Polymeric micelles based on amphiphilic oleic acid modified carboxymethyl chitosan for oral drug delivery of bcs class iv compound: Intestinal permeability and pharmacokinetic evaluation

Article abstract of DOI:10.1016/j.ejps.2020.105466

Chemical modification of chitosan derivatives with hydrophobic fatty acids to enhance their self-aggregation behavior is well established. Previously our group reported low molecular weight carboxymethyl chitosan (CMCS) which showed enhancement in apparent permeability of hydrophobic drug, tamoxifen. Further extension to this work, herein we synthesize a new polymer of oleic acid grafted low molecular weight carboxymethyl chitosan (OA-CMCS) for maneuvering biopharmaceutical performance of poorly water soluble drugs. This polymer was designed and synthesized via amidation reaction and well characterized by analytical tools like 1H-NMR and FT-IR spectroscopy. OA-CMCS conjugate easily self-organized into micelles like structure in an aqueous medium and showed a low critical micellar concentration of 1 μg/mL. Poorly water-soluble drug, docetaxel (DTX) was used as a model drug in this study. Optimization of variables resulted in the formation of spherical DTX loaded OA-CMCS micelles in the size range of 213.4 ± 9.6 nm with an entrapment efficiency of 57.26 ± 1.25percent. DTX loaded OA-CMCS micelles showed slow and sustained DTX release behavior in simulated body fluid during in vitro release study. The permeability of DTX loaded OA-CMCS micelles across the gastrointestinal tract were investigated by in vitro Caco-2 cells model. The apparent permeability of DTX loaded OA-CMCS micelles improved up to 6.57-fold in comparison to free DTX suspension which indicates the increase in paracellular absorption of DTX. Additionally, in vivo pharmacokinetic study demonstrates an increase in Cmax (1.97-fold) and AUC (2.62-fold) for DTX loaded OA-CMCS micelles compared to free DTX suspension. Hence, we propose OA-CMCS as a promising cargo to incorporate drugs for enhancement of biopharmaceutical performance.

Full text of DOI:10.1016/j.ejps.2020.105466

Journal Pre-proof
Polymeric micelles based on amphiphilic oleic acid modified
carboxymethyl chitosan for oral drug delivery of BCS class IV
compound: intestinal permeability and pharmacokinetic evaluation
Rahul Kumar Conceptualisation; Methodology; Investigation; Data curation; Validation ,
Arvind Sirvi Manuscript writing-original draft; Formal data analysis ,
Shamandeep Kaur Conceptualisation; data curation; Formal data analysis; Supervision ,
Sanjaya K. Samal Methodology (permeability study) ,
Sabyasachi Roy Methodology (HPLC method development) ,
Abhay T. Sangamwar Conceptualisation; Data review; Validation; Project administration; Writing-review and editing
PII:
S0928-0987(20)30255-4
DOI:
Reference:
https://doi.org/10.1016/j.ejps.2020.105466
PHASCI 105466
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
1 May 2020
11 July 2020
12 July 2020
Please cite this article as: Rahul Kumar Conceptualisation; Methodology; Investigation; Data curation; Validation ,
Arvind Sirvi Manuscript writing-original draft; Formal data analysis , Shamandeep Kaur Conceptualisation; data cu
Sanjaya K. Samal Methodology (permeability study) , Sabyasachi Roy Methodology (HPLC method development)
Abhay T. Sangamwar Conceptualisation; Data review; Validation; Project administration; Writing-review and editing
Polymeric micelles based on amphiphilic oleic acid modified carboxymethyl chitosan for oral drug
delivery of BCS class IV compound: intestinal permeability and pharmacokinetic evaluation, European
Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105466
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HIGHLIGHTS




Amphiphilic OA-CMCS conjugate was synthesized and characterized by analytical tools like
FTIR and 1H-NMR.
Hydrophobic modification of CMCS improves the encapsulation efficiency of hydrophobic drug,
docetaxel
Optimised polymeric micelles enhance paracellular absorption of docetaxel and reduced the drug
efflux.
Polymeric micelles demonstrate improved pharmacokinetic parameters for docetaxel compared to
free docetaxel suspension.

Title
Polymeric micelles based on amphiphilic oleic acid modified carboxymethyl chitosan for oral drug
delivery of BCS class IV compound: intestinal permeability and pharmacokinetic evaluation
Authors name and affiliations
Rahul Kumar#, Arvind Sirvi$, Shamandeep Kaur$, Sanjaya K. Samal$, Sabyasachi Roy#, Abhay T.
Sangamwar$,*
#Department of Pharmaceutical Technology (Formulations), National Institute of Pharmaceutical
Education and Research, Sector-67, S.A.S Nagar, Punjab-160062, India.
$ Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Sector-
67, S.A.S Nagar, Punjab-160062, India.
*Address for correspondence: Abhay T. Sangamwar, Department of Pharmaceutics, National Institute
of Pharmaceutical Education and Research, Sector-67, S.A.S Nagar, Punjab-160062, India. E-mail:
abhays@niper.ac.in, abhaysangamwar@gmail.com (A.T. Sangamwar); Tel: +91-0172 2214682; Fax:
+91-0172 2214692
ABSTRACT
Chemical modification of chitosan derivatives with hydrophobic fatty acids to enhance their self-
aggregation behaviour is well established. Previously our group reported low molecular weight
carboxymethyl chitosan (CMCS) which showed enhancement in apparent permeability of hydrophobic
drug, tamoxifen. Further extension to this work, herein we synthesize a new polymer of oleic acid grafted
low molecular weight carboxymethyl chitosan (OA-CMCS) for maneuvering biopharmaceutical
performance of poorly water soluble drugs. This polymer was designed and synthesized via amidation
1
reaction and well characterized by analytical tools like H-NMR and FT-IR spectroscopy. OA-CMCS
conjugate easily self-organized into micelles like structure in an aqueous medium and showed a low
critical micellar concentration of 1 µg/mL. Poorly water-soluble drug, docetaxel (DTX) was used as a
model drug in this study. Optimization of variables resulted in the formation of spherical DTX loaded
OA-CMCS micelles in the size range of 213.4 ± 9.6 nm with an entrapment efficiency of 57.26 ± 1.25%.
DTX loaded OA-CMCS micelles showed slow and sustained DTX release behavior in simulated body
fluid during in vitro release study. The permeability of DTX loaded OA-CMCS micelles across the
gastrointestinal tract were investigated by in vitro Caco-2 cells model. The apparent permeability of DTX
loaded OA-CMCS micelles improved up to 6.57-fold in comparison to free DTX suspension which

