Stealth recombinant human serum albumin nanoparticles conjugating 5-fluorouracil augmented drug delivery and cytotoxicity in human colon cancer, HT-29 cells

Article abstract of DOI:10.1016/j.colsurfb.2017.04.020

Background and objective 5-Fluorouracil (5-FU) is a first-line chemotherapeutic drug in colorectal cancer. However, intravenous administration of 5-FU at the dose of 7–12?mg/kg exhibits curbs like short half-life (20?min) and toxic side-effects on bone marrow cells. Therefore, in present investigation, 5-FU was conjugated to poly (ethylene glycol) anchored recombinant human serum albumin nanoparticles (5-FU-rHSA-PEG-NPs) to improve the pharmacokinetic and therapeutic profiles. Methods and results The mean particle size of 5-FU-rHSA-NPs was measured to be 44.3?±?5.8-nm, significantly (P??0.05) up to 48?h. However, addition of 20% v/v serum to PBS (pH?～?7.4) boosted the drug release. 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs released 78.26% and 48.9% of the 5-FU up to 48?h in presence of PBS (pH?～?7.4 and 20% serum) with significant difference (P?50 of 3.7-μM significantly (P?1/2) of 5.33?±?0.15-h significantly (P?a clinical product.

Full text of DOI:10.1016/j.colsurfb.2017.04.020

Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Stealth recombinant human serum albumin nanoparticles
conjugating 5-fluorouracil augmented drug delivery and cytotoxicity
in human colon cancer, HT-29 cells
Ankita Sharmaa, Amanpreet Kaura, Upendra Kumar Jaina, Ramesh Chandrab,c
,
Jitender Madana,∗
a Department of Pharmaceutics, Chandigarh College of Pharmacy, Mohali, Panjab, India
b Dr. B.R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
c Department of Chemistry, University of Delhi, Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Background and objective: 5-Fluorouracil (5-FU) is a first-line chemotherapeutic drug in colorectal cancer.
However, intravenous administration of 5-FU at the dose of 7–12 mg/kg exhibits curbs like short half-
life (20 min) and toxic side-effects on bone marrow cells. Therefore, in present investigation, 5-FU was
conjugated to poly (ethylene glycol) anchored recombinant human serum albumin nanoparticles (5-FU-
rHSA-PEG-NPs) to improve the pharmacokinetic and therapeutic profiles.
Received 23 November 2016
Received in revised form 6 April 2017
Accepted 10 April 2017
Available online 12 April 2017
Methods and results: The mean particle size of 5-FU-rHSA-NPs was measured to be 44.3 5.8-nm,
significantly (P < 0.05) lesser than 65.7 7.2-nm of 5-FU-rHSA-PEG-NPs. In addition, zeta-potential of
5-FU-rHSA-NPs was estimated to be −10.2 2.6-mV significantly (P < 0.05) lower than −25.8 3.5-mV
of 5-FU-rHSA-PEG-NPs. Moreover, both 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs were smooth, spherical
and regular in shape. In-vitro drug release analysis indicated that 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-
NPs separately released 10.9% and 9.23% of 5-FU in PBS (pH ∼ 7.4) with no significant difference (P > 0.05)
up to 48 h. However, addition of 20% v/v serum to PBS (pH ∼ 7.4) boosted the drug release. 5-FU-rHSA-NPs
and 5-FU-rHSA-PEG-NPs released 78.26% and 48.9% of the 5-FU up to 48 h in presence of PBS (pH ∼ 7.4
and 20% serum) with significant difference (P < 0.05). Furthermore, 5-FU-rHSA-PEG-NPs displayed the
IC50 of 3.7-␮M significantly (P < 0.05) lower than 6.8-␮M and 11.2-␮M of 5-FU-rHSA-NPs and 5-FU solu-
tion, respectively. One compartmental pharmacokinetic elements indicated that 5-FU-rHSA-PEG-NPs
demonstrated the half-life (t1/2) of 5.33 0.15-h significantly (P < 0.001) higher than 1.50 0.08-h and
0.30 0.09-h of 5-FU-rHSA-NPs and 5-FU solution, respectively.
Keywords:
5-Fluorouracil
Recombinant human serum albumin
Poly (ethylene glycol)
Conjugation
Cytotoxicity
Pharmacokinetic
Conclusion: 5-FU-rHSA-PEG-NPs tendered improved cytotoxicity and pharmacokinetic profile. Hence,
5-FU-rHSA-PEG-NPs must be further tested under stringent milieu for translating in to a clinical product.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
tion (US-FDA) has approved various anticancer drugs including
5-fluorouracil (5-FU), bevacizumab, avastin, capecitabine, oxali-
Despite noteworthy advances in cancer chemotherapy, cur-
rently used chemotherapeutic agents are unable to recover the
prognosis of advanced or recurrent colorectal cancer. The trans-
port of existing chemotherapeutic drugs by particulate or colloidal
nanocomposite has displayed lucrative advantages in terms of
improved penetration in tumour tissues, high bioavailability at the
site of action, augmented therapeutic index and reduced toxicity
to normal tissues [1]. United-States Food and Drug Administra-
platin and irinotecan for the treatment of colon cancer [2,3].
However, 5-FU is used as a first-line chemotherapeutic drug in
colorectal cancer, and instantly after surgery, is used as an adju-
vant therapy. Chemically, 5-FU (5-Fluoro-2,4-pyrimidinedione) is
an antimetabolite pyrimidine analogue that exhibits potent anti-
cancer activity against broad spectrum of solid tumours like
pancreas, liver, and colon [4–6]. Mechanistically, 5-FU hinders the
action of enzyme thymidylate synthase and consequently prohibits
the synthesis of pyrimidine thymidine, a nucleoside requisite for
DNA replication [7]. It has been reported that 5-FU metabolite, flu-
orouridine triphosphate (FUTP) disrupts the RNA processing and
functions in both human colon as well as breast cancer cells [8].
∗
Corresponding author.
E-mail address: jitenderpharmacy@gmail.com (J. Madan).
http://dx.doi.org/10.1016/j.colsurfb.2017.04.020
0927-7765/© 2017 Elsevier B.V. All rights reserved.

A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
201
Owing to erratic absorption and hampered bioavailability, 5-FU is
administered as intravenous (i.v) infusion at the dose of 7–12 mg/kg
for 4 days for successive chemotherapy [9]. However, 5-FU has cer-
tain curtails like short biological half-life (20 min) due to rapid
metabolism [10] and toxic side-effects on bone marrow cells [11].
Therefore, several attempts were undertaken to improve the deliv-
ery of 5-FU from both oral and parenteral route of administration.
Liposomes, microspheres, and polymeric nanoparticles have been
reported to perk up the therapeutic efficacy of 5-FU [12–15]. Nev-
ertheless, therapeutic efficacy of encapsulated 5-FU in nano/micro
carriers was impinged owing to superfluous release that conse-
quently delivered subtherapeutic pay load in vicinity of the tumour
cells. In addition, only few attempts have been embarked previ-
ously including 5-FU-lipid drug conjugate, 5-FU conjugated gold
nanoparticles, 5-FU-dendrimer conjugate, and dual drug conjugate
(imatinib and 5-FU) for improving the anticancer potential of 5-FU
in cancer cells [16–19].
albino male mice following i.v route of administration as per the
recommended dose-dosage regimen.
