The use of new quinazolinone derivative and doxorubicin loaded solid lipid nanoparticles in reversing drug resistance in experimental cancer cell lines: A systematic study

Article abstract of DOI:10.1016/j.jddst.2020.101569

The aim of this research is to study the feasibility of using carnauba wax as a new carrier for anticancer agents to deliver doxorubicin (DOX) as a model drug and a novel quinazolinone derivative (QZO-DER) to different types of normal and resistant cancer cell lines. A Box-Behnken design was implemented to investigate the influence of high melting point carnauba wax stabilized with cell membrane lipid (lecithin) and non-ionic biocompatible surfactant (span 60) in different concentration on particle size, entrapment efficiency of each drug and percent drug release. The solid lipid nanoparticles (SLNPs) were produced via the hot-melting homogenization technique. SLNPS particle size was from 16.58 ± 4 to 72.45 ± 1.21 nm and from 7.93 ± 1.67 to 174.31 ± 4.86 nm for DOX and QZO-DER respectively. Entrapment efficiency was from 51.78 ± 1.68% to 92.52 ± 2.47% and from 50.21 ± 1.8 to 82.95 ± 3.56% for DOX and QZO-DER respectively. While the percentage of release after 36 h was from 29.28 ± 3.89% to 78.08 ± 3.78% and from 37.5 ± 1.09 to 100 ± 1.25% for DOX and QZO-DER respectively. Selected formulations for DOX (OFX1 and OFX4) and QZO-DER (OFR4 and OFR6) were generated after validation of design. The in vitro anticancer activity was tested against both a panel of wild type and DOX-resistant human cancer cell lines. Cancer cell lines included colorectal cancer (HCT-116), breast cancer (MCF-7 and MDA-231), and lung adenocarcinoma (A549). Most of the tested SLNPs improved the efficacy of QZO-DER and DOX against the different cancer cell lines and have extended the spectrum to cover those accruing resistance during chemotherapy. QZO-DER loaded SLNPs exhibited the highest ability to reverse the drug resistance of MDA-231 cells compared to MDA-231/ADR cells was 19.7-fold, while DOX loaded SLNPs that showed reversal power was 1.8 fold for the same cells. Additionally, SLNPs showed a broad safety margin in normal cells. This study presented the use of SLNPs with carnauba wax as a potential therapeutic strategy to improve anticancer activity and overcome cancer resistance for clinical use.

Full text of DOI:10.1016/j.jddst.2020.101569

Journal Pre-proof
The use of new quinazolinone derivative and doxorubicin loaded solid lipid
nanoparticles in reversing drug resistance in experimental cancer cell lines: A
systematic study
Shahira F. El-Menshawe, Ossama M. Sayed, Heba A. Abou Taleb, Mina A. Saweris,
Dana M. Zaher, Hany A. Omar
PII:
S1773-2247(20)30027-7
DOI:
https://doi.org/10.1016/j.jddst.2020.101569
JDDST 101569
Reference:
To appear in: Journal of Drug Delivery Science and Technology
Received Date: 6 January 2020
Revised Date: 1 February 2020
Accepted Date: 6 February 2020
Please cite this article as: S.F. El-Menshawe, O.M. Sayed, H.A. Abou Taleb, M.A. Saweris, D.M. Zaher,
H.A. Omar, The use of new quinazolinone derivative and doxorubicin loaded solid lipid nanoparticles in
reversing drug resistance in experimental cancer cell lines: A systematic study, Journal of Drug Delivery
Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2020.101569.
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2020 Published by Elsevier B.V.

The Use of New Quinazolinone Derivative and Doxorubicin Loaded Solid Lipid
Nanoparticles in Reversing Drug Resistance in Experimental Cancer Cell Lines:
A Systematic Study.
a
a*
b
b
Shahira F. El-Menshawe , Ossama M Sayed , Heba A. Abou Taleb , Mina A. Saweris , Dana M.
c
c, d*
Zaher , Hany A. Omar
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef 62514, Egypt
b
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Nahda University,
Beni-Suef, Egypt
c
Sharjah Institute for Medical Research and College of Pharmacy, University of Sharjah, Sharjah
2
7272, United Arab Emirates
d
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef 62514, Egypt
*
Corresponding authors:
Ossama M Sayed, Ph.D., Department of Pharmaceutics and Industrial Pharmacy, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt email: usama.sayed@pharm.bsu.edu.eg
Hany A. Omar, Ph.D., Sharjah Institute for Medical Research and College of Pharmacy, University
of Sharjah, Sharjah 27272, United Arab Emirates email: hanyomar@sharjah.ac.ae
Word count: Abstract: 338; Manuscript: 5256;
Ref: 74; Figures 5
1

