw* at its N:Pmin than those

made with pK4 (Fig. 3). Grafting p(LL-b-EG) to pK

reduced Mw* by an order of magnitude when compared

to complexes made from their unmodified pK ana-

logs. A similarly dramatic difference was seen previ-

ously for pK42 and pK42-7k [20]. The presence of

p(LL-b-EG) could have reduced secondary polyion

interactions by restricting bound pK mobility (due to

pLL) or by sterically hindering the approach of other

molecules (due to pEG).

Size distributions of complexes made with modi-

fied and unmodified pK4 and pK14 polymer systems

resulted in much smaller and more homogenous

particles than seen previously when using pK42 and

pK42-7k [20]. This observation is consistent with

other studies that found that higher-molecular-weight

polylysine produces slightly larger complexes [28]. In

addition, time-resolved static light scattering studies

on complexes made with high-molecular-weight pK

have shown progressive aggregation [27]. Despite that

complexes made with pK14 and pK4 were close in

average size, they proved to be statistically different

(ANOVA, p < 0.01, Tukey HSD). This suggests that

pK4 complexes were less dense, particularly when

considering their Mw*. In addition, SEM imaging of

pK4 complexes proved to be difficult, which was

attributed to flattening or collapse of particles upon

drying and coating (Fig. 4). Terpolymer complexes

were larger (ANOVA, p < 0.01, Tukey HSD) than

their unmodified pK counterparts due to the presence

of grafted p(LL-b-EG). This trend was not apparent

previously when comparing pK42 and pK42-7k com-

plexes because of the relatively low degree of p(LL-b-

EG) substitution onto pK42 [20].

For effective transfection, internalized DNA–poly-

cation complexes must be able to balance between

maintaining plasmid protection until its deliverance

into the nucleus, where plasmid release is critical for

transcription. Agarose electrophoresis was used to

assess complex stability in the presence of excess

DNA at neutral (Fig. 5) and acidic pH (data not

shown). At 0 h, plasmid was released from all pK

complexes, indicating that resuspension in buffer

destabilizes the complexes. Previous assessment of

N:Pmin (Fig. 2) showed that condensation was com-

plete at a N:P of 1.5 for pK14 and pK42 terpolymers.

However, the ethidium bromide assay was performed

in ultrapure water and it has been shown that salt

solutions can induce polycation dissociation, particu-

larly when using low Mw polycations [29,30]. Com-

plexes made with pK42 showed the greatest resistance

to salt-induced dissociation at physiological pH. As

time increased, greater amounts of plasmid were

released, presumably induced by exchange with the

excess DNA bound on cellulose [3]. All terpolymers

showed greater stability in buffer when compared to

their respective pK control and, except for pK4-7k,

did not release any plasmid after 2 days of incubation.

These terpolymer compositions can prove to be useful

in protecting DNA from both extracellular and intra-

cellular degradation by providing stability at physio-

logical and acidic pH.

Longer-term investigation of complex stability in

physiological buffer showed that plasmid release was

affected by the amount of terpolymer used, pLL

length, and pK length (Fig. 6A). Upon resuspension,

complexes made with pK14 were easily destabilized

by salt and displayed a burst release of plasmid.

Complexes made with pK14-7k displayed better initial

and long-term stability, despite that an equal

(N:P= 1.2) or lesser (N:P= 0.62) amount of polylysine

was used. The controlled release of plasmid was likely

due to a combination of moderate pLL degradation and

salt destabilization of low-molecular-weight pK–

DNA interactions (Table 2). As expected, plasmid

release from terpolymer complexes was slower when

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almost twice as much terpolymer was used (N:P= 1.2

vs. 0.62). The effect of pK molecular weight on

terpolymer–DNA complex stability is illustrated by

comparing the degradation profiles of pK42-7k and

pK14-7k (N:P= 0.62). An equivalent amount of lysine

residues was used to make these complexes; however,

pK14-7k released significantly more plasmid over the

same time period. Complexes made with the longest

pLL segment, pK42-11k, maintained complete DNA

stabilization even after 6 weeks. Increasing the molec-

ular weight of pLL decreased its hydrolysis rate and,

combined with the tenacity of pK42, resulted in com-

plexes that showed no detectable plasmid release [31].

