Current Opinion in Solid State and Materials Science 12 (2008) 89–102

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Current Opinion in Solid State and Materials Science

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Synthetic polymeric vectors in gene therapy

Patit P. Kundu *, Vinay Sharma
Department of Polymer Science & Technology, University of Calcutta, 92, A. P. C. Road, Kolkata 700009, WB, India

a r t i c l e i n f o a b s t r a c t

Article history: The synthetic polymer vectors in gene therapy have opened a very prospective field for research in gene
Received 9 October 2008 therapy and the area in the use of these synthetic polymers as vectors in targeting oncogenes is develop-
Accepted 14 January 2009 ing very fast. With the completion of Human Genome Project, the list of genetic targets is growing very
fast. These diseases sparked the initiative to create such gene based therapeutics. The low levels of trans-
fection and expression of the gene held non-viral methods are at a disadvantage; however, recent
Keywords: advances in vector technology have yielded molecules and techniques with transfection efficiencies, sim-
Polymeric vectors
ilar to those of viruses. Amongst these synthetic polymers, polyethylenimine (PEI) and the poly(amido-
Gene therapy
Non-viral therapy
amine) (PAMAM) dendrimers and poly(b-amino ester) are used extensively as vectors in gene therapy.
pDNA Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Gene therapy may be defined as the treatment of human dis-
ease by the transfer of genetic material into specific cells of the
patient [1]. The advances in molecular biology and biotechnology
of the last 30 years have greatly enhanced the understanding of
the genetics of pathogenesis and have led to the identification of
numerous disease-causing genes. With the impending completion
of the Human Genome Project, the list of genetic disease targets is
likely to grow tremendously. It is not difficult to predict treatment
of genetic diseases such as hemophilia [2], muscular dystrophy
[3–5], or cystic fibrosis [6] through replacement of errant genes
within the affected cells. However, the gene therapy approaches
are also being developed for treatments of virtually all forms of
cardiovascular diseases [7], neurological diseases [8–10], infectious
diseases [11], wound healing [12], and cancer [13–15]. In these in-
stances, the delivered genes may be intended to augment naturally
occurring proteins (e.g growth factors or cytokines), to alter the
expression of existing genes facilitating a desired cellular or tissue
response, or to produce cytotoxic proteins or pro-drug-activating
enzymes, for example, to kill tumor cells in the cancer treatment
[13,15] or proliferating endothelial cells to inhibit restenosis
following balloon angioplasty [7,16]. The expression of viral genes
within certain tissues can also result in an efficient immune
response, leading to the development of DNA vaccines [17].
The gene carriers for these systems are of two kinds: one based
on recombinant viruses and another based on synthetic polymers.
The primary function of a virus is to efficiently carry its own gen-
ome from one host cell to another through (perhaps) hostile envi-
* Corresponding author.
E-mail address: ppk923@yahoo.com (P.P. Kundu).
ronments, enter the new target cell, navigate to the cell nucleus,
and initiate expression of its genome-albeit for the purpose of
self-replication. Viruses can be transformed into gene delivery
vehicles by removing part of the virus genome and replacing it
with a therapeutic gene. If done carefully, the resulting recombi-
nant virus will retain the functions essential for infection of spe-
cific target cells, but will be rendered incapable of replication
and, hence, will be nonpathogenic. The viruses evolved essentially
are sophisticated gene delivery vehicles and such recombinant
viral vectors are incredibly efficient.
Although viruses are incredibly efficient gene delivery agents,
synthetic vectors provide opportunities for improved safety, greater
flexibility, and more facile manufacturing. In general, synthetic
vectors are materials that bind electrostatically to DNA or RNA,
condensing the genetic material into nanometer-scale complexes
(a few tens to several hundred nanometers in diameter) that pro-
tect the genes and allow them to enter cells. Such materials have
included cationic peptides, proteins, polymers, and liposomes. Var-
ious synthetic vectors, including (diethylamino)ether (DEAE)-dex-
tran [18] and calcium phosphate [19], have been used extensively
for in vitro gene transfer studies since 1960s. However, develop-
ment of non-viral vectors for in vivo gene delivery, especially clin-
ical applications, has suffered from problems including toxicity,
low gene transfer efficiency, and in vivo instability. Despite the
high efficiency of viral vector in vitro, clinical trials are often lim-
ited by several concerns, e.g. toxicity, immunogenicity, inflamma-
tory properties, the limited size of the DNA, production and
packing problems, and the high cost. In addition, an overwhelming
immune reaction against adenovirus occurred in a patient at Penn-
sylvania University in 1999 [20,21] and a leukemia-like disease
were reported in a French patient in 2002 [22,23] As a result,
non-viral vector-mediated systems have become of interest,

1359-0286/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cossms.2009.01.005
90 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
because they are much safer, more cost-effective, and easier to
manufacture than viral vector systems.
Non-viral vectors for gene delivery are receiving increasing
attention for application in a broad variety of gene-mediated ther-
apies for humans. Chemical vectors are attractive to the pharma-
ceutical industry as alternatives to viral vectors, because of
compound stability and easy chemical modification. Furthermore,
the low cost and consistent standard of production (compared to
growth of viruses in bioreactors followed by purification), higher
bio-safety (less immunogenic as compared to viruses such as
adenoviruses), and a high flexibility (once a formulation has been
defined) make these compounds very attractive [24,25].
To design the optimal non-viral vector for particle-mediated
gene delivery, it must be able to partially fulfill a number of prede-
fined biological criteria depending on its specific therapeutic aim.
First, the vector must be able to transfer DNA molecules of varying
sizes. Second, the vector should be able to transducer dividing and/
or non-dividing cells, depending on the target cell type. Third, the
vector should target a specific cell type. Fourth, the vector should
have no or very low toxicity in vivo and avoid stimulating the
immune system. Fifth, depending on the therapeutic application,
the vector should be able to induce a sustained expression over a
defined period of time (optionally integrating into the host gen-
ome) and, finally, the vector must not transform the target cell.
2. Polymer characteristics required for gene delivery
For effective polyplex-mediated gene delivery, the cationic
polymer carrier has to fulfill a series of drug delivery functions in
the extracellular and intracellular transport of the DNA vector
(see Fig. 1). The polymer has to compact DNA into particles of
virus-like dimensions that can migrate through the blood circula-
tion into the target tissue, it has to protect the DNA from degrada-
tion and against undesired interactions with the biological
environment, to facilitate target cell binding and internalization
[26–30], ideally in a target cell specific manner [31,32], endosomal
escape [33], trafficking the cytoplasmic environment [34], and
localizing into the nucleus [35,36] as well as vector unpacking
[37]. In addition, the polymer should be nontoxic, non-immuno-
genic, and biodegradable. In reality, no polymer is able to carry
out all the extracellular and intracellular delivery functions; there-
fore additional functional elements have to be included into the
polyplex formulation (see Fig. 1).