Abstract:
The aim of this research is to study the feasibility of using carnauba wax as a new carrier for
anticancer agents to deliver doxorubicin (DOX) as a model drug and a novel quinazolinone
derivative (QZO-DER) to different types of normal and resistant cancer cell lines. A Box-Behnken
design was implemented to investigate the influence of high melting point carnauba wax stabilized
with cell membrane lipid (lecithin) and non-ionic biocompatible surfactant (span 60) in different
concentration on particle size, entrapment efficiency of each drug and percent drug release. The
solid lipid nanoparticles (SLNPs) were produced via the hot-melting homogenization technique.
SLNPS particle size was from 16.58 ± 4 to 72.45 ± 1.21 nm and from 7.93 ± 1.67 to 174.31 ± 4.86
nm for DOX and QZO-DER respectively. Entrapment efficiency was from 51.78 ± 1.68% to 92.52
±
2.47% and from 50.21 ± 1.8 to 82.95 ± 3.56% for DOX and QZO-DER respectively. While
percentage of release after 36 hours was from 29.28 ± 3.89% to 78.08 ± 3.78% and from 37.5 ±
.09 to 100 ± 1.25% for DOX and QZO-DER respectively. Selected formulations for DOX (OFX1
1
and OFX4) and QZO-DER (OFR4 and OFR6) were generated after validation of design. The in
vitro anticancer activity was tested against both a panel of wild type and DOX-resistant human
cancer cell lines. Cancer cell lines included colorectal cancer (HCT-116), breast cancer (MCF-7 and
MDA-231), and lung adenocarcinoma (A549). Most of the tested SLNPs improved the efficacy of
QZO-DER and DOX against the different cancer cell lines and have extended the spectrum to cover
those accruing resistance during chemotherapy. QZO-DER loaded SLNPs exhibited highest ability
to reverse the drug resistance of MDA-231 cells compared to MDA-231/ADR cells was 19.7-fold,
while DOX loaded SLNPs that showed reversal power was 1.8 fold for same cells. Additionally,
SLNPs showed a broad safety margin in normal cells. This study presented the use of SLNPs with
carnauba wax as a potential therapeutic strategy to improve anticancer activity and overcome cancer
resistance for clinical use.
2

Keywords: Carnauba wax; Lecithin; span 60; hot melting homogenization; resistant cancer cell
lines.
3

Graphical Abstract
4

Background:
Globally, cancer is the second major cause of mortality. In 2015, 17.5 million cases were
diagnosed with cancer, which resulted in 8.7 million deaths [1]. The most four common cancer
types worldwide are lung, prostate, breast, and colorectal cancer, which account for around 4 in
1
0 of all diagnosed cancers [2]. Therefore, cancer treatment stands at the forefront of the
medical field. Cancer treatment involves a range of one or more interventions such as surgery,
cryosurgery, immunotherapy, hormone therapy, radiotherapy, and chemotherapy.
However, current chemotherapeutic strategies fail to distinguish between healthy and cancerous
cells, which result in a limited therapeutic effect on cancer cells, and severe adverse effects on
healthy cells [3]. Since cancer cells acquire resistance to applied chemotherapeutic agents,
Multidrug resistance (MDR) represents a challenge for successful treatment [4].
One of the approaches to minimize the risk of systemic toxicity, potentiate the activity of
chemotherapeutic agents and overcome chemotherapeutics resistant is to design system in nano-
size as nanoparticles. Nanoparticles could achieve passive targeting for tumor, which is based
on the enhancement of permeability and retention effect as a result nanoparticles (NPs) might
preferentially extravasate into the tumor and retain in [5].
NPs can exhibit more superior anti-cancer activity than the corresponding free drug by
increasing the solubility of poorly water-soluble drugs and enhancing the drug uptake by cancer
cells [6, 7].
There are various types of lipid nanoparticles that have been used to formulate anticancer
agents, but more attention has been paid to solid lipid nanoparticles (SLNPs) [8]. One of the
SLNPs components is the lipid matrix, which remains solid at room and body temperature
compared to other lipid and polymeric nanocarriers [9]. SLNPs provide attractive features
including good stability, small particle size, and controlled drug release, in addition to the
5

elimination of the need for toxic organic solvents [10]. Furthermore, SLNPs were identified to
overcome MDR by passing through drug efflux transporters p-glycoproteins (P-gp), while
protecting the embedded drugs from the external biological environment and ultimately
enhancing the drug therapeutic efficacy [11]. The ability of SLNPs to overcome the P-gp role in
multidrug resistance in cancer therapy was investigated. SLNPs showed an enhancement in
apoptotic cell death through the increase in the cancer cell uptake of the drug [5, 12]. This
improved efficacy was reported in systems utilizing several anti-cancer drugs like paclitaxel, 5-
Fluorouracil, tamoxifen, anthracyclines, and others.
Carnauba wax or Brazilian wax is naturally obtained from a specific palm tree known as
Copernicia cerifera, a plant native of the northeast of Brazil. Carnauba wax has several
applications, as gelling, releasing and glazing agent. It has a high melting point (between 82.0-
8
5.5 °C) making it a good option to be used in pharmaceutical systems, such as in the
production of SLNPs [13, 14]. This type of lipid carriers has never been tested for it ability to
deliver chemotherapeutic agents to cancer cells.
“2-(4-methoxyphenyl)-3-((2,3,4-trihydroxybenzylidene)amino)quinazolin-4(3H)-one” is a new
Quinazolinone derivative (QZO-DER) (Figure 1) has shown to inhibit the action on
phosphodiesterase (PDE4) enzyme that has been demonstrated to play a role in the
angiogenesis, proliferation, and motility of multiple cancer types [15, 16]. Selective PDE4
inhibitors show antiproliferative activity against B-cells and T-cells. Based on this finding,
selective PDE4 inhibitors were considered as novel anticancer agents. This was further
supported by their antiproliferative effect against murine cancer cells [17].
Quinazolinone is heterocyclic compounds which contribute in a wide range of pharmaceutical
interest such as anti-inflammatory [18], anticancer [19], antimicrobial [20], anticonvulsant [21],
antimalarial [22], antihypertensive [23] and antileishmanial [24] and antihistaminic activity[25].
6

