Peptides 31 (2010) 184193

Contents lists available at ScienceDirect

Peptides
journal homepage: www.elsevier.com/locate/peptides

Review

Recent developments in liposomes, microparticles and nanoparticles

for protein and peptide drug delivery
Mei Lin Tan a, P.F.M. Choong a,b, C.R. Dass a,*
a
Department of Orthopedics, University of Melbourne, St Vincents Hospital Melbourne, PO Box 2900, Fitzroy 3065, Australia
b
Bone and Soft Tissue Sarcoma Service, Peter MacCallum Cancer Institute, Melbourne, Australia

A R T I C L E I N F O A B S T R A C T

Article history: Proteins and peptides are increasingly recognized as potential leads for the development of new
Received 18 August 2009 therapeutics for a variety of human ailments. Due to their relatively specic mode of action, proteins and
Received in revised form 1 October 2009 peptides can be administered at relatively low doses for therapeutic effects. As natural biological
Accepted 1 October 2009
products, these low doses reduce the risk otherwise caused by other small molecular drugs or larger
Available online 9 October 2009
charged molecules. Unfortunately, their therapeutic potential and clinical application is frequently
hampered by various obstacles to their successful delivery. This review discusses the recent
Keywords:
developments in the elds of liposome, microparticle and nanoparticle pertinent to protein and
Drug delivery systems
Proteins
peptide delivery covering those systems tested and/or validated in vivo.
Peptides 2009 Elsevier Inc. All rights reserved.
Therapy

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
2. Drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.1. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
2.2. Microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.3. Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

1. Introduction medicines (including proteins and peptides) relating to more than

100 diseases, including cancer, infectious diseases, autoimmune
The pursuit of the human genome revealed many answers, diseases, AIDS/HIV and related conditions. Among these, more
among them, ndings which indicate that more than half of our than 66 of these new medicines were derived from proteins or
30,000 genes encode proteins. The need for an understanding of peptides. These new drugs are in human clinical trials or under
this pool of unknown proteins subsequently led to the establish- review by the Food and Drug Administration [53]. Proteins and
ment of the Human Proteome Organization. As such, many of these peptides are increasingly recognized as potential leads for the
proteins have shown to be valuable reservoirs of drug candidates, development of new therapeutics. Due to their relatively specic
or serve as targets for protein or peptide-based drug research and mode of action, protein and peptide therapy need only in theory to
development (R&D). In 2008, The Pharmaceutical Research and be delivered in low doses. As natural biological products, these low
Manufacturers of Americas (PhRMA) report on Biotechnology doses reduce the risk otherwise caused by other small molecular
Medicines in Development, listed 633 new biotechnology drugs or larger charged molecules (example oligonucleotides).
Unfortunately, their therapeutic potential and clinical application
is frequently hampered by various obstacles to their successful
* Corresponding author at: Orthopedics Department, SVHM, L3, Daly Wing, 35
delivery [20,56]. This review aims to examine the various drug
Victoria Pde., Fitzroy 3065, Australia. Tel.: +61 3 9288 3990/3980; fax: +61 3 9416
3610.
delivery systems (DDSs) designed for the delivery of protein and
E-mail address: crispin.dass@svhm.org.au (C.R. Dass). peptide therapeutics for human disease therapy, with a focus on
URL: http://www.svhm.org.au DDSs that have been investigated in an in vivo setting.

0196-9781/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2009.10.002
 M.L. Tan et al. / Peptides 31 (2010) 184193
At a cellular level, the delivery of proteins and large peptides in
vivo can be hindered by their three-dimensional structure, spatial
occupation and hydrophilic/hydrophobic nature. Thus, diffusion
transport of these large pharmaceutical proteins is generally
slower unless specic transporters are available. Protein stability,
based on weak non-covalent interactions between secondary,
tertiary and quaternary structures of proteins, is also crucial to
prevent any disruptions that will destabilize the proteins [57].
These factors present proteins and peptides as highly vulnerable
molecules with short in vivo half-lives, due to degradation by
enzymes and proteases, either at the administration site or en-
route to the site of pharmacological action, resulting in poor
bioavailability. In certain cases, frequent administration at high
doses might be required to obtain therapeutic effects in vivo,
creating a risk for unexpected and unwanted side-effects such as
immune responses. In addition, during preparation of the protein/
peptide drug, manufacturing processes and environmental
factors may damage the proteins, reduce their biological activity,
induce aggregation, render the proteins immunogenic and lead to
their precipitation [20,57]. These processes include sterilization
and lyophilisation while the contributing environmental factors
are pH, ionic strength, temperature, high pressure, non-aqueous
solvents, metal ions, detergents, adsorption, agitation and
shearing.
For these reasons, a carrier or delivery system for therapeutic
proteins and peptides would be ideal. Drug delivery systems
(DDSs) can be advantageous to enhancing disease therapy by: (1)
enhancing protein/peptide solubility; (2) controlled release of
protein/peptide molecules; (3) sustained release instead of burst
release effect, avoiding undesirable side-effects; (4) improved
biodistribution of protein/peptides; and with improved technology
possibly (5) target the diseased tissue in vivo [2]. In an ideal setting,
protein/peptides administered through DDSs should decrease the
rate of drug clearance from the system, meaning that lower
volumes or concentrations of the drug can be administered.
Lowered volumes or concentrations of proteins/peptides could
also prove useful for certain proteins/peptides that are inherently
difcult to synthesize, extract, purify or become toxic at high
doses. The area under the time versus concentration curve also
increases, thus lengthening the treatment duration and possibly
decreasing treatment frequency [21]. In addition, the DDS has to be
able to bind the therapeutic protein/peptide in a fashion that does
not affect its bioactivity but hold it sufciently until the DDS
reaches its site of action and releases the protein/peptide in a
controlled, sustained manner. In this instance, DDSs formulated
with biocompatible and biodegradable materials would certainly
be advantageous so as to minimize any adverse host response to
the DDSs.
This review aims to present on-going research on drug delivery
systems for proteins and peptides in an informative manner.
Recent publications containing in vivo experimentation and strictly
belonging to either liposomal, microparticle or nanoparticle
delivery systems were chosen to be included in this review. It
is not intended for the authors to strictly criticize each article
but rather this review serves to update readers regarding new
developments in protein and peptide delivery systems while
providing suggestions for future advancements.
2. Drug delivery systems
2.1. Liposomes
First designed in the late sixties, liposomes are microscopic
vesicles composed of lipid membranes surrounding discrete
aqueous compartments [59]. Over the decades, various types of
liposome formulations have been made available, each differing in
185
their dimensions, composition, surface charge and structure, some
of which designed through a combination of specic types of
liposomes [15]. For the treatment of cancer, liposomal-DDSs have
been developed for small molecular drugs such as doxorubicin
(Caelyx1/Doxil1) and daunorubicin (DaunoXome1), though few
such DDSs have been formulated for protein and peptides.
