Enhanced in vivo tumour imaging by EDTA-bis-GNGR functionalized core shell CdSe:ZnS quantum dot: Synergistic effect of active passive targeting

Article abstract of DOI:10.1039/c5ra22989h

It has been shown in one of our earlier studies that the homodimeric N₂S₂ system enhances the biocompatibility of semiconductor core shell CdSe:ZnS quantum dots by lowering their toxicity and optimizing the steric ligand packing dens

Full text of DOI:10.1039/c5ra22989h

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Enhanced in vivo tumour imaging by EDTA-Bis-GNGR functionalized core shell CdSe:
ZnS quantum dot: synergistic effect of active passive targeting
Rashi Mathura*, Narmada Baga, Raunak Varshneya, Firasat Hussainb, Ankur Kaula, Neelam
Kumaria, Ramprakash Chauhanc, Shivani Singha, Sweta Singha, Anil. K. Mishraa*
aDivision of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and
Allied Science, Brig. S.K. Mazumdar Raod, Lucknow Road, Timarpur, Delhi – 110054
bDepartment of Chemistry, Delhi University, New Delhi-110054, India
cDepartment of Chemistry, SVGC Ghumarwin, Bilaspur, Himachal Pradesh-174021, India
*Corresponding Author: email: rashimathur07@gmail.com, akmishra63@gmail.com

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Abstract
It has been shown in one of our earlier studies that the homodimeric N2S2 system enhances the
biocompatibility of semiconductor core shell CdSe: ZnS quantum dots by lowering their toxicity
and optimizing the steric ligand packing density. In this study we functionalize the core shell
quantum dots with the same homodimeric ligand and add two moieties of GNGR (GlyꢀAsnꢀGlyꢀ
Asp) peptide which contain the NGR motif known to target the CD13 receptors in tumour
vasculature. The aim is to study the influence of active receptor based targeting by peptide and
passive targeting by QD nanoconjugate on tumour imaging. The core shell CdSe:ZnS quantum
dot were synthesised and conjugated with EDTAꢀBisꢀGNGR ligand. The docking studies show
high binding affinity of the synthesised quantum dot nanoconjugate to the CD13 receptor. The
GNGR peptide was synthesised on solid phase using Fmoc chemistry, followed by its
conjugation to EDTAꢀBisꢀCysteamine. The complete physicochemical characterisation of the
1
ligand was done using HNMR, 13CNMR and Mass. The comparative in vivo kinetics,
biodistribution and tumour targeting by native GNGR, EDTAꢀBisꢀGNGR and EDTAꢀBisꢀ
GNGRꢀQD was studied in murines after radiolabelling with99mTc. The changes observed in vivo
on comparing the three are very interesting. A seven fold increase in tumour uptake is seen after
nanoconjugation highlighting the synergistic effect of active passive targeting resulting in
enhanced tumour imaging. This study thus opens up a new area where the nanoplatforms can be
designed to get the best of both targeting and potential theranostic applications.
Keywords: ActiveꢀPassive Targeting, Radiolabelling, Scintigraphy

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1. Introduction
The use of quantum dots as optical probes in diagnostic, immunoassays and cell labelling has
been extensively explored1ꢀ3 but their translation into products of clinical relevance has been
limited due to the inherent toxicity of the core shell which contains metals like cadmium4ꢀ6. The
passivation of the core with a higher band gap inorganic material helps control the leaching out
of the core 7ꢀ8 however, the synthetic protocols use organic solvents which result in the formation
of hydrophobic dots which cannot be used directly for biological applications. Hence the focus
moved to the functionalization of these cadmium based core shell quantum dots with
biocompatible ligands like antibodies, small molecules and peptides 9ꢀ11. These ligands helped in
making the QD hydrophilic but resulted in reducing their core emissions due to steric hindrance.
Therefore, the need arose to design biocompatible ligands which can not only optimize the steric
packing density for effective targeting but also maintain the native core shell emissions.
Last decade saw an increased focus on development of molecules which can interact with the
overexpressed receptors in neoꢀangiogenesis which is very important for tumour growth and
proliferation. When panning through the peptide phage library it was found that the peptide with
NGR motif showed specific targeting towards the CD13 receptors found in neoangiogenic
tumour vasculature 12ꢀ13 and also target the αvβ3 integrins 14ꢀ16. Thus this tripeptide emerged as an
important sequence for use in nonꢀinvasive imaging. It has been reported earlier in literature that
the modification of these peptides in terms of their multimerisation, PEGylation and cyclization
can improve their tumour targeting efficiency by enhancing the target to nonꢀtarget ratio17ꢀ22
.
Earlier studies show that the cyclic form of NGR show better binding to the CD 13 receptors as
compared to the linear form. It was also observed that the targeting ability of the peptide to

