NIH Public Access

Author Manuscript
J Pharm Sci. Author manuscript; available in PMC 2009 November 1.
Published in final edited form as:
NIH-PA Author Manuscript

J Pharm Sci. 2008 November ; 97(11): 4794–4806. doi:10.1002/jps.21338.

Peptide-Mediated Lipofection Is Governed by Lipoplex Physical

Properties and the Density of Surface-Displayed Amines

JENNIFER C. REA1, ANNELISE E. BARRON2, and LONNIE D. SHEA1,3

1Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd. E156,
Evanston, Illinois 60208−3120
2Department of Bioengineering, Stanford University, W300B James H. Clark Center, 318 Campus Drive,
Stanford, California 94305−5440
3Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611

Abstract
NIH-PA Author Manuscript

Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical

properties to overcome rate-limiting steps to gene transfer. The objectives of this study were to
determine the regimes over which cationic peptides enhance lipofection and to investigate the
mechanism of action, such as increased cellular association resulting from changes in lipoplex
physical properties. Short, cationic peptides were incorporated into lipoplexes by mixing peptide,
lipid and DNA. Lipoplexes were characterized using gel retardation, dynamic light scattering, and
fluorescent microscopy, and the amount of surface-displayed amines was quantified by
fluorescamine. Size, zeta potential, and surface amines for lipoplexes were dependent on peptide/
DNA ratio. Inclusion of peptides in lipoplexes resulted in up to a 13-fold increase in percentage of
cells transfected, and up to a 76-fold increase in protein expression. This transfection enhancement
corresponded to a small particle diameter and positive zeta potential of lipoplexes, as well as increased
amount of surface-displayed amines. Relative to lipid alone, these properties of the peptide-modified
lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for
transfection with lipoplexes. The addition of peptides is a simple method of lipofection enhancement,
as direct chemical modification of lipids is not necessary for increased transfection.

Keywords
NIH-PA Author Manuscript

DNA delivery; gene delivery; gene vectors; lipoplexes; cationic lipids; peptides

INTRODUCTION
Lipoplexes have been used extensively for in vitro and in vivo gene delivery applications, and
are currently in clinical trials. Lipid-based vectors condense DNA and protect it from
degradation, promote membrane interaction and destabilize endosomes, and can have low
toxicity.1-5 Despite their widespread use, the efficiency of lipid-mediated gene transfection
still lags behind that of viral vectors. Lipoplex stability, cellular internalization, and
intracellular trafficking barriers can limit the efficacy of lipoplexes.6-8 Efforts to improve
lipid-based gene delivery have focused on modifying the cationic lipid structure, altering lipid
formulations, and adding polymers to lipid-DNA complexes to enhance transfection.9-15 In
particular, polymers or peptides with specific functionalities may be added to lipids to function

Correspondence to: Lonnie D. Shea (Telephone: 847−491−7043; Fax: 847−491−3728; E-mail: [email protected]).
 REA et al. Page 2

as modular units that can target a range of cellular processes.16 For example, bioactive peptides
for receptor targeting, endosomal destabilization, and nuclear localization have been utilized
to enhance lipofection.17-27
NIH-PA Author Manuscript

Bioactive peptides can influence lipofection through a sequence-specific response or by

imparting nonspecific changes in conformation or other physiochemical properties of the
lipoplex, such as zeta potential and average diameter. Increasing the amount of peptides within
lipoplexes initially results in an increase of particle size from less than 500 nm to up to 2000
nm, while further increasing the peptide loading beyond a certain threshold resulted in a
reduction in diameter to less than 500 nm.28,29 Despite the similar physical characteristics of
peptide/DNA lipoplexes at the high peptide regime (i.e., the region in which the lipoplex size
decreases from its maximum following the initial increase) compared to that of the low peptide
regime, to our knowledge gene delivery in the high peptide regime has not been widely
investigated. Relative to high molecular weight proteins, peptides typically have a lower
affinity for DNA,30 and their inclusion in the lipoplex may be dependent on the amount of
peptide added. For either bioactive or nonbioactive peptides, increasing the peptide quantity
may increase the incorporation of peptides into the lipoplexes, which may facilitate cellular
interactions through modified lipoplex surface properties.31

In this report, we investigate peptide-mediated lipofection to determine whether peptides can

interface specifically or nonspecifically with one or more steps in the gene transfer process.
NIH-PA Author Manuscript

We hypothesize that the physical properties of the lipoplex as well as the surface characteristics,
such as the density of surface-displayed amines, may be dependent on the amount of peptide
added to the lipoplexes and may affect transfection efficiency. Peptide-lipoplexes were formed
at peptide/DNA weight ratios ranging from 0 to 120 by adding cationic peptides to DNA prior
to complexation with lipids. The physiochemical properties of lipoplexes were characterized
by gel retardation, average diameter, zeta potential, lipid and peptide content, and the amount
of surface-displayed amines. Lipoplexes were visualized using fluorescence microscopy to
determine the extent of peptide and DNA colocalization within lipoplexes, and to determine
the extent of lipoplex localization to cells. In addition, the percentage of cells transfected,
protein expression and the quantity of DNA associated with cells were quantified. This report
investigates peptide addition as a simple method to manipulate the physical properties and
surface chemistry of lipoplexes, which can increase cellular association and transfection
efficiency. These studies add to the basic understanding of gene delivery and may contribute
to the design of modular gene delivery vectors.

MATERIALS AND METHODS

Materials
NIH-PA Author Manuscript

Plasmids encoding for luciferase and enhanced green fluorescent fusion protein (pEGFP-Luc)
with a CMV promoter were purified from bacteria culture using Qiagen (Valencia, CA)
reagents and stored in Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.4). Lipofectamine,
Lipofectamine 2000, fluorescamine, Oregon Green dye, tetramethylrhodamine (TAMRA), and
LysoTracker reagent were purchased from Invitrogen (Carlsbad, CA). Amino acids and resin
for peptide synthesis were purchased from Novabiochem (San Diego, CA). The remaining
peptide synthesis reagents were purchased from Applied Biosystems (Foster City, CA). All
other reagents were obtained from Fisher Scientific (Waltham, MA) unless otherwise noted.

