Synthesis, characterization, and in-vitro antitumor activity of the polyethylene glycol (350 and 1000) succinate derivatives of the tocopherol and tocotrienol isomers of Vitamin E

Article abstract of DOI:10.1016/j.ijpharm.2017.01.020

Vitamin E refers to a group of saturated tocopherol (T) isomers and the biologically more active unsaturated tocotrienol (T₃) isomers. PEGylated α-tocopherol, commercially known as Vitamin E TPGS, has been used as an emulsifier and therapeutic agent for children with vitamin E deficiency. Limited information, however, is available about the PEG conjugates of the tocotrienol isomers of vitamin E. The current work was therefore undertaken to synthesize and characterize the water soluble polyethylene glycol (PEG 350 and 1000) derivatives of T and T₃. Yield and the identity of the synthesized products were confirmed by ¹H NMR, mass spectroscopy, HPLC, and thermal analysis. The self-assembly of the PEGylated vitamin E isomers in water at critical micelle concentrations (CMC) was further confirmed by size, zeta, and Cryo-TEM image analysis. While stable at pH 7.4, PEG conjugates were found to rapidly hydrolyze at pH 1.2. Our data showed that PEGylated T₃ isomers were significantly more active as inhibitors for P-glycoprotein than PEGylated T. The in vitro cytotoxicity of the conjugates was also tested against a large panel of normal and tumorigenic cells. Of the conjugates, γ-T₃PGS 1000 and δ-T₃PGS 1000 were found to have the least toxicity against non-tumorigenic breast and pancreatic cell lines, which may be advantageous for its use as functional excipients in drug delivery. The results from the current work have demonstrated the feasibility of synthesizing PEGylated conjugates of vitamin E isomers and highlighted the potential use of these conjugates in drug delivery as functional and safer excipients especially for γ-T₃PGS 1000 and δ-T₃PGS 1000 conjugate.

Full text of DOI:10.1016/j.ijpharm.2017.01.020

International Journal of Pharmaceutics 519 (2017) 145–156
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Research paper
Synthesis, characterization, and in-vitro antitumor activity of the
polyethylene glycol (350 and 1000) succinate derivatives of the
tocopherol and tocotrienol isomers of Vitamin E
Ahmed Abu-Fayyada, Sami Nazzala,b,
*
a
College of Health and Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA
b
College of Pharmacy, Taipei Medical University, Taipei, Taiwan
A R T I C L E I N F O
A B S T R A C T
Article history:
Vitamin E refers to a group of saturated tocopherol (T) isomers and the biologically more active
Received 6 October 2016
Received in revised form 5 January 2017
Accepted 10 January 2017
Available online 16 January 2017
unsaturated tocotrienol (T3) isomers. PEGylated
a-tocopherol, commercially known as Vitamin E TPGS,
has been used as an emulsifier and therapeutic agent for children with vitamin E deficiency. Limited
information, however, is available about the PEG conjugates of the tocotrienol isomers of vitamin E. The
current work was therefore undertaken to synthesize and characterize the water soluble polyethylene
glycol (PEG 350 and 1000) derivatives of Tand T3. Yield and the identity of the synthesized products were
confirmed by 1H NMR, mass spectroscopy, HPLC, and thermal analysis. The self-assembly of the
PEGylated vitamin E isomers in water at critical micelle concentrations (CMC) was further confirmed by
size, zeta, and Cryo-TEM image analysis. While stable at pH 7.4, PEG conjugates were found to rapidly
hydrolyze at pH 1.2. Our data showed that PEGylated T3 isomers were significantly more active as
inhibitors for P-glycoprotein than PEGylated T. The in vitro cytotoxicity of the conjugates was also tested
Keywords:
Vitamin E
Vitamin E TPGS
Tocopherol
Tocotrienols
PEGylation
Characterization
Functional excipient
Anti-tumor activity
against a large panel of normal and tumorigenic cells. Of the conjugates, g-T3PGS 1000 and d-T3PGS 1000
were found to have the least toxicity against non-tumorigenic breast and pancreatic cell lines, which may
be advantageous for its use as functional excipients in drug delivery. The results from the current work
have demonstrated the feasibility of synthesizing PEGylated conjugates of vitamin E isomers and
highlighted the potential use of these conjugates in drug delivery as functional and safer excipients
especially for g-T3PGS 1000 and d-T3PGS 1000 conjugate.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
“Vitamin E TPGS” or simply TPGS (Guo et al., 2013). TPGS was
shown to be effective in reversing or preventing vitamin E
deficiency during chronic childhood cholestasis when given orally
and was therefore used in the treatment of children with vitamin E
deficiency (Sokol et al., 1993). TPGS has also been used as an
emulsifier and solubilizer in pharmaceutical products such as
Agenerase1 (Zhang et al., 2012; Highleyman, 1999) where TPGS
Vitamin E represents a family of eight related isomers that are
classified into tocopherols and tocotrienols. Each subgroup
consists of an
substitution on the chroman ring and the degree of conjugation in
their phytyl side chain (Sylvester et al., 2010). -Tocopherol, which
a, b, g and d isomer that differ in the methyl
a
was the first isomer to be identified in the 1920s was used as an
antioxidant (Evans and Bishop, 1922; Evans, 1962). Due to it its
poor aqueous solubility, the Eastman chemical company intro-
duced a water soluble derivative in the 1950s by chemically
increased the solubility of amprenavir in water from 36
mg/mL to
720 g/mL (Yu et al.,1999). TPGS and the free -tocopherol isomer
m
a
were also used to reformulate paclitaxel into an injectable
emulsion (TOCOSOLTM).
conjugating
a
-tocopherol with poly ethylene glycol (PEG 1000)
While TPGS has been primarily used as an excipient, it was
shown to have an anticancer activity against the MCF-7 and MDA-
MB-231 breast cancer cell lines (Neophytou et al., 2014). It was
postulated that TPGS can induce apoptosis by inhibiting phospho-
AKT and downregulating the anti-apoptotic proteins survivin and
Bcl-2 G1/S, and to induce cell cycle arrest by up-regulating P21 and
P27Kip1 proteins (Neophytou et al., 2014). TPGS has also been
using a succinate linker, which became commercially known as
* Corresponding author at: Department of Basic Pharmaceutical Sciences, School
of Pharmacy, College of Health and Pharmaceutical Sciences, University of Louisiana
at Monroe, 1800 Bienville Drive Monroe, LA 71201, USA.
E-mail address: nazzal@ulm.edu (S. Nazzal).
http://dx.doi.org/10.1016/j.ijpharm.2017.01.020
0378-5173/© 2017 Elsevier B.V. All rights reserved.

