Review
Poly(glycoamidoamine)s: a broad class
of carbohydrate-containing
polycations for nucleic acid delivery
Nilesh P. Ingle1, Brett Malone2 and Theresa M. Reineke1
1
Department of Chemistry and Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, VA 24061, USA
2
Techulon Inc., 2200 Kraft Drive, Blacksburg, VA 24060, USA
In the era of nucleic acid therapeutics, there is an urgent
need for non-viral delivery vehicles that can cross the
extracellular and intracellular barriers and deliver nucleic
acids to specific intracellular regions. This paper reviews
the development of a subclass of polymer-based delivery
vehicles termed poly(glycoamidoamine)s (PGAAs). The
general design of this family consists of carbohydrate
residues copolymerized with oligoethyleneamine units,
which have proven to be an effective motif that pro-
motes polyplex formation, efficient cellular internaliza-
tion, high gene expression and low cytotoxicity with
cultured cell lines and primary cell types. We then dis-
cuss the structure–property relationships of the PGAA
class of delivery vehicles and studies aimed at under-
standing the mechanisms involved in cellular internali-
zation and trafficking.
Intracellular delivery of nucleic acids
The intracellular delivery of nucleic acids has far-reaching
implications in both health-related research and thera-
peutic development. The ability to treat a disease target in
a specific and selective manner by utilizing the ever-grow-
ing genetic and epigenetic information obtained from
biomedical research could lead to novel research tools,
diagnostic methods and treatment strategies. However, to
advance this field toward this end goal, promoting delivery
into cells and being able to specifically target the delivery
to diseased cells is a major obstacle that needs to be
overcome. Negatively-charged nucleic acids are repelled
by the plasma membrane, which is typically coated with
sulfated glycosaminoglycans (GAGs) that are also nega-
tive in charge; therefore, a means of masking the charge on
the nucleic acid, protecting these sensitive biomacromo-
lecules from enzymatic degradation, and compacting
these large structures into small structures that can tra-
verse the cell membrane are the key hurdles to overcome to
make this therapeutic method feasible. Nucleic acid de-
livery vehicles perform this crucial function of transport.
However, the discovery of effective and benign methods of
polynucleotide delivery has emerged as the primary chal-
lenge to this field. Nucleic acid delivery methods are
continuously evolving to meet the many specialized
requirements in disease research and treatment, such
Corresponding author: Reineke, T.M. (treineke@vt.edu).
0167-7799/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2011.04.012 Trends in Biotechnology, September 2011, Vol. 29, No. 9
as the delivery of specific nucleic acid sequences that
can cause inhibition of human epidermal growth factor
receptor 2 (HER-2) in breast and ovarian cancer cells [1].
Although there are many variables that need to be consid-
ered in developing specific delivery methods, the clear
trend is toward more specificity and intent of function.
In other words, the special nature of nucleic acids is that
they have the ability to selectively target a gene in a cell
that is responsible for expressing a specific protein related
to a disease. A primary research aim for this field is to
increase the amount of nucleic acid delivered to the target
tissue at a low dose profile while maintaining a minimal
level of cellular toxicity and off-target effects. Herein, we
provide a brief review of a subclass of glycopolymer tech-
nologies available for polynucleotide delivery. First, we
discuss the requirements that are shaping the develop-
ment of delivery methods. Second, we examine structure–
property relationships of novel polymers termed poly(gly-
coamidoamine)s (PGAAs). These materials are created by
coupling a variety of mono-, di- and oligo-saccharide mono-
mers with oligoethyleneamine units to create a new family
of non-viral nucleic acid delivery vehicles. Finally, we
discuss recent progress in the understanding of the cellu-
lar interaction and intracellular trafficking behavior of
these materials.
Factors shaping development
Several key factors are contributing to the need for im-
proved delivery of nucleic acids. First, research tools and
therapies are more highly specialized and the payloads for
the delivery vehicles are more diverse. For example, plas-
mid DNA (pDNA), small interfering RNA (siRNA) and
microRNA have all become important components in the
arsenal of cellular modification for therapeutic and bio-
technology purposes [2]. In addition, the cells of interest for
certain diseases are sensitive and expensive. Primary cells,
such as cortical neurons or induced pluripotent stem cells
(IPSCs), are key to advanced cell therapies for many
indications. To be an effective long-term transfection solu-
tion, the intended carrier must be able to withstand the
rigors of clinical and regulatory protocol. Clinical solutions
require safe, non-toxic delivery approaches that can be
easily manufactured at a low cost and have demonstrated
low toxicity profiles.
443
Review
Structure and properties of nucleic acid delivery
vehicles
Non-viral delivery vehicles consist of complexes between
nucleic acids and various macromolecules such as poly-
mers (to form polyplexes) or lipids (to form lipoplexes).
Furthermore, multifaceted assemblies consisting of
nucleic acids and one or more polymer types and/or lipids
to form composite–polyplexes [3] are also being extensively