2. Materials and methods
2.1. Chemicals
5-FU (Mw ∼ 130.077) was obtained as a gift sample from
Arbro Pharmaceuticals, New Delhi, India. Recombinant human
serum albumin (rHSA; ␣-lysine amino acids ∼62, Mw ∼ 66.5 kDa,
Purity ∼ 96–99%) was purchased from Himedia, Mumbai, India.
Poly (ethylene glycol) monoamine (Mw ∼ 5000) was obtained
from
Sigma-Aldrich,
USA.
N-(3-dimethylaminopropyl)-N-
ethylcarbodiimide hydrochloride (EDAC.HCl) and N-hydroxy
succinimide (NHS) were purchased from Molychem, Mumbai,
India. All other chemicals used were of highest analytical grade.
Human serum albumin (HSA) with an average half-life of
19 days, is a biocompatible, biodegradable and non-immunogenic
biopolymer [20,21]. Recombinant human serum albumin (rHSA)
offers lucrative merits like avoidance of potential risk of con-
tamination by blood-derived pathogens and profuse supply [22].
Moreover, rHSA is amphiphilic in nature owing to surfactant
traits that prevents protein aggregation, stabilizes the conforma-
tional structure of therapeutic moiety and maintains its bioactivity
throughout the shelf-life of product [23]. Besides, rHSA is also
used as a cryoprotectant owing to high glass transition tem-
perature [24]. Recently, US-FDA has approved paclitaxel-rHSA
conjugate (Abraxane® ) for site-specific delivery. This product
offered improved tumour targeting due to enhanced permeation
and retention (EPR) effect as compared to free drug [25].
Several chemotherapeutic drugs have been encapsulated in
HSA nano/micro carriers for targeting tumour tissues through par-
enteral routes of administration [26–28]. Furthermore, albumin
based nanocarriers accumulate more frequently in tumour tissues
owing to EPR attribute [29,30]. The augmented EPR effect of albu-
min based nanocarriers may be ascribed to the fact that solid
tumours commonly enjoy an immature, highly porous vascula-
ture that is acted upon by vascular permeability enhancing factors
like nitric oxide. However, tumour vasculature lacks adequate
lymphatic drainage. As a consequence, macromolecules (>40 kDa)
accumulate within the tumour interstitium due to EPR effect.
Tumour is also a site for albumin catabolism. Tumours consume
albumin as a source of energy, split albumin into amino acids in
lysosomes that are subsequently exercised by cancer cells for accel-
erating their growth and progression [31]. This was also supported
by hypoalbuminiemia in cancer patients as a result of albumin
catabolism in tumour tissues [32].
PEGylation of nanoscaled drug delivery systems offered supe-
rior circulation half-life [33] and abridged toxicity of protein.
Polyethylene glycol (PEG) is advantageous owing to its low toxic-
ity, squat immunogenicity and high biocompatibility [34–36]. PEG
has capability to protect the protein against enzymatic degrada-
tion and engulfment by reticuloendothelial system (RES) [37,38].
The density of PEG overlays a stearic barrier on to the sur-
face of nanoparticles by means of its hydrophilic chains and
thereby reduces the opportunity of opsonization and phago-
cytosis of nanoparticles. Therefore, in present investigation,
initially 5-FU was conjugated to rHSA and later, 5-FU-rHSA was
chemically coupled to poly (ethylene glycol) monoamine (5-FU-
rHSA-PEG) for tailoring hydrophilic layer. Next, 5-FU-rHSA-PEG
nanoparticles (5-FU-rHSA-PEG-NPs) and unmodified nanoparticles
(5-FU-rHSA-NPs) were formulated by desolvation technique [39]
and characterized in vitro using various analytical, spectral and
biological techniques. The pharmacokinetic profile of 5-FU-rHSA-
PEG-NPs, 5-FU-rHSA-NPs and 5-FU solution was analyzed in Swiss
2.2. Cell culture and mediums
Human colon cancer cell line (HT-29) was maintained in 95%
air and 5% CO2 at 37 ◦C using Dulbecco’s Modified Eagle’s Medium
(DMEM) (Biologicals, Israel) supplemented with 10% fetal bovine
serum. All experiments were performed with asynchronous popu-
lation in exponential growth phase (24 h after plating) [40].
2.3. Synthesis of 5-fluorouracil coupled poly (ethylene) glycol
conjugated recombinant human serum albumin
2.3.1. Synthesis of 5-fluorouracil acetate
The synthesis of 5-fluorouracil acetate was instigated with alky-
lation that leads to the formation of an intermediate product,
5-flurouracil-methyl ester (Scheme 1) [41]. In brief, 5-FU (0.005 M,
0.65 gm) was dissolved in ␣-chloroacetic acid methyl ester (0.01 M,
1.8 gm) in addition to 100 mg of freshly prepared potassium car-
bonate. Subsequently, 40 ml of dimethyl formamide was added to
the reaction mixture followed by stirring at room temperature for
14–18 h. The completion of the reaction was verified by thin layer
chromatography (TLC) using 20% ethyl acetate: hexane mixture as
mobile phase. Finally, the mixture was poured into the crushed ice
and precipitates of 5-FU-CH2COOCH3 were filtered off, and dried.
The yield of 5-FU-CH2COOCH3 was estimated to be 78.4%. The
synthesis of 5-FU-CH2-COOCH3 was confirmed by 1H NMR spec-
troscopy using BRUKER DPX 300 MHz spectrophotometer. The 1H
NMR of 5-FU was also captured for comparison.
Next, hydrolysis of 5-FU-CH2COOCH3 was carried out to yield
5-FU-CH2COOH (Scheme 1) [42]. In an experiment, 1.0 g of 5-FU-
CH2COOCH3 was mixed with 10 ml of 10% NaOH solution and the
reaction mixture was stirred for 2–12 h at room temperature. The
completion of the reaction was verified by TLC using 20% ethyl
acetate: hexane as mobile phase. Finally, the solution was neutral-
ized with 4 N HCl and extracted with 50 ml of ethyl acetate and
50 ml of water. The end product was dried over sodium sulphate
followed by distillation that eventually supplied 5-FU-CH2COOH
(Scheme 1). The yield of 5-FU-CH2COOH was measured to be 67.8%.
The synthesis of 5-FU-CH2COOH was confirmed by 1H NMR spec-
troscopy, as specified earlier.
2.3.2. Synthesis of 5-fluorourcil coupled recombinant human
serum albumin
5-Fluorourcil coupled recombinant human serum albumin
(5-FU-rHSA) was synthesised by covalent-coupling method
(Scheme 1) [43]. Briefly, 5-FU-CH2COOH (0.188 g, 0.1 M), EDAC.HCl
(0.384 g, 0.2 M) and NHS (0.23 g, 0.2 M) were added to 100 ml of
phosphate buffer of pH ∼ 4.7. Following this, 0.665 g (100 ␮M) of
rHSA was added to the buffer and stirred overnight. Next day, 5-
FU-rHSA was purified by dialysis against distilled water for 8 h.

