Diamond & Related Materials 94 (2019) 172–185

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Diamond & Related Materials

journal homepage: www.elsevier.com/locate/diamond

Synthesis and characterization of nanodiamond-anticancer drug conjugates T

for tumor targeting
Sweta Garga, Ashish Garga, Nitendra K. Sahua, Awesh K. Yadavb,
⁎

a
RKDF College of Pharmacy, SRK University, Bhopal 462026, M.P., India
b
Drug Delivery and Nanotechnology Laboratory, Bhagyoday Tirth Pharmacy College, Sagar 470002, M. P., India

ARTICLE INFO ABSTRACT

Keywords: Cancer therapy had got a new approach as a nanodiamond-conjugated anticancer drug. In this work, we have
Nanodiamond introduced a carbon nanomaterial (nanodiamond), to bind with anticancer drug [Usnic Acid (UA), 5-
Sustained release Fluorouracil (5-FU) and Curcumin (CUR)] with the help of linker (ADH) for cancer drug delivery and therapy.
Drug delivery Nanodiamonds (ND) was initially carboxylated by the surface modification along the treatment with strong
Cytotoxicity
alkaline solution (H2SO4:HNO3) and then activated the carboxyl moiety of ND with the addition of EDC.
Conjugates
Molecular docking
Anticancer drugs were bound to the ND through a succession of chemical modifications by adipic acid dihy-
drazide (ADH). The ND-Drug conjugate was analyzed by Nuclear Magnetic Resonance (1H NMR) Spectroscopy,
Fourier Transform Infrared (FTIR) Spectroscopy and Mass Spectroscopy (MS), Atomic Force Microscopy (AFM),
Scanning Electron Microscopy (SEM), Particle size, Zeta potential, Drug release, SRB assay against MCF-7 and
Hep-G2 cells, Molecular docking, Pharmacophore mapping and DNA fragmentation. Spectroscopic analysis
confirms the conjugation of nanodiamond with different anticancer drug. AFM and SEM photomicrograph re-
presents the surface morphological features of ND-Drug conjugates. In- vitro investigation showed that ND-Drug
conjugates have slow and sustained drug release characteristics. In-vitro cytotoxicity studies, an enormous cy-
totoxic potential of ND-Drug conjugates were showed against cancer cell line. Docking and pharmacophore
mapping analysis were also suggested that the ND-Drug conjugates possess effective interaction with significant
proteins and pharmacophore mapping was suggested that ND-Drug conjugates were specifically matched with
hypo 1 model with good fit value. Above all findings were suggested that the ND-Drug conjugates may be a
potential inhibitor of MCF-7 and Hep-G2 cancer cells to act as a drug candidate. According to all these data it can
be confirm that the ND-Drug conjugates could be an effective agent for drug delivery and could be promising in
future for tumor targeting strategy.

1. Introduction [4]. As the targeted therapeutics is improving in-spite of it breast cancer

Chemotherapy is a process for treating cancerous diseases; but due
to improper selective recognition of cancerous and normal cells, there
are various side effects are seen in patients during the chemotherapy.
With the progress in nanotechnology, in the treatment of cancerous
disease, drugs conjugated or adsorbed into nanocarriers presented
various advantages related with free drugs, like controlled drug release,
altered drug biodistribution, prolonged drug circulation in the blood,
and anti-multidrug resistance; are now became keen topic for research
[1,2]. Nanoparticles have property to collect in the tumor tissue with
increased permeability and retention effect [3], which can achieve
passive targeting. Breast cancer is becoming lethal for women
throughout world, approximately 14% of all cancer deaths in female
patients sometime go through relapse because of drug resistance me-
chanism. Mostly happening drug resistant mechanism have the efflux of
drug from the cells, mediated primarily by trans-membrane adenosine
triphosphate (ATP) dependent pumps known ATP-binding cassette
(ABC) transporter proteins [5].
Nowadays, nanomaterial based on carbons like fullerenes and na-
notubes, are widely used in biological applications because of biolo-
gical, chemical and physical properties. NDs are getting attention as
they possess low toxicity high chemical stability, high affinity for bio-
molecules, and ease of surface functionalization [6–8]. NDs have shown
outstanding compatibility in biological environments at good ther-
apeutics concentrations [9]. NDs are chemically modified for treatment
of various cancers with water insoluble drug [10,11].

⁎
Corresponding author at: Drug Delivery and Nanotechnology Laboratory, Bhagyoday Tirth Pharmacy College, Sagar 470002, M. P., India.
E-mail address: aweshyadav@gmail.com (A.K. Yadav).

