Development of lipoprotein-drug conjugates for targeted drug delivery

Article abstract of DOI:10.1080/07391102.2020.1803964

Tumour targeting approaches used in cancer chemotherapy offers prolonged, localized, and protected drug interaction with the diseased tissue with minimal side effects and systemic toxicity, which are accountable for the failure of chemotherapy using conventional delivery systems. The purpose of the present study is to develop an anticancer targeted drug delivery system using synthesized lipoproteins with the integration of quality by design approach. Lipoprotein structures were designed, and quality by design approach was implemented to select variables for optimization. Further, the lipoproteins were synthesized and characterized by physicochemical properties. Physical composites of synthesized lipoproteins with the drug (tablets) were prepared and evaluated for post-compression parameters. Moreover, drug-lipoprotein chemical conjugates were synthesized and characterized for physicochemical properties, including cellular drug uptake and cytotoxicity study on HaCaT cancer cells. Synthesized lipoproteins showed good swelling capacity but poor flowability. Nuclear magnetic resonance and infrared spectroscopy of conjugates showed characteristic peaks. Tablets from all batches extended the drug release up to 12 h. All synthesized conjugates showed improved cellular drug uptake (up to 86.1%) and inhibition (87.39%) of HaCat cancer cells. These findings explored the possible use of synthesized lipoproteins in the development of anti-cancer drug formulation against HaCat cancer cells. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1803964

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Development of lipoprotein-drug conjugates for
targeted drug delivery
Manish S. Bhatia , Sujata P. Choudhari , Rakesh P. Dhavale & Vinod L.
Gaikwad
To cite this article: Manish S. Bhatia , Sujata P. Choudhari , Rakesh P. Dhavale & Vinod L.
Gaikwad (2020): Development of lipoprotein-drug conjugates for targeted drug delivery, Journal of
Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1803964
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1803964
Development of lipoprotein-drug conjugates for targeted drug delivery
a
a
a
b
Manish S. Bhatia , Sujata P. Choudhari , Rakesh P. Dhavale and Vinod L. Gaikwad
a
b
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur, India; Department of Pharmaceutics, BVDU
Poona College of Pharmacy, Pune, India
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 28 April 2020
Accepted 27 July 2020
Tumour targeting approaches used in cancer chemotherapy offers prolonged, localized, and protected
drug interaction with the diseased tissue with minimal side effects and systemic toxicity, which are
accountable for the failure of chemotherapy using conventional delivery systems. The purpose of the
present study is to develop an anticancer targeted drug delivery system using synthesized lipoproteins
with the integration of quality by design approach. Lipoprotein structures were designed, and quality
by design approach was implemented to select variables for optimization. Further, the lipoproteins
were synthesized and characterized by physicochemical properties. Physical composites of synthesized
lipoproteins with the drug (tablets) were prepared and evaluated for post-compression parameters.
Moreover, drug-lipoprotein chemical conjugates were synthesized and characterized for physicochemi-
cal properties, including cellular drug uptake and cytotoxicity study on HaCaT cancer cells. Synthesized
lipoproteins showed good swelling capacity but poor flowability. Nuclear magnetic resonance and
infrared spectroscopy of conjugates showed characteristic peaks. Tablets from all batches extended
the drug release up to 12 h. All synthesized conjugates showed improved cellular drug uptake (up to
KEYWORDS
Lipoprotein; drug-
lipoprotein conjugate;
targeted drug delivery;
immortalized human HaCaT
keratinocyte
8
6.1%) and inhibition (87.39%) of HaCat cancer cells. These findings explored the possible use of syn-
thesized lipoproteins in the development of anti-cancer drug formulation against HaCat cancer cells.
Abbreviations: ARG: L-Arginine; CCI: Carr’s Compressibility Index; DMEM: Dulbecco’s Modified Eagle’s
Medium; DMSO: Dimethyl sulfoxide; FBS: Fetal Bovine Serum; FTIR: Fourier Transform Infrared
Spectroscopy; HBSS: Hank’s Balanced Salt Solution; HLB: Hydrophilic-Lipophilic Balance; HPLC: High-
Performance Liquid Chromatography; LOD: Loss on Drying; PBS: Phosphate-buffered Saline; QbD:
Quality by Design; RT: Room Temperature; SER: L-Serine; SRB: Sulforhodamine B; TCA: Tri-chloroacetic
Acid; TYR: L-Tyrosine
1
. Introduction
heterogeneous biology of the growing tumours. Therefore,
the ultimate goal of cancer therapy is to improve the survival
time and quality of life of the patient by reducing the sys-
temic toxicity of drug/s being used. Anticancer drugs usually
lack important requisites such as unable to reach the target
site in the desired quantity and are not effective enough in
the tumour microenvironment (Rios-Doria et al., 2012). All
these factors together with the physiology of the tumour
result in the failure of chemotherapy. Conversely, conven-
tional drug delivery system requires a high dose of drugs
with frequent dosing attributed to its early biodegradation
associated toxic effects, fluctuations in drug level, and lack of
ability to attain therapeutic concentration.
The human body is composed of trillions of living normal
cells that grow by dividing into new cells and die in an
orderly manner by apoptosis, a programmed phenomenon
of self-destruction (Alison, 2001). If these cells show uncon-
trolled growth, commonly known as cancer, which is one of
the leading cause of death (Martinho et al., 2011). Cancer
represents an enormous biomedical challenge for targeted
drug delivery due to the several physiological barriers such
as vascular endothelial pores, heterogeneous blood supply,
acidic pH and architecture, etc. The use of differences in
these barriers between normal and cancerous cells in, either
way, could help in the selective delivery of drug/s to the
cancer cells.
Therefore, tumour-targeting approaches have been devel-
oped to improve efficacy and reduce toxicity by altering the
bio-distribution of anti-cancer drugs (Lee et al., 2008). An
Along with debulking surgery, chemotherapy has been
the main modality of treatment for cancer disease. However, ideal approach to targeted therapy comprises prior know-
its success rate remains low, primarily due to its severe side ledge of the vascular surface of the cancer cell as well as
effects on healthy cells, limited accessibility of drugs to the lowering the penetration of drugs inside a normal cell
tumour tissue, their intolerable toxicity, multi-drug resistance together with improving tumour access. Targeted drug deliv-
developed from repeated chemotherapies and the dynamic ery system is used to prolong, localize, and have protected
CONTACT Manish S. Bhatia
16013, Maharashtra, India
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
drmsbhatia13@gmail.com
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur
4

