Cell-Penetrating Magnetic Nanoparticles for Highly Efficient Delivery and Intracellular Imaging of siRNA

RNA interference is one of the most promising technologies for cancer therapeutics, while the development of a safe and effective small interfering RNA (siRNA) delivery system is still challenging. Here, amphipol polymer and protamine peptide were employed to modify magnetic nanoparticles to form cell-penetrating magnetic nanoparticles (CPMNs). The unique CPMN could efficiently deliver the eGFP siRNA intracellularly and silence the eGFP expression in cancer cells, which was verified by fluorescent imaging of cancer cells. Compared with lipofectamine and polyethyleneimine (PEI), CPMNs showed superior silencing efficiency and biocompatibility with minimum siRNA concentration as 5 nm in serum-containing medium. CPMN was proved to be an efficient siRNA delivery system, which will have great potential in applications as a universal transmembrane carrier for intracellular gene delivery and simultaneous MRI imaging.     

Oleyl-chitosan nanoparticles based on a dual probe for optical/MR imaging in vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.     

Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (相似文献   

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which generate significant susceptibility effects resulting in strong T2 and T*2 contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.     

Near-infrared imaging method for the in vivo assessment of the biodistribution of nanoporous silicon particles

In the development of new nanoparticle-based technologies for therapeutic and diagnostic purposes, understanding the fate of nanoparticles in the body is crucial. We recently developed a multistage vector delivery system comprising biodegradable and biocompatible nanoporous silicon particles (first-stage microparticles [S1MPs]) able to host, protect, and deliver second-stage therapeutic and diagnostic nanoparticles (S2NPs) on intravenous injection. This delivery system aims at sequentially overcoming the biologic barriers en route to the target delivery site by separating and assigning tasks to the coordinated logic-embedded vectors constituting it. In this work, by conjugating a near-infrared dye on the surface of the S1MP without compromising the porous structure and potential loading of S2NPs, we were able to monitor the in vivo distribution of S1MPs in healthy mice using an optical imaging system. It was observed that particles predominantly accumulated in the liver and spleen at the end of 24 hours. Further quantification of S1MPs in the major organs of the animals by elemental analysis of silicon using inductively coupled plasma-atomic electron spectroscopy verified the accuracy of in vivo near-infrared imaging as a tool for evaluation of nanovector biodistribution.     

Superparamagnetic iron oxide nanoparticles target BxPC-3 cells and silence MUC4 for the treatment of pancreatic cancer

PurposeSuperparamagnetic iron oxide nanoparticles (SPION) are excellent magnetic resonance imaging (MRI) contrast agents. Mucin 4 (MUC4) acts as pancreatic cancer (PC) tumor antigen and influences PC progression. Small interfering RNAs (siRNAs) are used as a gene-silencing tool to treat a variety of diseases.MethodsWe designed a therapeutic probe based on polyetherimide-superparamagnetic iron oxide nanoparticles (PEI-SPION) combined with siRNA nanoprobes (PEI-SPION-siRNA) to assess the contrast in MRI. The biocompatibility of the nanocomposite, and silencing of MUC4 were characterized and evaluated.ResultsThe prepared molecular probe had a particle size of 61.7 ± 18.5 nm and a surface of 46.7 ± 0.8mV and showed good biocompatibility in vitro and T2 relaxation efficiency. It can also load and protect siRNA. PEI-SPION-siRNA showed a good silencing effect on MUC4.ConclusionPEI-SPION-siRNA may be beneficial as a novel theranostic tool for PC.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