Quinazolines were the nucleus of marketed drugs as selective kinase inhibitors have the FDA
approval as anti-cancer such as Gefitinib V, Erlotinib VI, and Lapatinib VII, which showed
potent activity[26].
In 2013, Marwa F. Ahmed and Mahmoud Younis [19] were synthesized a novel 6,8-dibromo-
4
(3H)quinazolinone derivatives, these compounds were tested against breast carcinoma cell line
MCF-7, where two compounds, showed extraordinary low IC50 = 1.7 and 1.8 µg/mL if
compared to doxorubicin IC50 = 29.6 µg/mL. Hamdy M. Abdel-Rahman et. al.[27] were
synthesized a novel series of quinazolin-4(3H)-one/Schiff base hybrids as phosphodiesterase 4
inhibitors. One compound (QZO-DER) showed significant antiproliferative activity with IC =
5
0
0
.14, 0.08 and 0.32 µM in breast, lung, and colon cancer cell lines, respectively, compared to
doxorubicin which has IC = 0.11, 3.13 and 2.75 µM, respectively in the same cancer cell lines.
50
In addition, PDE4 inhibition has been proven to have potential chemotherapy against various
types of cancer. Therefore, it is of great interest to discover of new selective PDE4B inhibitors
with antineoplastic activity [28, 29].
Doxorubicin (DOX) (figure1), one of the anthracycline chemotherapeutic agents, has been used
for more than 30 years to treat a variety of human cancers. The mechanism of its anti-cancer
activity is mediated by its intercalation into DNA, inhibiting topoisomerase II, and preventing
the synthesis of DNA and RNA [30]. In addition to its anti-cancer activity, DOX has short and
long-term cardiac toxicity, Moreover, after an anthracyclines treatment course, many patients
initially achieve a complete remission; however, approximately 70% of the patients eventually
experience a disease relapse due to the development of MDR. Therefore, it is essential to
develop a drug delivery system to reduce systemic toxicity and improve the anticancer activity
of DOX [31, 32].
7

For this purpose, QZO-DER loaded SLN and DOX-loaded SLN formulations were produced to
enhance the cytotoxic effect of chemotherapy by reversing the resistance of multi-drug resistant
cancer cells though designing formulations that allow the drug to bypass efflux pump transport
using excipient could inhibit the function of P-gp. These excipients (or additives) offer
advantages of being safe, not being absorbed from the gut, pharmaceutically acceptable and
have a history of being incorporated in many parenteral and enteral formulations as solubilizing
or stabilizing agents.
Insert Figure (1)
Methods:
Materials
Soya lecithin (Phospholipon 90G) PhosphatidylCholine (PC) was a kind gift from Lipoid GmbH
(
Germany), Carnauba wax (CW) was a kind gift from Chemical Industries Development
CID) Company (Egypt), DOX was obtained from EIMC United Pharmaceuticals (Cairo, Egypt),
(
QZO-DER was obtained as generously gift sample from Medicinal Chemistry department, Faculty
of Pharmacy, Nahda University, Egypt. Span 60, Tween 80, N-butanol were purchased from Al
Naser company (Egypt).
Experimental design
A three-level three-factor Box-Behnken (BB) design was employed to statistically optimize the
formulation variables for preparing QZO-DER and DOX SLNPs, using Design Expert® statistical
software Ver. 7.0 (Stat-Ease, Inc., MN, USA).[33] Three independent variables were evaluated:
Carnauba Wax Percentage (X1), Phosphatidyl Choline Percentage (X2) and span 60 percentage (X3).
The selection of lipid moiety and surfactant was based upon preliminary screening. The Particle
8

size (Y1: PS), Encapsulation Efficiency (Y2: EE%) and Drug release (%) (Y3: %DR) were selected
as the dependent variables. The independent (low, medium and high levels) and dependent variables
are shown in Table 1.
Insert Table (1)
Preparation of QZO-DER / DOX loaded SLNPs
Commercial doxorubicin was purchased as hydrochloride salt to enhance its aqueous solubility.
However, for the effective incorporation of the drug in the hydrophobic SLNPs, the drug must be
hydrophobized. Chemical conversion of doxorubicin-HCl into its free base (hydrophobic) was
achieved through a chemical reaction with triethylamine (TEA) (0.5 ml) then doxorubicin free base
was extracted by chloroform (50 ml), chloroformic extract then dried under vacuum to obtain the
Crystals of DOX base [12].
SLNPs were successfully fabricated by the hot melting homogenization technique [8, 11, 34]. For
this study, two matrix lipid with lipids (phospholipon 90G, CW) were chosen along with the two
commonly surfactants (span 60, Tween 80) and all ingredients were carefully weighed and heated
above melting point by 5 to 10 ºC up to 85 ºC. Subsequently 10 mg of either (QZO-DER or DOX)
was added and dissolved completely to form a drug-lipid melt. The aqueous solution was composed
of hot distilled water heated to 80 ºC then slowly added to the drug-lipid melt while stirring with
maintaining the temperature to form coarse pre-emulsion. Additional homogenization was carried
out by using high-speed homogenizer (IKA T-25 ULTRA-TURAX Digital Homogenizer) at 15000
rpm for 15 min to form o/w emulsion. At room temperature, the final dispersion was cooled to
solidify nanoparticles forming SLNPs and the formulation was stored in tightly sealed tubes.
9