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.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Table 1. Combination codes for designed lipoprotein structures.
Lipoprotein code
Combination
Lipoprotein code
Combination
A
B
C
D
E
Oleoyl amide-ARG
Oleoyl amide-SER
Oleoyl amide-TYR
Oleoyl amide-ARG-SER-TYR
Oleoyl amide-ARG-TYR-SER
F
G
H
I
Oleoyl amide-SER-ARG-TYR
Oleoyl amide-SER-TYR-ARG
Oleoyl amide-TYR-ARG-SER
Oleoyl amide-TYR-SER-ARG
2.2.3.1.2. Preparation of protein structure. Structure of the 2.2.4.3. Synthesis of SER oleoyl amide. SER was reacted
B-Raf (PDB ID 4XV9) was downloaded from the free protein with oleoyl chloride in the presence of sodium hydroxide
database (www.rcsb.org, Zhang et. al. 2015). The protein under vigorous shaking until the odor of oleoyl chloride was
structure was further used for docking analysis by VLife MDS disappeared. The amide so formed was washed with water
4
.4 commercial software.
and recrystallized from ethanol.
2
.2.3.1.3. Docking analysis of designed lipoproteins.
2
.2.4.4. Synthesis of TYR oleoyl amide. TYR was reacted
Docking analysis was used to study the macromolecule and
micro-molecule interactions. It was carried out using the bio-
predicta module of the VLife MDS 4.4 to explore the docking
ability of the synthesized lipoproteins.
with oleoyl chloride in the presence of sodium hydroxide
under vigorous shaking until the odor of oleoyl chloride was
disappeared. The amide so formed was washed with water
and recrystallized from ethanol.
2
.2.4. Scheme of synthesis for selected lipoproteins
2
2
.2.5. Characterization of lipoproteins
.2.5.1. Organoleptic properties. The lipoproteins synthe-
A new acid-sensitive lipoprotein having micelle forming
property was proposed to be formed by a two-step reaction
involving amino acid and fatty acid. The first step of the syn-
thesis was the formation of fatty acid chloride, and the
second step was the reaction of the amino acid with fatty
acid chloride to form an amide. Such synthesized lipoprotein
is biodegradable and biocompatible.
sized were visually examined for appearance, color, and odor.
2
.2.5.2. Melting point. The melting point is used to identify
the product and also to detect the termination stage of the
reaction. The digital melting point apparatus was used to
determine the melting point of synthesized lipoproteins.
2
.2.4.1. Synthesis of oleoyl chloride from oleic acid.
2
.2.5.3. FTIR analysis. About 2 mg of synthesized lipopro-
Previous researchers have reported the preparation of fatty
acid chloride by acylation of fatty acid using thionyl chloride
or phosphorus chloride in the presence of an inert organic
solvent (Gibson, 2007).
teins sample was placed in between the pellets of previously
dried KBr at 120 C for 30 min, uniformly mixed with drug,
and kept in sample holder to record the FTIR spectra.
ꢀ
In the present study, the synthesis of oleoyl chloride
involves acylation reaction using thionyl chloride and
dichloromethane. Initially, oleic acid (2 g ± 1%) and dichloro-
methane (16 mL ± 2%) was added to the three-neck flask.
The solution was heated to reflux (39 C–41 C) for about 1 h
under continuous stirring. To this refluxing solution, a solu-
2
.2.5.4. pKa. pKa value is the negative logarithm of the
equilibrium coefficient of neutral and charged species at a
given pH. Dissociation or ionization constant of the drug is
important physicochemical property as it shows a strong
influence on formulation and pharmacokinetics. In the pre-
ꢀ
ꢀ
tion of thionyl chloride (10 g ± 1%) was added. Finally, the sent research, pKa for synthesized lipoproteins was deter-
reaction was kept under stirring with reflux for 3 h until the mined by the titration method using pH meter. An
brown-colored acid chloride solution (oleoyl chloride) was accurately weighed quantity of synthesized lipoprotein was
formed which was subsequently cooled, stored in brown dissolved in 100 mL of water. A probe of calibrated pH meter
bottles, and used without undue delay.
was immersed in this solution kept under continuous stirring,
and HCl was added dropwise from the burette. The titration
was continued until constant pH was reached. Further, the
titration curve of pH against mL of HCl was plotted for the
determination of the equivalence point and pKa.
2
.2.4.2. Synthesis of ARG oleoyl amide. An amide of amino
acid and fatty acid chloride was synthesized by using the
Schottan-Baumann reaction. The fatty acid chloride reacts
with amino acids in an alkaline environment to produce an
amide (Menalda, 2010; Vogel & Furniss, 1989).
2.2.5.5. Partition coefficient. The partition coefficient was
Arginine (1 g) was suspended in 20 mL of 5% sodium estimated by a simple shake flask method. A stock solution
hydroxide solution in a well-corked small conical flask. Further, of synthesized lipoprotein in octanol (organic phase, 10 mg
2
mL of oleoyl chloride (0.5 mL at a time) was added with con- in 110 mL) was prepared and mixed in predetermined ratios
stant shaking followed by cooling in water. The solution was with a specified volume of aqueous phase (water). The mix-
shaken vigorously for about 5–10 mins until the odor of oleoyl ture was vigorously shaken for 1 h and kept aside for 2 h at
chloride gets disappeared. Further, solid oleoyl chloride deriva- RT. Further, the two layers were separated with the help of
tive was filtered, and the residue was washed with cold water separating funnel, and the concentration of lipoproteins dis-
and subsequently recrystallized from ethanol.
tributed in layers was estimated spectrophotometrically (UV-

