Antiviral Chemistry & Chemotherapy 2010; 21:5370 (doi: 10.

3851/IMP1684)

Review
Nanoparticulate delivery systems for antiviral drugs
David Lembo1, Roberta Cavalli 2 *
1

Dipartimento di Scienze Cliniche e Biologiche, Universit degli Studi di Torino, Orbassano Torino, Italy
Dipartimento di Scienza e Tecnologia del Farmaco, Universit degli Studi di Torino, Torino, Italy

*Corresponding author e-mail: roberta.cavalli@unito.it

Nanomedicine opens new therapeutic avenues for

attacking viral diseases and for improving treatment
success rates. Nanoparticulate-based systems might
change the release kinetics of antivirals, increase their
bioavailability, improve their efficacy, restrict adverse
drug side effects and reduce treatment costs. Moreover,
they could permit the delivery of antiviral drugs to specific target sites and viral reservoirs in the body. These
features are particularly relevant in viral diseases where
high drug doses are needed, drugs are expensive and

the success of a therapy is associated with a patients

adherence to the administration protocol.
This review presents the current status in the emerging
area of nanoparticulate delivery systems in antiviral
therapy, providing their definition and description, and
highlighting some peculiar features. The paper closes
with a discussion on the future challenges that must be
addressed before the potential of nanotechnology can be
translated into safe and effective antiviral formulations
for clinical use.

Introduction
The global impact of viral infections, the development
of resistance to current drugs and the emergence of new
viruses all translate into the incessant scientific challenge
of drug discovery and formulation development. Over the
past 3 decades, many researchers have focused on developing new antivirals that are able to target important therapeutic processes. By 1990, just 5 drugs had been licensed
as antiviral agents [1], whereas approximately 20years
later more than 40 were on the market. Most of these
agents were developed for the treatment of HIV infection,
whereas others were active against various herpesviruses
(herpes simplex virus [HSV], varicella zoster virus [VZV]
and human cytomegalovirus [HCMV]), hepatitis B and C
viruses, and influenza A and B viruses.
In 2009, the global market for antiviral drugs reached
total sales of approximately USD 28 billion. Sales of
antivirals increased by approximately 20% from 2004
to 2006, and a continuing growth trend has been estimated until 2011. Moreover, the market is likely to witness even further future growth because of the existence
of unmet needs, expanding populations, better diagnostics, innovative drugs and new therapeutics; however,
developing a safe and effective antiviral drug is a difficult task, and the list of viral diseases for which antiviral therapies are available is still relatively short.
Several factors hinder the development of antiviral
drugs. Viruses are obligate intracellular parasites that
2010 International Medical Press 1359-6535 (print) 2040-2066 (online)

largely depend on the host cell biosynthetic machinery

for their replication; therefore, only a limited number
of virus-specific metabolic functions can be targeted
by antiviral drugs without harming the host. Ideally,
these targets are viral proteins essential for viral replication and pathogenesis that are sufficiently different
to any host protein to allow selectivity. Moreover, most
of these functions are specific for each virus, making it
difficult to develop broad-spectrum antivirals that are
active against diverse viruses that cause similar symptoms. The antivirals developed against some viruses (for
example, HSV and HIV) treat the acute disease but do
not cure the latent infection. This results in recurrent or
chronic diseases that require treatment for longer periods of time. These and other issues represent a major
challenge in antiviral research and development.
A second key challenge of antiviral therapeutics
regards the development of new drug formulations. This
involves changing the physicochemical and biopharmaceutical properties of antiviral molecules using technological strategies during the preparation of their dosage
forms. For example, the reformulation of an antiviral
drug already present on the market might be performed
in order to modify its bioavailability and pharmacokinetics. Further improvements to a therapy can also
be obtained through the use of innovative delivery systems for antiviral administration; for example, the use
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D Lembo & R Cavalli

of nanotechnology has led to the development of nanoparticulate carriers. Nanotechnological approaches can
be used to improve the design, formulation and delivery
of antiviral drugs.
This relatively new class of therapeutic nanomaterials, also called nanopharmaceuticals, displays unique
properties that arise because of their small sizes, high
surface-to-volume ratios and their modifiable surfaces.
Nanoparticulate carriers are able to incorporate small
molecules, as well as proteins and nucleic acids, thus
bestowing nanomaterials with a broad spectrum of prospective therapeutic applications and the potential to
target specific tissue sites where the antivirals are needed.
This review describes the current and future generations
of nanoparticulate delivery systems and their use as carriers for the transport of antiviral drugs.

Current antiviral therapies

The antiviral therapies currently approved are based on
the use of small molecular weight drugs or proteins that
stimulate the innate immune response (interferon). In
addition, an antisense oligonucleotide (fomivirsen) has
also been approved for the therapy of retinitis caused by
strains of HCMV resistant to conventional drugs [2].
Table 1 and Table 2 report the antiviral agents
present on the market and used in clinical practice.
The approved antiviral drugs for HIV infections are
summarized in Table 1 and other antiviral agents are
listed, according to the viral infection, in Table 2. As
shown, the majority of antiviral drugs are administered orally, although some are delivered via parenteral
(subcutaneous, intravenous and intravitreal) or topical routes.
Many antiviral drugs present problems that reduce
their efficacy, such as limited solubility, a short halflife or slow, incomplete or highly variable absorption.
Consequently, high doses and frequent administration
are required that, in turn, can negatively affect patient
compliance, causing severe side effects.
Many antivirals, such as the antiretrovirals acyclovir
and ganciclovir, show low bioavailability when administered orally. An adequate bioavailability (that is,
adequate absorption by the gastrointestinal tract that
depends on solubility and permeability) is fundamental
for the success of an antiviral. Good solubility and permeability are considered as markers of adequate oral
bioavailability and are essential prerequisites for antiviral drugs. Based on their solubility and permeability,
Amidon et al. [3] classified all the orally administered
drugs into four classes (I, II, III and IV) according to
decreasing solubility and permeability values using the
Biopharmaceutics Classification System (BCS). According to the BCS, a molecule is considered highly soluble when its highest dose solubilizes in 250ml of an
54

Table 1. Approved antiviral drugs for HIV infections

Drug class
and name

Route of
administration

Nucleoside reverse transcriptase inhibitors

Abacavir: 2-amino-6-cyclopropylaminopurin-
9-yl-2-cyclopentene
Didanosine: 2,3-dideoxyinosine
Emtricitabine: (-)--l-3-thia-2,3-dideoxy-
5-fluorocytidine
Lamivudine: (-)--l-3-thia-2,3-dideoxycytidine
Stavudine: 2,3-dideoxy-2,3-didehydrothymidine
Zalcitabine: 2,3-dideoxycytidine
Zidovudine: 3-azido-2,3-dideoxythymidine
Nucleotide reverse transcriptase inhibitors
Tenofovir disoproxil fumarate: bis(isopropoxy-
carbonyloxymethyl)ester of (R)-9-(2-phosphonylmethoxypropyl)adenine
Non-nucleoside reverse transcriptase inhibitors
Delavirdine
Efavirenz
Etravirine
Nevirapine
Integrase inhibitors
Raltegravir
Protease inhibitors
Amprenavir
Atazanavir
Darunavir
Fosamprenavir (a prodrug of amprenavir)
Indinavir
Lopinavir
Nelfinavir
Ritonavir
Saquinavir
Tipranavir
Fusion/entry inhibitors
Enfuvirtide (T-20)
Maraviroc

Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral

Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Oral
Subcutaneous
Oral

aqueous medium over the pH range 17.5 at 37C. By

contrast, a molecule is considered highly permeable
when the extent of intestinal absorption in humans is
>90% of the administered dose based on the mass-balance determination or in comparison to an intravenous
reference dose. Besides solubility and permeability,
other factors are also able to affect the oral bioavailability of an antiviral, including the action of intestinal
metabolizing enzymes, efflux transporters and food.
Consequently, oral absorption is variable and depends
on many conditions. The oral administration of an antiviral with a low or variable bioavailability thus requires
the use of higher doses and prolonged treatment durations in order to eradicate the virus. For example, most
of the HIV protease inhibitors are of high molecular
weight (>500 Da) and possess pH-dependent solubility
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Nanoparticulate drug delivery for antivirals

Table 2. Approved antiviral drugs for HBV, HCV, HSV, VZV,

HCMV and influenza virus infections

Drug name
Approved for HBV
Adefovir dipivoxil
Entecavir
Interferon-2b
Lamivudine
Pegylated interferon-2a
Telbivudine
Tenofovir disoproxil fumarate
Approved for HCV
Ribavirin
Pegylated interferon-a
Approved for HSV and VZV
Acyclovir

Brivudin
Famciclovir
Iodoxuridine (prodrug of penciclovir)
Penciclovir
Trifluridine
Valaciclovir
Approved for HCMV
Nucleoside DNA polymerase inhibitors
Ganciclovir

Cidofovir
Valganciclovir (prodrug of ganciclovir)
Non-nucleoside DNA polymerase inhibitors
Foscarnet
Antisense oligonucleotide-gene expression
inhibitors
Fomivirsen
Approved for influenza
M2 inhibitors
Amantadine
Rimantadine
Neuraminidase inhibitors
Oseltamivir
Zanamivir

Route of
administration

Oral
Oral
Subcutaneous
Oral
Subcutaneous
Oral
Oral
Oral
Subcutaneous
Intravenous, oral or
topical
Oral
Oral
Intravenous
Topical
Eye drops
Oral

Intravenous, oral or
intravitreal
Intravenous
Oral
Intravenous

Intravitreal

Oral
Oral
Oral
Inhalation

HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella

zoster virus.

(that is, they are more soluble at low pH) and high
lipophilicity, properties that each could adversely affect
oral bioavailability [4]. These properties are classified
as both III and IV, according to the BCS system. The
majority of nucleoside reverse transcriptase inhibitors
show good systemic absorption, although didanosine
(BCS class III) and zidovudine exhibit variable bioavailability. The bioavailability of the commercially available
dosage forms of antiretroviral drugs were recently summarized by Sharma and Garg [5] who showed that the
majority of these drugs undergo limited absorption.
Antiviral Chemistry & Chemotherapy 21.2

Acyclovir, used in different dosage forms to treat

HSV and VZV infections, has a low oral bioavailability (1520%) because of its slow and incomplete
absorption in the gastrointestinal tract (BCS class III);
high doses (up to 1,200mg/day) are therefore required
for this antiviral agent. Approximately 80% of the
administered dose of acyclovir is never absorbed. To
overcome this problem, derivatives and prodrugs have
been synthesised, such as valaciclovir, the l-valine ester
of acyclovir, and famciclovir, a prodrug of penciclovir,
which show improved oral absorptions in comparison
with the parent drug. Topical acyclovir therapy has
low efficacy because of the low penetration of acyclovir in the basal epidermis, and topical formulations of
the drug (ointments or creams) need to be applied 56
times per day.
The anti-HCMV drug ganciclovir represents another
example of an antiviral with very poor oral bioavailability (69%), requiring the daily administration of a
dose >1g. Moreover, the oral administration of other
antivirals is impossible; for example, foscarnet and
cidofovir require intravenous administration because
of their extremely low oral absorption and their gastrointestinal toxicity.
The intravitreal administration of ganciclovir and
fomivirsen were demonstrated to be more effective
than intravenous administration for the local treatment
of the posterior segment of the eye for some ocular
pathologies, including retinitis, but high doses or the
administration of several frequent doses are required,
and intraocular injections are poorly tolerated and run
associated risks.
Another problem of antiviral agents is that the
chronic treatment with such drugs can produce moderate levels of drug toxicity, which might lead to serious
complications in the patient. Moreover, prolonged antiviral therapy increases the likelihood that drug-resistant
strains of the virus will emerge [68].
To improve the therapeutic activity of antivirals
present on the market it is possible to change the conventional dosage forms. Radically modified formulation
of drug dosage forms, such as depot-like injectables,
modified release tablets and improved topical delivery
systems, have been developed and are currently under
investigation by many pharmaceutical companies for
their use in the administration of the antiviral drugs
already on the market. This type of approach can be
useful to increase the BCS score of antivirals, particularly if their solubility and dissolution rate are improved
with the reformulation.
Such new formulations of conventional dosage
forms, which can modify the residence time and reduce
the administered dose, aim at overcoming the problems of non-compliance brought about by side effects
associated with a drug and difficult dosing regimens.
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D Lembo & R Cavalli

An example is the development of long-acting interferons conjugated with poly(ethylene glycol) (PEG)
molecules and designed for weekly dosing instead of
the usual regimen of injections 3 per week. In addition to the modification of formulations, another
strategy now being pursued for combating viral infections is the design of novel nanodelivery systems for
drug administration.

Nanotechnologies to improve the delivery of

antiviral agents
Over the past 2 decades, nanotechnology solutions
have been developed to improve the delivery of active
molecules. Nanotechnology is the creation and utilization of materials and systems on the nanometre
scale (a nanometre is one-billionth of a metre). In
diagnostic and therapeutic fields, nano-scale strategies mainly consist of nanoparticles and nanoconstructs and are referred to as nanomedicines [9]. The
application of nanomedicine for the delivery of active
antiviral molecules by means of nanocarriers, above
all, aims at obtaining higher potency and lower toxicity in the patient.
It was recently estimated that the drug delivery
industry is currently worth approximately USD 80billion and a major component of this sum is devoted to
the design of controlled release and targeting systems.
Thus, the development of new methods for achieving
controlled release is a very attractive research area, both
in terms of the need to improve healthcare and from the
perspective of pharmaceutical companies to maintain
revenue and to ensure patent positions in both existing and new drugs. A report by Cientifica Ltd [10] estimated the nano-based drug delivery market to be worth
USD 3.4billion in 2007 and that it would increase to
approximately USD 26billion by 2012.
Nanodelivery systems, which mainly consist of nano
particulate systems (including nanoparticles, nanocapsules, vesicles, dendrimers, micelles and inorganic
Table 3. Possible nanocarriers for antiviral therapy
Types of nanocarriers
Micelles
Microspheres
Polymeric nanoparticles
SLN
NLC
Liposomes
Dendrimers
Vesicles
Cyclodextrin-based systems
Emulsions
NLC, nanostructured lipid carriers; SLN, solid lipid nanoparticles.
56

