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BIOMAC 4576 112

International Journal of Biological Macromolecules xxx (2014) xxxxxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Chitosan as a suitable nanocarrier material for anti-Alzheimer drug

delivery

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Jayrajsinh Sarvaiya , Y.K. Agrawal

Institute of Research and Development, Gujarat Forensic Sciences University, Gandhinagar, India

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a r t i c l e

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a b s t r a c t

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Article history:
Received 1 July 2014
Received in revised form 24 August 2014
Accepted 28 August 2014
Available online xxx

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Keywords:
Chitosan
Alzheimers disease
Brain targeting
Anti-Alzheimer drugs

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Contents

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Chitosan, a biocompatible natural polysaccharide is frequently reported carrier material in targeted drug
delivery to treat neurodegenerative disorders. Chitosan and its biodegradable products exert its bioactivities on nerve cells and blood brain barrier at the molecular level, which are benecial in anti-Alzheimer
therapy. Flexibility of surface modication, the ability to get attached with varieties of ligand molecules
and the formation of the stable nano complex in physiological condition make chitosan an adorable material for delivery of anti-Alzheimer drugs and siRNA to the brain. The success rate of nose to brain delivery
of anti-Alzheimer drugs enhances when chitosan used as a carrier material. This review covers direct and
indirect anti-Alzheimer effects of chitosan, surface modication strategies to augment permeation from
the bloodbrain barrier structure, different ligands reported for brain targeting of chitosan nanoparticles
containing anti Alzheimer drugs, blood compatibility and widely utilized chitosan nanoparticle fabrication techniques. Key intellectual claims are also condensed through patents to appraise chitosan as an
attractive polymer for brain targeted nanoformulation which is currently facing oversight by regulatory
agencies and manufacturers.
2014 Elsevier B.V. All rights reserved.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chitosan as a carrier material in anti-Alzheimer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Properties of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Biodegradability of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Chitosan modications for brain targeting and blood compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.
Chitosan modication for brain targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.
Chitosan modication for blood compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
synergistic roles of chitosan in anti-Alzheimer therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
SiRNA targeting to brain by chitosan nanocarrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fabrication options for brain targeted chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Ionic gelation/crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Micellization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Spinning disk processing technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Emulsication method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Surface modications with ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Brain targeting with chitosan nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Feasibility of non-invasive routes of administration by chitosan nanoparticle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author at: Institute of Research and Development, Gujarat Forensic Sciences University, DFS Campus, Sector 18A, Gandhinagar, Gujarat 382018, India.
Tel.: +91 9638344834/45.
E-mail addresses: jis 24484@yahoo.com, jayrajsinh.sarvaiya@gmail.com (J. Sarvaiya).
http://dx.doi.org/10.1016/j.ijbiomac.2014.08.052
0141-8130/ 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: J. Sarvaiya, Y.K. Agrawal, Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.08.052

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1. Introduction
Applications of polymeric nanocarrier have astonished in recent
years because of its prominent role in therapeutic interventions in
brain disorders. A successful therapy in neurodegenerative disease
requires central nervous system (CNS) rejuvenation or protection
though drug delivery. Alzheimers disease (AD) is the most fatal
neurodegenerative disorder with the modest progress in therapeutic inventions. Especially, 0.7 million aged Americans are
anticipated to die because of AD in year 2014, whereas people
suffering of this deadly disease is going to reach at 13.8 million
mark in the USA alone by the year 2050 [1]. The recent clinical
failures of several promising drug candidates in AD are spurring
a greater sense of urgency to investigate new targets, new drug
carriers and their interconnectivity. The prime reason behind therapeutic failure provocation of most of the agents in AD treatment
is blood brain barrier (BBB) wherein thin blood vessels are covered
by astrocyte foot processes in brain [24]. Vascular endothelium in
the brain is formed by comparatively tighter intercellular junctions
compared to other parts of the body. This structural uniqueness
makes BBB an impermeable hurdle for foreign particulate including
a drug substance and hinders its passage to amyloid rich brain part
[5,6]. BBB essentially inhibits the normal passage of objects more
than 12 nm in diameter and cellular endocytosis of objects with
more than 30 nm. Moreover, the extracellular space viscosity may
impede propagation of the object in a human brain. To overcome
these obstacles, nanocarrier drug delivery systems like liposomes,
polymeric nanoparticles and solid lipid nanoparticles are being
investigated vastly with prolic end results [710]. Among them,
chitosan owns certain characteristics and bioactivities that provide
it a slight edge over other nanocarrier materials.
Chitosan is a cationic polysaccharide which is extensively studied in brain scaffold preparations and spinal cord implantable
systems. Moreover, it has displayed multiple bioactivities in various investigations carried out in the last decade [1114]. It is
also widely employed in nanoparticle preparation for transportation of loaded drugs to targeted organs in case of cancer, diabetes,
lung diseases and CNS disorders [15]. Chitosan and its degradation products, at the molecular level, exhibits anti-Alzheimer
mechanisms majorly by prevention of phosphorylation of c-Jun
N-terminal kinase caused by -amyloid [16], inhibition of proinammatory cytokines and blockage of nitric oxide sythase [17].
Moreover, biodegradability, biocompatibility, exibility in surface
modication and ease of multiple preparation methods are added
advantages of chitosan, which makes it an attractive nanoshells
forming material to achieve successful delivery of drugs and nucleic
acids to brain interstices. The review is aimed to scrutinize reports
of the application of chitosan and its derivatives in nano drug delivery system targeting Alzheimers disease. Furthermore, different
biomarkers and pathological conditions of AD are also correlated
with bioactivities of chitosan in the present work.

