2. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1612 future science group
epithelium (e.g., IGF-I [29] and insulin [4,27,30]) or
via endocytosis [4,31]. However, uptake via these
routes does not lead to pharmacological effects
in vivo due to simultaneous protein metabolism.
Therefore attempts have been made to facilitate
absorption and protect against metabolism by
use of different delivery systems. Such delivery
systems have a number of barriers to overcome
in terms of the GI tract including:
n Mucus layer on epithelial cells;
n Unstirred water layer on epithelial cells;
n Low permeability of cell membranes and GI
tract to macromolecules;
n The presence of efflux pumps in cell
membranes;
n Tight junctions between epithelial cells;
n Chemical and enzymatic degradation/metab
olism of proteins in GI fluid and on the brush
borders and inside epithelial cells.
The mucus layer on epithelial cells is continuously
replenished every 12–24 h [32] and is a physical
diffusion barrier to drug absorption, particularly
macromolecules [1,5,33,34]. It can act to prevent
absorption due to the adhesive nature of mucin
(the major component of mucus) causing pro-
teins to be trapped in the mucus layer and there-
fore preventing their uptake/passage between
cells [1,35–37]. Amphiphilic structures such as
proteins may also interact with mucin via non-
specific hydrophobic interactions such as those
seen with bacteria and surfactants [34], further
reducing their ability to be absorbed. Cationic
proteins may also electrostatically interact with
the negatively charged groups on the side chains
of mucin [34,38], limiting their uptake.
The presence of an unstirred water layer on
epithelial cells is less significant in absorption,
but still can play a limiting role, particularly for
hydrophobic drugs. However, in the case of most
proteins they will readily pass across this layer
due to their hydrophilic nature and their absorp-
tion is mainly limited by their inability to diffuse
through lipid cell membranes [1].
There are many potential mechanisms of
drug absorption via either the paracellular or
transcellular routes (Figure 1) [1,3,4,34,39].
The paracellular route is attractive for proteins
due to the lack of enzymatic activity in the inter-
cellular spaces [1,4,8,9]. However, access to intercel-
lular spaces is limited by tight junctions between
cells. The integrity of these tight junctions is
maintained by a number of transmembrane
(occludin and claudin) and cytoplasmic proteins
(a family of zona occluding proteins, e.g., ZO-1)
and when these junctions are closed only very
small molecules, for example, water, may pass
through them [4,10,40]. Also found within the tight
junctions are cadherin glycoproteins, which rely
on maintenance of physiological concentrations of
calcium to preserve tight-junction integrity [4,10].
Therefore, paracellular transport is not normally
an option for macromolecules. As stated above, the
junctions found between cells only allow for the
passage of very small hydrophilic molecules [4,5].
Molecules of 10–50 Å have been shown to pass
between cells, although not usually more than
11.5 Å, due to the gap between cells being less
than a nanometer [3,10,34]. There is also a limit in
molecular weight of 200 g/mol via the paracellu-
lar route, much smaller than most peptides [1,7,39].
Therefore, for macromolecules to have a chance
to pass through tight junctions, they need to be
opened to quite a significant extent [4,10].
Similarly, although a large number of small-
drug molecules are absorbed via transcellular
passive diffusion, this is not possible for pro-
teins given their hydrophilicity, low log P values,
high-molecular weights and charged nature,
which mean they have very low absorption dif-
fusion coefficients [1,3,4,7,35,41]. The presence of
efflux pumps (e.g., P-glycoprotein [P-gp] and
multidrug resistance protein) in GI epithelial
cell membranes may also result in any protein
that is absorbed into cells being removed before
transport to the systemic circulation can be com-
pleted [1,3,4,9,39,42]. However, this tends to be more
of a problem with small molecules [39]. An excep-
tion to this being cyclosporin A, which has been
shown to be a substrate for P-gp [1,9,42].
Peyer’s patches are also be involved in trans
cellular absorption. These are lymphoid nodules
found primarily in the ileum of the small intes-
tines and are covered by a different type of epi-
thelial cells than the rest of the GI tract, called
microfold or M cells [4]. The patches are predom
inantly involved in mediating immune response
to pathogens in the GI tract and transporting
absorbed substances to the lymph nodes and
spleen. However, their structure (underdeveloped
villi and mucus layer) allows for nano-sized mate-
rials to easily pass through, usually of 200 nm
or less in diameter [4,43,44], while materials of
3000–5000 nm or more in diameter are trapped
within the patches [4,45]. However, they const
itute a limited part of the GI tract epithelium as
a whole, which limits their role in absorption of
therapeutic compounds [4,43,45].
4. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1614 future science group
delivery. Therefore a number of different technol-
ogies to improve oral bioavailability of proteins
have been developed.
Technologies to facilitate oral
protein delivery
Early steps in improving oral absorption involved
co-administration/co-formulation of proteins
with protease inhibitors, such as sodium glyco-
cholate, aprotinin, bacitracin, soybean trypsin
inhibitor, camostat mesilate [54,55] and absorp-
tion enhancers such as bile salts, salicylates, sur-
factants and chelating agents [56,57]. However,
these were found to have deleterious effects on
the GI tract wall, on normal digestion and the
pancreas itself [1,3–7,9,10].
Subsequently, encapsulation of proteins in
particulate structures of various sizes and mor-
phologies has become one of the most widely
used approaches to facilitate oral delivery [43,58–
62]. This concept is usually aimed at modifying
the pharmacokinetic profile and physicochemical
characteristics of the resultant construct, as well
as providing an outer polymeric membrane for
the entrapped protein, which can serve as a
protective coat from proteolytic enzymes [61,62].
Particulate carriers having different functional-
ities can be tailor-made by careful selection of
parent polymers that possess desirable proper-
ties such as enzyme inhibition, mucoadhesion,
absorption-enhancing or pH/environmentally
sensitive properties [63–69]. These delivery sys-
tems may also be functionalized by attachment of
various receptor-recognizable/targeting ligands
to preformed particles [64,70,71]. Particulate
carriers including liposomes [3,6,72], micropar-
ticles [3,6,7,72], and polymeric colloids including
polyelectrolyte nanoparticulates [3,52,53,73–79],
have been developed with varying degrees
of success.
For oral protein delivery, the use of liposomes
(tiny, vesicular self-assemblies consisting of
hydrophobic and hydrophilic domains formed
when phospholipids are dispersed in aqueous
media) is challenging due to the inability of lipid-
based systems to entrap sufficient quantities of
hydrophilic drugs resulting in problems such as
low GI tract stability, low drug-loading capacity
and leakage of entrapped drug [3,6,72,80].
Moreover, the fabrication of micropart
icles often requires the use of organic solvents,
high temperatures/pressures and hydropho-
bic polymers, which can denature proteins. In
addition, their use in oral protein delivery has
led to highly variable levels of protein in the
circulation [1,7,81–84].
In contrast, polymeric colloidal systems are
thought to be advantageous, as they can be more
quickly and completely absorbed due to their
large surface areas and nano-range sizes, as well
as protect proteins from degradation and improve
their in vivo stability [3,6,52,75,85,86]. However,
there can be a lack of control of protein release
and levels of protein loading can be low [3,53].
Polyelectrolyte complexes (PECs) are nor-
mally nanosized particulates that are composed
of polyelectrolyte polymers and a protein. Their
formation is driven by coulombic interactions,
that is, electrostatic interactions and hydrogen
bonding, as well as hydrophobic interactions in
some cases (Figure 2) [52,76,78,82,86,87]. They can
also involve prior copolymerization of oppo-
sitely charged polymers, which can then inter-
act electrostatically with each other and with a
protein [65,66].
PECs are able to form spontaneously (self
assemble) under benign conditions and can have
high levels of protein loading, which is advanta-
geous in comparison to some of the other delivery
technologies mentioned above [78,82,84,86,88–90].
Key Terms
Polyelectrolyte complex:
Polyelectrolyte
polymer–protein complex
whose formation is primarily
driven by coulombic forces.
Chitosan: Cationic
polysaccharide derived from
crustaceans used in a large
number of polyelectrolyte
complexes.
Thiomers: Polymers that
contain moieties with
thiol groups.
Caco-2: Colorectal cancer cell
line, one of the most common
in vitro cell models for the GI
tract.
Quaternary ammonium
moiety: Quaternary amine,
commonly produced by the
trimethylation of the primary
amines of chitosan.
Table 1. Various GI tract enzymes and their respective protein target sites.
Enzyme Target sites
Endopeptidases Nonterminal residues
Trypsin After arginine, lysine unless followed by proline
a-chymotrypsin After phenylalanine, tyrosine and tryptophan unless followed by proline; also
after asparagine, leucine, methionine and histidine
Pepsin Before leucine, phenylalanine, tyrosine and tryptophan unless preceded by
proline; can also cleave at many other sites
Elastase After glycine, alanine, serine and valine unless followed by proline
Exopeptidases Terminal residues
Carboxypeptidase A Aromatic/aliphatic hydrocarbon chain residues at the carboxy-terminal
Carboxypeptidase B Cationic residues at carboxy-terminal
Aminopeptidase Residues at amino-terminal
6. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1616 future science group
interpolymer complexes of polyanions and poly-
cations have been produced to try and mitigate
the problems of using polyanions or polycations
alone [2,84,90,103].
Electrostatically induced intermolecular com-
plexation occurring between polycationic and
polyanionic molecules present in aqueous solu-
tion leads to the formation of discrete interpoly-
mer complexes, which have been shown to be
suitable delivery systems for oral proteins [104,105].
Interpolymer complexes are often prepared by
mixing aqueous solutions containing oppositely
charged polymers at a specific pH resulting in
the formation of interpolymer PECs exhibit-
ing varied morphologies and functionalities as
dictated by their chemical composition [106,107].