A novel attempt to synthesize and deliver pro-apoptotic
membrane proteins through liposomes were attempted in human
colorectal carcinoma cells [34]. Natural liposomes derived from
spinach thylacoids were prepared through the evaporation/
resuspension technique, together with recombinant VDAC
(voltage-dependent anion channel), Bak and BakDBH3 protein,
producing LB (Bak liposomes), LBDBH3 (BakDBH3 liposomes), LV
(VDAC liposomes) and LVB (VDAC-Bak liposomes). Upon incuba-
tion of the liposomes with colorectal carcinoma cells, apoptosis
was induced within 24 h with the release of cytochrome c and
activation of caspases-3, -7, -9 and PARP. Thus, these results
highlight the potential of pro-apoptotic proteins integrated into
natural liposomes, presenting as candidates for cancer protein
therapy. However, in vivo efcacy still needs to be determined, as
the in vitro performance of a DDS does not necessarily predict its
performance in vivo.
Studies were also carried out to examine the local delivery of
therapeutic molecules encapsulated within liposomes as a
potential method to treat ocular inammation [8]. Using the
thin-lm hydration method, the authors formulated rhodamine-
conjugated pegylated (PEG) liposomes composed of phosphaidyl-
choline (PC), phosphatidylglycerol (PG), cholesterol (Chol), 1,2-
distearoyl-sn-glycero-3-phosphoethanil-amine-N-[methoxy-
poly-(ethyleneglycol)-2000] (PEG-DSPE) and phosphatidyle-tha-
nolamine-N-(lissamine rhodamine B sulfonyl; PE-Rh). Rhodamine-
conjugated liposomes loaded with vasoactive intestinal peptide
(VIP), an immunosuppressive neuropeptide [46], were injected
intravitreally into the eyes of rats for a study of its biodistribution.
Twenty-four hours following the intravitreal injection, VIP-Rh-Lip
uorescent VIP-containing liposomes as well as VIP-null uor-
escent liposomes, were detected mainly in the posterior segment
of the eye (vitreous, inner layer of the retina) and the conjunctiva
and episclera. Studies also determined that the liposomes were
internalized by activated retinal Muller glial cells, ocular tissue
resident macrophages and rare inltrating activated macrophages.
However, it was unclear as to whether both Rh-Lip and VIP-Rh-Lip
were internalized in a similar manner although some evidence
pointed to the presence of VIP still contained within the
conjunctiva 24 h after injection. Both Rh-Lip and VIP-Rh-Lip were
distributed to the cervical lymph nodes and internalized by
resident ED-3 positive macrophages adjacent to CD4 and CD8-
positive T lymphocytes. In addition, it was found that the T
lymphocytes in close contact with VIP-Rh-Lip-containing macro-
phages expressed VIP. The accumulation of liposomes within the
subcapsular sinus of the cervical lymph nodes following intravi-
treal injection in the conjunctiva was possible through the
conjunctival lymphatics. Thus, through this study, results showing
the detection of VIP in both macrophages and T cells in cervical
lymph nodes suggested that intravitreal injection of VIP-Rh-Lip
may increase ocular immune privileges by modulating the loco-
regional immune environment. In conclusion, these observations
suggested that these VIP-loaded liposomes could prove a promis-
ing therapeutic strategy to dampen ocular inammation by
modulating macrophage and T cell activation. As the focus of
the research was tuned towards the therapeutic and benecial
properties of the liposome, there was minimal physical character-
ization attempted. However, such characterizations should be
considered in the future as they can shed light for further
improvements on the liposome. In addition, the effects of a
dampened immune response require more thorough analysis so as
186 M.L. Tan et al. / Peptides 31 (2010) 184193
to prevent a lack of immunity when potential pathogens are
encountered.
In yet another report utilizing the VIP, a treatment for lung
diseases was described [23]. Although the administration of VIP
garnered positive therapeutic potential in human trials for
pulmonary artery hypertension, there is yet a VIP agonist in
clinical use [22]. This is attributed to the rapid proteolytic digestion
and inactivation of VIP by endopeptidases, thus reducing the
bioactivity of the peptide [35]. To enhance the local availability and
therapeutic effect of the peptide, a VIP depot formulation was
designed to provide a retarded release of the peptide, allowing the
peptide to be protected before its uptake by the appropriate
receptor [23]. The authors have previously formulated a uni-
lamellar liposome and were now developing this liposomal
formulation as an inhalable peptide carrier. VIP-loaded liposomes,
composed of polyethylene glycol-conjugated distearyl phospha-
tidyl ethanolamine (DSPE-PEG2000), lysosteryl-phosphatidylgly-
cerol (lyso-PG) and palmitoyl-oleoyl-phosphatidylcholine (POPC),
were synthesized by a thin-lm rehydration method to incorpo-
rate an aqueous solution of VIP. PEG also formed a PEG-shell which
protected surface-attached VIP peptides from enzymatic degrada-
tion in the airway and inhibited particle aggregation. VIP-loaded
liposomes were subsequently nebulized at room temperature,
resulting in liposomes in the spray mist of a mean diameter below
14 mm in size. The authors reported that there were no signicant
changes in size before and after nebulization, indicative of the
absence of degradation or aggregation of liposomes, and further
supporting the suitability of an inhalable VIP liposome. When the
VIP-loaded liposomes were tested biologically through relaxation
experiments on rat arteries, the liposomes allowed a delay in the
peptide release and produced greater vasorelaxation potential, in
comparison to free VIP peptides. In addition, the authors noted that
there was a smooth vasorelaxation response towards effect
saturation, indicative of a continuous and smooth peptide release
from the liposomes. Release of the VIP peptides were also
attributed to triggering by target cells, as the VIP-loaded liposomes
were stable without loss of peptide during storage. However,
concerning the applicability of stored VIP-loaded liposomes, the
current technology employed by the authors only allowed storage
of the liposomes for a maximum of 1 month before activity
declined. This VIP-loaded liposomal formulation has shown
potential towards the adaptation for the encapsulation of other
peptide drugs. In summary, the authors have formulated a VIP-
loaded liposome that increases peptide availability by (i) providing
protection of peptides from peptidases and other forms of
degradation and, (ii) prolonging the specic activity of the peptide
and indicative of a sustained substance release in the lungs [23].
The authors should proceed to examine the in vivo safety,
biocompatibility and biodegradability of these liposomes, given
the variety of chemicals and materials used in the liposome
formulation.
Liposome delivery of therapeutic proteins was also attempted
as a DDS for vaccination against genital Chlamydia infection [24].