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2. Chitosan as a carrier material in anti-Alzheimer therapy

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2.1. Properties of chitosan

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Chitosan is a cationic heteropolymer obtained from chitin,

a natural polysaccharide present in the exoskeleton of shrimp,
crustaceous, fungi and yeasts [18]. Structurally, Chitosan is a
linear chain of randomly present N-acetyl-d-glucosamine and dglucosamine units which are attached by beta (1,4)-glycosidic
bonds. Chitosan consists of an amino group at the C2 carbon after
deacetylation of chitin structure. When the degree of deacetylation and portion of nitrogen is more than 60% and 7% respectively,
chitin is called chitosan. DD (degree of deacetylation) ranges from

66% to 95% in marketed chitosan. Solubility of chitosan in acidic

water and interaction with negative charged substances are due
to protonation of amino group in water. Such distinctive nature of
chitosan among all polysaccharides enables it to form water soluble salts like HCl salt and carboxylate salt. These groups further
enhance the ability of chitosan to get soluble even in slightly acidic
condition.
Physical properties and associated chemical behaviors of chitosan are strongly governed by its molecular weight and degree of
deacetylation [19,20]. High molecular weight chitosan connes its
usage due to high viscosity and very low solubility in neutral aqueous solution whereas similar properties of low molecular weight
chitosan drastically changes to widen its applications in preparation of nanoparticles for drug targeting. Encapsulation efciency of
chitosan carrier increases as molecular weight decreases according to Yang et al. [21]. They reported high entrapment efciency
of low sized (less than 70 nm mean diameter) NPs prepared from
low molecular weight chitosan (55 kDa) in their work. Molecular weight reduction of high molecular weight chitosan can be
accomplished by chemical depolymerization through the use of
NaNO2 , hot dilute sulfuric acid [22] and strong acids specically
hydrochloric acid, phosphoric acid and sulfuric acid. Chitosan and
its derivatives exhibit variant properties, degradation behavior and
bioactivities because of the existence of conformational structural
disparity. Depending on the type of solvent, chitosan molecules
take up four different types of helical conformations which determine its bioactivities in neuroprotection [23].
2.2. Biodegradability of chitosan
Chitosan is transformed to oligomer before further conversion
into the N glucosamine unit in vivo. This degradation is catalyzed
by lysozymes [24], lipases, proteases, chitinase, chitin deacetylase, beta-N-acetylhexosaminidase [25,26], collagenase [27] and
chitosanase enzymes [28]. Lysozyme and chitisonase enzymes are
two major sources of chitosan biofate which are present in many
tissues of human body including brain. Chitinase is also present in
the brain with the noteworthy elevated level in CSF (cerebro-spinal
uid) during AD [29]. The level of chitinase during Alzheimers
disease is equivalent to its biomarker status consideration in context of AD diagnosis with 85.5% accuracy, observed in a clinical
trial in contrast to 78.4% and 77.6% accuracy observed in case of
-amyloid and tau respectively during the clinical study [30].
Chitosan biodegradation was observed to be faster when degree
of deacetylation (DD) is less than 70% and chitosan cytotoxicity is
lesser when molecular weight is low [31,32]. This emphasized on
DD as a degradation rate governing factor for chitosan nanoparticles. Other similar reports afrmed higher rate of degradation of
low molecular weight (Mw) chitosan compared to high molecular
weight chitosan [3335]. Crosslinking agent utilized in preparation of chitosan nanoparticles also inuences its degradation
rate depending on crosslinking mechanism. Ionic gelation cross
linked chitosan by use of TPP (Tri poly phosphate) degrades slower
than chemically cross linked chitosan nanoparticles by use of glutaraldehyde [36]. The effect of cross linkers is more pronounced
on high and medium Mw chitosan in comparison of low Mw
chitosan. Availability of amine group also inuences susceptibility of chitosan for enzymatic degradation by microbial enzymes
presenting less digestion of N-stearoylchitosan in gastrointestinal
tract compared to raw chitosan [37]. Biodistribution of chitosan
nanoparticle after I.V. (Intravenous) injection depends on surface
characteristics and size. Chitosan NPs with complete neutralization
of positive charge and more than 200 nm diameters were observed
to be taken up by the spleen and liver RES (Reticuloendothelial
system) during an investigation by Zhang et al. [38]. They concluded that hydrophobic modication of chitosan by incorporating

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N-octyl-O-sulphate moiety showed maximum accumulation in

kidney, liver and spleen followed by minimum brain uptake. In
one of the empirical outcome, Costa-Pinto et al. [39] reported
vascularization promotional activity of biodegraded product of chitosan when lysozyme mediated degradation was prominent. This
showed evidence of bioactivity exerted by biodegradation product
of chitosan.

nanoparticles, which can eventually help the nanoparticle to reach

at BBB in proportionally higher amount. Kulkarni et al. [49] noted
that nanoparticles with less than 100 nm size can cross BBB more
effectively than higher sized particles when chitosan surface is
modied by polyethylene glycol. Hence, numbers of chitosan structural modication approaches are available which can enhance BBB
permeation effectively.

2.3. Chitosan modications for brain targeting and blood

compatibility

2.3.2. Chitosan modication for blood compatibility

In AD, blood composition is altered, which includes damaged
red blood cell membrane structure and elevated level of oxidized
low density lipoprotein [50]. It is not yet studied whether nanoparticle composition exposed to blood of an AD affected person
can reveal informal hemo-behavior or not, but hemocompatibility studies of modied chitosan nanoparticles in healthy serum
has provided decent results and made it an acceptable nanocarrier material. Chemical modication of surface characteristics of
chitosan nanocarrier is almost a prerequisite for targeted delivery to the amyloid rich structure of the brain. This modication
also facilitates availability of nanoparticles in systemic circulation
for longer duration bypassing opsonization. As per USFDA and EU,
nanomedicine for blood infusion or injection required to study
extensively for its hemocompatibility. Though majority of the marketed nanoformulation causes an infusion reaction up to a certain
extent, modied chitosan nanoparticles hold the expectation to
act as a highly blood compatible nanocarrier. Brain targeting of
chitosan derived nanoparticles when administered or reached in
systemic circulation before reaching at AD affected brain parts, its
hemo incompatibility can be evaluated through thrombosis, coagulation, platelet activation, blood cell changes and complementary
activation, macrophage uptake of nanoparticles and rheological
alteration of blood [51]. Even after reaching to cerebral area,
nanoparticles gets exposed to blood plasma and blood component
in narrow brain capillary network formed by 100 billion capillaries
with a total of 650 km capillary lengths. The necessity of chitosan
hemocompatibility improvement was rst introduced by Malette
et al. [52] through his reports of red blood cell rouleaux formation
caused by reaction between the negatively charged red cell membrane and the cationic chitosan solution mediated crosslinkage or
re-polymerization. This phenomenon is observed to be increased
with decrease in molecular weight of chitosan due to more and
more protonated amine group becomes available in depolymerized chitosan [53]. In a similar work, Bentholon et al. [54] reported
suppression of complement activation by high molecular weight
chitosan compared to short chain chitosan. Empirical review of
the strategies for improvement of hemocompatibility of chitosan
was made by Balan et al. [55], who emphasized on chemical
modication of polymer and the association of chitosan with hemocompatibility enhancing capping agents. Xu et al. [56] described the
importance of the brain targeted succinic anhydride-conjugated
chitosan nanoparticle that can fulll dual objectives of impediment of cytokine production and RBC aggregation. Biocompatibility
and blood compatibility of N-octyl-O-glycol chitosan was established against FDA approved product-Taxol and it has proved that
amphiphilic modication is the ultimate advancement in deriving
chemically modied chitosan with blood compatible nature [57].
Chitosan derivatives with signicant improvement in blood compatibility, are represented in Fig. 2. Among which, alkylglyceryl
chitosan, N-trimethyl chitosan and PEGyalted chitosan have proved
its brain targeting efciency by crossing BBB.