However, tailoring the use of these interpoly-
electrolyte structures to surmounting the bar-
riers inherent in oral protein delivery involves
careful selection of complementary polycations
and polyanions, which interact optimally pro-
viding a robust network suitable for the incor-
poration and protection of proteins intended for
oral administration. The concept of interpolymer
complexation also conveniently creates a platform
where unique properties of different polymers
that have been shown to be capable of improving
oral protein bioavailability can be imparted into
a single delivery system, thereby potentiating the
efficacy of the resultant construct. Drug release
from the interpolyelectrolyte network is usually
mediated by decomplexation of the PECs at pH
conditions that reduce the electrostatic forces
between the interacting polyelectrolytes leading
to dissociation of the complex and release of the
incorporated protein.
Invariably, many research groups have
exploited the versatility of chitosan by complex-
ing it/its derivatives with various polyanions of
which the majority are anionic polysaccharides
of natural origin such as glucomannans and
mucopolysaccharides as well as other natural and
synthetic polymers [2,22,30,66,83,84,87,88,90].
As well as forming interpolymer PECs by
mixing chitosan and polyanions, these can also
be formed after prior copolymerization of poly-
electrolytes. This can counteract some of the
potential problems associated with PECs that rely
solely on coulombic interactions. These include
counterion effects and the high solubility of chi-
tosan in the stomach and of polyanions in the
lower GI tract, which can result in partial PEC
collapse and subsequent protein dumping, limit-
ing protection from degradation and promotion
of absorption [2,24–26,65,66,78,82,89,99–102,108,109].
Alginate–chitosan PECs
Alginate is an anionic polysaccharide extracted
from bacteria or algae and is made of residues of
mannuronicandguluronicacidofvaryingratios.It
exists as salt forms due to cooperative binding with
cations, for example, sodium alginate. Alginates
form gels when mixed with divalent cations (at
certain stoichiometric ratios), such as calcium,
due to their exchange with sodium, which then
results in inter-chain associations being formed
(dimerization of the alginate) primarily between
guluronic residues [2,22,24,30,83,100,108]. Alginates are
already used in a number of foods as a thicken-
ing agent/stabilizer and have been shown to be
both biocompatible and biodegradable. They are
approved for human use by the US FDA.
In terms of their use as oral delivery vehicles,
alginates have been shown to form strong mucoad-
hesive bonds when compared with other polymers
suchaschitosan [2,22,30,83].However,alginateshave
a pH-solubility problem in that although the gels
they form release very little protein in the stomach,
once they reach the lower GI tract they can rapidly
dissolve preventing controlled release of protein
as well as providing very limited protection from
degradation. In addition, protein electrostatic
interaction with alginates can cause detrimental
changes in protein conformation and loading of
proteins can be low [2]. Therefore, attempts have
been made to use alginate in concert with other
polymers to overcome these problems.
One such combination involves the use of algi-
nate with chitosan, which forms electrostatic inter-
actions between the carboxyl groups of alginate
and amino groups of chitosan [2,22,24,30,83,100,108].
This predominantly occurs between carboxyls
on mannuronic rather than guluronic residues of
alginate and aminos on chitosan, probably due to
the lower pKa
of mannuronic residues (3.38 com-
pared with 3.65 for guluronic) [110]. The potential
advantage of this combination is that it allows the
formation of a pH-sensitive gel network, which
can complex/load a protein for delivery. This
network does not entirely dissolve in the stom-
ach or intestines due to the presence of alginate
and chitosan, respectively. It can still swell in the
intestines, as alginate does on its own, but due to
the presence of chitosan does not completely dis-
solve there and so may allow for mucoadhesion
and controlled release to take place.
These PECs are produced by first forming
nuclei of alginate complexed with calcium and a
protein followed by addition of chitosan to form
PEC nanoparticles via crosslinking between chito-
san and alginate, as well as between each polymer
Key Term
Interpolymer complex:
Polyelectrolyte complex
consisting of two or more
oppositely charged polymers.
7. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1617future science group
and the protein (Figure 3) [22,30,83]. Pregelation
with calcium is thought to increase PEC stability
by providing an additional level of crosslinking
between alginate chains. Crucially these PECs
have been shown, via derivatized Fourier trans-
form IR and circular dichronism spectra, to have
no significant effect on insulin secondary struc-
ture and that insulin is stable in the PECs for up
to one month, suggesting that the protein is still
in its native conformation after complexation [22].
However, the limitation of these systems is that
polymer–polymer and polymer–insulin interac-
tions are also still pH dependent with insulin
association efficiency falling from 81 to 60% of
total insulin used in nanoparticle manufacture
when PEC solution pH was increased from 4.7
to 5.2 due to a reduction in charge density of
chitosan and insulin (pI = 5.3) [22]. This can lead
to low protein loading and cause burst release at
both low and high pH [2,7]. Indeed it was found
that alginate–chitosan PECs showed burst release
of insulin between 40–60% within 5 min in sim-
ulated gastric (pH 1.2) and simulated intestinal
fluid (pH 6.8) [22]. This was due to the variations
in charge density on chitosan, alginate and insu-
lin at these pH values resulting in weak interac-
tions between the three components of the PEC
and so uncontrolled release of insulin.
In addition to this, some precipitation of alg
inate and chitosan can still occur over the pH
range in the GI tract resulting in large increases
in particle size, which in turn could limit cellular
interactions and uptake due to the reduction in
PEC surface area. Sarmento and colleagues found
that decreasing pH from 4.7 to 4.2 resulted in
particle size increasing from 797 to 2858 nm,
as the reduction in pH reduces the ionization of
alginate (pKa
between 3.3 and 3.7, depending on
ratio of mannuronic to guluronic residues) [87].
Regardless of these problems, however, algi-
nate–chitosan PECs have been shown to be effec-
tive at reducing blood glucose levels in diabetic
rats in vivo [30]. Oral doses of 50 or 100 IU/kg
contained in alginate–chitosan PECs were shown
to gradually reduce glycemia over a 14 h period
to approximately 60% of initial values. This
O
HO
HO
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
O
O
O
-
-
-
-
-
-
-
-
O
O
O
O
O
HO
HO
OH
OH
O
OO
NH+
OHO
OH
HO
OH
HO
HO
n
n
HO
HO
HOHO
Guluronic residue
Calcium
crosslink
Deprotonated alginate
Electrostatic
interaction
Mannuronic residue
Protonated
chitosan
O
O
OH
O
OH
OH
OH
OH
OH
O
OO
O
O
O
O
O O
O
O
O
O
O
Ca2+
OH
OHOHOH
+
H3
N
+
H3
N
+
HN
HO
NH3
+
NH3
+
Figure 3. Polyelectrolyte complex of calcium-crosslinked alginate complexed with
chitosan.
8. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1618 future science group
compared favorably with a 2.5 IU/kg dose of
noncomplexed insulin given subcutaneously
(sc.), which reduced glycemia by more than 50%
within 2 h, but which returned to near initial
blood glucose levels after 8 h. It was also differ-
ent from an oral insulin solution and a physical
mixture of insulin and empty nanoparticles (both
50 IU/kg), which resulted in a maximum reduc-
tion in glycemia of only 20% after 6 h, which then
returned to near pretreatment levels rapidly [30].
This indicated that although some insulin
would have been released in the stomach and
overall control of release was probably poor (as
well as possible precipitation/dissolution of the
polymer complex), the PECs allowed insulin
to pass through the GI epithelium intact and
produce a hypoglycemic effect. This effect was
delayed compared with sc. insulin, as it would be
expected that the PECs adhere to the GI mucosa
and produce a prolonged release of insulin. As
well as this, the time taken for GI transit, trans-
port of insulin through the various barriers of
the GI tract epithelium, then the circulation
and finally to the liver before it could exert its
affect would take far longer than transit to the
circulation after sc. administration [4].
These results are promising in the search for
an oral insulin-delivery system in that it would
appear that the alginate–chitosan PECs are able
to protect insulin and facilitate its absorption to
some extent. It would also suggest a prolonged
control of blood glucose levels would be pos-
sible, which would reduce patient dosing as well
as potentially provide for a reduction in diabetic
complications.
However, unsurprisingly, the doses required to
elicit this effect are high compared with sc. doses
(a dose of 25 IU/kg given as an oral nanopar-
ticle produced a much reduced effect [maxi-
mum reduction of 25% glycemia after 8 h]).
Bioavailability of the 50 and 100 IU/kg doses
were 5.1 (equivalent to 7% of administered dose)
and 9.5 ng/ml.h, respectively, which although
much higher than oral insulin administered non-
complexed (2.1 ng/ml.h), could still be improved
in order to reduce dose sizes. It should also be
borne in mind that these relatively high doses are
required due to loss of insulin during GI transit
and transport across the GI epithelium due to
chemical and enzymatic breakdown. Therefore,
further work on these systems’ ability to protect
insulin from degradation could be investigated.
Additionally, although several uptake mecha-
nisms were postulated (absorptive endocytosis,
paracellular transport, Peyer’s patches) the exact
routes and mechanisms of uptake have not yet
been determined. If the mechanisms by which
these PECs facilitate insulin uptake could be fully
elucidated, it could indicate any further changes
to the PECs structure, which may aid uptake and
also minimize insulin loss during delivery.
Further alginate–chitosan PECs were devel-
oped by El-Sherbiny and colleagues involving a
carboxymethylchitosan(CMC)–sodium acrylate
copolymer, which could be complexed with cal-
cium-crosslinked alginate and a PEGylated form
of chitosan [99–102]. By grafting sodium acrylate
to the CMC, the number of carboxylic groups
within the PEC structure were increased. This
was done in order to limit the potential for swell-
ing, and therefore protein release, at gastric pH.
This is possible as the carboxylic groups can form
hydrogen bonds with each other and the hydrox-
yls of CMC as well as form strong ionic interac-
tions with calcium ions thereby minimizing the
ability of the PECs to swell at low pHs. The loss
of a large proportion of the amine groups in the
structure of chitosan due to carboxymethylation
and grafting of sodium acrylate further limits
ionization at low pH and therefore swelling due
to repulsion between protonated amino groups.