indicates the increase in paracellular absorption of DTX. Additionally, in vivo pharmacokinetic study
demonstrates an increase in Cmax (1.97-fold) and AUC (2.62-fold) for DTX loaded OA-CMCS micelles
compared to free DTX suspension. Hence, we propose OA-CMCS as a promising cargo to incorporate
drugs for enhancement of biopharmaceutical performance.
KEYWORDS: Carboxymethyl chitosan; Oleic acid; Hydrophobic modification; Polymeric micelles;
Permeability enhancement; Oral absorption
1. INTRODUCTION
Amphiphilic polymers attracted much interest in designing drug delivery systems for hydrophobic drugs,
due to their ability to self-assemble into nanostructures with hydrophobic core and a hydrophilic outer
shell in an aqueous medium. The hydrophobic core can accommodate hydrophobic drugs effectively,
whereas the hydrophilic shell can facilitate colloidal stability (Du et al., 2009; Hu et al., 2006). Because
of their unique feature of self aggregation, several amphiphilic co-polymers have been studied
extensively for pharmaceutical and biomedical application (Clare et al., 2012; Li et al., 2008; Qu et al.,
2018). Recently, Natural polysaccharides as biomaterials are mostly preferred for drug delivery
applications due to their versatile properties (Tian and Mao, 2012; Zhang et al., 2009).
Chitosan (CS), an abundant natural based polysaccharide produced by deacetylation of chitin. Chitosan is
a cationic copolymer of N-acetyl-D- glucosamine and D-glucosamine units, linked by β-1,4 glycosidic
linkages (Chatelet et al., 2001; Hejazi and Amiji, 2003). CS has been exhaustively studied for their
pharmaceutical and biomedical applications due to their fascinating properties such as biodegradability,
biocompatibility and non-toxicity (Shariatinia, 2018; Tian and Mao, 2012). Furthermore, this bioactive
polymer has several functional features like antibacterial, antioxidant, and wound healing activity
(Anitha et al., 2009; Muxika et al., 2017). Despite its unique properties, CS is not often used in oral
delivery of drugs due to its high viscosity, poor solubility under the physiological conditions and
uncontrollable drug release (Upadhyaya et al., 2014). To get control of these drawbacks, chemical
derivatisation of chitosan is carried out by several researchers (Jiang et al., 2006; Yuan et al., 2010). In
particular, carboxymethyl derivative of chitosan demonstrate outstanding physicochemical and biological
properties. It is also reported that CMCS shows mucoadhesion property for the micelle to cross complex
gastrointestinal (GI) milieu which is helpful in management of the GI side effects such as mucositis and
GI disturbance (Liang et al., 2008). Recently, low molecular weight carboxymethyl chitosan (LMW-

CMCS) has been identified as a suitable polymer due to its favourable characteristics like low viscosity,
high aqueous solubility as compared to conventional high molecular weight equivalents. It is reported
that LMW-CMCS is beneficial for oral absorption of hydrophobic drug as it reversibly open tight
junctions between intestinal epithelial cells in a Caco-2 cell monolayers (Jena and Sangamwar, 2016).
Furthermore, many investigations are carried out on grafting of modified chitosan in view of widening
their applications in oral drug delivery of hydrophobic drugs. The hydrophobic association of modified
chitosan with lipidic groups can form self-aggregate molecules in an aqueous medium (Desbrieres et al.,
1996). The lipophilic core provides good solubilisation and high drug loading capacity for hydrophobic
drugs. Lipid molecules like cholesterol (Yinsong et al., 2007), linolenic acid (Tan and Liu, 2009), stearic
acid (Guo et al., 2013), palmitic acid (Ramalingam and Ko, 2016) and oleic acid (Lee et al., 2011) have
been used for hydrophobic modification of chitosan derivatives. Additionally, some reports revealed that
unsaturated fatty acids-bearing micellar systems show a synergistic effect to an encapsulated active agent
in order to improve the effectiveness of treatment (Gao et al., 2019; Gao et al., 2018). Oleic acid (OA) is
long chain unsaturated fatty acid, considered as GRAS (Generally Recognized as Safe) by the U.S. Food
and Drug Administration (FDA). OA has been verified to improve intestinal absorption of hydrophobic
drug by binding to circulating lipoproteins, followed by intestinal lipid absorption pathway such as
chylomicron synthesis and lymphatic transport (Porter et al., 2007). Furthermore, OA stimulates
calmodulin and calmodulin-dependent protein kinase-mediated Ca2+ channels and rises intracellular
calcium levels and subsequently modulate the paracellular permeability (Kobayashi et al., 1996).
Previously, Li and research group have reported the synthesis and characterization of self-assembled
nanoparticles based on oleoyl-carboxymethyl chitosan and results revealed an enhancement in solubility,
and release behavior of rifampicin upon nanoparticle formation with oleoyl-carboxymethyl chitosan (Li
et al., 2011). Thus it seems appropriate to further explore and investigate the effect of oleic acid modified
CMCS on biopharmaceutical performance of poorly water-soluble drugs such as intestinal permeability
and pharmacokinetic properties.
In present work, we attempted to design and develop an oral formulation, which offers drug absorption in
a sustained manner and improve therapeutic efficacy of poorly water-soluble drug. Initially, we
synthesized a novel polymer backbone; an amphiphilic oleic acid grafted carboxymethyl chitosan (OA-
CMCS) via amidation reaction and characterized by analytical tools like 1H-NMR and FT-IR
spectroscopy. BCS class IV compound, docetaxel (DTX) was used as model hydrophobic drug in this
study. The co-solvent evaporation technique is used for formulating DTX-loaded OA-CMCS micelles.
Subsequently, the physicochemical characteristics of DTX-loaded OA-CMCS micelles such as micelle
size, morphology and stability in physiological body fluid and in vitro release were characterised. The
permeability investigation of optimized micelle was accomplished by Caco-2 cell transport model. In

