Extraction of urinary cell-free DNA by using triamine-modified silica particles for liquid biopsy.

The presence of approximately 200-bp cell-free DNA (cfDNA) in the urine has attracted attention as a biomarker for liquid biopsy. However, it is currently useful only for diagnoses of cancers in which a large amount of cfDNA is excreted in the urine. Therefore, the development of an efficient method for extracting cfDNA existing in small amounts in the urine is essential for diagnosing many other diseases. We examined the effect of particle size, small pore size (surface area), and surface modification of porous silica particles on the efficiency of DNA extraction. Our observations suggested that cfDNA could be captured by tertiary amine-modified particles and then removed from the particles by repeatedly washing with sodium bicarbonate (pH 11). Using this method with 30 mg of triamine-modified particles, we succeeded in extracting a few hundred nanograms of cfDNA from 15 mL urine. Furthermore, we could detect ~ 67 fg/mL caries DNA (211 bp) in 15 mL urine sample, suggesting that this method may be suitable for the extraction of genetic biomarkers for cfDNA-based liquid biopsy.

A Simple and Easy Method of Monitoring Doxorubicin Release from a Liposomal Drug Formulation in the Serum Using Fluorescence Spectroscopy.

Formulation of a drug as liposomes facilitates its delivery to the disease target. Rightly, liposomes are gaining popularity in the medical field. In order for the drug to show efficacy, release of the encapsulated drug from the liposome at the target site is required. However, the release is affected by the permeability of the lipid bilayer of the liposome, and it is important to examine the effect of the surrounding environment on the permeability. In this study, we showed the usefulness of fluorescence analysis, especially fluorescence fingerprint, for a rapid and simple monitoring of release of an encapsulated anticancer drug (doxorubicin) from its liposomal formulation (DOXIL). Our result indicated that the release is accelerated by the existence of membrane permeable ions, such as tris(hydroxymethyl)aminomethane, and blood proteins like albumin. Hence, monitoring of doxorubicin release by fluorescence analysis is useful for the efficacy evaluation of DOXIL in a biomimetic environment.

Fluorescence Tumor-Imaging Using a Thermo-Responsive Molecule with an Emissive Aminoquinoline Derivative.

We synthesized (2,4-trifluoromethyl-7-N-bis(2,5,8,11-tetraoxatridecane-13-yl)-aminoquinoline) TFMAQ-diEg4, an emissive aminoquinoline derivative that incorporated two tetraethyleneglycol chains into an amino group. TFMAQ-diEg4 showed fluorescence and thermo-responsive properties accompanied by a lower critical solution temperature (LCST), due to the introduction of the oligoethylene glycol chain. This thermo-responsive LCST behavior occurred at the border of a cloud point. Below and above the cloud point, self-assemblies of 6-7-nm nanoparticles and ~2000-nm microparticles were observed, in vitro. In addition, TFMAQ-diEg4 showed a high solubility, over 20 mM for aqueous solution, in vivo, which not only prevented thrombosis but also allowed various examinations, such as single intravenous administration and intravenous drips. Intravenous administration of TFMAQ-diEg4, to tumor-bearing, mice led to the accumulation of the molecule in the tumor tissue, as observed by fluorescence imaging. A subset of mice was treated with local heat around their tumor tissue and an intravenous drip of TFMAQ-diEg4, which led to a high intensity of TFMAQ-diEg4 emission within the tumor tissue. Therefore, we revealed that TFMAQ-diEg4 was useful as a fluorescence probe with thermo-responsive properties.

Water-Proton Relaxivities of Radical Nanoparticles Self-Assembled via Hydration or Dehydration Processes.

Nanoparticles capable of accumulating in tumor tissues are promising materials for tumor imaging and therapy. In this study, two radical nanoparticles (RNPs), denoted as 1 and 2, composed of self-assembled ureabenzene derivatives possessing one or two amphiphilic side chains were demonstrated to be candidates for metal-free functional magnetic resonance imaging (MRI) contrast agents (CAs). Because of the self-assembly behavior of 1 and 2 in a saline solution, spherical RNPs of sizes ∼50-90 and ∼30-100 nm were detected. In a highly concentrated solution, RNP 1 showed considerably small water-proton relaxivity values (r1 and r2), whereas RNP 2 showed an r1 value that was around 5 times larger than that of RNP 1. These distinct r1 values might be caused by differences in the self-assembly behavior by a hydration or dehydration process. In vivo studies with RNP 2 demonstrated a slightly enhanced T1-weighted image in mice, suggesting that the RNPs can potentially be used as metal-free functional MRI CAs for T1-weighted imaging.

