Lysine-Based C60-Fullerene Nanoconjugates for Monomethyl Fumarate Delivery: A Novel Nanomedicine for Brain Cancer Cells

Article abstract of DOI:10.1021/acsbiomaterials.7b01031

In the present study, water-soluble lysine-based C₆₀-fullerene nanoconjugates (CF-LYS-TEG-MMF) were synthesized using a biodegradable linker for the better delivery of monomethyl fumarate (MMF) employing Prato reaction. CF-LYS-TEG-MMF resulted

Full text of DOI:10.1021/acsbiomaterials.7b01031

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Controlled Release and Delivery Systems
Lysine-based C60-fullerene nanoconjugates for monomethyl
fumarate delivery: A novel nanomedicine for brain cancer cells
Manish Kumar, Gajanand Sharma, Rajendra Kumar, Bhupinder Singh, Om Prakash Katare, and Kaisar Raza
ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b01031 • Publication Date (Web): 30 Apr 2018
Downloaded from http://pubs.acs.org on May 9, 2018
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Lysine-based C -fullerene nanoconjugates for monomethyl fumarate
60
delivery: A novel nanomedicine for brain cancer cells
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2,3
Manish Kumar , Gajanand Sharma , Rajendra Kumar , Bhupinder Singh , Om
2
1,*
Prakash Katare and Kaisar Raza
1
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central
University of Rajasthan, Bandar Sindri, NH-8, Dist. Ajmer, Rajasthan, India-305
817
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University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced
Studies, Panjab University, Sector 14, Chandigarh, India-160 014
3
UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and
Nanocomposites, Panjab University, Sector 14, Chandigarh, India-160 014
*Corresponding Author
Dr. Kaisar Raza
Department of Pharmacy
School of Chemical Sciences & Pharmacy
Central University of Rajasthan
Bandar Sindri, Distt. Ajmer, Rajasthan, Indiaꢀ305 817
Eꢀmail: drkaisar@curaj.ac.in; razakaisar_pharma@yahoo.co.in
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Abstract
In the present study, water soluble lysineꢀbased C60ꢀfullerene nanoconjugates
(
CFꢀLYSꢀTEGꢀMMF) were synthesized using a biodegradable linker for the better
delivery of monomethyl fumarate (MMF) employing Prato reaction. CFꢀLYSꢀTEGꢀ
MMF resulted in enhanced cytotoxicity on neuroblastoma cells, meanwhile found
to be substantially biocompatible to erythrocytes. The designed nanoconjugate
exhibited a pHꢀbased drug release pattern, minimizing the leaching of drug at
plasma pH. However, the carrier offered maximum drug release at cancer cell pH
indicating huge promise in internalization of drug molecules at the site of target.
The pharmacokinetics of MMF in rodents was significantly improved in terms of
enhanced bioavailable drug fraction in the central compartment, reduced drug
clearance, elevated plasma concentrations and prolonged biological residence of
drug. Enhanced in-vitro efficacy in SHꢀSY5Y neuroblastoma cells, improved
erythrocyte compatibility, high drug loading and conducive pharmacokinetic
profile by CFꢀLYSꢀTEGꢀMMF offers a huge promise in brain drug delivery, dose
reduction and dosageꢀregimen alteration for the management of brain tumors
employing MMF.
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Key words: Neuroblastoma, Brain delivery, Pharmacokinetics, Prato reaction,
Linker, Prodrug, Bioavailability, C60 fullerene nanoconjugate
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Introduction
Often, it is observed that the agents which execute activity for primary brain
cancers during in vitro evaluation, seldom appear effective on the disease during
1
clinical evaluation . Numerous reasons have been hypothesized for such failures,
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including the failure of these agents to get access of the tough blood brain
barrier (BBB), which further result in subꢀtherapeutic concentrations at the
2
target site, i.e., brain . Furthermore, an understanding is also developing that
owing to their shorter plasmaꢀhalf lives, the therapeutic agents of lowꢀmolecular
mass, generally are unable to maintain the effective steady state concentrations
3
,4
within the cancerous cells
.
It is now wellꢀdocumented that both the biological features like specific
enzymes/transporters required, as well as the physicochemical properties of the
drugs like Hꢀbonding capability, ionization constant, molecular mass and
hydrophobicity, decide the BBB crossing and subsequent central nervous system
5
(CNS) permeation of the drugs. On the other hand, the endogenous water
soluble small polar molecules like saccharides and amino acids get an easy
access of BBB/CNS due to the wellꢀestablished transport systems like soluteꢀ
4
,6
carrier (SLC) transporters . The receptorꢀmediated endocytosis initially involves
endocytosis at the blood (luminal) side, which is a receptorꢀmediated step,
subsequently followed by intracellular movement of the endocytosed
particle/molecule. This endocytosed component is ultimately exposed to the
7
brain (abluminal) side of brain endothelial cell by exocytosis .