addition, the in vivo pharmacokinetic study was carried out for DTX-loaded OA-CMCS micelle in
Sprague Dawley (SD) rats using free DTX suspension as a control.
2. MATERIALS AND METHODS
2.1 Materials and animals
Carboxymethyl chitosan was purchased from Carbosynth Limited (Berkshire, UK). Low molecular
4
weight of CMCS v 5 x Da) was prepared as per previously reported method by our group
(Jena and Sangamwar, 2016). Docetaxel was obtained generously as a gift sample from Mac-Chem
Products Pvt. Ltd. (Mumbai, India). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC), pyrene, and dialysis membrane (molecular weight cutoff, MWCO 12 kDa) were purchased from
Himedia Laboratories Pvt. Ltd (Mumbai, India). N-hydroxysuccinimide (NHS) and Oleic acid were
procured from Sigma-Aldrich Chemicals Pvt. Ltd. (Bangalore, India) and Signet Chemical Corp. Ltd
(Mumbai, India), respectively. Hydrogen peroxide (30% w/v in water) was purchased from Loba Chemie
Pvt. Ltd (Mumbai, India). Pancreatin extrapure and Pepsin (3000 FCC U/mg) were obtained from Sisco
Research Laboratories Pvt. Ltd. (Mumbai, India). Absolute ethanol AR grade was purchased from
Changshu Honsheng Fine Chemical Co., Ltd. (Jiangsu, China). All Other organic solvents and chemicals
were used without further purification and were of analytical grade.
Healthy female Sprague Dawley (SD) rats weighing 180-200 g were used for in vivo pharmacokinetic
evaluation, all the animal study procedures were approved by the Institutional Animal Ethics Committee
(IAEC), National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Punjab
(IAEC/17/68; Dated 22-12-2017). Throughout the experiments, all the rats were kept in plastic cages and
maintained under standard laboratory conditions with a natural 12 h light and dark cycle.
2.2 Synthesis of OA-CMCS conjugate
The oleic acid- carboxymethyl chitosan conjugate (OA-CMCS) was produced by reaction between
carboxyl group of OA with the primary amino groups of low molecular weight CMCS, using EDC /NHS
as a coupling agent (Wang et al., 2019). Briefly, an ethanolic solution of OA, EDC and NHS (OA: EDC:
NHS~1:5:2 molar ratio) was prepared. The resulting reaction mixture was agitated for 30 min at
controlled room temperature condition to activate the carboxylic acid group of oleic acid. Subsequently,
the hydro-alcoholic (water: absolute ethanol ~9:1) solution of CMCS (1g, 0.045 mM) was prepared. The
ethanolic solution of OA was added dropwise into above CMCS solution and stirred overnight. The
resulting product was purified by using dialysis membrane (MWCO 12 kDa HIMEDIA®) against
distilled water for 3 days. Finally, OA-CMCS conjugate was obtained by freeze drying.

