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Review
. 2016 Sep 1:104:44-60.
doi: 10.1016/j.addr.2016.01.008. Epub 2016 Jan 22.

Imaging-guided delivery of RNAi for anticancer treatment

Junqing Wang  1 , Peng Mi  2 , Gan Lin  3 , Yì Xiáng J Wáng  4 , Gang Liu  5 , Xiaoyuan Chen  6
Affiliations
Review

Imaging-guided delivery of RNAi for anticancer treatment

Junqing Wang et al. Adv Drug Deliv Rev. .

Abstract

The RNA interference (RNAi) technique is a new modality for cancer therapy, and several candidates are being tested clinically. In the development of RNAi-based therapeutics, imaging methods can provide a visible and quantitative way to investigate the therapeutic effect at anatomical, cellular, and molecular level; to noninvasively trace the distribution; to and study the biological processes in preclinical and clinical stages. Their abilities are important not only for therapeutic optimization and evaluation but also for shortening of the time of drug development to market. Typically, imaging-functionalized RNAi therapeutics delivery that combines nanovehicles and imaging techniques to study and improve their biodistribution and accumulation in tumor site has been progressively integrated into anticancer drug discovery and development processes. This review presents an overview of the current status of translating the RNAi cancer therapeutics in the clinic, a brief description of the biological barriers in drug delivery, and the roles of imaging in aspects of administration route, systemic circulation, and cellular barriers for the clinical translation of RNAi cancer therapeutics, and with partial content for discussing the safety concerns. Finally, we focus on imaging-guided delivery of RNAi therapeutics in preclinical development, including the basic principles of different imaging modalities, and their advantages and limitations for biological imaging. With growing number of RNAi therapeutics entering the clinic, various imaging methods will play an important role in facilitating the translation of RNAi cancer therapeutics from bench to bedside.

Keywords: Cancer; Gene delivery; Molecular imaging; Nanocarrier; RNA interference; Theranostics; siRNA.

