Evaluation of BBB permeable nucleolipid (NLDPU): A di-C15-ketalised palmitone appended uridine as neuro-tracer for SPECT

Article abstract of DOI:10.1016/j.ijpharm.2019.04.074

Despite being in routine for onco-diagnostics for years, the applicability of nucleosidic molecular imaging probes is severely restricted in neurological applications due to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers utility for neuro-onco early diagnostics, suitable modification which enhances their BBB permeation needs investigation. Among various modifications, lipidization of nucleosides has been reported to enhance cellular permeability. Extending the concept, the aim was to exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based non-lipidic (NS_DAU)and di-C15-ketal appended lipidic (NL_DPU)ligands were conjugated to chelator, DTPA (DTPA-NS_DAU and DTPA-NL_DPU)using multi-step chemistry. The ligands were evaluated in parallel for comparative physical and biological parameters. Additionally, effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-specific protein binding were evaluated. Fluorescence quenching of BSA indicated appreciable interaction of DTPA-NL_DPU with protein only above 10 mM without inducing conformational changes. In addition, DTPA-NL_DPU was found to be haematocompatible and cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used for ^99mTc-complexation for SPECT imaging. Optimized ^99mTc-radiolabeling parameters resulted in quantitative (≥97%)labeling with good stability parameters in in-vitro serum and cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (^99mTc-DTPA-NL_DPU)was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal mice as compared to 0.06% ID/g uptake of ^99mTc-DTPA-NS_DAU at 5 min. Blood kinetics indicate biphasic profile and t1/2(distribution)46 min for ^99mTc-DTPA-NL_DPU. The preferential accumulation of ^99mTc-DTPA-NL_DPU in brain tumor intracranial xenograft indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this work presents the potential of the biocompatible nucleolipidic system for brain targeting and early diagnostics.

Full text of DOI:10.1016/j.ijpharm.2019.04.074

Accepted Manuscript
Evaluation of bbb permeable nucleolipid (nldpu): a di-c15-ketalised palmitone
appended uridine as neuro-tracer for spect
Swastika, Shubhra Chaturvedi, Ankur Kaul, Puja Panwar Hazari, Preeti Jha,
Sunil Pal, Sangeeta Lal, B Singh, Philippe Barthélémy, Anil K Mishra
PII:
S0378-5173(19)30345-X
https://doi.org/10.1016/j.ijpharm.2019.04.074
IJP 18322
DOI:
Reference:
International Journal of Pharmaceutics
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 December 2018
16 April 2019
27 April 2019
Please cite this article as: Swastika, S. Chaturvedi, A. Kaul, P. Panwar Hazari, P. Jha, S. Pal, S. Lal, B. Singh, P.
Barthélémy, A.K. Mishra, Evaluation of bbb permeable nucleolipid (nldpu): a di-c15-ketalised palmitone appended
uridine as neuro-tracer for spect, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/
j.ijpharm.2019.04.074
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EVALUATION
OF
BBB
PERMEABLE
NUCLEOLIPID (NLDPU): A DI-C15-KETALISED
PALMITONE APPENDED URIDINE AS NEURO-
TRACER FOR SPECT
Swastika,a,b Shubhra Chaturvedi,a Ankur Kaul,a Puja Panwar Hazari,a Preeti Jha,a Sunil Pal,a Sangeeta
Lal,c B Singh,b Philippe Barthélémy,d,* and Anil K Mishra a,*
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied
Sciences, Brig. S. K. Mazumdar Road, Delhi-110054, India
bDepartment of Chemistry, Banaras Hindu University, Varanasi, Uttar Pradesh, India
cSchool of Physical Sciences, Jawaharlal Nehru University, Delhi-110067
d INSERM U1212 and Université de Bordeaux, Bordeaux, F-33076, France
*Email: philippe.barthelemy@inserm.fr Phone No; 33 557 574 853
*Email: akmishra63@gmail.com Phone No: +011-23905117

ABSTRACT: Despite being in routine for onco-diagnostics for years, the applicability of
nucleosidic molecular imaging probes is severely restricted in neurological applications due
to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers
utility for neuro-onco early diagnostics, suitable modification which enhances their BBB
permeation needs investigation. Among various modifications, lipidization of nucleosides has
been reported to enhance cellular permeability. Extending the concept, the aim was to
exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to
cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based
non-lipidic (NSDAU) and di-C15-ketal appended lipidic (NLDPU) ligands were conjugated to
chelator, DTPA (DTPA-NSDAU and DTPA-NLDPU) using multi-step chemistry. The ligands
were evaluated in parallel for comparative physical and biological parameters. Additionally,
effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-
specific protein binding were evaluated. Fluorescence quenching of BSA indicated
appreciable interaction of DTPA-NLDPU with protein only above 10 mM without inducing
conformational changes. In addition, DTPA-NLDPU was found to be haematocompatible and
cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used
for 99mTc-complexation for SPECT imaging. Optimized 99mTc-radiolabeling parameters
resulted in quantitative ( 97%) labeling with good stability parameters in in-vitro serum and
cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (99mTc-DTPA-
NLDPU) was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal
mice as compared to 0.06% ID/g uptake of 99mTc-DTPA-NSDAU at 5 min. Blood kinetics
indicate biphasic profile and t1/2(distribution) 46 min for 99mTc-DTPA-NLDPU. The
preferential accumulation of 99mTc-DTPA-NLDPU in brain tumor intracranial xenograft
indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this
work presents the potential of the biocompatible nucleolipidic system for brain targeting and
early diagnostics.
KEYWORDS: nucleolipid, radiotracer, brain-imaging, BBB permeable, SPECT imaging,
nucleoside tracer.

1. INTRODUCTION
Imaging of metabolic alterations has been exploited extensively for the detection and
assessment of therapeutic intervention in diseases particularly, cancer (Di
Gialleonardo et al, 2016). The basis of metabolic molecular imaging is the metabolic
reprogramming in tumors, inflammation-infection and neurodegenerative diseases
(Demetrius et al., 2014; Levy and Bartosch , 2016; Gleeson and Sheedy, 2016).
Enhanced uptake of metabolites in uncontrolled proliferating cells, like tumors, leads
to a differential concentration in comparison to normal-cells which results in the
required contrast for imaging. Among the metabolic tracers, the glucose analogue,
fluorine labelled deoxy glucose (FDG), has been the major radiotracer for molecular
imaging using positron emission tomography (PET) with applications in oncology,
inflammation, and infections. FDG is preferentially retained at the diseased site due to
enhanced glucose requirement and intracellular entrapment, resulting in required
contrast for imaging (Alauddin, 2013). However, (1) the non-specific uptake in non-
tumor sites particularly at inflammation and infection sites, (2) uncorrelated uptake
with respect to cellular proliferation and (3) high background in certain tissues
associated with high glucose metabolism like the brain, limits FDG application in
oncological and neuro-oncology imaging, (Chen et al., 2005; Basu et al., 2009), in
spite of FDG being permeable to intact blood-brain barrier (BBB) (Saha, 2004).
Alternately, metabolic tracers based on nucleosides offered specificity and
quantitative correlation of the tracer uptake with the growth rate of tumor.
Nucleosides being the structural unit of nucleic acids have a high turnover in rapidly
3

