Design and development of nanomaterials for biomolecular detection and cancer therapy

Design and development of nanomaterials for
biomolecular detection and cancer therapy
Arunkumar Rengaraj
Supervised by
Yun-Suk Huh, PhD
1Department of Biological Engineering,
INHA University, Korea
Nano
Bio engineering
Analysis
Laboratory

Introduction
Nanomaterials for biomolecular detection
 Graphene-protamine conjugate for the sensing of heparin
 Graphene/NiO composite for Cholesterol sensing
NBA Nano/Bio engineering/Analysis LaboratoryL
Outline
Nanomaterials for cancer therapy
 NMOF/PEG~DOX for drug delivery and ROS therapy
 NCTP~DOX for drug delivery for cancer senescence
 PAMAM/5-FU conjugate for targeting E6&E7 oncoproteins
Conclusion

Part 2. Nanomaterials for biomolecular detection
Where is part 1

Biomolecular detection
Biomolecules Effect over body
keywordsuggest.org

Diseases related to metabolic disorders Diabetes cases in developing countries
https://medicalxpress.com http://blogs.reuters.com

Conventional Diagnosis method – Biochemical assay
 It is an analytical in vitro procedure used to detect, quantify and/or
study the binding or activity of a biological molecule.
Problems in current diagnosis methods
 In vitro procedure.
 Required more amount of time and money.
 Need expertise to do the diagnosis.
 Most of the methods are qualitative.
https://www.thermofisher.com

Advantage of electrochemical sensing
 Highly specific.
 Linear response, Tiny & biocompatible.
 Easy to use.
 Required only small sample volume.
Electrochemical detection
Electrochemical sensor Nanomaterial based Electrochemical sensor
Advantage of nanomaterial-enhanced
electrochemical sensing
 Larger surface area for functionalization.
 High conductivity.
 High Physicochemical stability.
O Valappil M, Alwarappan S, N Narayanan T. Atomic Layers in Electrochemical Biosensing
Applications-Graphene and Beyond. Current Organic Chemistry. 2015 Jun 1;19(12):1163-75.

GIVE OBJECTIVE OF YOUR WORK – INCLUDE OBJECTIVE SLIDE

Part 2.1. Reduced graphene oxide/gold-
protamine composite for electrochemical
detection of Heparin

TEM image a) GO b) GO/Au and SEM image c) GO d)
GO/Au .
Our strategy
Advantage
 GO/Au provides fast and direct electron transfer between
electrode and biomolecule.
 Au-NPs help for the to anchor protamine over the
biosensor.  Uniformly decorated gold on the surface of the GO.
Heparin sensor
Sensor fabrication Morphology of the composite
Give title

a) XRD b) Raman c)FTIR analysis for GO & GO/Au, d)XPS
survey data for GO/Au.
Spectroscopical analyses
 XRD confirms the presence of Au on GO.
 Raman spectrum shows the defect introduced in GO
due to Au incorporation (small change in ID/IG).
 What FT-IR tells
 XPS survey spectra reveals the binding of Au on the
GO.
Heparin sensorNanomaterials for biomolecular detection
Characterization
Inferences:

Heparin sensor
 GO, GO/Au produce very small lesser resistance as
compared to than that of bare GC.
 GO/Au-Protamine-Heparin shows higher resistance
(blocking of electron transfer).
Optimization
Conformation of the fabrication process Stability of the biosensor
 Good electrochemical stability – confirmed by liner
increase in oxidation and reduction potential.
 Irreversible interaction between protamine and
Heparin (Ks = 0.5 s-1 from Epa vs. log v plot)
Impedance analysis
Title missing

Heparin sensing using a) CV, c) DPV and e) Impedance.
Electrochemical analysis
Heparin sensor
 Heparin exhibited different detection limits in CV and DPV analysis
 CV analysis:
Detection limit: 1.5 nM to 0.25 µM and R2 = 0.999
 DPV analysis:
Sensing range: 0.2 V
Detection limit: 1.59 nM to 19 µM and R2 = 0.992
 Current decreases with increasing heparin concentration
 Different electrochemical methods exhibited different range of heparin detection.
 There is a decreasing current in each increase in concentration heparin.
 CV shows oxidation (0.4 V), reduction (0.2V) peaks .
 CV shows the detection limit (1.5 nM to 0.25 µM) with the R2 value of 0.999.
 In DPV the sensing range was set at 0.2V.
Title missing