4
M. S. BHATIA ET AL.
Visible spectrophotometer, Jasco V-630, Japan). The partition 2.2.5.10. Conductivity. Stock solution (50 mL) with the
coefficient (K) was calculated by using Equation (1),
desired molarity was prepared, and the electrode was placed
in it. The conductivity of the solution was determined by the
conductometer, which was previously calibrated with deion-
ized water.
K ¼ Caq=Corg
(1)
where, Caq and Corg are the concentrations of synthesized
lipoprotein in the aqueous phase and organic phases,
respectively.
2
.2.5.11. LOD. LOD is the loss of weight of the sample as % w/
w after removing water or any volatile matter under a specific
condition. For this, the pre-weighed sample in a petri dish was
placed in an oven at a specific temperature for a specific time.
After drying, the contents were re-weighed, and LOD was cal-
culated by using the following formula (Equation 6),
2
.2.5.6. HLB. Every surfactant has a distinct HLB number
which simplifies its selection in the formulations. Low HLB
value surfactants are lipophilic and used as W/O emulsifying
agents, however high HLB value surfactants are hydrophilic,
which are used as O/W emulsifying agents, solubilizing
agents as well as detergents. HLB value was calculated as
the ratio of saponification value (S) to acid value (A) of
recovered or unreacted acid with the synthesized lipopro-
teins (Equation 2).
ðW2 ꢁ W3Þ
LOD ¼
ꢂ 100
(6)
ðW2 ꢁ W1Þ
where W is the weight of the petri dish, W is the weight of
1
2
the sample and W is the weight of petri dish with sample
3
after drying.
HLB ¼ 20 ð1 ꢁ S=AÞ
(2)
The saponification number (S) indicates the average molecu-
lar weight of the compound. In a round bottom flask, 2 g of
synthesized lipoprotein was mixed with 20 mL of 0.5 M etha-
nolic NaOH and refluxed for 30 min. This mixture was then
titrated against 0.5 M HCl using phenolphthalein as an indi-
cator, and the endpoint was determined (b). Simultaneously,
blank titration (a) was performed (Equation 3).
2
.2.5.12. Angle of repose. A fixed funnel fixed cone method
was used to determine the angle of repose as an indicator of
the flowability of the powder. The lower tip of the funnel
was fixed at 2.5 cm from the flat surface, and the powder
was poured into the funnel to form a pile. Further, the
height of the tip (h) from the surface and diameter of the
pile (d) was measured and used to calculate the angle of
(3) repose by using the following formula (Equation 7),
S ¼ ða ꢁ bÞꢂ28:05 = weight of sample
Acid value (A) measures the amount of carboxylic acid group
in the test compound. For this, 0.2 g (W) synthesized lipopro-
tein was dissolved in 50 mL neutralized ethanol with the aid
of heat on a water bath. This solution was then titrated with
2h
d
Angle of repose ðhÞ ¼ tan ꢁ
1
(7)
2
.2.5.13. CCI. CCI is indicative of the flowability of the powder,
and it is calculated by using the following formula (Equation 8),
0
.1 N KOH using phenolphthalein as an indicator, and the
endpoint was determined (V) for the estimation of acid value
Equation 4).
A ¼ V ꢂ Normality of KOH ꢂ 56:1 = W
Tapped Density ꢁ BulkDensity
(
CCI ¼
ꢂ100
(8)
Tapped Density
(4)
2
.2.5.14. Hausner’s ratio. The Hausner’s ratio is also indica-
2
.2.5.7. Wettability. Wettability was determined by spread-
tive of the flowability. A Hausner’s ratio below 1.25 shows
good flowability, however, greater than 1.25 denotes poor
flowability. It is calculated by using the following formula
ing (surface energy) and contact ability (contact angle) of the
liquid over a solid surface. If sufficient contact is achieved
between the two phases, then physical attraction due to
intermolecular forces gets developed and the liquid remains
in contact with the surface.
(
Equation 9),
Tapped Density
Bulk Density
Hausner’s ratio ¼
(9)
2
.2.5.8. Swelling capacity. A small quantity of lipoprotein
(
W ) was mixed with deionized water in a beaker. After 20
1
2
.2.6. Preparation of tablets
mins, swollen lipoprotein was separated using filter paper
W ), and % swelling capacity was determined by using the
Initially, powder mass having a uniform particle size was
obtained by sieving all ingredients through sieve size 180
microns (ASTM #80). Further, ingredients were weighed sep-
arately (Table 2) from each batch and mixed thoroughly until
a homogeneous mixture (physical composite) was obtained.
(
2
following formula (Equation 5).
%
Swelling ¼ W2 ꢁ W1 = W1
(5)
2
.2.5.9. Viscosity. The viscosity of synthesized lipoprotein A total of nine batches of Vemurafenib tablets (A1 to T3,
was determined by using Brookfield viscometer, which meas- 600 ± 5 mg) were prepared as per composition given in
ures the force required to rotate the spindle in the fluid. Table 2 by direct compression method using a rotary tablet
Different concentrations (2.5, 5, and 10%) of all the three lip- press (CIP Machineries Pvt. Ltd., Ahmedabad, Gujrat, India)
oproteins solutions were tested for viscosity measurement having a 13-mm round flat-faced punch and die set for the
2
for 10 and 100 rpm at RT.
constant hardness of 4–5 Kg/cm .

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
a
Table 2. The composition of tablet formulations.
Batch Code
Role of Ingredient
Lipoprotein
Name of Ingredient
ARG oleate
SER oleate
TYR oleate
Vemurafenib
Microcrystalline cellulose
Methyl cellulose
Magnesium stearate
Talc
A1
A2
A3
S1
S2
S3
T1
T2
T3
50
–
–
240
q.s.
40
3
100
–
–
240
q.s.
30
3
150
–
–
240
q.s.
20
3
–
50
–
240
q.s.
40
3
–
100
–
240
q.s.
30
3
–
150
–
240
q.s.
20
3
–
–
–
–
–
–
50
240
q.s.
40
3
6
600
100
240
q.s.
30
3
6
600
150
240
q.s.
20
3
6
600
Drug
Diluent
Binder
Lubricant
Glidant
Total weight
a
6
600
6
600
6
600
6
600
6
600
6
600
All quantities are expressed in mg.
2
.2.7. Characterization of tablets
2.2.8. Preparation of
ical conjugate
a
drug-lipoprotein chem-
All batches of tablet formulations were characterized by sev-
eral post-compression parameters as discussed below.
Synthesized lipoproteins (Arginine oleate, Serine oleate,
Tyrosine oleate) were chemically conjugated with the
2
.2.7.1. Weight variation. A total of 20 tablets from each Vemurafenib. For this, a quantity of powder equivalent to
batch (A1 to T3) were weighed individually, and a % devi- one mole of re-chlorinated lipoprotein (Arginine oleate,
ation from the average weight was calculated.
Serine oleate, or Tyrosine oleate) was accurately weighed
and dissolved in sufficient quantity of alcohol. The result-
2
(
.2.7.2. Hardness. The hardness of tablets from each batch ing mixture was heated in a water bath to avoid the
A1 to T3) was determined using a Monsanto type hardness evaporation of alcohol (Solution-I). In another beaker, a
sufficient quantity of freshly prepared 0.2 M KOH solution
tester. The tablet was placed diametrically, and the pressure
was applied gradually until the tablet breaks (Kg/cm ).
2
was mixed with the one mole of Vemurafenib and
ꢀ
allowed to heat up to 60 C (Solution-II). Further, Solution
I and II were mixed at the same temperature, and the
product (drug-lipoprotein conjugate) was collected in cold
water after neutralizing the solution with HCl
2
.2.7.3. Friability. A pre-weighed (W ) sample of 20 tablets
i
from each batch was subjected to tumbling in Roche friabil-
ity tester for 4 mins at 25 rpm. After 100 revolutions, the tab-
lets were removed, dedusted, and reweighed (W ) to
(
Figures 1–4).
f
calculate the % friability (Equation 10).
2
2
.2.9. Characterization of the drug-lipoprotein chem-
ical conjugate
.2.9.1. Organoleptic properties. The chemical conjugate
Wi ꢁ W
f
%
Friability ¼
ꢂ100
(10)
Wi
was visually examined for color, odor, and appearance.
2
.2.7.4. Disintegration test. The disintegration test was per-
2
.2.9.2. Melting point. The melting point of the synthesized
formed on six tablets selected randomly from each batch as
per official procedure by using the USP disintegration test
apparatus and 0.1 N HCl as the medium. The test was con-
ducted for the specified time, and tablets were inspected for
complete disintegration in the media, and the time required
for disintegration was recorded.
drug-lipoprotein conjugate was determined by Thiele’s tube
method for estimation of its purity.
2
.2.9.3. FTIR. FTIR spectra of the synthesized lipoprotein
conjugates were recorded.
2
.2.9.4. Solubility. The solubility of the chemical conjugate
2
.2.7.5. In-vitro drug release study. The mechanism and
was determined in DMSO and chloroform.
rate of drug release from tablets were estimated using USP
Type II (paddle method) dissolution test apparatus
2
2
.2.9.5. Biological activity
.2.9.5.1. Cell preparation. Early passage of adherent culture
of immortalized human HaCaT keratinocytes was procured
which was subsequently replaced by phosphate buffer pH from National Center for Cell Sciences, Pune (Maharashtra,
(
Electrolab, TDT 08 L, Mumbai, Maharashtra, India). The dissol-
ution medium used was 900 mL of 0.1 N HCl for an initial 4 h,
ꢀ
2
7
.4, both maintained at 37 ± 0.5 C. The stirring rate of the India) in 75 cm culture flasks containing DMEM with high
paddle was kept constant at 100 rpm. Aliquots of 5 mL were glucose (4.5 g/L) as the base medium, supplemented with
withdrawn at predefined time points, and the same was 10% v/v heat-inactivated FBS and two mM L-glutamine. The
replaced with a fresh dissolution medium. The withdrawn antibiotic solution, 1% v/v (10000 lg/mL streptomycin and
samples were filtered using Whatman filter paper no. 41 and 10000 units/mL penicillin) was added in a flask and main-
ꢀ
filtrates were analyzed spectrophotometrically (UV-Visible tained at 37 C with 5% CO2 in a humidified incubator
spectrophotometer, Jasco V-630, Japan) at 271 nm after suit- (NuAire Inc., Purecell 5100, Plymouth, Minnesota, United
able dilution.
States). Before performing the assay, the cell line was

