Modulating the site-specific oral delivery of sorafenib using sugar-grafted nanoparticles for hepatocellular carcinoma treatment

Article abstract of DOI:10.1016/j.ejps.2019.104978

Globally, one in six deaths is reported due to cancer suggesting the critical need for development of advanced treatment regimens. In this study, solid lipid nanoparticles (SLN) were prepared and appended with polyethylene glycol (PEGylated) galactose and a multikinase inhibitor sorafenib (SRFB) was used as chemotherapeutic drug, for treating hepatocellular carcinoma (HCC). The nanoparticles were evaluated for in-vitro and in-vivo performances to showcase the targeting efficiency and therapeutic benefits of the sorafenib loaded ligand conjugated nanoparticles (GAL-SSLN). When compared with SRFB or Sorafenib loaded SLN, GAL-SSLN showed superior cytotoxicity and apoptosis in HepG2 (human hepatocellular carcinoma cells). In addition, in-vivo pharmacokinetics and real time biodistribution studies in BALB/c mice showed that the surface conjugation of nanoparticles with galactose resulted in better pharmacokinetic performance and targeted delivery of the nanoparticles to liver. Results indicated that GAL-SSLN showed promising attributes in terms of targeting sorafenib to liver and therapeutic efficacy.

Full text of DOI:10.1016/j.ejps.2019.104978

Accepted Manuscript
Modulating the site-specific oral delivery of sorafenib using
sugar-grafted nanoparticles for hepatocellular carcinoma
treatment
Lakshmi Tunki, Hitesh Kulhari, Lakshma Nayak Vadithe,
Madhusudana Kuncha, Suresh Bhargava, Deep Pooja,
Ramakrishna Sistla
PII:
DOI:
Article Number:
Reference:
S0928-0987(19)30241-6
https://doi.org/10.1016/j.ejps.2019.104978
104978
PHASCI 104978
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
6 April 2019
7 June 2019
25 June 2019
Please cite this article as: L. Tunki, H. Kulhari, L.N. Vadithe, et al., Modulating the site-
specific oral delivery of sorafenib using sugar-grafted nanoparticles for hepatocellular
carcinoma treatment, European Journal of Pharmaceutical Sciences, https://doi.org/
10.1016/j.ejps.2019.104978
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Modulating the site-specific oral delivery of sorafenib using sugar-grafted
nanoparticles for hepatocellular carcinoma treatment
Lakshmi Tunkia,c, Hitesh Kulharib, Lakshma Nayak Vadithea, Madhusudana Kunchaa, Suresh
Bhargavac, Deep Poojaa,*, Ramakrishna Sistlaa,*
aApplied Biology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad,
Telangana 500007, India
bSchool of Nano Sciences, Central University of Gujarat, Gandhinagar, Gujarat 302030, India
cCentre for Advanced Materials and Industrial Chemistry, School of Science, RMIT University,
Melbourne, VIC 3001, Australia
Corresponding Author
*Email: sistla@iict.res.in, sistla@csiriict.in (R.S.), d.dpooja00@gmail.com (D.P.)
CSIR-IICT communication no: IICT/Pubs./2019/027.
ABSTRACT
Globally, one in six deaths is reported due to cancer suggesting the critical need for development
of advanced treatment regimens. In this study, solid lipid nanoparticles (SLN) were prepared and
appended with polyethylene glycol (PEGylated) galactose and a multikinase inhibitor sorafenib
(SRFB) was used as chemotherapeutic drug, for treating hepatocellular carcinoma (HCC). The
nanoparticles were evaluated for in-vitro and in-vivo performances to showcase the targeting
efficiency and therapeutic benefits of the sorafenib loaded ligand conjugated nanoparticles
1

ACCEPTED MANUSCRIPT
(GAL-SSLN). When compared with SRFB or Sorafenib loaded SLN, GAL-SSLN showed
superior cytotoxicity and apoptosis in HepG2 (human hepatocellular carcinoma cells). In
addition, in-vivo pharmacokinetics and real time biodistribution studies in BALB/c mice showed
that the surface conjugation of nanoparticles with galactose resulted in better pharmacokinetic
performance and targeted delivery of the nanoparticles to liver. Results indicated that GAL-
SSLN showed promising attributes in terms of targeting sorafenib to liver and therapeutic
efficacy.
KEYWORDS
PEGylated galactose, Solid lipid nanoparticles, Sorafenib, Hepatocellular carcinoma,
Bioavailability, Targeting.
Graphical abstract
2

ACCEPTED MANUSCRIPT
ABBREVIATIONS
1HNMR, Proton nuclear magnetic resonance; DMEM, Dulbecco’s modified eagle medium;
DMSO, Dimethyl sulfoxide; DPBS, Dulbecco's phosphate buffered saline; DSC, Differential
scanning calorimetry; EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; EE,
Encapsulation efficiency; FBS, Fetal bovine serum; FTIR, Fourier transform infrared
spectroscopy; HPLC, High performance liquid chromatography; IC50, Half-maximal inhibitory
concentration; MTT, 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; NHS, N-
hydroxysuccinimide; PBS, phosphate buffer saline; SAB, Sodium acetate buffer; SD, Standard
deviation; VEGF, Vascular endothelial growth factor; SLN, Solid lipid nanoparticle; SRFB,
Sorafenib; SSLN, SRFB loaded SLN; GAL-SSLN, SRFB loaded PEGylated galactose
conjugated SLN; HepG2, human hepatocellular carcinoma cells; HCC, hepatocellular
carcinoma; IARC, International Agency for Research on Cancer; PEG, Polyethylene glycol;
CTLR, C-type lectin receptors; ASGPR, asialoglycoprotein receptor ligand; PDGF, platelet-
derived growth receptors; GMS, Glyceryl monostearate; BSLN, Blank SLN; C6, coumarin-6;
JC-1,
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine
iodide;
IAEC,
Institutional Animal Ethics Committee; CPCSEA, Committee for the Purpose of Control and
Supervision of Experiments on Animals; ACN, Acetonitrile; MeOH, Methanol; Cmax, maximum
plasma concentration; AUC, area under the curve; AUMC, area under the first moment curve
plasma; tmax, time to reach peak plasma concentration; t1/2, half-life; MRT, mean residence time;
DiR, 1, 1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide; ANOVA, Analysis of
variance.
3

