Novel PEGylated derivatives of α-tocopherol for nanocarrier formulations; synthesis, characterization and in vitro cytotoxicity against MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2021.127907

Despite numerous beneficial therapeutic effects namely antioxidant and anti-inflammatory activity, Vitamin E has limited clinical applications due to its low water solubility. Throughout the present work, α-tocopherol's new PEGylated derivatives alongside with polyethylene glycol 300 (α-TPGT³⁰⁰), 400 (α-TPGT⁴⁰⁰), and 1000 (α-TPGT¹⁰⁰⁰) were synthesized. A 1,2,3-triazole ring was utilized as a linker for the attachment of alpha tocopherol to the PEGs through a click reaction. The purified derivatives were characterized by the means of ¹H NMR, ¹³C NMR, mass spectroscopy, UV–vis and FT-IR methods. Synthesized derivatives’ capacity to produce self-assembly nanoparticles was evaluated employing the critical micelle concentration (CMC) values. The stability of the micelles was studied by size analysis. In vitro cytotoxicity of the products was investigated using MTT assay against MCF-7 breast cancer cells. The IC₅₀ value for TPGT¹⁰⁰⁰ after 24 h treatment was 15.0 ± 1.8 μM, whereas no significant cytotoxicity effect was observed following the treatment of MCF-7 cells by TPGT^{300, 400}. The present study showed that polymeric micelle TPGT¹⁰⁰⁰ possessed better physicochemical and biological properties including relatively lower CMC value, higher stability in FBS environment in addition to higher cytotoxicity against MCF-7 breast cancer cells compared to the lower molecular weight PEGylated derivatives. These results confirmed that increasing PEG chain length left a positive effect on the polymeric micelle properties and also improved the cytotoxicity effect of new PEGylated vitamin E derivatives.

Full text of DOI:10.1016/j.bmcl.2021.127907

Bioorg. Med. Chem. Lett. 40 (2021) 127907
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel PEGylated derivatives of α-tocopherol for nanocarrier formulations;
synthesis, characterization and in vitro cytotoxicity against MCF-7 breast
cancer cells
,
Niloofar Savadkouhia b, Zeinab Mazareib, Maryam Esmaeelzadeha, Peyman Salehia,
,
*
Hasan Rafatib
a Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran
b Department of Pharmaceutical Engineering, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Despite numerous beneficial therapeutic effects namely antioxidant and anti-inflammatory activity, Vitamin E
Nanocarriers
Vitamin E
has limited clinical applications due to its low water solubility. Throughout the present work, α-tocopherol’s new
PEGylated derivatives alongside with polyethylene glycol 300 (
α
-1TPGT300), 400 (
α
-TPGT400), and 1000
PEGylation
Click chemistry
Anti-cancer
(
α
-TPGT1000) were synthesized. A 1,2,3-triazole ring was utilized as a linker for the attachment of alpha
tocopherol to the PEGs through a click reaction. The purified derivatives were characterized by the means of 1H
NMR, 13C NMR, mass spectroscopy, UV–vis and FT-IR methods. Synthesized derivatives’ capacity to produce self-
assembly nanoparticles was evaluated employing the critical micelle concentration (CMC) values. The stability of
the micelles was studied by size analysis. In vitro cytotoxicity of the products was investigated using MTT assay
against MCF-7 breast cancer cells. The IC50 value for TPGT1000 after 24 h treatment was 15.0 ± 1.8 µM, whereas
no significant cytotoxicity effect was observed following the treatment of MCF-7 cells by TPGT300, 400. The
present study showed that polymeric micelle TPGT1000 possessed better physicochemical and biological prop-