proliferating cells viz., tumorigenic cells, proportional to the DNA synthesis
(Alauddin, 2013).
Figure 1: Well-known nucleoside-based probes for imaging with 18F-FLT dominating the
clinical application in onco-diagnostics: FLT:fluoro-labelled thymidine, FIAU or
Fialuridine:2'-fluoro-2'-deoxy-1beta-D-arabinofuranosyl-5-iodouracil, FMAU: 1-(2’-
Deoxy-2’-fluoro-β-D-arabinofuranosyl)thymine, FFEAU: (1-(2’-deoxy-2’-fluoro-beta-D-
arabinofuranosyl)-5-(2-fluoroethyl)uridine),
FEAU:2'-fluoro-5-ethyl-1-beta-D
arabinofuranosyluracil, FBAU-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-bromouracil,
FAC: 1-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)cytosine, FMAC: 1-(2′-deoxy-2′-
fluoro-β-d-arabinofuranosyl)-5-methylcytosine,
hydroxymethylbutyl)guanine, FHPG:
methyl)guanine, FHOMP: 6-((1-fluoro-3-hydroxypropan-2-yloxy)
methylpyrimidine-2,4(1H,3H)-dione)
FHBG:
9-((3-fluoro-1-hydroxy-2-propoxy)-
methyl)-5-
9-(4-fluoro-
3-
A gamut of nucleoside based radiotracers namely, derivatives of thymidine [18F-FLT],
uridine [124I/131I-FIAU, 11C/18F-FMAU, 18F-FFEAU, 18F-FEAU, 18F-FBAU], cytosine
18
18
[18F-FAC, F-FMAC], guanosine [18F-FHBG, F-FHPG] and modified pyrimidines
[FHOMP] have been developed for imaging of cancer (Figure 1, see caption for
abbreviations; Alauddin, 2013; Meyer et al., 2014; Pysz and Willmann, 2014) The
tracers varied in their ability to produce signals (tumor: background sensitivity),
metabolic degradation and bio-distribution. Despite being studied and used routinely
in clinics for oncologic diagnostics for past three-four decades, the utility of the
4

nucleosides probes (e.g. FLT) for delineation of low grade tumors having
uncompromised BBB is severely restricted due to low permeability across the intact
BBB (Chen et al., 2005). FLT (Chen et al., 2005; Alauddin, 2013; Meyer et al., 2014;
Pysz and Willmann, 2014), FIAU, (Pysz and Willmann, 2014; Langen and Galldiks,
2016; Tehrani and Shields et al., 2013), FHBG (Langen and Galldiks, 2016), FHPG,
(Langen and Galldiks, 2016) FEAU, FFEAU, (Miyagawa et al., 2008) FAC, FMAC,
(Schwarzenberg et al., 2011) and FBAU (Kao et al., 2002) are known not to cross the
BBB. In order to make the nucleosides radiotracers permeate through the intact BBB,
lipophilic modifications of nucleosides were reported (Chaturvedi and Mishra, 2016).
These lipophilic modifications included the introduction of di-benzoylation at 3’ and
5’ of FBAU (Kao et al., 2002) and vinylic substitution at 5’ position of uridine in
FIAU (Li et al., 2008). The results were promising with [76Br]-FBAU-3’, 5’-
dibenzoate having a brain uptake of 0.23%ID/g as opposed to brain uptake of only
0.08 %ID/g for [76Br]-FBAU. However, in case of the vinylic substitution in-spite of
enhanced lipophilicity, the brain accumulation of FIAU and vinylic-FIAU were
similar. Lipidization of drugs has also been one of the approaches, to improve the
cellular internalization and brain delivery (Zaro, 2015). Among promising lipid-drug
conjugates are palmitoyl conjugated oxytocin derivative (LOT-1) (Mizuno et al.,
2015) designed for enhance brain delivery, and docosahexanoic acid (DHA)-
conjugated paclitaxel (Bedikian et al., 2010) having enhanced tumor uptake and
improved pharmacokinetics. As an attempt to enhance cellular internalization,
nucleosides have also been appended with long alkyl chains, resulting in nucleolipids
(Rosemeyer, 2005; Gissot et al., 2008; Baillet et al., 2018). Initial leads in the
structural design of nucleolipids came from the existence of naturally occurring
conjugates like cytidine diphosphate diacylglycerol (CDP-diacylglycerol) in
5

biological systems. The early reports on the biomedical application of nucleolipids
include modification of 1-(beta)-D-arabinofuranosylcytosine, an anticancer drug, as
adamantine or palmitate derivatives to enhance cellular penetration and improve
efficacy ((Rosemeyer et al., 2005). The N4-acyl derivatives of Arabinosylcytosine
AraC, appended with C15-C22 saturated fatty acids, were shown to have better
antitumor activity attributed to the enhanced cellular internalization and stability
towards deaminases (Lansakara et al., 2012).
Figure 2 : Schematic representation of nucleoside internalizing through the blood-brain barrier
(BBB) 1. Natural nucleoside primarily is transported across the BBB through receptor- mediated
internalization 2. Modified nucleosides unrecognized by receptors rely on passive diffusion across
the BBB 3. Lipidic nucleosides similar to modified nucleosides rely on mechanisms of passive
diffusion, e.g. flip-flop across BBB.
Nucleosides permeate across cellular membrane through nucleoside transporters, whereas the
enhanced cellular internalization of nucleolipids is due to the lipid moiety which mediates
diffusion and flip-flop mechanism thereby accelerating the absorption of nucleolipids across
cellular membranes (Scheidt et al., 2004; Allain et al., 2012; Zhu et al., 2013; Figure 2)
Experimental evidence includes diffusion mediated cellular internalization of lipophilized
gemcitabine derivatives in nucleoside transporter-hENT1 deficient cell line ((Lansakara et al.,
2012; Baillet et al., 2018). Since lipidization is a well-established strategy for developing
BBB permeating drugs, we hypothesize that the nucleolipid may prove to be effective in
6

delivering nucleoside-based radiotracer across BBB wherein nucleolipids act as (a) carrier
molecule across BBB and also as (b) selective metabolite tracer due to the presence of
nucleoside. In addition to being brain impermeable, majority of nucleoside-based tracers are
halogen labelled [18F for PET imaging and 76Br/123I for single photon emission computed
tomography (SPECT) imaging]. 76Br or 123I labelled tracers are liable to undergo
dehalogenation as primary degradation mode thereby limiting biological use. 18F labelled
tracers have limited dehalogenation but the short half-life of 109.7 min and high operational
cost associated with 18F production using on-site cyclotron limit its applicability, especially in
remote areas. Metal-based tracers are attractive alternatives as the radionuclides for imaging
viz.,68Ga and 99mTc are generator based thereby having a lower economic burden which can
have higher societal implications. (Chaturvedi et al., 2017)
For biological application, the metal-based tracers require a strong chelator to be appended
with the ligand which results in stable metal complexes with low probability of transchelation
reaction with the biological molecules. The polyaminocarboxylic acid derived chelators like
diethylenetriaminepentaacetic acid (DTPA), undergo fast and high yielding metal
complexation. DTPA is a known chelator for 68Ga (PET), 99mTc (SPECT), 111In (SPECT) and
also Gd+3 (Magnetic Resonance Imaging (MRI)), Tb+3 and Eu+3 (Optical imaging) thereby
allowing operational versatility to the radiotracer by changing the metal ion. (Li et al.,1995;
Saha, 2004; Liu et al., 2008).
As an attempt to enhance the brain-penetrating ability of the nucleosides, we have evaluated
the potential of the uridine based nucleolipid (NLDPU) having two C15 chains for brain
penetration. NLDPU has been conjugated with DTPA which serves as the chelator for the
radionuclide 99mTc, thereby resulting in 99mTc-DTPA-NLDPU. For comparison, non-lipidic
uridine (NSDAU) derivative (99mTc-DTPA-NSDAU) has also been evaluated. The results
indicate enhanced brain penetrating properties of 99mTc-DTPA-NLDPU and its possible
7

