Active Tumor Permeation and Uptake of Surface Charge-Switchable Theranostic Nanoparticles for Imaging-Guided Photothermal/Chemo Combinatorial Therapy

Abstract

To significantly promote tumor uptake and penetration of therapeutics, a nanovehicle system comprising poly(lactic-co-glycolic acid) (PLGA) as the hydrophobic cores coated with pH-responsive N-acetyl histidine modified D-α-tocopheryl polyethylene glycol succinate (NAcHis-TPGS) is developed in this work. The nanocarriers with switchable surface charges in response to tumor extracellular acidity (pHe) were capable of selectively co-delivering indocyanine green (ICG), a photothermal agent, and doxorubicin (DOX), a chemotherapy drug, to tumor sites. The in vitro cellular uptake of ICG/DOX-loaded nanoparticles by cancer cells and macrophages was significantly promoted in weak acidic environments due to the increased protonation of the NAcHis moieties. The results of in vivo and ex vivo biodistribution studies demonstrated that upon intravenous injection the theranostic nanoparticles were substantially accumulated in TRAMP-C1 solid tumor of tumor-bearing mice. Immunohistochemical examination of tumor sections confirmed the active permeation of the nanoparticles into deep tumor hypoxia due to their small size, pHe-induced near neutral surface, and the additional hitchhiking transport via tumor-associated macrophages. The prominent imaging-guided photothermal therapy of ICG/DOX-loaded nanoparticles after tumor accumulation induced extensive tumor tissue/vessel ablation, which further promoted their extravasation and DOX tumor permeation, thus effectively suppressing tumor growth.

Keywords: chemotherapy; deep tumor penetration; photothermal therapy; surface charge transition; tumor hypoxia.

Scheme 1

Illustration of active tumor penetration and uptake of dual drug-loaded nanoparticles with pH_e-triggered surface charge transition for the imaging-guided photothermal/chemo combinatorial therapy.

Figure 1

(a) DLS particle size distribution profiles of nanoparticles in aqueous solution of pH 7.4. (b) TEM images of (i) pristine NHTPNs, (ii) ICG-loaded NHTPNs, (iii) DOX-loaded NHTPNs, (iv) ICG/DOX-loaded PNs, (v) ICG/DOX-loaded TPNs and (vi) ICG/DOX-loaded NHTPNs. Scale bars are 200 nm. (c) UV/Vis spectra of free ICG, ICG/DOX-loaded NHTPNs, TPNs and PNs in PBS.

Figure 2

(a) Normalized maximum absorbance of free ICG and various ICG-containing nanoparticles in PBS at 37 ^oC. (b) Time-evolved mean hydrodynamic diameters (D_h) of various cargo-loaded nanoparticles in PBS at 37 ^oC. (c) Temperature profiles and (d) thermal images of free ICG and ICG-containing nanoparticles (ICG concentration = 10 μM) in PBS with 808 nm NIR laser irradiation (1.0 W/cm²).

Figure 3

(a) Zeta potential of pristine and cargo-loaded nanoparticles in aqueous solutions. (b) DLS particle size distribution profiles of ICG/DOX-loaded NHTPNs in aqueous solutions. (c) Cumulative ICG release profiles of various cargo-loaded nanoparticles in PBS at 37 ^oC. (d) Cumulative DOX release profiles of ICG/DOX-loaded NHTPNs and TPNs in aqueous solutions of pH 7.4 and 6.0.

Figure 4

(a) Fluorescence images of ICG molecules from TRAMP-C1 cells incubated with free ICG and ICG/DOX-loaded TPNs and NHTPNs, respectively, under different pH conditions attained by IVIS. (b) Intracellular DOX amount of TRAMP-C1 cells incubated with various DOX formulations. (c) Flow cytometric histograms TRAMP-C1 cells treated with ICG/DOX-loaded TPNs and NHTPNs at pH 7.4 and 6.3 for 2 h (DOX concentration = 20 μΜ). (d) Fluorescence images of TRAMP-C1 cells incubated with ICG/DOX-loaded TPN and NHTPNs at pH 7.4 and 6.3 for 1.5 h (DOX concentration = 10 μΜ). Nuclei and F-actin cytoskeleton were stained with Hoechst and F-actin marker, respectively. Scale bar is 50 μm.

Figure 5

(a) Cell viability of TRAMP-C1 cells incubated with various formulations for 24 h with and without the 5-min NIR laser irradiation, followed by additional 24 h incubation. (b) Cell viability of TRAMP-C1 cells incubated respectively with ICG-loaded NHTPNs and ICG/DOX-loaded NHTPNs (ICG 5.0 μM and DOX 5.5 μM, if applicable) at pH 7.4 and 6.3.

Figure 6

(a) In vivo NIR fluorescence images of TRAMP-C1 tumor-bearing mice receiving intravenous injection of PBS and different ICG-containing formulations evaluated by IVIS. (b) NIR fluorescence images of the isolated major organs and tumors 48 h post-injection with PBS and different ICG-containing formulations. (c) Average ICG fluorescence intensities of individual organs and tumor from TRAMP-C1 tumor-bearing mice treated with different ICG-containing formulations (n = 5 per group).

Figure 7

(a) Intratumoral distribution of free DOX and ICG/DOX-loaded TPNs and NHTPNs in relation to the loci of angiogenic blood vessels examined by immunofluorescence microscopy. (b) Intratumoral distribution of ICG/DOX-loaded NHTPNs in relation to the loci of tumor hypoxia and TAM by IHC examination of contiguous tumor sections. (c) Intratumoral distribution of free DOX and ICG/DOX-loaded TPNs and NHTPNs in relation to the loci of tumor hypoxia examined by immunofluorescence microscopy. Tumor cell nuclei, angiogenic blood vessels, tumor hypoxia and TAM were stained with DAPI and CD31, HIF 1α and CD11b markers, respectively. Scale bars are 50 μm in (a) and 100 μm in (b and c).

Figure 8

(a) Temperature profiles and (b) infrared thermographic maps at the tumor sites of TRAMP-C1 tumor-bearing mice receiving various formulations and exposed to NIR laser irradiation of 1.0 W/cm² 6 h post-injection.

Figure 9

Fluorescence images of tumor sections from the mice bearing TRAMP-C1 tumor 48 h post-injection of various formulations (a) without and (b) with NIR irradiation. Cell nuclei and apoptotic cells were stained with DAPI (blue) and caspase-3 marker (green), respectively. Scale bars are 100 μm.

Figure 10

(a) Tumor growth inhibition profiles of the mice bearing TRAMP-C1 tumor injected with various formulations, followed by NIR laser irradiation (5 min, 1.0 W/cm²) 6 h post-injection or without any laser treatment (n = 4 per group). Morphology and size of the tumors of each group isolated from the sacrificed mice at day 15 (the end point) after the treatment. (b) Images of H&E-stained tumor sections harvested from the tumor-bearing mice receiving treatments. Scale bars are 100 μm.