Nanotechnology for drug and gene therapy: the importance
of understanding molecular mechanisms of delivery
Vinod Labhasetwar
Nanotechnology, although not a new concept, has gained
significant momentum in recent years. This stems partly from
the realization that nanosystems have significantly different
biological properties from large-sized systems (e.g. implants or
microparticles) that could be used effectively to overcome
problems in drug and gene therapy. In drug therapy, we face
the problems of inefficacy or nonspecific effects; hence,
nanosystems are being developed for targeted drug therapy. In
gene therapy using non-viral systems, the main issues are
relatively transient gene expression and lower efficiency than
viral vectors. Research efforts have focused on understanding
the barriers in gene delivery so that non-viral systems can be
developed that are as effective as viral systems in gene
transfection. Understanding the molecular mechanisms that
underlie the interactions of nanosystems with the cell, their
uptake properties and retention will be crucial for the
successful development of these systems.
Addresses
Department of Pharmaceutical Sciences, College of Pharmacy,
Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA
Corresponding author: Labhasetwar, Vinod (vlabhase@unmc.edu)
Current Opinion in Biotechnology 2005, 16:674–680
This review comes from a themed issue on
Pharmaceutical biotechnology
Edited by Ismail Kola and Daria Hazuda
Available online 2nd November 2005
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2005.10.009
Introduction
The idea that nanosystems have unique physical and
biological properties that might be used to overcome
the problems of gene and drug delivery has gained
interest in recent years. Nanosystems can be designed
with different compositions and biological properties.
Some of these systems, such as nanoparticles, dendrimers,
nanocages, micelles, molecular conjugates, liposomes and
so on, have been extensively investigated for drug and
gene delivery applications [1]. Rather than testing dif-
ferent systems, however, a more effective approach for
achieving drug delivery would be to rationally develop
nanosystems based on the understanding of their inter-
actions with the biological environment, target cell-sur-
face receptors, molecular mechanisms, and pathobiology
Current Opinion in Biotechnology 2005, 16:674–680
of the disease under consideration. In addition to knowl-
edge of the therapeutic dose and the duration of drug
therapy required at the disease site, a better understanding
of other key factors is also important (Box 1). These
include knowledge of target cell populations, cell-surface
receptors, changes that occur in the target tissue at the
molecular and cellular level with progression of the dis-
ease, the mechanism of drug action and its site of action
(intracellular versus extracellular). Another consideration
is whether a combination of drugs that work by different
pathways would be more effective than a single drug
therapy; for example, Sengupta et al. [2] recently demon-
strated that a nanoparticle formulation loaded with an anti-
angiogenic agent (combretastatin-A4) and a cancer che-
motherapeutic agent (doxorubicin) was more effective in
suppressing tumor growth than a single drug therapy.
In addition, we need to understand the barriers to drug and
gene therapy. These could be as simple as the instability of
therapeutic agents (e.g. proteins or genes) in the biological
environment or as complex as the inability to localize a
drug more effectively to the target cell population or to a
specific receptor. Reduced drug efficacy could also result
from other biological factors, such as genetic mutations
leading to changes in cell-surface receptors [3], the over-
expression of efflux pumps (e.g. P-glycoprotein) in
response to chemotherapy [4] or changes in signaling
pathways with the progression of disease (e.g. cancers
and restenosis) [5,6]. Several studies have demonstrated
excessive methylation of DNA with the progression of
cancer [7], and this is considered one of the reasons for the
failure of many antineoplastic agents (e.g. doxorubicin and
cisplatin) in drug-resistant cancers [8].
Although it is known that nanoparticles are taken up by
cells more efficiently than large-sized microparticles [9],
we have little understanding of their mechanism of
uptake, intracellular trafficking, retention, and sorting
pathways into different intracellular compartments. Intra-
cellular organelles, such as mitochondria, the nucleus,
endo-lysosomes and so on, form the site of action for many
therapeutic agents [10]. The cytoplasm itself is also the
target for certain drugs such as glucocorticoids, the recep-
tors of which are cytoplasmic. Small interfering RNAs
(siRNA), which are being investigated as a technique for
silencing the expression of disease-causing genes, also
require cytoplasmic delivery [11]. Hence, the efficient
intracellular delivery of nanoparticles and their retention
are crucial factors in enhancing the efficacy of the encap-
sulated therapeutic agent. Designing nanoparticles or
other nanosystems that could target drugs to a specific
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Box 1 Issues to consider in drug and gene delivery.
Localization to the target
Localization could be required to a specific organ, tissue, cells or
even to a specific intracellular compartment
Drug retention or duration of gene transfection
Factors to consider here include the required concentration of the
therapeutic and the duration of drug retention or gene expression
Barriers
Could be as simple as the instability of therapeutic agents in the
biological environment or as complex as the inability to localize a
drug more effectively to a specific target organ, tissue, cell or
intracellular receptor
The pathobiology of disease conditions
The progression of a disease can change key factors and alter the
drug target in some way. This issue needs to be considered
when developing delivery systems
Delivery system
The delivery system employed needs to meet several criteria
regarding biocompatibility and efficient release kinetics
intracellular compartment or receptor is a challenging
task, but such systems would not only reduce the non-
specific effect of the drug but could significantly potenti-
ate their efficacy. In this review I discuss the significance
of understanding the molecular mechanisms underlying
the interaction of nanoparticles with cells, their uptake
properties and retention for developing efficient nano-
systems for drug and gene delivery applications.
Nanotechnology for gene therapy
In addition to the efficient intracellular delivery of DNA
and its rapid escape from the degradative environment of
endosomes, the movement of DNA from the cytoplasm to
the nucleus remains one of the major barriers in gene
therapy. Without localization of DNA to the nucleus, no
transcription or ‘gene therapy’ can take place. Although
significant progress has been made in developing nano-
systems for the intracellular delivery of DNA, the nuclear
membrane remains the rate-limiting barrier to efficient
gene transfection [12]. All passive and active transport
into and out of the nucleus occurs through the nuclear
pore complex (NPC), which is present in the nuclear
membrane. The NPC has a pore-like, molecular sieve
function: molecules smaller than 40–50 kDa can diffuse
freely across, whereas larger proteins require a nuclear
localizing signaling (NLS) peptide to be specifically
targeted to the nucleus [13]. DNA is highly negatively
charged and, for better gene transfection, its charge is
neutralized using either cationic ions (e.g. Ca2+ or Mg2+)
or cationic polymers (e.g. poly-L-lysine). This results in
collapse of the DNA molecules into small particles and is
referred to as ‘counter-ion condensed’ DNA. Condensed
DNA is more stable in the body because it is protected
from degradation due to nucleases, enzymes present in
the body. Furthermore, the condensed DNA is taken up
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Nanotechnology for drug and gene therapy Labhasetwar 675
more efficiently by cells than uncondensed DNA. Even
when fully counter-ion condensed, a single plasmid mole-
cule condenses into a sphere of about 25 nm. Thus, the
size of DNA is considered to be the major limiting factor
in its low transfer rate into the nucleus [14]. Viruses that
replicate themselves in the nuclei have a special mechan-
ism that allows them to deliver their genome into the
nucleus (e.g. influenza virus and polyoma virus). Like-
wise, adenovirus and human immunodeficiency virus
(HIV) have their own mechanisms for carrying genetic
materials into the nucleus [15]; however, as non-viral
synthetic systems lack any such mechanisms, their effi-
cacy as a gene expression system is significantly lower
[16]. The instability of the delivered DNA is also an issue,
as its half-life is only 60–90 min in the cytoplasm [17]. On
the basis of our knowledge of viral systems, several
investigators have tried to mimic viral mechanisms to
develop efficient non-viral systems [18]. To develop such
systems, however, it will be important to learn more about
the way in which the currently used non-viral systems
interact with cells, their intracellular transport mechan-
isms, and the barriers encountered for the cellular and
nuclear delivery of DNA.
The problem of exocytosis
We have carried out a number of studies aimed at under-
standing the intracellular trafficking of biodegradable
nanoparticles. These nanoparticles were formulated
using poly DL-lactide co-glycolide (PLGA) or polylactide
(PLA) polymers with an encapsulated therapeutic agent
or gene of interest [19]. Using this system, we have
demonstrated the rapid escape of PLGA-based nanopar-
ticles from endosomes to the cytosol following their
cellular uptake, a desirable characteristic for gene deliv-
ery systems [20]. However, further studies showed that
a significant fraction of the nanoparticles remain in the
recycling endosomes and undergo exocytosis (i.e. they
escape from the cell without releasing the therapeutic
agent inside the cell [21]). This could be a major limiting
factor in nanoparticle-mediated gene delivery. Exocytosis
could be a general phenomena for most nanosystems, but
so far has only been reported for liposomes and the
PLGA-based nanoparticles described above [22].
Levels of gene expression
In gene delivery studies using nanoparticles, the intra-
cellular distribution of DNA (labeled with TOTO dye)
was investigated with and without encapsulation in nano-
particles in a breast cancer cell line (MDA-M435S). The
DNA used in the study comprised the wild-type gene for
p53 (wt-p53). The results demonstrated cellular uptake of
plasmid DNA (without nanoparticle encapsulation), but
the DNA (red color) was seen mainly in the perinuclear
area and its intracellular retention did not exceed beyond
three days post-transfection. With DNA-loaded nanopar-
ticles, however, the red color of DNA gradually increased
with the incubation time, suggesting its sustained release
Current Opinion in Biotechnology 2005, 16:674–680
676 Pharmaceutical biotechnology

