Drug-Polymer Interaction Studies of Cytarabine Loaded Chitosan Nanoparticles

Abstract

Assessment of possible incompatibilities between drug and excipients is an important
parameter of preformulation stage during the pharmaceutical product development of active
pharmaceutical ingredient (API). The potential physical and chemical interaction among the
components of a delivery system can affect the chemical nature, bioavailability, stability, and
subsequently therapeutic efficacy of drugs. In this study, ATR-FTIR spectroscopy was employed to
investigate the possible intermolecular interaction of Cytarabine with deacetylated chitosan and
tripolyphosphate in the resulting physical blends and crosslinked nanoparticulate system. Two
different strategies, physical blending and ionotropic gelation, were adopted to prepare binary or
tertiary mixtures and nanoparticulate formulation, respectively. The IR spectra of CB showed
characteristic peaks at 3438.27 cm-1 (primary amine), 3264.74 cm-1 (hydroxyl group) and 1654.98
cm-1 (C=O stretch in cyclic ring); CS at 3361.47 cm-1 (N-H stretching), 1646.18 cm-1 (C=O of Amide
I), 1582.36 cm-1 (C=O of Amide II), and sTPP at 1135.77 cm-1 (P=O). CS-sTPP chemical interaction
was confirmed from the shift in the absorption band of carbonyl groups (amide I, II) to 1634.66 cm-1
and 1541.17 cm-1 in blank chitosan nanoparticles, and 1636.87 cm-1, 1543.33 cm-1 in CSNP1 (2:6:1),
and at 1646.15 cm-1 and 1557.04 cm-1 in CSNP2 (1:3:1). The characteristic peaks of CB were also
present in chitosan formulation with a slight shift in the amino group at 3429.43 cm-1 and 3423.21
cm-1, in the hydroxyl group at 3274.54 cm-1 and 3270.73 cm-1, CSNP1 and CSNP2, respectively. The
findings counseled no significant interaction in IR absorption pattern of cytarabine functional groups
after encapsulation in CS-sTP

Full text

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1045
Drug-Polymer Interaction Studies of Cytarabine Loaded
Chitosan Nanoparticles
1Asadullah Madni*, 1Prince Muhammad Kashif, 1Imran Nazir, 2Nayab Tahir, 1Mubashar Rehman,
3Muhammad Imran Khan, 1Muhammad Abdur Rahim and 1Abdul Jabar
1Department of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur (63100), Punjab, Pakistan.
2College of Pharmacy, University of Sargodha, Sargodha (40100), Punjab, Pakistan
3Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture,
Faisalabad (38040), Punjab, Pakistan.
asadullah.madni@iub.edu.pk; asadpharmacist@hotmail.com*
(Received on 7th December 2016, accepted in revised form 7th November 2017)
Summary: Assessment of possible incompatibilities between drug and excipients is an important
parameter of preformulation stage during the pharmaceutical product development of active
pharmaceutical ingredient (API). The potential physical and chemical interaction among the
components of a delivery system can affect the chemical nature, bioavailability, stability, and
subsequently therapeutic efficacy of drugs. In this study, ATR-FTIR spectroscopy was employed to
investigate the possible intermolecular interaction of Cytarabine with deacetylated chitosan and
tripolyphosphate in the resulting physical blends and crosslinked nanoparticulate system. Two
different strategies, physical blending and ionotropic gelation, were adopted to prepare binary or
tertiary mixtures and nanoparticulate formulation, respectively. The IR spectra of CB showed
characteristic peaks at 3438.27 cm-1 (primary amine), 3264.74 cm-1 (hydroxyl group) and 1654.98
cm-1 (C=O stretch in cyclic ring); CS at 3361.47 cm-1 (N-H stretching), 1646.18 cm-1 (C=O of Amide
I), 1582.36 cm-1 (C=O of Amide II), and sTPP at 1135.77 cm-1 (P=O). CS-sTPP chemical interaction
was confirmed from the shift in the absorption band of carbonyl groups (amide I, II) to 1634.66 cm-1
and 1541.17 cm-1 in blank chitosan nanoparticles, and 1636.87 cm-1, 1543.33 cm-1 in CSNP1 (2:6:1),
and at 1646.15 cm-1 and 1557.04 cm-1 in CSNP2 (1:3:1). The characteristic peaks of CB were also
present in chitosan formulation with a slight shift in the amino group at 3429.43 cm-1 and 3423.21
cm-1, in the hydroxyl group at 3274.54 cm-1 and 3270.73 cm-1, CSNP1 and CSNP2, respectively. The
findings counseled no significant interaction in IR absorption pattern of cytarabine functional groups
after encapsulation in CS-sTPP complex, which projected the potential of chitosan nanoparticulate
system to entrap cytarabine.