Blood compatibility, blood stability and BBB epithelial surface adhesion are prerequisites for successful brain targeting by
nanocarrier material to treat neurodegenerative disorders.
2.3.1. Chitosan modication for brain targeting
Feasibility of surface modication of chitosan structure allows
delivery of brain targeted nanoparticles with desired features and
versatile characteristics. Moreover, modied chitosan is equally
efcient to cap surface of other nanocarrier materials like PLGA
(poly (lactic-co-glycolic acid)) and PLA (poly lactic acid) with a view
to enhance BBB permeation, cell compatibility and serum stability
[40,41]. The hydrophobic portion of chitosan derivative assists in
release of anionic ions which are attached with chitosan by polyplex formation. At the same time, genetic transfection modulation
and cellular adsorption on surface of neuronal cells are facilitated
by hydrophobic moieties of chitosan derivatives. Cationic chitosan
surface may get attached to albumin and results in aggregation of
blood cells after I.V. administration, but hydrophilic modication
of chitosan enhances blood stability of chitosan polyplex by prevention of albumin and chitosan complication. Here, the existence
of segregated nanoparticles allows enhanced permeation through
the blood brain barrier and neuronal cells.
Among all the reported modied chitosan derivatives (Fig. 1),
TMC(N-Trimethyl chitosan) is a vastly studied form for brain targeting with anti-Alzheimer and other neuroprotective drugs because
positive charged TMC and anionic sialic acid residues of glycoprotein present on blood brain barrier undergoes electrostatic
interactions. TMC is reported for its usefulness in surface modication of PLGA nanoparticles for brain targeting. TMC was covalently
coupled to the surface of PLGA nanoparticles (PLGANP) via a
carbodiimide-mediated linkage to act as surface modier for PLGA
nanospheres. The senile plaque and biochemical tests conrmed
the brain-targeted effects of TMC/PLGANP for delivery of Coenzyme Q10 and 6-coumarin [41] in the treatment of AD associated
neurodegenration. Furthermore, TMC nanoparticles prepared by
the ionic gelatin method can easily entrap FITC (uorescein isothiocyanate) which can be used for labeling purposes while the
study of brain targeted drug delivery in neurological disorders
[42], especially to demonstrate absorptive mediated transcytosis
as the transport mechanism. TMC also makes polyionic complex
with polyethylene glycol (PEG) and such nanoparticles possess
enhanced blood stability. These nanoparticles have also exhibited
its efciency to act as a carrier of SiRNA (small interfering RNA)
delivery to neuronal cells because of enhanced stability of TMC
polyplex under charge neutralizing biological environment [43,44].
In addition, Zwitter ionic chitosan derivatives, PEGylated chitosan and tween 80 coated chitosan [45] are also popular forms
of nanocarrier materials to persuade BBB permeation of neuroprotective agents. In a contrary result, chitosan nanoparticle
with unmodied surface delivered the drug across BBB found to
be equal to the amount of drug delivered by Tween 80 coated
chitosan nanoparticles [4648]. Though, the non ionic polysorbate surfactant in low concentration (0.5% w/V) can be used
to stabilize the chitosan nanoparticle formulation. This surfactant molecule contributes in the prevention of aggregation of

2.4. synergistic roles of chitosan in anti-Alzheimer therapy

AD is characterized by an extracellular deposition of senile
plaque formed by amyloid-beta peptide (A) in the brain. This
is a minute level steady state (2227 ng/day and 710 mg/year),

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Fig. 1. Various options of chitosan modication for preparation of brain targeted nanoparticles. Abbreviations: HA: Hyaluronic acid; LS: Lecithine; G: Carragenam polymer.

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deposition with the invisible symptomatic response initially [58].

Oligomer aggregation on neuronal end and on receptor surface is
causing brain inammation, synaptic dystrophy [58,59]. In addition, hyperphosphorylated tau protein associations are considered
to form neurobrillary tangles inside the nerve cell bodies eventually [60]. In continuation of this, the microtubules split up and
damage neuronal messenger system which later transforms in
the death of the nerve cells leading to dementia. Though molecular level mechanism contributing to neuronal damage and loss of
synaptic signaling is more or less inuenced by amyloid plaque and
Tau tangle formation in the brain, many other factors like vascular
damage, altered level of Neprilysin, Insulin, BACE 1 (beta-site APP
cleaving enzyme 1), RAGE (Receptor for Advanced Glycation Endproducts), neurosteroids, TNF (Tumor necrosis factor), metabolic
complications also contribute in the fate of the AD affected brain.
Moreover, lysosomal failure, inammation, hypoxia, less glucose
uptake by affected part, alteration in signaling proteins, circuitry
hyper-excitability and alterations in glutamate receptors [61,62]
are also pathological conditions to be countered. The disease
becomes more fatal because of low efciency of neural cells for
regeneration and restoration of neural elasticity in comparison of
cells of other organs due to regeneration constraining ability of
Nogo-A and NgR1 receptor proteins [63,64].
Chitosan and its biodegradation products resists progression of
various pathological conditions of brain in AD by direct or indirect mechanism of actions apart from its ability to act as a cationic
surface, which get attached with anionic epithelium of BBB electrostatically. Furthermore, brain targeting of drug by using chitosan is
appealing due to its ability to recognize and adhere to neural tissues selectively [65]. By such preferentially targeting of damaged
neuron, chitosan facilitates restoration of normal neural activities
and physiology which otherwise altered under inuence of AD and
compromised BBB (Fig. 3).
Few studies alluded dose dependent neuroprotective effects
[66] of chitosan oligosaccharides, a biodegradation product of