146
A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
shown to inhibit the function of the efflux-pump P-glycoprotein
(P-gp), which mediates multi-drug resistance (MDR) to cancer cells
by lowering intracellular drug accumulation. TPGS acts on P-gp, in
part, by rigidifying lipid bilayers of cell membrane and primarily by
inhibiting P-gp ATPase activity (Duhem et al., 2014).
from Natland International Corporation (Research Triangle Park,
NC). 10 cm d x 60 cm L LG-0000 chromatography column with a
fritted disc/PTFE stopcock was custom made by Wilmad-LabGlass
Inc. (Vineland, NJ). Ethyl Acetate (EtOAc) from Pharmco-AAPER
(Shelbyville, KY). Methoxy polyethylene glycols (mPEG 1000 and
350) were from INEOS Oxide (Antwerp, Belgium). Triethylamine
and succinic anhydride were from Alfa Aesar (Ward Hill, MA).
Toluene and Chloroform-d (CDCL3) were from Acros (Bridgewater,
NJ). Hexanes, AR1, p-Toluenesulfonic acid monohydrate (p-TsOH),
sodium sulfate anhydrous (Na2SO4) and sodium bicarbonate
(NaHCO3) were from Avantor (Center Valley,PA). Acetonitrile and
dichloromethane were from EMD Millipore (Temecula, CA). All
chemicals and solvents were of reagent grade or higher and were
used as supplied without further modification.
Although extensive research has been reported on the
tocopherol isomers of vitamin E including their PEG derivative
(TPGS), the tocotrienol isomers (T3) of vitamin E were only
discovered in the 1960s (Whittle et al., 1966; Pennock et al., 1964)
and it was not until the 1990s that the anticancer activity of this
class of molecules was identified (Pennock et al., 1964). Since then,
numerous studies have been reported on the formulation and
testing of the tocotrienol isomers against tumor cells in vitro and in
animal models (Aggarwal and Nesaretnam, 2012; Sen et al., 2006).
Tocotrienols display potent anti-proliferative, apoptotic and
autophagic effects against breast cancer cells. The anti-cancer
effect of tocotrienols were found to be associated with suppression
in growth factor receptor mitogenic signaling pathway and
inhibition of epithelial-to-mesenchymal transition in cancer cell
lines (Ahmed et al., 2016). The ability of tocotrienols to inhibit the
activation and signaling of a wide variety of membrane bound
receptors was recently explained (Alawin et al., 2016). It was found
2.2. Extraction of vitamin E isomers from TocotrolTM L50P
The individual tocopherol and tocotrienol isomers of vitamin E
were extracted from TocotrolTM L50P as follows. Approximately
500 gm of TocotrolTM was first chromatographed on open column
containing 1.5 kg silica gel. The column was flushed initially with
approximately 70 L n-hexane to remove non-vitamin E lipid
fractions. The column was then eluted with a gradient solvent
system composed of n-hexane and an increasing concentration of
ethyl acetate (0–12%). Fractions with pure hexane contained
that
g-tocotrienol accumulate in and disrupt the integrity of the
lipid raft domain within the plasma membrane of breast cancer
cells, and this disruption of lipid raft integrity was associated with
a reduction in receptor activation and signaling (Ahmed et al.,
2016). Based on the plethora of data on the antitumor activity of
the free tocotrienol isomers of vitamin E, it was our hypothesis that
primarily
a
-T. Increasing the EtOAc to 1% allowed for the elution of
-T3. Increasing EtOAc to 2% allowed for the separation of
-T3. The -T3 isomer appeared in fractions with >3 and up to
pure
pure
a
g
d
substituting the
a
-tocopherol isomer of vitamin E in TPGS with
12% EtOAc. Thin-layer chromatography (TLC) was performed on
silica gel 60 F254 pre-coated aluminum ALUGRAM1 sheets
(Macherey-Nagel Inc., Bethlehem, PA). After immersion in samples,
sheets were sprayed with 4-anisaldehyde reagent and observed
under UV light (254 and 366 nm) using UVGL-15 compact UV lamp
tocotrienols would have a higher pharmacological or antitumor
activity, especially against breast and pancreatic cancer, in addition
to serving as a solubilizer. Therefore, the overall aim of this study
was to compare and contrast between the PEGylated a-tocopherol
and PEGylated tocotrienol isomers of vitamin E. More specifically,
the objectives of the current study were to (1) design and
(UVP LLC, Upland, CA). Fractions rich in
were concentrated using Heidolph Laborota 4000 rotary
evaporator (Elk Grove Village, IL) to give yellow to orange ( -T)
and orange viscous oils ( -T3, -T3 and -T3). High performance
liquid chromatography (HPLC), mass spectroscopy (MS), and
proton nuclear magnetic resonance (1H NMR) were performed
to confirm the identity of the extracts as discussed in subsequent
subsections.
a-T, a-T3, g-T3 and d-T3
a
synthesize PEG conjugates of four vitamin E isomers;
a
d
-tocopherol
-tocotrienol
-T3), that have been isolated from TocotrolTM L50P, a palm oil
a
(
(
a
d
-T),
a
-tocotrienol (
a
-T3),
g
-tocotrienol (
g
-T3) and
a
g
d
fraction that contains approximately 43% tocotrienols. Two
molecular weight variants of mPEG were used to accomplish this
goal; mPEG 350 and mPEG 1000; (2) characterize the PEG
conjugates by HPLC, 1H NMR, mass spectroscopy, and thermal
analysis; and to analyze the self-assembled micelles of the
PEGylated isomers in water for particle size, zeta potential, critical
micelle concentration, and by Cryo-TEM microscopy; (3) test the
inhibitory effect of the PEGylated vitamin E isomers on P-
glycoprotein ATPase activity; and (4) evaluate the in-vitro
anticancer activity of the conjugates against the following panel
of cell lines: breast cancer (MCF-7 and MDA-MB-231), pancreatic
cancer (AsPC-1, BxPC-3, MIA-PaCa-2 and PANC-1), human epithe-
lial mammary gland (hTERT-HME), human pancreatic duct (hTERT-
HPNE-1), and the non-tumorigenic human mammary gland (MCF-
10). To the best of our knowledge this work, along with our
previous study (Abu-Fayyad et al., 2015), mark the first report on
the full characterization and in-vitro cytotoxicity evaluation of
PEGylated tocopherol and tocotrienol isomers of vitamin E.
2.3. Synthesis of the succinate derivatives of
a
-T, -T3 and
a
-T3,
g
d-T3
The method for the synthesis of
-T3 succinate and -T3 succinate was adapted from our previous
work (Abu-Fayyad et al., 2015). The general reaction scheme is
outlined in Fig. 1A. The individual -T, -T3, -T3 and -T3 isomers
a
-T succinate, a-T3 succinate,
g
d
a
a
g
d
(1.2 g) were first dissolved in 6 mL toluene. Equimolar amounts of
succinic anhydride were then mixed with the isomer solutions. The
mixtures were then stirred at 85 ꢀC in a paraffin oil bath. The
temperature was maintained using an IKA1 RCT heater supported
with an IKA1 ETS-D4 fuzzy digital thermometer (IKA1 works Inc.,
Wilmington, NC). The reaction was stopped after 9 h and cooled to
room temperature. Water was then added and the reaction
mixture was extracted with dichloromethane. The upper oily layer
was kept and the lower aqueous phase was further extracted with
dichloromethane. The combined oily layers were then washed
three times with 1 N HCL (7 mL each) and twice with water (8 mL
each). Following extraction, the collected oil layers was dried over
anhydrous Na2SO4, filtered, and concentrated with a rotary
evaporator. The concentrate was then mixed with Celite1 545
for further purification on column chromatography. After backing
the column with a silica gel slurry (230–400 mesh size), the
samples were eluted through the column with the aid of a gradient
ethyl acetate/hexane solution with an increasing ethyl acetate
2. Materials and methods
2.1. Materials
Vitamin E isomers for the current work were isolated from
TocotrolTM L50P, a viscous tocotrienol-rich fraction of palm fruit oil,
that contains approximately 43% tocotrienol isomers (Fuji Health
Science Inc., Burlington, NJ). Silica Gel with a 230–400 mesh size,
which is suitable for flash (low pressure) chromatography, was