By coating sodium acrylate–CMC–alginate with
PEGylated chitosan, this should offer ever greater
protection against premature protein release in
the stomach by forming strong electrostatic inter-
actions with CMC–sodium acrylate and alginate
(Figure 4) [100].
It was found that swelling was very low (less
than 5% increase in weight of PECs after 2 h) in
simulated gastric fluid (pH 2.1), but fairly exten-
sive (up to 40% after a further 8 h) in simulated
intestinal fluid (pH 7.4). Release of bovine serum
albumin (BSA) from these PECs was monitored
in the same buffers and was found to be lower
with those PECs coated with PEGylated chitosan
than those with no coating in simulated gastric
fluid (~10 compared with ~25% release after 2 h)
and fairly well controlled once transferred to sim-
ulated intestinal fluid (70–85% after a further
8 h). PECs coated with PEGylated chitosan also
had greater loading capacities than noncoated
forms (63–83% encapsulation for coated PECs
as compared with 31–47% for noncoated) [100].
From this, it can be seen that the additional
hydrogel coating allowed for the formation of
strong electrostatic interactions between it and
the other polymers in the PEC thereby gaining
greater control over swelling and release, but
also allowed for greater interaction with a model
protein to allow for higher rates of encapsulation.
11. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1621future science group
exact mechanisms and transporters involved in
uptake and translocation through cells are also
to be elucidated.
Poly(aspartic acid)–chitosan PECs
Poly(aspartic acid) is also a poly(carboxylic acid)
like alginate. However, it is synthetic rather
than derived from natural resources. It is pro-
duced by polycondensation of aspartic acid to
poly(succinimide) followed by hydrolytic ring-
opening. As it is based on an amino acid, the
polymer formed has a protein-like structure and
is water soluble and biodegradable [84,90].
Shu and colleagues formed PECs between
chitosan and thiolated polyaspartic acid with
a view to produce pH-sensitive swellable
nanoparticles for the oral delivery of insulin
(Figure 6) [90]. The potential advantage of these
PECs is that they may be more stable than other
chitosan–polyanion-based PEC. Previously, the
electrostatic interactions used to form PECs
based on chitosan and other polyanions were
weaker or stronger in different parts of the GI
tract due to the variation in pH, depending upon
their pKa
, and were generally unstable in vivo
due to salting-out effects [22,30,78,83,87,109].
However, the use of a thiolated polymer in
this case has not only allowed for electrostatic
interactions to take place, but for disulfide-bond
crosslinking, which increased the strength of
interactions and overall nanoparticle stability
at low pH (pH 1.2–4) and in increasing salt
concentrations in vitro.
Additionally, in vitro release studies dem-
onstrated that PECs, which relied solely upon
electrostatic interactions, released insulin very
quickly (~90% within 30 min) at pH 1.2,
while chemically crosslinked particles showed a
more controlled release at pH 1.2 (~40% after
120 min). Although after adjustment of the pH
to 6.8, the remaining insulin from crosslinked
nanoparticles was very rapidly released (~90%
after a further 30 min).
The rapid release in pH 6.8 is surprising,
given that disulfide-bond formation should be
increased at a pH above 5 due to the increasing
oxidation of the thiol groups on the PECs [32,63,94–
97,103]. The opposite appears to have happened
whereby the interpolymer interactions have
become so weak that the nanoparticles can no
longer retain insulin at increasing pHs. It may be
that the use of chitosan has resulted in a degree
of precipitation of the formulation at pH 6.8
given that it would be fairly insoluble at that pH.
As the disulfides do not form between chitosan
and aspartic acid, but only between aspartic
acid chains this would not solve the problem
of the lack of solubility of chitosan at higher
pH. This together with the loss of electrostatic
interaction between chitosan and aspartic acid
could have caused the burst release. A variation
on this formulation was also examined where a
low-molecular weight (6000 g/mol), water-solu-
ble chitosan was used instead and poly(aspartic
acid) was used without thiolation. In this study,
the polyelectrolytes were co-mixed with PEG
in order to provide additional sites for inter-
polymer interactions and, therefore, form more
stable PECs [84]. PEG was also used in order to
promote mucoadhesion by improving complex
chain flexibility and mobility as well as maintain
the secondary and tertiary conformation of BSA
when encapsulated.
The addition of PEG may have resulted in
interpolymer interaction between its ether group
and the carboxylic-acid group of the aspartic
acid as well as between the primary amines on
chitosan and the hydroxyl on PEG. As a result of
this, the interpolymer network was strengthened
and more compact nanoparticles were formed
(from a radius of approximately 175 nm with
no PEG to 125 nm at 30 mg/ml PEG). These
nanoparticles were also stable along a range of
salt concentrations with the particle radius not
exceeding 300 nm up to 0.025 mol/l NaCl.
This was in contrast to the previous study where
particle radius increased up to 400 nm at the
same salt concentration [90]. However it is dif-
ficult to attribute this increased stability solely
to the presence of PEG, given the number of
other variables that changed between the two
studies [84,90].
Regardless of the apparent increase in par-
ticle stability (uncontrolled), pH-dependent
release was again found. In vitro BSA release was
approximately 20% at pH 2.5, 60% at pH 1.2
and 80% at pH 7.4 (after 240 min). Again, it
can be seen that noncontrolled release occurred
at a higher pH, which would not be beneficial
in vivo. The reduced protonation of chitosan at
pH 7.4 would again, regardless of its increased
water solubility at low-molecular weight, result
in very loose and diffuse PECs at this pH causing
rapid and uncontrolled protein release.
In order to improve upon this delivery sys-
tem, quaternization and/or thiolation of chitosan
may also be required in order that electrostatic
forces between polymers are strengthened and
are pH independent as well as potentially allow-
ing the formation of disulfides between chitosan
12. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1622 future science group
and (thiolated) aspartic acid in order to further
strengthen the interpolymer network. In addi-
tion, conjugation of PEG to either polymer may
also facilitate greater complex stability, solubil-
ity, biocompatibility and mucoadhesion than the
weaker hydrogen bonding seen above between
PEG and chitosan [82,88,89,91,92]. Indeed, it has
been postulated that the counterion effects of
salt can be counteracted to a limited extent
by steric hindrance of quaternary ammonium
salt groups, grafted PEG and disulfide-bond
formation [82,89].
However, it must also be borne in mind that
the ability to control release of proteins is also
dictated by their charge which will also change
with pH. The ability of a protein to form elec-
trostatic interactions with a polymer will alter
drastically around their pI, as the charge on the
polymer goes from cationic to anionic or vice
versa [92]. Therefore any system that is to pro-
duce controlled release in the GI tract and avoid
release in the stomach, must not rely solely upon
electrostatic interactions between the polymer
and protein.
Poly(methacrylic acid) copolymer PECs
Poly(methacrylic acid) (PMMA) is another weak
acid, which has been used to complex with pro-
teins. However, as with other polyanions men-
tioned above, when used alone it is unable to
Electrostatic
interaction
HO
HO
HO
HO
HO HO
HO
HO
+
H3
N
+
H3
N
+
H3
N +
H3
N
+
HN
H2
H2 H2
H2
H2
CH2
CH2
CH2
CH2
CH2
CH2
H2
NH
+
OH
OH
OH
OH
OH
OHOH
O
O O
O
O O
CC
C C C
C = O
NH
NH
C = O
C
O
S
S
O
O
O
O
- - -
- - -
-
-
O
O O O O
O O
O O
O
O
O
O
O
O
O
n
x y
n
yx
H
N
H
N
H
N
H
N
H
N
H
N
O O
O
O
O
OH
Disulfide
crosslink
Ionised
poly(aspartic acid)
Protonated
chitosanNH3
+
NH3
+
Figure 6. Polyelectrolyte complex of disulfide crosslinked thiolated poly(aspartic acid)
complexed with chitosan.
13. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1623future science group
efficiently complex with proteins at the pH range
of the GI tract. Therefore attempts have been
made to produce more stable complexes by copo-
lymerizing with PEG. This increases complex
stability as the PEG ether groups and carboxyl
groups of PMMA can form hydrogen bonds to
produce an interpolymer complex [13,111–113].
PMMA-based nanoparticles have been pro-
duced by copolymerizing with an amine mod
ified PEG, which facilitated interpolymer ionic
crosslinking [13]. Individual PMMA–PEG par-
ticles were approximately 800 nm in diameter.
However, a high degree of aggregation occurred
at gastric pH resulting in the form
ation of
micron-sized particulates. At intestinal/physi-
ological pH, above the pKa
of PMMA, increased
interparticle repulsion occurred resulting in
smaller, discrete particles. This is very impor-
tant as smaller particles are more likely to inti-
mately interact with the GI tract epithelium in
terms of promoting both mucoadhesion and
absorption [13,88].
PMMA–PEG particles were able to demon-
strate ex vivo mucoadhesion for up to 3 h due to
both the predominantly anionic charge on the
particles at pH 7 and possibly the ability of PEG
to entangle with mucus glycoproteins. However,
longer lasting mucoadhesion would be desirable
to set up and maintain a protein concentration
gradient across the GI tract mucosa into the cir-
culation and so further particle size reduction
could be beneficial.
BSA was loaded into these particles with
encapsulation efficiencies of up to 80%. In vitro
release from these particles showed a very slow
and incomplete release at pH 1.2 (less than 10%
after 3 h) and relatively rapid release at pH 7.4
(~90% after 3 h). This pH-dependent release
could be attributed to the increase in swelling of
the particles at elevated pH due to the increased
ionization of carboxyl groups on PMMA caus-
ing repulsion between PMMA chains above its
pKa
[13]. Again, this is not ideal as although the
protein would not be released in the stomach, the
rapid release in the intestines would be unlikely
to result in the maintenance of therapeutic levels
and would most likely result in the requirement
of repeated dosing. The same study also utilized
the PMMA–PEG PECs to load insulin alone and
precomplexed to carboxymethyl-b-cyclodextrin
(Cb-CD). The Cb-CD was used to improve
insulin stability in the delivery system, by pre-
venting insulin hydrophobic intermolecular
interactions which can lead to aggregation and
a reduction in activity [21].
