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Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Improving the transfection efficiency of non-viral carriers by using cationic polymers is a useful approach to addressing several challenges in gene therapy, such as cellular uptake, endosomal escape, and toxicity. Among the various cationic polymers, polyamidoamine (PAMAM) dendrimers have been widely utilized because of the abundance of terminal functional groups, thereby enabling further functionalization and enhancing DNA condensation and internalization into cells. The combination of various functional groups is required for these PAMAM dendrimer derivatives to function appropriately for gene delivery. Herein, we synthesized PAMAM G2-HRChol by conjugating dipeptide (histidine-arginine) and cholesterol at different ratios (6% or 23%) on the surface of PAMAM dendrimer generation 2 (PAMAM G2). Both PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% have buffering capacity, leading to improved endosomal escape after entering the cells. PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers were condensed with pDNA to form nano-polyplexes at a weight ratio of 4 (polymer/pDNA). Polyplexes are positively charged, which facilitates cellular uptake. The transfection efficiency of PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23% dendrimers was similar to that of PEI 25 kDa under optimum conditions, and the cytotoxicity was much lower than that of PEI 25 kDa in HeLa cells. In addition, after apoptin gene transfection was performed, cell death ratios of 34.47% and 22.47% were observed for PAMAM G2-HRChol 6% and PAMAM G2-HRChol 23%, respectively. The results show that a suitable amount of cholesterol can improve gene transfection efficiency, and the PAMAM G2-HRChol 6% dendrimer could be a potential gene carrier in HeLa cells.

In this study, we aimed to synthesize PAMAMG3 derivatives (PAMAMG3-KRRR and PAMAMG3-HKRRR), using KRRR peptides as a nuclear localization signal and introduced histidine residues into the KRRR-grafted PAMAMG3 for delivering a therapeutic, carcinoma cell-selective apoptosis gene, apoptin into human primary glioma (GBL-14) cells and human dermal fibroblasts. We examined their cytotoxicity and gene expression using luciferase activity and enhanced green fluorescent protein PAMAMG3 derivatives in both cell lines. We treated cells with PAMAMG3 derivative/apoptin complexes and investigated their intracellular distribution using confocal microscopy. The PAMAMG3-KRRR and PAMAMG3-HKRRR dendrimers were found to escape from endolysosomes into the cytosol. The JC-1 assay, glutathione levels, and Annexin V staining results showed that apoptin triggered cell death in GBL-14 cells. Overall, these findings indicated that the PAMAMG3-HKRRR/apoptin complex is a potential candidate for an effective nonviral gene delivery system for brain tumor therapy in vitro.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease caused by motor neuron damage in the central nervous system, and it is difficult to diagnose early. Drosophila melanogaster is widely used to investigate disease mechanisms and discover biomarkers because it is easy to induce disease in Drosophila through genetic engineering. We performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate changes in phospholipid distribution in the brain tissue of an ALS-induced Drosophila model. Fly brain tissues of several hundred micrometers or less were sampled using a fly collar to obtain reproducible tissue sections of similar sizes. MSI of brain tissues of Drosophila cultured for 1 or 10 days showed that the distribution of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), was significantly different between the control group and the ALS group. In addition, the lipid profile according to phospholipids differed as the culture time increased from 1 to 10 days. These results suggest that disease indicators based on lipid metabolites can be discovered by performing MALDI-MSI on very small brain tissue samples from the Drosophila disease model to ultimately assess the phospholipid changes that occur in early-stage ALS.

Zaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central β-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.