inside the cells (Figure 1a). The slow but sustained
appearance of DNA inside the cells also explains the
steady increase in gene transfection observed with nano-
particles. The study demonstrated sustained activity with
wt-p53 DNA–nanoparticles as compared with the plasmid
alone (Figure 1b), thus complimenting the data with
TOTO-labeled DNA. The increase in p53 to b-actin
ratio (where b-actin acts as an internal control) suggests
an increase in wt-p53 gene expression with time, which
could result from the sustained release of DNA from
nanoparticles. As the DNA is encapsulated in nanoparti-
cles, it is also protected from degradation by nucleases.
Nanoparticles loaded with wt-p53 DNA demonstrated an
increase in antiproliferative activity with incubation time,
whereas plasmid DNA alone (Figure 1c) demonstrated
transient antiproliferative activity. For comparison, a
DNA–lipid complex (DNA–LipofectamineTM) was also
studied and, as with the plasmid DNA alone, was seen to
exhibit transient activity. In general, lipid–DNA com-
plexes demonstrated higher levels of gene transfection in
vitro than nanoparticle-mediated gene transfection, but
gene transfection with lipid complexes is transient,
whereas that with nanoparticles is more sustained [23].
The sustainability of gene expression
Based on the results of the above study, it appears that the
duration of gene expression is also crucial for sustaining
the therapeutic effect of the gene — in this case the