2.3 Characterizations of OA-CMCS conjugate
2.3.1 FT-IR and 1H-NMR
The chemical structure of OA-CMCS conjugate was characterized by FT-IR and 1H-NMR spectroscopy.
The FT-IR spectra of low molecular weight CMCS, Oleic acid and OA-CMCS conjugate were performed
using IR spectrophotometer (Perkin Elmer Corp, UK) with aid of KBr pellets. The scanning wavenumber
1
range was from 400 to 4000 cm-1. The H-NMR spectra were recorded in CDCl3 by employing NMR
spectrometer (Bruker 400 MHz UltraShieldTM, Germany) using tetramethylsilane as an internal standard.
2.3.2 Measurement of critical micelle concentration
To determine the critical micelle concentration (CMC) of OA-CMCS in aqueous solution, pyrene as a
fluorescent probe method was employed. All the measurements were performed by using fluorescence
spectrometer (Varian CARY-Eclipse, Victoria, Australia). In 10 mL volumetric flasks sequentially
definite amount of pyrene solution (6.0 x 10-6 mol/L) in acetone were prepared, followed by evaporation
of acetone under reduced pressure at 40 °C for 2 h. The various OA-CMCS conjugate suspensions
(concentration ranged from 10 ng/mL to 1 mg/mL), were added to each 10 mL volumetric flasks. The
solutions were kept on ultrasonic bath at 37 ºC for 24 h to attain equilibration. Emission spectra were
recorded using fluorescence spectrophotometer. The probe was excited at 333 nm, and the emission
spectra were obtained in the range of 345–500 nm. The peak intensities I1 (378 nm) and I3 (394 nm) were
used to calculate critical micelle concentration.
2.4 Preparation of polymeric micelles
Docetaxel (5 mg) was dissolved in 12 mL of ethanol. Meanwhile, the OA-CMCS conjugate (20 mg)
solution was prepared by dissolving in 30 mL of mixture of distilled water and ethanol (98:2, % v/v).
Then ethanolic solution of DTX was added into the polymeric solution in a dropwise fashion, followed
by sonication in a bath sonicator (WUC-9L, Wensar®, Mumbai, India) at 30 ± 0.5 °C for 20 min. The
dispersion was further subjected to vacuum (Rotavapor R-210, BUCHI Labortechnik AG, Switzerland)
to remove the traces of organic solvent. The resultant dispersion was centrifuged (Sigma 3K30, SIGMA
Laborzentrifugen GmbH, Germany) at 10,000 rpm for 10 min to remove insoluble DTX. The obtained
polymeric micelles were refrigerated for further use. The blank micelles were also prepared in a similar
manner, but without adding DTX.
2.5 Characterization of polymeric micelles
2.5.1 Micelle size and zeta potential
Dynamic light scattering (DLS) method was used to measure average micelle size and size distribution of
the blank and DTX loaded OA-CMCS micelles. The diluted colloidal solution of polymeric micelles

were prepared in deionized water and subjected to DLS analysis using Zeta sizer (Nano ZS, Malvern
Instruments, UK). The zeta potential of the micelles with the same concentration was determined in
deionized water solution by the Zetasizer.
2.5.2 Morphological observation
The morphological features of the DTX loaded OA-CMCS micelles were viewed under transmission
electron microscope (FEI Tecnai G2F20, Netherlands). Prior to investigation, the sample was negatively
stained with 1.5% w/v phosphotungstic acid and placed on copper grids with films.
2.5.3 Entrapment efficiency and drug loading
The entrapped drug in formulation was measured by HPLC (Waters 2695 Separations Module) with the
analytical column Phenomenex® C- 8 5 x 4 6 mm, 5 μm). The PDA detector (Waters 2696
photodiode array detector) was set at wavelength of 230 nm for DTX. The mobile phase, acetonitrile:
ammonium acetate buffer (65:35) was pumped in isocratic mode at a flow rate of 1.2 mL/min. briefly,
100 µL of micelles were accurately taken and volume was made up to 1 mL using acetonitrile to analyse
the drug content using HPLC. The entrapment efficiency (EE) and drug loading (DL) of DTX were
calculated by the following Eq. (1) and Eq. (2).
weight of in micelles
( )
Eq. (1)
Eq. (2)
weight of added
weight of in micelles
weight of polyme conjugate
( )
2.6 Stability of micelles in GI fluids
For evaluating the stability of DTX loaded OA-CMCS micelles in different physiological media i.e.
simulated gastric fluid (SGF pH 1.2) with or without pepsin, simulated intestinal fluid (SIF pH 6.8) with
or without pancreatin were taken. It was also assessed in the presence of phosphate buffer (pH 5.5). In
these medium μ of micella solutions we e diluted up to m and incubated fo h in SGF with o
without pepsin, and for 6 h in SIF with or without pancreatin and phosphate buffer media. The samples
were analysed for particle size and PDI subsequent to incubation.
2.7 In vitro drug release studies
In vitro release profiles of DTX from the DTX loaded OA-CMCS were examined by reported dialysis
bag method with some modification (Dhaundiyal et al., 2016). The solution of DTX loaded OA-CMCS
micelles (2 mL) was added to each dialysis bag (MWCO 14 kDa) and immersed in 25 mL release

mediums. The simulated gastric fluid (SGF pH 1.2) with or without pepsin, simulated intestinal fluid
(SIF pH 6.8) with or without pancreatin were used as release medium in this study. The tests were
conducted in water bath shaker (EQUITRON® Medica Instrument Mfg. Co., India), which was
maintained at 37 ± 0.5 °C and shaken horizontally at 100 rpm. At predetermined time intervals (0.5, 1, 2,
4, 6, 8, 12, 24, 48 and 72 h), sample (1 mL) of the release medium was collected and replaced with 1 mL
of fresh medium to maintain sink condition. The drug concentration in samples was assessed by HPLC
procedure.
2.8 In vitro permeability study
2.8.1 Cell culture
Caco-2 cells were procured from National Centre for Cell Science (NCCS), India and were grown in
tissue cultured flasks (75 cm2) and maintained in humidified 5% CO2 atmosphere at 37 °C. The
ulbecco’s modified Eagle’s medium DMEM) was used as growth medium, that were supplemented
with 100 IU/mL penicillin, 10% fetal bovine serum (FBS), and μg/m st eptomycin At every
alternate day, the growth medium was changed and growing cells were introduced to fresh media.
2.8.1 Permeability assay
Caco-2 cells were harvested and seeded in 96-well plates (Corning Incorporated Costor, Kennebunk,
USA) at a density of 75,000 cells per well then grown as a confluent monolayer by incubating at 37 ºC
in 5% CO2 humidified chamber for 21 days. The growth media were changed with fresh one at every
alternate day. The trans-epithelial electrical resistance (TEER) value was calculated every alternate day
so as to evaluate the monolayer integrity which was calculated by following Eq. (3).
( )
Eq. (3)
Wherein RM is the resistance of filter insert with cells, RB is the resistance of the filter alone and A is the
surface area in cm2. For permeability study, the TEER value should be g eate than 4 Ω cm2.
Before performing to the permeability assay, the cell monolayers were washed twice with phosphate
buffer saline (pH 7.4) to remove traces of culture media. Thereafter, the cells were equilibrated in HBSS
for 30 min at 37 °C. The transport studies of free DTX and DTX loaded OA-CMCS micelles were
conducted at apical to basolateral (A→B) and basolateral to apical (B→A) directions as per previously
reported method (Metre et al., 2018). For A to B t anspo t A→B), 5 μ ) and DTX loaded OA-
CMCS micelles (equivalent to 50 µM of DTX) in HBSS buffer with MES 2.5 mM, pH 6.5, were added
apically 5 μ ), and 6 μ of blank HBSS buffe containing HEPES 5 m , pH 7 4, was added to
basolateral side. For B to A t anspo t B→A), samples were placed in basolateral and concentration in
the apical side was measured. The P-gp efflux inhibition study was also performed in the presence of
verapamil (100 μ ) a well-known P-gp inhibitor. Samples were withdrawn from the apical and