To develop an efficient vector for mitochondria-targeted drug delivery, we synthesized triphenylphosphonium (TPP)-modified glycol chitosan polymeric microspheres that had a unique chemical structure with both lipophilic phenyl groups and cationic phosphonium. Notably, TPP can easily pass through the phospholipid bilayer of mitochondria, thereby resulting in specific accumulation of a combined drug molecule in the mitochondria due to the membrane potential between TPP and its membrane. Therefore, TPP has been widely used as a mitochondria-targeting moiety. Triphenylphosphonium-glycol chitosan derivatives (GC-TPP and GME-TPP) with two different degrees of substitution (11% and 36%) were prepared by amidation and Michael addition. The chemical structures of GC-TPP and GME-TPP were characterized by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy, and their sizes were measured via field emission scanning electron microscopy and dynamic light scattering. Cellular uptake through flow cytometric analysis and confocal microscopy confirmed that both GC-TPP and GME-TPP were well introduced into cells, targeting the mitochondria. In addition, cytotoxicity testing of the most common cell lines, such as HEK293, HeLa, NIH3T3, and HepG2, indicated the absence of polymer toxicity. To evaluate the carrier effectiveness of TPP for drug delivery, doxorubicin (Dox) was used as an anticancer drug. Confocal microscopy images showed that Dox-loaded GME-TPP accumulated inside cells more than Dox-loaded GC-TPP. The anticancer effects of Dox were also determined by MTT assay, apoptosis/necrosis assay, and three-dimensional spheroids. In summary, the results indicate that GC-TPP and GME-TPP microspheres possess great potential as effective drug delivery carriers.

The enhancement of surface wettability by hydrophilic polymer coatings has been of great interest because it has been used to address several technical challenges such as biofouling and surface fogging. Among the hydrophilic polymers, zwitterionic polymers have been extensively utilized to coat solid surfaces due to their excellent capability to bind water molecules, thereby forming dense hydration layers on the solid surfaces. For these zwitterionic polymers to function appropriately on the solid surfaces, techniques for fixing polymers onto the solid surface with high efficiency are required. Herein, we report a new approach to graft zwitterionic polymers onto solid substrates. The approach is based on the mussel-inspired surface chemistry and metal coordination. It consists of polydopamine coating and the coordination-driven grafting of the zwitterionic polymers. Polydopamine coating enables the versatile surface immobilization of catechols. Zwitterionic polymers are then easily fixed onto the catechol-immobilized surface by metal-mediated crosslinking reactions. Using this approach, nanometer-thick zwitterionic polymer layers that are highly resistant to bacterial adhesion and fog generation could be successfully fabricated on solid substrates in a substrate-independent manner.

Dendrimer micelles with glycyrrhizic acid (GA) were developed for anti-inflammatory therapy of acute lung injury (ALI). Cholesterol was conjugated to histidine- and arginine-grafted polyamidoamine (PamHR) for micelle formation. The cholesterol-conjugated PamHR (PamHRchol) was mixed with amphiphilic GA to produce PamHRchol/GA mixed micelles. The GA integrated into the micelles had two functions: it acted as an anti-inflammatory drug and facilitated intracellular gene delivery. The PamHRchol/GA micelles formed stable complexes with plasmid DNA. Integrating GA into the micelles increased their transfection efficiency. Confocal microscopy and flow-cytometry studies confirmed that the PamHRchol/GA micelles improved cellular uptake compared with PamHRchol. A competition assay with free GA suggested that the enhanced transfection efficiency of the micelles might be due to the interaction between GA and its receptor. In addition, GA has a membrane-destabilizing effect, and a chloroquine pretreatment assay confirmed that GA increased endosomal escape. Furthermore, the PamHRchol/GA micelles reduced tumor necrosis factor-α in lipopolysaccharide-activated Raw264.7 cells, suggesting a mechanism for its anti-inflammatory effects. To evaluate the therapeutic potential of the PamHRchol/GA micelles, the heme oxygenase-1 (HO-1) gene was delivered into the lungs of mice with ALI. The PamHRchol/GA micelles had higher gene delivery efficiency into the lungs than polyethylenimine (25 kDa, PEI25k) and the PamHRchol micelles. The combined effects of the HO-1 gene and GA produced effective anti-inflammation response in the lungs of the ALI animals. Therefore, the dual-function PamHRchol/GA micelles, which acted as an anti-inflammatory drug and a gene carrier, could be a useful therapy for inflammatory lung diseases.

