druk functional polymers for targeted delivery of nucleic acid drugs 2009 macromolecular bioscience

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Functional Polymers for Targeted Delivery of

Nucleic Acid Drugs

Hyejung Mok, Tae Gwan Park*

Introduction

Nucleic acid drugs, such as plasmid desoxyribonucleic acid

(DNA), antisense oligonucleotides and small interfering

ribonucleic acids(siRNA),have emerged as newtherapeutic

agents for the treatment of incurable diseases and genetic

disorders including cancer.[1–4] Such nucleic acid drugs are

expected to correct cellular malfunctions by expressing or

silencing specific genes related to the diseases. Although

many studies using non-viral vectors have reported

promising therapeutic effects of nucleic acid drugs

in vitro and in vivo, there are still several critical problems

to be solved prior to clinical applications. One of the major

barriers is the delivery issue: safe and efficient carriers are

highly desirable for specific gene expression or silencing at

desired cell/tissue sites. Since nucleic acid drugs are

negatively charged macromolecules with an extremely

lowextentofcellularuptake,variouscationicpolymersand

lipids have been utilized to form nano-complexes withthem via electrostatic interactions. The polyelectrolyte

complexescan be readily transported withinthe cells viaan

endocytic mechanism. They generally exhibit enhanced

cellular uptake with concomitantly increasing gene

transfection efficiency. To further improve cell-specific

therapeutic efficacy with reducing side effects, cationic

polymers and lipids were often conjugated with cell-

recognizable functionalmoietiesfor delivery of nucleic acid

drugs to thedesired targetsite.In contrast to cationic lipids,

a wide variety of cationic polymers can be molecularly

engineered to have multiple functional moieties in the

structure. For example, polyethylene glycol (PEG) is

routinely incorporated into cationic polymers for complex

stabilization in the blood stream, and various targeting

ligands such as folate, peptides, and antibodies are

conjugated for cell-specific delivery.[5–8] Nanosized poly-

electrolyte complexesprotect condensed nucleic acidsfrom

enzymatic degradation, while surface exposed targeting

ligands enableselective geneexpressioninvivo and invitro.

Stimuli-sensitive cationic polymers that deliver genes into

cells in response to externally modulated conditions were

alsoutilizedfortargeteddeliveryofgenes. [9–11]Triggeredby

external stimuli such as pH, temperature and magnetic

Review

H. Mok, T. G. Park

Department of Biological Sciences and the Graduate Program of

Nanoscience and Technology, Korea Advanced Institute of

Science and Technology, Daejeon 305-701, Republic of Korea

Fax: þ82 42 350 2610; E-mail: [email protected]

Cationic polymers have been chemically modified with a variety of targeting molecules such

as peptides, proteins, antibodies, sugars and vitamins for targeted delivery of nucleic acid

drugs to specific cells. Stimuli-sensitive polymers exhibiting different size, charge and con-

formation in response to physiological signals from

specific cells have also been utilized for targeted deliv-

ery. To achieve target-specific delivery of nucleic acids,

conjugation chemistry is critical to produce stable

nanosized polyplexes tethered with cell-recognizable

ligands for facile cellular uptake via a receptor-

mediated endocytic pathway. In this review, synthetic

strategies of functional cationic polymers with various

targeting ligands are presented.

Macromol. Biosci. 2009, 9 , 731–743

2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/mabi.200900044 731



field,site-specificgenetransfectioncouldbeachievedatthe

desired target site. Among many targeted polymeric

delivery systems reported in the literature, this review

mainly focuses on a few examples of cell-specific delivery

systems for nucleic acid drugs with an emphasis on various

types of cationic polymers, available targeting ligands,

conjugation strategies, and therapeutic applications.

Several examples of targeted delivery systems triggered

by external stimuli are also briefly introduced.

Cationic Polymers for Gene Delivery

Synthetic and natural cationic polymers have been

popularly used as non-viral carriers for gene therapy due

to their far less cytotoxicities as compared to that of viral

carriers. Poly(L-lysine) (PLL) is one of the most commonly

used cationic polymers for gene delivery [Figure 1(A)]. PLL

can effectively form nano-sized polyelectrolyte complexes

with nucleic acid drugs due to primary e-amine groups of

lysine residues. However, PLL has only the primary amine

groups in the backbone, which are insufficient for facil-

itating the endosome escape of polyelectrolyte complexesinto the cytosol area. To confer an endosome escape

property to PLL, endosome breaking peptides, such as

polyhistidine and KALA peptide, were also conjugated to

enhance gene delivery efficiency.[12,13]

Poly(ethylenimine)(PEI)showsfarsuperiorgenedelivery

efficiency to PLL due to its high charge density and good

buffering capacity, which enable the formation of more

compact polyelectrolyte complexes with nucleic acids

that have enhanced endosome escape ability. In particular,

branched PEI has primary, secondary, and tertiary

amine groups, providing sufficient buffering activity at

endosomal pH for facile endosome escape of the nucleic

acids by the ‘‘proton sponge’’ effect [Figure 1(B)].[14]

However, high molecular weight PEI shows severe cyto-

toxicityproblems,makingits clinical application difficultin

spite of its excellent delivery efficiency. Linear PEI, having

only the secondary amine groups in the backbone except

for the terminal primary amine groups, is known to have

much lower cytotoxicity than branched PEI, while its

transfection efficiency is similar to that of the branched

one [Figure 1(C)].[15] In some cases, linear PEI (22k) even

showed higher transfection efficiency than branched PEI

(25k) for plasmid DNA delivery.[16] To reduce the cytotoxi-

city of PEI concomitantly maintaining the delivery

efficiency, cleavable and biodegradable PEI polymers havebeen synthesized.[17]

Poly(b-amino ester)s are biodegradable cationic poly-

mers with ester linkages in the backbone, which facilitates

the decomplexationof polyelectrolyte complexesat low pH

endosomal conditions [Figure 1(D)]. Poly(b-amino ester)s

were prepared by conjugation of primary or secondary

amine monomers to various di-acrylate monomers via a

Michael-type addition reaction. According to previous

reports, a familyof poly(b-amino ester) derivatives showed

more enhanced gene transfection efficiency with reduced

cell cytotoxicity, compared to PEI, which might be

attributed to the cleavage of poly(b-amino ester) backbone

in an acidic condition.[18,19]

