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. 2019 Oct 12;9(24):7458-7473.
doi: 10.7150/thno.38081. eCollection 2019.

Orally Deliverable Nanotherapeutics for the Synergistic Treatment of Colitis-Associated Colorectal Cancer

Weidong Han  1 , Binbin Xie  1 , Yiran Li  1 , Linlin Shi  1 , Jianqin Wan  2 , Xiaona Chen  2 , Hangxiang Wang  2
Affiliations

Orally Deliverable Nanotherapeutics for the Synergistic Treatment of Colitis-Associated Colorectal Cancer

Weidong Han et al. Theranostics. .

Abstract

Purpose: Colitis-associated colorectal cancer (CAC) poses substantial challenges for effective treatment. Currently, there is a considerable need for the development of orally bioavailable dosage forms that enable the safe and effective delivery of therapeutic drugs to local diseased lesions in the gastrointestinal tract. Experimental Design: In this study, we developed orally deliverable nanotherapeutics for the synergistic treatment of inflammatory bowel diseases (IBDs) and CAC. Water-insoluble curcumin (CUR) and 7-ethyl-10-hydroxycamptothecin (SN38), which served as anti-inflammatory and cytotoxic agents, respectively, were chemically engineered into hydrophilic mucoadhesive chitosan for the generation of chitosan-drug amphiphiles. Results: The resulting amphiphilic constructs formed core-shell nanostructures in aqueous solutions and were orally administered for in vivo therapeutic studies. Using a preclinical CAC mouse model, we showed that the orally delivered nanotherapeutics locally accumulated in inflamed intestinal regions and tumor tissues. Furthermore, the therapeutic synergy of the combined nanotherapeutics in CAC mice was evaluated. Compared with their individual drug forms, combined CUR and SN38 nanoparticles yielded synergistic effects to alleviate intestinal inflammation and protect mice from ulcerative colitis. Notably, the combinatorial therapy demonstrated a remarkable tumor shrinkage with only ~6% of the total tumors exceeding 4 mm in diameter, whereas ~35% of tumors were observed to exceed a diameter of 4 mm in the saline-treated CAC mice. These data suggest a new and reliable approach for improving the treatment of IBD and CAC. Conclusions: Our results showed that bioadhesive chitosan materials can be used to produce colloidal-stable nanotherapeutics that are suitable for oral delivery. Both nanotherapeutics exhibited substantial accumulation in inflamed intestinal regions and tumor tissues and showed good synergy for treating CAC, warranting further clinical translation.