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A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
Scheme 1. Schematic representation of the steps involved in synthesis of 5-FU-rHSA-PEG.
The synthesis of 5-FU-rHSA was confirmed by FT-IR (Perkin Elmer,
Massachussetts, USA) spectroscopy and compared with spectrum
of 5-FU. Samples were prepared using KBr pellet (5 mg sample in
200 mg KBr) at a force of 40 psi for 5 min with a hydrostatic press.
Spectra were recorded in the range of 4000–400 cm−1 at a resolu-
added to the solution and stirred for next 24 h to yield 5-FU-rHSA-
PEG (Scheme 1). The synthesis of 5-FU-rHSA-PEG was confirmed
by FT-IR spectroscopy, as specified earlier.
2.3.5. SDS-PAGE analysis
tion of 4 cm−1
.
The SDS-PAGE assay was performed on 12% polyacrylamide gel
to determine the molecular mass of 5-FU-rHSA and 5-FU-rHSA-
PEG, respectively. In brief, rHSA, 5-FU-rHSA and 5-FU-rHSA-PEG
in equal concentration (100 ␮g/ml) were separately diluted in 1 M
Tris HCl buffer (pH ∼ 6.8) containing 10% SDS, 11.6% (v/v) glycerol
and 1% bromophenol blue with 1% ␤-mercaptoethanol. All samples
were heated for 10 min in a boiling water bath and later loaded on
to the gel to analyze the molecular mass.
2.3.3. Determination of conjugation efficiency of 5-fluorouracil to
recombinant human serum albumin
Conjugation efficiency was analyzed by Bradford assay method
[44]. For testing, 1 mg of rHSA or 5-FU-rHSA was added to 10 ml
of distilled water. From this stock solution, 2 ml of the sample was
withdrawn and added to a mixture of 1.5 ml of Bradford reagent
and 1.5 ml of distilled water. All test tubes were incubated at room
temperature for 10 min. Samples were filtered through 0.22-␮m
membrane filter (MDI, Ambala, India) and the absorbance of each
filtrate was measured at 595 nm using a UV/Visible spectropho-
tometer (1800, Shimadzu, Kyoto, Japan). The conjugation efficiency
was calculated from the following formula:
2.4. Preparation of poly (ethylene glycol) conjugated recombinant
human serum albumin nanoparticles coupling 5-fluorouracil
Poly (ethylene glycol) conjugated recombinant human serum
A of amines in rHSA − A of amines in conjugated rHSA × 100
(
)
% Conjugation efficiency = A of conjugated amines
A of amines in rHSA
Where A = absorbance
albumin nanoparticles coupling 5-fluorouracil (5-FU-rHSA-PEG-
NPs) were prepared by desolvation technique [39]. 50 mg of
5-FU-rHSA-PEG was dissolved in 25 ml of distilled water to form
the aqueous phase. Subsequently, acetone (5 ml) [45] was added to
the aqueous phase at the rate of 1 ml/min under constant stirring
at room temperature. Following desolvation process, 25% v/v glu-
traldehyde solution (50 ␮l) was included in the mixture to crosslink
the desolvated nanoparticles. Stirring was continued for next 24 h
at room temperature for efficient cross-linking. Later, tailored
2.3.4. Synthesis of poly (ethylene glycol) conjugated recombinant
human serum albumin coupling 5-fluorouracil
5-FU-rHSA was conjugated to poly (ethylene glycol) monoamine
(5-FU-rHSA-PEG) by covalent-coupling technique [43]. In brief,
0.2 g of 5-FU-rHSA was added to 20 ml of phosphate buffer
(pH ∼ 4.7) containing EDAC.HCl (0.152 g, 4 mM) and NHS (0.092 g,
4 mM). Following this, mixture was stirred at room temperature
for 4 h. In last, poly (ethylene glycol) monoamine (2 mM, 0.2 g) was

A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
203
nanoparticles were purified by centrifugation (Sorvall Ultracen-
trifuge, Thermo Fisher Scientific, Waltham, Massachusetts, United
States) at 30,000 rpm for 1 h and redispersed in distilled water.
Finally, nanoparticles dispersion was freeze-dried (Lark Technol-
ogy, Chennai, India) to get the fine powder. Correspondingly,
5-FU-rHSA-NPs were also formulated for comparative studies.
5-FU-rHSA-NPs, and 5-FU-rHSA-PEG-NPs for 72 h. In last, control
and treated cells were incubated with MTT dye at a concen-
tration of 5 mg/ml for 4 h at 37 ◦C. Subsequent to lysis of the
cells, formazon crystals were obtained, which were solubilised in
100 ␮l of dimethyl sulfoxide. The absorbance was read at 570 nm
using 630 nm as a reference wavelength by ELISA Reader (Tecan,
Switzerland). The results were expressed as IC50. The IC50 refers to
the concentration of drug required to kill 50% of the cells.
2.5. Characterization of nanoparticles
2.5.1. Particle size and zeta-potential
2.6.2. Pharmacokinetic analysis
Particle size and zeta-potential of nanoparticles were deter-
mined by Malvern Nano ZS (Malvern Instruments, Worcestershire,
UK). Each nanoparticle sample (5 mg) was separately suspended
in 10 ml of phosphate buffer saline (PBS, pH ∼ 7.4) and particle size
was measured. An electric field of 150 mV was applied to determine
the electrophoretic velocity of nanoparticles. All measurements
were carried out in triplicate (n = 3).
Pharmacokinetic analysis of 5-FU solution, 5-FU-rHSA-NPs, and
5-FU-rHSA-PEG-NPs was carried out using high-performance liq-
uid chromatography (HPLC) by determining the concentration of
5-FU in plasma. HPLC system of Agilent Technologies 1220 infinity
LC was used. The chromatographic separation was achieved in a
reverse phase C18 column (4.6 mm × 150 mm) packed with parti-
cles of 5 ␮m. The mobile phase was consisted of acetonitrile: water
(10: 90) mixture [51] that was filtered through 0.45-␮m mem-
brane filter (MDI, Ambala, India). The flow rate of mobile phase
was adjusted at 1 ml/min. The samples were detected at a wave-
length of 265 nm [49] and the detector was set at 0.005 absorbance
unit, full scale.
2.5.2. Transmission electron microscopy
Transmission electron microscopy (TEM, FTI Tecnai F20) was
used to visualize the nanoparticle shape. For this purpose, an aque-
ous dispersion of each nanoparticle sample was drop casted onto
a carbon coated copper grid, and the grid was air dried at room
temperature before loading it into the microscope which was main-
tained at a voltage of 80 kV.
The pharmacokinetic study was carried out according to guide-
lines of the Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), Ministry of Culture, Gov-
ernment of India. Swiss albino male mice weighing 20–25 g
were maintained on standard laboratory chow and water ad
libitum in a temperature and light-controlled environment at
Chandigarh College of Pharmacy, Mohali, Punjab, India. The Insti-
tutional Animal Ethics Committee (IAEC, Registration number:
1201/PO/Re/S/08/CPCSEA) had approved the study In short, 66 mice
were randomly subdivided into 22 groups of three animals each.
