RNA interference RNAi technology can specifically silence the expression of a target gene and has emerged as a promising therapeutic method to treat cancer. In the present study, we showed that natural halloysite nanotube HNT —assisted delivery of an active small interfering RNA siRNA targeting receptor-interacting protein kinase 4 RIPK4 efficiently silenced its expression to treat bladder cancer.
The siRNA markedly down-regulated RIPK4 expression in bladder cancer cells and bladder tumors, thus inhibiting tumorigenesis and progression in three bladder tumor models a subcutaneous model, an in situ bladder tumor model, and a lung metastasis model , with no adverse effects. Thus, we revealed a simple but effective method to inhibit bladder cancer using RIPK4 silencing, indicating a promising therapeutic method for bladder cancer.
For tumor eradication, targeted gene therapy has received increased research attention. Small interfering RNA siRNA has shown potential as a molecular approach to down-regulate specific gene expression in cancer cells 1 , 2. To silence genes that inhibit proliferation, promote apoptosis, or prevent multidrug resistance, siRNA delivered using nanocarriers has become a new focus for anticancer therapy 3.
Delivery of siRNA to specific tumor cells and tissues is necessary to limit off-target effects that could adversely affect the therapeutic index and the clinical application of siRNA 4. Targeting cancer using nanoparticle drug carriers usually comprises optimizing the biophysical properties of the carrier to enhance nonspecific permeation and increase accumulation in the tumor 5.
Small Interfering RNA
This is usually achieved by the biochemical targeting of cancer cell—specific, overexpressed surface receptors 6. For example, a phase 1 human trial data reported safe gene silencing in patients with melanoma using nanoparticle-mediated molecular targeting of cancer cells overexpressing the transferrin receptor 7.
In the past decade, siRNA-mediated delivery has become more widespread; however, the lack of efficient in vivo delivery vehicles, particularly for the systemic delivery into immune cells, remains a major challenge for translating siRNA into therapeutics 8 , 9. Recently, however, nanoparticle siRNA delivery vehicles have been developed that are highly potent, specific, and biocompatible 10 — However, developing clinical nanosystems to selectively deliver siRNA into both immune cells and cancer cells remains a challenge Materials such as lipids, polymers, dendrimers, polymeric micelles, and metallic core nanoparticles have been constructed to efficiently deliver siRNA 9 , 14 , Natural halloysite nanotubes HNTs; external diameter, 50 to 70 nm; lumenal diameter, 10 to 15 nm; and length, 1 to 1.
The application of HNTs for nucleic acid molecules could resolve the problems of inefficient use, easy decomposition, and toxic side effects of siRNA.
Research activities have focused on appropriate size regulation, smart lumen construction, easy coupling of surface modifications, and enhanced biocompatibility 16 — RIPK4 is overexpressed in skin, ovarian, cervical, and colorectal cancers 19 , Previously, we showed that RIPK4 is highly expressed in bladder cancer tissues and represents an independent prognostic marker for poor survival In addition, marked antitumor effects were observed after RIPK4 knockdown in bladder cancer The results showed that the HNT-encapsulated siRNA was more stable in serum, had an increased circulation lifetime in blood, was more readily taken up by bladder cancer cells, and accumulated in bladder cancer tumors.
Thus, the nanoparticle-mediated delivery system developed in the present study is a simple and effective method to inhibit bladder cancer tumorigenesis as a potential previously unidentified therapy for bladder cancer. Nanoparticles within the nm range should be suitable candidates as carriers to deliver siRNA into cells, according to previous studies 22 , Oversized or undersized halloysites would probably not be taken up by cells and would show ineffective siRNA loading.
The ends of the nanotubes were unblocked, and the lumen of the nanotubes was visible. Time-dependent release of the siRNA occurred under neutral conditions Fig. The isoelectric point was approximately 6.
The pH-dependent charged form will benefit the controlled release of siRNA and improve their resistance to degradation. The efficiency of siRNA binding was determined using electrophoretic mobility shift assays in agarose gels. The cytotoxicity of the HNTs was assessed using the 3- 4,5-dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT assay.
As shown in fig. At nM, the transfection did not change notably. Fluorescein green —labeled siRIPK4. To achieve cytoplasmic posttranscriptional gene silencing, siRNA escape from lysosomes after entering the cell is essential As shown in Fig. Two hours later, efficient codelivery of the siRNA into the cytoplasm was demonstrated by the punctate distribution in the T24 cell cytoplasm and at the periphery of the nuclei of the FAM-siRNA fluorescence intensity. In addition, at 4 and 8 hours, the fluorescence intensity was significantly enhanced.
Small interfering RNA (siRNA)
The results suggested that immediately after transfection, the hybrid nanoparticles are located in the endosomes or early lysosomes. After prolonged incubation, the FAM-siRNA were released from the lysosomes and showed marked fluorescence intensity in the cytoplasm. We then analyzed the colocalization of siRIPK4 with the lysosomes over incubation time quantitatively fig. S3A , which provided results that were consistent with the observations in fig.
Small interfering RNA
In addition, the ratio of the escape of siRIPK4 from the lysosomes increased from 34 to As expected, the RIPK4 protein level, as assessed using Western blotting, decreased dose dependently fig.
S5 and S6. S7 , which demonstrated that RIPK4 down-regulation caused apoptosis. In T24 cells treated with various formulations, the cell cycle of was examined using flow cytometry. Delivering therapeutic siRNA to the target tissue directly i.