As per scientific consensus, the inability to cross BBB, is the main reason behind
8
the underꢀdevelopment of CNS drugs . Nowꢀaꢀdays, the nanotechnologyꢀbased
drug delivery carriers with a potential to cross the BBB are emerging as an
indispensable tools to deliver the drugs in brain. Several types of nanoparticles
are being studied in biomedical research, such as magnetic nanoparticles, gold
7
,9,
nanoparticles, waterꢀsoluble fullerene derivatives and silica nanoparticles
Carriers like C60ꢀfullerene immobilized on silica surface have been studied and
10
the proꢀoxidant effect has been successfully reported.
The proꢀoxidant
properties of fullereneꢀcontaining composites under the influence of UV and Xꢀ
ray irradiation opens the perspective of further application in an important
1
1
domain of bioꢀnanotechnology, i.e., photodynamic therapy . As far as fullerenes
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are concerned, this third allotropic form of carbon (after graphite and diamond),
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2
was discovered in 1985 . Hydrophobicity, spherical shape of the molecule,
unusual redox properties allowing attachments of up to six electrons, and low
toxicity, have acted as the stimulators for biological investigations of this
surprising molecule. Applications of such unique molecule are halted by the
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exceptionally low solubility of these buckyballs in water , though these tiny
structures possess substantial permeability across lipid membranes. Henceforth,
waterꢀsoluble C60ꢀfullerene (CF) derivatives have attracted significant attention
due to broad range of biological activities including significant antioxidant and
radical scavenging properties as well as antimicrobial/antiviral activities coupled
14,15,16,17
with mild Pꢀgp inhibition
. In addition, fullerene derivatives can also
penetrate efficiently across the BBB, and can be used as drug delivery carriers
1
8, 19
for delivering the drugs to brain
. However, scientists are generally
concerned with the toxicity of the CFs and derivatives, which generally arises
due to the less explored aspects of these nanocarriers. There are numerous
reports which advocate the internalization of CFs in mammalian cells without
exhibiting any toxicity concerns. Its reported that aqueous C60 suspensions
prepared without any polar organic solvents showed no acute or subacute
toxicity in rodents, however, such systems were observed to offer liver
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protection against freeꢀradical damage. Higher doses of CFs, i.e., from 2500
mg to 5000 mg/kg have been administered intraꢀperitoneally to the rodents and
1
2
the researchers reported no signs of toxicity in the treatment groups . In
another preclinical study of acute oral administration of fullerenes in Spragueꢀ
Dawley rats at the dose of 2 g/kg, the researchers reported no evidences of
acute oral toxicity or genotoxicity and the measured outcomes of the treated
animal group were observed to be identical to that of the control animal group in
2
1
every studied aspect . Such tolerance at higher doses provide an evidence for
biological suitability of CFs. Availability of CFꢀbased marketed/clinical trial
products (NOVA C60 and LipoFullerene)
,
is further a testimony to the applicability
22,23,24.
of naïve CFs in the development of human usable products
The nonꢀ
covalent complex of CFꢀdrug (doxorubicin/cisplatin) has been reported to offer
2
5, 26
medicoꢀbiological synergy on its administration
. Therefore, fabrication of
fullereneꢀdrug hybrids can be a promising approach to alleviate the concerns
21
associated with brain delivery of neurologically active agents .
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Monomethyl fumarate (MMF), an active form of drug dimethyl fumarate, is
known to induce tumor cell lysis, preferably by the mechanisms involving
22
NKp46 . Though a biologically active, fumaric acid esters are reported to be
associated with concerns like gastrointestinal flushing, lower brain permeability,
23,24
and economic hurdles due to multiple dosing concerns
. Henceforth, the
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present study was aimed to synthesize of lysineꢀfunctionalized CFs using well
25,26
known Prato reaction
followed by the attachment of a biodegradable
hydrophilic linker to increase the aqueous solubility as well as the loading of
MMF. The developed nanoconjugate is expected to overcome these challenges
and show effective brain delivery of MMF to SHꢀSY5Y neuroblastoma cells of
human origin.