Poly(amidoamine) (PAMAM) dendrimers are the most

intensively studied cationic dendrimers for gene delivery

[Figure 1(E)]. PAMAMdendrimers havebeenpopularly used

as carriers for targeted delivery of anticancer drugs, as well

as nucleic acid drugs.[20] PAMAM wasprepared by stepwise

polymerization from an initiator core, such as ammonia

and ethylenediamine.[21] As the number of generations

increased, transfection efficiency was enhanced, but cell

cytotoxicity also increased.[21,22] The cytotoxicity of

PAMAM dendrimer generation 4 [molecular weight

H. Mok, T. G. Park

Hyejung Mok is currently a postdoctoral associate

in Professor Tae Gwan Park’s laboratory, Depart-

ment of Biological Sciences at Korea Advanced

Institute of Science and Technology (KAIST). She

received Ph.D. from KAIST under thesupervisionof

Professor Tae Gwan Park in 2008. She published 17papers during her Ph.D. study. Her main research

interests lie in novel siRNA and protein delivery

systems based on polymeric biomaterials.

Tae Gwan Park received a B.S. in chemical

technology from Seoul National University in

1980, an M.S. in biological sciences from Korea

Advanced Institute of Science and Technology in

1983, and a Ph.D. in bioengineering from the

University of Washington in 1990 under the

direction of Prof. Allan S. Hoffman. Following a

postdoctoral research associate experience

(1990–1991) at the Massachusetts Institute of

Technology in Prof. Robert Langer’s laboratory,he joined Temple University, School of Pharmacy

as an assistant professor. In 1995, he returned to

Korea and became a professor at the Korea

Advanced Institute of Science and Technology.

He received the Nanotechnology Innovative

Research Award, Korea (2006), and KAIST Research

Award, Korea (2007). More recently, he received

the 2009 Clemson Award for Contributions to the

Literature from the Society for Biomaterials. His

research interests include nanobiomaterial-based

drug delivery systems, gene therapy, and tissue

engineering. He has published over 203 papers in

SCI journals, received 30 domestic and foreign

patents, and licensed out several technologies.He currently serves as an editorial board member

of Bioconjugate Chemistry, J. Controlled Release,

Pharmaceutical Research, Macromolecular Bio-

science and J. Bioactive and Compatible Polymers.

732

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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/mabi.200900044



(MW)¼14215Da]wassimilartothatofPLL(MW ¼56 kDa).

The optimal generation number for efficient transfection

was dependent on the cell types.[21] After intravenous

injection of generation 9 PAMAM dendrimer/plasmid DNA

complexes at an N/P ratio of 5, significant gene expression

was observed only in the lung tissue, suggesting that

PAMAM dendrimer could be applied as an effective

pulmonary gene carrier.[23] PAMAM dendrimer (generation

7)/luciferase siRNA complexes exhibited up to 80% of

gene silencing effect at an N/P ratio of 10 in luciferase

expressing A549 cells.[24]

One of the naturally occurring cationic polymers useful

for gene carriers is chitosan.[25] Chitosan is a linear and

biodegradable polysaccharide composed of b-(1,4)-linked

D-glucosamin and N -acetyl-D-glucosamine. Chitosan is

known to be biocompatible and non-toxic because it can

be degraded into N -acetylglucosamine by lysozyme in the

body.[26] Water soluble chitosan with a low-molecular-

weight (22 kDa) especially exhibited better gene transfec-

tion efficiency and improved cell viabi-

lity for plasmid DNA delivery than PLL

(20kDa) in vitro,[25] suggesting that

chitosan could be a useful carrier candi-

date for gene therapy.

Cationic polymers and nucleic acidsform polyelectrolyte complex nanopar-

ticles mainly by electrostatic interac-

tions. The resultant nano-complexes,

polyplexes, can be easily aggregated or

disintegrated depending on environ-

mental conditions such as pH, salt

concentration and the presence of other

charged molecules. The stability issue of

polyplexes is particularly serious in the

bloodstream due to the non-specific

adsorption of serum proteins on the

surface. PEG has been commonly con-

jugated to cationic polymers to enhance

the stability of polyplexes in vitro and

in vivo.[27–29] PEGylated polycation/DNA

complexes showed reduced toxicity

while significantly prolonging blood

circulation time after intravenous

administration, compared to un-PEGy-

lated polycation/DNA complexes.[28]

PEGylated nanoparticles also exhibited

passive tumor targeting via the

enhanced permeability and retention

(EPR) effect.[30] Until now, various block

and graft copolymers with PEG havebeen synthesized and characterized for

gene delivery applications.[13,27–29] To

prepare an A/B-type PLL/PEG block copo-

lymer, N -carboxyanhydrideof z-protected

L-lysine was polymerized from a-methoxy-v-amino PEG as

an initiator.[27] The transfection efficiency of PLL/PEG block

copolymer/DNA complexes increased 6-fold, compared to

that of PLL/DNA complexes in 293cells.[27] Comb-type PEG-

grafted PLL copolymers were also prepared by reacting

primary amine groups(aand e aminogroups)ofPLLwithan

amine reactive succinimidyl succinate PEG derivative.[13]

While there was no significant difference in the transfec-

tion efficiency of plasmid DNA between PLL complexes

and PEG-graft-PLL complexes in serum deficient medium,

PEG-graft-PLL complexes showed a 6 times higher transfec-

tion efficiency in 10% serum containing medium than

PLL complexes.[13] This was attributed to reduced adsorp-

tion of serum proteins and enhanced stability of polyelec-

trolyte complexes via PEGylation in the serum medium.

Moreover, PEGylated PEI exhibited longer blood circulation

and more reduced acute toxicity than PEI by decreasing

non-specific accumulation in the liver after intravenous

administration.[28,29]

Functional Polymers for Targeted Delivery of Nucleic Acid Drugs

Figure 1. (A) Poly(L-lysine) (PLL), (B) branched PEI, (C) linear PEI, (D) poly(b-amino ester),and (E) poly(amidoamine) (PAMAM).


