Hepatocellular carcinoma-targeted nanoparticles for cancer therapy

Abstract

Nanocarriers, such as liposomes, have the potential to increase the payload of chemotherapeutic drugs while decreasing toxicity to non-target tissues; such advantageous properties can be further enhanced through surface conjugation of nanocarriers with targeting moieties. We previously reported that SP94 peptides, identified by phage display, exhibited higher binding affinity to human hepatocellular carcinoma (HCC) than to hepatocytes and other normal cells. Here, we confirm the tumor-targeting properties of SP94 peptide by near-infrared fluorescence imaging. Non-targeted PEGylated liposomal doxorubicin (LD) and SP94‑conjugated PEGylated liposomal doxorubicin (SP94‑LD) were compared by assessing pharmacokinetics, tissue distribution, and antitumor efficacy in xenograft-bearing mice, in order to investigate the effectiveness of SP94‑mediated targeting for cancer therapy. SP94‑LD demonstrated a significant increase in drug accumulation in tumors, while its plasma residence time was the same as its non-targeted equivalent. Consistent with this result, conjugation of targeting peptide SP94 enhances the therapeutic efficacy of liposomal doxorubicin in mouse models with hepatocellular carcinoma xenografts. Furthermore, combination targeted therapy exhibited a significant enhancement against orthotopic tumor growth, and markedly extended the survival of mice compared with all other treatments. Our study shows that SP94‑mediated targeting enhances antitumor efficacy by improving tumor pharmacokinetics and tissue distribution, allowing large amounts of antitumor drugs to accumulate in tumors.

Figure 1

Transmission electron microscopic images of SP94-LD and LD internalized by SK-HEP-1 cells. The SK-HEP-1 cells (1×10⁷ cells) were incubated with 10 µg/ml SP94-LD or LD at 37°C for 10 min, and then frozen and processed for TEM. Representative electron micrographs of the cells treated with SP94-LD (A) and LD (B) are shown. (C) Coated pit structures were visually identified by the presence of the electron-dense coating of the plasma membrane (arrows). (D) High magnification image of the coated pit structures, indicated by the boxed area in (C). (E) Image of SP94-LD in endosomes (arrows). (F) High magnification image of the endosomes, as shown in (E). (G) The late endosomes deliver their cargo, SP94-LD, to the lysosomes (arrows), resulting in the electron-dense, multivesicular appearance of late endosomes known as multivesicular bodies (MVBs). (H and I) High magnification images of the regions indicated by the boxed areas in (G). TEM photomicrographs were subjected to morphometric analysis with ImageJ software. (J) Vesicle number and area distribution of cells treated with SP94-LD or LD. (K) Statistical analysis of vesicle count per cell. (L) Statistical analysis of average area per vesicle. (M) Statistical analysis of total vesicle area per cell (n=16–18).

Figure 2

In vivo imaging of near-infrared, fluorochrome-labeled, HCC-targeted phage. (A) Mice bearing subcutaneous tumors (Mahlavu) were injected with HiLyte Fluor 750-labeled HCC-targeted phage (PC94) or HiLyte Fluor 750-labeled wild-type phage (Cp, control phage), and imaged at 0.1, 0.5, 6, 24, and 48 h after injection. (B) Quantification and kinetics of fluorochrome-labeled phage targeting. (C) Ex vivo fluorescence images of organs harvested 48 h after injection. (D) Fluorescence values from each organ.

Figure 3

Pharmacokinetic profiles of liposomal doxorubicin conjugated to targeting peptide SP94 in mice bearing hepatocellular carcinoma xenografts. NOD.CB17-Prkdc^scid/J mice bearing SK-HEP-1 tumors (four mice per time-point) received tail vein injections of either LD or SP94-LD at 2 mg/kg. At selected time-points post injection, mice were euthanized, and whole blood and various organs were excised and analyzed for doxorubicin auto-fluorescence signals. The levels of doxorubicin were quantified with a standard curve generated from the fluorescence emission of known amounts of doxorubicin (N=4). Doxorubicin concentrations in (A) plasma and (B) tumors are shown. (C) Time course of doxorubicin distribution in the indicated organs after i.v. administration of either LD or SP94-LD at 2 mg/kg. Data represent the means ± SEM of total doxorubicin in the brain, heart, lung liver, kidney and tumor.

