Latent Toehold-Mediated DNA Circuits Based on a Bulge-Loop Structure for Leakage Reduction and Its Application to Signal-Amplifying DNA Logic Gates.

DNA circuits based on successive toehold-mediated DNA displacement reactions, particularly entropy-driven DNA circuit (EDC) systems, have attracted considerable attention as powerful enzyme-free tools for dynamic DNA nanotechnology. However, background leakage (noise signal) often occurs when the circuit is executed nonspecifically, even in the absence of the appropriate catalyst DNA (input). This study designed and developed a new latent toehold-mediated DNA circuit (LDC) system that relies on a bulge-loop structure as a latent toehold toward leakage reduction. Furthermore, the number (size) of nucleotides (nt) in the bulge-loop is found to play a significant role in the performance (i.e., leakage, signal, and kinetics) of LDC systems. In fact, the signal rate for the LDC systems increased as the number of nt in the bulge-loop increased from 4 to 8, whereas the leakage rate of the LDC systems with bulge-loops of 7 nt or less was low, but the leakage rate of the LDC system with a bulge-loop of 8 nt increased significantly. Note that the LDC system with the optimal bulge-loop (7 nt) was capable of not only reducing the leakage but also accelerating the circuit speed without any signal loss, unlike methods of reducing the leakage by reducing the signal reported previously for the conventional EDC systems. These facts indicate that the 7 nt bulge-loop acts as a "latent" toehold for the DNA circuit system. By using the amplification function of output signals with an accelerated circuit and reduced leakage, our LDC system with a 7 nt bulge-loop could be applied directly and successfully to signal-amplifying DNA logic gates such as OR and AND gates, and thus, sufficient output signals could be obtained even with a small amount of input. These findings reveal that our LDC systems with a bulge-loop structure can replace the conventional EDC system and have enormous potential in the field of DNA nanotechnology.

Acceleration of DNA Hybridization Chain Reactions on 3D Nanointerfaces of Magnetic Particles and Their Direct Application in the Enzyme-Free Amplified Detection of microRNA.

Accelerated DNA hybridization chain reactions (HCRs) using DNA origami as a scaffold have received considerable attention in dynamic DNA nanotechnology. However, tailor-made designs are essential for DNA origami scaffolds, hampering the practical application of accelerated HCRs. Here, we constructed the semilocalized HCR and localized HCR systems using magnetic beads (MBs) as a simple scaffold to explore them for the enzyme-free miR-21 detection. The semilocalized HCR system relied on free diffusing one hairpin DNA and MBs immobilized with another hairpin DNA, and the localized HCR system relied on MBs coimmobilized with two hairpin DNAs. We demonstrated that the DNA density on MBs plays a critical role in HCR kinetics and limit of detection (LOD). Among semilocalized HCR systems, MBs with a medium DNA density showed a faster HCR and lower LOD (10 pM) than the diffusive (conventional) HCR system (LOD: 86 pM). In contrast, the HCR further accelerated for the localized HCR systems as the DNA density increased. The localized HCR system with the highest DNA density showed the fastest HCR and the lowest LOD (533 fM). These findings are of great importance for the rational design of accelerated HCRs using simple scaffolds for practical applications.

Simple Single-Legged DNA Walkers at Diffusion-Limited Nanointerfaces of Gold Nanoparticles Driven by a DNA Circuit Mechanism.