Catechol and/or pyrogallol groups are recognized as crucial for the formation of polyphenol coatings on various substrates. Meanwhile, studies on polyphenolic molecules that do not contain such groups are relatively rare. The key molecule in turmeric-based universal (i.e., substrate-independent) coatings is curcumin, which contains no catechol or pyrogallol groups. As chemically reactive hydroxyl groups would remain after curcumin coating, it is hypothesized that curcumin coating can serve as a reactive layer for controlling interfacial properties. In this study, a curcumin-based surface modification method is developed to graft polymer brushes from various substrates, including titanium dioxide, gold, glass, stainless steel, and nylon. α-Bromoisobutyryl bromide, a polymerization initiator, is introduced to the curcumin-coated substrates via esterification; subsequently, poly(oligo(ethylene glycol) methacrylate) (poly(OEGMA)) is grafted from the surfaces. Compared to the control surfaces, poly(OEGMA)-grafted surfaces significantly suppress bacterial adhesion by up to 99.4%, demonstrating their antibacterial properties. Considering its facile and versatile surface modification, curcumin-based polymer grafting can be an efficient method for controlling the chemical/physical properties of surfaces in a substrate-independent manner.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease caused by motor neuron damage in the central nervous system, and it is difficult to diagnose early.

Mitochondrial drug delivery systems require development of highly selective mitochondria-targeting carriers. In this study, we report that mitochondria targeting sequence (MTS)-hybrid cationic oligopeptide, MTS-H3R9, shows the dual role of a mitochondria targeting vector along with anticancer effect for cancer therapy. In cytotoxicity assays, MTS-H3R9 was shown to be more effective than MTS. MTS-H3R9 showed significant cell penetration and internalization activity compared to that of MTS along with more efficient escape from lysosome to the cytosol. We showed efficient targeting of MTS-H3R9 to mitochondria in HeLa cell line. Furthermore, we exhibited anticancer agent properties that mitochondrial-accumulated MTS-H3R9 caused cell death by reactive oxygen species generation and loss of mitochondrial membrane potential. MTS-H3R9 exhibited dramatically increased anticancer activity in 3D spheroids as well as in a 2D culture model. We demonstrated that MTS-H3R9 provides dual potentials both as a vehicle for targeted delivery and as a cancer treatment agent for therapeutic applications.

Catechols are prone to oxidative polymerization as well as complex formation with metal ions. These two features of catechols have played an important role in the construction of functional films on various surfaces. For example, marine antifouling films and antibacterial films were successfully prepared by oxidative polymerization and metal complexation of catechol-containing molecules, respectively. However, the effect of simultaneous metal complexation and oxidative polymerization on functional film formation has not yet been fully investigated. Herein, as a derivative of 3-(3,4-dihydroxyphenyl)-l-alanine (DOPA), we synthesized an ethylene glycol-derivatized DOPA (OEG-DOPA) and formed OEG-DOPA thin films based on (1) oxidative polymerization and (2) the complexation between catechol groups of OEG-DOPA and iron(III) (FeIII) ions. Either or both approaches were used for the film formation. OEG-DOPA film formation was characterized by ellipsometry, contact angle goniometry, field emission scanning electron microscopy, and X-ray photoelectron spectroscopy. Among the conditions used, the formation of a uniform film was only achieved with the dual cross-linking system of FeIII complexation and oxidation-induced covalent bond formation. Compared to the uncoated substrate and other OEG-DOPA films prepared under different conditions, the uniform OEG-DOPA film strongly inhibited bacterial adhesion, showing excellent antibacterial capability. We think that our surface-coating strategy can be applied to medical devices, tools, and implants where bacterial adhesion and biofilm formation should be prevented. This work can also serve as a basis for the construction of functional thin films for other catechol-functionalized materials.

Cancer cells divide uncontrollably due to their metabolic imbalance, resistance to mitochondria-mediated apoptosis, and ability to sustain telomere crisis by activating telomere reverse transcriptase. Therefore, mitochondria-mediated cell death has gained considerable attention as an alternative strategy to kill cancer cells. In the present study, an amphiphilic polymer composed of glycol chitosan (GC) and dequalinium (DQA), was synthesized via Michael addition reaction using a methyl acrylate linker and used to target mitochondria. DQA was selected as the mitochondria targeting moiety as well as the lipophilic component of polymer that will self-assemble into nanoparticles in aqueous solvent. GC-DQA nanoparticles were nontoxic compared to positive control when cell viability were assessed in both cancerous and non-cancerous cells. Mitochondria targeting and cell uptake was confirmed by confocal microscopy and flow cytometry, respectively. Curcumin was selected as the anticancer drug and while tested in vitro, the IC50 concentration of the micellar form was 10 μM in cancer cells. These results validate the promising potential of GC-DQA nanoparticles as an efficient mitochondria-targeting drug delivery system for cancer therapy.