Heparin sensorNanomaterials for biomolecular detection
Conclusion
 GO, Au-NPs provided large specific surface area, which greatly
increase the probability of Protamine-heparin interactions.
 The GO/Au played an important role in establishing a fast
electron transfer path to facilitate direct electron transfer.
 An affinity-sensor for heparin based on GO/Au-protamine was
proposed and exhibited low detection limit, good sensitivity, and
excellent selectivity.
Heparin sensing using a) stability, b) Selectivity
Sample Heparin concentration (µM) Recovery (%)
Added Founda
1 1.00 1.05 ± 0.05 105
2 5.00 4.80 ± 0.12 96
3 10.00 9.25 ± 0.23 93
4 15.00 12.98 ± 0.5 87
Heparin sensing using in FBS
Stability and selectivity
Title missing

Part 2.2. Electrodeposition of flower-like nickel oxide
on CVD-grown graphene to develop an
electrochemical non-enzymatic biosensor

Raman spectra of (a) graphene, (b) the NiO/graphene nanocomposite,
(c) the 2D band of graphene fitting d) XRD of the nanocomposite.
Our strategy
Advantages
 Non enzymatic : NiO (intermediate)
 Few layers graphene – High electron transfer rate
with low resistance.
 I2D/IG ratio of the as–synthesized graphene was calculated
as 2.1.
Cholesterol sensor
Layers of graphene and crystallinity of NiO
Figure caption Missing

SEM images, elemental mapping of the NiO/graphene
nanocomposite.
Characterization
Cholesterol sensor
Flower-like NiO nanostructures
XPS Survey, N2p, C1s, and O1s core–level spectra of the
nanocomposite.
 The survey spectrum exhibited C1s (285.0), O1s (531.8), and
Ni2p (851.9 eV).
Binding energy of graphene and NiO

CV curve for graphene and compositeat a scan rate of
100 mV/s in 1 M KOH.
Optimization and sensing of cholesterol
Cholesterol sensor
 NiO/graphene showed
 higher current due to the high electrocatalytic
activity of the NiO and
 large active surface area and better
Cholesterol sensing
 Linear increase in oxidation, reduction potential with
increase in concentration of cholesterol.
Higher electrical conductivity of NiO/graphene

Selectivity and stability of the sensor
Cholesterol sensor
(a) Choroamperometric data (b) plot of peak current
versus concentration, and (c) interference test of the
nanocomposite (20 mM cholesterol in the presence of 20
mM glucose, 20 mM AA, 20 mM UA, 20 mM DA, 40 mM
NaCl, 40 mM uracil, and 20 mM Trp)
Stability of the electrode
Sample Diluted
sample (mM)
Spiked
(mM)
Detected
(mM)
RSD (%) Recovery
(%)
1 0.2 0.5 0.67 2.14 95.7
2 0.13 0.3 0.42 1.32 97.7
3 0.08 0.2 0.275 1.87 98.2
4 0.05 0.15 0.204 1.19 102
Cholesterol detection in milk sample
Interference analysis

Conclusion
Cholesterol sensorNanomaterials for biomolecular detection
 We successfully fabricated a NiO/graphene nanocomposite by electrodeposition of NiO onto the surface of CVD-
grown graphene, which was then used to investigate the non–enzymatic detection of cholesterol.
 The composite electrode detected a range of cholesterol concentrations from 0.5 mM to 2 mM with high sensitivity.
Additionally, a low detection limit and rapid response time of 0.13 mM and 5 s, respectively, were observed.
 Moreover, interference materials (AA, UA, uracil, glucose, Trp, DA, and NaCl) exerted no significant effects on the
detection of cholesterol.
 The high sensitivity of cholesterol could be attributed to the excellent electrocatalytic activity of NiO and the large
specific surface area of the graphene.