Keywords: ATR-FTIR spectrometry, Chitosan, Cytarabine, Drug-polymer compatibility, Ionotropic gelation
method, Sodium tripolyphosphate
Introduction
The production of quality medicines is a
fundamental challenge for bulk drug and its suitable
delivery system. The assessment of excipients for
impurities and their chemical compatibility with drug
molecules is inevitable to assure the stability and
effectiveness of a delivery system [1]. In the
preformulation phase, drug-excipient compatibility
study signifies the chemical nature, bioavailability and
therapeutic safety of drug by evaluating the potential
physical and chemical interaction [2] Among the
various techniques, employed for assessment of
possible drug-excipient interactions, such as
differential scanning calorimetry (DSC),
thermogravimetry (TG), hot-stage microscopy (HSM),
X-ray diffractometry (XRD), diffuse reflectance
spectroscopy (DRS), self-interactive chromatography
(SIC), High Pressure Liquid Chromatography (HPLC),
thin layer chromatography (TLC), and liquid
chromatography (LC) with ultraviolet detection [3, 4],
FT-IR is most commonly employed, fast, robust and
less-expensive technique [5].
Since, the dispersive systems were
superseded by the much more powerful FT-IR
(Fourier-Transform-Infrared) spectrometers IR-
spectroscopy has developed into a widely used
routine analytical tool [6] The versatile and green
approach of ATR-FTIR technique has made for direct
analysis of possible physicochemical interactions
between active moiety and formulation components
[7] Furthermore, FTIR spectroscopy provides reliable
evidence of the appearance, modification, or
desertion of attribute peaks of various functional
groups such as C=O, C-H or N-H of the excipients
and drugs among physical mixtures and nanoparticles
formulations [8] This instrument allows measuring
all types of samples, whether they are solid, liquid or
gaseous [9]
In the clinical trial phase, the failure of a huge
number of medicaments to achieve encouraging
therapeutic outcomes is due to the lack of target
specificity, the frequent and large doses, and subsequent
*To whom all correspondence should be addressed.

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1046
adverse effects [10]. Furthermore, the drastic
physiological condition (gastric pH) and degradation of
biologically active agents hinder the distribution of drug
to a specific disease site [11] Therefore, the interesting
concept of micro and nanoparticles has developed to
overcome these disadvantages as a novel drug delivery
system. Primarily, the incorporation of active
pharmaceutical ingredients in micro/nano-particulate
matrix system is deliberating for their protection from
drastic in-vivo environment [12]. Moreover, the
controlled and sustained liberation of medicinal
substances, and conjugation or surface modification of
nanoparticles with targeting moiety also provides the
potential of treating site-specific release [12]. In these
nanoparticulate systems, a wide range of natural or
synthetic polymers (polysaccharides, polymethacrylates,
polyanhydrates, polyesters/ortho esters, poly lactic-co-
glycolic acid and polyphosphohazanes), lipids,
dendrimers, and surfactants can be employed to integrate
drugs by passive absorption and chemical conjugation
[13].