SLNPs characterization
Particle size analysis
The particle size was determined using photon correlation spectroscopy by Zetasizer 2000 (Malvern
Instruments Ltd, Malvern, UK)[33]. The SLNPs nanosuspension was not diluted before testing and
all measurements were done in triplicate using right-angle scattering at 25 ˚C.
Entrapment efficiency (EE%)
EE% has been obtained by the indirect method. The un-trapped drug was analyzed in the
supernatant after cooling centrifugation (Refrigerated SIGMA 3-16K centrifuge), as previously
described [33]. The concentration of QZO-DER and DOX in the aqueous phase was determined
using UV–visible spectrophotometer (SHIMADZU “UV 800”) at λmax 342.2 and 499 respectively.
Values of EE % were calculated using Eq (1).
ꢀ
ꢁꢂ ꢃ
ꢄꢅ
EE% =
ꢂꢆꢂꢇꢈꢈ
(Eq. 1)
Where Sf and Ss were the amounts of drug added in the formulation and amount of drug in the
supernatant, respectively.
In vitro release study:
The in vitro QZO-DER and DOX dissolution from SLNPs was performed as described before,
using (ERWEKA DT 720) dissolution tester (250 ml phosphate buffer saline, 5 ml samples,
o
temperature set to 37±0.5 C) [35]. The samples of each formula were withdrawn from the medium
and substituted with a fresh medium over a period of 36 h. The samples were then measured
1
0

spectrophotometrically at λ max 304.4 for QZO-DER and λ max 498 nm for DOX (SHIMADZU “UV
00”)
8
Formulation optimization:
The optimized three formulations for each QZO-DER and DOX were obtained using the Design
Expert® software by constraints on encapsulation efficiency percent of the SLNPs to obtain the
highest value, on drug release to obtain the highest value and on particle size to obtain the smallest
value. The recommended optimized six formulations were then prepared and evaluated in triplicate
to check the validity of the calculated optimal formulation factors and predicted responses given by
the software were chosen.
Transmission electron microscopy (TEM):
The particle morphology of the optimized SLNPs of QZO-DER both and DOX were examined
using a transmission electron microscope JEM-1400 (JEOL Ltd., Tokyo, Japan) as stated in
previous work[33].
Fourier-transform infrared spectroscopy (FTIR):
Briefly, as stated in previous work, both drugs (QZO-DER, DOX) and both optimum formulations
(OFR, OFX) were all evaluated using an FTIR Spectrophotometer (Shimadzu IR-345, Japan) in an
-1
inert atmosphere over a wave number range of 4000-400 cm [36].
Differential scanning calorimetry (DSC):
Shimadzu DSC-50 (Shimadzu Corporation, Kyoto, Japan) was used to determine the crystalline
properties of different materials and Formulations[36]. The analysis was carried out at a
1
1

temperature range of 0–250°C and the heating rate was 10°C/min, at a flow rate of 25 mL/min. The
DSC thermograms were recorded. The melting point and transition measurements were measured
by the supplied software.
Cell viability assay
Cell line and culture conditions:
Human colorectal cancer cell line
®
In this study, Human colorectal cancer cell line, HCT-116 (ATCC CCL-247™), was obtained as
previously described in Saber et al work [37].
Human breast cancer cell lines and Human lung carcinoma
Human breast cancer cell lines (MCF-7 and MDA-231) and Human lung carcinoma (A549) were
purchased from the European Collection of Cell Cultures (ECACC, UK). At Sharjah Institute for
Medical Research, University of Sharjah (UAE), cells were maintained in Roswell Park Memorial
Institute medium (RPMI: Sigma-Aldrich). The normal fibroblasts (F180) were kindly supplied by
professor Ekkehard Dikomey (University Cancer Center, Hamburg University, Hamburg, Germany)
and maintained in Dulbecco’s Modified Eagle Medium (DMEM: Sigma-Aldrich).
All incubations were done with media containing 10 % fetal bovine serum (FBS) (Sigma-Aldrich)
o
and 1 % penicillin/streptomycin (Sigma-Aldrich) at 37 C in a humidified atmosphere of 5 % CO .
2
Generation of MCF-7, MDA-231 and A549 resistant cells to doxorubicin
1
2

o
MCF-7, MDA-231, and A549 cells were seeded in T75 flasks and incubated overnight at 37 C.
Then cells were treated with the IC10 of doxorubicin (Sigma-Aldrich) for each cell line. The
survived population of cells was transferred to a new flask and treated with gradually increasing
concentrations of doxorubicin for 8 months to develop resistance. To maintain resistance, cells were
continuously treated with the IC of doxorubicin.
1
0
Human colorectal cancer cell line assay
Sulforhodamine B assay was used to evaluate cytotoxicity. Protocol conducted and interpretations
was according to Vichai et al [38]. SLNPs and free drug were added after 24 h incubation with
various concentrations and incubated at 37 °C for 48 h to determine their IC50s (the concentration
of the drug required to produce 50% cell growth inhibition).
Human breast cancer cell lines and Human lung carcinoma
Cell viability was assessed using MTT assay as described above. Briefly, MCF-7, MDA-231, A549,
4
and their generated resistant cells were seeded at 4 ×10 cells per well in 96 well flat-bottom plates
and incubated overnight. Then, cells were treated with different concentrations of doxorubicin,
Standard QZO-DER, OFR4, OFR6, OFX1 and OFX4 for 48 h. The used vehicle (DMSO) was used
as a negative control. After that, the media were replaced with 200 µl of media containing 0.5
o
mg/ml of MTT tetrazolium dye (Sigma-Aldrich, USA). Cells were incubated for 2 h at 37 C. The
formed formazan crystals were solubilized with 200 µl of DMSO per well. Absorbance was
measured at 570 nm using a microplate reader (Thermo Scientific, UK).
Reversal power calculation
The reversal power was calculated from Eq. (2):
1
3