Peptide Synthesis and Purification

An Applied Biosystems 433A peptide synthesizer was used to synthesize the SV40 T-antigen
nuclear localization sequence (NLS) and a scrambled peptide control. The SV40 peptide,
EGPKKKRKVG, containing the minimal SV40 T-antigen NLS, and the sSV40 peptide,

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 3

EKRGKVKPKG, a scrambled version of SV40, were synthesized using standard solid phase
methods and FastMocTM chemistry. The peptide was cleaved with a mixture of 90%
trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), 2.5% thioanosole, and 5% water for
NIH-PA Author Manuscript

1−2 h at room temperature, and then lyophilized. The crude peptide was analyzed for purity
by reversed-phase high-pressure liquid chromatography (RP-HPLC). The peptides were
purified by preparative RP-HPLC with a gradient of 0−20% acetonitrile with 0.1% TFA in
water with 0.1% TFA. Peptide molecular weight was confirmed using electrospray ionization
(ESI) mass spectroscopy and matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry at Northwestern University's Analytical Services
Laboratory. Peptide purity was analyzed using analytical RP-HPLC and the purity of both
peptides was determined to be >95%.

Gel Retardation Assay

DNA condensation by peptides was analyzed by gel electrophoresis. Peptide/DNA complexes
were formed by adding 22.5 μL of peptide solution containing varying amounts of peptide to
22.5 μL DNA solution (1.5 μg EGFP-Luc plasmid in TBS buffer), and the resulting solution
was mixed by gentle pipetting. Complexes were incubated for 20 min at room temperature.
For some samples, 5 μL of trypsin-EDTA solution was added to degrade the peptide after
complex formation. After complex formation, 10 μL of each sample were loaded onto a 1%
agarose gel in TBE buffer, which ran at 120 V for 30 min. DNA bands on the gel were visualized
under UV light by ethidium bromide staining.
NIH-PA Author Manuscript

Peptide-Lipoplex Formation
Lipofectamine (Invitrogen), a 3:1 (w/w) formulation of the polycationic lipid 2,3-dioleyloxy-
N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate
(DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE), was used at a 10:1
(w/w) ratio with DNA, within the manufacturer's recommended usage range. Peptide-
lipoplexes were formed by adding 22.5 μL of peptide solution containing varying amounts of
peptide to 22.5 μL DNA solution, and the resulting solution was then mixed by gentle pipetting.
The peptide/DNA mixture was allowed to incubate for 20 min at room temperature prior to
the dropwise addition of 45 μL of lipid solution. The resulting solution was mixed by gentle
pipetting and incubated for 20 min. Peptide-lipoplexes were formed in serum-free cell growth
media [Dulbecco's Modified Eagle Medium (DMEM), Invitrogen] at pH 7.4. For studies using
Lipofectamine 2000, the lipid formulation was used at a 1:3 ratio with DNA. Zeta potential
and z-average diameters were measured with a Zetasizer Nano ZS (Malvern, Worcestershire,
UK). All peptide/DNA ratios reported herein are by weight; the conversion between peptide/
DNA (w/w) ratio and peptide/DNA N/P ratio is 1 (w/w) ratio to 1.46 N/P ratio.
NIH-PA Author Manuscript

Free Lipid and Peptide after Lipoplex Formation

Complexes were formed with Lipofectamine, peptide, and DNA as described above. After
complex formation, complexes were filtered using a Millipore (Billerica, MA) Microcon spin
filter (MWCO 3000) to separate complexes from free lipid and peptide in solution. The
concentration of free lipid in the filtrate was measured by a colorimetric method using
ammonium ferrothiocynate, which readily forms a colored complex with phospholipids that is
soluble in chloroform.32 An aqueous ammonium ferrothiocynate solution was prepared by
dissolving 2.70 g ferric chloride hexahydrate and 3.04 g ammonium thiocyanate in 100 mL
deionized distilled water. Samples (20−40 μL) were added to chloroform and vigorously
mixed. The ammonium ferrothiocyanate solution was then added and the resulting mixture
was vigorously shaken for 1 min. After incubating at room temperature for 45 min, the
absorbance of the organic phase was measured at 488 nm. A standard curve was prepared using
known amounts of Lipofectamine.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 4

Free peptide in the filtrate was measured by dialyzing the samples using a Spectrum Labs
(Rancho Dominguez, CA) dialysis membrane with 1000 MWCO, lyophilizing, and then taking
the absorbance of samples resuspended in water at 220 nm using a Cary 50 UV/Vis
NIH-PA Author Manuscript

spectrophotometer (Varian, Inc., Palo Alto, CA). A standard curve was prepared using known
amounts of peptide.