Molecular imaging: Bridging the gap between neuroradiology and neurohistology

Historically, in vivo imaging methods have largely relied on imaging gross anatomy. More recently it has become possible to depict biological processes at the cellular and molecular level. These new research methods use magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared optical imaging, scintigraphy, and autoradiography in vivo and in vitro. Of primary interest is the development of methods using MRI and PET with which the progress of gene therapy in glioblastoma (herpes simplex virus-thymidine kinase) and Parkinson's disease can be monitored and graphically displayed. The distribution of serotonin receptors in the human brain and the duration of serotonin-receptor antagonist binding can be assessed by PET. With PET, it is possible to localize neurofibrillary tangles (NFTs) and beta-amyloid senile plaques (APs) in the brains of living Alzheimer disease (AD) patients. MR tracking of transplanted oligodendrocyte progenitors is feasible for determining the extent of remyelinization in myelin-deficient rats. Stroke therapy in adult rats with subventricular zone cells can be monitored by MRI. Transgene expression (beta-galactosidase, tyrosinase, engineered transferrin receptor) can also be visualized using MRI. Macrophages can be marked with certain iron-containing contrast agents which, through accumulation at the margins of glioblastomas, ameliorate the visual demarcation in MRI. The use of near-infrared optical imaging techniques to visualize matrix-metalloproteinases and cathepsin B can improve the assessment of tumor aggressiveness and angiogenesis-inhibitory therapy. Apoptosis could be detected using near-infrared optical imaging representation of caspase 3 activity and annexin B. This review demonstrates the need for neurohistological research if further progress is to be made in the emerging but burgeoning field of molecular imaging.     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Antibody-directed cell-type-specific delivery of siRNA

Over the past four years, chemically synthesized short interfering RNA (siRNA) has become the standard tool for specific silencing of gene expression in vitro. The most difficult task in transferring this technology to an in vivo setting is to develop appropriate delivery strategies. With this aim, Song et al. recently reported the development of antibody-protamine fusion proteins as vehicles for receptor-directed delivery of siRNA. When a mixture of siRNA targeting tumor-related genes was administered in this way, tumor growth was inhibited in an engineered melanoma model, demonstrating the therapeutic potential of this technology. However, several challenges remain to be overcome before targeted gene silencing can become a reality for patients.     

An integrated method for reproducible and accurate image-guided stereotactic cranial irradiation of brain tumors using the small animal radiation research platform

Preclinical studies of cranial radiation therapy (RT) using animal brain tumor models have been hampered by technical limitations in the delivery of clinically relevant RT. We established a bioimageable mouse model of glioblastoma multiforme (GBM) and an image-guided radiation delivery system that facilitated precise tumor localization and treatment and which closely resembled clinical RT. Our novel radiation system makes use of magnetic resonance imaging (MRI) and bioluminescent imaging (BLI) to define tumor volumes, computed tomographic (CT) imaging for accurate treatment planning, a novel mouse immobilization system, and precise treatments delivered with the Small Animal Radiation Research Platform. We demonstrated that, in vivo, BLI correlated well with MRI for defining tumor volumes. Our novel restraint system enhanced setup reproducibility and precision, was atraumatic, and minimized artifacts on CT imaging used for treatment planning. We confirmed precise radiation delivery through immunofluorescent analysis of the phosphorylation of histone H2AX in irradiated brains and brain tumors. Assays with an intravenous near-infrared fluorescent probe confirmed that radiation of orthografts increased disruption of the tumor blood-brain barrier (BBB). This integrated model system, which facilitated delivery of precise, reproducible, stereotactic cranial RT in mice and confirmed RT's resultant histologic and BBB changes, may aid future brain tumor research.     

Emerging concepts in molecular MRI

Molecular magnetic resonance imaging (MRI) offers the potential to image some events at the cellular and subcellular level and many significant advances have recently been witnessed in this field. The introduction of targeted MR contrast agents has enabled the imaging of sparsely expressed biological targets in vivo. Furthermore, high-throughput screens of nanoparticle libraries have identified nanoparticles that act as novel contrast agents and which can be targeted with enhanced diagnostic specificity and range. Another class of magnetic nanoparticles have also been designed to image dynamic events; these act as 'switches' and could be used in vitro, and potentially in vivo, as biosensors. Other specialized MR probes have been developed to image enzyme activity in vivo. Lastly, the use of chemical exchange and off-resonance techniques have been developed, adding another dimension to the broad capabilities of molecular MRI and offering the potential of multispectral imaging. These and other advances in molecular MRI offer great promise for the future and have significant potential for clinical translation.     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.     

