Nano-delivery Vehicle Based on Chlorin E6, Photodynamic Therapy, Doxorubicin Chemotherapy Provides Targeted Treatment of HER-2, Negative, ανβ3-positive Breast Cancer.

The prognosis for patients with HER-2 negative breast cancer is currently poor, largely due to the lack of efficacious targeted therapeutics. Photodynamic nanomaterial technologies have rapidly developed in recent years, but their anti-tumor effects are often limited by poor targeting, low transformation efficiency, toxicity, and other factors. Thus, we prepared a new type of nanoparticles (Ce6/Dox@NPs-cRGD, CDNR) with cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)) that target the ανβ3 receptor. We loaded those nanoparticles (NPs) with a combination of the doxorubicin (Dox) and photosensitizer chlorin E6 (Ce6) to test synergy between chemotherapy and photodynamic therapy (PDT) for the treatment of ανβ3 receptor positive and HER-2 negative breast cancer. Through analysis of the Fourier transform infrared and UV-vis spectra of these NPs, we found that Ce6 and Dox were successfully loaded into the CDNR. According to dynamic light scattering (DLS) analyses, CDNR particles had a diameter of 112.6 nm (polydispersity index 0.11), which was also confirmed via TEM characterization. The zeta potential was about -21.5 mV. Stability studies showed that CDNR particle size was stable in ddH2O, PBS, and DMEM + 5% FBS for 16 days. The drug loading content of Dox and Ce6 were 5.3 and 6.8 %, respectively. Release studies of CDNR showed that the slow release of Dox was accelerated with increasing GSH concentration, and there was no burst release effect. From studying the absorbance of 9,10-dimethylanthrancene (ABDA), we found that CDNR produces high levels of ROS after excitation with a 670 nm laser, and ROS production increased with increasing radiation time. CDNR was significantly taken up by MCF-7 cells at 6 h because of cRGD targeting. In a CCK8 test, the relative growth rate (RGR) of CDNR +670 nm laser for MCF-7 cells was less than 75% at 20 μg/mL after 24 h treatment and 15 μg/mL after 48 h treatment. We found that CDNR's effects on RGR were concentration dependent. Live-cell staining with a DCFH-DA kit and flow cytometry assay further supported that a CDNR +670 nm laser provided the maximum chemotherapy-PDT toxicity and production of intracellular ROS, and that cell death was mainly caused by necrosis and apoptosis. In vivo experiments showed that using the cRGD-targeting strategy, CDNR had a stronger affinity and increased half-life relative to Ce6/Dox@NPs in mice with MCF-7 xenograft tumors. Further, the Cmax of CDNR in the transplanted tumor occurred 8 hours post-injection (HPI) and there was still detectable signal at 24 HPI. In addition, MCF-7 bearing mice that were treated with CDNR +670 nm PDT at 8 HPI had a significantly decreased tumor volume (P <  0.05) and prolonged survival time compared to other groups. Thus, CDNR plus 670 nm PDT was associated with favorable anti-tumor activity with no appreciable impact on body weight or the major organs in mice, as determined by immunohistochemistry/immunofluorescence and hematoxylin-eosin staining. In conclusion, CDNR with 670 nm laser irradiation represents a promising new potential treatment paradigm for the management of breast cancers that are ανβ3-receptor positive and HER-2 negative.