ꢔꢕꢖꢔ
ꢗ
ꢉꢊꢋꢊꢌꢍꢎꢏꢂꢐꢑꢒꢊꢌꢓꢂ
Eq. (2)
ꢕꢖ ꢗ
where R was the IC value of drug solution against drug resistant cells, R was the IC value of
f
50
N
50
drug loaded SLN against drug resistant cells; S was the IC value of drug solution against drug
f
50
sensitive cells, S was the IC value of drug loaded SLN against drug sensitive cells.
N
50
Statistical Analysis of data:
All data regarding particle size, EE%, % and %DR were analyzed by Design Expert® and
differences at the P < 0.05 level considered significant.
Data from cell line experiments were subjected to analysis of variance (ANOVA) followed by
Fisher's PSLD test for multiple comparisons among groups, and differences at the P < 0.05 level
considered significant. All results were expressed as the mean ± SD.
Results
Effect of formulation variables on particle size
SLNPs particle size for both QZO-DER and DOX demonstrated the results in table (2). The effects
of X , X and X on the SLNPs particle size (Y ) can be illustrated through the surface response
1
2,
3
1
curves in figure (2). The combined effect of the X variables on Y of QZO-DER showed good
1
fitting according to the quadratic model (p<0.05) though logarithmic transformation according to
the following equation:
ꢥ
ꢥ
ꢘꢙꢚꢐꢛꢜꢛꢝꢞꢛꢇꢇꢟꢈꢛꢈꢠꢡꢂꢢ ꢟꢈꢛꢈꢇꢤꢢ ꢦꢈꢛꢞꢇꢢ ꢦꢈꢛꢈꢇꢨꢢ ꢢ ꢟꢈꢛꢈꢩꢩꢢ ꢢ ꢦꢈꢛꢪꢪꢢ ꢢ ꢟꢈꢛꢇꢩꢢ ꢦꢈꢛꢪꢢ ꢦ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
ꢈꢛꢨꢩꢢꢥ
(Eq. 3)
ꢧ
The combined effect of X variables on Y of DOX-SLNP showed good fit according to the
1
quadratic model (p<0.05) according to the following equation:
1
4

ꢥ
ꢐꢛꢜꢛꢝꢨꢫꢛꢈꢫꢟꢇꢪꢛꢠꢠꢂꢢ ꢟꢨꢛꢨꢩꢢ ꢦꢨꢛꢩꢢ ꢦꢞꢛꢇꢇꢢ ꢢ ꢟꢇꢩꢛꢫꢇꢢ ꢢ ꢦꢫꢛꢈꢩꢢ ꢢ ꢟꢠꢛꢨꢪꢢ ꢦ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
ꢥ
ꢇꢨꢛꢫꢤꢢ ꢦꢡꢛꢠꢢ
(Eq. 4)
ꢥ
ꢧ
Where X
1
is the percentage of Carnauba wax, X
2
is the percentage of Phospholipids and X is the
3
concentration of span 60.
From both equations (3, 4), it was noted that increasing the percentage of both carnauba wax and
PC lead to an increase in particle size, while increasing span 60 concentration lead to a significant
decrease (p<0.05) in particle size (from 174.31 to 7.93 nm in case of QZO-DER and from 72.45 to
1
2.1 nm in case of DOX).
Effect of formulation variables on encapsulation efficiency percent
The entrapment efficiency (EE%) data of the formulated SLNPs are shown in table (2). The
combined effect of the X , X and X on the EE% (Y ) of the QZO-DER and DOX SLNPs can be
1
2,
3
2
shown through the surface response curves in figure (2). ANOVA test for the observed EE% in
QZO-DER SLNPs data indicates that the quadratic model was significant and fitting for the data
(p<0.05). The combined effect of the independent variables on EE% of QZO-DER SLNPs showed
fitting according to the quadratic model according to the following equation:
ꢥ
ꢥ
ꢬꢬꢭꢝꢤꢪꢛꢫꢫꢟꢈꢛꢈꢠꢞꢂꢢ ꢦꢠꢛꢞꢤꢂꢢ ꢦꢈꢛꢡꢪꢂꢢ ꢦꢩꢛꢪꢫꢂꢢ ꢢ ꢟꢩꢛꢨꢞꢂꢢ ꢢ ꢦꢤꢛꢈꢤꢂꢢ ꢢ ꢦꢪꢛꢡꢨꢂꢢ ꢦꢩꢛꢪꢞꢂꢢ ꢦ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
ꢪꢛꢩꢩꢂꢢꢥ
(Eq. 5)
ꢧ
The combined effect of the independent variables on EE% of DOX SLNPs showed fitting according
to the quadratic model (p<0.05) according to the following equation:
ꢥ
ꢥ
ꢬꢬꢭꢝꢫꢇꢛꢩꢟꢈꢛꢈꢠꢡꢂꢢ ꢦꢨꢛꢠꢨꢂꢢ ꢦꢇꢛꢠꢪꢂꢢ ꢦꢪꢛꢠꢡꢂꢢ ꢢ ꢟꢤꢛꢤꢨꢂꢢ ꢢ ꢦꢇꢇꢛꢡꢞꢂꢢ ꢢ ꢦꢨꢛꢫꢞꢂꢢ ꢦꢤꢛꢨꢫꢂꢢ ꢦ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
ꢪꢛꢡꢞꢂꢢꢥ
(Eq. 6)
ꢧ
From both equations, there was a significant positive effect (p<0.05) of increasing the percentage of
carnauba wax on the entrapment efficiency of both drugs (from 46.42±1.78% to 76.94±2.78% for
1
5