Low molecular weight chitosan (CS), a natural
cationic and hydrophilic amino-polysaccharide
macromolecule, is being used in the novel delivery
systems because of distinctive biological attributes, such
as muco-adhesion, biodegradation, haemocompatibility,
non-toxicity, and antibacterial property [14]. CS based
nano-particles/fibers, hydrogels and membranes have
been formulated to embed bioactive moieties
(hydrophilic, hydrophobic and macromolecules), due to
the specific tissue targeting and, sustained/controlled
release [15] Ionic gelation technique, among multiple
preparation methods comprising complex coacervation,
ionotropic gelation, self-assembly method through
chemical modification, solvent evaporation/diffusion, and
emulsion-droplet coalescence, is preferred for its
convenience, relative simplicity, and the rid of organic
solvents and high temperatures [16]. Chitosan
nanoparticles (CSNPs), have been widely developed for
the treatment of various conditions. For example,
nanoparticles of 5-fluorouracil, paclitaxel, catechin,
doxorubicin, leucovorin, and pravastatin are used in
cancer therapy. Nanoparticles of various other
therapeutical agents such as, donepezil, rivastigmine,
tacrine, ampicillin, chlorhexidine, levofloxacin, and
vancomycin are utilized to treat Alzheimer disease. In
order to manage Parkinson’s disease, bromocriptine,
dopamine, pramipexole, and selegiline can be
encapsulated in nanoparticulate system. Numerous
antiviral agents (saquinavir, stavudine, tenofovir, and
zidovudine) are now encapsulated into chitosan
nanocarriers in order to combat HIV. Moreover, several
antioxidants (ascorbic acid, rutoside, salvia officinalis,
and satureja montana) can also be loaded in
nanoparticulate system [17].
We envisioned the present compatibility
investigation to determine possible interaction of
Cytarabine (CB) with CS nanoparticle formulation
components. All individual components as polymers and
drug, their physical mixture, and developed
Nanoparticulate formulations have been examined by
ATR-FTIR spectrometer. Based on this observation,
future studies can be planned to evaluate the effects of
formulation parameters on the response variables. The
2D-chemical structure of cytarabine, chitosan, and
sodium tripolyphosphate was prepared on Chemdraw 8.0
Pro CambridgeSoft Corporation, USA (Fig. 1).
Experimental
Materials
Cytarabine (C9H13N3O5, PubChem CID: 6253)
was purchased from Beijing Mesochem Technology Co.,
LTD (China). Chitosan low molecular weight
(C56H103N9O39, PubChem CID: 71853) was purchased
from Sigma-Aldrich Chemie GmbH (USA), Sodium
tripolyphosphate (Na5P3O10, PubChem CID: 24455) from
MSDS ScienceLab.com, Inc. (Texas) and Acetic acid,
glacial 100% (C2H4O2, PubChem CID: 176) Merck
Millipore (USA).
Preparation of Physical Mixtures
Physical blends were prepared by intimate
trituration of equimolar quantities (10 mg) of CB, CS,
sTPP in an agate mortar for about 10 minutes to attain a
homogenized mixture [18]. The abbreviations and
equivalent ratios of resultant samples are described in
Table-1.
Table-1: Chemical composition, equivalent ratio, and sample code for ATR-FTIR analysis.
Sr. No Sample code Chemical composition Equivalent ratio
1 CB Cytarabine 1
2 CS Chitosan 1
3 sTPP Sodium Tripolyphosphate 1
4 CB-CS Cytarabine + Chitosan 1:1
5 CB-sTPP Cytarabine + Sodium Tripolyphosphate 1:1
6 CS-sTPP Chitosan + Sodium Tripolyphosphate 1:1
7 CB-CS-sTPP Cytarabine + Chitosan + Sodium Tripolyphosphate 1:1:1
8 CSNPbChitosan Nanoparticles without Cytarabi ne -
9 CSNP1Chitosan Nanoparticles loaded with Cytarabi ne -
10 CSNP2Chitosan Nanoparticles loaded with Cytarabi ne -

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1047
Fig. 1: 2D structure of Cytarabine, Chitosan, and Sodium tripolyphosphate (Chemdraw 8.0).
Table-2: Chitosan based nanoparticles with and without the loading of cytarabine.
