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Journal of Materials Chemistry B

Graphene quantum dots conjugated albumin nanoparticles for targeted drug delivery andimaging of pancreatic cancer
Preeti Nigama*#, Shobha Waghmodeb,#, Michelle Louisc, Shishanka Wangnooc, Pooja
Chavanb, Dhiman Sarkara

Pancreatic cancer is considered to be the deadliest of all cancers due to its poor prognosis
and resistance to conventional therapies. In this study the potential of hyaluronic acidfunctionalized and green fluorescent graphene quantum dots (GQDs) labeled humanserum albumin nanoparticles for pancreatic cancer specific drug delivery and bioimaging
was explored. GQDs with their tunable fluorescence properties and biocompatibility haveattracted a lot of interest in recent years as compared to their metal semiconductor
counterparts. We adopted lawsone (2-hydroxy 1,4 naphthoquinone) as a novel, reducing
agent for synthesis of quantum dots and apart from excellent fluorescence of synthesized
GQDs, a good quantum yield of ~14% was also obtained. Gemcitabine, the most preferreddrug for pancreatic cancer treatment was encapsulated in albumin nanoparticles and it
was observed that our nanoformulation significantly enhanced the bioavailability andsustained release property of the drug to pancreatic cancer cells in-vitro. Meanwhile GQDs
mediated excellent bioimaging enhanced the efficacy of our system as drug delivery vehicle.
Pancreatic cancer originates from altered cells in pancreatic tissues. Adenocarcinoma whicharises within the exocrine component of the pancreas is the most common type of
pancreatic cancer. It possesses the worst mortality rate in all cancers and is the fourth mostcommon cause of cancer-related deaths in the United States and the eighth worldwide due to its
extremely poor prognosis in locally advanced or metastasized stage.1, 2 Moreover pancreaticcancer is resistant to most of the conventional therapies that exhibit only palliative effect withminimal impact on survival. Due to the enriched tumor stromal component and disorganizedvasculature of pancreatic cancer tissues, efficient delivery of therapeutic agents is quite difficult35