nanomaterials), have been designed to deliver small

molecular weight drugs, but they can also be exploited
for the delivery of macromolecules and biological therapeutics such as oligonucleotides [11] (Table3).
The miniaturization of materials often imparts novel
physicochemical properties. Specifically, as a particles
size decreases, a greater proportion of its atoms are
located on the surface relative to its core; thus, there is
an increase in the surface-area-to-volume ratio, often
rendering the particle more reactive. Nanocarriers
can be synthesized by various methods, such as selfassembly, vapour and electrostatic deposition, solvent
diffusion and solvent evaporation techniques, coacervation and nanomanipulation.
Using these nanocarriers it might be possible to
overcome many problems of antiviral drugs in conventional dosage forms; their use might help to control solubility and dissolution rates (improvement in
BCS score), increase drug bioavailability, protect sensitive drugs from degradation, reduce side effects and
ameliorate tissue drug tolerance. Moreover, this type
of nanotechnological approach provides the possibility of targeting specific biological sites either passively
or actively (Table 4). Because of their unique features,
such as size and lipophilicity, nanocarriers can target
drugs to specific tissues or organs, such as the liver
or the brain, while modifying nanocarrier surfaces
enables them to reach particular sites and deliver the
drug to specific cellular targets.
Nanodelivery systems can be applied for the local
or systemic delivery of antiviral drugs. With respect to
intravenous administration, they must be in the nanometre range in order to circulate in the bloodstream
without being retained by the pulmonary capillaries.
Specific strategies have been designed to overcome
their uptake by the reticolo-endothelial system (RES).
The most frequent approach to increase the longevity
of nanocarriers avoiding the RES uptake is to modify
their surface with certain hydrophilic polymers, such
as PEG.
The nanoparticulate systems also present characteristics that are very suitable for ocular, nasal and pulmonary administration routes. Nanocarriers could be useful for the selective delivery of antiviral drugs or small
interfering RNA (siRNA) to the nasal epithelia and lungs
Table 4. Summary of the advantages of nanocarriers
Key nanocarrier advantages
Improved bioavailability
Controlled release
Protection of drugs
Decrease the emergence of drug resistance
The overcoming of anatomical/cellular barriers
Specific targeting

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Nanoparticulate drug delivery for antivirals

in order to target viruses that infect the respiratory tract,

such as influenza viruses, respiratory syncytial virus and
rhinoviruses, to name just a few.
Promising compounds shown to have antiviral effects
in vitro, but that are not currently being administered
in vivo because of solubility and bioavailability problems, could be administered using nanocarriers that can
permit their administration; this can include peptides
and nucleic acid delivery [12]. Certain nanodelivery
systems might be suitable for the delivery of peptides
and proteins protecting them from degradation.
In the past few years, RNA interference (RNAi) has
emerged as a promising antiviral strategy that acts by
silencing the gene expression of human viral pathogens,
including that of influenza viruses, severe acute respiratory syndrome virus, flaviviruses, HIV, HCV and HBV
[1316]. Very recently, a study by DeVincenzo et al.
[17] provided a unique proof-of-concept for an RNAibased therapy in humans directed against respiratory
syncytial virus [17]. Nevertheless, there are still many
obstacles that impede the translation of RNAi into a
potential therapeutic platform, and the most important
obstacle regards the delivery of siRNA in vivo. Targeting the action of siRNA to specific tissues and cells
could minimize toxic side effects and improve their therapeutic efficacy. However, even if siRNA reach the correct cellular target, their size and their negative charge
make it difficult for them to cross the cell membrane,
and many primary cell types are highly recalcitrant to
siRNA uptake. Consequently, many delivery strategies
based on nanotechnology are currently under development to address these challenges [18]. In addition to the
advantages described above, the submicron size range
of these delivery systems can also render intracellular
uptake and transport of active compounds possible.
In particular, the delivery of macromolecules into the
cytoplasm is limited by their low membrane permeability and their degradation in the endosomal environment after uptake by endocytosis. Their incorporation
into a nanoparticulate system could promote cell internalization and protect the molecules from degradation.
This is an important feature because most antiviral
drugs, like nucleoside analogues, target viral functions
that are carried out within a cell. Various mechanisms
govern the entry of nanoparticulates into cells, including caveolae-mediated endocytosis, clathrin-mediated
endocytosis, phagocytosis and macropinocytosis [19].
Fluorescent-labelled nanoparticles can be used to study
particle uptake by cells and their cellular trafficking.
Another relevant feature of nanocarriers is the ability
to overcome the physiological barriers. Nanomedicine
is able to promote the delivery of drugs to the central
nervous system. Various studies using different nanocarriers report enhanced in vitro and in vivo bloodbrain
barrier (BBB) permeability and drug accumulation in
Antiviral Chemistry & Chemotherapy 21.2

the brain [20]. Nanocarriers can enhance brain delivery

by three major approaches: increasing the local drug
concentration gradient at the BBB by passive targeting,
allowing drug trafficking by non-specific or receptormediated endocytosis and blocking drug efflux transporters at the BBB. By selecting the components and the
formulation parameters it is possible to prepare nanocarriers with physicochemical properties that allow
delivery to the brain.
Three categories of nanocarriers have been investigated
for the delivery of antiretrovirals to the central nervous system: polymer/dendrimer-based, lipid-based and
micelle-based systems [20]. The bloodretinal barrier, the
anatomical barrier that protects the eyes, could also be
overcome using nanocarriers. Moreover, using nanotechnology-based systems it could be possible to reach anatomical compartments or cellular viral reservoirs that are
not easily accessible to drugs in their current dosage form.
For instance, the central nervous system, the cerebrospinal fluid, the lymphatic system, the macrophages and the
semen are almost completely inaccessible to drugs, and
are therefore compartments where HIV is harboured and
evolves independently despite a successful highly active
antiretroviral therapy [2123]. Suboptimal drug penetration into these compartments complicates the treatment
of HIV infection and the eradication of viral reservoirs
from the patient. Similar issues apply to herpesviruses,
which latently infect particular cells and tissues.
The administration of antivirals in nanoparticles might
affect the therapeutic efficacy inhibiting efflux transporters. Drug efflux transporters, such as P-glycoprotein
(P-gp) play an important role in limiting the transport
of xenobiotic molecules through various critical barriers
in the body. Many orally administered drugs must cross
the basolateral membrane in the intestinal epithelium to
reach the blood. P-gp could drive compounds from inside
the cells back into the intestinal lumen preventing their
absorption. In cancer cells P-gp enables the development
of resistance to anticancer drugs [24]. The activity of
efflux transporters, which expel drugs from cells, leads to
subtherapeutic drug concentrations. Indeed, P-gp inhibition represents one potential strategy for the improvement
of antiviral intestinal absorption. It has been previously
demonstrated that the absorption of acyclovir in vitro is
increased in the presence of P-gp-specific inhibitors, but
this inhibition can increase side effects [25,26]. Another
strategy is the use of nanoparticulate systems to deliver
the drug into the cells favouring the absorption.
Another advantage of nanoparticles is that multifunctional systems can be obtained by engineering their
surfaces. The advantageous characteristics resulting
from such modifications, including longevity, targetability and stimuli sensitivity, thus combine to produce multifunctional nanocarriers that can simultaneously perform more than one useful function [27].
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Such multifunctional nanocarriers could significantly

enhance the efficacy of many therapeutic protocols.
Another emerging area of research is the development
of integrated multifunctional nanosystems for diagnosis
and therapy. These novel systems, called theranostics,
are designed specifically for the simultaneous diagnosis
and treatment of cancer. The nanosystem must be able
to biomark cancer cells in order to achieve simultaneous
and targeted imaging and treatment [28]. In the future,
these integrated medical nanosystems could prove to be
useful for the molecular diagnosis, treatment and monitoring of viral infections at the cellular level.
Nanoparticles have similar nanometre dimensions
to viruses. This feature led several researchers to investigate the physical interaction of nanoparticles with
viruses and to explore whether this interaction could be
exploited as an antiviral strategy. Indeed, silver nanoparticles with mean particle diameters ranging from 10 to
50nm have been shown to inhibit infection by various
viruses including HIV, HBV, respiratory syncytial virus
and monkeypox virus [2933]. All of these studies concluded that the direct interaction between the nanoparticles and the virus was responsible for the antiviral activity observed. It seems that nanoparticles exert antiviral
activity at an early stage of viral replication, most likely
as a virucidal agent or as an inhibitor of viral attachment
and entry. Baram-Pinto et al. [34] further developed this
strategy by designing silver nanoparticles capped with
mercaptoethane sulphonate in order to target HSV and
to compete for its binding to cell-surface heparan sulphates. This strategy resulted in effective inhibition of
HSV type-1 infection in cell culture and led the authors
to propose capped nanoparticles as active ingredients of
topical microbicides for the prevention of viral infections
that depend on heparan sulphates for entry.

