Growth and Migration Blocking Effect of Nanaomycin K, a Compound Produced by Streptomyces sp., on Prostate Cancer Cell Lines In Vitro and In Vivo.

Since castration-resistant prostate cancer (CRPC) acquires resistance to molecularly targeted drugs, discovering a class of drugs with different mechanisms of action is needed for more efficient treatment. In this study, we investigated the anti-tumor effects of nanaomycin K, derived from "Streptomyces rosa subsp. notoensis" OS-3966. The cell lines used were LNCaP (non-CRPC), PC-3 (CRPC), and TRAMP-C2 (CRPC). Experiments included cell proliferation analysis, wound healing analysis, and Western blotting. In addition, nanaomycin K was administered intratumorally to TRAMP-C2 carcinoma-bearing mice to assess effects on tumor growth. Furthermore, immuno-histochemistry staining was performed on excised tissues. Nanaomycin K suppressed cell proliferation in all cell lines (p < 0.001) and suppressed wound healing in TRAMP-C2 (p = 0.008). Nanaomycin K suppressed or showed a tendency to suppress the expression of N-cadherin, Vimentin, Slug, and Ras in all cell lines, and suppressed the phosphorylation of p38, SAPK/JNK, and Erk1/2 in LNCaP and TRAMP-C2. In vivo, nanaomycin K safely inhibited tumor growth (p = 0.001). In addition, suppression of phospho-Erk1/2 and increased expression of E-cadherin and cleaved-Caspase3 were observed in excised tumors. Nanaomycin K inhibits tumor growth and suppresses migration by inhibiting epithelial-mesenchymal transition in prostate cancer. Its mechanism of action is related to the inhibition of phosphorylation of the MAPK signaling pathway.

Proapoptotic effect of nonthermal pulsed ultrasound on prostate cancer cells in a nude mouse model.

BACKGROUND: Ultrasound (US) can induce cell injury, and we have previously reported that adjusting the pulse repetition frequency (PRF) of ultrasound output can induce prostate cancer cell destruction without causing a rise in the temperature of the irradiated area. In this study, we examined the mechanism of nonthermal ultrasound cell destruction, which was not fully clarified in our previous reports. METHODS: In vitro, we evaluated postirradiation cells immediately after treatment and examined membrane disruption by proliferation assay, LDH assay, and apoptosis assay. In vivo, we injected mice with human LNCaP and PC-3 prostate cancer cells and evaluated the therapeutic effects of US irradiation by H-E staining and immunostaining. RESULTS: Proliferation assays showed inhibition at 3 h postirradiation independently of PRF and cell line (p < 0.05). Quantitative assessment of apoptosis/necrosis by flow cytometry showed widely varying results depending on cell type. LNCaP showed an increase in late apoptosis at 0 h independent of PRF (p < 0.05), while PC-3 showed no significant difference at 0 h. The LDH assay showed an increase in LDH independent of PRF in LNCaP (p < 0.05 respectively), but no significant difference in PC-3. In vivo, tumor volume was compared and a significant reduction was observed at 10 Hz for LNCaP (p < 0.05) and 100 Hz for PC-3 (p < 0.001) at 3 weeks after the start of irradiation. The excised tumors were evaluated with Ki-67, Caspase-3, and CD-31 and showed a significant treatment effect independent of cell type and PRF (p < 0.001 respectively). CONCLUSION: Examining the mechanism behind the therapeutic effect of US irradiation revealed that the main effect was achieved by apoptosis induction rather than necrosis.

Catechin-Albumin Conjugates: Enhanced Antioxidant Capacity and Anticancer Effects.

(+)-Catechin conjugated with human serum albumin (CT-HSA) was prepared and evaluated as a drug carrier bearing anticancer effects. It was found that 2.4 mol of CT was conjugate to 1 mol HSA. The CT-HSA has an antioxidant capacity of about 3.3 times the amount of CT in the conjugate. Intracellular incorporation of the CT-HSA was analyzed by fluorescence-activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM) measurements using fluorescein isothiocyanate (FITC)-labelled CT-HSA. The results indicated that the FITC-labelled CT-HSA was incorporated into HeLa cells in a concentration-dependent manner. The CT-HSA enhanced the binding of anticancer drugs (5-fluorouracil (5-Fu) and mitomycin C (MMC)) comparing with HSA, and the CT-HSA mixed with 5-Fu or MMC decreased significantly the HeLa cell viability as compared with the same concentration of each drug. In addition, intracellular reactive oxygen species (ROS) scavenging by the CT-HSA is likely to affect the anticancer effects. Thus, the CT-HSA enhanced anticancer drug efficacy in relation to controlling ROS-scavenging ability.