ACCEPTED MANUSCRIPT
1. Introduction
According to GLOBOCAN 2018 cancer statistics, International Agency for Research on
Cancer (IARC) estimated about 18.1 million new cancer cases and 9.6 million cancer deaths
across the world in 2018. Among these, liver or hepatocellular carcinoma would account for
8.2% of mortality rate (Bray et al., 2018). There is an increasing global burden of HCC and is
expected to increase to 1 million cases per year in the coming years. Sorafenib (SRFB) is a FDA
approved oral multikinase inhibitor and is used as first line chemotherapeutic treatment for
patients with advanced HCC (Llovet et al., 2018). SRFB inhibits tumor cell proliferation by
downregulating Ras oncogene activity and prevents angiogenesis via inhibiting vascular
endothelial growth factor (VEGF) and platelet-derived growth (PDGF) receptors thus the
progression of tumor is ceased (Wilhelm et al., 2006).
The major pitfall of conventional use of SRFB is its low aqueous solubility leading to
low oral bioavailability (~8.43%) and thus low therapeutic efficacy (Zhang et al., 2014). Further,
non-specific biodistribution of SRFB results in systemic adverse effects like gastrointestinal
disturbances, low blood flow through heart, heart attack, hand-foot skin reaction, alterations in
thyroid hormone levels, non-specific tissues uptake etc., affecting the quality life of patients and
disrupting the treatment which further lead to compromised treatment. This suggests the need for
development of an alternate delivery system that can take the edge off the above effects to large
extent if not completely (Cervello et al., 2017; Cheng et al., 2009).
The present advanced technology in the field of medicine is nanomedicine, which is in
boom in various fields of diagnosis, treatment and prevention. Nanomedicine claim to have
4

ACCEPTED MANUSCRIPT
inimitable physicochemical properties that can be used to improve both pharmacokinetic and
pharmacodynamic properties of the drug molecules (Wicki et al., 2015; Cai and Sheng, 2015).
The art of encapsulating the drug in carrier and then anchoring it with targeting ligand is one of
the current areas of research in cancer chemotherapy. This kind of approach is aimed at reducing
the adverse side effects by directing the drug to reach the target organ. In addition, nanoparticles
also release the encapsulated drug in controlled manner which helps in reducing the dose as well
as number of doses.
In recent years, various formulations of sorafenib loaded nanoparticles were prepared and
evaluated for anticancer efficacy both in-vitro and in-vivo. Yanhong Su et al., formulated
biocompatible micelles for SRFB delivery and showed better antitumor activity with prolonged
circulation time (Song et al., 2018). Xia sheng et al., prepared polymeric nanoparticles and
showcased that SRFB encapsulated in nanoparticles showed higher therapeutic efficacy than the
free SRFB (Sheng et al., 2017). In a study, anti-GPC3 antibody-tagged sorafenib loaded
nanoparticles were developed to inhibit the growth of liver xenograft tumours (Tang et al.,
2018). Melchiorre Cervello prepared nanoparticles loaded with sorafenib surface modified with
brush copolymer which showed enhanced permeability into tumor and thus, improved
therapeutic efficacy against Hep3B xenograft model in nude mice (Cervello et al., 2017).
In this present work, SRFB loaded solid lipid nanoparticles were formulated and to
impart selectivity to the nanoparticles towards HCC. Galactose, a C-type lectin receptors
(CTLR) binding ligand, was conjugated on the surface of nanoparticles. Asialoglycoprotein
receptors (ASGPR) are one among the highly expressed CTLR in liver as well as in HCC cells
and are known to least or minimally present on other cells (D’Souza and Devarajan, 2015).
Among the various carbohydrate receptor ligands like N-acetylgalactosamine, lactose, mannose,
5

ACCEPTED MANUSCRIPT
sialic acid, galactose etc, galactose is widely used as a targeting ligand against HCC because of
its high targeting affinity (Richard J. Stockert, 1995; Hashida et al., 2001; Li et al., 2016; Oh et
al., 2016; Kawakami and Hashida, 2014; Huang et al., 2018). Further, herein we synthesized and
used poly(ethylene glycol) conjugated galactose as a liver directing ligand. PEGylation of
nanoparticles with PEG having molecular weight of more than 2000 daltons offers the
advantages like impediment of particle aggregation, and escaping their uptake by
reticuloendothelial cells and thus, the increase in their circulation half-life (Ding et al., 2014;
Otsuka et al., 2003). Therefore, a liver targeting, biocompatible drug delivery system was
designed to improve the oral delivery and efficacy of SRFB in the form of SSLN and GAL-
SSLN to treat HCC.
2. Materials and methods
2.1 Materials
Glyceryl monostearate (GMS) was obtained from Alfa Aesar (Johnson Matthey
Chemicals, Hyderabad, India). Dialysis tubing (molecular weight cut off 12–14 and 2 kDa),
thiazolyl blue tetrazolium bromide (MTT), stearic acid, N—(3—dimethylaminopropyl)—N’—
ethylcarbodiimide (EDC), N—hydroxysuccinimide (NHS), poly(ethylene glycol) bis(amine)
average molecular weight of 3400, dimethyl sulfoxide (DMSO), Hoechst 33258 and annexin
V—FITC apoptosis detection kit were procured from Sigma–Aldrich (St., Louis, MO, USA).
Lecithin soy was acquired from Himedia (Mumbai, India). Sorafenib tosylate (SRFB) was a gift
from TherDose Pharma Pvt. Ltd., (Hyderabad, India). 1, 1′—Dioctadecyl—3,3,3′,3′—
6

ACCEPTED MANUSCRIPT
tetramethylindotricarbocyanine iodide (DiR) was procured from Biotium (Hayward, CA). High
performance liquid chromatography (HPLC) grade solvents such as chloroform, methanol and
acetonitrile were purchased from Merck limited (Mumbai, India). HepG2 human liver cancer
cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).
Dulbecco’s modified eagle’s media (DMEM), fetal bovine serum (FBS) and phosphate buffer
saline (PBS) were bought from Gibco, USA. Tween 80 and D (+) galactose were products of sd
Fine Chem Ltd. (Mumbai, India). Remaining chemicals and solvents were of analytical grade
and used further without any purification.
2.2 Synthesis of PEGylated galactose conjugate
For opening galactose ring molecules and to generate aldehyde group, D-galactose (8
µM) was dissolved in 0.1 M sodium acetate buffer (pH 4) and kept at ambient temperature for 4
h (Kumar et al., 2006). After that, ring opened galactose was reacted with PEG-bis (amine) in 1:2
molar ratio in 0.1 M sodium tetraborate buffer, stirred magnetically for two days at ambient
temperature. Finally, the formed PEGylated galactose was dialyzed using dialysis tubing of
molecular weight cutoff 2 kDa against MilliQ water to remove unreacted compounds,
lyophilized and stored at room temperature (Lim et al., 2000).
2.3 Characterization of PEGylated galactose conjugate
The formed conjugate was characterized using Fourier transform infrared analysis (FTIR)
analysis using a FTIR spectrophotometer (Perkin Elmer, UK). Samples were mixed with KBr
and compression pressed to form pellet. The pellet transmittance was measured by scanning
wave number over a range of 400 to 4000 cm-1. Proton NMR spectra of galactose, PEG-
7