Cell Culture and Transfection

Transfection studies were performed with NIH/3T3 and HEK293T cells (ATCC, Manassas,
VA) cultured at 37°C and 5% CO2 in DMEM supplemented with 1% sodium pyruvate, 1%
penicillin–streptomycin, 1.5 g/L NaHCO3, and 10% fetal bovine serum (FBS). For bolus
delivery, cells were seeded at a density of 15000 cells per well for NIH/3T3 cells and 20000
cells per well for HEK293T cells in 48-well plates (300 μL total volume) and cultured overnight
before complexes were added to the culture media. Complexes were formed as described
above, with 30 μL of the peptide-lipoplexes added per well. Transfection was characterized
by the number of transfected cells (GFP expression) measured 24 h post-transfection and the
extent of transgene expression (luciferase levels) measured 48 h post-transfection. Transfected
cells were visualized and manually counted using an epifluorescence microscope (Leica,
Bannockburn, IL) with a FITC filter and equipped with a digital camera. The percentage of
transfected cells was calculated as the number of EGFP-positive cells divided by total cell
number, which was determined by manual counting. The extent of transgene expression was
quantified by measuring the luciferase activity using the Luciferase Assay System (Promega,
NIH-PA Author Manuscript

Madison, WI). Cells were lysed and assayed for enzymatic activity 48 h after transfection. The
luminometer was set for a 3 s delay with signal integration for 10 s. Luciferase activity was
normalized to the amount of total protein in the sample, which was measured using a BCA
assay (Pierce, Rockford, IL) following the manufacturer's instructions. Transfection by
surface-bound lipoplexes was performed to determine the effect of particle density on
transfection efficiency. For surface-mediated DNA delivery, complexes were formed in
unsupplemented DMEM as described above, then allowed to deposit onto 48-well tissue
culture polystyrene plates for 24 h, with plates being washed twice with unsupplemented
DMEM before the seeding of cells to remove unbound lipoplexes.

Viability of the cell population was analyzed using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxyme-thoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium, inner salt (MTS) assay
(Promega), which reports on metabolic activity. Briefly, MTS reagent was added (20% of
culture volume) to the cultures, and after 1.5 h incubation, the absorbance at 490 nm was
measured with a spectrophotometer. The relative cell viability was reported as the absorbance
for the experimental condition divided by the absorbance for the control condition (no DNA
or transfection reagent), with absorbances corrected by subtracting the absorbance of the
reagents (without cells).
NIH-PA Author Manuscript

Colocalization of Fluorescently Labeled DNA and Peptide

EGFP-Luc plasmid was labeled with rhodamine using a Label-IT rhodamine-labeling kit
(Mirus, Madison, WI) according to the manufacturer's instructions. Briefly, DNA and Label
IT reagents were mixed and incubated at 37°C for 1 h. After incubation, DNA was precipitated
with 70% ethanol and resuspended in 10 μL Tris-EDTA (TE) buffer. Peptides were labeled
with TAMRA (5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester) or Oregon Green
(Invitrogen) according to the manufacturer's protocol. Briefly, peptides were mixed with
Oregon Green or TAMRA dissolved in DMSO and incubated with gentle agitation for 2 h in
the dark. The peptides were then dialyzed and lyophilized, and the lyophilized peptide was
resuspended in water to a concentration of 10 mg/mL.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 5

To determine if DNA and peptide colocalized, lipoplexes were formed with rhodamine-labeled
DNA and Oregon-Green-labeled peptides as described above. Complexes were immobilized
on tissue culture polystyrene (96-well plate; Corning, Corning, NY) immediately following
NIH-PA Author Manuscript

complex formation by incubation of DNA complexes (50 μL) with the substrate for 2 or 24 h
and were then visualized using fluorescence microscopy. To determine if DNA and peptide
colocalized with cells, lipoplexes were formed with rhodamine-labeled DNA or TAMRA-
labeled peptides. Lipoplexes were added to cells 24 h after cell seeding in 48-well plates. At
specified time points after transfection, cells were washed twice with PBS and then incubated
for 30 min with Lysotracker reagent. Cells and complexes were then visualized using
fluorescence microscopy at 2, 24, and 48 h.

Cellular Association of Lipoplexes

The amount of DNA associated with cells was monitored using plasmids radiolabeled with
α-32P dATP. Briefly, a nick translation kit (Amersham Pharmacia Biotech, Piscataway, NJ)
was used following the manufacturer's protocol with minor modifications. Cells were
transfected with radiolabeled DNA as described above and harvested from plates by
trypsinization. Following harvesting and washing with PBS, the amount of DNA associated
with cells was determined by immersing samples in 5 mL scintillation cocktail (Biosafe II,
Research Products International Corp., Mount Prospect, IL) for measurement with a
scintillation counter. The counts were correlated to DNA concentration using a standard curve
and results were reported as the amount of DNA measured per cell.
NIH-PA Author Manuscript

Detection of Accessible Primary Amines on Lipoplexes

A fluorescamine assay was used to determine the amount of primary amines accessible on the
peptide-lipoplexes.33 Complexes were formed with Lipofectamine, peptide and α-32P-labeled
DNA as described above. After complex formation, complexes were isolated by filtration using
a Millipore Microcon spin filter (MWCO 3000). The retentate was recovered and diluted in
1.4 mL assay buffer (100 mM boric acid–NaOH, pH 7.4) prior to rapid addition of 500 μL
0.01% fluorescamine (prepared in acetone). Samples were rapidly inverted 4−5 times and
incubated at room temperature for 10 min. Fluorescence was measured using a TBS-380 Mini-
Fluorometer (Turner Biosystems, Sunnyvale, CA) using λex 365 nm and λem 440−470 nm. A
standard curve was constructed using known amounts of peptide to correlate fluorescence
signal to amount of primary amines. The quantity of DNA associated with the lipoplexes was
determined by scintillation as previously described. The total number of primary amines
detected on lipoplexes was normalized by the mass of DNA in the sample.

Statistics
Statistical analysis was performed using JMP software (SAS Institute, Inc., Cary, NC).
NIH-PA Author Manuscript

Comparative analyses were completed using one-way ANOVA with Tukey post-tests, at a 95%
confidence level. Mean values with standard deviation are reported and all experiments were
performed in triplicate.