Curve fitting on plasmid release profiles for ter-

polymer–DNA complexes revealed very good linear

fits (R2z 0.86), which indicated approximately zero-

order release kinetics over the 6-week experiment (Fig.

6B). Longer experiments are necessary to determine

whether these initial kinetics are truly sustainable until

100% of the plasmid is released. The copious research

on modulating parameters to achieve zero-order drug

release from degradable profiles demonstrates that

controlled release of molecular therapies is desirable

for sustained levels of treatment. Similarly, zero-order

plasmid release rates enable constant dosage of DNA

with a single application that can extend otherwise

transient expression, since nonviral vectors do not

integrate plasmids into the host genome. Other gene

therapy constructs have also utilized this strategy by

making matrices of hydrolytically susceptible polyest-

ers for slow release of plasmid for continuous expres-

sion [16,18]. However, terpolymer–DNA complexes

presented in this work are unique from most delivery

assemblies in that their nanoscale size and potential for

ligand functionalization encourage potential internali-

zation so that progressive plasmid release occurs

intracellularly.

Electrophoretic analysis of plasmid released from

complexes revealed that as time progressed, the

relative amount of open circular plasmid increased

for samples that had detectable plasmid release (Fig.

7). The issue of additional bands not attributed to

supercoiled and open circular plasmid was clarified

by exposing day 42 samples to HindIII digestion

(Fig. 8). Restriction enzymes will cleave open cir-

cular and supercoiled DNA, as well as any multi-

mers, as demonstrated by digestion of uncomplexed

pEGFPLuc plasmid. Plasmid digestion revealed that

in the samples with detectable release, linearized

plasmid was present in samples at long time points.

The possible contribution of terpolymer degradation

and experimental conditions on plasmid damage

should be noted, but was not clearly separable under

these conditions. Despite that there was a decrease in

the amount of supercoiled plasmid released, a sig-

nificant amount of open circular plasmid was also

present, which is a competent form for transcription

[32].

5. Conclusions

This study has demonstrated that modulating the

relative amounts of each component poly(lactide-g-

(lactide-b-ethylene glycol)) terpolymers resulted in

significantly different polymer–DNA complex char-

acteristics. Increasing the number of p(LL-b-EG)

chains grafted to pK can interfere with binding to

DNA. However, the number of pK residues needed

for complete condensation can still be reduced by

obtaining a balance between pK length and p(LL-b-

EG) grafting ratios. Approximately zero-order plas-

mid release kinetics was observed from terpolymer–

DNA complexes and the rate of release can be

modulated according to the molecular weight of pK

and pLL, as well as by the total amount of terpoly-

mer used. These results suggest that terpolymer

composition can be optimized for different plasmid

release kinetics and potentially optimized expression

profiles.

References

[1] E. Marshall, Gene therapy. Second child in French trial is

found to have leukemia, Science 299 (5605) (2003) 320.

[2] J. Kaiser, Gene therapy. Seeking the cause of induced leuke-

mias in X-SCID trial, Science 299 (5606) (2003) 495.

[3] D.V. Schaffer, N.A. Fidelman, N. Dan, D.A. Lauffenburger,

Vector unpacking as a potential barrier for receptor-mediated

polyplex gene delivery, Biotechnology and Bioengineering 67

(5) (2000) 598–606.

[4] A.G. Ziady, T. Ferkol, D.V. Dawson, D.H. Perlmutter, P.B.

Davis, Chain length of the polylysine in receptor-targeted

gene transfer complexes affects duration of reporter gene ex-

pression both in vitro and in vivo, Journal of Biological

Chemistry 274 (8) (1999) 4908–4916.