ACCEPTED MANUSCRIPT
bis(amine) and PEGylated galactose conjugate were observed using BRUKER DPX 300MHz
spectrometer (Bruker, Karlsruhe, Germany). The samples were solubilized in D2O.
2.4 Preparation of solid lipid nanoparticles
Emulsification and solvent evaporation method was used to prepare SLN as reported
previously (Yuan et al., 2013). Briefly, 80 mg of GMS, 20 mg of stearic acid and 40 mg soy
lecithin were dissolved in 2 mL of chloroform, acting as lipidic organic phase, transferred to 10
mL of aqueous surfactant phase composed of 1.5% w/v of Tween 80. This dispersion was
homogenized (Ultra-turrax, Germany) at 11,000 rpm for 5 min. The formed coarse emulsion was
further comminuted using the force of sonic waves by probe sonicator (vibra Cell, Sonics, USA)
for 20 min. Finally, the obtained nanoemulsion was kept for stirring at 1000 rpm for 3 h on
magnetic stirrer for formation of nanoparticles through solvent evaporation process. SLN were
collected by centrifugation at 13,000 rpm for 45 min using centrifuge (Biofuge Stratos, Heraeus,
Germany) (Pooja et al., 2016b). For the preparation of SRFB loaded SLN (SSLN), 4 mg of
SRFB was dissolved in 0.2 mL of methanol and was incorporated into the organic lipidic phase.
2.5 Sorafenib encapsulation efficiency
Encapsulation efficiency of SRFB into SLN was determined by indirect method and
employing a HPLC system (Waters, USA). Supernatant obtained after centrifugation of
nanoparticle dispersion was used to determine the unencapsulated or free drug. The amount or
percentage of encapsulated SRFB in nanoparticles was calculated by means of the formula, %
encapsulation efficiency = [1-(Ds/Di)]×100, where Ds: amount of drug present in supernatant;
Di: initial amount of drug added to formulate the SSLN. Determination of SRFB content was
performed using reverse phase HPLC equipped with octadecylsilane (C18) column (Waters
8

ACCEPTED MANUSCRIPT
Reliant, 5 µm, 4.6 ×250 mm) maintained at a temperature of 25±5 °C and photodiode array
detector. A mobile phase containing mixture of acetonitrile and water (70:30, v/v), pumped at a
flow rate of 1.0 mL/min, injection volume 20 µL and detection was set at a wavelength of 265
nm.
2.6. Surface conjugation of PEGylated Galactose to SSLN using carbodiimide reaction
PEGylated galactose was conjugated with its free amine group onto carboxylic acid
group bearing SLN using carbodiimido coupling reaction (Pooja et al., 2016a). Briefly 50 mg of
lyophilized formulation was dispersed in PBS pH 7.4 and 1 mL each of 0.1 M EDC/NHS were
added and incubated for 2 h. SLN were then centrifuged at 13,000 rpm for 20 min to remove
EDC and NHS. The collected nanoparticles were then dispersed in PBS, PEGylated galactose
(50 mg) was added and conjugation was allowed by overnight incubation. The galactose
conjugated nanoparticles were separated by centrifugation at 13,000 rpm for 30 min.
2.7 Characterization of nanoparticles
Particle size, polydispersity index (PDI) and zeta potential were determined for the blank
solid lipid nanoparticles (BSLN), SSLN and GAL-SSLN using dynamic light scattering by
particle size analyzer Nano-ZS (Malvern Instrument Ltd., UK). DSC analysis was performed to
check the physical state of SRFB before and after encapsulation in SLN using DSC STAR ONE
(Mettler, Switzerland). Thermograms of free SRFB and SSLN were obtained by scanning the
samples (5 mg) sealed into aluminum pan from 30 °C to 300 °C at a speed of 10 °C/min, under
nitrogen environment. FTIR of SRFB, lyophilized SSLN and GAL-SSLN was performed using
FTIR spectrometer (Perkin Elmer, UK). Samples were prepared in KBr pellet and transmittance
was measured ranging from 400 to 4000 cm-1.
9

ACCEPTED MANUSCRIPT
2.8 In-vitro drug release
In-vitro drug release studies were carried using dialysis bag method (molecular weight
cutoff 12-14 kDa) and the release behavior of SRFB loaded formulations viz. SSLN and GAL-
SSLN were compared with free SRFB. Different release media like sodium acetate buffer (SAB)
pH 5.0, 0.1 N HCl pH 1.2, phosphate buffer pH 6.8 and saline (PBS) pH 7.4 were used for the
studies. SRFB, SSLN or GAL-SSLN, equivalent to 1 mg, were suspended in 1 mL of 0.5 %
Tween 80 solution, taken in dialysis bags and kept in 50 mL of drug release media containing
1% Tween 80 maintained at temperature of 37 ± 0.5 °C, were allowed to stir at 100 rpm. At
specified time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48 h), one mL of media was taken out and
replaced with fresh media to maintain the sink conditions. These samples were then used for
estimation of SRFB released into the media using HPLC instrument.
2.9 Anti proliferation assay
HepG2 human liver cancer cell were grown in DMEM (Dulbecco's Modified Eagle's
medium), supplemented with 10% fetal bovine serum (FBS) in CO2 incubator maintained at 37
°C and 5 % CO2. Cells after trypsinization were harvested and seeded at 1×104 cells/well in 96
well plate and allowed for adhesion by incubating for 24 h. After which the cells were treated
with SRFB formulations (SRFB, BSLN, SSLN, GAL-SSLN) at concentrations of 1, 5, 10, 25, 50
µg/mL and incubated for 48 h. Pure SRFB was dissolved in DMSO and further diluted with
DMEM medium for making up of various dilutions. Thiazolyl blue tetrazolium bromide (MTT)
at 5 mg/mL was added to each well at 10 µL volume and incubated further for 4 h, after which
the media was removed and DMSO was added at volume of 100 µL to each well and absorbance
was measured at 570 nm for the formed formazan crystals using multidetection microplate reader
10

ACCEPTED MANUSCRIPT
(Synergy-4, Biotek, USA). Untreated cells grown under similar conditions were used as control
for calculation of half-maximum inhibitory concentration (IC50) (Botta et al., 2007).
2.10 Nuclear fragmentation by Hoechst staining
HepG2 cells were seeded at a density of 10,000 cells over 18 mm cover slips and
incubated for 24 h. Cells were treated with SRFB, SLN or GAL-SSLN at 5 and 10 µg/mL
concentrations for 48 h. Hoechst 33258 was added to the cells at a concentration of 5 µg/mL and
incubated for 30 min at 37 °C. Cells were washed with PBS and were observed under fluorescent
microscope (Olympus microscope) to qualitatively determine the proportion of living and
apoptotic cells based on their relative fluorescence and nuclear fragmentation (Kumar et al.,
2017).
2.11 Annexin V-FITC assay
HepG2 (1×106) cells were seeded in six well plate and allowed to grow overnight. The
medium was then replaced with complete medium containing SRFB, SSLN and GAL-SSLN at 5
and 10 µg/mL concentrations. After 48 h of treatment, cells were harvested by trypsinization,
washed with PBS and centrifuged at 5000 rpm. Then the cells were stained with annexin V-FITC
and propidium iodide using the annexin V-FITC apoptosis detection kit. Flow cytometry was
used for analysis of apoptosis of the above treatments (Browne et al., 1991).
2.12 Cellular uptake study
Uptake of SLN formulations were studied by replacing the SRFB with a fluorescent
probe i.e. coumarin-6 (C6). About 1×106 cells were seeded in 6-wells plate and allowed for
adhering overnight in incubator. The cells were incubated with C6 alone, C6-SLN and C6-GAL
11