We developed polymer-based nanoparticle carriers for gene delivery, glycol chitosan (GC)-methyl acrylate-low molecular weight polyethylenimine (800Da PEI), GMP, and investigated their ability to deliver second mitochondria-activation of caspase (Smac) gene into cancer cell lines. We analyzed cytotoxicity level and gene transfection efficiency in GBL-14, U373-MG, MDA-MB-231, and dermal fibroblasts (HDF) cell lines. GMP exhibited significantly lower cytotoxicity level, as compared with PEI25KD. Also, it showed high transfection efficiency in these cell lines. We treated these cells with a complex of GMP and Smac, and investigated their facilitated cellular uptake by flow cytometry. Also, analyses using cell cycle distribution, Annexin V staining, JC-1, and caspase-3 activity, indicated that apoptosis was induced by GMP/FLAG-Smac combined with etoposide in MDA-MB-231 cell line. This study demonstrates that combining the GMP/FLAG-Smac complex with chemotherapeutic agents could potentially be an effective approach for cancer therapy.

Polyamidoamine (PAMAM) dendrimer is one of the cationic polymers widely used in nonviral vector-based gene therapy. As PAMAM comprises biodegradable peptide bonds, it shows biocompatibility and relatively low cytotoxicity. In comparison with polyethylenimine (PEI, 25 kDa) that shows high transfection efficiency, the native PAMAM dendrimer has limitations as a gene carrier. To improve the gene expression efficiency, we introduced KRTR peptides derived from the influenza B virus nucleoprotein, known as a nuclear localization signal (NLS), to PAMAM generation 3. We synthesized PAMAM-RTRK and PAMAM-HRTRK and evaluated the effects of the histidine residue on cytotoxicity. The transfection efficiency of PAMAM-RTRK and PAMAM-HRTRK was similar to that of PEI in HepG2 cells, while their gene expression capacities were much higher than capacity of PEI in A549 cells. PAMAM-HRTRK showed relatively lower cytotoxicity than PAMAM-RTRK in the two cell lines, but the transfection capacity was similar for the two polymers. These results imply that the combination of histidine and KRTR peptide may potentially result in a novel PAMAM-based vector that could be used for prolonged gene therapy through the reduction in cytotoxicity and enhancement of gene expression.

This work reports a facile and effective antibacterial coating for oxide substrates. As a coating material, a random copolymer, abbreviated as poly(TMSMA-r-PEGMA), was synthesized by radical polymerization of 3-(trimethoxysilyl)propyl methacrylate (TMSMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA). Polymeric self-assembled monolayers of poly(TMSMA-r-PEGMA) were formed on various inorganic oxide substrates, including silicon oxide, titanium dioxide, aluminum oxide, and glass, via the simple dip-coating process. The polymer-coated substrates were characterized by ellipsometry, contact angle measurements, and X-ray photoelectron spectroscopy. The bacterial adhesion on the polymer-coated substrates was completely suppressed compared to that on the uncoated substrates.

Polyamidoamine (PAMAM) dendrimers are biocompatible polymers utilized in multiple biomedical applications including tissue engineering, medical diagnosis, drug and gene delivery systems, and biosensors. Normally, high-generation PAMAM dendrimers are advantageous for use in gene therapy research because they have a relatively high transfection efficiency. A high-generation PAMAM dendrimer has a high charge density, which induces greater damage to the membranous organelles than that induced by a low-generation PAMAM dendrimer. In this study, we added NLS sequences derived from the human papillomavirus (HPV) type 11 E2 protein to the low-generation PAMAM generation 2 (PAMAM G2) dendrimer and simultaneously introduced histidine residues to reduce cytotoxicity. RKRARH-PAMAM G2 showed similar and high transfection efficiencies in Neuro-2A and NIH3T3 cell lines and relatively low cytotoxicities relative to that of polyethylenimine 25 kDa (PEI 25 kDa).

The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N-octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology.

Zaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central β-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.

