Hydrophobic Modifications of Cationic Polymers For Gene Delivery

Progress in Polymer Science 35 (2010) 11441162
Contents lists available at ScienceDirect
Progress in Polymer Science

journal homepage: www.elsevier.com/locate/ppolysci
Hydrophobic modications of cationic polymers for gene delivery

Zonghua Liu a , Ziyong Zhang a , Changren Zhou a,b , Yanpeng Jiao a,b,
a
b
Department of Materials Science and Engineering, Jinan University, Guangzhou 510632, China
Engineering Research Center of Articial Organ and Materials, Ministry of Education, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history:
Received 20 November 2009
Received in revised form 22 February 2010
Accepted 14 April 2010
Available online 22 April 2010
Keywords:
Polyethylenimine
Chitosan
Polylysine
Hydrophobic modication
Gene delivery
a b s t r a c t
Cationic polymers are the subject of intense research as non-viral gene delivery systems,
due to their exible properties, facile synthesis, robustness, and proven gene delivery efciency. Nevertheless, low transfection efciency and undesirable cytotoxicity remain the
most challenging aspects of these cationic polymers. To overcome the disadvantages, various modications have been made to improve their gene delivery efcacy. Among them,
hydrophobic modications of the cationic polymers are receiving more and more attention. Most studies have shown that incorporation of hydrophobic chains can improve
gene delivery efciency, mainly explained by hydrophobic interaction conferred to the
resulting amphiphilic polycation derivatives and by the enhanced cellular uptake by the
hydrophobic chains via the lipophilic cell membrane. This review discusses recent studies
on the hydrophobic modications of cationic polymers for gene delivery. The effects of the
hydrophobic modications are discussed in terms of critical issues in the gene delivery
process, such as gene encapsulation, adsorption to cell membrane, serum inhibition, gene
dissociation, cytotoxicity, and tissue-targeting. Moreover, various hydrophobic modications of the main cationic polymeric gene carriers (polyethylenimine, chitosan, polylysine,
etc.) are described with regards to the resulting gene delivery activity. The structurefunction relationships discussed here provide important information and insight for the
design of novel gene vectors.
2010 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of hydrophobic modications on gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Increased physical encapsulation of genetic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Promotion of complex charge inversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Enhanced adsorption to cell membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Alleviation of serum inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Facilitation of gene dissociation from polycation carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.
Effects on cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.7.
Targeting-specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrophobic modications of PEI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Linear alkyl chains for PEI modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Effects of chain length and DS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Fax: +86 20 85226663.

E-mail address: tjiaoyp@jnu.edu.cn (Y. Jiao).
0079-6700/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2010.04.007
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4.
5.
6.
7.
Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162
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3.1.2.
Effects of alkylation position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.3.
Explanation for reduced transfection efciency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Pluronic for PEI modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Cyclic hydrophobic molecules for PEI modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrophobic modications of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Linear alkyl chains for chitosan modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Cyclic hydrophobic molecules for chitosan modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrophobic modications of PLL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Linear alkyl chains for PLL modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Pluronic for PLL modications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrophobic modications of other polycations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Hydrophobic modications of poly(amidoamine) dendrimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Hydrophobic modications of DMAEMA copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.
Hydrophobic modications of dextran-spermine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Nomenclature
BMA
CMC
DOTAP
butylmethacrylate
critical micellar concentration
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium sulphate)
DPPC
dipalmitoyl-sn-glycero-3-phosphocholine
DS
degree of substitution
EDC
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
IPAAm N-isopropylacrylamide
MDMBzChC methylated
N-(4-N,Ndimethylaminobenzyl) chitosan chloride
MDMCMChC methylated
N-(4-N,Ndimethylaminocinnamyl) chitosan chloride
MPyMeChC methylated N-(4-pyridinylmethyl) chitosan chloride
NHS
N-hydroxysuccinimide
PAA
propylacrylic acid
pDMAEMA poly(2-N-(dimethylaminoethyl)
methacrylate)
PEG
poly(ethylene glycol)
PEI
polyethylenimine
PEO
poly(ethylene oxide)
PLL
poly(l-lysine)
PPAA
poly(propylacrylic acid)
PPO
poly(propylene oxide)
RBCs
red blood cells
TMChC N,N,N-trimethyl chitosan chloride
VEGF
vascular endothelial growth factor
1. Introduction
Gene therapy has been proposed as an epoch-making
curative method for both inherited and acquired genetic
diseases by its transfer of genetic materials, either RNA or
DNA, into specic human tissues or cells, to replace defective genes, substitute missing genes, silence unwanted
gene expression, or introduce new cellular biofunctions [1].
Interest in gene therapy has soared in recent years because
of its great promise in treating diseases ranging from inherited disorders to acquired conditions and cancers [2]. At
the center of gene therapy is gene transfer, the delivery
of genes into the desired cells, which is followed by gene
expression. Although many experiments strongly suggest
that the uptake of naked genes occurs, the process is too
slow for a telling biological effect [3]. In addition, naked
DNA or RNA is highly sensitive to serum nuclease digestion. An appropriate delivery system is necessary, as both
extracellular barriers (opsonins, phagocytes, extracellular
matrices, and degradative enzymes) and intracellular barriers (lack of proper recognition characteristics necessary
to direct intracellular transport, degradation within lysosomal compartments, and release from transport vesicles)
can prevent gene delivery, transcription, and translation
[46]. Therefore, gene therapy is only possible with an efcient carrier for protection and transportation.
Currently, developing a stable and efcient delivery
system is a major challenge for gene therapy. The optimal delivery strategy aims to improve the stability of
genes after their administration into the body, improve
gene pharmacokinetics and biodistribution, deliver genes
specically to the desired tissue site, reduce off-target
effects, facilitate the cellular uptake of genes within target cells, and promote efcient intracellular trafcking [7].
Gene delivery systems that are currently under investigation include both viral and non-viral carriers. While viral
carriers (retroviruses and adenoviruses, etc.) can produce
efcient expression of transgenes within cells of interest,
they also have several disadvantages, such as the possibility
of uncontrolled cell proliferation of transduced cells, high
immunogenicity, inammation of the transduced tissues,
random genomic integration, and severe limitations in the
size of foreign transgenes [8]. By contrast, non-viral carriers have low immunogenicity, greater adaptability, the
capacity to handle larger sizes of genes, and a potential for
large-scale manufacture. Nevertheless, the non-viral carriers have some problems in the areas of substantially low
gene expression and toxicity [9].
Non-viral carriers mainly consist of cationic lipids
and cationic polymers. To discriminate between them,
gene/cationic lipid complexes are termed lipoplexes and
1146
Table 1
Chemical structures of polycations for gene delivery.
PEI [16]
Chitosan [88]
gene/polycation complexes are termed polyplexes [10].

The cationic lipids have been extensively developed as
effective gene carriers in the form of liposomes [1113].
In addition, cationic polymers have also received attention for their potential use as non-viral gene delivery
agents [14]. Under certain conditions, the polycations
can self-assemble with negatively charged DNA or RNA
via electrostatic interactions, to condense the string-like
DNA or RNA into compact nanoparticles. The resulting polyplexes provide excellent protection for genes
from attack by extracellular nucleases and allow for
facile cellular uptake via an endocytic pathway. Polycations have some advantages over liposomes, due to
their: (i) relatively small size and narrow distribution, (ii)
high stability, and (iii) better exibility achieved simply
by varying the chemical composition, molecular weight
(Mw ), and architecture (linear, randomly branched, dendrimer, block, and graft copolymer, etc.) [4]. At present,
polyethylenimine (PEI, refers to branched PEI, unless otherwise noted) [1517], chitosan [1618], poly(l-lysine) (PLL)
[16,17], and poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) [16,17,19], among others, have been
developed for effective gene delivery. The chemical structures of PEI, chitosan, and PLL are shown in Table 1.
To date, the efcacy of cationic polymers for gene delivery is less than ideal, because of the low transfection
efciency and undesirable toxicity. To address these issues,
various modications have been explored to improve the
gene delivery efcacy [16,17]. Among them, the conjugation of hydrophobic segments to the polycations has
displayed promising results. As the rst step to entering
cells, the polyplexes must traverse through a hydrophobic
lipid-based plasma membrane. Lipid-containing carriers are suggested to have an enhanced compatibility
with the plasma membrane, and commercially available,
lipid-containing transfecting agents, INTERFERin and Lipofectamine 2000 are more efcient in delivering siRNA, in
comparison to jetPEI and Metafectene. Presumably, the
hydrophobic moieties could enhance the complexplasma
membrane interactions, and facilitate the endocytosis [7].
In addition, hydrophobic segments such as linear fatty
acids [20,21], lithocholic acid [20], cholesterol [2023], and
-tocopherol (Vitamin E) [24] have been conjugated to
siRNA to enhance gene delivery [25,26]. Toward this end,
cationic polymers have also been substituted with various
hydrophobic molecules to improve gene delivery activity
(see Tables 25 ). Like electrostatic forces, hydrophobic
PLL [16]
interactions play an important role in the gene delivery