These 22 groups were further categorized as group A, B, and C.
Though this gives us three mice per time point for analysis, but
to make sure that our systemic error is acceptable, we had ana-
lyzed each sample in triplicate (n=3) and no variation was seen
(at 95% confidence level). In group A, 5-FU infusion (12 mg/kg) [9]
was administered i.v. through tail vein of the mice. Group B and C
were administered 5-FU-rHSA-NPs (∼12 mg/kg of 5-FU) and 5-FU-
rHSA-PEG-NPs (∼12 mg/kg of 5-FU) respectively through i.v route.
The blood samples were collected from retro-orbital plexuses and
stored in polypropylene microcentrifuge tubes containing 100 ␮l of
sodium citrate (10% w/v), as an anticoagulant. Samples were cen-
trifuged at 4600 rpm for 15 min (Remi, Mumbai, India) and plasma
was collected. Plasma samples were stored at −20 ◦C until analyzed.
Plasma concentration data were analyzed with standard one-
compartmental method using WINNONLIN (Software version 4.1;
Pharsight, California, USA) software. The pharmacokinetic parame-
ters like ke (elimination rate constant, h−1), t1/2 (half life, h), AUClast
(area under the curve up to last sampling point, h ␮g/ml), AUCinf
(area under the curve up to infinite, h ␮g/ml), AUMC (h2 ␮g/ml),
MRT (mean residence time, h), CLTotal (total clearance rate, l/h),
and Vd (volume of distribution, l) were computed and compiled.
The area under the curve (AUClast) was calculated using the linear
trapezoidal rule up to the last sampling point with detectable levels
with extrapolation to infinity (AUCinf) by Eq. (1):
2.5.3. Powder x-ray diffraction pattern
The lattice structure of nanoparticles was validated by X-
ray diffractometer (X’Pert PRO, Panalytical Company, Almelo, The
Netherlands) using Ni-filtered, Cu K␣-radiation, voltage of 60 Kv
and a current of 50 mA. The scanning rate used was 1◦/min over
5◦ to 80◦ diffraction angle at 2ꢀ. The PXRD pattern of 5-FU, 5-FU-
rHSA-NPs, and 5-FU-rHSA-PEG-NPs was recorded. The crystal size
of 5-FU, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs was measured
using Scherer equation [46].
0.94ꢁ
ˇ
2
Dp =
CosꢀWhere Dp refers to average crystallite size,
ˇ = Line broadening in radians, ꢀ = Bragg angle ␭ = X-ray wavelength
2.5.4. In-vitro drug release
In vitro release of drug from tailored nanoformulations was mea-
sured using dialysis membrane technique [47]. Experimentally,
2 ml dispersion containing 12 mg of 5-FU or 472 mg of 5-FU-rHSA-
PEG (∼12 mg of 5-FU) or 439 mg of 5-FU-rHSA (∼12 mg of 5-FU)
was added to a dialysis bag (12 Kda pore size). The bags were
then suspended separately in 900 ml of PBS (pH ∼ 7.4) and PBS
(pH ∼ 7.4) containing 20% v/v fetal bovine serum that maintained
at 37 ◦C and 100 rpm, as recommended for dissolution testing of
parenteral products [48]. At 0.083, 0.25, 0.5, 1, 2, 4, 8, 16, 24, and
48 h, 5 ml of the sample was withdrawn and concurrently replaced
with fresh dissolution medium to maintain the sink condition. The
drug concentration was measured after filtration through 0.22 ␮m
membrane filter (MDI, Ambala, India) at 265 nm [49] by using a
UV/Visible spectrophotometer (1800, Shimadzu, Kyoto, Japan).
2.6. Comparative therapeutic efficacy testing of
5-FU-rHSA-PEG-NPs and 5-FU-rHSA-NPs
AUCinf = AUClast + C/ke
(1)
2.6.1. Standard cell proliferation assay
In vitro cytotoxicity was determined by MTT assay (3-
[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) [50].
Concisely, 7 × 103 HT-29 cells were plated in 200 ␮l of serum
DMEM suspended in each well of a 96-wells microtitre plate (Tar-
sons, India). Post incubation period of 24 h, the serum DMEM
was replaced with serum free-DMEM. Subsequently, HT-29 cells
were exposed to a gradient concentration of 2–16 ␮M of 5-FU,
ke represents the elimination rate constant and it was calculated
from the slope of the data points in final log linear part of the drug-
concentration-time curve by least square linear regression analysis.
The terminal disposition half-life (t1/2) was calculated using Eq. (2):
1
t
= 0.693/ke
(2)
2

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A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
the synthesis of 5-FU-CH2COOH (Scheme 1) was confirmed by 1
H
MRT (mean residence time) was calculated from the AUClast and
AUMC as seen in Eq. (3): AUMC was obtained from a plot of product
of plasma drug concentration and time (C.t.) vis-à-vis ‘t’ from zero
to infinity.
NMR that depicted the presence of H2C-COO- at 4.54 ppm (Singlet)
(Fig. 1). Next, FT-IR spectroscopy was used to confirm the synthe-
sis of 5-FU-rHSA biomacromolecule (Scheme 1, Fig. 1 and Suppl.
Table 1). The spectrum of 5-FU displayed peaks at 1081.90 cm−1
,
MRT = AUMC/AUClast
(3)
1239.83 cm−1, and 1348.43 cm−1 for fluorine, 1639.20 cm−1 for
C
O and 3526.57 cm−1 for −NH group. However, in spec-
Correspondingly, volume of distribution (Vd) was calculated
from (CLTotal)/ke, where CLTotal represents the total body clearance.
trum of 5-FU-rHSA, the peaks of fluorine were slightly shifted,
respectively at 1043.25 cm−1 and 1251.48 cm−1. In addition, the
CLTotal = Dose/AUCinf
Vd = CLTotal/ke
(4)
(5)
peaks for −NH
C
O and −COOH were appeared respectively at
1668.46 cm−1 and 1251.48 cm−1 (Fig. 1 and Suppl. Table 1). Cor-
respondingly, the synthesis of 5-FU-rHSA-PEG was also confirmed
by FT-IR spectroscopy (Scheme 1, Fig. 1 and Suppl. Table 1). The
spectrum of 5-FU-rHSA-PEG exhibited peaks at 1160.78 cm−1 and
2.7. Statistical analysis
1248.05 cm−1 for fluorine as well as 1688.29 cm−1 for −NH
C O
Results were expressed as mean S.D for n = 3 by using column
statistics in Graph Pad, Prism04 Software. Unpaired ‘t’ test was used
to calculate the statistical significant difference between mean val-
ues of two groups. One-way and two-way ANOVA tests were used
for comparison between different groups.
group. Hence, FT-IR spectra evinced that 5-FU persisted its charac-
teristics in protein matrix and steps of conjugation did not impact
the chemical stability of drug. In addition, Bradford assay method
[44] and SDS-PAGE were also implicated to determine the conjuga-
tion efficiency of drug to protein matrix. The conjugation efficiency
of 5-FU to rHSA was estimated to be 67.2%. The molecular weight
of 5-FU-rHSA and 5-FU-rHSA-PEG was determined respectively in
the range of 66–69 KDa and 71–73 KDa as compared to 66–67
KDa for rHSA alone (Suppl. Fig. 1). Hence, the tailored conjugates
(5-FU-rHSA or 5-FU-rHSA-PEG) contain 13–14 mM of 5-FU/mM of
rHSA.