application for brain imaging. Though we have evaluated the proposed system as SPECT
tracer, the ligand can find application as tracer for any of the molecular imaging modality-
PET/SPECT/MRI or optical imaging by virtue of the versatile nature of the chelator.
2. MATERIALS AND METHODS
2.1 Synthesis and characterization
Chemicals:
Uridine, tosylic acid, tosyl anhydride, triethyl orthoformate, sodium bicarbonate, triethyl
amine, chloroform, sodium azide, pyridine, dimethyl formamide, palladium on activated
charcoal 10%, dry methanol and DTPA bis anhydride were purchased from Sigma-Aldrich
Co., USA. Palmitone (16-hentraicontanone) was purchased from TCI Chemicals, India. Silica
gel mesh size 80-120 for column chromatography was procured from Merck, Germany.
Concentrated sulphuric acid, diethylenetriaminpentaacetic acid, stannous chloride, stannous
tartrate, sodium bicarbonate, 3-(4, 5- dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromide
(MTT), sulfo-rhodamine B (SRB) dye, glacial acetic acid, Trichloroacetic acid (TCA),
pyridine, Bovine serum albumin (BSA) were purchased from Sigma Aldrich, St Louis, MO,
USA. All other chemicals used were of the standard analytical grade. Glass wares used were
oven-dried.
Characterization:
13
1H and C- NMR spectra were recorded on Bruker Avance II 400 MHz and Bruker Avance
600 MHz using deuterated solvents procured from Merck, Germany at 298 K using 10-15 mg
compound. Chemical shifts are reported in ppm and referenced to the solvent residual peak.
Mass Spectroscopy data was acquired using electrospray ionization-mass spectrometry (ESI-
MS) on Agilent 6310 ion trap fitted with Agilent 1200 LC (M/S Agilent Germany).
Lyophilization was performed on M/S Labconco system.
8

2.1.1. Synthesis of DTPA-NSDAU
2’, 3’-O-Isopropylidene Uridine (1a)
The synthesis was carried as already reported with minor modifications (Levene and
Tipson,1934). In brief, uridine (2.042 mol, 500 mg) was dissolved in 20 mL of acetone
containing 4 Å molecular sieves. Sequential addition of anhydrous copper sulphate (5.10
mmol, 814.99 mg) and 0.025 mL of conc. sulphuric acid (diluted in 5 mL acetone) was
carried. The solution was stirred at room temperature for 48 h. The reaction solution was
filtered to remove copper sulphate, and the filtrate was evaporated under vacuum. The
compound was obtained as white foam. Percent yield 73.14% (TLC: Rf 0.5 in ethyl acetate)
1H-NMR (400 MHz; (CD3)2-SO) δH: 11.37 (1 H, brs, -NH), 7.78 (1 H, d, J ≈8 Hz : H-6),
5.81(1 H, d, J ≈2Hz : H-1’), 5.62 (1 H, dd, J ≈8 Hz: H-5), 4.87 (1 H, d: -CHuridine), 4.72 (1
H, d: -CHuridine ), 4.05 (1 H m: -CHuridine), 3.58 (2H, m : H-5’), 3.32 (1 H, br : -OH), 1.46-
1.26 (6 H; 2×-CH3)
13C-NMR (100 MHz; (CD3)2-SO) δc: 163.29, 150.40, 142.01, 113.02, 101.78, 91.20, 86.58,
83.76, 80.53, 61.32, 27.00, 25.20
MS (ESI-): m/z calcd for C12H16N2O6 [M]: 284.27: found 283 [M-H]-
5’-Azido-5’Deoxy-2’, 3’-O-Isopropylidene Uridine (2a)
2’,3’-O-Isopropylidene Uridine (1a) (1.76 mmol, 500 mg) was suspended in dichloromethane
(10 mL). Addition of pyridine (5 mL) dissolved the compound completely. The solution was
cooled in an ice-bath. Tosyl chloride (2.28 mmol, 435.36 mg) was added in small batches.
The reaction was left overnight on stirring. The solution was concentrated in vacuum and the
crude mixture obtained was used as such. The mixture was then reacted with sodium azide
(5.13 mmol, 334 mg) in 15 mL dimethylformamide (DMF) and refluxed for 8 h. After
evaporation under vacuum, yellow-colored oil was obtained. Purification on silica gel column
9

chromatography using hexane/ EtOAc lead to the desired compound as white solid (TLC: Rf
0.2 in hexane/ EtOAc 1:1).
1H-NMR (400 MHz; CDCl3 (0.3% TMS)) δH: 10.03 (1 H, brs, -NH), 7.241 (1 H, d, H-6),
5.71 (1 H, t, H-1’), 5.62 (1 H, J=2.4 Hz: H-5), 4.95 (1 H, m: -CHuridine), 4.74 (m, 1H: -
CHuridine), 4.17 (1 H, m: -CHuridine), 3.56 (2 H, m, 5’-CH2), 1.51-1.09 (6 H, 2 ×CH3)
13C-NMR (100 MHz; CDCl3 (0.3% TMS)) δc: 162.98, 149.50, 142.24, 114.84, 102.86, 94.62,
85.66, 84.28, 81.42, 52.32, 27.09, 25.23
MS (ESI-): m/z calcd for C12H16N2O6 [M]: 309.28: found 309.54
5’-Amino-5’Deoxy-2’, 3’-O-Isopropylidene Uridine (3a)
5’-Azido-5’Deoxy-2’,3’-O-Isopropylidene Uridine (2a) (1.61 mmol, 500 mg) was dissolved
in dry methanol (20 mL) under an inert atmosphere (nitrogen) in ice-bath. After brief stirring,
hydrogen gas was bubbled into it using long needle syringe. Palladium (10% activated on
charcoal) was added in a catalytic amount (137 mg, 1.29 mmol). Adequate precautions were
exercised to avoid contact of the catalyst with air. The reaction was stirred for 6 h and
allowed to attain room temperature. For separating palladium, the reaction mixture was
poured over celite bed and washed thrice with methanol. On drying the filtrate, light yellow
powder with a yield of 80% was obtained (TLC: Rf 0.3 in hexane/EtOAc 1:1).
1H NMR (400 MHz; CDCl3 (0.3% TMS)) δH: 9.7 (1 H, brs, -NH), 7.23 (1 H, d: H-6), 5.75 (1
H, d, J=8.4Hz: H-5), 5.59 (1 H, J=2Hz: H-1’), 4.93 (1 H, m: -CHuridine), 4.75(1 H, m: -
CHuridine), 4.18 (1 H, m: -CHuridine), 3.56 (2 H, m, 5’), 3.05(2 H, brs, -NH2), 1.5-1.18 (6
H, 2 ×CH3)
13C-NMR (100 MHz; CDCl3 (0.3% TMS)) δc:163.79, 150.12, 142.58, 114.68, 102.73, 84.30,
84.23, 82.07, 81.43, 44.14, 25.13, 24.59
MS (ESI-): m/z calcd for C12H16N2O6 [M]-: 283.28: found 282.9[M-H]-
10