Factors influencing DNA binding affinity which are inherent to
the chemical structure of the polymer (intrinsic properties) are:
(i) the number of charge groups per single polymer; (ii) the type
of charge groups (e.g. primary, secondary, quaternary amino
groups, amidine groups); (iii) the spacing of charge groups within
the polymer; (iv) the degree of branching in the polymer back-
bone; and (v) hydrophobicity of the cationic carrier. In addition,
Fig. 1. The extracellular and intra cellular transport of DNA vector.
the ‘‘external/extrinsic” conditions of the microenvironment, such
as: (vi) the ionic strength of the polyplex solution; (vii) the concen-
tration and (viii) positive/negative charge ratios of polymer and
DNA; and (ix) the technical process of polyplex formation (i.e.
kinetically vs. thermodynamically controlled process) strongly
influence the polyplex formation. The latter extrinsic factors pro-
vide some flexibility in selecting the most appropriate conditions
for polyplex formation. However, it has to be kept in mind that
finally upon administration the physiological environment will
dictate the stability and fate of polyplexes; therefore the proper
intrinsic polymer characteristics in DNA binding and polyplex for-
mation are the dominating issues. In regard to the required num-
ber of positive polymer charges, systematic studies demonstrated
that a minimum length of six to eight cationic amino acids (lysine,
arginine) is required to compact DNA into polyplex structures,
active in gene delivery in vitro [38].
The number is presumably higher when in vivo application is
considered. DNA binding can be driven by application of higher
polymer to DNA charge ratios. This can be nicely monitored by aga-
rose gel retardation assay or ethidium bromide exclusion assay
[39]. Depending on the size and affinity of the polycation, this
may also result in increased net positive polyplex charge, which
promotes cellular uptake and transfection efficiency. However, this
also results in toxicity, due to destabilization and loss of integrity
of cellular membranes, and the presence of excess free polycation.
In addition, the excess positive charge on the polycations may hin-
der the transfection efficiency because unpacking of the complexes
and release of the DNA is difficult to achieve if the binding is very
strong, which results in a low transfection efficiency [40]. The
influence of charge group type [41] and spacing [42] was evaluated
by Davis and colleagues in a carbohydrate-containing polycation
series. They demonstrated that both the distance between the
carbohydrate unit and the charge groups in the backbone, and also
the types of amino group (quaternary amines vs. amidine group)
are the primary factors that influence the carrier’s transfection effi-
ciency in vitro.
3. Polymers vectors
Many types of polymers have been specifically designed for use
as gene delivery vectors. In many cases, the polymers were de-
signed to address one of the perceived gene delivery barriers; for
example, DNA packaging and stability in vivo, biocompatibility,
and endosomal escape have been used as design criteria. The
results of such studies have been mixed, with some polymers per-
forming as well as, or even slightly better than, polyethylenimine
(PEI) and the poly(amidoamine) (PAMAM) dendrimers. The differ-
ent synthetic polymers used for non-viral delivery are discussed
hereunder.
3.1. Polyethylenimine (PEI) based vectors
Synthetic polymers have been used extensively in tissue engi-
neering, drug delivery, and gene therapy [43,44]. PEI is one of the
most used polymer for gene delivery [45–49]. Hornsby and
coworkers [50] covalently attached PEI to polylysine based films
using the bifunctional reagent Bis-(sulfosuccinimidyl) suberate
(BS3), which cross-links amino groups and was also attached to
the collagen-coated cell culture polystyrene. After attaching PEI
to the polymer films, they measured the amount of PEI attached
to the films. Plasmid pEGFP-N1 was used to test the transfection
from PEI-coated films. The plasmid contained the complementary
DNA (cDNA) for enhanced green fluorescent protein (GFP) under
the control of the cytomegalovirus promoter. It was observed that
the transfection continued for several days and only the covalently
attached PEI was capable of transfection.
 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Blessing et al. [51] synthesized a conjugate of PEI covalently
modified with epidermal growth factor (EGF) peptides. EGF recep-
tor is an attractive therapeutic target for tumor targeting due to its
high percentage in human carcinomas. They found it very useful
for the high transfection ability with lower DNA doses, which is
very essential for in vivo applications. These EGF containing DNA
complexes were specifically taken by the EGF receptor-mediated
endocytosis pathway. The reversibly cross-linked PEI was used
for non-viral gene transfer by Lee et al. [52]. They successfully
administered in vitro gene delivery of primary amines [dithi-
obis(succinimidylpropionate) (DSP) or dimethyl-3,30 -dithiobispro-
pionimidate.2HCl (DTBP)] cross-linked PEI to Chinese hamster
ovary (CHO) cells. The transfection efficiencies were evaluated in
CHO cells by using a luciferase reporter gene under a cytomegalo-
virus (CMV) promoter. The proposed transfection scheme using
reversibly cross-linked low molecular weight PEI is shown in
Fig. 2. The scheme also shows the endocytosis and subsequent
endosome release and the intracellular reduction of polymer disul-
fide bonds leads to the release of DNA for the nuclear uptake and
transcription. The cross-linking of low molecular weight PEI with
homobifunctional amine reactive cross-linking agents to form high
molecular weight conjugates resulted in effective transfection of
CHO cells in vitro. It is observed that the DSP–PEI conjugates out-
perform the DTBP-PEI conjugates at similar cross-linking ratios.
The star-block copolymers were reported via a facile synthetic
route using hexamethylene diisocyanate as linker between
poly(ethylene glycol) (PEG) and PEI blocks by Kissel and coworkers
[53]. They first characterized the block copolymers obtained via
NMR, FTIR, TGA, DSC, capillary viscometry, and size exclusion chro-
matography coupled with multiple angle laser light scattering
(SEC-MALLS). The copolymer–DNA complexes were also character-
ized via atomic force spectroscopy (AFM), dynamic light scattering
(DLS), laser Doppler (LDA), and DNA condensation assay tech-
niques. Kissel and coworkers [54] also synthesized copolymers of
PEI and N-(2-hydroxyethyl)-ehylene imine (HEEI) to study the
effect of the polymer structure on the physicochemical and biolog-
ical properties of DNA complexes. Polymers with a higher PEI con-
tents and hence higher branching yielded higher reporter gene
Fig. 2. The transfection scheme using reversibly cross-linked low molecular weight
PEI.
91
expression than polymers with more HEEI. There was increase in
transfection efficiency, which was correlated with higher ability
of DNA binding and condensation and formation of the small sized
complexes. It was also observed that the polymer structure also
influenced the basicity and protonation of copolymers and lead
to different DNA binding, complex size, cytotoxicity and transfec-
tion efficiency.
The improvement in the stability of the polycationic vectors by
dextran-grafted branched PEI was reported by Tseng and Jong [55].
Dextrans of molecular weight 10,000 (dex-10,000) and 1500 (dex-
1500) were used to produce various degrees of grafting on linear
and branched PEI, and the dextran-grafted polymers were used
to prepare DNA–polymer complexes. The changes in size and in
f-potential and the extent of DNA release after the exposure of
the complexes to bovine serum albumin (BSA) were used to evalu-
ate the stability of the complexes prepared at various ratios of DNA
to polymer. Only the use of dextran-grafted branched PEI was
found to be effective to improve the stability of the complexes in
the presence of BSA. Dex-10,000 was noted to provide a slightly
better shielding than dex-1500 against the aggregation caused by
BSA and helped to maintain the sizes within 200 nm and the
f-potentials close to neutral. Fig. 3A and B showed the extent of
DNA being condensed by the dex-g-PEI. When DNA was condensed
after the addition of polymer, the intercalating dye was excluded
out of the DNA double helix, causing a reduction in fluorescence
intensity [56]. The percentage of uncondensed DNA was calculated
as the fluorescence intensity after the addition of polymer divided
by that before the addition of polymer. When N/P ratio exceeded 6,
Fig. 3. Percentage (mean±SD; n = three independent preparations of complex) of
uncondensed DNA within the complexes prepared with dextran-grafted and
unmodified PEIs at different charge ratios. (A) The amount of uncondensed DNA
for using linear PEIs. The estimated degrees of grafting on each linear PEI are 4 (4)
and 10 (h) molecules of dex-1500, and one (N) and two (j) molecules of dex-
10,000, respectively. (B) The amount of uncondensed DNA for using branched PEIs.