3. Results
3.1. Synthesis and confirmation of 5-FU-rHSA and
5-FU-rHSA-PEG
In present investigation, 5-FU-rHSA and 5-FU-rHSA-PEG were
designed and synthesized (Scheme 1) for customizing the par-
enteral nanoformulations to achieve a long circulation half-life.
The 1H NMR spectrum of 5-FU-CH2OOCH3 displayed the peaks at
2.7768 and 2.7603 ppm, respectively for the protons of −CH2 and
−CH3 functional group (Scheme 1 and Fig. 1). On the other hand,
1H NMR spectrum of 5-FU template exhibited the peaks in the
range of 7.64-7.65 ppm for CH CF of 5-FU (Fig. 1). Following this,
3.2. Preparation and characterization of 5-FU-rHSA-NPs and
5-FU-rHSA-PEG-NPs
5-FU-rHSA-PEG-NPs and 5-FU-rHSA-NPs were prepared by
using desolvation method [39]. Acetone was used as desolvating
agent to precipitate the protein in aqueous phase [45]. The mean
Fig. 1. A) FT-IR spectroscopy of 5-FU, 5-FU-rHSA and 5-FU-rHSA-PEG scanned between 4000 and 400 cm−1 at a resolution of 4 cm−1; B) 1H NMR spectrum of 5-FU, 5-FU-
CH2COOCH3 and 5-FU-CH2COOH.

A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
205
Fig. 2. Particle size distributions, zeta-potential and transmission electron microscopy (TEM) of A) 5-FU-rHSA-NPs and B) 5-FU-rHSA-PEG-NPs. Scale bar ∼100 nm in TEM.
tively. However, the crystallite size may not be correlated with the
mean particle size of tailored nanoformulations.
3.3. In-vitro drug release analysis
In-vitro drug release study was carried out using dialysis mem-
brane technique [47]. 5-FU solution displayed a maximum release
of 99.5% in PBS (pH ∼ 7.4) at 0.5 h. On the other hand, 5-FU-
Fig. 3. Powder X-ray diffraction (PXRD) pattern of 5-FU, 5-FU-rHSA-NPs and 5-FU-
rHSA-PEG-NPs scanned between 5 and 80◦ at 2ꢀ.
rHSA-NPs and 5-FU-rHSA-PEG-NPs only released 10.9% and 9.23%,
respectively in PBS (pH ∼ 7.4) without any significant difference
(P > 0.05) (Fig. 4A–B). Continuation to this, addition of 20% v/v
serum to PBS (pH ∼ 7.4) further boosted the drug release. The tai-
lored nanoformulations, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs
released 78.26% and 48.9% of the 5-FU in 48 h, significantly (P < 0.05)
higher than the amount of drug released by 5-FU-rHSA-NPs and
5-FU-rHSA-PEG-NPs in PBS (pH ∼ 7.4) only (Fig. 4A–B).
particle size of 5-FU-rHSA-NPs was measured to be 44.3 5.8 nm,
significantly (P < 0.05) lesser than 65.7 7.2 nm of 5-FU-rHSA-PEG-
NPs (Fig. 2A–B). Correspondingly, zeta-potential of 5-FU-rHSA-NPs
was estimated to be −10.2 2.6 mV significantly (P < 0.05) smaller
than −25.8 3.5 mV of 5-FU-rHSA-PEG-NPs (Fig. 2A–B). TEM pho-
tomicrographs taken at the scale of 100 nm substantiated that both
5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs were smooth, spherical
and regular in shape (Fig. 2A–B). The structural architecture of tai-
lored nanoformulations was elucidated using PXRD technique. The
XRD pattern of 5-FU was noticed to be crystalline in nature (blue
arrow) (Fig. 3). In contrast, amorphous lattice was discerned for 5-
FU-rHSA-NPs owing to the presence of diffused peaks (red arrow).
Corresponding to 5-FU-rHSA-NPs, the amorphous nature was also
retained in 5-FU-rHSA-PEG-NPs due to the presence of brushes of
hydrophilic PEG polymer (green arrow) (Fig. 3). The crystallite size
of 5-FU was calculated to be 102.96 nm in comparison to 35.21 nm
and 57.62 nm of 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs, respec-
3.4. 5-FU-rHSA-PEG-NPs superiorly inhibited cell proliferation in
HT-29 cells
The cytotoxicity of 5-FU solution, 5-FU-rHSA-NPs and 5-FU-
rHSA-PEG-NPs was measured in HT-29 cells using standard cell
proliferation assay [50]. 5-FU-rHSA-PEG-NPs displayed the IC50 of
3.7 ␮M significantly (P < 0.05) lower than 6.8 ␮M of 5-FU-rHSA-NPs
and 11.2 ␮M of 5-FU solution, respectively (Fig. 5). Correspond-
ingly, there was significance (P < 0.05) difference between the IC50
of 5-FU-rHSA-NPs and 5-FU, respectively.

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A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
Fig. 4. In-vitro drug release of 5-FU solution, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs carried out in A) phosphate buffer saline (PBS, pH ∼ 7.4) and, B) PBS (pH ∼ 7.4, with
20% serum) at 37 ◦C and 100 rpm, as recommended for dissolution testing of parenteral products.
and 5-FU solution, respectively. Consistently, 5-FU-rHSA-PEG-NPs
reported the half-life (t1/2) of 5.33 0.15 h significantly (P < 0.001)
higher than 1.50 0.08 h and 0.30 0.09 h of 5-FU-rHSA-NPs
and 5-FU solution, respectively. Furthermore, 5-FU-rHSA-PEG-
NPs displayed the AUClast of about 966.78 18.94 ␮g.h/ml,
significantly (P < 0.001) higher than 323.72 12.49 ␮g·h/ml and
50.57 4.72 ␮g·h/ml of 5-FU-rHSA-NPs and 5-FU solution, respec-
tively (Table 1). Hence, in-vivo analysis substantiated the prolonged
release pattern and enhanced drug exposure of 5-FU from tailored
nanoformulations upon administration through i.v route. Corre-
spondingly, 5-FU-rHSA-PEG-NPs also demonstrated the superiority
over 5-FU-rHSA-NPs and 5-FU solution in terms of mean residence
Fig. 5. Percent cell viability analysis of HT-29, human colon cancer cells follow-
ing treatment with 5-FU, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs for 72 h using
standard cell proliferation assay.
time (MRT), volume of distribution (Vd) and total body clearance
(CLTotal) (Table 1).
