Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes.

BACKGROUND: Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly. RESULTS: In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses. CONCLUSIONS: Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.

Ions doped melanin nanoparticle as a multiple imaging agent.

BACKGROUND: Multimodal nanomaterials are useful for providing enhanced diagnostic information simultaneously for a variety of in vivo imaging methods. According to our research findings, these multimodal nanomaterials offer promising applications for cancer therapy. RESULTS: Melanin nanoparticles can be used as a platform imaging material and they can be simply produced by complexation with various imaging active ions. They are capable of specifically targeting epidermal growth factor receptor (EGFR)-expressing cancer cells by being anchored with a specific antibody. Ion-doped melanin nanoparticles were found to have high bioavailability with long-term stability in solution, without any cytotoxicity in both in vitro and in vivo systems. CONCLUSION: By combining different imaging modalities with melanin particles, we can use the complexes to obtain faster diagnoses by computed tomography deep-body imaging and greater detailed pathological diagnostic information by magnetic resonance imaging. The ion-doped melanin nanoparticles also have applications for radio-diagnostic treatment and radio imaging-guided surgery, warranting further proof of concept experimental.

Induced heat property of polyethyleneglycol-coated iron oxide nanoparticles with dispersion stability for hyperthermia.

Fe3O4 nanoparticles have been used for hyperthermia treatment in an attempt to overcome various problems. When using hyperthermia treamtment, it is critical to control the surface modification of the particles. Magnetic nanoparticles tend to aggregate due to strong magnetic dipole--dipole attractions. The particles then have a high surface area and are of larger sizes, posing serious practical limitations. The nanoparticles are used to generate maximum heat and to maintain a constant heating temperature using the minimum magnetic nanoparticles dosage. In this study, we investigated the effect of PEG coated onto Fe3O4 nanoparticles. We tested the dispersion stability and repetitive heating property of nanoparticles for different PEG concentrations under an AC magnetic field. The results confirmed that the nanoparticles on a colloidal system maintained the heating properties of repetitve inductive heating as PEG concentration increased with dispersion stability. The nanoparticles with superior dispersion stability will be appropriate for hyperthermia applications in cancer treatments.

Rapid detection and profiling of cancer cells in fine-needle aspirates.

There is a growing need for fast, highly sensitive and quantitative technologies to detect and profile unaltered cells in biological samples. Technologies in current clinical use are often time consuming, expensive, or require considerable sample sizes. Here, we report a diagnostic magnetic resonance (DMR) sensor that combines a miniaturized NMR probe with targeted magnetic nanoparticles for detection and molecular profiling of cancer cells. The sensor measures the transverse relaxation rate of water molecules in biological samples in which target cells of interest are labeled with magnetic nanoparticles. We achieved remarkable sensitivity improvements over our prior DMR prototypes by synthesizing new nanoparticles with higher transverse relaxivity and by optimizing assay protocols. We detected as few as 2 cancer cells in 1-microL sample volumes of unprocessed fine-needle aspirates of tumors and profiled the expression of several cellular markers in <15 min.

Gene Therapy Using Nanocarriers for Pancreatic Ductal Adenocarcinoma: Applications and Challenges in Cancer Therapeutics.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and its incidence is increasing. PDAC often shows resistance to several therapeutic modalities and a higher recurrence rate after surgical treatment in the early localized stage. Combination chemotherapy in advanced pancreatic cancer has minimal impact on overall survival. RNA interference (RNAi) is a promising tool for regulating target genes to achieve sequence-specific gene silencing. Here, we summarize RNAi-based therapeutics using nanomedicine-based delivery systems that are currently being tested in clinical trials and are being developed for the treatment of PDAC. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing has been widely used for the development of cancer models as a genetic screening tool for the identification and validation of therapeutic targets, as well as for potential cancer therapeutics. This review discusses current advances in CRISPR/Cas9 technology and its application to PDAC research. Continued progress in understanding the PDAC tumor microenvironment and nanomedicine-based gene therapy will improve the clinical outcomes of patients with PDAC.

One-shot dual gene editing for drug-resistant pancreatic cancer therapy.