researched in this field. All of these delivery techniques
being utilized for the delivery of nucleic acid drugs have
revealed promising characteristics. The structure and
unique function of these macromolecules allows for the
encapsulation, protection and stabilization of their nucleic
acid cargo. In addition, these delivery modalities can be
readily modified via synthetic methods to promote effective
delivery that is specific for the desired target [4]. The
surface of most cell types is negatively-charged [5], which
is mostly due to sulfated proteoglycans and negatively-
charged phospho-lipids displayed on the plasma mem-
brane surface [6]. Nucleic acids, the cargo of the carriers,
also have a net negative charge on their surface from the
phosphodiester backbone. For this reason, the molecules
used to complex nucleic acids, for the most part, are
cationic. This serves to facilitate electrostatic interactions
with nucleic acids, which ultimately drives the complexa-
tion and compaction of the polynucleotide cargo. In addi-
tion, the polycation vehicles also serve to charge neutralize
this macromolecular cargo to promote complex interaction
with the cell surface and internalization. Studies have
shown that with naked pDNA (and other nucleic acids),
delivery is marginal at best and yields poor results, in part
due to a charge-repulsion from the negative surface of cells
[7]. In this review, we will focus on polymeric routes of
nucleic acid delivery because of their complex structures
and unique ability to be synthetically modified for optimiz-
ing delivery.
Effect of surface charge
Polymers (usually polycations) are used as nucleic acid
delivery vehicles because they can assemble with various
sizes of polynucleotides (Figure 1) in aqueous solution and
compact their cargo into colloidal nanoparticle complexes.
This process neutralizes the polyanionic charge and,
in most cases, these complexes are overcharged, which
results in a net positive charge on the surface (typically
20–40 mV, as determined by Zeta potential measurements
[8,9]). The positive charge facilitates interaction of poly-
plexes and lipoplexes with the plasma membrane of the cell
and subsequent cellular uptake of these complexes [10,11].
In addition, these macromolecular materials also perform
a very important function of shielding the nucleic acid from
the detrimental extracellular and intracellular nucleases
that can degrade the cargo as the complexes traffic into and
through the cell [12]. The vehicle is also responsible for
promoting cell surface interaction, internalization, traf-
ficking and release of the cargo within the cell. Of course
the cargo type (i.e. pDNA, oligonucleotides or siRNA)
significantly determines the desired intracellular destina-
tion, whether it be the nucleus (pDNA or some oligo types)
or the cytoplasm (siRNA or other oligo forms), and thus this
factor also significantly dictates where the nucleic acid
444
Trends in Biotechnology September 2011, Vol. 29, No. 9
should be released from the vehicle. For example, pDNA
can be transported to the nucleus of cells by some non-viral
vehicles; however, the mechanisms of this process are still
unclear and under investigation [13,14].
Complexes between polymers and nucleic acids are
typically characterized by their charge or N/P ratio. This
number is defined by the molar ratio of the number of
nitrogen (N) or cationic groups in the polymer repeat unit
to the number of phosphate groups (P) on the nucleic acid
cargo that is used to form the nanoparticle complexes. For
in vitro transfection purposes, a positive N/P or charge
ratio is most commonly utilized because this promotes
strong and non-specific interaction of the polyplexes with
the surface of cultured cells. Also, in pure water, this
overall cationic surface (or Zeta) potential promotes colloi-
dal stabilization as a result of the charge repulsion of the
nanoparticle complexes. However, once introduced into
salt- or serum-containing media, these complexes rapidly
aggregate. For in vitro or ex vivo delivery methods, this is
not of concern because it has been found that a number of
nanoparticle sizes and shapes can be readily taken up by
cultured cells [15]. However, for in vivo purposes, this lack
of colloidal stability is of high concern and must be avoided
for systemic delivery success because it may cause aggre-
gation with blood components, facilitate rapid systemic
clearance by the reticuloendothelial system and, in turn,
significantly reduce dose and target specificity. To this end,
the amount of nucleic acid reaching the target cell site
(such as a tumor) is significantly reduced. The modification
of the polymers to avoid aggregation is briefly discussed
later in this review.
Effect of size and shape
The size and shape that these polyplexes form can vary
significantly based on the formulation conditions of the
polyplexes and the structure of the vehicle [16]. In general,
it is most desirable to obtain polyplexes of 50–100 nm to
promote cellular internalization via endocytosis; in vivo
use requires polyplexes near the smaller end of this range.
To this end, physicochemical characterization of the
polyplexes is very important in understanding polymer–
polynucleotide binding affinity, complex stability (in phys-
iological conditions), size, shape and colloidal behavior in
the media used for delivery (or in the blood for in vivo
applications) because these parameters can significantly
affect efficacy and side effects. There are many analytical
methods currently used to characterize polyplex formation,
and the field is generally in need of enhancing the knowl-
