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by a solvent extraction method, as supplied by the Regional mL) was added dropwise to the methoxy-PEG5000 solution. Aer
Center for Radiopharmaceuticals (northern region), Board of stirring for 72 h at room temperature, the reaction mixture was
Radiation and Isotope Technology (BRIT) (unit of BARC), concentrated and the product was precipitated in 20 mL of cold
Department of Atomic Energy, India. Human NSCLC cell line diethyl ether and further puried by washing thrice with diethyl
A549 was purchased from American Type Culture Collection ether. PEG–biotin was obtained as a light brown powder (yield:
(Rockville, MD, USA).
ꢄ95%).
Chemical characterization
Animal models
Nuclear magnetic resonance. 1H NMR and 13C NMR analysis
was performed on a Bruker Avance 400 MHz spectrometer in 5
mm diameter WILMAD® NMR tubes. 10 to 20 mg of the
compound was dissolved in 700 mL CDCl3 for evaluation.
Infrared spectroscopy. Functional groups were analyzed
using a Thermo Scientic Nicolet 8700 ATR instrument.
Mass spectroscopy. ESI mass spectra were recorded on an
Agilent Technologies 6310 Ion-Trap LCMS.
Healthy athymic mice (weighing between 20 and 25 g) and
albino New Zealand rabbits with body weights between 2.5 and
3 kg and with no prior drug treatment were used for the blood
kinetics, imaging and biodistribution studies. All animal
studies were carried out in accordance with the guidelines
compiled by CPCSEA (Committee for the Purpose of Control
and Supervision of Experiments on Animals), Ministry of
Culture, Govt. of India, and all the study protocols were rst
approved by the Institutional Animal Ethics Committee. Mice
and rabbits were housed at the in-house Experimental Animal
UV-Vis spectroscopy. UV-Vis spectra were recorded on
a Synergy 2 Multi-mode reader by BioTek Instruments, Inc.,
USA.
ꢁ
facility, under controlled temperature conditions of 22 C ꢂ 2
ꢁC, and were kept on a standard diet and water. All possible
steps were taken to abate the suffering of the animals at each
stage of the experiment.
Nanoparticle formation of BpT and Bp4eT loaded PEGylated
solid lipid nanoparticles (SLNs)
A549 xenogras. A549 cells were maintained in RPM1
medium supplemented with 10% (v/v) heat inactivated fetal
bovine serum, 2 mM L-glutamine and 100 U mLꢀ1 penicillin–
streptomycin in a humidied incubator at 37 ꢁC in 5% CO2. 100
mL PBS containing 2 ꢃ 106 A549 cells were subcutaneously
injected into the right ank of the athymic mice. Aer 7 to 10
weeks, the tumour volumes reached around 50 mm3 and the
mice models were ready for biodistribution and imaging
studies.
Stearic acid based solid lipid nanoparticles (SLNs) were previ-
ously optimized by our group, and the optimal formulation in
terms of size and polydispersity index (PDI) was used for
loading these iron chelators.34 In this work, the synthesized
PEG–biotin was incorporated in the SLN formulation instead of
the PEG used earlier. Briey, stearic acid was heated to 50 ꢁC (in
a water bath) and added to the organic phase comprising a 1 : 1
mixture of acetone and ethanol. An aqueous phase containing
the surfactant Tween-80 and the co-surfactant soya lecithin in
a 1 : 1 ratio was maintained at pH 1.1 with 1 N HCl in an ice
bath. The hot organic phase was quenched by pouring it into
the aqueous phase, maintained at 0 ꢁC, under constant stirring.
The resulting o/w emulsion was further sonicated in a bath
sonicator (Bandelin Sonorex RK52H, Germany) for 15 min and
a probe sonicator (VCX 750, Vibracell™, Sonics & Materials Inc.,
Newton CT, USA) for 5 min. The SLNs dispersions were puried
by centrifugation at 4 ꢁC (Eppendorf 5810R Centrifuge, Thermo
Fisher Scientic, USA) at 10 000 rpm, followed by water wash-
ings (ꢃ3) until the supernatant was completely neutral (pH 7.0).
The resulting formulation had the following composition (w/
v%): lipid 1.1%, PEG–biotin 0.125%, soya lecithin 2.5% and
Tween-80 2.5%. The drug loaded SLNs were similarly prepared
by adding BpT and Bp4eT drugs (0.1 to 1.1%) to the formula-
tion. These formulations were hence named BpT-PB-SLNs and
Bp4eT-PB-SLNs for BpT and Bp4eT loaded SLNs, respectively.
The SLN suspensions were lyophilized in a freeze-drier to obtain
them in dry powder form for further evaluation (Freezone 2.5
plus, Labconco Corporation, MO, USA).
Synthesis
Synthesis of 2-benzoyl pyridine thiosemicarbazone (BpT). 2-
Benzoyl pyridine thiosemicarbazone (BpT) was synthesized
according to a protocol by Richardson et al., with slight modi-
cations, using Schiff-base condensation.32 2-Benzoyl pyridine
(2.0102 g, 10.972 mmol) was dissolved in EtOH (20 mL). 80 mL of
glacial acetic acid was added to a 5% aqueous solution of thi-
osemicarbazide (1 g, 10.972 mmol) and reuxed for 8 to 10 h.
The mixture was cooled to 5 ꢁC and the yellow precipitate
formed was washed with a 1 : 1 mixture of EtOH : acetone fol-
lowed by washing thrice with diethyl ether to obtain BpT in the
form of yellow crystals (yield: ꢄ70%).
Synthesis of 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone
(Bp4eT). The synthesis of 2-benzoyl pyridine 4-ethyl-3-
thiosemicarbazone was performed by the same method as
BpT, by using 4-ethyl-3-thiosemicarbazide (500 mg, 4.19 mmol)
in place of thiosemicarbazide along with 2-benzoyl pyridine
(768.5 mg, 4.19 mmol). Similar processing resulted in pale
yellow shiny crystals of Bp4eT (yield: ꢄ72%).
Physico-chemical characterization
Synthesis of PEG–biotin conjugate (PB). The PEG–biotin
conjugate was prepared by Steglich esterication.33 In brief,
Dynamic light scattering and zeta potential. The dry powder
methoxy-PEG5000 (341.1 mg, 0.068 mmol) was dissolved in DMF of the SLN formulation was re-suspended in water to form
(15 mL). A solution of biotin (20 mg, 0.0818 mmol), DCC (35.18 a homogenous solution. This solution was used for the deter-
mg, 0.1704 mmol) and DMAP (8.33 mg, 0.068 mmol) in DMF (15 mination of the hydrodynamic diameter and zeta potential
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 61585–61598 | 61587