Recent Advances in pH-Sensitive Polymeric Nanoparticles for Smart Drug Delivery in Cancer Therapy.

BACKGROUND: Rapid developments in the field of nanoparticles during the last decades have led to its increased application in drug delivery approaches. The advantages of nano-based drug delivery systems include improved therapeutic efficacy of drugs and the reduction of side effects. OBJECTIVE: This review aims to highlight advances in stimuli-triggered drug delivery approaches using polymeric nanoparticles with a focus on pH-sensitive drug- and theranostic delivery systems. RESULTS AND CONCLUSION: Of the various organic/inorganic nanoparticles, polymeric nanoparticles have fulfilled an integral role in the advancement of drug delivery systems by virtue of the ease to incorporate and modify targeting moieties in combination with controlled drug release over prolonged periods. Furthermore, polymeric nanoparticles facilitate theranostic treatment by the incorporation of imaging agents in addition to therapeutics. Recently, stimuli-responsive polymeric nanoparticles emerged as smart drug carriers, in which drug release is affected by physical and/or chemical structural changes induced by a specific stimulus (e.g. pH, temperature, and specific enzymes). The use of these nanocomposites reduces premature drug release and maintains effective drug levels at the pathological target.

Antioxidant activity of levan coated cerium oxide nanoparticles.

Levan coated cerium oxide nanoparticles (LCNPs) with the enhanced antioxidant activity were successfully synthesized and characterized. Levan and their derivatives are attractive for biomedical applications attributable to their antioxidant, anti-inflammation and anti-tumor properties. LCNPs were synthesized using the one-pot and green synthesis system with levan. For production of nanoparticles, levan plays a role as a stabilizing and reducing agent. Fourier transform infrared spectroscopy (FT-IR) analysis showed that LCNPs successfully synthesized. The morphology and size of nanoparticles were confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). LCNPs have good water solubility and stability. The conjugation of levan with cerium oxide nanoparticles improved antioxidant activity. Moreover the level of ROS was reduced after treatment of LCNPs to H2O2 stimulated NIH3T3 cells. These results demonstrate that the LCNPs are useful for applying of treatment of ROS induced diseases.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance.

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X-/-) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X-/-) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the µ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.

Preparation of pyrenyl-based multifunctional nanocomposites for biomedical applications.

Nanocomposites are widely used to obtain an accurate diagnosis of, and to provide effective therapy for, a number of diseases, because they can be easily formulated by introducing therapeutic agents (e.g., drugs and genes) and imaging agents (e.g., magnetic nanocrystals). Furthermore, nanocomposites can be developed as all-in-one systems, which enable cancer diagnosis and therapy, as well as the simultaneous monitoring of drug behavior. In this protocol, we describe the synthesis of four pyrenyl-based polymers (pyrenyl polyethylene glycol (Py-PEG), pyrenyl dextran (Py-DEX), pyrenyl hyaluronan (Py-HA) and pyrenyl-conjugated heterofunctional PEG (pyrenyl PEG)) and their subsequent use in the preparation of multifunctional nanocomposites for different applications including multimodal imaging, targeted cancer detection and pH-sensitive drug delivery. Notably, these nanocomposites can be used to simultaneously perform multiple tasks--for example, delivering magnetic particles for early cancer detection by MRI, efficient cataloging of patient groups for personalized therapy and real-time monitoring of disease progress. Starting from the synthesis of pyrenyl-based polymers, this protocol can be completed in ∼15 d.

Simple, rapid detection of influenza A (H1N1) viruses using a highly sensitive peptide-based molecular beacon.

A peptide-based molecular beacon (PEP-MB) was prepared for the simple, rapid, and specific detection of H1N1 viruses using a fluorescence resonance energy transfer (FRET) system. The PEP-MB exhibited minimal fluorescence in its "closed" hairpin structure. However, in the presence of H1N1 viruses, the specific recognition of the hemagglutinin (HA) protein of H1 strains by the PEP-MB causes the beacon to assume an "open" structure that emits strong fluorescence. The PEP-MB could detect H1N1 viruses within 15 min or even 5 min and can exhibit strong fluorescence even at low viral concentrations, with a detection limit of 4 copies.

A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.

A scanometric antibody probe for facile and sensitive immunoassays.

We have developed a novel scanometric antibody probe for rapid, sensitive, and naked-eye-visible immunoassays. Using this probe, we clearly demonstrated the successful scanometric detection and identification of influenza A viruses on a microarray. In addition, the sensitivity of the scanometric immunoassay was comparable to that of the fluorescence-based method.

Photo-crosslinkable hydrogel-based 3D microfluidic culture device.

We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 µm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.

Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation.

Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for measuring alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to the hydrolysis of its phosphate groups by ALP. In addition, gold nanoparticles help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that the AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity.

A novel copper-chelating strategy for fluorescent proteins to image dynamic copper fluctuations on live cell surfaces.

