Systemic delivery of microRNA-34a for cancer stem cell therapy.

Although many potent conventional therapies are available to cancer patients, the spontaneous reappearance of tumors in the long term remains a significant concern with these therapies. This unfortunate phenomenon of therapeutic relapse may be caused by the lack of efficiency in eliminating the cancer stem cell (CSC) population in the tumor mass. For example, lung cancer accounts for the majority of cancerrelated deaths worldwide. Currently, chemotherapy and radiotherapy remain the basis of treatment against lung cancer because of their capability to reduce the whole tumor bulk. However, such reduction is nonspecific with regard to CSCs, resulting in development of relapse and resistance to treatment. Thus, use of conventional therapies for the treatment of lung cancer is limited. CSC-specific targeting has been introduced as an alternative, because of its potential to kill tumor-initiating cancer cells. The identification of CSC markers and their exploitation in targeted chemotherapy is an ultimate goal for CSC-based therapy. Recently, CD44 has been demonstrated to serve as an important marker of a subset population of CSCs in many tumors, including lung cancer. However, specific therapeutic targeting of CD44 in CSCs is still in its infancy. MicroRNA-34a (miR-34a), which is one of the most prominent endogenous miRNAs involved in the genesis and progression of human cancers, functions as a tumor suppressor and is commonly down-regulated in many human cancers. Enhanced expression of miR-34a in the lung inhibits tumor growth, owing to the down-regulation of the protein survivin in the tumor. Recently, miR-34a was reported to be an important regulator, inhibiting both CSC differentiation and metastasis by directly repressing the CSC marker CD44, suggesting that CD44 is a direct and functional target of miR34a. Thus, the involvement of miR-34a in suppressing CSC proliferation makes it a promising candidate for lung cancer therapy. Nevertheless, the therapeutic effect of nuclease-labile and hydrophilic molecules such as miRNAs is hampered by poor delivery systems. Therefore, it is important to develop an effective and functional drug-delivery system for miRNAs, which should have the two following key characteristics: 1) it protects encapsulated miRNAs from degradation by nucleases when the nucleic acids circulate in the blood; 2) it includes a cationic component that facilitates association with anionic miRNAs. Additional properties that are more generally applicable to delivery systems would include, but are not necessarily limited to: outstanding cellular-uptake efficiency both in vitro and in vivo, and accumulation of the drug at the correct tumor site. To date, use of viral vectors for gene delivery may be accompanied by an immune response, and direct evidence for the clinical applicability of polymer-based nanoparticles is still limited. To address these issues, solid lipid nanoparticles (SLNs) with the required properties (mentioned above) are beginning to emerge for delivery of many drugs, including gene-based drugs. Additionally, it remains unclear how to properly and efficiently deliver CSCmodulating macromolecules, such as miR-34a, into the lung for CSC therapy. In an effort to find an appropriate delivery method, we employed SLNs that contain dimethyldioctadecylammonium bromide (DDAB) to condense miRNAs in order to enhance cellular uptake and increase the localization of the drug in the cancerous lung, followed by repressing the CSC properties. The miSLNs-34a formulation (miR-34a-loaded DDABSLNs) was characterized, followed by in vitro and in vivo assessment of its enhanced stability and antitumor properties. Furthermore, the CD44 (CSC-like) phenotype was evaluated after intravenous injection of miSLNs-34a into B16F10CD44-bearing tumors in mouse lungs. Taken together, miR-34a is considered a new functional regulator of CD44, and to our knowledge, this is the first miR-34a delivery system to achieve efficient CSC therapy. Herein, DDAB-containing solid lipid nanoparticles were prepared by a film-ultrasonic method (see Experimental Section in the Supporting Information). MiRNA-loaded SLNs (miSLNs) were then prepared by coincubation of SLNs with miRNA at different ratios (DDAB/RNA, w/w). Figure 1 and Figure S1 in the Supporting Information show the size distributions and morphology of SLNs and miSLNs. The average size of miSLNs, having spherical morphology, was approximately 200 nm, which is within the injectable range for intravenous administration ( 200 nm). The entrapment efficiency of miSLNs was (96.44 0.17)%, and the RNA loading yield in miSLNs was (2.57 0.10) nmol per mg of lipids. In addition, Table S1 shows that SLNs can protect miRNA from degradation, and this protection ability was better than that of lipofectamine. We first determined the properties of B16F10-CD44 cells by sphere-formation assays in in vitro cellular studies and found that the B16F10-CD44 cells were CSC-like (Figure S2). An investigation of the cytotoxicity of the excipients [*] S. J. Shi, L. Han, Prof. T. Gong, Prof. Z. R. Zhang, Prof. X. Sun Key Laboratory of Drug Targeting and Drug Delivery Systems Ministry of Education, West China School of Pharmacy Sichuan University Chengdu No.17, Section 3, Renmin South Rd, Chengdu, 610041 (P.R. China) E-mail: xunsun22@gmail.com