Part 3.1. Nanomaterials for cancer therapy

 Drug delivery refers to approaches, formulations
technologies, and systems for transporting a
pharmaceutical compound in the body as needed to
safely achieve its desired therapeutic effect.
Drug delivery
Solution Suspension
Ointment Tablets
Routes of drug delivery
 Based on the condition of patient
 Requirement of drug dosage
http://www.singhvaid.com/tag/routes/

Drug delivery
Problems with the conventional drug delivery system Comparison between Conventional, controlled drug delivery system
https://www.studyblue.com/notes/note/n/modified-release-drug-delivery-
systems
 Inconvenient.
 Inactivation by gastric juice.
 Difficult to monitor.
 Metabolism before reaching target cell.
 Too many adverse reactions.
 Rapid clearance of targeted systems.

Drug delivery
Nano-formulation for the controlled drug delivery system
Sun W, Hu Q, Ji W, Wright G, Gu Z. Leveraging Physiology for Precision
Drug Delivery. Physiological Reviews. 2017 Jan 1;97(1):189-225..
Nanocarriers for drug delivery
Sagnella SM, McCarroll JA, Kavallaris M. Drug delivery: beyond active tumour targeting.
Nanomedicine: Nanotechnology, Biology and Medicine. 2014 Aug 31;10(6):1131-7.

Drug delivery
Cancer Cancer therapy
Oral cancer
http://www.fhshh.com
http://www.rahmacancercare.com http://www.universitycancercenters.com
Surgery: Infected cell of cancer removed.
Radiation: High-energy particles or waves, such as x-rays, gamma rays, electron
beams, or protons, to destroy or damage cancer cells.
Hormone therapy: Reduce or increase particular hormone in order to treat cancer.
Chemotherapy : Using a drugs to treat cancer cells.
Targeted therapy : Using monoclonal antibody to treat cancer.

Drug delivery
Nanocarriers for cancer therapy Our approach to utilize nanomaterials for cancer therapy
Conde J, Tian F, Baptista PV, de la Fuente JM. Nano-Oncologicals: New Targeting
and Delivery Approaches. Advances in Delivery Science and Technology. 2014:123-
60.
Porous nanoscale NH2-MIL-125 as a potential platform for
drug delivery, imaging and ROS therapy utilized Low Intensity
Visible light exposure system
Porous Covalent Triazine Polymer as a Potential Nanocargo
for Cancer Therapy and Imaging
PAMAM/5-flurouracil drug conjugate for targeting E6 and
E7 oncoproteins in cervical cancer: a combined
experimental/in silico approach

Part 3.2. Porous NH2-MIL-125 as an efficient nano-
platform for drug delivery, imaging and ROS therapy
utilized Low-Intensity Visible light exposure system
http://www.breastlink.com/breast-cancer-101

Cancer drug delivery
 MOFs are porous crystalline materials whose framework is constituted
of metal ions or metal ion clusters occupying nodal framework positions
coordinated by di-, tri- or multipodal organic ligands.
 Metal-organic interactions occur between metal and heteroatoms such
as nitrogen, oxygen, sulphur and phosphorus.
MOF- Drug deliveryNanomaterials for cancer therapy
Synthesis of NH2-MIL-125 through solvothermal method
What are MOF ? Fabrication of NMOF/PEG~ DOX

MOF- Drug delivery
Synthesis of NMOF/PEG under sonication at different
time interval (5-20min).
Morphology of MOF, NMOF and NMOF/PEG
PEG was strongly encapsulated into the MOF due to the hydrophobic
interaction of PEG with organic linkers in the MOF and (ii) ionic
interactions between the hydroxyl group of the PEG and Ti metal ions
in the MOF.
Sonication driven NMOF synthesis