It is challenging to diagnose patients with pancreatic ductal adenocarcinoma (PDAC) early on, and their treatment is often complex. Gemcitabine (GEM) is the first-line treatment for PDAC, but its efficacy is limited in most patients due to the GEM resistance from KRAS and P53 gene mutations. We describe the correction of a double gene mutation and therapeutic effect for the GEM resistant PDAC. Bio-available nanoliposomes (NL) possessing Cas9-ribonucleoproteins and adenine-base editors were developed to conduct KRAS and P53 mutation gene editing directly. NLs were conjugated with EGFR antibodies to tumor-specific delivery, and the anti-cancer effect was verified in vitro and in vivo Model. Our GEM-combinatorial therapeutic strategies using double gene editing systems with one-shot may be a potent therapy for PDAC, overcoming chemoresistance.

Ultrasound-activated particles as CRISPR/Cas9 delivery system for androgenic alopecia therapy.

Compared to a plasmid, viral, and other delivery systems, direct Cas9/sgRNA protein delivery has several advantages such as low off-targeting effects and non-integration, but it still has limitations due to low transfer efficiency. As such, the CRISPR/Cas9 system is being developed in combination with nano-carrier technology to enhance delivery efficiency and biocompatibility. We designed a microbubble-nanoliposomal particle as a Cas9/sgRNA riboprotein complex carrier, which effectively facilitates local delivery to a specific site when agitated by ultrasound activation. In practice, we successfully transferred the protein constructs into dermal papilla cells in the hair follicle of androgenic alopecia animals by microbubble cavitation induced sonoporation of our particle. The delivered Cas9/sgRNA recognized and edited specifically the target gene with high efficiency in vitro and in vivo, thus recovering hair growth. We demonstrated the topical application of ultrasound-activated nanoparticles for androgenic alopecia therapy through the suppression of SRD5A2 protein production by CRISPR-based genomic editing.

[Sentinel lymph node mapping of the stomach using fluorescent magnetic nanoparticles in rabbits].

BACKGROUND/AIMS: Sentinel lymph node (SLN) mapping of the stomach cancer using available techniques is limited by unpredictable lymphatic drainage patterns and skip metastasis. The aim of this study was to test the feasibility of gastric SLN mapping using fluorescent magnetic nanoparticles (FMNP) of uniform nano-size. METHODS: Biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness with 60 nm thickness were used as model nanomaterials. Gastric lymphatic mapping was performed by injecting 100 microL of either FMNP or isosulafan blue subserosally. Gastric injections (n=7) were made into the body, approximately 5 cm from the lesser curvature of rabbits. Sentinel lymph nodes were visualized using fluorescent nanoparticle detection system. RESULTS: In 7 rabbits, it was demonstrated that FMNP quickly and accurately detected sentinel lymph nodes. Injection into the stomach resulted in identification of a retrogastric lymph node. Histological analysis in all cases confirmed the presence of nodal tissue. CONCLUSIONS: FMNP can be a potential alternative to existing tracers in the detection of SLN in this animal experiment.

Synthesis of ultrasound contrast agents: characteristics and size distribution analysis (secondary publication).

PURPOSE: The purpose of this study was to establish a method for ultrasound (US) contrast agent synthesis and to evaluate the characteristics of the synthesized US contrast agent. METHODS: A US contrast agent, composed of liposome and sulfur hexafluoride (SF6), was synthesized by dissolving 21 µmol 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC, C40H80NO8P), 9 µmol cholesterol, and 1.9 µmol of dihexadecylphosphate (DCP, [CH3(CH2)15O]2P(O)OH) in chloroform. After evaporation in a warm water bath and drying for 12-24 hours, the contrast agent was synthesized using the sonication process by the addition of a buffer and SF6 gas. The size distribution of the bubbles was analyzed using dynamic light scattering measurement methods. The degradation curve was evaluated by assessing the change in the number of contrast agent bubbles using light microscopy immediately, 12, 24, 36, 48, 60, 72, and 84 hours after synthesis. The echogenicity of the synthesized microbubbles was compared with commercially available microbubbles (SonoVue, Bracco). RESULTS: contrast agent was synthesized successfully using an evaporation-drying-sonication method. Most bubbles had a mean diameter of 154.2 nm and showed marked degradation 24 hours after synthesis. Although no statistically significant differences were observed between SonoVue and the synthesized contrast agent, a difference in echogenicity was observed between the synthesized contrast agent and saline (P<0.01). CONCLUSION: We successfully synthesized a US contrast agent using an evaporation-dryingsonication method. These results may help future research in the fields of anticancer drug delivery, gene delivery, targeted molecular imaging, and targeted therapy.