Encapsulation efficiency was high at
approximately 78% (insulin alone) and 73%
(insulin–Cb-CD) and insulin was shown to
still be biologically active after encapsulation
using an ELISA. However, insulin release from
the particles was very rapid at pH 7.4 (in effect,
dose dumping) with approximately 70% release
within 60 min regardless whether insulin was
encapsulated alone or complexed to Cb-CD.
The more rapid release of insulin compared
with BSA may in large part be due to its much
smaller-molecular weight compared with BSA
(5800 compared with 66,000 g/mol). The pore
size of the swollen particles may be such that
insulin can pass easily through them, while the
larger BSA requires more time in order to be able
to diffuse through the interpolymer complex.
Therefore, more precise control over swelling
(and hence pore size) is required for delivery of
smaller proteins.
One way to achieve this may be by increas-
ing the molecular weight of the PEG used.
Higher-molecular weight PEG could result in
greater interpolymer interactions, thereby reduc-
ing swelling and therefore pore size. However,
this reduction in pore size could also limit insu-
lin loading. Increasing PEG molecular weight
in PECs from 400 to 1000 has been shown to
reduce in vitro insulin release (from 41 to 39%
after 2 h) and also reduced insulin encapsulation
(from 38 to 32%) [112]. A reduction in insulin
loading may be necessary to provide greater con-
trol of insulin release. However, the reductions
in release seen here are quite small and so other
options need to be considered, perhaps including
the use of considerably higher molecular weight
PEGs, for example, ≥10000 g/mol.
Other work with PMMA–PEG copolymers
has involved the incorporation of chitosan
into PECs. Sajeesh and Sharma copolymerized
PMMA, chitosan and polyether (a copolymer
of PEG and polypropylene glycol) [21]. In this
case the electrostatic interaction was between the
primary aminos on chitosan and the carboxyl
groups on PMMA while polyether formed hydro-
gen bonds with PMMA. The choice of polyether
rather than PEG alone was due to the potential
for polyethers to promote absorption across epi-
thelial barriers via a surfactant-like effect on cell
membranes (as with the Pluronics®
) [21]. This
PEC was complexed with insulin either alone or
after insulin was again complexed with a hydro-
philic derivative of b-cyclodextrin (2-hydroxy-
propoyl b-cyclodextrin [HPbCD]) to increase
its stability in the formulation.
14. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1624 future science group
Encapsulation efficiency of insulin was approx-
imately 85% regardless of whether insulin was
used alone or as part of a CD complex indicating
a lack of interaction between HPbCD and the
interpolymer complex. This is borne out by the
similarity in in vitro release from particles, which
carried insulin alone or as part of a HPbCD com-
plex; pH-dependent release was demonstrated
with approximately 15% release within 2 h at
pH 1.2, and 75–80% release at pH 7.4 over the
same time regardless of the presence of HPbCD.
The lack of control of release at pH 7.4 had
still not been overcome by these further formula-
tion changes, and would suggest that while the
use of a CD ensures that insulin remains active
when formulated it does nothing to aid control
of insulin release as it cannot interact with the
polymers present in the PEC.
This study was also able to show that these
PECs can show some concentration-depen-
dent trypsin inhibitory activity when using
N-a-benzoyl-l-arginine ethyl ester (BAEE)
and casein as low- and high-molecular weight
substrates, respectively. Greater protection was
offered to casein than BAEE over a PEC concen-
tration range of 0.25–1% (w/v), which may be
due to differences in substrate molecular weight
(24,000 g/mol for casein compared with approx
imately 300 g/mol for BAEE). As the studies were
conducted at pH 7.6, the PECs would have been
swollen and so release of BAEE would have been
even more rapid than insulin, while casein release
could have been much slower. This difference in
free and encapsulated substrate could have meant
that as more casein was encapsulated for longer,
then a physical barrier to trypsin binding, as well
as any potential chelation of metal ions by the
carboxyl groups of PMMA, would have provided
greater protection to it than BAEE. However,
the protective effects seen are modest and only
reached at most to approximately 40% inhibition
of trypsin at 1%(w/v) PEC concentration.
The lack of a substantial inhibitory effect
may be due to the lack of ‘free’ carboxyls on the
PMMA after copolymerization. Electrostatic
interactions and hydrogen bonding within the
PEC between polymers and also the encapsu-
lated protein maintain the PEC structure in
solution. Therefore it may be that very few car-
boxyls were not already interacting with other
PEC components and so very few of them were
available to chelate metal ions.
This would seem to be confirmed by a simi-
lar study, which showed that carbopol 974 had a
greater inhibitory effect on trypsin activity against
casein than PMMA–chitosan–PEG PECs; 80%
inhibition compared with 50% inhibition at a
concentration of 1%(w/v), respectively [88].
Carbopols are weakly crosslinked hydrogels of
acrylic acid and alkyl ethers and, as such, may
have a fewer number of carboxyls involved in
crosslinking than these PMMA-based PECs.
However, it would be useful to compare PMMA
alone with crosslinked versions to confirm this.
In addition, attempts to reduce the degree of
interaction between these polymers, perhaps by
varying the molar ratios used for polymerization
or molecular weights of the polymers, would
provide greater insight into this. However, a
reduction in interaction between polymers used
in these systems would probably result in even
faster release of encapsulated proteins due to fur-
ther increases in pore size on swelling or perhaps
collapse of PECs altogether on increases in pH.
Further studies in this area have been con-
ducted to try and improve control of protein
release and enzyme inhibition of PMMA–chi-
tosan–PEG nanoparticles by conjugation of cys-
teine to the surface of these PECs [65,66]. As with
other thiomers mentioned above, the presence
of these thiol groups should confer additional
properties to the PECs including covalent muco-
adhesive bond formation and greater chelation
of metal cations to facilitate both tight-junction
opening and enzyme deactivation.
These PMMA–chitosan–PEG thiomers are
different in that the surface of the PECs is thio-
lated and not the interior. To date, most thiomers
have relied on disulfide crosslinking within PECs
to form stable particles as the pH rises to five
and above [32,63,94–97,103]. However, as mentioned
above, this reduces the number of ‘free’ thiol
groups available for mucoadhesion and enzyme
inhibition. One way to improve these properties
may be to attach thiol-containing moieties to the
surface of PECs instead, which could maximize
the ‘free’ thiol content of the formulation. It
has been found, however, that disulfides do still
form between surface thiol groups. Sajeesh et al.
found that thiol content of thiolated PMMA–
chitosan–PEG PECs increased from 506 to
1214 µmol/100 mg of PECs after treatment with
sodium borohydride to cleave disulfides [65].
The potential to form disulfides between par-
ticles should also be considered, which would
promote particle aggregation, particularly at ele-
vated pHs, such as in the lower GI tract. If this
were to occur, intimate contact between particles
and the GI mucosa could be reduced as well as
the ability to promote mucoadhesion, enzyme
15. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1625future science group
inhibition and mucosa permeability, thus, limit-
ing the ability to both protect a loaded protein
and facilitate its absorption. Limiting disulfide
formation may be achieved by the presence of
other functional groups or ligands, which could
increase steric hindrance between thiols, limit-
ing their ability to conjugate, such as quaternary
ammonium moieties or PEG [82,89].
Although no electron microscopy was per-
formed on the thiolated PMMA–chitosan–PEG
PECs in this study, particles would appear to be
aggregated at pH 7, given that sizes increased with
the number of thiol groups grafted to the particle
surface. PECs with no thiol groups registered a
hydrodynamic diameter of 1030 nm, while those
with 506 µmol of thiol groups/100 mg of PECs
were 1900 nm. Indeed increased aggregation of
the thiolated compared with nonthiolated PECs
appeared to be confirmed by optical microscopy
in a later study [66].
Importantly, the thiolated particles showed
little cytotoxicity when analyzed using an MTT
assay with even the highest thiol content PECs
(506 µmol of thiol groups/100 mg of PECs) at
the highest PEC concentration (2 mg/ml) show-
ing approximately 85% of Caco-2 cells remained
viable after 6 h incubation. This indicates that
any increase in permeability of cells caused by
these PECs should not be due to cellular damage,
or at least very low levels of damage, which may
be reversible.
These PECs were also shown to facilitate
transport of a macromolecular marker (fluores-
cein isothiocyanate-dextran 4000 g/mol [FD4])
across Caco-2 cell monolayers. This ability
was increased with the number of thiol groups
attached to the PECs and was shown to be depen-
dent upon the concentration of calcium in the
media used. As calcium concentration increased
the apparent permeability of FD4 decreased,
which would indicate that the transport of
FD4 across cell layers was due to calcium chela-
tion by the PECs, that is, it primarily involved
paracellular transport.
This was confirmed by the differences in
TEER found when Caco-2 cells were incubated
with thiolated PECs in the presence and absence
of calcium. In the absence of calcium TEER val-
ues dropped to 40% of their initial values within
30 min, compared with only 60% of initial val-
ues when calcium chloride concentration was
10 mM.
TEER reduction and concomitant transport
of FD4 was relatively similar for thiolated and
nonthiolated PECs at 0 mM calcium chloride,
as calcium concentrations increased, thiolated
PECs were able to reduce TEER and promote
FD4 transport to a greater extent than nonthio-
lated forms. This would suggest that thiol groups
are more effective chelators of metal cations than
carboxyl groups and so their use may be highly
advantageous in attempts to facilitate macromole-
cule absorption. However, when transport of FD4
across ex vivo rat intestinal tissue was determined
the presence of thiol groups appeared to make
little difference with apparent permeability of
FD4 being 7.4 × 10-7
and 7.9 × 10-7
cm�����������/s for non-
thiolated and thiolated PECs, respectively. This
highlights the limitations of using Caco-2 cells
as an in vitro model for the GI tract epithelium.
