Photodynamic Therapy Induced Enhancement of Tumor Vasculature Permeability Using an Upconversion Nanoconstruct for Improved Intratumoral Nanoparticle Delivery in Deep Tissues

Abstract

Photodynamic therapy (PDT) has recently emerged as an approach to enhance intratumoral accumulation of nanoparticles. However, conventional PDT is greatly limited by the inability of the excitation light to sufficiently penetrate tissue, rendering PDT ineffective in the relatively deep tumors. To address this limitation, we developed a novel PDT platform and reported for the first time the effect of deep-tissue PDT on nanoparticle uptake in tumors. This platform employed c(RGDyK)-conjugated upconversion nanoparticles (UCNPs), which facilitate active targeting of the nanoconstruct to tumor vasculature and achieve the deep-tissue photosensitizer activation by NIR light irradiation. Results indicated that our PDT system efficiently enhanced intratumoral uptake of different nanoparticles in a deep-seated tumor model. The optimal light dose for deep-tissue PDT (34 mW/cm(2)) was determined and the most robust permeability enhancement was achieved by administering the nanoparticles within 15 minutes following PDT treatment. Further, a two-step treatment strategy was developed and validated featuring the capability of improving the therapeutic efficacy of Doxil while simultaneously reducing its cardiotoxicity. This two-step treatment resulted in a tumor inhibition rate of 79% compared with 56% after Doxil treatment alone. These findings provide evidence in support of the clinical application of deep-tissue PDT for enhanced nano-drug delivery.

Keywords: Drug delivery.; EPR; Nanoparticles; Photodynamic therapy; Upconversion.

Figure 1

Synthesis and characterization of c(RGDyK)-SOC-UCNP-ZnPc (R-SuZn): (A) Experimental design of the synthetic process of c(RGDyK)-SOC-UCNP-ZnPc; (B) Morphology and size distribution of R-SuZn as demonstrated by TEM and DLS analysis; (C) Representative images of c(RGDyK)-SOC-UCNP-ZnPc (left) and c(RGDyK)-SOC-UCNP (right) under ambient light; (D) UCL emission spectra of c(RGDyK)-SOC-UCNP, c(RGDyK)-SOC-UCNP-ZnPc (100 μg/mL) and c(RGDyK)-SOC-UCNP- ZnPc (400 μg/mL).

Figure 2

ZnPc loading capacity, encapsulation efficiency, and stability in PBS and in vitro: (A) Loading capacity at different ZnPc concentrations; (B) Encapsulation efficiencies at different ZnPc concentrations; (C) Change in ZnPc release into PBS with different pH values (5.7, 7.4 and 8.0); (D) Photostability of R-SuZn after 120min exposure to light (980nm); (E) PC-3 cells uptake of R-SuZn; colocalization of ZnPc and UCNPs is indicated.

Figure 3

Enhanced tumor uptake of liposome-ICG and liposomal-Rhodamine facilitated by PDT: (A) Experimental strategy to evaluate enhanced tumor uptake of liposome-ICG in bilateral tumor models by PDT, in the absence (a) or presence (b) of a pork tissue barrier; (B) The tumor:skin ratio in superficial tumor models 24hr after 660 nm light; (C) The tumor: skin ratio in deep-seated tumor models treated with 660 nm light or 980 nm light; (D) Determination of tumor uptake of liposomal Rhodamine by laser confocal microscopy in superficial and deep-seated tumor models treated with 660nm light or 980 nm light.

Figure 4

Enhanced tumor uptake of HSA-ICG and gold nanoparticles after PDT treatment: (A) Tumor uptake of HSA-ICG after PDT treatment in superficial and deep-seated tumor models with 660 nm or 980 nm light irradiation; (B) Dark field images of gold nanoparticles accumulation in tumor tissues after PDT treatment in superficial and deep-seated tumor models with 660 nm or 980 nm light irradiation.

Figure 5

Effect of light fluence rate and injection interval after PDT on enhanced uptake of nanoparticles: (A) Varying tumor uptake of liposome-ICG with altered light fluence rate in deep-seated tumor models; (B) Tumor: skin ratios of liposome-ICG in mice treated with different light fluence rate; (C) Laser confocal microscopy images of tumor tissues with differing Doxil uptake under NIR light at various light fluence rate; (D) Impact of light fluence rate on intratumoral doxorubicin concentration; (E) Effect of injection interval of nanoparticle after PDT treatment on doxorubicin uptake in tumor tissues.

Figure 6

Therapeutic efficacy of the two-step strategy following deep-tissue PDT and Doxil injection: (A) Representative images of dissected tumors across different treatment groups; (B) Tumor volume at 14 days in different treatment groups; (C) Comparison of body weights of mice across different treatment groups over the 14-day time course; (D) H&E staining of histological sections of tumor tissues and cardiac tissue after different treatments.