2
M. S. BHATIA ET AL.
drug interaction with the diseased tissue, thereby preventing (Poona Chemical Laboratory, Pune, Maharashtra, India);
the untoward effect on the healthy tissue. This results in an Dulbecco’s Modified Eagle’s Medium, L-glutamine,
improved pharmacokinetic profile with reduced toxicity, mul- Streptomycin-penicillin solution, Fetal bovine serum, Trypsin-
tidrug resistance as well as the reduced formation of EDTA, Phosphate buffered saline solution, Hank’s Balanced
degraded compounds by providing in-vitro and in-vivo pro- Salt Solution, Sodium pyruvate, Potassium dihydrogen phos-
tection with the help of drug carriers (Upadhyay, 2018). phate and Tris-base (Himedia Laboratories, Mumbai,
Recently, several targeting approaches such as passive tar- Maharashtra, India); Tri-chloroacetic acid, Sulforhodamine B
geting, active targeting, tumour vasculature targeting, trig- and Triton-X 100 (Alfa aesar/Thermo Fisher Scientific India
gered release, multifunctional nanocarriers are under Pvt. Ltd., Mumbai, Maharashtra, India); High Performance
investigation (Batrakova et al., 2006; Bildstein et al., 2011; Liquid Chromatography grade acetonitrile (Qualigens Fine
Dass & Choong, 2006; Du et al., 2011; Francis et al., 2004; Ng Chemicals Pvt. Ltd./Thermo Fisher Scientific India Pvt. Ltd.,
et al., 2011). Previous researchers have reported the role of Mumbai, Maharashtra, India); and Tissue culture flask
2
efflux transporters in cellular delivery of anticancer drugs (75 cm ) and flat bottom polystyrene 96 well-tissue culture
(
Gaikwad & Bhatia, 2013). Moreover, the use of low-density plate (Tarsons Products Pvt. Ltd., Mumbai, Maharashtra,
lipoproteins (Almer et al., 2015; Harisa & Alanazi, 2014; India) were purchased. All other ingredients and chemicals
Upadhyay, 2018), nanoparticles (Chuang et al., 2020; Hu used were of analytical grade or higher.
et al., 2010; Sinha et al., 2006), organic nanoscale carriers
coupled with ligands (Shi et al., 2009) have been studied pre-
2
.2. Methods
viously for targeted delivery of the anticancer drugs for
improved therapeutic efficacy, pharmacokinetics, and longev-
ity as well as reduced side effects.
2
2
.2.1. Characterization of drug
.2.1.1. Organoleptic properties. The organoleptic study of
QbD approach gives a better scientific understanding of
critical process parameters and material attributes during the
product development process that significantly affects the
quality target product profile (Nadpara et al., 2012). The
desired product quality can be achieved by the integration
of prior knowledge and the result of studies using the design
of experiments along with the use of quality risk manage-
ment throughout the life of the product (Jain, 2014). It
improves manufacturing efficiency with a reduction in cost,
waste, and product rejection (Gaikwad et al., 2012, 2017).
The purpose of the present research was to develop a
drug delivery system by QbD approach using synthesized lip-
oproteins for targeting cancer cells. The lipoprotein struc-
tures were designed from three amino acids (arginine, serine,
and tyrosine), and the QbD approach was integrated for the
selection of variables for optimization through virtual screen-
ing of lipoprotein structures. Further, the lipoproteins were
synthesized and characterized by different physicochemical
properties. The synthesized lipoproteins were used to pre-
pare physical composite (tablets) as well as a chemical
conjugate with the drug. Prepared tablets were characterized
for different post-compression parameters, including in-vitro
dissolution study. However, synthesized drug-lipoprotein
chemical conjugates were characterized by different physico-
chemical properties, including cellular drug uptake and cyto-
toxic assay using immortalized human HaCaT keratinocyte
cancer cell line culture.
Vemurafenib was done by visual analysis.
2
.2.1.2. Melting point. The melting point of Vemurafenib
was determined by Thiele’s tube method.
2
.2.1.3. UV-Visible spectroscopy. Stock solution (10 mg/mL)
of Vemurafenib in methanol was prepared and scanned
spectrophotometrically (UV-Visible spectrophotometer, Jasco
V-630, Japan) within the range of 200–400 nm using metha-
nol as a blank. The wavelength for absorption maxima (kmax
)
so obtained was used for further studies. Moreover, the
same procedure was followed to determine the kmax in 0.1 N
HCl and phosphate buffer pH 7.4. The calibration curve of
Vemurafenib was prepared in methanol, 0.1 N HCl, and phos-
phate buffer pH 7.4 separately.
2
.2.1.4. Solubility study. The solubility of Vemurafenib was
determined in DMSO, methanol, ethanol, dichloromethane,
and distilled water. The drug was added to an excess of solv-
ent in a vial, tightly closed, and kept aside for 6 h. The solu-
tion was filtered using Whatman filter paper, and the filtrate
was evaporated to determine the weight of residue.
2
.2.1.5. LOD. The LOD of the Vemurafenib was determined
as per the procedure described earlier.
2
.2.2. Design of lipoprotein structures
Lipoprotein structures were designed, virtually keeping a
combination of the three selected amino acids (Table 1).
2
. Materials and methods
.1. Materials
Vemurafenib was supplied as a gift sample by Cipla Ltd 2.2.3. QbD approach in the selection of variables targeted
2
(
Mumbai, Maharashtra, India). Oleic acid, L-Arginine, L-Serine, for optimization
L-Tyrosine, Thionyl chloride (Molychem, Mumbai, 2.2.3.1. Virtual screening of lipoproteins.
Maharashtra, India); Dichloromethane (Loba Chemie, 2.2.3.1.1. Dataset preparation. Molecule builder module of
Mumbai, Maharashtra, India); Sodium hydroxide (Finar VLife MDS 4.4 commercial software was used to draw the 2D
Limited, Mumbai, Maharashtra, India); Potassium hydroxide structures of the designed lipoproteins.