basolateral side at 15, 30, 60, 90 and 120 min and volume withdrawn was replaced with blank buffer
solution each time. The apparent permeability coefficients, Papp (cm/s), for both A→B and B→A studies
were calculated from following Eq. (4).
Papp = (dQ/dT)/C0A
Eq. (4)
Wherein dQ/dT is the slope obtained by best fit line of cumulative transported amount Vs time, A is the
surface area (cm2) of the filters or inserts and C0 is the initial drug concentration (µM) in donor
compartment. The efflux ratio (ER) was calculated from the following Eq. (5).
ER = Papp (B-A)/Papp (A-B)
Eq. (5)
2.9 Pharmacokinetic study
Prior to the experimentation, Sprague Dawley rats randomly distributed into two groups with five rats in
each group. Group I received oral administration of free DTX suspension (DTX suspended in 0.25% w/v
sodium carboxymethyl cellulose) and group II received an oral administration of DTX loaded OA-CMCS
micelles in double distilled water, at a dose equivalent to 10 mg/kg body weight of DTX. The blood
samples were collected from the tail vein at predetermined time intervals of 0.5, 1, 2, 4, 6, 8, 12, 24, 48
and 72 h after administration of the drug and transferred to heparinized tubes. The blood samples were
centrifuged at 11,000 rpm for 10 min and then separated plasma were stored at -20 °C for HPLC
analysis.
2.9.1 HPLC analysis
To determine plasma drug concentrations, approximately μ of plasma was mixed with 5 μ of
simvastatin μg/m ) solution inte nal standa d) and vortexed for 1 min. Thereafter, acetonitrile
(requi ed volume) and methanol 5 μ ) were added as protein precipitating agent. The mixture was
vortexed for 5 min, followed by centrifugation for 10 min at 6000 rpm. Lastly, 40 µL of supernatant was
injected into the HPLC system after being filtered th ough 5 μm membrane filter. Analysis was
performed by isocratic elution using stationary phase (Agilent eclipse XDB-C18, 5 μm, 250 × 4.6 mm)
and mobile phase (55% v/v acetonitrile and 45% v/v water) at flow rate of 0.8 mL/min. The plasma
calibration plot for docetaxel was represented in Fig. S1 and different chromatograms of blank plasma,

blank plasma spiked with an internal standard and actual plasma sample taken from rats was summarized
in Fig. S2.
2.10 Statistical analysis
The data are represented as the mean ± standard deviation (SD) and the difference between two groups
was analysed via one way ANOVA test. The p value < 0.05 indicated statistical significance in results.
3. RESULT AND DISCUSSION
3.1 Synthesis and characterization of OA-CMCS conjugate
Detailed synthetic route of OA-CMCS conjugate was summarized in Fig. 1. The OA-CMCS conjugate
was synthesized via amide bond formation between carboxyl groups of OA with amine group of CMCS.
The chemical structure of newly synthesized conjugate was analyzed using FT-IR represented in Fig. 2.
In the CMCS spectrum, the both characteristic bands of symmetric and asymmetric vibration of carboxyl
group (-COO-) were apparent at 1432.12 cm-1 and 1624.15 cm-1 respectively. The IR spectra of OA
exhibit absorbance peaks at 2922.68 cm-1 and 2853.73 cm-1 assigned to alkane stretching (CH2-CH2) and
the absorbance at 1706.66 cm-1 is attributed to C=O stretching (acid). The presence of characteristic
peaks at 1638.02 cm-1 in the conjugate clearly demonstrates the amide bond formation between CMCS
and OA (El-Sherbiny, 2010; Wang et al., 2019). The structure of the conjugate was further confirmed by
1H-NMR spectra and spectrum for CMCS, OA and OA-CMCS conjugate were shown in Fig. 3. The
peaks were between δ 3 3 to δ 3 9 attributed to the presence of ring methine protons of CMCS. The
characteristic peak of CMCS were observed at δ 1.96 and δ 4 8 ppm which were attributed to the acetyl
protons substituted on the C2 and the C6 positions, respectively (Chen and Park, 2003; Yinsong et al.,
2007). The spectrum of oleic acid in CDCl3 at 4 Hz showed peaks at δ values at 5.3 ppm
corresponding to the olefinic protons (alkene protons) and the corresponding signals of the rest of the
p otons, below δ 3 Additionally, two methylene p otons adjacent to the unsatu ation we e obse ved at
δ ppm and the te minal methyl g oup p otons appea ed at δ 8 to 9 (Lee and Na, 2020; Sharma
and Singh, 2017). The appearance of characteristics olefinic protons peak (δ value at 5.3 ppm) of oleic
acid in OA-CMCS conjugate spectra evident that grafting of oleic acid on carboxymethyl chitosan. Also,
there was an overlap of methylene proton adjacent to double bond with the acetoamido group protons
1
singlet at δ 96 ppm), esulting into b oadening of the peak in H-NMR spectra of OA-CMCS
conjugate . The results of FT-IR and 1H-NMR spectrum suggest the successful conjugation between OA
and CMCS.