Table 1
4. Discussion
One-compartmental pharmacokinetic analysis of 5-FU, 5-FU-rHSA-NPs and 5-
FU-rHSA-PEG-NPs administered to male mice through intravenous route of
administration at the dose equivalent to 12 mg/kg of 5-FU.
Nanomedicines may become the game changers if these are
systematically designed, optimized and characterized with critical
parameters. In present investigation, 5-FU was primarily conju-
gated to rHSA (5-FU-rHSA) and later tailored biomacromolecule
was tethered with PEG (5-FU-rHSA-PEG) by covalent-coupling
method [43] for shaping the stealth character (Scheme 1, Fig. 1
and Suppl. Table 1). Large molecular weight (20–50 kDa) PEG
has been proven useful for extending the blood circulation time,
although it enhances the size of nanoparticles. However, large
size nanoparticles are complicated to clear from the kidney and
preferably, deposited by the liver. Hence, to avoid liver clearance,
attachment of low molecular weight PEG (2000–5000 Da) has been
advocated to extend the in vivo half-life of nanoparticles. In addi-
tion, chain length, shape, and density of PEG also influence the
extent of hydrophilicity and phagocytosis [52]. The density of PEG
in the range of 0.5-5% w/w was optimized to be able to remark-
ably reduce the amount of protein as compared to unmodified
nanoparticles [53]. Hence, it is apparent that 5-FU-rHSA-PEG holds
compelling evidences to be used for customizing the nanoparti-
cles (Fig. 2A–B). Further, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs
were prepared by desolvation technique using acetone as desol-
vating agent [39,45]. Using molecular dynamics techniques, we
reported that acetone is a favourable desolvating agent in compar-
ison to ethanol, as it generates smaller size nanoparticles of protein
( < 100 nm) with uniform size distribution (Fig. 2A–B) [45]. Sur-
face charge is a vital physicochemical element that influences the
stability and fate of nanoparticles in physiological milieu. Usually,
the surface charge of ≥ 25 mV is desirable for achieving a sta-
Nanoformulations
Parameters
5-FUa,*
5-FU-rHSA-NPsa,* 5-FU-rHSA-PEG-NPsa,*
Ke (h−1
t1/2 h−1
)
2.303 0.105 0.46 0.08
0.13 0.04
5.33 0.15
0.30 0.09
1.50 0.08
AUClast (␮g h/ml) 50.57 4.72
323.72 12.49
393 20.32
0.03 0.008
0.065 0.052
644.39 8.72
1.63 0.42
966.78 18.94
1582.93 33.25
0.007 0.001
0.05 0.011
4155.081 25.68
2.62 0.21
AUCinf (␮g h/ml)
70.28 8.95
0.17 0.04
0.073 0.06
CL (Lh−1
Vd (L)
)
AUMC (␮g h2/ml) 13.51 2.63
MRT (h−1
)
0.26 0.05
Note: 5-FU: 5-Fluorouracil, 5-FU-rHSA-NPs: 5-Fluoruracil conjugated recombi-
nant human serum albumin nanoparticles, 5-FU-rHSA-PEG-NPs: 5-Fluorouracil
conjugated poly (ethylene glycol) anchored recombinant human serum albumin
nanoparticles.
a
Each experiment was carried out in triplicate (n = 3).
P < 0.001 (One-way ANOVA test).
*
3.5. 5-FU-rHSA-PEG-NPs exhibited longer circulation half-life in
preclinical analysis
The pharmacokinetic elements of tailored nanoformulations
were analyzed in vivo. The pharmacokinetic parameters and
plasma concentration-time profile curves were calculated, com-
piled and analyzed using one-compartmental parameters (Table 1
and Fig. 6). 5-FU-rHSA-PEG-NPs documented 0.13 0.04 h−1 of
elimination rate constant (ke) significantly (P < 0.001) lower
than 0.46 0.08 h−1 and 2.303 0.105 h−1 of 5-FU-rHSA-NPs

A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
207
Fig. 6. Plasma-concentration (␮g/ml) vis-à-vis time profile (h) curve of 5-FU solution, 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs administered through i.v route in Swiss albino
male mice at the dose of 12 mg/kg of 5-FU along with representative chromatograms.
ble dispersion. High positive or negative surface charge generates
larger repulsive forces, whereas repulsion between nanoparti-
cles with identical electric charge prevents aggregation and thus
ensures easy redispersion [54,55]. Moreover, surface charge also
determines various physiological activities of nanoparticles like
circulation time, metabolism, clearance, and immune response
[55,56]. Therefore, owing to the presence of negative surface charge
(Fig. 2A–B), a highly stable and efficacious nanosuspension of 5-
FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs may be expected in both
in vitro and in vivo testing conditions. Binding of plasma pro-
teins is an opening mechanism for RES to recognize the injected
nanoparticles that consequently causes a huge loss of the admin-
istered dose. Macrophages in liver (Kupffer cells) recognize the
opsonized nanoparticles via scavenger receptors. Liver, spleen and
bone marrow are the major RES organs responsible for the fate
of nanoparticles. All macrophages reported a net negative sur-
face charge. On the other hand, activated macrophages bear a
lower zeta-potential and a higher isoelectrophoretic point than
resident and elicited macrophages. This may be attributed to
higher density of sialic acid residues on the surface of activated
macrophages [56,57]. Hence, owing to electrophoretic repulsion
between negatively charged 5-FU-rHSA-PEG-NPs and negatively
charged macrophages, a delay in the fate of nanoparticles and pro-
longed plasma half-life may be anticipated.
Nanoparticles shape directly persuades the cellular uptake
[58]. The cellular uptake of nanoparticles >100 nm has fol-
lowed the order of nanorods > nanospheres > nanocylinders and
nanocubes [58–60]. Interestingly, nanospheres were endocytosed
more rapidly than nanorods when nanoparticles size was <100 nm
[59,60]. Therefore, owing to spherical shape of 5-FU-rHSA-NPs and
5-FU-rHSA-PEG-NPs (Fig. 2A–B), a higher cellular uptake may be
anticipated in cancer cells. Furthermore, PXRD confirmed the amor-
phous lattice of 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs (Fig. 3).
Generally, due to asymmetrical structural lattice, the amorphous
phase demands minimal energy and consequently offers higher
solubility and bioavailability of drugs [36]. Hence, sub-cellular par-
ticle size, negative surface charge, spherical shape and amorphous
lattice attributes of 5-FU-rHSA-NPs and 5-FU-rHSA-PEG-NPs, were
found to be decisive for delivering through i.v route of admin-
istration for offering prolonged release and improved circulation
half-life.
