Polymer conjugated graphene-oxide nanoparticles impair nuclear DNA and Topoisomerase I in cancer

Cancer chemotherapy had been dominated by the use of small molecule DNA damaging drugs. Eventually, the emergence of DNA damage repair machinery in cancer cells has led to combination therapy with the DNA topology controlling enzyme, topoisomerase I inhibitor along with DNA impairing agents. However, integrating multiple drugs having diverse water solubility and hence bio-distribution effectively for cancer treatment remains a significant challenge, which can be addressed by using suitable nano-scale materials. Herein, we have chemically conjugated graphene oxide (GO) with biocompatible and hydrophilic polymers [polyethylene glycol (PEG) and ethylene-diamine modified poly-isobutylene-maleic anhydride (PMA-ED)], which can encompass highly hydrophobic topoisomerase I inhibitor, SN38. Interestingly, these sheet structured GO-polymer-SN38 composites self-assembled into spherical nanoparticles in water after complexing with a hydrophilic DNA damaging drug, cisplatin. These nanoparticles showed much improved colloidal stability in water compared to their drug-loaded non-polymeric counterpart. These SN38 and cisplatin laden GO-polymer nanoparticles were taken up by HeLa cancer cells through clathrin-dependent endocytosis to home into lysosomes within 6 h, as confirmed by confocal microscopy. A combination of gel electrophoresis, flow cytometry, and fluorescence microscopy showed that these nanoparticles damaged nuclear DNA and induced topoisomerase I inhibition leading to apoptosis and finally improved HeLa cell death. These self-assembled GO-polymer nanoparticles can be used for strategic impairment of multiple cellular targets involving hydrophobic and hydrophilic drugs for effective combination therapy.

by TCS Leica SP8 machine. FACS analysis was performed using BD FACS Calibur TM flow cytometer.
For quantitative reaction of maleic anhydride with the primary amine, the reaction mixture was concentrated roughly up to one fifth of the original volume using rotavapor system under a reduced pressure after 3 hours of reaction. Further, the concentrated solution was left overnight at 60 °C under stirring conditions. Finally, THF was completely evaporated and the resultant polymer was washed with cold diethyether and dried under vacuum to yield a yellowish powder (9, Scheme 1).
The compound was further dissolved in 3 mL dicholoromethane (DCM) and kept in an ice bath, into which 1.5 mL of TFA was added dropwise. The reaction mixture was stirred at room temperature for 3h. Finally, the solvent was evaporated under vacuum and remaining viscous liquid was washed with cold diethyether to get poly (isobutylne-alt-maleic anhydride)-ehtylenediamine conjugate (10) which was dried under vacuum. Yield = 80% over 2 steps.
Further, SN-38 was dissolved in minimum amount of DMSO and reacted with GO-PEG (3) and GO-PMA-ED (11) in a 1:0.5 weight ratio in water for 24h followed by dialysis against distilled water for 1 day to remove DMSO and also centrifugation to remove unbound SN38 to obtain GO-PEG-SN38 (4) and GO-PMA-ED-SN (12) composites. [2] The obtained GO-PEG-SN38 and GO-PMA-ED-SN38 were then reacted with aquated cisplatin (CDDP) (5mg/mL) in 1:5 weight ratio in water for 24 h at room temperature. Excess aquated cisplatin was removed by dialysis for 6-8 hrs against distilled water which yielded the GO-PEG-SN38-CDDP (6) and GO-PMA-ED-SN38-CDDP (13).

Characterization.
FT-IR: Fourier transform infrared (FTIR) spectroscopy was performed using a NICOLET 6700 FTIR from Thermo Scientific.

Estimation of size, shape and morphology by FESEM, and AFM:
The morphology of GO-PEG-SN38-CDDP and GO-PIMA_Ed-SN38-CDDP nanoparticles was observed using field-emission scanning electron microscopy (FESEM) and atomic force microscopy, by spotting the samples on a silicon wafer and mica sheet respectively and imaged using Carl Zeiss Ultraplus scanning electron microscope at an operating voltage of 4KV and Nanowizard Atomic force microscope. [3] Raman Spectroscopy: Raman spectra for GO-PEG-SN38-CDDP and GO-PMA-NP were recorded with Lab RAM HR 800 (Horiba scientific) instrument using laser excitation wavelength of 532 nm with 50X objective.

Quantification of drug loading in nanoparticles:
Loading of the individual drugs SN38 (387nm), Cisplatin (707 nm) in GO-PEG-NP and GO-PMA-NP was estimated by UV-visible spectroscopy and the drug loading efficiency was calculated as [4] : Drug encapsulation efficiency (%) = Amount of drug loaded in nanoparticle X 100 Total amount of drug used S4 Ratio of loading of SN38:CDDP in GO-PEG-SN38-CDDP is 1:0.8 and in GO-PMA-ED-SN38-CDDP is 1:0.97.
Fluorescence Spectroscopy. Steady state fluorescence of for fluorescent drug SN38 was recorded using a Flouromax-4 (HORIBA scientific, USA) the emission spectra for SN38 was recorder at λmax = 560nm.