RESULTS
Physiochemical Properties of Peptide-Lipoplexes
Condensation of DNA by cationic peptides was investigated by gel retardation (Fig. 1). As the
weight ratio of peptide to DNA was increased from 10 to 120, DNA migration was increasingly
inhibited for both intact (SV40) and scrambled-sequence (sSV40) NLS peptides. However,
even at the highest peptide/DNA ratio of 120, the peptides did not completely inhibit DNA
migration. The intensity of each band was similar, indicating that complexation did not exclude

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 6

ethidium bromide intercalation. Incubation of the peptide/DNA complex with trypsin, which
degrades the peptide, allowed the DNA to migrate unhindered.
NIH-PA Author Manuscript

The size and zeta potential of peptide-lipoplexes were dependent on peptide/DNA ratio. At
peptide/DNA ratios of 40 and below, the peptide-lipoplexes maintained an average diameter
of less than 500 nm for both the SV40 and the sSV40 peptides (Fig. 2A). For peptide/DNA
ratios of 60−100, the average diameter of the peptide-lipoplexes was greater than 500 nm,
increasing up to 1500 nm. Further increasing the peptide/DNA ratio above 108 resulted in a
decrease in the diameter of the lipoplexes to less than 500 nm. The trends observed with particle
size were comparable to zeta potential. At low and high peptide/DNA ratios (i.e., <60 and
>100), the zeta potential of the peptide-lipoplexes was positive, which is desirable for cell
membrane interactions, but at an intermediate peptide/DNA ratio between 60 and 100, the zeta
potential of the complexes was negative (Fig. 2B). Thus, although the peptides do not
completely condense DNA as seen by gel retardation, the peptides affect the size and zeta
potential of the lipoplexes.

Subsequent experiments were performed to determine if peptides were incorporated into the
lipoplexes at several peptide/DNA ratios. The fluorescence images support the size results
obtained by dynamic light scattering; lipoplexes formed without peptide (Fig. 3A) were
noticeably smaller than lipoplexes formed at a peptide/DNA ratio of 80 (Fig. 3B). Peptides
colocalized with DNA at both a peptide/DNA ratio of 80 (Fig. 3B) and 108 (Fig. 3C). Similar
NIH-PA Author Manuscript

fluorescence images were obtained for both the sSV40 peptide and the SV40 peptide (data not
shown).

Free Lipid and Peptide after Complex Formation

The amount of free lipid and peptide in solution were measured to further investigate the
observations for the physical properties of the peptide-lipoplexes. The quantity of free lipid
increased as the peptide/DNA ratio increased from 0 to 80 (Fig. 4A). Further increasing the
sSV40 peptide/DNA ratio to 120 resulted in a decrease in free lipid. At a peptide/DNA ratio
of 80, the percentage of free lipid was approximately 60%, which differs significantly from
other peptide/DNA ratios. Additionally, the lipoplex diameter was maximal at this condition
among all the conditions tested, while the zeta potential was minimal. The percentage of free
sSV40 peptide increased with an increase in the peptide/DNA ratio (Fig. 4B). Nearly all of the
peptide is included in the lipoplex at low peptide/DNA ratios, while at higher ratios, an
increased amount of peptide is excluded from the complex. The highest percentage of excluded
peptide was approximately 20%, which occurred at a peptide/DNA ratio of 108. Similar results
were obtained with the SV40 peptide (data not shown).

Peptide Content and Lipofection Efficiency

NIH-PA Author Manuscript

Lipoplexes formed at peptide/DNA ratios of 108 and above significantly increased both the
percentage of transfected cells and reporter protein expression in NIH/3T3 mouse fibroblasts
compared to lower peptide/DNA ratios with both the SV40 peptide and the sSV40 peptide
(Fig. 5A and B). This trend was also observed with HEK293T human embryonic kidney cells
(data not shown). The addition of cationic peptide increased the percentage of transfected NIH/
3T3 cells up to 13-fold and protein expression 76-fold. Fluorescence microscopy experiments
using lipoplexes formed with TAMRA-labeled peptide and rhodamine-labeled DNA indicated
colocalization of peptide and DNA within cells (peptide/DNA, w/w, ratios of 20 and 100) at
2, 24, and 48 h (data not shown). For high peptide/DNA ratios, the transfection efficiency is
comparable to the transfection efficiency of using lipids alone with more than twice the amount
of DNA (Fig. 5C). For Lipofectamine 2000, the addition of peptide at a peptide/DNA ratio of
108 increased the percentage of transfected NIH/3T3 cells 1.9-fold and protein expression by
14.9-fold (data not shown). Although lipoplexes without any peptide or with small peptides

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 7

amounts demonstrated positive zeta potential and small particle size, they did not transfect as
well as the lipoplexes with peptide/DNA ratios of 108 and 120, which had similar physical
characteristics independent of sequence. Thus, the size and zeta potential of the complexes are
NIH-PA Author Manuscript

not the only factors that contribute to the increased transfection efficiency.

The order of addition of transfection reagents on transfection enhancement was also

investigated. Transfection enhancement was not observed when lipids were added to DNA
before the addition of SV40 peptide at any of the peptide/DNA ratios tested (Fig. 6). Similar
results were obtained with the sSV40 peptide (data not shown). The preformed lipid/DNA
complex may have excluded the peptides due to the peptides’ short length and low relative
affinity compared to the lipids, whereas the addition of peptides before lipids to DNA resulted
in a significant increase in transfection efficiency (at peptide/DNA ratios at 108 and above)
presumably because the peptides were included in the lipoplexes.