Targeted delivery of antiviral agents

The concept of targeted drugs was first suggested
by Paul Ehrlich in 1906 who postulated the magic
bullet theory. One century after this intuition, targeted drug delivery by functionalized nanocarriers
has become one of the most attractive and promising
areas of research in nanomedicine. However, it should
be pointed out that some key challenges must be
addressed before achieving quantitative delivery and
targeting in vivo [35]. Site-specific drug delivery could
be obtained with different types of nanocarriers.
The majority of studies performed to date have
focused on developing systems that improve the biodistribution of anticancer drugs and their accumulation in
specific tissues. Three distinct strategies exist for drug
targeting: direct injection to a specific site, passive targeting and active targeting. Passive targeting means the
nanoparticulate carrier can reach a given organ by the
58

virtue of its intrinsic properties, such as particle size

or lipophilicity, whereas active targeting involves the
presence of a homing device that guides the carrier to
its target site. Passive targeting associated with nanocarrier size permits the penetration of nanoparticles
into tumour tissues because of the presence of leaky
vasculature. This effect referred to as the enhanced
permeability and retention (EPR) effect results in
nanoparticle accumulation within the tumours as demonstrated by Maeda et al. [36] (Figure 1). Because of
their small sizes and surface characteristics, nanoparticles can be taken up by the lymphatic tissue in the gut
(that is, the Peyers patches containing M cells) after
oral administration.
Lymphatic targeting has increased the amount of
attention directed at nanopharmaceuticals because of
the prospect of directly targeting lymphocytes with
immunomodulators, resident HIV viruses with antiviral agents and disseminated tumour metastasis [37].
In stark contrast to molecularly dissolved drugs, nanocarriers can be designed for targeting the lymphatic circulation. With regards to injectable systems, although
the particles must be large enough to drain, preferentially through the lymphatics, they must also be small
enough to diffuse through the interstitial space away
from the injection site. Sizes in the range of 10100nm
are optimal. Moreover, hydrophilic nanoparticles clear
more rapidly than hydrophobic nanoparticles following interstitial injection.
To date, lymphatic uptake has been widely investigated in relation to the oral administration of
medicines. Desai et al. [38] studied the influence of
poly(d,l-lactide-co-glycolide) nanoparticle sizes on gastrointestinal uptake in rats. Depending on the nanoparticle size, the Peyers patch tissue showed a 2200-fold
higher uptake than non-patch tissue. The use of nanoparticle systems for oral drug delivery to the lymphatic
system is rendered possible because of physiological
particulate uptake mechanisms in the gut, especially
the transcellular pathway involving vesicular transport
through the M cells of Peyers patches. Nanoparticles
are taken up by M cells in a size-dependent manner
and transported to lymphocytes in the form of vesicles. The lymphatic absorption of a drug can prevent
its systemic metabolism by the liver and permit targeting to the lymphatic system. This peculiar particulate
behaviour permits the lymphatic system to be reached
and could be exploited to target viral reservoirs held
within this compartment.
Active targeting can be accomplished by different
strategies all consisting of surface modifications, in
particular via a specific ligand-receptor-like mechanism. The primary strategy uses monoclonal antibodies
raised against specific cells or tissues. Other molecules,
such as sugars, polymers, proteins, vitamins, lectins
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Nanoparticulate drug delivery for antivirals

Figure 1. Schematic representation of passive targeting of tumour tissues associated with the enhanced permeability and
retention effect

Role of cutoff size

300700 nm

Normal vasculature

Leaky vasculature
Drug/drug carrier penetrates
into interstitium

Tumour-specific vascular pathophysiology, with defective architecture and impaired lymphatic drainage, provides an increased permeability to macromolecules and
nanoparticulates. This phenomenon has become a gold standard for the delivery of drugs to solid tumours.

and aptamers, can also be used as homing devices as

depicted in Figure2 [39].
Another approach to target specific body areas or
intracellular compartments is the use of stimuli-sensitive
nanocarriers. This strategy exploits either intrinsically
abnormal pH, redox and temperature values of pathological sites and intracellular organelles (that is, the endosomes) or externally applied stimuli, such as a magnetic
field, temperature and ultrasounds. All of these stimuli
are expected to dissolve, to modify or to guide the sensitive nanocarriers, resulting in the release of the loaded
drug in a particular region, such as tumours, inflammation sites, infarcts or endosomes [27].
pH-sensitive nanocarriers are of particular interest in
the area of therapeutic applications. The concept of pHsensitive systems emerged from the knowledge that certain enveloped viruses (for example, the influenza virus)
lose their envelope in the acidic environment of the
endosomal lumen thereby infecting the cells, and that
some pathological tissues, as in tumours, inflammations
and infections, exhibit a relatively more acidic environment than normal tissues. Different classes of pH-sensitive systems have been proposed, such as liposomes,
polymeric micelles and nanogels [9]. These pH-sensitive
carriers can promote the intracellular release of the
encapsulated drug when the pH changes. pH-sensitive
liposomes are stable at physiological pH levels (7.4) but
become unstable and fusogenic at acidic conditions (that
is, in a lysosomal environment), releasing their aqueous
content in the intracellular compartment.
Antiviral Chemistry & Chemotherapy 21.2

External stimuli can also be used in combination

with labelled nanocarriers that are externally guided
(for example, by a magnetic field) or with specific delivery systems activated by the application of a physical
stimulus, such as temperature or ultrasounds.
In magnetic drug delivery, an external magnet is used
to guide the drug-loaded nano- or microparticles to the
targeted organ and to hold them there. The carrier is
therefore magnetically concentrated in the target organ,
but the subsequent release of the drug is a passive process affected by the properties of the particulate system.
By contrast, the use of ultrasound permits the activation
of the drug release at the site of action. Unlike the various
targeted systems developed for anticancer therapy, few
examples have been reported in the literature until now
for targeted antiviral therapy. Most of these are listed in
Table 5 [4051] and mainly concern liposomes or nanoparticles designed for the HIV treatment. An example of
an external stimulus approach is that of magnetic microspheres containing interferon to achieve targeting using
an external magnetic field [46].
Macrophages can act as a virus reservoir and sustain
replication of HIV [52]. Macrophage targeting using
nanoparticulate systems can be a therapeutic strategy
because macrophages easily phagocytose foreign nanoparticles [53]. The size, composition and surface properties of nanoparticles can all affect macrophage uptake.
For example, polyhexylcyanoacrylate nanoparticles
with a diameter of approximately 200nm were found to
be the most useful for targeting antiviral substances to
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Figure 2. Schematic representation of a functionalized nanoparticle

Targeting
molecule

Cell penetrating
peptide

Matrix

Drug
Image contrast
agent

Magnetic probe
Poly(ethylene glycol)

Poly(ethylene glycol) chains act as stealth agents. Targeting molecules can be attached to the surface of the functionalized nanoparticle as depicted. The
presence of the contrast agent could illuminate the interaction of the nanoparticle with a target cell. The magnetic probe could permit the nanoparticle
localization using an external magnetic field.