We designed and prepared a single-legged DNA walker that relies on the creation of a simple diffusion-limited nanointerface on a gold nanoparticle (DNA/PEG(+)-GNP) track co-modified with fluorescence-labeled hairpin DNA and poly(ethylene glycol) (PEG) containing a positively charged amino group at one end. The movement of our single-legged DNA walker is driven by an enzyme-free DNA circuit mechanism through cascading toehold mediated DNA displacement reactions (TMDRs) using fuel hairpin DNAs. The acceleration of TMDRs was observed for the DNA/PEG(+)-GNP track through electrostatic interaction between the positively charged track and negatively charged DNAs, resulting in the acceleration of the DNA circuit and amplification of the fluorescence signal. Furthermore, the DNA/PEG(+)-GNP track allowed autonomous and persistent movement of a walker DNA strand on the same GNP track, because the intraparticle DNA circuit occurred preferentially by preventing diffusion of the negatively charged free walker DNA strand from near the positively charged tracks into solution through electrostatic interaction. Based on comparative study of kinetics of TMDRs and DNA walking behaviors, it is to be noted that the DNA/PEG(+)-GNP track showed the fastest DNA circuit reaction (walking rate) and the largest number of steps taken by the walker DNA strand compared to other GNP tracks with varying nanointerfaces that differ in terms of their type of charges (no and negative charges), density of positive charges, and number of hairpin DNAs per GNP track. These facts reveal that the positive charges on the GNP track play an important role in the acceleration of the DNA circuit, as well as the successful walking motion of the single-legged DNA strand. By using the fluorescence signal amplification functions, our single-legged DNA walker could be applied directly and successfully to enzyme-free miRNA-detection systems. The miRNA-detection system provided higher discrimination of other mismatched miRNAs and higher sensitivity (the lowest LOD: 4.0 pM) when compared to other miRNA-detection systems based on other GNP tracks without positive charges. Unlike existing single-legged DNA walkers, our single-legged DNA walkers do not require complex processes, such as immobilization of the walker DNA strand on the tracks and precise adjustment of the sequence of walker DNA. Therefore, our strategy, based on the creation of diffusion-limited nanointerfaces, has enormous potential for the applications of single-legged DNA walkers to biosensors, bioimaging, and computing.

Dynamically Programmed Switchable DNA Hydrogels Based on a DNA Circuit Mechanism.

Biological stimuli-responsive DNA hydrogels have attracted much attention in the field of medical engineering owing to their unique phase transitions from gel to sol through cleavage of DNA cross-linking points in response to specific biomolecular inputs. In this paper, a new class of biological stimuli-responsive DNA hydrogels with a dynamically programmed DNA system that relies on a DNA circuit system through cascading toehold-mediated DNA displacement reactions is constructed, allowing the catalytic cleavage of cross-linking points and main chains in response to an appropriate DNA input. The dynamically programmed DNA hydrogels exhibit a significant sharp phase transition from gel to sol in comparison to another DNA hydrogel showing noncatalytic cleavage of cross-linking points due to synchronization of the catalytic cleavage of cross-linking points and the main chains. Further, the sol-gel phase transitions of the DNA hydrogels in response to the DNA input are easily tunable by changing the cross-linking density. Additionally, with a structure-switching aptamer, DNA hydrogels encapsulating PEGylated gold nanoparticles can be used as enzyme-free signal amplifiers for the colorimetric detection of adenosine 5'-triphosphate (ATP); this detection system provides simplicity and higher sensitivity (limit of detection: 5.6 × 10-6 m at 30 min) compared to other DNA hydrogel-based ATP detection systems.

Comparative Study of DNA Circuit System-Based Proportional and Exponential Amplification Strategies for Enzyme-Free and Rapid Detection of miRNA at Room Temperature.

Because circulating microRNAs (miRNAs) have been recognized as a new class of blood-based biomarkers for various diseases, a significant challenge has been the development of point-of-care testing (POCT) systems based on detection of circulating miRNAs directly from serum. A promising approach to POCT systems is considered to be the development of enzyme-free and isothermal detection systems. Here, two types of DNA circuit system based on proportional and exponential amplification strategies were constructed using double-stranded DNA-modified magnetic beads (dsDNA-MBs) and their performances for detection of miRNA were studied comparatively. Both proportional and exponential amplification DNA circuit systems enabled the detection of target miRNA (miR-141) at room temperature without the need for additional enzymes because miR-141 acted as a catalyst for successive toehold-mediated DNA displacement reactions. A significant increase in the noise fluorescence signal was observed for the exponential amplification DNA circuit system because of the leakage (undesired DNA displacement reaction) revealed by the kinetic study on each DNA displacement reaction. Nevertheless, the exponential amplification DNA circuit system showed a lower limit of detection (LOD: 46 pM) and shorter assay time (15 min) compared to those of the proportional amplification DNA circuit system (LOD: 103 pM at 180 min). It is most likely that the exponential amplification DNA circuit system enabled amplification of both the signals and target miR-141, whereas the proportional amplification DNA circuit system enabled amplification of the signals alone. In addition, the exponential amplification DNA circuit system was able to discriminate between mismatched base sequences in miR-200 family members and specifically detect miR-141 even in the presence of serum. These findings are important for the rational design for POCT systems.