erties including relatively lower CMC value, higher stability in FBS environment in addition to higher cytotox-
icity against MCF-7 breast cancer cells compared to the lower molecular weight PEGylated derivatives. These
results confirmed that increasing PEG chain length left a positive effect on the polymeric micelle properties and
also improved the cytotoxicity effect of new PEGylated vitamin E derivatives.
Vitamin E belongs to a family of eight-membered lipid-soluble
compounds divided into two main subgroups known as tocopherols (T)
and tocotrienols (T3). Vitamin E plays a vital role in quenching free
radicals when used together with other antioxidant compounds such as
vitamin C and beta–carotene. It has been proven that vitamin E is
essential for the function of nerve and muscle cells. The anti-
inflammatory effect of vitamin E by inhibition of lipoxygenase enzyme
leads to the inhibition of inflammatory intermediates like leukotri-
enes.1,2 Vitamin E leaves a therapeutic effect on a variety of illnesses
such as cancer,3 heart diseases, and the last but not the least the Alz-
heimer’s disease.4 However, vitamin E’s low aqueous solubility has
confined its clinical applications. To overcome the mentioned drawback,
succinate and characterized as D-alpha tocopherol polyethylene glycol
1000 succinate (vitamin E TPGS) has been synthesized and marketed
extensively. TPGS has an effective role in enhancing the solubility and
absorption of many poor water-soluble drugs. The tocol ester TPGS1000
is a water-soluble form of vitamin E with an amphiphilic structure that
can self-assemble to form micelles.5
Micelles are well known nanocarriers extensively used in pharma-
ceutical industries for drug delivery and targeting purposes. Regarding
the high loading capacity and the possibility of targeting the surface of
this nanocarrier, micelles can be considered as effective carriers for drug
delivery.6 Polyethylene glycol (PEG) is one of the most universal poly-
mers hired for the fabrication of nanocarriers based on its unique
properties such as biocompatibility, biodegradability, and non-toxicity.
a
polyethylene glycol (PEG) chain attached to the α-tocopherol
* Corresponding author at: Department of Pharmaceutical Engineering, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin,
1983963113 Tehran, Iran.
E-mail address: H_Rafati@sbu.ac.ir (H. Rafati).
1
α
-Tocopherol polyethylene glycol triazole
https://doi.org/10.1016/j.bmcl.2021.127907
Received 14 November 2020; Received in revised form 15 February 2021; Accepted 21 February 2021
Available online 6 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
Scheme 1. Synthesis of PEGylated derivatives of vitamin E.
The surface PEGylation of nanoparticles also leads to the enhancement
of the nanoparticles half- life in vivo, aggregation reduction, facilitation
of water solubility, plus reduction of opsonization by the immune system
cells.7,8,9
reactions.10
The Huisgen 1,3-dipolar cycloaddition reaction is a recognized click
reaction which is also termed as the [3+2] azide-alkyne cycloaddition,
whose responsibility is to fuse two unsaturated reactants including
azides and alkynes to afford five-membered heterocycles.11 1,2,3-Tria-
zole rings are nitrogen-containing heterocycles able to form hydrogen
bonds and leading to improved solubility and ability to interact with the
biomolecules. They are highly stable against metabolic degradation
Click chemistry, introduced by K. Barry Sharpless in 1998, was fully
describes in 2001 for the first time. Click chemistry has several advan-
tages such as very fast reaction, simple to use, easy purification of the
products, and finally high yield of the products due to selectivity of the
2

