RESEARCH ARTICLE

Evaluation of multimeric siRNA conjugates for efficientprotamine-based delivery into breast cancer cells

Hyundong Yoo • Hyejung Mok

Received: 10 December 2013 / Accepted: 16 February 2014

� The Pharmaceutical Society of Korea 2014

Abstract Despite the preferable properties of well-defined

cationic peptides for small interfering RNA (siRNA) deliv-

ery, their application as siRNA carriers remains limited due

to their poor binding affinity with short-chain RNAs. In this

study, we investigated the feasibility of a novel strategy for

circumventing this limitation, by assessing the utility of

multimeric conjugates of siRNA for improving the binding

affinity of siRNAs with cationic peptides and the extent of

intracellular delivery. Protamine, a natural and arginine-rich

peptide, was used to produce stably condensed polyelectro-

lyte complexes (PECs) with multimeric siRNAs (multi-

siRNA) with a size of 120 nm while conventional siRNA/

protamine particles are over 500 nm. The formulated multi-

siRNA/protamine PECs showed greatly enhanced stability,

intracellular uptake, and biocompatibility compared to

conventional, monomeric (mono)-siRNA/protamine parti-

cles. With the addition of chloroquine, multi-siRNA/prot-

amine PECs successfully inhibited target gene expression in

MDA-MB-435 cells, a breast cancer cell line, even in the

presence of serum protein. This study demonstrates that

multi-siRNA conjugates greatly facilitate the formulation of

nano-sized protamine-based carriers and significantly

improve intracellular delivery in vitro compared to common

siRNAs, and therefore may provide a platform for the design

of peptide-based siRNA delivery systems for in vivo

applications.

Keywords Functional peptide � Protamine � Multimeric

siRNA � Charge density � Gene silencing

Introduction

Peptide-based carriers have received attention as promising

candidate materials for the delivery of therapeutic genes,

drugs, and proteins due to their favorable characteristics,

e.g., well-defined structure, ease of synthesis, reproduc-

ibility, and biocompatibility (Huang et al. 2013; Yewale

et al. 2013). Thus, diverse functional peptides, such as

fusogenic peptides and cell-penetrating peptides, have been

intensively studied as potential carriers for therapeutic

genes such as small interfering RNAs (siRNAs) (Kumar

et al. 2008; Meade and Dowdy 2007; Scholz and Wagner

2012). Peptide carriers allow enhanced delivery of nucleic

acids by forming nano-sized particles via noncovalent

interactions (mainly electrostatic interactions) as well as

attaching to nucleotides via direct chemical conjugation

(Kim and Kim 2009; Meade and Dowdy 2007). Negatively

charged nucleic acids can form nano-sized polyelectrolyte

complexes (PECs) with cationic peptides, which is popular

formulation method due to its simple processes and the

favorable translocation of PECs into the cytoplasm. The

biological performances of formulated PECs, e.g. intra-

cellular uptake, transfection efficiency, and cytotoxicity,

depend on their physicochemical properties including size,

compactness, and stability, which are closely associated

with the intensity of the electrostatic interactions between

the electrolytes (Fischer et al. 2004, 2003). The strong

electrostatic interactions of molecules with high charge

density and molecular weight usually generate densely

packed PECs, allowing successful intracellular delivery.

For example, plasmid DNA can form PECs with insuffi-

ciently charged cations such as low molecular weight linear

polyethyleneimine, while antisense ODN and siRNAs

cannot form nanoparticles under the same conditions.

Accordingly, one of the major challenges in the design of

H. Yoo � H. Mok (&)

Department of Bioscience and Biotechnology, Konkuk

University, Seoul 143-701, Republic of Korea

e-mail: hjmok@konkuk.ac.kr

123

Arch. Pharm. Res.