edge base of how the polymer structure influences polyplex
characteristics. Generally, the polyplexes are first charac-
terized for their binding to nucleic acids via gel electropho-
resis shift assays, which reveals the N/P ratio of complex
neutralization and is generally a qualitative assessment of
polyplex formation [17,18]. Dynamic light scattering (DLS)
and Zeta potential measurements are typically completed
to assess the general nanoparticle hydrodynamic radius
and surface charge, respectively. These two techniques are
the primary methods of characterizing nanoparticle size
and charge and are also used to monitor polyplex colloidal
stability upon the addition of salt and serum media. How-
ever, a drawback of these techniques is that they do not
(Figure_1)TD$IG][ Review
GlycofectTM
OH OH O
R
O OH OH
N
H
( N
)
4
N
H
Trehalose polycations
N N O OH
N HO OH O H
O N
HO OH N N
HO O N N
H H
Figure 1. Polycations can readily assemble with polyanionic nucleic acids and form colloidal complexes termed polyplexes that facilitate cellular uptake and entry. The
two examples of glycopolymers developed by Reineke et al. that are displayed have shown high efficiency for delivering nucleic acids into cells. Adapted, with permission,
from [17,44].
discriminate different morphologies of polyplexes (rod-like
or torroidal) because most DLS instrumentation assumes
that the complexes are spherical in nature. For this reason,
direct visualization techniques, such as transmission elec-
tron microscopy (TEM), scanning electron microscopy
(SEM) and atomic force microscopy (AFM) are also used
in conjunction with light scattering to observe general
polyplex size and shape [19]. Many other characterization
tools are currently in use such as isothermal titration
calorimetry and multi-angle laser light scattering; indeed,
this subfield of analytical characterization of polyplex
substructure is certainly important and in need of further
research and development [20–22].
Structure and bioactivity relationships of nucleic acid
delivery vehicles
A primary challenge in this field is finding materials that
promote both safe and effective delivery efficiency because
some of the most effective delivery vehicles tend to damage
the cell, cause high cell death and/or can illicit an immune
response [23]. Also, understanding the structure–bioactiv-
ity relationships is key to the development of successful
vehicles because many seemingly trivial attributes within
identical chemical structures of the polymer repeat unit
such as different counterions [24,25], linear versus
branched [26,27] and the polymer length [16,28–30] can
play a large role in the observed toxicity and efficiency. To
examine this, many creative structures continue to be
developed and examined for their structure–activity rela-
tionships, and some of these systems are summarized in
Table 1. For the purposes of this review, we focus on the
structure–activity studies that our group has completed on
a subclass of structures we have designed and developed
termed PGAAs.
Linear monosaccharides
PGAAs (Figure 2) were designed to embody a multifunc-
tional structure platform consisting of carbohydrate
Trends in Biotechnology September 2011, Vol. 29, No. 9
+
+ +
pDNA
+ +
+ +
14
Polymer Plasmid DNA
O
N
1-4
55 or 77
+ + +
+ +
+ +
+
++ + ++
+
Polyplex
TRENDS in Biotechnology
groups and oligoethyleneamines (between one and six)
that are linked together by amide bonds [31]. These struc-
tures were originally designed to encompass the most
promising and active structural attributes of the two most
commonly-studied polymeric delivery vehicles, polyethyle-
neimine (PEI), a highly active yet toxic polymer, and
chitosan, a benign polysaccharide that generally yields
low delivery efficiency. The first example of this strategy
was examined by creating a series of 16 structures by
copolymerizing monosaccharide monomers, L-tartarate
(T), D-glucarate (D), meso-galactarate (G) and D-manna-
rate (M) with four different oligoethyleneamine monomers
containing between one and four ethyleneamines (four
examples are shown in Figure 2) [17,32,33]. These glyco-
polymers were found to bind and compact pDNA into
polyplexes and successfully deliver pDNA into various cell
types without toxic effects. Interestingly, these PGAAs
have been found to rapidly hydrolyze under physiological
conditions, which probably contributes to their nontoxic
nature [31]. The number and stereochemistry of the hy-
droxyl groups was found to alter the efficiency, cell surface
binding and internalization, and the mechanisms of intra-
cellular trafficking; the galactarate polymers with four
ethyleneamine units (G4), in general, yielded the highest
efficiency with most cell types [7,34]. In addition, the
biological effect of increasing the number of ethylenea-
mines to five and six and the effects of linear versus
branched analogs of these PGAA structures have also been
investigated. In general, increasing the number of ethyle-
neamines does not significantly enhance transfection effi-
ciency whereas it does increase toxicity. Linear analogs
were also found to be more toxic than branched analogs of
the same molecular weight and chemical structure [26].
Polymer G4 has recently been licensed by Techulon and is
being sold in a novel formulation under the trade name
GlycofectTM; it shows high activity in delivering pDNA to
many primary cell types without causing high toxicity,
which is a drawback of other leading transfection reagents
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Review Trends in Biotechnology September 2011, Vol. 29, No. 9