chitosan by demonstrating its ability to restrict microglia inammation and oxidative damage to nerve cells [6771]. In this context,
the DD and the degree of polymerization of chitosan oligosaccharide required to be >95% and 26 respectively, whereas the
concentration of the polymer as a therapeutic agent is required to
be 10400 g/ml in the AD affected area. Restriction in the activities
of ROS (reactive oxygen species), NO (nitric oxide), NF-kB (nuclear
factor-kappa B), AP-1 (activator protein-1), MAPK (mitogen activated protein kinase) are among the direct measures contrived
when the optimum concentration of chitosan is present in biouid.
Other anti-inammatory response of chitosan is mediated through
BACE inhibition activity which restricts pro inammatory actions
of RAGE in Alzheimers disease [66,72]. RAGE receptors are over
expressed in epithelial cells of AD affected blood vessels and
primarily responsible for the inux of plasma amyloid peptide
to brain through the BBB. Rat et al. [73] showed multi target
potent neuroprotective polypeptide like PACAP (pituitary adenylate cyclase-activating polypeptide) and glial derived neurotropic
factor can be delivered to the brain by complexing with chitosan.
Both the polypeptides are acting as ligands for RAGE (transporter
receptor for advanced glycation end products) and receptor of glial
cell line-derived neurotrophic factor respectively [74].
The brain targeting mechanisms of chitosan nanoparticles are
not only limited to its molecular level interaction on the cerebrovascular endothelial cell surface, but extended to close junction
part between endothelial cells also. Chitosan is reported to enhance
paracellular permeability of molecules by tempting redistribution
of tight junction protein ZO-1 (Zonula Occudens) [75] which is a
major component protein in tight junction in BBB. This bioactivity
of chitosan resembles molecular mechanism of nicotine as reported
by Brian et al. [76], they have depicted increase in in vivo BBB permeation by nicotine is mediated by redistribution of tight junction
protein. Though the expression of ZO-1 is suppressed in AD affected
BBB [77], still interaction of chitosan with this protein encourage brain targeting without surface modication of nanoparticles.

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Fig. 2. Types of chitosan derivatives with proved hemocompatibility in contrast to unmodied chitosan.

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Alzheimers disease progression enhances BBB disruption correlated with faulty clearance from brain and across the BBB primarily
determines amyloid- retention in the brain, causing the formation of neurotoxic amyloid oligomer strand the promotion of
brain and cerebrovascular amyloidosis. Enhanced level of ROS and
inammation in AD affected brain is promoted by the accumulation of amyloid-beta in cerebral arteries leads to micro hemorrhage
and neurovascular alterations in AD. This vascular damage can be
countered by use of chitosan as a carrier material because chitosan is reported to promote wound healing and neovascularization
[78]. In contrast, few investigations have also proved inhibition
of angiogenesis activity of chitooligosaccharide, but such studies
were carried out to see the effect of chitosan in angiogenesis of
cancer.
One of the major pathological mechanisms in AD is exhibited
by the dysfunctional homeostasis of metals by the blood brain
barrier. Leaky blood vessels contribute in abnormal accumulation
of iron, copper and zinc in AD affected brain with progression
of the disease [79,80]. Chitosan conjugated chelating agents have
been evaluated in other than anti-Alzheimer purpose with promising outcomes [81,82]. These studies endorse chitosan as a carrier
material for metal chelating therapy in Alzheimers disease. Consequently, the nanoparticle-chelator system possess ability to
facilitate not only metal binding of chelating agent in affected brain
part, but also elimination of metal complexed chelator from the
brain parenchyma. This is possible if apolipoprotein E is used as
ligand on chitosan nanoparticle with a view of receptor mediated
endocytosis.

Along with the aforementioned dual application of chitosan as a

bioactive, biodegradable supramolecule and material for nanocarrier in brain deliver, use of chitosan in Alzheimers disease is also
encouraged by reports of successful drug targeting to brain due to
its ability to deliver siRNA (small interfering RNA), proteins, peptides, biologically derived drugs and compatibility with numbers
of ligands.
2.5. SiRNA targeting to brain by chitosan nanocarrier
SiRNA (small interfering RNA) strand with 2025 base pair
length and with negative charge is mainly used as a therapeutic
agent when over activity of enzymes and certain proteins are the
root causes of the disease. Epigenetic modications and michondrial metabolism alterations are encompassed among key reasons
in many neurodegenerative diseases, including of Alzheimers disease in aged person. Several lines of evidence have shown that
the therapeutic activity of siRNA has a potential for the treatment
of neurodegenerative diseases by selectively suppression of disease linked genes [83,84]. More specically, BACE 1, APP (Amyloid
precursor protein), PS1 (presenilin 1) and PS2 genes over expressions are predominant in AD due to genetic mutation caused by
methylation of DNA and alteration of miRNA [85].
SiRNA therapy in Alzheimers disease essentially requires suitable nanocarrier which can protect siRNA during its passage from
blood vessels and successfully deliver a genetic load in targeted
neuronal cells [86]. Positively charged chitosan fullls the prerequisites of siRNA containing nanoparticles by its ability to cross

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Fig. 3. Molecular Targets of chitosan and its biodegradation units in pathological conditions of AD: (1) Leptin serum level enhanced by chitosan and cascade events inhibits
CNS inammation. (2) Insulin level enhanced by chitosan. (3) Tight junction protein redistribution achieved by chitosan facilitates access of drug loaded nanocarrier to
nerve tissues. (4) Chitosan makes complex with metal ions and protects neurons from source of free radicals for nerve damage and amyloid beta plaque formation. (5) BACE
inhibition by chitosan prevent peptide production. (6) EPCP (Endothelial protein C receptor) down regulation by chitosan prevent inammatory response of nerves and
nerve apoptosis. (7) Anticoagulant effect of chitosan prevent blood clots in amyloid saturated blood in brain hemisphere.

Fig. 4. Covalent binding between chitosan and siRNA: in chitosan nanocomplex formation for brain transfection. N/P ratio in nanocomplex determines siRNA stability and
release during drug targeting.