5’-DTPA-5’Deoxy-2’, 3’-O-Isopropylidene Uridine (4a)
5’-Amino-5’Deoxy-2’, 3’-O-Isopropylidene Uridine (3a) (500 mg, 1.766 mmol) was
dissolved in 5 ml anhyd DMF and stirred. To this, solution of DTPA bis anhydride (630 mg,
1.766 mmol) in anhyd DMF (10 mL) was added drop-wise. The pH of the reaction mixture
was adjusted to 8 using triethylamine and the contents were refluxed at 80° C for 48 h. The
progress of the reaction was monitored by TLC in ethyl acetate and 0.2% methanol. The
compound with a yield 54 %was obtained as white solid after precipitating in acetone and
diethyl ether (TLC: Rf 0.43 in ethyl acetate and 0.2% methanol)
1H-NMR (400 MHz; (CD3)2-SO) δH: 10.54 (brs : 4x-COOH DTPA), 7.94 (brs: -NHDAU),
7.66 (1H, brs, -NHamide), 7.30 (1H, s: H-6), 5.73-5.62 (2H : H-1’, H-5), 4.82-3.73 (4H: -
CHuridine), 3.19 (4H, br, 5’-H, 1X-CH2,DTPA), 2.89-2.79(m, 8H, 4X-CH2,DTPA),1.89
(8H, s, 4X-CH2,DTPA), 1.06 (6H, 2X-CH3)
13C-NMR (100 MHz; (CD3)2-SO) δc: 174.46, 168.63, 163.47, 163.36, 158.64, 154.13,
151.51, 144.16, 140.41, 135.33, 130.14, 113.31, 103.01, 87.04, 86.12, 85.56, 71.75, 56.16,
55.26, 36.35, 25.54.
MS (ESI-): m/z calcd for C26H38N6O14 [M]-: 658.24: found 656.1 [M-2H]-
2.1.2 Synthesis of DTPA-NLDPU
2’, 3’-O-16-hentriacontanyliden-uridine (1b)
The compound was synthesized as per the already mentioned protocol (Gissot et al., 2008).
Uridine (5.54 mmol, 1.35 g) was dissolved in 20 ml of tetrahydrofuran. Sequential addition of
16-hentriacontanone (1.11 mmol, 500mg), triethylorthoformate (0.933 ml, 5.54 mmol, 822.51
mg) and tosylic acid monohydrate (TsOH.H2O: 1.11 mmol, 211.1 mg) was carried, and the
11

solution was refluxed for 24 h. After 24 h, the reaction was quenched by addition of 2 ml of
triethylamine. The reaction solution was then poured over ice and sodium bicarbonate (4 g)
and stirred briefly. The compound was extracted in dichloromethane. The yellow colored
organic layer was collected and dried over sodium sulfate. After evaporation under reduced
pressure, crude solid (yield 62%) was obtained. Further, purification on silica column with
hexane/ EtOAc gave the desired product as white solid. (TLC: Rf 0.4 in hexane/ EtOAc 1:1).
1H-NMR (400 MHz; CDCl3 (0.3% TMS)) δH 9.13 (1 H, brs, -NH), 7.33 (1 H, d, J 8 Hz: H-
6), 5.76 (1 H, d, J 8Hz: H-5), 5.54 (1 H, dd, J 2 Hz: H-1’), 5.02 (1 H, d: -CHuridine), 4.95 (1
H, d: -CHuridine), 4.27 (1 H, m: -CHuridine), 3.91 (2H, dd : H-5’), 2.90 (1 H, br : -OH),
1.74-1.26 (56 H; Lipidic chain)
13C-NMR (100 MHz; CDCl3 (0.3% TMS)) δc: 163.07, 150.26, 143.08, 118.45, 102.60, 96.35,
87.22, 83.83, 80.29, 62.70, 31.90, 29.82, 29.68, 29.64, 29.56, 29.34, 24.16, 23.56, 22.662,
14.091
5’-Azido-5’Deoxy-2’, 3’-O-16-hentriacontanyliden-uridine (2b)
Compound 1(b) (1, 1.18 mmol, 800 mg) was tosylated using tosyl chloride (1.77 mmol, 354
mg) in pyridine (5 mL) and dichloromethane (10 mL) overnight at ice-cold conditions. After
evaporation under reduced pressure, the crude compound was obtained which was taken
further without purification. The tosylated compound was then reacted with sodium azide
(2.406 mmol, 156 mg) in 15 mL dimethylformamide (DMF) and refluxed for 8 h. The
reaction mixture was evaporated, and the yellow-colored compound was obtained. Further
purification on silica gel column chromatography using hexane-EtOAc leads to the desired
compound as white solid (yield 86%, Rf 0.6 in hexane: EtOAc 2:1).
12

1H-NMR (400 MHz; CDCl3 (0.3% TMS)) δH: 9.32 (br, 1H, -NH), 7.20 (1H, d, H-6), 5.68
(1H, d, 1’), 5.56 (1H, d, H-5, J= 6Hz), 4.942 (1 H, d: -CHuridine), 4.72(1 H, d: -CHuridine),
4.155 (1 H, d: -CHuridine), 3.535 (2H, m, 5’), 1.71-0.88 (56 H)
13C-NMR (100 MHz; CDCl3 (0.3% TMS)) δc: 163.00, 149.85, 142.66, 118.73, 102.79,
95.28, 86.096, 84.48, 52.41, 36.90, 31.90, 29.79, 29.337, 24.11, 22.67, 13.76
MS (ESI-): m/z calcd for C40H71N5O5 [M-]: 701.5: found 701.2
5’-Amino-5’Deoxy-2’, 3’-O-16-hentriacontanyliden-uridine (3b)
Compound 2(b) (0.541 mmol, 380mg) was dissolved in dry methanol under an inert
atmosphere (Nitrogen) in ice- bath. After brief stirring, hydrogen gas was bubbled into it.
Palladium (10% activated on charcoal) was added in a catalytic amount (34 mg, 0.324
mmol). Adequate precautions were exercised to avoid contact of the catalyst with air. The
reaction was stirred for 6 h and allowed to attain room temperature. The reaction mixture was
poured over celite bed and washed with methanol. On drying the filtrate, the white powdery
product was obtained. Further purification by column chromatography on silica gel using
hexane: EtOAc as eluent, gave the desired product as white solid (Rf 0.43 in hexane: EtOAc
1:1).
1H-NMR (400 MHz; CDCl3 (0.3% TMS)) δH :9.30 (1H, brs, -NH), 7.17 (1H, s, H-6), 5.75
(1H, H-1’), 5.54 (1H, d, H-5), 4.93 (1H, m: -CHuridine), 4.74 (m, 1H: -CHuridine), 4.20 (1
H, m: -CHuridine), 3.51(2H, m, 5’), 1.65 (2H, brs, -NH2),1.34-0.83 (56 H, Lipidic chain)
13C- NMR (100 MHz; CDCl3 (0.3% TMS)) δc: 141.33, 139.27, 117.96, 114.07, 102.30,
94.01, 85.89, 85.27, 81.21, 78.87, 69.96, 58.59, 50.57, 37.02, 36.96, 33.83, 32.11, 31.94,
29.17, 14.13
MS (ESI+): m/z calcd for C40H73N3O5 [M] +: 676.02: found 676.8[M] +, 690.8 [M+Na]+
5’-DTPA-5’Deoxy-2’, 3’- O-16-hentriacontanyliden-uridine (4b)
13