Formulation code Cytarabi ne(mg) Chitosan(mg) SodiumTripoly-phosphate(mg) Stirring rate (rpm) Stirring time (h)
CSNPb- 60 10 700 2
CSNP120 60 10 700 2
CSNP220 60 20 700 2
Synthesis of Blank Chitosan Nanoparticles
The ionotropic gelation method was
employed to develop CS nanoparticles according to
the method reported by Calvo et al. [19] Briefly, 10
ml sTPP aqueous solution (1mg/ml) added to 20 ml
of 1% aqueous acetic acid solution containing CS (3
mg/ml) to produce blank nanoparticles under
constant stirring (700 rpm) at ambient temperature
(Table-2). The resulted nanoparticle suspension was
ultra-centrifuged using Sigma 1-14 (Sigma
Laborzentrifugen GmbH, Germany), and washed
three times with distilled water to evacuate the
unentrapped drug, and lyophilized in the freeze dryer
(CHRIST Alpha 1-2 LD plus, UK) for 24h [20]. The
interaction (inter- and intramolecular) between the
high degree of positively charged amino groups of
CS and the anionic groups of sTPP resulted from the
formation of CS based NPs [21]. The mechanism of
cross-linking described as illustration in Fig. 2 [22,
23].
Preparation of Chitosan Nanoparticles Loaded with
Cytarabine
Cytarabine loaded chitosan nanoparticles
were fabricated employing ion gelation method
reported by Kalam et al. [24] CB was dissolved into
20 ml of aqueous acetic acidic solution (1%)
containing CS before the addition of 10 ml of sTPP
(0.1%). Thereafter, the formulation was stirred
magnetically at 700 rpm for 2h, at room temperature.
Two formulations (CSNP1 and CSNP2) were
fabricated as mentioned in Table-2.
ATR-FTIR Instrumentation
Attenuated total reflection- Fourier
transform infrared (ATR-FTIR) spectroscopic
technique was employed to analyze
physical/chemical interaction between pure
components and developed formulations [25].
Chemical images of pure drug, polymer, cross linker,
their physical blends, and nanoparticles (with and
without drug) were acquired by the FTIR
spectroscopic imaging approach using ATR-FTIR
spectrometer (Bruker tensor 27, Germany). All
spectral images were collected using OPUS (v6.5)
FT-IR data collection software over a spectral range
of 4000-400 cm-1 with 16 co-added scans at a
resolution of 4 cm-1. The standard sample cell in the
FTIR is a Pike Miracle single-bounce attenuated total
reflectance (ATR) equipped with a ZnSe single
crystal. Prepared samples were placed directly on the
small crystal spot, and the arm rotated over and
turned down to press the sample down onto the
crystal face to get better contact [26].

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1048
Fig. 2: Schematic mechanism of nanoparticles preparation by crosslinking of chitosan with sodium
tripolyphosphate using ionotropic gelation (Chemdraw 8.0).
Entrapment Efficiency and Drug Release Profile
The entrapment efficiency of CB was
determined by centrifugation method. Nanoparticles
were separated from aqueous environment of
nanoparticulate system by the ultracentrifugation at
14000 rpm for 30 min. The clear superintend was
further diluted 100 times with phosphate buffer saline
(pH 7.4). The unentrapped drug was measured from
diluted solution by UV spectrophotometer at λmax of
CB (272 nm). Furthermore, standard curve was
constructed in PBS solution (pH 7.4) by plotting
graph between CB concentrations of 1 mg/ml to 15
mg/ml and UV absorbance, to calculate the amount
of entrapped drug in nanoparticulate system. The
calibration curve equation (y = 0.0414x - 0.0056) at
R² = 0.9996 was obtained from MS Excel 2010 and
regressed as standard for the quantitative analysis
[27].