Materials and Methods
Materials:
Tetraethylene glycol (TEG) (LD50: 10000 mg/kg to 34,760 mg/kg in rats),
monomethyl
diphenyltetrazolium bromide (MTT) were purchased from M/s SigmaꢀAldrich,
New Delhi, India. Tetrahydrofuran (THF), 1,2ꢀdichloro benzene,
dichloromethane, triethyl amine, sodium chloride, buffer ingredients and
paraformaldehyde were procured from M/s CDH Pvt Ltd., New Delhi, India. C60
fumarate
(MMF)
and
3ꢀ(4,5ꢀdimethylthiazoleꢀ2ꢀyl)ꢀ2,5ꢀ
ꢀ
fullerenes (CFs) were obtained from M/s M/s BuckyUSA, Houston, USA. pꢀ
toluene sulphonyl chloride (TsCl), N,N′ꢀdicyclohexyl carbodiimide (DCC) and 4ꢀ
(dimethylamino)pyridine (DMAP) were supplied by M/s Spectrochem Pvt. Ltd,
Mumbai, India. Lysine (LD50: 10000 mg/kg in rats) and dialysis membrane were
bought from M/s Himedia laboratories Co., Ltd., Mumbai, India. National
Institute of Immunology, New Delhi was the source for SHꢀSY5Y cell lines.
Distilled water was employed throughout the studies. The chemicals/reagents
were used as such, without any further purification.
Methods
Synthesis of lysine-functionalized C60-fullerene
(1)CFs were functionalized using well known [3+2] cycloaddition reaction of
fullerene chemistry, i.e., Prato reaction. In brief, 30 mL of 1,2ꢀdichlorobenzene
was employed to dissolve CFs (60 mg). To this purple solution,
paraformaldehyde (10 equivalents) and lysine (5 equivalents) were added while
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stirring at 155°C for 48 h (till the colour changes to brown). Solvent was
evaporated to get darkish brown solid using distillation. The solid was dispersed
in methanol and centrifuged to remove the unreacted lysine and
paraformaldehyde, and dried under vacuum to fetch the lysine functionalized
2
5
fullerenes (2) (CFꢀLYS), (%Yield: 90%) (Figure 1) .
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Synthesis of tosyl-tetraethylene glycol (Ts-TEG)
(3) TEG (1.0 equivalent) and TEA (3.0 equivalents) were mixed in DCM (15 mL)
at 0°C. TsCl (1.0 equivalent) in DCM (15 mL) was added slowly over 30 min to
the mixture. The reaction mixture was stirred at 0°C for 2 h, followed by
overnight stirring at room temperature. It was washed subsequently with 100
mL of 3% hydrochloric acid and 100 mL of brine, and then extracted with DCM
(100 mL). The extraction process was performed thrice and the separated
organic layer was pooled. This pooled organic phase was dried over Na
2
SO and
4
further by vacuum. The solvent was evaporated to fetch the product of viscous
nature (4) (TsꢀTEG). Silica gel chromatography using petroleum ether and ethyl
acetate (40 %v/v) as the mobile phase was employed to further purify the
product (%Yield: 65.55%). The synthetic scheme of this product is depicted in
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Figure 1 .
Conjugation of tetraethylene glycol to the fullerenes (CF-LYS-TEG-OH)
4
(5 equivalents) and K
2
CO (10 equivalents) were added to the dispersion of
3
CFꢀLYS (100 mg, 1 equivalent) in 20 mL acetonitrile, and the reaction mixture
was stirred at 110°C for 96 h. Thin layer chromatography (TLC) was employed
to monitor the extent of the reaction. Rotatory evaporation (Rꢀ215, M/s Buchi,
Flawil, Switzerland) was performed to remove the solvent, after the completion
of reaction, and washed with water, then dried to fetch the (5) CFꢀLYSꢀTEGꢀOH,
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as shown in Figure 1 (%Yield: 95.27%)
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Figure 1: Schematic representation of the synthetic protocol employed for the
synthesis of the desired CFꢀLYSꢀTEGꢀMMF nanoconjugate
Preparation of MMF conjugated linker-tethered fullerene (CF-LYS-TEG-
MMF)
(6) MMF (4 equivalents), DCC (9 equivalents) and DMAP (1 equivalent) was
added to the dispersion of 5 (1 equivalent) in THF (5 mL) at 0°C for halfꢀanꢀhour
and further stirred for 24 h at the ambient temperature. Progress of the reaction
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was monitored using TLC. Solvent was evaporated and the residue was washed
twice with THF to remove unreacted reactants and washed several times with nꢀ
hexane to remove 1,3ꢀdicyclohexylurea from the MMF conjugated linkerꢀtethered
fullerene, i.e., (7) CF-LYS-TEG-MMF (%Yield: 80%). The synthetic scheme of
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this product has been depicted in Figure 1 .