2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.mbs-journal.de 733



Conjugation Strategies for Targeting Ligandsfor Receptor/Ligand Interaction-MediatedDelivery of Nucleic Acid Drugs

Cationic polymers with multiple functional groups were

covalentlyor non-covalently conjugatedwithcell recogniz-able targeting ligands, such as peptides, proteins, anti-

bodies, sugars, vitamins and chemicals. Specific interac-

tions between ligands and receptors on the cellular

membrane can mediate receptor-mediated endocytosis of

polyelectrolyte complexes via a clathrin-dependent or a

clathrin-independent mechanism, resulting in much

enhanced cellular uptake.[31] As listed in Table 1, various

targeting ligands have been directly conjugated to cationic

functional polymers for efficient delivery of therapeutic

genes to specific cells.

Four different strategies for immobilizing targeting

ligandsontothesurfaceofDNA/polymernano-complexes

are described in this paper: ligand-polymer conjugate;

ligand-linker-polymer conjugate; ligand-nucleic acid

conjugate; ligand-linker-nucleic acid conjugate (Figure 2).

Various ligand molecules are commonly conjugated to

cationic functional polymers with and without a linker

polymer (spacer). To expose targeting ligands onto the

outer surface of polyplexes while maintaining the

complex stability, the targeting ligand was terminally

tethered to the distal end of a hydrophilic linker polymer,

such as PEG, and the ligand-PEG conjugate was covalently

linked to cationic polymers. Direct conjugation of target-ing ligands to therapeutic short chain oligodeoxynucleo-

tide and siRNA nucleic acids instead of circular plasmid

DNA often exhibited target-specific cellular uptake even

without using carrier polymers, although this could

partially impair the activity of nucleic acid drugs during

the conjugation process.[32] It should be noted that

nucleic acid-ligand conjugates cannot easily escape from

endosome to cytosol after receptor mediated endocytosis,

resulting in limited therapeutic effects.[33] Therefore, to

facilitate the endosome escape, carrier polymers should

also be required to conjugate with various fusogenic

moieties that destabilize the cell membrane at acidic

pH. PEG was also chemically conjugated to oligonucleo-

tide and siRNA via a cleavable linkage so that the

PEG-nucleic acid conjugate might form a more stable

polyelectrolyte complex than the naked one in serum

containing medium.

H. Mok, T. G. Park

Table 1. A list of representative targeted polymeric delivery systems for nucleic acids.

Targeting ligand Polymer Target receptor Target cell/tissue Cell line Ref.

peptide RGD peptide PEI integrin receptor an

b3 h uman melanoma cell Mewo cell [36]PEI-PEG [37]poly(b-amino ester) [38]

LHRH peptide siRNA-PEG LHRH receptor tumor tissue A2780 [40]PAMAM [41]

protein lactoferrin PAMAM-PEG lactoferrin receptor brain brain capillaryendothelial cell(BCEC)

[42]

PEI bronchial epithelial cell BEAS-2B cell [43]transferrin PAMAM-PEG transferrin receptor brain BCEC, in vivo [42][45]EGF biotin-PEG EGF receptor tumor tissue A431 cell [8]RAP (receptor-associatedprotein)

PLL, PDL LDL receptor human hepatoma cell HepG2 cell [48]

antibody anti-JL1 monoclonal antibody PLL JL-1 human leukemia T cell Molt 4 cell [52]chimeric anti-EGF receptorantibody (cetuximab)

PAMAM EGF receptor tumor tissue [53]

anti-HER-2 antibody

(Herceptin, Trastuzumab)

PEI human EGF receptor tumor tissue Sk-Br-3 cell [5]

SKOV-3 cells [16]anti-CD90 antibody

(Trastuzumab)

PEI CD90 fibroblast WI26SV40 [16]

anti-GAD antibody PEI-PEG GAD islet cell MIN6 cell [7]sugar galactose, lactose PEI-PVP asialoglycoprotein

receptorhepatoma cell HepG2 cell [54]

siRNA-PEG HuH-7 cell [56]mannose PLL mannose receptor macrophage cell,

dendritic cellprimarymacrophagecell, in vivo

[57][59]

chitosan RAW264.7 [58]vitamin folate PLL-PEG folate receptor tumor tissue KB cell [6]

PEI-PEG [61–63]ODN-PEG [64]

734

Macromol. Biosci. 2009 , 9 , 731–743




Targeting Ligand-MediatedDelivery of Nucleic Acid Drugs

Peptides

Arg-Gly-Asp (RGD) PeptideThe three-amino-acid sequence peptide

RGD has been extensively used as a cell-

recognizable ligand for integrin receptor

an b3/a

n b5 targeting.[34–37] In particular,

integrin receptor an b3/a

n b5 has been

known tobe specifically over-expressed in

angiogenic endothelial cells of the tumor

tissue, which enables the RGD peptide to

be used as a tumor targeting ligand.

Linear RGD(RGDC) and cyclic RGD(cRGD)

with one (CGRGDSPC) or two disulfide

bonds (ACDCRGDCFCG) have beenemployed for cell-specific gene delivery.

Among these, cRGD peptide with two

disulfide bonds showed 200-fold and 20-

fold higher potency than linear RGD and

cRGD with one disulfide bond, respec-

tively.[35] A thiol group of cysteine

modified RGD peptide (linear RGDC)

was used to conjugate to carrier poly-

mers.[36] Primary amine groups of PEI

were activated with a heterofunctional

crosslinker of N -succinimidyl-3-(2-pyri-

dyldithio)propionate (SPDP), and the

SPDP-activated PEI was reacted with

the thiol group of cysteine modified

RGD peptide via a disulfide exchange

reaction to prepare a RGD/PEI conjugate

[Figure 3(A)]. The resultant RGD/PEI

conjugate with a substitution degree of

4.6% showed 50 times higher gene

expression of luciferase plasmid than

that of the unmodified PEI for Mewo cells

(human melanoma cells). However, no

significant difference in luciferase gene

expression wasobservedin A549 cells,an

integrin receptor an b3 negative cell.Cysteine-modified RGD peptide was also

conjugated directly to poly(b-amino

ester) via a disulfide bond for integrin

receptor targeting.[38] An N-terminal

amine group of cRGD peptide without a

cysteine residue was also used for the

conjugation reaction with carrier poly-

mers or linker polymers.[37] The terminal

amine group of cRGD was activated with

a heterofunctional PEG, NHS/PEG/VS


Figure 2. Schematic illustration for targeted delivery of polymer/nucleic acid complexesusing four kinds of strategy, polymer-ligand conjugate, polymer-linker-ligand conju-gate, nucleic acid-ligand conjugate, and nucleic acid-linker-ligand conjugate, via recep-tor mediated endocytosis.