DOI 10.1007/s12272-014-0359-8

peptide-based carriers is caused by their lower molecular

weight and poorer spatial charge density compared to those

of high molecular synthetic/natural polymers and lipids,

which hinders firmly compacted complexation with nucleic

acids. Loosely fabricated PECs can be easily dissociated by

competitive molecules, limiting their intracellular delivery

and potential in vivo applications. Thus, the development

of novel strategies for the design of peptide-based stable

PECs that do not elicit cytotoxicity is highly desirable.

Recently, functional peptides have been linked via chem-

ical bonds to generate oligomeric peptides with reducible

linkages. This approach appears efficient, as the peptides

were found to provide efficient delivery of genes into cells

and facilitate appropriate biological activity in vitro and

in vivo without noticeable cytotoxicity, compared to

common peptides (Kiselev et al. 2013; Mok and Park 2008;

Won et al. 2011). It is conceivable that oligomeric peptides

might interact and be complexed with genes via strong

ionic interactions in the extracellular space, while allowing

easy release of incorporated genes for biological process-

ing in the reductive cytoplasm, due to the dissociation of

reducible linkages.

As an alternative approach, the structure of nucleic acids

can be modified to facilitate optimal ionic interactions with

peptide carriers. In our previous study, siRNAs were

multimerized via chemical conjugation, which has the

potential to significantly improve their binding affinity to

cationic polymers and the extent of intracellular delivery

(Lee et al. 2012; Mok et al. 2010). Here, we present a

comparative evaluation of multi-siRNA conjugates and

common monomeric siRNAs for use as peptide-based

carrier systems, in terms of particle formulation, intracel-

lular delivery, and gene suppression of siRNAs. In this

study, a natural and arginine-rich peptide, a protamine, was

selected for the condensation of siRNAs. Protamine-based

siRNA complexes were characterized by gel electropho-

resis and dynamic light scattering (DLS). The extent of

intracellular delivery of siRNAs/protamine complexes was

visualized using confocal microscopy. In addition, bio-

logical activities, including target gene suppression and

cell viability of peptide-based particles, were assessed

quantitatively using anti-green fluorescence protein (GFP)

siRNAs for stably GFP-expressing MDA-MB-435 cells.

Materials and methods

Materials

Conventional siRNAs and modified siRNAs with a thiol-

group at the distal 30 end were purchased from Bioneer Co.

(Daejeon, Republic of Korea). The siRNA sequences were as

follows: GFP sense strand siRNA, 50-GCAAGCUGACC

CUGAAGUUdTdT-30; antisense strand siRNA, 50-AACUUCAGGGUCAGCUUGCdTdT-30 (Mok et al. 2010).

Protamine, diethylpyrocarbonate (DEPC), heparin (MW:

12 kDa), and chloroquine were purchased from Sigma (St.

Louis, MO). KALA peptide (WEAKLAKALAKALAKHL

AKALAKALKACEA) was purchased from Peptron Inc.

(Daejeon, South Korea). POPOTM-3 iodide was purchased

from Invitrogen (Carlsbad, CA). Dulbecco’s Modified Eagle

Medium (DMEM) medium, penicillin/streptomycin (P/S),

and fetal bovine serum (FBS) were obtained from Gibco

BRL (Grand Island, NY). Cell Counting Kit-8 (CCK-8) was

purchased from Dojindo Laboratories (Kumamoto, Japan).

All other chemicals and reagents were of analytical grade.

Preparation of multi-siRNA

Multi-siRNAs were prepared as described in our previous

study, with slight modifications (Mok et al. 2010). Briefly,

free thiol groups were generated at the 30 end of sense and

antisense single strand siRNAs (25 nmol) by overnight

incubation with a reducing agent, 2 M dithiothreitol (DTT)