The estimated degree of grafting on each branched PEI are 5 (N), 22 (j), and 45 ()
molecules of dex- 10,000, and 10 (4), 42 (h), and 76 (}) molecules of dex-1500,
respectively. In both panels the solid and dashed lines represent the unmodified
and dextran-grafted PEIs, respectively.
92 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102

the percentages of uncondensed DNA approached different asymp-

totes, irrespective of the forms of polymer.
The extent of condensation, however, varied with different
forms of polymers. Branched PEI condensed DNA more efficiently
than linear PEI, presumably because of the strong interactions be-
tween DNA and the tertiary amines on branched PEI. The grafting
of dextran promoted the ability of DNA condensation of linear
PEI, for example, a reduction in the percentage of uncondensed
DNA from 30% to 15% at N/P ratio of 6 (Fig. 3A). For dex-g-linear
PEI, the various degrees of grafting and the different molecular
weights of dextran used in this study showed roughly the same ex-
tent of DNA condensation. For branched PEI, the grafting of dextran
slightly decreased percentage of uncondensed DNA at N/P ratios
below 2, but had little effect on the promotion of DNA condensa-
tion at other N/P ratios (Fig. 3B). For those branched PEIs that
grafted more than 40 molecules of either dex-1500 or dex-
10,000, the extents of DNA condensation decreased in comparisons
with the use of unmodified branched PEI.
Davis et al. [57] studied the cyclodextrin modified PEI polymers
for gene delivery. Linear and branched poly(ethylenimines), lPEI,
and bPEI, respectively, grafted with b-cyclodextrin are prepared
to give CD-lPEI and CD-bPEI, respectively, and were investigated
as in vitro and in vivo non-viral gene delivery agents. The in vitro
toxicity and transfection efficiency were sensitive to the level of
cyclodextrin grafting. The cyclodextrin-containing polycations,
when combined with adamantane-poly(ethylene glycol) (AD-
PEG) conjugates, form particles that were stable at physiological
salt concentrations. PEGylated CD-lPEI-based particles generated
an in vitro gene expression equal to or greater than lPEI as mea-
sured by the percentage of Enhanced Green Fluorescent Proteins
(EGFP) expressing cells. Tail vein injections into mice of 120 lg
of plasmid DNA formulated with CD-lPEI and AD-PEG did not
revealed observable toxicities, and both nucleic acid accumulation
and expression were observed in liver.
Pack et al. [58] studied the effect of acetylation of PEI on the
gene delivery through weakened polymer–DNA interactions. The
effects of increasing the degree of acetylation elucidated the source
of the higher gene delivery efficiency. Despite a reduced buffering
capacity, gene delivery activity continued to increase (up to 58-fold
in HEK293) with acetylation of up to 57% of primary amines but
decreased at higher degrees of acetylation. Characterization of
polymer–DNA interactions showed that acetylated polymers
would bind less strongly to DNA. Further, a fluorescence resonance
Fig. 4. In vitro transfection efficiency. Polyplexes of plasmid DNA (pGL3) with
energy transfer assay showed that increasing acetylation caused unmodified or acetylated PEI were used to transfect (A) MDA-MB-231, (B) C2C12,
polyplexes to unpackage inside cells to a higher degree than poly- and (C) HEK293 cells. Luciferase activity in the cell lysates is reported as relative
plexes formed with unmodified PEI. Overall, the data suggested light units (RLU) normalized by the mass of total protein in the lysate. (N) 3; error
that the increased gene delivery activity might be attributable to bars represent standard deviation.

an appropriate balance between polymer buffering capacity and

strength of polymer/DNA interactions.
The transfection efficiency of the acetylated polymers was
tested on three model cell lines: MDA-MB-231 human breast car-
cinoma cells, HEK293 human embryonic kidney cells, and C2C12
mouse myocytes (Fig. 4). Cells, transfected with the luciferase-
encoding plasmid (pGL3) in the absence of any polymer, exhibited
negligible luciferase expression. Unmodified PEI mediated rela-
tively efficient gene delivery in each of the cell lines. The highest
luciferase expression consistently occurred using 2:1 (w:w) PEI:D-
NA in all three cell lines, and higher mass ratios had little effect
(1:1 and lower mass ratios exhibited statistically significant reduc-
tion of gene delivery activity; not shown in the figure). In another
publication, the same group reported that gene delivery efficiency
increased with the degree of acetylation up to 43% [59]. That trend
appeared to continue up to 57% acetylation, but gene delivery de-
creased with further acetylation in all three cell lines. Specifically,
the transfection efficiency of PEI-Ac57 (3:1 w:w) was approxi-
mately 47-, 58-, and 30-fold higher than unmodified PEI (2:1
w:w) in MDA-MB-231, HEK293, and C2C12 cells, respectively. At
higher degrees of acetylation, gene delivery efficiency decreased,
but the optimal polymer/DNA ratio increased (e.g., 4:1 w:w was
optimal for PEI-Ac96 in all three cell lines and 5:1 w:w was the
best ratio tested for PEI-Ac100 in MDA-MB-231 and C2C12 cells).
The optimal degree of acetylation suggests that there may be a bal-
ance between two competing effects of acetylation.
Bae et al. [60] studied the tumor specific gene delivery system
using pH sensitive sulfonamide/PEI polymers. Plasmid DNA was
complexed with polyethyleneimine (PEI) and further with a pH
sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine)
(PSD)-block-PEG (PSD-b-PEG), to obtain nano particles. The shield-
ing/deshielding of nano particles was tested along with cell viabil-
ity and transfection efficiency at physiological and tumor pH. The
nano particles composed of DNA/PEI/PSD-b-PEG were 300 nm in
size and showed low cytotoxicity and transfection at pH 7.4 due
to shielding of PEI by PSD-b-PEG. The PSD-b-PEG bound to PEI/
DNA complex decreased the interaction of PEI positive charges
 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Fig. 5. Targeting based on difference in pH: (a) shows formation of the nanoparticle
complex through charge–charge interaction between DNA, cationic polymer (PEI),
and PSD-b-PEG; (b) shows the complex shielded at physiological pH and deshielded
at cancer pH.
with cells and reduced the cytotoxicity by 60%. At pH 6.6, the nano
particles showed high cytotoxicity and transfection, indicating
PSD-b-PEG detachment from the nano particles and permit PEI to
interact with cells. PSD-b-PEG was able to distinguish the small dif-
ference in pH. The polymeric nano particle formed through electro-
static attraction was designed in such a way that the final particle
remained neutral. The neutral particle had the least interaction
with the body and would be more stable compared to anionic
and cationic particles, during systemic circulation. The central idea
of this design was that when the particles experience a decrease in
pH as they extravasated into tumor tissue due to enhanced perme-
ability and retention effect, the sulfonamide groups would lose
their charge and got detached from the carrier complex. The
detachment of sulfonamide from the complex exposed pCMV-
Luc-DNA condensed with cell transfecting PEI, to act on cancer
cells. The scheme is shown in Fig. 5.