Figure 3. (A) RGD conjugated PEI, (B) LHRH- PEG-siRNA conjugate.





( N -hydroxysuccinimide/PEG/vinylsulfone), in anhydrous

dimethylformamide (DMF) for 2 h to minimize the activity

loss of vinyl sulfone groups after the reaction.The resulting

cRGD/PEG/VS conjugate was grafted to primary amine

groups of PEI. The cRGD/PEI conjugate exhibited that one

RGD substitution per PEI molecule might be sufficientenough to show good cell binding affinity to the integrin

receptor an b3/a

n b5.[37]

LHRH

Luteinizing hormone-releasing hormone (LHRH) has been

known to be a good tumor targeting ligand because

LHRH receptor is commonly over-expressed in various

cancer cells including prostate, breast, and ovarian

cancer cells.[39,40] Instead of the native LHRH peptide

(Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly), LHRH analogs

(Gln-His-Trp-Ser-Tyr-DLys-Leu-Arg-Pro or Pyr-His-Trp-Ser-

Tyr-DLys-Leu-Arg-Pro-Gly), in which the glycine residue at

the position 6 was replaced by D-lysine, have been used as a

commercially available therapeutic peptide drug (Leupro-

lide acetate) to treat prostate cancer and endometriosis.

Since the N-terminal amine group and the C-terminal

carboxylic acid group of LHRH analog are blocked by

pyroglutamation and amidation, respectively, the e-amino

group of the lysine residue in the backbone was conjugated

to cationic polymers.[40] For direct conjugation of LHRH

peptide to PAMAM, the amino groups of PAMAM were

modified with carboxylic acid groups via ring-opening

acylation with glutaric anhydride, and these carboxylic

acid groups were then conjugated with the e amine groupof D-Lys6-LHRH via 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide hydrochloride (EDC) chemistry.[41] When

using a linker polymer, PEG, for the conjugation of LHRH,

PEG with a terminal carboxylic acid group was used for the

conjugation to siRNA.[40]Afterblockinganaminegroupofa

heterobifunctional PEG derivative, NH2–PEG–COOH, using

SPDP, theremnant carboxylic acid group wasconjugated to

the e amine group of D-Lys6 of the LHRH peptide using EDC.

The resulting SPDP activated PEG/LHRH was reduced to

produce a free thiol group. An amine group at the terminal

5’-sense double strand siRNA was activated with SPDP and

reacted with thiol/PEG/LHRH to prepare a siRNA/PEG/

LHRH conjugate [Figure 3(B)]. PEI (25k) was used as a core

condensing polymer for siRNA delivery. Gene silencing

effects by siRNA/PEG/LHRH/PEI and siRNA/PEG/PEI were

analyzed in twotypes of human ovarian cancercells, A2780

(LHRH receptor over-expressing cell) and SK-OV-3 (LHRH

receptor deficient cell). While similar gene silencing

extents by siRNA/PEG/LHRH/PEI and siRNA/PEG/PEI were

shown in SK-OV-3 cells, siRNA/PEG/LHRH/PEI exhibited a

20% higher gene silencing effect than siRNA/PEG/PEI in

A2780 cells, which was attributed to LHRH receptor

mediated targeting.

Proteins

Lactoferrin and Transferrin

Lactoferrin, an iron-binding glycoprotein, is a globular

protein with a molecular weight of 80kDa, which can bind

ferricionsreversibly.Lactoferrincouldbeusedasalungandbrain targeting ligand because lactoferrin receptors are

dominantly expressed in brain capillaries and bronchial

epithelial cells.[42,43] It is known that lactoferrin-mediated

transcytosis occurredin BBB(blood brainbarrier),openinga

new possibility for brain delivery of nanoparticulate gene

carriers.[42] To prepare a lactoferrin-PEI conjugate, vicinal

hydroxyl groups of N -acetylneuraminic acid in lactoferrin

were oxidized to form amine-reactive aldehyde groups,

which were then conjugated to primary amine groups of

b-PEI (MW¼25 kDa). While the lactoferrin/PEI (lactoferrin:

PEI¼1: 17.2 molar ratio)conjugates showed a 5-fold higher

transfection efficiency than PEI for bronchial epithelial

BEAS-2B cells, no significant difference in transfection

efficiency was observed for alveolar epithelial cells, A549

cells. Transferrinis alsoa glycoprotein that transportsferric

ions, which is over-expressed about ten-fold e for tumor

cells as compared to normal cells.[44] Primary amine

groups of transferrin were used to conjugate with cationic

polymers. PAMAM-PEG-targeting ligand conjugates

were prepared using a heterobifunctional PEG derivative

[a-maleimidyl-v- N -hydroxylsuccinimidylpoly(ethylene

glycol) (NHS-PEG-MAL), MW¼3400Da).[42,45] After pri-

mary amine groups of PAMAM were conjugated to NHS-

PEG-MAL, the resulting conjugate was reacted with

thiolated transferrin that was prepared by using a Traut’sreagent, to form PAMAM-PEG-transferrin conjugate. The

extentofluciferasegeneexpressioninbrainusingPAMAM-

PEG-transferrin as a carrier was about two-fold higher than

when PAMAM or PAMAM-PEG was used after intravenous

injection. To compare the targeting activity of lactoferrin

to that of transferrin, PAMAM-PEG-lactoferrin was also

prepared based on a similar reaction procedure. The amine

groups of lactoferrin were converted to thiol groups using

N -succinimidyl-S-acetylthioacetate (SATA), which were

conjugated to PAMAM-PEG. PAMAM-PEG-lactoferrin

exhibited 2 times higher gene transfection efficiency

thanPAMAM-PEG-transferrininbraincapillaryendothelial

cells (BCEC) in vitro and in the brain after intravenous

injection in vivo. Thus, itcan bededuced thatlactoferrin isa

little more efficient than transferrin for brain targeting of

nucleic acid drugs.