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
A
B
Fig. 1. FT-IR spectrum of A: PEG-300-azide, B: TPGT300
.
Fig. 2. 1H NMR spectrum of TPGT300
.
3

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
Fig. 3. 13C NMR spectra of TPGT300
.
compared to the other compounds.12 A wide assortment of therapeutic
applications have been reported for triazoles due to their diverse bio-
logical activities including antimicrobial, antiviral, anti-inflammatory,
analgesic, anticancer, antifungal, and anticonvulsant activities. In this
regard, these particular scaffolds attract considerable attention in me-
dicinal chemistry.13
vitamin E’s low bioavailability, PEGylation of α-tocopherol was per-
formed by click reaction utilizing the triazole function as a linker. The
novel derivatives of vitamin E were synthesized by using monoazide
PEGs with different molecular weights (i.e. 300, 400, and 1000 g/mol)
and propargylated vitamin E through 1,2,3-triazole linker in the pres-
ence of copper (II) sulfate as a catalyst and sodium ascorbate as a
reducing agent. The synthetic pathway is exhibited in Scheme 1. The
products were characterized by 1HNMR and FT-IR spectra. FT-IR spec-
trum of PEGylated derivative (300 g/mol) is presented in (Fig. 1). Ac-
cording to this spectrum, the azide group signal disappeared in 1206
cmꢀ 1. The presence of the signals in 1100, 1253, 1621, and 1738 cmꢀ 1
The objectives of the current study were to (a) synthesize new
PEGylated
α-tocopherol derivatives utilizing polyethylene glycol 300
(
α
-TPGT300), 400 (
α
-TPGT400), and 1000 (
α
-TPGT1000) by a click reac-
tion; (b) fabricate nano-structured micelles from PEGylated derivatives,
and characterize the designed micelles by DLS and evaluate the micelles’
stability in different media; (c) assess the biological activity of the
–
–
–
are correlated to the C O bond, C N bond, C C double bond, and
–
–
PEGylated
α-tocopherol derivatives by in vitro cytotoxicity assay against
C N double bond stretching, respectively, that confirmed the formation
–
human breast cancer MCF-7 cell line.
of triazole ring. 1HNMR of PEGylated 300 g/mol derivative (Fig. 2)
demonstrated a unique signal in 7.88 ppm that is correlated to the
To overcome poor water solubility and limited absorption plus
4