An Efficient Particle-Based DNA Circuit System: Catalytic Disassembly of DNA/PEG-Modified Gold Nanoparticle-Magnetic Bead Composites for Colorimetric Detection of miRNA.

An efficient particle-based DNA circuit system for a new colorimetric miRNA assay is designed and devised based on a catalytic disassembly strategy through a target miRNA-triggered DNA circuit mechanism. The new particle-based DNA circuit system shows a rapid color change as well as significant improvement of sensitivity without need for other enzymes or instruments.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

Ultrasensitive detection of DNA and RNA based on enzyme-free click chemical ligation chain reaction on dispersed gold nanoparticles.

An ultrasensitive colorimetric DNA and RNA assay using a combination of enzyme-free click chemical ligation chain reaction (CCLCR) on dispersed gold nanoparticles (GNPs) and a magnetic separation process has been developed. The click chemical ligation between an azide-containing probe DNA-modified GNP and a dibenzocyclooctyne-containing probe biotinyl DNA occurred through hybridization with target DNA (RNA) to form the biotinyl-ligated GNPs (ligated products). Eventually, both the biotinyl-ligated GNPs and target DNA (RNA) were amplified exponentially using thermal cycling. After separation of the biotinyl-ligated GNPs using streptavidin-modified magnetic beads, the change in intensity of the surface plasmon band at 525 nm in the supernatants was observed by UV/vis measurement and was also evident visually. The CCLCR assay provides ultrasensitive detection (50 zM: several copies) of target DNA that is comparable to PCR-based approaches. Note that target RNA could also be detected with similar sensitivity without the need for reverse transcription to the corresponding cDNA. The amplification efficiency of the CCLCR assay was as high as 82% due to the pseudohomogeneous reaction behavior of CCLCR on dispersed GNPs. In addition, the CCLCR assay was able to discriminate differences in single-base mismatches and to specifically detect target DNA and target RNA from the cell lysate.

Radiosensitization of tumor cells through endoplasmic reticulum stress induced by PEGylated nanogel containing gold nanoparticles.

High atomic number molecules, such as gold and platinum, are known to enhance the biological effect of X-irradiation. This study was aimed to determine the radiosensitizing potential of PEGylated nanogel containing gold nanoparticles (GNG) and the cellular mechanism involved. GNG pretreatment increased the levels of reproductive cell death and apoptosis induced by X-irradiation. GNG accumulated in cytoplasm and increased the expression of endoplasmic reticulum (ER) stress-related protein. GNG suppressed the repair capacity of DNA after X-irradiation by down-regulating DNA repair-related proteins. Our results suggest that GNG radiosensitized cells by enhancing apoptosis and impairing DNA repair capacity via ER stress induction.

Enzyme-free fluorescent-amplified aptasensors based on target-responsive DNA strand displacement via toehold-mediated click chemical ligation.

A new target-responsive DNA strand displacement system via toehold-mediated click chemical ligation was designed and prepared for enzyme-free fluorescent-amplified aptasensors. The aptasensors significantly amplified fluorescent signals in response to targets based on target recycling processes.

Pharmacokinetics of core-polymerized, boron-conjugated micelles designed for boron neutron capture therapy for cancer.

Core-polymerized and boron-conjugated micelles (PM micelles) were prepared by free radical copolymerization of a PEG-b-PLA block copolymer bearing an acetal group and a methacryloyl group (acetal-PEG-b-PLA-MA), with 1-(4-vinylbenzyl)-closo-carborane (VB-carborane), and the utility of these micelles as a tumor-targeted boron delivery system was investigated for boron neutron capture therapy (BNCT). Non-polymerized micelles (NPM micelles) that incorporated VB-carborane physically showed significant leakage of VB-carborane (ca. 50%) after 12 h incubation with 10% fetal bovine serum (FBS) at 37 °C. On the other hand, no leakage from the PM micelles was observed even after 48 h of incubation. To clarify the pharmacokinetics of the micelles, (125)I (radioisotope)-labeled PM and NPM micelles were administered to colon-26 tumor-bearing BALB/c mice. The (125)I-labeled PM micelles showed prolonged blood circulation (area under the concentration curve (AUC): 943.4) than the (125)I-labeled NPM micelles (AUC: 495.1), whereas tumor accumulation was similar for both types of micelles (AUC(PM micelle): 249.6, AUC(NPM micelle): 201.1). In contrast, the tumor accumulation of boron species in the PM micelles (AUC: 268.6) was 7-fold higher than the NPM micelles (AUC: 37.1), determined by ICP-AES. Thermal neutron irradiation yielded tumor growth suppression in the tumor-bearing mice treated with the PM micelles without reduction in body weight. On the basis of these data, the PM micelles represent a promising approach to the creation of boron carrier for BNCT.

Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid.

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.

Efficient siRNA delivery based on PEGylated and partially quaternized polyamine nanogels: enhanced gene silencing activity by the cooperative effect of tertiary and quaternary amino groups in the core.

For the development of an siRNA delivery system using polyion complexes (PICs) based on PEGylated nanogel consisting of a cross-linked poly[2-(N,N-diethylaminoethyl) methacrylate] (PEAMA) gel core and tethered poly(ethylene glycol) (PEG) chains, quaternary ammonium groups were introduced in the polyamine gel core to enhance the binding ability with siRNA and the stability of the PICs. Consequently, the quaternization of the polyamine core of the nanogel facilitated the binding ability with siRNA at a low N/P ratio, and the stability against polyanion displacement was enhanced as the degree of quaternization (DQ) of the nanogel increased. Although the installation of the positively charged quaternary ammonium moieties in the core of the nanogel resulted in the increment of the xi-potential of the PICs (e.g. + 23 mV for DQ=100%), the cytotoxicity was reduced with the increase of DQ presumably due to the hydrophilic character of the quaternary ammonium groups. The installation of quaternary ammonium groups in the core of the nanogel enhanced the endogenous gene silencing activity against the survivin gene in human hepatocarcinoma (HuH-7 cells), especially, the partly quaternized polyamine nanogel (DQ=10%) showed the highest gene silencing ability among the quaternized polyamine nanogels, including the tertiary amine nanogel. The cellular uptake analysis of the Rhodamine B-labeled Q-nanogel/fluorescein-labeled siRNA complex revealed that the quaternization of PEAMA moieties enhanced the cellular uptake level of fluorescein-labeled siRNA with the increase in DQ, whereas the cellular uptake of the Rhodamine B-labeled Q-nanogels was almost of the same level regardless of the DQ value, indicating that significant cellular uptake of the fluorescein-labeled siRNA is most likely due to the enhancement of the binding ability with siRNA in the serum-containing medium. Note that the endosomal escape efficiency was reduced with increase in the DQ value due to the decrease in the buffering capacity (tertiary amino groups) of the PEAMA core. On the basis of these results, the ratio of quaternary ammonium groups to tertiary amino groups in the core of the nanogel plays a pivotal role in the achievement of significant gene silencing through enhanced cellular uptake (quaternary ammonium groups) and subsequent endosomal escape (tertiary amino groups).

Large payloads of gold nanoparticles into the polyamine network core of stimuli-responsive PEGylated nanogels for selective and noninvasive cancer photothermal therapy.

A biocompatible photothermal nanomedicine based on a PEGylated nanogel containing gold nanoparticles (GNPs) in a cross-linked network core of stimuli-responsive poly[2-(N,N-diethylamino)ethyl methacrylate] (PEAMA) gel for cancer photothermal therapy (PTT) was prepared through the reduction of Au(iii) ions without any reducing agents. The influence of the reduction conditions, such as pH, temperature, and N/Au ratio (molar ratio of the amino groups in the PEGylated nanogel to the Au(iii) ions), on the formation of the GNPs in the stimuli-responsive PEAMA gel core (reducing environment) was also studied. Note that the PEGylated nanogel containing GNPs prepared at pH 6, 60 degrees C and N/Au = 1 (PEGylated GNG (1)) was found to have the highest GNP-loading capacity with a diameter of about 8 nm, as observed by TEM; viz., about 27 GNPs formed in a single PEAMA gel core. PEGylated GNG (1) showed a remarkable photothermal efficacy (DeltaT = 7.7 degrees C) under irradiation with Ar ion (Ar(+)) laser (514.5 nm) at a fluence of 39 W cm(-2) for 6 min (14 kJ cm(-2)). Note that PEGylated GNG (1) showed non-cytotoxicity in the absence of irradiation with Ar(+) laser (480 microg mL(-1): > 90% cell viability), whereas pronounced cytotoxicity (IC(50) = 110 microg mL(-1)) was observed for PEGylated GNG (1) under irradiation with Ar(+) laser at a fluence of 26 W cm(-2) for 5 min (7.8 kJ cm(-2)), because of the heat-generation from the GNPs in the cells, which resulted in selective and noninvasive cancer PTT. Thus, PEGylated GNG (1), which has a high GNP-loading capacity, would be a promising nanomedicine for cancer PTT.

