M I N I R E V I E W

Minireview: Delivering the Code: Polyplex Carriers

for Deoxyribonucleic Acid and Ribonucleic Acid
Interference Therapies

R. James Christie, N. Nishiyama, and K. Kataoka

Department of Materials Engineering (R.J.C., K.K.), Graduate School of Engineering and Center for

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NanoBio Integration (N.N., K.K.), University of Tokyo, Tokyo 113-8656, Japan; Center for Disease
Biology and Integrative Medicine (N.N., K.K.), Graduate School of Medicine, University of Tokyo, Tokyo
113-0033, Japan; and Core Research for Evolutional Science and Technology (K.K.), Japan Science and
Technology Agency, Saitama 332-0012, Japan

Nucleic acid-based therapies offer great potential for treatment of a variety of diseases in-
cluding cancer by modulating protein expression with DNA or small interfering RNA. However,
realization of their full therapeutic potential is currently limited due to an inability to reach
the target site in an active form. Identification of delivery barriers such as stability in circulation,
resistance to degradation and entrapment in subcellular vesicles has led to development of
sophisticated multifunctional synthetic polymers for forming ionic complexes with nucleic
acids and also providing performance-enhancing features. The most promising designs com-
prise features to help increase stability in circulation and also contain functionality to aid in
endosome escape of nucleic acid cargo after cellular internalization. (Endocrinology 151:
466 – 473, 2010)

ene and RNA interference (RNAi) therapies present a
G great opportunity for the treatment of intractable dis-
eases such as genetic disorders, degenerative diseases,
infections, and malignancies. Such therapies offer se-
quence-specific activity on the transcriptional (DNA) or
translational [small interfering RNA (siRNA)] level.
However, a major obstacle limiting their practical ap-
plication is the lack of safe and efficient methods for
their delivery. Recombinant viral vectors including ret-
rovirus, lentivirus, adenovirus, and adeno-associated
virus show high efficiency for introducing genetic ma-
terials into cells; however, their clinical use might be
limited by inherent risks by possible contamination of
wild-type viruses and severe immune reactions after re-
peated administration (1, 2). Additionally, viral vectors
are difficult to produce on a large scale due to compli-
cated manufacturing processes in special facilities al-
though efforts to improve the process continue (3). In
contrast, nonviral synthetic vectors composed of cat-
ionic lipids or cationic polymers termed polyplexes and
lipoplexes, respectively, are attractive alternatives due
to their safety for clinical use, simplicity of preparation,
and easy large-scale production. The major disadvan-
tage of synthetic vectors is a relatively lower transfec-
tion efficiency compared with their viral counterparts
(4). Therefore, enormous effort has been devoted to
improving the transfection efficiency of synthetic vec-
tors. In addition, there is a strong impetus for the de-
velopment of synthetic vectors for systemic administra-
tion, which is particularly important for the practical
use of siRNA. In this minireview, we will discuss several
key factors and considerations for the development of
nucleic acid delivery systems focusing on polyplexes
rather than lipoplexes because of the variety of chemical
designs available for optimization and their advanta-
geous features for use as pharmaceutical products, in-
cluding reproducibility of preparation and stability for
long-term storage.

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: DSP, Dithiobis(succinimidyl propionate); EPR, enhanced permeability and
Printed in U.S.A. retention; PAsp(DET), poly{N-关N-(2-aminoethyl)-2-aminoethyl兴aspartamide}; pDNA, plasmid
Copyright © 2010 by The Endocrine Society DNA; PEG, poly(ethylene glycol); PEI, poly(ethylenimine); PHPMA, poly关N-(2-hydroxypropyl)
doi: 10.1210/en.2009-1045 Received September 3, 2009. Accepted November 17, 2009. methacrylamide兴; pKa, acid dissociation constant; PLL, poly(L-lysine); PSAO, poly(silamine);
First Published Online December 23, 2009 RISC, RNA-induced silencing complex; RNAi, RNA interference; siRNA, small interfering RNA.