Pun and Burke [61] studied the extracellular barriers to in vivo
PEI and PEGylated PEI polyplex-mediated gene delivery to the
liver. The unpackaging of polyplexes by serum proteins, soluble
glycosaminoglycans, and an extracellular matrix extract was dem-
onstrated by a YOYO-1 fluorescence quenching assay. Additionally,
exposing polyplexes to serum or proteoglycans before in vitro
transfection caused a decreased cellular uptake of DNA. Lastly,
PEI polyplexes and PEGylated PEI polyplexes were injected into
the portal vein of mice, and the intrahepatic distributions of
labeled DNA and polymer were assessed by confocal microscopy.
PEI polyplexes delivered DNA to the liver, but extensive vector
unpackaging was observed, with PEI primarily colocalized with
the extracellular matrix. PEGylated polyplexes mediated less
DNA delivery to the liver, possibly due to premature vector
unpackaging in the blood. It was observed that both the blood
and the extracellular matrix have significant extracellular barriers
to polyplex-mediated in vivo gene delivery to the liver.
3.2. Polyamidoamine (PAMAM) based vectors
Polyamidoamine (Starburst) dendrimers are spheroidal, cascade
polymers that can be synthesized from an ammonia or ethylenedi-
amine core by successive addition of methyl acrylate and ethylene-
diamine [62]. The size of the polymer, and importantly the surface
charge, are controlled by varying the number of generations in the
synthesis. Haensler and Szoka [63] originally reported the use of
PAMAM dendrimers for gene delivery. Uekama et al. [64,65] re-
ported the conjugates of polyamidoamine with cyclodextrin. The
starburst polyamidoamine dendrimer conjugates with a-, b-, and
93
c-cyclodextrins (CDE conjugates), expecting the synergistic effect
of dendrimer and cyclodextrins (CyDs) were synthesized [64].
The CyDs were covaletly bound in a 1:1 molar ratio (confirmed
by 1H NMR) to the dendrimer. The agarose gel electrophoretic
studies confirmed that the CDE conjugates formed the complexes
with plasmid DNA (pDNA) and protected the degradation of pDNA
by DNase I in the same manner as dendrimer. CDE conjugates
showed a potent luciferase gene expression, especially in the den-
drimer conjugate with a-CyD (R-CDE conjugate), which provided
the greatest transfection activity (approximately 100 times higher
than those of dendrimer alone and of the physical mixture of den-
drimer and a-CyD) in NIH3T3 and RAW264.7 cells. In addition, the
gene transfer activity of a-CDE conjugate was superior to that of
lipofectin. The enhancing gene transfer effect of a-CDE conjugate
might be attributable to not only increasing the cellular associa-
tion, but also changing the intracellular trafficking of pDNA. These
studies indicated that a-CDE conjugate, had great advantages as
non-viral vectors, i.e., an easy preparation, superior transfection
efficiency, and less cytotoxicity.
Griffiths et al. [66] studied the mechanism of poly(amidoamine)
as endosomolytic polymer and correlated the physicochemical and
biological properties. Bioresponsive poly(amidoamine)s (PAA)s are
currently under development as endosomolytic polymers for
intracellular delivery of proteins and genes. Small-angle neutron
scattering (SANS) was used to systematically investigate the pH-
dependent conformational change of an endosomolytic polymer,
the PAA ISA 23. The radius of gyration of the ISA23 was determined
as a function of pH and counterion, the aim being to correlate
changes in polymer conformation with membrane activity as-
sessed using a rat red blood cell hemolysis assay. With decreasing
pH, the ISA23 radius of gyration increased to a maximum
(Rg  80 Å) at around pH 3, before subsequently decreasing once
more. At high pH and high ionic strengths, the polymer was nega-
tively charged and adopts a rather compact structure (Rg < 20 Å),
presumably with the dissociated carboxylic groups on the exterior
of the polymer coil. At low pH, the coil again collapses (Rg < 20 Å),
presumably due to the effects of the high ionic strength. It was
concluded that the nature of the salt form had no direct bearing
on the size of the polymer coil, but it did indirectly determine
the prevailing pH and, hence, polymer conformation. Pulsed-gradi-
ent spin-echo NMR measurements were in good agreement with
the SANS estimates of the radius of gyration.
Szoka et al. [67] used degraded PAA dendrimers for in vitro gene
delivery. They transfected cultured cells using complexes between
DNA and spherical cationic polyamidoamine polymers (Starburst
dendrimers) that consist of primary amines on the surface and ter-
tiary amines in the interior. A 50-fold increase in the transfection
activity of the dendrimers was observed by heat treatment in a
variety of solvolytic solvents, e.g., water or butanol. This treatment
induced significant degradation of the dendrimer at the amide
linkage, resulting in a heterodisperse population of compounds
with molecular weights ranging from the very low (<1500 Da) to
several tens of kilodaltons. The compound facilitating transfection
was the high molecular weight component of the degraded prod-
uct and was denoted as a fractured dendrimer. Transfection activ-
ity was related both to the initial size of the dendrimer and the
degree of degradation. Fractured dendrimers exhibited an increase
in apparent volume as measured by an increase in the reduced
viscosity upon protonation of the terminal amines as pH was
reduced from 10.5 to 7.2, whereas intact dendrimers did not re-
duced. Dendrimers with defective branching were also synthesized
and also showed improved transfection activity compared to that
of the intact dendrimers. For a series of heat-treated dendrimers,
a correlation between transfection activity and the degree of flex-
ibility was also observed. The transfection activity (relative to the
untreated dendrimer) of acetic anhydride titrated dendrimers (fifth
94 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Fig. 6. Transfection activity (relative to the untreated dendrimer) of acetic
anhydride titrated dendrimers (fifth generation ammonia core). Complexes were
formed and added to CV-1 cells cultured in 96-well trays as described in the
Experimental procedures. The pCMV-bGal plasmid DNA was held constant at 0.6 lg
per well, and the ratio of all dendrimer terminal groups (modified or not) to DNA
phosphates was varied from 0.5 to 24. The maximum activity level, regardless of
terminal group to phosphate ratio used, is reported. Control sample is the
unmodified dendrimer; the 0% sample underwent the same treatment as the other
samples, but in the absence of acetic anhydride. Data reported are the mean and
range of duplicate wells in one run which was representative of three similar
experiments.
generation ammonia core) are shown in Fig. 6. The primary amine
content of the fifth generation ammonia core dendrimer was var-
ied by direct reaction with acetic anhydride. Ninhydrin analysis
showed nearly stoichiometric addition by acetic anhydride to ter-
minal amines. Transfection by these amine-titrated dendrimers
showed a significant decrease in activity for a 25% reduction in
available terminal amines.
Complete abolishment of activity was seen at 50% reduction,
even in the presence of sufficient excess positive charge to neutral-
ize the DNA. Thus, if all primary amines were assumed to be pro-
tonated at physiological pH, a high surface charge density was
necessary for dendrimer transfection. However, the terminal
amine contents in highly active, fractured dendrimers that had
been dialyzed and lyophilized to remove low molecular weight
components following heat treatment were significantly lower
than their intact counterparts, which were not active in transfec-
tion (Tables 1–2). Therefore, a high number of terminal amines
were required for transfection by dendrimers, other physical prop-
erties of the polymer also significantly contributed to their activity.