Unlike the GI epithelium, Caco-2 cells do not
have a mucus layer and their TEER values are
markedly higher [114]. This is potentially a major
flaw with the use of Caco-2 cells as a model and
as such HT-29 cells may be more appropriate in
that their TEER values are in line with GI epi-
thelial cells and they produce a mucus layer and
M cells [26,78,114]. In particular the presence of a
mucus layer can have marked effects on PEC inti-
mate contact with mucosal membranes and tight
junctions resulting in a major reduction in the
ability of PECs to facilitate protein absorption.
The presence of thiol groups may result in disul-
fide formation between PECs and cysteine resi-
dues in mucus glycoproteins, which could mark-
edly inhibit their ability to reach and interact
with cell membranes [65]. Co-cultures of these cell
lines could provide an in vitro model, which more
closely mimics the intestinal epithelia, as together
they would produce monolayers which had both
absorptive and mucus secreting cells [26,114].
However, it should be noted that Caco-2 has been
shown to be a useful model many times over and
has indeed generated in vitro data that correlates
well with in vivo findings [114].
In a later study by the same group, insulin was
loaded into the PECs rather than FD4 [66]. It
was notable that in vitro insulin degradation by a
mixture of trypsin and a-chymotrypsin (10 U/ml
each) was prevented to a greater extent with non-
thiolated PECs than thiolated versions. After
60 min, thiolated PECs had only approximately
10% of their initial insulin load nondegraded,
while nonthiolated PECs had approximately 25%
nondegraded. This is surprising given that one
expected advantage of thiolated PECs is their
increased ability to protect proteins from enzy-
matic breakdown [32,63,94–97,103]. Again this may
refer back to the propensity for surface-function-
alized PECs to form disulfides which in turn
16. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1626 future science group
may limit their ability to chelate metal cations
which are responsible for enzyme activity [66]. It
should also be noted that regardless of PEC struc-
ture, total insulin degradation occurred within
90 min incubation, which suggests that neither
formulation may offer enough protection in vivo.
Conversely however, in vivo studies using dia-
betic rats demonstrated that blood glucose levels
were reduced by both thiolated and nonthiolated
PECs (with a dose of 50 IU/kg). Thiolated PECs
demonstrated a greater reduction of approxi-
mately 40% of initial glucose levels within 2 h
of administration compared with approximately
a 10% reduction for nonthiolated PECs. Blood
glucose levels in rats treated with thiolated
PECs gradually increased over the next 8 h, but
a reduction in glucose levels of approximately
10–20% was still maintained after 10 h. The
greatest reduction in glucose levels by nonthio-
lated PECs was seen after 6 h (20–30% reduc-
tion), which was quickly followed by a return to
pretest glucose levels within the next 2 h.
This highlights the difference in cellular
interaction of the PECs when thiol groups are
present. Even with the apparent crosslinking
between particles, enough ‘free’ thiols may be
present to rapidly chelate metal cations and inter-
act with tight-junction proteins to promote insu-
lin absorption. This could subsequently lead to
rapid insulin uptake and so a fairly rapid and sub-
stantial decline in blood glucose. The increased
ability of the thiolated PECs to adhere to the GI
tract mucosa may also explain their ability to
maintain this effect for a prolonged period. This
adherence to the GI tract wall could allow for an
insulin concentration gradient to be set up across
the epithelium into the circulation.
The prolonged reduction in glucose levels
found here is highly desirable in the treatment
of diabetes as it could maintain glucose control
without having to administer a large number of
doses per day, while potentially providing more
prolonged glycemic control than current sc.
dosing. However, as with the studies discussed
above, the reduction in glucose seen with PECs
is less than with a dose of insulin given sc. (70–
80% reduction within 90 min) and for PEC oral
formulations to be effective they must reduce
glucose levels to a great extent as well as main-
tain that effect over many hours (the accepted
gold standard for any insulin-dependent dia-
betes treatment is a reduction in glycosylated
hemoglobin levels to less than 6.5–7% [115]).
Therefore, although surface thiolation may
appear attractive in theory, in practice the very
high reactivity of thiol groups (after oxida-
tion) means that crosslinking between particles
becomes inevitable, thus limiting the ability of
these groups to aid enzymatic protection, muco-
adhesion and protein absorption. Further work in
this area should look at attempts to limit particle
crosslinking and reduce particle size to the nano-
range to improve both enzymatic protection as
well as increase absorption.
Future perspective
Oral protein delivery has still not been achieved
and although progress has been made, a great
deal of work remains to be done. The use of
PECs seems to allow for both protection against
degradation and promotion of absorption to a
limited extent. However, bioavailability of insu-
lin through the oral route is still much lower than
that of conventional parenteral delivery, requir-
ing the use of more than ten-times the sc. dose
of insulin to achieve and maintain hypoglycemia
in animal models.
There has been a general trend towards the use
of chemically modified chitosan either alone or in
combination with other polymers to form PECs.
As part of this, the use of thiomers is highly
advantageous in both promotion of absorption
and protection against enzymatic breakdown of
insulin. The use of PEG has also been a feature
of the research discussed in this review due to its
many potential benefits in terms of mucoadhe-
sion, maintenance of active protein conformation
and PEC stability. However, neither the use of
thiomers or the incorporation of PEG has so far
lead to a substantial breakthrough in oral pro-
tein delivery as they still do not provide for large
quantities of bioactive protein to pass across the
GI tract.
Interpolymer complexes seem to hold great
potential due to their ability to produce pH-
dependent release and mucoadhesion. However,
they too have their limitations in terms of effi-
cient insulin delivery to the circulation due
to continued loss of insulin during GI transit
through premature release and lack of complete
protection against degradation. This seems to be
primarily due to the limitation of utilizing PECs,
which rely solely on pH-dependent, coulombic
interactions for their formation (Table 2).
Therefore in future studies, it may be bene
ficial to form interpolymer complexes via chemi-
cal crosslinks, which are not affected by the ionic
strength and changes in the pH of GI fluid.
However, this could lead to other issues in regards
to whether favorable protein conformations could
17. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1627future science group
be maintained and how proteins could be released
on passage through the GI epithelium.
In order to achieve high oral insulin bioavail-
ability, more work needs to be done to look at
developing polymers that have specific binding
sites for insulin (or other proteins), which pro-
tect their degradation target sites, maintain their
native conformation and also efficiently inter-
act with GI epithelial cell membranes and tight
junctions to rapidly and completely facilitate
absorption.
With this in mind, it is crucial to develop
better screening techniques for PECs to deter-
mine their uptake routes and exact mechanisms
of interaction with cell membranes and tight-
junction proteins in order that these structure
can be further fine tuned to aid protein trans-
port and translocation. This will require much
greater collaboration between polymer chemists,
human (gut) physiologists, biochemists and
pharmacologists to systematically develop PEC
systems from the ground up. Until this is done,
it is unlikely oral bioavailability of proteins can
be increased (reproducible, rapid and sustained
glycemic control can be achieved) to the point
where these delivery systems are commercially
viable alternatives to the status quo.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a finan-
cial interest in or financial conflict with the subject matter
or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or
options, expert testimony, grants or patents received or
pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.
Table 2. Summary of polymers used in interpolymer complexes.
Polymers Important findings Ref.
Chitosan and calcium-
crosslinked alginate
Lack of control over insulin release at intestinal pH
in vitro
Insulin stable in PECs for up to 1 month
Insulin bioavailability in diabetic rats of 7%
[22,30,83,87]
Carboxymethylated chitosan-
acrylate, calcium crosslinked
alginate and PEGylated chitosan
Coating of PECs with PEGylated chitosan and
removal of majority of primary amines from chitosan
structure offers control of protein release over pH
range of GI fluid
[99–102]
Chitosan, calcium-crosslinked
alginate, dextran sulfate,
PEG/poloxamer and albumin
Protection of insulin against pepsin degradation
in vitro
Insulin bioavailability in diabetic rats of 13%
Insulin appears to be absorbed transcellularly
[24–26,108]
Thiolated poly(aspartic acid)
and chitosan
Lack of control over release at intestinal pH in vitro
Slower release at pH 1.2 when thiolated form of
aspartic acid used
[84,90]
Poly(aspartic acid), chitosan
and PEG
Complexes more compact and stable on increases in
salt concentration when PEG incorporated into PEC
PMMA–PEG copolymer Lack of control over release at intestinal pH in vitro [13,21,65,66,88]
PMMA–PEG–chitosan copolymer
and Cb-CD insulin
Lack of control over release at intestinal pH in vitro
Cb-CD maintains insulin biological activity after
complexation with copolymer
Thiolated PMMA–Chitosan–PEG
copolymer
Presence of thiol groups increases transport of FD4
across Caco-2 monolayer
Thiol groups appear to increase chelation of calcium
in vitro thereby increasing reduction in Caco-2 TEER;
Thiol groups make no appreciable difference in FD4
transport across ex vivo mucosa
Carboxylic groups rather than thiols offer increased
protection against trypsin and a-chymotrypsin
in vitro
Thiolated PECs able to reduce glycemia by 40%
within 2 h of administration. Nonthiolated PECs
reduce glycemia by only 10% in same time.
Cb-CD: Carboxymethyl-b-cyclodextrin; FD4: Fluorescein isothiocyanate-dextran 4000 g/mol; PEC: Polyelectrolyte complex;
PEG: Polyetyhlene glycol; PMMA: Poly(methacrylic acid); TEER: Transepithelial electrical resistance.
18. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1628 future science group
Executive summary
Current status of oral insulin delivery
„„ Proteins are becoming an increasingly popular option for treatment, due to high specificity and low toxicity.
