Perspectives for Improving the Tumor Targeting of Nanomedicine via the EPR Effect in Clinical Tumors.

Over the past few decades, the enhanced permeability and retention (EPR) effect of nanomedicine has been a crucial phenomenon in targeted cancer therapy. Specifically, understanding the EPR effect has been a significant aspect of delivering anticancer agents efficiently to targeted tumors. Although the therapeutic effect has been demonstrated in experimental models using mouse xenografts, the clinical translation of the EPR effect of nanomedicine faces several challenges due to dense extracellular matrix (ECM), high interstitial fluid pressure (IFP) levels, and other factors that arise from tumor heterogeneity and complexity. Therefore, understanding the mechanism of the EPR effect of nanomedicine in clinics is essential to overcome the hurdles of the clinical translation of nanomedicine. This paper introduces the basic mechanism of the EPR effect of nanomedicine, the recently discussed challenges of the EPR effect of nanomedicine, and various strategies of recent nanomedicine to overcome the limitations expected from the patients' tumor microenvironments.

Citation: Tight Junction Protein Expression-Inducing Probiotics Alleviate TNBS-Induced Cognitive Impairment with Colitis in Mice.

A leaky gut is closely connected with systemic inflammation and psychiatric disorder. The rectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) induces gut inflammation and cognitive function in mice. Therefore, we selected Bifidobacterium longum NK219, Lactococcus lactis NK209, and Lactobacillus rhamnosus NK210, which induced claudin-1 expression in TNBS- or lipopolysaccharide (LPS)-stimulated Caco-2 cells, from the fecal bacteria collection of humans and investigated their effects on cognitive function and systemic inflammatory immune response in TNBS-treated mice. The intrarectal injection of TNBS increased cognitive impairment-like behaviors in the novel object recognition and Y-maze tests, TNF-α, IL-1ß, and IL-17 expression in the hippocampus and colon, and LPS level in the blood and feces, while the expression of hippocampal claudin-5 and colonic claudin-1 decreased. Oral administration of NK209, NK210, and NK219 singly or together decreased TNBS-impaired cognitive behaviors, TNF-α and IL-1ß expression, NF-κB+Iba1+ cell and LPS+Iba1+ cell numbers in the hippocampus, and LPS level in the blood and feces, whereas BDNF+NeuN+ cell and claudin-5+ cell numbers and IL-10 expression increased. Furthermore, they suppressed TNBS-induced colon shortening and colonic TNF-α and IL-1ß expression, while colonic IL-10 expression and mucin protein-2+ cell and claudin-1+ cell numbers expression increased. Of these, NK219 most strongly alleviated cognitive impairment and colitis. They additively alleviated cognitive impairment with colitis. Based on these findings, NK209, NK210, NK219, and their combinations may alleviate cognitive impairment with systemic inflammation by suppressing the absorption of gut bacterial products including LPS into the blood through the suppression of gut bacterial LPS production and alleviation of a leaky gut by increasing gut tight junction proteins and mucin-2 expression.

Lactobacillus rhamnosus and Bifidobacterium longum alleviate colitis and cognitive impairment in mice by regulating IFN-γ to IL-10 and TNF-α to IL-10 expression ratios.

Gut lactobacilli and bifidobacteria on the immune homeostasis. Therefore, to understand the mechanism in vivo, we selected human fecal Lactobacillus rhamnosus NK210 and Bifidobacterium longum NK219, which strongly suppressed the IFN-γ to IL-10 expression (IIE) ratio in lipopolysaccharide-stimulated macrophages. Thereafter, we examined their effects on the endotoxin, antibiotics, or antitumor drug-stimulated immune imbalance in mice. Intraperitoneal injection of lipopolysaccharide and oral gavage of ampicillin increased IFN-γ and TNF-α expression in the spleen, colon, and hippocampus, while IL-10 expression decreased. However, intraperitoneal injection of cyclophosphamide suppressed IFN-γ, TNF-α, and IL-10 expression. LPS exposure induced splenic natural killer cell cytotoxicity against YAC-1 cells (sNK-C) and peritoneal macrophage phagocytosis against Candida albicans (pMA-P) activities, while cyclophosphamide and ampicillin treatments suppressed sNK-C and pMA-P activities. However, LPS, ampicillin, cyclophosphamide all increased IIE and TNF-α to IL-10 expression (TIE) ratios. Oral administration of NK210 and/or NK219 significantly reduced LPS-induced sNK-C, pMA-P, and IFN-γ expression, while cyclophosphamide- or ampicillin-suppressed sNK-C and pMA-P activities, cyclophosphamide-suppressed IFN-γ, TNF-α, and IL-10 expression, and ampicillin-suppressed IL-10 expression increased. Nevertheless, they suppressed LPS-, ampicillin-, or cyclophosphamide-induced IIE and TIE ratios, cognitive impairment, and gut dysbiosis. In particular, NK219, but not NK210, increased the IIE expression ratio in vitro and in vivo, and enhanced sNK-C and pMA-P activities in normal control mice, while cognitive function and gut microbiota composition were not significantly affected. These findings suggest that NK210, Lactobacillus sp, and NK219, Bifidobacterium additively or synergistically alleviate gut dysbiosis, inflammation, and cognitive impairment with immune imbalance by controlling IIE and TIE ratios.