process, and the introduction of hydrophobic chains can
affect not only the interaction with the plasma membrane,
but interactions at most steps during the whole gene delivery process.
In this review, we discuss the hydrophobic modications that have crucial effects to the gene delivery process.
By understanding the role played by the hydrophobic
chains, along with the specic hydrophobic modications in the common polycations, new and more effective
designs of gene carriers may be possible.
2. Effects of hydrophobic modications on gene
delivery
Studies have shown that lipid components can signicantly alter some of the physicochemical and biological properties of polycations used in gene delivery
[27]. Specically, the key steps involved in polyplexmediated gene delivery include compacting genes, binding
to the cell surface, cellular uptake, escape from the
endosomallysosomal network, translocation to the cell
nucleus, and vector unpacking. The hydrophobic segments
that are conjugated to polycations may inuence some of
these continuous steps, as discussed below.
2.1. Increased physical encapsulation of genetic
materials
Besides electrostatic forces, hydrophobic interactions
may also contribute to the complex formation between
genes and hydrophobized polycations, as the incorporation of hydrophobic moieties might cause a cooperative
binding of genetic materials [28]. In a study of siRNA delivery with hydrophobically oleic and stearic acid-modied
PEIs, Alshamsan et al. [7] proposed that electrostatic interactions might not be the only mechanism by which the
modied PEIs form complexes with siRNA. Based on their
zeta potential results, the expected increase in the net surface charge proportional to the PEI ratio was observed
only with the PEI/siRNA complexes. With the modied
PEIs/siRNA, and despite the increasing PEI ratio in the formulation, the variability in surface charge was attributed
to the relatively exible three-dimensional conformation
of the grafted fatty acids. The exibility of the fatty acids
is able to create a non-uniform surface charge distribution on the particles, leading to the unpredictable response
1147
Table 2
Chemical structures of hydrophobic molecules for PEI conjugations.
Hydrophobic molecules
Structures of activated hydrophobic molecules
Ref.
Carboxylic acid NHS ester
[67]
1-Iodododecane 1-iodohexadecane
[48]
Palmitoyl chloride
[35]
Fatty acyl chloride
[7,31]
1,1 -Carbonyldiimidazole activated Pluronic
[38,7678]
Cyanuric chloride activated PPOPEO
[72]
Folate NHS ester
[5457]
PEI-poly(-benzyl l-glutamate)
[49]
Cholesteryl chloroformate
[7983,85]
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Table 2 (Continued )
Ref.
Aldehyded PEG-cholesterol ether
[84]
Cholest-5-en-3-oxyethane tosylate
[42]
Dexamethasone mesylate
[6264]
in an electric eld. Although a reduction in zeta potential

with increasing PEI content was not expected, it could be
explained by the incremental increases in the non-cationic
fatty acid content. Therefore, the exibility of the aliphatic
fatty acids was suggested to allow for physical encapsulation of siRNA, which could explain the superior condensing
and protective effect of the modied PEIs over native PEI,
despite the variable zeta potentials [7].
In addition, Masotti et al. [27] found that acylation
reduced the basicity of PEI (with the higher the percentage of substitution, the less basicity in the resulting
molecule), caused by conversion of some amino groups
from primary to secondary. Nevertheless, the reduced
basicity did not interfere with polyplex formation. No signicant differences were seen in the binding ability of
acylated PEI derivatives with respect to native PEI, likely
due to the strongly basic nature of PEI [27]. Moreover, the
presence of a hydrophobic poly(propylene oxide) (PPO)
block in the Pluronic-polycation conjugates enhanced the
association with DNA, which was strongly exothermic. In
contrast, the DNA interaction with the PPO-free conjugate was much weaker and signicantly less exothermic
[29]. The results of atomic force microscopy revealed that
stearyl-octaarginine, but not octaarginine, can completely
condense DNA into stable complexes, which can be highly
adsorbed to the cell surface and then highly internalized
[30].
Nevertheless, Neamnark et al. [31] reported that
the linear lipid substitution in PEI led to a reduced
tendency of the polymers to form complexes with
plasmid DNA. Reduced availability of free amine
groups and/or the steric hindrance due to lengthy
aliphatic chains were given as likely explanations for this

observation.
2.2. Promotion of complex charge inversion
Kuhn et al. [28] studied complex formation involving DNA and cationic amphiphilic molecules. As the
amphiphile was added to the DNA solution, cooperative
binding of the amphiphile to the DNA molecules took place.
At a transition point, a large fraction of the DNA charge was
neutralized by the condensed amphiphile. If the density of
the amphiphile was increased beyond this point, a charge
inversion of the DNA became possible. The density of the
amphiphile needed to reach the charge inversion depended
strongly on the hydrophobicity of the amphiphile. In particular, for sufciently hydrophobic amphiphiles, such as
some cationic lipids, the charge inversion could occur at
extremely low densities. In principle, the charge inversion
could allow the amphiphile/DNA complexes to approach
the negatively charged cell membrane, permitting the
transfection to take place [28].
2.3. Enhanced adsorption to cell membrane
Adsorption of amphiphiles to red blood cells (RBCs),
as a model of the mammalian cell membrane, has been
widely investigated [32], and hydrophobic interaction is
known to play a key role in the binding of amphiphilic
compounds to biological and articial lipid membranes.
Moreover, the adsorption can induce subsequent endocytosis. For instance, when monomeric poly(ethylene glycol)
(PEG)-cholesterol is added to cells, it immediately becomes
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Table 3
Chemical structures of hydrophobic molecules for chitosan conjugations.
Ref.
Dodecyl bromide
[91,92]
Hexadecyl bromide
[93]
EDC activated stearic acid
[98]
Palmitic acid NHS ester
[93]
(2-Dodecen-1-yl)succinic anhydride
[9497]
4-Dimethylaminocinnamaldehyde,
4-dimethylaminobenzaldehyde and
4-pyridinecarboxaldehyde
[99,100]
EDC activated deoxycholic acid
[101104]
5-Cholanic acid NHS ester
[105]
Cholesteryl chloroformate
[106]
distributed in the cell membrane. In nucleated cells, very

high concentrations of PEG-cholesterol induced planarization of membrane invaginations, including caveolae and
clathrin-coated pits. In nucleated cells, stress appears
to be accommodated by endocytic sequestration of the
expanded surface area [33,34].
Generally, the uptake of polyplexes into cells is said to
be by adsorptive endocytosis [5]. In this way, the hydrophobic modications of polycations should enhance their
adsorption to the cell membrane, and facilitate adsorp-
tive endocytosis [6]. Fig. 1 shows the cellular entry of

hydrophobized polycation/gene complex through nonspecic adsorptive endocytosis. Incani et al. [35] found
that the impartment of palmitic acids to PEI and PLL led
to a higher binding of the hydrophobized polymers to
bone marrow stromal cells, in comparison to native polymers, in a degree of substitution (DS)-dependent way [35].
PEI-bearing pendant hexyl or dodecyl chains interacted
strongly with phospholipids, whereas the unmodied
polymer did not [36]. The presence of long, lipophilic sub-
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Table 4
Chemical structures of hydrophobic molecules for PLL conjugations.
Ref.
Myristoyl chloride, Palmitic acid NHS

ester, Oleic acid NHS ester
[35,44,107,108]
4-Nitrophenyl chloroformate activated