ACCEPTED MANUSCRIPT
SLN at a concentration of 5 µg/mL. At specific time intervals of 1, 2 and 4 h the cells were
observed for the uptake of nanoparticles using fully automated confocal laser-scanning
microscopy (Olympus FluoView FV10i) (Kadari et al., 2017).
2.13 Determination of mitochondrial membrane potential (∆Ψm)
HepG2 (1×106 cells/well) cells were cultured in six well plates. After seeding, cells were
treated with SRFB, SSLN and GAL-SSLN at 5 and 10 µg/mL concentrations. After 48 h of
treatment, cells were collected by trypsinization and washed with PBS followed by resuspending
in JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide) and incubated
at 37 °C for 15 min. Cells were then subjected to flow cytometric analysis on a flow cytometer in
the FL1, FL2 channel to detect mitochondrial membrane potential (Chakravarti et al., 2012).
2.14 Plasma pharmacokinetic study
In-vivo pharmacokinetic studies were performed in BALB/c mice; animal experimental
procedure was permitted by the Institutional Animal Ethics Committee (IAEC) of the CSIR-
Indian Institute of Chemical Technology, Hyderabad under the approval number (IICT/39/2017).
Studies performed on mice were according to the guidelines of the Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA, Government of India). A week
prior to performance of the studies animals were acclimatized in a biosafe house which is
maintained at an ambient temperature of 22 ± 2 °C and relative humidity of 50−60% under a
normal 12 h/12 h light/dark cycle.
One day before the experimentation animals were fasted overnight with water availability
ad libitium. In total 36 animals were grouped randomly into three groups i.e. one for SRFB
12

ACCEPTED MANUSCRIPT
suspension and others for SSLN and GAL-SSLN. The suspension of SRFB was prepared by
dispersing the drug into 2% gum acacia whereas SLN formulations were prepared by dispersing
them in normal saline. SRFB suspension or SLN formulations were administered to mice at a
dose of 25 mg/Kg body weight through per oral gavage. Blood samples of approximate volume
0.3 mL were withdrawn at predetermined time points of (0.5, 1, 1.5, 2, 4, 6, 8 and 24 h) from
retro-orbital plexus. Plasma was collected from blood samples after centrifugation at 2000 × g
for 10 min. Collected plasma was immediately preserved at – 80 °C until further analysis using
HPLC.
2.15 Plasma processing and analysis
For pharmacokinetic estimation of SRFB by HPLC, plasma samples were thawed to
reach room temperature from which 90 μL of plasma aliquot was taken, to this 10 µL of erlotinib
(1 mg/mL) was added as an internal standard and vortexed for 90 sec. Then, a volume of 400 μL
of precipitating agent [ACN:MeOH (1:1 v/v)] was added, again vortexed for 120 sec, and then
centrifuged at 10,000 rpm for 10 min at 22 °C temperature. The obtained supernatant was
filtered through 0.2 μm filter and a volume of 20 μL was injected to the HPLC system for
analysis at 265 nm. The pharmacokinetic parameters like maximum plasma concentration (Cmax),
area under the curve (AUC), area under the first moment curve plasma (AUMC), time to reach
peak plasma concentration (tmax), half-life (t1/2), clearance and mean residence time (MRT) were
determined by employing drug concentration versus time curve using PK solver, a menu-driven
add-in program for Microsoft Excel which is used for dealing with fundamental problems in
pharmacokinetic and pharmacodynamic data analysis (Zhang et al., 2010).
2.16 Real time biodistribution studies using IVIS system
13

ACCEPTED MANUSCRIPT
In-vivo imaging was carried out in BALB/c mice. To assess the fate of plain and targeted
SLN after per oral administration, drug was replaced with real-time near infrared fluorophore
(NIRF)-1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) dye. Before the
experiment, mice were fasted overnight and the next day DiR loaded formulations i.e., ligand
free (DiR-SLN) and PEGylated galactose conjugated SLN (DiR-GAL SLN) were administered
to mice through per oral route at 100 µL volume at dose of 0.5 mg/Kg of DiR. Distribution of the
nanoparticles into various organs was monitored through fluorescence at regular, predetermined
time points of 2, 4, 6, 8, 10, 24, and 48 h by anaesthetizing the mice using ketamine xylazine
injection. After the last time point of the study i.e., 48 h, mice were sacrificed to reveal the
fluorescence of various organs viz brain, heart, lungs, liver, kidneys, pancreas and spleen, using
IVIS spectrum in-vivo imaging system (Caliper, MA, USA) (Song et al., 2018).
2.17 Statistical analysis
All the experiments were performed in triplicate (n=3), data was complied, analyzed
using Graph Pad prism software (version 5), and data was expressed as mean ± standard
deviation (SD). Comparison between groups were performed by using one way ANOVA
applying Tukey's multiple comparison test and two way ANOVA through Bonferroni’s post-test
with (*p < 0.05, **p < 0.01 and ***p < 0.001). A value of p > 0.05 represents not significant.
3. Results
3.1 Synthesis and characterization of PEGylated galactose conjugate
The schematic representation of the conjugate synthesis is shown in Figure 1. Ring
opened galactose having aldehyde functional group gets involved in bonding with one of the
14

ACCEPTED MANUSCRIPT
amine groups in PEG bis (amine) and leads to formation of schiffs base (-N=CH-) which gets
reduced to secondary amine (-NH-CH2-) and both exist in equilibrium. PEGylated galactose
formation was confirmed by the formation of schiffs base. The peak at 1415 cm-1 confirms the
formation of schiffs base. The peaks at 1644 and 1562 cm-1 are due to stretching vibration of the
imine (-C=N-H) group (Figure 2(A)) (Jain et al., 2015).
1
In H-NMR, the signals for pure galactose were obtained at δ 4.7 ppm (D2O), 4.15 ppm
(‒CH2OH), 3.75, 3.61, 3.48 and 3.20 ppm (‒CH2 of ring), 2.79 ‒ 1.90 ppm (‒OH). The
characteristic peaks for pure PEG bis (amine) could be observed at δ 7.30 ppm (CDCl3), 1.45 to
2.2 ppm (‒NH2), 2.62 to 2.91 ppm (α-CH2 of NH2) and 3.61 to 4.53 ppm (β-CH2). The chemical
1
shift values shifted in PEGylated galactose conjugate can be clearly seen in H-NMR spectra
(Figure 2(B)). The conjugate showed chemical shift values at δ 4.7 ppm (D2O), 4.12 to 3 ppm (‒
CH2 of ring, β-CH2 of PEG), 2.78 to 1.72 and 1.0 to 0.96 ppm (α-CH2 of NH2 and ‒OH of
galactose ring). The appearance of chemical shift values at δ 3.14 to 3.02 ppm and 1.02 to 0.96
ppm confirmed the successful conjugation of galactose and PEG bis (amine).
3.2 Preparation of SRFB loaded solid lipid nanoparticles (SSLN and GAL-SSLN)
SLN were formulated by emulsification and solvent evaporation method where initial oil
in water (O/W) emulsion is formed under homogenization. Inner organic phase was comprised of
lipidic components like glyceryl monosterate for providing the solid matrix of SLN, stearic acid
for bringing up the terminal functional carboxylic group (-COOH) and soy lecithin as an internal
emulsifier. The formed coarse emulsion after homogenization was converted to nanoemulsion
using sonic waves where they break up the larger oil globules into smaller size droplets after
which the formed nanoemulsion was solvent evaporated for hardening and size reduction of the
15