6
M. S. BHATIA ET AL.
Figure 1. Chemical conjugation of synthesized lipoproteins (Arginine oleate, Serine oleate, Tyrosine oleate) with Vemurafenib.
Figure 2. Chemical conjugate-A of ARG oleate with Vemurafenib.
observed to obtain nearly 80% confluency; then culture added and mixed uniformly to obtain a homogeneous cell
media was removed from the flask. The cells were briefly suspension. Cell concentration was counted by a haematocy-
rinsed with 10 mL PBS (pH 7.4) to remove left residual media tometer chamber under a microscope, using a 1:1 mixture of
and approximately 0.25% w/v trypsin-EDTA was added cell suspension and 0.4% w/v trypan blue solution. The cell
4
evenly to cover the confluent cell layer thus the cells will be concentration was adjusted to a density of 1 ꢂ 10 cells per
dissociated from the flask surface. When the cells start to dis- well. This cell concentration was used for cellular uptake
sociate, ten volumes of culture medium containing FBS was study and cytotoxic assay.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 3. Chemical conjugate-B of SER oleate with Vemurafenib.
Figure 4. Chemical conjugate-C of TYR oleate with Vemurafenib.
2
.2.9.5.2. Cellular uptake study. The HaCaT cells (at 80% con- with multiwavelength detector) at 271 nm for the estimation
fluency) were harvested with 0.25% w/v trypsin-EDTA solution of drug content in the cell. The HPLC system was equipped
5
and cultured in a sterile 96 well plate at a density of 2 ꢂ 10
with a model PU 2080 pump, UV detector (Czerny turners
cells/well and incubated overnight for 24h in a humidified mount monochromator with deuterium lamp as a light
incubator to reach 90% confluent layer (Derakhshandeh et al., source), and Rheodyne manual injector. Borwin Version 1.5
software was used for data acquisition and analysis. The chro-
2
011). After incubation, the medium was removed, and the
matographic condition was set as below: analytical column
cells were washed with 200lL HBSS and equilibrated for 1 h in
an incubator. HBSS was removed and 200 lL (100lg/mL)
drug-lipoprotein conjugate batches, 300 lL (100lg/mL)
Vemurafenib and standard drug 5-fluorouracil (positive control)
were added to each well. DMSO was added to separate wells
(
4.6mm ꢂ 150 mm, ProdigyTM C18, 5 lm ODS-3 100 Å); mobile
phase: 0.1% trifluoroacetic acid in water/acetonitrile (40/60%v/
v); flow-rate: 1.0 mL/min; injection volume, 20lL; temperature:
ꢀ
3
5 C and a 20mL loop. The regression analysis was performed
to compare the HPLC results with the analytical profile of pure
drug (calibration curve plotted using peak height correspond-
ing to 1, 2, 4, 8, 16, 32, 64 and 128 mg/mL Vemurafenib
concentrations) for quantitation of cellular uptake of the
Vemurafenib. A one-way ANOVA was performed to compare
the uptake parameters between the Vemurafenib-lipoprotein
conjugate and free drug (at p < 0.05).
as a negative control. The plate was kept in an incubator for
ꢀ
1
2h at 37 C in a humidified incubator. After incubation, the
sterilized micropipette wells were washed three times with ice-
cold PBS (pH 7.4) to remove the drug solutions. The cells were
exposed to a 50lL solution of 0.5% v/v Triton-X 100 for
3
0min. Further, 200 lL of mobile phase containing 0.1% v/v
trifluoroacetic acid in water/acetonitrile (50/50% v/v) was
added to the samples and centrifuged at 11000 rpm for 20min 2.2.9.5.3. Cytotoxicity study using the SRB assay. For cyto-
in a refrigerated centrifuge (Remi Elektrotechnik Ltd., Mumbai, toxicity assay, 100 lL of HaCaT cell suspension per well
5
Maharashtra, India). The supernatants obtained thereof were (obtained from the density of 2 ꢂ 10 cells/mL) was seeded in 96
analyzed by HPLC (Reversed-Phase, Jasco MD-2010, Japan, well tissue culture plates. DMSO (negative control) was added in