The major outer membrane protein (MOMP) is a target of both
humoral and cellular immune responses during infections in
humans and is a leading vaccine candidate. Despite the success of
native MOMP vaccines [41,42], recombinant MOMP, MOMP
peptides or MOMP DNA have not yielded expected protection as
delivery systems could not retain MOMPs strong cell-mediated
immunity and thus results in a dramatic inuence on the vaccines
efcacy [40,50]. Using a recently developed cationic liposome
formulation 1 (CAF01) [14], the authors compared the efcacy of
MOMP/CAF01 or MOMP adjuvanted with alum (T helper cells type
2-promoting aluminium hydroxide) by mouse immunizations
followed by a Chlamydia muridarum antigen challenge six weeks
later. To further investigate the cell-mediated immunity mechan-
ism involved upon MOMP vaccination, investigators carried out
their studies on normal C57BL/6 mice as well as CD4+ T cells
depleted C57BL/6 mice. The results showed that mice immunized
with MOMP/CAF01 had a clear protective effect over mice treated
with saline or MOMP/alum, boasting signicantly fewer bacteria
on days seven and 14 after infection. Mice vaccinated with MOMP/
alum displayed a strong anti-MOMP humoral response with high
IgG1 titers, low levels of IFN-g and tumor necrosis factor (TNF)-a,
and a slight reduction in Chlamydia load. On the other hand, mice
vaccinated with MOMP/CAF01 displayed high titers of IgG2b, IFN-
a and reduced vaginal Chlamydia load. Additionally, MOMP/CAF01
vaccinated mice showed a reduced infection-resolution time
compared to saline-treated mice. Also, the results showed that
vaccine-mediated protection against Chlamydia was dependent on
CD4+ T cells. Having demonstrated that the MOMP/CAF01 vaccine
effect was mediated primarily by CD4+ T cells, the authors also
showed that similar levels of infection protection were provided by
vaccination with rMOMP/CAF01, verifying that vaccination pro-
tection was not dependent on MOMP conformation. In conclusion,
this study showed that a vaccine based on natively refolded as well
as recombinant MOMP administered through the CAF01 delivery
system induced a CD4+ T cell-dependent immune response that
efciently protects against vaginal antigen challenge with C.
muridarum.
For diabetes mellitus, the major peptide used for treatment is
insulin. Despite its importance in diabetes management, conven-
tional insulin therapies are limited by conditions such as
retinopathy, nephropathy thickened capillary basement mem-
branes and cardiovascular complications [16]. In addition, effective
insulin delivery could be limited by high elimination rates
resulting in a short duration of drug contact with its absorption
sites and consequently a low bioavailability. Despite the avail-
ability of commercial pre-lled insulin, these treatment options
are cumbersome to the patients and might not effectively mimic
physiological patterns of insulin secretion and action. In an
attempt to address these issues, the authors investigated the
applicability of insulin-containing multivesicular liposomes
(MVLs), with the addition of novel chitosan and carbopol coating
(CS/P-MVLs) as sustained release protein delivery systems via the
nasal and ocular routes [26]. As a result, the authors obtained
insulin-loaded MVLs of 2634 mm with a high protein loading
between 58% and 62%. The in vivo applicability of these liposomes
were tested through drop-style nasal and ocular administrations in
STZ-induced diabetic rats and compared to non-coated MVLs,
conventional liposomes and free insulin solution. In STZ-induced
diabetic rats, CS-MVLs effectively reduced the blood glucose levels
by 35% up to 48 h after nasal administration, compared to a
marginal reduction of 22% by non-coated MVLs up to 12 h after
administration. A comparison between chitosan-(CS-MVLs) and
carbopol-(CP-MVLs) coated MVLs showed a maximum of 65%
reduction in blood glucose levels and 55% reduction, respectively,
at 8 h. Upon ocular administration, the CS-MVL carrier showed
better blood glucose reduction levels of up to 30% reduction at 24 h
compared to CP formulation and other tested formulations. This
superior effect observed in both nasal and ocular administrations
has been mainly attributed to the prolonged residence of the
mucoadhesive MVLs in the mucosa as well as the protective effect
of the MVLs against enzymatic attacks of the peptide drug. In
conclusion, nasal administration of CS-MVLs has shown a slightly
better hypoglycaemic prole as compared to the ocular route.
Nonetheless, these experiments have proved to be very promising
to show effective delivery of insulin to the systemic circulation via
non-invasive routes, and will be further investigated and devel-
oped for improved transmucosal insulin formulations.
Investigating another peptide drug, calcitonin, Werle and
Takeuchi aimed to study the efcacy of liposomes coated with
 M.L. Tan et al. / Peptides 31 (2010) 184193
the polymer-protease inhibitor conjugate chitosan-aprotinin for
oral peptide delivery in rats [60]. The calcitonin-containing
chitosan-aprotinin multilamellar vesicles (MVL) liposome con-
jugate prepared ranged from 3 to 4.5 mm in diameter and was
loaded with at least 75% of the drug. Formulations of chitosan-
coated MLVs, chitosan-aprotinin coated MLVs and free calcitonin
were administered intragastrically into fasting rats which were
tested for their blood calcium levels at pre-determined time-
points. The results showed a signicant decrease of blood calcium
levels in chitosan-aprotinin-coated MLVs after 30 min of admin-
istration as compared to chitosan-coated MLVs. The authors
speculated that this effect was most likely mediated by the
protease-inhibitory activity of chitosan-aprotinin, as the two
formulations did not differ markedly in terms of drug-loading and
mucoadhesive properties. Additionally, in rats treated with
chitosan-coated MLVs, calcium levels returned to the initial
readings after 24 h, whereas calcium levels remained decreased
after 24 h in rats treated with chitosan-aprotinin coated MLVs.
Comparing the blood calcium concentrationtime curve (AAC) of
both liposomes and calcitonin solution, chitosan-coated MLVs
increased the AAC about 11-fold above calcitonin solution, while
chitosan-aprotinin MLVs yielded a 15-fold increase. This result
again suggested that the efcacy of chitosan-coated MLVs can be
improved through the covalent attachment of specic protease
inhibitors such as aprotinin to the polymer. Thus, it was concluded
that in order for the liposome to achieve a more pronounced effect
in vivo, an optimized liposomal chitosan-aprotinin delivery system
which displays a maximal amount of covalently-bound aprotinin
without exhibiting decreased mucoadhesive properties must be
designed.
Looking at the various liposomal delivery systems presented
above for different proteins and peptides, there were almost no
two similar formulation methods or materials. This could be
attributed to the difference in protein/peptide structure, charge,
stability, solubility and other properties. When more becomes
understood about the biochemistry of the protein or peptide, other
factors such as the intended administration route for a desired
treatment and also the action site of the drug should be taken into
consideration. However, these varied formulations would not be
helpful to researchers seeking a liposome suitable for their
therapeutic of interest. A more suitable approach would be for
researchers to rst characterize their therapeutic of interest for
properties such as structure, surface charge and solubility before
identifying a formulation method which is most appropriate. To
prevent unwarranted side-effects and negative interactions with
the host, researchers should also opt for safer materials that are
natural, biocompatible, biodegradable and less likely to incite an
adverse reaction in the host. In addition, most liposomal-DDSs
were formulated using complex techniques requiring specialized
equipment or chemicals that might be unreachable for others.
