Targeted delivery of irinotecan to colon cancer cells using epidermal growth factor receptor-conjugated liposomes

Abstract

Background: CPT-11 (irinotecan) is one of the most efficient agents used for colorectal cancer chemotherapy. However, as for many other chemotherapeutic drugs, how to minimize the side effects of CPT-11 still needs to be thoroughly described.

Objectives: This study aimed to develop the CPT-11-loaded DSPE-PEG 2000 targeting EGFR liposomal delivery system and characterize its targeting specificity and therapeutic effect on colorectal cancer (CRC) cells in vitro and in vivo.

Results: The synthesized liposome exhibited spherical shapes (84.6 ± 1.2 nm to 150.4 nm ± 0.8 nm of estimated average sizes), good stability, sustained release, and enough drug loading (55.19%). For in vitro experiments, SW620 cells treated with CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome showed lower survival extended level of intracellular ROS production. In addition, it generated an enhanced apoptotic cell rate by upregulating the protein expression of both cleaved-caspase-3 and cleaved-caspase-9 compared with those of SW620 cells treated with free CPT-11. Importantly, the xenograft model showed that both the non-target and EGFR-targeted liposomes significantly inhibited tumor growth compared to free CPT-11.

Conclusions: Compared with the non-target CPT-11-loaded DSPE-PEG₂₀₀₀ liposome, CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome treatment showed much better antitumor activity in vitro in vivo. Thus, our findings provide new assets and expectations for CRC targeting therapy.

Keywords: Colorectal cancer; DSPE-PEG2000; EGFR; Irinotecan (CPT-11); SW620 cell.

Fig. 1

The morphology and particle size of CPT-11-loaded DSPE-PEG₂₀₀₀ liposome. A The morphology of CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome; B CPT-11-loaded DSPE-PEG₂₀₀₀ liposome; C the particle size of CPT-11-loaded DSPE-PEG₂₀₀₀ liposome targeting EGFR. CPT-11-Lipo: CPT-11-loaded DSPE-PEG₂₀₀₀ liposome; EGFR-Lipo-CPT-11: CPT-11-loaded DSPE-PEG₂₀₀₀ liposome coated with anti-epidermal growth factor receptor (EGFR) antibody

Fig. 2

The physical stability and cumulative release profile of CPT-11-loaded DSPE-PEG₂₀₀₀ liposome in vitro. A The physical strength was measured by the particle sizes of CPT-11-loaded DSPE-PEG₂₀₀₀ liposome at the 1st day and 14th day after preparation; B the cumulative release profile of the prepared liposome

Fig. 3

In vitro anticancer effects. SW620 and CW-2 cell lines expressed the highest and lowest levels of EGFR (A and B). Under treatments with free CPT-11, liposomal CPT-11 (Lipo-CPT-11) or EGFR-Lipo-CPT-11 for 24 h, SW620 cells [highest EGFR-expressing cell lines] showed a significant reduction of cell viability while the CW-2 cell lines (lowest EGFR-expressing cell lines) were less affected (C and D)

Fig. 4

In vitro cytotoxicity of free irinotecan and the antitumor activity of CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome. A Cell viability of HCT116, SW620, CW-2 and LoVo colon cancer cells using CCD18Co normal primary cells to control CRC toxicity. B In vitro cytotoxicity of free CPT-11 at the concentration of 1, 5, 10, 50 and 100 μg/mL; C the antitumor activity of CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome in vitro; D survival rate of cancer cells treated with free CPT-11, CPT-11-Lipo and EGFR-Lipo-CPT-11, compared with the normal primary cells (control), generated the lowest (**p < 0.01, ***p < 0.001)

Fig. 5

ROS formation induced by CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome. A Fluorescent intensity of CPT-11, CPT-11-Lipo, EGFR-Lipo-CPT-11 and control cells. Original magnification 100×. scale bar: 100 µm. B The scale bar representation of the fluorescent intensity. C Mean fluorescent intensity (MFI) of CPT-11, CPT-11-Lipo, EGFR-Lipo-CPT-11 and control cells; D intracellular ROS level of CPT-11, CPT-11-Lipo, EGFR-Lipo-CPT-11 and control cells (**p < 0.01, ***p < 0.001)

Fig. 6

The apoptosis effect of CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome in vitro. A Flow cytometry was performed to assess the apoptosis effects of CPT-11, CPT-11-Lipo, EGFR-Lipo-CPT-11, and control; B–D Western blot results showing the protein levels of cleaved-caspase-3 and cleaved-caspase-9 from four different groups mentioned above and their gray value (**p < 0.01, ***p < 0.001)

Fig. 7

In vivo tumor inhibition effects of CPT-11-loaded DSPE-PEG₂₀₀₀ targeting EGFR liposome in the SW620 xenograft model. A The tumor volume of nudes treated with CPT-11, CPT-11-Lipo, EGFR-Lipo-CPT-11 and control every 3 days for 24 days; B the tumor mass of nudes treated with four different treatment groups mentioned above (*p < 0.05, **p < 0.01). CPT-11, irinotecan; Lipo-CPT-11, CPT-11 loaded liposome; EGFR-Lipo-CPT-11; CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome