Gene-editing by CRISPR-Cas9 in combination with anthracycline therapy via tumor microenvironment-switchable, EGFR-targeted, and nucleus-directed nanoparticles for head and neck cancer suppression.

Head and neck cancer (HNC) has a high incidence and a poor prognosis. Epirubicin, a topoisomerase inhibitor, is a potential anthracycline chemotherapeutic for HNC treatment. HuR (ELAVL1), an RNA-binding protein, plays a critical role in promoting tumor survival, invasion, and resistance. HuR knockout via CRISPR/Cas9 (HuR CRISPR) is a possible strategy for the simultaneous modulation of the various pathways of tumor progression. Multifunctional nanoparticles modified with pH-sensitive epidermal growth factor receptor (EGFR)-targeting and nucleus-directed peptides were designed for the efficient delivery of HuR CRISPR and epirubicin to human tongue squamous carcinoma SAS cells and SAS tumor-bearing mice. The pH-sensitive nanoparticles responded to the acidic pH value as a switch to expose the targeting peptides. The cellular uptake and transfection efficiency of these nanoparticles in SAS cells increased via EGFR targeting, ligand-mediated endocytosis, and endosomal escape. These nanoparticles showed low cytotoxicity towards normal oral keratinocyte NOK cells. CRISPR/Cas9 was transported into the nucleus via the nuclear directing peptide and successfully knocked out HuR to suppress proliferation, metastasis, and resistance in SAS cells. The multiple inhibition of EGFR/ß-catenin/epithelial-mesenchymal transition pathways was mediated through modulating the EGFR/PI3K/mTOR/AKT axis. The co-treatment of epirubicin and HuR CRISPR in SAS cells further facilitated apoptosis/necroptosis/autophagy and caused cancer cell death. In combination with HuR CRISPR nanoparticles, the efficacy and safety of epirubicin nanoparticles against cancer in SAS tumor-bearing mice improved significantly. Collectively, these nanoparticles showed a tumor pH response, active EGFR targeting, and nuclear localization and thus offered a combinatorial spatiotemporal platform for chemotherapy and the CRISPR/Cas gene-editing system.

Mitochondrion-Directed Nanoparticles Loaded with a Natural Compound and a microRNA for Promoting Cancer Cell Death via the Modulation of Tumor Metabolism and Mitochondrial Dynamics.

Mitochondrial dysfunction may cause cancer and metabolic syndrome. Ellagic acid (abbreviated as E), a phytochemical, possesses anticancer activity. MicroRNA 125 (miR-125) may regulate metabolism. However, E has low aqueous solubility, and miR-125 is unstable in a biological fluid. Hence, this study aimed to develop nanoparticle formulations for the co-treatment of miR-125 and E. These nanoparticles were modified with one mitochondrion-directed peptide and a tumor-targeted ligand, and their modulating effects on mitochondrial dysfunction, antitumor efficacy, and safety in head and neck cancer (HNC) were evaluated. Results revealed that miR-125- and E-loaded nanoparticles effectively targeted cancer cells and intracellular mitochondria. The co-treatment significantly altered cellular bioenergetics, lipid, and glucose metabolism in human tongue squamous carcinoma SAS cells. This combination therapy also regulated protein expression associated with bioenergenesis and mitochondrial dynamics. These formulations also modulated multiple pathways of tumor metabolism, apoptosis, resistance, and metastasis in SAS cells. In vivo mouse experiments showed that the combined treatment of miR-125 and E nanoparticles exhibited significant hypoglycemic and hypolipidemic effects. The combinatorial therapy of E and miR-125 nanoparticles effectively reduced SAS tumor growth. To our best knowledge, this prospective study provided a basis for combining miRNA with a natural compound in nanoformulations to regulate mitochondrial dysfunction and energy metabolism associated with cancer.

PEG-coated nanoparticles detachable in acidic microenvironments for the tumor-directed delivery of chemo- and gene therapies for head and neck cancer.