[35]. Figure 3 shows the expression profile and human
dermal fibroblast cell (HDFn) morphology for G4 compared
to Lipofectamine [35].
Larger carbohydrate-based monomers
In addition to these original structures that incorporate
linear monosaccharides, larger carbohydrate-based mono-
mers have been used to synthesize glycopolymer delivery
vehicles with structures similar in nature to the original
linear monosaccharide structures but with some chemical
differences. Both trehalose [16,17,36] and b-cyclodextrin
monomers [37] have been incorporated into new related
architectures by our research team to further investigate
the effects of carbohydrate size and polymer length
(Figure 4).
These structures were created by polymerizing, via the
click reaction (azide–alkyne cycloaddition), a diazide–car-
bohydrate monomer (either b-cyclodextrin or trehalose)
with a set of dialkyne–ethyleneamine monomers that con-
tain between one and four secondary amines. This poly-
merization reaction forms a 1,2,3-triazole linkage between
the monomers and allowed a variety of polymers with
different molecular weights to be created. This structural
versatility allowed the assessment of how structural
changes affect both toxicity and delivery efficiency. In
general, the trehalose and b-cyclodextrin families of struc-

Table 1. Nucleic acid delivery vehicles based on carbohydrate and synthetic polymeric derivatives
Abbreviation Chemical name Refs.
Carbohydrate-based polymers
PGAAs
D1–D6 Poly(D-glucaramidoamine)s [17,31,32]
G1–G6 Poly(galactaramidoamine)s; trade name for G4: Glycofect TM [17,31]
M1–M6 Poly(D-mannaramidoamine)s [17,31]
T1–T6 Poly(L-tartaramidoamine)s [31,48]
O4 Poly(oxalamidopentaethylenehexamine) [31,43]
S4 Poly(succinamidopentaethylenetetramine) [31,43]
A4 Poly(adipamidopentaethylenetetramine) [31,43]
Disaccharide-containing polymers
Tr1–Tr4 Trehalose and a dialkyne-pentaethylene tetramine monomer [44]
AP2, AP3 D-trehalose amidine-based comonomers [49]
Glycopolymer chelates
Eu3a, Eu3b Lanthanide chelate with europium [50]
Gd3a, Gd3b Lanthanide chelate with gadolinium [50]
Cyclodextrin polymers
Cd1–Cd4 b-cyclodextrin ‘click’ polymers [37]
Gd4, Gd10 Gadolinium conjugated b-cyclodextrin [51,52]
Eu4, Eu10 Europium conjugated b-cyclodextrin [51,52]
QP4 Quaternary ammonium polycation conjugated b-cyclodextrin [53]
Tf–PEG–AD Transferrin and adamantane conjugated poly(ethylene glycol) [54]
6a–6d; 7a–7d g-Cyclodextrin series [18]
CD–IPEI, CD–lPEI+AD–PEG, CD–bPEI+AD–PEG Linear and branched poly(ethylenimine) grafted with b-cyclodextrin and conjugated [55]
with adamantane–poly(ethylene glycol)
a, b and g CDE conjugates Poly(amidoamine) dendrimer conjugates with a, b and g cyclodextrin [56]
AP1–CD, AP2–CD, IM1–CD to IM3–CD, Pyridylamino, alkylimidazole, methoxyethylamino or primary amine group [57]
AM1–CD, ME1–CD modified cyclodextrins
Hist–bCDP6 Histidylated–b cyclodextrin polymer [58]
bCDP4–bCDP8, bCDP10 b-Cyclodextrin-containing polycations [59]
paCD Polycationic amphiphilic cyclodextrins [60]
Malonic-based polymers
Ma-x-y-Gal-z: Ma, malonic groups; x, spacer length between polymer backbone and triazole [61]
Ma-1-3-Gal-40, Ma-1-2-Gal-18, Ma-1-2-Gal-13, ring; y, spacer length between triazole ring and carbohydrate; Gal, b-D-galactose;
Ma-1-6-Gal-37, Ma-1-6-Gal-22, Ma-1-6-Gal-09, Glu, b-D-glucose; Man, a-D-mannose; z, degree of carbohydrate substitution
Ma-1-9-Gal-44, Ma-1-9-Gal-24, Ma-1-9-Gal-08,
Ma-1-3-Glu-10, Ma-1-3-Man-22
Chitosan-based polymers
Chitosan–g–PEI Poly(ethylenimine) grafted on chitosan [62]
Gal–PEG–chitosan–g–PEI Galactosylated poly(ethylene glycol)–chitosan–g–poly(ethylenimine) [63]
TMC Trimethylchitosan [64]
Schizophyllan-based polymers
SPG–DNA–polycation ternary complex Schizophyllan–DNA–poly(ethylenimine) [65]
Dextran-based polymers
DEAE–dextran 2-Diethylaminoethyl–dextran [66]
DDMC 2-Diethylaminoethyl–dextranmethyl methacrylate graft copolymer [66]
PAA–DS–DNA Poly(allylamine)–dextran sulfate–DNA [67]