Polyamidoamine (PAMAM) dendrimer is an extensively studied polymer in the biomedical research because of its low polydispersity, distinct molecular structure, and surface functionalities. Generally, a high-generational PAMAM dendrimer is used for gene delivery because transfection efficiency is dependent on charge density; however, an increase in charge density induces disruption of the cellular membrane, and damage to the membrane results in cytotoxicity. In this study, we selected PAMAM generation 2 to reduce the cytotoxic effect and conjugated RRILH and RRLHL sequences, nuclear localization signals (NLS) derived from herpesviridae to PAMAM generation 2. The transfection efficiency of RRILH-PAMAM G2 and RRLHL-PAMAM G2 was similar to that of polyethylenimine (PEI) in Neuro2A, HT22, and HaCaT cells, whereas their transfection efficiency was much higher than that of PEI in NIH3T3 cells. RRILH-PAMAM G2 showed relatively lower cytotoxicity than did RRLHL-PAMAM G2 in all cell lines, but the transfection capacity of the two polymers was similar. Our study shows that low-generational PAMAM dendrimer conjugated with NLS sequences has potential as an alternative to PEI in gene delivery.

Polyamidoamine dendrimer has been studied as an efficient gene carrier. Due to its anti-inflammatory properties, polyamidoamine is a useful gene carrier, especially for inflammatory diseases. However, the commonly used polyamidoamine generation 6 dendrimer (PG6) has higher cytotoxicity than low-molecular weight polyamidoamines, which limits its applications. Therefore, early-generation polyamidoamine dendrimers, such as generation 2 (PG2), have been investigated as an alternative to PG6, although PG2 has a lower transfection efficiency. In this study, to improve gene delivery efficiency, histidine and arginine were conjugated on the primary amines of PG2, synthesizing PG2HR. The gene delivery efficiency of PG2HR was higher than that of PG2 or of PG2 conjugated with only arginine (PG2R), which may be due to higher cellular uptake and endosomal escape of the plasmid DNA (pDNA)/PG2HR complex. In addition, PG2HR had lower cytotoxicity than polyethylenimine (25 kDa, PEI25k), PG2, and PG2R. Mechanism studies showed that PG2HR delivered pDNA into the cells mainly by clathrin-independent endocytosis and partly by macropinocytosis. The therapeutic potential of PG2HR-mediated gene delivery was evaluated in middle cerebral artery occlusion (MCAO)-reperfusion stroke animal models. Heme oxygenase-1 (HO-1) plasmid was delivered into the brain by local injection. The results showed that PG2HR had higher gene delivery efficiency in the brain than did PEI25k, PG2, or PG2R. Furthermore, compared to the pHO-1/PEI25k, pHO-1/PG2, and pHO-1/PG2R complexes, the pHO-1/PG2HR complex had reduced apoptosis levels and infarct sizes in ischemic brains. Therefore, because of its low cytotoxicity and high gene delivery efficiency, PG2HR may be useful for gene therapy of inflammatory diseases including ischemic stroke.

Although a variety of non-viral gene delivery systems have been developed, they still suffer from low efficiency and specificity. Herein, we present the assembly of a dendrimer complex comprising a DNA cargo and a targeting moiety as a new format for targeted gene delivery. A PAMAM dendrimer modified with histidine and arginine (HR-dendrimer) was used to enhance the endosomal escape and transfection efficiency. An EGFR-specific repebody, composed of leucine-rich repeat (LRR) modules, was employed as a targeting moiety. A polyanionic peptide was genetically fused to the repebody, followed by incubation with an HR-dendrimer and a DNA cargo to assemble the dendrimer complex through an electrostatic interaction. The resulting dendrimer complex was shown to deliver a DNA cargo with high efficiency in a receptor-specific manner. An analysis using a confocal microscope confirmed the internalization of the dendrimer complex and subsequent dissociation of a DNA cargo from the complex. The present approach can be broadly used in a targeted gene delivery in many areas.