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desired site is greatly influenced by the two flanking moieties. This lead to several modifications
in the structure of NGR23ꢀ28
.
M. Oosterndrop et.al in 2008 reported the synthesis of cNGR labelled paramagnetic CdSe/ZnS
quantum dots which was the first documentary proof for the in vivo application of cNGR as a
target ligand for nonꢀinvasive imaging29. In this study the streptavidin modified quantum dots
were simultaneously conjugated with biotinꢀtagged cNGR and biotinꢀtagged Gd(III)ꢀDTPA.
Both MRI and two photon laser scanning microscopy (TPLSM) showed that the cNGRꢀpQD to
be highly specific towards surface active angiogenic endothelial cells as compared to the normal
cells and tissues both in vitro and in vivo. The dual modified contrast agent was highly selective
and exhibited threefold higher MRI contrast in tumour than the nonꢀconjugated QDs. Thus the
ability of this peptide to target angiogenesis/ neovasculature has prompted us to modify the
flanking moieties of the NGR motif to design a molecule which can be used for specific imaging
utilizing the platform of quantum dots for enhanced uptake (nanotechnology based tumour
vasculature targeting including active and passive targeting)16.
Here we have synthesised a linear tetrapeptide GNGR (GlyꢀAsnꢀGlyꢀAsp) and conjugated it to a
homodimeric N2S2 ligand which is an EDTAꢀBisꢀcysteamine. In one of our earlier study30 we
have shown that this bis ligand very effectively controls the in vivo toxicity caused due to the
leaching out of the Cd+2 ions by providing a chelating effect. In this study we have tried to
evaluate that if a specific targeting moiety is attached to this ligand then will there be an in vivo
synergistic effect of active passive targeting or will only one of the mechanism predominate.
Each of the bis ligand has two targeting moieties i.e., GNGR peptide attached to it and many
such conjugates are attached to the quantum dot surface. This type of system should be able to
optimize the steric ligand packing while maintaining the emission of the core. The grafting of

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this ligand on the surface of the core shell CdSe:ZnS quantum dots thus makes it a very
interesting system to study as it has the potential to be also used as a theragnostic nanoplatform.
2. Experimental
2.1 Materials
Ethylenediamine tetraacetic (EDTA) anhydride, diisopropyl carbodimide (DIC), dimethoxytrityl
chloride (DMTꢀCl), triethylamine (Et3N), dimethyl aminopyridine (DMAP), pyridine,
cysteamine hydrochloride, trifluoroacetic acid (TFA), dichloromethane (DCM), acetonitrile
(ACN), tetrahydro furan (THF), rink amide resin, FmocꢀGlyꢀOH, FmocꢀArg(Pbf)ꢀOH, Fmocꢀ
Asn(Trt)ꢀOH, dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA), acetonitrile
(HPLC grade), dry diethyl ether, stannous chloride dehydrated (SnCl2 .2H2O) were used from
SigmaꢀAldrich. HOBt was obtained from Hi Media, INDIA. All the other reagents used were of
analytical grade and as received. 99mTc was procured from Regional Centre for
Radiopharmaceuticals (Northern Region), Board of Radiation and Isotope Technology (BRIT),
Department of atomic energy, India were used. Abbreviations Fmoc: 9ꢀ
fluorenylmethoxycarbonyl Boc: tertꢀButyl carbamates Trt: triphenylmethyl, pbf:
pentamethyldihydrobenzofuran 5ꢀsulfonyl Arg: Arginine, Gly: Glycine, Asp: Aspargine
2.2 Methods
The synthesized materials are characterized by NMR measurements were carried out on a Bruker
400 MHz system. Mass spectroscopic analysis was performed using an Agilent 1100 System
coupled with LC operation in ESI (Electro Spray Ionization) positive/ negative mode. UVꢀVis
scanning was done on a UVꢀVis spectrophotometer from Molecular devices SpectraMaxꢀMꢀIIꢀE.
Gamma ray spectrometer (type GRS23C) (Electronics Cooperation of India Pvt. Ltd.,) was used

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for determining the amount of activity (γꢀemitter) in the samples. Gamma scintillation camera
(HAWKEYE) was used for imaging of animals. Analytical RPꢀHPLC was performed on Jasco
HPLC system. A Phenomenex Jupiter 5ꢁ ꢀ Cꢀ18 300 Ȧ (250 mm x 4.60mm, 5ꢁ) was used. The
solvent system consisted of 0.1% TFA in H2O (solvent A) and 0.1% TFA in CH3CN: H2O
(90:10) (solvent B). Elution was achieved by applying a linear gradient from 100% A to 70% B
in 35 min, at a flow rate of 1 ml/min. Peptide peaks were detected spectrophotometrically (223
nm).
2.3 Computational Methodology
The computational protocol involved the flexible docking assisted analysis of dynamic
interactions of peptidic ligands with Aminopeptidase N (APN) also known as CD13. All the
computational studies were performed with the molecular modelling software, Schrödinger,
LLC, New York, NY, 2014 maestro 9.731.
The atomic coordinates of crystal structure of CD13, 4FYS has been retrieved from RCSB PDB
and employed for docking analysis having resolution of 2.01Å32. The Human CD13, 4FYS, is
dimeric zinc containing metallopeptidase and coꢀcrystalized with peptidic substrate, angiotensin
IV. The crystal structure was preꢀprocessed by deleting the heteroꢀatoms, addition of hydrogen
atoms and assignment of bond orders and correct atom types. Further, monomeric moiety was
retained for optimization and refinement to obtained processed 3D structure of CD13 to employ
in docking analysis. For docking analysis, the coꢀcrystalized peptidic substrate, angiotensin IV
and the linear and cyclic NGR peptide has been used as references with respect to our designed
molecule EDTAꢀBisꢀGNGR. On symmetry basis we have considered only a monomeric unit of
EDTAꢀBisꢀGNGR for computational studies. All these four molecules were minimized and
optimized to obtain the lowest possible energy conformers in the force field OPLS2005 without

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any constraints by using the Ligprep v2.9 module. These conformers were generated in
protonation states at pH=7.0 ± 2.0 with retained specified chirality’s.
The optimized CD13 receptor, 4FYS and five low energy conformers of ligands were subjected
to Flexible docking analysis by Induced Fit docking (IFD) analysis. IFD analysis was included to
allow the flexibility to the ligands and receptor to visualize the binding pose and interactions
dynamically at the active site of CD13, 4FYS. IFD studies involves the highꢀprecision Glide XP
scoring to predict the binding mode of the ligands. Docking postꢀprocessing was done including
analysis of key interactions and binding affinity analysis.
2.4 Solid phase synthesis of (Gly-Asn-Gly-Arg) GNGR tetrapeptide
The Fmoc protected rink amide resin (200 mg) was swelled in 4 mL DCM for 60 min and
drained. The Fmoc was then deꢀprotected using 20% (v/v) solution of piperidine in DMF for 20
min. The first amino acid was coupled to the resin by using 4 (eq) of FmocꢀArg (pbf)ꢀOH
relative to the resin capacity and 4.0 equivalents (eq) of HOBT and DIC each relative to amino
acid and 2 mL of DMF, the mixture was shaken for 2 h. Then the mixture was washed with DMF
(3×25mL), MeOH (3×25mL), and DCM (3×25mL) and dried in vacuum. The Fmoc group was
deꢀprotected and the corresponding Nꢀdeꢀprotected peptide ꢀNH2ꢀ Arg (pbf)ꢀOHꢀCONHꢀRink
amide, a mixture of 4 (eq.) of FmocꢀGlyꢀOH relative to the resin and 4.0 equivalents of HOBT
and DIC relatives to amino acid and 2 mL of DMF was added. The resin solution was left for
shaking for 2 h at room temperature. Further the mixture was dried and washed with DMF
(3×25mL), MeOH (3×25mL), and DCM (3×25mL) respectively. Then in the next step Fmoc ꢀ
Asn (Trt)ꢀOH was coupled with the deꢀprotected ꢀNH2ꢀGlyꢀNHCOꢀArg (pbf)ꢀCONHꢀRink
amide to form Fmoc ꢀAsn (Trt)ꢀCONHꢀGlyꢀNHCOꢀArg (pbf)ꢀCONHꢀRink amide. Further
washing of the resin and respective deꢀprotection was carried out as described above. Finally,