8
M. S. BHATIA ET AL.
cells supplemented with DMEM, and 100 lL of supplemented Table 3. The solubility of Vemurafenib in different solvents at RT.
DMEM was added to peripheral wells of the plate (no cells) as Solvent
blank and subsequently allowed to attach overnight in an incu- DMSO
Vemurafenib Solubility (mg/mL)
50
4.10
Methanol
bator. After 24h incubation, the culture media was decanted,
Ethanol
3.8
and the cells were rinsed with 150 lL of culture media. The Dichloromethane
HaCaT cell line was then incubated with Vemurafenib-ARG ole-
1.5
0.0095
Distilled water
ate, Vemurafenib-SER oleate, and Vemurafenib-TYR oleate (every
1
0lg/mL). After incubation, 100 lL cold TCA (10% w/v) was 3.1.3.2. Calibration curve in 0.1 N HCl. The calibration curve
added gently to each well without removing the cell culture of absorbance against concentration was constructed in
ꢀ
supernatant and incubated at 4 C for 1 h. The plate was washed 0.1 N HCl and found to be linear within the range of
with triple distilled water, air-dried, and 100 lL SRB solution 10–50 lg/mL at 271 nm obeying Beer Lambert’s law. The
(
0.057% w/v) was added to each well and left for 30min at RT. slope, intercept, and the correlation coefficient was observed
as 0.00213, 0.00218, and 0.9995, respectively.
Further, the plate was quickly rinsed four times with 1% v/v
acetic acid to remove the unbound dye and allowed to air dry at
RT. After complete air drying, 200 lL of 10mM Tris base solution
3
.1.3.3. Calibration curve in phosphate buffer. Calibration
curve of absorbance against concentration was plotted in
phosphate buffer pH 7.4 and found to be linear within the
range of 10–50 lg/mL at 225 nm obeying Beer Lambert’s law.
The slope, intercept, and the correlation coefficient was
observed as 0.00426, ꢁ0.0012, and 0.992, respectively.
(
pH 10.5) was added to each well and shaken for 5 min on a gyr-
atory shaker to solubilize protein-bound dye. The assay relies on
the ability of SRB to bind to protein components of cells that
have been fixed to tissue-culture plates by TCA (Vichai &
Kirtikara, 2006). The amount of dye extracted from stained cells
proportional to the cell mass was measured as optical density at
5
10 nm by using a microplate reader (Bio-Rad Laboratories India
3
.1.4. Solubility study
Pvt. Ltd., Gurgaon, Haryana, India). These results were compared
with the standard inhibitor used as a positive control (5-fluorour-
acil, 20lg/mL). The cytotoxicity or % killing of cells by
Vemurafenib-lipoprotein conjugates was calculated by using
the following formula (Equation 11). A one-way ANOVA was
used to compare the % cytotoxicity between the different treat-
ment groups (at p < 0.05).
Solubility studies indicated poor solubility of Vemurafenib in
water (0.0095 mg/mL) compared to other solvents (Table 3).
3
.1.5. LOD
The LOD of Vemurafenib was found to be not more
than 1%.
ꢀ
ꢁ
Mean ODsample
Mean ODday0
%
Cytotoxicity ¼ 100 –
ꢂ 100
(11)
3
.2. Design of lipoprotein structures and QbD approach
in selection of variables targeted for optimization
Lipoprotein structures designed with a combination of the
three amino acids were screened virtually by the QbD
approach for the selection of the variables that are targeted
for optimization.
3
. Results and discussion
3.1. Characterization of drug
3
.1.1. Organoleptic properties
Vemurafenib was found to be colorless, white, and crystal-
line powder.
3
.2.1. Virtual screening of lipoprotein
Dataset was prepared using optimized 3D structures of the
designed lipoprotein structures. Further, protein structures
were prepared, refined, and subsequently processed for the
docking analysis.
3
.1.2. Melting point
The melting point of Vemurafenib was observed within the
ꢀ
ꢀ
range of 269–274 C (reported melting point 272–274 C).
3
.2.1.1. Docking analysis of designed lipoproteins.
Docking analysis was performed to explore the binding
potential of the designed, chemically conjugated lipoproteins
with the selected target . Amino acid substituted lipoproteins
3
.1.3. UV-visible spectroscopy
UV-Visible spectra of Vemurafenib in methanol, phosphate
buffer pH 7.4, and 0.1 N HCl showed absorbance maxima
(lipoamino acids) A, B, and C showed significant binding
(kmax) at 271 nm, 225 nm, and 271 nm respectively.
interactions than multi-amino containing lipoproteins (D, E,
F, G, H, I). Table 4 summarizes the docking score and signifi-
cant binding interactions of the conjugated lipoproteins.
Docking analysis indicated that the increase in the molecular
3
.1.3.1. Calibration curve in methanol. The calibration
curve of absorbance against concentration was constructed
in methanol and found to be linear within the range of weight of the molecule was due to the increase in the size of the
0–50 lg/mL at 271 nm obeying Beer Lambert’s law. The peptide. The positive docking score of the chemically modified tri-
slope, intercept, and the correlation coefficient was observed peptide lipoproteins offered an unfavorable orientation of these
as 0.014, 0.017, and 0.9732, respectively. molecules for binding with the selecetd target B-Raf gene.
1

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
However, a mono amino acid containing lipoproteins have signifi- 3.3.2. Synthesis of ARG oleoyl amide, SER oleoyl amide,
cant binding energy than the other six lipoproteins. Docking
and TYR oleoyl amide
interaction of chemical conjugates A, B, and C are shown below Synthesis of ARG oleoyl amide, SER oleoyl amide, and TYR
(Figures 5–8).
oleoyl amide from oleoyl chloride was carried out by the
method reported by Schotten – Baumann reaction.
3.3. Synthesis of lipoproteins
Novel drug carriers were designed by reacting oleic acid
with either arginine, serine, or tyrosine. The synthesis of lipo-
3
.4. Characterization of lipoproteins
proteins was done in two steps. Oleic acid and amino acids 3.4.1. Organoleptic properties
were used as a basic framework for imparting hydrophobicity All synthesized lipoproteins were characterized by organolep-
and polarity to the lipoprotein, respectively. All the synthe- tic properties (Table 5).
sized molecules were characterized by spectroscopic and
physicochemical properties.
3
.4.2. Melting point
The melting of TYR oleoyl amide occurred at high tempera-
3
.3.1. Synthesis of oleoyl chloride from oleic acid
ꢀ
ꢀ
ture (194–196 C) than ARG oleoyl amide (74–76 C) and SER
Synthesis of oleoyl chloride from oleic acid was carried out
by the method reported by Gibbson et al. (Gibson, 2007).
The synthesized oleoyl chloride was observed as a yellowish-
brown oily liquid with aromatic odor showed a boiling point
ꢀ
oleoyl amide (68–70 C) attributed to the higher number of
Carbon atoms and a branched structure.
ꢀ
within the range of 192–194 C.
3
.4.3. FTIR analysis
Table 4. Binding energy and binding interactions of chemically conjugated
lipoproteins.
FTIR spectra of synthesized ARG oleoyl amide (Figure 9)
showed characteristic peaks at 1520.06 cm (N–H bending),
ꢁ
1
Chemically Lipoprotein Code
Docking score
ꢁ1
ꢁ1
ꢁ1
1
607.38 cm (C ¼ C), 1683.55 cm (C ¼ O), and 2847.38 cm
A-VER
B-VER
C-VER
D-VER
E-VER
F-VER
G-VER
H-VER
I-VER
ꢁ657.9
ꢁ551.0
ꢁ453.1
120.2
532.1
496.2
876.5
968.2
326.1
(C–H); SER oleoyl amide (Figure 10) showed characteristic
ꢁ1
ꢁ1
peaks at 1590.02 cm (N–H bending), 1616.06 cm (C ¼ C),
ꢁ1
ꢁ1
1
698.02 cm
(C ¼ O), and 2847.38 cm
(C–H); TYR oleoyl
ꢁ
1
amide (Figure 11) showed characteristic peaks at 1510.95 cm
ꢁ1
ꢁ1
(N–H bending), 1606.41 cm
(C ¼ C), 1701.87 cm
(C ¼ O),
ꢁ
1
and 2847.38 cm (C–H).
Figure 5. Docking interaction of Vemurafenib-ARG oleate chemical conjugate ‘A’.