Highly selective organic transistor biosensor with inkjet printed graphene oxide support system.

Most of the reported field effect transistors (FETs) fall short of a general method to uniquely specify and detect a target analyte. For this reason, we propose a pentacene-based FET with a graphene oxide support system (GOSS), composed of functionalized graphene oxide (GO) ink. The GOSS with a specific moiety group to capture the biomaterial of interest was inkjet printed on the pentacene FET. It provided modular receptor sites on the surface of pentacene, without alteration of the device. To evaluate the performance of a GOSS-pentacene FET biosensor, we detected the artificial DNA and circulating tumor cells as a proof-of-concept. The mobility of the FET dramatically changed upon capturing the target biomolecule on the GOSS. The FET exhibited high selectivity with 0.1 pmoles of the target DNA and a few cancer cells per detection volume. This study suggests a valuable sensor for medical diagnosis that can be mass produced effortlessly at low-cost.

Fattigation-platform theranostic nanoparticles for cancer therapy.

A new conceptual nanoparticle consisting of a silica-coated iron oxide magnetic core and a fattigation-based biocompatible shell with oleic acid and hydrophilic protein (gelatin). The prepared particle can be a useful theranostics platform material for diagnostic imaging and as a drug delivery system. Oleic acid and gelatin were conjugated on the silica-coated magnetic nanoparticle surface to provide three primary functionalities: 1) enhancing biocompatibility and solubility in aqueous solution and providing the ability to incorporate hydrophobic chemical drugs into the shell for delivery, 2) improving treatment-response magnetic monitoring as a diagnostic agent with low nanotoxicity, and 3) increasing anticancer efficacy owing to the controlled release of the incorporated drug in cells and in an animal model. We prepared magnetic-silica nanoparticles with super-paramagnetic properties, which are utilized as a T2-weighted magnetic resonance imaging agent. After formation of an oleic acid-gelatin shell, the prepared materials exhibited high loading capacity for a hydrophobic anticancer drug (paclitaxel). Our particle platform system exhibited higher therapeutic efficacy and lower toxicological effects in vitro and in an in vivo cancer model than a clinically available chemo-drug (Taxol®). Our findings strongly suggest that this nanoparticle system can serve as a platform for cancer therapy by the incorporation of chemical drugs.

Toxicity and tissue distribution of magnetic nanoparticles in mice.

The development of technology enables the reduction of material size in science. The use of particle reduction in size from micro to nanoscale not only provides benefits to diverse scientific fields but also poses potential risks to humans and the environment. For the successful application of nanomaterials in bioscience, it is essential to understand the biological fate and potential toxicity of nanoparticles. The aim of this study was to evaluate the biological distribution as well as the potential toxicity of magnetic nanoparticles to enable their diverse applications in life science, such as drug development, protein detection, and gene delivery. We recently synthesized biocompatible silica-overcoated magnetic nanoparticles containing rhodamine B isothiocyanate (RITC) within a silica shell of controllable thickness [MNPs@SiO2(RITC)]. In this study, the MNPs@SiO2(RITC) with 50-nm thickness were used as a model nanomaterial. After intraperitoneal administration of MNPs@SiO2(RITC) for 4 weeks into mice, the nanoparticles were detected in the brain, indicating that such nanosized materials can penetrate blood-brain barrier (BBB) without disturbing its function or producing apparent toxicity. After a 4-week observation, MNPs@SiO2(RITC) was still present in various organs without causing apparent toxicity. Taken together, our results demonstrated that magnetic nanoparticles of 50-nm size did not cause apparent toxicity under the experimental conditions of this study.