Compound 4 (0.512 mmol, 350 mg) was dissolved in 5 ml anhyd DMF and stirred. To this, a
solution of DTPA bis-anhydride (218.9 mg, 0.612 mmol) in anhyd.DMF (10 mL) was added
drop wise. The pH of the reaction mixture was adjusted to 8 using triethylamine and refluxed
at 80° C for 48 h. The progress of the reaction was monitored by TLC in ethyl acetate and
0.2% methanol. The compound was obtained as white solid after precipitating in acetone and
characterised.
1H-NMR (400 MHz; (CD3)2-SO) δH :11.40-10.30 (brs : 4x-COOHDTPA), 7.73 (2H, m: H-6,
-CONH),6.045(1H, d: H-1’) 5.71 (1H, brs, -NHamide), 5.59 (2H: H-5), 4.96 (m, 1H: -
CHuridine), 4.68 (1 H: -CHuridine), 4.57 (1H: -CHuridine), 4.28 (1H: -CHuridine), 3.90 (2H,
br, 5’-H), 3.45 (10H, 5-CH2,DTPA), 2.89-2.99 (m, 8H, 4X-CH2,DTPA), 1.21 (48 H, 2X-
CH2 , 24 CH2 -Lipidic Chain), 0.82-0.81 (6H, 2X-CH3)
13C-NMR (100 MHz; (CD3)2-SO) δc :178.70, 176.30, 174.70, 172.64, 160.28, 154.80,
149.81, 143.63, 142.46, 139.42, 115.79, 79.23, 60.04, 45.77, 39.99, 31.40, 30.81, 29.53,
29.11, 28.90, 28.81, 22.21, 17.70, 14.06
MS (ESI-): m/z calcd for C54H94N6O14 [M]: 1050.3: found 1050.8
2.2 Physicochemical characterization
UV Spectroscopy:
Varying concentrations of ligands (DTPA-NSDAU and DTPA-NLDPU) were prepared in
Millipore water, and the UV spectra (180-700 nm) were recorded on Synergy 2 Multi-mode
reader (M/S BioTek Instruments, USA). For comparison, compounds DTPA-NSDAU and
DTPA-NLDPU were analyzed under similar conditions. In all calculations, the water
background was subtracted from the sample.
Potentiometric titrations:
Direct potentiometric titrations were performed for 0.1 M ligands (DTPA-NSDAU and DTPA-
NLDPU) against 0.01 M Tetrabutylammonium hydroxide (TBAOH) using a Metrohm ion
14

analysis potentiometer fitted with a pH meter-electrode system, calibrated for pH 4, 7 and 9
before performing the experiment.
2.3 Protein binding assessment by Fluorescence quenching:
Fluorescence studies:
In order to study the protein binding by fluorescence quenching, fluorescence characteristics
of the ligands (DTPA-NSDAU and DTPA-NLDPU) were established. Fluorescence emission
spectrum of varying concentrations (0.5 mM- 12.5 mM) of the ligands was recorded from
300-700 nm on Synergy 2 Multi-mode reader (M/S BioTek Instruments, USA) with
excitation at 280 nm.
Fluorescence Quenching:
The experiment was conducted as reported previously with minor modifications (Mingxiong
et al., 2013). 0.1 mM Bovine Serum Albumin (BSA) stock solution, solutions and dilutions of
ligands (DTPA-NSDAU and DTPA-NLDPU) were prepared in 1X Tris buffer solution. In a 96
well black opaque plate, 100 L of BSA was incubated with different concentrations of the
ligands. The final volume in each well was 200 L. The solutions were mixed thoroughly by
gentle pipetting. The mixture was allowed to stand for 5 min. The fluorescence spectrum
from 300-700 nm was recorded using Synergy 2 Multi-mode reader (M/S BioTek
Instruments, USA) with excitation at 280 nm. 100 L BSA with 100 L buffer served as
negative control which gave the maximum fluorescence intensity. The experiments were
conducted at room temperature, and Tris buffer background was subtracted.
Each measurement was carried in triplicates. The data obtained was fitted in the following
graphs for the evaluation of parameters. In order to establish the quenching mechanism of the
ligands on BSA, Stern-Volmer equation was used for analysis.
15

퐹표
퐹
= 1 + 퐾푠푣[푄]
where: Fo= Fluorescence intensity of pure BSA (negative control) : F= Fluorescence
intensity of BSA + analog: Ksv= biomolecular quenching constant: [Q]=molar concentration
of quencher (ligand)
Binding constant and number of binding site
The binding constant and the number of binding sites were evaluated using the equation:
퐿표푔퐹푂 ‒ 퐹 = 푛퐿표푔[푄]
+LogK
퐹
where: Fo= Fluorescence intensity of pure BSA (negative control): F= Fluorescence intensity
of BSA+ analog: K=binding constant: [Q]=molar concentration of quencher (analog): n=
number of binding sites
For the calculation of parameters, the data was plotted as Log ((Fo-F)/F) against
concentration of quencher Log [Q]. K and n were calculated from the intercept and the slope
respectively.
2.4 Toxicity Assessment:
2.4.1 Haemo-compatibility using haemolysis activity:
Haemolytic assessment of the ligands (DTPA-NSDAU and DTPA-NLDPU) was done using red
blood cells (RBC) from a heparinized human blood samples following previously reported
protocols with minor modifications (Dumoga et al., 2017). In order to separate the RBCs
from the plasma, the samples were centrifuged at 3000 rpm for 10 min at 4°C. The RBC
pellet was washed thrice with saline (pH 7.4) to decolorize and suspended in 50 mL of
phosphate buffered saline (pH 7.4) to form an RBC suspension. Aliquots of RBCs (1.8 mL)
were added with 0.2 mL of the 2.5 mM ligands to have a final concentration of 0.25 mM.
After thorough, gentle mixing, the samples were incubated for required time at 37°C. For
16

