Professional Documents
Culture Documents
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
5.
6.
7.
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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
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Table 1
Chemical structures of polycations for gene delivery.
PEI [16]
Chitosan [88]
PLL [16]
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Table 2
Chemical structures of hydrophobic molecules for PEI conjugations.
Hydrophobic molecules
Ref.
[67]
1-Iodododecane 1-iodohexadecane
[48]
Palmitoyl chloride
[35]
[7,31]
[38,7678]
[72]
[5457]
PEI-poly(-benzyl l-glutamate)
[49]
Cholesteryl chloroformate
[7983,85]
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Table 2 (Continued )
Hydrophobic molecules
Ref.
[84]
Cholest-5-en-3-oxyethane tosylate
[42]
Dexamethasone mesylate
[6264]
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Table 3
Chemical structures of hydrophobic molecules for chitosan conjugations.
Hydrophobic molecules
Ref.
Dodecyl bromide
[91,92]
Hexadecyl bromide
[93]
[98]
[93]
(2-Dodecen-1-yl)succinic anhydride
[9497]
4-Dimethylaminocinnamaldehyde,
4-dimethylaminobenzaldehyde and
4-pyridinecarboxaldehyde
[99,100]
[101104]
[105]
Cholesteryl chloroformate
[106]
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Table 4
Chemical structures of hydrophobic molecules for PLL conjugations.
Hydrophobic molecules
Ref.
[35,44,107,108]
[111]
[52,53]
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
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Table 5
Chemical structures of hydrophobically modied polycations.
Polyamidoamine-triamcinolone acetonide conjugate [66]
Poly(N-isopropylacrylamide
(IPAAm)-co-2-DMAEMA-co-butylmethacrylate (BMA)) [43]
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(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-
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