solution, at pH 8.0. Following the deprotection process, the

reactant was purified three times with a desalting column

(MWCO 7 k) to remove excess DTT and then dried using a

speed-vac. The resulting sense and antisense single-strand

siRNAs with free thiol groups were dissolved in 25 lL of

PBS solution and reacted with dithiobismaleimidoethane

(DTME, 50 nmol) overnight at room temperature with stir-

ring (850 rpm). The resulting dimeric sense and antisense

single strands were hybridized together via hydrogen bond-

ing, resulting in multi-siRNAs. The multi-siRNAs and

common mono-siRNAs (1 lg) were loaded onto 1 % aga-

rose gels and 15 % polyacrylamide gels for 45 min of gel

electrophoresis at 180 V. The siRNAs in the gels were

stained with ethidium bromide (EtBr) and visualized using a

UV-trans-illuminator. The resulting gel images were used

for quantitative analysis of multi-siRNAs using Image J

software (National Institute of Health, USA; http://rsb.info.

nih.gov/ij/) according to previous study (Mok et al. 2010).

Preparation of siRNA/protamine complexes

To prepare PECs, 1 lg of mono- and multi-siRNAs in

DEPC-treated deionized water (DEPC-DW) was mixed

with predetermined amounts of protamine and KALA

peptides by pipetting at weight ratios of 0, 0.1, 0.5, 1, and 2

at room temperature and incubated for 25 min. The PECs

prepared were loaded onto 15 % acrylamide gels and gel

electrophoresis was performed for 45 min. Migration of

each RNA was visualized using a UV trans-illuminator

after EtBr staining.

H. Yoo, H. Mok

123

For the competition assay, varying amounts of heparin

(MW: 17,000–19,000) were added to each PEC (prot-

amine/siRNA weight ratio = 2) at heparin/RNA weight

ratios of 0, 1, 2, 5, and 10 for 30 min. The free siRNAs

released from PECs into the solutions were examined by

polyacrylamide gel electrophoresis.

To prepare PECs, each RNA (10 lg) in DEPC-DW was

complexed with protamine (20 lg) for 25 min at room

temperature. The hydrodynamic sizes of the PECs con-

taining mono- and multi-siRNAs were determined by DLS

(ZEN 3690, Malvern Instruments Ltd., Malvern, UK).

Intracellular uptake of siRNA/protamine complexes

Donated human breast cancer MDA-MB-435 cells stably

expressing GFP (MDA-MB-GFP) were maintained in

DMEM supplemented with 10 % FBS, 100 units/mL

penicillin, and 100 lg/mL streptomycin at 37 �C in a

humidified atmosphere of 5 % CO2. Cells were plated on

4-well chamber slides at a density of 2 9 105 cells/well for

24 h prior to transfection. To stain RNA, 3 lL of POPOTM-

3 iodide dyes (1 mM) was incubated with the RNA (10 lg)

for 1 h at room temperature. After intercalation, POPO-3-

labeled siRNAs were purified by ethanol precipitation.

After dissolving POPO-3-labeled siRNA in DEPC-DW, the

concentration of siRNA in solution was measured using a

Nanodrop spectrophotometer (Thermo Scientific, Wil-

mington, DE). Labeled mono- and multi- siRNAs (1 lg)

were mixed with protamine (12 lg) for 25 min and then

added to each well of a chamber slide. After 4 h of incu-

bation, cells were washed three times with fresh PBS

solution at room temperature and fixed with 3.7 % form-

aldehyde solution in PBS at 48C. Cells were visualized

using confocal microscopy (FV-1000 spectral, Olympus,

Japan).

Cell viability assay

MDA-MB-GFP cells were plated on 96-well plates at a

density of 5 9 103 cells/well for 24 h prior to transfection.

Branched PEI (MW 25 k), linear PEI (MW 25 k), and

protamine were administered at predetermined polymer

concentrations in 10 % serum-containing medium for 5 h.

After replacing the medium with fresh 10 % serum media,

cells were incubated for a further 24 h before cell viability

was assessed by CCK-8 assay, according to the manufac-

turers’ instructions.