1
0
M. S. BHATIA ET AL.
Figure 6. Docking interaction of Vemurafenib-SER oleate chemical conjugate ‘B’.
Figure 7. Docking interaction of Vemurafenib-TYR oleate chemical conjugate ‘C’.
3
.4.4. pKa
compound (SER oleoyl amide). The pKa values obtained
The pKa of synthesized lipoprotein was determined by the for arginine, serine, and tyrosine oleoyl amide were indica-
titration method using pH meter (Table 6). The pKa was tive of chances to undergo hydrophilic/hydrophobic
used as an index to express the acidity of weak acids changes within the pathophysiological micro-environmen-
where low pKa value indicates the acidic nature of the tal conditions.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 8. Docking interaction of chemical conjugates ‘D’ to ‘I’.
Table 5. Organoleptic properties of synthesized lipoproteins.
Observation
Property
ARG oleoyl amide
SER oleoyl amide
TYR oleoyl amide
Nature
Color
Odor
Solid
Buff yellow
Aromatic
Solid
Buff yellow
Aromatic
Solid
Buff yellow
Aromatic

1
2
M. S. BHATIA ET AL.
Figure 9. FTIR spectrum of synthesized ARG oleoyl amide.
Figure 10. FTIR spectrum of synthesized SER oleoyl amide.
Figure 11. FTIR spectrum of synthesized TYR oleoyl amide.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Table 6. Physicochemical properties of synthesized lipoproteins.
Lipoprotein
pKa (at RT)
Partition coefficient
Saponification Value
Acid Value
HLB Value
ARG oleoyl amide
SER oleoyl amide
TYR oleoyl amide
6.92
6.56
6.74
0.033
0.169
0.0130
28
33.6
28
29.12
34.72
32.48
0.8
0.8
2.8
Table 7. The wettability, swelling capacity, and viscosity of synthesized lipoproteins.
ꢀ
Lipoprotein
Wettability [Contact angle ( )]
Swelling capacity (%)
Viscosity (cP) at 100 rpm
5 %
2
.5 %
10 %
ARG oleoyl amide
SER oleoyl amide
TYR oleoyl amide
30.2
23.4
18.7
75
62.5
50
2.84
14
52
3.94
27
64
14
48
105
Table 8. Conductance, and LOD of synthesized lipoproteins.
associated ability to swell that allows retention of an
adequate volume of water within its structure without its dis-
solution in water.
Conductance
Lipoprotein
Surface energy
(ms/cm)
LOD (%)
ARG oleoyl amide
SER oleoyl amide
TYR oleoyl amide
64.39
67.41
69.21
0.4
0.3
0.4
162
165
168.05
3
.4.9. Viscosity
Table 9. Micromeritics of synthesized lipoproteins.
The viscosity of synthesized lipoproteins was determined by
using Brookfield viscometer at 100 rpm at RT. TYR oleoyl
amide showed the highest viscosity at all the concentrations
tested (Table 7) attributed to its high HLB and good
water solubility.
The angle of
repose ( )
ꢀ
Lipoprotein
CCI (%)
Hausner’s ratio
ARG oleoyl amide
SER oleoyl amide
TYR oleoyl amide
9.3
11.59
15.18
20
14.28
20
0.8
0.85
0.8
3
.4.10. Conductivity
The conductance for all lipoproteins was observed between
.3 and 0.4 ms/cm (Table 8).
3
.4.5. Partition coefficient
The partition coefficient, i.e. the ratio of the concentration of
synthesized lipoproteins partitioned between two immiscible
phases (octanol-water) after achieving equilibrium at RT,
helps in the determination of the lipophilicity. It gives an
idea about the use of lipoprotein as a carrier and to facilitate
the transport of the drug across the lipid bilayers to the tar-
get site. SER oleoyl amide and TYR oleoyl amide showed the
highest (0.169) and lowest (0.0130) partition coefficients,
respectively (Table 6).
0
3
.4.11. LOD
LOD for all lipoproteins was observed between 162 to
68.05% (Table 8).
1
3
.4.12. Angle of repose
All lipoproteins showed the angle of repose within passable
3
.4.6. HLB
range as indicative of considerable flowability in order of
The HLB values (ratio of saponification value to the acid ARG oleoyl amide > SER oleoyl amide > TYR oleoyl amide
value) of synthesized lipoproteins are given in Table 6. TYR (Table 9).
oleoyl amide showed high HLB value as indicative of good
water solubility. These results were in good agreement with
partition coefficients.
3
.4.13. CCI
CCI indicated poor flowability for all lipoproteins (Table 9).
3
.4.7. Wettability
The wettability of synthesized lipoproteins was determined
3
.4.14. Hausner’s ratio
in terms of contact angle and surface energy by automated Hausner’s ratio for all lipoproteins was found within 0.8 to
instruments (Table 7). TYR oleoyl amide showed a low con- 0.85 (< 1, Table 9) indicating high packability and hence
ꢀ
tact angle (18.7 ) as indicative of good wettability and poor flowability of the powder bed.
water solubility.
3
.5. Preparation and characterization of tablets
3
.4.8. Swelling capacity
Swelling capacity indicates the ability to absorb water at a Tablets prepared using Vemurafenib, synthesized lipoprotein,
given atmospheric condition. ARG oleoyl amide showed the and other excipients were characterized for different post-
highest swelling capacity (Table 7) due to its hydrophobicity compressional parameters.