3.2 Measurement of critical micelle concentration
The CMC of the conjugate was determined by using pyrene as a probe in fluorescence spectrometry. As
pyrene is a hydrophobic molecule, preferentially partition into the interior of the hydrophobic domains.
As the polarity of the media decline, the ratio decreases and when the concentration exceeds a certain
level it changes considerably, indicating the partitioning of pyrene between the aqueous and hydrophobic
domains. The fluorescence intensity changes due to the change in solvent polarity surrounding pyrene
molecules. Fig. 4 depicts a typical plot of intensity ratio (I378/I394) vs. log C of OA-CMCS. The CMC
value of OA-CMCS conjugate was found to be 1 µg/mL. The lower CMC value of OA-CMCS conjugate
indicates micelle formation at low concentration and stability in highly diluted environment (Du et al.,
2009).
3.3 Characterization of polymeric micelles
3.3.1 Micelle size distribution and zeta potential
The micelle size and zeta potential value of the blank micelles and DTX loaded OA-CMCS micelles are
shown in Table 1. It is observed that DTX loaded OA-CMCS micelles have increased micelle size due to
the incorporation of DTX. However, zeta potential of blank micelles and DTX loaded OA-CMCS
micelle do not show significant change indicating stability after DTX incorporation. Fig. 5A shows
micelle size distribution graph for DTX loaded OA-CMCS micelles. The polydispersity index of blank
micelles and DTX loaded OA-CMCS micelle, estimated by Zetasizer (Nano ZS, Malvern Instruments,
UK), were fairly low (~0.2), which suggested homogenous size distribution of micelles.
3.3.2 Morphological characterisation
The TEM images of DTX loaded OA-CMCS micelle was represented in Fig. 5B. The spherical and
homogenous size distribution was observed for DTX loaded OA-CMCS micelle which proves self
aggregation formation in aqueous media.
3.3.3 Entrapment efficiency and drug loading
The percent encapsulation efficiency (EE) and drug loading (DL) are significant factor for delivery of
hydrophobic drugs. The EE (%) and DL (%) of DTX loaded OA-CMCS micelle determined by HPLC is
shown in Table 2. The feed concentration ratio of the drug to polymer altered the amount of entrapped
drug in micellar system. The encapsulation efficiency of DTX loaded OA-CMCS micelle was increased
from 35% to 57% along with increase in the weight ratio of DTX to OA-CMCS from 1:2 to 1:4. Further
increasing the drug feeding ratio (1:4 to 1:8), did not increased encapsulation efficiency. The maximum
EE (%) and DL (%) were observed when the feed ratio of DTX to the OA-CMCS polymer was 1:4.

3.4 Stability of micelles in GI fluids
The GI stability of DTX loaded OA-CMCS micelles were investigated in different dilution medium such
as SGF and SIF with or without enzymes to mimic the in vivo gastrointestinal conditions. As shown in
Fig. 6, micelle size of DTX loaded micelles shows slight differences when formulation is incubated in
SGF without pepsin and SGF with pepsin enzyme for 2 h. The zeta potential of micelle was shifted from
-28.266 ± 1.00 mV to +6.11 ± 1.71 mV due to the cationic nature of unreacted amino groups of CMCS
that were protonated at acidic pH (Ubaidulla et al., 2007). However, the mean micelle size was increased
when incubated in SIF and SIF containing pancreatin for 6 h. That might be ascribed to the formation of
loose aggregates due to catalytic action of amylase on CMCS (Fattahi et al., 2012). The PDI values of
micelles did not differ markedly from their initial values after incubation in different medium which
indicates stability of micelles in gastrointestinal environments.
3.5 In vitro drug release studies
In vitro release profiles of DTX from the DTX loaded OA-CMCS micelles were examined by dialysis
bag method in different release mediums. It was observed that the pH value of release medium alter the
release kinetic of DTX loaded OA-CMCS micelle. As shown in Fig. 7A, the DTX loaded OA-CMCS
micelle indicated faster release in first 0.5 h, approximately 11.7 ± 0.30 % of the DTX was rapidly
released into acidic media of SGF pH 1.2 with or without pepsin and then achieved plateau after 12h.
After the initial burst release, micelles displayed sustain release behaviour and only 65.45 ± 0.92% of
encapsulated drug released from micelles over 72 h. The initial rapid drug release in acidic environment
could be contributed to formation of loose micelle structure, due to the stronger protonation of free amino
groups of CMCS in SGF medium (pH 1.2) and released non-encapsulated drug portion (Ubaidulla et al.,
2007). However, the release profile of micelles in SGF with pepsin (pH 1.2) shows similar sustained
release pattern and only about 66.25 ± 1.04 % release over 72 h. In Fig. 7B, the DTX loaded OA-CMCS
micelles exhibited slow and sustained release characteristics in SIF without pancreatin, (pH 6.8), i.e.
released only about 9.96 ± 0.33 % within 0.5 h, and about 60.80 ± 1.60 % within 12 h and then attained
plateau over 24 h. The DTX loaded OA-CMCS micelles shows burst release in SIF (pH 6.8) with
pancreatin release media, about 18.19 ± 0.46 % of the DTX was released from within first 0.5 h and
nearly about 73.31 ± 2.32 % of encapsulated DTX was released in 72 h. The initial burst release of DTX
is ascribed to the loose aggregates formation due to catalytic action of amylase on CMCS in presence of
pancreatin (Fattahi et al., 2012).
3.6 Permeability assay
In order to evaluate permeability, Caco-2 cell transport experiment was performed and the Papp of DTX
and DTX loaded OA-CMCS micelle from A→B transport and B→A efflux were compared (Table 3).
As compa ed to cont ol, the abso ptive t anspo t A→B) of in loaded OA-CMCS