466 endo.endojournals.org Endocrinology, February 2010, 151(2):466 – 473

Endocrinology, February 2010, 151(2):466 – 473
Nucleic Acid Therapies
Although DNA and siRNA therapies both offer sequence-
specific specificity, their mechanism of action, duration of
therapeutic effect, and factors affecting polyplex design
are quite different. DNA-based therapies generally use
plasmid DNA (pDNA) purified from Escherichia coli cul-
tures and consist of thousands of base pairs, resulting in an
unusually large and highly charged therapeutic agent. The
therapeutic goal in this case is to produce therapeutic pro-
teins, and thus, DNA must be delivered into the nucleus
(5). This presents quite a challenge for treatment of non-
dividing cells but is less of a barrier in cancer cells due to
rapid proliferation and dissolution of the nuclear mem-
brane during cell division. Once the DNA is delivered into
the nucleus, expression of the target protein can last for
weeks or longer, depending on the cell type targeted and
carrier used (6, 7). Due to the size of therapeutic DNAs,
formation of complexes is a dramatic event with conden-
sation and collapse of single plasmids into nanoparticles,
depending on the conditions (8).
siRNA-based therapies use double-stranded RNA mol-
ecules 21–23 bp long with 1–2-base overhangs to induce
RNAi and prevent protein expression (9). In this ap-
proach, the delivered siRNA molecule binds to a multi-
protein complex in the cell cytoplasm to form a RNA-
induced silencing complex (RISC), which results in
degradation of mRNA strands with sequences compli-
mentary to the delivered siRNA (10). This strategy has two
advantages; first, the therapeutic target is in the cyto-
plasm, which eliminates the need for nuclear delivery; sec-
ond, the RISC complex has potential to cleave many
mRNAs once activated and the effect can be observed for
days in rapidly dividing cells and weeks in nondividing
cells (11). The overall effect of siRNA therapies is shorter-
lived than DNA-based counterparts because the RISC
complex and residual siRNAs will eventually be degraded.
However, RNAi has been observed for over 90 d in kidney
tissue by transfecting a plasmid encoding for expression of
siRNA (12). Formation of polyplexes is generally more
difficult with siRNAs than with pDNAs because of their
smaller size and rigidity. Additionally, formation of par-
ticles that are tens on nanometers in diameter requires the
assembly of multiple individual siRNA and polycation
polyplexes. To overcome this, polycations are often de-
signed with additional stabilizing features such as hydro-
phobic moieties and chemical cross-links, which will be
discussed in more detail in the following sections. Alterna-
tively, the siRNA can be modified to contain stabilizing
chemistries such as modification of the sugar backbone, base
structures, or addition of cholesterol which is relatively sim-
ple to accomplish as siRNA is prepared synthetically (13).
endo.endojournals.org 467
Several potential drawbacks reside in clinical application
of nucleic acids. Although pDNA and siRNA are less immu-
nogenic than viral vectors, they can potentially activate the
innate immune system through recognition by the corre-
sponding Toll-like receptors, leading to production of in-
flammatory cytokines and a rapid removal of the transfected
cells (14, 15). The immunostimulatory effect by pDNA is
known to come from unmethylated CpG dinucleotides in
specific sequence contexts, called the CpG motif, which
binds to Toll-like receptor-9 (16). Therefore, the use of CpG-
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free plasmid might impair the immune responses.