Self-Assembly Behavior of Emissive Urea Benzene Derivatives Enables Heat-Induced Accumulation in Tumor Tissue.

In this study we describe the construction of a system composed of thermally responsive molecules that can be induced to accumulate in tumor tissues by heating. EgX molecules consisting of an urea-benzene framework and oligoethylene glycol (OEG) functional groups with an emissive aminoquinoline formed nanoparticles (NPs) ∼10 nm in size at 23 °C with a fluorescence quantum yield of 7-10%. At higher temperatures, additional self-assembly occurred as a result of OEG dehydration, and the NPs grew to over 1000 nm in size; this was accompanied by low critical solution temperature behavior. EgXs accumulated in tumor tissues of mice at a body temperature of around 33-35 °C, an effect that was accelerated by external heating around the tumor to approximately 40 °C as a result of increased particle size and enhanced retention in tissue. These EgX NPs can serve as a tool for in vivo monitoring of tumor progression and response to treatment.

Electroinduced Delivery of Hydrogel Nanoparticles in Colon 26 Cells, Visualized by Confocal Fluorescence System.

BACKGROUND: Nano-scale drug delivery systems (nano-DDS) are under intense investigation. Nano-platforms are developed for specific administration of small molecules, drugs, genes, contrast agents [quantum dots (QDs)] both in vivo and in vitro. Electroporation is a biophysical phenomenon which consists of the application of external electrical pulses across the cell membrane. The aim of this study was to research electro-assisted Colon 26 cell line internalization of QDs and QD-loaded nano-hydrogels (polymersomes) visualized by confocal microscopy and their influence on cell viability. MATERIALS AND METHODS: The experiments were performed on the Colon 26 cancer cell line, using a confocal fluorescent imaging system and cell viability test. RESULTS: Electroporation facilitated the delivery of nanoparticles in vivo. We demonstrated increased voltage-dependent delivery of nanoparticles into cells after electrotreatment, without significant cell viability reduction. CONCLUSION: The delivery and retention of the polymersomes in vitro is a promising tool for future cancer treatment strategies and nanomedcine.

Passive and electro-assisted delivery of hydrogel nanoparticles in solid tumors, visualized by optical and magnetic resonance imaging in vivo.

The present study describes a development of nanohydrogel, loaded with QD(705) and manganese (QD(705)@Nanogel and QD(705)@Mn@Nanogel), and its passive and electro-assisted delivery in solid tumors, visualized by fluorescence imaging and magnetic resonance imaging (MRI) on colon cancer-grafted mice as a model. QD(705)@Nanogel was delivered passively predominantly into the tumor, which was visualized in vivo and ex vivo using fluorescent imaging. The fluorescence intensity increased gradually within 30 min after injection, reached a plateau between 30 min and 2 h, and decreased gradually to the baseline within 24 h. The fluorescence intensity in the tumor area was about 2.5 times higher than the background fluorescence. A very weak fluorescent signal was detected in the liver area, but not in the areas of the kidneys or bladder. This result was in contrast with our previous study, indicating that FITC@Mn@Nanogel did not enter into the tumor and was detected rapidly in the kidney and bladder after i.v. injection [J. Mater. Chem. B 2013, 1, 4932-4938]. We found that the embedding of a hard material (as QD) in nanohydrogel changes the physical properties of the soft material (decreases the size and negative charge and changes the shape) and alters its pharmacodynamics. Electroporation facilitated the delivery of the nanohydrogel in the tumor tissue, visualized by fluorescent imaging and MRI. Strong signal intensity was recorded in the tumor area shortly after the combined treatment (QD@Mn@Nanogel + electroporation), and it was observed even 48 h after the electroporation. The data demonstrate more effective penetration of the nanoparticles in the tumor due to the increased permeability of blood vessels at the electroporated area. There was no rupture of blood vessels after electroporation, and there were no artifacts in the images due to a bleeding.

Thermoactivatable polymer-grafted liposomes for low-invasive image-guided chemotherapy.