Table 5. Targeted delivery systems developed for antiviral drugs

Drug

Virus

Nanodevice

siRNA
HCV
Cationic liposomes
AZT
HIV
Albumin nanoparticles
Protease inhibitor HIV
Pegylated liposomes

siRNA
HIV
Immunoliposomes
Nosiheptide
HBV
Recombinant HDL
Acyclovir
HBV
Recombinant HDL
Interferon
Magnetic microspheres
Saquinavir
HIV
Nanoparticles
gp120 Folding
HIV
Liposomes
inhibitor
Interferon-
Nanoparticles
Indinavir
HIV
Immunoliposomes

Protease inhibitor HIV
Liposomes

Targeting

Targeted tissue

In vivo studies Reference

Apolipoprotein A1
Transferrin
Monoclonal antibody
against gp120
Antibody against LFA1
Recombinant HDL
Recombinant HDL
External magnetic field
Transferrin
CD4 antigen

Liver
Brain
HIV-positive cells

Yes
Yes
No

Kim et al. [40]

Mishra et al. [41]
Clayton et al. [42]

Lymphocytes
Liver
Liver

Brain
HIV-positive cells

Yes
Yes
Yes
No
No
No

Kim et al. [43]

Feng et al. [44]
Feng et al. [45]
Zhou et al. [46]
Mahajan et al. [47]
Pollock et al. [48]

Digalactosyl diacyl glycerol

Antibodies against human and
murine HLA-DR and CD4 antigen
CD4 antigen

Liver
Lymphoid tissues

No
Yes

Chiellini et al. [49]

Gagn et al. [50]

Lymphocytes

Yes

Dzgnes et al. [51]

AZT, zidovudine; HDL, high-density lipoprotein; HLA, human leukocyte antigen; LFA1, lymphocyte function-associated antigen 1; siRNA, small interfering RNA.
60

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Nanoparticulate drug delivery for antivirals

macrophages [54]; the same study demonstrated a good

level of nanoparticle incorporation in macrophages
obtained from HIV-infected patients. Additional targeting moieties can be added to nanoparticles to enhance
the level of macrophage uptake. Mannan-coated nanoparticles containing didanosine were found to undergo
greater targeting to macrophages by exploiting mannosyl receptor-mediated endocytosis.
Drug targeting using surface-modified nanocarriers is
a strategy that permits delivery at the organ or even cell
level. It was recently shown that the intracellular distribution of nanoparticles could be controlled by coupling TAT peptide or cell penetrating peptides to the

Table 6. Summary of possible surface modifications of

nanoparticle-based systems for targeting purposes
Surface modification
Presence of surface charge
Surface coating
PEG coating
Antibody binding
Antibody fragment conjugation
CD4-derived peptide conjugation
Mannose conjugation
Galactose conjugation
Transferrin
Apolipoprotein
Recombinant HDL

Figure 3. Structure of a liposome and schematic

representation of possible drug incorporation

Hydrophobic drug

Aqueous
solution

Hydrophobic tail
Hydrophilic head

Antiviral Chemistry & Chemotherapy 21.2

Overview of particulate carriers

The need for the development of new formulations for
HIV, HBV, HCV and HSV antiviral treatments has been
the major driving force in antiviral research. In this
review, an up-to-date summary of the new formulations
of HIV drugs will not be given because this task was
recently completed by Sosnik et al. [56]. Moreover bioconjugate systems and films have not been considered
because this review was focused on particulate delivery
systems. An overview of the most studied drug delivery
systems proposed for use in antiviral therapies is, however, reported below.

Liposomes

HDL, high-density lipoprotein; PEG, poly(ethylene glycol).

Hydrophilic drug

nanoparticle surface in order to facilitate endosomal

escape. This is particularly important for drugs that act
within the cytosol or that must reach the nucleus [55].
The various nanoparticle surface modification strategies used for targeting purposes are listed in Table6.

Liposomes were the first vesicular carriers, proposed

by Gregoriadis [57], to be used as drug delivery systems. Liposomes are lipid concentric vesicles in which
an aqueous volume is completely enclosed in a lipid
bilayer composed mainly of phospholipids and cholesterol (Figure3). Liposomes can vary in diameter, from
20 to 30nm up to microns, depending on their chemical composition and the preparation method used.
Structurally, they can be classified as either small unilamellar vesicles or large unilamellar vesicles. They are
able to encapsulate hydrophilic drugs within their inner
aqueous phase and lipophilic drugs within their lipid
bilayers. Liposomes are recognized as foreign matter by
RES. Because HIV resides in macrophages, liposomes
have been studied as promising carriers for anti-HIV
drugs [58].
The liposome surface can be modified for different
purposes. The incorporation of PEG molecules into the
liposome bilayer prevents its interaction with plasma
proteins and can consequently retard the recognition
and removal of liposomes by RES. Functionalization of
the liposome surface in order to achieve specific targeting has also been studied. These vesicular carriers also
present certain disadvantages, such as poor stability
both in vitro and in vivo, low encapsulation efficiency
and high cost of production.
Some liposomal formulations are in clinical practice
for the intravenous administration of anticancer or antifungal drugs. Liposomal formulations for cancer therapy
currently on the market are Doxil (pegylated liposomal
doxorubicin; Ortho Biotech Products, Bridgewater, NJ,
USA), Myocet (non-pegylated liposomal doxorubicin;
Cephalon, Frazer, PA, USA) and DaunoXome (nonpegylated liposomal daunorubicin; Nextar, Boulder,
61

D Lembo & R Cavalli

CO, USA); Ambisome (Gilead Sciences, Foster City,

CA, USA) is a liposomal formulation of amphotericin.
A vaginal liposomal delivery system for acyclovir has
been designed for the local treatment of genital herpes
[59]. A bioadhesive hydrogel consisting of Carbopol
974P was used as vehicle for the liposome containing
acyclovir. In vitro release studies showed that the system can be considered for the vaginal sustained release
of acyclovir. Liposomes have also been studied for the
oral administration of interferon- [60], the intravitreal
administration of antisense oligonucleotides using liposomes for the treatment of CMV have been designed
[61] and positively charged liposomes for the topical
administration of acyclovir have been prepared [62].
The in vitro corneal penetration and in vivo corneal
absorption of acyclovir from acyclovir-loaded liposomes have been investigated. The extent of absorption with positively charged liposomes was higher than
those from negatively charged liposomes. These were
probably able to bind to the corneal surface, leading
to an increased residence time favouring the acyclovir
absorption [63]. Chetoni et al. [64] confirmed the liposomal efficacy for the ocular delivery of acyclovir.
Another form of vesicle proposed for drug delivery is
the niosome, a vesicle similar to a liposome but formed
with non-ionic surfactant instead of lipids [65]. The
incorporation of acyclovir into liposomes and niosomes
was recently compared [66]: niosomes were found to
perform as better carriers for acyclovir because of their
superior loading and slower release of the drug compared to that obtained with liposomes. Acyclovir-loaded
in niosomes consisting of Span 60 (Merck, Frankfurt,
Germany), cholesterol and dicetylphosphate have been
investigated to improve the oral bioavailability of the
drug. In vivo studies revealed that the niosomal dispersion enhanced, by >2-fold, the oral bioavailability of
acyclovir in relation to the free solution [67].