Formation of Polyplexes
Polyplexes are formed through the electrostatic interac-
tion between negatively charged nucleic acids and cationic
polymers. Typical examples of cationic polymers used for
polyplex formation are shown in Fig. 1. In each case, nu-
cleic acids are incorporated into nanoparticles with a size
of several tens to hundreds of nanometers (17, 18). For
successful transfection by systemic delivery, the poly-
plexes should be stable under extracellular conditions to
prevent the release of cargo at off-target sites and protect
the nucleic acids from enzymatic degradation. The stabil-
ity of polyplexes depends on the chemical structure, mo-
lecular weight, and three-dimensional structure of cat-
ionic polymers as well as the mixing ratio of cationic
polymers to nucleic acids. Because cationic amines are of-
ten used as the complexing agent the mixing ratio is typ-
ically referred to as the N to P ratio where N represents
amines in the polymer and P represents phosphates in the
nucleic acid; however, this ratio may also be denoted as the
r value. Generally, cationic polymers having more than
several tens of cationic units are required to form stable
polyplexes suitable for gene and siRNA delivery. In the
case of PEGylated pDNA complexes, complex stability
increases with increased polycation length (19). Poly-
plexes may form with nucleic acids at excess cationic mix-
ing ratios with low acid dissociation constant (pKa) poly-
cations (pKa ⬃6 – 8), which may result in a polyplex with
an overall positive charge. Polyplexes with excess positive
charge may preferentially interact with negatively charged
cell surfaces, leading to increased cellular uptake. How-
ever, such cationic polyplexes easily form large aggregates
due to the salting-out effect and electrostatic interaction
with negatively charged serum proteins such as albumin.
Such large aggregate formation presents a serious problem
for systemic gene and siRNA delivery due to undesired effects
such as embolization of the lung capillaries after iv injection
(20). Therefore, the surface of polyplexes are typically mod-
ified with hydrophilic and biocompatible polymers such poly-
(ethylene glycol) (PEG) or poly关N-(2-hydroxypropyl)methac-
468 Christie et al. Minireview
FIG. 1. Synthetic components found in nonviral polyplex delivery systems for nucleic acid therapies.
rylamide兴 (PHPMA) for in vivo applications because such
modifications increase the colloidal stability and water sol-
ubility and also prevent aggregation and interaction with
serum proteins by a steric repulsion effect (18, 21–25). PE-
Gylation is the most common method of improving the cir-
culation property of colloidal nanoparticles (26). Polyplexes
formed with PEGylated polycations results in further assem-
bly into nanometer-sized higher ordered micelle structures,
with the charge-neutralized nucleic acid and polycation con-
tained in the core, which is surrounded by a PEG corona.
Reaching the Target Site
As mentioned previously, systemic administration of ther-
apeutic polyplexes is desirable for practical application of
a variety of treatments and especially for cancer therapies
because direct intratumoral injection is not always avail-
able. In the case of tumor targeting after iv injection, cir-
culating polyplexes may accumulate in solid tumors with-
out the aid of tissue-specific ligands. This phenomenon is
explained by the microvascular hyperpermeability to circu-
lating macromolecules and the impaired lymphatic drainage
in solid tumors and is termed the enhanced permeability and
retention (EPR) effect (27). The EPR effect appears to be
universally observed in malignant tumors because exper-
imental tumors were reported to show the vascular cutoff
size of several hundred nanometers, regardless of the tu-
mor origin and inoculation sites (28, 29). Note that the
EPR effect is also important for the tumor-specific accu-
mulation of drug vehicles modified with targetable ligands
because tumor cells are generally located outside of the
vasculature and the targetable ligands recognize the tumor
Endocrinology, February 2010, 151(2):466 – 473
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cells after the extravasation process. Thus, the EPR effect
is currently a strategic basis for designing tumor-tar-
getable nanocarriers.
To maximize the benefits of the EPR effect, polyplexes
must be long circulating so that they may have multiple
passes through the tumor tissue and thus a greater poten-
tial for accumulation. In this regard, PEGylation (19) as
well as coating polyplex surfaces with water-soluble
(PHPMA) (30) has been shown to increase circulation
time as it helps to prevent aggregation and interactions
with serum components and reduces uptake by the reticu-
loendothelial system (21). Polyplex surface-modification
also helps prevent nuclease attack of polyplexes, as sim-
ple poly(L-lysine) (PLL)-pDNA complexes are degraded
within 10 min in blood (24). However, care must be taken
when designing PEG or PHPMA-modified polyplexes.
Modification of poly(ethylenimine) to generate graft co-
polymers may not impart prolonged circulation, as graft-
ing groups onto the polymer backbone can result in in-
creased steric repulsion in the polyplex core as well as
reduced cationic charge thus destabilizing the polyplex
(31–33). Thus, using block type copolymers or taking
other steps to ensure that only the surface of the polyplex
is modified may result in superior structures (Fig. 1).