Figure 4

Conjugation of targeting peptide SP94 to liposomal doxorubicin selectively enhances drug delivery to tumor cells in vivo. NOD.CB17-Prkdc^scid/J mice bearing SK-HEP-1 tumors (four mice per time-point) received tail vein injections of either LD or SP94-LD at 1 mg/kg. At selected time-points post-injection, mice were euthanized. Various organs were excised after perfusion with PBS, and analyzed for auto-fluorescent doxorubicin signals. The levels of doxorubicin were quantified with a standard curve generated from the fluorescence emission of known amounts of doxorubicin (N=4). Doxorubicin concentrations in (A) plasma and (B) tumors are shown. (C) Tissue concentrations of doxorubicin in mice administered with either LD or SP94-LD at selected time-points after injection.

Figure 5

In vivo antitumor effects of liposomal doxorubicin conjugated to SP94 in mice bearing human hepatocellular carcinoma xenograft tumors. Mice bearing SK-HEP-1 were treated with 1 mg/kg SP94-LD, 1 mg/kg LD, 1 mg/kg FD, or PBS. All compounds were injected twice weekly via tail vein. (A) Tumor volume of mice in each group at the indicated times (n=6 in each group). (B) At the end of the treatment period, tumor weight was measured. (C) Mean body weight of mice in each treatment group. (D) Frozen sections of tumor tissues from each treatment group were stained with TUNEL (green) and DAPI (blue) to visualize apoptotic tumor cells. (E) TUNEL-positive cells in each treatment group. (F) Sections were stained with anti-CD31 antibodies to visualize tumor blood vessels (red), and counterstained with DAPI (blue). (G) Analysis of blood vessels in tumor tissues stained with anti-CD31 antibody (N=3).

Figure 6

Antitumor capacity of liposomal doxorubicin conjugated to targeting peptide SP94 in animals with advanced hepatocellular carcinoma. (A) NOD. CB17-Prkdc^scid/J mice were injected with 5×10⁶ SK-Hep-1-Luc cells expressing luciferase. Tumor growth was examined by monitoring bioluminescence using the IVIS 200 Imaging system. All animals were kept under a constant supply of isoflurane using an automated anesthesia machine attached to an imaging device. After the total flux of bioluminescence reached 9.5×10⁹ p/s, mice were treated with 1 mg/kg SP94-LD, 1 mg/kg LD, 2 mg/kg FD, or PBS. All compounds were injected twice weekly via tail vein. (B) Vital tumor cells monitored by bioluminescence quantification. (C) Body weight of each group. P-values were calculated by t-test. ^*P<0.05.

Figure 7

The combination of doxorubicin and vinorelbine in HCC cells. (A) Dose-response matrix for the effect of doxorubicin and vinorelbine in HCC cells. (B) Single-agent and combination responses determined by an MTT viability assay in Mahlavu cells. The landscapes of the combination responses for doxorubicin and vinorelbine based on (C) Loewe model and (D) HSA model.

Figure 8

Establishment of orthotopic models of hepatocellular carcinoma, and the therapeutic potential of combination therapy. NOD.CB17-Prkdc^scid/J mice were orthotopically implanted with SK-HEP-1-luc cells, and treated with vehicle (PBS), FD (1 mg/kg) + FV (2 mg/kg), LD (1 mg/kg) + sLV (2 mg/kg), SP94-LD (1 mg/kg) + SP94-sLV (2 mg/kg), or sorafenib (30 mg/kg), at 4 days after tumor inoculation. All compounds were injected every other day via tail vein. (A) Tumor growth was monitored based on bioluminescence, using the IVIS 200 Imaging system. (B) Kaplan-Meier survival analysis showing the probability of survival for all subjects. (C) Body weight. (D) Kaplan-Meier survival plot. (E) Median survival of each treatment group. (F) Survival analysis by log-rank (Mantel-Cox) test showing the probability of survival for all subjects (N=8).