Hydrotropic Hydrogels Prepared from Polyglycerol Dendrimers: Enhanced Solubilization and Release of Paclitaxel.

Polyglycerol dendrimers (PGD) exhibit unique properties such as drug delivery, drug solubilization, bioimaging, and diagnostics. In this study, PGD hydrogels were prepared and evaluated as devices for controlled drug release with good solubilization properties. The PGD hydrogels were prepared by crosslinking using ethylene glycol diglycidylether (EGDGE). The concentrations of EGDGE and PGDs were varied. The hydrogels were swellable in ethanol for loading paclitaxel (PTX). The amount of PTX in the hydrogels increased with the swelling ratio, which is proportional to EGDGE/OH ratio, meaning that heterogeneous crosslinking of PGD made high dense region of PGD molecules in the matrix. The hydrogels remained transparent after loading PTX and standing in water for one day, indicating that PTX was dispersed in the hydrogels without any crystallization in water. The results of FTIR imaging of the PTX-loaded PGD hydrogels revealed good dispersion of PTX in the hydrogel matrix. Sixty percent of the loaded PTX was released in a sink condition within 90 min, suggesting that the solubilized PTX would be useful for controlled release without any precipitation. Polyglycerol dendrimer hydrogels are expected to be applicable for rapid release of poorly water-soluble drugs, e.g., for oral administration.

Inhibition of Melittin Activity Using a Small Molecule with an Indole Ring.

We investigated d-amino acids as potential inhibitors targeting l-peptide toxins. Among the l- and d-amino acids tested, we found that d-tryptophan (d-Trp) acted as an inhibitor of melittin-induced hemolysis. We then evaluated various Trp derivatives and found that 5-chlorotryptamine (5CT) had the largest inhibitory effect on melittin. The indole ring, amino group, and steric hindrance of an inhibitor played important roles in the inhibition of melittin activity. Despite the small size and simple molecular structure of 5CT, its IC50 was approximately 13 µg/mL. Fluorescence quenching, circular dichroism measurements, and size-exclusion chromatography revealed that 5CT interacted with Trp19 in melittin and affected the formation of the melittin tetramer involved in hemolysis. Molecular dynamics simulation of melittin also indicated that the interaction of 5CT with Trp19 in melittin affected the formation of the tetramer.

Cellular Therapy Using Epitope-Imprinted Composite Nanoparticles to Remove α-Synuclein from an In Vitro Model.

Several degenerative disorders of the central nervous system, including Parkinson's disease (PD), are related to the pathological aggregation of proteins. Antibodies against toxic disease proteins, such as α-synuclein (SNCA), are therefore being developed as possible therapeutics. In this work, one peptide (YVGSKTKEGVVHGVA) from SNCA was used as the epitope to construct magnetic molecularly imprinted composite nanoparticles (MMIPs). These composite nanoparticles were characterized by dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), isothermal titration calorimetry (ITC), Brunauer-Emmett-Teller (BET) analysis, and superconducting quantum interference device (SQUID) analysis. Finally, the viability of brain endothelial cells that were treated with MMIPs was measured, and the extraction of SNCA from CRISPR/dCas9a-activated HEK293T cells from the in vitro model system was demonstrated for the therapeutic application of MMIPs.

Synthesis and Hydrogelation of Star-Shaped Graft Copolypetides with Asymmetric Topology.