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
A
B
C
Fig. 4. Particle size distribution diagram of micelles, A: TPGT1000, B: TPGT400, C: TPGT300
.
5

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
1.6
1.4
1.2
1
250
200
150
100
50
0.8
0.6
0.4
0.2
0
-1.8 -1.7 -1.6 -1.5 -1.4 -1.3 -1.2 -1.1 -1 -0.9 -0.8 -0.7 -0.6 -0.5
Log concentration of polymeric micelle
0
0
7
14
Time (day)
30
60
Fig. 5. Polymeric micelle size stability after 2 months storage at 4 ◦C.
Fig. 6. CMC values for polymeric micelle systems in water.
hydrogen of the triazole ring. Singlet signal in 4.85 ppm represents
methylene protons of the propargyl group. Methylene protons attached
to the triazole ring appeared in the triplet form with an integral of 2 in
4.60 ppm. In Fig. 3, the 13C spectrum of TPGT300 displayed six carbon
signals in 117.57, 122.98, 126.07, 127.96, 147.87, and 148.24 ppm that
in conformity with the DLS result. In PEGylated derivatives of 300 and
400 g/mol, an aggregation of the micelles was observed in TEM mi-
crographs. For DLS analysis, sample solutions of the synthesized com-
pounds were diluted with deionized water prior to particle size
measurement in order to retain the particle count in the range of 20–200
kcps. However, for TEM analysis, sample solutions of particles were
dropped on a carbon-coated copper grid and dried subsequently. Once
the water in preparation medium was removed, a close contact of
nanoparticles in TEM analysis was observed (probably through forma-
tion of loose aggregates or flocculates). The mean particle size of indi-
vidual particles within the flocculates is in harmony with the results of
DLS analysis.
contributed to phenyl group of α-tocopherol and also showed two car-
bon signals in 123.79 and 144.74 ppm that respectively contributed to
methine and quaternary carbon of triazole ring. These indicative peaks
confirmed formation of the product. Full proton and carbon assignment
from TPGT300, 400, 1000 was mentioned within the supplementary data.
The size and size distribution of PEGylated derivatives were together
measured employing dynamic light scattering (DLS) and the relative
diagrams are presented in (Figs. 4 and 5). As can be observed, the mean
particle size of the prepared micelles ranges from 100 nm to 200 nm. No
significant changes in the micelle size or size distribution were recorded
subsequent to storing the micelles at 4 ◦C for a period of two months,
which confirms the high stability of the polymeric micelles.
The effect of FBS on the particle size alternates the polymeric mi-
celles at 37 ◦C within a period of 0–72 h and was monitored by DLS
(Fig. 8). The results displayed that polymeric micelles of PEGlayted
derivatives 300 and 400 g/mL were stable for 24 h and then an
enhancement in the size of the polymeric micelle was observed. How-
ever, the polymeric micelle TPGT1000 was stable for 48 h. It can be
proposed that the increase of the PEG chain in TPGT1000 positively in-
fluences the stability of the polymeric micelle structure in a simulated
biological environment of the body. Probably increasing the PEG chain
length reduces the protein adsorption on the surface of the polymeric
micelle via creating spatial hindrance and as a result reduces the micelle
degradation and protein corona.19
The CMC values of the polymeric micelles have an essential effect on
their stability, both in vitro and in vivo.14 Within this study, iodine had a
task as a hydrophobic probe to evaluate the CMC values of the polymeric
micelles TPGT with different PEG weights of (PEG 300, 400, and 1000 g/
mol). Solubilized I2 distinction to participate in the hydrophobic section
of TPGT, leads to the transformation of I-3 to I2 from the surplus potas-
sium iodide (KI) in the solution. To determine the CMC values of poly-
meric micelles, the ultraviolet absorbance of KI/I2 at 366 nm profile
were plotted versus the concentration of polymeric micelles. According
to Fig. 6, the results showed that increasing the molecular weight of PEG
in polymeric micelles produced a profound reduction in the CMC values.
The relatively lowness of CMC of the polymeric micelles TPGT1000, is
able to improve the micelles’ stability as well as boosting resistance
against degradation upon dilution by blood circulation in the body,
which is significant for effectively delivering to diverse types of tu-
mors.15,16 The hydrophilic sections of the amphiphilic molecules and the
size of PEG have an effective part in micelles’ function. Longer PEG
chain in TPGT1000 compared to TPGT300, 400 provides higher HLB 2value
The cytotoxicity effects of the polymeric micelles were investigated
against MCF-7 cells. Fig. 9 shows the viability of MCF-7 cells after 24 h
treatment with the polymeric micelles using various PEG chains (300,
400, 1000 g/mL). As shown in Fig. 9, the polymeric micelle of TPGT1000
exhibited higher cytotoxicity and inhibited the cell viability about
65.09 ± 1.13% in 25 µM concentration with an IC50 value of 15 ± 1.8
µM. As reported in previously published studies, free α-tocopherol has
no significant cytotoxicity effect against MCF-7 cells in 25 µM.20 Another
report mentions that the IC50 value of TPGS1000 against MCF-7 breast
cancer cells is 26.75 ± 1.07 µM.21 According to these results, conjuga-
tion of PEG 1000 to α-tocopherol result in better cytotoxicity against
that leads to a relatively low CMC in TPGT1000 17,18
.
MCF-7 cells compared to free α-tocopherol. The higher degree in cyto-
The TEM images showed that the polymeric micelles of TPGT1000
toxicity might be correlated to the ability of TPGT1000 to generate 3ROS,
which consequently induces apoptotic cell death in more effective
were spherical (Fig. 7) within the size range of 100–200 nm which was
manner than that of
α
-tocopherol.22 Polymeric micelles with PEGs 300
and 400 g/mL showed no significant cytotoxicity effect against MCF-7
2
3
Hydrophilic–lipophilic balance
Reactive oxygen species
6

