This presentation deals with the brief study of functionalized graphene oxide nanoparticles for the delivery of model anticancer drug. It represents benefits of targeted drug delivery over the conventional drug delivery, It is one of the remingtons journal club seminar which is part of masters degree progam in pharmacy.

1. Remington’s Journal Club 2020-21
H R Patel Institute of Pharmaceutical Education and Research, Shirpur, 425405 (M S).
Ananya Deba, Vimala R.b,*
Journal of Drug Delivery Science and Technology
Elsevier Publication
Impact factor: 3.981
Camptothecin loaded Graphene Oxide nanoparticle Functionalized with
Polyethylene glycol and Folic acid for Anticancer drug delivery
Mr. Gaurav Shriram Patil
M Pharm II Year
Dept. of Pharmaceutics
HRPIPER, Shirpur.
Dr. G. B. Patil
Assistant Professor
Dept. of Pharmaceutics
HRPIPER, Shirpur.
Presented by- Guided by-
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2. Abstract
Introduction
Significance/Rationale of Research work
Methodology (Materials and Methods)
Results and Discussions
Conclusions
References
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Table of content

3. List of Abbreviations
Abbreviations Read as
CPT Camptothecin
GO Graphene oxide
FA Folic acid
PEG Polyethylene glycol
XRD X-ray diffraction
SEM Scanning electron microscopy
TEM Transmission electron microscopy
FT-IR Fourier transform infrared spectroscopy
MCF-7 Michigan Cancer Foundation-7
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
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5. Abstract
• Graphene oxide nanomaterials are widely used to achieve effective cancer treatment.
• In this study, a novel graphene oxide mediated drug delivery system was synthesized by combining
anticancer drug camptothecin (CPT) to the large surface area of graphene oxide by π-π stacking.
• Initially graphene oxide was synthesized by hummer's method and to obtain a more precise drug
delivery, the system was loaded onto polyethylene glycol (PEG) decorated with folic acid.
• The resultant conjugate was characterized by Fourier Transform Infrared spectroscopy (FTIR), X-
Ray Diffraction (XRD) analysis, UV-visible Spectrophotometery, Scanning Electron Microscopy
(SEM) and Transmission Electron Microscopy (TEM).
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6. • The graphene-oxide -polyethylene glycol-folic acid - camptothecin (GO-PEG-FA-CPT) drug
delivery system showed a pH dependent drug release as observed by UV analysis.
• The anticancer activity of the synthesized drug delivery system was studied by MTT assay using
MCF-7 breast cancer cell lines. The conjugate showed enhanced anticancer activity and thus could
be used as a potential candidate for drug delivery.
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7. Introduction
• Cancer is a life threatening disease which leads to abnormal growth of cells with the `potential to invade other
parts of the body.
• The major cause of cancer related deaths in economically developed countries are due to the adoption of
lifestyle which includes physical inactivity, smoking, drinking, and westernized diets [1].
• The presently available chemotherapeutic drugs are low molecular weight agents with high pharmacokinetic
volume of distribution both of which contributes to their cytotoxicity[2].
• These drugs lack specificity and cause significant damage to noncancerous cells. In this scenario
nanomaterials could be good allies to give more specific cancer treatment effectively reducing undesirable
side effects and accurate diagnosis and successful therapy.
• The commonly available nanoparticles involved in drug delivery are liposome mediated, dendimers, carbon
nanotubes, gold nanoparticles etc.
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8. • Allotropes of carbon viz. Carbon nano tubes (CNT), multiwalled carbon nanotbes (MWCNT), nanohorns,
nanodiamonds and a newly developed allotrope of carbon “graphene” has gained tremendous attention in the
scientific community due to its brilliant features.
• Graphene has a wide number of biomedical applications due to its large surface area, electrostatic
interactions, thermal and electrical conductivity and easy functionalization due to the presence of π-π
interaction [3].
• In order to impart solubility to the drug, prevent aggregation of blood cells and increase the biocompatibility
and bioavailability of the drug, graphene oxide is conjugated with various polymers [4–6].
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9. • PEG is considered as a smart polymer and is extensively preferred by the pharmaceutical companies for drug
delivery due to its enhance bioavailability, non-immunogenic and highly soluble properties.
• Further the overexpression of folate receptors on cancer cells has been exploited to achieve a precise drug
delivery by decorating the drug carrier with folic acid [7].
• The nontoxic, non-immunogenic and stable properties of folic acid makes it a suitable candidate for
conjugation with nanocomposites [8].
• The present study focuses on obtaining targeted medication delivery utilising camptothecin as a model drug
that targets topoisomerase-I, thereby stabilising the cleavable complex between DNA and topoisomerase I
[9–12].
• Graphene oxide conjugated nanoparticle were synthesized and characterized by UV, XRD, FTIR, SEM and
TEM. Cellular toxicity has been investigated using MTT assay and RBC rupture by GO was analyzed using
hemolysis assay.
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10. Rationale of research
• Conventional drug delivery systems and treatment approaches of have several limitations, including low
solubility of small molecules, rapid metabolism and elimination of drugs, failure to attain desired target site
concentration , cytotoxicity etc.
• In the present scenario many conventional anticancer drugs lack of specificity and cause significant damage to
healthy cells.
• To address above limitations the use of nanomaterials in cancer therapy has led to some significant edge
research during the last few years.
• To overcome these issues at some extent, authors was hypothesized the PEGlyated GO nanoparticles for the
effective and site specific delivery of drug to the cancerous cells.
• Thus the following researched worked had been undertaken to fabricate PEGlyated graphene oxide loaded
with anticancer drug upon administration for the potential treatment of breast cancer.
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11. Methodology (Materials and methods)
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Sr. No List of the materials Materials procured from
1 Graphite powder Sigma Aldrich
2 Ethanol Sigma Aldrich
3 N-(3-dimethylaminopropyl-N-
ethylcarbodimide) hydrochloride
Sigma Aldrich
4 Polyethylene glycol bis (amine) Sigma Aldrich
5 N-Hydroxysuccinimide Sigma Aldrich
6 Folic acid Sigma Aldrich
Materials:
Methods:
1. Synthesis of Graphene oxide [13-14]
2. PEG ylation of graphene oxide (GO-PEG) [15-16]
3. Loading of drug
4. Characterization