Cellular uptake of magnetic nanoparticle is mediated through energy-dependent endocytosis in A549 cells.

Biocompatible silica-overcoated magnetic nanoparticles containing an organic fluorescence dye, rhodamine B isothiocyanate (RITC), within a silica shell [50 nm size, MNP@SiO2(RITC)s] were synthesized. For future application of the MNP@SiO2(RITC)s into diverse areas of research such as drug or gene delivery, bioimaging, and biosensors, detailed information of the cellular uptake process of the nanoparticles is essential. Thus, this study was performed to elucidate the precise mechanism by which the lung cancer cells uptake the magnetic nanoparticles. Lung cells were chosen for this study because inhalation is the most likely route of exposure and lung cancer cells were also found to uptake magnetic nanoparticles rapidly in preliminary experiments. The lung cells were pretreated with different metabolic inhibitors. Our results revealed that low temperature disturbed the uptake of magnetic nanoparticles into the cells. Metabolic inhibitors also prevented the delivery of the materials into cells. Use of TEM clearly demonstrated that uptake of the nanoparticles was mediated through endosomes. Taken together, our results demonstrate that magnetic nanoparticles can be internalized into the cells through an energy-dependent endosomal-lysosomal mechanism.

Ultrasound-Guided Delivery of siRNA and a Chemotherapeutic Drug by Using Microbubble Complexes: In Vitro and In Vivo Evaluations in a Prostate Cancer Model.

OBJECTIVE: To evaluate the effectiveness of ultrasound and microbubble-liposome complex (MLC)-mediated delivery of siRNA and doxorubicin into prostate cancer cells and its therapeutic capabilities both in vitro and in vivo. MATERIALS AND METHODS: Microbubble-liposome complexes conjugated with anti-human epidermal growth factor receptor type 2 (Her2) antibodies were developed to target human prostate cancer cell lines PC-3 and LNCaP. Intracellular delivery of MLC was observed by confocal microscopy. We loaded MLC with survivin-targeted small interfering RNA (siRNA) and doxorubicin, and delivered it into prostate cancer cells. The release of these agents was facilitated by ultrasound application. Cell viability was analyzed by MTT assay after the delivery of siRNA and doxorubicin. Survivin-targeted siRNA loaded MLC was delivered into the xenograft mouse tumor model. Western blotting was performed to quantify the expression of survivin in vivo. RESULTS: Confocal microscopy demonstrated substantial intracellular uptake of MLCs in LNCaP, which expresses higher levels of Her2 than PC-3. The viability of LNCaP cells was significantly reduced after the delivery of MLCs loaded with siRNA and doxorubicin (85.0 ± 2.9%), which was further potentiated by application of ultrasound (55.0 ± 3.5%, p = 0.009). Survivin expression was suppressed in vivo in LNCaP tumor xenograft model following the ultrasound and MLC-guided delivery of siRNA (77.4 ± 4.90% to 36.7 ± 1.34%, p = 0.027). CONCLUSION: Microbubble-liposome complex can effectively target prostate cancer cells, enabling intracellular delivery of the treatment agents with the use of ultrasound. Ultrasound and MLC-mediated delivery of survivin-targeted siRNA and doxorubicin can induce prostate cell apoptosis and block survivin expression in vitro and in vivo.

Boiling Method-Based Zinc Oxide Nanorods for Enhancement of Adipose-Derived Stem Cell Proliferation.