In-vitro drug release profile of Cytarabine
from chitosan nanoparticles was evaluated by dialysis
tube method. The weighed amount of nanoparticulate
system was filled in dialysis tube and was immersed
in 400 ml of PBS at pH 7.4. The temperature of
medium was maintained at 37 ± 0.5 ºC and stirred at
100 rpm in the USP paddle assembly attached with
PTFC-2/8 Fraction Collector auto-sampler (Pharma
Test, Hainburg, Germany). During the 24 h drug
release study, subsequent series of 5 ml aliquots were
pulled back from dialysis solution at predetermined
time intervals (1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18,
20, 22 and 24 h) and substituted with equal volume of
fresh PBS to sustain sink conditions. Samples were
adequately diluted, filtered and analyzed by UV-
spectrophotometer (IRMACO GmbH, Geesthacht,
Germany) at 272 nm. The released amount of CB
was calculated from the regression equation and
cumulative drug release (%) was calculated.
Results and Discussion
ATR-FTIR is recognized analytical
technique for the analysis of functional group, phase
separation behavior, and different types of bonding
forces in pharmaceutical ingredients. Considering
mid-infrared spectral modifications in the molecular
band strength and frequency shifts are acknowledged
measures for the occurrence and intensity of inter-
intramolecular forces [28]. Generally, pharmaceutical
entities comprise particular bonds and characteristic
functional groups that vibrate independently at their
equilibrium position and weekly interact with each
other, without the influence of any electromagnetic
(EM) radiation effect [29]. After the application of an
infrared (IR) radiation through a sample, the
transitions among vibrational and rotational power
levels result in the alteration of dipole moment,
because of molecular absorption of IR [30].
Moreover, IR absorption is specific to unique
molecular vibration frequency, and the resulting
spectrum epitomizes the molecular
transmission/absorption and generates a precise
fingerprint of sample [31].

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1049
Fig. 3: FTIR fingerprint of individual components (a) cytarabine, (b) chitosan, and (c) sodium
tripolyphosphate.
Table-3: Functional groups and characteristic peaks of pure ingredients.
Samples Functional Groups with Characteristic Pea ks References
CB N-H (3438.27 cm-1 & 3356.33 cm-1), O-H (3264.74 cm-1), C-H (293 5.62 cm-1), C=O (1654.98 cm-1), N-H (1576.07 c m-1), C-N
(1473.25 cm-1), C-O-H (1279.18 cm-1), and C-O-C (1109.10 cm-1 & 1069.08 cm-1). [5, 32 33]
CS N-H & O-H (3361.47 cm-1), C-H (2878.38 cm-1), O-H (2362.21 cm-1), C=O (1646.18 cm-1 & 1582.36 cm-1), CH 3 (1374.26
cm-1), C-O-C (1149.85 cm-1), C-O (1059.02 cm-1)[6, 35, 40]
STPP P=O (1135.77 cm-1) and P-O (891.19 cm-1 & 734.44 cm-1) [5, 9]
Therefore, the elucidation of structure–
property relations of pure components with their
physical blend, and nanoparticle formulations were
analyzed by ATR-FTIR spectrometer and interpreted
to indicate the possible interactions and
incompatibilities.
FT-IR spectra of pure components
The characteristic bands of cytarabine,
deacetylated chitosan, and sodium tripolyphosphate
are presented in Fig. 3 and the locations of
characteristic band values are given in Table-3. FTIR
spectra of CB, as described in Fig. 3(a), revealed the
characteristic peak of primary amine (N-H) at
3438.27 cm-1 and 3356.27 cm-1 (asymmetric and
symmetric stretch, respectively), broad absorption
peak at 3264.74 cm-1 disclosed the presence of O-H
stretching. The characteristic peak at a wave number
of 2935.62 cm-1 showed C-H asymmetrical stretch;
the sharp peaks at 1654.98 cm-1 and 1576.07 cm-1
indicated C=O stretching (aromatic) and N-H
bending (secondary amide), respectively. C-N
stretching, C-O-H bending and C-O-C stretching
(ether) were also observed at 1473.25 cm-1, 1279.18
cm-1 and 1109.10 cm-1, 1069.08 cm-1 respectively.
Other researchers in their study, indicating the purity
and identity of CB, also reported similar
characteristic peaks of pure cytarabine [32, 33].