1
4
M. S. BHATIA ET AL.
Table 10. Post-compression evaluation of tablet formulations.
2
Weight variation
Property
Hardness (Kg/cm )
Friability (%)
2
Disintegration (min)
2
Batch Code
1
2
3
1
2
3
1
3
1
3
A (ARG oleoyl amide)
S (SER oleoyl amide)
T (TYR oleoyl amide)
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
5
5
5
4.5
5
5
5
5
4
1
0.8
0.6
0.8
0.6
0.7
0.9
0.8
0.8
12
10
10
10
13
13
13
13
10
Figure 12. In-vitro drug release study from Vemurafenib-synthesized lipoprotein tablets.
3
.5.1. Weight variation
kinetics was shown by batch A1 with the highest t90%
All tablet formulations showed weight variation, not more (708.9 min, Table 11) amongst all the nine formulations
than 1% (Table 10).
tested. This was attributed to the highly hydrophobic nature
of ARG oleoyl amide that allows limited inside entry of dis-
solution media and hence controlled the diffusion of the
drug outside the matrix for an extended period.
3
.5.2. Hardness
All tablet formulations showed hardness within the defined
Amongst SER oleoyl amide formulations, S1 and S3 fol-
lowed Korsmeyer –Peppas model with super case-II transport
as drug release mechanism (release exponent > 1.0).
However, formulation S2 followed Hixson–Crowell model for
drug release with high t90% (702.5 min, Table 11) indicating
extended drug release low wettability by dissolution media.
TYR oleoyl amide containing formulations showed faster
drug release amongst all the nine formulations tested. All
formulations (T1, T2, and T3) showed Korsmeyer –Peppas as
a best-fit model with a super case-II transport mechanism for
drug release (release exponent > 1.0). Batch T2 showed the
faster release of the drug (t90% ¼ 417.7 min, Table 11)
amongst all the nine batches attributed to the lower wetting
time associated faster disintegration of the tablet.
2
limits of 4–5 Kg/cm (Table 10).
3
.5.3. Friability
Friability of all tablets was observed within the acceptable
limits of less than 1% (Table 10).
3
.5.4. Disintegration test
All batches showed concentration-dependent effect lipopro-
teins on the disintegration time of tablets (Table 10). Longer
time required for disintegration indicates poor wettability of
the ARG oleoyl amide.
3
.5.5. In-vitro drug release study
3
.6. Preparation and characterization of drug-lipoprotein
chemical conjugate
All tablet formulations indicated slow release in acidic media;
however, fast release in alkaline media (Figure 12).
Formulations containing ARG oleoyl amide, A2, and A3 In the chemical conjugation process synthesized lipoprotein
batches followed Korsmeyer –Peppas kinetics with a super (ARG oleate, SER oleate, or TYR oleate) was chemically
case-II transport mechanism for drug release (release expo- bonded with the Vemurafenib and characterized for different
nent > 1.0). However, extended drug release by zero-order parameters as discussed below.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
15
a
Table 11. In-vitro drug release kinetics from tablet formulations.
Korsmeyer–Peppas model
Zero-order
model (r)
First-order
model (r)
Higuchi Matrix
model (r)
Hixson–Crowell
model (r)
Batch Code
(r)
(n)
A best fit kinetic model
t
90% (min)
A1
A2
A3
S1
S2
S3
T1
T2
T3
a
0.9504
0.9281
0.9407
0.9562
0.9378
0.9208
0.9220
0.8939
0.9039
0.8771
0.7776
0.9328
0.8599
0.9328
0.9234
0.8385
0.7950
0.8624
0.8188
0.7796
0.8304
0.8211
0.8348
0.8146
0.7818
0.7480
0.7597
0.9166
0.8486
0.9438
0.9042
0.9437
0.9332
0.8812
0.8603
0.8850
0.9440
0.9836
0.9525
0.9837
0.9413
0.9408
0.9713
0.9627
0.9787
1.7446
2.6451
2.9021
1.7776
2.9509
3.0823
1.9727
3.4627
3.2791
Zero order
708.9
604.2
531.5
643.7
702.5
531.5
421.0
417.7
456.6
Korsmeyer–Peppas
Korsmeyer–Peppas
Korsmeyer–Peppas
Hixson–Crowell
Korsmeyer–Peppas
Korsmeyer–Peppas
Korsmeyer–Peppas
Korsmeyer–Peppas
Where ‘r’ and ‘n’ denotes correlation coefficient and release exponent, respectively.
Table 12. Organoleptic properties of drug-lipoprotein chemical conjugates.
Observation
Property
Conjugate A (Vemurafenib-ARG oleate)
Conjugate B (Vemurafenib-SER oleate)
Conjugate C (Vemurafenib-TYR oleate)
State
Color
Odor
Solid
Solid
Solid
Slight yellowish white
Aromatic
Slight yellowish white
Aromatic
Slight yellowish white
Aromatic
Figure 13. FTIR spectrum of Vemurafenib-ARG oleate chemical conjugate ‘A’.
ꢁ
1
ꢁ1
ꢁ1
3
.6.1. Organoleptic properties
1458.39 cm (C ¼ C), 1637.05 cm (C ¼ O), and 2902.26 cm
Organoleptic properties of chemical conjugates were sum- (C–H); chemical conjugate ‘B’ (Figure 14) showed principal
ꢁ
1
ꢁ1
marized in Table 12.
.6.2. Melting point
peaks at 1485.62 cm (N–H bending), 1637.47 cm (C ¼ C),
ꢁ1
ꢁ1
1
707.87 cm (C ¼ O), and 2917.11 cm (C–H); chemical conju-
ꢁ1
3
gate ‘C’ (Figure 15) showed principal peaks at 1485.44 cm
ꢁ1
ꢁ1
Melting points for drug-lipoprotein conjugates ‘A’, ‘B’, and ‘C’ (N–H bending), 1637.66 cm
were observed as 138–140 C, 190–192 C and 170–172 C and 2853.19 cm (C–H).
(C ¼ C), 1708.29 cm
(C ¼ O),
ꢀ
ꢀ
ꢀ
ꢁ1
respectively.
3
.6.3. FTIR
3.6.4. Solubility
FTIR spectra of synthesized chemical conjugate ‘A’ (Figure 13) All synthesized drug-lipoprotein chemical conjugates were
ꢁ
1
showed principal peaks at 1414.31 cm
(N–H bending), completely soluble in DMSO and chloroform.

1
6
M. S. BHATIA ET AL.
Figure 14. FTIR spectrum of Vemurafenib-SER oleate chemical conjugate ‘B’.
Figure 15. FTIR spectrum of Vemurafenib-TYR oleate chemical conjugate ‘C’.
3
3
.6.5. Biological activity
estimated by HPLC analysis. The use of cold PBS for washing
.6.5.1. Cellular uptake study. The Vemurafenib content the cells lead to complete removal of Vemurafenib-lipoprotein
accumulated in immortalized human HaCaT keratinocytes was conjugate and Vemurafenib solution adhered to the cell

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
17
Table 13. Intracellular concentration and % cellular uptake of Vemurafenib from drug-lipoprotein conjugates in cell lysate
samples by HPLC analysis.
Intracellular Drug
Concentration (mg/mL)
Cellular Drug
Uptake (%)
a
Drug-lipoprotein conjugate
Vemurafenib
23.7 ± 0.11
17.2 ± 0.16
16.6 ± 0.19
17.1 ± 0.12
79.1
86.1
82.8
85.7
Vemurafenib-ARG oleate
Vemurafenib-SER oleate
Vemurafenib-TYR oleate
a
Indicates average ± standard deviation (n ¼ 3).
Figure 16. HPLC chromatograms for estimation of HaCaT intracellular drug concentration from pure Vemurafenib solution and drug-lipoprotein conjugates
Vemurafenib-ARG oleate, Vemurafenib-SER oleate, and Vemurafenib-TYR oleate).
(
Table 14. Anticancer activity (% cytotoxicity) of 5-fluorouracil and Vemurafenib-
lipoprotein conjugates on immortalized human HaCaT keratinocytes.
3
.6.5.2. Cytotoxicity study using SRB assay. The in-vitro
cytotoxic assay of Vemurafenib-lipoprotein conjugates was
performed on immortalized human HaCaT keratinocyte can-
cer cell line to determine its potency. The % cytotoxicity of
the compounds was calculated by comparing the results
obtained from Vemurafenib-lipoprotein conjugate solution as
a test (10 mg/mL each) with that of the 5-fluorouracil solution
as standard (20 mg/mL). All synthesized drug-lipoprotein con-
jugates showed better inhibition of cancer cells compared to
a positive control (Table 14). However, TYR oleoyl amide
showed the highest inhibition (87.39%) than other
lipoproteins.
Compound
-flurouracil (positive control)
Vemurafenib-ARG oleate
Vemurafenib-SER oleate
Vemurafenib-TYR oleate
Concentration (mg/mL)
% Inhibition
5
20
10
10
10
47.82
76.52
86.08
87.39
membrane after incubation. The cells exposed to Vemurafenib-
lipoprotein conjugate showed pronounced accumulation than
cells exposed to pure Vemurafenib solution in 12 h. This was
attributed to the ability of lipoprotein-associated Vemurafenib
to bypass the vesicular drug transport with net effect as
improved intracellular delivery or uptake of the drug by
tumour cells. Moreover, the drug-lipoprotein conjugate pro-
Moreover, Vemurafenib-lipoprotein conjugates showed a
profound cytotoxic effect on HaCat cells compared to pure
Vemurafenib solution. This was attributed to the prepared
vides a continuous dynamic balance between cellular uptake lipoprotein conjugates which allow drug uptake by tumour
and exocytosis after incubation for an extended time. The cells through binding with receptors present over the
intracellular concentration and
%
cellular uptake of tumour cell surface. After uptake, the drug-lipoprotein conju-
Vemurafenib from lipoprotein conjugates in HaCaT cells was gate gets processed in endosome whereby lysosome fuses
observed within the range of 16.6 mg/mL to 17.2 mg/mL (loaded to endosome for the release of drugs through enzymatic
amount ¼ 100 mg/mL) and 82.8% to 86.1%, respectively (Table cleavage or pH environment (Runquist & Havel, 1991). This
1
3 and Figure 16). The improved % cellular uptake of results in the accumulation of maximum drug amount from
Vemurafenib from lipoprotein conjugates compared to the lipoprotein conjugate inside the tumour cells. These results
pure drug solution was attributed to the presence of amino indicated the valuable use of synthesized lipoproteins in the
acid that greatly influences the lipidic environment and enhan- anti-cancer drug formulation to be used in the management
ces the drug permeation through cancer vasculature.
of skin cancer disease.