QZO-DER and from 51.78±1.68% to 92.52±2.47% for DOX), while increasing the percentage of
PC and span 60 lead to a significant decrease in EE% (p<0.05) [39].
Effect of formulation variables on drug release percent
Release profiles of QZO-DER and DOX from their SLNP formulations are shown in figures (3).
The combined X , X and X the %DR (Y ) of the QZO-DER and DOX SLNPs are be shown
1
2
3
3
through the surface response curves in figure (2). The combined effect of the X variables on %DR
Y ) of QZO-DER SLNPs showed fitting (p<0.05) according to the quadratic model according to
(
3
the following equation:
ꢥ
ꢥ
ꢥ
ꢭꢮꢉ ꢝꢫꢈꢛꢡꢞꢦꢞꢛꢞꢠꢂꢢ ꢟꢠꢛꢩꢂꢢ ꢟꢠꢛꢈꢪꢂꢢ ꢟꢞꢛꢠꢫꢂꢢ ꢢ ꢟꢠꢛꢪꢂꢢ ꢢ ꢦꢈꢛꢠꢪꢂꢢ ꢢ ꢟꢇꢈꢛꢞꢡꢂꢢ ꢟꢪꢛꢈꢞꢂꢢ ꢦꢨꢛꢨꢡꢂꢢ
ꢧ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
(Eq. 7)
The combined effect of X variables on Y of DOX SLNPs showed fitting according to the quadratic
3
model (p<0.05) according to the following equation:
ꢥ
ꢥ
ꢭꢮꢉꢝꢩꢪꢛꢈꢞꢦꢇꢛꢠꢪꢂꢢ ꢟꢩꢛꢪꢞꢂꢢ ꢟꢠꢛꢤꢞꢂꢢ ꢟꢪꢂꢢ ꢢ ꢟꢠꢛꢇꢩꢂꢢ ꢢ ꢦꢈꢛꢠꢂꢢ ꢢ ꢟꢇꢞꢂꢢ ꢟꢇꢈꢛꢠꢂꢢ ꢦ
ꢣ
ꢥ
ꢧ
ꢣ
ꢥ
ꢣ
ꢧ
ꢥ
ꢧ
ꢣ
ꢥ
ꢇꢛꢇꢩꢂꢢꢧꢥ
(Eq. 8)
Increasing carnauba wax percentage had a negative effect of %DR and lead to slower drug release
from SLNPs. These finding came in accordance with previous literature, which showed slower
release with increased EE%. [40-44]. Both PC and span 60 enhanced the release of both drugs from
SLNPs (78.08% and 100% for QZO-DER and DOX respectively, p<0.05) due to their solubilizing
and surface active action [45-47].
Insert Figure (2)
Insert Table (2)
Insert Figure (3)
1
6

Release kinetics of the different SLNPs formulations of both QZO-DER and Dox are shown in table
3). Most of the formulations showed a release pattern fitting Kross-Peppas model of release with
(
the exception of F4 and its center points (7,8,9,12) which showed more fitting in the first-order
model, F1 and F14 which Fitted Hixson model and F2, F6 and F13 which fitted Higuchi model.
Kross-Peppas models apply to different pharmaceutical dosage forms, where the dissolution occurs
in planes which are parallel to the particle surface if the particle dimensions decreases
proportionally, in such a way that the initial geometrical form keeps constant all the time [48].
Table (3) shows the expected and observed values for the optimized formulations of both DOX and
QZO-DER. All observed responses didn’t significantly vary from the expected values (<30% RSD)
(p>0.05). This indicated the validity of the design for predicting optimized formulations.
Insert Table (3)
Table (4) represents the ANOVA data generated from the experimental design software regarding
the significance of the effect of each variable and their interactions on the resulted responses. The
2
table also shows the regression data represented by the values of adjusted and predicted R for each
variable. The table shows good linearity of the models as shown from the high levels of correlation
coefficients. The high p-values of the lack of fit indicate the validity of the design.
Insert Table (4)
Release profiles of selected formulations of both QZO-DER SLNPs and DOX-SLNPs are shown in
2
figure (3) while the release kinetics equations from fitting release data and its R is shown in table
(3). From the illustrated data, it was shown that half of the formulations (3 formulations) were
obeying Higuchi release kinetics while the other three was following either Kross-Peppas or first
order kinetics. Statistical analysis of data showed good fitting of models and reasonable precision.
Transmission electron microscopy (TEM)
1
7

Transmission electron microscope photographs of SLNPs of QZO-DER and DOX are shown in
figure (4-c and d). Photographs showed spherical particles of distinct borders.
Insert Figure (4)
Differential Scanning Calorimetry (DSC)
Figure (4-a) shows the DSC thermograms of both QZO-DER, DOX, carnauba wax,
phosphatidylcholine, span 60 and their physical mixtures and freeze-dried formulations. It was
o
obvious that the melting peaks for QZO-DER and DOX at 252 and 220 C respectively were absent
in both physical mixtures and freeze-dried formulations due to their presence as amorphous states in
consequence for the presence of other excipients [49-51].
Fourier transform infrared spectroscopy (FTIR)
-1
For the IR spectrum of QZO-DER, peaks assigned to cyclic amide C=O stretch at 1635 cm and
-1
N=C stretch of the Schiff's base, -OH stretch of phenolic characteristic peaks at 2920 and 3408 cm
-1
were observed (figure 4-b). In the IR spectrum of DOX, the peak at 1037 and 2925 cm confirm the
-1
presence of C-O stretch and C-C-H Aliphatic long-chain, and the peak at 3433 cm shows –OH
-1
stretch, C=O stretch of ketone was observed at 1733 cm (Figure 4-b). Marker peaks for DOX and
QZO-DER were found in their mixtures and freeze formulations that indicate there is no interaction
between drug and excipients.
Formula OFR 4, OFR 6 and OFX 4, OFX 1were selected based upon their EE%, particle size and
release patterns to study their cytotoxic effect on different types and strains of cancer cell lines
Cytotoxicity assays
The anti-cancer activity of the generated solid lipid nanoparticles with their corresponding controls
was evaluated using either MTT cell viability assay or SRB assay against human cancer cell lines
1
8