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the blood brain barrier and effective gene transfection in neuronal

cells. Positively charged chitosan forms nanosized polyplex by ionic
interaction with a negatively charged siRNA strand under acidic
condition as depicted in Fig. 4. Under alkaline and neutral physiological conditions, spontaneous polyplex is retained by hydrogen
bonding and hydrophobic interactions as evident by gel electrophoresis [87]. ChitosansiRNA nanocomplex makes hindrance
against serum nucleases degradation of siRNA. Studies suggest that
siRNA remains fully protected up to 7 h in 50% serum during ex
vivo study. Complete degradation of siRNA was noted only after
48 h of the incubation time with serum in contrast to 30 min life
of free siRNA in 5% serum containing medium [88]. Formation of
corona around chitosan nanoparticles during blood capillary transportation also makes barrier a between entrapped gene interfering
material and its degrading enzymes present in the blood.
Alzheimers disease requires chronic treatment and slow degradation of chitosan is an added advantage in delivering the genetic
content to target cells for longer duration because the majority of gene silencing effects are observed for a short span of
time by other rapid degrading nanocarrier materials. Moreover,
SiRNApolyethylenimine complex suffers from issues of genetic
material stability in nanocomplex and readily dissociation at the
cellular site of action [89]. When fully deacetylated chitosan with
molecular weight from 34 kDa to 140 kDa was used to deliver
siRNA to various cell lines during in vitro experiments, about
90% gene silencing efciency was resulted. SiRNA transfection to
BBB by chitosan nanocarrier have shown hampered p-glycoprotein
expression when siRNA entrapped nanoparticles were exposed to
the immortalized rat endothelial cell line RBE4 [90]. The results
of these in vitro study opens up avenues for in vivo evaluation
with a view to enhanced drug permeation through BBB by altering
gp-glycoprotein efux system, especially from the brainstem and
the superior temporal cortex sites of the brain where P-gp receptors are over expressed in Alzheimers disease [91]. Level of TNFR I
(Tumor necrosis factor receptor I) is elevated in all neurodegenerative diseases with active participation in neuronal cell apoptosis. At
the same time expression of TNFR II (Tumor necrosis factor receptor II) [92], which is a neutopreotective domain, decreased in AD
disease. Kim et al. [93] reported signicant reduction in neuronal
damage by suppressing blood and brain TNF-alpha production by
use of anti-TNF-alpha-siRNA IV injection. RVG peptide utilized for
brain targeting of systemic administered siRNA have shown to protect siRNA in blood serum but no enhancement of bioavailability
in brain in comparison of unmodied siRNA. This circumstance
can be improved by the use of chitosan as nanocarrier for transfection of same genes. In vivo brain imaging revealed the ability
of trimethyl chitosan nanoparticles tagged with RVG peptide to
enhance bioavailability of siRNA in brain [94,95]. TNF gene silencing in anti inammatory condition was also achieved by cysteine
peptides attached on chitosan nanoparticles, though drug targeting
in this case was arthritis [96,97]. Moreover, nucleic acid delivery to
neuronal cell is possible by TMC micellar nanospheres due to its
exceptional pH stability and slow degradation in vivo.
Success of genetic transfection by chitosan nanocarrier in neurodegenerative diseases is dependent on different formulation
parameters and properties of chitosan grade. Consideration of
such factors like N/P (nitrogen/phosphorous) ratio, pH and DD
overcomes inherent concerns of siRNA delivery to brain like detachment of siRNA from chitosan skeleton, insufcient cellular uptake
and inadequate transportation to the cell nucleus, degradation of
siRNA during blood circulation and in extracellular space. As of
2014 two antisense drugs have been approved by the U.S. Food
and Drug Administration, Fomivirsen (Vitravene) and Mipomersen
(Kynamro). Recently Alnylam Pharmaceuticals announced their
positive progress in clinical trials of siRNA therapy by use of GalNac
(a monomer of chitosan) conjugation with siRNA. The hopeful

regulatory approval of RNA-based therapy for CNS is still a vision for

the future, but chitosan has been proposed as a reliable alternative
for comparatively high toxic carriers like PEI (polyethyleneimine)
and viral vectors used in several IND products.
3. Fabrication options for brain targeted chitosan
nanoparticles
Chitosan as a nanocarrier material possesses excellent exibility to make drug entrapped nanoshell by various methods.
Comparatively requirements of lesser harsh conditions required
to fabricate chitosan in nano form supersede its selection in brain
targeted nanodelivery of proteins, enzymes and siRNA particularly. Ionic gelation and in situ crosslinking, micellization, spinning
disk technology and emulsication methods are the most in focus
techniques of chitosan nanoparticles formation containing targeted
neuroprotective substances in Alzheimers disease as scrutinized
below. Necessity of neuronal cell targeted ligands, stability of
nanoshell in normal and altered homeostatic condition.
3.1. Ionic gelation/crosslinking
Chitosan nanoparticles are prepared by ionic-gelation (physical crosslinking) or covalent crosslinking (chemical crosslinking)
requires mild processing condition and mere polyionic cross linkers as an essential adjuvant. Though covalent cross-linking provides
high drug entrapment efciency, the later type of cross-linking possesses much more sustained drug release mechanism due to its pH
and enzyme stability in vivo. Inorganic ions, such as Fe(CN)6 4 ,
Fe(CN)6 3 , citrate and calcium ions are also used frequently for
ionic gelation of chitosan other than the use of TPP (Tri poly phosphate). TPP has been widely exploited for gelation purpose, but
recent studies suggest that pyrophosphate as an ionic crosslinker
provides more colloidal stability to chitosan nanoparticles than
TPP [98,99]. Chitosan nanoparticles prepared by use of TPP displayed brain targeting efciency by both in vivo blood brain
barrier permeation studies and ex vivo neuronal uptake studies
in case of various anti-Alzheimers therapeutic agents like Thymoquinone [100], Rivastigmine [101], Venlafaxine [102], Tacrine
[103], Cyclophosphamide [104], Caspase inhibitors [105], Estradiol
[106]. NPs prepared by this method are also found to be more pertinent in FITC (uoroscein isothiocyanate) labeling of nanoparticle
in order to study internalization and cellular uptake imagine by
confocal micrography.
Chitosan easily forms poly electrolyte complex with hyaluronic
acid, chondroitin sulfate [107109], genipin [110112], epichlorhydrin, diethyl squarate, Hexamethylene-1, 6-diaminocarboxysulphonate, dialdehydes like glutaraldehydea and glyoxal, diisocyanates, epoxides, triuoroacetic acid, glycerol phosphate, oxalic
acid, tannic acid, pyrophosphate and Sodium hexa meta phosphate also. Among these, glutaraldehyde and glyoxal are reported
to exert neurotoxicity and cytotoxicity respectively in different
studies leading to a strangled preference for their use in brain
targeted chitosan nanoparticle preparation. Moreover, PEG diacrylate, oxidized beta cylcodeztrine, scleroglucan, telechelic-poly vinyl
alcohol and PEG derived dialdehydes are also used as crosslinkers in chitosan matrix, but they require polymerization initiators
like Sodium cyanoborohydrine and 2,2-dimethoxy-s-phenyl acetophenone which are not utterly studied for their safety concerns
in human.
3.2. Micellization
Shao et al. [113] reported ability of micellar structure to inhibit
aggregation of beta amyloid peptide and its conversion into neurotoxic clusters due to structural resemblance between micelles