1
8
M. S. BHATIA ET AL.
for transport and delivery of therapeutic and imaging agents.
Nanomaterials, 10(5), 906. https://doi.org/10.3390/nano10050906
4
. Conclusions
We have developed a novel drug delivery approach by Dass, C. R., & Choong, P. F. M. (2006). Targeting of small molecule anti-
cancer drugs to the tumour and its vasculature using cationic lipo-
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chemically and physically conjugating the lipoprotein struc-
ture simulating moiety with Vemurafenib for cancer cell tar-
geting. Different amino acids were used as building blocks
Derakhshandeh, K., Hochhaus, G., & Dadashzadeh, S. (2011). In-vitro cel-
for a hydrophilic moiety of synthesized lipoprotein structures,
and the hydrophobic part was limited to oleic acid. Nine dif-
ferent lipoproteins were computationally designed and virtu-
ally assessed for their binding affinity with B-Raf protein.
QbD approach was used to identify three lipoproteins as
potential candidates for drug targeting. All the synthesized
lipoproteins on agitation in drug solutions were found to
produce micellar structures simulating the transportability of
lipoproteins. The amide bond between the hydrophobic and
lular uptake and transport study of 9-nitrocamptothecin PLGA nano-
particles across Caco-2 cell monolayer model. Iranian Journal of
Pharmaceutical Research: IJPR, 10(3), 425–434.
Du, Y.-Z., Cai, P., Ping Liu, X., Xi Li, X., Xuan Huang, Y.-Q., Yi-Qiang Ye, F.-
Y., Feng-Ying Chen, H., Yuan, F.-Q., & Hu, F.-Q. (2011). RGD peptide-
mediated chitosan-based polymeric micelles targeting delivery for
integrin-overexpressing tumor cells. International Journal of
Nanomedicine, 6, 3499–3508. https://doi.org/10.2147/IJN.S26670
Francis, M. F., Cristea, M., & Winnik, F. M. (2004). Polymeric micelles for
oral drug delivery: Why and how. Pure and Applied Chemistry, 76(7–8),
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hydrophilic moieties is a stable connection that shows excep- Gaikwad, V., Gujar, V., Rao, P., & Chaudhari, P. (2012). Formulation and
tional resistance to hydrolysis. The synthesized lipoproteins
act as cationic surfactant by protonation of the amino group
in acidic or neutral solution. In-vitro dissolution studies per-
formed for Vemurafenib-lipoprotein physical composites (tab-
lets) showed extended drug release profiles (up to 12 h). The
presence of amide proton in the NMR spectrum confirmed
the chemical interaction for the formation of Vemurafenib-
lipoprotein chemical conjugate. The drug-lipoprotein conju-
gates screened for in-vitro anticancer activity indicated
evaluation of niosomal in-situ gel for ophthalmic use. American
Journal of PharmTech Research, 2(6), 863–882.
Gaikwad, V. L., & Bhatia, M. S. (2013). Polymers influencing transportabil-
ity profile of drug. Saudi Pharmaceutical Journal: SPJ: The Official
Publication of the Saudi Pharmaceutical Society, 21(4), 327–335. https://
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Gaikwad, V. L., Bhatia, N. M., Singhvi, I., Mahadik, K. R., & Bhatia, M. S.
(
2017). Computational modeling of polymeric physicochemical prop-
erties for formulation development of a drug containing basic func-
tionality. Journal of Pharmaceutical Sciences, 106(11), 3337–3345.
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improved cellular drug uptake and inhibition of immortalized Gibson, S. (2007). Process for the preparation of saturated or unsaturated
primary fatty amines (Patent No. United States Patent 7705186).
Harisa, G. I., & Alanazi, F. K. (2014). Low density lipoprotein bionanopar-
human HaCaT keratinocytes compared to 5-fluorouracil
(
standard). Therefore, the lipoproteins and its chemical conju-
ticles: From cholesterol transport to delivery of anti-cancer drugs.
Saudi Pharmaceutical Journal: SPJ: The Official Publication of the Saudi
Pharmaceutical Society, 22(6), 504–515. https://doi.org/10.1016/j.jsps.
2013.12.015
gates synthesized with the drug in the present study could
be used as a potential component in the development of
targeted drug delivery system against cancer cells.
Hu, C.-M J., Aryal, S., & Zhang, L. (2010). Nanoparticle-assisted combin-
ation therapies for effective cancer treatment. Therapeutic Delivery,
1
(2), 323–334. https://doi.org/10.4155/tde.10.13
Acknowledgements
Jain, S. (2014). Quality by design (QBD): A comprehensive understanding
of implementation and challenges in pharmaceuticals development.
International Journal of Pharmacy and Pharmaceutical Sciences, 6,
29–35.
Authors are thankful to the Principal, Dr. H. N. More, Bharati Vidyapeeth,
College of Pharmacy, Kolhapur, Maharashtra, India for providing neces-
sary facilities required to carry out this research work and Dr. Sandip
Patil, Adarsh College of Pharmacy, Vita, Maharashtra, India for carring Lee, E. S., Gao, Z., & Bae, Y. H. (2008). Recent progress in tumor pH tar-
out biological activity
geting nanotechnology. Journal of Controlled Release: Official Journal
of the Controlled Release Society, 132(3), 164–170. https://doi.org/10.
1016/j.jconrel.2008.05.003
Disclosure statement
Martinho, N., Damg ꢀe , C., & Reis, C. P. (2011). Recent advances in drug
delivery systems. Journal of Biomaterials and Nanobiotechnology,
The authors declare no competing financial interest.
02(05), 510–526. https://doi.org/10.4236/jbnb.2011.225062
Menalda, F. A. (2010). Quantitative investigation of the Schotten-
Baumann reaction. Recueil Des Travaux Chimiques Des Pays-Bas,
49(10), 967–995. https://doi.org/10.1002/recl.19300491006
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