including MCF-7, MCF-7 resistant, MDA-231, MDA-231 resistant, A549, A549 resistant, and
HCT-116 cells. Their safety was evaluated in normal cells (F180). The determined IC50 values and
reversal power values are presented in table (5).
HCT-116 responded to the treatment with standard doxorubicin with IC of 10 µM. Interestingly,
5
0
the treatment with OFX1 and OFX4 increased the efficacy of DOX with IC of 6.5 µM and 7 µM,
50
respectively. In addition, OFR4 and OFR6 showed an induced anti-cancer effect with IC of 8 µM
5
0
and 6.32 µM, respectively, compared to the IC of QZO-DER (28.3 µM, p<0.05).
5
0
In addition, Standard doxorubicin exhibited an anti-cancer effect against MCF-7 cells with IC of
5
0
1
.12 µM. The developed OFX1 did not improve the Standard doxorubicin anticancer activity with
IC of 1.12. Interestingly, OFX4 showed a promising potency with IC of 0.66 µM (p<0.05).
5
0
50
MCF-7 cells didn’t respond to the treatment with Standard QZO-DER. However, OFR4 and OFR6
improved the activity with IC of 2.9 µM and 6.12 µM respectively (p<0.05).
5
0
On the other hand, MCF-7 resistant cells were not responsive to Standard doxorubicin and the
treatment with OFX1, and OFX4 had a considerable increase in the anti-cancer activity with IC of
5
0
2
.88, 4.9 µM. Standard QZO-DER showed more potent activity in MCF-7 resistant cells compared
to MCF-7 cells (p<0.05). Interestingly, OFR6 showed an increase in potency compared to Standard
QZO-DER with IC of 1.68 µM (p<0.05). However, OFR4 did not have a pronounced difference
5
0
with IC of 2.85 µM (p>0.05).
5
0
Moreover, Standard doxorubicin exhibited an anti-cancer activity against A549 cells with IC of
5
0
6
.69 µM, while the developed OFR4 and OFR6 did not show promising activity (p>0.05). Similar
to the observed response in MCF-7 cells, Standard QZO-DER had no distinct effect in A549 while
OFX4 demonstrated IC of 5.9 µM. A slight increase in the effect was observed after the treatment
5
0
with OFX1 (p<0.05).
1
9

Following the same anti-cancer activity pattern in MCF-7 resistant cells, no effect was observed for
Standard doxorubicin in A549 resistant cells. However, OFX4 showed promising activity with IC50
of 3.5 µM. Moreover, A549 resistant cells were more sensitive to the treatment with Standard QZO-
DER compared to OFR4 and OFR6 (p<0.05).
Furthermore, MDA-231 was not responsive to Standard doxorubicin. Interestingly, OFX1, OFX4
showed a potent anti-cancer activity with IC50 of 4.8, and 4.9 µM, respectively (p<0.05). In
addition, OFR6 showed an increase in the effect with IC of 3.78 µM (p<0.05) compared to the
5
0
Standard QZO-DER. while OFR4 did not indicate a distinct improvement in the anti-cancer activity
p>0.05).
(
In MDA-231 resistant cells, OFR4 and OFR6 improved the anti-cancer activity (p<0.05) of
Standard QZO-DER. Interestingly, OFX1 and OFX4showed higher anti-cancer potency compared
to standard doxorubicin with IC of 6.2, and 5 µM respectively (p<0.05).
5
0
The reversal power values of the different SLNPs of both actives showed a considerable ability of
the formulations to reverse the resistance of different cell line while maintaining the safety towards
the normal fibroblast lines.
The safety profiles of all tested compounds were evaluated on F180 cells. The cells viability was
not affected after treatment with all compounds with IC more than 10 µM (p>0.05).
5
0
Insert Table (5)
Insert Figure (5)
Discussion:
SLNs are a category of remarkable drug delivery systems that have been investigated in the
biomedical field for several years. One of the main explanations for the speed flourishing of SLNs
2
0