Subcellular Barriers
Nucleic acids are impermeable to cell membranes; there-
fore, the polyplex delivery systems are internalized by
endocytosis, which ultimately results in localization
into endosome and lysosome compartments. Therefore,
the polyplexes and therapeutic cargo must escape from
Endocrinology, February 2010, 151(2):466 – 473
these compartments into the cytoplasm to circumvent hy-
drolytic and enzymatic degradation of the nucleic acid cargo
so that they may reach their subcellular site of activity intact
(34, 35). Thus, endosomal escape is a key step for intracel-
lular gene and siRNA delivery. In this regard, several cationic
polymers with a pKa value between physiological and ly-
sosomal pH, including poly(ethylenimine) (PEI) (36),
poly(amidoamine) (37), poly(histidine) (38), poly{N-关N-
(2-aminoethyl)-2-aminoethyl兴aspartamide} [PAsp(DET)] (39)
are known to facilitate the endosomal escape of poly-
plexes. Behr and colleagues (40) hypothetically explained
this activity by the proton sponge effect, in which proto-
nation of amines in the polymer within acidic endosomal
compartments may cause an increase in ion osmotic pres-
sure, therefore disrupting the endosomal membrane. This
effect is well supported by several experimental results and
is an important basis for polyplex design. Thus, the above-
mentioned cationic polymers have been widely studied as
efficient gene and siRNA carriers; however, their in vivo
use is severely restricted by several factors. First, poly-
plexes formed from cationic polymers with a compara-
tively low pKa require an excess amount of cationic poly-
mers to provide enough stability for efficient gene and
siRNA transfection. However, such polyplexes contain
free cationic polymers, which substantially contribute to
cytotoxicity as well as efficient transfection. After systemic
administration, free polymers will gradually be removed
from the polyplexes and thus exert a cytotoxic effect. Sec-
ond, as mentioned before, PEGylation of polyplexes is
commonly used to improve their circulation property.
However, modification of the polyplexes with a dense
layer of high-molecular-weight PEG often dramatically
decreases their in vitro transfection efficiency, which may
not be explained by simply a decrease in internalization
amount (41, 42). It is likely that PEGylation may lessen the
endosomal escape ability of the polyplexes. It is assumed
that direct interaction of cationic polymers with the en-
dosomal membrane may be essential for endosomal es-
cape of polyplexes in addition to the proton sponge effect.
In the case of gene delivery, pDNA must be further
transported into the nucleus. However, the polyplexes can-
not pass through the nuclear membrane because the nuclear
pore complex allows only the passage of small molecules.
When cancer cells are transfected with polyplexes, pDNA
can enter the nucleus due to the breakdown of the nuclear
membrane during cell division. In contrast, transport across
the nuclear membrane is essential for gene transfection to
nondividing cells. Introduction of the nuclear localization
signal peptides into polyplex systems has been attempted to
circumvent the nuclear transport barrier (43, 44).
After the polyplexes reach the target site, they are re-
quired to release nucleic acids. It is likely that competitive
endo.endojournals.org 469
binding anionic components such as genomic DNA,
mRNA, and cytosolic proteins may facilitate the release of
nucleic acids from the polyplexes. Also, chemical modifi-
cation of cationic polymers has been attempted to improve
the intracellular release of nucleic acids from the poly-
plexes, such as degradable polymer backbones (45).
Polyplex Stabilization: Disulfide
Cross-Linking
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Additional polyplex stability may be obtained by cross-
linking the core; this may be more critical for siRNA sys-
tems than pDNA systems due to the much-reduced size of
the polyanion (plasmid DNA contains thousands of neg-
ative charges, whereas siRNA contains only ⬃50). One
common method to increase complex stability in the case of
pDNA is to simply increase the polycation chain length, thus
increasing its electrostatic attractive force and cooperative
binding capability to pDNA (23). However, increasing the
polycation length can also lead to overstabilization of the
complex as well as increased toxicity. Cross-linking poly-
plexes in a reversible fashion would allow stability at off-
target sites, followed by degradation and release of nucleic
acids at the desired site of activity.