micelle was enhanced by 6.57-fold which suggesting that polymeric micellar system could enhance the
oral absorption of DTX. Impact of active transport was studied by carrying out the transport studies in
the opposite direction, B→A (Metre et al., 2018). The B→A Papp coefficient for DTX was higher than the
A→B Papp coefficient indicating that DTX as a substrate of P-gp. In the presence of verapamil, DTX
shows significant decrease in B→A Papp coefficient which suggests effect of P-glycoprotein suppression.
There is insignificant difference between the A→B and B→A Papp values for DTX loaded OA-CMCS
micelles, implying the suppression of P-gp. The mean efflux ratio for DTX, DTX plus verapamil and
DTX loaded micelles were calculated and presented in Fig. 8. DTX loaded micelles shows significant
decrease in efflux ratio by 5.5-fold as compare to free DTX. The results herein found that DTX loaded
micelles enhance the permeation of drug across epithelium. Two effects can be considered: Firstly,
CMCS possess negative charges thus make strong binding capacity to Ca2+ from adherens junctions. This
property could deprive the divalent ions from extracellular matrix and increase the paracellular
permeability of the epithelium (Jena et al., 2017). Secondly, OA has permeation enhancing and P-gp
inhibition activity which might be a reason for enhanced permeation of DTX from micelles (Kobayashi
et al., 1996; Komarov et al., 1996).
3.7 Pharmacokinetic study
The plasma concentration-time curve of docetaxel following oral administration of docetaxel suspension,
DTX loaded OA-CMCS micelles to Sprague-Dawley rats is shown in Fig. 9 and the related
pharmacokinetic parameters are illustrated in Table 4. As shown in Fig. 9, DTX suspension has shown
limited maximum drug plasma concentration (Cmax) of 0.0708±0.0122 µg/mL and area under curve
(AUC0-72 h) of 1.8168±0.4272 µg.h/mL. As compared to DTX-suspension, Cmax and AUC (0-72h) was 1.97
fold and 2.62 fold higher for DTX loaded OA-CMCS micelle, respectively. From the Fig. 9 it is also
evident that the time required to reach the maximum plasma concentration (tmax) was increased from 6 h
for DTX-suspension to 8 h for DTX loaded OA-CMCS micelle (formulation), which might be due to
sustained release of drug from the micelle. Similarly, MRT of the micellar system was 2.12 fold greater
than that of the DTX-suspension and the elimination half-life (t1/2) increased from 31.25 h (DTX-
suspension) to 60.92 h (DTX loaded OA-CMCS micelle), thus 1.94 times increase was observed which
indicates that drug remains in the body for longer period of time to show its action. These results
revealed that encapsulation of DTX in OA-CMCS micelles significantly enhanced the intestinal
absorption of DTX. The inference from the pharmacokinetic parameters that could be drawn is that
CMCS even at highly diluted gastrointestinal conditions achieved stable configuration. Also, it increased
the permeability of the drug through mucosal epithelium increasing intestinal absorption via paracellular
absorption. The in vivo metabolism was delayed by micelles by protecting the drug from enzymatic as
well as chemical degradation (Song et al., 2011).

4. CONCLUSION
In this study, we have synthesized amphiphilic conjugate based polymeric micelles composed of LMW
CMCS and OA for encapsulation of DTX and enhancing its oral bioavailability. OA-CMCS conjugate
was prepared by the modification of CMCS with OA via amidation reaction and characterized by using
spectroscopic techniques. OA-CMCS conjugate confirmed that it can easily self-organized into micelles
like structure in an aqueous medium and showed a low critical micellar concentration of 1 µg/mL. The
DTX was encapsulated in OA-CMCS conjugate by co-solvent evaporation method. The developed
micelle was proven to be homogenous spherical shape with uniform size range (~215 nm). Furthermore,
the DTX loaded OA-CMCS micelles showed excellent stability in simulated body fluid of different pH.
In vitro release study showed that DTX loaded OA-CMCS micelles exhibited delayed release profile in
simulated body fluids. Furthermore, the permeation study results demonstrated that DTX loaded
polymeric micelles showed augmented intestinal absorption behaviour with decreased efflux ratio in
comparison of free DTX. In addition, pharmacokinetic studies indicated that DTX loaded OA-CMCS
micelles showed significant improvement in oral absorption as compared to DTX-suspension. Overall,
the results of this investigation demonstrate that amphiphilic carboxymethyl chitosan derivatives could
be a promising carrier for poorly water soluble drug for enhancing biopharmaceutical performance.
Moreover, OA-CMCS might be potential carrier for oral absorption of P-gp substrate molecules.
Author Contributions
Rahul Kumar: Conceptualisation, methodology, investigation, data curation, validation
Arvind Sirvi: Manuscript writing-original draft, formal data analysis,
Shamandeep Kaur: Conceptualisation, data curation, formal data analysis, supervision
Sanjaya K. Samal: Methodology (permeability study)
Sabyasachi Roy: Methodology (HPLC method development)
Abhay T. Sangamwar: Conceptualisation, data review, validation, project administration, writing-review
and editing
Acknowledgement
Authors would like to acknowledge Director, NIPER S.A.S. Nagar for providing necessary facilities and
infrastructure. Shamandeep Kaur is grateful to CSIR, India for providing research fellowship. Authors
also would like to acknowledge Ms. Harshita Tanwar for valuable help.
Note
The authors declare no competing financial interest.