[5] P. Erbacher, A.C. Roche, M. Monsigny, P. Midoux, The re-

S. Park, K.E. Healy / Journal of Controlled Release 95 (2004) 639–651 651

GEN

ED

ELIV

ER

Y

duction of the positive charges of polylysine by partial gluco-

noylation increases the transfection efficiency of polylysine/

DNA complexes, Biochimica et Biophysica Acta 1324 (1)

(1997) 27–36.

[6] P. Erbacher, A.C. Roche, M. Monsigny, P. Midoux, Glycosy-

lated polylysine/DNA complexes: gene transfer efficiency in

relation with the size and the sugar substitution level of gly-

cosylated polylysines and with the plasmid size, Bioconjugate

Chemistry 6 (4) (1995) 401–410.

[7] A. Maheshwari, R.I. Mahato, J. McGregor, S. Han, W.E.

Samlowski, J.S. Park, S.W. Kim, Soluble biodegradable poly-

mer-based cytokine gene delivery for cancer treatment, Mo-

lecular Therapy 2 (2) (2000) 121–130.

[8] Y.B. Lim, S.O. Han, H.U. Kong, Y. Lee, J.S. Park, B. Jeong,

S.W. Kim, Biodegradable polyester, poly[alpha-(4-aminobu-

tyl)-L-glycolic acid], as a non-toxic gene carrier, Pharmaceut-

ical Research 17 (7) (2000) 811–816.

[9] C.H. Ahn, S.Y. Chae, Y.H. Bae, S.W. Kim, Biodegradable

poly(ethylenimine) for plasmid DNA delivery, Journal of

Controlled Release 80 (1–3) (2002) 273–282.

[10] H. Petersen, T. Merdan, K. Kunath, D. Fischer, T. Kissel,

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biode-

gradable polyethylenimine derivative with an advantageous

pH-dependent hydrolytic degradation for gene delivery, Bio-

conjugate Chemistry 13 (4) (2002) 812–821.

[11] M.A. Gosselin, W. Guo, R.J. Lee, Efficient gene transfer using

reversibly cross-linked low molecular weight polyethyleni-

mine, Bioconjugate Chemistry 12 (6) (2001) 989–994.

[12] C. Pichon, E. LeCam, B. Guerin, D. Coulaud, E. Delain, P.

Midoux, Poly[Lys-(AEDTP)]: a cationic polymer that allows

dissociation of pDNA/cationic polymer complexes in a reduc-

tive medium and enhances polyfection, Bioconjugate Chem-

istry 13 (1) (2002) 76–82.

[13] F. Tang, J.A. Hughes, Use of dithiodiglycolic acid as a tether

for cationic lipids decreases the cytotoxicity and increases

transgene expression of plasmid DNA in vitro, Bioconjugate

Chemistry 10 (5) (1999) 791–796.

[14] Y. Park, K.Y. Kwok, C. Boukarim, K.G. Rice, Synthesis of

sulfhydryl cross-linking poly(ethylene glycol)-peptides and

glycopeptides as carriers for gene delivery, Bioconjugate

Chemistry 13 (2) (2002) 232–239.

[15] M. Ouyang, J.S. Remy, F.C. Szoka Jr., Controlled template-

assisted assembly of plasmid DNA into nanometric particles

with high DNA concentration, Bioconjugate Chemistry 11 (1)

(2000) 104–112.

[16] L.D. Shea, E. Smiley, J. Bonadio, D.J. Mooney, DNA delivery

from polymer matrices for tissue engineering, Nature Biotech-

nology 17 (6) (1999) 551–554.

[17] J.H. Jeong, T.G. Park, Poly(L-lysine)-g-poly(D,L-lactic-co-gly-

colic acid) micelles for low cytotoxic biodegradable gene de-

livery carriers, Journal of Controlled Release 82 (1) (2002)

159–166.

