Lysine-Based C60-Fullerene Nanoconjugates for Monomethyl

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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 C60-fullerene nanoconjugates for monomethyl fumarate delivery: A novel nanomedicine for brain cancer cells Manish Kumar1, Gajanand Sharma2, Rajendra Kumar3, Bhupinder Singh2,3, Om Prakash Katare2 and Kaisar Raza1,* 1

Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandar Sindri, NH-8, Dist. Ajmer, Rajasthan, India-305 817 2

3

University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Studies, Panjab University, Sector 14, Chandigarh, India-160 014

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, India-305 817 E-mail: [email protected]; [email protected]


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Abstract 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-TEGMMF resulted in enhanced cytotoxicity on neuroblastoma cells, meanwhile found to be substantially biocompatible to erythrocytes. The designed nanoconjugate exhibited a pH-based 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 SH-SY5Y neuroblastoma cells, improved erythrocyte compatibility, high drug loading and conducive pharmacokinetic profile by CF-LYS-TEG-MMF offers a huge promise in brain drug delivery, dose reduction and dosage-regimen alteration for the management of brain tumors employing MMF. 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 clinical evaluation1. Numerous reasons have been hypothesized for such failures, including the failure of these agents to get access of the tough blood brain barrier (BBB), which further result in sub-therapeutic concentrations at the target site, i.e., brain2. Furthermore, an understanding is also developing that owing to their shorter plasma-half lives, the therapeutic agents of low-molecular mass, generally are unable to maintain the effective steady state concentrations within the cancerous cells3,4. It is now well-documented that both the biological features like specific enzymes/transporters required, as well as the physicochemical properties of the drugs like H-bonding capability, ionization constant, molecular mass and hydrophobicity, decide the BBB crossing and subsequent central nervous system (CNS) permeation of the drugs.5 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 well-established transport systems like solutecarrier (SLC) transporters4,6. The receptor-mediated endocytosis initially involves endocytosis at the blood (luminal) side, which is a receptor-mediated step, subsequently

followed

by

intracellular

movement

of

the

endocytosed

particle/molecule. This endocytosed component is ultimately exposed to the brain (abluminal) side of brain endothelial cell by exocytosis7. As per scientific consensus, the inability to cross BBB, is the main reason behind the under-development of CNS drugs8. Now-a-days, the nanotechnology-based 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 nanoparticles, water-soluble fullerene derivatives and silica nanoparticles7,9, Carriers like C60-fullerene immobilized on silica surface have been studied and the pro-oxidant effect has been successfully reported.10 The pro-oxidant properties of fullerene-containing composites under the influence of UV and Xray irradiation opens the perspective of further application in an important domain of bio-nanotechnology, i.e., photodynamic therapy11. As far as fullerenes


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are concerned, this third allotropic form of carbon (after graphite and diamond), was discovered in 198512. 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 exceptionally low solubility of these buckyballs in water13, though these tiny structures possess substantial permeability across lipid membranes. Henceforth, water-soluble C60-fullerene (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 with mild P-gp inhibition14,15,16,17. In addition, fullerene derivatives can also penetrate efficiently across the BBB, and can be used as drug delivery carriers for delivering the drugs to brain18,

19

. 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 protection against free-radical damage.20 Higher doses of CFs, i.e., from 2500 mg to 5000 mg/kg have been administered intra-peritoneally to the rodents and the researchers reported no signs of toxicity in the treatment groups12. In another preclinical study of acute oral administration of fullerenes in SpragueDawley 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 every studied aspect21. Such tolerance at higher doses provide an evidence for biological suitability of CFs. Availability of CF-based marketed/clinical trial products (NOVA C60 and LipoFullerene), is further a testimony to the applicability of naïve CFs in the development of human usable products22,23,24. The noncovalent complex of CF-drug (doxorubicin/cisplatin) has been reported to offer medico-biological synergy on its administration25,

26

. Therefore, fabrication of

fullerene-drug hybrids can be a promising approach to alleviate the concerns associated with brain delivery of neurologically active agents21.



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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 NKp4622. Though a biologically active, fumaric acid esters are reported to be associated with concerns like gastrointestinal flushing, lower brain permeability, and economic hurdles due to multiple dosing concerns23,24. Henceforth, the present study was aimed to synthesize of lysine-functionalized CFs using well known Prato reaction25,26 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 SH-SY5Y neuroblastoma cells of human origin. Materials and Methods Materials: Tetraethylene glycol (TEG) (LD50: 10000 mg/kg to 34,760 mg/kg in rats), monomethyl

fumarate

(MMF)

and

3-(4,5-dimethylthiazole-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) were purchased from M/s Sigma-Aldrich, New

Delhi,

India.