A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
147
Fig. 1. Synthesis scheme of the succinate derivatives (A) and the PEGylated derivatives of the vitamin E isomers (B).
fraction from 10% to 45%. After concentration, collected fractions
afforded approximately 1 g of each succinate derivative: -T and
-T3 (white solids) and -T3 and -T3 (yellow viscous liquids).
two times. The combined organic layers were washed with
NaHCO3 solution (7 mL) three times and brine (5 mL) for two
times, then dried over anhydrous Na2SO2. The final mixture was
concentrated using a rotary evaporator to afford approximately
a
a
g
d
2.4. Synthesis of the
a
-T,
a
-T3,
g
-T3, and
d
-T3 PEG conjugates
600 mg of each PEGylated isomer (
-T3PGS 350 and -T3PGS 1000, -T3PGS 350, 1000
-T3PGS 350, and -T3PGS 1000) as brownish yellow solid for
mPEG 1000 and semi-solid materials for mPEG 350.
a
-TPGS 350,
a
-TPGS 1000,
a
d
a
g
g-T3PGS 1000
The synthesis method was modified from Lipshutz et al. (2011)
and Abu-Fayyad et al. (2015) using terminally methylated PEGs
with molecular weights of approximately 350 (mPEG 350) and
1000 (mPEG1000). The reaction scheme is outlined in Fig. 1B.
mPEG 1000 was selected to be comparable to the commercially
available TPGS. mPEG 350 was selected to observe the effect of PEG
chain length on the physicochemical characteristics and in vitro
cytotoxicity of the conjugates. It was reported that the length of the
PEG moiety could affect the therapeutic and physicochemical
properties of PEGylated conjugates (Mao et al., 2006).
d
2.5. 1H NMR analysis of the PEGylated
a-T, a-T3, g-T3, and d-T3 isomers
1H NMR study was carried out to confirm the PEGylation of the
isomers. Conjugates were solubilized in CDCL3 and then 0.7 mL of
each solution was transferred to NMR tubes for measurement.
High resolution spectra were obtained using a JEOL Eclipse NMR
spectrometer and analyzed by the DeltaTM NMR Data Processing
Software (JEOL USA Inc., Peabody, MA). All spectra were collected
at a spinning frequency of 400 MHz at 20 ꢀC with chemical shifts
An equimolar mixture of
succinate, or
a-T succinate, a-T3 succinate, g-T3
d
-T3 succinate (ꢁ 400 mg) with mPEG 1000 or mPEG
350 in toluene was refluxed for 8 h using a Dean-Stark trap
(Chemglass Life Sciences Inc., Vineland, NJ). The mixture was then
left to cool at room temperature. Saturated NaHCO3 was then
added and the mixture was extracted with dichloromethane for
reported in ppm (d). The collection of spectra was one-dimensional
with 64 scans of 16 K points over 20 ppm and a recycle delay of 5s.
To confirm the stability of the conjugates, NMR tubes were re-
tested after one year of storage in the refrigerator.