MOF- Drug delivery
Characterization and properties of MOF, NMOF, NMOF/PEG and NMOF/PEG-DOX. a)
XRD patterns, b) BET analysis for the surface area of the materials, c) TGA analysis,
and (d) UV-Vis absorbance and photoluminescence spectra (insert) of NMOF/PEG u
nder different excitation.
 XRD shows there is no changes in crystallinity.
 BET surface area of NH2-MIL-125 was 1540 m2 g−1 with a pore size
of 0.63 nm.
 After sonication, the surface area of NMOF increased to 1720 m2
g−1.
 After the PEGylation process, there was a considerable decrease in
surface area to 155 m2g−1.
 The UV absorption spectrum (Fig. 1d) of MOF shows the absorbance
at approximately 340 nm, attributed to the n–π* transition of the
nitrogen lone pair electron in 2-aminoterephthalic acid.
Crystalline, high surface area, biocompatible NMOF

Biocompatibility & Nanocarrier tracking
MOF- Drug delivery
 NMOF/PEG exhibited less toxicity than MOF, NMOF
at all four concentrations (10-200 µg/ml).
Our approach
Low-intensity visible light system for cancer treatment
http://hospitals.jefferson.edu

Drug adsorption, release and ROS induction by NMOF/PEG
MOF- Drug delivery
NMOF/PEG a) Drug (DOX) adsorption, b) pH sensitive & photosensitive
drug release c) ROS level at different concentrations of NMOF/PEG upon
constant irradiation and d) Cell viability (MCF-7) of NMOF/PEG-DOX with
and without irradiation.
 Drug molecules interacting with the nanoparticle matrix by the hydrophobic
and H-bonding interactions between the DOX and organic linker of the
nanoparticles.
 The observed DOX loading was saturated of 200µg/mg on NMOF/PEG.
 The adsorption of drug molecules into the NMOF/PEG was characterized by
FT-IR, BET surface area, and DLS.
 50µg/ml NMOF/PEG/DOX (10 µg of DOX-loaded in NMOF/PEG) killed
more than 80% of MCF-7 in the absence of irradiation.

MOF- Drug delivery
Optical microscopy images of MCF-7 cells under different conditions: (a)
untreated, (b) NMOF/PEG (c) DOX-loaded NMOF/PEG, and (d) incubated
with DCFDA of DOX-NMOF/PEG after irradiation. The left column shows
the bright field images. The central 2 columns are the fluorescence images
of NMOF/PEG and DOX channels. The right column presents overlay
images of the bright field and fluorescence images. The fluorescence
images were acquired by fluorescence microscopy under an excitation
range of 330–380nm and 450–490nm for the NMOF/PEG and DOX
channels, respectively.
Laser confocal imaging

 We successfully synthesized NMOF/PEG and used it as both a potential photosensitizer and pH-responsive nanocarrier
for cancer therapy and imaging.
 NMOF/PEG with pH-dependent drug releasing properties was suitable for drug delivery over cancer cells.
 The NMOF/PEG-DOX with ROS induction exhibited a higher cytotoxic effect on cancer cells than the NMOF/PEG-
DOX and DOX alone.
 The NMOF/PEG–DOX complex features a DOX-loading capacity of 200 µg/mg due to its high specific surface area, π-π
stacking and hydrophobic interaction.
 Our results indicate the potential for use of NMOF/PEG as a low-toxic and biocompatible 2D nanomaterial for cancer
diagnosis and therapy.
Conclusion
MOF- Drug deliveryNanomaterials for cancer therapy

Part 3.3. Porous Covalent Triazine Polymer as a
Potential Nanocargo for Cancer Therapy and
Imaging
http://medimyth.blogspot.kr/2013_12_01_archive.html

NCTP- SenescenceNanomaterials for cancer therapy
What are COF ?
 The CTP is a sub-class of POPs, it has high nitrogen contents and free
lone pair of electrons, which provide high electron density in their
network.
 Covalent triazine polymer (CTP) was successfully synthesized via the
Friedel-Crafts reaction.
 These free electrons interact easily with drug compounds, making
electrophilic substitution easier.
Strategy employed to synthesize NCTP-DOX
 The CTP was broken into NCTP using sonication process.