Thus, researchers should consider using simpler, less complex
chemicals that might additionally prove to be less toxic to the host.
Another concern might be due to the size of liposomes, which are
typically around or above the micrometer range as the larger the
DDS, the higher the possibility of triggering an opsonization effect
by the macrophages or other components of the immune system
[38]. Largely-sized liposomes might also encounter various
barriers within the hosts endothelial surfaces and various other
barriers such as the bloodbrain barrier.
2.2. Microparticles
Generally, microparticles can be classied as particles between
1 and 1000 mm. Initial experiments with microparticles started in
the early seventies and have since studied extensively with many
different materials and polymers. As yet, there are very few
187
microparticle drug delivery formulations approved for clinical use
[64,65] although more clinical trials are currently underway
[12,39]. The following articles present various microparticles
designed for peptides and proteins delivery.
Using a synthesized peptide with the major T cell epitope of Ole
e 1, the main allergen of olive pollen, Marazuela et al. evaluated
whether intranasal administration of peptide containing-poly(-
lactide-co-glycolide) (PLGA) microparticles (MP) were able to
prevent mice from allergic sensitivity to the whole protein [37].
Peptide-PLGA microparticles prepared by the solvent evaporation
double emulsion method were mostly spherical and measured
0.8 mm in diameter. This was encouraging to the authors as other
studies have shown that microparticles smaller than 10 mm had a
higher uptake by M cells of the nasal associated lymphoid tissues
(NALT), implying that the PLGA MP used in the study was suitable
for M cells uptake and translocation to lymphoid organs to initiate
antigen-specic immune responses. Also, with a peptide-loading
capacity of around 3 mg/mg, the quantity was considered high
enough for in vivo administration of the peptide without the need
for large concentrations of MP or increased MP doses. Although the
authors were initially concerned that the peptides integrity might
be compromised by the use of harsh organic solvents in the solvent
evaporation method, results showed that the MP preparation
process did not signicantly affect the peptides potency. It was
reported that the PLGA MP had an initial burst release of 74%
peptide followed by a slow and sustained release over several
weeks up to 28 days. This property of the MP was an advantage as it
had previously been reported that an initial burst release of antigen
could stimulate the immune system to mucosal vaccines, and then
allow the MPs slow continuous release of peptide to provide a long
lasting immune response [1].
In this study, the authors found that three consecutive doses of
3 mg peptide in PLGA MP were enough to prevent subsequent
sensitization to the whole Ole e 1 allergen. Peptide-PLGA MP pre-
treatment signicantly reduced IgE Ab levels and specic IgG1 Ab,
while increasing IgG2a Ab levels, as opposed to less signicant
results observed in sham-treated mice. Intranasal pre-treatment
with peptide-PLGA MP also resulted in a decrease in IL-5 and IL-10
levels although there were no signicant changes in IFN-g levels.
Importantly, prophylactic treatment with peptide-PLGA MPs
prevented lung inammation. It was inferred that a strong Th1-
type response was present with both the increase of IgG2a Ab and
the high levels of IFN-g. Thus, the use of PLGA MP containing
appropriate antigen can be considered when required for
establishing a specic Th1-type response in humans. A signicant
nding from this report was that through the intranasal
administration of the major T cell epitope of Ole e 1 peptide,
PLGA MP formed a protective effect against sensitization to the
whole allergen. Although the observed ndings might not be
directly translated to humans, this model addressed the immu-
nological effects most commonly experienced in clinical features of
allergy, posing the PLGA MP carrier system with a good potential
for peptide-based vaccines.
In another study, the authors sought to evaluate gelatine
microparticles for the controlled release of bone morphogenetic
protein-2 (BMP-2) [44]. To begin, the authors hypothesized that
decreasing gelatine cross-linking and using collagenase-contain-
ing PBS would result in faster degradation of gelatine and
consequently higher BMP-2 release. Also, it was hypothesized
that higher loadings of BMP-2 in the gelatine microparticles would
result in higher burst release after 24 h. PLGA microparticles,
commonly studied for BMP-2 controlled release, were used as a
side-by-side comparison to gelatine microparticles. Both micro-
particles were loaded with BMP-2 by the diffusion/adsorption
method and tested for various parameters that affected BMP-2
release from microparticles. The optimized parameters were then
188 M.L. Tan et al. / Peptides 31 (2010) 184193
used for in vivo testing to evaluate the release of BMP-2 from
composite scaffolds of gelatine microparticles within a porous
poly(propylene fumarate) (PPF) scaffold. PLGA microparticles had
an average diameter of 2542 mm, depending on the molecular
weight of PLGA, while gelatine microparticles were sieved for a
diameter of 50100 mm. In general, gelatine microparticles
displayed minimal burst release effect (<10%) at 24 h followed
by a moderate or slow release over a period up to 28 days, with
gelatine microparticles incubated in collagenase-containing PBS
exhibiting higher release rates and BMP-2 release. For both high
and low molecular weight PLGA microparticles, a moderate burst
release was observed followed by sustained release over 28 days.
To examine the in vivo release of BMP-2 from gelatine micro-
particles, composite scaffolds with 10 and 40 mm gelatine
microparticles were tested in a subcutaneous mouse model. At
each time point where scaffolds were retrieved, a brous capsule
with extensive neovascularisation was observed surrounding the
composites, although no ectopic bone formation was observed.
Both 10 and 40 mm composites showed signicantly lower burst
and cumulative release of BMP in comparison to PPF controls. In
addition, observations on higher cross-linking resulting in lower
BMP-2 release were also noted in vivo. In conclusion, the authors
correctly hypothesised that decreasing gelatine cross-linking and
addition of collagenase would result in degradation of gelatine and
consequently higher BMP-2 release. Interestingly, the hypothesis
of higher loaded doses of BMP-2 resulting in higher burst release
was not observed, instead an opposite effect of decreased release
with higher BMP-2 doses was seen. Finally, both the 10 and 40 mm
composite scaffolds of BMP-2 gelatine microparticles showed
minimal burst release followed by sustained release over 28 days,
indicating that this system can be used for the controlled delivery
of BMP-2, and thus provide a controlled sustained delivery system
which more closely mimics the growth factor prole during
natural bone healing.
In a subsequent study by the same author, vascular endothelial
growth factor (VEGF) was investigated as the polypeptide of
interest to be delivered by gelatine microparticles [43]. Similarly,
parameters such as the effects of gelatine cross-linking, growth
factor dose and release medium on VEGF release kinetics were
examined in vitro and in vivo, before forming composite scaffolds of
gelatine microparticles within a porous PPF scaffold. Focusing on
the microparticles in vivo characteristics, it was interesting to note
that there was no cross-linking effect on release rates for burst and
continued release which was unlike in vitro observations.