https://doi.org/10.1016/j.diamond.2019.03.008
Received 27 October 2018; Received in revised form 11 March 2019; Accepted 12 March 2019
Available online 16 March 2019
0925-9635/ © 2019 Elsevier B.V. All rights reserved.
S. Garg, et al.
Currently, nanomaterials are coming as effective tools for increasing
drug delivery and imaging. In recent work it has shown that nanodia-
mond based drug carriers are used to conquer chemoresistance in many
types of cancers [12].
To examine new therapeutic approach in target drug delivery, we
evaluate the delivery of anticancer drug adsorbed onto NDs for tar-
geting to breast cancer cells. The NDs has ability to bind broad spec-
trum of anticancer drug substance by covalent and non-covalent
linking, due to its uniquely faceted surfaces, which provide release of
drug in sustained and controlled manner for enhancing its therapeutic
efficacy. More prominently, NDs also shows their outstanding chemical
and physical features like, biocompatibility, stability, improvement of
therapeutic efficiency etc. and it has been used to deliver the anticancer
drug, nucleic acid, insulin and many other therapeutic molecules for
effective and prolonged release features [13–16]. DOX conjugated NDs
system also has been demonstrate the effective tumor targeting efficacy
[11].
In current research work, we investigate the effectively of ND-Drug
conjugates against breast and liver cancer cell lines, and also explore
the drug retention in cancer cell lines. The anticancer drug substance
bind effectively with the surface modified nanodiamond via ADH. The
resultant complex, ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR were
further characterized by different assay. Importantly, we show that ND-
Drug conjugates enhance sensitivity of resistant of drug in breast and
liver cancer cells, possibly mediated via increased drug retention in the
tumor cells. Additionally, it also possess the prolonged and sustained
drug release mechanism from ND-Drug conjugates. This work suggests
the use of nanodiamond as a promising and effective drug delivery
platform for anticancer drug against tumor cells.
2. Materials and methods
2.1. Materials
Nanodiamond powder (ND), (+)-Usnic acid (UA) and Adipic acid
dihydrazide (ADH) were procured from Sigma Aldrich (USA). 5-
Fluorouracil (5-FU) was obtained as generous gift sample from Neon
Laboratories limited, Mumbai (MS), India. Curcumin (CUR) was ac-
quired from Central Drug House (CDH), Delhi, India. N-hydro-
xysuccinimide (NHS), dialysis membrane, 1-ethyl-3-(3-dimethylami-
nopropyl) carbodiimide hydrochloride (EDC-HCl), N,N′-Dicyclohexyl
Carbodiimide (DCC), Pluronic F-68 were purchased from HiMedia la-
boratories, Mumbai, India. Acetone, ethanol, concentrated HNO3, sul-
phuric acid, hydrochloric acid, isopropyl alcohol and acetonitrile pur-
chased from Merck Limited, Mumbai, India. Other chemicals which are
consumed are of investigative chemical grade and used as brought.
2.2. Methodology
2.2.1. Synthesis of carboxylated ND (ND-COOH)
The commercially available synthesized nanodiamond powder
having particle size of < 10 nm, were carboxylated by following the
standard procedure [17]. Primarily the obtained nanodiamond powder
was treated with the mixture of H2SO4 and HNO3 (3:1 v/v) at room
temperature for 48 h and then diluted with the addition of deionized
water and centrifuged at 900 rpm for 30 min to separate out the ND
particles. The obtained particles were rinsed by using deionized H2O.
Then obtained solution was again centrifuged at 900 rpm to separate
out the ND particle, which was further heated at 90 °C for 2 h in 0.1 M
NaOH solution. Then ND sample was heated again with 0.1 M HCl for
2 h at 90 °C, and washed by using deionized water to solution became
weakly acidic. The obtained carboxylated-NDs were separated and
dried under vacuum for further procedure [17].
2.2.2. Synthesis of activated nanodiamond
Firstly, 50 mg of ND powder was dispersed in 10 mL of distilled
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Diamond & Related Materials 94 (2019) 172–185
water (5 mg/mL) and then 250 mg of EDC-HCl was added with con-
tinuous stirring (Remi, Mumbai, India) for 12 h and maintain the pH 5.8
with the addition of 0.1 N HCl. The EDC-HCl provides the effective
activation of the carboxyl moiety of the NDs end group which enhances
the attachment of NDs with another group of next chemical compound
(-NH2 moiety of the adipic acid dihydrazide). Subsequently addition of
125 mg of NHS into nanodiamond dispersion with constant rate of
stirring and maintain the pH 5.8 with the addition of 0.1 N HCl.
2.2.3. Synthesis of ADH conjugated nanodiamond (ND-ADH-NH2)
In activated NDs dispersion the 250 mg of ADH (five fold excess)
was added with constant stirring for 6 h for attachment of amine moiety
of ADH with the activated carboxyl moiety of NDs and formulation of
ND-ADH-NH2 was done by carbodiimide conjugation.
2.2.4. Synthesis of different ND-Drug conjugates
To synthesize the different ND-Drug conjugates, the different an-
ticancer drugs (UA, 5-FU and CUR) were conjugated with activated NDs
and ADH conjugates ND moiety (ND-ADH-NH2). Primarily, all the an-
ticancer drugs were dissolved separately into different solvent system
according to their solubility parameters. UA (50 mg) was dispersed in
10 mL of acetone, in this same pattern the 5-FU (50 mg) and CUR
(50 mg) was separately dispersed in 10 mL of acetone and ethanol
medium individually, then the prepared drug solution with their spe-
cified solvent system were activated with the addition of DCC and NHS.
Surfactant (Pluronic F-68) medium of different concentration (0.25%,
0.5% and 1%) was formed by dissolving Pluronic-F-68 in aqueous
medium. 10 mL of different drug dispersion (UA, 5-FU and CUR) and
ND-ADH-NH2 conjugate (5 mg/mL) was added in drop wise manner in
alternating sequence into separate Pluronic-F-68 (surfactant) medium
with continuous stirring for 12 h to formulate drug conjugated nano-
diamond i.e. ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR con-
jugates. All the reaction mixtures were dialyzed to remove unreacted
ND, ADH, UA, 5-FU and CUR from all the formulated ND-Drug con-
jugates. The obtained different ND-drug was separated by membrane
filter (0.45 μm) and centrifuged at 15,000 rpm for 30 min (Remi,
Mumbai, India). After centrifugation discarded the supernatant and
lyophilized and preferred for subsequent analysis.
2.3. Characterization parameters of different ND-drug conjugates
All the different ND-Drug conjugates (ND-ADH-UA, ND-ADH-5-FU
and ND-ADH-CUR conjugates) were prepared and authenticated by
different spectroscopic instrumental methods i.e. nuclear magnetic re-
sonance (1H NMR) (Bruker AvII-400), Mass (ESI-MS) (Waters-UPLC-
TQD) and Fourier transform infrared (FTIR) (8400S, Shimadzu) spec-
troscopic technique. FTIR is most common technique used to determine
the different functional group present in the prepared nanodiamond
conjugates.
2.4. Particle morphology, particle size characteristic and surface charge
(zeta potential)
The size of ND-drug conjugates and the surface charge character-
istics of synthesized different ND-Drug conjugates were done by
HORIBA SZ-100 series, (Horiba Scientific, Kyoto, Japan). The surface
characteristics of prepared ND-Drug conjugates were examined by
Variable Pressure Field Emission Scanning Electron Microscope (Supra
55, VP FE-SEM, Carl Zeiss). The application was done at various pres-
sure 2-133 Pa with accelerating voltage 0.1 to 30 kV. The surface
morphology of formulated different ND-Drug conjugate were also be
analyzed with Atomic Force Microscopic method (AFM) (Alpha300RA
AFM, WITec, Germany). For taking AFM photomicrograph of the dif-
ferent ND-Drug dispersion was spread on glass substrate on AC mode.
S. Garg, et al.
2.5. Differential scanning calorimetry (DSC) analysis
The significant physical characteristics pattern of synthesized dif-
ferent ND-Drug conjugates was analyzed by DSC instrument (DSC-60,
Kyoto, Japan). The instrument work on the heat flux type principle. To
obtaining the DSC thermogram, about 5–10 mg of prepared conjugate
sample were placed into the aluminium pan and run the instrument at a
scanning temperature range from 0 °C to 400 °C with the heating rate
10 °C in inert atmosphere.
2.6. Drug loading efficiency of different ND-drug conjugates
The loading proficiency of different synthesized ND-Drug con-
jugates by HPLC system. Different anticancer bioactives (UA, 5-FU and
CUR) conjugated nanodiamond were separately dispersed in 10 mL of
PBS (pH 7.4) medium then the conjugates dispersed medium was filled
in centrifuge tube and then centrifuged at 15000 rpm in cooling cen-
trifuge (C-24BL, Remi, Mumbai, India) for 5 min. Then after cen-
trifugation the supernatant were taken out and analyzed by using high
performance liquid chromatography (HPLC) (Shimadzu, Japan). The
HPLC system consist reverse phase Cosmosil C18 column
(4.6 mm × 250 mm, 5 μm, China) with UV detector was used to analyse
the entrapment efficiency of different prepared drug ND conjugates.
The temperature of column should be maintained at 30 °C. In case of
nanodiamond conjugates containing UA (ND-ADH-UA), for detection of
UA content, methanol: phosphate buffer (pH 7.4) at (70:30 v/v) was
taken as mobile phase with the flow rate 0.8 mL/min and detection of
UA was done at 245 nm [18]. For 5-FU-nanodiamond conjugate (ND-
ADH-5-FU), acetonitrile: acetate buffer (pH 4.4) at ratio of 15:85 was
carried out as mobile phase with the flow rate of 0.8 mL/min, and the
drug (5-FU) was detected at 260 nm [19]. Finally for the conjugate (ND-
ADH-CUR), the mobile phase was acetonitrile: glacial acetic acid %)
(60:40, v/v) with the flow rate of 1.0 mL/min. The column temperature
was adjusted at 30 °C and the typical injection volume was 20 μl and
detection of CUR was done at 430 nm using UV detector [20,21]. The
amount of drug loaded in ND was calculated from unbound drug in the
supernatant, the loading efficiency of ND-Drug conjugates was ana-
lyzed.
2.7. In-vitro release profile
The in-vitro drug release characteristics of the prepared ND-Drug
conjugated were performed by modified dissolution technique. All the
prepared conjugates were placed separately in dialysis bag (HiMedia
Lab Limited, Mumbai, India) and dipped into the separate container
containing 10 mL of phosphate buffer saline (PBS) having different pH
range (pH 7.4, 6.5 and 5.5) and the medium was stirred with the rate of
