Preparation and tumor cell model based biobehavioral evaluation of the nanocarrier system using partially reduced graphene oxide functionalized by surfactant

Abstract

Background

Currently, surfactant-functionalized nanomaterials are tending toward development of novel tumor-targeted drug carriers to overcome multidrug resistance in cancer therapy. Now, investigating the biocompatibility and uptake mechanism of specific drug delivery systems is a growing trend, but usually a troublesome issue, in simple pharmaceutical research.

Methods

We first reported the partially reduced graphene oxide modified with poly(sodium 4-styrenesulfonate) (PSS) as a nanocarrier system. Then, the nanocarrier was characterized by atomic force microscope, scanning electron microscope, high-resolution transmission electron microscope, ultraviolet–visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy, X-Ray powder diffraction, and Raman spectroscopy. Epirubicin (EPI) was attached to PSSG via π–π stacking, hydrogen bonding, and physical absorption to form conjugates of PSSG–EPI. The adsorption and desorption profiles, cytotoxicity coupled with drug accumulation, and uptake of PSSG and PSSG–EPI were evaluated. Finally, the subcellular behaviors, distribution, and biological fate of the drug delivery system were explored by confocal laser scanning microscope using direct fluorescence colocalization imaging and transmission electron microscopy.

Results

The partially reduced graphene oxide sheets functionalized by surfactant exhibit good dispersibility. Moreover, due to much less carboxyl groups retained on the edge of PSSG sheets, the nanocarriers exhibit biocompatibility in vitro. The obtained PSSG shows a high drug-loading capacity of 2.22 mg/mg. The complexes of PSSG–EPI can be transferred to lysosomes in 2 hours through endocytosis, then the drug is released in the cytoplasm in 8 hours, and ultimately EPI is delivered into cell nucleus to exhibit medicinal effects in 1 day.

Conclusion

The comprehensive exploration of the biological uptake mechanism of functional graphene-mediated tumor cell targeting model provides a typical protocol for evaluation of drug delivery system and will benefit the discovery of new surfactant-modified nanocarriers in nanomedicine.

Keywords:

• biocompatibility
• cytotoxicity
• drug delivery
• controlled release
• fluorescence colocalization
• epirubicin

Supplementary materials

Figure S1 Schematic illustration of the PSSG preparation with 12-hour reduction.

Abbreviations: GO, graphene oxide; PSSG, PSS-decorated nanographene; PSS, poly(sodium 4-styrenesulfonate); h, hours.

Figure S2 FTIR spectral monitoring of the PSSG preparation at different reduction time.

Notes: Spectra of PSSG were measured with 3-hour (a), 4-hour (b), and 12-hour (c) reduction, respectively. The characteristic peak at 1,735 cm^–1 in carboxyl groups disappeared in (c) only. GO (d) was used as control.

Abbreviations: FTIR, Fourier transform infrared; GO, graphene oxide; PSSG, PSS-decorated nanographene; PSS, poly(sodium 4-styrenesulfonate).

Figure S3 Standard calibration curve of EPI fluorescence in water solutions.

Abbreviation: EPI, epirubicin.

Figure S4 Calibration of relative fluorescence intensity of EPI in different solvents.

Notes: To exclude the fluorescent quantum yields of EPI dissolved in ethanol and other buffer solutions, the released EPI from PSSG–EPI (20 μg/mL) in the supernatants was also measured with a fluorescence spectrometer. EPI was dissolved at concentration of 20 μg/mL in water, ethanol, and buffer solutions with pH 7.4, 4.6, and 2.0. Left Y-axis shows the relative fluorescence percentages of EPI in different solvents referring to the EPI in water (control group). Right Y-axis shows the corresponding correction factors of different solvents.

Abbreviations: EPI, epirubicin; PSSG, PSS-decorated nanographene; PSS, poly(sodium 4-styrenesulfonate).

Acknowledgments

The authors acknowledge the financial assistance from National Natural Science Foundation of China (21275105), China Postdoctoral Science Foundation (2013M531961), National Recruitment Program of Global Experts (NRPGE), and the Hundred Talents Program of Sichuan Province (HTPSP). The authors also appreciate the professional operation guidance for CLSM from Olympus engineers.

Disclosure

The authors report no conflicts of interest in this work.