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List of Figures
Figure 1 Synthesis scheme of Oleic acid-Carboxymethyl chitosan conjugate.
Figure 2 Infra-red spectra of CMCS, Oleic acid, and OA-CMCS Conjugate.
Figure 3 1H-NMR spectra of CMCS, Oleic acid, and OA-CMCS Conjugate.
Figure 4 Intensity ratio plot of I378/I394 of pyrene versus log C for OA-CMCS conjugate.
Figure 5 Physicochemical characterization of micelles: A) micelle size distribution of micelles size
distribution, and B) TEM image of DTX loaded OA-CMCS micelles.
Figure 6 Stability of DTX loaded OA-CMCS micelles in different gastrointestinal dilution medium (Data
are means ± standard deviation (n=3).
Figure 7 In vitro release of DTX loaded OA-CMCS micelles in (A) SGF, pH 1.2 with or without pepsin,
and (B) SIF, pH 6.8 with o without panc eatin at 37 ± ℃ ata ep esents mean ± S n =
3).
Figure 8 Mean efflux ratio of DTX, DTX plus verapamil and DTX loaded OA-CMCS micelle.
Figure 9 Plasma concentration vs time profile of DTX after oral administration of 10 mg/kg dose of DTX
suspension and DTX loaded OA-CMCS micelles. (Each data represented as mean+ standard
deviation of 5 rats)

Figure 1
HO
O
O
Oleic acid
EDC, NHS
Ethanol
RT, 30 min
O
N
O
O
OCH2COOH
O
Ethanol/Water
RT, 24h
O
n
NH2
HO
O
HOOCH2CO
O
NH
OH
O
Oleic acid-CMCS Conjugate
Fig. 1 Synthesis scheme of Oleic acid-Carboxymethyl chitosan conjugate.

Figure 2
Fig. 2 Infra-red spectra of CMCS, Oleic acid, and OA-CMCS Conjugate

Figure 3
Fig. 3 1H-NMR spectra of CMCS, Oleic acid, and OA-CMCS Conjugate

Figure 4
Fig. 4 Intensity ratio plot of I378/I394 of pyrene versus log C for OA-CMCS conjugate.

Figure 5
B
A
Fig. 5 Physicochemical characterization of micelles: A) micelle size distribution of micelles size
distribution, and B) TEM image of DTX loaded OA-CMCS micelles.

Figure 6
350
300
250
200
150
100
50
0.5
0.4
0.3
0.2
0.1
0
Particle size (nm)
PDI
0
SGF pH-1.2 SGF pH-1.2 SIF pH-6.8 SIF pH-6.8
Phosphate
without
pepsin
with pepsin
without
pancreatin
with
pancreatin
buffer pH-5.5
Fig. 6 Stability of DTX loaded OA-CMCS micelles in different gastrointestinal dilution medium (Data are
means ± standard deviation (n=3).

Figure 7
B
A
Fig. 7 In vitro release of DTX loaded OA-CMCS micelles in (A) SGF, pH 1.2 with or without pepsin, and
B) SIF, pH 6 8 with o without panc eatin at 37 ± ℃ ata ep esents mean ± S n = 3)

Figure 8
7
6
5
4
3
2
1
0
DTX
DTX + Verapamil
DTX loaded PMs
Fig. 8 Mean efflux ratio of DTX, DTX plus verapamil and DTX loaded OA-CMCS micelle.

Figure 9
Fig. 9 Plasma concentration vs time profile of DTX after oral administration of 10 mg/kg dose of DTX
suspension and DTX loaded OA-CMCS micelles. (Each data represented as mean+ standard
deviation of 5 rats)

List of Table
Table 1 Characterization of blank and DTX loaded OA-CMCS micelles
Table 2 Effect of feed weight ratio (DTX-polymer) on physicochemical parameters of DTX loaded OA -
CMCS micelles
Table 3 Apparent permeability coefficient of DTX, DTX plus verapamil, and DTX loaded OA-CMCS
micelles. All values are means ± S.D. (n=3)
Table 4 Pharmacokinetic parameters of DTX suspension and DTX loaded OA-CMCS micelles upon
single oral administration at dose equivalent to 10 mg/kg body weight of DTX to Sprague-
Dawley rats. All values are means ± S.D (n=5).

Table 1
Table 1; Characterization of blank and DTX loaded OA-CMCS micelles
Micelles
Micelle Size (nm)
PDI
Zeta potential (-mV)
140.05±5.52
213.40 ± 9.68
0.207±0.013
0.191 ± 0.068
36.7±2.64
Blank micelle
34.23 ± 3.35
DTX loaded OA-CMCS
micelle