To assess the cytotoxicity of the siRNA/protamine

complexes, two types of siRNAs were mixed with prot-

amine at weight ratios of 0, 3, 6, and 12 for 25 min. The

formulated siRNA/protamine PECs were added to the cells

at an siRNA concentration of 290 nM in 6 % serum-con-

taining medium and the mixtures were incubated for 5 h.

Next, the medium was replaced and cells were incubated

for a further 24 h. Cell viability was assessed using the

CCK-8 assay according to manufacturer’s protocol.

Gene inhibition assay

Cells were seeded on 24-well plates at a density of 5 9 104

cells/well for 24 h prior to transfection. The siRNA/prot-

amine complexes were administered to cells at an siRNA

concentration of 290 nM in 6 % serum-containing medium

with and without chloroquine (50 lM) and incubated for

5 h. The medium was replaced with fresh 10 % serum-

containing medium and cells were incubated for a further

2 days. To obtain cell lysates, cells were treated with PBS

solution with 1 % Triton X-100 and centrifuged to remove

cell debris. The amount of GFP expression was measured

using a spectrofluorophotometer (Molecular Devices,

Sunnyvale, CA) at excitation and emission wavelengths of

480 and 520 nm, respectively. To observe GFP gene

expression level, cells were seeded on 4-well chamber slide

at a density of 5 9 104 cells/well. Next day, The multi-

siRNA/protamine PECs were transfected to cells (siRNA

concentration of 290 nM) in 6 % serum-containing med-

ium with and without chloroquine (50 lM) and incubated

for 5 h. The medium was replaced with fresh 10 % serum-

containing medium and cells were incubated for a further

2 days. Cells were washed with PBS two times and treated

with 3.7 % formaldehyde solution in PBS for fixation. The

cells were visualized using confocal microscopy.

Results

Formulation of protamine/multi-siRNA PECs

siRNAs that had been thiol-functionalized at both 30-ends

were chemically conjugated alone to form multimeric cross-

linked siRNAs conjugates via disulfide bonds, using similar

methods published in a previous study (Mok et al. 2010). The

prepared multi-siRNAs were examined by gel electropho-

resis using both agarose and acrylamide gels, as shown in

Fig. 1a. As expected, the multi-siRNAs showed obviously

retarded mobility in both agarose and acrylamide gels

because their molecular weight was higher than that of

mono-siRNAs. However, the decreased mobility could be

fully recovered in the presence of the reducing agent, DTT,

because of cleavage of internal disulfide linkages in multi-

meric siRNAs, as shown in our previous study (Mok et al.

2010). The difference in gel mobility for common siRNAs

and multimeric siRNAs was much greater in polyacrylamide

Evaluation of multimeric siRNA conjugates for breast cancer cells

123

gels than in agarose gels. It is well known that the pore size is

larger in 1 % agarose gels ([360 nm) than acrylamide gels

(*70–130 nm), which may result in a difference in resolu-

tion between the two gel types (Heuer et al. 2003; Pernodet

et al. 1997). Using gel images, the conjugation yield of

multimeric siRNAs was quantitatively analyzed using the

Image J program. Approximately 80 % of thiol-modified

siRNAs were successfully connected via disulfide bonds.

As potential peptide carriers, two types of peptides

were assessed in terms of complexation properties with

RNAs via ionic interactions. Protamine, a natural cationic

peptide with a molecular weight of *4 kDa, is a well-

known DNA-condensing peptide with membrane-translo-

cating ability (Brewer et al. 1999; Reynolds et al. 2005).

In addition, protamine is also clinically available peptide

as a heparin-neutralizing agent (Makris et al. 2000).