Figure 1

Nanoparticles for gene delivery. (a) Intracellular DNA delivery. A single dose of DNA (either alone as a plasmid or encapsulated in nanoparticles)
was added to cells and observed under a confocal microscope over several days. DNA was labeled with TOTO and appears red, whereas the
nanoparticles contained a fluorescent dye (6-coumarin) and appear green. Yellow areas result from the co-localization of released DNA (red) and
nanoparticles (green). (b) Reverse transcriptase–PCR data for cells transfected with the wt-p53 gene. In this analysis, b-actin was used as an
internal control. Lane 1, molecular weight markers; lane 2, p53 DNA-loaded nanoparticles; lane 3, p53(–ve) DNA-loaded nanoparticles; lane 4,
p53 DNA only; lane 5, b-actin for p53 DNA-loaded nanoparticles; lane 6, b-actin for p53(–ve) DNA-loaded nanoparticles; lane 7, b-actin for
p53 DNA. (c,d) The antiproliferative activity of wt-p53 DNA on the breast cancer cell line MDA-M435S. The cells were treated with either
(c) wt-p53 plasmid DNA or wt-p53 DNA-loaded nanoparticles or (d) a DNA–LipofectamineTM complex. Cell proliferation was measured using a
mitogenic assay (Promega). Antiproliferative activity was compared with the respective controls. Control nanoparticles and medium had similar
growth curves, suggesting that the observed antiproliferative effect was due to wt-p53 DNA encapsulated in nanoparticles. (Figures reproduced
with permission from [23]).