446
 Review Trends in Biotechnology September 2011, Vol. 29, No. 9

Table 1 (Continued )
Abbreviation Chemical name Refs.
Lysine-based polymers
PLL Poly(L-lysine) [68,69]
PEG–poly(Lys) Poly(ethylene glycol)-conjugated poly(lysine) [70]
cRGD–PEG–P(Lys-SH) Thiolated c(RGDfK)–poly(ethylene glycol)–block–poly(L-lysine) [71]
PDL Poly(D-lysine) [69]
Polyethylenimine-based polymers
pLPEI Short linear poly(ethylenimine) [72]
PEI–CD b-cyclodextrin-conjugated poly(ethylenimine) [73]
bPEI Branched poly(ethylenimine) [74]
Polymethacrylate-based polymers
Poly(BMDO-co-DMAEMA) 5,6-Benzo-2-methylene-1,3-dioxepane-co-N,N-dimethylaminoethyl methacrylate [75]
p(CBMA)–EE Poly(carboxybetaine methacrylate–ethyl ester [39]
PAEMA Poly[N-(2-aminoethyl)methacrylamide trifluoroacetate] [76,77]
PAHPMA Poly(3-amino-2-hydroxypropyl methacrylate) [77]
PAEAEMA Poly(2-(2-aminoethylamino)ethyl methacrylate) [77]
PAEAHPMA Poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) [77]
PDMAEMA Poly(2-dimethylamino) ethyl methacrylate [78]

[(Figure_2)TD$IG]

(i) OH OH O (ii)

Luciferase Gene Expression (RLU/mg)

R O 10
D4 N H 10
N N O4 N
N
N
O OH OH H 4 H H 4 H
O
12 16 9
10

OH OH O O H 8
R 10
G4 N S4 N
N
N
N N H 4 H
O OH OH H H O 11
4
11 107

OH OH O
O H 106
M4 R N
N A4 N N
N N O H 4 H
O OH OH H 4 H 11 105
Y EI ine 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30
14 NL P
O Jet tam
NA fec D4 G4 M4 T4 A4 S4 O4
pD po
OH O Li
T4 R
N
N N
O OH H 4 H
14

TRENDS in Biotechnology

Figure 2. (i) Novel poly(glycoamidoamine) (D4, G4, M4 and T4) and poly(amidoamine) (O4, S4 and A4) polymers for non-viral nucleic acid delivery. (ii) Luciferase gene
expression (RLU/mg protein) in human adenocervical carcinoma (HeLa) cells. Transfections were completed in reduced serum media (OptiMEM). The molecular weights of
the polymers shown and examined in this study are as follows: D4 = 4.9 kDa, G4 = 4.6 kDa, M4 = 5.6 kDa, T4 = 4.8 kDa, A4 = 3.8 kDa, S4 = 3.3 kDa, O4 = 4.5 kDa. Adapted, with
permission from [31].
[(Figure_3)TD$IG]

DNA only G4 (GlycofectTM) Lipofectamine 2000

TRENDS in Biotechnology

Figure 3. GlycofectTM (a novel formulation of G4) yields significantly higher green fluorescent protein (GFP) gene expression in human dermal fibroblast cells (HDFn) after
48 hours post transfection. Both the differential interference contrast images (DIC) (top row) and wide field microscopy images (bottom row) are displayed. Adapted, with
permission from [35].

447
(Figure_4)TD$IG][ Review Trends in Biotechnology September 2011, Vol. 29, No. 9

(i) (ii) OH
O
HO O
O O OH
OH HO OH
O OH HO O
HO
N N O OH O OH O O O
N HO OH O O N HO H
O H N OH N N
N N N N N
HO OH N N HO
H 1-4 H
HO O N N O OH OH O N
H 1-4 H OH HO O
N
nw O nw
HO O O
(a) (b) (c) (d) HO

Key:
(iii) 1010 (iv) Jet-PEI Cd349 Cd447
(a) 1010
(a) Cd246 Cd146

RLU/mg of protien
RLU/mg protien

109
108 108
107
106 106
Key:
9a 9b
105
9c Jet-PEI
104 104
0 5 10 15 20 25 0 5 10 15 20 25
N/P Ratio N/P Ratio
Transfection in Opti-MEM Transfection in Opti-MEM

Key:
1010 1010 Jet-PEI Cd493 Cd4200
(b) (b) Cd447 Cd427
RLU/mg of protien

109
RLU/mg protien

108 108
107

106 106
Key: 105
9a 9b
9c Jet-PEI
104 104
0 5 10 15 20 25 0 5 10 15 20 25
N/P Ratio N/P Ratio
Transfection in DMEM Transfection in Opti-MEM
TRENDS in Biotechnology