ACCEPTED MANUSCRIPT
SLN. Similarly, for making of SSLN, SRFB was dissolved in methanol and added to organic
phase. For surface grafting of PEGylated galactose on the formed SSLN, EDC/NHS coupling
reaction was used to form an amide between carboxylic group of SLN and amine group of
PEGylated galactose.
3.3 Physicochemical characterization of nanoparticles
The average particle size of BSLN, SSLN and GAL-SSLN were 59.31±2.99, 68.36±3.14
and 111.00±6.99 nm respectively (Table 1). The surface conjugation of galactose on SLN
brought an increase of nearly 50 nm in size. All formulations showed narrow polydispersity
index and good zeta potential in the range of 0.209±0.022, 0.228±0.012, 0.354±0.024 and -
20.9±1.41, -20.13±1.35, -19.8±1.11 mV respectively for BSLN, SSLN and GAL-SSLN
formulations. Analytical HPLC chromatography for sorafenib showed the peak at retention time
of 6.7 min. The encapsulation efficiency of SSLN and GAL-SSLN was found to be similar
(97±1.5 and 95±1.8 respectively) indicating the no or minimal loss of drug during galactose
surface conjugation to SSLN.
FTIR analysis of the SRFB showed characteristic peaks at 1724, 1458, 1183 and 937 cm-1
which were also observed in SSLN and GAL-SSLN indicating the encapsulation of SRFB into
both the formulations. In addition, the carboxylic acid group peak observed at 1730 cm-1 in
SSLN while amide peaks at 1647 and 1561 cm-1 are observed in GAL-SSLN, confirming the
formation of amide bond between the nanoparticles and the ligand. DSC analysis of pure SRFB
showed peak at 243 °C corresponding to its melting point. The absence of this endothermic peak
in SSLN indicated the encapsulation of drug in amorphous form and homogenous dispersity of
the drug in the lipid matrix (Figure 3(A-C)).
16

ACCEPTED MANUSCRIPT
3.4 In-vitro drug release profile
In-vitro drug release studies were performed for pure SRFB, SSLN and GAL-SSLN in
different media like 0.1 N HCl (pH 1.2), phosphate buffer pH 6.8, SAB pH (5.0) and PBS pH
7.4, representing the pH conditions of stomach, small intestine, tumor microenvironment and
circulation fluid respectively to reveal the release blueprint of drug from SLN after oral
administration. Pure drug solution showed almost complete release (> 95%) in all media within 8
h whereas the SRFB release from SLN showed only 25-40% release for the same 8 h time in
different media suggesting the sustained release pattern of SRFB from the lipid matrix (Figure
4).
3.5 Induction of anti proliferation
Cell viability studies were performed against HepG2 cells for various formulations viz
SRFB, BSLN, SSLN and GAL-SSLN to explore the cytotoxicity behavior of the SRFB in free as
well as encapsulated form. The half inhibitory concentrations (IC50) of all the formulations are
shown in Table 2 and cell viability versus concentration graph shown in Figure 5. Both SSLN
and GAL-SSLN showed IC50 values of 9.77±1.47 and 6.57±0.58 respectively in comparison
with SRFB showing 9.48±0.45 ug/mL.
3.6 SRFB loaded nanoparticles induced apoptosis in human liver cancer cells
Apoptosis is one of the major pathways that lead to the cell death. Chromatin
condensation and fragmented nuclei are known as the classic characteristics of apoptosis. It was
considered of interest to investigate the apoptosis inducing effect of SRFB, SSLN and GAL-
SSLN by Hoechst 33258 staining in HepG2 cells. Cells were treated with formulations at 5 and
10 µg/mL concentrations for 48 h. Results indicated that nanoparticle formulations showed
17

ACCEPTED MANUSCRIPT
potent effect on nuclear condensation and fragmentation when compared with untreated control
cells and the results also demonstrated that GAL-SSLN was better than SSLN in exerting the
cellular apoptosis (Figure 6).
The apoptotic effect of SRFB, SSLN and GAL-SSLN was further evaluated by annexin
V–FITC/propidium iodide dual staining assay to examine the externalization of
phosphatidylserine and to comprehend whether it is due to physiological apoptosis or nonspecific
necrosis (Konstantinov and Berger, 1999). In this study, HepG2 cells were treated with SRFB,
SSLN and GAL-SSLN for 48 h at 5 and 10 µg/mL concentrations to examine the apoptotic
effect. It was observed that these formulations showed significant apoptosis against HepG2 cells
as shown in Figure 7. Results indicated that SRFB, SSLN and GAL-SSLN showed 26.19, 24.48
and 33.03 % of apoptosis at 5 µg concentration whereas they showed 58.21, 48.56 and 63.29 %
of apoptosis at 10 µg concentration respectively. These results clearly demonstrated that GAL-
SSLN was effective in inducing higher cellular apoptosis compared to SSLN or SRFB alone.
3.7 Cellular uptake study
Fluorescent nanoparticles (C6-SLN and C6-GAL SLN) were prepared by loading C6 in
place of sorafenib to determine the uptake of SLN by HepG2 cells. After the incubation times of
1, 2, and 4 h with C6 alone, C6-SLN and C6-GAL SLN the green signal of C6 in the cells was
observed and it was found that at all the time points C6-GAL SLN showed higher uptake than
C6-SLN. Further, the uptake of nanoparticles was also time dependent as the intensity of green
fluorescence was increased at each subsequent time point. These results clearly indicated that the
presence of ASGPR on HepG2 cells aids in augmented uptake of C6-GAL SLN in comparison to
C6-SLN (Figure 8).
18