100 rpm at 37 ± 0.5 °C. As the same manner the unmodified drug
solution (CUR solution in ethanol, UA and 5-FU solution in acetone)
was also placed separately in the dialysis bag then dipped into different
pH PBS media as mentioned above. At a definite time interval an
adequate quantity of the medium was taken out and replaced with the
same quantity of the fresh PBS medium to maintain the sink condition.
The collected medium was centrifuged at 5000 rpm for 15 min, subse-
quently the supernatant was taken out from the centrifuge tube and
analyzed by HPLC system as previously described to carry out the
amount of percentage cumulative drug released from different ND-Drug
conjugates [22].
2.8. Hemolytic toxicity
For hemolytic activity, whole human blood was amassed and col-
lected in a collection vial from authorized pathology centre as denoted
in Bhadra et al. [23]. Firstly human blood was centrifuged at
10,000 rpm for 15 min for complete separation of RBC and plasma. The
plasma was discarded and RBC was taken for further procedure. The red
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Diamond & Related Materials 94 (2019) 172–185
blood corpuscles (RBCs) (1 mL) was incubated separately with 10 mL of
phosphate buffer solution pH 7.4 (taken as 100% hemolytic standard).
In this hematocrit solution the different ND-Drug conjugates and un-
modified anticancer drugs, were added separately on hematocrit solu-
tion. The collection tube was allowed to stand for 1–2 h at 37 °C, after
that the ND-Drug conjugates in hematocrit mixture was centrifuged at
5000 rpm for 10 min, and then the absorbance was taken of supernatant
at 540 nm to optimize the effect of ND-Drug conjugates and plain
(unmodified) drugs against RBCs, which was useful to predict the
percentage hemolysis.
2.9. SRB (sulforhodamine B) assay
Initially the cell line were grown in the RPMI 1640 medium con-
taining fetal bovine serum (FBS 10%) and L-glutamine (2 mM) further
cell were inoculated on 96 well microtiter well plate containing 100 μL
plating densities. After it microtiter plates were incubated at tempera-
ture 37 °C, 5% CO2, 100% relative humidity and 95% air for 24 h before
addition of different formulations (i.e. different ND-Drug conjugates,
ND, plain anticancer drugs). Firstly conjugates were dissolved in di-
methyl sulfoxide at 100 mg/mL further diluted to 1 mg/mL using dis-
tilled and kept frozen before use. Aliquots for frozen concentrate di-
luted to 100 μg/mL, 200 μg/mL, 400 μg/mL and 800 μg/mL during the
addition of different conjugates with total medium consisting test ar-
ticles. Final concentration of 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL
was prepared by adding 10 μl of different dilutions in microtiter wells
consisting 90 μl of medium. Plates were incubated after compound was
added at standard conditions for 48 h and assay was performed with
cold trichloroacetic acid (TCA) and incubated at 4 °C for 60 min further
the supernatant was discarded and plates were cleaned. After than all
the wells were treated with Sulforhodamine B (SRB) solution (50 μl) at
0.4% (w/v) in 1% acetic acid and incubated at room temperature for
30 min. The plates were dried and the absorbance was noted at 540 nm
with 690 nm reference wavelength. Percent growth of cell was de-
termined against control wells by plate-by-plate basis and further cal-
culated as: [Ti/C] × 100%. Finally the morphology of MCF-7 and Hep-
G2 cells was determined by the light microscope [24,25].
2.10. Molecular docking studies
To further elucidate the binding mechanism of the different ND-
Drug conjugates, molecular docking studies implying the aforemen-
tioned anticancer drug target protein, human DNA topoisomerase II
was carried out. The aim of the molecular docking study is to predict
the ligand binding affinity in the effective sites of the targeting protein
afterwards their stable complex formation and hence estimating the
inhibitory potential of the ligand to their target protein. The target
protein DNA topoisomerase II was prepared by retrieving the high re-
solution crystal structure of Human DNA topoisomerase II in complex
with DNA and etoposide (PDB ID: 1T8I, resolution 3.0 Å) from PDB
database. The protein is prepared using DS prepare protein protocol
after removing all the bound crystal water molecules and heteroatoms
including ligands with the subsequent addition of hydrogen atoms and
further energy minimized using CHARMm force field through the ap-
pliance of different DS minimization algorithms until the protein sa-
tisfies with a convergence gradient of 0.001 kcal/mol. After energy
minimisation, active site of the protein was selected based on the ligand
binding location of etoposide and an active site sphere was defined with
a radius of 10 Å respectively. The 2D chemical structures of the ND-
Drug conjugates compounds (ND-ADH-UA, ND-ADH-5-FU, ND-ADH-
CUR) are sketched using ACD/ChemSketch (12.0) and preparation of
the ligands with constraint parameters accompanying their 3D con-
version is done using prepare ligands module and catalyst algorithm of
DS respectively. The compounds are then minimized with Smart
Minimizer method followed by Steepest Descent method using
CHARMm force field in DS till converging to a resolution of 0.001 kcal/
S. Garg, et al.
Fig. 1. Graphical representation of Conjugation pattern of ND-Drug conjugates and proposed hypothesis of tumor targeting strategy of ND-Drug conjugate.
mol. The molecular docking procedure is carried out by LibDock, a site-
featured directed docking algorithm of DS which estimates the appro-
priate binding conformations of ND-Drug conjugates in the defined
active site of human DNA topoisomerase II (PDB ID: 1T8I). Scoring
function of the LibDock calculates the binding affinity score or docking
score (LibDock score) of protein-ligand complex. Also the possible
binding energies, hydrogen bonding and various interaction poses are
calculated. The top ranked docked complexes of each compound are
selected on the basis of LibDock Score. Binding poses with highest
LibDock score and lowest binding energy are preferred as the best pose
and further binding interactions of the best pose for each compound are
analyzed. Docking procedure was validated by redocking the inbuilt
ligand etopside with the human DNA topoisomerase II binding site. ND-
Drug conjugates docking results are examined in comparison to that of
the etopside in terms of their hydrogen bond formations and docking
scores [26,27].
2.11. Ligand based pharmacophore modelling
ND-Drug conjugates were further evaluated for their chemical fea-
tures important for binding, by generating ligand based pharmacophore
model (Common feature pharmacophore model) using HipHop module
of Catalyst in DS. Based on the atom-types present in the ligand mo-
lecule under investigation, it identifies the spatial arrangements of
functional groups frequently observed in the components, responsible
for its various biological activities. In the process of pharmacophore
hypothesis generation, initially conformers for the energy minimized
ND-Drug conjugates compounds are generated using Diverse Conformer
Generation protocol with Fast conformer generation option of DS.
Other parameters, like the maximum number of 255 conformers and an
energy threshold value of 20 kcal/mol above the global energy
minimum are chosen during conformation.
Generation Feature Mapping protocol is used to identify the features
such as hydrogen bond acceptor (HBA), hydrogen bond donor (HBD),
hydrophobic feature (HY), positive ionizable and negative ionizable
features in order to create reliable pharmacophore sites. Finally, the DS
Common Feature Pharmacophore Generation protocol is executed with
default value of 2 for principal value, 0 for maximum omit feature value
and 2.97 Å for the minimum inter-feature distance. Ten pharmacophore
hypotheses are generated and the best one was selected based on the
ranking score which further analyzed for compound mapping. The fit
values and its aligned pharmacophore features are the suitable criteria
to select the best mapped compound [26,27].
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Diamond & Related Materials 94 (2019) 172–185
2.12. ADMET prediction
The pharmacokinetic and toxicity of ND-Drug conjugates are
screened using the DS ADMET descriptor module in order to determine
the overall drug toxicity risks as well its pharmacokinetic features. The
ADMET properties include blood brain barrier (BBB), water solubility,
CYP2D6 binding, plasma protein binding, CYP2D6 binding and in-
testinal absorption. In addition, AlogP98 and PSA_2D were used in
plotting the confidence ellipses. Analysis of the results is based on the
standard parameters according to the software limitations [26].
2.13. DNA fragmentation analysis
Genomic DNA with low-molecular-weight was extracted as detailed
previously [28,29]. A breast cancer cell (MCF-7) was firstly treated with
ND-Drug conjugate (ND-ADH-CUR) then cell was lysed in a buffer so-
lution consisting 5 mM EDTA (ethylene diamine tetra acetic acid),
5 mM of pH 7.5 Tris HCl and 0.5% Triton X-100 intended for an about
30 min on ice. The prepared lysate media were vortexed by vortex
shaker and then centrifuged at 30,000 for about 20 mins. The frag-
mented DNA present in the supernatant was reacted with RNAse, after
then treated with proteinase K digestion, and added on the mixture of
phenol, chloroform and isoamyl alcohol in different volume con-
centration (25:24:1) extraction and isopropanol precipitation. The se-
paration of DNA was carried out through agarose gel (1.5%), and then
stained by using ethidium bromide solution (0.1 μg/mL) and inter-
pretation of fragmentation can be visualized under UV source.
2.14. Statistical analysis
Final research outcome were showed as mean ± SD. All processes
were performed thrice.
3. Result and discussion
The main objective behind the current study was to synthesize
different ND-Drug conjugates for improved the targeting toward tumor
cell as well as enhancing the potency of anticancer drug to provide
maximum therapeutic effect. The surface of nanodiamond can be
modified by many functional groups for providing higher stability of
conjugates. The structure with several functional moiety depicted by
Ansari et al., 2016 [30]. Graphical abstract (Fig. 1) represent the pro-
posed tumor targeting strategy of ND-Drug conjugates.
S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