Figure 4. (i) Trehalose and b-cyclodextrin polymers for non-viral nucleic acid delivery. (a) The trehalose carbohydrate unit that helps to increase cell viability, solubility and
stability in serum-containing media. (b) Heterocycle-amide groups to increase binding affinity. (c) An oligoethyleneamine unit to aid electrostatic binding of the polymer
with the phosphodiester backbone of the nucleic acids and enhance cellular uptake. (d) The degree of polymerization that can be tuned easily to optimize delivery
performance for various applications. (ii) b-Cyclodextrin-based polymers with similar structure to the trehalose polymers (i) have also been created and examined. (iii) Gene
expression (RLU/mg protein) for trehalose polymers with ethyleneamines units = 1, where the degree of polymerization (n) = 34, 2 (n = 39), and 3 (n = 40) at: 0 (DNA only), 3,
5, 7, 10, 15 and 25 N/P ratios. (iv) Gene expression (RLU/mg protein) for b-cyclodextrin polymers at: 0, 5, 7, 10, 15, 20 and 25 N/P ratios. The control is Jet-PEI (N/P = 5), and
the cell type used was human adenocervical carcinoma (HeLa) cells. Abbreviation: RLU, relative light units. Adapted, with permission from [37,46].

tures followed similar biological trends with both HeLa With similar structural features to these carbohydrate-
and H9c2(2-1) cells. As the number of ethyleneamines based polymers, our research team has also created com-
increased, the intracellular delivery of pDNA and trans- plimentary structures termed ‘click clusters’ that contain
gene expression increased. However, having either three or seven ethyleneamine arms coupled to a b-cyclodextrin
four ethyleneamines in the repeat units (with both fami- core via triazole linkage chemistry. Unlike the previous
lies) yielded similar results for toxicity and transfection polymer-based systems that have a dispersity index asso-
efficiency. Interestingly, in some cases the structures with ciated with the molecular weight, these macromolecules
three ethyleneamines yielded higher efficiency than four are completely monodisperse, as supported by mass spec-
(transgene expression similar to Jet-PEI). A major differ- trometry data. As published, these structures facilitate
ence between these structures and the original monosac- nucleic acid binding and compaction [38] (Figure 5). In-
charide PGAAs is that polyplexes formed at low N/P ratios terestingly, these macromolecules were similar to the b-
(N/P = 5–7) and facilitated high gene expression, whereas cyclodextrin polymers in that the structures that contain
high N/P ratios of 20–30 were needed to promote high gene three and four secondary amines both yielded high trans-
expression levels with the original set of monosaccharide fection results. However, unlike the b-cyclodextrin poly-
PGAAs. As the polymer molecular weight increased, the mers that yield high gene expression at low N/P ratios, a
delivery efficiency increased significantly, yet with the cost much higher N/P ratio of 20–50 was required of the click
of also increasing the toxicity [16,17,36]. clusters to observe similar high transfection efficiency

448
(Figure_5)TD$IG][ Review Trends in Biotechnology September 2011, Vol. 29, No. 9

106

Relative light units (RLU)

(i) (ii) (a) (In HeLa cells)
105
H2N
X NH 104
2
NH
X 103
NH

HN HN 102
O N N
N N O 101
H2N N N O O
NH N N H
O O N 100
O HO OH N NH 9a 9b 9c 9d 9e

ct
X

NA

EI
N 2
OH

erfe
O

t-P
N O OH HO

pD
H O H

Je
HOO X

Sup
N N O OH
N OH HO N N
OH HO O N 106
O OH HO

Relative light units (RLU)

HO O (b) (In H9C2 cells)
O NH
O N O O O 105
N
NH N N N
HN 104
O N HN
NH 103
H2N X NH2
X
102
HN X=0-4
101
X
NH2 100
9a 9b 9c 9d 9e

ct
I
NA

-PE

erfe
pD

Jet

Sup
Key: N/P 5 N/P 10 N/P 20 N/P 50

TRENDS in Biotechnology

Figure 5. (i) Schematic structures of the b-cyclodextrin ‘click cluster’ delivery vehicles. (ii) Gene expression for analogues 9a (x = 0), 9b (x = 1), 9c (x = 2), 9d (x = 3) and 9e
(x = 4) in human adenocervical carcinoma (HeLa) cells and rat cardiomyoblast H9C2(2-1) cells in OptiMEM at N/P ratios of 5, 10, 20 and 50. The controls are pDNA, Jet-PEI (N/
P = 5) and Superfect (N/P = 5). Abbreviation: RLU, relative light units. Adapted, with permission from [38].