Ligand Proteins

The epidermal growth factor (EGF) receptor is known to be

greatly over-expressed in tumor tissues about 100-fold

higher than normal tissues.[44] EGF could be internalized

within the cells via EGF receptor-mediated endocytosis by

H. Mok, T. G. Park

736

Macromol. Biosci. 2009 , 9 , 731–743




forming dimer complexes. Thus, EGF has been popularly

usedasagoodtargetingmoietyfortumortissues.EGFhasa

small molecular weight about 6 kDa (53 amino acids) with

three internal disulfide bonds.[46,47] Amine groups of EGF

have been commonly used for the conjugation reaction

with polymers in order to avoid interaction with intramo-lecular disulfide bonds. Because EGF has two lysine

residues, three amine groups (two e-amino groups of

Lys28 and Lys 48, and the a amino group of N-terminal)

could be used for the conjugation.[47] According to our

previous studies, only the N-terminal mono-PEGylated EGF

fully maintained its activities such as cell proliferation and

receptor binding as much as the native EGF. Thus, the

polymer conjugation site of EGF is an important issue for

targeting. To minimize the loss of targeting activity by

green fluorescent protein (GFP) through non-specific

conjugation of EGF to polymers, EGF could be physically

immobilized onto polymer/nucleic acid complexes via

biotin/streptavidin interaction.[8] N -terminal mono-PEGy-

lated and biotinylated EGF (biotin-PEG-EGF) was specifi-

cally separated by size-exclusion chromatography. After

preparing PEI/plasmid DNA complexes, streptavidins were

coatedonto thecomplexesvia ionic interaction. Then, biotin-

PEG-EGF was immobilized onto streptavidin-PEI-DNA

complexes via biotin-streptavidin interaction. PEI/plasmid

complexes coated with N-terminal specifically PEGylated

EGF exhibited significantly higher transfection efficiency

than those with non-specifically PEGylated EGF (multi-

PEGylated EGF). Luciferase gene expression by biotin-PEG-

EGF/streptavidin-PEI-DNA complexes was about ten fold

higher than that byLipofectamine andPEI forA431,a humanepidermoid carcinoma cell line, highly expressing EGF

receptor.

For the delivery of nucleic acid drugs via a low-density

lipoprotein (LDL) receptor-mediated pathway, a receptor-

associated protein (RAP) to be used as a targeting ligand

was also conjugated to cationic polymers. RAP (39–

44kDa) is known to be expressed in various tissues such as

liver and brain, and it shows binding affinity to LDL. RAP-

LDL complexes are internalized into cells via receptor-

mediated endocytosis.[48,49] The expression level of LDL

receptors on the cell membrane markedly increased in

rapidly dividing cells such as malignant cells. RAP proteins

containing a C-terminal cysteine residue instead of a

heparin binding domain were cloned and expressed in the

E. coli system. Purified cysteine-modified RAP protein was

reacted with SPDP (heterofunctional crosslinker) activated

poly(D-lysine) (PDL) or PLL to prepare a PDL-RAP or PLL-RAP

conjugate. PDL-RAP conjugate showed enhanced gene

transfection efficiency compared to that of PDL and PLL-

RAP, probably because plasmid DNA complexes with PDL-

RAP were able to be readily internalized within human

hepatoma HepG2 cells via LDL receptor mediated endocy-

tosis. In addition, PDL-RAP/DNA complexes were more

resistant to degradation both in the lysosomal compart-

ment and in the cytosolic condition than PLL-RAP/DNA

complexes.

Antibodies

Antibodies (150kDa) composed of twolarge heavy chains

and two small light chains have been used as a good

targeting ligand due to their superior specificity towards

target molecules. In particular, increasing number of

therapeutic monoclonal antibodies approved by FDA has

shown their values for targeted therapy in cancer, immune

diseases, and heart diseases.[50] Besides whole antibodies,

genetically engineered low-molecular-weight fragments,

such as single chain variant fragment (scFv) (25kDa),

dibody (55 kDa), minibody (80 kDa), and scFv-Fc (105 kDa),

have been conjugated to chemical drugs for targeted

delivery.[51] The major challenge for antibody conjugation

to polymers is to synthesize a homogeneous conjugate

population while maintaining its antigen binding activity.

Thus, site-specific conjugation of antibodies with polymers

is a critical issue especially because high molecular weight

antibodies have many reactive functional groups for

conjugation. Non-specific antibody conjugation might be

apt to damage the target binding ability partially or

completely.

For conjugating polymers to antibodies without impair-

ing an antigen binding region, the glycosylated part has

been used as an optimal site for conjugation.[52,53]

Previously, a carbohydrate-directed antibody modification

method was reported for the preparation of PLL-antibodyconjugate.[52] Anti-JL1 antibody, a monoclonal antibody

against JL1 antigen (stage II-specific human T cell lympho-

cyte differentiation antigen), was used to target human

leukemia T cells. Two hydroxyl groups in the sugar ring of

anti-JL1antibodywereconvertedtoaldehydegroupsbythe

periodate oxidation reaction via the hydrazide group of a

heterofunctional crosslinker, 3-(2-pyridyldithio)propionyl-

hydrazide (PDPH) [Figure 4(A)]. To prepare a thiolated PLL,

thee-aminogroupofPLLwasmodifiedwithsuccinimidyl-3-

(2-pyridyldithio)propionate(SPDP), and dithiothreitol(DTT)

was treated to the conjugate. The resulting 2-pyridyl

disulfide groups of antibody were reacted to thiolated PLL

via a disulfide linkage to prepare an anti-JL1 antibody-PLL

conjugate (Ab-PLL). The Ab-PLL conjugate exhibited a good

targeting effect for plasmid DNA delivery. The extent of

galactosidase gene expression by DNA/Ab-PLL complexes

(about 300 nm in diameter at a polymer/DNA weight ratio

of 5) was about 10 times higher than that by DNA/PLL

complexes, and about 2 times higher than that by the

commercial reagent Lipofectin for Molt 4 cells, human

leukemia T cells. A chimeric anti-EGF receptor monoclonal

antibody Cetuximab (IMC-C225), a brain targeting moiety,

was also oxidized in a similar method to convert the sugar






hydroxyl groups of Cetuximab to aldehyde groups.[53] For

conjugating an antibody to a PAMAM dendrimer of

generation 5, amine groups of PAMAM were activated

with SPDP and reduced by DTT treatment. The resultant

thiol modified PAMAM was activated with N -(k-maleimi-

doundecanoic acid)hydrazide (KUMH), a heterofunctional

crosslinker, and then reacted with aldehyde groups of

oxidized Cetuximab to prepare a Cetuximab-conjugated

PAMAM.