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and biological membranes and surface charge of micelles. An FDA

approved anti-Alzheimer drug, Rivastigmine was also evaluated
by Lu et al. [114] for its usefulness against intracellular oxidative
stress after loading in polymeric micelles. They substantiated proof
of concept in drug loaded micelle carriers ability in protection of
cells against AB-induced injury. Dynamically polymer micelles with
surface loaded Tat peptide, after intra nasal administration demonstrated successful delivery of nucleic acid to brain tissues in various
research reports [115]. The natural afnity of beta amyloids interaction with micellar structure is implicit from amyloid staining by
ThT (Thioavin T) micelles. This staining is a result of hydrophobic
interaction and the role of positive charge on Thiavin T molecule,
in spite of incessant speculations for exact mechanism behind it
[116,117]. These all emphasize on utilization of chitosan micelles
in drug delivery of anti-Alzheimer agent and prevention of amyloid
beta aggregation.
Amphiphilic chitosan (e.g.: stearoyl, palmitoyl and octanoyl chitosan) derivatives self-assemble into nanoszied micelles depending
on the type of solvent used. Further, outer surface of the micelle
can be lmed by cross linker to form the structure of nanoshell
to enhance integrity of the nanostructure. This method also bears
merit of requiring mild processing parameters. The efcacy of
Propofol with micellar formulation was observed to be more than
the marketed injectable product conrming BBB permeability of
chitosan micelle [118]. Mechanism behind micelle permeability
through BBB is not yet clear, but inhibition of efux pumps is speculated as one of the causes [119]. Chitosan micelles have gained
attention in CNS targeted dosage form design due to its ability
to improve solubility of hydrophobic drugs, more stability than
low molecular weight surfactant micelles, ability to overcome drug
resistance developed by target cells and comparatively a lower
critical association concentration [120,121] required. PEG and PCL
(polycaprolactone) block co-polymer derived micelles faces problem of stability in biological uid due to less resistance to dilution
and dissolution because of higher critical micelle concentration
range of co-polymers. In contrast, phase behavioral disparity in case
of chitosan graft copolymer micelle is observed by high stability in
physiological condition due to low critical micelle concentration
value [122].
3.3. Spinning disk processing technology
Principle of conventional ionotropic gelation is extensively
utilized in advanced technologies of chitosan nanoparticle preparation like SDT (spinning disk processing technology). SDT is
considered a preferred one for large scale production of chitosan
nanoparticles due to its distinctive merits, including remarkable
size and shape control of nanoparticles, small sized nanoparticle generation compared to conventional methods and control on
agglomeration of particles by formation of stable nanoparticles
with high zeta potential (>45 mv) [123,124]. Manufacturing parameters to be controlled are temperature, disk surface property, speed
of mixing and the rate of introduction of reactants in SDT whereas
formulation parameters like concentration of chitosan, acidity of
solvent (acetic acid concentration), concentration of cross linker
plays a critical role in the overall quality of the product. Concentration of Chitosan solution from 0.25% w/v to 0.5% w/v impart an
enormous effect on particle size with more than tenfold increase in
size from initial 20 nm particles while SDT method was used with
1000 rpm disk speed as stated by Loh et al. [125]. In a separate
work, they mentioned efciency of SDT to prepare monodispersed
chitosan nanoparticles of 2025 nm size and importance of ultrane particles in high cell [126]. Moreover, augmented paracellular
pathway and facilitation of the cell nucleus targeting of chitosan
nanospheres observed by confocal microscopy in their work signify
utilization of the SDP method in SiRNA therapy in neurodegerative

diseases. SDP method is also useful to develop bottom up principle based solventnon solvent nanoprecipitation. This concept was
utilized to prepare curcumin nanoparticles with narrow size distribution and enhanced water solubility [127] which can be utilized
further for chitosan nanoparticle delivery. Huanbutta et al. [128]
demonstrated multi-step, spinning disk processing for preparation
of chitosan nanoparticles loaded with diclofenac sodium. The stepwise procedure was able to coat pH responsive acrylate co-polymer
around aggregation of 10 nm chitosan nanoparticles. The end result
with 88% drug entrapment efciency from the utilized method and
high systemic outreach of chitosan nanoparticles possess proof of
concept in non-invasive, oral delivery of nanomedicine for brain
targeting. In spite of the feasibility of multistep processing and ne
tunability of coated nanoparticle characteristics attained by SDT,
the technique is yet to be veried and modied in the preparation
of complex composite nanoparticles with ligand tagging.
3.4. Emulsication method
The emulsion solvent diffusion method of preparing chitosan
nanoparticles was derived from method utilized by Niwa et al. [129]
for the preparation of PLGA-based nanoparticles. This method is
specically utilized for preparation of chitosan nanoparticles containing hydrophobic drugs. The general methodology involved in
preparation of chitosan nanoparticles by emulsication requires
the addition of an organic phase containing the drug to chitosan
aqueous solution and a stabilizer under stirring [75]. Further,
o/w emulsion formed by rst step is exposed to high pressure
homogenization followed by removal of organic solvent. Chitosan
nanospheres containing F-Ab (sub fragments of amyloid beta), prepared by emulsication method yielded end product with sphere
size of 15.23 10.97 nm, was successfully utilized as a nano-carrier
for vaccination in Alzheimers disease [130]. This result was in line
of other opinions giving attention to usefulness for chitosan in regulation of cellular and humoral immunity and microphage activity
[131133]. In general, investigators highlights inclusion of chemical crosslinking of chitosan as an essential step after preparation of
chitosan dispersion is emulsied with surfactants. Lacunae in the
complete removal of harmful solvents (e.g.: acetone, chloroform) at
the end of the process and addition of toxic crosslinker (e.g.: gluturaldehyde) is a matter of enormous concern in the selection of this
technique of nanoparticle preparation aimed with brain delivery.
4. Surface modications with ligands
Brain hemisphere access for chitosan nanoparticles is possible
through many pathways at blood brain barrier, including transcellular and paracellular diffusion, receptor mediated transcytosis,
ion-channel activated transport, facilitated diffusion, active efux
transport and carrier mediated transcytosis. Genes of Alzheimers
disease express its multiple activities in microvascular damage and
compromised blood brain barrier. This enhances the chances of
the paracellular pathway of chitosan nanoparticle transmission to
brain parenchyma and nerve cells. Apart from this late diseased
condition, receptor mediated and carrier mediated transcytosis
is the major pathway for BBB permeation [134]. Ligand tagged
nanocarrier design accomplishes outreach of therapeutic substance
in AD affected parts of the brain more prominently than untagged
chitosan nanocarrier. Currently multi-ligand approach is also gaining recognition to make enhanced use of the complex nature of
neurodegenerative diseases in terms of up regulation of certain
receptors on brain epithelial cells. Ligands have been substantiated for their efcacy to get linked with chitosan nanoparicles and
to act as a Trojan horse which can bypass the degradation effect
of lysosomes of cytoplasm during movement from luminal side to