thus it is imperative to discover innovative strategies for targeted drug delivery to treat thislethal disease.
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In recent decades nanotechnology has shown great potential in the field of medicine with vast
applications in diagnostic and therapeutic agents for cancer targeting and imaging.6-8 It is a wellknown fact that chemotherapy exhibits massive side effects due to indiscriminate distribution of
anticancer drug to cancerous and healthy cells. To minimize this, researchers from all over theworld are contributing to develop nano-devices for cancer detection and delivering antineoplastic
drugs to the cancer cells. Recently Food and Drug Administration approval of albumin-taxol
nanoformulation (Abraxane-ABI 007), for the treatment of breast cancer, has boosted theresearch for the development of nanoparticle based targeted drug delivery systems.
The present study demonstrates a novel nanoformulation containing GQDs conjugatedgemcitabine loaded human serum albumin (HSA) nanoparticles based targeted delivery andimaging system for pancreatic cancer. Albumin is the most abundant protein in our body andnanoformulations based on albumin are free from immunogenic and hemolytic problems.
Moreover it is a well known fact that HSA can infiltrate tumor cells via gp60 pathway 9 thatmakes it an excellent drug delivery vehicle. Gemcitabine (Gem) is the most preferred drug forthe treatment of pancreatic cancer but unfortunately it exhibits trivial impact on lifespan due toits inadequate cellular uptake and small half life.10, 11 To overcome this dilemma, generally highand frequent doses of Gem are to be administered to the patient that often translates into systemic
toxicity and resistance towards the drug hence outshining the drug’s persuasive pharmacologicaleffects. Hong et al. discovered that CD-44 positive cells are responsible for gemcitabine
resistance in pancreatic cancer cells. CD-44 is a pancreatic cancer stem cell surface marker
which is highly expressed on human pancreatic adenocarcinoma cells and is responsible forproliferation and reconstitution of resistant pancreatic cancer cells.11 As a remedial application,
targeted therapy against CD-44 cells can conquer drug resistance in the treatment of pancreaticcancer. Hyaluronic acid (HA) is a natural polysaccharide and a major component of theextracellular matrix and synovial fluids of the body. It binds to various cancer cells that overexpress CD44, an HA receptor.12,13 HA was utilized as a targeting moiety of the HSA-Gem
conjugates for resistant pancreatic cancer therapy while green fluorescent GQDs were used forbioimaging of cancer cells.
Bioimaging or visualization of living cells is very important for medical diagnostic purposes.
Previous studies have shown the extensive use of many fluorescent agents for this purpose, on
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the other hand photobleaching and high cost of these compounds opens a gateway to find their
better substitutes. Quantum dots-nano-entities made of semiconductor materials (cadmium, zinc,selenium) are an attractive option to be explored as bio-illuminators for living cells. Although
they possess stable fluorescence, toxicity of cadmium and other semiconductor material is a
cause of concern that restrains their biological application.14,15 Graphene quantum dots (GQDs)
on the other hand provide a much better option for their application as bioimaging agents due totheir excellent photoluminescent, conductive and biocompatible properties.16,17 In this study, thepotential of GQDs conjugated human serum albumin nanoparticles for bioimaging and targeted
drug delivery was explored and to the best of our knowledge, this is the first report coupling both
HA and GQDs for pancreatic cancer specific nanoformulation.
The main objective of this study was to synthesize graphene quantum dots and to conjugate themwith HA functionalized HSA-NPs for preparing a novel and efficient nano-delivery vehicle forpancreatic cancer specific drug delivery and bioimaging. HSA (molecular weight 65 kDA) is aplenteous protein in human blood plasma with a blood concentration of about 50 mg ml-1 and it
comprises of 585 amino acids along with 17 disulfide bridges.18 For the synthesis of albumin
nanoparticles a well established desolvation method was adopted. Basic mechanism of theprocess includes condensation of albumin and its interaction with ethanol and glutaraldehyde.
Prior to final synthesis, different parameters (pH, glutaraldehyde concentration, crosslinkingtime) were optimized to prepare an efficient nanoformulation. pH of the solution plays a very
important role in size control of HSA nanoparticles during the process because of its effect on
coagulation of HSA molecules. The isoelectric point (pI) of HSA is about 4.7 and at pH range
near its isoelectric point, coagulation increases due to enhanced protein-protein interaction which
in turn results into larger particle size. While at pH values much higher than the pI, electrostatic
repulsive conditions prevent interactions between protein molecules and protein-solvent
interaction decreases the coagulation and fine HSA nanoparticles may be formed.19 After
working with different pH ranging from pH=7 to 10, it was concluded from the experimental
data that at pH 9, particles of smallest size (56-250 nm) were obtained.
HSA possesses 59 free amines but while crosslinking with glutaraldehyde, residual surface
amine level shrinks significantly therefore before nanoparticles synthesis, HSA was conjugatedwith dimethyl maleic anhydride (DMMA) to create amine-protected HSA as reported
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elsewhere.20,21 DMMA was detached from the surface amines via hydrolysis after synthesizing
HSA-NPs. SEM images revealed the spherical morphology of HSA nanoparticles (Fig. 1) withmean particle diameter of approximately 150 nm (ranging from 56-250nm) and good dispersion.
DLS histogram presents the size distribution of nanoparticles Fig. S1 (supplementary
information). Nanospheres exhibit chemotherapeutic advantages of biodegradability andenhanced permeability and retention effect (EPR) for cancer therapy. Thus, these are the mostapplied form of drug delivery vector. Earlier studies have also shown that phagocytosis of tumor
cells depends on size of nanoparticles and nanoparticles with 100-200 nm diameters exhibit
preferential accumulation on tumor cells than healthy cells.22,23 To confirm the functionalizationof HA on HSA-NPs FT-IR spectroscopy was performed. As indicated in Fig. 2(i), apart from thecharacteristic peak of HA at 3322 cm-1 due to O-H stretching, a peak corresponding to amidebond (CO-NH) at ~1680 cm -1 in the spectrum of HA-HSA confirms the modification of HSANPs with HA. Thermo Gravimetric analysis (TGA) revealed three step decomposition patternsfor HSA nanoparticles (Fig. 3(i)) where as HA modified HSA exhibited an additional
decomposition step as shown in Fig. 3 (ii). Specifically, for HA modification ~36.24% weightloss occurred indicating a successful functionalization of HA on albumin nanoparticles.
Graphene quantum dots due to their photoluminescence and biocompatibility have attracted a lotof interest in their bioimaging applications as compared to their semiconductor counter parts.
The two prime approaches for synthesizing GQDs can be classified as bottom-up and top down
methods and various procedures have been reported based on these techniques for synthesis of
GQDs.24-26. In bottom up approach, quantum dots are synthesized from a carbon precursor
containing conjugated carbon atoms where as top down methods involve cutting large graphenesheets. Although bottom up approach provides a precise control over size and morphology, thesynthesis procedure is quite tedious and complicated. With regard to this, top down approaches
are a better alternative with their simple methodology and mass production. GQDs were preparedvia hydrothermal reduction of Graphene oxide (GO) with Lawsone and were further
characterized for their optical properties. Hydrothermal approach of synthesis was adopted
because of its simplicity, versatility and scalability. This is the first report demonstrating thesynthesis of GQDs with Lawsone as a reducing agent. The synthesized particles exhibited
excellent green fluorescence. Apart from the characteristic peak at 230 nm due to π- π* transition
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of C=C domains, UV-Vis spectrum of GQDs also showed an absorption peak at 340 nm (Fig.
S2). The peak at 340 nm represents the uniform sp2 clusters in GQDs.25 An excitation dependent
PL spectra was obtained from GQDs and two peaks at 430 nm and 540 nm were observed with
340 nm excitation whereas for 360-480 nm excitation, PL spectra was dominated by only one
peak at 540 nm, responsible for green fluorescence of GQDs (Fig. 4). Zhu et. al. have also
reported similar observations, expected to arise because of quantum size effects and surface
defects due to variations in synthesis procedures.24 Quantum yield of GQDs was ~14% andcalculated as per the method described by Zhu et. al. at 360 nm excitation wavelength using
9,10-Bis (phenylethynyl) anthracene in cyclohexane (QY=1) as reference compound.24 Fig. 5
shows TEM image of GQDs with an average diameter of ~5 nm while the supplementary fig. S3
represents the XRD patterns of samples before and after the hydrothermal reduction. The GO
exhibited a sharp peak at 11.5o, which completely disappeared in reduced graphene sheets and acharacteristic peak at 26.6o represents the reduction of GO to quantum dots. The FTIR spectrumof GQDs revealed the presence of C=O, O-H, epoxy/ether groups, which makes them watersoluble. After conjugation of GQDs with HSA, apart from the characteristic peaks, a peak at
1670 cm-1 which corresponds to amide bond (CO-NH) formation between the carboxy groups of
GQDs and amino groups of HSA-NPs was observed (Fig. 2 (ii)). Zeta potential of blank HSA
nanoparticles, HA-HSA and HSA-HA-GQDs was determined via dynamic light scattering (DLS)
and found to be -27.78, -31.45 and -33.21 mV respectively. HA is negatively charged at
physiological pH as indicated from the zeta potential analysis. The high surface charge indicateseffective water suspension and is advantageous for storage and administration of nanoparticles.
As previously reported, anionic nanocarriers are more preferred for targeted drug delivery thantheir cationic counterparts due to nonspecific binding of cationic vehicles to different cells after
systemic administration. Moreover electrostatic repulsion among negatively charged
nanospheres also prevents their aggregation to evade inactivity27-29. Considering these facts, it
can be concluded that our graphene quantum dots conjugated system has the potential to be
developed as an efficient drug delivery tool. UV-Vis analysis revealed that approximately 91%
drug was encapsulated within the albumin nanoparticles (Fig. S4). Summarized details of sizeand other physiochemical properties of HSA-NPs and quantum dots are shown in Table S1.