To study the self-assembly and hydrogel formation of the star-shaped graft copolypeptides with asymmetric topology, star-shaped poly(L-lysine) with various arm numbers were synthesized by using asymmetric polyglycerol dendrimers (PGDs) as the initiators and 1,1,3,3-tetramethylguanidine (TMG) as an activator for OH groups, followed by deprotection and grafting with indole or phenyl group on the side chain. The packing of the grafting moiety via non-covalent interactions not only facilitated the polypeptide segments to adopt more ordered conformations but also triggered the spontaneous hydrogelation. The hydrogelation ability was found to be correlated with polypeptide composition and topology. The star-shaped polypeptides with asymmetric topology exhibited poorer hydrogelation ability than those with symmetric topology due to the less efficient packing of the grafted moiety. The star-shaped polypeptides grafted with indole group on the side chain exhibited better hydrogelation ability than those grafted with phenyl group with the same arm number. This report demonstrated that the grafted moiety and polypeptide topology possessed the potential ability to modulate the polypeptide hydrogelation and hydrogel characteristics.

Combined Treatment with Ultrasound and Immune Checkpoint Inhibitors for Prostate Cancer.

Background: Ultrasound (US) is mostly used for diagnostic purpose but could be used for cancer treatments with a US intensity or frequency fitted to such a purpose. Prostate cancer (PC) has the highest prevalence in the urological field, but indications for immune checkpoint inhibitors (ICIs) for PC are limited to very few cases. In this study, we compared the antitumor effect of US irradiation alone with the combined use of US and ICIs in vitro and in vivo. Methods: PC cell line TRAMP-C2 cells were used in our experiments. TRAMP-C2 cells were irradiated with US with pulse repeated frequencies (PRF) of 1, 10, and 100 Hz. Cell proliferation was evaluated by MTS assay and apoptotic cells were analyzed using flow cytometry. To verify the antitumor effect of US irradiation on PC in vivo, we conducted animal experiments using mice. TRAMP-C2-bearing mice were irradiated with US with PRF of 10 and 100 Hz. Three weeks after the start of US irradiation, anti-PD-1 antibody was administered to the mice. Finally, mice were sacrificed and tumors were collected. Immunohistochemical (IHC) analyses were assessed for cleaved caspase-3 and CD3 in tumor cell extracts. Results: Cell proliferation assays showed that 1 and 10 Hz US significantly inhibited cell survival (p < 0.0001). In addition, US irradiation induced apoptosis at 1, 10, and 100 Hz (p = 0.0129, p = 0.0150, and p = 0.0017, respectively). In animal experiments, a significant tumor growth inhibitory effect was observed at 10 and 100 Hz, and 100 Hz + ICIs (p < 0.05, respectively). Hematoxylin−eosin (H−E) staining showed a significant increase in the necrotic area of the tumor at 100 Hz and 100 Hz + ICIs (p < 0.05, respectively). In addition, under IHC staining the expression level of cleaved caspase-3 and the number of CD3-positive cells increased at 100 Hz (p < 0.05, respectively). Conclusion: US irradiation induced apoptosis in cells and reduced cell viability. In vivo tumor growth was suppressed by combined treatment with US irradiation and ICIs. Further research on immune system activation will lead to less invasive and more efficient treatments for PC.

Size Dependency of Selective Cellular Uptake of Epigallocatechin Gallate-modified Gold Nanoparticles for Effective Radiosensitization.

The high incidence and mortality of cancer make it a global health issue. However, conventional cancer therapies have several disadvantages, especially serious side effects due to low selective toxicity to cancer cells. Gold nanoparticles (AuNPs) are an excellent drug carrier, enhance drug delivery efficiency, and hold promise for photothermal and radiation therapies. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic antioxidant constituent of green tea, has a potent antitumor effect, and binds specifically to the 67 kDa laminin receptor, which is overexpressed on the surface of several cancer cell lines such as HeLa and MDA-MB-231 cells. We synthesized EGCG-modified AuNPs (EGCG-AuNPs) using ratios (nEGCG/ngold) from 1:2 to 10:1 and evaluated their size, morphology, stability, antioxidant ability, cytotoxicity, cellular uptake, and uptake mechanisms in vitro in comparison with the conventional AuNPs prepared by using citrate as the reducing agent (citrate-AuNPs). In HeLa cells, EGCG-AuNPs (10:1) (135 nm diameter, sea-urchin-like shape) exhibited the highest cellular uptake. Conversely, EGCG-AuNPs (1:2) (39 nm diameter, spherical shape) were preferentially taken up by MDA-MB-231 cells. Cellular uptake of EGCG-AuNPs toward normal cells (NIH3T3 cells) was found to be in a nonspecific manner, and the amount of uptake was suppressed. X-ray irradiation after cellular uptake of EGCG-AuNPs (1:2) in MDA-MB-231 cells significantly enhanced irradiation-induced cell death. These findings suggest enhanced cellular uptake of EGCG-AuNPs with a 39 nm diameter and their potential use in combinatorial therapeutics of EGCG-AuNPs for breast cancer.