Fig. 2. 1H NMR spectra of (a) ND-ADH-UA, (b) ND-ADH-5-FU and (c) ND-ADH-CUR.

176
S. Garg, et al.
Fig. 3. AFM image of (a) ND-ADH-UA (b) ND-ADH-5-FU and (c) ND-ADH-CUR and SEM image of (d) ND-ADH-UA, (e) ND-ADH-5FU and (f) ND-ADH-CUR.
3.1. Spectroscopic analysis (1H NMR, Mass and FTIR)
The 1H NMR spectra of synthesized different ND-Drug conjugates
was illustrated in Figure (Fig. 2). In 1H NMR spectrum of ND-Drug
conjugates different distinctive peak was obtained. In the spectrum of
ND-ADH-UA (Fig. 2a), the proton assignment of ND was obtained at
1.02 ppm (2H, S) and 1.04 ppm (2H, S), shows presence of alkyl group,
the peak at 2.17 (1H, S) shows hydroxyl moiety (R-OH). The proton
assignment of ADH existed at 8.7 ppm shows presence of amine (1H, S),
1.11 ppm (2H, S), 1.57 ppm (2H, S), 1.89 ppm (2H, S), 1.90 ppm (2H,
S), 1.92 ppm (2H, S), 1.94 ppm (2H, S) and the proton assignment of UA
was obtained at 1.59 ppm (3H, S), 1.796 ppm (3H, S), 2.168 ppm (3H,
S), 2.303 ppm (3H, S), 3.608 ppm (3H, S), 6.31 ppm (4H, S), 11.3 ppm
(1H, S). Beside of these proton assignment there are additional major
peak was observed at 8.6 ppm (1H, S) and 8.7 ppm (1H, S), justify the
presence of amide, which formed due to carbodiimide conjugation of
carboxyl moiety of ND and amine group of ADH. All the proton as-
signment justifies the presence ND, UA and ADH in the conjugation of
ND-ADH-UA. In the spectrum of ND-ADH-5-FU (Fig. 2b), the proton
assignment at 8.5 ppm (1H, S), and 8.6 ppm (1H, S) was obtained which
shows the presence of amide bond. The different proton assignment of
ND was found at 1.02 ppm and 1.04 ppm (2H, S), shows presence of
secondary alkyl group (R2CH2), 2.18 (1H, S) shows presence of hy-
droxyl. The proton assignment of ADH obtained at 1.51 (2H, S), 1.92
(2H, S), 2.16 (2H, S) and at 8.89 ppm (1H, S). The proton assignment of
5-FU was obtained at ppm 7.58 ppm (1H, S) and 7.60 ppm (1H, S),
11.39 (1H, S), 10.64 (1H, S). In the spectrum of ND-ADH-CUR (Fig. 2c),
the proton assignment of CUR was obtained at 3.91 ppm (3H, S),
6.05 ppm (1H, T), 6.69 ppm (1H, S), 6.84 ppm (1H, S), 7.09 ppm (1H,
S), 7.32 ppm (1H, S), 7.4 ppm (1H, S) and 9.69 ppm (1H, S). The proton
spectrum of ND was obtained 1.02 ppm and 1.04 ppm (2H, S), shows
presence of secondary alkyl group (R2CH2) and at 2.17 ppm (1H, S)
shows hydroxyl moiety (ROH). The proton assignment of ADH was
obtained at 1.54 ppm (1H, S), 1.89 ppm (1H, S), 1.90 ppm (1H, S),
2.16 ppm (2H, S) and at 8.89 ppm (1H, S). Among these proton as-
signment there are additional major peak was observed at 8.6 ppm (1H,
S) and 8.7 ppm (1H, S), justify the presence of amide, which formed due
to carbodiimide conjugation of carboxyl moiety of ND and amine group
of ADH. All the proton assignment justifies the presence ND, CUR and
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Diamond & Related Materials 94 (2019) 172–185
ADH in the conjugation of ND-ADH-CUR conjugates.
Mass spectrometry (MS) is an analytical technique used for analyse
the characteristics features of individual molecules. The ESI MS spec-
trum of different ND-Drug conjugates were shown in Fig. (Fig. 1S). The
ESI MS spectrum of ND-ADH-UA, spectrum of UA was obtained at m/z
345 and MS spectrum of ADH was found at m/z 104, m/z 115, m/z 142,
m/z 143 and m/z 174 (Fig. 1S a and 1S b). The ESI MS spectrum of ND-
ADH-5-FU, spectrum of 5-FU was occur at m/z 130 and MS spectrum of
ADH was depicted at m/z 104, m/z 115, m/z 128, m/z 143, m/z 144, m/
z 160 and m/z 174 (Fig. 1S c and 1S d). In case of ND-ADH-CUR, the ESI
MS spectrum of CUR was found at m/z 245, m/z 369, m/z 370, m/z 391,
m/z 392, m/z 407, m/z 408 and m/z 759 whereas at m/z 103, m/z 104,
m/z 115, m/z 129, m/z 142, m/z 143, m/z 160 and m/z 174 was the
obtained spectrum of ADH (Fig. 1S e and 1S f). The distinctive peak of
different carbon clusters of nanodiamond particles obtained in the ESI
MS spectra in all the ND-Drug conjugates at various intensity, which
reveal the presence of nanodiamond in all the conjugates [31,32].
The FTIR spectra of different drug ND conjugates were shown in
Fig. 2S. The conjugate ND-ADH-UA, displayed distinctive peak at
675 cm−1, 840 cm−1, 927 cm−1, 2875 cm−1 and 2947 cm−1 shows
CeH stretch, at 1263 cm−1 attribute to presence of C-O-C stretching, at
1595 cm−1 has a predominantly mixed C]C stretch and C]O stretch,
at 1651 cm−1 displayed the presence of C]O stretch, a broad spectrum
at 3209 cm−1 and 3431 cm−1 shows the presence of NeH stretch and a
band at 3566 cm−1 confirms the presence of OeH stretch (Fig. 2S a).
The major peak of ND-ADH-5-FU (Fig. 2S b), was obtained at 669 cm−1,
927 cm−1, 2875 cm−1 and 2947 cm−1 shows presence of CeH stretch,
1263 cm−1 peak assigned to the C-O-C stretching, peak at 1564 cm−1
and 1595 cm−1 attribute to presence of C]C stretch, at 1633 cm−1
peak shows the C]O stretch, at peak 3211 cm−1 in the spectrum shows
the presence of OeH stretch and at 3452 cm−1, NeH stretching of the
conjugates occur. In the case of ND-ADH-CUR, peak at 1276 cm−1
verified the presence of presence of C-O-C stretching, a typical peak at
1595 cm−1 has a predominantly mixed C]C stretch and C]O stretch,
at 3479 cm−1 shows the presence of NeH stretching and the 3207 and
3778 cm−1attribute to presence of OeH stretch, CeH stretch of was at
2947 cm−1 and 2974 cm−1 (Fig. 2S c).
S. Garg, et al.
Table 1
Ingredients and concentration using in the formulation of ND-Drug conjugates.
ND-drug Nanodiamond Distilled Usnic acid 5-Fluorouracil
conjugates concentration water (mg) (mg)
(mg) (mL)
ND-ADH-UA 50 10 50 – –
ND-ADH-5FU 50 10 – 50
ND-ADH- CUR 50 10 – –
3.2. Surface characteristics
The surface characteristics like shape, size and texture of different
ND-Drug conjugates was examined by using atomic force microscopic
technique (Alpha300RA AFM, WITec, Germany). The prepared dif-
ferent ND-Drug conjugates having nanometric size range as monitored
by AFM image (Fig. 3a, b and c). The photomicrograph shows the
uniform arrangement as well as similar height of the different ND-drug
conjugates. The photomicrograph obtained from Field Emission Scan-
ning Electron Microscope (Supra 55, VP FE-SEM, Carl Zeiss), also re-
presents the distribution pattern of ND-Drug conjugates (ND-ADH-UA;
ND-ADH-5-FU; ND-ADH-CUR) (Fig. 3 d, e and f). The SEM image
confirms the rounded and prickly shape characteristics of the prepared
different ND-Drug conjugates. Summarizing, we have shown that the