A PAMAM-PEG-PAMAM based triblock copolymers were re-
ported as carrier for gene delivery [68]. PAMAM dendrimer is an
efficient gene carrier itself, but it is associated with certain prob-
lems, such as low water solubility and considerable cytotoxicity.
Table 1
Polymer characteristics associated with dendrimers heated to the point of highest transfection activity.a
Dendrimer Heating Measured relative Computed % amide Initial Computed final Initial
time (h)b amine contentc bonds cleaved molecular (kD) molecular (kD) molecule molecule
5-TAEA 8 0.93 13 21 18.3
6-TAEA 10 0.83 35 43 28.5
6-EDA 12 0.76 50 58 28.9
8-EDA 20 0.62 70 233 70.1
a
Dendrimers of various initial size were heat treated, dialyzed against water, then assayed for primary amine content. The amine content for each dendrimer, relative to
that of its intact counterpart, was used to determine the extent of degradation based on the fractured dendrimer model, and thus, the model parameters.
b
Heating time at which maximum activity was observed.
c
Amine content by weight relative to the corresponding intact dendrimer as determined by ninhydrin assay.
The introduction of PEG to engineer a nontoxic and highly transfec-
tion efficient polymeric gene carrier was analyzed (because PEG is
known to convey water solubility and biocompatibility to the con-
jugated copolymer). The copolymer was composed of a PEG core
and two PAMAM substructures on both sides. It was able to self-
assemble with plasmid DNA forming a compact polyplex, which
showed greatly enhanced water solubility compared to the PA-
MAM dendrimer itself. The polyplex thus formed was found to
have a spherical shape with a nanosize appropriate to gene deliv-
ery and a narrow size distribution. The copolymer had little cyto-
toxicity in mammalian cells, low interaction with serum, and
high transfection efficiency comparable to that of PEI in 293 cells.
Li et al. [69] used G5 Polyamidoamine (PAMAM) dendrimers for
gene delivery (from G0 to G10 generation, the molecular weight
and the number of surface active units of PAMAM dendrimers in-
crease exponentially, and their diameters increase linearly). They
isolated plasmid DNA ensuring an OD 260/280 ratio above 1.8
and incubated these components (PAMAM & plasmid DNA) for
1 h at room temperature in different weight ratios. The binding
assay was analyzed by agarose gel electrophoresis. These studies
indicated that these G5 PAMAM dendrimers could be used to
mediate efficient, nonspecific in vitro transfer of genetic material
into eukaryotic cells and in vivo transfer. PAMAM dendrimers have
sufficient surface amine groups, which make them feasibly interact
with DNA, form complexes through their charge-based interac-
tions, and transfer DNA to cells efficiently both in vitro and in vivo.
3.3. Acrylate based polymer vectors
The group of Hennink et al. [70–73] synthesized and evaluated
poly(2-(dimethylamino) ethyl methacrylate) (pDMAEMA) for gene
transfer. The polymer forms positively charged DNA polyplexes,
which can be used successfully for in vitro transfection of different
cell lines, including COS-7 and OVCAR-3 cells. HMW forms of the
polymer (>300 kDa) were able to condense DNA effectively into
particles of 150–200 nm, whereas LMW pDMAEMA forms large
complexes (size 0.5–1.0 micrometer). Like other cationic polymers,
pDMAEMA is cytotoxic, therefore, copolymers of DMAEMA with
methyl methacrylate (MMA), ethoxytriethylene glycol methacry-
late (triEGMA), or N-vinyl-pyrrolidone (NVP) were also evaluated.
A copolymer with 20 mol% of MMA showed reduced transfection
efficiency but an increased cytotoxicity. A copolymer with triEGMA
(48 mol%) showed both a reduced transfection efficiency and a
reduced cytotoxicity, whereas a copolymer with NVP (54 mol%)
showed an increased transfection efficiency but a decreased
cytotoxicity as compared to pDMAEMA. There data indicated that
aggregate formation of positively charged polyplexes with blood
components followed by trapping of the polyplex aggregates in
the lung capillaries is probably responsible for a preferential lung
uptake and transfection.
Henninck and colleagues [74] also synthesized a new polymer,
with two tertiary amine groups in each monomeric unit, poly-
(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-
Computed final Computed net Computed Computed
molecule flexibility (vol/wt) % defects
96 77 69 6.9 5
192 107 80 7.6 10
256 97 57 7.5 13
1024 190 41 7.4 17
 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Table 2
Calculated parameters for dendrimers deliberately synthesized with defectsa.
% Defects Measured relative Theor. relative Computed %
amine contentb amine contentc defects incorporatedd
0 1 1 0
8 0.9 0.85 6 116
17 0.76 0.7 13
33 0.13 0.42 36
a
Proportion of defective monomer (N,N-dimethylacrylamide) added during the synthesis.
b
Amine content by weight relative to the intact dendrimer as determined by ninhydrin assay.
c
Theoretical values as determined by the proportion of defects introduced at each generation (e.g., the number of primary amines per molecule for the 8% 6-TAEA defective
dendrimer is 192_ (0.92).
d
Calculated parameter value.
e
Determined by size exclusion chromatography.
amino]-ethyl ester) (pDAMA). pDAMA has a low toxicity, but also
very low transfection activity. The addition of a membrane-disrup-
tive peptide considerably increased the transfection efficiency. This
indicated that the pDAMA polyplexes alone were not able to medi-
ate escape from the endosomes via the proton sponge mechanism,
which implied that the proton sponge hypothesis was not always
applicable for polymers with buffering capacity at low pH.
Brushed polymers composed of a backbone of poly(hydroxy-
ethyl methacrylate) (pHEMA) onto which poly(2-(dimethylamino)
ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable
linker were synthesized and evaluated as non-viral gene delivery
vectors [75]. Both pDMAEMA and pHEMA polymers with con-
trolled molecular weights and narrow distributions were synthe-
sized by controlled atom transfer radical polymerization (ATRP)
(Fig. 7). The azide initiator was used to ensure complete and mono-
azide functionalization of the pDMAEMA polymer chains. Click
reaction between pHEMA with alkyne side groups and the azide
end group in the pDMAEMA resulted in a high-molecular-weight
polymer composed of low-molecular-weight constituents via an
easily degradable carbonate ester linker. The length of the pDMA-
EMA grafts as well as the number of grafts of the brushed pHEMA–
pDMAEMA can be easily varied. At physiological conditions (pH 7.4
and 37 °C), the brushed polymer degraded by hydrolysis of the car-
bonate ester with a half-life of 96 h. The molecular weights of the
formed degradation products was very close to that of the starting
pDMAEMA, which was likely below the renal excretion limit
(<30 kDa). It was shown that the degradable brushed pHEMA–
pDMAEMAs were able to condense plasmid DNA into positively
charged nanosized particles. The resulting polyplexes were able
to transfect cells efficiently in the presence of the endosomal mem-
brane disrupting INF-7 peptide, and all these degradable polymers
showed lower cellular toxicity compared to a high-molecular-
weight pDMAEMA reference. On the other hand, the low-molecu-
lar-weight pDMAEMA used for the grafting to pHEMA was neither
able to condense the structure of DNA nor able to transfect cells.