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A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
2.6. Mass spectroscopic analysis of the PEGylated
-T3 isomers
a
-T,
a
-T3,
g
-T3, and
2 mL sample of each stock solution of the PEGylated isomer was
then added to the pyrene vials. The glass vials were then capped
and incubated overnight at room temperature while shaking at
200 rpm. Samples from each vial were then examined by
fluorescence spectroscopy using a K2 multi-frequency cross-
correlation phase and modulation fluorimeter (ISS, Inc., Cham-
paign, IL). The emission spectra of pyrene were recorded between
360 and 410 nm (Ex = 340 nm). CMC values were estimated from
the plot of the fluorescence intensity of samples at 390 nm versus
the logarithmic concentration of the PEGylated isomers.
d
Mass spectroscopic analysis of the PEGylated isomers was
carried out using a JEOL AccuTOFTM time-of-flight mass spectrom-
eter (JEOL Ltd., Tokyo, Japan). The AccuTOF analyzer performs mass
measurement of compounds of high molecular weight using ion
optics and an ADC-based digitizer to deliver a dynamic range
exceeding 4 orders of magnitude. The mass spectrometer was
equipped with orthogonal spray electrospray ionization (ESI) ion
source. Before analysis, the PEGylated isomers were dissolved in
HPLC grade methanol. A 50 mL sample was then injected through a
2.10. Particle size and zeta potential analysis of the self-assembled
Rheodyne 6-port valve injector. The mass spectrometer was
operated in positive-ion mode (ESI + ve) with an applied needle
voltage of 2000 V. The atmospheric pressure interface potential
was set to the following values: orifice 1 = 55 V, ring lens
voltage = 5 V, and orifice 2 = 6 V. The detector voltage was set to
1900 V. Orifice 1 temperature was adjusted to 80 ꢀC with dissolving
temperature at 250 ꢀC. Nebulizing gas flow was set at 2 L/min rate
whereas the desolvation gas (N2) flow was adjusted to 5 L/min rate.
PEGylated
a-T, a-T3, g-T3, and d-T3 isomers in water
The mean particle size of PEGylated isomers in water was
measured at 25 ꢀC and a 90ꢀ laser light scattering by photon
correlation spectroscopy (PCS) using a NicompTM380 ZLS submi-
cron particle size analyzer (Particle Sizing System, Port Richey, FL).
Individual samples were diluted with deionized water (DI) in order
to avoid multiple scattering and to achieve a scattering intensity of
300 kHz. The number-weighted mean diameter of the particles
was calculated based on Stokes–Einstein law by curve fitting of the
correlation function. Zeta-potential of the samples was measured
by the same instrument using the zeta option.
2.7. HPLC analysis of the PEGylated a-T, a-T3, g-T3, and d-T3 isomers
The PEGylation of the isomers was further confirmed by HPLC. A
SpectraSystem HPLC system fitted with a UV/Visible variable
wavelength detector was used for analysis (Thermo Electron Inc.,
2.11. Cryo-Transmission electron microscopy (Cryo-TEM) imaging of
the self-assembled PEGylated
water
San Jose, CA). Briefly, the PEGylated conjugates (
-TPGS 1000, -T3PGS 350 and -T3PGS 1000, -T3PGS 350, 1000
-T3PGS 1000 -T3PGS 350 and -T3PGS 1000) were dissolved in
the mobile phase, which consisted of 90% v/v acetonitrile and 10%
a-TPGS 350,
a-T, a-T3, g-T3, and d-T3 isomers in
a
a
a
g
g
d
d
Cryo-TEM imaging of the PEGylated isomers was carried out in
order to visualize the structure of the self-assemblies in water. 3
sample of each PEGylated isomer dissolved in water at
concentration above CMC ( > 170 mg/ml) was applied to glow-
discharged 200 mesh Quantifoil R2/1 grids (Electron Microscopy
Sciences, Hatfield, PA), which were previously washed with
chloroform and ethanol. The grids were then blotted and plunged
into liquid ethane. The frozen grids were then transferred to a
Gatan 622 cryo-holder and observed in an FEI Tecnai F20 electron
microscope operated at 200 kV with magnifications from 29,000 x
to 80,000 x and defocus settings of 4–6 Lm (FEI Company,
Hillsboro, OR). Images were collected under low-dose conditions
on a Gatan Ultrascan 4000 CCD camera.
v/v water. 20 m
L samples were then injected into KinetexTM C18
m
a
L
(5
m
L, 150 Â 4.6 mm) analytical column, pre-heated to 40 ꢀC
(Phenomenex Inc., Torrance, CA). The flow rate of the mobile
phase was adjusted to 1.5 mL/min. The PEGylated isomers were
detected at 285 lmax. Data acquisition and analysis was performed
using ChromQuestTM chromatography software version 4.2
(Thermo Electron Corp., San Jose, CA).
2.8. Thermal analysis of the PEGylated
isomers
a-T, a-T3, g-T3, and d-T3
The thermal analysis of the isomers conjugated to mPEG 1000
was performed using a TA 2920 modulated differential scanning
calorimeter (DSC, TA Instruments-Waters LLC, New Castle, DE).
Since the mPEG 350 conjugates were semi solid at room
temperature they were not subjected to thermal analysis.
2.12. In-vitro hydrolysis study of the PEGylated
-T3 isomers
a-T, a-T3, g-T3, and
d
Accurately weighted samples (3.98 mg
a-TPGS 1000, 6.60 mg
Stability against hydrolysis is critical for applications requiring
a
-T3PGS 1000, 3.00 mg -T3PGS 1000, and 4.73 mg d
g
-T3PGS 1000)
were hermitically sealed in aluminum pans and heated from 0 ꢀC to
80 ꢀC at a rate of 10 ꢀC/min. The onset and peak of the melting
endotherms were measured with the aid of the universal analysis
2000 software, V 4.2 (TA Instruments-Waters LLC, New Castle, DE).
the use of the PEGylated isomers in aqueous media. Since the T and
T3 isomers were conjugated to the PEG moiety via an ester linkage,
PEGylated products were expected to undergo hydrolysis on
exposure to acidic or alkaline aqueous conditions. The stability of
the conjugates was therefore tested in different buffer solutions.
Briefly, the conjugates were dissolved in HCl (pH 1.2), phosphate
buffer (pH 7.4) and boric acid buffer (pH 10) to a final concentration
of 2 mg/ml. These pH values were intended to mimic the
conditions that were used for testing the stability of the
commercial vitamin E TPGS Vitamin E TPGS (2015). Samples were
incubated for 20 h in an oven at 37 ꢀC while continuously shaking
at 100 RPM. At specific time points throughout the incubation
2.9. Determination of the critical micellar concentration (CMC) of the
PEGylated
a-T, a-T3, g-T3, and d-T3 isomers
The CMC of the PEGylated isomers in water was determined
using pyrene as a fluorescence probe as previously reported (Abu-
Fayyad et al., 2015). Before analysis, 0.05 to 0.67 mg/ml stock
solutions of
350, and 0.02 to 1.00 mg/ml sock solutions of
-T3PGS 1000, -T3PGS 1000 and -T3PGS 1000 were prepared by
dissolving the conjugates in water. Before testing, 250 L of pyrene
solubilized in acetone at a concentration of 4.69 g/ml was added
to glass vials and flushed with N2 gas then dried under vacuum. A
a
-TPGS 350,
a
-T3PGS 350,
g
-T3PGS 350 and
d-T3PGS
-TPGS 1000,
period, 100 mL samples were withdrawn and diluted to 1 mL with
a
the mobile phase and analyzed by HPLC as described earlier. The
percentage of the conjugate hydrolyzed was calculated by taking
the ratio of the AUC of the free isomers to the free isomer
standards.
a
g
d
m
m

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149
2.13. Evaluation of the P-glycoprotein (P-gp) ATPase inhibitory activity
Base medium (Incell Corporation LLC, San Antonio, TX) supple-
mented with 5% fetal bovine serum, 10 ng/ml human recombinant
EGF, 5.5 mM D-glucose (1 g/L), and 750 ng/ml puromycin. All other
media were supplemented with 10% fetal bovine serum,1% insulin,
and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA). For all
cell lines, 5000 cells/well were seeded into 96-well plates and
incubated at 37 ꢀC with 5% CO2. After overnight incubation the
P-gp is an ATP-dependent drug efflux pump that plays an
important role in multi-drug resistance and drug bioavailability.
The effect of the PEGylated vitamin E isomers on the P-gp ATPase
activity was carried out by the Pgp-GloTM assay (Promega
Corporation, Madison, WI). The Pgp-GloTM assay detects the
effects of compounds on recombinant human P-gp in a cell
membrane fraction. The assay relies on the ATP-dependent light-
generating reaction of firefly luciferase where an increase in
luminescence is indicative of the inhibitory effect of compounds on
the P-gp ATPase enzyme. PEGylated vitamin E isomers at a final
culture medium was replaced with 50
containing the PEGylated isomers at concentrations ranging from
0.5 to 25 M for breast cancer and human kidney cell lines and
0.028 to 66.7 M for pancreatic cell lines. After 72 h of incubation
the medium was replaced with 20 L of CellTiter-Blue reagent
mL of fresh medium
m
m
m
concentration of 10 to 100
m
M in assay buffer were added to the
(Promega Inc. Madison, WI). Plates were allowed to incubate at
37 ꢀC for 1 h and the absorbance at 570 nm was measured using a
BioTek Synergy HT multi-mode microplate reader (BioTek,
Winooski, VT). Cell viability was calculated as the percentage of
cells remaining viable in reference to the untreated cells. The 50%
inhibitory concentration (IC50) values were determined by non-
linear regression curve fit analysis using GraphPad Prism 5
(GraphPad Software, La Jolla, CA). Statistical significance was
determined by one-way ANOVA followed by Tukey post hoc
analysis. A difference of P < 0.05 was considered to be statistically
significant.
wells of white 96-well plate containing 20 mL
a
(0.5 mM)
verapamil, which was required to activate P-gp ATPase (Collnot
et al., 2007). The reaction was initiated by adding Mg ATP (25 mM)
to each well. The plate was placed on a shaker for 5 min and then
incubated for 40 min at 37 ꢀC. The reaction was stopped by adding
50 mL of the ATP detection reagent. After addition, the plate was
left at room temperature for 20 min to allow for the luminescent
signal to develop. Luminescence was measured using a Synergy 2
Multi-Mode BioTek plate reader (BioTek Instruments, Inc.
Winooski, VT).
2.14. In vitro cytotoxicity of the PEGylated
isomers
a
-T,
a
-T3,
g
-T3, and
d
-T3
3. Results and discussion
3.1. Extraction of vitamin E isomers from TocotrolTM L50P
The individual vitamin E isomers were isolated in multigram
The in vitro cytotoxicity of the PEGylated isomers was evaluated
against MCF-7 and MDA-MB-231 (breast cancer) and AsPC-1,
BxPC-3, MIA-PaCa-2 and PANC-1 (pancreatic cancer) cell lines.
Conjugates were also tested against two immortalized cell lines;
human epithelial mammary gland (hTERT-HME) and human
pancreatic duct (hTERT-HPNE-1), and on non-tumorigenic human
mammary gland (MCF-10) cells. Cell lines were purchased from
ATCCTM (Manassas, VA). MCF-7, MDA-MB-231, MIA-PaCa-2, and
PANC-1 cells were maintained in DMEM medium (Invitrogen,
Carlsbad, CA). AsPC-1 and BxPC-3 cells were maintained in 1640-
GlutaMAXTM RPMI medium (Life Technologies, Carlsbad, CA).
TERT-HME and MCF-10A cells were maintained in MEGMTM
Mammary Epithelial Cell Growth Medium (Lonza Inc., Allendale,
NJ). hTERT-HPNE cells were maintained in a 75% DMEM/25% M3
quantities (25 g a-T, 20 g a-T3, 25 g g-T3, and 15 g d-T3). The purity
of the isolated isomers was >90% as confirmed by TLC and HPLC
analysis. The 1H NMR spectra of the free isomers was in agreement
with the reported values in the literature.
3.2. Synthesis and characterization of the succinate ester and the
PEGylated derivatives of the
a-T, a-T3, g-T3 and d-T3 isomers
The synthesis scheme of the succinate and PEGylated deriv-
atives of vitamin E isomers is outlined in Fig.1A and B, respectively.
The succinate derivatives of vitamin
E isomers were first
synthesized by ring-opening reflux reaction of succinic anhydride
Fig. 2. HPLC chromatograms showing the peaks and retention times associated with PEGylated vitamin E derivatives with mPEG 350 for -TPGS 350,
a
a-T3PGS 350, g-T3PGS
350 and -T3PGS 350 (A), and PEGylated vitamin E derivatives with mPEG 1000 for -TPGS 1000, -T3PGS 1000, -T3PGS 1000 and d-T3PGS 1000 (B).
d
a
a
g