prepared ND-Drug conjugates displayed flake-like structure with pre-
ferred crystal orientation as well as its also clearly depicted that the
particles are certainly not all the same. The photomicrograph from SEM
and AFM, it was displayed that some particles show clearer sharp edges
than others, according to these finding we assume, however, that sharp
edges on that length scale would be quite reactive [33,34].
3.3. Zeta potential determination, particle size analysis and drug
entrapment proficiency
According to the result depicted in Table 1, the particle size eva-
luation of different ND-Drug conjugates (Fig. 3S) were carried out using
particle size analyzer and the particle size of ND-Drug conjugates were
observed 114.32 nm in case of ND-ADH-UA (Fig. 3Sa), 81.6 nm in case
of ND-ADH-5-FU (Fig. 3Sb) and in the case of ND-ADH-CUR (Fig. 3S c),
the obtained particle size was 39.85 nm. All the data obtained from
particle size analysis, it was confirm that the ND-drug conjugates
having nanometric size range. Zeta potential of ND-Drug conjugates
was obtained to be −10.6 mV, −11.2 mV and − 14.3 mV in case of
ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR respectively (Fig. 3Sd,e
and f). The negative value of the zeta potential analysis of ND con-
jugates may be due to the carboxyl moiety of the nanodiamond. In the
charge particle having increased zeta potential, it causes formation of
more stable particles due to higher repulsive interaction. It was ob-
served that the lower negative zeta potential may increase the stability
of the system [35,36]. The loading efficiency of unconjugated (un-
bound) anticancer drug measured by HPLC and found to
85.3 ± 0.25%, 88.14 ± 1.38% and 93.8 ± 0.85% in case of ND-
ADH-UA, ND-ADH-5FU and ND-ADH-CUR respectively (Table 1).
3.4. Differential scanning calorimetry (DSC) analysis
DSC thermograms of different ND-Drug conjugates are shown in Fig
(Fig. 4S). In case of ND-ADH-UA (Fig. 4S a), the distinctive peak of UA
revealed at 204.7 °C, whereas the peak of ADH was obtained at
180.32 °C. At the same way at 284.55 °C, the peak of 5-FU was acquired
and at 185.82 °C, the peak of ADH was noticed, in case of ND-ADH-5-FU
(Fig. 4S b). In the ND-ADH-CUR (Fig. 4S c), the distinctive peak of
curcumin (CUR) was spotted at 183 °C, whereas the peak of ADH in ND-
ADH-CUR, obtained at 181 °C. In all the different ND-drug conjugates,
the small tiny peak of nanodiamond was attained at 228.2 °C, 228 °C
and 227.5 °C in the case of ND-ADH-UA, ND-ADH-5-FU, ND-ADH-CUR
178
Diamond & Related Materials 94 (2019) 172–185
Curcumin Acetone/ Particle size Zeta potential % Drug loading
(mg) Ethanol (nm) efficiency
(mL)
10 114.32 ± 0.5 −10.6 mV 85.3 ± 0.25
– 10 81.6 ± 1.15 −11.2 mV 88.14 ± 1.38
50 10 39.85 ± 1.18 −14.3 mV 93.8 ± 0.85
respectively, which due to the oxidation of ND at the temperature
228 °C [37]. The entire DSC curve signifying the presence of anticancer
drug in crystalline nature as well as in stable form inside the nano-
diamond.
3.5. In-vitro drug release pattern
The sustained release nature of drug from different ND-Drug con-
jugate system illustrated in graph (Fig. 4). It is recognized that well-
organized release of drug from a drug delivery system is essential for
therapeutic action of most of the anticancer drug conjugated formula-
tions. Assimilation of acidic environment between the carrier and drug
capable the liberation of an anticancer drug from the carrier (ND) into
the cancer cell (having slightly acidic environment (pH 6.5)) then after
endocytosis phase in the endosomes consisting pH range 5–6 and ly-
sosomes (pH 4–5) of cancer cells. For this circumstances, the percentage
release of drug from ND-Drug conjugates was performed at 37 °C under
simulated physiological conditions (phosphate-buffered saline, pH 7.4)
and an acidic environment (phosphate-buffered saline, pH 5.5, an
acidic endosome environment and pH 6.5, a simulated tumor environ-
ment) to analyse the feasibility of ND-Drug conjugates as an drug de-
livery system for anticancer drug. As depicted in Fig. 4 (a), the amount
and rate of drug released from ND-Drug conjugates were dependent on
the pH of the medium. ND-Drug conjugates displayed a rapid release
pattern of anticancer drugs (UA, 5-FU and CUR) at pH 5.5 then pH 6.5
and then at pH 7.4. The release rate of drug from different ND-Drug
conjugates (ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR) after 96 h
was found to be 33.5%, 35.1% and 36.3% respectively at pH 5.5,
whereas 22.8%, 24.5% and 26.4% was found in case of ND-ADH-UA,
ND-ADH-5-FU and ND-ADH-CUR at pH 6.5. 14.6%, 15.8% and 17.7%
of drug release was obtained at pH 7.4 for ND-ADH-UA, ND-ADH-5-FU
and ND-ADH-CUR correspondingly. Drug conjugated with ND via
amide bond which is relatively more stable than physical absorption,
thus slowing down the release of anticancer drug from ND conjugates
[38,39]. This is due to because of surface modification of ND via car-
bodiimide conjugation between of the carboxyl group of nanodiamond
with the amine group of adipic acid dihydrazide and formation of
crosslinked core-shell micelle, which having less solubility, may pro-
mote sustained release [30]. The result also suggests that the amount of
anticancer drugs (UA, 5-FU and CUR) released from ND-Drug con-
jugates was enhanced by an acidic environmental condition. The data
also has been suggested by various researchers, that acidic pH condition
triggered the cleavage of amide bond [40–43]. The slow rate of antic-
ancer drug (UA, 5-FU and CUR) release from ND-Drug conjugates was
calculated at pH 7.4, which mimics the physiological environment of
the bloodstream and establish that reduced amount of anticancer drugs
is liberated from conjugates in the blood circulation. It was assume that
ND-Drug conjugates would adhere preferentially in the site of tumor
cell via enhanced permeability and retention (EPR) effect. Fig. 4 (b),
also represents the release of unmodified anticancer drug (UA, 5-FU and
CUR), and the plateaus by approximately 30 mins at pH 7.4, 6.5 and 5.5
respectively, with a highest release of 96.8%, 99.3% and 94.3% at
pH 7.4, the release 97.2%, 99.6% and 94.7% occurs at pH 6.5, and
97.5%, 99.8% and 95.4% at pH 5.5 in case of UA, 5-FU and CUR cor-
respondingly. In contrast with ND-Drug conjugates, the free or un-
modified anticancer drugs (UA, 5-FU and CUR) cause damage cells in a
S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

a
45
40 ND-ADH-UA (5.5)

% Cumulative drug release

35 ND-ADH-5FU (5.5)
ND-ADH-CUR (5.5)
30
ND-ADH-UA (6.5)
25 ND-ADH-5FU (6.5)
20 ND-ADH-CUR (6.5)
ND-ADH-UA (7.4)
15
ND-ADH-5FU (7.4)
10
ND-ADH-CUR (7.4)
5
0
0 hrs 1 hrs 2 hrs 4 hrs 8 hrs 12 24 36 48 60 72 84 96
hrs hrs hrs hrs hrs hrs hrs hrs

Time (hrs)

b
100

5FU (5.5)
80
% Cumulative drug release

5FU (7.4)

5FU (6.5)

60 UA (5.5)

UA (6.5)

40 UA (7.4)

CUR (5.5)

CUR (6.5)
20
CUR (7.4)

0
0 hrs 0.5 hrs 1 hrs 1.5 hrs 2 hrs 2.5 hrs 3 hrs
Time (hrs)

Fig. 4. (a) Drug release profile of ND-Drug conjugates (ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR).
(b) Drug release profile of Unmodified (UA, 5˗FU and CUR) at different pH conditions.

normal physiological environment, causing serious side effects. (different ND-Drug conjugates and anticancer drugs). The result of
percentage growth inhibition of cell is illustrated in Fig. (Fig. 5a and b).
3.6. Hemolytic toxicity study Which revealed that higher concentration of sample ND-Drug con-
jugates inhibit the cell growth. Furthermore, the cell viability also gets
The hemotoxic effect of the prepared ND-Drug conjugates was es- declined with increase in the concentration of sample. ND-Drug con-
timated by hemolytic toxicity study. The different ND-Drug conjugates jugates formulations were experiential to be cytotoxic to a greater
(ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR) exhibited hemolytic amount with the concentration between 10 and 80 μg/mL. The Figure
toxicity up to, 7.15 ± 1.19%, 6.19 ± 0.5% and 4.5 ± 1.19% in- also states the cytotoxic effect of ND and unmodified anticancer drugs
dividually. Whereas the plain drug (UA, 5-FU and CUR) represents (UA, 5-FU and CUR). Fig. 5c, showed normal nuclei having flattened
hemolytic toxicity 59.5 ± 1.2%, 56.19 ± 1.5% and 54.21 ± 0.5% and smooth morphology in normal culture conditions (control) in case
respectively. There was decline in hemolytic toxicity by different ND- of positive control typical changes in morphology occurs, and different
Drug conjugates in compare to plain drug, caused due to delayed re- ND-drug conjugates formulation in which membrane bulging and de-
lease of encapsulated drug molecules in the nanodiamond conjugates. tached from the surface were noticed in MCF-7 and Hep-G2 cell lines.
The repression of hemotoxicity of drug can be linked among other si- The finding concluded that cytotoxic effect of optimized ND-Drug
milar studies described previously [44]. conjugates discovered to have greater inhibitory effect.

3.7. SRB (sulforhodamine B) assay 3.8. Molecular docking analysis

The in-vitro cytotoxicity screening of ND-Drug conjugates in human Molecular docking study was performed for newly designed dif-
breast cancer cellline (MCF-7) and human hepatoma celline (Hep-G2) ferent ND-Drug conjugates, ND-ADH-5-FU, ND-ADH-CUR, ND-ADH-UA
was established by SRB assay. The result obtained by the assay affirm using the DNA topoisomerase II (PDB ID: 1T8I) enzyme as receptor
dose dependent assessment of cytotoxicity in which the cellular bioa- molecule with the aid of Libdock module of DS. Binding affinity eva-
vailability decreased with increasing the concentration of sample luation and inhibitory potential of these compounds were measured

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S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

a Growth Curve: Human

Breast Cancer Cell Line MCF-7

100

ND-ADH-UA

% Control Growth
ND-ADH-5FU
ND-ADH-CUR
ND
50 UA
5-FU
CUR

0
10µg/ml 20µg/ml 40µg/ml 80µg/ml

Concentration ( g/ml)

200
b Growth Curve: Human
Hepatoma Cell line Hep-G2

150
%Control Growth

ND-ADH-UA
ND-ADH-5FU
100 ND-ADH-CUR
ND
UA
5-FU
50 CUR

0
10µg/ml 20µg/ml 40µg/ml 80µg/ml
Concentration ( g/ml)

Fig. 5. Cell-line study of ND-ADH-UA, ND-ADH-5FU and ND-ADH-CUR against (a) MCF-7 and (b) Hep-G2 cells.
(c) Effect of ND-drug conjugates (a) ND-ADH-UA, (b) ND-ADH-5FU, and (c) ND-ADH-CUR on cell viability and cell morphology of MCF-7 and Hep-G2 cell lines by
SRB assay.

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S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

Table 2 interaction diagrams obtained in docking of different ND-Drug con-

Analysis of docking scores, binding energies and no of hydrogen bonds of the jugates with DNA topoisomerase II (PDB ID: 1T8I) are shown in Fig. 6
different ND conjugates. and the interaction data are tabulated in Table 3. Apart from the hy-
Name LibDockScore H-BOND Binding energy drogen bonding interactions, hydrophobic bonding was also observed.
Docking analysis of three ND-Drug conjugates with the human topoi-
ND-ADH-UA 174.17 5 2108.58 somerase II, revealed that the compound ND-ADH-CUR is showing a
ND-ADH-5FU 200.294 5 1076.42
high docking score of 226.644 with six hydrogen bond interactions
ND-ADH-CUR 226.644 6 2277.35
followed by ND-ADH-5-FU and ND-ADH-UA having the docking scores
of 200.294 and 174.17 with five hydrogen bond interactions. From the
through LibDock docking score and H-bond interactions. Further the compound ND-ADH-UA, five hydrogen bonds formed between ND
docking reliability was validated by comparing with that of the co- compound with ARG364, TGP11 and DC111 (C:TGP11:H2-ND-ADH-
crystallized ligand etopside. Docking results showed that LibDock UA:O21; C:TGP11:H3-ND-ADH-UA:O21; A:ARG364:HH12-ND-ADH-
program successfully docked all the three ND-Drug conjugates into the UA:O2; A:ARG364:HH21 - ND-ADH-UA:O32; ND-ADH-UA:H89 -
binding site of DNA topoisomerase II with high LibDock scores that are D:DC112:O2 with a distance of hydrogen bond at 2.397000; 1.790000;
in the range of co-crystallized ligand etopside. Results also revealed that 1.611000; 2.440000; 2.433000 Å respectively shown as Fig. 6 (a and b).
docked compounds were found to be having similar interactions as of The compound ND-ADH-5-FU formed 5 hydrogen bond with ARG364,
co-crystallized ligand etopside. The high LibDock score of the ligand THR718 and TGP11 as shown in Fig. 6 (c and d). Two hydrogen bond
pose with least binding energy was taken into account for the predic- are formed between the nucleotide ARG364 and the ND-ADH-5-FU
tion of the best ligand binding conformation. Table 2 depicts the Lib- (A:ARG364:HH21 - ND-ADH-5FU:O2 and A:ARG364:HH22 - ND-ADH-
Dock scores, number of hydrogen bonds (H-bonds) and binding en- 5FU:O2) with H-bond distance of 2.436000 and 1.049000 Å and two
ergies for ND-Drug conjugates. The binding modes and ligand–protein different hydrogen bond also observed between the THR718 and ND-
ADH-5FU (A:THR718:HG1 - ND-ADH-5-FU:O40 and ND-ADH-5-

Fig. 6. Receptor-ligand hydrogen bonding interactions of (a, b) ND-ADH-UA, (c, d) ND-ADH-5FU and (e, f) ND-ADH-CUR with active site residues (Green dotted lines
are indicating hydrogen bonds), ligand molecule represented as Ball-stick. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

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S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

Table 3
Docking analysis of the intra and intermolecular hydrogen bonds of the ND-Drug conjugates compounds.
Ligand name Interacting Residues Interacting atoms H-Distance

ND-ADH-UA ARG364, C:TGP11:H2 - ND-ADH-UA:O21 2.397000

THR718, C:TGP11:H3 - ND-ADH-UA:O21 1.790000
ASP533, A:ARG364:HH12 - ND-ADH-UA:O21 1.611000
ASN722 A:ARG364:HH21 - ND-ADH-UA:O32 2.440000
DC112, ND-ADH-UA:H89 - D:DC112:O2 2.433000
DA113,
TGP11, C:TGP11:N3 - ND-ADH-UA:H108 1.992000
DT10, C:TGP11:H4 - ND-ADH-UA:H92 1.515000
DG12 A:ARG364:HH11 - ND-ADH-UA:H80 1.527000

ND-ADH-5FU ARG364, A:ARG364:HH21 - ND-ADH-5FU:O2 2.436000

ASN722, A:ARG364:HH22 - ND-ADH-5FU:O2 1.049000
THR718, A:THR718:HG1 - ND-ADH-5FU:O40 1.776000
ASP533, ND-ADH-5FU:H53 - C:TGP11:O6 2.223000
DA113, ND-ADH-5FU:H85 - A:THR718:OG1 1.859000
TGP11,
DT10, ND-ADH-5FU:C1 - A:ARG364:HH22 2.013000
DG12 ND-ADH-5FU:H85 - A:THR718:HG1 1.398000
ND-ADH-5FU:O2 - A:ARG364:NH2 1.976000
ND-ADH-5FU:C31 - C:TGP11:H2 1.960000

ND-ADH-CUR ARG364, A:ARG364:HH22 - ND-ADH-CUR:O56 2.108000

THR718, ND-ADH-CUR:H90 - A:THR718:OG1 2.165000
ASN722, ND-ADH-CUR:H89 - C:DG12:OP1 2.021000
ASP533, ND-ADH-CUR:H90 - C:DG12:OP1 2.280000
DA113, ND-ADH-CUR:H112 - C:TGP11:S5' 2.071000
TGP11, ND-ADH-CUR:H116 - D:DA113:O4' 2.279000
DT10,
DG12 C:DG12:C5' - ND-ADH-CUR:H85 2.036000
A:ARG364:NH2 - ND-ADH-CUR:H116 1.979000
A:ARG364:HH22 - ND-ADH-CUR:H116 1.442000

FU:H85 - A:THR718:OG1) with a hydrogen bond distance of 1.776000

and 1.859000 Å respectively and one hydrogen bond formed between Table 4
53rd hydrogen atom of ND-ADH-5-FU and 6th oxygen atom of TGP111 Chemical feature compositions for the ten hypotheses generated from three ND-
with a hydrogen bond distance of 2.223000 Å (ND-ADH-5-FU:H53- Drug conjugates.
C:TGP11:O6). From the docking conformation of the compounds, it was S. No. Hypothesis Ranking score ranging Direct Hit Partial Hit Fit value
exposed that compound ND-ADH-CUR create six hydrogen bonds with
ARG364, THR718, DG12, TGP11 and DA113 as shown in Fig. 6 (e and 1 HDAAAA 75.344 111 000 6
2 HDAAAA 68.479 111 000 6
f). A single hydrogen bond is formed between 22nd hydrogen atom of
3 HAAAAA 67.879 111 000 6
residue ARG364 with oxygen atom (56th) of the compound ND-ADH- 4 HHDAAA 65.918 111 000 6
CUR (A: ARG364: HH22 - ND-ADH-CUR:O56) with 2.108000 Å dis- 5 HAAAAA 64.529 111 000 6
tance of hydrogen bond. Between the hydrogen atom of compound ND- 6 HAAAAA 64.529 111 000 6
ADH-CUR and the oxygen atom of the residue THR718 (ND-ADH-CUR: 7 HHAAAA 63.861 111 000 6
8 HDDAA 57.429 111 000 5
H90 - A:THR718:OG1) a single hydrogen bond was formed with a
9 HDAAA 56.829 111 000 5
distance of 2.165000 Å. Between the compound and the nucleotide 10 HHDAA 55.737 111 000 5
guanine (DG12). DG12 interacted with 89 hydrogen atom of the com-
pound (ADH-CUR:H89 - C:DG12:OP1) and the other with the 90 hy-
drogen atom of the compound (ND-ADH-CUR:H90 - C:DG12:OP1) with ionizable. The HipHop algorithm computes ten common pharmaco-
a hydrogen bond distances of 2.021000 Å and 2.280000 Å respectively, phore hypotheses having 75.344 to 55.737 (ranking scores ranging) and
in this was two hydrogen bond occurs between compound and the maximum fit values ranging from 6 to 5 that are tabulated in Table 4.
nucleotide guanine. Another nucleotide guanine (TGP11) interacted Analysis of ranking scores and fit values of these hypotheses revealed
with the 112 hydrogen atom of the compound (ND-ADH-CUR: H112 - that hypothesis is selected as most trustworthy pharmacophore hy-
C:TGP11:S5’) with a distance of 2.071000 Å. Nucleotide adenine pothesis (Fig. 7a) containing one hydrogen bond donor, four hydrogen
(DA113) formed a single hydrogen bond with the 116 hydrogen atom of bond acceptor and one hydrophobic feature. Thus the hypothesis 1 is
the compound (ND-ADH-CUR: H116 - D: DA113:O4’) with a distance of follow to investigate the fit value of the compound for their enormous
H-bond with 2.279000 Å. Some non-bonded interactions are obtained features. All the prepared ND-Drug conjugates were mapped on the
with nucleotide guanine and ARG364. hypothesis 1 and ranked of the compound on hypo 1 based on the fit
value. The compound presumed fit values from exact hypothesis Hypo 1
3.9. Ligand based pharmacophore modelling are shown in Table 5. Along the high fit value of 6 compound ND-ADH-
CUR goes well in the pharmacophore. The feature mapping of the
Common feature pharmacophore model was built using a set of ND- compound ND-ADH-UA, ND-ADH-5-FU and ND-ADH-CUR on the con-
Drug conjugates with their diverse conformational structures using sidered pharmacophore Hypothesis 1 is shown in Fig. 7b, c and d re-
HipHop algorithm of DS Catalyst program. The features selected for this spectively.
run were hydrogen bond acceptor (HBA), hydrogen bond donor (HBD),
and hydrophobic feature (HY), positive ionizable and negative