Tetrahydrofuran

(THF),

1,2-dichloro

benzene,

dichloromethane, triethyl amine, sodium chloride, buffer ingredients and paraformaldehyde were procured from M/s CDH Pvt Ltd., New Delhi, India. C60fullerenes (CFs) were obtained from M/s M/s BuckyUSA, Houston, USA. ptoluene sulphonyl chloride (TsCl), N,N′-dicyclohexyl carbodiimide (DCC) and 4(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 SH-SY5Y 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 1,2-dichlorobenzene 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 fullerenes (2) (CF-LYS), (%Yield: 90%) (Figure 1)25. 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 Na2SO4 and further by vacuum. The solvent was evaporated to fetch the product of viscous nature (4) (Ts-TEG). 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 Figure 127. Conjugation of tetraethylene glycol to the fullerenes (CF-LYS-TEG-OH) 4 (5 equivalents) and K2CO3 (10 equivalents) were added to the dispersion of CF-LYS (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 (R-215, 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) CF-LYS-TEG-OH, as shown in Figure 1 (%Yield: 95.27%)28



Figure 1: Schematic representation of the synthetic protocol employed for the synthesis of the desired CF-LYS-TEG-MMF nanoconjugate Preparation of MMF conjugated linker-tethered fullerene (CF-LYS-TEGMMF) (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 half-an-hour 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 nhexane to remove 1,3-dicyclohexylurea from the MMF conjugated linker-tethered fullerene, i.e., (7) CF-LYS-TEG-MMF (%Yield: 80%). The synthetic scheme of this product has been depicted in Figure 129. Characterization and Evaluation Studies Fourier Transform Infrared Spectroscopy (FT-IR) Functional group identification of the compounds was done by fourier transform infrared spectroscopy (FT-IR). The samples were mixed with potassium bromide and pallets were prepared using hydraulic press. The FT-IR spectra were recorded in the desired wave number range using FT-IR 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 deuterated chloroform (CDCl3), and 1H-NMR 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 zeta-potential of the undiluted samples. The morphology of CF-LYS and CFLYS-TEG-MMF was visualized by field emission scanning electron microscopy (FE-SEM)(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 MNIT, and photographs were clicked at suitable magnification(s)30. 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 RP-HPLC method (LC-2010C HT, M/s Schimadzu Co. Ltd., Chiyoda-ku, Tokyo, Japan). Mobile phase was consisted of water and acetonitrile (60:40 v/v), which was pumped at the rate of 1 mL min-1 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 drug conjugated on to the 100 parts of the CF-LYS-TEG-OH13. In-vitro Release Studies The release of MMF from the CF-LYS-TEG-MMF nanoconjugate was determined by dialysis technique. Naïve MMF and CF-LYS-TEG-MTX 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 protocol31. 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 on healthy human blood32. 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 anticoagulant

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 CF-LYS (equivalent to the carrier required to load 1 mg of MMF) and 2.7 mg of CF-LYS-TEG-MMF 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. UV-Visible spectroscopy with an λmax of


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415 nm was employed to analysed the clear supernatant of each test tube, and the percent haemolysis was determined using the Equation 113.

................. (1)

Ds, Dnc and Dpc are the absorption of sample, positive control and negative control, respectively. In-vitro Cytotoxicity assay A 5*103 SH-SY5Y cells were cultured in 96-well microplate, with 5% CO2 at 37

.

Cells were allowed to adhere and grow for overnight before treatment. The test samples, i.e., MMF and CF-LYS-TEG-MMF, were added in the well plates in various equivalent concentrations of MMF (1 to 100 µM). The concentrations of CF-LYS were used as the equivalent concentrations of CF-LYS-TEG-MMF. 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 the IC50 values in the set experimental conditions33.

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 (200-250 g; 4-6 weeks old) and divided into two groups of four animals each. Group 1 rats received plain MMF, whereas Group 2 was administered with CF-



LYS-TEG-MMF. The route of administration followed was intra-venous through the tail vein of rats. The doses equivalent to 5 mg/kg of MMF34 were aseptically prepared in “water for injection”. At pre-determined time points, the blood sampling was performed from the retro-orbital 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 performed using RP-HPLC23. Results and Discussion Spectroscopic Characterization: Synthesis of Ts-TEG was confirmed using FT-IR as shown in Figure 2(A). Free hydroxyl group of TS-TEG shown a broad peak at 3447.33 cm-1. The stretching of methylene (–CH2-) was observed at 2890.11 cm-1 and the respective bending occurred at 1454.31 cm-1. (S=O) of Ts-TEG exhibited a asymmetric stretch at 1356.63 cm-1 and symmetric stretch at 1189.90 cm-1. Presence of several peaks between 1000 cm-1 to 750 cm-1 corresponded to S-O stretch. Figure 2(B) depicts the FT-IR spectrum of CF-LYS-TEG and confirmed the successful synthesis of the same. As a broad peak at 3435.57 cm-1 corresponded to the free –OH of the TEG chain, whereas the stretching vibrations of methylene (CH2-) occurred at 2890.11 cm-1. The synthesis of the final conjugate, i.e., CFLYS-TEG-MMF was also confirmed from FT-IR results, as depicted in Figure 2(C). Stretching vibration from >C=C< of MMF was exhibited at 1645.64 cm-1, whereas the stretching of =C-H merged with the broad peak of few –hydroxyl groups on CF-LYS-TEG. A strong peak at 1732.60 cm-1 confirmed the formation of ester linkage between CF-LYS-TEG and MMF. Spectrum of CF-LYS-TEG-MMF also exhibited a broad peak at 3425.10 cm-1, ascribable to the the presence of few unreacted hydroxyl group of CF-LYS-TEG. Formation of Ts-TEG was further confirmed by