Polyplexes have been prepared to contain disulfide
cross-links that allow for environment sensitive cleavage
by reducing agents or disulfide-specific enzymes. This
strategy is especially attractive for targeted degradation of
polyplexes in the intracellular environment because the
glutathione concentration (a natural disulfide reducing
agent) is about 50 –1000 times higher inside cells com-
pared with the extracellular environment (46). Disulfide
bonds in cross-linked PEI have been shown to cleave after
a few hours in live HeLa cells, whereas a disulfide-con-
taining dye was effectively reduced to the free thiol form
in RAW 264.7 murine macrophage cell lysate in a similar
time frame (47, 48). Disulfide cross-linked carriers are also
attractive due to their ease of preparation, several amine-
reactive thiol-modification reagents are available that can
preserve the charge of the parent polymer (iminothiolane)
or reduce the charge of the parent polymer [N-succinimi-
dyl 3-(2-pyridyldithio)propionate (SPDP) and dithio-
bis(succinimidyl propionate) (DSP)], allowing for further
fine-tuning of polyplex properties.
Thiol-modified polycations have shown the ability to
increase the efficacy of nucleic acid delivery systems due to
stabilization of the polyplex by disulfide formation. Poly-
plexes prepared with thiol-modified PEG-block-PLL co-
polymers have shown both increased transfection using
pDNA and increased knockdown using siRNA. In the case
of pDNA, improved transfection was observed in cultured
293T cells using polyplexes prepared with SPDP-modified
470 Christie et al. Minireview
PEG-block-PLL copolymer, with approximately 1 order
of magnitude increase relative to non-cross-linked parent
polymer (49). Gene silencing in cultured Huh7 cells was
observed at nanomolar siRNA concentrations after treat-
ment with polyplexes formed from iminothiolane-modi-
fied PEG-block-PLL, with essentially no knockdown ob-
served using noncross-linked polyplexes prepared from
unmodified PEG-block-PLL (50). The in vitro knockdown
efficacy was dependent on the degree of iminothiolane
modification; however, low iminothiolane modification
(⬃14%) resulted in the best performance.
Furthermore, disulfide cross-linked PEI polyplexes have
also shown enhanced performance. Cross-linked polyplexes
prepared with pDNA and DSP-modified PEI homopolymer
prevented release of complexed pDNA after challenge with
the anionic exchange reagent heparin or high salt concen-
tration (51). DSP modification of branched PEI-graft-PEG
significantly improved the circulation time of pDNA poly-
plexes; however, a relatively high cross-linking degree (50%)
was needed (52).
Triblock Copolymers
The use of multiblock copolymers as complexing agents
allows for introduction of different functionality onto one
macromolecule. Specifically, A-B-C type multiblock co-
polymers have been prepared as advanced polyplex for-
mation agents with the polymer designed to contain dis-
tinctly different regions in the backbone, each designed to
serve a specific purpose. Several designs use A-B-C type
block copolymers comprising a PEG segment for biocom-
patibility and extended blood circulation on one end, a
polycation on the other end for nucleic acid complexation,
and various segments in the middle containing hydropho-
bic, pH buffering, or endosome disrupting chemistries.