Pluronic
[111]
Folate NHS ester
[52,53]
stituents on PEI would therefore be expected to strengthen

the interaction of PEI/DNA complexes with cell membrane, enhance the endocytosis of complexes, and, in
turn, increase the transfection efciency. Also, stearyloctaarginine, but not octaarginine, was found to completely
condense DNA into stable complexes that could be highly
adsorbed to the cell surface, with high internalization [30].
Nevertheless, Masotti et al. found that hydrophobic chemical grafting, regardless of the percentage of substitution,
did not affect the ability of polyplex to migrate (translocate)
into cells [27].
Recently, in a study of polymer adsorption to RBCs,
Liu et al. [37] measured the adsorption of a hydrophobized hyperbranched polyglycerol-PEG copolymer to RBCs
(used as a mammalian cell membrane model). The results
showed that, at 1 mg/ml in saline, the amount of binding
of the stearoyl chains bearing copolymer to the RBCs was
1.7 105 molecules/cell, which is 2 orders of magnitude
higher than that of the unhydrophobized copolymer. The
stearoyl chains played a key role in the high adsorption of
the hydrophobized copolymer. Microscopically, the binding of the hydrophobized copolymer to the cell membrane
caused a marked change in cell morphology.
2.4. Alleviation of serum inhibition
Fig. 1. Cellular entry of hydrophobized polycation/gene complex through

non-specic adsorptive endocytosis.
Serum is rich in anionic proteins that can interact with

positively charged polycations. These interactions result
in the adsorption of serum proteins on polyplexes, and
their subsequent, diminished endocytosis and gene expression. Serum was found to inhibit the transfection efciency
of cationic carriersthe higher percentage of serum, the
more inhibition. In addition, the conjugation of polycations with amphiphilic Pluronic resulted in copolymers
that were capable of forming stable complexes in serum
[38]. Furthermore, the addition of free Pluronic to PEI/DNA
complexes counter-acted the reduction of serum-mediated
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Table 5
Chemical structures of hydrophobically modied polycations.
Polyamidoamine-triamcinolone acetonide conjugate [66]
Fluorphore-conjugated poly(amidoamine) [116]
Dodecyl-polyamidoamine (generation 4) [117]
Polyamidoamine bearing phenylalanine or leucine [118]
pDMAEMA-Pluronic conjugate [40]
Poly(N-isopropylacrylamide
(IPAAm)-co-2-DMAEMA-co-butylmethacrylate (BMA)) [43]
DMAEMA-PAA-BMA copolymer [121]
N-Oleyl-dextran-spermine conjugate [41123]
gene transfer [39]. The free Pluronic also dramatically

increased the transfection activity of the Pluronic L92pDMAEMA conjugate and reduced the serum-mediated
inhibition of DNA transfer. As little as 0.005% Pluronic
resulted in a great increase in transfection activity, due to
the shielding of the conjugate/DNA complexes from the
serum. The shielding may be the result of the protective
effect of a steric barrier that Pluronic assembled with the
complexes toward the complexserum interaction [40].
In a study by Eliyahu et al., the transfection activity
of dextran-spermine conjugate decreased with increasing serum concentration in a concentration-dependent
manner, reaching 95% inhibition at 50% serum in cell

growth medium. In contrast, oleyl derivatives of the
dextran-spermine conjugate actively transfected at 50%
serum. Moreover, higher transfection activity was achieved
using higher amounts of the oleyl moiety in derivatives.
Enhanced transfection of N-oleyl-dextran-spermine in
serum-rich medium is likely due to the interference of the
hydrophobic oleyl moieties in the DNA approach toward
the cationic polymer. The interference may cause a weaker
association of DNA with N-oleyl-dextran-spermine, making the subsequent dissociation of DNA from the complex
easier [41]. In addition, the greater serum compatibility of
1152
PEI-cholesterol-based lipopolymers might be explained by

cholesterol grafting, as the presence of cholesterol moieties could prevent serum proteins from dissociating the
lipopolymer/DNA complexes [42].
The serum inhibition of gene delivery could be
attributed to the effects of each serum component. As mentioned in Section 2.3, in the study of polymer adsorption to
RBCs, Liu et al. [37] also studied the effect of plasma or two
plasma components (albumin or brinogen) on the amount
of adsorption. Interestingly, plasma could wash away most
of the bound hydrophobized polymers from RBCs. Moreover, albumin (but not brinogen) was found to be the
main plasma component that caused the marked desorption of the bound polymers from the cell membrane. The
effect of albumin on the desorption was suggested to be
due to the hydrophobic interaction between the hydrophobic pockets of albumin and the hydrophobic stearoyl chains
of the modied polymer. In the same way, with high concentration (3050 mg/ml) and with hydrophobic pockets,
albumin could also play a key role in the serum inhibition of gene delivery. From the above results, we infer
that albumin could bind the hydrophobized cationic polymers through hydrophobic interactions, thus, reducing the
adsorption of these gene carriers to the cell membrane.
This could also explain the above results where the addition of free Pluronic alleviated the serum inhibition of gene
transfer. When free Pluronic was added, albumin could
bind the free Pluronic and hence bind fewer hydrophobized polycations. In this way, the free hydrophobized
polycations/gene complex could adsorb to the cell membrane without interference from albumin. Other serum
components may also be affecting gene delivery in similar
ways.
2.5. Facilitation of gene dissociation from polycation
carriers
The contrary needs for tight complex formation outside
a cell, and easy dissociation of the complex inside the cell,
is a challenge for non-viral vectors. The hydrophobic interaction between hydrophobic units is expected to inhibit
the dissociation of carrier/gene complexes to a much lesser
degree than would the ionic interactions between cationic
carrier and anionic gene. The favorable characteristics of
the hydrophobic units may lead to a higher transfection
efciency, compared to polymer systems that only use ionic
interactions [43]. When PLL was partially derivatized with
fatty acyl chains of varying length, researchers reported
an additional increase in transfection efciency (approx.
1.32-fold). The enhanced expression activity could conceivably be due to decreased electrostatic interactions
between the acylated PLL and DNA, thereby allowing the
internalized DNA to dissociate from the acylated PLL and
enter the nucleus more easily [44]. In addition, Park et
al. [45] reported that the gene delivery efciency of PEI
(25 kDa) increased upon acetylation, by as much as 21-fold
with 43% modication of the primary amines. Further characterization of the polymer/DNA interactions showed that
acetylated PEIs were bound less strongly to DNA, to suggest that the increased transfection may be the result of
enhanced dissociation of polyplexes inside cells [46].
2.6. Effects on cytotoxicity

Fischer et al. investigated in vitro cytotoxicity of polycations to determine the inuence of polycation structure on
cell viability and hemolysis [47]. Among the polycations
tested, PEI and PLL were found to be the most toxic. The
magnitude of the cytotoxic effects, for all polycations, was
found to be time- and concentration-dependent. The Mw ,
type of amino functions, and the cationic charge density
of the polycations were conrmed as key parameters for
the interaction with the cell membrane, and consequently,
for causing cell damage. Furthermore, characterization of
the cell death induced by the PEI and PEI/DNA complexes
suggested a necrotic type of death [47]. To date, the mechanism of cytotoxicity caused by polycations is not yet fully
understood.
The effects of hydrophobic modications of polycations on cytotoxicity have not been investigated in detail.
Whether cytotoxicity is increased or decreased by the
incorporation of hydrophobic chains is still controversial.
More information about the structure-toxicity relationship
is necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems. Thomas
et al. [48] studied the cytotoxicities of unalkylated and
alkylated PEIs, and found that the alkylated PEIs displayed no or reduced cytotoxicity, compared to the parent
PEI. Tian et al. [49] also reported reduced cytotoxicity of PEI after introducing a biocompatible hydrophobic
poly(-benzyl l-glutamate) moiety, and hypothesized that
the poly(-benzyl l-glutamate) block shielded the high
positive charge density on primary amine groups of
PEI.
Some authors have reported negative effects of
hydrophobic modications, in terms of cell toxicity, in
comparison to the parent polymers. Eliyahu et al. [41]
found that the incorporation of an oleyl moiety to a
dextran-spermine conjugate resulted in increased cytotoxicity, when compared to dextran-spermine. Neamnark et
al. [31] reported an increased cytotoxicity of PEI 2kDa as
a result of lipid substitution, which was expected from
the design of polymers with improved cell interactions.
Similarities in the toxicities of modied PEIs, for both
primary bone marrow stromal cells and 293T cells, suggests that the cytotoxicities were not cell-specic, and
likely involved common mechanisms, such as disruption
of the plasma membrane [31]. Caution should thus be
taken when introducing hydrophobic chains so as to minimize undesired effects and maximize desired effects. In
some cases, as little as 1 lipid per PEI was sufcient to
signicantly improve the delivery capability of the native
PEI [31].
From these results, and from considering some of the
known interactions of lipid-containing substances with the
cell membrane, we infer that the cytotoxicity induced by
hydrophobic modications depends on the DS, hydrophobicity, and/or the chemical structure of the incorporated
hydrophobic segments. From a review on membrane
actions of anesthetics and tranquilizers [32], the adsorption of these lipophilic substances at certain levels could
cause hemolysis. In addition, the RBC membrane becomes
seriously crenated in solutions with a high concentration of
hydrophobized polymers (PEG-cholesterol [34] or stearoyl