1

H NMR (Figure 3). Peaks

between 3.69 ppm to 3.06 ppm were ascribed to the oxygen-linked protons of methylene group, –CH2OCH2-and -CH2OH. Intrinsic peaks between 7.31 ppm to 7.77 ppm were ascribed to –CH group of aromatic ring35. Formation of CF-LYSTEG-MMF conjugate was confirmed by appearance of additional peaks between 6.07 ppm to 6.70 ppm, ascribable to the protons of -CH=CH- group, exclusively present in MMF, as depicted in Figure 436.


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Figure 2: Figure showing the observed FT-IR spectra of various compounds: (A) Ts-TEG; (B) CF-LYS-TEG; (C) CF- LYS-TEG-MMF



Figure 3: 1H-NMR spectrum of the intermediate Ts-TEG in CDCl3

Figure 4: 1H-NMR spectrum of CF-LYS-TEG-MMF in d6-DMSO

Physicochemical Characterization The physicochemical properties of the intermediates and the final conjugate has been shown in Table 1. Understanding of the zeta-potential and particle size of the nanosystems can help in predicting the fate at the biological level37. 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 surpass rapid renal clearance38. 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 zeta-potential values can be attributed to the conjugation of protonated amino groups of lysine with the tosyl-TEG. On the other hand, the mean particle size and zeta potential values of final CF-LYS-TEG-MMF 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 FE-SEM images of the CF-LYS and CF-LYS-TEG-MMF nanoconjugate have been presented in the Figure 5A and Figure 5B, respectively. Though nothing can be clearly vouched from the FE-SEM images, still the absence of any visible contaminant is vivid. The findings are in close consonance

with the

published FE-SEM results of such systems39.

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 CF-LYSTEG-MMF nanoconjugate can offer an effective therapeutic drug delivery option for MMF37.

Figure 5: Microphotographs of FE-SEM images of: (A) CF-LYS (10,000X) and (B) CF-LYS-TEG-MMF (10,000X)



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Table 1: Tabular Representation of Log P and aqueous solubility of different samples. S. No 1 2 3

Sample TEG Ts-TEG CF-LYS-TEG-MMF

Log P -0.95 1.27 -0.40

Aqueous Solubility 5 mg/mL 1.25 mg/mL 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

Sample

Particle size (nm)

PDI

1.

CF-LYS

81.00 ± 2.25

0.100

+26.10 ± 1.44

2.

CF-LYS-TEG-OH

124.29 ± 2.76

0.147

-5.36 ± 1.40

3.

CF-LYS-TEG-MMF

137.6 ± 1.86

0.206

-12.20 ± 1.72

(mV)

1.1 In-vitro Drug Release: The drug release behaviour of naïve MMF and CF-LYS-TEG-MMF 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). CF-LYS-TEG-MMF nanoconjugate offered pH-dependent drug release, reasonably due to the presence of ester linkage between CF-LYS-TEG and MMF, whereas naïve MMF showed no significant pH dependency40. The discriminative drug release profile in two different pH values carriers a promising advantage for the designed system, intended to be administered by intra-venous 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 cells41. Moreover, we believed that once internalized inside target cells, the ester linkage of CF-LYS-TEG-MMF could also be preferentially hydrolysed to release MMF by the endo/lysosomal esterases coupled with acid calatyled hydrolysis due to acidic pH of the cancer cell41. 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 non-swellable matrices for the drug; hence, Higuchi profile is expected from such carriers and the present findings are in consonance with the previous published reports33.

Figure 6: In-vitro % cumulative MMF release data of MMF and CF-LYS-TEG-MMF 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 CF-LYS-TEG-MMF nanoconjugate (4.56%), whereas MMF was observed to induced maximum hemolysis (9.86%), followed by CF-LYS (7.71%). The difference from the data of pure drug was statistically significant

at

p

Lysine-Based C60-Fullerene Nanoconjugates for Monomethyl

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