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Characterization and Evaluation Studies
Fourier Transform Infrared Spectroscopy (FT-IR)
Functional group identification of the compounds was done by fourier transform
infrared spectroscopy (FTꢀIR). The samples were mixed with potassium bromide
and pallets were prepared using hydraulic press. The FTꢀIR spectra were
recorded in the desired wave number range using FTꢀIR spectrometer (Spectrum
two, M/s Perkin Elmer Co., Waltham, Massachusetts, USA).
Nuclear Magnetic Resonance Spectroscopy (NMR)
Saturated solutions of the conjugate and intermediates were prepared using
1
deuterated chloroform (CDCl
3
), and HꢀNMR were recorded on ASCEND 500WB
NMR Spectrometer (M/s Bruker Bio Spin Corporation, Indiana, USA).
Particle Size, PDI, Zeta Potential and Morphology
The values of average hydrodynamic diameter and polydispersity index (PDI) of
various systems were determined using Malvern Zetasizer (M/s Malvern,
Worcestershire, UK). For the sample preparation, each sample was diluted with
deionised water, followed by ultrasonic dispersion. The result was compiled from
three consecutive recordings of the same sample and the average was reported
as the final result. Malvern Zetasizer was further employed to record the values
of zetaꢀpotential of the undiluted samples. The morphology of CFꢀLYS and CFꢀ
LYSꢀTEGꢀMMF was visualized by field emission scanning electron microscopy
(FEꢀSEM)(NOVA NANO 450 FEI, Netherlands), which is installed at Materials
Research Centre, Malaviya National Institute of Technology (MNIT), Jaipur,
Rajasthan, India. The samples were prepared as per the standard protocol of
3
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MNIT, and photographs were clicked at suitable magnification(s) .
Extent of Drug Conjugation and Drug Loading
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Drug conjugation was determined using the washings of Section 2.2.4.
Subsequent to evaporation of the solvents, the unreacted residue was
reconstituted in the mobile phase and quantified by RPꢀHPLC method (LCꢀ2010C
HT, M/s Schimadzu Co. Ltd., Chiyodaꢀku, Tokyo, Japan). Mobile phase was
consisted of water and acetonitrile (60:40 v/v), which was pumped at the rate of
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ꢀ
1
1
mL min and the equipment was set to withdraw 20 µL as the sample for
analysis at 216 nm on Oyster BDS premium C18 HPLC column (M/s Merck
Specialities Pvt. Ltd., Mumbai, India). Drug loading was reported as the parts of
1
3
drug conjugated on to the 100 parts of the CFꢀLYSꢀTEGꢀOH .
In-vitro Release Studies
The release of MMF from the CFꢀLYSꢀTEGꢀMMF nanoconjugate was determined
by dialysis technique. Naïve MMF and CFꢀLYSꢀTEGꢀMTX conjugate were packed
separately in dialysis bags, and dialyzed against two pH values i.e., phosphate
buffered solutions (PBS) of pH 7.4 and pH 5.0, separately as previously reported
31
protocol . The obtained data was analysed for the drug release kinetics
employing various kinetic models.
Ex-vivo Hemolysis Studies
Hemocompatibility of prepared systems was examined employing the protocols
3
2
on healthy human blood . The study was executed after the necessary approval
from the Institutional Ethical Committee, Central University of Rajasthan, India.
In brief, 1 mL of blood was sampled from healthy human volunteer into an antiꢀ
coagulant coated test tube. Erythrocytes were separated employing
centrifugation at 2000 rpm. The erythrocytes were washed thrice with normal
saline and again dispersed in normal saline. The formulations under test, i.e., 1
mg of Naïve MMF, 1.7 mg of CFꢀLYS (equivalent to the carrier required to load 1
mg of MMF) and 2.7 mg of CFꢀLYSꢀTEGꢀMMF conjugate (equivalent to 1 mg of
loaded MMF) were mixed with 1 mL of the 1 % erythrocyte dispersion.
Analogously, the control sample was prepared by dispersing equivalent amount
of erythrocytes in saline (negative control) as well as double distilled water
(positive control), separately. All the samples were kept in dark for 1 h at 37 °C.
Subsequent to that, the samples were centrifuged at 2000 × g for 300 sec to
fetch with the transparent supernatant. UVꢀVisible spectroscopy with an λmax of
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415 nm was employed to analysed the clear supernatant of each test tube, and
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3
the percent haemolysis was determined using the Equation 1 .