Copper is indispensable in most aerobic organisms although it is toxic if unregulated as illustrated in many neurodegenerative diseases. To elucidate the mechanisms underlying copper release from cells, a membrane-targeting reporter which can compete with extracellular copper-binding molecules is highly desirable. However, engineering a reporter protein to provide both high sensitivity and selectivity for copper(ii) has been challenging, likely due to a lack of proper copper(ii)-chelating strategies within proteins. Here, we report a new genetically encoded fluorescent copper(ii) reporter by employing a copper-binding tripeptide derived from human serum albumin (HSA), which is one of the major copper-binding proteins in extracellular environments. Optimized insertion of the tripeptide into the green fluorescent protein leads to rapid fluorescence quenching (up to >85% change) upon copper-binding, while other metal ions have no effect. Furthermore, the high binding affinity of the reporter enables reliable copper detection even in the presence of competing biomolecules such as HSA and amyloid beta peptides. We also demonstrate that our reporter proteins can be used to visualize dynamic copper fluctuations on living HeLa cell surfaces.

Organ-specific distribution of gold nanoparticles by their surface functionalization.

The behavior and fate of intravenously (i.v.) injected nanoparticles (NPs) can be controlled by several physicochemical factors including size, shape and surface charge. To evaluate the role of surface charge on distribution of NPs, we used neutral-charged 15-nm-sized polyethylene glycol-coated gold nanoparticles (AuNP(PEG)) as a core NP and carboxyl or amine groups were conjugated to AuNP(PEG) to generate negative (AuNP(COOH)) or positive AuNP (AuNP(NH2)), respectively. Each type of AuNP was i.v. injected into mice (1 mg kg(-1)) and the concentration of Au was measured in different organs at 30 min, 4, 24 h, 7, 14 days, 1, 3 and 6 months post-injection. The organ distribution also showed the higher deposition rate depending on their functional groups: AuNP(PEG) for mesenteric lymph node, kidney, brain and testis; AuNP(COOH) for liver; AuNP(NH2) for spleen, lung and heart. The blood circulation time and the major excretion route were different depending on their functional groups. In conclusion, functional groups conjugated on the surface of AuNPs produce differences in blood kinetics, organ distribution and elimination pattern which can be important information for directing NPs to specific organs or improving the kinetic properties.

Self-assembled levan nanoparticles for targeted breast cancer imaging.

We report on the targeted imaging of breast cancer using self-assembled levan nanoparticles. Indocyanine green (ICG) was encapsulated in levan nanoparticles via self-assembly. Levan-ICG nanoparticles were found to be successfully accumulated in breast cancer via specific interaction between fructose moieties in levan and overexpressed glucose transporter 5 in breast cancer cells.

Detection of UV-induced mutagenic thymine dimer using graphene oxide.

In this paper, we report for the first time that graphene oxide (GO) can interact with mutagenic DNA but not intact DNA. After UV-irradiated fluorophore-linked DNA containing thymine repeats was mixed with GO, a decrease in fluorescence was observed in a time-dependent manner. In contrast, no fluorescence change was observed with intact DNA, indicating that UV irradiation of DNA resulted in the formation of mutagenic bases. Because GO is known to act as a fluorescence quencher, the decreased fluorescence implies adsorption of the UV-irradiated DNA onto GO. It appears that the decreased fluorescence might result from the greater accessibility of hydrophobic methyl groups and phenyl rings of thymine dimers to GO and from deformed DNA structures with less effective charge shielding under salt-containing conditions. Using this affinity of GO for mutagenic DNA, we could detect UV-irradiated DNA at concentrations as low as 100 pM. We were also able to analyze the ability of phototoxic drugs to catalyze the formation of mutagens under UV irradiation with GO. Because our method is highly sensitive and feasible and does not require the pretreatment of DNA, we propose that it could accelerate the screening of potential phototoxic drug candidates that would be able to sensitize mutagenic dsDNA.

Live imaging of cellular dynamics using a multi-imaging vector in single cells.

Real-time monitoring of cellular dynamics in living organisms is highly challenging. We developed a multi-imaging vector based on 2A peptides. Live imaging of subcellular compartments can be performed following the transfection of cells with another vector, the multi-labeling vector, which contains localization signals and various fluorescent protein variants.

Inhibition of adenylyl cyclase type 5 prevents L-DOPA-induced dyskinesia in an animal model of Parkinson's disease.