Adipose-derived stem cells (ASCs) are typically expanded to acquire large numbers of cells for therapeutic applications. Diverse stimuli such as sphingosylphosphocholine and vitamin C have been used to increase the production yield and regenerative potential of ASCs. In the present study, we hypothesized that ZnO nanorods have promising potential for the enhancement of ASC proliferation. ZnO nanorods were prepared using three different methods: grinding and boiling at low temperature with and without surfactant. The physicochemical properties of the nanorods such as their crystallinity, morphology, size, and solvent compatibility were evaluated, and then, the ability of the synthesized ZnO nanorods to enhance ASC proliferation was investigated. Scanning electron microscopy images of all of the ZnO powders showed rod-shaped nanoflakes with lengths of 200-500 nm. Notably, although ZnO-G produced by the grinding method was well dispersed in ethanol, atomic force microscopy images of dispersions of both ZnO-B from boiling methods and ZnO-G indicated the presence of clusters of ZnO nanorods. In contrast, ZnO-B was freely dispersible in 5% dextrose of water and dimethyl sulfoxide, whereas ZnO-G and ZnO-M, produced by boiling with ethanolamine, were not. All three types of ZnO nanorods increased the proliferation of ASCs in a dose-dependent manner. These results collectively suggest that ZnO nanorods have promising potential for use as an agent for the enhancement of ASC proliferation.

Development of a novel microbubble-liposome complex conjugated with peptide ligands targeting IL4R on brain tumor cells.

Gas (SF6)-filled microbubbles (MBs) were prepared by emulsion and solvent-evaporation method. The prepared MBs were further conjugated with doxorubicin (Dox)-loaded nano-sized liposome and peptide ligands to interleukin-4 receptor (IL4R) for targeting brain tumor cells. The final MB-liposome (Dox)-IL4R targeting peptide ligand [MB-Lipo (Dox)-IL4RTP] had a spherical structure with the mean size of 1,500 nm. The MB-Lipo (Dox)­IL4RTP exhibited cellular uptake in U87MG brain tumor cells (a brain tumor cell line expressing strongly IL4R) with frequency ultrasound energy suggesting that MB-Lipo (Dox)­IL4RTP provided effective targeting ability for brain tumor cells. In addition, WST-1 assay results showed that MB-Lipo (Dox)­IL4RTP inhibited the proliferation of U87MG cells IL4R­dependently. This was confirmed by western blotting of γH2AX, phospho (Ser15)-p53, p53 and p21 which are signal transduction proteins involved in DNA damage response and cell cycle arrest. Taken together, these results indicate that MB-Lipo (Dox)-IL4RTP represents a promising ultrasonic contrast agent for tumor-targeting ultrasonic imaging.

Optimization of ultrasound parameters for microbubble-nanoliposome complex-mediated delivery.

PURPOSE: The aim of this study was to identify the optimal ultrasound (US) parameters for gene and drug delivery. METHODS: In order to target SkBr3, which is a breast cancer cell overexpressing the Her2 receptor, trastuzumab (Herceptin) was used. Micobubble-nanoliposome complex (MLC) was mixed with trastuzumab and stored overnight. Finally, MLC was combined with Her2Ab. A US device equipped with a 1-MHz probe was used for delivery to the cell. Several parameters, including intensity (w/cm(2)), time (minutes), and duty cycle (%), were varied within a range from 1 w/cm(2), 1 minute, and 20% to 2 w/cm(2), 2 minutes, and 60%, respectively. A confocal laser scanning microscope (CLSM) was used to confirm the delivery of MLC to the cells after US treatment. RESULTS: MLC with fluorescent dyes and trastuzumab was synthesized successfully. By delivering MLC with Her2Ab to cells, the targeting effect of trastuzumab with MLC was confirmed by CLSM. The cell membranes showed green (fluorescein isothiocyanate) and red (Texas red) fluorescence but treatments with MLC without Her2Ab did not show any fluorescence. Optimal conditions for US-mediated delivery were 1 or 2 w/cm(2), 2 minutes, and 60% (uptake ratio, 95.9% for 1 w/cm(2) and 95.7% for 2 w/cm(2)) for hydrophobic materials and 2 w/cm(2), 2 minutes, and 60% (uptake ratio, 95.0%) for hydrophilic materials. CONCLUSION: The greater the strength, duty cycle, and period of US application within the tested range, the more efficiently the fluorescent contents were conveyed.

Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells.

Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 µg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application.

Enhancement of cancer specific delivery using ultrasound active bio-originated particles.

A hybrid multifunctional particle comprising of a microbbule (MB), liposome (Lipo), and an Fe ion chelated melanin nanoparticle (MNP(Fe)) was applied for ultrasound mediated cancer targeting as a theranostic agent.