chains-bearing polymer [37]). Liu et al. [37] also showed
that low amphiphilic polymer concentration (lower than
1 mg/ml, or low adsorption of 104 molecules/cell) would
not cause RBC crenation or hemolysis. Thus, optimization
of the DS, hydrophobicity, and/or chemical structures of
hydrophobic segments of the polycations is necessary to
avoid introducing cytotoxicity while improving the efciency of gene delivery.
2.7. Targeting-specicity
The targeted delivery of genes to desired cells and
tissues could greatly improve gene expression in the
target tissue, minimize uptake by non-target tissues
and potential systemic toxicity, and reduce the dosage
required to achieve a desired therapeutic effect. Specic
interaction between a ligand and its receptor generally
enhances the cellular uptake by receptor-mediated endocytosis [25,50]. Moreover, cell-specic ligands, including
aptamers, antibodies, growth factors, sugar molecules,
vitamins, transferrin, and hormones, have been used to
confer cell specicity in gene delivery systems.
Coincidently, two common cell-specic ligands (folate
and glucocorticoid) are also hydrophobic molecules. The
folate receptor is a glycophospholipid-anchored membrane protein responsible for internalizing folic acid and
folate analogs, and is over-expressed in a wide variety
of human tumors, including >90% of ovarian carcinomas.
Also, the normal tissue distribution of the folate receptor
is highly restricted, making it a useful marker for targeted
drug delivery to tumors. Folate has been conjugated to PLL
[5153] and PEI [5457].
The glucocorticoid receptor is an intracellular receptor, regulating gene expression in the presence of specic
ligands. In the presence of glucocorticoid, the receptor
binds to the ligand and the receptor-ligand complex is
translocated into the nucleus [58]. Nuclear pore complexes
have been reported to be dilated by the glucocorticoid
receptor in the presence of dexamethasone (chemical
structure shown in Table 2.), a potent glucocorticoid steroid
[59,60]. This dilation effect is considered to be favorable for the translocation of polyplexes into the nucleus,
and gene transport into the nucleus was found to be
facilitated when dexamethasone was conjugated to polycations such as poly(amidoamine) dendrimer [61] and PEI
[6264]. Five glucocorticoids (betamethasone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone),
with different structures and potencies, have also been
conjugated to PEI [65], and the same authors synthesized
conjugates of poly(amidoamine)-triamcinolone acetonide
(a glucocorticoid of high potency) (chemical structure
shown in Table 5.) for efcient translocation of pDNA into
the nucleus [66].
In these studies, the enhanced transfection efciency
was attributed to the specic ligand-receptor-mediated
endocytosis. Little analysis was performed on the potential effects of the hydrophobicity of these ligands on gene
delivery. We speculate that these hydrophobic ligands contributed to not only ligand-receptor-mediated endocytosis
but also to other important effects, as discussed above.
1153
3. Hydrophobic modications of PEI

PEI, a linear or branched synthetic polycation, is one
of the most effective, commercially available gene delivery polymer and has been used both in vitro and in vivo
[15,16]. It is an attractive carrier for intracellular gene
delivery because of its well-established ability to condense nucleic acids via electrostatic interaction between
anionic DNA or RNA and the cationic primary, secondary,
and tertiary amines of the polymer. In addition to its
DNA-condensing properties, this polymer possesses significant endosomolytic activity because of its strong buffering
capacity at acidic endosomal pH (known as the protonsponge effect). Due to the presence of primary (25%),
secondary (50%), and tertiary (25%) amino groups that are
amenable to diverse and selective chemical modications,
PEI can be easily functionalized by a variety of hydrophobic modications to optimize its gene delivery activity.
The hydrophobic molecules used for PEI modications are
shown in Table 2.
3.1. Linear alkyl chains for PEI modications
Fatty acids and linear alkyl chains can be readily activated and conjugated to polycations. Several factors such
as alkyl chain length, DS, and alkylation position can also
affect the gene delivery efciency of modied polycations.
3.1.1. Effects of chain length and DS
Forrest et al. [45] found that acetylated PEI with DS
of 43% induced up to a 21-fold enhancement in transfection efciency in the C2C12 cell line and an 8-fold
transfection increase in the MDA-MB-231 cell line, compared to unmodied PEI. The authors also found that gene
delivery activity continued to increase (up to 58-fold in
HEK293) with acetylation of up to 57% of the primary
amines, but decreased with higher amounts of acetylation [46]. Doody et al. [67] conjugated acetate, butanoate,
and hexanoate to PEI (25 kDa), and found that substitution of primary amines generally increased the transfection
efciency, relative to unconjugated PEI, but increasing
DS beyond 25 mol% generally decreased the transfection efciency from the optimum. Additionally, increasing
hydrocarbon length generally decreased the transfection
efciency [67]. Nimesh et al. [68] prepared various acylated PEI derivatives (750 kDa) by treating them with acetic,
propionic, and butyric anhydrides, which were further noncovalently cross-linked to obtain nanoparticles. Among
the samples studied on the COS-1 cell line, the highest
expression level was found with the 30% propionoylated
derivative (i.e., 12-fold for nanoparticles prepared from PEI
750 kDa). The transfection efciency was found to depend
on the acyl chain length and the degree of acylation. In a
myristate-conjugated PEI series, Kim et al. [69] reported
that decreasing the number of myristate substituted on PEI
increased the transfection.
Incani et al. [35] designed PEI-palmitic acid conjugates for gene carriers for bone marrow stromal cells. The
palmitic acid substitution did not improve the capacity of
PEI to act as a DNA carrier. The authors further evaluated
oleic acid- and stearic acid-modied PEI derivatives for
1154
siRNA delivery in vitro [7]. With B16 melanoma cells, the

modied PEIs were superior or comparable to some commercially available transfection agents (3-fold increase of
siRNA delivery, compared to the parent PEI; 5-fold greater
delivery than that of jetPEI and Metafectene; delivery comparable to that of Lipofectamine 2000; 1.6-fold decrease in
delivery, in comparison to INTERFERin). In addition, stearic
acid-modied PEI displayed a superior ability over oleic
acid-modied PEI to condense, protect, and deliver siRNA,
possibly because of the chemical structure of stearic acid.
The free-rotation property of the saturated carbon atoms
in stearic acid would be expected to give the molecule
more exibility to move inward or project outward from
the complex [7].
Despite these results, it is still too early to draw conclusions about the specic effects of alkyl chain length or DS on
gene delivery efciency. Moreover, the alkyl chain length
and DS may also affect other properties of hydrophobized
polycations, such as cytotoxicity, biocompatibility, and in
vivo circulation time. Optimal chain length and DS should
be selected by considering overall effects of hydrophobic
modications on the gene therapy.
3.1.2. Effects of alkylation position
To clarify the structure-activity relationship, Thomas
et al. [48] alkylated PEIs with two Mw (25 or 2 kDa) with
dodecyl or hexadecyl substitution. Partial alkylation of the
primary amino groups with dodecyl and hexadecyl halides
markedly decreased the transfection efciency of PEI25
(Mw = 25 kDa) in the absence or presence of serum. Nevertheless, the quaternization of the tertiary amino groups of
the PEI25 with hexadecyl iodide resulted in higher transfection efciency, compared to that of the hexadecylated
PEI25 on the primary amino groups, and in the presence of
serum, an even greater efciency was found in comparison
to the native PEI25.
These results indicate that the position of alkylation
(primary vs. tertiary nitrogen) has a profound effect on the
transfection efciency. Thomas et al. [48] proposed that, in
PEI, with its tertiary amines quaternized, the hydrophobic hexadecyl substituents were expected to be located
toward the interior of the polycation. In contrast, in PEI,
with its primary amino groups alkylated, the hydrophobic substituents should be situated on the periphery of the
polymer and, to avoid the thermodynamically costly exposure to water, should undergo clustering. These features
may be responsible for the observed differences in transfection efciency.
In contrast to PEI25, primary amine alkylation of PEI2
(Mw = 2 kDa) dramatically increased the transfection efciency [48]. Moreover, in the presence of serum, dodecyland hexadecyl-PEI2s were 400 and 550 times, respectively,
more efcient than PEI2 itself.
3.1.3. Explanation for reduced transfection efciency
Some hydrophobic modications of PEI with linear alkyl
chains were reported to cause reduced transfection efciency. Brissault et al. [70] showed that, when all of the
amino nitrogens of a linear PEI were substituted with
propyl groups, the transfection efciency was strongly
reduced, compared to that of the native PEI, which may
have been at least partially due to an increased cytotoxicity