evaluation of time-dependent haemolytic action of the compound, the samples were evaluated
at different time points (1 h, 2 h, 3 h and 4 h) wherein after the required time interval, the
samples were centrifuged at 3000 rpm at 4°C for 10 min. The end point was the destruction
of the RBSs, qualitatively evident from the extent of red coloration and quantitatively
measurable by measuring the optical density of the supernatant at 540 nm using Synergy 2
Multi-mode reader (M/S BioTek Instruments, USA). Positive and negative control samples
contained Triton X-100 (2% v/v) and saline respectively. In Triton X-100 treated RBCs,
intense red coloration due to free haemoglobin was observed which indicated maximum
haemolysis, also evident by the maximum absorbance at 540 nm. Percent haemolysis was
calculated by the following equation:
퐴푏푠 푠푎푚푝푙푒,휆540푛푚
퐴푏푠(푡푟푖푡표푛휒 ‒ 100,휆540푛푚)
%퐻푎푒푚표푙푦푠푖푠 =
= 100
2.4.2 Cyto-toxicity using cell line:
Cell Culture: Human embryonic kidney (HEK) cell lines were procured from National Centre
for Cell Science (NCCS) Pune, India. The cell line was cultured in high glucose DMEM
supplemented with 10% fetal bovine serum (FBS) and antibiotics: 1% (v/v) penicillin-
streptomycin. The cell line was maintained in humidified 5% CO2 incubator at 37º C. Cells
were routinely sub-cultured twice a week using 0.05% trypsin in 0.02% EDTA.
SRB assay was performed to find the cytotoxicity of the compound DTPA-NLDPU using HEK
cell lines. The procedure was adopted as reported previously with minor modifications
(Khurana et al., 2015). Cells were seeded at the density of 5000 cells per well in 96 well
microtiter plate. Cells were treated with 0.02 mL of varying concentration (0.0001 μM-1000
μM) of DTPA-NLDPU for different time intervals (24, 48 and 72 h). After the incubation for
intended time, the wells were emptied, and the cells were fixed with 100 μL of ice cold 10%
(w/v) trichloroacetic acid (TCA) for 1 h at 4°C. This was followed by four washings of the
17

well with water. Plates were then air dried and 100 μL of 0.05 % SRB solution was added in
the well for 30 min for staining. Plates were quickly rinsed four times with 1% (v/v) acetic
acid to remove excess unbound dye and air dried. Bound protein stain was solubilised using
200 μL of 10 mM Tris base solution (pH 10.5), and the plate was shaken on the gyratory
shaker for 5 to 10 min. In the positive control, the cells were treated with HBSS (Hank’s
Balanced Salt Solution) buffer instead of the compound and treated exactly similar as
described above. The color intensity was measured fluorometrically at excitation 488 nm and
emission at 585 nm on Biotek Synergy H4 hybrid multiplate reader. The surviving fraction
was calculated and plotted against the concentration range (0.001 μM-1000 μM) as a function
of time.
2.5 Radiolabelling and stability parameters:
99mTc-pertechnetate was procured from Board of Radiation and Isotope Technology (BRIT),
Delhi, India. Since 99mTc is a gamma emitting radionuclide (Eγ=140.5 keV), adequate
precautions were taken while it’s handling. Radioactive counts were recorded on the well
counter (Caprac R Capintec, USA) calibrated for 99mTc energy.
2.5.1 Radiolabelling
The ligands (DTPA-NSDAU and DTPA-NLDPU) were radiolabelled with 99mTc using the
standard procedure with slight alterations(Spies et al.,2007)and various radiolabeling
parameters were optimized to attain high labelling efficiency and radiochemical purity. The
parameters include stannous tartrate concentration and incubation time. In brief, following
procedure was followed. Freshly prepared stannous tartrate (0.43 mg/mL in 10 % acetic acid)
was added to 300 µl of (4a and 4b) (2.8 nmol). The pH was adjusted to 7.0 with 1 M
Na2CO3. 99mTc-pertechnetate (380±10 MBq) was added and incubated for 1 h at room
temperature. Ascending instant thin-layer chromatography using silica gel-coated fiber sheets
18

(ITLC-SG) (Gelman Science Inc., Ann Arbor, MI, USA) were used for evaluating labeling
efficiency and radiochemical purity. Radiolabeling efficiency was confirmed by thin layer
chromatography (TLC) wherein free pertechnetate with an Rf of 0.9–1.0, migrates to the top
portion of the strip and the labelled complex (99mTc-DTPA-NSDAU or 99mTc-DTPA-NLDPU
)
and 99mTc-colloids if any remains at the bottom when the mobile phase is 100% acetone.
99mTc-colloids were estimated by running ITLC in pyridine: acetic acid: water combination
(3:5:1.5) wherein the radiolabeled complex and free pertechnetate migrated at the top leaving
colloids at the bottom. The radioactivity in different regions of the TLC strip was determined
by the gamma counter.
2.5.2 Log P value determination:
The lipophilicity parameter (log P) value was determined as the ratio of the concentration of
compound between n-octanol and water (pH 7.4). The logarithmic value of the partition
coefficient is defined as Log P. Briefly the analog was dissolved in 1 mL water and
solubilized. To this an equal volume of n–octanol was added and the mixture was shaken
vigorously on the vortex shaker for 10 min. The mixture was laid aside so that the layers
separate. After proper separation of the layers, the concentration of the compound was
measured in 100 L of the two layers. The experiments were performed in triplicates.
For unlabelled ligands, DTPA-NSDAU and DTPA-NLDPU, the lipophilicity parameter was
evaluated by measuring the absorbance at 260 nm for both layers.
퐴푏푠휆260,푂푐푡푎푛표푙
표푔푃 = 퐿표푔( 퐴푏푠휆260,푊푎푡푒푟
Lipophilicity parameter= L
)
For the radiolabelled complex (99mTc-DTPA-NSDAU and 99mTc-DTPA-NLDPU), approximately
7.2 ± 1.3 MBq of the labelled compound was diluted in PBS, pH 7.4. To this, equal volume
of 1-octanol was added. After vortexing and separation, as above, radioactive counts were
measured in 100 L of the two layers using a well-type gamma counter, and the following
equation was used:
19

퐶표푢푛푡푠(푂푐푡푎푛표푙)
Lipophilicity parameter =
퐿표푔 푃 = 퐿표푔[ 퐶표푢푛푡푠(푊푎푡푒푟)
]
In Vitro Serum Stability:
The radiolabelled compound was assessed for in-vitro stability through serum stability assay.
Freshly collected human serum was incubated with 0.1 mL of radiolabelled compound (0.3
MBq) at 37 °C for varying time points. After desired incubation time (viz., 0.5-24 h), the
samples were spotted on an ITLC-SG. ITLC-SG was analysed as described previously in the
Radiolabelling section.
2.5.3 Cysteine challenge studies:
For challenge studies using cysteine, samples comprised of 0.5 mL of the radiolabelled
complex (0.3 MBq, 0.5 mg) in PBS mixed with 1 mL of cysteine solution of varying
concentration (25, 50, 100 mM in water). After incubation of 2 h at 37°C, aliquots from the
samples were spotted on an ITLC-SG strip. The strip was developed in 0.1 M PBS wherein
the complex was at the bottom whereas radiolabelled cysteine had an Rf of 0.73. The ITLC
was analyzed as described previously in the radiolabeling section.
2.6 In Vivo Evaluation and Pharmacokinetics
Experimental Animals and Ethical statement:
The studies have been carried on female Balb/c mice (aged 2 months, weighing 25 ± 2 g) and
New Zealand albino rabbits (weighing 2-3 kg). The animals were housed in the INMAS
experimental animal house facility in sterile cages and pathogen-free conditions. They were
fed ad libitum with standard food and water, maintained at 22-25°C with a 12 h day and night
cycle. The experiments have been performed as per the guidelines of INMAS Institutional
Animal ethics committee (Reg no.8/GO/a/99/CPCSEA). New Zealand rabbits were used for
blood clearance studies. BALB/c mice were used for imaging and bio-distribution studies.
Intracranial brain mice U87-MG xenograft:
20