Transplantation of fecal microbiota from patients with inflammatory bowel disease and depression alters immune response and behavior in recipient mice.

Gut dysbiosis is closely associated with the occurrence of inflammatory bowel disease (IBD) and psychiatric disorder. Here, to understand the difference of gut microbiota composition and physiological effect between IBD patients with (IBD/D+) or without depression (IBD/D-), we analyzed the fecal microbiota composition of patients with IBD with (/D+) or without depression (/D-) and healthy volunteers (HVs) and examined the effects of these fecal microbiota transplantations (FMTs) on the occurrence of systemic inflammation and anxiety/depression in mice. FMTs from patients with IBD/D+ or IBD/D- caused IBD-like colitis in the transplanted mice: they increased the myeloperoxidase activity, IL-1ß and IL-6 expression, and NF-κB+/CD11c+ cell population in the colon. Transplantation of the IBD/D+ patient feces (IBD/D+-F) caused IBD-like colitis more strongly than that of IBD/D--F. FMTs from patients with IBD/D+ also caused anxiety-/depression-like behaviors, increased the NF-κB+/Iba1+ and lipopolysaccharide (LPS)+/Iba1+ cell populations, and decreased the BDNF+/NeuN+ cell population in the hippocampus. They increased LPS levels in the blood. FMTs from patients with IBD/D- caused anxiety-like, but not depression-like, behaviors. α-/ß-diversities and composition of gut microbiota in IBD-F were different from those of HV feces (HV-F). The Enterobacteriaceae and Enterococcaceae populations and LPS levels were higher in the IBD-F than in the HV-F. The Enterococcaceae population was higher in IBD/D+-F vs. IBD/D--F. However, the transplantation of HV-F into mice previously transplanted with IBD/D+-F significantly reduced depression-like behaviors, NF-κB+/Iba1+ and LPS+/Iba1+ cell populations in the hippocampus, LPS levels in the feces and blood, and IL-1ß expression in the colon. These findings suggest that the outbreak of depression/anxiety may be dependent on the systemic inflammation with a leaky gut through the gut dysbiosis-attributable overproduction of bacterial LPS and suppression of tight junction protein expression in patients with IBD.

Combined treatment with auranofin and trametinib induces synergistic apoptosis in breast cancer cells.

Auranofin is a gold complex used as an anti-rheumatic agent and may act as a potent anticancer drug against breast tumors. Trametinib is a specific mitogen-activated protein kinase inhibitor, approved for the treatment of metastatic melanoma. The aim of this study was to examine the synergistic effects of auranofin and trametinib on apoptosis in MCF-7 human breast cancer cells. The combination treatment inhibited cancer cell proliferation and induced cell cycle arrest at the sub-G1 phase and apoptosis via poly (ADP-ribose) polymerase cleavage and caspase-3/7 activation. It is noteworthy that this treatment significantly increased p38 mitogen-activated protein kinase (MAPK) phosphorylation to induce mitochondrial stress, subsequently promoting cancer cell apoptosis through release of apoptosis-inducing factor. Further data demonstrated that combined treatment significantly induced increase in nuclear translocation of AIF. These results indicated that activation of the p38 MAPK signaling pathway and mitochondrial apoptosis may contribute to the synergistic consequences in MCF-7 cells. Collectively, our data demonstrated that combined treatment with auranofin and trametinib exhibited synergistic breast cancer cell death and this combination might be utilized as a novel therapeutic strategy for breast cancer.

PEG-PLA-Coated and Uncoated Radio-Luminescent CaWO4 Micro- and Nanoparticles for Concomitant Radiation and UV-A/Radio-Enhancement Cancer Treatments.