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is widely used as a therapeutic choice for the treatment of patients with Parkinson's disease. However, the long-term use of L-DOPA leads to the development of debilitating involuntary movements, called L-DOPA-induced dyskinesia (LID). The cAMP/protein kinase A (PKA) signaling in the striatum is known to play a role in LID. However, from among the nine known adenylyl cyclases (ACs) present in the striatum, the AC that mediates LID remains unknown. To address this issue, we prepared an animal model with unilateral 6-hydroxydopamine lesions in the substantia nigra in wild-type and AC5-knock-out (KO) mice, and examined behavioral responses to short-term or long-term treatment with L-DOPA. Compared with the behavioral responses of wild-type mice, LID was profoundly reduced in AC5-KO mice. The behavioral protection of long-term treatment with L-DOPA in AC5-KO mice was preceded by a decrease in the phosphorylation levels of PKA substrates ERK (extracellular signal-regulated kinase) 1/2, MSK1 (mitogen- and stress-activated protein kinase 1), and histone H3, levels of which were all increased in the lesioned striatum of wild-type mice. Consistently, FosB/ΔFosB expression, which was induced by long-term L-DOPA treatment in the lesioned striatum, was also decreased in AC5-KO mice. Moreover, suppression of AC5 in the dorsal striatum with lentivirus-shRNA-AC5 was sufficient to attenuate LID, suggesting that the AC5-regulated signaling cascade in the striatum mediates LID. These results identify the AC5/cAMP system in the dorsal striatum as a therapeutic target for the treatment of LID in patients with Parkinson's disease.

Ultra-specific zeptomole microRNA detection by plasmonic nanowire interstice sensor with Bi-temperature hybridization.

MicroRNAs (miRNAs) are emerging new biomarkers for many human diseases. To fully employ miRNAs as biomarkers for clinical diagnosis, it is most desirable to accurately determine the expression patterns of miRNAs. The optimum miRNA profiling method would feature 1) highest sensitivity with a wide dynamic range for accurate expression patterns, 2) supreme specificity to discriminate single nucleotide polymorphisms (SNPs), and 3) simple sensing processes to minimize measurement variation. Here, an ultra-specific detection method of miRNAs with zeptomole sensitivity is reported by applying bi-temperature hybridizations on single-crystalline plasmonic nanowire interstice (PNI) sensors. This method shows near-perfect accuracy of SNPs and a very low detection limit of 100 am (50 zeptomole) without any amplification or labeling steps. Furthermore, multiplex sensing capability and wide dynamic ranges (100 am-100 pm) of this method allows reliable observation of the expression patterns of miRNAs extracted from human tissues. The PNI sensor offers combination of ultra-specificity and zeptomole sensitivity while requiring two steps of hybridization between short oligonucleotides, which could present the best set of features for optimum miRNA sensing method.

Photophysical properties and singlet oxygen generation efficiencies of water-soluble fullerene nanoparticles.

As various fullerene derivatives have been developed, it is necessary to explore their photophysical properties for potential use in photoelectronics and medicine. Here, we address the photophysical properties of newly synthesized water-soluble fullerene-based nanoparticles and polyhydroxylated fullerene as a representative water-soluble fullerene derivative. They show broad emission band arising from a wide-range of excitation energies. It is attributed to the optical transitions from disorder-induced states, which decay in the nanosecond time range. We determine the kinetic properties of the singlet oxygen ((1)O2) luminescence generated by the fullerene nanoparticles and polyhydroxylated fullerene to consider the potential as photodynamic agents. Triplet state decay of the nanoparticles was longer than (1)O2 lifetime in water. Singlet oxygen quantum yield of a series of the fullerene nanoparticles is comparably higher ranging from 0.15 to 0.2 than that of polyhydroxylated fullerene, which is about 0.06.

Selection of aptamers for mature white adipocytes by cell SELEX using flow cytometry.

BACKGROUND: Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. METHODS AND RESULTS: We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. CONCLUSIONS: These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.

A signal-on fluorescent assay for DNA methyltransferase activity using a methylation-resistant endonuclease.

A simple, rapid, and signal-on fluorescent assay was developed for activity analysis of DNA methyltransferase and for screening of its inhibitors based on a methylation-resistant endonuclease and SYBR Green I.

In vivo imaging of islet transplantation using PLGA nanoparticles containing iron oxide and indocyanine green.

PURPOSE: We determined whether poly(lactic-co-glycolic acid) nanoparticles would be a useful reagent for the successful monitoring of isolated islets by magnetic resonance imaging and optical imaging systems, without clinically relevant toxicity in vitro or in vivo. METHODS: We used iron oxide for MR imaging and a cyanide dye approved by the Food and Drug Administration (indocyanine green) for optical imaging and estimated the in vivo detection of transplanted pancreatic islets. RESULTS: The poly(lactic-co-glycolic acid) nanoparticles were associated with the islets in vitro and were successfully detected by 4.7 T (MR) and optical imaging, without other toxic effects. When labeled islets were transplanted under the mouse kidney capsule, in vivo T2/ T2*-weighted scans with 4.7 T MR detected as few as 300 labeled islets by 4 weeks. Optical in vivo imaging revealed indocyanine green fluorescence by 2 and 4 days after transplantation of islets containing 250 and 500 µg/mL poly(lactic-co-glycolic acid) nanoparticles, respectively. These results were further supported by the immunohistochemical results for insulin and iron in the recipient mouse kidney and pancreas. CONCLUSIONS: Taken together, these data indicate that poly(lactic-co-glycolic acid) nanoparticles may be used to label transplanted islets and may be imaged with in vivo MR and optical imaging systems.