Enhanced photo/chemo combination efficiency against bladder tumor by encapsulation of DOX and ZnPC into in situ-formed thermosensitive polymer hydrogel

Abstract

Background: Chemotherapy after transurethral resection is commonly recommended for bladder cancer. However, studies have shown that chemotherapy solely can hardly decrease progression rates of bladder cancer. The combination of chemotherapeutic agents with photo-dynamic therapy (PDT), a new promising localized therapy, may become a workable strategy for combating bladder cancer. This study reports the combination of doxorubicin (DOX)-based chemotherapy and zinc phthalocyanine (ZnPC)-based PDT using in situ-formed thermal-responsive copolymer hydrogel.

Materials and methods: The copolymer was synthesized by polymerization of 3-caprolactone, 1,4,8-trioxa[4.6]spiro-9-undecanone and poly(ethylene glycol) and was abbreviated as PCL-PTSUO-PEG. The thermal-responsive nanoparticles (TNPs) were prepared by the nanoprecipitation technology. The thermal-responsive hydrogel was formed after 37°C heating of TNP solution. The size, morphology and dynamic viscosity of hydrogel were detected. The in vitro drug release profile of TNP/DOX/ZnPC was performed. Cell uptake, cell inhibition and ROS generation of TNP/DOX/ZnPC were studied in 5637 cells. The in vivo antitumor activity of TNP/DOX/ZnPC was evaluated in nude mice bearing 5637 cells xenograft.

Results: TNP/DOX and TNP/ZnPC had an average diameter of 102 and 108 nm, respectively. After being heated at 37°C for 5 minutes, TNP/DOX and TNP/ZnPC solution turned uniform light red and dark green hydrogel. ZnPC encapsulation designed by TNP could significantly improve its aqueous solubility to 1.9 mg/mL. Cell inhibition showed that the best cell inhibition was found, with cell viability of 18.5%, when the weight ratio of DOX and ZnPC encapsulated in the TNP reached about 1:5. TNP/DOX/ZnPC generated relative high level of ROS with 4.8-fold of free ZnPC and 1.6-fold of TNP/ZnPC. TNP/DOX/ZnPC showed only 8-fold of relative tumor growth without obvious toxicity to the mice.

Conclusion: Thermosensitive thermal-responsive hydrogel reported in this contribution are promising in situ-formed matrix for DOX- and ZnPC-based photo/chemo combination treatment for bladder cancer therapy.

Keywords: bladder cancer; chemotherapy; combination therapy; hydrogel; photodynamic therapy; thermo-sensitive.

Figure 1

The schematic illustration of photo/chemo combination therapy via in situ-formed thermal-sensitive polymer hydrogel (TNP^GEL) in a xenograft tumor model. Abbreviations: DOX, doxorubicin; TNP, thermal-responsive nanoparticle; TNP/DOX, TNP encapsulated with DOX; TNP^GEL, Hydrogel formed by 37°C heating in vitro or formed peritumor in nude mice; TNP/ZnPC, TNP encapsulated with ZnPC; ZnPC, zinc phthalocyanine.

Figure 2

Particle size of (A) TNP and (B) TNP/DOX/ZnPC and SEM morphology of TNP^GEL (A inset) and TNP/DOX/ZnPC^GEL (B inset) after 37°C treatment. Note: The scale bar was 100 nm for all the SEM images. Abbreviations: TNP, thermal-responsive nanoparticle; TNP/DOX/ZnPC, mixture of equivalent mole of TNP/DOX and TNP/ZnPC; ZnPC, zinc phthalocyanine; SEM, scanning electron microscope.

Figure 3

Optical images of blank TNP hydrogel and hydrogels after DOX and/or ZnPC encapsulation (A) and in vitro cumulative drug release from TNP/DOX/ZnPC hydrogel within 5 weeks with TNP/DOX:TNP/ZnPC of 1:5 (B). 1, 2, 3 and 4 in (A) means blank TNP, TNP/DOX, TNP/ZnPC and TNP/DOX/ZnPC, specifically. Abbreviations: DOX, doxorubicin; TNP, thermal-responsive nanoparticle; ZnPC, zinc phthalocyanine; TNP/DOX/ZnPC, mixture of equivalent mole of TNP/DOX and TNP/ZnPC.

Figure 4

Cellular uptake of free drugs and TNP/DOX/ZnPC at the DOX concentration of 17.8 µg/mL and ZnPC concentration of 20 µg/mL against 5637 cells. Note: The scale bar was 25 µm for all images. Abbreviations: DOX, doxorubicin; TNP, thermal-responsive nanoparticle; ZnPC, zinc phthalocyanine; TNP/DOX/ZnPC, mixture of equivalent mole of TNP/DOX and TNP/ZnPC.

Figure 5

(A) In vitro cellular killing efficiency of TNP/DOX/ZnPC with laser irradiation at different weight ratios of DOX and ZnPC determined by MTT assay at 24 hours. Notes: Concentrations of TNP were equal to the amount of TNP in corresponding drug-loaded group. (B) Cellular ROS generation assay of free ZnPC and TNP/DOX/ZnPC with weight ratio of DOX:ZnPC=1:5 in 5637 cells at 4 hours postincubation. Laser irradiation was 100 J/cm², 660 nm, 120 seconds. (n=6, ***P<0.001.) Abbreviations: DOX, doxorubicin; TNP, thermal-responsive nanoparticle; ZnPC, zinc phthalocyanine; TNP/DOX/ZnPC, mixture of equivalent mole of TNP/DOX and TNP/ZnPC.

Figure 6

In vivo 5637 cells xenograft inhibition analysis. Notes: Relative tumor volume (A) and survival percentage (B) after different treatments at day 0 with DOX and/or ZnPC was administrated at a total dose of 20 mg/kg body weight (n=6, **P<0.01). Abbreviations: DOX, doxorubicin; TNP, thermal-responsive nanoparticle; ZnPC, zinc phthalocyanine.