Folate-Targeted Nanoliposomal Chemophototherapy

Abstract

Light-responsive liposomes have been developed for the on-demand release of drugs. However, efficient delivery of chemotherapeutic drugs to tumor for cancer theranostics remains a challenge. Herein, folic acid (FA), an established ligand for targeted drug delivery, was used to decorate light-sensitive porphyrin-phospholipid (PoP) liposomes, which were assessed for FA-targeted chemophototherapy (CPT). PoP liposomes and FA-conjugated PoP liposomes were loaded with Doxorubicin (Dox), and physical properties were characterized. In vitro, FA-PoP liposomes that were incubated with FA receptor-overexpressing human KB cancer cells showed increased uptake compared to non-targeted PoP liposomes. Dox and PoP contributed towards chemophototherapy (CPT) in vitro, and PoP and FA-PoP liposomes induced cell killing. In vivo, mice bearing subcutaneous KB tumors treated with PoP or FA-PoP liposomes loaded with Dox, followed by 665 nm laser treatment, had delayed tumor growth and improved survival. Dox delivery to tumors increased following laser irradiation for both PoP and FA-PoP liposomes. Thus, while Dox-FA-PoP liposomes were effective following systemic administration and local light irradiation in this tumor model, the FA targeting moiety did not appear essential for anti-tumor responses.

Keywords: chemophototherapy; doxorubicin; folate; folic acid; liposome; photodynamic therapy.

Figure 1

Drug loading and triggered release of Dox from PoP and FA-PoP liposomes. (A) Fluorescence emission spectra of PoP in PoP or FA-PoP liposomes. (B) Dox fluorescence from liposome fractions collected from gel filtration (G75 Sephadex) column. (C) Serum stability of Dox-loaded PoP or FA-PoP liposomes incubated in 50% FBS. (D) Light-triggered release of Dox from liposomes incubated in 50% FBS. Laser irradiation was provided by a 665 nm laser diode.

Figure 2

Cellular binding and uptake of FA-PoP liposomes. (A) PoP mean fluorescence intensity (MFI) of PoP and FA-PoP liposomes incubated with KB cells and measured with flow cytometry. Liposomes were prepared with varying percentages of FA, as indicated. (B) Representative PoP uptake histogram of PoP and FA-PoP samples incubated with KB cells. (C) Cellular uptake of liposomes measured by fluorometric analysis of cell lysate. Cells were incubated with increasing concentrations of PoP or FA-PoP liposomes at the indicated PoP concentration. (D) Uptake of liposomes (40 nM PoP concentration) in folate-receptor positive KB cells and folate-receptor negative A549 cells incubated for 45 min. (E) Microscopy images of KB cells incubated with indicated liposomes (40 nM PoP basis) for 45 min. PoP was read at 420/665 exc./em. For folate-blocking studies (indicated with excess FA in the figure legend), 500–1000 fold excess FA was used.

Figure 3

In vitro chemophototherapy of Dox-FA-PoP liposomes. (A) Dark toxicity of Dox-PoP and Dox-FA-PoP liposomes at indicated concentrations incubated with KB cells for 45 min. (B) Cell viability of cells incubated with liposomes followed by laser irradiation at 10 J cm⁻². Laser irradiation studies were carried out using a 665 nm laser. Statistical difference compared to control cells with * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.005.

Figure 4

In vivo evalution of FA-PoP liposomes. Tissue distribution of Dox-PoP and Dox-FA-PoP liposomes injected in nude mice (4 mg kg⁻¹ Dox) bearing dual KB tumors sacrificed after (A) 4 h, or (B) 24 h, n = 4 mice per group. An unpaired T-test was performed for laser-irradiated samples. NS indicated not significant by t-test. Tumors were treated with laser at 250 J cm⁻² 1 h after i.v. injection via the tail vein. For tumor inhibition studies, nude mice bearing KB tumor were injected with 3 mg kg⁻¹ and irradiated with a 665 nm laser at 400 J cm⁻². (C) Kaplan–Meier survival curves for mice injected with indicated liposomes. (D) Tumor volume of nude mice injected with indicated liposomes. (E) Tumor surface temperature monitored during laser treatment.

Figure 5

Fluorescence imaging of tumor slices obtained from KB tumor-bearing nude mice treated with Dox-PoP or Dox-FA-PoP with or without laser irradiation. Mice were sacrificed 4 h after injection.