Polyplex micelles prepared with pDNA and triblock
copolymers have indeed shown promising results. Such
micelles comprised of A-B-C type block copolymers with
an endosome buffering or endosome disrupting B segment
have been prepared from PEG-block-poly[(3-morpholin-
opropyl)aspartamide] (PMPA) block-PLL (53), PEG-block-
poly(silamine) (PSAO)-block-poly关2-(N, N-dimethylamino)
ethyl methacrylate兴 (PAMA) (54) and PEG-block-
PAsp(DET)-block-PLL (55). In vitro transfection experi-
ments revealed that incorporation of the endosome dis-
rupting feature improved transfection efficacy by about 1
order of magnitude for PEG-block-PMPA-block-PLL and
PEG-block-PAsp(DET)-block-PLL and about 0.5 order
of magnitude in the case of PEG-block-PSAO-block-
PAMA micelles (the PEG-block-PSAO-block-PAMA
block copolymer also contained a lactose cell targeting
moiety on the distal end of the PEG segment). Further-
Endocrinology, February 2010, 151(2):466 – 473
more, introduction of hydroxychloroquine (an agent
known to disrupt lysosomes) did not drastically increase
transfection efficiency, indicating that the triblock copol-
ymer was sufficient for facilitating the lysosome escape of
pDNA. Another report showed that incorporation of a
hydrophobic segment between the PEG and polycation
block similar in length to the polycation block signifi-
cantly improved the stability and resistance of pDNA
complexes to enzymatic degradation; however, transfec-
tion efficacy was about 30% lower than the diblock co-
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polymer when tested in cultured cells (56). Whereas this
increased stability may be beneficial for the circulation
properties, it comes at a cost on the cellular level as the
increased stability also inhibits release of cargo.
Triblock copolymers have also been used for prepara-
tion of effective siRNA polyplexes. One recent formula-
tion used a poly(propylene sulfide) hydrophobic middle
block in between PEG and a short cationic polypeptide
(either K9 or Tat) to form complexes on the order of one
to several hundreds of nanometers in size (57). This study
showed that polyplex efficacy could be fine-tuned by
changing the length of the hydrophobic segment in the
polymer as well as the type of polycationic peptide used to
complex with siRNA. A remarkable amount of gene
down-regulation was achieved by using a short 5-unit
poly(propylene sulfide) block with the K9 peptide as the
polycation segment in cultured cells at picomole siRNA
concentrations. Yet another study used a triblock copol-
ymer with a long degradable 100-unit poly(␧-caprolac-
tone) hydrophobic segment in between PEG and a short
12-mer polycation (58). This block copolymer formed mi-
celles with siRNA via a slightly different mechanism than
discussed thus far because the long hydrophobic segment
allowed for formation of micelles before the introduction
of siRNA. Introduction of siRNA resulted in electrostactic
binding of siRNA as a middle layer in between the PEG
corona and the hydrophobic core. This formulation ex-
hibited modest gene silencing of 40 –70% at picomole con-
centrations in cultured HEK293 cells.
PEG-Detachable Systems
As mentioned previously, PEGylation of polyplexes pro-
vides many benefits for improved in vivo performance;
however, PEGylation often decreases the in vitro perfor-
mance of polyplex carriers. This has lead to preparation of
PEG-detachable polyplex systems that provide the desired
stealth properties at off-target sites followed by degrada-
tion and removal of the PEG layer at the desired site of
activity. Once the PEG layer is removed, the polyplex can
better release entrapped cargo by a variety of mechanisms
such as competitive binding with naturally occurring an-
Endocrinology, February 2010, 151(2):466 – 473
ionic components such as genomic DNA, mRNA, and cy-
tosolic proteins. For cancer therapies, PEG detachment
can occur in the extracellular tumor environment by ex-
ploiting the slightly lower pH of tumor tissue (⬃pH 5.7–
7.8) (59). One such carrier has been developed that uses
PEG-block-poly(sulfadimethoxane) to form a ternary
complex with a positive charge-excess DNA and PEI (60).