What is Small Interfering RNA?
In various mouse xenograft models, direct intratumoral injection of siRNA to achieve local delivery has been reported. Polyethyleneimine loaded with siRNA inhibited tumor growth after intratumoral injection into mice bearing glioblastoma xenografts To prevent the recurrence of superficial bladder cancer postoperatively without inducing serious side effects induced by systemic administration, intravesical instillation of chemotherapy drugs e.
Therefore, the challenges associated with the systemic delivery of siRNA may be avoided in bladder cancer. Figure 3A shows that 0. F Weight of tumors dissected from mice after treatment.
We wondered if HNT nanoparticle—coated siRIPK4 could pass through the bloodstream to reach tumor sites, and whether they are stable in circulation. The biodistribution of the complexes in mice was detected by the Xenogen IVIS Lumina system after 6 hours of injection. S9 and S Thereafter, the IHC was used to analyze the expression of the proliferation-related gene Ki to evaluate whether down-regulation of RIPK4 inhibited tumor tissue cell proliferation.
siRNA Library Screening and Anticancer Drug Target Discovery
To determine tumor cell apoptosis, the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick end labeling TUNEL assay was used. The rat in situ bladder cancer model was established successfully.
The weights of the rat bladders and the number of rat bladder lesions differed among the treatment groups table S1. Pulmonary metastasis nodules were counted in the excised lungs of each group Fig.
Small interfering RNA-based molecular therapy of cancers
N, normal lung; M, lung metastatic tumor. Quantitative assessment of pulmonary metastatic nodules in the T24 tumor—bearing mice right. The mammalian innate immune system is potently activated by free siRNA duplexes, leading to systemic in vivo inflammation resulting from the induction of high levels of inflammatory cytokines This inflammatory response to siRNA could result in significant in vivo toxicity. We investigated the in vivo toxicity of the siRIPK4 complexes at various doses.
S12 Similar changes to those observed by Western blotting were seen upon immunohistochemical staining of subcutaneous tumor tissues fig. Down-regulated RIPK4 expression inhibits bladder cancer proliferation and progression.
Gene silencing has been proposed as a treatment for human diseases, with siRNA as the most promising silencing mechanism. However, the intracellular delivery of siRNA to the specific tissues and organs expressing the target gene is the major challenge to their use in humans 7.
Two practical considerations must be taken into account: the siRNA itself stability and specificity and its delivery system In the bloodstream, serum nucleases degrade naked RNA, which can lead to stimulation of the innate immune system In the present study, we showed that HNT-based delivery of siRNA increased their serum stability and decreased their off-target effects while maintaining their silencing ability.
The application of HNTs to deliver nucleic acid molecules could resolve the problems of inefficient use, easy decomposition, and toxic side effects However, delivery issues still inhibit the production of safe, robust, and efficient siRNA-based treatments Despite their small size, siRNA transit across the cell membrane is hampered by their negative charge and hydrophilicity.
Furthermore, siRNA are rapidly eliminated from circulation before reaching the diseased tissue 34 , Thus, modifications and delivery systems for siRNA can have intended effects targeted gene silencing and unintended effects, such as immune stimulation, toxicity, and off-target silencing For in vivo delivery, many materials have been explored, including lipids, polymers antibodies, peptides, aptamers, and small molecules. To minimize the side effects of conventional nontargeted chemo- and small RNA therapeutics, we hypothesized that the physiochemical properties of the materials alone could endow selective delivery to specific cells.
We identified selective HNTs that efficiently transfected bladder cancer cells in vitro and in vivo. We demonstrated that they could be used to develop cancer cell—specific drug carriers. The present study established the feasibility of this approach to increase on-target drug activity.
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It also showed that HNT carriers exhibit cell type—dependent delivery. We showed that HNTs could deliver siRNA efficiently and specifically into bladder cancer cells to achieve targeted gene silencing.
The efficiency of siRNA uptake and gene knockdown using fusion protein complexes could be affected by the affinity of the antibody, the cell surface variations in the density of the receptor, the efficiency of receptor internalization and endocytosis, and their subsequent release from endosomes. Further study is needed to determine the mechanisms governing the release of the complexed siRNA from the endosomes, their entry into the cytoplasm, and the cellular location of RNA-induced silencing complex formation and activity, which are crucial bottlenecks for intracellular delivery Generally, in early-phase clinical studies, liposomes and polymer-based particles for the systemic delivery of anticancer siRNA are retained in the liver or excreted through kidney filtration, and thus do not concentrate in tumors We hypothesized that the diameters of the siRIPK4 and HNT particles were small enough such that the complex can cross the hepatic sinusoidal endothelium and glomerular filtration barrier to access the malignant tissue.
First, the purification process should be improved to eliminate potential contaminants, such as endotoxins. Second, optimization of the active targeting antibody, siRNA, and dosing regimen is required in rodents and nonhuman primates.
Third, the trafficking pathway allowing the siRNA to exit the endosomes and enter the cytoplasm should be determined. The significance of the present study lies in the selective delivery of siRIPK4 to bladder tumors. The results demonstrate the potential of siRNA to treat patients with bladder cancer.
HNT aqueous solution preparation: 1. The siRNA loading efficiency was evaluated by gel electrophoresis of the supernatant. The suspensions were reserved for analysis.