„„ Oral delivery is preferential for patients and clinicians.
„„ A number of oral insulin formulations are in clinical trials at the moment.
Barriers to oral protein delivery
„„ Proteins are almost completely degraded in the GI tract due to enzymatic and chemical effects.
„„ Proteins cannot pass across epithelial layers unaided due to their large size, molecular weight and charged nature.
„„ There are numerous routes to pass across the gastrointestinal epithelium both para- and trans-cellularly. However, these are not
accessible to proteins when administered alone.
Technologies to facilitate oral protein delivery
„„ Numerous attempts have been made to promote absorption and prevent degradation of protein in the GI tract that have proved
unsuccessful due to problems in manufacturing of dosage forms and/or toxic side effects on administration.
„„ Polyelectrolyte complexes potentially offer a way to benignly encapsulate (via coulombic and hydrophobic interactions) and
deliver proteins.
Chitosan polyelectrolyte complexes
„„ Chitosan in its native state is ineffective in oral protein delivery due to its weakly basic nature.
„„ Modified forms of chitosan have been produced via trimethylation of primary amines or attachment of thiol-containing ligands. These
forms have shown improved ability to facilitate insulin absorption and improve protection in the intestines.
Interpolymer polyelectrolyte complexes
„„ Polyelectrolyte complexes formed from mixing (or copolymerization) of oppositely charged polymers has been achieved. Use of
polyelectrolyte mixtures can overcome some of the problems of using polycations/anions alone, namely premature release and polymer
precipitation.
„„ These have primarily been produced using mixtures of alginate and chitosan. However other polyanions have been used with chitosan
including poly(aspartic acid) and poly(methacrylic acid).
„„ Some of these systems have an ability to produce sustained hypoglycemic effects in rat models.
„„ Further work is required to maximize the efficiency of these systems, particularly in their ability to facilitate insulin absorption.
References
Papers of special note have been highlighted as:
n of interest
nn of considerable interest
1 Hamman JH, Enslin GM, Kotzé AF. Oral
delivery of peptide drugs: barriers and
developments. BioDrugs 19(3), 165–177
(2005).
nn Detailed review of various barriers to oral
protein delivery and technologies to
overcome them.
2 George M, Abraham TE. Polyionic
hydrocolloids for the intestinal delivery of
protein drugs: alginate and chitosan – a
review. J. Control. Release 114, 1–14 (2006).
3 Morishita M, Peppas NA. Is the oral route
possible for peptide and protein drug delivery?
Drug Discov. Today 11(19–20), 905–910
(2006).
4 Woitiski CB, Carvalho RA, Ribeiro AJ,
Neufeld RJ, Veiga F. Strategies toward the
improved oral delivery of insulin
nanoparticles via gastrointestinal uptake and
translocation. BioDrugs 22(4), 223–237
(2008).
5 Peppas NA, Carr DA. Impact of absorption
and transport on intelligent therapeutics
and nanoscale delivery of protein
therapeutic agents. Chem. Eng. Sci. 64,
4553–4565 (2009).
6 Park K, Kwan IC, Park K. Oral protein
delivery: current status and future prospect.
React. Funct. Polym. 71, 280–287 (2011)
nn Informative review of the status of oral
protein delivery and delivery systems
undergoing clinical trials.
7 Singh R, Singh S, Lillard JW. Past, present,
and future technologies for oral delivery of
therapeutic proteins. J. Pharm. Sci. 97(7),
2497–2523 (2008).
n Review of a number of technologies with
potential to facilitate oral protein delivery.
8 Woodley F. Enzymatic barriers for Gl
peptide and protein delivery. Crit. Rev. Ther.
Drug Carrier Syst. 11, 61–95 (1994).
nn Detailed review of enzymatic barriers to
oral protein delivery.
9 Mahato RI, Narang AS, Thoma L, Miller
DD. Emerging trends in oral delivery of
peptide and protein drugs. Crit. Rev. Ther.
Drug Carrier Syst. 20(2–3), 153–214
(2003).
nn Highly detailed and comprehensive review
of all barriers to oral protein delivery and
perspective various technologies to
overcome those problems.
10 Peppas NA, Kavimandan NJ. Nanoscale
analysis of protein and peptide absorption:
Insulin absorption using complexation and
pH-sensitive hydrogels as delivery vehicles.
Eur. J. Pharm. Sci. 29, 183–197 (2006).
11 Liu H, Wang Y, Li S. Advanced delivery of
ciclosporin A: Present state and perspective.
Expert Opin. Drug Deliv. 4, 349–358
(2007).
12 van Kerrebroeck P, Rezapour M, Cortesse A,
Thüroff J, Riis A, Nørgaard JP.
Desmopressin in the treatment of nocturia: a
double-blind, placebo-controlled study. Eur.
Urol. 52(1), 221–229 (2007).
13 Sajeesh S, Sharma CP. Novel polyelectrolyte
complexes based on poly(methacrylic
acid)-bis(2-aminopropyl)poly(ethylene
glycol) for oral protein delivery. J. Biomater.
Sci. Polym. Ed. 18, 1125–1139 (2007).
19. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1629future science group
14 Lee Y-H, Sinko PJ. Oral delivery of salmon
calcitonin. Adv. Drug Deliv. Rev. 42(3),
225–238 (2000).
15 Kidron M, Dinh S, Menachem Y et al. A
novel per-oral insulin formulation: proof of
concept study in non-diabetic subjects.
Diabet. Med. 21, 354–357 (2004).
16 Morçöl T, Nagappan P, Nerenbaum L,
Mitchell A, Bell SJD. Calcium
phosphate–PEG–insulin–casein (CAPIC)
particles as oral delivery systems for insulin.
Int. J. Pharm. 277, 91–97 (2004).
17 Clement S, Dandona R, Still JG, Kosutic G.
Oral modified insulin (HIM2) in patients
with type 1 diabetes mellitus: results from a
phase I/II clinical trial. Metabolism 53(1),
54–58 (2004).
18 des Rieux A, Ragnarsson EG, Gullberg E,
Préat V, Schneider YJ, Artursson P.
Transport of nanoparticles across an in vitro
model of the human intestinal follicle
associated epithelium. Eur. J. Pharm. Sci.
25(4–5), 455–465 (2005).
19 Skyler JS, Krischer JP, Wolfsdorf J et al.
Effects of oral insulin in relatives of patients
with type 1 diabetes: The Diabetes
Prevention Trial – Type 1. Diabetes Care
28(5), 1068–1076 (2005).
20 Cilek A, Celebi N, Tirnaksiz F. Lecithin-
based microemulsion of a peptide for oral
administration: preparation,
characterization, and physical stability of
the formulation. Drug Deliv. 13, 19–24
(2006).
21 Sajeesh S, Sharma CP. Cyclodextrin-insulin
complex encapsulated polymethacrylic acid
based nanoparticles for oral insulin delivery.
Int. J. Pharm. 325, 147–154 (2006).
22 Sarmento B, Ferreira D, Jorgensen L, van de
Weert M. Probing insulin’s secondary
structure after entrapment into alginate–
chitosan nanoparticles. Eur. J. Pharm.
Biopharm. 65, 10–17 (2007).
23 Bayat A, Larijani B, Ahmadian S, Junginger
HE, Rafiee-Tehrani M. Preparation and
characterization of insulin nanoparticles
using chitosan and its quaternized
derivatives. Nanomed. Nanotechnol. Biol.
Med. 4, 115–120 (2008).
24 Woitiski CB, Veiga F, Ribeiro AJ, Neufeld
RJ. Design for optimization of nanoparticles
integrating biomaterials for orally dosed
insulin. Eur. J. Pharm. Biopharm. 73, 25–33
(2009).
25 Woitiski CB, Neufeld RJ, Veiga F, Carvalho
RA, Figueiredo IV. Pharmacological effect
of orally delivered insulin facilitated by
multilayered stable nanoparticles. Eur.
J. Pharm. Sci. 41, 556–563 (2010).
26 Woitiski CB, Sarmento B, Carvalho RA,
Neufeld RJ, Veiga F. Facilitated nanoscale
delivery of insulin across intestinal membrane
models. Int. J. Pharm. 412, 123–131 (2011).
27 Florence AT. Issues in oral nanoparticle drug
carrier uptake and targeting. J. Drug Target.
12(2), 65–70 (2004).
28 Löbenberg R, Amidon GL. Modern
bioavailability, bioequivalence and
biopharmaceutics classification system. New
scientific approaches to international
regulatory standards. Eur. J. Pharm.
Biopharm. 50, 3–12 (2000).
29 Bastian SE, Walton PE, Ballard FJ, Belford
DA. Transport of IGF-I across epithelial cell
monolayers. J. Endocrinol. 162, 361–369
(1999).
30 Sarmento B, Ribeiro A, Veiga F, Sampaio P,
Neufeld RJ, Ferreira D. Alginate–chitosan
nanoparticles are effective for oral insulin
delivery. Pharm. Res. 24(12), 2198–2206
(2007).
31 Agarwal V, Khan MA. Current status of the
oral delivery of insulin. Pharm. Technol. 10,
76–90 (2001).
32 Guggi D, Kast CE, Bernkop-Schnürch A.
In vivo evaluation of an oral salmon
calcitonin-delivery system based on a
thiolated chitosan carrier matrix. Pharm. Res.
20(12), 1989–1994 (2003).
33 Desai MA, Mutlu M, Vadgama P. A study of
macromolecular diffusion through native
porcine mucus. Experientia 48, 22–26 (1992).
34 Norris DA, Puri N, Sinko PJ. The effect of
physical barriers and properties on the oral
absorption of particulates. Adv. Drug Deliv.
Rev. 34(2–3), 135–154 (1998).
35 Washington N, Washington C, Wilson CG.
Physiological Pharmaceutics. Barriers to Drug
Absorption (2nd Edition). Washington N,
Washington C, Wilson CG (Eds). Taylor and
Francis, London, UK (2002).