Cellular Association of DNA

Cellular association of lipoplexes was subsequently quantified to investigate the mechanism
of transfection enhancement. At a peptide/DNA ratios of 108, a significantly higher amount
of DNA was associated to the NIH/3T3 cells compared to peptide/DNA ratios of 0, 20, and 80
for both intact NLS peptide and scrambled NLS peptide (Fig. 7). The inclusion of peptide with
lipoplexes increased DNA association by up to threefold compared to lipoplexes without
peptide.
NIH-PA Author Manuscript

Lipofection Efficiency of Surface-Immobilized Lipoplexes

We subsequently tested the hypothesis that the increased cellular quantities may result from
the peptide-lipoplexes having a higher density than lipoplexes alone, which could increase
delivery due to rapid settling of the lipoplexes onto the cells. The average diameter of lipoplexes
without peptide was similar to the average diameters of lipoplexes with a high amount of
peptide. Given that the peptide colocalizes with the lipoplexes, the peptide-containing
lipoplexes may have a higher density than the lipoplexes without peptide. To minimize the
influence of lipoplex density on transfection, lipoplexes were deposited onto tissue culture
plates 24 h prior to the addition of NIH/3T3 cells, which reduces mass transport limitations
affected by particle density.34 At a peptide/DNA ratio of 120, a significantly higher amount
of luciferase expression was observed compared to lipid alone for surface-bound lipoplexes
(Fig. 8). Similar results were obtained with SV40 peptide (data not shown). Thus, lipofection
is enhanced with the addition of peptide when complexes are administered to cells by either
bolus delivery or surface-mediated delivery, suggesting that complex density is not responsible
for increased transfection.
NIH-PA Author Manuscript

Quantification of Surface Amines on Peptide-Lipoplexes

We subsequently tested the hypothesis that increasing the amount of peptide added to the
lipoplexes not only affects average size and zeta potential, but also increases the amount of
peptide displayed on the surface, which could be observed through the number of accessible
amine groups. The amount of primary amines on the lipoplex surface was quantified using a
fluorescamine assay and normalized to the total DNA. As the sSV40 peptide/DNA (w/w) ratio
increased above 20, the primary amines on the lipoplex surface also increased (Fig. 9). A
peptide/DNA ratio of 80 had the highest amount of detectable surface amines, indicating that
the number of surface amines does not directly correlate with the amount of peptide added.
Similar results were obtained with SV40 peptide (data not shown).

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 8

DISCUSSION
This report investigates transfection with peptide-lipoplexes formed at varying peptide/DNA
NIH-PA Author Manuscript

weight ratios and the influence of the peptides on the lipoplex's physical properties. At peptide/
DNA ratios below 60 or above 108, the average diameters of the complexes were less than 500
nm and the zeta potential was positive, while at peptide/DNA ratios between 60 and 100, the
average diameters of the complexes were greater than 500 nm, increasing to up to 1500 nm
with negative zeta potentials. Peptide-lipoplexes containing either intact or scrambled NLS
peptides at peptide/DNA ratios of 108 and above significantly increased both the percentage
of transfected cells and reporter protein expression compared to peptide/DNA ratios of 80 and
below, with increases up to 13-fold for percent transfection and 76-fold for luciferase
expression of NIH/3T3 cells. Comparable transfection efficiencies were achieved with less
than half the amount of DNA with peptide-lipoplexes compared to lipoplexes without peptide.
An increased cellular association of DNA was observed at peptide/DNA ratios above 108
compared to lower peptide/DNA ratios. In addition, inclusion of peptides in lipoplexes did not
increase cytotoxicity (data not shown), consistent with literature reports on low molecular
weight peptides relative to large proteins.35 Finally, the physical properties of the peptide-
lipoplexes and surface primary amines on the lipoplexes contributed to increased cellular
association and subsequent gene expression. The results obtained herein were similar with both
the SV40 and sSV40 peptides, indicating that sequence specificity was not responsible for the
observed results. The approach to form peptide-lipoplexes used herein is a more robust
NIH-PA Author Manuscript

approach to form modular complexes relative to approaches based on synthesis of a single

molecule with multiple domains, as different peptides can easily be substituted into the
lipoplex.

We hypothesize that an increased quantity of peptide may be necessary to achieve significant

loading of peptide in the lipoplex due to the relatively low affinity of the peptide for DNA.
Earlier studies of poly-L-lysine (PLL) with lipids showed that PLL enhanced lipid-mediated
gene delivery at lower polycation/DNA ratios than with peptides, most likely because PLL is
a much larger molecule and has higher relative affinity to the DNA compared to the peptides.
29 The short length and relatively small number of charges allows only weak association
between the peptide and the DNA relative to high molecular weight proteins and polymers. At
intermediate peptide amounts, the lipoplexes have a large diameter and a negative zeta
potential, indicative of a disrupted structure that is perhaps due to the weakly bound peptides
interfering with lipoplex formation. A disrupted structure is consistent with the free lipid data;
at a peptide/DNA ratio of 80, there is significantly more free lipid than at peptide/DNA ratios
of 0, 20, and 108. At higher peptide amounts, the peptide and lipids form smaller particles with
positive zeta potentials, suggesting that the peptides and lipids may form ordered structures
that shield the negative charge of DNA. Although the peptide-lipoplexes formed at a peptide/
NIH-PA Author Manuscript

DNA ratio of 80 have detectable amines on the surface, these lipoplexes still have a negative
zeta potential. Zeta potential does not indicate surface potential, but reflects the effective charge
of the particles compared to the bulk solution. Thus, although some peptide-lipoplexes have
detectable amines on the surface, the overall charge of the particle in solution may still be
negative, which was observed for the peptide-lipoplexes formed at a peptide/DNA ratio of 80.
The negative zeta potential and large particle diameter of the lipoplexes formed at a peptide/
DNA (w/w) ratio of 80 likely contribute to the relatively low transfection. Interestingly, the
addition of peptides prior to the addition of lipids did not prevent the peptides’ ability to be
displayed on the surface of the lipoplexes. In addition, the order of addition of the peptides and
lipids was critical to enhanced lipofection; lipid addition prior to peptides likely excludes the
small peptides from the lipoplex.

