Abstract
Abstract
Background
Betulinic acid (BA) has been well investigated for its antiproliferative and mitochondrial pathway-mediated apoptosis-inducing effects on various cancers. However, its poor solubility and off-target activity have limited its utility in clinical trials. Additionally, the immune modulatory role of betulinic acid analogue in the tumor microenvironment (TME) is largely unknown. Here, we designed a potential nanotherapy for colorectal cancer (CRC) with a lead betulinic acid analogue, named as 2c, carrying a 1,2,3-triazole-moiety attached to BA through a linker, found more effective than BA for inhibiting CRC cell lines, and was chosen here for this investigation. Epithelial cell adhesion molecule (EpCAM) is highly overexpressed on the CRC cell membrane. A single-stranded short oligonucleotide sequence, aptamer (Apt), that folds into a 3D-defined architecture can be used as a targeting ligand for its specific binding to a target protein. EpCAM targeting aptamer was designed for site-specific homing of aptamer-conjugated-2c-loaded nanoparticles (Apt-2cNP) at the CRC tumor site to enhance therapeutic potential and reduce off-target toxicity in normal cells. We investigated the in vitro and in vivo therapeutic efficacy and anti-tumorigenic immune response of aptamer conjugated nanotherapy in CRC-TME.
Methods
After the characterization of nanoengineered aptamer conjugated betulinic acid nanotherapy, we evaluated therapeutic efficacy, tumor targeting efficiency, and anti-tumorigenic immune response using cell-based assays and mouse and rat models.
Results
We found that Apt-2cNP improved drug bioavailability, enhanced its biological half-life, improved antiproliferative activity, and minimized off-target cytotoxicity. Importantly, in an in vivo TME, Apt-2cNP showed promising signs of anti-tumorigenic immune response (increased mDC/pDC ratio, enhanced M1 macrophage population, and CD8 T-cells). Furthermore, in vivo upregulation of pro-apoptotic while downregulation of anti-apoptotic genes and significant healing efficacy on cancer tissue histopathology suggest that Apt-2cNP had predominantly greater therapeutic potential than the non-aptamer-conjugated nanoparticles and free drug. Moreover, we observed greater tumor accumulation of the radiolabeled Apt-2cNP by live imaging in the CRC rat model.
Conclusions
Enhanced therapeutic efficacy and robust anti-tumorigenic immune response of Apt-2cNP in the CRC-TME are promising indicators of its potential as a prospective therapeutic agent for managing CRC. However, further studies are warranted.
Graphical abstract
Funder
Department of Biotechnology, Govt. of India
Publisher
Springer Science and Business Media LLC
Reference82 articles.
1. Betulinic Acid Triggers CD95 (APO-1/Fas)- and p53-independent Apoptosis via Activation of Caspases in Neuroectodermal Tumors1|Cancer Research|American Association for Cancer Research. [cited 2022 Sep 18]. Available from: https://aacrjournals.org/cancerres/article/57/21/4956/503713/Betulinic-Acid-Triggers-CD95-APO-1-Fas-and-p53.
2. Fulda S, Scaffidi G, Susin SA, Krammer PH, Kroemer G, Peter ME, et al. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem. 1998;273:33942–8.
3. Wick W, Grimmel C, Wagenknecht B, Dichgans J, Weller M. Betulinic acid-induced apoptosis in glioma cells: a sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing. J Pharmacol Exp Ther. 1999;289:1306.
4. Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, et al. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett. 2002;175:17–25.
5. Ji ZN, Ye WC, Liu GG, Hsiao WLW. 23-Hydroxybetulinic acid-mediated apoptosis is accompanied by decreases in bcl-2 expression and telomerase activity in HL-60 Cells. Life Sci. 2002;72:1–9.