The aim of this work was to study the comparison of anti-oxidative activity in a single serving size of commercial coffees and teas. Commercial regular coffees and teas, including, brand regular coffees (BCA, BCB, BCC, BCD, and BCE), green tea (GTA, GTB, GTC, and GTD), black tea (BTA, BTB, and BTC), pu-erh tea (PTA, PTB, and PTC), chamomile tea (CTA, CTB, and CTC), peppermint tea (PA, PB, and PC), polygonatum odoratum tea (POTA, POTB, and POTC), and jujube tea (JTA, JTB, and JTC) were assayed for the levels of ascorbic acid, caffeine, total content of polyphenols and flavonoids, and ability to scavenge free radicals, using two in vitro antioxidant assays. The scavenging abilities of BCA and BCC were 664.91 ± 48.87 mg ascorbic acid equivalent/serving size and 624.36 ± 16.18 mg ascorbic acid equivalent/serving size, respectively. The four beverage samples (BCA, BCC, GTD, and BTA) significantly reduced the production of reactive oxygen species (ROS) and intracellular oxidative stress induced by H2O2. These results suggest that the beverages possess significant radical scavenging ability, which may be due to the presence of antioxidants. Furthermore, the significant reducing level of ROS evidences the potential antioxidant effects of these beverages in human cells.

Peptide nanostructure has been widely explored for drug-delivery systems in recent studies. Peptides possess comparatively lower cytotoxicity and are more efficient than polymeric carriers. Here, we propose a peptide nanorod system, composed of an amphiphilic oligo-peptide RH3F8 (Arg–His3–Phe8), as a drug-delivery carrier. Arginine is an essential amino acid in typical cell-penetration peptides, and histidine induces endo- and lysosomal escape because of its proton sponge effect. Phenylalanine is introduced to provide rich hydrophobicity for stable self-assembly and drug encapsulation. The self-assembled structure of RH3F8 showed nanorod-shaped morphology, positive surface charge, and retained formation in water for 35 days. RH3F8, labeled with Nile Red, showed high cellar uptake and accumulation in both cytoplasm and nucleus. The RH3F8 nanorods demonstrated negligible cytotoxicity, as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and hemolys...

Ordered porous biocompatible polymer films with controllable pore dimensions have vast application potential in biotechnology. In this article, a large-scale, ordered honeycomb biocompatible poly(lactic acid) film that is fabricated using a simple, scalable solution-immersion phase separation method is demonstrated. The entire preparation process is conducted in normal air without additional humidity or surfactants required. Notably, methanol, which is purposely employed to form a ternary polymer-solvent-nonsolvent solution, is critical because it not only induces phase separation and guarantees a highly ordered structure but it also assists in effectively controlling the surface morphology of the structured film. The ordered honeycomb poly(lactic acid) film produced using this approach demonstrates good adhesion and proliferation of NIH3T3 cells as evidence for use in future biomedical applications.

Malignant glioma is the most common and aggressive form of primary brain tumor in adults. In this study, we describe the efficacy of nonviral gene delivery carriers, histidine- and arginine- or histidine- and lysine-grafted polyamidoamine (PAMAM) dendrimers (PAMAM-H-R and PAMAM-H-K), in delivering a therapeutic and a tumor-selective killer gene, apoptin, using human glioma cells (U87-MG) and newborn human dermal fibroblast cells. We analyzed transfection efficiency using luciferase and a plasmid DNA encoding for enhanced green fluorescent protein and assessed cell viability in both cells. The results show that transfection efficiency of PAMAM-H-R and PAMAM-H-K was greatly increased compared with that of native PAMAM. Moreover, among PAMAM derivatives, cytotoxicity of PAMAM-H-K was very low. We treated both cells with complexes of PAMAM-H-R or PAMAM-H-K and apoptin and analyzed their cellular uptake by flow cytometry and localization by confocal microscopy. Furthermore, cell cycle distribution, caspase 3 activity assay, and JC-1 analysis showed cell death induced by apoptin in U87-MG cells. The present study demonstrates that a PAMAM-H-R/apoptin complex is an effective gene carrier system in glioma cell culture.