In the FTIR fingerprint of CS, as in Fig.
3(b), the overlapped characteristic peak of N-H and
O-H stretching at 3361.47 cm-1, and absorption band
at 2878.38 cm-1 depicted asymmetrical stretching
vibrations of C-H. The further spectral interpretation
demonstrated a broad absorption peak of O-H
(stretching) overlapped with C-H at the wave number
of 2362.21 cm-1 (which occurs in the presence of a
carbonyl group). C=O stretching of amide I at
1646.18 cm-1 and amide II at 1582.36 cm-1 confirmed

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1050
the presence of N-acetylated groups of CS. The wave
numbers at 1374.26 cm-1, 1149.85 cm-1 and 1059.02
cm-1 and ensured the presence of CH3 (asymmetrical
deformation), C-O-C bridge (asymmetrical
stretching) and C-O (stretching), respectively. Others
also reported all the absorption bands of functional
groups in chitosan [34, 35]. IR spectral analysis of
sTPP, as explained in Fig. 3(c), showed strong P=O
stretching vibrations at 1135.77 cm-1, and P-O
stretching at 891.19 cm-1 and 734.44 cm-1. Similar
characteristic peaks were observed in a study
presented by Lima et al. [36].
FT-IR Spectrum of Physical Blends
Any possible interaction in the physical
mixtures of Cytarabine and formulation excipients
has been examined and depicted in Fig. 4. The binary
blend of drug with polymer (CB-CS), as presented in
Fig. 4(a), confirmed all the identical peaks with
imperceptible shift and indicating the lack of
interaction between functional groups. IR spectra of
cytarabine with a cross-linker, as shown in Fig. 4(b),
depicted no significant shift in characteristic peaks
compared to the individual components (Fig. 3)
demonstrating the absence of interactions. The
incompatibility of chitosan with sodium
tripolyphosphate also studied and the absence of
linkage between –NH2 and –P-OH was observed in
their physical mixture.
Similarly, the IR spectral analysis of drug,
polymer, and cross-linker also confirmed the
presence of all individual attribute peaks in their
physical mixture without any virtual shift, as
illustrated in Fig. 4(d). This part of the investigation
has confirmed the compatibility of the drug with its
formulation components before the development of
nanoparticles. Locations of characteristic band values
are summarized in Table-4.
FT-IR Spectrum of Chitosan Nanoparticles
Fig. 4: FTIR fingerprint of physical blends: (a) CB-CS, (b) CB-sTPP, (c) CS-sTPP, and (d) CB-CS-sTPP.

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1051
Table-4: Functional groups and characteristic peaks of physical blends.
Physical blends Characteristic bands with functional groups
CB-CS 3435-3263.85 cm-1 (N-H & O-H), 2886.19 cm-1 (C-H), 2359.57 c m-1 (O-H), 1654.63 cm-1 (C=O), 1576.90 cm-1 (N-H), 1473.25 c m-1 (C-
N), 1372.79 cm-1 (-CH3), 1279.57 cm-1 (C-O-H), and 1149-1068.94 cm -1 (C-O-C).
CB-sTPP 3438.16-3264.58 cm-1 (N-H & O-H), 2937. 26 cm-1 (C-H), 1655.20 cm-1 (C=O), 1577.01 cm-1 (N-H), 1474.06 cm-1 (C-N), 1279.57 cm-1
(C-O-H), 1136.98 cm-1 (P=O), 1109.98-1069.48 cm-1 (C-O-C), and 735.37 cm-1 (P-O).
CS-sTPP 3356.32 cm-1 (N-H & O-H), 2871.14 cm-1 (C-H), 2359.91 cm-1 (O-H), 1645.26 cm-1 (C=O), 1583.36 cm-1 (C=O), 1374.81 cm-1 (-CH3),
1138.38 cm-1 (P=O), 1056.92 cm-1 (C-O) and 734.58 cm-1 (P-O).