The sulfonamide groups on the PEG-block-PSDM copol-
ymer are anionic at physiological pH but become charge
neutral at pH 6.6, thus eliminating the electrostatic at-
traction to the positively charged PEI-DNA complex lead-
ing to removal of PEG from the polyplex exterior. The
ternary complex indeed showed higher transfection effi-
ciency at pH 6.6 than pH 7.4 in cultured A2780 adeno-
carcinoma cells despite significant cytotoxicity, the use of
a less toxic polycation in this system could result in even
higher efficacy. Alternatively, carriers can be designed that
release PEG after cellular internalization using pH-sensi-
tive hydrazone and acetal linkages or by disulfide reduc-
tion. Covalent acetal and hydrazone linkages can be de-
signed to be stable at pH 7.4 but labile at about pH 5,
which is convenient for site-specific degradation in acidic
lysosome compartments after cellular uptake of poly-
plexes. Such complexes have been prepared by postmodi-
fication of PLL/pDNA complexes to generate PLL-hydra-
zone-PEG polyplexes that exhibited 2 orders of magnitude
higher transfection efficacy in vitro and 1 order of mag-
nitude higher transfection efficacy in vivo compared with
complexes containing a stable PEG layer (61). Modifica-
tion of PEI with PEG bound via an acetal linkage also
improved the in vitro transfection performance in both
Renca-EGFR and K562 cells by an order of magnitude com-
pared with nondegradable PEGylated polyplexes (62). Sim-
ilar success has been observed with polyplexes formed
with PEG-block-PAsp(DET) and pDNA, in which PEG is
attached via a disulfide linkage. The PEG-detachable sys-
tem exhibited 2–3 orders of magnitude better transfection
efficiency than the nondetachable counterpart in HeLa
cells (63). Although the above-mentioned PEG-detachable
systems exhibit orders of magnitude increases in transfec-
tion efficacy in vitro compared with non PEG-detachable
systems, their efficacy is generally about 1 order of mag-
nitude lower than non-PEGylated polyplexes prepared
with homopolymer.
Summary
Nonviral nucleic acid delivery vehicles using multifunc-
tional polymers represent a great improvement compared
with their simple polycation precursors, but much work
remains to produce a universal nucleic acid carrier for in
vivo use. Knowledge of the specific barriers responsible for
endo.endojournals.org 471
reduced efficacy has led to creative use of many different
chemistries to produce more effective carrier systems.
PEGylation of polyplexes protects nucleic acids from deg-
radation, reduces nonspecific interactions with serum
components and allows for extended circulation. Polyca-
tions with buffering capacity between about pH 5–7.4 can
improve efficacy of delivery systems by destabilizing ly-
sosomal compartments, facilitating release of nucleic ac-
ids into the cytoplasm. Finally, reversible cross-linking of
the polyplex core can provide increased stability of the
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carrier structure. In essence, these advanced polyplex sys-
tems have become nanodevices, equipped with specific
functionalities at the molecular level.
As work progresses in this area, however, the need for
balance of carrier properties and the chemistries that fa-
cilitate them has become quite clear, and it is unlikely that
one single chemistry will produce a delivery vehicle with
all of the desired properties. For example, although
PEGylation improves the circulation properties of nucleic
acid polyplexes, it also lowers efficacy on the cellular level
due to reduced cellular uptake and interactions with
charged cellular components that facilitate release of
cargo. Although polycations containing functional groups
with pKa values between the physiological and lysosomal
pH have a buffering effect that improves lysosome escape,
they form less stable complexes due to a lower ability to
maintain a strong positive charge. On the other hand,
polycations with a high pKa form stable complexes with
nucleic acids but cannot facilitate endosome escape and
they are often toxic. Additionally, optimal conditions for
polyplex formation and resulting efficacy are often differ-
ent for siRNA and DNA.
Development of a universal carrier for nucleic acid ther-
apies will likely see much progress in the near future. Al-
though carrier features must be specific for DNA and
siRNA, once effective carriers are developed for each, they
will likely be universal for any DNA or siRNA sequence
because carrier-nucleic acid interactions are primarily
based on the phosphate backbone and are not base se-
quence specific. This multidisciplinary area of research has
used and will continue to use developments in cellular and
molecular biology, chemistry, and medicine to produce
advanced carriers with improved efficacy. Such advances
will help to realize the full potential of nucleic acids as an
effective therapeutic modality.
Acknowledgments
Address all correspondence and requests for reprints to: Dr.
Nobuhiro Nishiyama, Center for Disease Biology and Integrative
Medicine, or Professor Kazunori Kataoka, Department of
Materials Engineering, Graduate School of Medicine, Univer-
472 Christie et al. Minireview Endocrinology, February 2010, 151(2):466 – 473

sity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656,
Japan. E-mail: nishiyama@bmw.t.u-tokyo.ac.jp; or kataoka@bmw.
20.
t.u-tokyo.ac.jp.
Disclosure Summary: The authors have nothing to disclose.
21.
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