12. 1. Synthesis of Graphene oxide 2. PEGlyation of Graphene oxide
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Purified graphene oxide
GO-COOH
Dissolved in 100 ml DIW & ultrasonicated
Nano graphene oxide
PEG lyation achieved by addition NaOH
and Chloroacetic acid to GO soln &
sonicated for 3 h
GO-PEG
1. EDC was added to above soln and
sonicated for the five minutes
2. PEG-NH2 was added at
last & stirred for 24 h
0.5% folic acid was added
to soln for target
specification

13. 3. Loading of the drug:
• Anticancer drug Camptothecin (CPT) 20 mg was loaded onto GO-PEG-FA complex, simply by mixing CPT to GO-
PEG-FA solution followed by overnight stirring.
• The unbound CPT was removed by washing at 9000 rpm at room temperature for 20 min and filtration by 10 KDa
filter paper.
• The resultant GO-PEG-FA complex was re-suspended and stored at 4o C [13].
4. Characterization:
• The chemical structure of the composite was analyzed by Fourier transform infrared spectroscopy ( IR Affinity-1,
Shimadzu FT-IR spectrophotometer) and UV-visible spectrophotometry.
• Pure phase identification of the synthesized GO and its composite was achieved by the X-ray diffractometer (XRD)
(Bruker, D8 Advanced Germany)
• Morphological characterization was done by the Scanning eletron microscope and transmission electron microscope.
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14. Results and discussions
Synthesis, Functionalization and Characterization:
• GO nanoparticles where successfully synthesized by modified hummer’s method and the obtained
GO was converted into nano graphene oxide by sonication.
• Functionalization of GO was carried out using PEG and FA, lastly CPT was loaded onto the
complex and finally GO-PEG-FA-CPT complex was obtained.
• The formed complexes (GO, GO-PEG, GO-PEG-FA and GO-PEG-FA-CPT) was subjected to
variety of evaluation parameters.
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Fig. 1. Showing UV visible absorption of GO GO-PEG, GO-PEG-FA and GO-PEG-FA-CPT. The loading of CPT was evidenced by a strong absorption peak centered at 365 nm.
1. GO shows peak at 230 nm
2. GO-PEG shows peak at 287 nm
3. Addition of Folic acid indicated hump in the peak
4. GO-PEG-FA-CPT gives peaks at 380 and 365 nm resp.
1. UV-Visible spectroscopy:

16. 2. XRD study:
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Fig. 2. Showing XRD pattern of GO, GO-PEG, GO-PEG-FA and GO-PEG-FA-CPT.
Characteristic peak of GO can be
visible at 11.09o (111) d=1.14 Å
GO-PEG peaks obtained at 18.74o,
22.82o,31.28o and 45.6o
GO-PEG-FA peaks obtained at
18.72o, 23.2o,31.2o and 45.1o
GO-PEG-FA-CPT peaks obtained
at 19.36o, 23.47o,31.9o and 45.61o
and 56.7o

17. 3. FT-IR study:
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Fig. 3. Fourier transform infrared (FTIR) spectra of GO [17] and GO-PEG.
In graphene oxide the peaks observed at
• 3352.58 cm-1 (O-H) deformations,
• 1722.43 cm-1 (C=O),
• 1599 cm-1 (C=C),
• 1369.46 cm-1 (C-O) and 1039.63 cm-1 (C-O)
Whereas, GO-PEG the peaks were observed at
• 3350.35 cm-1 (O-H),
• 2231.93 cm-1 (C=N),
• 1639 cm-1 (NH-CO-),
• 1367.53 cm-1 (C-O)
These are the signature functional groups of PEG. Which
indicates successful conjugation of PEG on GO.