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Figures

Fig. 1
Fig. 1
The workflow of developing RNAi therapeutics and translating them for clinical applications. There are mainly four steps for this, including: targeting gene selection, compound screening, clinical evaluation and market release. Imaging methods could assist the development of RNAi therapeutics at every step in the RNAi therapeutics development processes, including the marking for gene selection, tracing the small RNA sequences for pharmacokinetics study, evaluating the gene silencing efficacy, as well as diagnosing tumors and monitoring the therapeutic effects.
Fig. 2
Fig. 2
(a) Schematic structure of ALN-VSP formulation: The siRNA lipid nanoparticle is composed of cationic or ionizable lipid with cholesterol and encapsulated with two different siRNAs to target KSP and VEGF genes. The nanoparticles are stabilized by surface binding of PEGylated lipids. (b) CT images demonstrated a complete response of ALN-VSP in an endometrial cancer patient with multiple liver metastatic tumors (black arrows). (c) DCE-MRI images from another patient with three metastatic pancreatic neuroendocrine tumors in the right lobe of the liver (white arrows). A significant change of tumor blood flow was observed during the treatment at 0.7 mg/kg (indicated in red). Figures adapted with permission from [11].
Fig. 3
Fig. 3
Schematic illustration of nanocarrier-mediated delivery of RNAi therapeutics: The biological barriers for gene silencing in cancer treatment include reaching the circulation, crossing the vascular barrier, cellular uptake and endosomal escape. Following systemic administration in patients, the RNAi therapeutics could be transported to the blood vessels in tumor tissues. The gene vectors could escape from the sinusoidal and fenestrated capillaries, and retain in the tumor regions. The gene vectors could be taken by cancer cells through endocytosis or ligand-mediated intracellular transport and then transferred into the cytoplasm to silence target genes for cancer therapy.
Fig. 4
Fig. 4. Optical fluorescence imaging
(a) A schematic illustration of the basic principle of in vivo fluorescence imaging. First, a fluorescently labeled RNAi therapeutics is injected into mouse body, and the subject is illuminated by an excitation light at specific wavelength, resulting an excitation of fluorescent molecules and subsequent emission light of different wavelength. The emitted light is filtered and then detected by CCD image sensor. The fluorescence signals and animal photo are finally converted to a single detailed image. The figures on right show using fluorescence imaging to trace polymeric micelles incorporated with dye labeled siRNA in the mice body. (b) Schematic illustration of polymeric micelle structure formed by self-assembly of block copolymers with fluorescent dye labeled siRNA. (c) Ear-lobe dermis snap-shots of blood circulation of two types of micelles after intravenous injection for 1 and 10 min. (d) Micro distribution of micelles incorporated with Cy5-labeled siRNA at 24 h post-injection. (red: siRNA; green: tumor cell nuclei; purple: other cell nuclei). Figures adapted with permission from [110].
Fig. 5
Fig. 5. Optical bioluminescence imaging
(a) A schematic illustration of the basic principle of in vivo bioluminescence imaging. In this case, mouse with luciferase (Luc) labeled cancer cells that express luciferase is required. After injecting the enzyme substrate luciferin into the mouse body, the bioluminescence light is generated when the luciferin molecules interact with luciferase via enzyme-catalyzed oxidation. Finally, the emitted light is detected by a cooled CCD camera and the image is produced by computer. (b) The plasmids used for transfection into mouse liver. (c) Representative images (upper image) of mice co-transfected with luciferase plasmid pGL3-control without siRNA, with luciferase siRNA or with unrelated siRNA. The results indicate that the luciferase expression was specifically suppressed by siRNA-mediated inhibition in adult mice, but the unrelated siRNAs had no effect. Another result from (c) (lower image) shows the gene silencing of luciferase expression by functional shRNAs (pShh1-Ff1). These outcomes demonstrated that the plasmid-encoded shRNAs can induce an effective and specific RNAi response in vivo. Adapted with permission from [122].
Fig. 6
Fig. 6. Positron emission tomography (PET) and single photon emission computed tomography (SPECT)
(a) A schematic illustration of the basic principles of in vivo PET imaging. In this technique, the radioactive isotopes labeled RNAi therapeutics is injected into animals. Positrons are emitted from the isotopes associating with electrons, which cause annihilation and subsequent production of two gamma (γ) rays. The two high-energy γ rays are travelling at 180° from each other. Then, the γ rays are received by detector array with electrical signals, and finally converted into tomographic images. (b) Another schematic illustration of the basic principle of in vivo SPECT imaging. Firstly, the radioactive isotopes-labeled RNAi therapeutics is administered into the mouse to emitγ rays. The γ rays produced by isotopes in SPECT do not travel in opposite directions, instead, are collected by detector array that rotates around animals, while any diagonally incident γ rays are filtered by collimator. Theγ rays received by detector array are converted and reconstructed into to-mographic images.
Fig. 7
Fig. 7
Micro-PET/CT images of nude mice bearing with HeLa cervical tumor xenografts in the right rear leg at 6 h after intravenous injection of folic acid (F-PEG-HAuNS-siRNA(DOTA-64Cu) or PEG-HAuNS-siRNA(DOTA-64Cu). Tumors were marked by arrowheads. Figure adapted with permission from [133].
Fig. 8
Fig. 8. Magnetic resonance imaging (MRI)
(a) A schematic illustration of the basic principle of in vivo MR imaging. In general, a MRI scanner consists of three types of coils: the first coil provides a strong homogenous magnetic field, the second coil generates the varying strength of magnetic field in X, Y and Z directions to encode the spatial position of MR signal and the third coil produces the radio frequency to alter the magnetic dipoles of protons in the subject, generating MR signals to be detected and reconstructed into MR image by computer. MR contrast agents can be labeled onto RNAi therapeutics to track their distribution in vivo through T1 and T2 signal enhancement. (b) Typical contrast agents for MRI, including low-molecular-weight paramagnetic compound, T1 CA-loaded vehicle, magnetic nanoparticle, T1-T2 compounds hybrid nanoparticle and T1 compounds hybrid magnetic nanoparticle. (c) Representative T1-weighted MR images (top) and quantitative T1 maps (down) of a tumor (400 mm3) before and after intravenous injection of siRNA-incorporated nanoplex at the dose of 300 mg/kg. Figures adapted with permission from [143].
Fig. 9
Fig. 9. Ultrasound imaging
(a) A schematic illustration of the basic principle of in vivo ultrasound imaging. Firstly, the ultrasound contrast agent that encapsulates RNAi therapeutics is injected into animals, then, high frequency sound waves are sent and penetrated into the subjects, while the time intervals of subsequent reflection of sound waves is recorded by a transducer. The detected signals are converted and constructed into images. A coupling medium is usually used between the contact surface of transducer and subject for sound wave transmission. (b) Typical contrast agents for ultrasound, such as liposome, nanobubble, nanodroplet and microbubble. (c) Ultrasound-triggered destruction of siRNA-incorporated microbubbles. Ultrasound could disrupt the contrast agents with high acoustic pressure to release RNAi agents when delivered to the blood vessels of the tumor region. Then, the released RNAi agents enter into tumor cells to silence target gene.
Fig. 10
Fig. 10
Multimodality in vivo imaging of RNAi by using MRI and optical imaging. (a) Schematic illustration of the multi-functional nanocarriers with core of magnetic nanoparticles, and surface conjugated with Cy5.5, GFP siRNA (siGFP) and membrane translocation peptides (MPAP). (b) In vivo MR imaging of mice bearing with subcutaneous LS174T human colorectal adenocarcinoma tumors (arrows) before and after administration of the multifunctional nanocarriers, indicating significant drop of T2-weighted contrast enhancement in tumor regions after injection of the contrast agents. (c) A high-intensity NIRF signal in the tumor confirmed the successful delivery of the nanocarriers, while the left mouse with white light, middle one with NIRF and right one with color-coded overlay. Adapted with permission from [175].
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