Stimuli-responsive smart nanogels for cancer diagnostics and therapy.

This article discusses stimuli-responsive poly(ethylene glycol) (PEG)-coated (PEGylated) nanogels and their biomedical applications. Preparation and characterization of stimuli-responsive PEGylated nanogels composed of a crosslinked poly(2-[N,N-diethylamino]ethyl methacrylate) (PEAMA) core and PEG tethered chains are initially described. Stimuli-responsive PEGylated nanogels show unique properties and functions in synchronizing with the reversible volume phase transition of the PEAMA core in response to the extracellular pH (7-6.5) of a tumor environment as well as endosomal/lysosomal pH (6.5-5.0) and temperature. We list several biomedical applications of stimuli-responsive PEGylated nanogels, including (19)F magnetic resonance spectroscopic imaging (MRS/I) probe to visualize acidosis (tumor tissue), intracellular drug and siRNA delivery, antennas for cancer photothermal therapy and apoptosis probe for monitoring response to cancer therapy. Thus, stimuli-responsive PEGylated nanogels can be utilized as smart nanomedicines for cancer diagnostics and therapy.

A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo.

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of both pDNA and siRNA. Modification of a MEND with polyuethylene glycol, i.e., PEGylation, is a potential strategy for in vivo delivery of MENDs to tumor tissue. However, PEGylation also inhibits both uptake and endosomal escape of MENDs. To overcome these limitations, we developed a PEG-peptide-DOPE (PPD) that can be cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, to further improve the silencing activity of encapsulated siRNA, we modified the PPD-MEND with a pH-sensitive fusogenic GALA peptide (GALA/PPD-MEND). First, we determined the GALA and PPD content that would optimize the synergistic functions of GALA and PPD. The most efficient gene silencing activity was achieved when GALA and either conventional PEG-lipid or PPD were used to modify the MEND at a molar ratio of 1:1. In this case, the silencing activity was comparable to that achieved when using a MEND that had not been modified with PEG (unmodified MEND). Furthermore, in vivo topical administration revealed that optimized PPD/GALA-MENDa resulted in more efficient gene silencing compared with unmodified MENDs. Collectively, data demonstrate that introduction of both of a pH-sensitive fusogenic GALA peptide and PPD into the MEND facilitates nanoparticle endosomal escape, thereby enhancing the efficiency of siRNA delivery and gene silencing.

Enhanced cytoplasmic delivery of siRNA using a stabilized polyion complex based on PEGylated nanogels with a cross-linked polyamine structure.

A novel siRNA delivery system using a polyion complex (PIC) based on PEGylated polyamine nanogels composed of a chemically cross-linked poly[2-(N,N-diethylaminoethyl)methacrylate] (PDEAMA) core and surrounded by PEG tethered chains is described. The nanogel formed PIC spontaneously through electrostatic interaction upon mixing with siRNA. The nanogel/siRNA complex was characterized by a gel retardation assay, size and ζ-potential measurements, and gene silencing activity using a cultured cell line. The nanogel/siRNA complexes showed higher polyanion exchange tolerability compared with the noncross-linked PEG-b-PDEAMA/siRNA complexes, indicating that the three-dimensionally cross-linked structure of the nanogel enhanced the stability of the PIC. Furthermore, the nanogel/siRNA complex was observed to undergo a remarkable enhancement of the gene silencing activity against the firefly luciferase gene expressed in HuH-7 cells at low N/P ratios (N/P = 2), whereas the noncross-linked PEG-b-PDEAMA/siRNA complexes showed negligible gene silencing activity. Moreover, confocal fluorescence microscopy revealed an efficient endosomal escape capability for the transportation of siRNAs into the cytoplasm, presumably due to the buffering effect of the PDEAMA core. Therefore, the PIC of siRNA with cross-linked polyamine nanogel is a potentially effective siRNA carrier for the development of in vivo therapeutic applications of siRNA.