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Table 1
Lingands utilized for delivery of chitosan nanoparticles in anti-Alzheimer therapy.
Ligands

Bridge for surface

functionalization

Reference

Magnevist (MRI contrast

agent)
Transferrin monoclonal
antibodies

Conjugation

[104]

Chitosan-PEG-BiotinSteptavidin
linkage
Palmitoylated glycol chitosan
by using dimethylsuberimidate
mediated covalent linkage
Ion complexation with Fatty
acid-modied OCMCh
(O-carboxymethyl chitosan)
Nanocomplex formation

[105]

Direct adsorption on chitosan

nanospheres

[130]

Covalent attachment to
chitosan followed by
nanoparticle formation
MAL-PEG-NHS linker

[139]

Transferrin

Insulin and Insulin like

growth factor
Intramembranous
fragments of A
(Vaccination)
Glutathione

[137]

[138]

[137]

Rabiesvirus glycoprotein
[140]
29-Cys peptide
Other potential brain targeting ligands reported in brain targeting studiesa are
Tat Peptide, non-toxic analog of diphtheria toxin and tetanus toxin,
Enkephalins, Thiamine, Leptin and Angiopep.
a

Not yet extensively studied in anti-Alzheimer therapy involving chitosan

nanoparticles.

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the abluminal side. Other than listed, neuronal cell specicity of

nanoparticles is enhanced by RDP (a novel peptide with 39 amino
acids, resembling RVG) [135,136] as a ligand. Transcytosis receptor,
transferring-not predominantly present on cell surfaces affected by
AD as in case of cancer cell, but still reported to enhance nanocarrier transport facilitation through the BBB. Chitosan is observed to
affect receptor protein redistribution in the close junction of BBB
which ultimately affects adsorptive endocytosis of drug containing carrier through clathrin and caveolin mediated pathway. Brain
and neuronal cell specic ligands tagged successfully on chitosan
nanoparticles are summarized in Table 1.

5. Brain targeting with chitosan nanoparticles

5.1. Feasibility of non-invasive routes of administration by
chitosan nanoparticle
Nose to brain targeting of therapeutic macromolecules has
emerged as a viable approach for treatment of neurological disorders. Intranasal drug delivery for treatment of AD is recommended
strongly by many investigators with evidences from preclinical
studies. Recent success in clinical trials of intranasal therapy of
insulin for Alzheimers disease has demonstrated an acute increase
in the level of insulin in the AD affected brain. Activation of insulin
like growth factor receptor and anti-amyloidogenic mechanism
of administering insulin can reverse cognitive impairment in AD
affected persons [141143]. Yu et al. [144] rst reported enhanced
bioavailability of insulin by use of chitosan solution (1.5% w/v) due
to its absorption enhancing capacity when solution is administered
by the intranasal route. Chitosan solution as aqueous vehicles has
shown 14 fold increase in the bioavailability of the brain targeted
NGF (nerve growth factor) peptide via the nasal route during in
vitro and in vivo experiments. Bovine olfactory epithelium was
used as a barrier in these experiments. Further, Nearly 34% drop in
trans-olfactory epithelial electrical resistance was observed by use
of 0.25% w/v chitosan solution, showing permeability enhancement
of olfactory epithelium [145].