Current Opinion in Biotechnology 2005, 16:674–680 www.sciencedirect.com


antiproliferative effect of p53 protein. When developing
gene expression vectors, it is therefore important to
understand the level and duration of gene expression
required in the target tissue. This of course will depend
to a large extent on the disease condition, and whether it
is feasible to repeatedly administer the vector to sustain
gene expression without causing toxicity. In this regard,
PLGA/PLA nanoparticles with encapsulated DNA are
advantageous because they can protect the encapsulated
DNA and release it slowly as the polymer matrix breaks
down, thus sustaining gene expression [24]. It seems
feasible to tune the level and duration of gene expression
using PLGA/PLA nanoparticles by modulating the DNA
release rates. Gene expression mediated by nanoparticles
probably occurs through the interaction of the released
DNA with certain cellular proteins in the cytosol, which
then carry the DNA to the nucleus [25–27]. Alternatively,
the DNA could be transported to the nucleus during cell
division when the nuclear envelop is more permeable
[28]. In certain disease conditions, a low level of gene
expression for a sustained duration could be more desir-
able than high levels of transient gene expression. For
example, to induce neovasculature in ischemic (oxygen-
deprived) tissue, a low but sustained expression of growth
factors is considered more effective so that the new blood
vessels formed are matured and functional [29]. Thus, for
a particular disease condition, a crucial issue is to deter-
mine at what level and for how long the expressed protein
is required in the target tissue to achieve the desired
therapeutic outcome.
Surface properties of nanoparticles
The PLGA/PLA nanoparticles discussed above are anio-
nic at physiological pH. But, how could modulating the
surface charge of the nanoparticle affect gene transfec-
tion? The role of the cationic charge of DNA in complex
with DNA-condensing polymers is often discussed in the
context of particle size and gene transfection, but its
effect on the intracellular trafficking of complexes and
their interactions with intracellular transporters is poorly
understood. A few studies have reported an active trans-
port pathway for non-viral gene delivery systems.
Recently, Suh and colleagues [30,31] demonstrated
the active motor-protein-driven transport of polyethyle-
nimine-amine–DNA nanocomplexes (150 nm in dia-
meter) on microtubules (MTs) towards the nucleus,
and showed perinuclear accumulation of the complex
within minutes after transfection. Transport of this com-
plex by random thermal motion is estimated to take 8.7 h
to cover a distance of 10 mm through the cytosol, which is
significantly slower than what was actually observed.
Although the above study nicely demonstrated the
MT-mediated transport pathway, the authors did not
determine what specific characteristic(s) of the complex
are responsible for this active transport process. The
involvement of MTs has also been reported in the intra-
cellular transport of cationic liposomes [32]. Park et al. [33]
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Nanotechnology for drug and gene therapy Labhasetwar 677
have recently formulated low molecular weight prota-
mines that are structurally similar to the HIV–TAT
protein transduction peptide and have studied their gene
transfection efficiency. These protamines formed com-
plexes with DNA that were about 120 nm in diameter and
had a zeta potential (a measure of surface charge) of
+30 mV. These complexes demonstrated similar trans-
cellular localization behavior and kinetics to DNA–TAT
complexes, and efficiently transferred DNA into the
nucleus in a short time period. In fact, in their earlier
studies using liposomes, Zelphati and Szoka [34] demon-
strated that the positive charge on the complex is required
for oligonucleotide delivery to the nucleus, thus high-
lighting the role of the surface charge of a carrier system in
nuclear delivery. Similarly, in studies with gold nanopar-
ticles conjugated to different TAT peptides, Tkachenko
et al. [35] demonstrated that a highly cationic TAT
peptide results in the nuclear delivery of gold particles,
thus substantiating the significance of charge on cellular
transport processes. It would be interesting to determine
the intracellular transport mechanism of cationic systems.
The challenge, however, is to develop a cationic nano-
system that is efficient in gene transfection, but at the
same time is non-toxic and stable in the presence of blood
or other body fluids. In a recent article by Mooney, the
role of the rigidity of the cell adhesion substrate (i.e.
molecules such as oligopeptides that interact with the cell
membrane) on the level of gene transfer and expression
has been discussed [36,37]. Thus, understanding the
physicochemical interactions between the carrier system
and cell surface and intracellular transport mechanisms
could be crucial for designing efficient non-viral gene
expression vectors.
Nanotechnology for drug delivery
Drug dose and duration
In addition to their role in gene therapy, nanoparticles
have been investigated for applications in drug delivery.
A recent study suggested that the dose and duration of a
drug’s availability at the intracellular site of action deter-
mine its therapeutic efficacy [38]. In this work, PLGA and
PLA-based nanoparticles were formulated to encapsulate
dexamethasone, a glucocorticoid with an intracellular site
of action. Dexamethasone is a chemotherapeutic agent
that has an antiproliferative effect. The drug binds to
cytoplasmic receptors and the subsequent drug–receptor
complex is transported to the nucleus resulting in the
expression of certain genes that control cell proliferation.
Two formulations of drug-loaded nanoparticles were
tested for their antiproliferative activity in vascular
smooth muscle cells; the formulations differed only in
their ability to release different doses of the drug
(Figure 2a). The nanoparticle formulation that released
a higher dose of the drug for a prolonged period com-
pletely inhibited the proliferation of cells with a single-
dose treatment over a period of about two weeks, whereas
nanoparticles that released a lower dose of the drug for a
Current Opinion in Biotechnology 2005, 16:674–680
678 Pharmaceutical biotechnology