Currently, there is great interest in the development of ways to achieve the benefits of radiation treatments with reduced negative effects. The present study demonstrates the utilization of radio-luminescent particles (RLPs) as a means to achieve radio-sensitization and enhancement and their ability to affect head- and neck-cancer-cell cultures (in vitro) and xenografts (in vivo). Our approach utilizes a naturally abundant radio-luminescent mineral, calcium tungstate (CaWO4), in its micro or nanoparticulate form for generating secondary UV-A light by γ ray or X-ray photons. In vitro tests demonstrate that unoptimized RLP materials (uncoated CaWO4 (CWO) microparticles (MPs) and PEG-PLA-coated CWO nanoparticles (NPs)) induce a significant enhancement of the tumor-suppressive effect of X-rays and γ rays in both radio-sensitive- and radio-resistant-cancer models; uncoated CWO MPs and PEG-PLA-coated CWO NPs demonstrate comparable radio-sensitization efficacies in vitro. Mechanistic studies reveal that concomitant CaWO4 causes increased mitotic death in radio-resistant cells treated with radiation, whereas CaWO4 sensitizes radio-sensitive cells to X-ray-induced apoptosis and necrosis. The radio-sensitization efficacy of intratumorally injected CaWO4 particles (uncoated CWO MPs and PEG-PLA-coated CWO NPs) is also evaluated in vivo in mouse head- and neck-cancer xenografts. Uncoated CWO MPs suppress tumor growth more effectively than PEG-PLA-coated CWO NPs. On the basis of theoretical considerations, an argument is proposed that uncoated CWO MPs release subtoxic levels of tungstate ions, which cause increased photoelectric-electron-emission effects. The effect of folic acid functionalization on the in vitro radio-sensitization behavior produced by PEG-PLA-coated CWO NPs is studied. Surface folic acid results in a significant improvement in the radio-sensitization efficiency of CaWO4.

Nano carriers that enable co-delivery of chemotherapy and RNAi agents for treatment of drug-resistant cancers.

Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints (Gottesman et al., 2002; Holohan et al., 2013). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1, encoded by the ATP-Binding Cassette Sub-Family B Member 1 (ABCB1) gene)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means that the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.

The potential and advances in RNAi therapy: chemical and structural modifications of siRNA molecules and use of biocompatible nanocarriers.

Small interfering RNA (siRNA) has attracted great attention as a potential new drug due to its highly sequence-specific gene silencing ability and generality in therapeutic target. However, the medical applications of siRNA have been severely hindered by the lack of an optimal systemic delivery methodology. This poor delivery performance of siRNA is mainly caused by its inherent physicochemical properties including short and stiff structure, low charge density and vulnerability to nuclease cleavage. Thus, the successful development of efficient systemic delivery platform for siRNA is a fundamental requirement necessary to bring siRNA-based drugs to the market. Herein, we describe some siRNA delivery methods based on the chemical and structural modifications of delivery materials and siRNA itself to carry siRNA therapeutics safely to the targeted place without adverse effects. This review particularly explains the latest progress of chemically and structurally modified siRNA polymer (poly-siRNA)-based delivery systems. The stable and compact siRNA polyplexes, which are formed by poly-siRNA and different types of biocompatible materials, can enhance serum stability and target delivery efficiency in vitro and in vivo. In addition, this review provides specific information on poly-siRNA delivery systems from basics to therapeutic applications in different animal disease models.

MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine.

Mesenchymal stem cells (MSCs) have attracted much attention in regenerative medicine owing to their apparent usefulness as multi-potent replacement cells. The potential of MSC therapy can be further improved by transforming MSCs with therapeutic genes that maximize the efficacy of gene therapy and their own therapeutic ability. Since most conventional transfection methodologies have shown marginal success in delivering exogenous genes into primary cultured cells, efficient gene transfer into primary MSCs is a prerequisite for the development of MSC-based gene therapy strategies to achieve repair and regeneration of damaged tissues. Herein, facially amphipathic bile acid-modified polyethyleneimine (BA-PEI) conjugates were synthesized and used to transfer hypoxia-inducible vascular endothelial growth factor gene (pHI-VEGF) in MSCs for the treatment of rat myocardial infarction. Under the optimized transfection conditions, the BA-PEI conjugates significantly increased the VEGF protein expression levels in rat MSCs, compared with traditional transfection methods such as Lipofectamine™ and branched-PEI (25 kDa). Furthermore, the prepared pHI-VEGF-engineered MSCs (VEGF-MSCs) resulted in improved cell viability, particularly during severe hypoxic exposure in vitro. The transplantation of MSCs genetically modified to overexpress VEGF by BA-PEI enhanced the capillary formation in the infarction region and eventually attenuated left ventricular remodeling after myocardial infarction in rats. This study demonstrates the applicability of the BA-PEI conjugates for the efficient transfection of therapeutic genes into MSCs and the feasibility of using the genetically engineered MSCs in regenerative medicine for myocardial infarction.