from the propyl substitution. Further, to combine polymer
properties with the lipid type of carriers [71], PEI (25 kDa)
was rst quaternized by methylation and then acylated,
using pendant palmitic acid chains, to create amphiphilic
PEI variants, which formed nanoparticles or vesicles. The
modications improved the materials biocompatibility
markedly but also reduced transfection efciency.
To explore the reasons for the reduced transfection,
Masotti et al. [27] conducted a physicochemical and
biological study of several hydrophobized PEI (25 kDa)
derivatives with three different hydrophobic chains (C12,
C14, and C16) and three fatty acid residues (lauroyl-,
myristoyl-, and palmitoyl-) at different DS. The authors
found that, the higher the DS, the smaller was the basicity of the resulting derivatives, due to conversion of some
amino groups from primary to secondary (and likely from
secondary to tertiary, in the highly substituted derivatives). In any case, the reduced basicity did not interfere
with the polyplex formation process, likely due to the
strong base nature of PEI (as a proton sponge). Hence, the
acidbase properties suggested that, once the complexes
penetrate into cells and the endosome enlarges, the DNA
release mechanism should be similar. In addition, all of the
derivatives efciently bound DNA even at low N/P ratios,
and no signicant differences with respect to PEI were
observed. The higher-substituted PEI derivatives displayed
similar biological properties (toxicity, translocation ability,
and transfection efciency) with respect to the lowersubstituted forms. Moreover, even a small DS (2% or 3%) was
sufcient to signicantly alter some of the physicochemical and biological properties. For example, a small DS (3%)
was enough to decrease the transfection efciency of these
compounds. Hydrophobic grafting to the PEI polyplexes
conferred them with a great stability against competitive
exchange reactions by polyanions. These results indicate
that the relative stability of hydrophobic PEI polyplexes is
likely correlated with their transfection efciency in vitro.
3.2. Pluronic for PEI modications
Pluronic , also termed Poloxamer or poly(ethylene
oxide)b-poly(propylene oxide)b-poly(ethylene oxide)
block copolymers (PEOPPOPEO) is a kind of amphiphilic
non-ionic water soluble polyether. Pluronic block copolymers are recognized as pharmaceutical excipients, listed
in the US and British Pharmacopoeia. Its amphiphilicity
stems from the hydrophobic PPO block and the hydrophilic
PEO blocks. Pluronic can facilitate the formation of polyplexes with a core-shell structure, in which the shells are
composed of PEO chains and ionized amino groups, while
the cores are comprised of PPO blocks and neutralized
PEI/DNA complexes [29]. It was reported that the presence
of the hydrophobic PPO block in Pluronic tended to enhance
the stability of the complex between polycation and DNA
[72]. The PPO chains in the Pluronic molecules are known
to interact with the lipid membrane to induce structural
rearrangements. Furthermore, due to the presence of PPO
chains, Pluronic molecules are able to translocate within
cells, while the homopolymer PEO does not exhibit this
ability. The sticky elements, which, in essence, are simi-
lar to fusogenic sequences in viruses, allow for the effective

delivery of genetic material within a cell [73]. The application of Pluronic in gene delivery has been reviewed by
Lemieux et al. [74], Kabanov et al. [73], and Bromberg et al.
[75].
To improve the interaction with the cell membrane,
Kabanov et al. [38] synthesized a Pluronic 123-g-PEI (2 kDa)
conjugate for gene transfer. The Pluronic 123-g-PEI system
was less stable in dispersion, probably due to the effect of
the insoluble PPO chains in the Pluronic 123 moiety, but
could be stabilized in dispersion by the addition of excess
Pluronic 123. The transfection activity of the conjugate was
much lower than that of PEI (25 kDa) or of Superfect, but
it exhibited high transfection activity in vitro when mixed
with free Pluronic 123 (9:1, w/w) by forming small and stable complexes with DNA. Both copolymer components, the
Pluronic 123-g-PEI and free Pluronic 123, appeared to be
essential for the high transfection activity, because, when
formulated with the DNA separately, these components
did not improve the transfection [38]. The effect was also
reported by Bromberg et al. [40] that Pluronic P123 significantly increased the level of transfection when added to
a Pluronic L92-pDMAEMA conjugate composition, resulting in 1.7- and 1.3-fold higher expressions, in comparison
to the effects of Pluronic L92-pDMAEMA alone or Lipofectamine, respectively. In addition, some dependency was
seen on the order of adding the block copolymer components: the gene expression was the same or even slightly
increased when corresponding amounts of Pluronic 123
was rst added to cells for 1015 min, and followed by
the addition of the DNA and Pluronic 123-g-PEI complex
[38]. Kabanovs group further found that the free Pluronic
component of this gene delivery system enhanced DNA
uptake and expression primarily as a result of optimization of the size and morphology of the polyplex. The free
Pluronic did not appear to contribute signicantly, alone, to
enhance DNA uptake or transgene expression, or to protect
from nuclease degradation. The authors also pointed out a
potential limitation of this system for in vivo applications,
in the insufcient level of protection of plasmid DNA from
nuclease degradation [76].
Pluronic 123-g-PEI was shown to modify the biodistribution of plasmid DNA toward the liver [77]. Pluronic
123-g-PEI-formulated plasmids, carrying the gene encoding for the murine ICAM-1 molecule, were injected i.v.
into transgenic ICAM-1-decient mice. Pluronic 123-g-PEI
induced a dose-dependent expression of ICAM-1 in the
liver. Furthermore, this expression of ICAM-1 induced neutrophil invasion in the liver, while no such invasion was
observed in mice injected with formulated control plasmids or naked DNA. These results suggested that Pluronic
123-g-PEI allowed for the functional transgene expression in the liver following i.v. injection [77]. In addition,
Belenkov et al. [78] grafted PEI to Pluronic to overcome the
limitation that PEI/DNA complexes were often poorly soluble. The results clearly showed that the copolymers/Ku86
antisense oligonucleotide complexes were highly stable in
aqueous dispersions, even upon complete neutralization of
charge. These complexes retained transfection ability in the
presence of serum. This was an obvious improvement compared with the native PEI, which formed with DNA either
1155
insoluble complexes or soluble complexes that were highly

sensitive to media composition [78].
Bromberg et al. [72] attached PPOPEO polyether to
PEI and guanidinylated the conjugate. The resulting guanidinylated PEI-polyether conjugates efciently complexed
plasmid DNA. The chemical modication of the PEI structure by the polyether facilitated the interaction with DNA,
probably because of the enhanced solubility of the polyplexes that made the amino groups of the copolymer
more accessible to nucleotides. The polyplexes possessed
enhanced colloidal stability in the presence of serum proteins, indicating the steric repulsion of the serum proteins
by the PEO segments of the polyether in the outer shell
of the polyplex particles. In vitro studies demonstrated
improved transfection efciency of the conjugates. The
guanidinylation and the conjugation with the polyether
enhanced the interaction of conjugates with genetic material and reduced the cytotoxicity of the polyplexes with a
L929 cell line, due to the presence of PEO chains, which
partially shielded the positive charges of the polyplexes
[72].
3.3. Cyclic hydrophobic molecules for PEI modications
Cholesterol is a naturally occurring lipidic steroid found
in the cell membrane, and is transported in blood plasma
and metabolized in the body of all animals. It is an essential
component of the mammalian cell membrane, where it is
required to establish proper membrane permeability and
uidity.
To combine the advantages of cholesterol and PEI,
Mahato et al. [79] synthesized water-soluble lipopolymers
by conjugating cholesterol to PEI (1.8 kDa) for enhanced
delivery of the interleukin-12 gene. The lipopolymers
formed micelles in water with a critical micellar concentration (CMC) of 1.43 mg/ml. Unlike PEI, the lipopolymers
likely interacted with the gene through both ionic and
hydrophobic interactions. Moreover, micellization of the
lipopolymers may concentrate a positive charge on the
surface, and thus, PEI of high Mw may not be required.
The lipopolymer/pDNA (immune modulating cytokine,
interleukin-12 expression plasmid) complexes, or the
lipopolymer itself, were non-toxic to CT-26 colon adenocarcinoma or to 293 T human embryonic kidney
transformed cells when formulated at an N/P ratio of
10/1 or less. In contrast, PEI (25 kDa)/pDNA complexes
were toxic to these cells. The lipopolymer/pDNA complexes
demonstrated higher transfection efciency in both CT-26
and 293 T cells, in comparison to PEI 25 kDa- or PEI 1.8 kDabased formulations [79,80]. The authors also investigated
the organ distribution and anti-tumor response in vivo after
intratumoral administration of the interleukin-12 expression plasmid in complex with the lipopolymer. The organ
distribution data demonstrated an enhanced retention of
the complexes within tumors and limited accumulation in
other organs for up to 96 h [81]. The lipopolymer was also
examined for potential applications delivering siRNA that
targeted vascular endothelial growth factor (VEGF) in vitro
and in vivo. The lipopolymer was complexed with siRNA
designed to inhibit human VEGF expression, or with scrambled siRNA as a control. The lipopolymer protected siRNAs
1156
from enzymatic degradation in serum-conditioned media.