Micelles
Micelles are colloidal structures (with particle diameters
normally within the 5 to 100nm range) belonging to a
group of association or amphiphilic colloids (molecules
that consist of two clearly distinct regions with opposite affinities towards water), which form spontaneously at certain concentrations and temperatures from
amphiphilic molecules or surfactants. At low concentrations in an aqueous medium, such amphiphilic molecules exist separately; however, as their concentration is
increased, aggregation takes place, although only within
a rather narrow concentration interval. The concentration of a monomeric amphiphile at which micelles appear
is called the critical micelle concentration, whereas the
temperature below which amphiphilic molecules exist
as unimers and above which they appear as aggregates is called the critical micellization temperature.
62

Figure 4. Schematic illustration of a micelle

Hydrophobic drug

Aqueous solution

Hydrophobic tail
Hydrophilic head

The formation of micelles is driven by the decrease of

free energy in the system because of the removal of its
hydrophobic fragments from the aqueous environment
and the re-establishment of a hydrogen bond network in
water. The hydrophobic fragments of amphiphilic molecules form the core of a micelle, whereas hydrophilic
fragments form the micelles shell. When used as drug
carriers in aqueous media, micelles are able to solubilize poorly soluble lipophilic agents within its core, and
polar molecules can be adsorbed onto the micelles surface [68] (Figure 4).
Polymeric micelles are nanostructures used to
improve aqueous solubility, intestinal permeability and
site targeting of several drugs. Compared to conventional surfactant-based micelles, polymeric micelles
are composed of block copolymers and show greater
stability in vivo. Micelles of PEG-polylactide copolymer surface modified with galactose units can interact
with lectins [69]. Lectin receptors are present on HIV
viral reservoirs, such as T-lymphocytes and macrophages; thus, these copolymer micelles can be used as
an approach for targeting viral reservoirs.
Block-copolymers of polyethylene-oxidepolypropylene-oxide, known as Pluronics (BASF, Florham Park,
NJ, USA), have been proposed to enhance the intestinal permeability of antiretroviral drugs. Amphiphilic
molecules can also be used to obtain self-assembled
nanoparticles in water. Self-assembled delivery systems
using cholesteryl derivatives as prodrugs for antiviral
therapy have also been studied. Cholesteryl derivatives
of acyclovir have been synthesised and show a typical
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Nanoparticulate drug delivery for antivirals

Figure 5. Schematic morphologies of the two types of particulate material

Drug

Drug

Core

Matrix

Inner cavity

Capsule shell

(A) Core-shell nanomicrocapsule or reservoir and (B) matrix nanomicroparticle.

amphiphilic structure with the lipid as hydrophobic tail

and the antiviral nucleoside as the polar head [70]. Selfassembled drug delivery systems have also been designed
to obtain nanoparticles from amphiphilic molecules. A
lipid derivative of acyclovir has been synthesized showing the ability to form nanoparticles that were rapidly
removed from blood circulation by macrophage uptake
after their injection in rabbits [71].

Microspheres
Microspheres are particulate carriers within the micron
size range and are generally constituted of biodegradable polymers. They could be monolithic-type (matrixtype) or reservoir-type (capsular), the latter of which
are called microcapsules (Figure 5). A wide range of
techniques has been developed for their preparation
to date. Different microsphere formulations have been
studied as drug delivery systems for antivirals. Specifically, biodegradable particles could be suitable for antiviral administration via the intraocular route. Poly-d,llactide and poly(d,l-lactide-co-glycolide) microcapsules
have been prepared by the spray-drying technique, and
in the case of acyclovir were found to achieve high
encapsulation efficiency. The microspheres were tested
in vivo by intravitreal administration in rabbits and
showed a prolonged release of acyclovir [72]. Microspheres of poly(d,l-lactide-co-glycolide) have been proposed for the intravitreal administration of acyclovir
that aim at sustaining the release of the drug in order
to minimize the dose as much as possible [73]. These
Antiviral Chemistry & Chemotherapy 21.2

acyclovir-loaded microspheres were prepared by the

solvent evaporation method and a factorial design was
applied to reduce particle sizes to values suitable for
injection through a 27G needle and to increase drug
loading. The same author proposed the successful
application of biodegradable microspheres of poly(d,llactide-co-glycolide) containing the combination of
acyclovir and vitamin A palmitate to treat herpes
simplex and EpsteinBarr viruses. Acyclovir loading
increased when vitaminA palmitate was added to the
microspheres and the in vitro acyclovir release was subsequently prolonged for 50 days [74].
Semi-interpenetrating polymer networks of acrylamide grafted onto dextran and chitosan were prepared using an emulsion cross-linking method, with
glutaraldehyde as the cross-linker for the encapsulation of acyclovir [75]. Microspheres of approximately
300 m were obtained showing prolonged release
kinetics of acyclovir.
To increase the oral bioavailability of acyclovir,
mucoadhesive microspheres have been investigated as
gastroretentive delivery systems. Dhaliwal et al. [76]
evaluated different polymers and showed that the thiolated chitosan mucoadhesive microspheres improved
the acyclovir oral bioavailability because of the
enhanced retention in the upper gastrointestinal tract.
Recently, mucoadhesive acyclovir-loaded microspheres
were developed using ethylcellulose as matrix and
Carpobol 947 (Lubrizol, Wicklife, OH, USA) as the
mucoadhesive polymer, with the purpose of improving
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D Lembo & R Cavalli

oral bioavailability of acyclovir [77]; the results of a

mucoadhesion study [77] showed a prolonged residence
time of the drug in rat gastrointestinal tracts.
Polymeric microspheres were also designed for the
topical application of acyclovir in order to increase the
drug concentration in the basal epidermis, which is the
site of HSV infections [78]. The microspheres increased
the retention of the drug in comparison with a drug
suspension, and consequently allowed a decrease of the
topical administration of acyclovir. A delayed release of
acyclovir was also obtained by cross-linked malonylchitosan microspheres obtained by coacervationphase
separation [79]. The same research group also proposed
acyclovir-loaded chitosan microspheres obtained by the
spray-drying technique [80]. Microspheres loaded with
interferon have been proposed for oral delivery [60].
Microspheres have also been proposed for use in sustained delivery systems for vaccines.

Nanoparticles
Nanoparticles are solid colloidal particles <1 micron
in diameter and can be created using polymers, lipids,
proteins or other substances, such as inorganic materials. They can have a matrix-like or capsule-like structure (Figure 5) as described above for microspheres,
obtaining nanoparticles and nanocapsules, respectively.
The active molecules can be dissolved or encapsulated
within the nanoparticles. Because of their small sizes
they can be administered intravenously. As for liposomes, opsonization of nanoparticles in the blood can
be prevented by the presence of hydrophilic moieties on
their surface, such as PEG chains. These are known as
stealth nanoparticles [81].
Polymeric nanoparticles are formulated using either
natural or synthetic polymers with a high level of biocompatibility to reduce cytotoxicity and maximize tissue compatibility. The only polymers that have been
approved by the US Food and Drug Administration for
human use are poly-d,l-lactic acid (PLA), polyglycolic
acid, poly(lactic-co-glycolic acid), poly-e-caprolactone
and poly(methylmethacrylate). Nanospheres made of
PLA containing acyclovir were prepared by a nanoprecipitation process. To obtain PEG-coated nanospheres
1,2-distearoyl--phosphatidylethanolamine was also
incorporated. In vitro experiments were carried out
to compare the ocular pharmacokinetics of the two
types of acyclovir-loaded nanospheres compared with
an aqueous suspension of the free drug. A PEG-coated
microsphere significantly improved the aqueous level
of acyclovir and could be suitable for the treatment of
ocular viral infections.
Poly(hexylcyanacrylate) nanoparticles were prepared by an emulsion polymerization process as antisense oligonucleotide carriers for antiviral therapy
[82]. Cidofovir, a nucleotide analogue active against
64

