Phthalocyanine-loaded graphene nanoplatform for imaging-guided combinatorial phototherapy

Abstract

We report a novel cancer-targeted nanomedicine platform for imaging and prospect for future treatment of unresected ovarian cancer tumors by intraoperative multimodal phototherapy. To develop the required theranostic system, novel low-oxygen graphene nanosheets were chemically modified with polypropylenimine dendrimers loaded with phthalocyanine (Pc) as a photosensitizer. Such a molecular design prevents fluorescence quenching of the Pc by graphene nanosheets, providing the possibility of fluorescence imaging. Furthermore, the developed nanoplatform was conjugated with poly(ethylene glycol), to improve biocompatibility, and with luteinizing hormone-releasing hormone (LHRH) peptide, for tumor-targeted delivery. Notably, a low-power near-infrared (NIR) irradiation of single wavelength was used for both heat generation by the graphene nanosheets (photothermal therapy [PTT]) and for reactive oxygen species (ROS)-production by Pc (photodynamic therapy [PDT]). The combinatorial phototherapy resulted in an enhanced destruction of ovarian cancer cells, with a killing efficacy of 90%–95% at low Pc and low-oxygen graphene dosages, presumably conferring cytotoxicity to the synergistic effects of generated ROS and mild hyperthermia. An animal study confirmed that Pc loaded into the nanoplatform can be employed as a NIR fluorescence agent for imaging-guided drug delivery. Hence, the newly developed Pc-graphene nanoplatform has the significant potential as an effective NIR theranostic probe for imaging and combinatorial phototherapy.

Keywords:

• graphene nanosheets
• phthalocyanine
• photothermal therapy
• photodynamic therapy
• theranostic

Supplementary materials

Fourier transform infrared-attenuated total reflectance (FTIR-ATR) studies

FTIR-ATR measurements of the freeze-dried samples were conducted using a Nicolet iS5 FTIR Spectrometer equipped with iD7-ATR (Thermo Fisher Scientific, Waltham, MA, USA). The FTIR-ATR spectra were acquired within the 4,000–400 cm⁻¹ spectral region. Background spectra of a clean attenuated total reflectance (ATR) surface were acquired prior to each sample measurement, using the same acquisition parameters.

FTIR-ATR spectroscopic measurements were performed to confirm the presence of poly(ethylene glycol) (PEG) and luteinizing hormone-releasing hormone (LHRH) after functionalization of low-oxygen graphene (LOGr). Figure S1 shows the comparison of the FTIR-ATR spectral features of nonmodified LOGr and LOGr functionalized first with polypropylenimine generation 4 (PPIG4) dendrimer loaded with phthalocyanine (Pc) derivative (Pc-PPIG4), and finally with PEG and LHRH peptide. As expected, nonmodified LOGr showed barely noticeable peaks at ~3,400, 2,100, 1,580, and 1,250 cm⁻¹ (Figure S1), possibly corresponding to C-C stretch and oxygen-related groups, such as hydroxyl and epoxy groups. When modified with Pc-PPG4, the ATR spectrum showed distinguished peaks related to the PPIG4 dendrimer structure (Figure S1A and B) as well as to Pc structure. After further functionalization with PEG and LHRH peptide, Figure S1A revealed the spectrum where the corresponding numbers of peaks identifying to PEG and LHRH structures were observed (Figure S1B). In particular, characteristic bands of PEG at ~2,880 (C-H) and ~1,100 (C-O-C) cm⁻¹ were clearly observed in the ATR spectrum of the final (LOGr-Pc-LHRH) freeze-dried sample (Figure S1A). In addition, presence of LHRH peptide was also confirmed via quite strong bands at ~3,270 (O-H, N-H) and ~1,650 (C=O) cm⁻¹.

Figure S1 FTIR-ATR characterization of LOGr-Pc-LHRH.

Notes: (A) FTIR-ATR spectra of nonmodified LOGr (black line), LOGr-Pc-PPIG4 (red line), and LOGr-Pc-LHRH (green line). (B) FTIR-ATR spectra of controls: Pc (red line), PPIG4 (blue line), PEG (gray line), and LHRH (green line).

Abbreviations: FTIR-ATR, Fourier transform infrared-attenuated total reflectance; LHRH, luteinizing hormone-releasing hormone; LOGr, low-oxygen graphene; Pc, phthalocyanine; PEG, poly(ethylene glycol); PPIG4, polypropylenimine generation 4.

Acknowledgments

This work was supported by the Pharmaceutical Research and Manufacturers of America Foundation, the Medical Research Foundation of Oregon, the College of Pharmacy at Oregon State University, and the National Science Foundation (grant number CBET 1438493). We are thankful to Dr B Dolan and A Palmer of the College of Veterinary Medicine, Oregon State University, for their help with the flow cytometry analysis.

Disclosure

The authors report no conflicts of interest in this work.