P. Goyal et al.
Carbohydrate Research 500 (2021) 108255
demonstrated as efficient drug delivery systems in vitro and in vivo.
Using this hydrophobic-hydrophilic polymer-ligand combination,
several stimuli-responsive amphiphiles have been created which are
responsive to pH, enzyme, light, temperature, etc. Recently, Zhang et al.
have fabricated a reduction sensitive drug delivery system for tumor
sites using pluronic F68 and cholesterol linked through a disulfide
linkage. By taking advantage of high concentration of glutathione at
tumor sites, the entrapped doxorubicin (DOX) in the micelles was
released in a sustained manner over a longer period of time. The pro-
jected system was not only fairly stable but also found to be almost
non-toxic to cells [27]. For target-specific delivery of therapeutics, these
conjugates require further derivatization to incorporate such properties,
which becomes cumbersome and a time consuming step. In order to
avoid such tedious multi-step processes, several polysaccharides have
been shown to possess targeting ability. A single-step coupling of hy-
drophobic drugs/ligands with such polysaccharides results in amphi-
philic conjugates capable of entrapping therapeutics and delivering
them in a site-specific manner. Dextran is one of the most commonly
used polymers, which possesses colon-targeting ability [28,29]. By
taking advantage of this property of dextran, we anticipated that an
amphiphilic conjugate of dextran-cholesterol would self-assemble in
aqueous medium entrapping a drug molecule of choice and deliver it at a
particular site in a controlled manner thereby improving the bioavail-
ability of the drug at that site. The resulting formulation would be
biocompatible, biodegradable and remain in the system for longer
duration due to hydrophilic shell comprising of glucose chains. There-
fore, in the present study, we have selected dextran and conjugated it
with cholesteryl hemisuccinate, prepared by the reaction of cholesterol
with succinic anhydride, using a condensing reagent, dicyclohex-
ylcarbodiimide (DCC), in the presence of 4-dimethylaminopyridine
(DMAP), to obtain cholesteryl-dextran (Chol-Dex) amphiphiles. Two
formulations (5 and 10% substitution of cholesteryl units, Chol-Dex-5
and Chol-Dex-10) were subjected to self-assembly followed by entrap-
ment of two drugs, metronidazole and rifampicin. Under different con-
ditions, the release profiles of the drugs were obtained and
cytocompatibility of the formulations was evaluated in vitro.
evaporator. Finally, the syrupy residue of cholesterol hemisuccinate was
left in a vacuum desiccator overnight (Yield = ~92%).
2.2. Synthesis of cholesteryl-dextran (Chol-Dex) conjugate
The cholesterol hemisuccinate (CS) was conjugated with dextran in
two different ratios. Dextran (1 g, 6.17 mmol) was dissolved in dry DMF
(25 ml) and added cholesterol hemisuccinate (149 mg, 0.3 mmol for 5%
substitution) followed by dicyclohexylcarbodiimide (93 mg, 0.45 mmol)
and DMAP (55 mg, 0.45 mmol). The reaction was allowed to stir over-
night at an ambient temperature and then kept in a refrigerator for 2h.
The precipitate was removed by filtration after washing with DMF (2
ml). The filtrate was concentrated on a rotary evaporator to one-third of
the volume and diethyl ether (100 ml) was added to precipitate the
cholesteryl-dextran (Chol-Dex). The solid was recovered, dispersed in
water and subjected to dialysis in a dialysis bag (MWCO 12 kDa) for 24 h
with intermittent change of water (4 х 6h). The dialyzed material was
concentrated to obtain the Chol-Dex-5 in ~82%. Similarly, Chol-Dex-10
with 10% substitution was prepared. Both the preparations were char-
acterized spectroscopically and degree of substitution was determined.