HCMV and smallpox virus, was encapsulated in

poly(isobutyl cyanoacrylate) nanocapsules with an
aqueous core by Hillaireau et al. [83].
Acyclovir-Eudragit nanoparticles were prepared
using different charge density Eudragit (Rhm, Darmstadt, Germany), copolymers of poly(ethylacrylate,
methacrylate and chlorotrimethyl methacrylate), and
the formulation bioavailability was assessed in human
volunteers compared with commercial products. The
polymeric nanoparticles increase the oral bioavailability and prolonged the activity of acyclovir [84].
The majority of lipid nanoparticles can be classified as
either solid lipid nanoparticles (SLN) or nanostructured
lipid carriers (NLC) and they are made from lipids that
are solid at body temperature with a mean diameter
generally within the 50500nm range. SLN were first
introduced by Gasco [85], and Mller and Lucks [86].
SLN possess several advantageous properties, including the solid state of their particle matrix, their ability
to protect chemically-labile ingredients against chemical
decomposition and their potential use in the modulation
of drug release. Upon parenteral administration of SLN,
improved bioavailability, targeting and enhanced cytotoxicity against cancer cells have been observed [81].
NLC are composed of a solid lipid matrix with a
certain liquid lipid phase content. SLN and NLC might
be able to overcome the problems of membrane stability and drug leaching that are associated with liposomes
and conventional emulsions [87,88]. Acyclovir-loaded
SLN have been prepared showing a good incorporation
[89], and those with diameters of approximately 400nm
showed greater in vitro activity than free drug [90]. Adefovir dipivoxil has been formulated in SLN composed of
monostearin for use in HBV therapy.
Protein nanoparticles have also been developed as
drug carriers. Albumin nanoparticles for ganciclovir
delivery were prepared by coacevation and chemical
cross-linking with glutaraldehyde. Depending on the
step in which the glutaraldehyde was added in the preparation method, nanoparticles between 200400 nm
with a different drug incorporation and release profile
were obtained [91].
Albumin nanoparticles have been studied as a
delivery system for antisense oligonucleotides [92].
Phosphodiester and phosphorothioate oligonucleotides
were adsorbed onto the surface or incorporated into the
matrix of nanoparticles. The antiviral activity was evaluated in fibroblasts infected with HCMV. Both types
of nanoparticle formulations protected the oligonucleotides from enzymatic degradation, thus increasing
their antiviral activity. Hillaireau et al. [83] encapsulated cidofovir in poly(isobutyl cyanoacrylate) nanocapsules with an aqueous core for the encapsulation of
azidothymidine-triphosphate and cidofovir showing a
high incorporation efficiency of the two drugs. Cationic
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Nanoparticulate drug delivery for antivirals

gelatin nanoparticles increased the immunostimulatory

effects of CpG oligonucleotides [93].

Dendrimers
Dendrimers are perfectly ordered, nanostructured
polymers, characterized by a branching structure
emanating from a central core (Figure6). Their small
sizes (<100 nm in diameter) and the possibility of
binding targeting ligands to them renders dendrimers attractive for use in drug delivery [94]. They have
been proposed as carriers for DNA, siRNA and antiviral drugs. Acyclovir-terminated thiophosphate dendrimers have been synthesised [95]: dendrimer drug
polyconjugates that are soluble in water and that can
act as a macromolecular prodrug of acyclovir have
been obtained. Peptide-derivatized dendrimers have
been found to inhibit HCMV replication by blocking
virus binding to cell-surface heparan sulphates [96].
VivaGel, a dendrimer-based formulation developed
by Starpharma (Melbourne, Australia) with activity
against HIV and HSV [97] has successfully completed
Phase I clinical trials [98] and is expected to be available on the market soon as a microbicide for the prevention of sexually-transmitted HSV infections.

Emulsion-based delivery systems

Emulsions are heterogeneous systems generally consisting of an aqueous phase and an oil phase. In oil-inwater emulsions, the oil is dispersed as droplets within
the water; the reverse is also possible and is called a

water-in-oil emulsion. Emulsions with droplet sizes

<100 nm are called microemulsions and are transparent systems. These microemulsions differ fundamentally from emulsions. Microemulsions are thermodynamically stable systems displaying indefinite stability,
whereas emulsions are merely kinetically stabilized and
thermodynamically instable, which means that emulsions will tend to separate into oil and water phases.
Microemulsions are systems consisting of water, oil,
surfactants and cosurfactants, and they are useful for
drug delivery because of their capacity to solubilize
both water-soluble and oil-soluble compounds.
The potential applications of emulsions and microemulsions include their use as carriers for drugs with
poor water solubility, sustained-release systems and sitespecific drug delivery achieved by binding ligands for
various cell-surface receptors to the particle surface.
Emulsion formulations can be used as vehicles for
delivering antivirals within the human body. A microemulsion drug delivery system of acyclovir has been
designed with the aim of improving oral bioavailability.
An in vitro intraduodenal diffusion and in vivo study
revealed an increased bioavailability of approximately
13% after the oral administration of an acyclovir microemulsion formulation compared with that achieved
with commercially available tablets [99]. Positively
charged microemulsions have been formulated for the
topical application of acyclovir. A two-fold increase in
acyclovir accumulation was obtained in comparison
with a drug suspension as control [100].

Figure 6. Schematic illustration of the synthesis of a fourth-generation dendrimer

A. Branched
monomer
Surface
subunit

B. Focal point
activation

Wedge

Growth of
hypercore
Core

Antiviral Chemistry & Chemotherapy 21.2

Hypercore

Fourth-generation dendrimer

65

D Lembo & R Cavalli

Hydrogels
Nano- and microhydrogels (that is, nanogels and
microgels) form another type of particulate polymeric
material. They are formed of cross-linked polymeric
networks ranging in size from 10 to 1,000nm swollen
by a good solvent. Compared with soluble polymers,
they show different properties, such as rheological
behaviour, resistance against degradation, high drug
loading and the possibility of carrying structural residues sensitive to external stimuli, such as pH, temperature, light and redox reactions. Hydrophilic natural or
synthetic polymers can be transformed in particulate
systems using different technological processes, such
as emulsion polymerization, chemical cross-linking and
physical cross-linking [101]. Thermosensitive hydrogels have been designed using a carboxymethylderivative of scleroglucan for topical application of antivirals
[102]. Ferruti et al. [103] developed nanohydrogels by
cross-linking copolymers based on poly(amidoamine)s
(PAAs) and - or -cyclodextrin (CD), capable of simultaneously solubilizing and selectively delivering drugs
without the formation of covalent polymerdrug linkages. These constructs form nanogel in aqueous systems with high swelling capacity [103]. The PAA/CD
nanoparticles can have a CD content of 1035% on
a weight-to-weight basis. They showed the capacity to
incorporate molecules, such as drugs and proteins, and
then slowly release them. Acyclovir-loaded PAA/CD
nanoparticles showed an incorporation of the drug of
approximately 15%, which is released with sustained
kinetics in vitro.