Background: Head and neck cancer (HNC) is a major cause of morbidity and mortality and has a poor treatment outcome. Irinotecan, a topoisomerase-I inhibitor, induces cell death by decreasing the religation of double-strand DNA. However, epithelial-mesenchymal transition (EMT), therapy resistance, and systemic toxicity caused by available antineoplastic agents hinder the efficacy and safety of HNC treatment. Chemotherapy combined with gene therapy shows potential application in circumventing therapy resistance and EMT. miR-200 exerts a remarkable suppressing effect on EMT-associated genes. Herein, liposomes and solid lipid nanoparticles (SLNs) modified with a pH-sensitive, self-destructive polyethylene glycol (PEG) shell and different peptides were designed as irinotecan and miR-200 nanovectors to enhance tumor-specific accumulation. These peptides included one ligand targeting the angiogenic tumor neovasculature, one mitochondrion-directed apoptosis-inducing peptide, and one cell-penetrating peptide (CPP) with high potency and selectivity toward cancer cells. Methods: Physicochemical characterization, cytotoxicity analysis, cellular uptake, regulation mechanisms, and in vivo studies on miR-200- and irinotecan-incorporated nanoparticles were performed to identify the potential antitumor efficacy and biosafety issues involved in HNC treatment and to elucidate the underlying signaling pathways. Results: We found that the cleavable PEG layer responded to low extracellular pH, and that the CPP and targeting peptides were exposed to improve the uptake and release of miR-200 and irinotecan into HNC human tongue squamous carcinoma (SAS) cells. The apoptosis of SAS cells treated with the combinatorial therapy was significantly induced by regulating various pathways, such as the Wnt/ß-catenin, MDR, and EMT pathways. The therapeutic efficacy and safety of the proposed co-treatment outperformed the commercially available Onivyde and other formulations used in a SAS tumor-bearing mouse model in this study. Conclusion: Chemotherapy and gene therapy co-treatment involving pH-sensitive and targeting peptide-modified nanoparticles may be an innovative strategy for HNC treatment.

pH-Responsive PEG-Shedding and Targeting Peptide-Modified Nanoparticles for Dual-Delivery of Irinotecan and microRNA to Enhance Tumor-Specific Therapy.

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA-200 (miR-200) has been reported to inhibit metastasis in cancer cells. Herein, pH-sensitive and peptide-modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR-200, respectively. These peptides include one cell-penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria-targeting peptide. The peptide-modified nanoparticles are further coated with a pH-sensitive PEG-lipid derivative with an imine bond. These specially-designed nanoparticles exhibit pH-responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR-200 by SLN further increases the cytotoxicity of irinotecan-loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/ß-catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC-bearing mice, the in vivo results further indicate that irinotecan and miR-200 in pH-responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate ß-catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH-responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.

Improving the anticancer effect of afatinib and microRNA by using lipid polymeric nanoparticles conjugated with dual pH-responsive and targeting peptides.

BACKGROUND: The emergence of resistance to chemotherapy or target therapy, tumor metastasis, and systemic toxicity caused by available anticancer drugs hamper the successful colorectal cancer (CRC) treatment. The rise in epidermal growth factor receptor (EGFR; human epidermal growth factor receptor 1; HER1) expression and enhanced phosphorylation of HER2 and HER3 are associated with tumor resistance, metastasis and invasion, thus resulting in poor outcome of anti-CRC therapy. The use of afatinib, a pan-HER inhibitor, is a potential therapeutic approach for resistant CRC. Additionally, miR-139 has been reported to be negatively correlated with chemoresistance, metastasis, and epithelial-mesenchymal transition (EMT) of CRC. Hence, we develop a nanoparticle formulation consisting of a polymer core to carry afatinib or miR-139, which is surrounded by lipids modified with a targeting ligand and a pH-sensitive penetrating peptide to improve the anticancer effect of cargos against CRC cells. RESULTS: Our findings show that this formulation displays a spherical shape with core/shell structure, homogeneous particle size distribution and negative zeta potential. The prepared formulations demonstrate a pH-sensitive release profile and an enhanced uptake of cargos into human colorectal adenocarcinoma Caco-2 cells in response to the acidic pH. This nanoparticle formulation incorporating afatinib and miR-139 exhibits low toxicity to normal cells but shows a better inhibitory effect on Caco-2 cells than other formulations. Moreover, the encapsulation of afatinib and miR-139 in peptide-modified nanoparticles remarkably induces apoptosis and inhibits migration and resistance of Caco-2 cells via suppression of pan-HER tyrosine kinase/multidrug resistance/metastasis pathways. CONCLUSION: This study proposes a multifunctional nanoparticle formulation for targeted modulation of apoptosis/EGFR/HER/EMT/resistance/progression pathways to increase the sensitivity of colon cancer cells to afatinib.