Kataoka et al. [76] synthesized polyion complex (PIC) micelles of
acetal-poly(ethylene glycol)-poly(2-(dimethylamino) ethyl meth-
acrylate) (acetal-PEG-PAMA) block copolymer and pDNA and stud-
ied their physicochemical properties and gene transfer efficiency.
They observed that with the increasing N/P ratio to unity, acetal-
PEG-PAMA cooperatively formed complex micelles with pDNA
through electrostatic interaction, allowing pDNA to condense effec-
tively. Dynamic light scattering measurements revealed that the
PIC micelle at N/P P3 had a constant size of approximately
90–100 nm. Eventually, it was also observed that the acetal-PEG-
PAMA/pDNA micelles underwent no precipitation even after long-
term storage for more than 1 month at all N/P ratios. The PIC
micelles were stable even in the presence of excess polyanions,
poly(vinyl sulfate), in contrast to polyplexes based on the PAMA
homopolymer, yet this stabilization effect was highly dependent
on the N/P ratio to reach a plateau at N/P = 3–4. Furthermore, the
pDNA in the micelle was adequately protected from DNase I attack.
95
Theor. 1° Theor. % Theor. Molecular Hydrodynamic Theor. flexibility
amines/moleculec amides remainingc weight (kD)c diameter (Å)e parameter (vol/wt)c
192 100 43.2 64 5
69 30.7 59 7
63 53 20.4 56 10.6
17 22 9.2 45 23.5
The transfection ability of the PIC micelles toward 293 cells was
remarkably enhanced with an increasing N/P ratio as high as 25.
The f-potential of the micelles with a high N/P ratio was an appre-
ciably large positive value, suggesting a non-cooperative micelle
formation. This deviated micellar composition with an excess cat-
ionic nature as well as the presence of free acetal-PEG-PAMA may
play a substantial role in the enhanced transfection efficiency of
the PIC micelle system in the high N/P ratio (25) region.
Wagner et al. [77] synthesized gene carriers based on hexane-
diol diacrylate linked oligoehylenimine and correlated their struc-
ture with biological properties. They synthesized two polycations
by applying different reaction temperatures, 20 °C (LT-OEI-HD)
and 60 °C (HT-OEI-HD). Their structural properties were analyzed
by NMR, FTIR, and SEC/MALLS (size exclusion chromatography
coupled with multi-angle laser light scattering detection). Reaction
temperature strongly influenced molecular weight and ester/amide
ratio and thus resulted in polycations with different biological
activities and degradation profiles. LT-OEIHD was an ester-based
polycation of 8.7 kDa which degraded rapidly at pH 7 and pH 9,
respectively. HT-OEIHD had a molecular weight of 26.6 kDa, was
mainly based on amides, and degraded more slowly than LT-OEIHD.
Both polymers mediated gene transfer as efficiently as linear PEI of
22 kDa in two cell lines while being less toxic at their optimal
conjugate/plasmid (C/P) ratios. LT-OEI-HD needed higher C/P ratios
for gene delivery; however, it was significantly less toxic than
HT-OEI-HD.
Too and coworkers [78] correlated transfection barriers and bio-
physical properties of cationic polymethacrylates quantitatively to
the amounts of cellular accumulation of pDNA and to the expres-
sion of mRNA by quantitative real-time polymerase chain reaction
(real-time PCR). They used three polymers namely poly(dimethyl-
amino) ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA
copolymer, and PEO-b-poly(diethylamino) ethyl methacrylate
(PEO-b-PDEA) copolymer. Despite their similar chemical structures,
the transfection efficiencies were significantly different. PEO-
b-PDEA copolymer was significantly less efficient as gene carrier
as compared to both PDMA and PEO-b-PDMA. Correlations between
cytotoxicity, cellular uptake of plasmid DNA, expression levels of
transgene, and protein, and the physical properties of the polymers
were observed. With the PEO-b-PDEA studies, cytotoxicity was due
primarily to the excess of polymers that did not participate in the
DNA binding.
Block copolymers poly(2-(dimethylamino) ethyl methacrylate)-
b-poly(polyethylene glycol methacrylate) (PDMAEMA-b-P(PEGMA))
were synthesized via reversible addition fragmentation chain
transfer polymerization (RAFT) [79]. The cross-linked micelles
were found to have no toxic effects on the cell line L929, while
PDMAEMA strongly inhibited cell growth, leading to substantial
cell death. The acid degradable methacrylamide based non-viral
gene carriers were reported by Kwon et al. [80]. These nanoparti-
cles based carriers showed efficient DNA release upon hydrolysis,
and enhanced in vitro and in vivo transfection efficiency. It was also
96 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Fig. 7. (a) ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) using N3-functionalized initiator in dichlorobenzene (DCB); (b) pHEMA with side chain of alkynes; (c)
Click chemistry to form degradable brushed pHEMA–pDMAEMA.
observed that the highly versatile nanoparticles can be used for
DNA vaccination via size-dependent targeting of phagocytic cells.
The properties of these nanoparticles such as acid degradability,
surface charges, and types of cargo (e.g., siRNA, miRNA, and oligo-
deoxynucleotides) can easily be tuned for specific applications, and
the targeting and imaging modalities can also be conjugated with
these nanoparticles to achieve targeted delivery, as well as com-
bined imaging and gene therapy.
3.4. Poly(amino-esters) based polymer vectors
Prof. Langer’s group at MIT developed a large library of poly-
(b-amino esters) (PBAEs) based on parallel synthesis using differ-
ent classes of amines and diol-diacrylates, and these were tested
for gene delivery applications [81–83]. By using high-throughput
synthesis techniques, they created libraries of over 2200 structur-
ally unique poly(b-amino esters) (PBAEs). Diacrylate monomers
(letters) and amine monomers (numbers) used to synthesize
PBAEs. are shown in Fig. 8. PBAEs are hydrolytically degradable,
condense plasmid DNA at physiological pH, and are readily synthe-
sized via the conjugate addition of primary or secondary amines to
diacrylates (Fig. 9) [82–84]. An initial screen of model polymers
identified these materials as potential gene carriers and demon-
strated that structural variations could have a significant impact
on DNA binding and transfection efficacies [82,85–87].
The amino-esters based polymer vector has numerous advanta-
ges over other polymer vectors such as (1) diamine and diacrylate
monomers are inexpensive, commercially available starting mate-
rials, (2) polymerization in a single synthetic step reaction, and (3)
no byproducts are generated during polymerization. Poly(b-amino
esters) (PBAEs) are promising compared with PEI due to their
biodegradability via hydrolytically degradable ester groups, their
reduced cytotoxicity, their ability for triggered DNA release within
the cell, and their potential for structural diversity [88]. Initial
studies of this class of polymer showed that by varying synthesis
conditions the polymer molecular weight can be varied from
2000 to 50,000 Da [89]. In physiological conditions, these polymers
have a degradation half-life on the order of hours, but this is slo-
wed considerably at a pH of 5 or when the polymers electrostati-
cally condense DNA and form nanoparticles [81–83].
Investigative studies on the proton sponge mechanism and
endosomal release by Akinc et al. [83,84] also revealed that PBAEs
 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102 97

Fig. 8. Diacrylate monomers (letters) and amine monomers (numbers) used to synthesize PBAEs.