(a) FTIR, (b) BET, (c) zeta potential, and (d) PL analysis of NCTP.
NCTP- Senescence
 The bands at 1610, 1519, and 1369 cm-1 were assigned to C=C, C=N,
and C-N stretching vibrations of the triazine rings in the polymer
network, respectively.
 The BET surface area of NCTP was 1220 m2/g, which was evaluated
from the 0.01 < P/P0 < 0.05 region of the adsorption curve.
 The first weight loss (around 10%) step in the TGA curve occurred at
below 120°C and was attributed to the loss of water. A second rapid
major mass loss occurred at 350–600 ᵒC and was assigned to the
decomposition of NCTP.
 Fluorescence at 700nm upon excitation at 350nm
Spectroscopic analysis

SEM and TEM analysis of CTP (a, d) before sonication, (b, e)
after sonication, and (c, f) after filtration.
Morphology of CTP, NCTP Biocompatibility of NCTP
Cytotoxic study of the NCTP (a) COS-7 and (b) HeLa cells.
 No significant loss of cell viability was observed, even
though we increased the NCTP concentration to 500 mg/mL.

(a) Loading efficiency of DOX, (b) pH induced controlled
drug release, and (c) mechanism for pH induced drug
release.
Drug adsorption, pH sensitive drug release Confocal imaging
(a) Optical microscopic images, (b) confocal fluorescence microscopy
images, and (c) overlay of HeLa cells before (i) and after (ii, iii) being
incubated for 24 h with NCTP and NCTP-DOX.

Cytotoxicity and Bio-TEM
Relative cell viability of (a) COS-7 and (b) HeLa cells treated
with free DOX and NCTP-DOX at various concentrations,
and (c) Bio-TEM images of (i) control and (ii) NCTP-DOX
treated HeLa cells after 6 h.
Cancer senescence
SA β-galactosidase assay for NCTP-DOX (a) before staining, (b) after
staining and protein expression of (c) DOX and (d) NCTP-DOX on
HeLa cells.

 We successfully synthesized NCTP and used it as both a potential photosensitizer and pH-responsive nanocarrier
for cancer therapy and imaging.
 NCTP with pH-dependent drug releasing properties were suitable for drug delivery over cancer cells.
 The NCTP-DOX exhibited a higher cytotoxic effect on cancer cells than free DOX and induced strong senescence
at lower concentration.
 The NCTP–DOX complex features a DOX-loading capacity of 200 mg/g due to its high specific surface area, π-π
stacking and hydrophobic interaction.
 Our results indicate the potential for use of NCTP as a low-toxic and biocompatible 2D nanomaterial for cancer
diagnosis and therapy.
Conclusion

Part 3.4. PAMAM/5-flurouracil drug conjugate for
targeting E6 and E7 oncoproteins in cervical
cancer: a combined experimental/in silico
approach
Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S.
Human papillomavirus and cervical cancer. The Lancet. 2007 Sep
14;370(9590):890-907.

PAMAM/5-FU for targeting E6 & E7Nanomaterials for cancer therapy
Our approach
Cervical cancer caused by HPV infection
http://www.mun.ca/bi
ology/desmid/brian/BI
OL2060/BIOL2060-
24/CB24.html
 HPV virus produce E6 & E7 protein inside the host.
 E6 & E7 protein inhibit P53,pRb protein in human and cause cancer.
 5-FU widely used to target E6 & E7 protein.
 The interaction between drug and oncoprotein is week.
 To increase avidity, we utilized Polymer/Drug conjugate (PAMAM/5-
FU)

Protein, ligand retrieval and preparation Molecular docking
PAMAM/5-FU for targeting E6 & E7

Molecular docking analysis. (a, d) conformational structure (b,
e) interaction profile, and (c, f) RMSD plot of E6 and E7
protein over 5-FU.
Interaction between drug (5-FU) With oncoproteins (E6,E7)
Interaction between Polymer/drug (PAMAM/5-FU) With
oncoproteins (E6,E7)
Molecular docking analysis. (a, d) conformational structure (b, e)
interaction profile, and (c, f) RMSD plot of E6 and E7 protein over
PAMAM/5-FU conjugate.

S. No
Dendrimer
generations
0.01M PAMAM dendrimer
Ethylene
diamine (mL)
Methyl
Acrylate
(mL)
1 0.5G 0.67 7.21
2 1.0G 5.36 -
3 1.5G - 14.42
4 2.0G 10.71 -
5 2.5G - 28.85
PAMAM/5-FU for targeting E6 & E7Nanomaterials for cancer therapy
Synthesis of different generation of PAMAM Characterization
(a) FTIR spectrum, (b) 1HNMR spectrum, (c) TEM image of 1.5G
PAMAM, and (d) viscosity of different generations of PAMAM.