18. 4. Scanning electron microscopy (SEM) :
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Fig. 4. SEM images showing (a) graphene oxide (GO) and (b) polyethylene glycol loaded graphene oxide (GO-PEG).
Scanning electron microscopy images (fig 4a)
shows typical wrinkled and crumpled structure
Width: 10.5 mm
As PEG incorporated on GO it results into the
matrix like structure as shown in fig 4b.
Width: 11.5 mm

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TEM images of GO was found to be
multilayered and wrinkled.
Size of the particles: <100 nm at
accelerating voltage of 200 KV
The conjugation of PEG to GO led to an
increase in the size of nanoparticles from
100-200 nm when viewed under
accelerating voltage of 200 KV fig
5. Transmission electron microscopy (TEM):
Fig. 5. TEM images and SAD pattern showing (a) graphene oxide (GO) and (b) polyethylene glycol loaded graphene oxide (GO-PEG).

20. 6. Drug loading and drug release :
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Fig. 6. Drug release of GO and GO-PEG at (a) pH 5.3 and (b) pH 7.4
Drug loading efficiency was calculated to be 45%
calculated using formula :
Loading efficiency LE= total amount of CPT
unbound/ total amount of CPT *100
Drug release from Drug release %
(at pH 5.3)
Drug release %
(at pH 7.4)
GO-CPT 72.6% 50.3%
GO-PEG-FA-CPT 51% 43%
Conclusion: The conjugation of GO with PEG and FA has
led to a controlled drug release. This was evident from the
difference in the drug release noticed with the conjugated and
non-conjugated graphene oxide nanoparticle which are
depicted in Fig. 6a and b. [17]

21. 7. In vitro cytotoxicity studies: Performed using MTT assay[11]
• In vitro cell viability of GO, GO-PEG, and GO-PEG-FA
were tested against breast cancer cell llines (MCF-7)
• Viability: 95% at concentration of 100 ug/ml indicating low
toxicity of GO, GO-PEG, and GO-PEG-FA against MCF-7
cell lines (fig. 7a)
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• To check chemotherapeutic activity GO-PEG-FA complex
was conjugated with CPT and inhibition on growth of MCF-7
cell lines was observed at various concentrations fig 7b.
• At 20 ug/ml % of inhibition rate of CPT complex found to be
15.12% and at same conc. the % of inhibition of GO-PEG-
FA-CPT complex was found to be 37.7%
• At 100 ug/ml 53.5% of inhibition rate was observed for free
CPT , whereas for GO-PEG-FA-CPT complex it is around
79.92% [17].
Fig. 7. (a) Cell viability of MCF-7 cells with different concentrations of GO, GO-PEG and GO-PEGFA. (b)Percentage inhibition of MCF-7 cells for 24 h after treatment with GO-PEG-FA CPTand free CPT.

22. 8.Hemolysis Assay:
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• The ability of GO and GO-PEG to hydrolyze red
blood cells (RBC) was compared.
• The RBC membranes were incubated with
different concentrations of GO and GO-PEG.
• Both GO and GO-PEG shows a dose dependent
hemolytic activity.
• To minimize the hemolytic activity, the large
surface area of GO was masked with PEG.
• GO PEG showed less hemolytic activity in
comparison to GO alone thus indicating that PEG
mask the hemolytic activity of GO (fig 8). [5]
Fig. 8. Percentage hemolysis of RBCs incubated with different concentrations of GO and GO-PEG.
Hemolysis %= (ODsample -ODnegative control )/ ODpositive control -ODnegative control )*100

23. Conclusion
• In summary, authors have synthesized of an efficient drug delivery system. GO-PEG-FA loaded with
anticancer drug and its cytotoxicity has been tested against breast cancer cell lines (MCF-7).
• Initially GO nanoparticles were synthesized and conjugated with PEG to increase its biocompatibility and
impart solubility to the drug. Hemolysis assay was carried out to check the biocompatibility of GO and GO-
PEG, and the results show decreased hemolysis after conjugation of GO with PEG.
• Thus the in vitro studies demonstrate the feasibility of employing functionalized GO as potential drug
delivery agent in cancer therapy.
• The present study is a part of a brief screening of polymers which can be used for the synthesis of a
nanocarrier involving graphene oxide for the delivery of anti-cancer drug. Further studies will be carried out
to demonstrate the efficiency of drug delivery by the nanocarrier under in vivo conditions.
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