Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors

Abstract

Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors.

Figure 1

Schematic of the liposome: 100 nm liposomes were prepared containing both maleimide and amine functionalized PEG chains. The maleimide group was used to attach the peptide through the thiol group on a cystine. The amine was used to conjugate a fluorescent AlexaFluor 750 to the liposome. Therapeutic liposomes were loaded with doxorubicin.

Figure 2

Targeted liposomes (TL) localized in the irradiated tumors. Intensity of the AlexaFluor 750 correlates to the local concentration of liposomes. (A) Nude mice bearing LLC tumors on both hind limbs received 3Gy irradiation on the right tumor only (indicated by the arrow). Enhanced TL accumulation could be detected 5 hrs after the liposome injection and persisted through 24 hrs post injection. By 50 hrs post injection the differential accumulation was no longer evident. Non-targeting liposomes (nTL) did not accumulate in either the irradiated or the untreated tumors. (B) NIR images of the TL and nTL within C57BL/6 mice bearing LLC tumor on both hind limbs that received 3Gy irradiation on the right tumor only. The Radiance (photons/sec/cm²) from the tumor regions (circled) was quantified 24 hrs post liposome injection. (C) Quantification of the liposome accumulation within tumors. A significant accumulation difference was detected between the irradiated and non-irradiated tumors treated with the TL. The nTL controls did not accumulate in either tumor. * p<0.05, n=5, the Student t-test.

Figure 3

TL loaded with doxorubicin increased the circulation life and intratumoral accumulation of doxorubicin. (A) By measuring the fluorescent signature of doxorubicin, the circulation half life is increased nearly 7-fold when encapsulated in a TL (TL-Dox) as opposed to administrated as a free drug (free Dox). (B) The ratio of drug within the tumor to those within the serum increases over time for TL-Dox within irradiated tumors. There are minimal concentration differences between the tumor and plasma compartment for TL-Dox in non-irradiated tumors or free Dox. (C) Direct imaging of the tumor illustrates the increased doxorubicin fluorescence in tumors receiving both irradiation and TL-Dox.

Figure 4

Proliferation and apoptosis within tumor tissues. (A) Proliferation of cancer cells within tumor tissues was detected with in vivo incorporation of BrdU. The proliferating cells were stained as red and nuclei as blue with DAPI. (B) Quantification of the proliferating cells. More than 100 cells were counted in each of 10 independent fields to calculate ratio of the BrdU-positive cells. Average +/− standard deviation were shown. (C) Apotosis detected with TUNEL assay. The apoptotic cells were indicated with arrows. (D) Quantification of the apoptotic cells. Scatter plot showed the number of the apoptotic cells in 10 independent fields, median +/− interquartile were presented. * p<0.05, n=10.

Figure 5

Therapeutic effect of the radiation-guided tumor-targeted drug delivery. Fold change of the (A) LLC and (B) H460 tumors in the treatment course. The TL-Dox was compared to the nTL-Dox and Free Dox to show the TL-Dox was more efficient to improve tumor growth control efficacy of ionizing radiation. (C) Blood vessel function. TL-Dox plus IR resulted in more efficient blood vessel shutdown within H460 tumors. Functional blood vessels were detected by intravenously administrated lectin (green) while tumor-associated blood vessels (red) were stained with antibody against the Von Willebrand Factor (vWF). The cell nuclei (blue) were stained with DAPI. For each of the treatment groups, two representative images (C) and fractions of the functional blood vessels (D) from 10 independent fields were shown. (E) H&E staining to show the necrotic cells within the tumor tissues. The necrosis areas were marked with dashed lines in the representative images. Highlight of the necrotic areas are shown in the images of high magnification in the lower panel. Fractions of the necrotic area within the imaged fields (F) showed the significant effect of the combined treatment with TL-Dox and IR on tumor cell necrosis. ** p<0.1; * p<0.05, n=10, the student’s t-test.