CB-CS-sTPP 3438-3264.47 cm-1 (N-H & O-H), 2936-2887.69 cm-1 (C-H), 2359.56 cm-1 (O-H), 1654.48 cm-1 (C=O), 1576.32 cm-1 (N-H), 1471.99 cm-
1 (C-N), 1372.43 cm-1 (-CH3), 1279.30 cm-1 (C-O-H), 1137.32 cm-1 (P=O), 1109-1069.18 cm-1 (C-O-C) and 736 cm-1 (P-O).
Fig. 5: FTIR spectra of CS nanoparticles: (a) CSNPb, (b) CSNP1, and (c) CSNP2.
The chitosan-sodium tripolyphosphate
matrix system is resulted due to interaction of amino
group (CS) and phosphate (sTPP) [37], as explained
in Fig. 2. Different chitosan nanoparticulate
formulations have been developed by the ionotropic
gelation method from different molar ratios of
formulation components (Table-2) to scrutinize the
possible interaction and compatibility of components
in the formulation. ATR based FTIR spectrum of
chitosan nanoparticles, as revealed in Fig. 5(a), the
individual chitosan spectra, a broad absorption peak
of the hydroxyl group (O-H) was observed at 3241.12
cm-1 specifying the enhanced hydrogen bonding. A
characteristic peak of O-H stretching (overlay C-H)
was perceived with a negligible shift in the wave
number from 2362.21 cm-1 to 2358.04 cm-1. The
absorption band of C=O of amide I at 1646.18 cm-1
was shifted at 1634.66 cm-1, indicating a possible
interaction with sTPP. Moreover, the bending
vibration of amide II at 1582.36 cm-1 (representing
deacetylated group) was also shifted to 1541.17 cm-1
after the ionic interaction with sTPP. This
conjugation (between ammonium and phosphoric
ions) decreases the solubility of chitosan and
accountable for the separation of CS nanoparticles
from the solution [16].
To compare the possible interaction of drug
with polymer-crosslinker complex, two different

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1052
formulations (CSNP1 and CSNP2) were developed
with different molar ratios of CB, CS and sTPP
(2:6:1 and 1:3:1). Fig. 5(b) and (c) represents IR
spectra of CB nanoparticles, which disclosed the
notable attenuation of absorption spectra with a
substantial decrease in strength of absorption peaks,
signifying the existence of interaction and entrapment
of CB in CS-sTPP matrix system [38]. The
characteristic peaks of primary amine (N-H)
symmetrical and asymmetrical at 3438.27 cm-1 and
3356.27 cm-1 of CB were shifted to lower
wavenumber in both formulations at 3429.43 cm-1
(CSNP1) and 3423.21 cm-1 (CSNP2) with the absence
of N-H asymmetrical peak, representing the
interaction between CB and chitosan. Small
differences were noticed at the wavenumber 3264.74
cm-1 (-OH stretching) which were shifted to
wavenumber 3274.54 cm-1 (CSNP1) and 3270.73 cm-
1 (CSNP2), and absorption peak at 2935.62 cm-1 (C-H
asymmetrical stretching) which is shifted to 2920.03
cm-1 (CSNP1) and 2886.15 cm-1 (CSNP2). The
spectral peaks of interaction between CS and sTPP
were also detected in CSNP1 and CSNP2 at 1636.87
cm-1, 1543.33 cm-1 and 1646.15 cm-1, 1557.04 cm-1,
respectively. These are the indicators of CS-sTPP
conjugation and interaction. The C-O-H (bending)
vibration peaks of CB were also observed in the
nanoparticle system at 1423.25 cm-1 and 1421.72 cm-
1 for CSNP1 and CSNP2 respectively, ensuring the
loading of the drug [39]. Moreover, Cytarabine has
three further strong absorption bands at 1279.18 cm-1,
1109.10 cm-1 and 1069.08 cm-1, which were
overlapped with chitosan absorption bands (perceived
at the same position in CSNP formulations).