Figure 2

Nanoparticles for drug delivery. (a) In vitro release of dexamethasone from two nanoparticle formulations (A and B). The formulations differed
only in their ability to release different doses (high or low) of the drug. In both cases, drug release is from 600 mg of nanoparticles. Data are
mean values  the standard deviation (n = 3). (b) The inhibition of vascular smooth muscle cell proliferation with dexamethasone in solution
and encapsulated in nanoparticle formulations. Cell growth was followed using a mitogenic assay, where absorbance is proportional to the
number of viable cells. Data are mean values  the standard deviation (n = 6). (Figures reproduced with permission from [38]).

shorter duration demonstrated only partial and transient
inhibition of cell proliferation (Figure 2b). In this study,
the antiproliferative effect of the drug-loaded nanoparti-
cles correlated well with the intracellular drug levels and
drug retention.
For many drugs it is also necessary for the drug carrier
system to escape from the endosomal compartment and
enter the cytoplasm [39]. For example, paclitaxel is a
potent anticancer agent that acts by binding to MTs [40]
and therefore needs to be in the cytoplasmic compart-
ment. Doxorubicin is another anticancer agent that acts
through intercalation with the nuclear DNA, it also needs
to escape the endosomal vesicles and enter the cytosol for
its subsequent diffusion into the nucleus.
Surface properties of nanoparticles
The surface properties of nanosystems are thought to play
a crucial role in interactions with cells and the intracel-
lular transport machinery and can significantly affect the
efficacy of the encapsulated drugs. For example, pacli-
taxel-loaded nanoparticles were shown to exhibit greater
efficacy when they were conjugated to transferrin (Tf)
ligand, both in vitro in breast and prostate cancer cell lines
(MCF-7 and PC-3) and also in a murine model of prostate
cancer [41]. The greater efficacy of the Tf-conjugated
nanoparticles was found to result from their greater cel-
lular uptake and reduced exocytosis relative to unconju-
gated nanoparticles [42]. Thus, the Tf-conjugated
nanoparticles retained higher doses of the drug intracel-
lularly and for a sustained period of time. The intracel-
lular transport and retention characteristics of Tf-
conjugated nanoparticles following their cellular uptake
via Tf receptors could differ from those of unconjugated
nanoparticles because of differences in their uptake
mechanism. Tf-conjugated nanoparticles are taken up
by cells via Tf receptors, whereas unconjugated nano-
particles are taken up by the non-specific endocytic path-
way, thus influencing nanoparticle uptake, intracellular
retention, and the therapeutic efficacy of the encapsu-
lated drug. Furthermore, in a separate study, it was
demonstrated that the sustained intracellular drug reten-
tion mediated by Tf-conjugated nanoparticles can par-
tially overcome drug resistance [42]. Further studies to
investigate the potential of various ligands like Tf could
prove useful in the development of more efficient deliv-
ery systems.
Conclusions
As we make progress in different areas of nanotechnology
for biomedical applications, it is necessary to understand
the molecular mechanisms involved in the delivery of
these nanosystems at the cellular and molecular level.
Such knowledge could help us to overcome the barriers to
effective drug and gene delivery. A multidisciplinary
approach involving expertise from many different fields,
including drug delivery, polymer chemistry, physics, bio-
chemistry, engineering and so on, is expected to elucidate
the various aspects of nanotechnology in the future.
Eventually, these nanosystems will be developed for
various clinical applications, raising the issue of their
safety; developing biocompatible nanosystems is there-

Current Opinion in Biotechnology 2005, 16:674–680 www.sciencedirect.com


fore crucial. In general, the outlook for nanotechnology
for various biomedical applications is both exciting and
promising.
Acknowledgements
The author acknowledges his laboratory members (S Prabha, J Panyam,
W Ma, and S Sahoo) for the data that have been reviewed in this article.
Grant support from the Nebraska Research Initiative (Gene Therapy
Program), the Department of Defense, and the National Institutes of
Health (R01 EB003975) is appreciated.
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