However, the authors were still able to determine that increased
gelatine cross-linking resulted in lower VEGF release, indicating
that by varying cross-linking extent, this protein delivery system
can be used for the controlled delivery of VEGF in vivo. Although
the authors examined the bioactivity of VEGF after in vitro release,
an in vivo study was not performed. Thus the DDS was not
thoroughly tested for its efcacy and would required further
investigations before recommendations are made.
In another study for diabetes treatment, insulin-loaded
microparticles were formulated for oral insulin delivery and its
effectiveness was tested through reduction of initial blood glucose
levels in diabetic rabbits [7]. Insulin-loaded microparticles were
prepared with mucin and sodium alginate combined at different
ratios based on a method of polymer coacervation and diffusion
lling at low temperatures of 30 and 5 8C, respectively. The
authors speculated that this formulation concept of mucin and
sodium alginate cross-linking would enhance the adsorption of
insulin from the gastro-intestinal tract (GIT) through the provision
of a protective environment for insulin and also improved
mucoadhesion of insulin microparticles. Although the low
temperatures required for this method could possibly be a
deterrent to this formulation method, it was argued that low
formulation temperatures stabilized the microparticles by the
formation of crystallites through hydrogen bonds among amine,
amide, carboxylic and hydroxyl groups in the mucin and sodium
alginate polymer networks [61]. Additionally, the low temperature
helped minimize the incompatibilities of peptides with the organic
solvents so that mucin was not denatured by acetone. No
signicant trend was observed in insulin-loaded microparticle
sizes with sodium alginate, mucin, sodium alginatemucin (3:1)
and sodium alginatemucin (1:3) microparticles, which were 310,
230, 260 and 680 mm, respectively. Subsequently, the insulin-
loaded microparticles prepared with different sodium alginate
mucin ratios were lled into hard gelatine capsules with each
capsule containing insulin equivalent to 100 IU of soluble insulin.
Diabetic rabbits were orally administered with capsules containing
insulin-loaded microparticles, while distilled water, insulin solu-
tion and subcutaneous administered insulin acted as controls.
Blood glucose levels were taken hourly and the percentage
reduction of initial glycaemia was used as evidence of insulin
absorption. As expected, distilled water did not cause a reduction
in blood glucose levels while orally administered insulin solution
resulted in a slight drop in blood glucose levels (30%) during the
rst 2 h after administration and returning to normal by the third
hour. In comparison, insulin-loaded microparticles produced
better blood glucose lowering effect. Among the three different
insulin-loaded microparticles (mucin, sodium alginate and mucin
sodium alginate), microparticles prepared with 1:3 mucin:sodium
alginate produced maximum blood glucose reduction (>70%) 5 h
after oral administration that was comparable to subcutaneously
administered insulin. The authors postulated that this could be due
to synergism between sodium alginate and mucin that confers
insulin protection or enhance absorption in the GIT. In conclusion,
the results obtained from this study demonstrated the effective-
ness of mucinated sodium alginate microparticles as a drug
delivery system for oral insulin delivery, and thus indicating the
possibility of designing an optimal drug delivery system using the
right combination of chemicals and formulation technique for the
oral delivery of insulin towards effective control of blood glucose.
Although positive and encouraging results have been shown in this
study, more work closely examining the formulation materials,
conditions and the efcacy of this potential DDS is required. A
concern for oral insulin capsules would be the palatability of the
drug and its effect on patient compliance, in addition, biodegrad-
ability and excretion of these mucinsodium alginate capsules and
its metabolites within a reasonable time is imperative, as insulin
treatment is mostly long-term and highly repetitive.
Having biodegradable and biocompatible characteristics,
hydroethlystarch (HES) was traditionally used in humans as
articial colloids for intravascular volume replacement [28,33].
Based on these characteristics, the authors developed and
characterized HES microparticles suitable for antigen delivery
systems, through the examination of its in vivo biocompatibility
and biodegradability, shelf-life stability, loading capacity, con-
trolled release and localization of loaded proteins [17]. In this
subsequent study, HES microparticles were accessed for their
ability to induce an immune response in mice against the bovine
serum albumin (BSA) protein in comparison with an aluminium
hydroxide (alum) adjuvant [3]. Firstly, HES microparticles were
prepared using the interfacial cross-linking method designed
previously [36]. Briey, HES was emulsied in cyclohexane, stirred
with terephtaloyl chloride, diluted with chloroform/cyclohexane
before washing in a series of ethanol and water and nally
lyophilized. HES microparticles without BSA loaded measured
between 4 to 15 mm with an average diameter of 8 mm. BSA was
loaded into HES microparticles through incubation and immedi-
ately used for inoculation into mice on days 1 and 21. Each group of
mice was immunized with saline, BSA solution, BSA in alum or
 M.L. Tan et al. / Peptides 31 (2010) 184193
BSA-HES microparticles, either through the intraperitoneal (ip) or
subcutaneous (sc) route. Blood samples were then collected
approximately every four days until 77 days after the rst
immunization. No primary immune response was induced by all
immunization doses delivered ip, although BSA-HES microparti-
cles elicited secondary response through induction of anti-BSA IgG
after the second immunization, with a maximal titer at day 38.
However, ip immunization with BSA-HES microparticles appeared
less efcient than BSA/alum. Upon sc immunization, antibody
response was detected in all three groups following the rst
injection. At this stage, BSA-HES microparticles and BSA/alum were
more efcient than free BSA. After the booster dose, high antibody
titers were observed in BSA/alum mice until day 77. However,
there was no signicant difference in antibody titers between mice
immunized with BSA-HES microparticles and free BSA. Therefore,
the sc route of administration for vaccination against BSA resulted
in a stronger IgG response as compared to ip administration. To
determine whether a Th1- or Th2-mediated immune response was
induced following BSA-HES microparticle immunization, titers of
IgG1 and IgG2a were quantied through an ELISA assay. The ip
immunization with BSA-HES microparticles elicited a high IgG1
than IgG2a titer; however SA/alum elicited a higher IgG1/IgG2a
ratio and was more efcient in triggering a Th2-type immune
response. Unfortunately, sc administered BSA-HES microparticles
only resulted in IgG1 and IgG2a responses comparable to free BSA,
while BSA/alum induced both a sustained IgG1 response and
increasing IgG2a response from days 30 to 77. Interestingly, mice
immunized ip with BSA-HES microparticles had increased levels of
IFN-g and IL-4. Similarly, mice injected sc with BSA-HES
microparticles induced higher IFN-g levels. In conclusion, this
study demonstrated the following: importance of the choice of the
route of administration and BSA-HES microparticles can induce
both a Th1 and Th2 immune response which is critical for the
control of malignant cells as well as infectious pathogens. Thus,
HES microparticles may be considered a suitable drug delivery
system for antigens to generate vaccines.