Amine groups of antibodies have also been used as

polymer conjugation sites. For EGF (growth factor) receptor

mediated delivery, anti-HER-2 (human epidermal growth

factor receptor-2) antibody (Herceptin, Trastuzumab) was

conjugated to PEI using a homofunctional crosslinker.[5]

Primary amine groups of PEI were

activated with dithiobis(succinimidyl-

propionate) (DSP) and subsequently

reacted with primary amine groups of

Herceptin. Although the PEI-Ab conju-

gate was prepared by a non-specificamine group mediated reaction, the

resultant HER2-PEI conjugate showed a

significantly higher gene transfection

efficiency of about 20-fold as compared

to PEI alone only for the HER2 over-

expressing cell line, Sk-Br-3, but not for

theHER2deficientcellline,MDA-MB-231

cell. However, direct and non-specific

conjugation of polymers to the amine

groupsof antibodies could often damage

the binding activities significantly.[16] In

order to compare thebinding affinitiesof

PEI-Ab conjugates, three different PEI-Ab

conjugates were prepared by different

coupling procedures.[16] Monoclonal

antibodies (anti-CD90 antibody, Trastu-

zumab) were covalently conjugated to

PEI using two different heterobifunc-

tional crosslinkers, SPDP and N -succini-

midyl - 4-(maleimidomethyl) cyclohex-

ancarboxylate (SMCC), respectively.

First, amine groups of an antibody

reacted with SPDP to prepare 2-pyridyl

disulfide groups. Branched PEI (b-PEI,

25k) was activated with SPDP andreduced using ethanethiol to prepare a

thiolated PEI. The thiolated PEI were

reacted with the 2-pyridyl disulfide

groups of the antibody to produce a

PEI-antibody conjugate via a cleavable

disulfide bond. Secondly, thiol groups of

the reduced antibody were used as a

coupling site of PEI. Thiolated antibody

reactedwith amine groupsof b-PEI using

SMCC via a non-cleavable linkage [Figure 4(B)]. Third,

antibodies were also non-covalently bound to PEI

pre-conjugated with an immunoglobulin affinity ligand, 3-

{2-[2-(vinylsulfonyl)ethylthio]ethyl}quinazoline-

2,4-(1 H ,3 H )dione (IBFB 110001). To prepare a PEI-IBFB

110001 conjugate, 3-(2mercaptoethyl)quinazoline-2,4-

(1 H ,3 H )dione) (IBFB 211239) treated with triethylamine

and divinylsulfone was reacted with b-PEI in ethanolic

solution. The PEI-IBFB 110001 conjugate was bound to the

antibody via a non-covalent fashion. While antibody-PEI

conjugates using SPDP exhibited non-specific transfection

efficiency for plasmid DNA, those using SMCC or IBFB

110001 showed target specific transfection in vitro. In

particular, the IBFB 110001 mediated PEI-antibody

H. Mok, T. G. Park

Figure 4. Antibody-polymer conjugates. (A) PLL-antibody conjugate, (B) antibody-PEIconjugate, and (C) antibody-PEG-PEI conjugate.

738

Macromol. Biosci. 2009 , 9 , 731–743




conjugate demonstrated the highest transfection efficiency

only forthe targetcells. This was likelyto be because amine

groupsintheFabregionoftheantibodywereblockedbyPEI

to a greater extent, losing antigen-antibody binding

capacity.

Free thiol groups of antibodies after reduction are alsogood functional sites for conjugation. Anti-GAD antibody,

specifically targeting GAD (glutamic acid decarboxylase)

that is only detected in pancreatic islet cells, was used for

target specific gene delivery. Thiol groups of reduced Fab

fragment in the GAD antibody reacted with amine groups

of PEI using a heterofunctional PEG (NHS-PEG-VS, N -

hydroxysuccinimide-PEG-vinylsulfone) to prepare a PEI-

PEG-Fab conjugate [Figure 4(C)].[7] The extent of luciferase

gene expression by the resultant PEI-PEG-Fab was over 3

times higher than that by PEI-PEG for GAD expressing

mouse insulinoma cells (MIN6 cells). The enhanced gene

expression was competitively inhibited by the addition of

freeGADantibody,provingthatGADantibodyspecificgene

expression occurred.

Sugars

Galactose

Galactose, a monosaccharide, has been used as a targeting

moiety for hepatoma cell targeting via asialoglycoprotein

receptors-mediated endocytosis.[54,55] Lactose, composed of

b-D-galactose and b-D-glucose through a b-1,4-glycosidic

linkage, could be also used as a targeting moiety for

hepatocyte targeting due to its terminal galactose. Galac-tose moieties were incorporated into amine groups of

polymers via a reductive amination reaction by the

addition of sodium cyanoborohydride.[54,55] Using lactose,

a galactose moiety was conjugated to the amine group of

PEI or amine functionalized PEG of poly(DMAEMA-NVP)-

block-PEG for specific targeting of plasmid DNA to

asialoglycoprotein receptor of hepatocytes. Galactosylated

PEI-poly(vinylpyrrolydone) showed about a 10-fold higher

transfection efficiency of luciferase plasmid DNA than that

of PEI for hepatoma HepG2 cells.[54] For targeted delivery of

siRNA, lactose was conjugated to the terminal end of PEG-

siRNA.[56] Usinglactose-PEG-acrylate, a thiolfunctionalized

siRNA (5’-modified sense strand siRNA) was conjugated to

the terminal of PEG via a Michael-type reaction. After the

conjugation reaction, unmodified antisense siRNA was

annealed to lactose-PEG-sense siRNA. The resulting lactose-

PEG-siRNA was acid-cleavable due to the presence of an

internalb-thiopropionate linkage. PLL (8.3 kDa) was used as

a core condensing polymer. Before complexation, the

e-amine group of PLL was thiolated using 2-iminothiolane

to form disulfide-crosslinked polyelectrolyte complexes

with lactose-PEG-siRNA. While naked siRNA/PLL showed

only 20% of gene silencing effect, lactose-PEG-siRNA/PLL

complexes exhibited 60% gene silencing effect for HuH-7

cells (human hepatoma cells).