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HSA also exhibits excellent ligand binding capacity, stability in pH variations, noncovalent
reversible binding properties and biocompatibility that makes it an ideal vector for transport andrelease of drugs to the cell surface. It consists of three homologous domains each comprises of
two helical sub-domains with stable binding sites responsible for high affinity binding of various
drugs at different therapeutic concentrations.30, 31 HA functionalized Gem-HSA nanoformulation
is more advantageous over other systems because of its specific targeting towards pancreaticcancer, sustained release of GEM and low toxicity to healthy cells. In our analysis, for
gemcitabine loading, we amalgamated both the techniques (direct drug loading and physicaladsorption at the surface of nanoparticles) to prepare nanoformulation with better encapsulation
efficiency and release profile. Both the methods possess their own benefits and shortcomings i.e.
direct loading suffers from the disadvantage of low encapsulation and loading efficiency incomparison to physical adsorption, but exhibits better sustain release properties than the latterone. The in vitro release profile of gemcitabine showed about 18 hrs sustained release at 37 0 C
in PBS (Fig. 6) and keeping in mind the high water solubility and short half life of gemcitabine
due to its rapid metabolism, our HA functionalized nanoformulation is rather an improvement forpancreatic cancer specific drug delivery. A biphasic release pattern was obtained for gemcitabinewith initial burst release followed by a slow release pattern up to ~18-20 hrs, the mechanism ofwhich can be explained by drug leaching or detachment in first phase of fast release due tophysically adsorbed drug on the surface of nanoparticles while first order diffusion controlled
behavior was responsible for slow release of the drug.
Panc-1 cell lines are rich in CD-44, a receptor for Hyaluronic acid and responsible forchemotherapy resistant pancreatic cancer cells. To predict the cellular uptake of HSA-NPs and
HA functionalized NPs, GQDs were tagged with the nanoparticles. A strong green fluorescence
was observed inside the cells resulting from the efficient uptake of HA-HSA-NPs by Panc-1
cancer cells as shown in Fig. 7 (iii). On the other hand, unfunctionalized HSA-NPs exhibited
weak fluorescence signal, indicating that a lesser amount of nanoparticles in comparison to HAHSA-NPs are internalized into the cancer cells due to absence of the target moiety for CD-44
positive Panc-1 cells as exhibited in Fig. 7 (ii). It is clear evidence that HA-HSA-NPs canefficiently target CD-44 over expressing pancreatic cells via HA mediated endocytosis pathway.