Thermogelling chitosan-g-(PAF-PEG) aqueous solution as an injectable scaffold.

The present study reports on a thermogelling poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) grafted chitosan (CS-g-(PAF-PEG)) system, focusing on phase diagram, transition mechanism, and in vivo gel duration. The sol-to-gel transition temperature decreased from 27 to 11 °C as the concentration increased from 4.0 wt % to 9.0 wt %. The polymer formed micelles with 10-50 nm in diameter at 10 °C and formed large aggregates ranging from hundreds to thousands of nanometers in size as the temperature increased from 10 to 35 °C, suggesting that an extensive molecular aggregation might be involved in the sol-to-gel transition. To study the transition mechanism on a molecular level, we investigated pH, circular dichroism spectra, and (13)C NMR spectra of the CS-g-(PAF-PEG) aqueous solution as a function of temperature. As the temperature increased, deprotonation of the chitosan and dehydration of the PEG were suggested, whereas the α-helical secondary structure of PAF was slightly changed in the sol-to-gel transition temperature range of 10-50 °C. A gel was formed in situ after injecting the CS-g-(PAF-PEG) aqueous solution into the subcutaneous layer of rats. About 60-70% of the gel was eliminated in 1 week, and the remaining gel was completely cleared from the implant site in 14 days. The results indicate the potential of CS-g-(PAF-PEG) as a promising short-term carrier for pharmaceutical agents.

Cell therapy for skin wound using fibroblast encapsulated poly(ethylene glycol)-poly(L-alanine) thermogel.

As a new application of a thermogel, a poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with Matrigel for cell proliferation and was significantly better than Matrigel for collagen types I and III formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the cell-encapsulated gel was formed in situ. Compared with the phosphate buffered saline treated system and the cell-free PEG-L-PA thermogel, the cell-encapsulated PEG-L-PA thermogel not only accelerated the wound closure but also improved epithelialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. The results demonstrate the potential of thermogels for cell therapy as an injectable tissue-engineering scaffold.

Biodegradable thermogels.

All living creatures respond to external stimuli. Similarly, some polymers undergo conformational changes in response to changes in temperature, pH, magnetic field, electrical field, or the wavelength of light. In one type of stimuli-responsive polymer, thermogel polymers, the polymer aqueous solution undergoes sol-to-gel transition as the temperature increases. Drugs or cells can be mixed into the polymer aqueous solution when it is in its lower viscosity solution state. After injection of the solution into a target site, heating prompts the formation of a hydrogel depot in situ, which can then act as a drug releasing system or a cell growing matrix. In this Account, we describe key materials developed in our laboratory for the construction of biodegradable thermogels. We particularly emphasize recently developed polypeptide-based materials where the secondary structure and nanoassembly play an important role in the determining the material properties. This Account will provide insights for controlling parameters, such as the sol-gel transition temperature, gel modulus, critical gel concentration, and degradability of the polymer, when designing a new thermogel system for a specific biomedical application. By varying the stereochemistry of amino acids in polypeptides, the molecular weight of hydrophobic/hydrophilic blocks, the composition of the polypeptides, the hydrophobic end-capping of the polypeptides, and the microsequences of a block copolymer, we have controlled the thermosensitivity and nanoassembly patterns of the polymers. We have investigated a series of thermogel biodegradable polymers. Polymers such as poly(lactic acid-co-glycolic acid), polycaprolactone, poly(trimethylene carbonate), polycyanoacrylate, sebacic ester, polypeptide were used as hydrophobic blocks, and poly(ethylene glycol) and poly(vinyl pyrrolidone) were used as hydrophilic blocks. To prepare a polymer sensitive to pH and temperature, carboxylic acid or amine groups were introduced along the polymer backbone. The sol-gel transition mechanism involves changes in the secondary structures of the hydrophobic polypeptide and in the conformation of the hydrophilic block. The polypeptide copolymers were stable in the phosphate buffered saline, but the presence of proteolytic enzymes such as elastase, cathepsin B, cathepsin C, and matrix metallopreoteinase accelerated their degradation. We also describe several biomedical applications of biogradable thermogel polymers. One subcutaneous injection of the insulin formulation of thermogel polypeptide copolymers in diabetic rats provided hypoglycemic efficacy for more than 16 days. The thermogels also provided a compatible microenvironment for chondrocytes, and these cells produced biomarkers for articular cartilage such as sulfated glucoaminoglycan (sGAG) and type II collagen. The thermogels were also used as a fixing agent for in situ cell imaging, and cellular activities such as endocytosis were observed by live cell microscopy.