Mannose

Glycoproteins with mannose, glucose, fucose and

N -acetylglucosamine units could be recognized by man-nose receptor-overexpressing cells such as macrophages

and dendritic cells and readily internalized via mannose

receptor-mediated endocytosis.[57] Because mannose has

been highlyexpressed on thesurfaces of bacteria and yeast,

mannose receptorsare over-expressed in thecellsrelated to

innate immune response.[58] Thus, mannose has been used

as a targeting moiety into macrophages and dendriticcells,

especially for the delivery of DNA vaccines.[59] Mannose

moieties were conjugated to amine groups of polymers

such as PLL and chitosan using mannopyranosylphenyl

isothiocyanate.[57,58] Vaccination of DNA encoding ovalbu-

minusingmannosylatedPLLcouldinduceboththeCD8and

CD4 T-cell immune response and antibody response, while

DNA complexed with naked PLL showed negligible effects

in vivo. The resulting induction of the immune response by

DNA/mannosylatedPLLcomplexescouldalsoinhibittumor

growth significantly in a tumor mice model.[59]

Folate

It has been known that folate receptor, a glycosylpho-

sphatidylinositol-anchored glycoprotein, is over-expressed

in several tumor tissues.[60] Folate (folic acid), a vitamin B9

(MW441Da), binds to the folate receptor with a high

binding affinity of K d109 M.[61] Thus, folate has been

considered as a useful targeting moiety for tumor or cancercell specific gene delivery. Although folate has two a and g

carboxylic acidgroupsavailablefor polymer conjugation, it

is known that only polymer conjugates viathe g -carboxylic

acid group maintain binding affinity to the folate

receptor.[61–63] For targeted delivery of plasmid DNA via

folate receptor mediated endocytosis, folate was conju-

gated at the PEG terminal of PEG-PLL.[6] After activating the

carboxylic acid group of folate using dicyclohexylcarbodii-

mide (DCC)/ N -hydroxysuccinimide (NHS), the terminal

amine group of NH2PEGCOOH (MW¼3 400) was

reacted to prepare a folate-PEG-COOH conjugate, which

was coupled to the a amino group of the e-amine-blocked

PLL [poly(e-CBZ-L-lysine), MW¼1 000]. After the de-block-

ing, the resultant folate-PEG-PLL conjugate formed stable

polyelectrolyte complexes with plasmid DNA with a size of

100nm. However, folate-PEG-PLL complexes showed

marginally enhanced transfection efficiency due to the

insufficient charge density and lack of endosome escaping

ability. To improve transfection yield, PEI (MW¼25000Da)

was additionally added to form polyelectrolyte complexes.

Folate-PEG-PLL/PEI/plasmid DNA complexes showed sig-

nificantly enhanced gene transfection efficiency, not for

A549 cells, folate-receptor deficient cells, but for KB cells,






folate receptor over-expressing cells.[6] The luciferase

expression of folate-PEG-PLL/PEI/luciferase plasmid DNA

complexes was about 10 times higher than that of

Lipofectamine or PEI for KB cells. Using a similar conjuga-

tion scheme, folate-PEG grafted PEI (folate-PEG-PEI) was

prepared for folate receptor targeted delivery of siRNA,antisense oligodeoxynucleotides (ODN), and plasmid

DNA.[64–66] DCC/NHS activated carboxylic acid group of

folate and then it was coupled with the amine group of a

heterofunctionalNH2PEGCOOHderivative.Then,amine

groups of PEI were conjugated to the activated carboxylic

acid group of folate-PEGCOOH by DCC/NHS [Figure 5(A)].

The relative transfectionlevel of plasmid DNAusing folate-

PEG-PEI was 12 times higher than commercially available

carriers such as Lipofectamine and PEI for folate receptor

positive cell line, KB cells.[64] GFP gene expression was

reduced below20% by anti-GFP siRNAplasmidDNA/folate-

PEG-PEI complexes, which was 2 times more efficient than

that by Lipofectamine and PEI.[65] Folate-PEG-PEI carriers

were utilized fortargeteddeliveryof notonly plasmid DNA,

but also oligonucleotide such as antisense ODN and

siRNA.[66] The gene silencing effect by siRNA or ODN/

folate-PEG-PEI complexeswas over 2 times higherthan that

by siRNAor ODN/PEI complexesfor thefolate receptor over-

expressing cell line, KB cells.

Folate was also conjugated to the terminal of antisense

ODN-PEG [Figure 5(B)].[67] Ag carboxylic acidgroup of folate

was transformed to a thiol group through a DCC/NHS

reaction with cystamine. An amine group of 50

- end aminemodified antisense ODN and thiol group of folate was

conjugated to the terminal of heterobifunctional NHS-PEG-

maleimide(NHS-PEG-MAL). The gene silencingefficiency of

ODN-PEG-folate/PEI,PEI,andLipofectaminecomplexeswas

20% for A549 cells (folate receptor deficient cells), while

they exhibited over 80% for KB cells (folate receptor over-

expressing cells).

Targeted Delivery of Nucleic Acid Drugs viaEnvironmentally Triggered Signals

The cell-specific delivery of nucleic aciddrugs usingcationic

carriers conjugatedwith a proper targeting ligand has often

not been accomplished as desired because of a limited

number of targeting ligands available for the cell recogni-

tion. Thus, environmental signal triggered delivery of

nucleic acids has been attempted as an

alternative option for targeted delivery

without employing cell-specific target-

ing ligands. Various stimuli-sensitive

cationic polymershave beensynthesized

and utilized for the targeted delivery of

nucleic acid drugs in vitro and in vivo

(Table 2). Physical properties of DNApolyplexes, such as size, surface charge

and surface shielding, can be changed

due to the response to environmental

signals secreted from specific cells, lead-

ing to enhanced cellular uptake into the

desired tissue (Figure 6).