Effect of Branching Degree of Dendritic Polyglycerols on Plasma Protein Adsorption: Relationship between Hydration States and Surface Morphology.

This study focuses on dendritic glycerols and investigates the construction of biocompatible surfaces by understanding how differences in the branching of these molecules change the interactions with the biological components. The two molecules, polyglycerol dendrimer (PGD), which has a completely branched structure, and hyperbranched polyglycerol (HPG), which has an incompletely branched structure, are compared and the differences in branching are evaluated. It is shown that PGD has a little bit more intermediate water than HPG, which reflects the differences in the branching. The effect of surface state on the adsorption of the plasma proteins, human serum albumin (HSA), fibrinogen (Fib), and fibronectin (FN), is discussed by modifying a glass surface using these molecules with different hydration states. The adsorption of HSA decreases to several percent for HPG and 10% for PGD compared to unmodified substrate. Although the adsorption of Fib decreases to 5% for HPG, an increase to 150% is observed for PGD. Since this specific Fib adsorption observed only onto PGD is suppressed in the cases of a mixed solution of HSA and Fib or sequentially using HSA solution and then Fib solution, it is thought that the Vroman effect is suppressed on the PGD-modified surface. Furthermore, when AFM measurements are performed in PBS to understand the surface roughness, PGD is found to be more highly non-uniform. Because of this, the nanometer scale roughness that is significantly observed only on the PGD-modified surface is thought to have an effect on the characteristic adsorption properties of Fib. Thus, although both PGD and HPG with different branching have intermediate water, the proportion differs between PGD and HPG. Therefore, it is found that differences occur in the plasma protein adsorption mechanisms depending on the coordinates and density of hydroxyl groups within the molecules.

Development of endodontic sealers containing antimicrobial-loaded polymer particles with long-term antibacterial effects.

OBJECTIVE: The objective of this study is to prepare new dental resins with a long-lasting antimicrobial activity. Specifically, this study evaluates an approach for controlling infection in root canals using sealers containing polyhydroxyethyl methacrylate trimethylolpropane trimethacrylate (polyHEMA/TMPT) particles loaded with cetylpyridinium chloride (CPC). In addition, the physical properties of sealers containing CPC-loaded polyHEMA/TMPT particles (CLP) are determined. METHODS: PolyHEMA/TMPT particles with 10 (10%-CLP) and 25wt.% CPC (25%-CLP) with different particle sizes were fabricated and incorporated in HEMA-based sealers. CPC-release profiles were evaluated over 14 days of immersion in water, followed by 14 days of storage and 14 days of water immersion. The antibacterial activity of these sealers against Enterococcus faecalis in dentinal tubules was assessed using a root-canal-infection model. Their sealing abilities were evaluated by fluid filtration and physical properties were tested according to the ISO 6876 standard. The long-term antibacterial activity of the cured sealer containing 25%-CLP (∼21µm particle diameter) was re-assessed after 1 year of storage. RESULTS: After 28 days of immersion, 25%-CLP exhibited a higher and sustained CPC release unlike 10%-CLP. Residual bacteria in root dentinal tubules were eradicated by obturation with 25%-CLP-containing sealers. The incorporation of 25%-CLP (∼21µm) had no adverse effects on the sealing ability and physical properties of the sealer and resulted in long-term antibacterial activity. SIGNIFICANCE: The incorporation of CPC-loaded particles in HEMA resins yielded endodontic sealers with long-term bactericidal activity against E. faecalis in root canals. These sealers can potentially be used to prevent recurrent apical periodontitis.