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After confirming the cellular uptake of nanoparticles, the anti-cancerous efficacy ofnanoformulations was analyzed in-vitro via MTT assay. Panc-1 cells were incubated with HSAGem, HSA-HA-Gem and free gem. As shown in Fig. S5, HA-HSA-Gem formulation was themost effective one due its targeted internalization in CD-44 expressing PANC-1 cells. MTT dataalso revealed that Albumin and HA-HSA nanoparticles were quiet biocompatible and showedalmost no toxicity to panc-1 cells with excellent cell viability 91-89 % respectively at different
concentrations (5-20 µg/ml). Moreover the prepared GQDs were also biocompatible even at highconcentrations (350-400 µg/ml). It can be concluded that our hyaluronic acid modified HSA-NPs
has a theranostic potential to be an efficient nano-vector for treatment of pancreatic cancer, thedeadliest in all cancers. Apart from improvising our system for prolonged release we are further
focusing on in-vivo experimentations to check the proficiency of this formulation so that it canbe developed like “Abraxane” to cure drug resistant pancreatic cancer.
Conclusion
Hyaluronic acid and graphene quantum dots functionalized HSA nanoparticles were prepared
and applied for bioimaging and targeted delivery of gemcitabine to pancreatic cancer. Due to therapid metabolism and short half life of gemcitabine its efficacy gets reduced when administeredin free form but with our nanoformulation we not only attained sustained release but also got
notable effect on bioavailability of gem to cancer cells in vitro as exhibited by MTT assay.
Presence of HA as targeting moiety on HSA-NPs is the essence of better efficiency of gem on
resistant pancreatic cancer cells while GQDs exhibited excellent bioimaging potential.
Experiments are being carried out to further improve this system for in vivo analysis.
Acknowledgement
This work was supported by The Department of Science & Technology, Government of India.
Author is grateful to Dr. Satish B. Ogale, Chief Scientist, National Chemical Laboratory forproviding necessary guidance and help during the synthesis process of nanomaterials.
Notes & ReferencesaCombichem Bioresource Centre, National Chemical Laboratory, Pune India.
Department of Chemistry, Garware College, Pune Indiac
Department of Microbiology, Fergusson College, Pune, India.
#
Equal first authorb

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19. S. Ko and S. Gunasekaran, J. Microencapsul., 2006, 23, 887-898.
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Figure 1: SEM image of HSA nanoparticles.

Figure 2: (i) FTIR spectra of (a) Hyaluronic acid, (b) HSA-Hyaluronic acid functionalized nanoparticles;(ii) FTIR of (a) GQDs; (b) HSA-GQD functionalized nanoparticles.

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Figure 3: TGA curve of (a) Albumin nanoparticles, (b) Hyaluronic acid functionalized albumin
nanoparticles (Ramp rate: 100 C/ min in nitrogen environment).

Figure 4: PL spectrum of aqueous solution of graphene quantum dots at excitation wavelengthof 360-480 nm (a) 360 nm, (b) 400 nm, (c) 420 nm, (d) 440 nm, (e) 460 nm, (f) 480 nm.
(Experiments were performed at room temperature (~25 0 C).

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Figure 5: TEM of graphene quantum dots (average particles size ~5 nm).

Figure 6: in-vitro release profile of gemcitabine from HSA nanoparticles in PBS (pH=7.0) at 370 C.

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Figure 7: Fluorescence imaging of Panc-1 cells after 24 hrs incubation in presence of GQDs conjugated
HSA nanoparticles in DMEM media with 10 % FBS in CO2 incubator at 37 0C; (i) Cells without staining(control), (ii) HSA nanoparticles with GQDs only, (iii) HSA NPs functionalized with HA and GQDs.

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