.
................ (1)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
s
D , Dnc and Dpc are the absorption of sample, positive control and negative control,
respectively.
In-vitro Cytotoxicity assay
3
A 5*10 SHꢀSY5Y cells were cultured in 96ꢀwell microplate, with 5% CO
2
at 37
.
Cells were allowed to adhere and grow for overnight before treatment. The test
samples, i.e., MMF and CFꢀLYSꢀTEGꢀMMF, were added in the well plates in
various equivalent concentrations of MMF (1 to 100 µM). The concentrations of
CFꢀLYS were used as the equivalent concentrations of CFꢀLYSꢀTEGꢀMMF. The
Untreated cells were used as control to calculate the % viability. Well plates
were subjected to incubation for 24 h at 37
. After 24 h incubation, 10 µL
(5mg/mL) of MTT solution was added to every well, and mixed gently. These
plates were further incubated for 4 h and the purple coloured formazan crystals
were observed.. These purple colored crystals were dissolved in dimethyl
sulphoxide and scanned for the optical density at 570 nm. Percentage viability
was calculated using Equation 2. The optical density was employed to calculate
33
the IC50 values in the set experimental conditions .
Pharmacokinetic Studies
After due approval from the Institutional Animal Ethics Committee of Panjab
University, Chandigarh, the animal studies were executed, that too with a strict
adherence to the ethical guidelines. The animals used were unisex Wistar rats
(200ꢀ250 g; 4ꢀ6 weeks old) and divided into two groups of four animals each.
Group 1 rats received plain MMF, whereas Group 2 was administered with CFꢀ
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LYSꢀTEGꢀMMF. The route of administration followed was intraꢀvenous through
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the tail vein of rats. The doses equivalent to 5 mg/kg of MMF were aseptically
prepared in “water for injection”. At preꢀdetermined time points, the blood
sampling was performed from the retroꢀorbital plexus of the animal. Prior to
plasma separation, one drop of 0.1 N HCl was added to induce the hydrolysis of
the conjugated drug for easy detection. The analysis of plasma samples was
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performed using RPꢀHPLC .
Results and Discussion
Spectroscopic Characterization:
Synthesis of TsꢀTEG was confirmed using FTꢀIR as shown in Figure 2(A). Free
ꢀ
1
hydroxyl group of TSꢀTEG shown a broad peak at 3447.33 cm . The stretching
ꢀ
1
of methylene (–CH
2
ꢀ) was observed at 2890.11 cm and the respective bending
ꢀ
1
occurred at 1454.31 cm . (S=O) of TsꢀTEG exhibited a asymmetric stretch at
ꢀ
1
ꢀ1
1
356.63 cm and symmetric stretch at 1189.90 cm . Presence of several peaks
ꢀ
1
ꢀ1
between 1000 cm to 750 cm corresponded to S-O stretch. Figure 2(B)
depicts the FTꢀIR spectrum of CFꢀLYSꢀTEG and confirmed the successful
ꢀ
1
synthesis of the same. As a broad peak at 3435.57 cm corresponded to the
free –OH of the TEG chain, whereas the stretching vibrations of methylene (ꢀ
ꢀ
1
CH
2
ꢀ) occurred at 2890.11 cm . The synthesis of the final conjugate, i.e., CFꢀ
LYSꢀTEGꢀMMF was also confirmed from FTꢀIR results, as depicted in Figure
ꢀ
1
2
(C). Stretching vibration from >C=C< of MMF was exhibited at 1645.64 cm ,
whereas the stretching of =C-H merged with the broad peak of few –hydroxyl
ꢀ1
groups on CFꢀLYSꢀTEG. A strong peak at 1732.60 cm confirmed the formation
of ester linkage between CFꢀLYSꢀTEG and MMF. Spectrum of CFꢀLYSꢀTEGꢀMMF
ꢀ
1
also exhibited a broad peak at 3425.10 cm , ascribable to the the presence of
few unreacted hydroxyl group of CFꢀLYSꢀTEG.
1
Formation of TsꢀTEG was further confirmed by H NMR (Figure 3). Peaks
between 3.69 ppm to 3.06 ppm were ascribed to the oxygenꢀlinked protons of
methylene group, –CH
2
OCH
2
ꢀand ꢀCH
2
OH. Intrinsic peaks between 7.31 ppm to
35
7
.77 ppm were ascribed to –CH group of aromatic ring . Formation of CFꢀLYSꢀ
TEGꢀMMF conjugate was confirmed by appearance of additional peaks between
.07 ppm to 6.70 ppm, ascribable to the protons of ꢀCH=CHꢀ group, exclusively
6
3
6
present in MMF, as depicted in Figure 4 .