(comparable to the commercial controls) with HeLa and
H9c2(2-1) cells [38].
Cellular interactions of nucleic acid delivery vehicles
Surface proteins on plasma membrane
As discussed earlier, the cell surface derives its negative
charge from glycoprotein-based structures termed GAGs
[34]. Primarily, there are six different types of GAGs:
heparin (H), heparan sulfate (HS), dermatan sulfate
(DS), chondroitin sulfate C (CSC), chondroitin sulfate A
(CSA) and hyaluronate (HA), with decreasing anionic
charge density from the former (H) to the later (HA), where
CSC = CSA [34]. To promote cellular entry, PGAA poly-
plexes are formulated by combining the cationic polymers
with anionic nucleic acids that have an overall positive
charge due to the higher stoichiometric ratio of amines.
The cationic polyplexes interact with anionic GAGs, and
this interaction is in part driven via electrostatic interac-
tions, which trigger subsequent internalization via endo-
cytosis. Our research has shown that the presence of GAGs
on the cell surface is necessary to promote cellular inter-
nalization because studies with GAG-deficient CHO cells
(pgsA-745 cell line) show a dramatic decrease in PGAA
polyplex uptake compared to CHO cells expressing normal
GAG levels. It was found that HS yields the highest
binding affinity to the PGAA polyplexes with HA coming
in a close second. Interestingly, if polyplex solutions were
exposed to excessive concentrations of the various GAGs
free in solution, HA bound to the surface of the polyplexes
to form ternary complexes, but HS dissociates the polyplex.
However, if transfections were completed with CHO cells
lacking only HS on the surface (but retaining the other
GAG structures), the internalization was unaffected,
which demonstrates that the other GAGs are as important
in the uptake of PGAA polyplexes. As a final note, although
charge certainly plays a role in polyplex internalizations
(desulfating the GAGs on CHO cells nearly abolished
PGAA polyplex uptake), the internalization was not solely
charge-dependent. The GAG with the lowest anionic den-
sity, HA, revealed the highest binding affinity to the PGAA
polyplexes. Likewise, CSA yielded a higher PGAA polyplex
binding affinity than CSC despite the fact that the two
structures have the same sulfation density (but different
sulfation sites on the polysaccharide structure). From
these studies, it was concluded that the carbohydrate
residues within the PGAA structures are probably playing
a role in promoting GAG binding via H-bonding [34], and
these effects are still widely being studied in our group.
Internalization pathways
After the polyplexes adhere to the GAGs on the outer
surface of cellular plasma membrane, the next step in
their transit route is trafficking across the plasma mem-
brane into the cell. The interaction of polyplexes with cell
surface receptors triggers an uptake mechanism called
endocytosis. Endocytosis is a general term that encom-
passes several mechanisms of active intracellular trans-
port such as clathrin, caveolae-mediated endocytosis,
macropinocytosis and some other lesser-known active
transport pathways. Our research team has examined both
the internalization pathways and the routes that lead to
nuclear delivery for gene expression with a combination of
both confocal microscopy and pharmacological inhibitor
experiments. Interestingly, the PGAA polyplexes were

449
 Review Trends in Biotechnology September 2011, Vol. 29, No. 9

found to be internalized into cells by a combination of these ing of PGAA polyplexes because their knockdown resulted
pathways primarily involving caveolae, whereas about in a significant drop in uptake (80%) (Figure 6). The
80% of PGAA polyplex internalization was inhibited with knockdown of dynamin resulted in a 99% reduction in gene
cellular exposure to filipin III (Figure 6). However, if the expression [7]. This work also revealed that caveolae
cells were exposed to chlorpromazine (clathrin inhibitor) or appears to be the primary route that leads to nuclear
dimethylameloride (macropinocytosis inhibitor), only ap- delivery of pDNA complexed with the PGAAs for transgene
proximately 50% and 20% internalization knockdown was expression because inhibiting both clathrin and macropi-
observed, respectively, for the PGAA polyplexes. [7]. Actin, nocytosis results in a negligible decrease in gene expres-
a primary intracellular highway, and dynamin, a protein sion. Actin and dynamin were also found to play very large
essential for pinching off endocytic vesicles from the plas- roles in nuclear delivery of pDNA with the PGAAs because
ma membrane to promote internalization, were also found knockdown of these key proteins decreased gene expres-
to be essential in both cellular internalization and traffick- sion [7].
[(Figure_6)TD$IG]

PGAA DNA Polyplex

+
OH OH O H +
H3CO N H + +
N N
O OH OH H 4 H + +
n

Macropinocytosis Clathrin Caveolae Direct membrane

penetration

Key: Actin
Dynamin-1
Cholesterol
Nucleus
Caveolin-1 (for gene expression)

Proteoglycan

(a) Endocytosis pathways for PGAA nucleic acid delivery vehicles

100.0 108
90.0
Luciferase expression (RLU)

#
107
80.0
Decrease in Cy5-pDNA
fluorescence (%)

70.0 # ##
106
60.0
*
∧ ∧
50.0 105
# # # #
40.0
104
30.0 **
#
20.0 103
10.0
102
0.0
Cells only DNA only G4 D4 M4 T4 Jet-PEI
G4 D4 M4 T4 Jet-PEI
Key: Untreated Clathrin Caveolae
Key: Clathrin Caveolae Macropinocytosis Macropinocytosis Macropinocytosis Actin
Macropinocytosis Actin Dynamin Dynamin