Similarly to liposome formulation, microparticle DDSs are
formulated with a wide variety of formulation materials and
methods. It is crucial to design DDSs that are both compatible with
the protein/peptide drugs as well as the host. Once again, the size
of these DDSs would also contribute to the overall efcacy of the
drug. As with the concerns about opsonization of liposomes above,
microparticles might also encounter similar problems. Thus,
nanoparticles being on a much smaller scale, might appear to be
a better DDS.
2.3. Nanoparticles
Moving onto a nano-scale, another promising therapeutic
carrier, glycol chitosan modied with hydrophobic analogs, has
been studied extensively due to its ability to absorb drugs with
high avidity within its hydrophobic inner cores and allow for their
prolonged, sustained release. Glycol chitosan nanoparticles have
also been reported to display fast cellular and tissue internalization
into tumors [13]. Additionally, chitosan and its derivatives are
biodegradable, biocompatible and poorly immunogenic [25]. Thus,
in this study, hydrophobically modied glycol chitosan (HGC)
nanoparticles were used as a drug delivery system for the
enhancement of anti-angiogenic and anti-tumor effects of an
RGD peptide in a solid tumor model [30]. RGD peptides were
recently developed to target the avb3 integrin on angiogenic
endothelial cells and have since shown to be valuable in tumor
therapy and imaging although their anti-tumor effects are less
promising. This was attributed to the relatively small RGD peptide
analogs which were rapidly cleared from the circulation. RGD-
containing HGC nanoparticles (RGD-HGC) were tested in in vivo
189
angiogenesis assays while the time-course and sustainability of
peptide release from RGD-HGC nanoparticles were examined
using a non-invasive optical imaging system. HGC nanoparticles
were rst prepared through the conjugation of glycol chitosan with
hydrophobic 5b-cholanic acid, followed by the encapsulation of
RGD by solvent evaporation. RGD peptides were efciently loaded
into HGC nanoparticles (85%) and displayed a spherical nanopar-
ticle structure measuring around 335 nm in diameter. There was a
burst release of RGD peptide (78%) from HGC nanoparticles within
one day but residual peptide (22%) showed a reasonable sustained
release over the next seven days. RGD peptides conjugated to
uorophore Cy5.5 were also encapsulated into HGC nanoparticles
for optical imaging. To examine the in vivo time-dependent drug
release prole of RGD-HGC nanoparticles in tumors, RGD-Cy5.5-
HGC nanoparticles and RGD-Cy5.5 were intravenously (iv) or
intratumorally (it) injected into B16F10 melanoma-bearing mice.
Results showed that RGD peptides injected it produced a stronger
uorescence signal within the tumor interstitial as compared to
mice injected with RGD peptides iv, although mice injected with
RGD-Cy5.5-HGC nanoparticles it showed the highest uorescence
intensity within the tumor. In addition, RGD-Cy5.5 released from
HGC nanoparticles co-located with CD31-positive microvessels,
indicating that RGD peptide bound strongly to the angiogenic
microvessels in tumor tissues. Comparisons of uorescence
intensities between tumor and normal tissues also showed that
RGD-HGC nanoparticles allowed the maintenance of a high
concentration of RGD peptide in the tumor, as compared to free
RGD. The anti-tumor efcacy of free RGD and RGD-HGC
nanoparticles were studied in the melanoma-bearing mice, where
free RGD peptides were injected iv and it while RGD-HGC
nanoparticles were injected it every two days. After twelve days
of RGD treatment, tumor growth in all mice were retarded, with
the RGD-HGC treated mice boasting signicantly smaller tumor
sizes than other treated groups. H&E staining further conrmed the
signicant reduction in tumor tissues and CD31 immunostaining
showed a large decrease (> 70%) in CD31-positive microvessels. In
conclusion, the anti-angiogenic effects of RGD-HGC nanoparticles
were superior to free RGD peptides as observed by the marked
tumor growth inhibition through suppression of tumor angiogen-
esis. This could be attributed to the protective effect of HGC
towards RGD against enzymatic degradation and also towards the
accumulation of RGD within the tumor. Thus, the RGD-HGC
nanoparticle model has shown great potential and could be further
investigated for anti-angiotherapy for local and regional tumor
therapy.
The bloodbrain barrier (BBB), made up of tight continuous
circumferential junctions between the brain micro-vessel
endothelial cells, forms a barrier against water-soluble molecules
and possibly affecting those with molecular weight above 500 Da,
posing many challenges for the delivery of drugs to the central
nervous system (CNS). This also constitutes a major impediment to
therapy as drugs are unable to reach the target organs at
therapeutic concentrations. Recently, nanoparticles were exam-
ined as a possibility for delivering drugs to the brain. In this report,
the authors sought to evaluate the brain transport of large
molecular weight drug-loaded nanoparticles by a microdialysis
sampling technique [11]. The peptide of interest was neurotoxin-I
(NT-I), an analgesic peptide with limited permeability across the
BBB while the peptide carrier consisted of the polymer PLA, a
biodegradable polyester approved for clinical use by the Food and
Drug Administration agency. NT-I nanoparticles prepared using
the double emulsication solvent evaporation method yielded
particles about 65 nm in diameter, and an encapsulation efciency
of about 35%. Rats were subsequently administered either NT-I
nanopartices or free NT-I solution intranasally (in) or intravenously
(iv). Prior to treatment administration, a brain microdialysis probe
190 M.L. Tan et al. / Peptides 31 (2010) 184193
was inserted into the pre-prepared right olfactory bulb of the brain
and sampling began 30 min after treatment. Brain microdialysate
samples were collected and analyzed for NT-I levels. Results
showed that brain delivery of NT-I was enhanced with PLA
nanoparticles administered via both in and iv routes. In addition, in
administration of NT-I PLA nanoparticles most signicantly
increased NT-I concentrations in the brain. Thus, with nanopar-
ticles as peptide carriers and delivery systems, in administration of
CNS drugs (proteins and peptides) can be improved for brain
transport.