VEGF production was efciently inhibited by the complexes, while the complexes of lipopolymer with scrambled
siRNA did not show this inhibitory effect. The lipopolymer/siRNA complexes reduced the VEGF production by
40%, when compared to unmodied PEI 1.8 kDa. Moreover,
the complexes reduced tumor volume by 55% at 21 days,
and by 65% at 28 days, when compared to controls [82]. In
the above studies, cholesteryl chloroformate was directly
linked to PEI through primary amines, which are important for efcient DNA condensation. Further, the primary
amines of PEI were rst blocked with benzyloxycarbonyl to
allow conjugation to occur through the secondary amines.
Also, only one cholesterol molecule was grafted onto each
PEI molecule, thus leaving enough space for steric interactions of the PEIs primary amines with the DNA. The CMCs
of cholesterol-PEI 1.8 kDa and cholesterol-PEI 10 kDa were
0.497 and 1.33 mg/ml, respectively. The acidbase titration
indicated a high buffering capacity of the lipopolymers,
with a range of pH from 5 to 7, indicating the possibility of
efcient endosomal release. Efcient condensation of the
plasmid DNA could be achieved using these lipopolymers.
The in vitro transfection of the lipopolymers in Jurkat cells
showed high levels of gene expression with little toxicity
[83].
By considering the efcient unpacking of a viral protein
shell, a novel, cholesterol-tethered bioresponsive PEI was
specially designed via a disulde-containing cross-linker.
The cholesterol lipid was added to increase the permeability of a gene vector through the cell membrane. The
cholesterol-tethered polycation could effectively induce
DNA condensation and form spherical particles with
diameters of about 200 nm at an N/P ratio of 10/1.
At a glutathione concentration of 3 mM (within the
range of intracellular concentrations), the polyplexes were
unpacked due to the bioresponsive cleavage of the disulde bonds. The in vitro experiment indicated that the
polyplexes had efcient transfection to the HEK293T cells
[84]. Additionally, linear PEI 25 kDa was modied with
cholesterol in three different geometries: linear-shaped,
T-shaped, and a combined linear-/T-shaped. These complexes were also effective in protecting the pDNA for up to
180 min in the presence of DNase I. B16-F0 cells, transfected
with linear- and linear-/T-shaped PEI-cholesterol conjugates showed protein expression levels that were higher
than that of the linear PEI alone, and twice that of PEI,
but without any signicant loss in cell viability. The different rate of transfection of the linear PEI-cholesterol/pDNA
complexes was due, in part, to conformational changes
from the point of complex formation to the interaction
with the plasma membrane. These conformation changes
provided protection for unprotonated secondary amines in
the linear PEI backbone by hydrophobic protection of the
cholesterol moiety termed, unprotonated reserves [85].
Furthermore, Tian et al. [49] synthesized a watersoluble copolymer of PEI with hydrophobic poly(-benzyl
l-glutamate) segments at the chain ends, which had much
lower toxicity than PEI, effectively condensed pDNA into
particles, and signicantly protected pDNA from enzymatic
degradation. The copolymer/DNA tended to form smaller
nanoparticles than those formed by PEI/DNA, as a result
of the hydrophobic segments compressing the particles in

aqueous solution. The in vitro transfection efciency of the
copolymer/pDNA complexes was improved in HeLa cells,
Vero cells, and 293T cells, in comparison to that of PEI
25 kDa [49].
4. Hydrophobic modications of chitosan
Chitosan is a natural cationic polysaccharide consisting
of 14 linked N-acetyl-d-glucosamine and d-glucosamine
subunits. It is derived from chitin by alkaline hydrolysis,
which is obtained from the shells of crustaceans, cuticles
of insect, and the cell wall of fungi and yeasts. Chitosan has
been widely studied in the eld of biomaterials in virtue
of its biocompatibility, biodegradability, bioadhesiveness,
non-toxicity, non-immunogenicity, and antibacterial and
antifungal bioactivity [86]. In particular, chitosan is well
known to adhere to mucosal surfaces and can transiently
open tight junctions between epithelial cells. In practice, chitosan has been used to deliver proteins, vaccines,
drugs, and genes [87,88]. Due to the presence of numerous hydroxyl and amine groups, chitosan can be chemically
modied to improve its bioactivity [18,89]. As shown in
Table 3, various hydrophobic molecules have been used to
create hydrophobic modications of chitosan to improve
its gene delivery functions.
4.1. Linear alkyl chains for chitosan modications
Thanou et al. [90] synthesized trimethylated chitosan from oligomeric chitosan (520 monomer units) and
compared it with DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium sulphate) for gene delivery.
Trimethylated chitosan/DNA complexes were shown to
transfect COS-1 cells, but to a lesser extent than
DOTAP/DNA lipoplexes. The trimethylated derivatives
appeared to be superior to the native chitosan. The presence of serum did not affect the transfection efciency
of the trimethylated chitosan/DNA complexes, whereas
serum decreased the efciency of the DOTAP/DNA complexes. Both DOTAP/DNA lipoplexes and trimethylated
chitosan/DNA complexes resulted in a decreased transfection efciency in Caco-2 cell cultures, in comparison to
COS-1 cells; however, trimethylated chitosan was superior
to DOTAP.
Alkylated chitosans were prepared by modifying chitosan with alkyl (butyl, octyl, dodecyl, and hexadecyl)
bromide [91,92]. The complex formation between alkylated chitosans and DNA required a relatively smaller
amount of alkylated chitosans compared to that of chitosan. Chitosan and alkylated chitosans could cause the
fusion of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
multilamellar vesicles and membrane destabilization. Furthermore, by introducing alkyl side chains, alterations in
the topological structure of DPPC were more evident. The
transfection efciency of chitosan and alkylated chitosans
into C2C12 cell lines depended on the hydrophobicity of
the chitosan. With a longer alkyl side chain, the transfection efciency increased, leveling off after the number
of carbons in the side chain exceeded eight. The higher
transfection efciency of the alkylated chitosans was pre-
sumably due to the increased entry into cells, facilitated