Use of chitosan and its derivatives like Chitosan-PLGA conjugate, in nanoparticle preparation for nose to brain targeting in
Alzheimers disease has been considered and demonstrated in various studies, exploring three distinct pathways through which
nanoparticles can reach to brain [146151]. Among which, olfactory and trigeminal nerve pathway originating in the brain and
terminating in the nasal cavity provide opportunity to deliver
therapeutic agents directly to central nervous system, representing the most persistent route in terms of non-invasive and direct
brain delivery of nanoparticles, [151,152] which can bypass BBB
as illustrated in Fig. 5. Fazil et al. [101] reported three fold higher
concentration of Rivastigmine in the brain after intranasal delivery in comparison of I.V. administration, on the basis of confocal
laser scanning microscopy and rhodamine-123 as a marker. Drug
particles loaded in nanocarriers like PLA provides more than 1.5
fold increase in brain accumulation of intranasal administered peptide drug when nanocarrier is coated with chitosan. Vaka et al.
[153] reported ability of carnosic acid loaded chitosan nanoparticles mediated up regulation of nuerotrophins. This study draws
attention to the requirement of lesser dosage, frequency of drug
loaded chitosan nanocarrier in comparison of IV injection of a
drug to attain therapeutic goal due to the efciency of chitosan
carrier to aggregate in the olfactory mucosal region after intra
nasal administration. In a separate study, concentration of estradiol was also observed to be higher in CSF and lower in blood after
intranasal administration of chitosan-estradiol nanoparticles [106].
Mistry et al. [154] and Shah et al. [155] concluded in their work
that nanoparticles with only less than 100 nm in size can undergo
olfactory axonal transport otherwise chitosan particles get transported to brain via a transcellular pathway because of adhesion
between chitosan nanoparticles and extracellular mucus. Similarly, nanoemulsion with globule size of less than 60 nm exhibits
mucoadhesiveness, high diffusion coefcient and remain devoid of
cliliary toxicity.
I.V. and intranasal are preferred route for delivery of therapeutic agent containing nanocarrier to the affected parts of the brain in
AD but still oral route is frequently explored with various nanocarriers for CNS drug delivery. Latasa et al. [156] prepared QAPGC
(quaternary ammonium palmitoyl glycol chitosan) nanoparticles
for delivery of therapeutic peptide to the brain. They identied
3% of the drug was able to reach in systemic circulation and brain
30 min after oral administration. Additionally, QAPDC was able to
protect the peptide from enzymatic degradation in GIT. GIT to brain
delivery is apparently evident by high permeability of chitosan and
Estradiol through Caco-2 cell lines. It was observed that permeability through colon cells is lowered when decrease in molecular
weight of chitosan. In a similar study, high molecular weight chitosan (22230 kDa) showed more cell toxicity in comparison of low
molecular weight (3.813 kDa) chitosan, demonstrating less than
70% cell viability at the end of the study when the former type of the
chitosan is used [157]. Thus, it is critical to select chitosan with suitable physical property in preparation of anti-Alzheimer containing
nano carrier to be delivered by oral route.
All of the drugs summarized above have efcacy to provide
relief from pathological and symptomatic conditions of AD. Hence
it is unbiased to say that chitosan nanoparticles alleviate effectiveness of anti-AD drugs in non-invasive approaches also against well
established IV administration approach of brain targeting.

6. Regulatory aspects
Chitosan is approved as a dietary supplement and food additive by regulatory agencies of the USA, Japan, Italy, Portugal and
England. In spite of several evidences of its safety as a pharmaceutical ingredient [158], chitosan is yet to be approved as a

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Fig. 5. Popular routes of administration and transport pathways of brain targeted chitosan nanoparticles: (A) Intranasal route and (B) I.V. route.
Table 2
List of patents featuring chitosan as a nanocarrier material for delivery of drugs with anti-Alzheimer activities.
Therapeutic
agent studied

Nanocarrier design

Prominent features of invention

Patent application number

Statins

Nanoemulsion (I.N.) for

inhalation; Chitosan-Statin
Conjugation via amide linkage
TMC-PEG graft co-polymer
micelles with RVG as ligand

Requires less than half of the drug

than oral and IV route

WO2014028587 A1

Acetylcholine receptor mediated

transfection of SiRNA to neuro-2a
cells achieved.
Nanoplex able to prevent
neurobrillary tangle formation by
streamlining tau functioning
Chitosan and its derivatives
stabilized nanospheres from
aggregation. Use of air bubble as
detection enhancing substance.
Drug loading is signicantly high in
dextran sulphate crosslinked
nanoparticles than TPP crosslinked
nanoparticles
Transferrin, insulin, Insulin like
growth factor and polysorbate-80
combinations as ligands. Patents
for degree of acetylation and low
molecular weight of carrier system
Trehalose as cryoprotectant in
formulation and chitosan as
permeation enhancer.
Use of cholesterol, stearic acid,
myristic, lauric. Capric, palmitic
acid as linker for hydrophilic drug
Chemical modication with
repetitive heating and cooling of
chitosan suspension
-Use of chitosan chloride or
glutamate salts in preparation of
nanoparticles. Zeta potential value
near 1mv.
Divalent metal like Mg accelerates
nose to brain delivery of CNS drug
containing chitosan nanocarrier.

CN103182087A

Nucleic acid

siRNA

Nanocomplex by ionic pairing

of siRNA and chitosan

Alzheimers disease
diagnostic agents

PLGA core enveloped by TMC

forming core-shell
nanoparticle

Hydralazine

Chitosan-TPP and
ChitosanDextran sulphate
nanoparticles

Drugs for CNS

Diversed nano carrier systems

Various hydrophobic drugs

Chitosan-poly--glutamic acid
conjugates forming micelles

Leucine-Enkephalin

Quarternary palmitoyl glycol

chitosan micelles

Curcumin

Modied chitosan
nanoparticles

Anti-inammatory agent

Chitosan salt-Hyaluronic acid

nanoparticle by ionic gelation
method

Proteins, drugs, genes

Chitosan reverse micelles

coated by crosslinkers which
can bind to divalent metal

US20120315322A1

US8449915 B1

US20110052713 A1

US20060051423 A1

US7879313 B1

WO2010100479 A1

CA2732635 A1

US20140038894 A1

WO2013040295 A2

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pharmaceutical excipient due to its non utility in new products

which are under regulatory approval process. Clinical trial advancement of GalNac (N-acetyla galactosamine) complexed siRNA and
chitosan carrier entrapped insulin delivery to brain for Alzheimers
disease treatment has encouraged possibility of its commercial
approval application in the future, which may insist regulatory
authorities to consider chitosan as a safe excipient in parenteral
and nasal drug delivery. Intellectual properties claimed by different
inventors in the context of application of chitosan in brain targeted
nanocarrier containing anti-Alzheimer drugs are listed in Table 2.
7. Conclusion
Use of chitosan and its derivatives as a nanocarrier material possesses high end result expectations in delivery of anti-Alzheimer
drugs to the brain. Multiple bioactivities of chitosan and its degradation products itself supports neuroprotective activities by acting
on numerous biomolecules present in the brain. Further, the proactive efcacy of chitosan to cross BBB also favors its selection in brain
targeting. Sufcient evidences are available for chitosan hemocompatibility and biocompatibility enhancement by various surface
modication approaches for its application in parenteral and nasal
drug delivery with view of brain targeting. It was also revealed
that enhanced expression of several biomolecules in AD affected
brain directly and indirectly linked with bioactivities of chitosan
units. Feasibility of different ligands to bind with chitosan and
its derivatives also allows targeting of neuronal cells in case of
Alzheimers disease therapy. Overall review implies chitosan as a
suitable nanocarrier material for delivery of anti-Alzheimer drug
delivery due to its inherent physicochemical properties, bioactivities and processing exibility.
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Please cite this article in press as: J. Sarvaiya, Y.K. Agrawal, Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.08.052

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