The increasing quantity of peptides within the lipoplexes increases the amount of accessible
amines, which is most likely from an increase in the amount of peptides on the lipoplex surface.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 9

This increase of peptides on the lipoplex surface could facilitate cellular interactions. Cellular
association of DNA has been determined to be a rate-limiting step for lipofection.36 Size, zeta
potential, and density have also been implicated as important factors for cellular internalization
NIH-PA Author Manuscript

of lipoplexes;25-27 however, these factors cannot explain observations for transfection with
lipoplexes containing significant quantities of peptide, as size and zeta potential were similar
for low and high peptide amounts while transfection enhancement was only observed at high
peptide/DNA ratios. Thus, the increased quantity of cell-associated DNA reported herein is
due to both favorable lipoplex physical properties (i.e., small average diameter and positive
zeta potential) and an increased amount of peptides at the surface of the lipoplex, which could
promote cellular association.37 For liposomes modified by stearylated peptides, the peptide
density on the surface of the liposome dictated transfection behavior; higher densities of
peptides on the surface resulted in increase cellular uptake and protein expression.37 In the
study presented herein, the increase in cell-associated DNA was modest compared to the
increase in transfection efficiency at peptide/DNA (w/w) ratios above 80. Thus, the addition
of peptide may affect cellular processes other than increased association and uptake. For
example, the addition of peptides may protect the DNA from degradation or impart enhanced
intracellular trafficking due to changes in endocytic uptake. Note that polyplexes, such as PEI/
DNA complexes, are not limited by cellular association but by intracellular trafficking. The
SV40 and sSV40 peptides added in large quantities do not enhance PEI-mediated transfection
(data not shown), possibly because cell association is not the limiting step for PEI vectors.
NIH-PA Author Manuscript

Despite the inability to fully condense DNA, these cationic peptides contributed to the
physiochemical properties, cellular association, and transfection efficiency of the lipoplexes.
Complete condensation of DNA by peptides had been proposed as necessary for effective
transfection,27 and cationic peptides condense DNA efficiently with a minimum of eight or
more positively charged amino acids.30 However, the peptide sequences used here contained
five positively charged residues and were incorporated into the lipoplex. The colocalization of
the peptides with the DNA may allow for the peptides to protect the DNA from intracellular
degradation, despite the fact that the DNA was not fully condensed. In addition, the short
peptides may be beneficial to facilitate vector unpacking; “looser” particles may result in higher
transfection efficiencies than “tighter” particles.35,38,39 Shorter polycations can have a higher
probability of dissociating from DNA than longer polycations and lipids, as shown by a
thermodynamic model as well as experimentally, thus permitting higher expression rates in
vitro. Thus, complete precondensation by peptides prior to the addition of lipid is not necessary
for enhanced transfection, as shown in this report, and may in fact be detrimental to the efficient
release of DNA within the cell.

Both the scrambled and intact NLS sequences enhanced lipofection to a similar extent and
resulted in peptide-lipoplexes with comparable physiochemical properties. NLSs, such as the
NIH-PA Author Manuscript

minimal SV40 T-ag NLS, have previously been used to transfect cells in vitro,40 with several
studies indicating a specific enhancement due to peptide sequence, while others report that
peptide sequence has minimal effect. The results reported herein indicate that physiochemical
properties of the complexes at high peptide/DNA ratios, rather than sequence specificity, is
the main contributing factor of enhanced transfection with peptide-lipoplexes. Our results do
not preclude an effect due to the sequence. The peptide influences the physiochemical
properties of the peptide-modified lipoplexes to increase cellular internalization, which is
reportedly the rate-limiting step for lipoplexes. By overcoming cellular internalization, another
step in the transfection process (e.g., endosomal escape, vector unpacking) is now rate-limiting.
If the rate-limiting step is not nuclear localization, then an effect due to the peptide's nuclear
localization ability would not be observed.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 10

CONCLUSION
This report describes enhancement of lipofection by the incorporation of peptides and indicates
NIH-PA Author Manuscript

that the mechanism of lipofection enhancement is an increased cellular association of the

lipoplexes. Cell association is a rate-limiting step for lipofection, and this increased cell
association correlated with physical properties favorable for cell uptake (e.g., size, zeta
potential) and an increased density of surface primary amines. High peptide/DNA ratios are
necessary to achieve peptide presentation on the surface of the lipoplex. Peptide/DNA ratios
exceeding 108 enhance lipofection despite the inability of the peptide to completely condense
DNA. These results suggest a robust mechanism through which lipoplexes can be modified,
and illustrates the potential of incorporating modular components into lipoplexes for enhanced
gene delivery.

ACKNOWLEDGMENTS
Support for this research was provided in part by the NIH [R01 EB003806-01 (AEB, LDS) and RO1 GM066830
(LDS)], the Institute for BioNanotechnology in Medicine (IBNAM) at Northwestern University, and a Ford Foundation
Predoctoral Fellowship (JCR). MALDI-TOF and ESI mass spectrometry were performed in the Analytical Services
Laboratory at Northwestern University. We would like to thank Zain Bengali, Angela Pannier, Tiffany Houchin-Ray,
Jaclyn Shepard, Nate Brown, Jill Millstone, Savka Stoeva, and Jae-Seung Lee for technical assistance.