by hydrophobic interactions and the easier unpacking
of DNA from the alkylated chitosan carriers, caused by
the hydrophobicity-induced weakening of the electrostatic
attraction between cargo and carriers [92]. In addition,
Uchegbu et al. [93] hydrophobically modied glycol chitosan with palmitoyl or hexadecyl, and in some cases,
additional N-methyl quaternary ammonium groups. Cytotoxicity studies showed that the biocompatibility of glycol
chitosan was adversely affected by the combination of a
palmitoyl group and depolymerisation, but biocompatibility was subsequently restored with the introduction of
N-methyl groups. Degree of polymerisation was found to be
the most important controller of the transfection efciency.
To impart oligochitosans with a surfactant-like structure and properties, Zhang et al. [9496] prepared
hydrophobized N-[2(3)-(2-dodecenyl)succinoyl chitosans
to improve their efciency as non-viral transfection vectors. The chitosan derivatives interacted with negatively
charged lipid liposomes, thus mimicking the internal layer
of the plasmid membrane. Unlike cationic surfactants,
the chitosan derivatives exhibited difculties in lateral
and transverse diffusion with respect to the membrane,
resulting in their low toxicity. Moreover, the prevailing
interaction of the chitosan derivative with the inner leaet
of the biomembrane would lead to a destabilization of
the endosomal membrane with liberation of DNA into the
cytoplasm [95,96]. The chitosan derivative with 3 mol%
substitution showed high transfection efciency in vivo
via systemic administration. Its efciency was even better than that of PEI, which is a rather efcient vector for in
vivo applications. Moreover, only the chitosan derivative
(3 mol%)/pDNA complexes were efciently internalized,
mainly through endocytosis [97].
A stearic acid-grafted chitosan oligosaccharide was
synthesized by a 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC)-mediated coupling reaction [98],
which formed micelles by self-aggregation in aqueous
solution. The CMC of the derivative with DS of 15.4%
was about 0.035 mg/ml. Due to the cationic property, the
micelles were studied as gene carriers. The transfection
of the derivative was not hindered by the presence of
10% fetal bovine serum, which showed a remarkable
enhancement effect. The optimal transfection efciency
of the micelles in A549 cells was about 15%, which is
higher than that of chitosan oligosaccharide (about 2%),
and comparable to that of LipofectamineTM 2000 (about
20%) [98].
4.2. Cyclic hydrophobic molecules for chitosan
modications
Three kinds of methylated chitosan derivatives, containing different aromatic moieties: i.e., methylated N(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4pyridinylmethyl) chitosan chloride (MPyMeChC), were
synthesized by two steps (reductive amination followed by
methylation). The transfection efciencies of these complexes were investigated using human hepatoma cells
1157
(Huh 7 cells), with comparisons to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efciencies was MPyMeChC > MDMBzChC > TMChC > MDMCMChC
[99]. The results indicated that the improved gene transfection of MDMBzChC was due to the hydrophobic
group (N,N-dimethylaminobenzyl) substitution on chitosan, which promoted the interaction and condensation
with DNA, as well as N-quaternization, which increased
chitosan water solubility and enhanced gene expression
[100].
Chitosan oligosaccharides were also chemically modied with hydrophobic deoxycholic acid [101104],
5-cholanic acid [105], and cholesterol [106]. The
hydrophobized chitosan derivatives formed nano-sized
self-aggregates (core-shell structure) in aqueous environments. They were characterized as being efcient gene
delivery systems.
5. Hydrophobic modications of PLL
PLL is a homo-polypeptide of the essential amino acid
l-lysine. Due to its cationic property, PLL has been widely
used as a non-viral gene carrier since the primary -amine
groups of lysine in PLL, which are protonated in a physiological environment, can interact electrostatically with
negatively charged DNA or RNA to form polyelectrolyte
complexes [16]. As shown in Table 4, many hydrophobic
molecules have been used for the hydrophobic modications of PLL to improve its gene delivery functions.
5.1. Linear alkyl chains for PLL modications
To design effective gene carriers for bone marrow
stromal cells, PLL was substituted with palmitic acid via
amide linkages [35]. Depending on the reaction conditions, PLL was substituted with 13.416.2 palmitic acids
per polymer chain. The substituted PLL displayed a slightly
lower binding efciency towards plasmid DNA but demonstrated a much improved cell penetration. The cell binding
of PLL-palmitic acid was particularly enhanced, resulting in a higher percentage of cells displaying signicant
polymer uptake. The delivery of plasmid DNA into the
bone marrow stromal cells was also signicantly increased
with PLL-palmitic acid, compared to PLL. The transfection efciency of PLL-palmitic acid was signicantly higher
(ve-fold), compared to unmodied PLL, showing that
palmitic acid substitution on PLL provided an effective carrier with an efciency equivalent to that of an adenoviral
carrier [35]. The authors also compared the effectiveness of
PLL-palmitic acid conjugates to LipofectamineTM 2000 for
plasmid delivery to bone marrow stromal cells [107]. PLLpalmitic acid conjugates delivered plasmid DNA to 80% of
the cells, achieving a maximum transfection efciency of
22%. This was signicantly higher than LipofectamineTM
2000-mediated transfection, with an efciency of 11%
under most optimal conditions. The PLL-palmitic acid conjugates were also used to deliver plasmid DNA into human
skin broblasts in vitro [108]. Compared with PLL, PLLpalmitic acid conjugates delivered plasmid DNA into a
higher proportion of the cells. PLL-palmitic acid conjugates
were found to have a higher transfection efciency, com-
1158
pared to that of PEI, Lipofectamine 2000, or an adenovirus

that were investigated.
To explore structure-function relationships for gene
delivery, Abbasi et al. [109] synthesized lipid-substituted
PLLs by N-acylation of PLL with NHS esters of fatty acids
(NHS-X, where X = C8, C14, C16, C18, C18:1, C8:2). The
extent of lipid substitution was critical in reducing complex
dissociation in response to a heparin challenge and also correlated positively with intracellular plasmid DNA delivery.
PLL could not deliver plasmid DNA into cells, but PLL substituted with lipids (longer than caprylic acid) transformed
the polymer into an efcient DNA carrier. The transfection
of skin broblasts was most effective with PLLs substituted
with myristic and stearic acids. The authors also found
that carriers with high Mw and high lipid substitution had
increased cellular uptake, and generated signicant gene
expression with relatively low toxicity in bone marrow
stromal cells [110]. Myristic, palmitic, and stearic acidsubstituted PLLs gave the most effective DNA delivery,
but this was likely because of the high substitution ratios
obtained with these lipids (10 lipids/PLL). Even though
other lipids were not effective, it was speculated that they
could also be effective if sufciently substituted on PLL.
5.2. Pluronic for PLL modications
To develop a gene delivery vector with low cytotoxicity and high efciency, Jeon et al. [111] synthesized
PLL-g-Pluronic with Pluronic as a conjugating molecule, to
overcome the low transfection efciency of PLL. The highest transfection efciency among the vectors tested, was
achieved at a 1:1 weight ratio of polymer:DNA, and a 3fold increase in transfection efciency was achieved by
treatment with a lysosomotropic agent, chloroquine. Compared to unmodied PLL, PLL-g-Pluronic (specically at the
1:1 weight ratio of polymer:DNA) showed an approximate
2-fold increase in transfection efciency with a similar
cytotoxicity [111].
6. Hydrophobic modications of other polycations
Besides the above-mentioned polycations, other polycations have been used for gene delivery, including
polyamidoamine dendrimer [17,112], pDMAEMA copolymers [17], spermine, etc. The hydrophobic modications
of these polycations are shown in Table 5.
6.1. Hydrophobic modications of poly(amidoamine)
dendrimer
Poly(amidoamine) is a commercially available cationic
dendrimer. Due to its minimal cytotoxicity over a wide
range of concentrations, poly(amidoamine) dendrimer has
been evaluated for gene therapy [17,112,113]. To transform
nearly neutral polyamidoamine-OH (hydroxyl-terminated
polyamidoamine) to be an efcient gene carrier, Lee et al.
[114] introduced internal quaternary ammonium salt to
the tertiary amine of polyamidoamine-OH dendrimers by
methylation to provide binding sites for negatively charged
plasmid DNA. The quaternization not only increased the
ability to bind to DNA but also improved the cytotox-
icity of the carrier. In addition, Vuillaume et al. [115]

synthesized a series of linear and permanently charged
poly(amidoammonium) salts by chemical modication of
a parent poly(amidoamine), containing two tertiary amino
groups per structural unit: one incorporated into the main
chain and the other xed at the end of a short bismethylene
spacer. The permanent charges were introduced through
a quaternization reaction involving iodomethane or 1iodododecane as an alkylating agent. Only the complexes
formed from polycations with a quaternized backbone
were able to generate signicant gene expression, which
was putatively attributed to a better dened toroidallike morphology, and a higher stability. The incorporation
of dodecane side-chains on highly charged polycations
severely amplied the cytotoxicity and, in return, dramatically affected the transfection level.
To determine the sub-cellular biodistribution of
poly(amidoamine) dendrimer/nucleic acid complexes, Yoo
et al. [116] conjugated the dendrimers with uorescent
dye Oregon green 488, and surprisingly, found that the
conjugation with the uorophore signicantly enhanced
the dendrimers abilities as a delivery agent. As a possible explanation, the relatively hydrophobic uor moieties
may have enhanced the ability of the dendrimer to disrupt
the endosomal membrane, thus allowing for more trafc
to the cytosol and nucleus. Additionally, Takahashi et al.
[117] designed polyamidoamine dendron-based cationic
lipid by introducing two dodecyl chains, which could transfect cells efciently with the synergistic effect of endosome
buffering and membrane fusion with the endosome, both
promoting the plasmid DNA transfer from endosome to
cytosol. While the dodecyl-polyamidoamine (generation
1) did not transfect CV1 cells, the lipoplexes containing the dodecyl-polyamidoamine (generations 24) could
induce transfection of the cells, and their activity was elevated with increasing generations of the dendron. Thus,
coupling of small, relatively hydrophobic molecules to
poly(amidoamine) dendrimers may provide a useful means
for enhancing their capabilities as gene delivery agents.
Kono et al. [118] designed poly(amidoamine) dendrimers with phenylalanine or leucine residues at their
chain ends. As a consequence, efcient gene transfection was achieved through the synergistic effects of the
proton sponge effect, induced by the dendrimer tertiary amines, and the hydrophobic interaction caused by
the hydrophobic amino acid residues in the dendrimer
periphery. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by the reaction
of the amine-terminated poly(amidoamine) G4 dendrimer
and l-phenylalanine, using 1,3-dicyclohexylcarbodiimide
as the condensing reagent. The transfection activity of
these phenylalanine-modied dendrimers were evaluated
in CV1 cells (an African green monkey kidney cell line),
and the activity increased concomitantly with the increasing number of terminal phenylalanine residues, except
for the dendrimer with 64 phenylalanine residues, which
was poorly water soluble and hardly formed a complex
with DNA at neutral pH. Under weakly acidic conditions;
however, the dendrimer with 64 phenylalanine residues
formed a complex with DNA, which demonstrated highly
efcient transfection. In contrast, the attachment of l-
leucine residues was unable to improve the transfection