Tumor tissue is well known to have

an acidic extracellular environment at

about pH5.8–7.4, which has been used

as an external signal for tumor target-

ing of various nanoparticles.[9,68]

Endosomal pH is also acidic at pH 5.5,

while cytosolic pH is neutral.[69] Mod-

ification of cationic polymers with a pH-

sensitive moiety allows the tumor cell

targeted or endosome-targeted delivery

of nucleic acid drugs. Poly(methacryloyl

sulfadimethoxine) (PSD), a pH-respon-

sive polymer, has a negative charge

at physiological conditions (pH¼7.4),

while it becomes neutral at acidic

pH6.6.[9] A terminal carboxylic acid

group of mPEG was activated with

H. Mok, T. G. Park

Figure 5. (A) folate-PEG-PEI conjugate and (B) folate-PEG-antisense ODN conjugate.

740

Macromol. Biosci. 2009 , 9 , 731–743




DCC/NHS and sequentially conjugated to the end of PSD.

Plasmid DNA/PEI complexes with a positive surface charge

were coated with negatively charged PSD-PEGconjugate at

neutral pH via ionic interactions. Physical immobilization

of PSD-PEG onto DNA/PEI complexes reduced surface

charge and cytotoxicity of the complexes. However, PSD-

PEG can be detached from the DNA/PEI complexes at

pH¼6.6 due to the charge reversion from negative to

positive charge. This reversion could mediate enhanced

transfection efficiency and cytotoxicity at acidic pH. This

gene delivery system viaan acidicpH-sensitive mode could

be applied to the tumor tissue specific gene therapy. More

recently, PLL conjugated with citraconic anhydride was

utilized as pH-sensitive surface charge reversal materials

for enhanced cellular uptake of quantum dots, adenovirus,

and nucleic acids at acidic pH.[68]

For temperature triggered gene delivery, a copolymer of

PEI and poly(NIPAM-VP) was used as a carrier polymer.[10]

Compared to PEI/DNA complexes, poly(NIPAM-VP)-block-

PEI was prepared by radical polymerization of N -isopropy-

lacrylamide (NIPAM) and 1-vinyl-2-pyrrolidinone (VP) from

PEI as a starting block. Poly(NIPAM-VP)-block-PEI/DNA

complexes at an N/P ratio of 6 exhibited reduced

cytotoxicity. While PEI/DNA complexes showed high

transfection efficiency regardless of hyperthermia, poly-

(NIPAM-VP)-block-PEI/DNAcomplexes exhibited hightrans-fection efficiency only in a hyperthermia condition by

forming large aggregates which make them more effective

for endosome escape. While poly(NIPAM-VP)-block-PEI/

DNAcomplexesare stabilizedby hydrophilic copolymers at

37 8C, the complexes were aggregated above 40 8C due to a

phase transition of poly(NIPAM) from hydrophilic to

hydrophobic property.The poly(NIPAM-VP)-block-PEIdeliv-

ered a 10-fold higher amount of plasmid DNA to tumor

tissue underthe hyperthermia conditionthan branched PEI

(25kDa) in a mouse model.[70] Moreover, hyperthermia

induced gene delivery by poly(NIPAM-VP)-block-PEI, which

significantly reduced non-specific delivery of plasmid DNA

to normal tissues such as lung, heart, liver and kidney.

A magnetic field could be also used as a targeting signal

for gene delivery.[11] To prepare magnetic field sensitive

nanoparticles, superparamagnetic nanoparticles (MNP)

were loaded into poly(D,L-lactide) (PLA) nanoparticles by

using an emulsification-solvent evaporation method.

Magnetite loaded PLA nanoparticles were coated with PEI

via ionic interactions to complex plasmid DNA. PEI coated

magnetite-PLA nanoparticles showed an enhanced serum

stabilityof plasmid DNA. A magneticfield (15min exposure

to a magnetic field, 500 G) specifically triggered gene


Table 2. Stimuli-sensitive targeted delivery systems for nucleic acids.

Targeting

signal

Stimuli Polymer Target

cell/tissue

Cell line Ref.

pH sensitive low pH (pH6.6) PEI/poly(methacryloylsulfa-

dimethoxine)-PEG

tumor cell A2780 (human ovarian

cancer cell)

[9]

temperature

sensitive

hyperthermia (42 8C) poly(NIPAM-VP)-block-PEI tumor cell mouse tumor model

(neuroblastoma

Neuro2A cells)

[10]

magnetic field m agnet PEI-coated magnetite-PLA

nanoparticle

A10 (rat aortic smooth

muscle cell), BAEC

[11]

Figure 6. Targeted delivery of nucleic acid drugs in response toenvironmental signals.





expression of plasmid DNA for A10 cells (rat aortic smooth

muscle cells) and bovine aortic endothelial (BAEC) cells.

Conclusion

A variety of carrier polymers have been proposed for

targeted delivery of nucleic acids, and some of them

exhibited exciting invivo results. For clinical applications of

nucleic acid drugs, efficient and targeted delivery issues

should be addressed by molecular engineering of carrier

polymers functionalized with targeting moieties. Many

conjugation strategiesto targetligandsfor carrier polymers

have been proposed to achieve maximum delivery

efficiency and targeting effect. Recent development of

smart multifunctional polymer carriers with enhanced

complex stability, prolonged circulation in the blood

stream, improved cellular uptake at a target site, facile

endosome escape, and minimal hindrance for intracellulargene processing could provide opportunities for clinical

applications of therapeutic nucleic acids.

Acknowledgements: This study was supported by the National

Research Laboratory project and KOSEF grant (R31-2008-000-10071-0) from the Ministry of Education, Science and Technology ,and the Nanomedicine Center grant from the Ministry of Health

and Welfare, Republic of Korea.

Received: January 29, 2009; Revised: April 11, 2009; Accepted:April 15, 2009; DOI: 10.1002/mabi.200900044

Keywords: bioengineering; conjugated polymers; drug deliverysystems; functionalization of polymers; structure-propertyrelations

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