[18] Y. Capan, B.H. Woo, S. Gebrekidan, S. Ahmed, P.P. DeLuca,

Preparation and characterization of poly (D,L-lactide-co-glyco-

lide) microspheres for controlled release of poly(L-lysine)

complexed plasmid DNA, Pharmaceutical Research 16 (4)

(1999) 509–513.

[19] W.T. Godbey, K.K. SW, A.G. Mikos, Size matters: molecular

weight affects the efficiency of poly(ethylenimine) as a gene

delivery vehicle, Journal of Biomedical Materials Research 45

(1999) 268–275.

[20] S. Park, K.E. Healy, Nanoparticulate DNA packaging using

terpolymers of poly(lysine-g-lactide-b-ethylene glycol), Bio-

conjugate Chemistry 14 (2003) 311–319.

[21] G.T. Hermanson, Bioconjugate Techniques, Academic Press,

San Diego, 1996.

[22] P.S. Kuhn, M.C. Barbosa, Y. Levin, Effects of hydrophobicity

in DNA surfactant complexation, Physica A 283 (1–2) (2000)

113–118.

[23] S. Bhattacharya, S.S. Mandal, Interaction of surfactants with

DNA. Role of hydrophobicity and surface charge on interca-

lation and DNA melting, Biochimica et Biophysica Acta 1323

(1) (1997) 29–44.

[24] D.C. Rau, V.A. Parsegian, Direct measurement of the inter-

molecular forces between counterion-condensed DNA double

helices, Biophysical Journal 61 (1992) 246–259.

[25] M.A. Wolfert, E.H. Schacht, V. Toncheva, K. Ulbrich, O.

Nazarova, L.W. Seymour, Characterization of vectors for

gene therapy formed by self-assembly of DNA with synthetic

block co-polymers, Human Gene Therapy 7 (17) (1996)

2123–2133.

[26] D.Y. Kwoh, C.C. Coffin, C.P. Lollo, J. Jovenal, M.G. Ba-

naszczyk, P. Mullen, A. Phillips, A. Amini, J. Fabrycki,

R.M. Bartholomew, S.W. Brostoff, D.J. Carlo, Stabilization

of poly-L-lysine/DNA polyplexes for in vivo gene delivery

to the liver, Biochimica et Biophysica Acta 1444 (2) (1999)

171–190.

[27] E. Lai, J.H. van Zanten, Monitoring DNA/poly-L-lysine poly-

plex formation with time-resolved multiangle laser light scat-

tering, Biophysical Journal 80 (2) (2001) 864–873.

[28] M.A. Wolfert, L.W. Seymour, Atomic force microscopic anal-

ysis of the influence of the molecular weight of poly(L)lysine

on the size of polyelectrolyte complexes formed with DNA,

Gene Therapy 3 (3) (1996) 269–273.

[29] T. Reschel, C. Konak, D. Oupicky, L.W. Seymour, K. Ulbrich,

Physical properties and in vitro transfection efficiency of gene

delivery vectors based on complexes of DNA with synthetic

polycations, Journal of Controlled Release 81 (1–2) (2002)

201–217.

[30] A.L. Parker, D. Oupicky, P.R. Dash, L.W. Seymour, Method-

ologies for monitoring nanoparticle formation by self-assem-

bly of DNA with poly(L-lysine), Annals of Biochemistry 302

(1) (2002) 75–80.

[31] S.M. Li, S. Anjard, I. Rashkov, M. Vert, Hydrolytic degrada-

tion of PLA/PEO/PLA triblock copolymers prepared in the

presence of Zn metal or CaH2, Polymer 39 (22) (1998)

5421–5430.

[32] H. Weintraub, P.F. Cheng, K. Conrad, Expression of trans-

fected DNA depends on DNA topology, Cell 46 (1) (1986)

115–122.