can successfully buffer the endosomal compartment, similarly to
PEI. A library of 140 PBAEs composed of 7 diacrylate monomers
and 20 amine monomers was synthesized in parallel and screened
for gene delivery efficacy [82,83]. In general, the best performing
complexes were found to have effective diameters smaller than
250 nm and positive f-potentials in 10 mM HEPES buffer. It was
determined that the majority of PBAE/DNA particles were limited
by poor cell uptake, two of the polymers had high uptake and med-
iated gene delivery 4- to 8-fold higher than PEI, comparable to the
efficacy of Lipofectamine 2000. These studies demonstrated the
utility of using parallel synthesis and screening of a polymer
library to identify novel gene delivery polymers with efficacy
98 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
Fig. 9. Synthesis of PBAEs by conjugate addition of amines to diacrylates.
greater than PEI. PBAEs that were poor transfection agents failed
for a variety of reasons [83].
Some polymers were simply not sufficiently water soluble,
while others were unable to electrostatically bind DNA tightly en-
ough to prevent its movement during gel electrophoresis. The
polymers which could bind DNA, many of them formed particles
with low cellular uptake. Some particles caused too high toxicity
even they had sufficient cellular uptake. Two leading PBAEs were
further optimized by varying polymer molecular weight, polymer
end group, and polymer to DNA weight ratio [90–92]. Interestingly,
it was also discovered that polymers terminated with diacrylate
monomers were unable to deliver DNA to cells, unlike the nearly
identical polymers that were terminated with amine monomers in-
stead. Zugates et al. [93,94] used one step reaction to conjugate 12
new amino monomers and three diacrylate terminated base poly-
mers. These newly synthesized amino-esters were studied for the
effect of the modification on the properties of these polymers for
gene delivery. In another study, the end groups were modified by
the two-step approach; the first step involved preparation of an
acrylate-terminated polymer followed by the second step of post
polymerization amine-capping to generate end-functionalized
polymers [95]. It is observed that the in vitro transfection levels
can be increased by 30% and the optimal polymer/DNA ratio low-
ered 5-fold by conjugation of the appropriate end group. The most
effective modifications were made by grafting primary diamine
molecules to the chain end.
Prof. Langer’s lab also used the best performing amino-ester
based polymer (C32) for gene therapy in the treatment of cancer
[96]. The best performing amino-ester polymer (C32) was tested
in mice for toxicity and DNA delivery after intratumor and intra-
muscular injection. C32 delivered DNA intratumorally 4-fold better
than one of the best commercially available reagents, jetPEI (poly-
ethyleneimine), and 26-fold better than naked DNA. In the same
lab, poly amino-ester containing microparticles were used to in-
crease the efficiency of the non-viral genetic vaccines [97]. These
formulations generated an increase of 3–5 orders of magnitude
in transfection efficiency and were potent activators of dendritic
cells in vitro. When these were used as vaccines in vivo, these
microparticle formulations, unlike conventional formulations, in-
duced antigen-specific rejection of transplanted syngenic tumor
cells.
Park et al. [98] synthesized biodegradable cross-linked poly-
(b-amino ester) (CLPAE) by Michael addition of pentaerythritol tri-
acrylate and N,N-dimethylethylenediamine and modified with
aminohexanoic acid and lysine to CLPAE-Ahx and CLPAE-Lys,
respectively, for a gene delivery system. The polymers showed
minimal cytotoxicity on 293 cells due to their biodegradability
and biocompatibility. Transfection efficiencies of CLPAEAhx and
CLPAE-Lys were comparable to that of PEI in 293 cells and C2C12
cells. Additionally, high transfection of CLPAEAhx on primary rat
aorta vascular smooth muscle cells (SMC) and primary mouse
embryonic fibroblast (MEF) cells showed a potential for a gene
delivery system on primary cells, restenosis treatment of human
SMC, and MEF cells. Park and coworkers [99] also compared the
transfection efficiency of the network poly(amino ester), (n-PAE),
with PEI. There was a marked difference in the cytotoxicity of
the polymers. The majority of PEI-transfected cells were granu-
lated and dead, whereas most of the cells transfected with n-PAE
were viable and healthy. In addition to this, the n-PAE-mediated
transfection was also very efficient in the presence of serum.
3.5. Poly(ethylene glycol) based polymer vectors
PEG is very useful because of its ease of preparation, relatively
low cost, controllable molecular weight and the ability to link it to
lipids or protein (including antibody) by a variety of methods. PEG
is the polymer of choice for protein modification because it possesses
several favorable properties such as the lack of immunogenicity,
antigenicity, and toxicity, and a high solubility in water and in many
organic solvents. PEG is also approved by the FDA for human use.
Common reasons for the PEGylation of a drug are to reduce its excre-
tion by the kidneys, to avoid or reduce its degradation by proteolytic
enzymes and/or hydrolytic media, to enhance its water solubility
(for highly hydrophobic molecules), to reduce its reticuloendothelial
(RES) clearance, and to reduce its immunogenicity and antigenicity
(mainly for peptides and proteins) [100–105]. Furthermore, for
small drugs, polymer conjugation may yield improved and more
convenient biodistribution, selected cellular uptake [106–108] or,
through tailor-made chemistry, a triggered drug release or targeting
into specific organs or cells [109].
Modification of polyplexes with poly(ethylene glycol) (PEG), typ-
ically grafted onto the polymer as a brush, can stabilize polyplexes
against salt, protein, and complement-induced inactivation
[110,111]. The increased stability due to PEGylation is presumed to
result from steric effects, leading to a decreased particle–particle
and particle–protein interactions. The effect depends on the PEG
molecular weight, the grafting density, and the method of attach-
ment of PEG to the polymer [110,112]. Langer et al. [113] synthe-
sized PEG-polyhistidine conjugates to evaluate the material as
potential gene delivery vehicles. Two conjugate architectures
(comb-shaped and linear A–B block copolymers) were synthesized
and formulated with plasmid DNA. The complexes were character-
ized with respect to DNA complexation capacity, hydrodynamic
diameter, f-potential, in vitro cytotoxicity and transfection capacity
in a model cell line. PEG content of the conjugate significantly influ-
enced the hydrodynamic diameter of the DNA: conjugate composite
in aqueous solution. For comb-shaped conjugates, steric hindrance
attributed to PEG led to a direct relationship between the PEG con-
tent and the complex size. Both architectures could condense plas-
mid DNA into complexes with hydrodynamic diameters <150 nm.
Complexation of DNA with the PEG-polyhistidine conjugates re-
sulted in nanocomposites with negative zeta potentials that re-
tarded DNase I mediated hydrolysis, and all conjugates showed a
low cytotoxicity to macrophages cultured in vitro. The transfection
efficiency was approximately equivalent to DNA-polylysine
complexes.
