Development of biocompatible and VEGF-targeted paclitaxel nanodrugs on albumin and graphene oxide dual-carrier for photothermal-triggered drug delivery in vitro and in vivo

Abstract

In this study, we performed the characterization and synthesis of biocompatible and targeted albumin and graphene oxide (GO) dual-carrier paclitaxel (PTX) nanoparticles for photothermal-triggered tumor therapy. PTX absorbed on GO nanosheets as cores were coated with human serum albumin (HSA), following surface conjugation with monoclonal antibodies (mAb) against vascular endothelial growth factor (VEGF; denoted as mAbVEGF) via polyethylene glycol linker to form targeted nanoparticles (PTX-GHP-VEGF). The spherical nanoparticles were 191±5 nm in size with good stability and biocompatibility. GO functioned as the first carrier and a near infrared absorber that can generate photothermal effects under 5-minute 808-nm laser irradiation to thermal trigger the release of PTX from the second carrier HSA nanoparticles. The mechanism of thermal-triggered drug release was also investigated preliminarily, in which the heat generated by GO induced swelling of PTX-GHP-VEGF nanoparticles which released the drugs. In vitro studies found that PTX-GHP-VEGF can efficiently target human SW-13 adrenocortical carcinoma cells as evaluated by confocal fluorescence microscopy as well as transmission electron microscopy, and showed an obvious thermal-triggered antitumor effect, mediated by apoptosis. Moreover, PTX-GHP-VEGF combined with near infrared irradiation showed specific tumor suppression effects with high survival rate after 100 days of treatment. PTX-GHP-VEGF also demonstrated high biosafety with no adverse effects on normal tissues and organs. These results highlight the remarkable potential of PTX-GHP-VEGF in photothermal controllable tumor treatment.

Keywords: graphene oxide; human serum albumin; paclitaxel; photothermal-triggered tumor therapy; tumor targeting.

Figure 1

Synthesis and application schematic. Note: PTX-GO-HSA-PEG-VEGF (PTX-GHP-VEGF) nanoparticles synthesis schematic and its photothermal-triggered drug release application. Abbreviations: PTX, paclitaxel; GO, graphene oxide; HSA, human serum albumin; PEG, polyethylene glycol; VEGF, vascular endothelial growth factor; NIR, near infrared; mAb, monoclonal antibodies; GHP, GO-HSA-PEG.

Figure 2

Morphology characterization. Notes: SEM (A) and TEM (B) images of PTX-GHP-VEGF. Particle size (C) and zeta potential (D) distribution of PTX-GHP-VEGF. Abbreviations: SEM, Scanning electron microscopy; TEM, transmission electron microscopy; PTX, paclitaxel; VEGF, vascular endothelial growth factor; GHP, GO-HSA-PEG.

Figure 3

Spectra and stability characterization. Notes: (A) The absorbance spectra of free PTX, GO, and PTX-GHP-VEGF. (B) The changes of absorbance intensity at 230 nm of PTX-GHP-VEGF over 15 days. (C) The change of average diameter of GO and PTX-GHP-VEGF over 15 days. (D) The PDI change of PTX-GHP-VEGF in water and saline over 4 weeks. Abbreviations: PTX, paclitaxel; GO, graphene oxide; VEGF, vascular endothelial growth factor; PDI, polydispersity index; Abs, absorbance; GHP, GO-HSA-PEG.

Figure 4

Photothermal effect and thermal stability. Notes: (A, B) Photothermal heating curves and the corresponding thermal images of water, GO, and PTX-GHP-VEGF solution at the same concentrations of GO under 808-nm laser irradiation (1 W/cm²). (C) UV–vis absorption spectra of PTX-GHP-VEGF solution before and after irradiations of 808-nm laser for 5 minutes, and (D) their corresponding TEM images. Abbreviations: PTX, paclitaxel; GO, graphene oxide; VEGF, vascular endothelial growth factor; TEM, transmission electron microscopy; NIR, near infrared; Abs, absorbance; UV, ultraviolet; vis, visible; GHP, GO-HSA-PEG.

Figure 5

Drug release. Notes: (A) Release kinetics of PTX from PTX-GHP-VEGF in PBS buffer (pH =7.4) in the absence and presence of 808-nm NIR laser irradiation. (B) The diameter change of PTX-GHP-VEGF under various temperature treatments. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; PBS, phosphate buffer; NIR, near infrared; GHP, GO-HSA-PEG.