Following this, in vitro release study of 5-FU from 5-FU-rHSA-
NPs and 5-FU-rHSA-PEG-NPs was performed in PBS (pH ∼ 7.4) in
presence and absence of serum (Fig. 4A–B). Conjugation of 5-FU
with stealth protein biomacromolecule prevented the superfluous
release of drug in PBS (pH ∼ 7.4) as compared to free 5-FU. Hence,
5-FU would not produce toxicity to normal tissues owing to erratic
biodistribution. However, addition of serum to PBS (pH ∼ 7.4) pro-
moted the prolonged release of 5-FU from 5-FU-rHSA-NPs. Human
serum contains proteolytic enzyme, chymase. It is well know
that albumin is a substrate for chymase. Chymase hydrolyzes the
albumin uniquely and the resulting fragments retained disulfide-
linkage likely between Cys-75 and Cys-90 [61]. Thus, gradual
degradation of rHSA in 5-FU-rHSA-NPs offered constant release of
5-FU for prolonged period of time. However, owing to the pres-
ence of higher negative surface charge on 5-FU-rHSA-PEG-NPs, the
rate of degradation was slower in comparison to 5-FU-rHSA-NPs
that subsequently suppressed the release of 5-FU. Therefore, we
may correlate and predict the longer in vivo circulation half-life of
5-FU-rHSA-PEG-NPs (Fig. 4A–B).
The therapeutic potential of 5-FU solution, 5-FU-rHSA-NPs and
5-FU-rHSA-PEG-NPs was investigated in HT-29 cells and expressed
in terms of IC50 value (Fig. 5). The IC50 of 5-FU-rHSA-PEG-NPs
was estimated to be ∼3.02 and ∼1.83 folds lower than the 5-FU
solution and 5-FU-rHSA-NPs, respectively. Hypoxia-inducible fac-
tor (HIF-1␣) protein is over-expressed in many human cancers
and is a major cause of resistance to drugs. HIF-1␣ up-regulation
decreases the effectiveness of several anticancer agents includ-
ing 5-FU in HT-29 cells, because it induces the expression of drug
efflux transporters (P-gp), alters DNA repair mechanisms and mod-
ifies the balance between pro- and antiapoptotic proteins [62].
Previous reports indicated that PEG inhibits the ATPase enzyme
that is involved in the functioning of P-gp efflux pump proteins,
thus modulates the transport of therapeutic moiety [63,64]. Hence,
we propose that the lower IC50 and enhanced cytotoxicity of 5-
FU-rHSA-PEG-NPs may be attributed to greater endocytosis of
nanoparticles by HT-29 cells in comparison to 5-FU solution and
5-FU-rHSA-NPs, respectively.
In last, pharmacokinetic parameters of 5-FU, 5-FU-rHSA-NPs
and 5-FU-rHSA-PEG-NPs were assessed and compared (Table 1 and
Fig. 6). 5-FU-rHSA-PEG-NPs displayed the superior pharmacoki-
netic profile in comparison to 5-FU-rHSA-NPs and 5-FU solution,

208
A. Sharma et al. / Colloids and Surfaces B: Biointerfaces 155 (2017) 200–208
respectively. We monitored ∼17.76 folds increment in the cir-
culation half-life of 5-FU when injected as 5-FU-rHSA-PEG-NPs
as compared to 5-FU solution. Correspondingly, other pharma-
cokinetic parameters like AUClast and MRT were also improved
upon administration of 5-FU-rHSA-PEG-NPs (Table 1). AUClast is
an imperative parameter to measure the drug exposure. Notable in
these results was approximately ∼2.98 folds increase in the AUClast
by 5-FU-rHSA-PEG-NPs over the 5-FU-rHSA-NPs and ∼19.11 folds
hike over the 5-FU solution in scheduled dose-dosage regimen.
Moreover, 5-FU-rHSA-PEG-NPs displayed higher MRT in plasma
that confirmed the stealth character and permitted the nanofor-
mulation to circulate for longer period of time by avoiding RES
uptake.
[18] S.M. Lee, Y.S. Bala, W.K. Lee, N.J. Jo, I. Chung, J. Biomed. Mater. Res. A 101
(2013) 2306.
[19] M.S. Matlapudi, A. Moin, R. Medishetti, K. Rajendra, A.M. Raichur, B.R. Kumar,
Curr. Drug Deliv. 12 (2015) 782.
[20] T. Peters, All About Albumin: Biochemistry, Genetics and Medical
Applications, Academic Press Limited, San Diego, CA, 1996.
[21] S. Sebak, M. Mirzaei, M. Malhotra, A. Kulamarva, S. Prakash, Int. J. Nanomed. 5
(2010) 525.
[22] K. Kobayashi, Biologicals 34 (2006) 55.
[23] W. Ohtani, Y. Nawa, K. Takeshima, H. Kamuro, K. Kobayashi, T. Ohmura, Anal.
Biochem. 256 (1998) 56.
[24] C.J. Hunt, Transfus. Med. Hemother. 38 (2011) 107.
[25] K. Taguchi, K. Yamasaki, H. Seo, M. Otagiri, Pharmaceutics 7 (2015) 255.
[26] O. Akbal, E. Erdal, T. Vural, D. Kavaz, E.B. Denkbas, Artif. Cells Nanomed.
Biotechnol. 17 (2016) 1.
[27] Q.L. Thao, H.J. Byeon, C. Lee, S. Lee, E.S. Lee, Y.W. Choi, H.G. Choi, E.S. Park, K.C.
Lee, Y.S. Youn, Pharm. Res. 33 (2016) 615.
[28] E.J. Truter, A.S. Santos, W.J. Els, Cell Biol. Int. 25 (2001) 51.
[29] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Control. Release 65 (2000)
271.
5. Conclusion
[30] H. Maeda, Biconjugate Chem. 21 (2010) 797.
[31] G. Stehle, H. Sinn, A. Wunder, H.H. Schrenk, J.C. Stewart, G. Hartung, W. Maier-
Borst, D.L. Heene, Crit. Rev. Oncol. 26 (1997) 77.
5-FU-rHSA-PEG-NPs and 5-FU-rHSA-NPs were successfully
customized to offer prolonged release through i.v route of admin-
istration. In vitro and in vivo testing parameters indicated that
5-FU-rHSA-PEG-NPs and 5-FU-rHSA-NPs release the drug in sus-
tained fashion in physiological mediums. The therapeutic efficacy
and pharmacokinetics of 5-FU-rHSA-PEG-NPs were estimated to be
superior than 5-FU-rHSA-NPs under a set of biological techniques.
Therefore, 5-FU-rHSA-PEG-NPs formulation may be a potential
candidate for testing under a set of stringent parameters for trans-
lating in to a clinical product.
[32] A.M. Merlot, D.S. Kalinowski, D.R. Richardson, Front. Physiol. 5 (2014) 299.
[33] J.M. Harris, R.B. Chess, Nat. Rev. Drug Discov. 2 (2003) 214.
[34] T. Sawa, J. Wu, T. Akaike, H. Maeda, Cancer Res. 60 (2000) 666.
[35] G. Pasut, F.M. Veronese, Prog. Polym. Sci. 32 (2007) 933.
[36] J. Madan, R.S. Pandey, U.K. Jain, O.P. Katare, R. Aneja, A. Katyal, Colloids Surf. B:
Biointerfaces 107 (2013) 235.
[37] J. Madan, R.S. Pandey, V. Jain, O.P. Katare, R. Chandra, A. Katyal, Nanomedicine
9 (2013) 492.