Yin et al. [114] reported the synthesis of poly(ethylene glycol)-
poly(n-butyl cyanoacrylate) nanocapsules with oil core via mini-
 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102
emulsion polymerization. The nanocapsules were synthesized
through miniemulsion polymerization of butylcyanoacrylate (BCA)
with PEG as initiator. The particle size and the f-potential of nano-
capsules were influenced by the PEG content in the polymerization
system. Fourier transform infrared (FTIR) spectra and 1H NMR dem-
onstrated the chemical coupling between PEG and poly(butylcyano-
acrylate) (PBCA). Thermal characteristics of the copolymer were
investigated by differential scanning calorimetry (DSC). The encap-
sulation efficiency increased concurrently with the increase of PEG
content in the system. The hemolytic assay and the cytotoxicity
measurement showed that the PEG coating could significantly
reduce the hemolytic potential and cytotoxicity of the nanocapsules.
Nagasaki and coworkers [115] prepared a novel ABC triblock copoly-
mer for constructing a pH-responsive and targetable non-viral gene
vector. The copolymer, lactosylated poly(ethylene glycol)-block-
poly(silamine)-block-poly[2-(N,N-dimethylamino)ethylmethacry-
late] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene
glycol) (A-segment), a pH-responsive polyamine segment (B-seg-
ment) and a DNA-condensing polyamine segment (C-segment).
The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid
DNA (pDNA) to form three-layered polyplex micelles with a
PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO
inner shell, and a lactosylated PEG outer shell, as confirmed by 1H
NMR spectroscopy. Under physiological conditions, the Lac-PEG-
PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number
of amino groups in the copolymer/number of phosphate groups
in pDNA) ratio above 3 were found to be able to condense pDNA,
thus adopting a relatively small size (<150 nm) and an almost neu-
tral surface charge (f+5 mV). The micelle underwent a pH-in-
duced size variation (132.6 nm for pH 7.4 and 181.8 nm for pH
pH 4.0). This is due to the conformational changes (globule-rod
transition) of the uncomplexed PSAO chain in response to pH, lead-
ing to swelling of the free PSAO inner shell at lowered pH while
retaining the condensed pDNA in the PAMA/pDNA PIC core. Fur-
thermore, the micelles exhibited a specific cellular uptake into
HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP)
receptor-mediated endocytosis and achieved a far more efficient
transfection ability of a reporter gene compared to the Lac-PEG-
PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles com-
posed of the diblock copolymers and pDNA.
Li et al. [116] synthesized acid labile block copolymers based on
poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacry-
late) segments connected through a cyclic ortho ester linkage.
PEG-a-PDMAEMA condensed with plasmid DNA formed polyplex
nanoparticles with an acid-triggered reversible PEG shield. The
pH-dependent shielding/deshielding effect of PEG chains on the
polyplex particles were evaluated by f-potential and size measure-
ments. At pH 7.4, polyplexes generated from PEG-a-PDMAEMA
exhibited smaller particle size, lower surface charge, reduced inter-
action with erythrocytes, and less cytotoxicity compared to PDMA-
EMA-derived polyplexes. At pH 5.0, f-potential of polyplexes
formed from PEG-a-PDMAEMA increased, leveled up after 2 h of
incubation and gradual aggregation occurred in the presence of
bovine serum albumin (BSA). In contrast, the stably shielded
polyplexes formed by DNA and an acid-stable block copolymer,
PEG-b-PDMAEMA, did not change in size and f-potential in 6 h.
In vitro transfection efficiency of the acid-labile copolymer greatly
increased after 6 h incubation at pH 5.0, approaching the same
level of PDMAEMA, whereas there was only slight increase in effi-
ciency for the stable copolymer, PEG-b-PDMAEMA.
3.6. Other polymer vectors
There are various other synthetic polymer vectors which are
currently being tested for gene delivery research such as poly(4-
vinylimidazole) [117], poly(vinyl alcohol) [118], polyallylamine
99
derivatives [119], aliphatic ionenes [120], polyvinyl derivatives
[121], etc. These polymers showed promising properties for gene
delivery. Poly(4-vinylimidazole) was studied as gene carrier by
Cho et al. [117] and they found that this polymer showed high
transfection efficiency through the proton sponge mechanism of
imidazole groups. Kissel and coworkers [118] reported the bio-
physical and transfection studies of an amine modified poly(vinyl
alcohol) for gene delivery. The resulting polymers were character-
ized using NMR, thermogravimetric analysis (TGA), and gel perma-
tion chromatography (GPC). Atomic force microscopy (AFM),
dynamic light scattering photon correlation spectroscopy (PCS),
and f-potential were used to investigate polyplexes of DNA with
PVA copolymers. These studies suggested an influence of the poly-
cation structure on the morphology of condensed DNA in polyplex-
es. Significant differences were observed by changing both the
degrees of amine substitution and the structure of the PVA back-
bone, demonstrating that both electrostatic and hydrophobic inter-
actions affected DNA condensation. DNA condensation measured
by an ethidium bromide intercalation assay showed a higher de-
gree of condensation with pDNA with increasing degrees of amine
substitution and more hydrophobic functional groups. These find-
ings were in line with transfection experiments, in which a good
uptake of these polymer–DNA complexes was noted with little
endosomal escape. Co-administration of chloroquine resulted in
an increased endosomal escape and higher transfection efficien-
cies, due to disruption of the endosomal membrane.
Boussif et al. [119] used polyallylamine derivatives for gene
delivery. They found that its ability to mediate gene transfer into
cells increased by several orders of magnitude. Transfection effi-
ciency was found to be dependent on the substitution level of ami-
no groups and reached a highest levels in the presence of
lysosomotropic and/or fusogenic agents. At optimal conditions,
glycolylated PAM was shown to be as efficient as the linear PEI
of 22 kDa. Langer et al. [120] used aliphatic ionenes as gene deliv-
ery agents and elucidated the structure-function relationship. The
ionene fractions and their polyelectrolyte complexes (PEC) with
DNA were studied using physicochemical and biological methods.
Ionene polymers were shown to possess low cytotoxicity (minimal
viability of the P388D1 murine macrophage cells was 80%). Degree
of polymerization (DP) and charge density of the ionenes were
shown to be factors of effective control of PEC dissolution in
water-salt solutions. Rolland et al. [121] used polyvinyl derivatives
as novel interactive polymers for controlled gene delivery to mus-
cle. A pDNA, containing a cytomegalovirus (CMV) promoter and a
galactosidase reporter gene (CMV-beta-gal), was injected either
in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvi-
nyl alcohol (PVA) solutions. The interactions between PVP and
pDNA were assessed by dynamic dialysis, isothermal titration cal-
orimetry (ITC), and fourier transformed infrared (FTIR) spectros-
copy. The interaction between PVP and pDNA was found to be an
endothermic process governed by hydrogen bonding and results
in protection of pDNA from extracellular nucleases.
4. Conclusions and future prospective
The gene therapy is presenting a promising approach for the
treatment of genetic diseases. Over the past decade and at present,
there has been continuous research to find better non-viral vectors
to overcome the limitations of viral vectors. There are many syn-
thetic polymers, which are being used and assessed for their appli-
cations in gene therapy. One of these is the poly(b-amino ester)
based vectors. Prof. Langer’s group has developed the library of
these newly synthesized poly(b-amino ester) based vectors. With
an increase in the understanding of different diseases, the clinical
applications will potentially expand into areas such as neurological
100 P.P. Kundu, V. Sharma / Current Opinion in Solid State and Materials Science 12 (2008) 89–102

disorders, tissue regeneration, cancer, monogeneic diseases, vascu- [32] Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML. Transferrin–polycation
conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci USA
lar disease, and infectious diseases. The non-viral vectors represent
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