Lectin-Mediated pH-Sensitive Doxorubicin Prodrug for Pre-Targeted Chemotherapy of Colorectal Cancer with Enhanced Efficacy and Reduced Side Effects

Abstract

Doxorubicin (DOX) has been clinically used as a broad-spectrum chemotherapeutic agent for decades, but its clinical application is hindered by the lack of tumour specificity, severe cardiotoxicity and haematotoxicity. Pre-targeted strategies are highly tumour-specific, therapeutic approaches. Herein, a novel pre-targeted system was constructed, aiming to enhance anticancer efficacy of DOX and maximally reduce its side effects. Methods: The DOX prodrug (bDOX) was first synthesized by conjugating DOX with mini-PEGylated (mPEGylated) biotin through a pH-sensitive bond. During the pre-targeted treatment, avidin was first administrated. After an optimized interval, bDOX was second administrated. The nontoxic prodrug bDOX was eventually transformed into the toxic anticancer form (DOX) by a pH-triggered cleavage specifically in tumour cells. The drug efficacy and side effect of the two-step, pre-targeted treatment were fully compared with free DOX in vitro and in vivo. Results: The prodrug bDOX was quite stable under neutral conditions and nearly nontoxic, but was immediately transformed into the toxic anticancer form (DOX) under acidic conditions. Compared to free DOX, the pre-targeted bDOX exhibited a higher cellular uptake by human colorectal tumour cells (LS180 and HT-29 cells). In vivo evaluation performed on LS180 xenograft animal model demonstrated that the pre-targeted bDOX achieved a much more significant tumour inhibition than free DOX. The largely decreased, unwanted bystander toxicity was demonstrated by changes in body weight, cardiomyocyte apoptosis, blood routine examination and splenic pathological changes. Conclusion: The high therapeutic efficacy, together with the minimal side effects, of this easily synthesized, pre-targeted system exhibited immense potentiality for the clinical application of DOX delivery.

Keywords: avidin; biotin; chemotherapy; doxorubicin prodrug; pre-targeted strategy.

Figure 1

Illustration of lectin-mediated pre-targeted bDOX for cancer treatment. (A) The distribution and elimination of avidin and bDOX. (B) The cellular entry process of pre-targeted bDOX. (C) The structure of bDOX.

Figure 2

Acid-sensitive DOX release from bDOX. (A-C) HPLC characterization of bDOX hydrolysis after dissolving bDOX in PB buffer solutions with varying pH for different times. Retention time was ~ 23 min for DOX and ~ 25 min for bDOX. (D) Quantification of released DOX from bDOX with varying pH for different times. The values represent mean ± SD (n = 3).

Figure 3

Chemotherapeutic effect of bDOX in vitro. Relative cell viability of (A) HT-29 , (B) LS180 and (C) HEK293 after incubating with un-pre-targeted bDOX (bDOX in graph), DOX or pre-targeted bDOX (pre-bDOX in graph) at different concentrations for 48 h. Log M represents the logarithmic concentration of DOX (mmol/L). *** p < 0.001. The values represent mean ± SD (n = 3). (pre-bDOX: pre-targeted bDOX).

Figure 4

In vitro cellular uptake. (A) Confocal laser scanning microscopy (CLSM) images of cellular uptake of bDOX, free DOX and pre-targeted bDOX in HEK293 , LS180 , and HT-29 cells. The scale bar represents 25 μm. (B) Fluorescence intensity percentages of the treated cells. The values represent mean ± SD (n = 7). * p < 0.05; ** p < 0.01. (pre-bDOX: pre-targeted bDOX).

Figure 5

Subcellular localization of pre-targeted bDOX performed on HT-29 cells. Lysosomes were stained by LysoTracker^TM Deep Red and colored in green; nuclei were stained by Hoechst 33342 and colored in blue. The pretargeted bDOX was colored in red. The scale bar represents 25 μm. (pre-bDOX: pre-targeted bDOX).

Figure 6

Biodistribution of avidin and pre-targeted bDOX. (A) Biodistribution of ^99mTc-biotin injected at different times (1 h, 2 h, 4 h, 5 h and 6 h) after the injection of avidin. The values represent mean ± SD (n = 4). (B) Bright-field (upper) and DOX fluorescence (below) images of tumour and major organs ex vivo after injecting free DOX or pre-targeted bDOX. The scale bar represents 1 cm. (C) Quantification of Figure 6B, exhibiting average relative DOX fluorescence intensity of tumour, heart and liver. The values represent mean ± SD (n = 3). (D) CLSM images of DAPI-stained frozen sections showing drug accumulation in tumour and heart tissues after injecting PBS, un-pre-targeted bDOX, free DOX or pre-targeted bDOX. Blue colour represents DAPI-stained nuclei, and pseudo-red colour represents fluorescence from DOX or bDOX. The scale bar represents 50 μm. * p < 0.05. (pre-bDOX: pre-targeted bDOX).

Figure 7

In vivo evaluation of chemotherapeutic effects and side effects. (A) Representative bioluminescence images of LS180-bearing BALB/c nude mice on different days after various treatments, showing the rate of tumour growth. The mice in each group are shown in Figure S15. (B) Quantification of the relative tumour fluorescence intensity in Figure 7A and Figure S15. The values represent mean ± SD (n = 5). (C) A photograph of tumours collected from different groups after therapy (day 10). Note that tumours from one mouse were stacked into a pile. The scale bar represents 2 cm. (D) Average tumour weight per mouse for different groups, weighed and calculated from Figure 7C. The values represent mean ± SD (n = 5). (E) Relative mouse body weight of various groups during therapy. The values represent mean ± SD (n = 5). Changes in blood routine examination parameters: (F) WBC and (G) MO of various groups during therapy. The values represent mean ± SD (n = 5). TUNEL-stained frozen section of (H) tumour and (I) heart tissues after the treatments. Compared to free DOX, increased tumour cellular apoptosis and decreased cardiomyocyte apoptosis were exhibited after a treatment with pre-targeted bDOX. The scale bar represents 25 μm. (J) H&E-stained tumour and spleen tissue sections after the treatments. Compared to free DOX, increased tumour necrosis and decreased spleen damage were exhibited after a treatment with pre-targeted bDOX. The scale bar represents 100 μm. * p < 0.05; ** p < 0.01; *** p < 0.001. (MO: monocytes; ns: no significance; pre-bDOX: pre-targeted bDOX; WBC: white blood cells).