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S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

Fig. 7. (a) Generated common feature-based ligand

pharmacophore model (hypothesis 1) showing the
hydrogen bond acceptors (green), hydrogen bond
donor(pink) and the hydrophobic feature (light
blue). Alignment of the compound (b) ND-ADH-UA,
(c) ND-ADH-5FU and (d) ND-ADH-CUR on the
common feature pharmacophore model. (For inter-
pretation of the references to colour in this figure
legend, the reader is referred to the web version of
this article.)

Table 5 Ellipses indicate the absorption model at 95% and 99% confidence limit
Predicted fit values of ND-Drug conjugates from the hypothesis Hypo 1. to the BBB and intestinal absorption models. The plot of polar surface
Name Fit Value Pharmprint
area and ALogP for ND-Drug conjugates are represented in Fig. 8.

ND-ADH-UA 3.612 ‘111111’ 3.11. DNA fragmentation analysis

ND-ADH-5FU 2.438 ‘111111’
ND-ADH-CUR 6 ‘111111’
3.10. ADMET prediction
ADMET studies of ND-Drug conjugates predicted using ADMET
descriptor module of DS to provide insight into the pharmacokinetic
property of the compounds. All the standard parameters are examined
and given in the Table 6. With the parameters of Discovery Studio,
analysis with standard value for human intestinal absorption in level 0,
for solubility level 3 and level 4, for non-inhibitory property level 0
with CYP450 2D6, for BBB penetration level 3 and for non-toxicity level
0 were filtered for getting drug like compounds. From the analysis, all
the three ND conjugates, showed good results of the ADMET para-
meters. ADMET descriptors, the 2D polar surface area in A2 per com-
pound is plotted against their consonant estimated atom-type partition
coefficient (ALogP98). The space covered by the ellipse is a prophecy of
excellent absorption without any violation of ADMET properties.
To assess the apoptosis mechanism of tumor cell by ND-Drug con-
jugates, the DNA fragmentation analysis was performed. The cell was
treated with ND-Drug conjugate for about 48 h and then isolated the
DNA and analyzed by agarose gel electrophoresis. After 48 h incuba-
tion, the cell treated with drug showed high number of DNA ladder
formation. In the result obtained from agarose gel electrophoresis of
MCF-7 cells, internucleosomal fragmentation was observed in MCF-7
cells after 48 h of with ND-Drug conjugate treatment (Fig. 9 and
Table 7). These finding suggested that ND-Drug conjugate (ND-ADH-
CUR) is an effective inducing agent of apoptosis against MCF-7 cell
lines.
4. Conclusion
The different ND-Drug conjugates were to be fabricated to reduce
the amount of drug and to be provide maximum therapeutic effect.
These ND-Drug conjugates shows sustained release of anticancer drug

Table 6
Predicted ADMET properties of ND-Drug compounds.
Name AlogP98 PSA_2D BBB level Absorption level Solubility level Hepatotoxicity CYP2D6 PPB level

ND-ADH-UA 4.753 205.606 4 3 3 0 0 1

ND-ADH-5FU 2.946 197.439 4 3 3 0 0 1
ND-ADH-CUR 7.471 178.448 4 3 3 0 0 2

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S. Garg, et al. Diamond & Related Materials 94 (2019) 172–185

Fig. 8. Plot of polar surface area (PSA) versus ALogP for ND-Drug conjugates showing the 95% and 99% confidence limit ellipses corresponding to the blood brain
barrier (BBB) and intestinal absorption.

Fig. 9. Agarose Gel Electrophoresis of ND-Drug conjugates nanocomplex (DNA Fragmentation study). 1: Marker, 2: Control, 3: ND-ADH-UA (100 μM), 4: ND-ADH-
5FU (100 μM), 5: ND-ADH-CUR (50 μM), 6: ND-ADH-CUR (100 μM).

Table 7
Agarose gel electrophoresis of ND-Drug Complex.
S. No Well
1 1
2 2
3 3
4 4
5 5
6 6
due to the hydrophilic layer of nanodiamond over the drug en-
capsulated in it. The zeta potential studies also revealed the higher
Sample
stability of nanodiamond conjugates because of their higher negative
surface charge. Cytotoxicity analysis revealed that ND-Drug conjugates
Marker showed potent cytotoxic effect against MCF-7 and Hep-G2 cancer cell
Control lines. After performing all the analysis it was accomplished that the ND-
ND-ADH-UA (100μM)
ND-ADH-5FU (100μM)
Drug conjugate system possess as potent agent for anticancer drug de-
ND-ADH-CUR (50μM) livery vehicle. As result obtained from docking and pharmacophore
ND-ADH-CUR (100μM) mapping analysis it was also suggest that the ND-Drug conjugates
possess effective interaction with significant proteins and the result
obtained from pharmacophore mapping also specify that the prepared

184
S. Garg, et al.
ND-Drug conjugates were specifically matched with hypo 1 model with
good fit value. This finding may suggest that the ND-Drug conjugates
may be a potential inhibitor of MCF-7 and Hep-G2 cancer cells to act as
a drug candidate. According to all these data it can be confirm that the
ND-Drug conjugates could be an effective agent for drug delivery and
could be promising in future for tumor targeting strategy.
Acknowledgments
The author acknowledges SAIF-CDRI (Lucknow, India) for facil-
itating the 1H NMR spectroscopy and ESI-MS analysis. The author put
across his cordial thanks to SAIF-MGU, Kottayam, India for permitting
the AFM facility and Diya lab, Mumbai for providing SEM facility and
DSC analysis facility. The author express thanks to SAIL SOPS RGPV
situated in Bhopal, India for providing FTIR, particle size determination
and zeta potential facilities. The author expresses his sincere thanks to
Advanced Centre for Treatment, Research and Education in Cancer
(ACTREC), Tata Memorial Centre (Navi Mumbai), India for granting in-
vitro SRB assay for anti-cancer activity analysis of conjugates. The au-
thor also express his thanks to Dr. Sreenivas Enaganti, Averin Biotech
(Hyderabad), for providing Molecular docking, pharmacophore map-
ping and ADMET study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.diamond.2019.03.008.
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