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Figure 2: Figure showing the observed FTꢀIR spectra of various compounds:
(A) TsꢀTEG; (B) CFꢀLYSꢀTEG; (C) CFꢀ LYSꢀTEGꢀMMF
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Figure 3: HꢀNMR spectrum of the intermediate TsꢀTEG in CDCl
3
0
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Figure 4: HꢀNMR spectrum of CFꢀLYSꢀTEGꢀMMF in d
6
ꢀDMSO
Physicochemical Characterization
The physicochemical properties of the intermediates and the final conjugate has
been shown in Table 1. Understanding of the zetaꢀpotential and particle size of
3
7
the nanosystems can help in predicting the fate at the biological level . In
general, nanoparticles possessing the particle size below 200 nm are considered
ideal for prolonged circulation due to the propensity to avoid liver uptake and
3
8
surpass rapid renal clearance . The linkage with biodegradable linker and
attachment of anticancer agent was reflected by the changes in the values of
particle size and zeta potential of the designed nanoparticles, as conspicuous
from Table 2. After attachment of biodegradable linker, the particle size of
functionalized fullerene significantly increased from 81.00 ± 2.25 nm to 124.29
±
2.76 nm (p < 0.05), and the zeta potential substantially decreased from to
26.10 ± 1.44 to ꢀ5.36 ± 1.40 mV (p < 0.05). The increase in the average
+
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hydrodynamic diameter provided the inference of the hydrophilic linker and the
reduction in zetaꢀpotential values can be attributed to the conjugation of
protonated amino groups of lysine with the tosylꢀTEG. On the other hand, the
mean particle size and zeta potential values of final CFꢀLYSꢀTEGꢀMMF conjugate
were observed to be 137.6 ± 1.86 nm and ꢀ12.20 ± 1.72 mV, respectively.
Further enhancement in surface negative charge can be ascribed to the MMF
moieties containing electron rich oxygen containing groups, as already depicted
in Figure 1. The FEꢀSEM images of the CFꢀLYS and CFꢀLYSꢀTEGꢀMMF
nanoconjugate have been presented in the Figure 5A and Figure 5B,
respectively. Though nothing can be clearly vouched from the FEꢀSEM images,
still the absence of any visible contaminant is vivid. The findings are in close
1
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consonance with the published FEꢀSEM results of such systems . The
conjugation efficiency of the employed process was observed to be > 95% and
the drug loading of the designed nanoconjugate was observed to be of the order
of 36.67 ± 2.94%. The results of micromeritics with smaller particle size, low
PDI values, narrow particle size distribution, moderate zeta potential, coupled
with higher drug conjugation and appreciable drug loading inferred that CFꢀLYSꢀ
TEGꢀMMF nanoconjugate can offer an effective therapeutic drug delivery option
3
7
for MMF .
Figure 5: Microphotographs of FEꢀSEM images of: (A) CFꢀLYS (10,000X) and
(B) CFꢀLYSꢀTEGꢀMMF (10,000X)
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Table 1: Tabular Representation of Log P and aqueous solubility of different
samples.
0
1
2
3
4
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S. No Sample
Log P
ꢀ0.95
1.27
Aqueous Solubility
5 mg/mL
1.25 mg/mL
1
2
3
TEG
TsꢀTEG
CFꢀLYSꢀTEGꢀMMF
ꢀ0.40
0.71 mg/mL
Table 2: Tabular Representation of Particle Size, PDI, Zeta Potential. Data are
presented as mean ± SD (n = 3)
S. No.
Zeta Potential
(mV)
Particle size
nm)
Sample
PDI
(
1.
2.
3.