REFERENCES
NIH-PA Author Manuscript

1. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen
M. Lipofection—A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci
USA 1987;84:7413–7417. [PubMed: 2823261]
2. Lasic DD, Templeton NS. Liposomes in gene therapy. Adv Drug Deliver Rev 1996;20:221–266.
3. Woodle MC, Scaria P. Cationic liposomes and nucleic acids. Curr Opin Colloid In 2001;6:78–84.
4. Templeton NS. Cationic liposome-mediated gene delivery in vivo. Biosci Rep 2002;22:283–295.
[PubMed: 12428905]
5. Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control
Release 2006;116:255–264. [PubMed: 16914222]
6. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to
gene-transfer by a cationic lipid. J Biol Chem 1995;270:18997–19007. [PubMed: 7642560]
7. Carriere M, Tranchant I, Niore PA, Byk G, Mignet N, Escriou V, Scherman D, Herscovici J.
Optimization of cationic lipid mediated gene transfer: Structure-function, physico-chemical, and
cellular studies. J Liposome Res 2002;12:95–106. [PubMed: 12604043]
8. Tranchant I, Thompson B, Nicolazzi C, Mignet N, Scherman D. Physicochemical optimisation of
plasmid delivery by cationic lipids. J Gene Med 2004;6:S24–S35. [PubMed: 14978748]
9. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL.
NIH-PA Author Manuscript

Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J
Biol Chem 1994;269:2550–2561. [PubMed: 8300583]
10. Mack KD, Walzem RL, Lehmann-Bruinsma K, Powell JS, Zeldis JB. Polylysine enhances cationic
liposome-mediated transfection of the hepatoblastoma cell line Hep G2. Biotechnol Appl Biochem
1996;23:217–220. [PubMed: 8679107]
11. Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene
Ther 1997;4:961–968. [PubMed: 9349433]
12. Cheung CY, Murthy N, Stayton PS, Hoffman AS. A pH-sensitive polymer that enhances cationic
lipid-mediated gene transfer. Bioconjug Chem 2001;12:906–910. [PubMed: 11716680]
13. Wang L, MacDonald RC. New strategy for transfection: Mixtures of medium-chain and long-chain
cationic lipids synergistically enhance transfection. Gene Ther 2004;11:1358–1362. [PubMed:
15215886]
14. Koynova R, Wang L, Tarahovsky Y, MacDonald RC. Lipid phase control of DNA delivery. Bioconjug
Chem 2005;16:1335–1339. [PubMed: 16287225]

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 11

15. Narang AS, Thoma L, Miller DD, Mahato RL. Cationic lipids with increased DNA binding affinity
for nonviral gene transfer in dividing and nondividing cells. Bioconjug Chem 2005;16:156–168.
[PubMed: 15656587]
NIH-PA Author Manuscript

16. De Laporte L, Cruz Rea J, Shea LD. Design of modular non-viral gene therapy vectors. Biomaterials
2006;27:947–954. [PubMed: 16243391]
17. Ma H, Zhu J, Maronski M, Kotzbauer PT, Lee VMY, Dichter MA, Diamond SL. Non-classical nuclear
localization signal peptides for high efficiency lipofection of primary neurons and neuronal cell lines.
Neuroscience 2002;112:1–5. [PubMed: 12044466]
18. Simoes S, Slepushkin V, Gaspar R, de Lima MCP, Duzgunes N. Gene delivery by negatively charged
ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides. Gene Ther
1998;5:955–964. [PubMed: 9813667]
19. Ou Z, Geiger T, Ou JS, Ackerman AW, Oldham KT, Pritchard KA. AP-4F, antennapedia peptide
linked to an amphipathic alpha helical peptide, increases the efficiency of lipofectamine-mediated
gene transfection in endothelial cells. Biochem Biophys Res Commun 2003;305:605–610. [PubMed:
12763037]
20. White RE, Wade-Martins R, Hart SL, Frampton J, Huey B, Desai-Mehta A, Cerosaletti KM,
Concannon P, James MR. Functional delivery of large genomic DNA to human cells with a peptide-
lipid vector. J Gene Med 2003;5:883–892. [PubMed: 14533197]
21. Renigunta A, Krasteva G, Konig P, Rose F, Klepetko W, Grimminger F, Seeger W, Hanze J. DNA
transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis.
Bioconjug Chem 2006;17:327–334. [PubMed: 16536462]
NIH-PA Author Manuscript

22. Subramanian A, Ranganathan P, Diamond SL. Nuclear targeting peptide scaffolds for lipofection of
nondividing mammalian cells. Nat Biotechnol 1999;17:873–877. [PubMed: 10471928]
23. Aronsohn AI, Hughes JA. Nuclear localization signal peptides enhance cationic liposome-mediated
gene therapy. J Drug Target 1997;5:163–169. [PubMed: 9606006]
24. Ritter W, Plank C, Lausier J, Rudolph C, Zink D, Reinhardt D, Rosenecker J. A novel transfecting
peptide comprising a tetrameric nuclear localization sequence. J Mol Med 2003;81:708–717.
[PubMed: 14574456]
25. Schwartz B, Ivanov MA, Pitard B, Escriou V, Rangara R, Byk G, Wils P, Crouzet J, Scherman D.
Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated
gene transfer in vitro and in vivo. Gene Ther 1999;6:282–292. [PubMed: 10435113]
26. Murray KD, Etheridge CJ, Shah SI, Matthews DA, Russell W, Gurling HMD, Miller AD. Enhanced
cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the
adenovirus core. Gene Ther 2001;8:453–460. [PubMed: 11313824]
27. Bremner KH, Seymour LW, Logan A, Read ML. Factors influencing the ability of nuclear localization
sequence peptides to enhance nonviral gene delivery. Bioconjug Chem 2004;15:152–161. [PubMed:
14733595]
28. Tagawa T, Manvell M, Brown N, Keller M, Perouzel E, Murray KD, Harbottle RP, Tecle M, Booy
F, Brahimi-Horn MC, Coutelle C, Lemoine NR, Alton EWFW, Miller AD. Characterisation of LMD
virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu (mu)
NIH-PA Author Manuscript

and plasmid DNA. Gene Ther 2002;9:564–576. [PubMed: 11973632]