Figure 6

In vitro biocompatibility. Notes: (A) Cell viability of SW-13 cells after treatment with GHP-VEGF at different concentrations for 24 hours. (B) Hemolysis ratio of RBCs after 2 hours incubation with PTX-GHP-VEGF at different concentrations. The inset shows the photograph of RBCs exposed to deionized water, PBS, and PTX-GHP-VEGF solution with different concentrations followed by centrifugation. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; PBS, phosphate-buffered saline; RBCs, red blood cells; GHP, GO-HSA-PEG.

Figure 7

Celluar uptake. Notes: (A) Confocal fluorescence images of SW-13 cells after incubation with free FITC, GHP-VEGF, and PTX-GHP-VEGF labeled by FITC. Green and blue colors represented FITC fluorescence and DAPI-stained cell nuclei, respectively. (B–E) Flow cytometry analysis and quantitative cellular uptake of SW-13 cells toward control, GHP-VEGF, and PTX-GHP-VEGF. (F, G) Bio-TEM images of SW-13 cells after incubation with PBS and PTX-GHP-VEGF for 3 hours. Red arrows indicated the presence of PTX-GHP-VEGF. Scale bar represents 20 µm (A), and 2 µm (F, G). Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TEM, transmission electron microscopy; DAPI, 6-diamidino-2-phenylindole; GHP, GO-HSA-PEG.

Figure 8

In vitro cytotoxicity. Note: Cell viabilities of SW-13 cells after incubation with different concentrations of PTX, GHP-VEGF, PTX-GHP, and PTX-GHP-VEGF for various times with (A–C) or without (D–F) 808-nm laser irradiation (1 W/cm²) for 5 minutes. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; GHP, GHP, GO-HSA-PEG.

Figure 9

Apoptosis detection. Notes: (A–D) The calcium AM/PI dual staining images of SW-13 cells after treatment by PBS, PBS + NIR, PTX-GHP-VEGF, and PTX-GHP-VEGF + NIR, respectively. (E–H) Flow cytometry analysis of SW-13 cells treated by PBS, PBS + NIR, PTX-GHP-VEGF, and PTX-GHP-VEGF + NIR, respectively. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; PBS, phosphate-buffered saline; PI, propidium iodide; FITC, fluorescein isothiocyanate; GHP, GO-HSA-PEG.

Figure 10

In vivo NIR thermal imaging. Notes: (A) The thermal images of tumor bearing mice post-tail vein injection of saline, PTX-GHP, and PTX-GHP-VEGF at 0, 12, 24 and 36 hours under 5-minute NIR irradiation (808 nm, 1 W/cm²), respectively. (B) The temperature statistical results of tumor regions of tumor bearing mice post-tail vein injection of saline, PTX-GHP, and PTX-GHP-VEGF at 0, 12, 24 and 36 hours under 5-minute NIR irradiation. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; GHP, GO-HSA-PEG.

Figure 11

In vivo anticancer. Note: The relative tumor volume (A) and survival rate (B) of tumor bearing mice after tail vein injection with control (saline), free PTX, GHP-VEGF + NIR, PTX-GHP + NIR, PTX-GHP-VEGF, and PTX-GHP-VEGF + NIR. Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; GHP, GO-HSA-PEG.

Figure 12

In vivo biocompatibility. Notes: (A) Body weight of 4T1 tumor bearing mice after various treatments. Blood biochemistry (B) and blood cells count (C, D) of mice at days 1 and 30 posttreatment with saline (control) and PTX-GHP-VEGF. (E) H&E-stained tissue sections of major organs, including the heart, liver, spleen, lung, and kidney from mice treated with saline (control) or PTX-GHP-VEGF at day 1 and day 30 (magnification: 100×). Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; H&E, hematoxylin and eosin; WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; PLT, platelets; AST, aspartate transaminase; ALT, alanine transaminase; GHP, GO-HSA-PEG.

Figure 12

In vivo biocompatibility. Notes: (A) Body weight of 4T1 tumor bearing mice after various treatments. Blood biochemistry (B) and blood cells count (C, D) of mice at days 1 and 30 posttreatment with saline (control) and PTX-GHP-VEGF. (E) H&E-stained tissue sections of major organs, including the heart, liver, spleen, lung, and kidney from mice treated with saline (control) or PTX-GHP-VEGF at day 1 and day 30 (magnification: 100×). Abbreviations: PTX, paclitaxel; VEGF, vascular endothelial growth factor; NIR, near infrared; H&E, hematoxylin and eosin; WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; PLT, platelets; AST, aspartate transaminase; ALT, alanine transaminase; GHP, GO-HSA-PEG.