CFꢀLYS
81.00 ± 2.25
124.29 ± 2.76
137.6 ± 1.86
0.100
0.147
0.206
+26.10 ± 1.44
ꢀ5.36 ± 1.40
ꢀ12.20 ± 1.72
CFꢀLYSꢀTEGꢀOH
CFꢀLYSꢀTEGꢀMMF
1.1 In-vitro Drug Release:
The drug release behaviour of naïve MMF and CFꢀLYSꢀTEGꢀMMF nanoconjugate
was examined in PBS of two different pH values, i.e., 7.4 and 5.0. These two
selected conditions acted as in-vitro models to plasma and cancer cell
environment, respectively. The release profile in both the conditions is depicted
in Figure 6. The developed nanoconjugate showed the significantly faster
release of MMF in PBS of pH 5.0, i.e., around 95.20% release in 48 h. On the
other hand, substantially lower MMF amounts were released in the PBS of pH
7
.4, i.e., 28.83 % in 48 h (p < 0.05). Percent drug release of naïve MMF was
found to be 84.43% at pH 5 and 97.6% at pH 7.4 (p < 0.05). CFꢀLYSꢀTEGꢀMMF
nanoconjugate offered pHꢀdependent drug release, reasonably due to the
presence of ester linkage between CFꢀLYSꢀTEG and MMF, whereas naïve MMF
4
0
showed no significant pH dependency . The discriminative drug release profile
in two different pH values carriers a promising advantage for the designed
system, intended to be administered by intraꢀvenous route. The nanoconjugate
assured minimal premature drug leakage to the site of administration, i.e.,
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plasma and maximal drug delivery at the site of action, i.e., cancer cells .
Moreover, we believed that once internalized inside target cells, the ester linkage
of CFꢀLYSꢀTEGꢀMMF could also be preferentially hydrolysed to release MMF by
the endo/lysosomal esterases coupled with acid calatyled hydrolysis due to acidic
4
1
pH of the cancer cell .
1
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The results of drug release kinetics modelling have been shown as Table 1 of
supplementary data. From the analysis of the data, it was inferred that the
release pattern of the drug from both the studied systems exhibited Higuchi
release profile at cancer cell pH as well as plasma pH. Since CFs can be
considered to be nonꢀswellable matrices for the drug; hence, Higuchi profile is
expected from such carriers and the present findings are in consonance with the
33
previous published reports .
Figure 6: In-vitro % cumulative MMF release data of MMF and CFꢀLYSꢀTEGꢀMMF
nanoconjugate at different pH values. Data are presented as mean ±
SD (n = 3). Asterisk (*) indicates the statistical significant difference
at each time point of the labelled release profile from that of the
MMF release in pH 7.4 at p < 0.05.
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Hemolytic Studies:
Since the biocompatibility of the drug delivery vehicle is of great importance for
clinical application, it becomes indispensable to assess the toxic effects of the
developed system on the mammalian cells, especially the red blood cells (RBCs).
The hemolytic potential of various samples was therefore explored. There was no
substantial hemolytic activity by CFꢀLYSꢀTEGꢀMMF nanoconjugate (4.56%),
whereas MMF was observed to induced maximum hemolysis (9.86%), followed
by CFꢀLYS (7.71%). The difference from the data of pure drug was statistically
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significant at
p
<
0.05. The studies unequivocally demonstrated the
biocompatibility of the novel nanoconjugate, indicating the suitability of the
same by i.v. route. This decline in hemolysis is possibly due to the conjugation
33
of free carboxylic group of MMF with the functionalized CFs .
In-vitro Cytotoxicity studies:
To investigate the cytotoxicity of CFꢀLYSꢀTEGꢀMMF nanoparticles on the
proposed target, i.e., neuroblastoma cells, in vitro cytotoxicity assay on SHꢀSY5Y
cells was performed. Cell viability was determined after the incubation with free
MMF, CFꢀLYSꢀTEG and CFꢀLYSꢀTEGꢀMMF conjugate at different micromolar
concentrations and results are portrayed in Figure 7. The nanocarrier itself, i.e.,
CFꢀLYS
,
without MMF was observed to nonꢀcytotoxic, even at higher
concentrations (≥ 140 µM). Plain MMF was observed to offer an IC50 value of 112
µM, which substantially reduced to 21.6 µM in the final nanoconjugate, i.e., CFꢀ
LYSꢀTEGꢀMMF (around 5ꢀfolds enhancement in efficacy; p < 0.05). Surprisingly,
the cargo vehicle for MMF did not offer any cytotoxicity, but the derived
nanoconjugate with MMF was found to be highly effective. This can be plausibly
due to the better penetration of CFꢀbased nanocarrier and further mild inhibition
of Pꢀgp efflux, which might have enhanced the intracellular concentrations of
12, 43
MMF, resulting in significantly higher cancer cell cytotoxicity
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against SHꢀSY5Y cells. Each cross bar indicates average value ± SD (n=3).
Asterisk (*) indicates the statistical significant difference of the labelled group
from the MMF group at p < 0.05.