Gadolinium-rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging

A novel dual-labeled nanoparticle for use in labeling and tracking cells in vivo is described. We report the construction and characterization of these gadolinium-rhodamine nanoparticles. These particles are constructed from lipid monomers with diacetylene bonds that are sonicated and photolyzed to form polymerized nanoparticles. Cells are efficiently labeled with these nanoparticles. We have inoculated labeled tumor cells subcutaneouosly into the flanks of C3H mice and have been able to image these labeled tumor cells via MRI and optical imaging. Furthermore, the labeled tumor cells can be visualized via fluorescent microscopy after tissue biopsy. Our results suggest that these nanoparticles could be used to track cells in vivo. This basic platform can be modified with different fluorophores and targeting agents for studying metastisic cell, stem cell, and immune cell trafficking among other applications.     

Gold nanoclusters as novel optical probes for in vitro and in vivo fluorescence imaging

Fluorescent probes play an important role in the development of fluorescence-based imaging techniques for life sciences research. Gold nanoclusters (AuNCs) are a novel type of fluorescent nanomaterials which have attracted great interest in recent years. Composed of only a few atoms, these ultrasmall AuNCs exhibit quantum confinement effects and molecule-like properties. Fluorescent AuNCs have an attractive set of features including ultrasmall size, good biocompatibility and photostability, and tunable emission in the red to near-infrared spectral region, which make them promising as fluorescent labels for biological imaging. Examples of their application include live cell labeling, cancer cell targeting, cellular apoptosis monitoring, and in vivo tumor imaging. Here, we present a brief overview of recent advances in utilizing these emissive ultrasmall AuNCs as optical probes for in vitro and in vivo fluorescence imaging.     

Theranostics: combining imaging and therapy

Employing theranostic nanoparticles, which combine both therapeutic and diagnostic capabilities in one dose, has promise to propel the biomedical field toward personalized medicine. This review presents an overview of different theranostic strategies developed for the diagnosis and treatment of disease, with an emphasis on cancer. Herein, therapeutic strategies such as nucleic acid delivery, chemotherapy, hyperthermia (photothermal ablation), photodynamic, and radiation therapy are combined with one or more imaging functionalities for both in vitro and in vivo studies. Different imaging probes, such as MRI contrast agents (T(1) and T(2) agents), fluorescent markers (organic dyes and inorganic quantum dots), and nuclear imaging agents (PET/SPECT agents), can be decorated onto therapeutic agents or therapeutic delivery vehicles in order to facilitate their imaging and, in so doing, gain information about the trafficking pathway, kinetics of delivery, and therapeutic efficacy; several such strategies are outlined. The creative approaches being developed for these classes of therapies and imaging modalities are discussed, and the recent developments in this field along with examples of technologies that hold promise for the future of cancer medicine are highlighted.     

Efficient and gentle siRNA delivery by magnetofection

Abstract

Magnetic force combined with magnetic nanoparticles recently has shown potential for enhancing nucleic acid delivery. Achieving effective siRNA delivery into primary cultured cells is challenging. We compared the utility of magnetofection with lipofection procedures for siRNA delivery to primary and immortalized mammalian fibroblasts. Transfection efficiency and cell viability were analyzed by flow cytometry and effects of gene knockdown were quantified by real-time PCR. Lipofectamine 2000 and magnetofection achieved high transfection efficiencies comparable to similar gene silencing effects of about 80%; the cytotoxic effect of magnetofection, however, was significantly less. Magnetofection is a reliable and gentle alternative method with low cytotoxicity for siRNA delivery into difficult to transfect cells such as mammalian fibroblasts. These features are especially advantageous for functional end point analyses of gene silencing, e.g., on the metabolite level.     

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.