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FmocꢀGlyꢀOH was coupled with deꢀprotected –NH2ꢀAsn (Trt)ꢀCONHꢀGlyꢀNHCOꢀArg (pbf)ꢀ
CONHꢀRink amide in the similar way to get Fmoc–GlyꢀCONHꢀAsn (Trt)ꢀCONHꢀGlyꢀNHCOꢀ
Arg (pbf)ꢀCONHꢀRink amide. The resin bounded peptide was finally deꢀprotected with TFA
cocktail (TFA/TIS/H2O/EDA (9.5/0.2/0.2/0.1 v/v). Throughout the reaction the progress of
amino acid coupling and deꢀprotection of Fmoc group was monitored by the colour change of
ninhydrin in the Kaiser test33 and purified using HPLC.
2.5 Synthesis of Cysteamine DMT
This was followed by the synthesis of dimethoxytrityl protected cysteamine. 1.2 (eq) of DMT
chloride (10mmol) was added to cysteamine hydrochloride (8.84mmol) in presence of catalyst
DMAP (0.00084mmol) and Et3N in neat pyridine as solvent in a 100 ml round bottom flask. The
reaction mixture was stirred at room temperature for 72 h under inert atmosphere. The
completion of the reaction was confirmed by TLC (DCM: MeOH 9:1). The solvent was
evaporated to dryness. Then the solution was diluted with DCM and extracted with chilled water
repeatedly to remove any trace amount of pyridine. The resultant reaction mixture in DCM was
then dried with sodium sulphate, filtered and evaporated to dryness. The obtained compound was
sticky and dark brown in colour with a yield of 80%. 1H NMR (CDCl3, 400 MHz): δ (ppm), 2.35
(t, 2H, ꢀCH2ꢀS), 2.63 (t, 2H, ꢀCH2ꢀNH2), 3.79 (s, 6H, CH3ꢀO), 6.79ꢀ7.44 (m, 13H, phe nyl ring),
13C NMR (CDCl3, 100 MHz): δ (ppm), 36.44 (ꢀCꢀS), 41.14 (CꢀN), 55.24 (CꢀO), 65.62 (Cꢀ3Ph),
113.11, 126.53, 128.39, 129.42, 130.70, 137.33, 145.56, 158.06 (carbons of phenyl ring).
2.6 Synthesis of EDTA-Bis-cystemaine-DMT
The second step shows the conjugation of DMT protected cysteamine with EDTA. This was
carried out by adding cysteamineꢀDMT (1.319mmol) to EDTA anhydride (2.638mmol) in

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presence of Et3N (384.39µl) in ACN as solvent under reflux condition at 700oC under inert
atmosphere for 48 h. The completion of the reaction was checked by TLC in DCM: MeOH (9:1).
After completion of the reaction the solvent was evaporated to dryness and the desired
compound was extracted with DCM: H2O. The organic layer was dried with sodium sulfate and
then evaporated to complete dryness. The obtained brown coloured compound was then further
precipitated with diethyl ether. The precipitate found was then repeatedly washed with diethyl
1
ether and dried to give a light brown coloured powder with a yield of 75%. H NMR (CDCl3,
400 MHz): δ (ppm), 2.37(m, 4H, ꢀCH2ꢀS), 3.02(m, 8H, ꢀCH2ꢀNH, NꢀCH2ꢀCH2ꢀN), 3.79(m, 20H,
13
ꢀCH2ꢀCOOH, ꢀCH2ꢀCONH, CH3ꢀO), 6.64ꢀ7.35(m, CH2 of phenyl ring). C NMR (CDCl3, 100
MHz): δ (ppm), 31.69 (ꢀCꢀS), 38.60(CꢀCONH), 45.59 (NꢀCꢀCꢀN), 56.49 (CꢀO), 65.60, 65.68,
65.97 (Cꢀ3Ph, ꢀNꢀCꢀCOOH, ꢀNꢀCꢀCONH), 113.14, 126.62, 127.94, 130.67, 137.14, 145.37,
158.00 (C of phenyl ring) 168.79 (COꢀNH), 170.00 (COꢀOH). m/z (ESI) calculated for
C56H62N4O10S2 was 1014.39 (M)+ and found 1013. 6 (MꢀH+).
2.7 Solid phase synthesis of EDTA-Bis-GNGR ligand
The prepared Fmoc–GlyꢀCONHꢀAsn (Trt)ꢀCONHꢀGlyꢀNHCOꢀArg (pbf)ꢀ CONHꢀRink amide
was deꢀprotected in 20% (v/v) solution of piperidine in DMF for 20 min. Then in the reaction
mixture 4 equivalents of the synthesized DMT protected EDTAꢀBisꢀCysteamine relative to the
resin capacity and 4 equivalents of HOBT and DIC relative to the ligand was added along with 2
ml of DMF and left for stirring for 2 h. The coupling was checked for completion with the help
of Kaiser test. The reaction mixture was then dried and washed with DMF (3×25mL), MeOH
(3×25mL), and DCM (3×25mL) respectively. Deꢀprotection and cleavage from the resin were
carried out using a trifluoroacetic acid mixture (TFA/TIS/H2O/ EDA (9.5/0.2/0.2/0.1 v/v/v/v/v/))
for 2 h at room temperature. After deꢀprotection, the solutions were concentrated and the