[38] J. Madan, N. Dhiman, S. Sardana, R. Aneja, R. Chandra, A. Katyal, Anticancer
Drugs 22 (2011) 543.
[39] K. Langer, S. Balthasar, V. Vogel, N. Dinauer, H. von Briesen, D. Schubert, D. Int,
J. Pharm. 257 (2003) 169.
[40] Y. Niv, B. Schwartz, Y. Amsalem, S.A. Lamprecht, Anticancer Res. 15 (1995)
2023.
Appendix A. Supplementary data
[41] K.K. Devadoss, R.P. Subramaniam, Tetrahedron Lett. 53 (2012) 3230.
[42] M. Li, Z. Liang, X. Sun, T. Gong, Z. Zhang, PLoS One 9 (2014) e112888.
[43] K. Kaur, R.K. Sodhi, A. Katyal, R. Aneja, U.K. Jain, O.P. Katare, J. Madan, Colloids
Surf. B: Biointerfaces 132 (2015) 225.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.04.
020.
[44] M.M. Bradford, Anal. Biochem. 72 (1976) 248.
[45] J. Madan, S.R. Gundala, Y. Kasetti, P.V. Bharatam, R. Aneja, A. Katyal, U.K. Jain,
Anticancer Drugs 25 (2014) 704.
References
[46] H.P. Klung, L.E. Alexander, X-Ray diffraction procedures for polycrystalline
and amorphous materials, 2nd ed., John Wiley and Sons, New York, 1954.
[47] P.K. Gupta, C.T. Hung, D.G. Perrier, J. Pharm. Sci. 76 (1987) 141.
[48] R. Puri, R.K. Bhatia, R.S. Pandey, U.K. Jain, O.P. Katare, J. Madan, Drug Dev. Ind.
Pharm. 42 (2016) 2020.
[1] J. Prados, C. Melguizo, R. Ortiz, G. Perazzoli, L. Cabeza, P.J. Alvarez, F.
Rodriguez-Serrano, A. Aranega, Anticancer Agents Med. Chem. 13 (2013)
1204.
[2] O. Glehen, F. Kwiatkowski, P.H. Sugarbaker, D. Elias, E.A. Levine, M. De
Simone, R. Barone, Y. Yonemura, F. Cavaliere, F. Quenet, M. Gutman, A.A.K.
Tentes, G. Lorimier, J.L. Bernard, J.M. Bereder, J. Porcheron, A. Gomez-Portilla,
P. Shen, M. Deraco, P. Rat, J. Clin. Oncol. 22 (2004) 3284.
[3] M.H. Cohen, J. Gootenberg, P. Keegan, R. Pazdur, Oncologist 12 (2007) 356.
[4] T. Ogiso, N. Noda, N. Asai, Y. Kato, Jpn. J. Pharmacol. 26 (1976) 445.
[5] H. Masuda, T. Ogiso, N. Noda, Y. Kato, Jpn. J. Pharmacol. 28 (1978) 175.
[6] W.B. Parker, Y.C. Cheng, Pharmacol. Ther. 48 (1990) 381.
[7] D.B. Longley, D.P. Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003) 330.
[8] B. Daniel, D. Longley, D. Paul Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003)
330.
[9] M. Aprahamian, C. Michel, W. Humbert, J.P. Devissaguet, C. Damge, Biol. Cell
61 (1987) 69.
[10] R.B. Diasio, Z. Lu, J. Clin Oncol. 12 (1994) 2239.
[11] R.L. Schilsky, J. Hohneker, M.J. Ratain, L. Janisch, L. Smetzer, V.S. Lucas, S.P.
Khor, R. Diasio, D.D. Von Hoff, H.A. Burris, J. Clin. Oncol. 16 (1998) 1450.
[12] G. Pasut, D. Paolino, C. Celia, A. Mero, A.S. Joseph, J. Wolfram, D. Cosco, O.
Schiavon, H. Shen, M. Fresta, J. Control. Release 199 (2015) 106.
[13] W. Cheng, R.G. Compton, Angew Chem. Int. Ed. Engl. 53 (2014) 13928.
[14] Q.Y. Bao, N. Zhang, D.D. Geng, J.W. Xue, M. Merritt, C. Zhang, Y. Ding, Int. J.
Pharm. 477 (2014) 408.
[49] I.A. Alvi, J. Madan, D. Kaushik, S. Sardana, R.S. Pandey, A. Ali, Anticancer Drugs
22 (2011) 774.
[50] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[51] A.C. de Mattos, N.M. Khalil, R.M. Mainardes, Braz. J. Pharm. Sci. 49 (2013) 117.
[52] J.J.F. Verhoef, T.J. Anchordoquy, Drug Deliv. Transl. Res. 3 (2013) 499.
[53] R. Gref, M. Luck, P. Quellec, M. Marchand, E. Dellacherie, S. Harnisch, T. Blunk,
R.H. Muller, Colloids Surf. B: Biointerfaces 18 (2000) 301.
[54] S. Patila, A. Sandberg, E. Heckert, W. Self, S. Sea, Biomaterials 28 (2007) 4600.
[55] N. Seetapan, P. Bejrapha, W. Srinuanchai, U.R. Ruktanonchai, Micron 41
(2010) 51.
[56] Y. Qiu, Y. Liu, L.M. Wang, L.G. Xu, R. Bai, Biomaterials 31 (2010) 7606.
[57] A. Varki, P. Gagneux, Ann. N. Y. Acad Sci. 1253 (2012) 16.
[58] B. Damascelli, G. Cantu, F. Mattavelli, P. Tamplenizza, P. Bidoli, E. Leo, F. Dosio,
A.M. Cerrotta, G. Di Tolla, L.F. Frigerio, F. Garbagnati, R. Lanocita, A. Marchiano,
G. Patelli, C. Spreafico, V. Ticha, V. Vespro, F. Zunino, Cancer 92 (2001) 2592.
[59] X. Zhu, B.D. Silva, X. Zou, B. Shen, Y. Sun, W. Feng, F. Li, RSC Adv. 4 (2014)
23580.
[60] S. Joshi, J.A. Ellis, C.W. Emala, J. Neuroanaesthesiol Crit. Care 1 (2014) 108.
[61] W.W. Raymond, S.W. Ruggles, C.S. Craik, G.H. Caughey, J. Biol. Chem. 278
(2003) 34517.
[62] J. Chen, Z. Ding, Y. Peng, F. Pan, J. Li, L. Zou, Y. Zhang, H. Liang, PLoS One 9
(2014) e98882.
[15] A. Lamprecht, H. Yamamoto, T. Hirofumi, Y. Kawashima, J. Control. Release 90
(2003) 313.
[16] N. Ashwanikumar, N.A. Kumar, S.A. Nair, G.S. Kumar, Acta Biomater. 10 (2014)
4685.
[63] M. Werle, J. Pharm. Sci. 97 (2008) 60.
[64] M. Werle, Pharm. Res. 25 (2008) 500.
[17] S.S. Agasti, A. Chompoosor, C.C. You, P. Ghosh, C.K. Kim, V.M. Rotello, J. Am.
Chem. Soc. 131 (2009) 5728.