36 Serra L, Domenech J, Peppas NA.
Engineering design and molecular dynamics
of mucoadhesive drug delivery systems as
targeting agents. Eur. J. Pharm. Biopharm.
71(3), 519–528 (2009).
37 Bhalodia R, Basu B, Garala K, Joshi B,
Mehta K. Buccoadhesive drug delivery
systems: a review. Int. J. Pharm. Biosci. 1(2),
1–32 (2010).
38 Strous GJ, Dekker J. Mucin-type
glycoproteins. Crit. Rev. Biochem. Mol. Biol.
27, 57–92 (1992).
39 Liu Z, Wang S, Hu M. Chapter 11: Oral
absorption basics: pathways, physico-
chemical and biological factors affecting
absorption. In: Developing Solid Oral Dosage
Forms: Pharmaceutical Theory and Practice.
Qiu Y, Chen Y, Liu L, Zhang GGZ (Eds).
Academic Press, London, UK (2009).
40 Salama NN, Eddington ND, Fasano A.
Tight junction modulation and its
relationship to drug delivery. Adv. Drug
Deliv. Rev. 58(1), 15–28 (2006).
41 Fix AJ. Oral controlled release technology for
peptides: status and future prospects. Pharm.
Res. 13, 1760–1764 (1996).
42 Wacher VJ, Salphati L, Benet LZ. Active
secretion and enterocytic drug metabolism
barriers to drug absorption. Adv. Drug Deliv.
Rev. 46, 89–102 (2001).
43 Jung T, Kamm W, Breitenbach A, Kaiserling
E, Xiao JX, Kissel T. Biodegradable
nanoparticles for oral delivery of peptides: is
there a role for polymers to affect mucosal
uptake? Eur. J. Pharm. Biopharm. 50(1),
147–160 (2000).
44 Yin L, Ding J, He C, Cui L, Tang C, Yin C.
Drug permeability and mucoadhesion
properties of thiolated trimethyl chitosan
nanoparticles in oral insulin delivery.
Biomaterials 30(29), 5691–5700 (2009).
45 van der Lubben IM, Verhoef JC, van Aelst
AC, Borchard G, Junginger HE. Chitosan
microparticles for oral vaccination:
preparation, characterization and
preliminary in vivo uptake studies in murine
Peyer’s patches. Biomaterials 22(7), 687–694
(2001).
46 Marks DL, Gores GJ, LaRusso NF. Hepatic
processing of peptides. In: Peptide-Based
Drug Design: Controlling Transport and
Metabolism. Taylor MD, Amidon GL. (Eds).
American Chemical Society, Washington,
DC, USA (1995).
47 Swaan PW. Recent advances in intestinal
macromolecular drug delivery via
receptor-mediated transport pathways.
Pharm. Res. 15, 826–834 (1998).
48 Schilling RJ, Mitra AK. Degradation of
insulin by trypsin and alpha-chymotrypsin.
Pharm. Res. 8(6), 721–727 (1991).
49 Zhou XH. Overcoming enzymatic and
absorption barriers to non-parenterally
administered protein and peptide drugs.
J. Control. Release 29, 239–252 (1994).
50 Calceti P, Salmaso S, Walker G,
Bernkop-Schnurch A. Development and
in vivo evaluation of an oral insulin–PEG
delivery system. Eur. J. Pharm. Sci. 22,
315–323 (2004).
51 Aoki Y, Morishita M, Asai K, Akikusa B,
Hosoda S, Takayama K. Region-dependent
role of the mucous/glycocalyx layers in
insulin permeation across rat small intestinal
membrane. Pharm. Res. 22(11), 1854–1862
(2005).
20. Review |Thompson & Ibie
Therapeutic Delivery (2011) 2(12)1630 future science group
52 Thompson CJ, Tetley L, Uchegbu IF, Cheng
WP. The complexation between novel comb
shaped amphiphilic polyallylamine and
insulin – towards oral insulin delivery. Int.
J. Pharm. 376, 46–55 (2009).
53 Thompson CJ, Tetley L, Cheng WP. The
influence of polymer architecture on the
protective effect of novel comb shaped
amphiphilic poly(allyl amine) against
in vitro enzymatic degradation of insulin –
towards oral insulin delivery. Int. J. Pharm.
383, 216–227 (2010).
54 Yamamoto A, Taniguchi T, Rikyuu K et al.
Effects of various protease inhibitors on the
intestinal absorption and degradation of
insulin in rats. Pharm. Res. 11(10),
1496–1500 (1994).
55 Narayani R, Rao KP. Hypoglycemic effect of
gelatin microspheres with entrapped insulin
and protease inhibitor in normal and
diabetic rats. Drug Deliv. 2(1), 29–38
(1995).
56 Mesiha M, Plakogiannis F, Vejosoth S.
Enhanced oral absorption of insulin from
desolvated fatty acid-sodium glycocholate
emulsions. Int. J. Pharm. 111(3), 213–216
(1994).
57 Eaimtrakarn S, Rama Prasad YV, Ohno
T et al. Absorption enhancing effect of
labrasol on the intestinal absorption of
insulin in rats. J. Drug Target. 10(3),
255–260 (2002).
58 Hodges G, Carr EA, Hazzard RA, Carr KE.
Uptake and translocation of microparticles
in small intestine. Digest. Dis. Sci. 40(5),
967–975 (1995).
59 Spangler RS. Insulin administration via
liposomes. Diabetes Care 13(9), 911–922
(1990).
60 des Rieux A, Fievez V, Garinot M, Schneider
YJ, Préat V. Nanoparticles as potential oral
delivery systems of proteins and vaccines: a
mechanistic approach. J. Control. Release
116(1), 1–27 (2006).
61 Damgé C, Michel C, Aprahamian M,
Couvreur P, Devissaguet JP. Nanocapsules as
carriers for oral peptide delivery. J. Control.
Release 13(2–3), 233–239 (1990).
62 Damgé C, Vranckx H, Balschmidt P,
Couvreur P. Poly(alkyl cyanoacrylate)
nanospheres for oral administration of
insulin. J. Pharm. Sci. 86(12), 1403–1409
(1997).
63 Dünnhaupt S, Barthelmes J, Hombach J,
Sakloetsakun D, Arkhipova V, Bernkop-
Schnürch A. Distribution of thiolated
mucoadhesive nanoparticles on intestinal
mucosa. Int. J. Pharm. 408(1–2), 191–199
(2011).
64 Hartig SM, Greene RR, DasGupta J et al.
Multifunctional nanoparticulate
polyelectrolyte complexes. Pharm. Res.
24(12), 2353–2369 (2007).
65 Sajeesh S, Bouchemal K, Sharma CP,
Vauthier C. Surface-functionalized
polymethacrylic acid based hydrogel
microparticles for oral drug delivery. Eur.
J. Pharm. Biopharm. 74, 209–218 (2010).
66 Sajeesh S, Bouchemal K, Sharma CP,
Vauthier C. Thiol-functionalized
polymethacrylic acid-based hydrogel
microparticles for oral insulin delivery. Acta
Biomaterialia 6, 3072–3080 (2010).
67 Qiu Y, Park K. Environment-sensitive
hydrogels for drug delivery. Adv. Drug Deliv.
Rev. 53(3), 321–339 (2001).
68 Ramkissoon-Ganorkar C, Liu F, Baudys M,
Kim SW. Modulating insulin-release profile
from pH/thermosensitive polymeric beads
through polymer molecular weight. J. Control.
Release 59(3), 287–298 (1999).
69 Takeuchi H, Matsui Y, Yamamoto H,
Kawashima Y. Mucoadhesive properties of
carbopol or chitosan-coated liposomes and
their effectiveness in the oral administration
of calcitonin to rats. J. Control. Release
86(2–3), 235–242 (2003).
70 Chalasani KB, Russell-Jones GJ, Jain AK,
Diwan PV, Jain SK. Effective oral delivery of
insulin in animal models using vitamin
B12
-coated dextran nanoparticles. J. Control.
Release 122(2), 141–150 (2007).
71 Wood KM, Stone G, Peppas NA. Wheat
germ agglutinin functionalized complexation
hydrogels for oral insulin delivery.
Biomacromolecules 9, 1293–1298 (2008).
72 Soppimath KS, Aminabhavi TM, Kulkarni
AR, Rudzinski WE. Biodegradable polymeric
nanoparticles as drug delivery devices.
J. Control. Release 70, 1–20 (2001).
73 Cui F, Shi K, Zhang L, Tao A, Kawashima Y.
Biodegradable nanoparticles loaded with
insulin–phospholipid complex for oral
delivery: preparation, in vitro characterization
and in vivo evaluation. J. Control. Release 114,
242–250 (2006).
74 Fan YF, Wang YN, Fan YG, Ma JB.
Preparation of insulin nanoparticles and their
encapsulation with biodegradable
polyelectrolytes via the layer-by-layer
adsorption. Int. J. Pharm. 324, 158–167
(2006).
75 Simon M, Behrens I, Dailey LA, Wittmar M,
Kissel T. Nanosized insulin-complexes based
on biodegradable amine-modified graft
polyesters poly[(vinyl-3-(diethylamino)-
propylcarbamate-co-(vinyl acetate)-co-(vinyl
alcohol)]–graft–poly(l-lactic acid): protection
against enzymatic degradation, interaction
with Caco-2 cell monolayers, peptide
transport and cytotoxicity. Eur. J. Pharm.
Biopharm. 66(2), 165–172 (2007).
76 Thompson CJ, Ding C, Qu X et al. The effect
of polymer architecture on the nano
self-assemblies based on novel comb-shaped
amphiphilic poly(allylamine). Colloid Polym.
Sci. 286, 1511–1526 (2008).