Human brain tumor xenografts were developed using human primary glioblastoma cell line
(U87-MG) as reported. (Ozawa and James., 2010). A serum- free single U87-MG cell
suspension (1×105 in 30 µL of saline) was used for intracranial cell implantation in ketamine-
xylazine anaesthetized athymic mice using use a small animal stereotactic frame. The
injection depth was controlled to have an injection at 3 mm from the underside of the skull.
After injection using Hamilton syringe 27 gauge, the skull was cleaned and the scalp was
sutured. Proper post-operative care including pain relief with diclofenac was carried. The
mice were monitored to regain normal activity and growth for 1 week. Visual inspection of
the intracranial xenograft in mice revealed no signs of inflammation, which indicated no
leakage of the cell line in the cranium of the mice. Histopathological studies of formalin-
fixed mice brain slices ascertained the tumor growth.
2.6.1 Blood kinetics studies:
Blood Kinetics studies are carried in normal New Zealand rabbits. The complexes (99mTc-
DTPA-NSDAU: 6 MBq, 1.5 mg in 1 mL and 99mTc- DTPA-NLDPU: 7.5 MBq, 1.5 mg in 1 mL)
were administered intravenously through the dorsal ear vein of the animal. From the other
ear’s vein, blood samples were collected at different time intervals. Activity associated was
counted using well shape gamma counter (Caprac R Capintec, USA). The radioactivity in
blood circulation after decay correction was expressed as a percentage of the total activity in
blood volume, taking 7.2% of the total body weight as the total weight of the blood. The
logarithm of the percent radioactivity vs. the time post-injection was used to evaluate the
distribution and elimination parameters. The rate constants for distribution and elimination (α
and β respectively) were evaluated by the slope of the regression line. The intercepts were
used to evaluate Co. The half-life corresponding to the distribution and elimination phases
were calculated as per the following.
21

1
0.693
푡
2,푑푖푠푡푟푖푏푢푡푖표푛 =
훼
0.693
1
푡 2,푒푙푖푚푖푛푎푡푖표푛 =
훽
2.6.2Bio-evaluation
2.6.2.1 Bio-distribution in Normal Mice:
The biodistribution studies of 99mTc-DTPA-NSDAU and 99mTc- DTPA-NLDPU was performed
in BALB/c mice under similar condition in order to have a comparative evaluation. In order
to have a time-dependent profile, bio-distribution studies were carried at different time points.
Mice were taken in triplicates in 4 different groups depending upon the different time points
(viz., 5 min, 15 min, 60 min and 120 min). For each time point, 20 μL of the radiolabelled
complexes: 99mTc-DTPA-NSDAU (10.3 ± 3.7 MBq) and 99mTc-DTPA-NLDPU (11.1 ± 2.7 MBq)
was carefully injected into the tail vein of mice. At intended time post-injection, mice were
sacrificed through the cardiac puncture, and different organs and tissues were dissected out
using the dissection kit. The organs of interest were removed, made free from adhering
tissues by rinsing in saline, collected in RIA vials and weighed. The radioactivity was
calculated by measuring radioactive counts in the gamma well counter. Uptake of the
radiotracer in each tissue was calculated and expressed as a percentage injected dose per
gram of the tissue (% ID/g) and corrected for 99mTc decay.
2.6.2.2 SPECT Imaging in Xenograft
SPECT imaging of the xenograft was carried in dual head camera after injecting 100 μL of
the radiolabelled complexes: 99mTc-DTPA-NSDAU (35 ± 1.8 MBq) and 99mTc-DTPA-NLDPU
(34 ± 2.2 MBq) into the tail vein of athymic mice. Semiquantitative analyses were assessed
using Amide 1.0.4.
22

3. RESULT AND DISCUSSION
Design of the Nucleolipid
The work aims to explore the potential of nucleolipid as imaging agent capable of penetrating
BBB. Uridine was conjugated with two chains of C15 saturated alkyl chain using 16-
hentriacontanone or palmitone at the 2’-3’ position of uridine using ketal linkage.
Figure 3: General design of ligands showing various units with their respective roles and general
structure of ligands DTPA-NSDAU and DTPA-NLDPU
For an effective flip-flop to take place the alkyl chain should be comparable to the
phospholipids forming unilamellar vesicles, and the membrane phospholipids are in the range
C14-C22, (Spector, et al.,1985) C15 alkyl chain was selected. In the past, palmitate (C15) has
been used to study fatty acid uptake and metabolism in the brain (Robinson et al.,1992), as
well as conjugated with drugs to improve cellular internalization or brain delivery along with
improved pharmacokinetics. Examples include palmitoyl conjugated oxytocin (LOT-
1:Mizuno et al., 2015) with improved efficacy and oligonucleotide GRN 163 (GRN163L:
Herbert et al., 2005) with the ability to internalize into cells without transfection facilitators.
In order to modify the nucleolipid as a SPECT tracer, the nucleolipid had to be functionalized
with chelator so that it can be loaded with 99mTc metal-radionuclide. The acyclic chelator,
DTPA was preferred asit is an excellent chelator for 99mTc and reported extensively for the
development of various 99mTc-based radiopharmaceuticals.8 99mTc-DTPA, pertechnetate is
23

commonly used as a radiotracer for renal functioning, gastro-emptying, and imaging of the
brain with the compromised BBB (Zolle, et al.,2007). Thus, an overall scheme (Scheme 1)
was designed in which nucleoside conjugated with lipid was appended with DTPA to be later
radiolabelled with 99mTc and evaluated as brain targeting tracer. In order to see the effect of
the lipidic chain, the non-lipidic nucleoside was also appended to DTPA and evaluated
similarly as lipidic-nucleoside-DTPA conjugate. The general design of ligands is shown in
Figure 3.
3.1 Synthesis and Characterization:
The synthesis scheme is depicted in Scheme 1. Synthesis of NSDAU (1a) was carried as
reported (Levene and Tipson, 1934). Briefly, acetone was conjugated to uridine as ketal in
the presence of copper sulfate and conc. sulphuric acid. Synthesis of NLDPU (1b) involved
appending the lipid chain of ketone fatty acid, palmitone (16-hentriacontanone), to uridine
with the formation of ketal linkage (Taïb et al., 2012). The appearance of peaks in 1H- NMR
at  1.4-1.2 for the methyl protons and at 114-118 ppm in 13C-NMR for the ketal carbon
confirmed the formation of the products. The 5’ position was modified as an azide using two-
step reaction which involved activation of the hydroxyl using tosyl followed by azidation.
The introduction of azide resulted in the up-field shift (from c  61 to c  52 ppm) of the 5’
carbon in 13C-NMR. For both ligands, the introduction of the amino group at 5’ position was
carried by catalytic reduction of the 5’-azide using 10% palladium on activated charcoal.
DTPA conjugation with NSDAU and NLDPU was carried as reported. (Chauhan et al., 2014).
The yield of formation of DTPA-NSDAU (4a) and DTPA-NLDPU (4b) was 78% and 64%
respectively. The appearance of DTPA characteristic peaks from 30-40 ppm for DTPA-alkyl
and 170-180 ppm for DTPA-COOH confirmed the formation. DTPA anhydride tends to
conjugate with two moieties resulting in bisamide-DTPA derivatives. In-spite of taking an
excess of NLDPU, bisamide-DTPA derivative could not be synthesized possibly because of the
24