Keywords: anti-inflammation; colitis-associated colorectal cancer; cytotoxic nanoparticle; oral delivery; self-assembly.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
A schematic of the self-assembly of chitosan-drug conjugates to form nanotherapeutics (i.e., nCUR and nSN38) and oral administration of the nanotherapeutics for CAC treatment. (A) Therapeutic SN38 and CUR agents were individually tethered to carboxylated chitosan by a hydrolyzable linkage. The formed chitosan-drug conjugates self-assembled into stable colloidal and bioadhesive nanotherapeutics suited for oral delivery. (B) After oral administration by drinking water containing the therapeutics ad libitum, the nCUR and nSN38 nanotherapeutics were tightly adhered to intestinal villi and efficiently accumulated in the inflamed colon tissues and tumors. Subsequently, chemically unmodified anti-inflammatory CUR and cytotoxic SN38 agents can be released to impair colitis and tumor growth, respectively.
Figure 2
Figure 2
Self-assembled nCUR inhibits inflammatory cytokines in macrophages. (A and B) mRNA expression levels of proinflammatory cytokines were assessed when Raw264.7 cells (A) and bone marrow-derived macrophages (BMDMs) (B) were incubated with CUR and nCUR for 12 h followed by exposure to LPS for 3 h. (C and D) mRNA expression levels of IL-1β, TNF-α, iNOS, and MCP-1 were assessed when Raw264.7 cells (C) and BMDMs (D) were treated with vehicle, SN38, SN38 combined with CUR and SN38 combined with nCUR for 6 h. The data were normalized to GAPDH and then presented as values relative to the control values. (E) Raw264.7 cells were stimulated with LPS for 12 h in the presence of CUR and nCUR. Three independent staining tests were performed, and the data are presented as the mean ± SD. N.S. indicates no significant difference; *p < 0.05, **p < 0.01, and ***p < 0.001, as determined by two-way ANOVA followed by Bonferroni's post hoc test.
Figure 3
Figure 3
Specific accumulation of nCUR nanotherapeutics in inflamed colons. (A) Representative ex vivo fluorescence images for the evaluation of nanoparticle distribution. C57BL/6 mice were given drinking water containing 3% DSS ad libitum to induce intestinal inflammation for 7 days. Mice with colitis were orally administered Cy5.5-labeled nCUR (termed Cy5.5-nCUR), and healthy mice were also included as controls. At 6 and 24 h postadministration, the major organs were excised for ex vivo NIR imaging. (B and C) Ex vivo imaging (B) and quantification of fluorescence intensities (C) of colons excised from each group of mice. Confocal microscopy images (D) and fluorescence intensities (E) of colorectal tissue sections harvested at 6 and 24 h after oral administration. The red and blue signals indicate Cy5.5 and nuclei counterstained with DAPI, respectively. The data are presented as the mean ± SD. *p < 0.05, **p <0.01, and ***p < 0.001, as determined by two-way ANOVAfollowed by Bonferroni's post hoc test.
Figure 4
Figure 4
Treatment with nCUR alleviates colitis at the early stage of CAC. (A) A schematic overview of the DSS-induced colitis model in C57BL/6 mice. The mice were injected with AOM followed by supplementation with 3% DSS in water for seven days. For anti-inflammatory treatment, nCUR was given daily in water by drinking ad libitum. The mice were euthanized on day 19 after AOM injection. (B) Treatment with nCUR improved survival in mice with DSS-induced fatal colitis (n = 7 mice in each group). Kaplan-Meier survival curves were compared by the log-rank test. (C) Weight loss was monitored throughout the therapeutic studies. (D) The disease activity index (DAI) was evaluated on day 19 after AOM injection. DAI is the summation of the stool consistency index, fecal bleeding index, and weight loss index. (E) Colons were photographed, and (F) colon lengths were measured at the end of the therapeutic studies. (G) Representative H&E staining of the mouse colon. The green arrows and dotted circles indicate crypts and leukocyte infiltration, respectively. (H) Relative expression of inflammatory cytokines in the murine colon on day 19 after the induction of colitis in mice. The data are presented as the mean ± SD. N.S. indicates no significant difference; *p < 0.05, **p < 0.01, and ***p < 0.001, as determined by two-way ANOVA followed by Bonferroni's post hoc test.
Figure 5
Figure 5
Tumor-specific delivery of the nanotherapeutics in a CAC mouse model. (A) Representative ex vivo fluorescence images for the evaluation of nanoparticle distribution in the major organs. C57BL/6 mice were intravenously injected with AOM followed by ad libitum administration of a 2% solution of DSS in drinking water for three cycles. This protocol allowed nearly all mice to develop tumors in the colonic tissues after 70 days. The mice with tumors were orally administered Cy5.5-labeled nSN38 (termed Cy5.5-nSN38). A solution containing free Cy5.5 was also orally administered as a reference. (B and C) Ex vivo imaging of the whole colons (B) and tumors (C) excised from CAC mice at 6 and 24 h postadministration. (D) Quantification of fluorescence intensities of colorectal tumors. Confocal microscopy images (E) and fluorescence intensities (F) of colorectal tissue sections harvested at 6 and 24 h after oral administration. The red and blue signals indicate Cy5.5 and nuclei counterstained with DAPI, respectively. The data are presented as the mean ± SD. *p < 0.05 and **p <0.01, as determined by two-way ANOVA followed by Bonferroni's post hoc test.
Figure 6
Figure 6
The combination of nCUR/nSN38 nanotherapeutics prevents the tumorigenesis and growth of CAC in vivo. (A) The establishment of an inflammation-driven colon cancer model in C57BL/6 mice and the therapeutic protocols of nCUR/nSN38. The mice received a single injection of AOM via the tail vein followed by supplementation with three cycles of 2% DSS. (B) Representative images of murine colons. (C-E) Tumor numbers per mouse (C), tumor diameters (D), and tumor size distribution (E) were measured in each group. (F) H&E staining of tumor morphology. The green arrows and dotted circles indicate crypts and leukocyte infiltration, respectively. The data are presented as the mean ± SD (n =12). *p < 0.05 and **p < 0.01, as determined by two-way ANOVA followed by Bonferroni's post hoc test.
Figure 7
Figure 7
The combination of nCUR/nSN38 inhibits the growth of CAC by inducing cell cycle arrest and apoptosis of tumor cells. (A) TUNEL analysis of the excised tumors from each mouse group at the end of the therapeutic studies. (B) Quantification of apoptotic cells by counting TUNEL-positive cells. (C) Immunohistochemical staining of Ki-67 using paraffin-embedded tumor sections and quantification of positive Ki-67 staining. (D) The expression of indicated proteins isolated from the colonic epithelia of healthy mice and tumors of CAC mice after various treatments. The tumors excised from CAC mice without any therapeutic treatments were included as controls. *p < 0.05 and **p < 0.01, as determined by two-way ANOVA followed by Bonferroni's post hoc test.
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