Background Recently, a combination of photodynamic therapy (PDT) and photothermal therapy (PTT) to generate reactive oxygen species (ROS) and heat to kill cancer cells, respectively has attracted considerable attention because it gives synergistic effects on the cancer treatment by utilizing the radiation of nontoxic low-energy photons such as long wavelength visible light and near IR (NIR) penetrating into subcutaneous region. For the effective combination of the phototherapies, various organic photosensitizer-conjugated gold nanocomplexes have been developed, but they have still some disadvantages due to photobleaching and unnecessary energy transfer of the organic photosensitizers. Results In this study, we fabricated novel inorganic phototherapeutic nanocomplexes (Au NR–TiO2 NCs) by conjugating gold nanorods (Au NRs) with defective TiO2 nanoparticle clusters (d-TiO2 NP clusters) and characterized their optical and photothermal properties. They were observed to absorb a broad range of visible light and near IR (NIR) from 500 to 1000 nm, exhibiting the generation of ROS as well as the photothermal effect for the simultaneous application of PDT and PTT. The resultant combination of PDT and PTT treatments of HeLa cells incubated with the nanocomplexes caused a synergistic increase in the cell death compared to the single treatment. Conclusion The higher efficacy of cell death by the combination of PDT and PTT treatments with the nanocomplexes is likely attributed to the increases of ROS generation from the TiO2 NCs with the aid of local surface plasma resonance (LSPR)-induced hot electrons and heat generation from Au NRs, suggesting that Au NR–TiO2 NCs are promising nanomaterials for the in vivo combinatorial phototherapy of cancer.

Stabilized plasmid-lipid particles (SPLPs) have been developed to overcome the low stability issue of cationic liposomes, however, SPLPs that are too stable result in unsatisfactory transfection efficiency. In this article, we prepared enzyme-responsive SPLPs (eSPLPs) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG-GLFG-K-(C16)2, a PEG lipid with an enzymatically-cleavable linker (glycine-phenylalanine-leucine-glycine, GFLG). eSPLPs were successfully prepared with plasmid DNA (pDNA) encapsulation efficiency of over 80%, using the detergent dialysis method. The PEG shell stabilized eSPLPs and maintained a hydrodynamic diameter of around 200nm. Although typical SPLPs were relatively intact in endosomal condition, the PEG shell of eSPLPs was cleaved following the degradation of the GFLG linker by cathepsin B in the endosome. Then, eSPLPs collapsed and induced endosomal disruption triggering the controlled release of the encapsulated pDNA into cytoplasm. Owing to the enzyme-responsive destabilization, eSPLPs showed a 10 to 100-fold higher transfection efficiency than control SPLPs, which was confirmed using luciferase assay. These results suggest that eSPLPs might be promising candidates for practical use as gene delivery systems, with both stability and high transfection efficiency for future in vivo applications.

In this study, the efficacy of the high-mobility group box-1 box A (HMGB1A)/heparin complex was evaluated for the treatment of acute lung injury (ALI). HMGB1A is an antagonist against wild-type high-mobility group box-1 (wtHMGB1), a pro-inflammatory cytokine that is involved in ALIs. HMGB1A has positive charges and can be captured in the mucus layer after intratracheal administration. To enhance the delivery and therapeutic efficiency of HMGB1A, the HMGB1A/heparin complex was produced using electrostatic interactions, with the expectation that the nano-sized complex with a negative surface charge could efficiently penetrate the mucus layer. Additionally, heparin itself had an anti-inflammatory effect. Complex formation with HMGB1A and heparin was confirmed by atomic force microscopy. The particle size and surface charge of the HMGB1A/heparin complex at a 1:1 weight ratio were 113nm and -25mV, respectively. Intratracheal administration of the complex was performed into an ALI animal model. The results showed that the HMGB1A/heparin complex reduced pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, more effectively than HMGB1A or heparin alone. Hematoxylin and eosin staining confirmed the decreased inflammatory reaction in the lungs after delivery of the HMGB1A/heparin complex. In conclusion, the HMGB1A/heparin complex might be useful to treat ALI.

The exceptional mechanical properties of the byssus—the fibrous holdfast of mussels that provides underwater adhesion—have potential applications in medicine and technology. The catechol–Fe3+–catechol interaction underlies the unique properties of mussel byssus and has emerged as a tool for developing functional hybrid materials such as pH-responsive, self-healing gels. Herein, the construction of functional alginate (Alg) film on a solid substrate inspired by mussel byssus is reported. The approach consists of spin-coating-assisted deposition of Alg catechols onto a solid substrate and their subsequent crosslinking via catechol–Fe3+–catechol interactions. This yields robust and multilayered Alg films that are resistant to protein adsorption and suppress bacterial adhesion. This method can be used to create antibacterial films for coating implanted medical devices.