Cytotoxicity of different generations of PAMAM nanoparticles,
(b) drug adsorption study, (c) drug release behaviour of 1.5G
and 2.5G PAMAM nanoparticles, and (d) stability of PAMAM/5-
FU formulation at a different temperature.
Biocompatibility, drug adsorption and drug release
Interaction of PAMAM/5-Fu with HeLa cell line
Confocal imaging of (a) DAPI stained HeLa cell, (b) FITC-P
AMAM/5-FU (c) FITC–PAMAM/5-FU in HeLa cell, and (d) FIT
C–5-FU in HeLa cell.

(a) Cytotoxicity analysis of 5-FU and PAMAM/5-FU conjugate
over HeLa cells, (b) Western blotting to evaluate the effects
of 5-FU and PAMAM/5-FU conjugate over tumor suppressor
proteins
Inhibition on tumor suppressor proteins(E6 &E7),
hemocomtability Conclusion
 Molecular docking analysis of PAMMA-5FU with the oncoproteins (E6
and E7) revealed excellent downregulation activities
 1.5G and 2.5G PAMAM for the encapsulation of 5-FU for controlled drug
delivery.
 Hematological analysis of the animals confirmed that the PAMAM/5-FU
exhibited fewer side effects than 5-FU.

Conclusion
 Nanomaterials became important components in bioanalytical devices and drug delivery.
 Since they clearly enhance the performances in terms of sensitivity, detection limits in sensors and showed enhanced
drug delivery performance with other functionalities (Bioimaging, combinational therapy).
 Furthermore, the combination of different nanomaterials, organic-inorganic increase, even more, the performances of
biosensors, drug delivery.
 Due to the vast number of different nanomaterials all with its own specific properties, only a few examples could be
demonstrated in this thesis here by emphasizing the principal advantages of such materials.

Publications
1. Reduced graphene oxide /Gold-protamine composite for electrochemical detection of Heparin. (Under preparation)
2. Arunkumar Rengaraj, Pillaiyar Puthiaraj, Nam Su Heo, Hoomin Lee, Soonjo Kwon, Wha-Seung Ahn,b and Yun Suk Huha. “Porous nanoscale NH2-MIL-
125 as a potential platform for drug delivery, imaging, and ROS therapy utilized Low-Intensity Visible light exposure system”. (Under Review).
3. Arunkumar Rengaraj, Subbiah Balaji, Yuvaraj Haldorai, Dhanusha Yesudhas, Nam-Soo Heo, Soonjo Kwon, Sangdun Choi, Young-Kyu Han, N. Hema
Shenpagam and Yun Suk Huh. “PAMAM/5-flurouracil drug conjugate for targeting E6 and E7 oncoproteins in cervical cancer: a combined experimental/in
silico approach”. RSC Advances, (2017).
4. Arunkumar Rengaraj , Pillaiyar Puthiaraj, Yuvaraj Haldorai, Nam Su Heo, Seung-Kyu Hwang, Young-Kyu Han, Soonjo Kwon, Wha-Seung Ahn, Yun Suk
Huh. “Porous Covalent Triazine Polymer as a Potential Nanocargo for Cancer Therapy and Imaging." ACS applied materials & interfaces (2016): 8947-8955.
5. Arunkumar Rengaraj, Yuvaraj Haldorai, Cheol Hwan Kwak, Seungbae Ahn, Ki-Joon Jeon, Seok Hoon Park, Young-Kyu Han, Yun Suk Huh.
“Electrodeposition of flower-like nickel oxide on CVD-grown graphene to develop an electrochemical non-enzymatic biosensor”, J. Mater. Chem. B, 2015,
3, 6301-630.

Group members
Yun Suk Huh’s group
Phone : +82-32-860-9177/ Fax : +82-32-872-4046
E-mail : yunsuk.huh@inha.ac.kr
Inha University

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