is their ability to effectively deliver both hydrophilic and hydrophobic drugs. Doxorubicin as model
anti – cancer drug has a major drawback which is the development of resistance from cancer cells of
different origin. The goal of this research was to study the feasibility of incorporating QZO-DER and
DOX into novel lipid nanoparticles and its efficacy in eradicating different varieties of cancer cell
lines. Using carnauba wax as a lipid carrier could be of great potential for further studies.
Changing the ratio of incorporated ingredients forming theses SLNPs greatly affected the particle
size, EE%, and %drug release. Increasing lipid composition lead to an increase in particle size due
to increased viscosity, which came in accordance with previous literature [40, 41, 43, 52-54]. On
the other hand, increasing span 60 concentration did not show enough reduction in particle size
until reaching a concentration of 0.2%, which was followed by a significant reduction in particle
size. This could be attributed to the low HLB nature of span 60 and the combined HLB values
required to emulsify carnauba wax (HLB=15) which could be achieved at the level of 0.2% span 60
combined with the fixed amount of added tween 80 [45, 47, 55]. Mixing two or more surfactants
tends to stabilize the lipid dispersion and reduce the size [42, 56]. Use of PC alone as sole
emulsifier may not be enough to stabilize the SLNPs owing to the difference of the HLB between
PC (HLB=4) and the lipid core (HLB=15). Therefore the combination of different surfactants will
lead to a stable dispersion system [42, 56].
The positive effect of increasing the concentration of carnauba wax on the EE% of both drugs came
in accordance with previous literature, which indicated the compatibility of drugs with carnauba
wax as lipid carrier [40, 41, 43, 52-54]. The negative effect of PC on the EE% of both drugs could
be attributed to the increased permeability of the particles and increased drug solubilization and
leakage of particles [45, 47, 55].
The nature of excipients considered to be a cardinal factor affecting drug release. The hydrophobic
excipients used, the slower will be the drug release [57-59]. One way to change the release rate is
2
1

adding hydrophilic or hydrophobic excipients [58, 59]. Increasing carnauba wax content in the
formulation lead to more slower release than low carnauba wax particles due to the hydrophobicity
and high melting point of carnauba wax, which came in accordance with previous literature [60].
Such slow release will ensure the availability of the drugs within the nanoparticulate system for the
delivery within cancer cells and increase the fraction of drug reaching intracellular domain through
lytic effect[61]. Drug burst in some formulations may be attributed to the presence of drugs in the
outer shell of the SLNPs which could happen during the formulation step where the lipid would
rapidly solidify and as the drug solution becomes supersaturated upon cooling it would be
concentrated in the outer shell of the SLNPs [62-64]. Other explanation is the effect of increasing
the amount of phosphatidylcholine decreased the interfacial tension between the lipid moiety and
the aqueous phase and lowering the melting point of the lipid moiety[65].
The complete disappearance of DOX and QZO-DER peaks in DSC thermograms might be due to
the complete miscibility within the lipid components. This was previously mentioned in literature
that disappearance of the melting peak is due to loss of crystallinity and increased drug
thermodynamic activity[66]. These findings could augment the high entrapment efficiency into the
SLNPs.
Particle size is one of the critical formulation characteristics which will alter the cellular uptake
efficiency that controls the adhesive strength between nanoparticles and cellular surface[67].
Spherical particles which have a size below 200 nm tend to accumulate in tumor tissues due to
increased permeability and retention [68]. Incorporating both DOX and QZO-DER in SLNPs
significantly affected their behavior towards different types of cell lines. Regarding DOX,
incorporation into nanocarriers greatly increased the uptake and activity against a drug-resistant cell
line that could be attributed to the by-passing of the efflux P-gp system in these resistant strains.
2
2

Incorporating DOX and QZO-DER into carnauba wax lipid carriers lead to increased permeability
into cancer cells either the normal or resistant types. It was well known that the incorporated tween
8
0 and PC as excipients in the formulation of SLNPs greatly inhibits the function of P-gp and thus
offering the active inside the cells in high concentrations[69, 70]. Incorporation of phospholipids
had a significant effect on the uptake and permeation of epirubicin in the work done by Yu-Li Lo
[
70]. These findings could be noticed from the increase of the reversal power of some of the
formulations. The different tested solid lipid nanoparticles have shown a significant improvement in
the anticancer activity against the resistant cells, as OFR6 in MCF-7, OFR4 in MDA-231 and OFX4
in A549 as was shown from the reversal power of each formula against the cell lines. As there are
several mechanisms of cancer cells’ resistance, and the most dominant mechanism is the over-
expression of MDR pumps, these nanoparticles might overcome this challenge by ensuring a
successful drug delivery.
Additionally, the enhanced effect could be attributed to the sustained release of the drug from the
carnauba wax matrix in high concentrations within the cellular targets [71]. Our findings came in
accordance with previous findings and literature [72-75]. Lipid SLN can enhance the drug transport
into cancer cells by increasing cellular uptake both in sensitive and resistant cancer cells. As low
intracellular drug level is the major aspect in multi-drug resistant cells when the anticancer agent
was administrated, the drug concentration increase in the target cells is the key point for reversing
the multi-drug resistance. Carnauba wax through its high entrapment efficiency for both actives
managed to present them inside the target cells in reasonable high concentrations. Thus, the cellular
drug uptake result of anticancer drug loaded SLN was consistent with its drug resistance reversal
activity. In addition, SLNPs components did not show any toxic effect on the normal fibroblast
F180 cell line, which indicated the safety of the lipid’s compositions.
Conclusion
2
3

The present work focuses the spotlight on the potential use of carnauba wax as a novel carrier for
cancer chemotherapeutic agents like the traditional doxorubicin and the novel quinazolinone
derivative. The use of cell membrane lipid and surfactant resulted in significant effects on the
particle size, entrapment efficiency and release percent. This research article also presented the
potential use of SLNPs as an efficient carrier for anticancer drugs and revealed an interesting effect
on both normal and resistant strains of different cell lines. The present study paves the way for
further studies on the effectiveness of these SLNPs in in-vivo cancer animal models.
Compliance with ethical standards
Conflict of interest. The authors declare that they have no conflict of interest.
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