steric hindrance. The inability to form bisamide-DTPA derivative, however, acted more
favorably. Attachment of two units of nucleolipid would have resulted in very high molecular
weight ligand. It is well documented that the chelating ability of the monoamide-DTPA is
better than DTPA-bis-derivative. Monoamide-DTPA metal complexes are known to have
better pharmacokinetics and in vitro/in vivo stability (Ardestani et al.,2010; Achilefu et
al.,2000).
Scheme 1: Reaction conditions (i) DTPA-NSDAU: (R=-CH3) acetone, copper sulfate,
conc. sulphuric acid, stirring 48 h; DTPA-NLDPU
:
(R=-(CH2)14-CH3) 16-
hentriacontanone, tosylic acid, triethyl orthofomic acid, THF, reflux, 48 h (ii) tosyl
chloride, pyridine-DCM (1:2), ice-cold, o/n (iii) sodium azide, DMF, reflux, 8 h (iv) Pd
(10% on activated charcoal), H2. (v) DTPA-anhydride, triethylamine, DMF, 80°C, 48 h
(vi) Na[99mTcO4], stannous tartrate.
3.2 Physicochemical characterization:
The physicochemical characterization of DTPA-NSDAU and DTPA-NLDPU was based on UV-
Vis and potentiometric titrations.
In order to study the effect of lipidization, we compared the UV-Vis spectra of DTPA-NSDAU
and DTPA-NLDPU. It is well known that the high and characteristic absorption of modified
25

nucleic acids and nucleosides in ultraviolet light (at λmax 260 nm) is due to the absorption of
their purine and pyrimidine components (Blackburn et al., 2006). The DTPA-NSDAU and
DTPA-NLDPU, both showed an absorption maxima (λmax) at 260 ± 2 nm (Figure S1)†. The
similar molar extinction coefficients of DTPA-NSDAU and DTPA-NLDPU indicated that the
long alkyl chain did not impede the extent of interaction of the light with the chromophore.
Potentiometric titrations of DTPA-NSDAU and DTPA-NLDPU were carried to study the effect
of the lipidic chain. Since we had a ketal linkage, the titration was carried from pH 3-12.
DTPA titration is well reported with six inflection points (1.6, 1.8, 2.5, 4.31, 8.5 and 10.5:
(Bonin et al., 2016)
Two acid dissociations exist for uridine viz., at 9.4 and 12.52 (Fox et al., 1959). The
inflection points of DTPA-NSDAU and DTPA-NLDPU were found to be similar (Table 1).
Table 1: Determination of pKa for DTPA-NSDAU and DTPA-NLDPU
Ligand
pK1
4.61
5.24
pK2
pK3
pK4
DTPA-NSDAU
DTPA-NLDPU
6.531
6.979
9.568
9.248
11.991
10.826
3.3 Non-Specific Protein Binding using Florescence spectroscopy:
One of the problems encountered with highly lipophilic compound is protein binding leading
to non-specific binding and background signal. In blood, the serum albumin (SA) interacts
with the ligand and affects the bioavailability and toxicity of the ligand. Fluorescence
quenching based on the decrease of the intrinsic tryptophan fluorescence of albumin due to
increase in molecular interactions with the ligand has been used to study the protein binding
effects of ligand (Mingxiong et al., 2013).
Fluorescence of the analogues (DTPA-NSDAU and DTPA-NLDPU) was compared with that of
bovine serum albumin (BSA). When excited at 280 nm, BSA exhibits fluorescence in the
26

region 340-350 nm, characteristic for tryptophan of BSA. No fluorescence was observed at
340 nm for the analogues (DTPA-NSDAU and DTPA-NLDPU) when excited at 280 nm (Figure
S2)†.
The ligands were incubated with BSA and extent of fluorescence quenching was measured
(Figure 4). As 90% of signal was retained, no appreciable fluorescence quenching was
observed for DTPA-NSDAU or DTPA-NLDPU up-to a concentration of 1 mM indicating the
suitability of the DTPA-NLDPU for diagnostics at micro-molar concentration.
As no shift in emission wavelength was observed for DTPA-NSDAU or DTPA-NLDPU, it can
be concluded that the analogues did not have any preferential interaction with tryptophan of
BSA and did not affect the conformation of BSA. The pattern of the extent of quenching
differed significantly for the analogues at higher concentration. We attribute this effect to two
factors (a) stearic hindrance and (b) increase in the lipophilic environment. At lower
concentration, the quenching extent of DTPA-NLDPU was more governed by stearic hindrance
than hydrophobic interactions; hence the extent was not very different from that of DTPA-
NSDAU. However, with an increase in concentration and associated lipophilicity, the extent of
quenching is more for DTPA-NLDPU due to the effect of hydrophobic interactions. Overall,
the quenching extent was greater for DTPA-NLDPU as evident by the higher bimolecular
quenching constant. The apparent association constants and the number of binding sites (n)
are listed in Table 2.
Table 2: Fluorescence quenching parameters for DTPA-NSDAU and DTPA-NLDPU
KSV (x103M-1)
0.02
K
N
DTPA-NSDAU
DTPA-NLDPU
6.23
10.08
1.45
0.9
0.09
3.4 Toxicity assessment:
3.4.1 Haemo-compatibility:
27

The diagnostic radiotracer is intended to be administered intravenously. Thus, RBCs happen
to be first to interact with the ligand. The lysis of RBCs can be an indicator of the blood
compatibility of the ligand. Lipophilic drugs or ligands are known to interact with the
proteins present in the membranes of erythrocytes (Hinderling et al., 1997) and the extent of
interaction in the form of haemolysis can indicate the toxicity of the ligand. It was observed
that neither DTPA-NSDAU nor DTPA-NLDPU induce any appreciable haemolysis up to 4 h
with 1.7% haemolysis for DTPA-NSDAU and 2% haemolysis for DTPA-NLDPU. These values
are comparable with the percent hemolysis of the negative control (1.4%). (Figure 5a)
28

Figure 4: Fluorescence quenching of BSA with ligands : Fluorescence spectra of BSA with
experiment with(a) DTPA-NSDAU and (b) DTPA-NLDPU spectra: (c,e and d,f) Stern-Volmer plots
for DTPA-NSDAU and DTPA-NLDPU respectively. The ex 280 nm and the emission spectra were
recorded from 300-700 nm
Figure 5: (a) Haemolysis study for DTPA-NSDAU and DTPA-NLDPU in comparison with saline
(negative control) and Triton X-100 (positive control). Saline treatment of RBC’s shows no
haemolysis and Triton X-100 treated RBC’s have 100% haemolysis. The data shows no
appreciable haemolytic activity of both the compounds DTPA-NSDAU and DTPA-NLDPU up to 4 h.
(b) In vitro cytotoxic evaluation of DTPA-NLDPU using SRB assay. Percent surviving fraction
plotted against concentration for different treatment time. HEK cells were seeded in 96-well cell
plate at a cell density of 5 × 103. After an intended time of incubation of 24, 48 and 72 h, SRB
analysis was performed. The percentage survival was calculated relative to untreated cells and
shown as percent survival ± SD (n = 3).
3.4.2 Cytotoxicity evaluation:
29