activity of the parent dendrimer, probably because of
the relatively lower hydrophobicity of this amino acid.
The phenylalanine-modied dendrimer exhibited a higher
transfection activity and a lower cytotoxicity than did some
widely used transfection agents. Thus, the phenylalaninemodied dendrimers were considered as promising gene
carriers [118].
6.2. Hydrophobic modications of DMAEMA copolymers
pDMAEMA is a water-soluble cationic polymer, which
can interact electrostatically with DNA to form stable polyelectrolyte complexes [17]. The tertiary amine groups of
pDMAEMA are partially protonated in a physiological solution because the average pKa of the amine groups is 7.5.
The transfection efciency and cytotoxicity of pDMAEMA
can be adjusted by copolymerization of DMAEMA and the
hydrophobic monomer units.
A water soluble terpolymer, N-isopropylacrylamide
(IPAAm)-co-DMAEMA-co-butylmethacrylate (BMA) was
synthesized by radical polymerization, and its efciency for
in vitro gene transfection was evaluated [43]. Transfection
efciency of a series of the copolymers, containing 20 mol%
of DMAEMA, varied with the hydrophobic BMA content.
The transfection efciency of the copolymers was low with
0, 2, and 5 mol% of BMA, but was about 2-fold higher with
10 mol% of BMA, when compared to the pDMAEMA control
homopolymer [43].
In an effort to mimic the viral endosomal escape mechanisms that trigger membrane destabilization at acidic
pH, polymers possessing pH-sensitive chemical functionalities, such as carboxylate groups, have been explored.
Poly(propylacrylic acid) (PPAA) undergoes a hydrophilicto-hydrophobic transition at endosomal pHs, mediating
membrane disruption [119]. The conformational shift is
triggered by the gradual protonation of carboxylic acid
residues along the polymer backbone and can be tuned
to occur at specic pHs by using copolymerization with
hydrophobic monomers [120]. Convertine et al. [121]
synthesized new diblock copolymers for siRNA delivery.
The diblock copolymers were composed of a positively
charged block of DMAEMA to mediate siRNA condensation, and a second endosomal-releasing block, composed of
DMAEMA and propylacrylic acid (PAA), in roughly equimolar ratios, together with BMA. These carriers became
sharply hemolytic in endosomal pH regimes, with increasing hemolytic activity seen as the percentage of BMA
in the second block was systematically increased. The
siRNA-mediated knockdown of a model protein, namely
glyceraldehyde 3-phosphate dehydrogenase, in HeLa cells
generally followed the hemolytic activity trends, with the
most hydrophobic second block (the highest BMA content)
exhibiting the best knockdown [121].
6.3. Hydrophobic modications of dextran-spermine
Spermine, a naturally occurring linear polyamine, is
involved in cellular metabolism and is found as a polycation
at physiological pH. In chromatin, tetra-amine spermine
helps to package DNA into cellular nucleus by neutral-
1159
ization of the polyanionic phosphate backbone charges,

bringing about DNA condensation, and assists in controlling DNA conformations [122]. In the eld of gene therapy,
spermine is often used as the DNA-binding portion of gene
vectors [6].
Hydrophobization of dextran-spermine was achieved
by treating the polymer with N-hydroxysuccinimide
derivatives of cholesterol and fatty acids, including C8C18
saturated fatty acids and oleic acid [123]. Although
hydrophobized polycations showed altered transfection characteristics, when compared to the unmodied
dextran-spermine, only the oleate-modied polycations
showed a signicant increase in the transfection efciency.
The improvement in cell transfection was attributed to
oleate residues, which likely increased the stability and
the uptake of polycation/DNA complexes [123]. To overcome the obstacle of serum inhibition, oleyl derivatives
of dextran-spermine (which form micelles in aqueous
phase) were synthesized at 1, 10, and 20 mol% (oleyl
moiety to -NH2 of the polymer) to form N-oleyldextranspermine [41]. Polyplexes that were based on
N-oleyl-dextran-spermine transfected well in medium
containing 50% serum; however, the transfection resulting
from the water-soluble dextran-spermine-based polyplexes decreased with increasing serum concentrations in
cell culture, in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium.
Preliminary results in zebra sh and mice demonstrated
the potential of N-oleyl-dextran-spermine for use as an
efcient non-viral vector for in vivo transfection [41].
7. Perspectives
From the above discussion, hydrophobic modications
of polycations have demonstrated promising efcacy in
gene delivery. Nevertheless, to take advantage of the
hydrophobic modications in preparing useful gene carriers, some key issues need to be considered, in particular:
(1) the physicochemical characteristics of hydrophobized polycation derivatives and polyplexes; (2) effects of
hydrophobic modication on each step of gene delivery;
(3) intracellular trafcking of hydrophobized polyplexes;
and (4) metabolism and elimination of the hydrophobized
polycations; among others. Moreover, biodegradable or
bio-responsive hydrophobic molecules are worth considering for their potential in creating polycation modications.
Acknowledgements
This work was nancially supported by the National
Natural Science Foundation of China (grant No. 50903039),
the National Natural Science Foundation of Guangdong
Province of China (grant No. 9451063201002459), Introduction of Talent Foundation of Jinan University of China,
and Sci-tech Innovation Foundation of the 211 Project
for Biomaterials and Tissue Engineering, Jinan University
of China.
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Hydrophobic Modifications of Cationic Polymers For Gene Delivery

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Progress in Polymer Science 35 (2010) 11441162

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Progress in Polymer Science

Hydrophobic modications of cationic polymers for gene delivery

Corresponding author. Fax: +86 20 85226663.

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

gene/polycation complexes are termed polyplexes [10].

interactions play an important role in the gene delivery

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Structures of activated hydrophobic molecules

Carboxylic acid NHS ester

Fatty acyl chloride

1,1 -Carbonyldiimidazole activated Pluronic

Cyanuric chloride activated PPOPEO

Folate NHS ester

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Structures of activated hydrophobic molecules

Aldehyded PEG-cholesterol ether

in an electric eld. Although a reduction in zeta potential

aliphatic chains were given as likely explanations for this

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Structures of activated hydrophobic molecules

EDC activated stearic acid

Palmitic acid NHS ester

EDC activated deoxycholic acid

5-Cholanic acid NHS ester

distributed in the cell membrane. In nucleated cells, very

tive endocytosis [6]. Fig. 1 shows the cellular entry of

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Structures of activated hydrophobic molecules

Myristoyl chloride, Palmitic acid NHS

4-Nitrophenyl chloroformate activated

Folate NHS ester

stituents on PEI would therefore be expected to strengthen

Fig. 1. Cellular entry of hydrophobized polycation/gene complex through

Serum is rich in anionic proteins that can interact with

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

Fluorphore-conjugated poly(amidoamine) [116]

Dodecyl-polyamidoamine (generation 4) [117]

Polyamidoamine bearing phenylalanine or leucine [118]

pDMAEMA-Pluronic conjugate [40]

DMAEMA-PAA-BMA copolymer [121]

N-Oleyl-dextran-spermine conjugate [41123]

gene transfer [39]. The free Pluronic also dramatically

manner, reaching 95% inhibition at 50% serum in cell

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

PEI-cholesterol-based lipopolymers might be explained by

2.6. Effects on cytotoxicity

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

hydrophobized polymers (PEG-cholesterol [34] or stearoyl

3. Hydrophobic modications of PEI

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

siRNA delivery in vitro [7]. With B16 melanoma cells, the

have been at least partially due to an increased cytotoxicity

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

lar to fusogenic sequences in viruses, allow for the effective

insoluble complexes or soluble complexes that were highly

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

from enzymatic degradation in serum-conditioned media.

of the hydrophobic segments compressing the particles in

Z. Liu et al. / Progress in Polymer Science 35 (2010) 11441162

sumably due to the increased entry into cells, facilitated