Cell-Encapsulating Hydrogel Puzzle: Polyrotaxane-Based Self-Healing Hydrogels.

Slide-ring hydrogels using polyrotaxanes have been developed as highly tough soft materials. However, they have never been used as biomaterials because of the lack of biocompatibility. Meanwhile, self-healing hydrogels are expected to improve fatigue resistance and extend the period of use. However, owing to the lack of high mechanical strength, they are limited in their use as biomaterials. Here we first developed a biocompatible self-healing/slide-ring hydrogel using glycol chitosan and a water-soluble polyrotaxane. We obtained excellent mechanical toughness and biocompatibility to promote the proliferation of human umbilical vein endothelial cells (HUVECs) encapsulated in the hydrogel. Owing to the rapid self-healing property, the cell-encapsulating gels adjusted arbitrarily, maintaining good cell proliferation function. Therefore, slide-ring hydrogels enable the use of biomaterials for soft-tissue engineering.

Amphiphilic Block Copolymers Bearing Hydrophobic γ-Tocopherol Groups with Labile Acetal Bond.

High concentrations of γ-tocopherol (γTCP) tend to show antioxidant, anti-inflammatory, and anticancer effects. In this study, we prepared polymer micelles under acidic conditions with a controlled release of γTCP due to the decomposition of pendant acetal bonds. First, a precursor diblock copolymer composed of poly(ethylene glycol) (PEG) and acrylic acid (AA) was prepared. This was followed by the synthesis of an amphiphilic diblock copolymer (PEG54-P(AA/VE6/γTCP29)140), incorporated into hydrophobic γTCP pendant groups attached to the main chain through an acetal bond. The prepared PEG54-P(AA/VE6/γTCP29)140 was further dispersed in water to form polymer micelles composed of hydrophobic cores that were generated from a hydrophobic block containing γTCPs and hydrophilic shells on the surface. Under acidic conditions, γTCP was then released from the core of the polymer micelles due to the decomposition of the pendant acetal bonds. In addition, polymer micelles swelled under acidic conditions due to hydration of the core.

Tuned cell attachments by double-network hydrogels consisting of glycol chitosan, carboxylmethyl cellulose and agar bearing robust and self-healing properties.

Herein we present a tuned cell attachment on self-healable double network hydrogel bearing dynamic covalent bonds and hydrogen bonds. Agar formed first network, while glycol chitosan and oxidized carboxylmethyl cellulose formed second network in the resultant double network hydrogel. Because of the simple one-pot preparation, the hydrogel can be injected by using syringes. The moduli of the hydrogel were improved compared to that of the parent single-network. The hydrogel exhibited self-healing ability without need for heating or cut surface treatment. The incorporation of agar in the double network induced the enhanced protein adsorption, and the following cell attachments were governed by the adsorbed protein states. Therefore, the double network hydrogel holds great potential for applications in various biomedical applications.

Amphiphilic Copolymer of Polyhedral Oligomeric Silsesquioxane (POSS) Methacrylate for Solid Dispersion of Paclitaxel.

Suitable polymers for the homogeneous formulation of drug/polymer mixtures should be selected to correct the structural and physicochemical nature with a rapid dissolution rate. This study aimed to evaluate a copolymer prepared by the radical polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and a polyhedral oligomeric silsesquioxane (POSS) methacrylate bearing an ethyl (C2H5) group (MPC-ran-C2H5-POSS) as a carrier for the solid formulation of paclitaxel (PTX). A single-phase homogeneous formulation of PTX with the mixture of the MPC-ran-C2H5-POSS and polyvinylpyrrolidone (PVP) was prepared by a solvent method. The formulation of MPC-ran-C2H5-POSS/PVP/PTX enhanced the dissolution rate and the dissolved amount (approximately 90% within 40 min) without precipitation. The X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) analysis confirmed the presence of PTX as an amorphous state. The amphiphilic nature of the MPC-ran-C2H5-POSS contributed to enhancing the aqueous solubility of PTX. The new formulation is applicable for solid dispersion technique via the supersaturation of PTX in an aqueous media.