KALA peptide (MW: 3130) is a cationic fusogenic pep-

tide that allows pH-dependent membrane destabilization

for efficient intracellular gene delivery (Mok and Park

2008; Wyman et al. 1997). Using two kinds of functional

peptides, the affinity of peptides to siRNAs was com-

paratively evaluated via a gel retardation assay after

incubation of KALA and protamine with two types of

siRNAs, as shown in Fig. 1b, c. Interestingly, despite the

similarity in molecular weight, protamine’s binding

capacity with both mono- and multi-siRNAs was superior

to that of KALA. More than half of the common siRNAs

remained free at a KALA/siRNA weight ratio of 2, which

indicates that PECs were not produced under those con-

ditions. However, siRNAs successfully interacted and

condensed with protamine peptides at a protamine/siRNA

weight ratio of 2, and no free siRNA was observed in the

gel. It should be noted that more than 60 % of protamine

is composed of arginine, while 23.3 % of KALA peptides

are cationic lysines (of the total 30 amino acids, 7 are

lysines). This suggests that the relatively small portion of

cationic amino acids in KALA peptides could provide

different affinity with siRNAs to protamine. Thus, in this

study, protamine was selected as a carrier peptide due to

its high density of cationic amino acids per single peptide

and favorable condensing capability with siRNAs. In

addition, two types of siRNAs, mono- and multi-siRNA,

were comparatively evaluated for complexation with

carrier peptides. Figure 1c shows that multi-siRNAs were

completely complexed with protamine and no free multi-

siRNAs remained at a protamine/siRNA weight ratio of 1,

while mono- siRNAs remained free under the same con-

ditions. This result indicates that multi-siRNA structures

allow much more improved condensation with cationic

peptides than conventional siRNA structures, probably

because of their high spatial charge density and flexible

internal spacer (Lee et al. 2012).

Characterization of protamine/multi-siRNA PECs

To assess whether multi-siRNAs can form PECs with

protamines firmly, particle size and stability were exam-

ined, as shown in Fig. 2. Particle sizes of protamine/siRNA

PECs were measured using DLS (Fig. 2a). The sizes of the

PECs that common mono- and multi-siRNA formed with

protamines were 511.2 ± 313.2 and 119.3 ± 66.8 nm,

respectively. The multi-siRNA PECs were much smaller in

diameter (*4.3-fold) than those formed with mono-siR-

NAs, probably due to strong ionic interaction and com-

paction with protamine. Particle stability was investigated

by performing gel electrophoresis after incubation of the

competitive anionic polymer, heparin, with protamine

PECs (Fig. 2b). After completion of interactions between

siRNAs and protamine at a weight ratio of 2, different

amounts of heparin were administered to determine the

minimum amounts of heparin required for dissociation of

siRNA/protamine complexes. The release of free siRNAs

from siRNA/protamine complexes was observed with an

increasing ratio of heparin to siRNA. Figure 2b shows that

a fivefold greater concentration of heparin was needed for

decomplexation of multi-siRNA/protamine particles than

for that of mono-siRNA/protamine particles.

a b c

Fig. 1 a Gel electrophoresis of common and multimeric siRNAs

using (left panel) 1 % agarose gel and (right panel) 15 % acrylamide

gel. b–c Gel retardation assays after incubation of two kinds of

siRNAs (common siRNA and multimeric siRNA) with peptide

carriers at various peptide/siRNA weight ratios using b KALA and

c protamine

H. Yoo, H. Mok

123

Intracellular uptake of protamine/multi-siRNA PECs

To compare the extent of intracellular delivery of fabri-

cated PECs, two types of siRNAs with protamine were

administered to MDA-MB-GFP cells after fluorescence

labeling of siRNAs with POPO-3 and were visualized by

confocal microscopy (Fig. 3). Because free POPO-3 was

completely removed during the ethanol precipitation pro-

cess, background fluorescence signal was negligible when

cells were treated with dye-labeled siRNAs alone. How-

ever, strong red fluorescence was observed in cells treated

with multi-siRNA/protamine PECs due to the excellent

intracellular uptake, while cells incubated with mono-siR-

NA PECs had much weaker fluorescence intensity, as

shown in Fig. 3. This result indicates that mono-/protamine

complexes were taken up less efficiently than multi-/prot-

amine complexes, which may be attributed to their large

size and relative instability.