In another work further investigating pathologies involving the
brain and the BBB, for the treatment of seizures, an endogenous
neuropeptide, thyrotropin-releasing hormone (TRH) is known to
have anticonvulsant effects in animal seizure models and clinical
trials involving intractable epilepsies [32,55]. TRHs bioactivity and
bioavailability however is hindered by rapid tissue metabolism
and the BBB. In this study, the authors designed a direct nose-to-
brain delivery of neuropeptides in sustained release biodegradable
nanoparticles (NPs) as a mode of therapy which enhances CNS
neuropeptide bioavailability [31]. In the interests of space, only
one aspect of this work directly related to nanoparticulate delivery
systems of proteins will be presented here. Using the kindling
model of temporal lobe epilepsy, the authors investigated whether
the intranasal administration of polylactide nanoparticles (PLA-
NPs) containing TRH (TRH-NPs) can impede kindling development
in terms of behavioral stage, after-discharge duration (ADD) and
clonus duration. PLA NPs were formulated together with or
without TRH by the solvent evaporation method using a double
emulsion process. TRH NPs and empty PLA NPs measured 108 and
102 nm, respectively, demonstrating a uniformity of size. Two
groups of rats were pre-treated with either TRH-NPs or empty NPs
seven days before kindling. Kindling stimulations were initiated on
the eighth day, along with intranasal administration of NPs and
continued daily until the rats were fully kindled (permanently
epileptic) or up to 20 stimulations. As compared to free
administration of TRH peptides or empty NPs, positive results
were observed in the TRH-NP group due to: (1) the ADD was
signicantly attenuated from the 10th to 13th stimulations; (2) the
number of stimulations required to reach the rst stage V seizure
and to become fully kindled was signicantly prolonged; (3) the
onset to clonus was signicantly delayed and suppressed overall
clonus duration during stage V seizures. Initial safety proles were
also positive given that there were no observable behavioral
changes or signicant weight change over the course of the study,
an indirect indication of a non-effect on the pituitary-thyroid
hormone axis. The levels of TRH in the brain after intranasal
delivery were not determined due to the technical difculty
involved, though they remain a subject for further research. In
conclusion, the authors believed that the data presented are the
rst direct proof of concept for efcacious use of intranasal
administration of sustained release anticonvulsant neuropeptide
NPs as a viable new means for suppressing temporal lobe seizures
and other intractable seizures. In future, TRH-NPs may provide an
alternative treatment for seizures that could signicantly impede
or even prevent epileptogenesis, however, prior to that, more
pharmacokinetic and pharmacotherapeutic research is necessary
to optimize this intranasal neuropeptide carrier system.
In yet another condition that is difcult in its treatment, spinal
cord injury (SCI) has the potential of destroying the axonal nerve
bers and triggering inammation that might lead to neuronal cell
death. A potential therapeutic treatment for SCI could occur
through neuroprotection by the administration of exogenous
neurotrophic growth factors to improve axonal outgrowth [54].
One such polypeptide growth factor, the glial cell line derived
neurotrophic factor (GDNF) is a transforming growth factor (TGF)-
b super family member which functions as a trophic factor for
dopaminergic neurons and nondopaminergic neurons, including
spinal cord motorneurons and peripheral ganglia [36]. Previous
studies for GDNF treatment have been administered through factor
infusion, cell-based transplantation or gene therapy, with results
showing its potential as an agent for CNS treatment [9,10,19,52].
To tap into the therapeutic potential of GDNF, the authors
hypothesized that a continuous supply of GDNF would give
greater efcacy to neural restoration of the injured spinal cord, and
thus designed a delivery system for the efcient delivery and
sustained release of GDNF in the injured animals [58]. Firstly, the
authors determined whether nano-scaled PLGA nanoparticles
could serve as carriers for GDNF by using uorescently-labeled
latex nanoparticles to investigate on the effects of particle size on
transport within the spinal cord. Intraspinal injection of the
nanoparticles was chosen so as to avoid a lack of efcacy that might
result during systemic administration and the resistance of the
bloodbrain barrier. Subsequently, PLGA-GDNF nanoparticles
were formulated and their therapeutic effect on neuronal bers
was evaluated in SCI rats. In the rst part of the study, latex
nanoparticles measuring 20 and 100 nm in diameter were injected
into the lumbar section L1 and imaged by a uorescence reader.
20 nm nanoparticles were largely located in the neuronal bers of
the thoracic and cervical spinal sections of rats, while 100 nm
nanoparticles were accumulated mainly in the central canal or at
the injection site. These results suggested that 20 nm nanopar-
ticles could be easily uptaken by neurons at the injection site and
transported through the healthy spinal cord by retrograde axonal
transport. A similar test conducted on SCI rats showed that the
transportation of nanoparticles was interrupted. In the next part
of the study, PLGA nanoparticles encapsulating GDNF were
synthesized by the double emulsion solvent evaporation method.
The resultant nanoparticles measured approximately 200 nm and
had a sustained release of GDNF over seven days. PLGA-GDNF
nanoparticles were injected into the injured spinal cords of SCI
rats and their effect was evaluated through the assessment of the
rats hindlimb locomotor function every 23 days up to 31 days.
Based on the Basso Beattie Bresnahan (BBB) locomotor rating
scale, PLGA-GDNF rats were given a score of 3.7 at week one as
compared to the score of PLGA rats at 1.3, and only showed slight
movement of one or two joints. Over subsequent weeks, BBB
scores in PLGA-GDNF rats increased signicantly over PLGA rats
or bolus GDNF rats. These ndings strongly suggested for the
continuous release of GDNF from PLGA nanoparticles to the
contused spinal cord leading to better locomotor recovery as
compared to other treatment groups. Physically, PLGA-GDNF
treated rats also afforded weight-supported plantar steps while
rats in other treatment groups only showed slight movements in
the joints. Immunouorescence further supported the nding by
showing elongated intact neuronal ber bundles with neurola-
ment-positive staining in the lesion center of PLGA-GDNF rat
spinal cord, while only ne fragmented neuronal bers were
observed in PLGA-treated mice. This was a highly positive sign for
spinal recovery treatment as effective neuroprotection was an
important factor in enhancing hindlimb locomotion recovery in
SCI rats. Hence, although the size of PLGA-GDNF nanoparticles
were larger than that suitable for retrograde axonal transport,
results demonstrated that GDNF loaded in PLGA was released
over time and remained bioactive in the injured spinal cord,
which results in an increased neuronal survival and better
hindlimb locomotion in SCI rats. Through further studies on the
safety, biocompatibility, shelf-life and efcacy of these nano-
particles, the potential of this treatment could be extended to
humans who are suffering the devastating effects of spinal cord
injury.