ACCEPTED MANUSCRIPT
3.8 Effect of SRFB loaded nanoparticles on the mitochondrial membrane potential (∆Ψm)
The maintenance of mitochondrial membrane potential (∆Ψm) is important for
mitochondrial integrity and bioenergetic function (Gonda et al., 2008). Mitochondrial changes,
including loss of ∆Ψm are key proceedings that take place during drug induced apoptosis. To
learn the apoptosis inducing effect of SRFB, SSLN and GAL SSLN mitochondrial membrane
potential (∆Ψm) changes were detected using the fluorescent probe JC-1 (5,5,6,6-tetrachloro
1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide-5 mg/mL). The red fluorescence and green
fluorescence were detected at FL-2 and FL-1 channel, respectively. During apoptosis ∆Ψm
decreases and is measured by decrease in the ratio of red fluorescence (FL-2) to green
fluorescence (FL-1). After 48 h of treatment, it was observed that the ∆Ψm of HepG2 cells is
reduced in case of all formulations but the reduction was the highest with GAL-SSLN (Figure
9).
3.9 Plasma Pharmacokinetic study
To study the in-vivo pharmacokinetic response of various prepared formulations i.e.
SRFB suspension, SSLN and GAL-SSLN were administered to BALB/c mice. Figure 10 shows
the graph plotted between mean plasma concentration and time profiles for three formulations.
The data of different pharmacokinetic parameters were analyzed by non-compartmental analysis
using PK solver and are presented in Table 3. The maximum plasma concentration Cmax of pure
SRFB, SSLN and GAL-SSLN was 6.75±1.07, 25.22±0.50 and 28.90±0.13 µg/mL, respectively.
In case of SRFB suspension, drug was not detectable in plasma after 8 h whereas of the drug
released from both SSLN and GAL-SSLN was detectable up to 24 h. The reason can be
explained that pure SRFB is having low solubility and thus gets eliminated from the body
19

ACCEPTED MANUSCRIPT
whereas the drug loaded into SLN showed enhanced plasma concentration because the drug gets
protected into solid lipid matrix and gets absorbed well through payer’s patches present in M
cells (Pooja et al., 2016a). This enhanced absorption in turn leads to improved AUC (an indicator
of extent of absorption) where SSLN (272.05±30.60 µg/mL*h) and GAL-SSLN (363.09±32.74
µg/mL*h) showed significantly higher values (p<0.001) when compared with free SRFB.
Clearance of SRFB was significantly reduced in case of both SSLN (7.8 fold) and GAL-SSLN
(18.3 fold) because of increased circulation time. In particular, GAL-SSLN showed drastic
decrease in clearance that can be accounted to the presence of PEG as a spacer molecule between
galactose and SLN. PEGylation offers numerous advantages and is well known to extend the
circulation time of nanoparticles (Yuan et al., 2013). Results obtained from the pharmacokinetic
analysis confirm the improved drug delivery efficiency of the GAL-SSLN when compared to
free SRFB.
3.10 Real time biodistribution of nanoparticles
The targeting ability of the synthesized conjugate towards liver was explored by in-vivo
biodistribution studies in BALB/c mice using a non-invasive imaging system. Near infrared
active dye (DiR)-loaded nanoparticles (DiR-SLN and DiR-GAL SLN) were prepared and
administered orally to the animals. In general, SLN have natural tendency to go and lodge in
lungs with the increase in time after oral administration (Pooja et al., 2016a). A similar kind of
distribution was observed in this study where unconjugated DiR-SLN were observed in the lungs
of the treated animals. In contrary, GAL-mediated, targeted nanoparticles were observed in liver
even after 48 h of incubation. Ex-vivo fluorescence performed at the end of 48 h showed strong
fluorescence intensity in liver in case of DiR-GAL SLN whereas the plain DiR-SLN showed
affinity towards lungs. This clearly indicates that the SLN conjugated with galactose showed
20

ACCEPTED MANUSCRIPT
targeting towards liver whereas the unconjugated SLN showed towards lungs (Pooja et al 2016a;
Du et al., 2010; van Rijt et al., 2014) (Figure 11(A, B)).
4. Discussion
The drawbacks of SRFB were subdued in the present study wherein SRFB loaded and
PEGylated galactose conjugated SLN were formulated with high drug encapsulation efficiency.
Nanoparticles in the size range of 10-150 nm were known to be as successful delivery vehicles
that will also ensure extended circulation time and tumor accumulation. Both SSLN and GAL-
SSLN were formulated below 150 nm that will support in increasing circulation time and tumor
assembling (Steichen et al., 2013). Drug release patterns of both SSLN and GAL-SSLN showed
similar biphasic release indicating the initial burst release followed by persistent release which
was in well agreement with our previous findings using hydrophobic drugs loaded into SLN
(Pooja et al., 2016a; Pooja et al., 2015a; Pooja et al., 2015b). The cytotoxicity effect of
formulations, clearly indicated that BSLN was non-toxic and cells incubated with BSLN showed
good proliferation at all concentrations indicating that the employed lipid carriers are
biocompatible and nontoxic. Moreover, the anticancer efficacy of SRFB was improved upon
encapsulation in GAL-SSLN indicating that conjugation of galactose in PEGylated form on SLN
improved its cytotoxic effect by means of increasing the penetrability of SRFB through receptor
mediated endocytosis (Ding et al., 2014; Ma et al., 2018; Huang et al., 2018).
The intracellular uptake studies of the C6-GAL SLN showed enhanced intracellular
localization in comparison to C6-SLN and C6. This study reveals the fact that PEG molecules act
like a spacer between the galactose and SLN also helps in binding with head groups of
phospholipids chain of cell membranes which aid in more penetration (Yamazaki and Ito, 1990).
21

ACCEPTED MANUSCRIPT
In essence, the cellular uptake study was well in accordance with the cytotoxicity results which
indicate that ligand conjugated SLN showed higher uptake and hence, increased cytotoxicity in
respect to unconjugated SLN (Ding et al., 2014; Ma et al., 2018; Huang et al., 2018).
For pharmacokinetic studies, pure SRFB was compared with both SSLN and GAL-
SSLN. The prolonged retention of SRFB encapsulated inside both SSLN and GAL-SSLN in
blood circulation also enhances its chance of reaching to targeting cells thereby extracting the
maximum benefit at the given concentration. Moreover, this enhanced bioavailability of the drug
through GAL-SSLN also helps in reducing the dose in given dosage regimen. Real time
biodistribution studies clearly confirmed that the formulation i.e. DiR-GAL SLN was
successfully targeted to liver since galactose residues were recognized by specific ASGPR
present on liver. In addition, the PEGylation of the galactose showed increase in the circulation
half-life and stability of the SLN.
5. Conclusion
SRFB encapsulation into SLN and use of PEGylated galactose as aiming ligand aid in
improving various parameters like cytotoxicity, intracellular uptake and apoptotic activities on
HepG2 cells. Although in-vivo studies were performed in normal BALB/c mice, results
showcased the ability of the GAL-SSLN to deliver SRFB to liver and thus minimizing the non-
specificity. Further, at the same time the newly developed ligand conjugated SLN displayed
increased circulation half-life and thus, enhancing the chances of reducing the dose as well as
number of administrations thereby an improvement in the patience compliance. Overall, the oral
bioavailability of the SRFB can be improved by encapsulating it in SLN and grafting it with
22