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peptides were precipitated by cold diethyl ether. The white precipitate of the peptides was
washed with cold diethyl ether and characterized by mass spectroscopy (ESI) m/z calculated for
C42H74N20O16S2 is 1178.50 and found to be 1175.9 corresponding to [Mꢀ2H+].
2.8 Synthesis of EDTA-Bis-GNGR-QD
After the synthesis by phase transfer extraction the EDTAꢀBisꢀGNGR ligand was appended over
the surface of red light emitting quantum dots. The reaction was taken in a solvent mixture of
THF: H2O (1:1). The quantum dots were precipitated from the preꢀexisting solvent CHCl3, by
the addition of methanol, dried and added to the reaction mixture containing the EDTAꢀBisꢀ
GNGR ligand. The reaction was left for stirring overnight at room temperature under inert
atmosphere. The surface passivation and completion of the reaction was confirmed by the change
in colour of the reaction mixture. The reaction mixture was evaporated to dryness and repeatedly
washed with chloroform to remove the uncoated quantum dots present. The final compound was
dissolved in small quantity of water and lyophilized to obtain the solid compound.
2.9 Radiolabelling
The bioꢀdistribution and pharmacokinetics of GNGR peptide, EDTAꢀBisꢀGNGR ligand, and
EDTAꢀBisꢀGNGRꢀQD nanoꢀconjugate was studied after radiolabelling with 99mTc using SPECT
imaging. An aqueous solution of GNGR peptide (1mg/ml), EDTAꢀBisꢀGNGR ligand (1mg/ml),
and EDTAꢀBisꢀGNGRꢀQD nanoꢀconjugate (1mg/ml) was taken in a shielded vial and stannous
chloride was added. Then the pH of the resulting solutions was adjusted to 6.5ꢀ7.0 with addition
of 0.5M NaHCO3. Further all the mixture was passed through 0.22 µm filters into three different
ꢀ
sterile glass vials. Freshly eluted 99mTcO4 (93.7MBq) was added to each vial and the resulting
complex was incubated for 15 min at room temperature for optimum radiolabelling yield. For all

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the complexes the radiolabelling efficiency was estimated chromatographically using ITLCꢀSG
as the stationary phase and 100% acetone as the mobile phase. The absence of free and
ꢀ
unbounded Na99mTcO4 was evident from the paper chromatography.
2.10 In vitro serum stability studies
The metabolic stability of the GNGR peptide, EDTAꢀBisꢀGNGR ligand, and EDTAꢀBisꢀGNGRꢀ
QD nanoꢀconjugate was ascertained in vitro in freshly collected serum from healthy animals. The
collected serum was prepared by allowing blood collected to clot for 1hr at 37°C in a humidified
incubator maintained at 5% carbon dioxide and 95% air. The samples were then centrifuged and
the serum was filtered through a 0.22 ꢁm filter into sterile plastic culture tubes. 100 ꢁL of 99mTc
labelled formulations were incubated respectively in 900 ꢁL of this serum (in duplicate) at 37°C
and analysed to check for any dissociation of the complex by ITLC using silica gel strips and
0.9% NaCl aqueous solution (saline) as developing solvent. The change in labelling efficiency
was monitored over a period of 24 hr.
Animal Handling and Preparation for in vivo Studies: All animal experiments and study
protocols were approved by the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Government of India, New Delhi. Animal handling and
experimentation were carried out as per the guidelines of the Institutional Animal Ethics
Committee. Male spargueꢀdawly (SD) rats of age 6ꢀ8 weeks and Balb/C mice are given food and
water ad libtium and housed in a 12h/12 h light and dark cycle.
2.11 Blood Kinetics and Bio-Distribution

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Blood clearance of 99mTcꢀlabeled nanoꢀconjugates was studied in healthy SD rats (n = 3) 100ꢁCi
of the radiolabelled complex in saline was administered intravenously through the tail vein of the
rats. Blood samples were collected using the retro orbital procedure. The Blood was collected
from this area in anesthetized rats using a microꢀhematocrit tube at different time intervals
ranging from 5 min to 24 h. Persistence of activity in the circulation was calculated as percentage
injected dose per whole blood, assuming a total blood volume as 7% of the body weight. The
radioactivity of the precipitate and supernatant was measured using a wellꢀtype gamma
spectrometer.
Tumour bearing Balb/c mice with EAT grafted tumour on the left forelimb were used. The mice
were administered 3.7 MBq of 99mTc labelled GNGR peptide, EDTAꢀBisꢀGNGR ligand, and
EDTAꢀBisꢀGNGRꢀQD nanoꢀconjugate, respectively, through the tail vein (i.v.). At 1, 2, 4 and
24 h post injection, the animals (n = 3) for each compound were euthanized and blood was
collected by cardiac puncture into preꢀweighed tubes. The mice were then dissected and different
organs (heart, lungs, liver, spleen, kidneys, stomach, intestine, muscle (normal and tumour) were
removed, weighed and their radioactive counts was taken with the help of a gamma counter.
Uptake of the radio labelled compound into each organ was measured per gram of the
tissue/organ and expressed as percentage injected dose per gram organ weight. The radioactivity
remaining in the tail (point of injection) was also measured and taken into account while
calculating the radioactivity.
2.12 Scintigraphic studies
Scintigraphic studies were performed on a gamma camera. 100ꢁL of the radiolabeled GNGR
peptide, EDTAꢀBisꢀGNGR ligand, and EDTAꢀBisꢀGNGRꢀQD nanobioconjugate, respectively,

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injected through tail vein of Balb/c mice. The tumour was inoculated in the left forelimb of the
mice using the EAT cell line.
3. Results and Discussion
3.1 Flexible Docking Analysis for EDTA-Bis-GNGR with Aminopeptidase-N (CD13)
The human CD13 structure, 4FYS in presence of a peptidic substrate, angiotensin IV shows its
binding with key interactions at substrate binding and catalytic site. Induced fit docking (IFD)
studies of peptidic substrate, angiotensin IV, linear & cyclic NGR (cꢀNGR) peptide and the
designed molecule EDTAꢀBisꢀGNGR was performed with 4FYS. The binding pose and
geometry of the ligands at the site were screened on the basis of Glide XP score and further
docking postꢀprocessing properties were obtained. The Glide XP score for angiotensin IV, linear
NGR, cNGR peptide and designed molecule EDTAꢀBisꢀGNGR were found to be ꢀ11.389, ꢀ
9.772, ꢀ11.527 and ꢀ9.953 respectively. The flexible docking resulted in comparable Gꢀscore of
EDTAꢀBisꢀGNGR to other reference molecules. Ala353 has been considered as the key
interacting residue as it is able to interact with all the reference molecules via hydrogen bonding.
Existence of similar interaction in designed molecule EDTAꢀBisꢀGNGR justify its ability to
identify the correct substrate binding site. Secondly the range of hydrogen bond distance for
EDTAꢀBisꢀGNGR with Ala353 is of 2.16 Ao which is quite comparable with the bond distance
of 2.67Ao for angiotensin, 2.31Ao for cNGR and 1.70Ao for linear NGR. This range of hydrogen
bonding validates the good receptor binding affinity of EDTAꢀBisꢀGNGR.
The common interacting residues in case of coꢀcrystalized ligand angiotensin and proposed
ligand EDTAꢀBisꢀGNGR are found to be Ala 353, Glu 380, Arg381, Thr384, His388, Glh418,
Tyr419 and Arg 442 but apart from this EDTAꢀBisꢀGNGR is also capable to interact with some
additional residues like Gly352, Asn438 and Glu 696. These large number of contributions

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should be able to result in good binding pose (Fig. 1a and 1b). Hence, overall the flexible
docking results provide us an insight into the ability of interaction and recognition of EDTAꢀBisꢀ
GNGR at substrate binding site with good receptor affinity towards CD13. Molecular docking
therefore predicts that the conjugation of GNGR with EDTAꢀ Bisꢀcysteamine shows a good
potential for targeting upregulated CD13 in angiogenic blood vessels.
3.2 Synthesis of EDTA-Bis-GNGR ligand
It is difficult to introduce peptide directly to the EDTAꢀBisꢀcysteamine ligand due to the
interference of –SH group or EDTAꢀBisꢀcysteamineꢀQD nanoꢀconjugate due to the complexity
of purification and characterization. Therefore, a different synthetic strategy was designed and
executed. As per the scheme shown in (Fig. 2) first the solid phase synthesis of GNGR peptide
was done on rink amide resin using Fmoc chemistry. The peptide is purified by HPLC and is
characterized using HRMS (ESIꢀTOF). GNGR retention time was Rt = 3.9 min. The molecular
mass of the pure GNGR were determined by TOF HRMS m/z = found: 402.5496 [M],
Calculated: 402.2 (S1)
The simultaneous synthesis of Cysteamine – dimethoxytrityl chloride (1, Fig.3) was confirmed
1
by the presence of characteristic multiplets in the H NMR in the range of 6.5 to 7.4 ppm
corresponding to the protons of phenyl ring and the singlet at 3.9 ppm due to the protons of
methoxy (OꢀCH3) group (S2). The ESI mass spectrum also shows two peaks at 402.0 and 303.2
corresponding to the [M+Na+] and fragmented dimethoxytrityl cation respectively (S3), the
carbonyl carbon (ꢀC=O). peaks at 168.79 and 170.00 ppm in 13C NMR spectrum (2, Fig.3)
confirms the synthesis of the newly formed amide and acid groups respectively as shown in (S4).
The final step of conjugation of peptide with the final structure of EDTAꢀBisꢀGNGR ligand was
done using solid phase synthesis (3, Fig. 3) and was confirmed by ESI mass spectrum with