Pharmacokinetics Studies:
To investigate the pharmacokinetics of MMF in rodents, a single intravenous
injection of free MMF and CFꢀLYSꢀTEGꢀMMF conjugate was administered in the
respective animal group. After the intravenous injection, the rats treated with
the CFꢀLYSꢀTEGꢀMMF conjugate showed initial higher blood circulating levels of
MMF, compared with the group receiving free MMF. At every time point, the
respective plasma concentration of MMF from the conjugate was significantly
higher than that from the naïve drug (p < 0.05). In addition, as the CFꢀLYSꢀ
TEGꢀMMF nanoconjugate significantly improved the pharmacokinetic parameters
of MMF in comparison to the free MMF, as indicated by 18 times reduced total
body clearance and 4.6 times enhancement in bioavailable drug (AUC), however
the elimination rate and the biological halfꢀlife remained same (Table 2). The
result also proved the idea of the fullereneꢀbased nanoꢀsized drug delivery
systems that would prevent the rapid uptake by the reticuloendothelial system,
enhance the blood persistence, and prolong the blood circulation time, while
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
reducing the systemic toxicity . However, authors find no pharmacokinetic
studies reported for MMF in rodents; however, a few manuscripts have
4
2, 44
quantified MMF in rats, after oral administration of dimethyl fumarate
.
Table 3: Tabular Representation of Various Pharmacokinetics Parameters
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S. No. Parameter
Unit
MMF
CF-LYS-TEG-MMF
30868.090
0.163
ꢀ
1
1
ng.mL .h 6710.150
ꢀ
1
2
3
4
5
K
h
0.165
0.979
0.162
4.191
V
d
L
L/h
H
0.214
Cl
0.035
t
½
4.262
Conclusions
The present studies present a fullereneꢀbased novel nanoconjugate, loaded with
a biocompatible linker and MMF. The developed system offered a pHꢀdependent
drug release with substantial cancer cell permeability and appreciable hemoꢀ
compatibility. The designed nanomedicine enhanced the efficacy of MMF in
neuroblastoma cells, meanwhile substantially enhanced the plasmaꢀaccessible
amounts as well as in-vivo circulation time of the drug. The preclinical outcomes
of the developed nanocarrier offer a promise of safe and effective delivery of
MMF to neuroblastoma cells for potential applications in conditions like brain
tumors.
Acknowledgements
University Grant Commission, New Delhi, India is highly acknowledged for
Rajeev Gandhi National Fellowship to Mr. Manish Kumar (F1ꢀ17.1/2014ꢀ
1
5/RGNFꢀ2014ꢀ15 STꢀRAJꢀ73879/(SAꢀIII/Website).
Supporting Information: Release kinetics data provided.
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49) Liang, X.; Meng, H.; Wang, Y.; He, H. Metallofullerene Nanoparticles
Circumvent Tumor Resistance to Cisplatin by Reactivating Endocytosis.
Proc. 2010, 107(16), 7449ꢀ7454, DOI: 10.1073/pnas.0909707107.
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50) Kumar, P.; Sharma, G.; Kumar, R.; Malik, R.; Singh, B.; Katare, O. P.;
Raza, K. VitaminꢀDerived Nanolipoidal Carriers for Brain Delivery of
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0.1021/acschemneuro.7b00041 .
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Figure
No.
Legends
Schematic representation of the synthetic protocol
employed for the synthesis of the desired CFꢀLYSꢀTEGꢀMMF
nanoconjugate
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Figure showing the observed FTꢀIR spectra of various
compounds: (A) TsꢀTEG; (B) CFꢀLYSꢀTEG; (C) CFꢀ LYSꢀTEGꢀ
MMF
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HꢀNMR spectrum of the intermediate TsꢀTEG in CDCl
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HꢀNMR spectrum of CFꢀLYSꢀTEGꢀMMF in d
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Microphotographs of FEꢀSEM images of: (A) CFꢀLYS
10,000X) and (B) CFꢀLYSꢀTEGꢀMMF (10,000X).
(
In-vitro % cumulative MMF release data of MMF and CFꢀ
LYSꢀTEGꢀMMF nanoconjugate at different pH values. Data
are presented as mean ± SD (n = 3)
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In vitro cytotoxicity data of CFꢀLYSꢀTEG, MMF, and CFꢀLYSꢀ
TEGꢀMMF against SHꢀSY5Y cells. Each cross bar indicates
average value ± SD (n=3)
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“For Table of Contents Use Only”
Lysine-based C60-fullerene nanoconjugates for monomethyl fumarate
delivery: A novel nanomedicine for brain cancer cells
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Katare and Kaisar Raza
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