Biocompatibility of protamine/multi-siRNA PECs

To determine whether protamines are biocompatible in cells,

MDA-MB-GFP cells were treated with various concentra-

tions of protamines and cell viability was evaluated using a

CCK-8 assay (Fig. 4a). In this experiment, two well-known

cationic polymers for gene delivery, branched PEI (bPEI)

and liner PEI (LPEI), were also administered to cells for

comparative evaluation of their biocompatibility. The rela-

tive cell viabilities after treatment with bPEI and LPEI at a

concentration of 80 lg/mL were 8.6 ± 0.2 and

14.7 ± 0.1 %, respectively, indicating severe cytotoxicity.

However, treatment with protamine produced a significantly

lower cytotoxic effect. In addition, cell viability was exam-

ined using protamine PECs with two types of siRNAs using

mono- and multi-PECs at various protamine/siRNA weight

ratios (Fig. 4b). Interestingly, only mono-siRNA based

PECs showed obvious cytotoxicity at a protamine/siRNA

weight ratio of 12. The relative cell viabilities of cells treated

with common siRNA PECs were 78.2 ± 7.3 %. Consider-

ing that protamine/siRNA complexes prepared using the

conventional formulation process, with a size of over

500 nm, resulted in*80 % of the cell viability observed in a

a bFig. 2 a Diameter of

protamine/siRNA PECs at a

peptide/siRNA weight ratio of 2

in DW. b Competition assay for

protamine/siRNA PECs in the

presence of varying amounts of

heparin

Fig. 3 Confocal microscopic images of intracellular siRNAs after

complexation with protamine in MDA-MB-435 cells. siRNAs were

stained with the red fluorescence dye POPO-3

Evaluation of multimeric siRNA conjugates for breast cancer cells

123

previous study, this result appears consistent (Kundu et al.

2012). The observed pattern may be attributed to the fact that

free protamines that could not participate in complexation

with siRNAs elicit cell toxicity because of the poor binding

affinity between common siRNAs and protamines.

Gene silencing in GFP expressing MDA-MB-435 cells

Using multi-siRNA/protamine PECs with more improved

biocompatibility, the extent of gene suppression after

treatment was quantitatively evaluated in Fig. 5. Unex-

pectedly, multi-siRNA/protamine PECs showed negligible

GFP gene inhibition under serum conditions, despite their

excellent intracellular delivery. In previous studies, endo-

somal escape and intracellular uptake have been considered

crucial determinants for successful biological activity of

siRNAs after transfection (Nguyen and Szoka 2012; Tseng

et al. 2009). Thus, chloroquine (CQ), a chemical agent that

enables endosomal escape, was added to the media with

siRNA/protamine complexes, and gene suppression was

examined. The extent of GFP gene expression after treat-

ment with multi-siRNA/protamine complexes at a prot-

amine/siRNA weight ratio of 12 was 97.8 ± 9.3 and

67.1 ± 6.3 % in the absence and presence of 50 lM of

CQ, respectively, for 5 h. This result shows that addition of

the endosomal escape moiety resulted in successful deliv-

ery of protamine/multi-siRNA complexes and induction of

notable gene inhibition without cytotoxicity to the MDA-

MB-435 cells. However, incubation of PECs with CQ for

12 h was too toxic to evaluate gene suppression. The GFP

gene suppression by multi-siRNA/protamine complexes

was also observed by confocal microscopy. Figure 5b

shows that multi-siRNA/protamine complexes successfully

inhibited target GFP gene expression in the presence of

CQ.

50

60

70

80

90

100

110

120

Weight ratios (protamine/siRNA)

Cel

l via

bili

ty (

%)

0 20 40 60 80 100 120 140 1600

20

40

60

80

100

Carrier conc. (µg/mL)

Cel

l via

bili

ty (

%)

a

b

Multi

Mono

bPEILPEIprotamine

0 3 6 12

n.s.