ACCEPTED MANUSCRIPT
PEGylated galactose. These results may open up an area for development of PEGylated
galactose grafted nanoparticles for targeting hepatocellular carcinoma.
FIGURES
Figure 1: Schematic representation for synthesis of PEGylated galactose from ring opened
galactose and PEG bis amine.
Figure 2: Characterization of PEGylated Galactose conjugate by A) FTIR B) NMR.
Figure 3: Physicochemical characterization of different sorafenib formulations. A) Particle size
and Zeta potential of blank SLN (BSLN), sorafenib loaded SLN (SSLN) and galactose
conjugated sorafenib loaded SLN (GAL-SSLN) measured by DLS. B) FTIR of sorafenib and
GAL-SSLN. c) DSC thermograph of sorafenib and SSLN.
Figure 4: In-vitro drug release studies of sorafenib (SRFB), sorafenib loaded SLN (SSLN) and
galactose conjugated sorafenib loaded SLN (GAL-SSLN) in A) sodium acetate buffer (SAB) pH
5.0 B) phosphate buffer saline (PBS) pH 7.4, C) 0.1N HCl pH 1.2 and D) phosphate buffer pH
6.8
Figure 5: Cell viability versus concentration curve on HepG2 cells incubated with different
formulations viz., blank SLN (BSLN), sorafenib (SRFB), sorafenib loaded SLN (SSLN) and
Galactose conjugated sorafenib loaded SLN (GAL-SSLN) after 48 h of incubation.
Figure 6: Hoechst staining on HepG2 cells for sorafenib (SRFB), sorafenib loaded SLN (SSLN)
and Galactose conjugated sorafenib loaded SLN (GAL-SSLN) after 48 h of incubation. (A:
Control, B and C: SRFB at 5 and 10 ug, D and E: SSLN at 5 and 10 ug, F and G: GAL-SSLN at
5 and 10 ug)
Figure 7: Annexin V-FITC apoptosis assay on HepG2 cells for sorafenib (SRFB), sorafenib
loaded SLN (SSLN) and Galactose conjugated sorafenib loaded SLN (GAL-SSLN) after 48 h of
23

ACCEPTED MANUSCRIPT
incubation. (A: Control, B and C: SRFB at 5 and 10 ug, D and E: SSLN at 5 and 10 ug, F and G:
GAL-SSLN at 5 and 10 ug)
Figure 8: Cellular uptake studies for native coumarin-6 (C6) (5 µg/ml), coumarin-6 loaded SLN
(C6-SLN) and coumarin-6 loaded galactose conjugated SLN (C6-GAL SLN) (5µg/ml) in HepG2
cells at different time intervals of 1, 2 and 4 h by confocal microscopy at 40X magnification.
Figure 9: Mitochondrial membrane potential (∆Ψm) on HepG2 cells for sorafenib (SRFB),
sorafenib loaded SLN (SSLN) and Galactose conjugated sorafenib loaded SLN (GAL-SSLN).
Formulations triggers mitochondrial injury. Drop in membrane potential (∆Ψm) was assessed by
JC-1 staining. Samples were then subjected to flowcytometry analysis on a FACScan (Becton
Dickinson) in the FL1, FL2 channel to detect mitochondrial potential. (A: Control, B and C:
SRFB at 5 and 10 ug, D and E: SSLN at 5 and 10 ug, F and G: GAL- SSLN at 5 and 10 ug)
Figure 10: Mean plasma drug concentration of sorafenib (SRFB), Sorafenib loaded SLN
(SSLN), galactose conjugated sorafenib loaded SLN (GAL-SSLN) at various time points of 0.5,
1, 2, 4, 6, 8, 24 h after per oral administration. Data represented as mean ± SD (n = 3, *
represents comparison of SRFB with SSLN, # represents comparison of SRFB with GAL-SSLN
and */ # p < 0.05, **/ ## p < 0.01 and ***/### p < 0.001)
Figure 11: The in-vivo fluorescence real time imaging and biodistribution studies of DiR loaded
solid lipid nanoparticles (DiR-SLN) and galactose conjugated DiR loaded solid lipid
nanoparticles (DiR-GAL SLN) after oral administration in BALB/c mice. (DiR: 1,11-
Dioctadecyl-3,3,31,31-tetramethylindotricarbocyanine iodide) A) In-vivo fluorescence images of
the mice at different time points. B) ex-vivo fluorescence images of vital organs (heart, liver,
spleen, pancreas, lung, brain and kidney) at 48 h post injection.
TABLES
24

ACCEPTED MANUSCRIPT
Table 1. Physicochemical characterization of empty solid lipid nanoparticles (BSLN), sorafenib
loaded nanoparticles (SSLN) and galactose conjugated SSLN (mean ± standard deviation; n = 3).
Formulation
Particle size
(d.nm)
Polydispersity Zeta potential
Drug encapsulation
efficiency (%)
index
(mV)
BSLN
SSLN
59.31±2.99
68.36±3.14
111±6.99
0.209±0.022
0.228±0.012
0.354±0.024
-20.9±1.41
-20.13±1.35
-19.8±1.11
-
97±1.5
95±1.8
GAL-SSLN
Table 2. Half maxillary inhibitory concentration (IC50) values of sorafenib (SRFB), SRFB
loaded nanoparticles (SSLN) and galactose conjugated SSLN (GAL-SSLN) against HepG2
human hepatocellular carcinoma cell line after 48 h of incubation.
Formulation
IC50 at 48h (μg/ml)
SRFB
BSLN
9.48±0.45
> 100 (193.70±1.46)
9.77±1.47a
SSLN
GAL-SSLN
6.57±0.58*
a
* represents comparison between SRFB vs SSLN and GAL-SSLN. represents insignificant
difference in comparison with SRFB and *p < 0.05, **p < 0.01 and ***p < 0.001.
Table 3. Pharmacokinetic parameters of sorafenib (SRFB), sorafenib loaded SLN (SSLN),
galactose conjugated sorafenib loaded SLN (GAL-SSLN).
25

ACCEPTED MANUSCRIPT
PK Parameter
Unit
SRFB
SSLN
GAL-SSLN
µg/ml
h
6.75±1.07
1.17±0.28
32.59±2.98
45.88±3.87
25.22±0.50***
4.00±0.00***
28.90±0.13***, ##
6.00±0.00***, ###
C max
t max
µg/ml*h
µg/ml*h
µg/ml*h2
h
272.05±30.60***
357.20±25.35***
363.09±32.74***, #
598.92±36.28***, ###
AUC 0-t
AUC 0-α
AUMC 0-α
MRT
326.10±23.78 6723.35±52.36** 14896.37±496.08***, ##
6.58±0.58
0.55±0.04
4.36±0.20
16.65±3.64**
0.07±0.004***
14.07±0.06***
26.49±1.58***, #
0.03±0.01***, a
16.75±1.09***, #
(µg/ml)/h
h
Clearance
t1/2
Cmax = peak plasma concentration, tmax = time to reach peak plasma concentration, AUC = area
under the curve, AUMC = area under the first moment curve, MRT = mean residence time, t1/2
=
plasma half life. Data represented as mean ± SD, n = 3 (* represents comparison of SRFB with
a
SSLN, # represents comparison of SRFB with GAL-SSLN, indicates insignificant difference
and */ # p < 0.05, **/ ## p < 0.01 and ***/### p < 0.001).
ACKNOWLEDGMENT
Authors thank Director, CSIR-Indian Institute of Chemical Technology, Hyderabad for
providing the necessary facilities and support. Authors are grateful to M/s Therdose Pharma for
providing sorafenib as gift sample. Lakshmi Tunki is thankful to the Director of the IICT-RMIT
Research Centre for providing PhD scholarships.
Author Contributions
26