Furthermore, CB showed physical
interaction with CS in nanoparticles, which may be
due to the electrostatic forces, but no additional
characteristic peak or shifting of bands was observed
in the physical mixtures or nanoparticulate
formulations. This analysis ensures the entrapment of
Cytarabine in biocompatible polymer (chitosan)
matrix and its accessibility to fabricate the drug
delivery system for biological actions.
Entrapment Efficiency and Drug Release Profile
Table-6 describes the encapsulation
efficiency of nanoparticle formulations at constant
drug content. CSNP1 demonstrated high entrapment
efficiency of Cytarabine into nanooparticles was
75.24%, while CSNP2 showed low by 64.41 %.
Formulation CSNP1 showed more sustained
discharge of Cytarabine from nanoparticulate system
whereas drug release from formulation CSNP2 was
less sustained (Table-6). The values of drug release
(%) after 12 hours and 24 hours were 51.65% and
82.84% respectively from CSNP1, and 72.34% and
89.64% from CSNP2 respectively. Nanoparticulate
formulation CSNP1 revealed high EE with sustained
liberation of drug from nanoparticles following 24
hour release period (Fig. 6).
Fig. 6: Drug release behaviour of Cytarabine from
chitosan nanoparticulate system (a) CSNP1,
and (b) CSNP2.
Table-5: Characteristic bands and functional groups of chitosan nanoparticulate system.
Formulations Characteristic bands with functional groups
CSNPb
3241.12 cm-1 (N-H & O-H), 2358.04 cm-1 (C-H), 1634.66 cm -1, 1541.17 cm-1 and 1375.98 cm-1 (ammonium a nd phosphoric ion
conjugation)
CSNP1
3429.43 cm-1 (N-H), 3274.54 cm-1 (O-H), 2920.03 cm-1 (C-H), 2293.28 c m-1 (O-H), 1636.87 cm-1 & 1543.33 cm-1 (ammonium a nd
phosphoric ion conjugation) and 1423.25 cm-1 (C-O-H).
CSNP2
3423.21 cm-1 (N-H), 3270.73 cm-1 (O-H), 2886.15 cm-1 (C-H), 2282.36 c m-1 (O-H), 1646.15 cm-1 (C=O of amide I), 1557.04 cm-1
(ammonium and phosphoric ion conjugation) a nd 1421.72 cm-1 (C-O-H)
Table-6: Entrapment efficiency and drug release behaviour of CSNP1 and CSNP2.
Formulation code Entrapment Efficiency (%) Drug release after 12hr (%) Drug release after 24hr (%)
CSNP175.24 51.65 82.84
CSNP264.41 72.34 89.64
Conclusion Chitosan nanoparticle formulations alone and

Asadullah Madni et al., J.Chem.Soc.Pak., Vol. 39, No. 06, 2017 1053
in combination with cytarabine were successfully
prepared using the ionic gelation technique. Among the
pre- and post-formulations studies, ATR-FTIR has
appeared as a fundamental technique to find out
interactions among the formulation components and
compatibility to be included in a drug delivery system.
The findings from this study have revealed the absence
of possible chemical interaction or incompatibility
between Cytarabine and chitosan-tripolyphosphate
matrix system. Physical state of the cytarabine was
modified, which corresponds to the drug crystallinity
whereas formulation is thrashing its sharpness at its
characteristic peaks. Absence of detectable crystalline
domains in cytarabine-loaded nanoparticulate clearly
indicates that drug remained intact and dispersed
completely in the formulation, thus modifying the
nanoparticles to a disordered-crystalline phase. The
analysis of various physical mixtures revealed a slight
shift in band/peaks of different functional groups and
characteristic peaks of cytarabine were observed at the
same wavenumber and intensity points. However, ATR-
FTIR spectra of the nanoparticulate formulations
revealed the successful entrapment of cytarabine in CS
nanoparticles. Furthermore, the compatibility
investigations of cytarabine with excipients pretended
the absence of precarious substances/excipients.
Therefore, it is established that cytarabine can be
successfully entrapped the CS nanoparticulate
formulations and provide guide for future compatibility
assessment of CB.
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