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in warm toluene and trimethylamine with the individual isomers.
The resulting succinate derivatives were then put through a
purification process as described in the methods section to give
approximately 1.41 gm products (ꢁ98% yield). As shown in
Fig. S1A–D in the Supplementary file, the ethanyl protons of
succinate appeared at 2.87–2.90 ppm (m, 4H, COCH2CH2CO). The
Conjugates were further analyzed by HPLC. Several HPLC
methods for analysis of vitamin E TPGS were reported in literature
(Kong et al., 2011). In the current work the optimum separation of
the mPEG 350 and mPEG1000 conjugates were achieved when the
HPLC column was preheated to 40 ꢀC. As expected, PEGylated
conjugates showed broad peaks (Fig. 2A and B) due to the broad
molecular weight distribution of the ethoxy moieties of PEG chain.
However, the PEGylated conjugates peaks were sufficiently
separated. As shown in Fig. 2A, the mPEG 350 conjugates with
their lower molecular weight were eluted at longer retention times
(tR) when compared to the mPEG 1000 conjugates with larger
molecular weights (Fig. 2B). Also, conjugates of the more
aromatic proton of
g-T3 appeared at 6.55 ppm (Fig. S1C). The two
d
-T3 aromatic protons appeared at 6.55 and 6.65 ppm (Fig. S1D).
Carbon chain signals of the succinate derivatives of vitamin E
isomers were located at 0.85-2.2 ppm (Fig. S1A–D, Supplementary
file). Reflexing the succinate derivatives with mPEG 350 and mPEG
1000 in warm toluene with p-TsOH as a catalyst gave the desired
PEGylated products: 1.14 gm (ꢁ95% yield) of the mPEG 1000
conjugates and 646 mg (ꢁ95%) yield of the mPEG 350 conjugates.
The purity of the PEGylated vitamin E derivatives was, on average,
96% as determined by HPLC analysis. The 1H NMR of the mPEG 350
and mPEG 1000 conjugates are shown in Figs. S2 and S3 in the
Supplementary file, respectively. The appearance of the peak at
4.23 ppm for all products (m, 2H), which is attributed to the ester
formation between the hydroxyl group of mPEG and carboxyl
group of the succinate, confirm the PEGylation reaction. The
chemical shift of the mPEG 350 [m, 24H, (CH2CH2O)6] and mPEG
1000 [m,92H, (CH2CH2O)24] ethoxyl protons were located at 3.5–
3.7 ppm for all conjugates as shown in Fig. S2A–D and Fig. S3A–D,
respectively. The terminal methoxy groups of the mPEG 350 and
mPEG 1000 conjugates were located at 3.36 ppm (3H, OCH3) as
shown in Fig. S2A–D and Fig. S3A–D, respectively. The PEGylation
of the isomers was further confirmed by time-of-light mass
spectroscopy. The average molecular weights of the PEGylated
vitamin E isomers with mPEG 350 were observed as peaks at m/z of
hydrophilic vitamin E isomer (d-T3) were separated first, followed
by the
g
-T3,
a
-T3, and then
a
-T conjugates.
3.3. CMC of the PEGylated
a
-T,
a-T3, g-T3, and d-T3 isomers
Amphiphilic molecules usually self-assemble above their CMC
in dispersion media to form micelles. The micelles-forming
property of the PEGylated vitamin E isomers was evaluated by
measuring their CMC in water. Pyrene was used as a fluorescent
probe since it incorporates into the core of micelles at concen-
trations above their CMC, which translates into an increase in
fluorescence intensity (La et al., 1996). At low conjugate concen-
trations pyrene remains outside in the aqueous media with low
intensity. Fig. 3A and B shows the fluorescence intensity of pyrene
as a function of the concentration of the PEGylated conjugates in
water. As shown in the figure, the CMCs decreased with a decrease
in the hydrophobicity of the isomer. The CMC of the most
hydrophobic isomer
0.061, 0.067 and 0.100 mg/ml for
-T3PGS 350, respectively (Fig. 3A). As shown in Fig. 3B, the CMC
values were 0.059, 0.072, 0.130 and 0.170 mg for -TPGS 1000,
-T3PGS 1000, -T3PGS 1000 and -T3PGS 1000, respectively.
a-TPGS 350 was 0.033 mg/ml followed by
874, 870, 872 and 855 for
and -T3PGS 350, respectively (Fig. S4A–D, Supplementary file).
The PEGylated vitamin E isomers with mPEG 1000 showed peaks at
m/z of 1492, 1456, 1472 and 1458 for -TPGS 1000, -T3PGS 1000,
-T3PGS 1000 and -T3PGS 1000, respectively (Fig. S5A–D,
a-TPGS 350,
a-T3PGS 350,
g
-T3PGS 350
a
-T3PGS 350, -T3PGS 350 and
g
d
d
a
a
a
a
g
d
g
d
Isomers conjugated with mPEG 350 had lower CMC as compared to
isomers conjugated to mPEG 1000. The impact of PEG chain length
Supplementary file). The average molecular weights of the
PEGylated isomers were in agreement with the expected values.
Fig. 3. Critical micelle concentration (CMC) measurements of PEGylated vitamin E derivatives with mPEG 350 for
a-TPGS 350, a-T3PGS 350, g-T3PGS 350 and d-T3PGS 350 (A),
and PEGylated vitamin E derivatives with mPEG 1000 for -TPGS 1000, -T3PGS 1000, -T3PGS 1000 and -T3PGS 1000 (B). This study was carried out using pyrene as a
a
a
g
d
hydrophobic fluorescence probe. The fluorescence intensity of pyrene was collected at the excitation wavelength of 340 nm and the emission was reported at wavelength of
390 nm. The fluorescence intensity was plotted as a function of logarithmic concentration of micelles.