Efficient short interference RNA delivery to tumor cells using a combination of octaarginine, GALA and tumor-specific, cleavable polyethylene glycol system.

We recently developed a multifunctional envelope-type nano device (MEND) for efficient nucleic acid delivery. Here, we report on the development of an octaarigine (R8)-modified MEND encapsulating small interfering RNA (siRNA) with a tumor-specific, cleavable, polyethylene glycol (PEG)-lipid (PPD). We first determined the optimal concentration of R8 and pH-sensitive fusogenic peptide (GALA) on the lipid envelope of MEND (R8/GALA-MEND). Then, we examined the combination of optimized R8/GALA-MEND with a PEG-lipid. When a conventional PEG-lipid was used, the R8/GALA-MEND failed to knockdown expression of the target gene. On the other hand, PPD-modified R8/GALA-MEND exhibited efficient silencing activity to the level of the PEG-unmodified R8/GALA-MEND. In addition, we compared a R8/GALA-MEND with a MEND composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) that is a conventional cationic lipid used as a lipoplex component. The knockdown ability of the R8/GALA-MEND was much higher than that of the DOTAP-based MEND at the dose that is commonly employed in in vitro siRNA transfection. These results demonstrate that the R8/GALA-MEND is a promising delivery system for the transfer of siRNA to tumor cells.

Structure and activity assay of nanozymes prepared by the coimmobilization of practically useful enzymes and hydrophilic block copolymers on gold nanoparticles.

Enzyme/polymer/gold nanoparticle hybrids, called "nanozymes", were prepared and structurally analyzed by dynamic light scattering (DLS), ultraviolet-visible spectroscopy, and zeta-potential and transmission electron microscopy (TEM) measurements, which showed that the nanozyme particles were mainly composed of a single gold nanoparticle, on whose surface the enzyme and polymer were coimmobilized. This kind of structure resulted in the high dispersion stability of the nanozyme under various conditions, accompanied by improved thermal stability of the enzyme.

Completely dispersible PEGylated gold nanoparticles under physiological conditions: modification of gold nanoparticles with precisely controlled PEG-b-polyamine.

A novel water-soluble, biocompatible polymer, poly(ethylene glycol)-block-poly((2-N,N-dimethylamino)ethyl methacrylate) (PEG-b-PAMA), possessing controlled molecular weight with a narrow molecular weight distribution, was synthesized by the atom-transfer radical polymerization (ATRP) method. PEG-b-PAMA having a short PAMA chain length was successfully synthesized under suitable polymerization conditions. Gold nanoparticles (GNPs) were modified using PEG-b-PAMA prepared under a variety of PEGylation conditions. Under alkaline conditions (pH >10) and an [N]/[GNP] ratio of more than 3300, the PEGylated GNPs (PEG-GNPs) showed complete dispersion stability, avoiding coagulation. The amino groups of the PAMA segment of the block copolymers were completely deprotonated above pH 10. This means that PEG-b-PAMA interacted with the GNP surface via multipoint coordination of the tertiary amino groups of PAMA, not electrostatically. The effect of the number of amino groups in the PAMA segment on GNP surface modifications was investigated by zeta potential and dynamic light scattering (DLS) measurements. When the PEG-GNPs were prepared in excess polymer solution, almost the same diameter was observed regardless of the PAMA chain length. After the PEG-GNPs were purified by centrifugation, the zeta potentials of all PEG-GNPs were shielded to almost 0 mV, indicating the effective modifications of the GNP surface by PEG-b-PAMA regardless of the chain length. However, the particle size and particle size distribution of the purified PEG-GNPs were strongly affected by the PAMA chain length. PEG-GNPs with longer PAMA segments underwent coagulation after purification, whereas PEG-GNPs with shorter PAMA segments increased their dispersion stability. The experimental results of the thermal gravimetric analysis confirmed that the PEG density on the GNP surface increased as the AMA units decreased to 3. Thus, the dispersion stability depended significantly on the PEG density on the GNP surface. GNPs modified with PEG-b-PAMA having short AMA units showed excellent dispersion stability under a variety of pH conditions. The excellent dispersion stability of the obtained PEG-GNP was also confirmed both in bovine serum albumin (BSA) solution and 95% human serum.