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molecular ion peak at 1175 due to [Mꢀ2H+] as shown in (S5). The synthesis of EDTAꢀBisꢀ
GNGRꢀQD (4 Fig. 3) nanobioconjugate was carried out by phase transfer extraction of the
hydrophobic quantum dots. The size of the quantum dot nanobioconjugate was evaluated by
transmission electron microscopy and it has been found to be less than 20 nm as shown in (Fig.
4).
3.3 In vitro serum stability studies
All the synthesized compounds GNGR peptide, EDTAꢀBisꢀGNGR ligand, and EDTAꢀBisꢀ
GNGRꢀQD nanoꢀconjugate, were labelled with 99mTc for studying the radiolabelling efficiency.
All the labelling parameters such as pH, concentration of reducing agents (SnCl2), temperature
etc. were standardized to achieve the maximum labelling efficiency. The proteolytic degradation
of the radiolabeled nanobioconjugate was determined in mice serum in vitro. Instant thin layer
chromatography of the prepared serum showed that the 99mTcꢀGNGR, 99mTcꢀEDTAꢀBisꢀGNGR,
and 99mTcꢀEDTAꢀBisꢀGNGRꢀQD nanoꢀconjugate, remained sufficiently stable during incubation
at 37°C with the serum. A maximum of 5ꢀ6% of radioactivity degraded after 24 h of incubation
advocating a high in vitro stability of almost 94ꢀ95% of all the radiolabelled complexes for up to
24 h.
3.4 Blood kinetics studies
The blood kinetics data clearly differentiates the pharmacokinetics of the peptide, quantum dot
conjugated and nonꢀconjugated ligands as shown in (Fig. 5). Where the native 99mTc GNGR
shows the presence of only 4% activity in blood circulation in the first 15 min post injection,
nearly 16% and 20% activity was found for the 99mTcꢀ EDTAꢀBisꢀGNGR ligand and 99mTcꢀ
EDTAꢀ BisꢀGNGRꢀQD conjugate. The circulation time was longer in case of quantum dot

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conjugated system which may be due to the reduced opsonisation which is dependent on the
surface chemistry and can lead to the decrease in nonꢀspecific uptake in the RES system. As per
the result the native GNGR peptide has a very high clearance from the blood to tissues and also
shows a fast clearance from the body. We had seen a similar behaviour in our previous studies
where the circulation of the conjugate increases after attaching the quantum dot to the EDTAꢀ
Bisꢀcysteamine.30 Here also in contrast to the native GNGR peptide and EDTAꢀBisꢀGNGR,
EDTAꢀ BisꢀGNGRꢀQD nanobioconjugate show higher in vivo retention which is required for a
good diagnostic imaging agent
3.5 Biodistribution and Scintigraphic Studies
The quantification of the tumour uptake of all the synthesized 99mTc radiolabelled compound was
carried out by in vivo biodistribution and scintigraphy studies. In our previous work we had seen
that EDTAꢀBisꢀCysteamine shows a charge dependent clearance from the body however, as soon
as it is conjugated to the Quantum dots the pharmacokinetics changed thereby indicating the role
of size in targeting.30 Here we have seen that the radiolabelled peptide (99mTcꢀGNGR) shows
maximum accumulation in liver 1, 4 and 24 h of post injection (29.46%ID/gm, 25.96%ID/gm,
7.47%ID/gm), followed by spleen (19.42%ID/gm, 14.085ID/gm, 2.14%ID/gm) respectively as
shown in (Fig. 6a). As the activity was not present in kidney after several hours of post
injection, the route of excretion was expected to be hepatobiliary. At the concentration of
administration, the uptake of the radiolabelled peptide was not high in the tumour approximately
0.98 %ID/gm and hence the tumourꢀtoꢀnonꢀtumour ratio i.e., 2.71% ID/gm was also not
significant. This can be attributed to the linear structure of the peptide and also due to the fact
that the native peptide shows a very fast clearance from the body only 0.52% ID/gm present 4 h
post injection. The higher uptake in liver and spleen also contributes in decreasing the tumourꢀtoꢀ