In situ thermal gelling polypeptide for chondrocytes 3D culture.

In the search for a cell-instructive or cell-interactive artificial extracellular matrix, synthetic hydrogels have been extensively investigated to apply three-dimensional (3D) cell culture and tissue engineering. Here, we are reporting a reverse thermal gelling l/dl-polyalanine block copolymer aqueous solution for chondrocyte 3D culture. The polymer aqueous solution undergoes sol-to-gel transition as the temperature increases, thus forming a 3D cell encapsulating scaffold in situ at 37 °C. In particular, the fraction of the ß-sheet structure of the polyalanine dictated the population and thickness of fibrous nanostructure of the hydrogel, which in turn affected the proliferation and protein expression of the encapsulated chondrocytes. As an injectable tissue engineering system of chondrocytes, very promising results were confirmed for nude mice, using the current polypeptide aqueous solution. This paper not only provides important clues in designing an artificial extracellular matrix but also proves the significance of thermal gelling polypeptide as a minimally-invasive tissue engineering scaffold.

Reverse thermal gelation of PAF-PLX-PAF block copolymer aqueous solution.

The aqueous solution of poly(L-Ala-co-L-Phe)-poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol)-poly(L-Ala-co-L-Phe) block copolymers (PAF-PLX-PAF) in a concentration range of 6.0-10.0 wt % underwent sol-to-gel transition as the temperature increased from 10 to 50 degrees C. Circular dichroism spectra, hydrophobic dye solubilization, dynamic light scattering, and transmission electron microscopy image of the polymer suggest that the polymers form micelles in water, where the hydrophilic (PLX) blocks form a shell and the hydrophobic (PAF) blocks form a core of the micelle. Circular dichroism, FTIR, and (13)C NMR spectra suggest that sol-to-gel transition accompanies partial strengthening of the beta-sheet structure of PAF and a decrease in molecular motion of the PLX. The sol-to-gel transition temperature could be controlled by varying the molecular weight of PAF and PLX blocks, the ratio of Ala to Phe, and the corresponding secondary structure of the polypeptide.

Temperature-sensitive biodegradable poly(ethylene glycol).

We report here that the incorporation of several disulfide bonds along poly(ethylene glycol) (PEG) gives temperature sensitivity, as well as biodegradability to PEG. To synthesize a PEG with temperature sensitivity in a physiologically important range (20-40 degrees C), PEGs with molecular masses of 400 and 600 Da were randomly coupled by disulfide bonds. As the mol ratio of PEG (400 Da) disulfide to PEG (600 Da) disulfide increased from 40:60 to 60:40, the cloud point of the polymer aqueous solution decreased from 35 degrees C to 27 degrees C. The disulfide bonds between PEGs were degraded in the presence of a thiol-containing biomolecule of glutathione in a thiol-concentration-dependent manner.

Reverse thermal organogelation of poly(ethylene glycol)-polypeptide diblock copolymers in chloroform.

Most organogel formation has been observed at low temperatures and the gel melts as the temperature increases. As an opposite case to traditional organogels, we report a reverse thermal gelation of methoxy-aminopoly(ethylene glycol)-homopolypeptide block-copolymer chloroform solutions that undergo a sol-to-gel transition as the temperature increases. A series of methoxy-aminopoly(ethylene glycol)-homopolypeptide block copolymers showing reverse thermal gelation in chloroform was synthesized and we prove that the secondary structure of the polypeptide determines the morphology of the organogel.

Enzymatically degradable temperature-sensitive polypeptide as a new in-situ gelling biomaterial.

We are reporting a poly (ethylene glycol)-block-poly(alanine-co-phenyl alanine) (PEG-PAF) aqueous solution that undergoes sol-to-gel transition as the temperature increases. The sol-to-gel transition was observed at as low a concentration as 3.0-7.0 wt.%. Micellar aggregation accompanying small conformational changes of the peptide from random coils to beta-sheets is suggested as the sol-to-gel transition mechanism of the PEG-PAF aqueous solution. The PEG-PAF is stable in phosphate buffered saline, however, it degraded in the subcutaneous layer of rats. In vitro study showed that proteolytic enzymes such as cathepsin B, cathepsin C, and elastase that are present in the subcutaneous layer of the mammalian tissue might be responsible for the degradation of the polymer in rats. As a feasibility study of this material, a single shot of an aqueous insulin formulation (13.8 mg insulin/kg) showed a hypoglycemic effect over 18 days in rats. The current functional polypeptide may be very promising as an in-situ gelling system for tissue engineering, cell/stem cell therapy, and drug delivery.