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
(B)
(A)
(C)
Fig. 7. TEM micrographs of polymeric micelles (A) TPGT1000, (B) TPGT 300, (C) TPGT400
.
500
400
300
200
100
0
0
6
20
24
48
72
Time(h)
Fig. 8. Stability of polymeric micelles in 50% FBS.
cells below the concentration of 100 µM. It can be postulated that
increasing the PEG chain length in polymeric micelle TPGT1000 probably
influences the interaction with the membrane protein receptors or
protein channels and also facilitates the cell entrance via the endocytosis
process and resultantly induced apoptosis.23,24,25
novel PEGylated derivatives of
α
-tocopherol and investigated the
physicochemical and biological effect of these compounds. The results
implied that TPGT1000 put on display better physicochemical properties
such as relatively lower CMC value and higher stability in FBS envi-
ronment. What’s more, TPGT1000 showed better biological effect, as a
result of its higher cytotoxicity against MCF-7 cells compared to that of
In this paper, we reported the synthesis and characterization of tree
7

N. Savadkouhi et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127907
TPGT1000
org/10.1016/j.bmcl.2021.127907.
References:
120
100
80
1
2
3
4
5
6
7
8
9
Azzi A, Ricciarelli R, Zingg JM. Non-antioxidant molecular functions of α-tocopherol
(vitamin E). FEBS Lett. 2002;519:8–10.
60
Constantinides PP, Han J, Davis SS. Advances in the use of tocols as drug delivery
vehicles. Pharm Res. 2006;23:243–255.
40
Das Gupta S, Suh N. Tocopherols in cancer: An update. Mol Nutr Food Res. 2016;60:
1354–1363.
20
Frank J. Beyond vitamin E supplementation: An alternative strategy to improve
vitamin E status. J Plant Physiol. 2005;162:834–843.
0
Guo Y, Luo J, Tan S, Otieno BO, Zhang Z. The applications of Vitamin e TPGS in drug
delivery. Eur J Pharm Sci. 2013;49:175–186.
control
5
10
15
20
25
El-Say KM, El-Sawy HS. Polymeric nanoparticles: promising platform for drug
delivery. Int J Pharm. 2017;528:675–691.
Concentration (µM)
Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving
nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2016;99:28–51.
Kolate A, Baradia D, Patil S, Vhora I, Kore G, Misra A. PEG - A versatile conjugating
ligand for drugs and drug delivery systems. J Control Release. 2014;192:67–81.
TPGT300
TPGT400
120
100
80
´
El-Hammadi MM, Delgado AV, Melguizo C, Prados JC, Arias JL. Folic acid-decorated
and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-
fluorouracil. Int J Pharm. 2017;516:61–70.
10 Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a
few good reactions. Angew Chem. Int Ed. 2001;40:2004–2021.
60
11 Nwe K, Brechbiel MW. Growing applications of “click chemistry” for bioconjugation
in contemporary biomedical research. Cancer Biother Radiopharm. 2009;24:289–302.
12 Moses JE, Moorhouse AD. The growing applications of click chemistry. Chem Soc Rev.
2007;36:1249–1262.
40
13 Zhou HC, Wang Y. Recent researches in triazole compounds as medicinal drugs. Curr
Med Chem. 2012;19:239–280.
20
14 Saxena V, Hussain MD. Poloxamer 407/TPGS mixed micelles for delivery of
gambogic acid to breast and multidrug-resistant cancer. Int J Nanomed. 2012;7:713.
15 Xu Q, Ensign LM, Boylan NJ, et al. Impact of surface polyethylene glycol (PEG)
density on biodegradable nanoparticle transport in mucus ex vivo and distribution in
vivo. ACS Nano. 2015;9:9217–9227.
0
control
20
40
60
80
100
Concentration (µM)
16 Lai SK, Wang YY, Hanes J. Mucus-penetrating nanoparticles for drug and gene
delivery to mucosal tissues. Adv Drug Deliv Rev. 2009;61:158–171.
17 Barakat Y, Gendy T, Ibrahim V. Surfactants with natural hydrophobic tails HLB-CMC
relationship of polyoxyethylenated cholesterol. Aust J Basic Appl Sci. 2008;2:
372–380.
Fig. 9. Diagrams of MCF-7 cancer cell viability at various polymeric micelle
concentrations after 24 h treatment.
TPGT300, 400. From the results presented in this study, it can be proposed
that increasing the PEG chain length in polymeric micelles can improve
the stability, solubility and therapeutic effects of the synthesized vitamin
E derivatives.
18 Sadoqi M, Lau-Cam CA, Wu SH. Investigation of the micellar properties of the
tocopheryl polyethylene glycol succinate surfactants TPGS 400 and TPGS 1000 by
steady state fluorometry. J Colloid Interface Sci. 2009;333:585–589.
19 Pozzi D, Colapicchioni V, Caracciolo G, et al. Effect of polyethyleneglycol (PEG)
chain length on the bio-nano- interactions between PEGylated lipid nanoparticles
and biological fluids: From nanostructure to uptake in cancer cells. Nanoscale. 2014;
6:2782. https://doi.org/10.1039/c3nr05559k.
Declaration of Competing Interest
20 Alqahtani S, Simon L, Astete CE, et al. Cellular uptake, antioxidant and
antiproliferative activity of entrapped a -tocopherol and c-tocotrienol in poly (lactic-
co-glycolic) acid (PLGA) and chitosan covered PLGA nanoparticles (PLGA-Chi).
J Colloid Interface Sci. 2015;445:243–251.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
21 Neophytou CM, Constantinou C, Papageorgis P, Constantinou AI. polyethylene glycol
succinate (TPGS) induces cell cycle arrest and apoptosis selectively in Survivin-
overexpressing breast cancer cells D-alpha-tocopheryl. Biochem Pharmacol. 2014;89:
31–42.
Acknowledgments
22 Youk H, Lee E, Choi M, et al. Enhanced anticancer efficacy of α-tocopheryl succinate
by conjugation with polyethylene glycol. J Control Release. 2005;107:43–52.
23 Batrakova EV, Han HY, Miller DW, Kabanov AV. Effects of pluronic P85 unimers and
micelles on drug permeability in polarized BBMEC and Caco-2 cells. Pharm Res.
1998;15:1525–1532.
This work has been supported by Shahid Beheshti University
Research Council and the authors gratefully acknowledge the support
provided by MPDRI. Authors duly acknowledge the help of Dr. M. Bar-
arjanian in this study.
¨
24 Shen Z, Ye H, Kroger M, Li Y. Aggregation of polyethylene glycol polymers
suppresses receptor-mediated endocytosis of PEGylated liposomes. Nanoscale. 2018;
10:4545–4560.
Disclosure statement
25 Sahay G, Batrakova EV, Kabanov AV. Different internalization pathways of
polymeric micelles and unimers and their effects on vesicular transport. Bioconjug
Chem. 2008;19:2023–2029.
No potential conflict of interest was reported by the authors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
8