77 Cheng WP, Thompson CJ, Ryan SM, Aguirre
T, Tetley L, Brayden DJ. In vitro and in vivo
characterisation of a novel peptide delivery
system: amphiphilic polyelectrolyte–salmon
calcitonin nanocomplexes. J. Control. Release
147, 289–297 (2010).
78 Thompson CJ, Cheng WP. Chemically
modified polyelectrolytes for intestinal
protein and peptide delivery. In: Peptide and
Protein Delivery. Chris van der Walle (Ed.).
Academic Press/Elsevier, San Diego, CA,
USA (2011).
nn Comprehensive perspective on the use of
modified forms of chitosan and amphiphilic
polymers in oral protein delivery.
79 Thompson CJ, Cheng WP, Gadad P et al.
Uptake and transport of novel amphiphilic
polyelectrolyte-insulin nanocomplexes by
Caco-2 cells – towards oral insulin. Pharm.
Res. 28, 886–896 (2011).
80 Gowthamarajan K, Kulkarni GT. Oral
insulin – fact or fiction? Resonance 8(5),
38–46 (2003).
81 van de Weert M, Hennink WE, Jiskoot W.
Protein instability in poly(lactic-co-glycolic
acid) microparticles. Pharm. Res. 17,
1159–1167 (2000).
82 Mao S, Bakowsky U, Jintapattankit A, Kissel
T. Self-assembled polyelectrolyte
nanocomplexes between chitosan derivatives
and insulin. J. Pharm. Sci. 95, 1035–1048
(2006).
83 Sarmento B, Ribeiro A, Veiga F, Ferreira D,
Neufeld RJ. Insulin-loaded nanoparticles are
prepared by alginate ionotropic pre-gelation
followed by chitosan polyelectrolyte
complexation. J. Nanosci. Nanotechnol. 7,
283–2841 (2007).
84 Shu S, Zhang X, Teng D, Wang Z, Li C.
Polyelectrolyte nanoparticles based on
water-soluble chitosan-poly(l-aspartic
acid)-polyethylene glycol for controlled
protein release. Carbohydr. Res. 344,
1197–1204 (2009).
85 Desai MP, Labhasetwar V, Walter E, Levy RJ,
Amidon GL. The mechanism of uptake of
biodegradable microparticles in Caco-2 cells
is size dependent. Pharm. Res. 14(11),
1568–1573 (1997).
86 Bayat A, Dorkoosh FA, Dehpour AR et al.
Nanoparticles of quaternized chitosan
21. The oral delivery of proteins using interpolymer polyelectrolyte complexes | Review
www.future-science.com 1631future science group
derivatives as a carrier for colon delivery of
insulin: ex vivo and in vivo studies. Int.
J. Pharm. 356(1–2), 259–266 (2008).
87 Sarmento B, Ferreira D, Veiga F, Ribeiro A.
Characterization of insulin-loaded alginate
nanoparticles produced by ionotropic
pre-gelation through DSC and FTIR studies.
Carbohydr. Polym. 66, 1–7 (2006).
88 Sajeesh S, Sharma CP. Novel pH responsive
polymethacrylic acid-chitosan-polyethylene
glycol nanoparticles for oral peptide delivery.
J. Biomed. Mater. Res. B Appl. Biomater. 76B,
298–305 (2006).
89 Sun W, Mao S, Mei D, Kissel T. Self-
assembled polyelectrolyte nanocomplexes
between chitosan derivatives and enoxaparin.
Eur. J. Pharm. Biopharm. 69, 471–425
(2008).
90 Shu S, Wang X, Zhang X, Zhang X, Wang Z,
Li C. Disulfide cross-linked biodegradable
polyelectrolyte nanoparticles for the oral
delivery of protein drugs. New J. Chem. 33,
1882–1887 (2009).
91 Mao S, Shuai X, Unger F, Wittmar M, Xie X,
Kissel T. Synthesis, characterization and
cytotoxicity of poly(ethylene glycol)-graft-
trimethyl chitosan block copolymers.
Biomaterials 26, 6343–6356 (2005).
92 Mao S, Germershaus O, Fischer D, Linn T,
Schnepf R, Kissel T. Uptake and transport of
PEG-grsft-trimethyl-chitosan copolymer–
insulin nanocomplexes by epithelial cells.
Pharm. Res. 22, 2058–2068 (2005).
93 Chen F, Zhang Z, Yuan F, Qin X, Wang M,
Huang Y. In vitro and in vivo study of
N-trimethyl chitosan nanoparticles for oral
protein delivery. Int. J. Pharm. 349, 226–233
(2008).
94 Bernkop-Schnürch A. Thiomers: a new
generation of mucoadhesive polymers. Adv.
Drug Deliv. Rev. 57, 1569–1582 (2005).
95 Albrecht K, Bernkop-Schnürch A. Thiomers:
forms, functions and applications to
nanomedicine. Nanomedicine 2(1), 41–50
(2007).
96 Föger F, Schmitz T, Bernkop-Schnürch A.
In vivo evaluation of an oral delivery system
for P-gp substrates based on thiolated
chitosan. Biomaterials 27(23), 4250–4255
(2006).
97 Sakloetsakun D, Iqbal J, Millotti G, Vetter A,
Bernkop-Schnürch A. Thiolated chitosans:
influence of various sulfhydryl ligands on
permeation-enhancing and P-gp inhibitory
properties. Drug Dev. Ind. Pharm. 37(6),
648–655 (2011).
98 Hu Y, Jiang X, Ding Y, Ge H, Yuan Y, Yang
C. Synthesis and characterization of
chitosan-poly(acrylic acid) nanoparticles.
Biomaterials 23, 3193–3201 (2002).
99 El-Sherbiny IM. Synthesis, characterization
and metal uptake capacity of a new
carboxymethyl chitosan derivative. Eur.
Polym. J. 45, 199–210 (2009).
100 El-Sherbiny IM. Enhanced pH-responsive
carrier system based on alginate and
chemically modified carboxymethyl chitosan
for oral delivery of protein drugs: preparation
and in vitro assessment. Carbohydr. Polym. 80,
1125–1136 (2010).
101 El-Sherbiny IM, Abdel-Bary EM, Harding
DRK. Preparation and in vitro evaluation of
new pH-sensitive hydrogel beads for oral
delivery of protein drugs. J. Appl. Polym. Sci.
115(5), 2828–2837 (2010).
102 El-Sherbiny IM, Elmahdy MM. Preparation,
characterization, structure and dynamics of
carboxymethyl chitosan grafted with acrylic
acid sodium salt. J. Appl. Polym. Sci. 118(4),
2134–2145 (2010).
103 Perera G, Greindl M, Palmberger T,
Bernkop-Schnürch A. Insulin-loaded
poly(acrylic acid)-cysteine nanoparticles:
stability studies towards digestive enzymes in
the intestine. Drug Deliv. 16, 254–260
(2009).
104 Thurow H, Geisen K. Stabilization of
dissolved proteins against denaturation at
hydrophobie interfaces. Diabetologia 27(2),
212–218 (1984).
105 Rahman NA, Mathiowitz E. Localization of
bovine serum albumin in double-walled
microspheres. J. Control. Release 94(1),
163–175 (2004).
106 Lowman AM, Cowans BA, Peppas NA.
Investigation of interpolymer complexation in
swollen polyelectolyte networks using
solid-state NMR spectroscopy. J. Polym. Sci. B
Polym. Phys. 38(21), 2823–2831 (2000).
107 Lankalapalli S, Kolapalli VRM.
Polyelectrolyte complexes: a review of their
applicability in drug delivery technology.
Indian J. Pharm. Sci. 71(5), 481–487 (2009).
108 Woitiski CB, Neufeld RJ, Ribeiro AJ, Veiga F.
Colloidal carrier integrating biomaterials for
oral insulin delivery: influence of component
formulation on physicochemical and
biological parameters. Acta Biomater. 5,
2475–2484 (2009).
109 Jintapattanakit A, Junyaprasert VB, Mao S,
Sitterberg J, Bakowsky U, Kissel T. Peroral
delivery of insulin using chitosan derivatives:
a comparative study of polyelectrolyte
nanocomplexes and nanoparticles. Int.
J. Pharm. 342, 240–249 (2007).
110 Dupuy B, Arien A, Minnot AP. FT-IR of
membranes made with alginate/polylysine
complexes. Variations with the mannuronic or
guluronic content of the polysaccharides.
Artif. Cells Blood Substit. Immobil. Biotechnol.
22(1), 71–82 (1994).
111 Foss AC, Peppas NA. Investigation of the
cytotoxicity and insulin transport of
acrylic-based copolymer protein delivery
systems in contact with Caco-2 cultures. Eur.
J. Pharm. Biopharm. 57, 447–455 (2004).
112 López JE, Peppas NA. Effect of poly (ethylene
glycol) molecular weight and microparticle
size on oral insulin delivery from P(MAA-g-
EG) microparticles. Drug Dev. Ind. Pharm.
30(5), 497–504 (2004).
113 Besheer A, Wood KM, Peppas NA, Mäder K.
Loading and mobility of spin-labeled insulin
in physiologically responsive complexation
hydrogels intended for oral administration.
J. Control. Release 111, 73–80 (2006).
114 Wood KM, Stone G, Peppas NA. The effect
of complexation hydrogels on insulin
transport in intestinal epithelial cell models.
Acta Biomaterialia 6, 48–56 (2010).
115 American Diabetes Association. Standards of
medical care in diabetes – 2009. Current
criteria for the diagnosis of diabetes. Diabetes
Care 32(Suppl. 1), S6–S12 (2009).
116 Sugano K, Kansy M, Artursson P et al.
Coexistence of passive and carrier-mediated
processes in drug transport. Nat. Rev. Drug
Discov. 9, 597–614 (2010).
117 Bae KH, Moon CW, Lee Y, Park TG.
Intracellular delivery of heparin complexed
with chitosan-g-poly(ethylene glycol) for
inducing apoptosis. Pharm. Res. 26(1),
93–100 (2009).