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nonꢀtumour contrast. This is also evident from the scintigraphic image of 99mTc radiolabelled
GNGR peptide 4 h post injection shown in (Fig. 7a). However, 99mTc radiolabelled EDTAꢀBisꢀ
GNGR ligand shows a different distribution pattern. Here the conjugate shows a comparatively
lower uptake in liver 1, 4 and 24 h of post injection i.e., 14.25 %ID/gm, 15.91 %ID/gm, 8.69
%ID/gm and spleen i.e., 6.85 %ID/gm, 10.24 %ID/gm, 4.15 %ID/gm as shown in (Fig. 6b).
However, an increased uptake was observed in the kidney, stomach and intestine indicating a
partial renal clearance along with the hepatobiliary route. There is an expected increase in the
tumour uptake just after 4 h post injection i.e. 4.02 %ID/gm which increases gradually over 24 h.
This may be because the 99mTcꢀEDTAꢀBisꢀGNGR ligand has a comparatively increased
circulation time in blood stream as evident from the radioactivity present in blood after 4 h of
post injection i.e. 3.08 %ID/gm. This might be giving ample time for the radiolabelled conjugate
to reach the site of interest and of course the bivalent effect of BisꢀGNGR also plays an
important role in this higher uptake. Therefore, we can infer that here the increased accumulation
at tumour site can be attributed basically to the active targeting of the BisꢀGNGR ligand.
Interestingly the tumourꢀtoꢀmuscle ratio increases from 1.81 %ID/gm to 4.00 %ID/gm from 4h
to 24 h post injection. The increased uptake in tumour is clearly visible in the scintigraphic
image shown in (Fig. 7b).
However, when the 99mTcꢀEDTAꢀBisꢀGNGR is conjugated to CdSe/ZnS coreshell quantum dots,
a very interesting and different biodistribution pattern is observed. There is a dramatic decrease
in the uptake at 1, 4, and 24h post injection in the liver (i.e., 3.82 %ID/gm, 5.47 %ID/gm, and
0.50 % ID/gm) and spleen (i.e., 2.23%ID/gm, 7.11%ID/gm, and 1.12% ID/gm) respectively as
shown in (Fig. 6c). This may be due to the fact that the kind of surface modification that has
been done in this study might reduce the chance of opsonisation, a critical factor responsible for

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the high nonꢀspecific accumulation of nanoparticles in MPS organs. This decrease significantly
impacts the tumourꢀtoꢀnonꢀtumour contrast. Hence 4 h post injection the nanobioconjugates
shows the highest uptake of 19.51%ID/gm activity in tumour (Fig.7c). The respective tumour to
muscle ratio is also significantly high i.e. 7.85 %ID/gm 24h post injection. This can be explained
by the increased circulation time of the quantum dot nanobioconjugate in blood stream than the
other radiolabelled compounds. Activity in blood 1 hr post injection of of 99mTcꢀEDTAꢀBisꢀ
GNGRꢀQD is 4.91 %ID/gm, 1.49 %ID/gm for 99mTcꢀEDTAꢀBisꢀGNGR ligand, and
0.49%ID/gm for 99mTcꢀ GNGR nanobioconjugate respectively.
This increased uptake can be explained due to the fact that in addition to the bivalent effect of
BisꢀGNGR, there is large loading of EDTAꢀ BisꢀGNGR ligand over the CdSe/ZnS quantum dot
surface which increases their specificity towards tumour site due to active targeting. As
discussed in the introduction the multimerisation of the peptide is affective in improving the
tumour homing affinity significantly. At the same time presence of quantum dot results in their
passive targeting due the leaky vasculature of tumour microenvironment and the poor lymphatic
drainage system through the EPR effect (enhanced permeation and retention). As a result, greater
than 7ꢀfold increase in tumour uptake of 99mTcꢀEDTAꢀ BisꢀGNGRꢀQD nanobioconjugate can be
seen as compared to the native GNGR. This observation clearly highlights the impact of
synergistic effect of active and passive targeting in tumour imaging.
4. Conclusions
To summarise we have seen that the molecular docking predicts that the conjugation of GNGR
with EDTAꢀBisꢀCysteamine has shown the potential for targeting tumours with upregulated
CD13 receptors. We have been able to synthesise a very effective system based on a nano
platform for enhancing the tumour to nonꢀtumour ratio. It is also evident that the quantum dots

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can play a novel role in changing the biodistribution and the pharmacokinetics of the biological
molecules. The steric ligand packing density is optimized by using the homodimeric system
which in addition to the bivalent ligand limits the opsonisation process resulting in enhanced
circulation time to make the targeting effective. The QD nanobioconjugates show a synergistic
effect of active (due to specific biomolecules) and passive targeting (due to the size and charge
of the QD) resulting in enhancing the accumulation of the imaging probes at the desired sites.
The use of a chelating system like EDTA also endows this nanoconjugate with the possibility for
186,188
therapy by replacing the 99mTc with
effective in targeted theranostics.
Re which shows similar chemistry and hence can be
5.
Acknowledgement
The authors would like to thank Director INMAS, for his constant support and encouragement in
carrying out this work. We are also thankful to Delhi University, India for providing
characterization facilities. One of the authors Narmada Bag is thankful to University Grants
Commission, for the financial support.

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6.
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