Hydrophobic Nature of Methacrylate-POSS in Combination with 2-(Methacryloyloxy)ethyl Phosphorylcholine for Enhanced Solubility and Controlled Release of Paclitaxel.

Amphiphilic copolymers consisting of 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and hydrophobic monomers are known as biomaterials for the administration of poorly water-soluble drugs such as paclitaxel (PTX). However, the hydrophobic monomers to be copolymerized with MPC have not been optimized for PTX solubilization and its dosage forms. Here, we show the enhanced PTX solubility by only an MPC-based amphiphilic copolymer using a polyhedral oligomeric silsesquioxane (POSS) methacrylate (MA) bearing an ethyl (C2H5) group as a vertex group. MPC was copolymerized with POSS methacrylates bearing different vertex groups of ethyl (C2H5), hexyl (C6H13), and octyl (C8H17) via radical polymerization. We found that the strong interaction between C2H5-POSS and PTX contributed to the slow release of PTX without any burst release. The C2H5-POSS-MA MPC copolymer was internalized into the cultured HeLa cells, which was confirmed by using a fluorescein-4-isothiocyanate (FITC)-labeled PTX, and the PTX-dissolved copolymer induced cell death. We anticipate that the C2H5-POSS-MA MPC copolymer is a good solubilizer bearing a controlled release function for PTX.

A Supramolecular Hydrogel Based on Polyglycerol Dendrimer-Specific Amino Group Recognition.

Dendrimer-based supramolecular hydrogels have gained attention in biomedical fields. While biocompatible dendrimers were used to prepare hydrogels via physical and/or chemical crosslinking, smart functions such as pH and molecular control remain undeveloped. Here, we present polyglycerol dendrimer-based supramolecular hydrogel formation induced by a specific interaction between the polyglycerol dendrimer and an amino group of glycol chitosan. Gelation was achieved by mixing the two aqueous solutions. Hydrogel formation was controlled by varying the polyglycerol dendrimer generation. The hydrogel showed pH-dependent swelling; strongly acidic conditions induced degradation via dissociation of the specific interaction. It also showed unique l-arginine-responsive degradation capability due to competitive exchange of the amino groups of glycol chitosan and l-arginine. These polyglycerol dendrimer-based supramolecular characteristics allow multimodal application in smart biomaterials.

Temperature-induced recovery of a bioactive enzyme using polyglycerol dendrimers: correlation between bound water and protein interaction.

Enzyme application has gained importance over the past decade in bioprocess, biomedical, and pharmaceutical fields. We found that polyglycerol dendrimers (PGDs), which are biocompatible molecules, can recover alcohol dehydrogenase (ADH) from aqueous solution under elevated temperature. A low concentration of PGD (5 wt.%) is sufficient for the recovery of high enzymatic activity, although a high concentration (25-75 wt.%) of glycerol is generally required to stabilize ADH. The enzymatic activity of ADH in suspension with PGDs is over 60% but it is only 10% in that with glycerol. The results of osmolarity and spin-lattice relaxation time (T1) of water measurements in the presence of PGDs suggest that increased amounts of bound water to PGD molecules trigger aggregation along with the direct interaction with ADH. PGDs therefore represent good potential additives for direct recovery of enzymes from aqueous solutions.

An injectable and self-healing hydrogel for spatiotemporal protein release via fragmentation after passing through needles.

A dynamic hydrogel formulated by mixing a glycol chitosan (GC) and an oxidized dextran (Odex) were studied for protein-controlled release in conjunction with the hydrogel fragmentation. A series of injectable dynamic hydrogels were derived from GC and Odex upon simple mixing without the addition of chemical crosslinking agents. The gelation readily took place at physiological pH and temperature. The influence of the concentration of GC and Odex on the gelation time, mechanical properties, water content, in vitro degradation were investigated. The Odex/GC hydrogels showed good self-healing ability under physiological conditions and kept the dynamic Schiff-base linkage at over 2 wt %. The release kinetics of a model protein (bovine serum albumin) was found to be controlled by changing the needle size upon injection, attributed to modulation of apparent size and shape of the fragmented hydrogels even in the self-healed state. Therefore, the GC-based injectable and dynamic hydrogels are expected to be a promising platform for protein delivery system and various biomedical applications.