A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
151
on CMC was in agreement with data reported for comparable
products in the literature (Mukerjee and Mysels, 1972).
The ability of the PEGylated isomers to self-assemble at low
CMC is advantageous for the solubilaztion and emulsification of
drugs without the dilution effect after injection in the blood
1000, g-T3PGS 1000 and d-T3PGS 1000, respectively. The higher
surface charge of the mPEG 350 conjugates could be attributed to
the lower shielding effect of the mPEG 350 moiety (Malhotra et al.,
2013). Cryo-TEM imaging of the PEGylated isomers confirmed their
size and shape. As shown in Fig. 4A, PEGylated isomers with mPEG
350 formed larger micelles (average size 60 nm) whereas
PEGylated isomers with mPEG 1000 formed smaller particles
(Fig. 4B) with an average size of 10 nm. Larger size for the short
mPEG 350 conjugates was expected due the inverse relationship
between the particle size and PEG molecular weight (Lipshutz
et al., 2011; Fang et al., 2005).
circulation. The CMC of the PEGylated isomers were <170
which is less than the CMC of common surfactants, such as Tween
20 with a CMC around 300 g/ml (Lo, 2003).
mg/ml,
m
3.4. Physical characterization of PEGylated
isomers
a-T, a-T3, g-T3, and d-T3
Thermal analysis of the conjugates showed that the melting
PEGylated vitamin E isomers self-assemble in aqueous media
due to their intermolecular interaction to minimize surface free
energy. The outermost corona of the PEGylated self-assembled
structures consists of PEG moiety whereas the phytyl side chain of
the vitamin E isomer form the core. As shown in Table 1, the
average sizes of the PEGylated vitamin E isomers with mPEG 350
points of mPEG 1000 conjugates was 39.51 ꢀC, 38.48 ꢀC, 36.08 ꢀC
and 42.37 ꢀC for
a-TPGS 1000,
a-T3PGS 1000, g-T3PGS 1000 and
d-T3PGS 1000 (Fig. 5), respectively. The melting points of mPEG
1000 conjugates was comparable to the reported melting point of
the commercial vitamin E TPGS (41 ꢀC) (Abu-Fayyad et al., 2015)
and the melting point of mPEG 1000 (37–40 ꢀC) (I Sciencelab.com,
2005). The mPEG 350 conjugates were liquid/semi-solid at room
temperature and therefore their melting point was not measured.
were 72.3, 68.6, 61.6 and 61.7 nm for
-T3PGS 350, respectively. The size of the isomers conjugated with
mPEG 1000 were 10.7, 11.2, 10.8 and 10.7 nm for -TPGS 1000,
-T3PGS 1000, -T3PGS 1000 and -T3PGS 1000, respectively. The
zeta potential of the PEGylated isomers with mPEG 350 was À22.2,
À26.5, À17.9, and À19.6 for -T3PGS 350, -T3PGS 350 and -T3PGS
350, respectively. Isomers conjugated with mPEG 1000 had zeta
a-T3PGS 350, g-T3PGS 350 and
d
a
a
g
d
3.5. In-vitro hydrolysis of the PEGylated
isomers
a-T, a-T3, g-T3, and d-T3
a
g
d
The in vitro hydrolysis study has demonstrated that the
conjugates were unstable at pH 1.2 HCl (Fig. 6), Approximately
potential of À15, À14.2, À11, and À13 for
a-TPGS 1000, a-T3PGS
Table 1
Physical characteristics of PEGylated vitamin E isomers;
and their self-assemblies in water.
a-TPGS 350,
a-T3PGS 350,
g-T3PGS 350,
d-T3PGS 350,
a-TPGS 1000,
a-T3PGS 1000,
g
-T3PGS 1000 and
d-T3PGS 1000
Conjugate
Particle Size (nm)a
Zeta potential
CMC
(mg/ml)
Melting point (ꢀC)
HPLC tR
(min)b
a
(j
)
a
a
a
a
-TPGS 350
72.3 Æ 7.7
10.7 Æ 2.5
68.6 Æ 8.2
11.2 Æ 1.8
61.6 Æ 7.3
10.8 Æ 8.3
61.7 Æ 9.1
10.7 Æ 7.5
À22.2 Æ 0.9
À15.0 Æ 1.2
À26.5 Æ 2.2
À14.2 Æ 3.2
À17.9 Æ 0.3
À11.0 Æ 0.8
À19.6 Æ 3.2
À13.0 Æ 1.8
0.033
0.059
0.061
0.095
0.067
0.130
0.100
0.170
–
8
6.3
7
5.1
6.4
4.4
5.6
3.7
-TPGS 1000
-T3PGS 350
-T3PGS 1000
39.51
–
38.48
–
g
g
-T3PGS 350
-T3PGS 1000
36.08
–
d
d
-T3PGS 350
-T3PGS 1000
42.37
a
Each value represents the mean Æ SD for triplicate samples.
b
tR: Retention time.
Fig. 4. (A) Cryo-TEM images of PEGylated vitamin E derivatives with mPEG 350 for (1)
a-TPGS 350, (2)
a-T3PGS 350, (3)
g-T3PGS 350 and (4)
d-T3PGS 350 (100 nm scale) and
(B) PEGylated vitamin E derivatives with mPEG 1000 for (1) -TPGS 1000, (2) -T3PGS 1000, (3)
a
a
g-T3PGS 1000 and (4) d
-T3PGS 1000 (100 nm scale).