Fig. 4 Cell viability after incubation with a varying amounts of

cationic polymers and b protamine/siRNA complexes for MDA-MB-

435 cells (*p \ 0.05)

a

b

Fig. 5 a The extent of GFP gene suppression by multi-siRNA/

protamine PECs with and without CQ (50 lM) in 10 % serum-

containing medium for GFP-expressing MDA-MB-435 cells. b Con-

focal microscopy images of GFP-expressing MDA-MB-435 cells

after treatment of multi-siRNA/protamine PECs with and without CQ

(50 lM)

H. Yoo, H. Mok

123

Discussion

Currently, protamine has received attention as a promising

material for formulation of nucleic acid-based drugs

in vitro and in vivo because of a relatively low price

compared to synthetic functional peptides, clinical avail-

ability, efficient translocation through cellular membranes,

and high affinity to nucleic acids (Choi et al. 2010; Kundu

et al. 2012). In this study, we evaluated multi-siRNA-based

ionic complexes with protamine as a new carrier for ther-

apeutic siRNAs. Our results exhibited that multi-siRNA

structures are superior to common siRNA structures for the

formulation and efficient intracellular uptake of protamine-

based PECs. Figure 2a shows that biocompatible prot-

amine formed small compact nanoparticles with multi-

siRNAs, with a size of 120 nm. Considering that particles

smaller than 200 nm possess great advantages in terms of

intracellular endocytosis in vitro and passively targeted

delivery in vivo via the enhanced permeability and reten-

tion (EPR) effect, peptide formulation using multimeric

siRNAs appears to be a promising strategy for efficient

siRNA delivery (Decuzzi et al. 2009). In addition, Fig. 2b

shows that multi-siRNA-based ionic complexes with

protamine were stable against exterior competitive poly-

electrolytes, while mono-siRNA/protamine complexes

were only loosely formed. Multi-siRNAs/protamine PECs

were not easily dissociated by anionic molecules probably

due to their strong ionic interactions. In addition, it was

also demonstrated that protamine-based multi-siRNA PECs

with a narrow size distribution and firm compaction pro-

vide high intracellular uptake as well as excellent target

gene inhibition using anti-GFP siRNAs. Notably, multi-

siRNA/protamine PECs showed excellent gene suppression

without cycotoxicity, which may be favorable for in vivo

applications. It should be also noticed that all transfections

were performed in the presence of serum proteins to con-

sider nonspecific interference of intracellular delivery by

serum proteins in vivo. In our previous study, multi-siR-

NAs showed superior condensation and delivery efficiency

with synthetic polymeric carriers, LPEIs, which had a low

charge. This study clearly demonstrated that clinically

available natural peptide protamine that pose less safety

concerns could replace high molecular weight cationic

polymers like LPEIs (25 k). However, for applying the

current protamine-based multi-siRNA delivery system

in vivo, further studies will be necessary to optimize

protamine carriers with endosomal escape moieties.

Conclusion

In conclusion, to our knowledge, this is the first study to

report that multi-siRNAs are favorable for incorporation in

peptide carriers and subsequent intracellular delivery

in vitro. Fabricated multi-siRNA/protamine PECs with a

size of 120 nm showed greatly improved intracellular

uptake and biocompatibility, compared to conventional

siRNA/protamine particles. In addition, the multi-siRNA/

protamine particles effectively suppressed target gene

expression in the presence of serum proteins and CQ

without cytotoxicity for the MDA-MB-435 breast cancer

cells. Thus, a serious issue in peptide-siRNA particle for-

mulation, that is, the poor binding affinity of siRNAs to

cationic peptides, could be overcome by using multi-siR-

NA conjugates, thereby providing a potential platform

technology for the design of peptide-based siRNA delivery

systems for in vivo applications.

Acknowledgments This study was supported by a grant from the

National R&D Program for Cancer Control, Ministry for Health and

Welfare, Republic of Korea (1220050).

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