(b) Uptake inhibition (c) Endocytosis inhibition

TRENDS in Biotechnology

Figure 6. (a) Different endocytosis mechanisms that are commonly examined as transfection routes for polyplexes. (b) Decrease in cellular uptake at 2.5 hours post
transfection for the PGAA polyplexes. All values are significantly different from pDNA at P < 0.05; except when marked by #. (c) Luciferase gene expression for the PGAA
polyplexes at 48 hours post transfection in cells inhibited for various endocytosis pathways. All values are significantly different from uninhibited control at P < 0.001;
where, *: P < 0.001; **: P < 0.005; ^: P < 0.1; and #: not significant. Inhibition method: Various routes involved in polyplex uptake were knocked down in the cultured cells
using the following inhibitors: chlorpromazine for clathrin; filipin III for caveolae; dimethylamiloride (DMA) for macropinocytosis; dynasore for dynamin; cytochalasin D to
depolymerize actin. Phorbol myristate acetate (PMA) was used to stimulate macropinocytosis. Experiments were performed with human adenocervical carcinoma (HeLa)
cells. Adapted, with permission, from [7].

450
Review
Perinuclear release of nucleic acid
Many further investigations are ongoing to better under-
stand their transport pathways and the release mecha-
nisms of the polymer from the nucleic acid. It should be
noted that escape of the endosomal/lysosomal pathway is
still currently thought to be an important step in the
overall delivery process of polyplexes to facilitate nucleic
acid delivery and drug efficacy [39]. Although much work
has been completed to understand this process, much work
still remains because the trafficking pathway and delivery
efficiency is dependent on both the structures of the poly-
mer and the nucleic acid. For example, as discussed above,
for pDNA, the nucleic acid must be transported to and
enter the nucleus to promote transgene expression. How-
ever, for siRNA delivery, the polyplexes must escape these
membrane-bound vesicles and enter the cytoplasm for
transport to the RNA-induced silencing complex (RISC)
for knockdown of mRNA expression [40]. The high trans-
fection efficiency of some commercial vehicles such as
polyamidoamine (PAMAM) dendrimer and PEI has been
attributed to the ability of these polymers to escape the
endosomes. This escape mechanism may be due to the
presence of a large number of positively-charged amines
that are thought to cause a ‘proton sponge’-like effect
resulting in endosomal swelling, rupture and ultimately
polyplex release [39,41].
After the polyplex reaches its destination within the
cell, it should also dissociate to expose the nucleic acid to
perform its therapeutic effect [42]. This dissociation can be
influenced by several factors such as pH, ionic strength of
the surrounding cytoplasmic fluid, temperature [42] and
the N/P ratio [31]. In particular, many systems are
designed to degrade under biological conditions to promote
nucleic acid release. Through a comparative degradation
analysis of the PGAA polymers, it was discovered that they
rapidly degrade under physiological temperature and pH
[31]. Interestingly, at acidic pH 5.0, the M4 and T4 poly-
mers degrade slower than the G4 and D4 polymers [31].
This degradation is currently attributed to hydrolysis of
the amide bond, which is unusual because amides are
normally hydrolytically stable under these mild conditions
[31]. This preliminary study found that the synergistic
presence of both the hydroxyl groups (from the carbohy-
drate unit) and the amines promote the rapid hydrolysis
event. Polymer hydrolysis was shown to aid gene expres-
sion because models with related structures that contain
alkyl chains in place of the carbohydrates (A4 and S4) do
not rapidly degrade under the experimental conditions and
were found to yield much lower gene expression profiles
(Figure 2).
Both fundamental and applied work on these PGAA
vehicles is still ongoing in our lab. For example, we are
interested in understanding how the polymer structure
affects the nucleic acid binding affinity, polyplex stability
and nucleic acid release. Fundamental studies have been
performed to reveal that the binding affinity of the polymer
to the nucleic acid is driven by a combination of both
electrostatics and H-bonding, and the presence of the
carbohydrates enhance the H-bonding interaction over
models lacking hydroxyls [43]. Furthermore, the stereo-
chemistry of the hydroxyl groups within the polymer
Trends in Biotechnology September 2011, Vol. 29, No. 9
structures play a role in the binding affinity, and polymer
G4 was found to yield the highest binding affinity to pDNA
[43]. This effect has also been studied with the trehalose
polymers; interestingly, increasing the length of the oli-
goethyleneamine units within this subclass of structures
was found to promote direct interactions within the
grooves of pDNA [16,44–46].
Concluding remarks
The field of non-viral nucleic acid delivery has the poten-
tial to yield significant advances in biotechnology research
and therapeutic development. As discussed herein, glyco-
polymer-based vehicles have been developed and continue
to be thoroughly studied for their bioactivity and mecha-
nistic properties. Our research team found that carbohy-
drate-based PGAA polymers have the ability to bind and
compact nucleic acids into polyplexes that facilitate effec-
tive intracellular pDNA delivery with low toxicity. Poly-
mer G4 is currently being sold in a novel formulation
under the name GlycofectTM; it shows exceptional pDNA
delivery efficiency with many primary cell types. This
exciting class of polymers has unique degradation profiles,
therefore they may also be promising for controlled release
applications [47].
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