Cyclodextrin-based delivery systems

CDs are cyclic oligosaccharides derived from starch
and shaped like truncated cones because of the chair
conformation of the glucopyranose units. The most
common CDs are -, - and -CDs, possessing six,
seven and eight glucopyranose units, respectively. CDs
are able to form non-covalent inclusion complexes (or
hostguest complexes). CD complexes are mainly used
in the pharmaceutical field to improve aqueous solubility, bioavailability and stability of drugs.
The CD complexation of acyclovir and ganciclovir
has been studied in order to increase the drugs solubilities [104]. The in vitro antiviral activity of ganciclovir
against several strains of HCMV was enhanced by complexation with -CD [105]. Derivatives of CDs have
been synthesised to improve the complexation capacity
and the technological properties of parent CDs.
A new PAA copolymer with -CD was obtained by
polyaddition reaction of 6-deoxy-6-amino--CD (-CDNH2) and 2-methylpiperazine to 2,2-bis(acrylamido)
acetic acid in aqueous medium. This -PAA/CD copolymer bears -CD units along the macromolecular chain,
is water-soluble and non-cytotoxic. This copolymer
66

showed a good acyclovir complexation capacity. The

antiviral activity of the acyclovir-PAA/CD complex
was evaluated against HSV in cell cultures exhibiting a
higher antiviral activity than the free drug [106].
Amphiphilic CD derivatives are of much interest for
pharmaceutical applications because of their ability to
self-organize in water [107]. Sulphated and non-sulphated amphiphilic -, - and -CDs show the ability
to complex with acyclovir [108].
Novel polymeric nanoparticles based on a -CDpoly(4-acryloylmorpholine) monoconjugate (-CDPACM), a tadpole-shaped polymer in which the -CD
ring is the hydrophilic head and the PACM chain the
amphiphilic tail, were prepared by the solvent injection technique. Acyclovir-loaded nanoparticles were
obtained from inclusion complexes of acyclovir with
-CD-PACM. The antiviral activity of acyclovir loaded
into -CD-PACM nanoparticles against two clinical
isolates of HSV type-1 was evaluated in comparison
with that of both the free drug and the soluble -CDPACM complex. When carried in the nanoparticles,
acyclovir showed an enhanced antiviral activity compared with the other formulations tested [109]. Acyclovir nanoparticles with other types of amphiphilic
CDs have been designed.
Nanoparticles made of amphiphilic perfluoroalkyl
-CD have been prepared for the transport of acyclovir [110]. These fluorinated amphiphilic CD nanoparticles encapsulated acyclovir with an efficiency of about
40% and allowed its sustained release. Nanosponges
are a new class of material consisting of nanoporous
solid particles. Recently, CD-based nanosponges have
been prepared [111,112]. They are nanoparticles with a
rather spherical shape consisting of hyper cross-linked
CDs ( or ) nanostructured in a tridimensional network. Nanosponges can be synthesised as neutral or
acid, and can be swellable according to the agent used
as cross-linker. The cross-linking-to-CD ratio can be
varied to improve the drug loading.
Nanosponges showed the capacity of encapsulating
various types of molecules in their structure based on
the formation of inclusion and non-inclusion complexes [113]. The acyclovir loading capacity in carboxylated nanosponges was approximately 60% w/w
and the in vitro release profile showed a prolonged
release over time. The acyclovir loaded in nanosponges
exhibited a higher antiviral activity than the free drug
evaluated against HSV in cell cultures [114].

Nanocrystals
Nanocrystals of pure drugs can improve their dissolution rate and bioavailability. They can be formulated as
aqueous nanosuspensions in the presence or absence of
stabilizers, such as non-ionic surfactants. Nanocrystals
are suitable for nanoparticulate formulation of drugs
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Nanoparticulate drug delivery for antivirals

with properties such as an insolubility in both water

and oil, a high melting point, a high log P and high
dose. Following the NoyesWhitney equation, progressive size reduction of the drug particles leads to an
increase in the surface area resulting in an increased
dissolution rate. Additionally, particle size reduction
results in a decrease of the diffusion layer thickness
surrounding the particles and an increased concentration gradient between the surface of the particle and
bulk solution, which facilitates particle dissolution by
increasing dissolution velocity. Therefore, nanosizing
is a suitable approach for increasing bioavailability of
those drugs where dissolution is the rate-limiting step
in systemic absorption. Van Eerdenbrugh et al. [115]
investigated the dissolution and in vitro absorption of
a poorly water soluble non-nucleoside reverse transcriptase inhibitor, loviride (water solubility 0.1mg/l),
after nanonization. Nanocrystals can be obtained by
different technological processes.
The media-milling technology, developed by MeriskoLiversidge et al. [116], is a proprietary wet milling
technology (Nanocrystal) of the Elan Corporation
(Athlone, Ireland), and there are at least four oral dosage forms currently in the market containing pure drug
nanoparticles (that is, Rapamune; Wyeth, Princeton,
NJ, USA).

Conclusions and future perspectives

The nanomedicine approach opens new therapeutic
strategies for attacking viral diseases and for improving
treatment success rates. Innovative nanomedicine solutions are expected to have great effects in the treatment
as well as the eradication of infectious diseases. Their
role could be important in prevention, early diagnosis,
more effective drug delivery systems, specific targeting
and personalized therapy (Table7).
In particular, nanoparticulate-based systems could
improve the efficacy of antivirals, restrict adverse drug
side effects and reduce treatment costs. These features
are particularly relevant in viral diseases where high drug
doses are needed, drugs are expensive and the success
of a therapy is associated with patient adherence to the
administration protocol. This latter issue is very important in viral treatments where complicated or chronic regimens are common. Nanotechnology can reduce intake
frequency and shorten the time of treatments, potentially
rendering the treatment more cost-effective.
In addition, nanomedicine can enhance the effectiveness of approved antiviral drugs and extend their
applicability by overcoming limitations, such as low
bioavailability and cellular barriers. However, several
important issues must be addressed before the potential
of nanotechnology can be translated into safe and effective antiviral formulations for clinical use.
Antiviral Chemistry & Chemotherapy 21.2

Table 7. Role of nanomedicine in antiviral therapy

Role of nanomedicine
Increased bioavailability
Improved antiviral delivery
Targeting specific body sites
Control of adverse side effects
Monitoring of antiviral therapy
Viral diagnostics
Patient compliance
Nanomicrobicides in prevention therapy
Personalized therapies

Nanotoxicology is a relatively new discipline and

requires further development [117119]. A deep
exploration of the toxicological and the bioelimination aspects of nanocarriers is required to ensure safe
manufacture and use of nanomaterials. Note, however,
that the riskbenefit assessment of an antiviral formulation should be based on more stringent criteria than
that for anticancer formulations, for example, because
many viral infections are not life-threatening.
Virologists should be directly involved in the development of antiviral nanocarriers. Besides discovering
new antiviral molecules to be delivered by nanocarriers, virologists should also be addressing other important matters; for instance, the selective targeting of a
nanocarrier to infected tissues requires the identification of molecules or functions that are differentially
expressed or carried out by a virus-infected cell. Ideally, this knowledge should be provided for each virus
against which an antiviral drug is available. So far, we
do not know whether it is possible to exploit passive
targeting for antiviral therapy in a way analogous to
that in anticancer therapy. Nanocarriers loaded with
anticancer drugs could be passively targeted to a
tumour because of the enhanced EPR effect. Virologists should explore the possibility that virus-infected
tissues or cells could be more susceptible (or recalcitrant) to nanodelivery systems.
From a technological point of view, the main objectives of future antiviral therapy research will be the
identification of new technologies for the characterization of nanoscale materials, the development of nanodelivery systems devoid of cytotoxicity and with high
biocompatibility and biodegradability, the functionalization of nanocarriers for effectively targeting specific
sites of viral infection in order to reduce drug-related
toxicity in other tissues, the design of molecules that, as
well as acting as the active carriers, also possess intrinsic
antiviral therapeutic properties (for example, dendrimers and metallic nanoparticles), the optimization and
scale-up of the production procedures in good manufacturing practice (GMP), the development of regulatory
guidelines suitable for nanocarriers, the development of
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D Lembo & R Cavalli

cost-effective nanotechnology-based formulations and,

finally, making them available to developing countries.
In conclusion, the main ethical and scientific challenges
of research into antiviral nanomedicines are to produce
safe and high quality therapies at reasonable costs

Acknowledgements
This research was supported by grants from Regione
Piemonte (Ricerca Finalizzata 2008-bis and 2009). We
thank Agnese Bisazza and Andrea Civra for their excellent assistance.

Disclosure statement
The authors declare no competing interests.

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Received 3 June 2010; accepted 27 July 2010; published online 3 November 2010

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