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A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
75%
-TPGS 350 were hydrolyzed after 20 h of incubation (Fig. 6). The
larger molecular weight conjugates were less susceptible to
hydrolysis at pH 1.2 with 37% -T3PGS 1000, 30% -T3PGS 1000,
28% -T3PGS, and 24% -T3PGS 1000 hydrolyzed after 20 h
incubation (Fig. 6). The most hydrophilic -T3 conjugates were
hydrolyzed first followed by the -T3, -T3 and -T isomers in the
d-T3PGS 350, 65% g-T3PGS 350, 55% a-T3PGS 350 and 43%
a
d
g
a
a
d
g
a
a
order of their hydrophilicity, which was in agreement with the
reported observations that enhanced polarity could improve
hydration for the esters (Markovic et al., 2011). Due to their
smaller size, however, isomers conjugated to mPEG 1000 had a
slower hydrolysis rate. It was reported that an increase in PEG
molecular weight will reduce particle size and impede the
hydrolysis process (Li et al., 2008).
The mPEG 350 conjugates were also sensitive to hydrolysis at
pH 10 (Fig. 6) with 45%, 35%, 24% and 20% of
d-T3PGS 350, g-T3PGS
350, -T3PGS 350 and -TPGS 350, hydrolyzing by the end of the
a
a
incubation period, respectively. The mPEG 1000 conjugates were
somewhat stable at pH 10 with less than 19% hydrolysis observed
after 20 h incubation (Fig. 6). All conjugates were stable at pH 7.4
with less than 15% hydrolyzed after 20 h (Fig. 6).
Fig. 5. DSC thermograms showing the melting endotherms of PEGylated vitamin E
3.6. Evaluation of the P-glycoprotein (P-gp) ATPase inhibitory activity
derivatives with mPEG 1000 for
-T3PGS 1000.
a-TPGS 1000, a-T3PGS 1000, g-T3PGS 1000 and
d
P-gp efflux pump is extensively distributed and expressed in
the intestinal epithelium as well as in the renal proximal tubular
Fig. 6. In-vitro hydrolysis of PEGylated vitamin E isomers in HCl solution, pH 1.2 (A), pohosphate buffer, pH 7.4 (B) and boric acid buffer, pH 10 (C). Samples were analyzed by
HPLC where% hydrolyzed was calculated from the AUC of the samples against the free isomer standards.

A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
153
cells and the epithelial cells of the blood-brain barrier. It plays an
3.7. In vitro cytotoxicity of the PEGylated
isomers
a-T, a-T3, g-T3, and d-T3
important role in conferring resistance to many chemotherapeutic
agents and to reduce the oral bioavailability of drugs (Guo et al.,
2013). When tested using the Pgp-GloTM assay (Fig. 7), all
PEGylated isomers were found to significantly inhibit P-gp ATPase
activity (P-value < 0.05) when compared to verapamil only treated
test samples (Fig. 7A and B). Verapamil was added to the test
samples as an activator since it was reported that inhibitors would
only exert activity on P-gp ATPase when incubated with known
activators (Collnot et al., 2010).
Due to the potential of using the PEGylated isomers of vitamin E
as vehicles for drug delivery, the anticancer activity of the
conjugates was tested against a wide range of breast and
pancreatic cell lines. Overall PEGylation was found to affect the
biological activity of the isomers as follows.
3.7.1. Breast cell lines
When compared to
a
-TPGS 1000, PEGylated
a
-T3 and
g
-T3, with
The cytotoxic activity of the conjugates was tested against MCF-
7 and MDA-MB-231 breast tumor cells (Figs. 8 and 9). MCF-7 are
invasive breast ductal carcinoma cells whereas MDA-MB-231 are
triple negative metastatic cells from the plural effusion origin. Both
cell lines were found to be sensitive to all conjugates to a different
degree as deduced from their IC50 values (Table 2). Of the
either mPEG 350 and 1000 side chain were found to be significantly
more active as inhibitors for P-gp ATPase (P value < 0.05, Fig. 7).
This effect, however, was dose independent, which was also
reported for vitamin E TPGS (Collnot et al., 2010). The inhibition of
P-gp ATPase activity by the PEGylated vitamin E isomers may
represent a promising approach to overcome chemotherapy
resistance due to P-gp-overexpression. For example, it was
reported that vitamin E TPGS can increase the cytotoxicity of
doxorubicin, vinblastine and paclitaxel due to its P-gp inhibitory
activity (Dintaman and Silverman, 1999). It is worth noting that
one mechanism by which vitamin E TPGS inhibits P-gp is by
rigidifying the cell membrane. A recent study found that the free
compounds, the mPEG 1000 conjugates of the
a isomers of
tocopherol and tocotrienol were found to be statistically more
potent (P value < 0.005) than the other conjugates against both cell
lines (Figs. 8 and 9, Table 2). It was also observed that isomers
conjugated to mPEG 1000 were more potent than those conjugated
to mPEG 350 (Figs. 8 and 9, Table 2). To evaluate the safety of the
conjugates and to examine whether their activity is specific against
tumor cells, conjugates were tested against the MCF-10A cells
(Fig. S6, Supplementary file), a non-tumorigenic epithelial cell line,
and hTERT-HME cells (Fig. 10), which were derived from a patient
undergoing reduction mammoplasty surgery and the cells were
immortalized by infection with a retrovirus. Conjugates were
found to be less toxic against the MCF-10A (Fig. S6, Supplementary
file, Table 2). However, when they were tested against the hTERT-
unmodified
g-T3 mediates its anticancer activity through the
localization and disruption of the integrity of the plasma
membrane’s lipid raft (Alawin et al., 2016), which indicates that
PEGylated vitamin E isomers may also localize and disrupt the
integrity of lipid rafts.
Fig. 7. Inhibitory effects of PEGylated vitamin E derivatives with mPEG 350 (A) and PEGylated vitamin E derivatives with mPEG 1000 (B) in comparison to sodium
orthovanadate (Na3VO4) and vitamin E TPGS on verapamil-stimulated P-gp ATPase activity. Test samples contained verapamil (0.2 mM) and PEGylated vitamin E isomers (10–
100 m
M) or sodium orthovanadate (Na3VO4) (0.25 mM) dissolved in Pgp-GloTM assay buffer. The reaction was initiated by the addition of MgATP. The plate was mixed for
5 min on plate shaker, then incubated for 40 min at 37 ꢀC. ATP detection reagent was then added and luminescence was measured. Values are reported as the mean Æ SD for
triplicate samples.

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A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
Fig. 8. Cell viability following treatment with free unmodified and PEGylated vitamin E isomers against the MCF-7 human breast cancer cell line. Cells were treated for 72 h.
Values reported are the mean Æ SD for triplicate samples.
HME cells, the PEG conjugates of the
tocotrienol isomers were found to be the least toxic (Fig. 10,
Table 2). Conjugates of the -tocopherol and -tocotrienol isomers
g
-tocotrienol and
d
-
less extent the d-tocotrienol, could be regarded as safer excipients
for drug delivery due to their lower activity against tumor cells
when compared to the free isomers (Figs. 8 and 9) and their lower
cytotoxicity against the normal hTERT-HME cells when compared
a
a
were highly toxic against the immortalized cells (Fig. 10, Fig. S6,
Supplementary file, Table 2). These results indicate that the activity
the PEGylated
-T3PGS 1000 against the non-tumorigenic breast hTERT-HME
cells was 15.5 M while the IC50 value of -TPGS 1000 or -T3PGS
1000 against the same cell line was less than 6.2 M (Table 2).
a isomers of vitamin E (Fig. 10). The IC50 value of
observed by the PEG conjugates of the
specific, which may limit their potential use in drug delivery.
Consequently, PEG conjugates of the -tocotrienol isomer, and to
a
isomers might be non-
g
m
a
a
g
m
Fig. 9. Cell viability following treatment with free unmodified and PEGylated vitamin E isomers against the MDA-MB-231 human breast cancer cell line. Cells were treated for
72 h. Values reported are the mean Æ SD for triplicate samples.