29. Gao X, Huang L. Potentiation of cationic liposome-mediated gene delivery by polycations.
Biochemistry 1996;35:1027–1036. [PubMed: 8547238]
30. Plank C, Tang MX, Wolfe AR, Szoka FC. Branched cationic peptides for gene delivery: Role of type
and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum Gene
Ther 1999;10:319–332. [PubMed: 10022556]
31. Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, Kishida H, Kudoh M, Mishina Y, Kataoka
K, Yamada M, Harashima H. Octaarginine-modified multifunctional envelope-type nanoparticles
for gene delivery. Gene Ther 2007;14:682–689. [PubMed: 17268535]
32. Stewart JCM. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal
Biochem 1980;104:10–14. [PubMed: 6892980]
33. Read ML, Etrych T, Ulbrich K, Seymour LW. Characterisation of the binding interaction between
poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery
vectors. FEBS Lett 1999;461:96–100. [PubMed: 10561503]

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 12

34. Bengali Z, Pannier AK, Segura T, Anderson BC, Jang JH, Mustoe TA, Shea LD. Gene delivery
through cell culture substrate adsorbed DNA complexes. Biotechnol Bioeng 2005;90:290–302.
[PubMed: 15800863]
NIH-PA Author Manuscript

35. Vaysse L, Arveiler B. Transfection using synthetic peptides: Comparison of three DNA-compacting
peptides and effect of centrifugation. Biochim Biophys Acta 2000;1474:244–250. [PubMed:
10742605]
36. Varga CM, Tedford NC, Thomas M, Klibanov AM, Griffith LG, Lauffenburger DA. Quantitative
comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene
delivery processes. Gene Ther 2005;12:1023–1032. [PubMed: 15815703]
37. Khalil IA, Kogure K, Futaki S, Harashima H. High density of octaarginine stimulates
macropinocytosis leading to efficient intracellular trafficking for gene expression. J Biol Chem
2006;281:3544–3551. [PubMed: 16326716]
38. Schaffer DV, Fidelman NA, Dan N, Lauffenburger DA. Vector unpacking as a potential barrier for
receptor-mediated polyplex gene delivery. Biotechnol Bioeng 2000;67:598–606. [PubMed:
10649234]
39. Akita H, Tanimoto M, Masuda T, Kogure K, Hama S, Ninomiya K, Futaki S, Harashima H. Evaluation
of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with micro (mu)
and NLS-micro by cytoplasmic and nuclear microinjection: A comparative study with poly-L-lysine.
J Gene Med 2006;8:198–206. [PubMed: 16285003]
40. Dean DA, Strong DD, Zimmer WE. Nuclear entry of nonviral vectors. Gene Ther 2005;12:881–890.
[PubMed: 15908994]
NIH-PA Author Manuscript
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 13
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 1.
Electrophoretic mobility of DNA with SV40 peptide (A) and sSV40 peptide (B). Lane
assignments correspond to peptide/DNA weight ratios. The last lane (labeled trp) corresponds
to complexes formed at a peptide/DNA ratio of 120 that were incubated with trypsin.
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 14
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 2.
Average diameter (A) and zeta potential (B) of peptide-lipoplexes formed with SV40 and
sSV40 peptides. All values are reported as mean±standard deviation.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 15
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 3.
Fluorescence microscopy images of peptide-lipoplexes. Rhodamine-labeled DNA was mixed
with Oregon Green-labeled sSV40 peptide and subsequently mixed with Lipofectamine.
Images are as follows: fluorescence microscopy image of Lipofectamine/DNA complexes
without peptide (A); overlaid fluorescence microscopy images of lipoplexes with sSV40
peptide, peptide/DNA ratio = 80 (B); overlaid fluorescence microscopy images of lipoplexes
with sSV40 peptide, peptide/DNA ratio = 108 (C). Scale bar = 20 μm.

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 16
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 4.
Percentage of free lipid and peptide remaining after complex formation. The percentage of free
lipid (A) and peptide (B) remaining after complex formation was calculated as a function of
peptide/DNA (w/w) ratio. All values are reported as mean±standard deviation (* p < 0.05, ***
p < 0.001).

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 17
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 5.
Transfection at varying peptide/DNA ratios and DNA amounts. Percentage of cells transfected
(A) and luciferase expression (B) of NIH/3T3 cells transfected with peptide-lipoplexes, 0.5
μg DNA per well, 10:1 lipid/DNA ratio (w/w), with varying amounts of SV40 and sSV40
peptide added. Percentage of NIH/3T3 cells transfected as a function of DNA added per well
(C); the SV40 peptide/DNA ratio was 108 (w/w) and the lipid/DNA ratio was 10 (w/w). All
values are reported as mean±standard deviation (*p < 0.05, **p < 0.01, ***p < 0.001).

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 18
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 6.
Percent transfection of NIH/3T3 cells by alternating the addition of lipid and SV40 peptide to
DNA. Lipofectamine was added to DNA prior to peptide (solid bars) or after peptide (dashed
bars). All values are reported as mean±standard deviation (*** p < 0.001).
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 19
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 7.
Cellular association of DNA. Radiolabeled DNA (0.5 μg per well) was complexed with varying
amounts of SV40 and sSV40 peptides and Lipofectamine (10:1, w/w, lipid/DNA) for
subsequent addition to NIH/3T3 cells. Amount of DNA associated was determined using a
scintillation counter. All values are reported as mean±standard deviation (*** p < 0.05, **p <
0.01, ***p < 0.001).
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 20
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 8.
Transfection by surface-bound lipoplexes. Luciferase expression of NIH/3T3 cells transfected
with peptide-lipoplexes immobilized to tissue-culture polystyrene, 1.25 μg DNA per well, 10:1
lipid/DNA ratio (w/w), with sSV40 peptide added. All values are reported as mean±standard
deviation (*p < 0.05).
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.

 REA et al. Page 21
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Figure 9.
Accessible amines on peptide-lipoplexes. Amines on the lipoplex surface were quantified and
normalized to the amount of DNA. All values are reported as mean±standard deviation, with
different letters denoting significant difference between values (p < 0.05).
NIH-PA Author Manuscript

J Pharm Sci. Author manuscript; available in PMC 2009 November 1.