Developments of anti-cancer treatments have been especially
difcult for researchers as chemotherapeutic drugs are often toxic
 M.L. Tan et al. / Peptides 31 (2010) 184193
to both cancer cells as well as normal cells. This was also true of the
naturally occurring tetrapeptide tubulysin [47]. Isolated from
strains of myxobacteria, tubulysin has demonstrated potent
antiproliferative activity against multiple cancer cell lines even at
nmol/L and pmol/L concentrations [48]. Tubulysin act as
antimitotic agents, causing depolymerization of cell microtubules
and triggering apoptosis. Tubulysin also worked in tumors
exhibiting multidrug resistance phenotypes [29]. However,
tubulysin is also highly toxic to healthy cells and has low
solubility in solvents. Thus, in this report, the authors synthesized
a thiol derivative of tubulysin A (TubA) and conjugated TubA to a
b-cyclodextrin-based polymer (CDP), forming CDP-TubA nano-
particles [49]. The CDP-TubA nanoparticles were synthesized in a
four step synthesis process resulting in particles of 100 to 130 nm
in diameter with a minimal release of TubA (<5%) over 72 h. The
anti-tumor activity of TubA was subsequently evaluated in
subcutaneous HT29 human colon carcinoma xenografts estab-
lished in female athymic nude mice. As a comparison, free TubA
was administered through three weekly doses of 0.1 mg/kg, which
unfortunately was higher than the maximum tolerated dose and
led to 50% mortality. In contrast, three weekly doses of CDP-TubA
nanoparticles at 3 mg/kg were well tolerated with minimal body
weight loss and signicant tumor growth delay. At the conclusion
of the experiment, there were three 90-day survivors, six partial
tumor regressions, three complete tumor regressions and one
tumor-free survivor. Even at 50% of the maximum tolerated dose
of CDP-TubA, the treatment was signicantly more effective in
terms of tumor size reduction and survival, as compared to
chemotherapeutic drugs vinblastin and paclitaxel at equitoxic
and maximum tolerated dose, respectively. The efcacy of CDP-
TubA nanoparticles were further evaluated in human H460 non-
small cell lung carcinoma xenografts. Treatment with CDP-TubA
at 3 mg/kg and 6 mg/kg (50% and 100% maximum tolerated dose,
respectively) resulted in a signicant tumor growth delay and
reduction in tumor sizes. Tumor growth curves also showed that
there was tumor stasis for approximately two weeks after the nal
dose of 6 mg/kg CDP-TubA indicative of its prolonged tumour
growth suppression ability and prolonged release of active drug
within the tumors. As expected, treatment of mice with free TubA
did not result in a signicant tumour growth delay or any
reduction in tumour burden. In conclusion, the thiol derivative of
tubulysin A, TubA conjugated to CDP forming CDP-TubA, has
shown equal or superior efcacy at tumor regression compared
with conventional chemotherapy drugs vinblastine and paclitaxel
but with reduced toxicity. This study also rst tested this novel
analog of tubulysin A, TubA, for anti-tumor activity against
established tumors and provides the path for further studies with
CDP-TubA nanoparticles. In addition, the authors could have
further probed for the side-effects of CDP-TubA on blood and
accessory organs for a thorough analysis of its improved
therapeutic index.
Bone morphogenetic proteins (BMPs) are a group of secreted
proteins which exerts an osteoinductive effect, of which BMP-2 has
been shown to induce osteogenesis [6]. Due to its potency at
stimulating bone growth at implantation sites, BMPs are an
alternative for autogenous bone grafting in bone healing [27,51].
Clinically, BMP is utilized for the treatment of bone fractures and
spinal fusion procedures [5,45]. Bone formation is induced when
BMP-2 is present locally at the site of administration and also
limited to the time when BMP is present. However, its efcacy is
reduced due to the fast clearance rate and diffusion rate of BMP-2
from the site of administration [17]. Hence, a carrier which
maintains BMP-2 at the treatment site was required. One of the
most effective carriers used recently is the type 1 bovine
absorbable collagen sponge (ACS), which although has excellent
properties as a carrier, has inherent problems with the control
191
release of BMP-2, often suffering from high initial burst release and
inability to provide sustained release of BMP-2. In a previous work
by the authors, BMP-2 containing bovine serum albumin (BSA)
nanoparticles (NPs) with a polyethylenimine (PEI) coating were
prepared and analyzed accordingly in an in vitro system [63]. In
this study, PEI-coated BMP-2 containing BSA NPs were evaluated in
a subcutaneous implant model to examine the pharmacokinetics
of BMP-2 release and also to assess their osteoinductive activity
[62]. Firstly, a comparison was made between PEI-coated and non-
coated BMP-2 NPs to examine the effect of PEI. Non-coated BMP-2
NPs experienced signicant burst release (>70%) as compared to
PEI-coated NPs (2030%) after one day. At the end of seven days,
about 4050% of BMP-2 remained in PEI-coated NPs while less
than 15% of BMP-2 remained in non-coated NPs. This was expected
as the PEI coating was expected to delay BSA degradation from the
NPs and thus allow the retarded release of BMP-2 from the BSA
matrix. The osteoinductive activity of BMP-2 NPs was then
assessed by subcutaneous implantation in rats. Higher ALP activity,
osteocalcin levels and calcium deposits were all observed in non-
coated BMP-2 NPs as compared to PEI-coated BMP-2 NPs,
suggesting of an undesired effect of PEI on the osteoinductive
activity of BMP-2, even when free BMP-2 was administered along
with PEI-coated BMP-2 NPs. To determine if altering the
concentration of BMP-2 could reduce the negative effects of PEI,
NPs were loaded with different concentrations of BMP-2. As before,
both ALP levels and calcication results showed that non-coated
NPs had the highest activity as compared to PEI-coated NPs and
free BMP-2. It appeared that the NP formulation process did not
adversely affect the osteoinductive activity of BMP-2, however, the
inclusion of PEI during NP formulation produced cytotoxic effects
that were detrimental to BMP-2 bioactivity. This observation was
further conrmed when little signs of bone formation were
observed with the delivery of PEI-coated NPs together with the
addition of free BMP-2. In conclusion, PEI-coated NPs had reduced
burst release and sustained delivery of BMP-2, however, PEI
adversely interacted with BMP-2 causing BMP-2 to lose its
osteoinductive activity. The results also showed a dose-dependent
increase in bone formation with increased BMP-2 loading into NPs.
Hence, the authors believed that the toxicity of PEI can be reduced
by optimizing the BMP-2 loading and retaining the osteoinductive
activity of BMP-2.
At rst glance, nanoparticle DDSs were used to explore
difcult diseases (spinal cord injury, seizures and brain tumors)
involving the penetration of the bloodbrain barrier, the positive
results itself attesting to the potential of this carrier system
over liposomes or microparticles. However, much more can be
done when formulating DDSs to produce carriers that are
compatible with protein and peptide therapeutics. Not forgetting
carriers that are compatible with the host to minimize host
rejection.
3. Future directions
With the advancement in protein and peptide technology,
more is being understood about various proteins and their roles
in human health and disease. For proteins with therapeutic
value, improving technologies also mean that they could be more
easily extracted, puried or synthesized for therapeutic purposes.
Unfortunately, a downside to protein and peptides are their
susceptibility to proteases and other factors. Thus, DDSs were
envisioned to provide protection to proteins/peptides until they
have reached their site of action. However, after a decade of
protein/peptide DDS research; there is still a lack of good systems
available. Thus, for more efcient protein and peptide DDSs,
researchers must consider the following: (1) safety and biocom-
patibility of the DDS; (2) material and host toxicity; (3)
192 M.L. Tan et al. / Peptides 31 (2010) 184193
encapsulation efciency; (4) retention or enhancement of the
natural therapeutic property of the protein/peptide; (5) tissue-
specic action; (6) uncomplicated preparation procedure and
administration; (7) cost-effective and not cost-prohibitive; (8)
biodegradability; (9) controlled, sustained release of therapeu-
tics.
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