A. Abu-Fayyad, S. Nazzal / International Journal of Pharmaceutics 519 (2017) 145–156
155
Table 2
IC50 values of PEGylated vitamin E isomers against a panel of cell lines.
Cell line
IC50
(mM)
a-TPGS 350
a-TPGS 1000
a-T3PGS 350
a-T3PGS 1000
g-T3PGS 350
g
-T3PGS 1000
d-T3PGS 350
d-T3PGS 1000
MCF-7
25.4
9.3 */a
9.5 */a
16.6a
5.7
24.6*
16.3*
15.0*
12.3
17.3/a
9.1*/a
10.5*/a
18.1a
6.2
29.6
18.6*
20.3
7.9*
19.1
16.1a
18.3a
10.0*/a
8.2*/a
15.7a
9.1
32.1
44.1
22.9
40.2
24.0**
MDA-MB-231
MCF-10A
hTERT-HME
AsPC-1
BxPC-3
MIA-PaCa-2
PANC-1
16.1a
9.6*/a
12.6a
10.7*/b
19.4a
15.5**/b
35.6**
45.3
13.0b
27.1**/a
7.5
–
27.4**/a
8.2
–
18.8a
18.7b
22.5**/a
13.4**/a
–
–
–
–
–
–
–
–
–
–
–
–
–
–
28.2
27.3
29.5**
–
–
hTERT-HPNE
–
7.9*
–
14.0
Anti-tumor activity: * significantly more active than the other conjugates against the same cell line (P value < 0.05).
Toxicity per cell line: ** significantly lower toxicity against the same cell line than other conjugates (P value < 0.05).
a,b
Toxicity per conjugate:
significant difference in cytotoxicity per conjugate when comparing tumorigenic to non-tumorigenic cells (P value < 0.05).
Fig. 10. Cell viability following treatment with free unmodified and PEGylated vitamin E isomers against the non-tumorigenic hTERT-HME human breast cells. Cells were
treated for 72 h. Values reported are the mean Æ SD for triplicate samples.
While the underlining molecular mechanisms by which PEGylated
vitamin E isomers mediate their anti-cancer activity were not
investigated in this study, it is probable that PEGylated vitamin E
isomers induce G1/S cell cycle arrest and apoptosis as reported for
vitamin E TPGS against breast cancer cells (Neophytou et al., 2014).
Since the mPEG 350 conjugates were found to be less active
than their mPEG 1000 counterparts, they were excluded from
subsequent testing against the pancreatic cell lines. It might be
worth noting that the concentration range of TPGS that has been
frequently used due to their resistance to current chemotherapy
(Deer et al., 2010). AsPC-1 cells were isolated from metastasized
adenocarcinoma of the pancreas head. BxPC-3 cells were derived
from non-metastasized adenocarcinoma of the pancreas body.
MIA-PaCa-2 cells were derived from the body and tail of the
pancreas. PANC-1 pancreatic cells represent a tumor of ductal
origin.
lines than the other conjugates (P < 0.05) (Fig. S7–S10, Supple-
mentary file, Table 2). -TPGS 1000 was also found to be
a-TPGS 1000 was found to be more toxic against these cell
a
used for paclitaxel delivery was between 0.067 and 0.67
m
M
significantly more toxic than the other conjugates against the
immortalized pancreatic hTERT-HPNE cells (P < 0.05) (Fig. S11,
(Constantinides et al., 2000), which is lower than the IC50 values
observed in this study. This indicates that the conjugates may have
a tolerable toxicity profile if used for drug delivery within this
concentration range.
Supplementary file, Table 2). On the other hand,
g-T3PGS 1000 and
d-T3PGS 1000 were found to be the least toxic conjugate against
the pancreatic tumor cells and had lesser toxicity against the
hTERT-HPNE cells when compared to the other conjugates
(P < 0.05) (Fig. S7–S11, Supplementary file, Table 2). These results
confirm what was observed against breast tumorigenic and non-
3.7.2. Pancreatic cell lines
The mPEG 1000 conjugates were tested against several
pancreatic cell lines (Figs. S7–S11, Supplementary file, Table 2).
AsPC-1, BxPC-3, MIA-PaCa-2, and PANC-1 cell lines have been
tumorigenic cells, where the
g-T3PGS 1000 and d-T3PGS 1000 were
found to be the least toxic conjugates. The exact mechanism by

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which the conjugates exert their effect is not fully understood and
is beyond the scope of the current study.
conformational flexibility, intracellular ATP levels, and role of time and site of
access. Mol. Pharm. 7, 642–651.
Constantinides, P.P., Lambert, K.J., Tustian, A.K., Schneider, B., Lalji, S., et al., 2000.
Formulation development and antitumor activity of a filter-sterilizable
emulsion of paclitaxel. Pharm. Res. 17, 175–182.
4. Conclusion
Deer, E.L., Gonzalez-Hernandez, J., Coursen, J.D., Shea, J.E., Ngatia, J., et al., 2010.
Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39, 425–435.
Dintaman, J.M., Silverman, J.A., 1999. Inhibition of P-glycoprotein by D-alpha-
tocopheryl polyethylene glycol 1000 succinate (TPGS). Pharm. Res. 16, 1550–
1556.
Duhem, N., Danhier, F., Préat, V., 2014. Vitamin E-based nanomedicines for anti-
cancer drug delivery. J. Controlled Release 182, 33–44.
Evans, H.M., Bishop, K.S., 1922. On the existence of a hitherto unrecognized dietary
In the current study, PEGylated tocopherol (T) and tocotrienols
(T3) isomers of vitamin E were synthesized and fully characterized.
Succination and subsequent PEGylation were confirmed by 1H
NMR, HPLC and mass spectroscopy. Due to the differences in the
hydrophilicity of the isomers and the differences in the molecular
weight of the PEG 350 vs 1000, conjugates were readily identified
and isolated. These conjugates were water soluble and were found
to assemble into submicron micelles. PEGylated isomers were also
shown to have some antitumor activity against both breast and
pancreatic tumor cells. mPEG 1000 conjugates were more active
against breast cancer cell lines than the mPEG 350 products.
Nonetheless, conjugates also exhibited toxicity against normal
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least toxicity against the cell lines, which may be advantageous for
their use as functional excipients in drug delivery. The results from
the current work have demonstrated the feasibility of synthesizing
PEGylated conjugates of vitamin E isomers and highlighted the
potential use of these conjugates in drug delivery as functional and
safer excipients especially for
conjugates.
g-T3PGS 1000 and d-T3PGS 1000
Acknowledgements
This work was supported by an Institutional Development
Award (IDeA) from the National Institute of General Medical
Sciences of the National Institutes of Health under grant number
P20GM103424. Drs. Jennifer L. Carroll and Ana-Maria Dragoi from
LSU Health Sciences Center-Shreveport are acknowledged for their
assistance with the in-vitro cell culture studies, and Dr. Robert
Cody from JEOL USA, Inc. for his assistance with mass spectrometry
analysis. Acknowledgment is also extended to Dr. Terje Dokland
and Ms. Cynthia Rodenburg for Cryo-TEM imaging. The EM work
was carried out in the Cryo-EM core facility, Department of
Microbiology, at the University of Alabama at Birmingham.
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Reference Data, National Bureau of Standards of NSRDS-NBS 36, Washington,
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Appendix A. Supplementary data
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