2.3. Self-assembly of Chol-Dex conjugates
The process of self-assembly of amphiphilic Chol-Dex (5 and 10%
substituted) was carried out by dialysis method. Chol-Dex (~2 mg) was
dissolved in DMSO (100 μl) and water (900 μl) was added gradually with
continuous stirring. The solution was vortexed for 5 min and left for 4 h
at RT. Then the solution was poured in dialysis tubing (MWCO 1.0 kDa)
and dialyzed against water for 6 h to get rid of organic solvent. The
dialyzed solution was lyophilized to obtain Chol-Dex nanocomposites as
solid material.
2.4. DLS measurements
To measure the average size and polydispersity index (PDI) of Chol-
Dex nanocomposites and drug-embedded nanocomposites, ~1.0 mg of
the material was dispersed in 1.0 ml of Milli Q water and vortexed for 2
min. The dispersion was sonicated for 5 min and subjected to mea-
surement of size on Zetasizer Nano-ZS (Malvern Instruments, UK). The
values are mean of three independent measurements in triplicates.
Stability of Chol-Dex nanomicelles, dispersed in Milli Q water, was also
examined by measuring the variation in the hydrodynamic diameter up
to 48 h.
2. Materials and methods
Dextran (MW 40,000), N,N-diisopropylethylamine (DIPEA), 4-dime-
thylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dul-
becco’s Modified Eagle’s medium (DMEM), metronidazole (MZ) and
rifampicin (RF) were purchased from Sigma-Aldrich Chemical Co.
(USA). Dialysis membrane (MW = 500–1000 Da) was procured from
Spectrum Labs, USA. Other chemicals and reagents used in the present
study were obtained from local vendors.
2.5. Transmission electron microscopy (TEM)
This technique was used to find out the actual size of the self-
assembled micellar nanocomposites in dryform. The dispersion solu-
1H NMR spectra of cholesterol hemisuccinate and cholesteryl-
dextran conjugates, dissolved in DMSO‑d6, were carried out on JEOL-
EXCP 400 MHz spectrometer, Japan. Chemical shifts (δ) are expressed
in ppm. FTIR spectra of the synthesized conjugates (~2.0 mg) were
recorded using PerkinElmer BX Series spectrophotometer, USA with the
following scan parameters: scan range, 4000 to 500 cmꢀ 1; number of
scans, 16; resolution, 4.0 cmꢀ 1; interval, 1.0 cmꢀ 1; unit %T.
tion (~5 μl of 1.0 mg/ml) was dropped on carbon-coated copper grids
and negatively stained with uranyl acetate (1%, 5 μl). These grids were
initially air-dried and then kept in a desiccator for vacuum drying.
Subsequently, the grids were subjected to investigation under HR-TEM
(200 kV) and images were captured.
2.6. Drug loaded Chol-Dex nanomicelles
2.1. Synthesis of cholesterol hemisuccinate
In this study, two hydrophobic drugs, metronidazole and rifampicin,
were used to entrap in the nanomicelles. The drugs have been encap-
sulated in amphiphilic molecules of both the formulations, Chol-Dex-5
and Chol-Dex-10, to check their entrapment efficiency. To achieve the
encapsulation of the drugs, the projected formulations and drugs were
taken in a w/w ratio of 10:2. Briefly, Chol-Dex-5 (~20 mg) and the drug
(~4 mg) were dissolved in DMSO (2.4 ml) to make a clear solution and
then Milli Q water (~21.6 ml) was added dropwise with continuous
vortexing over the period of 5 min. The homogenous solution was
incubated for 2 h and then subjected to dialysis against water in 1.0 kDa
Cholesterol (2.0 mmol, 775 mg), succinic anhydride (3.0 mmol, 300
mg), DIPEA (3.0 mmol, 510 μl) and DMAP (1.0 mmol, 122 mg) were
dissolved in 1,2-dichloroethane (8 ml) and stirred for 24 h at room
temperature. The progress of the reaction was monitored on TLC using
chloroform: methanol (9:1, v/v). After completion of the reaction, the
reaction mixture was further diluted with dichloroethane (30 ml) and
washed with 10% aqueous citric acid (2 × 20 ml) followed by washings
with distilled water (2 × 20 ml). The organic layer was collected, dried
over anhydrous sodium sulphate, filtered and concentrated on a rotary
2