The objective of this study was to develop a thermotriggered, polymer-based liposomal drug carrier with an activatable magnetic resonance imaging (MRI) contrast property for monitoring the release of substances and for localized tumor therapy. The multimodal thermoactivatable polymer-grafted liposomes (MTPLs) were tested to investigate whether the accumulation of MTPLs in colon-26 grafted tumors could be visualized in vivo using MRI and optical imaging, whether MTPLs induce signal enhancement, reflecting the release of their contents, after triggering by short-term heating (42.5°C for 10 minutes) 9 hours after MTPL administration (late-phase triggering), and whether MTPLs can provide a sufficient antitumor effect. The imaging and therapeutic properties of MTPLs were tested both in vitro and in vivo (BALB/c nude mice: heated group with MTPLs (n = 5), nonheated group with MTPLs (n = 5), heated group with doxorubicin-free MTPLs (n = 5), nonheated group with manganese-free MTPLs (n = 5), and kinetics observation group (n = 3); N = 23). Through in vivo MRI and fluorescent imaging, the MTPLs were shown to have significantly accumulated in the grafted colon-26 tumors 8 hours after administration. Delayed thermotriggering (9 hours after administration) caused MR signal enhancement, reflecting the release of their contents, after a short exposure to tolerable heat. In addition, significant antitumor effects were observed after treatment. The proposed polymer-based activatable MTPLs with a "delayed thermotrigger" provide a promising technology for cancer theranostics that allows minimal adverse effects and rapid interactive therapy.

Lymph node mapping using quantum dot-labeled polymersomes.

The present study was designed to investigate whether poly-ion complex hollow vesicles (polymersomes), based on chemically-modified chitosan, are appropriate for lymph node mapping in the context of their application in the development of theranostic nanosized drug delivery systems (nano-DDS). The experiments were performed on Balb/c nude mice (colon cancer-grafted). The mice were subjected to anesthesia and quantum dot (QD(705))-labeled polymersomes (d-120 nm) were injected intravenously via the tail vein. The optical imaging was carried out on Maestro EX Imaging System (excitation filter: 435-480 nm; emission filter: 700 nm). A strong fluorescent signal, corresponding to QD(705) fluorescence, was detected in the lymph nodes, as well as in the tumor. A very weak fluorescent signal was found in the liver area. The half-life of QD(705)-labelled polymersomes was 6 ± 2 hours in the bloodstream and 11 ± 3 hours in the lymph nodes. The data suggest that polymersomes are very promising carriers for lymph node mapping using QD as a contrast agent. They are useful matrix for development of nano-formulations with theranostic capabilities.

Gene regulation by intracellular delivery and photodegradation of nanoparticles containing small interfering RNA.

Although the use of small interfering RNA (siRNA) is a promising technique for gene regulation, spatiotemporal control of the effects of siRNA must be achieved if the technique is to be safe and practical. Here, a method for spatiotemporal regulation of genes with nanoparticles containing siRNA is reported. The siRNA is encapsulated in photodegradable nanoparticles that are internalized to SKOV3-Luc cells, where the siRNA is released from the nanoparticles by UV irradiation for 30 s. The encapsulated siRNA only shows no gene-silencing effects, but release of the siRNA upon UV radiation leads to sequence-specific silencing of the luciferase gene in the cells. These results indicate that photodegradable siRNA-containing nanoparticles can be useful for time- and space-dependent regulation of gene expression in cells.

Delivery, stabilization, and spatiotemporal activation of cargo molecules in cells with positively charged nanoparticles.

Positively charged photodegradable nanoparticles that simultaneously encapsulated various compounds including small and large molecules were prepared. The nanoparticles were internalized to the cell by endocytosis and were stable within the cells for at least 4 days. The encapsulated molecules were released into the cytosol using light stimuli.

Shishicrellastatins, inhibitors of cathepsin B, from the marine sponge Crella (Yvesia) spinulata.

Two dimeric steroid derivatives, shishicrellastatin A (1) and B (2), have been isolated as cathepsin B inhibitors from the marine sponge Crella (Yvesia) spinulata. Their structures were determined by interpretation of spectroscopic data. Shishicrellastatins inhibit cathepsin B with an IC(50) value of 8 µg/mL each.