ACCEPTED MANUSCRIPT
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
Authors declare no conflict of interest for this manuscript.
REFERENCES
Botta, M., Armaroli, S., Castagnolo, D., Fontana, G., Pera, P., Bombardelli, E., 2007. Synthesis
and biological evaluation of new taxoids derived from 2-deacetoxytaxinine J. Bioorg. Med.
Chem. Lett. 17, 1579–1583. https://doi.org/https://doi.org/10.1016/j.bmcl.2006.12.101
Bray, F., Ferlay, J., Soerjomataram, I., 2018. Global Cancer Statistics 2018ꢀ: GLOBOCAN
Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries 394–424.
https://doi.org/10.3322/caac.21492
Browne, L.J., Gude, C., Rodriguez, H., Steele, R.E., Bhatnager, A., 1991. Fadrozole
hydrochloride: a potent, selective, nonsteroidal inhibitor of aromatase for the treatment of
estrogen-dependent
disease.
J.
Med.
Chem.
34,
725–736.
https://doi.org/10.1021/jm00106a038
Cai, L.-T., Sheng, Z.-H., 2015. Advances in cancer nanomedicine. Cancer Biol. Med. 12, 141–
142. https://doi.org/10.7497/j.issn.2095-3941.2015.0049
Cervello, M., Pitarresi, G., Volpe, A.B., Porsio, B., Balasus, D., Emma, M.R., Azzolina, A.,
Puleio, R., Loria, G.R., Puleo, S., Giammona, G., 2017. Nanoparticles of a
polyaspartamide-based brush copolymer for modified release of sorafenib: In vitro and in
vivo
evaluation.
J.
Control.
Release
266,
47–56.
27

ACCEPTED MANUSCRIPT
https://doi.org/10.1016/j.jconrel.2017.09.014
Chakravarti, B., Maurya, R., Siddiqui, J.A., Kumar Bid, H., Rajendran, S.M., Yadav, P.P.,
Konwar, R., 2012. In vitro anti-breast cancer activity of ethanolic extract of Wrightia
tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid. J.
Ethnopharmacol. 142, 72–79. https://doi.org/https://doi.org/10.1016/j.jep.2012.04.015
Cheng, A.-L., Kang, Y.-K., Chen, Z., Tsao, C.-J., Qin, S., Kim, J.S., Luo, R., Feng, J., Ye, S.,
Yang, T.-S., Xu, J., Sun, Y., Liang, H., Liu, J., Wang, J., Tak, W.Y., Pan, H., Burock, K.,
Zou, J., Voliotis, D., Guan, Z., 2009. Efficacy and safety of sorafenib in patients in the
Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised,
double-blind,
placebo-controlled
trial.
Lancet
Oncol.
10,
25–34.
https://doi.org/10.1016/S1470-2045(08)70285-7
D’Souza, A.A., Devarajan, P. V, 2015. Asialoglycoprotein receptor mediated hepatocyte
targeting — Strategies and applications. J. Control. Release 203, 126–139.
https://doi.org/https://doi.org/10.1016/j.jconrel.2015.02.022
Ding, Y., Liang, J.J., Geng, D.D., Wu, D., Dong, L., Shen, W. Bin, Xia, X.H., Zhang, C., 2014.
Development of a liver-targeting gold-PEG-galactose nanoparticle platform and a structure-
function
study.
Part.
Part.
Syst.
Charact.
31,
347–356.
https://doi.org/10.1002/ppsc.201300120
Du, B., Yan, Y., Li, Y., Wang, S., Zhang, Z., 2010. Preparation and passive target of 5-
fluorouracil solid lipid nanoparticles. Pharm. Dev. Technol. 15, 346–353.
https://doi.org/10.3109/10837450903246390
28

ACCEPTED MANUSCRIPT
Gonda, K., Tsuchiya, H., Sakabe, T., Akechi, Y., Ikeda, R., Nishio, R., Terabayashi, K., Ishii, K.,
Matsumi, Y., Ashla, A.A., Okamoto, H., Takubo, K., Matsuoka, S., Watanabe, Y.,
Hoshikawa, Y., Kurimasa, A., Shiota, G., 2008. Synthetic retinoid CD437 induces
mitochondria-mediated apoptosis in hepatocellular carcinoma cells. Biochem. Biophys. Res.
Commun. 370, 629–633. https://doi.org/https://doi.org/10.1016/j.bbrc.2008.04.008
Hashida, M., Nishikawa, M., Yamashita, F., Takakura, Y., 2001. Cell-specific delivery of genes
with
glycosylated
carriers.
Adv.
Drug
Deliv.
Rev.
52,
187–196.
https://doi.org/https://doi.org/10.1016/S0169-409X(01)00209-5
Huang, K.-W., Lai, Y.-T., Chern, G.-J., Huang, S.-F., Tsai, C.-L., Sung, Y.-C., Chiang, C.-C.,
Hwang, P.-B., Ho, T.-L., Huang, R.-L., Shiue, T.-Y., Chen, Y., Wang, S.-K., 2018.
Galactose Derivative-Modified Nanoparticles for Efficient siRNA Delivery to
Hepatocellular
Carcinoma.
Biomacromolecules
19,
2330–2339.
https://doi.org/10.1021/acs.biomac.8b00358
Jain, A., Kesharwani, P., Garg, N.K., Jain, A., Jain, S.A., Jain, A.K., Nirbhavane, P.,
Ghanghoria, R., Tyagi, R.K., Katare, O.P., 2015. Galactose engineered solid lipid
nanoparticles for targeted delivery of doxorubicin. Colloids Surfaces B Biointerfaces 134,
47–58. https://doi.org/https://doi.org/10.1016/j.colsurfb.2015.06.027
Kadari, A., Gudem, S., Kulhari, H., Bhandi, M.M., Borkar, R.M., Kolapalli, V.R.M., Sistla, R.,
2017. Enhanced oral bioavailability and anticancer efficacy of fisetin by encapsulating as
inclusion complex with HPβCD in polymeric nanoparticles. Drug Deliv. 24, 224–232.
https://doi.org/10.1080/10717544.2016.1245366
Kawakami, S., Hashida, M., 2014. Glycosylation-mediated targeting of carriers. J. Control.
29