Search

Abstract Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating ‘artificial’ targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating ‘artificial’ targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.

Abstract Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Abstract Background Although a large amount of evidence has revealed that amyloid β (Aβ), especially Aβ oligomers, protofibrils, and pyroglutamated Aβs, participate primarily in the pathophysiological processes of Alzheimer’s disease, most clinical trials of anti-Aβ antibody therapy have never acquired successful efficacy in human clinical trials, partly because peripheral administration of antibody medications was unable to deliver sufficient amounts of the molecules to the brain. Recently, we developed polymeric nanomicelles capable of passing through the blood–brain barrier that function as chaperones to deliver larger amounts of heavy molecules to the brain. Herein, we aimed to evaluate the efficacy of newly developed antibody 6H4 fragments specific to Aβ oligomers encapsulated in polymeric nanomicelles on the development of Alzheimer’s disease pathology in Alzheimer’s disease model mice at the age of emergence of early Alzheimer’s disease pathology. Results During the 10-week administration of 6H4 antibody fragments in polymeric nanomicelles, a significant reduction in the amounts of various toxic Aβ species, such as Aβ oligomers, toxic Aβ conformers, and pyroglutamated Aβs in the brain was observed. In addition, immunohistochemistry indicated inhibition of diameters of Aβ plaques, Aβ-antibody immunoreactive areas, and also plaque core formation. Behavioral analysis of the mice model revealed that the 6H4 fragments-polymeric nanomicelle group was significantly better at maintaining long-term spatial reference memory in the probe and platform tests of the water maze, thereby indicating inhibition of the pathophysiological process of Alzheimer’s disease. Conclusions The results indicated that the strategy of reducing toxic Aβ species in early dementia owing to Alzheimer’s disease by providing sufficient antibodies in the brain may modify Alzheimer’s disease progression.

Nanotherapy has potential utility in cancer, particularly in targeted delivery of therapeutics. Here the authors demonstrate delivery of tranilast loaded micelles to improve the reprogramming of cancer associated fibroblasts and monitor tumour stiffness to predict responses.

Abstract Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL‐12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL‐12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL‐12‐based nanocytokine (Nano‐IL‐12) accumulate and release IL‐12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano‐IL‐12‐mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI‐resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.

Large-area craniofacial defects remain a challenge for orthopaedists, hastening the need to develop a facile and safe tissue engineering strategy; osteoconductive material and a combination of optimal growth factors and microenvironment should be considered. Faced with the unmet need, we propose that abundant cytokines and chemokines can be secreted from the bone defect, provoking the infiltration of endogenous stem cells to assist bone regeneration. We can provide a potent mRNA medicine cocktail to promptly initiate the formation of bone templates, osteogenesis, and subsequent bone matrix deposition via endochondral ossification, which may retard rapid fibroblast infiltration and prevent the formation of atrophic non-union. We explored the mutual interaction of BMP2 and TGFβ3 mRNA, both potent chondrogenic factors, on inducing endochondral ossification; examined the influence of in vitro the transcribed polyA tail length on mRNA stability; prepared mRNA nanomedicine using a PEGylated polyaspartamide block copolymer loaded in a gelatin sponge and grafted in a critical-sized calvarial defect; and evaluated bone regeneration using histological and μCT examination. The BMP2 and TGFβ3 composite mRNA nanomedicine resulted in over 10-fold new bone volume (BV) regeneration in 8 weeks than the BMP2 mRNA nanomedicine administration alone, demonstrating that the TGFβ3 mRNA nanomedicine synergistically enhances the bone’s formation capability, which is induced by BMP2 mRNA nanomedicine. Our data demonstrated that mRNA-medicine-mediated endochondral ossification provides an alternative cell-free tissue engineering methodology for guiding craniofacial defect healing.

Spinal cord injury (SCI) is a debilitating condition that can cause impaired motor function or full paralysis. In the days to weeks following the initial mechanical injury to the spinal cord, inflammation and apoptosis can cause additional damage to the injured tissues. This secondary injury impairs recovery. Brain-derived neurotrophic factor is a secreted protein that has been shown to improve a variety of neurological conditions, including SCI, by promoting neuron survival and synaptic plasticity. This study treated a mouse model of contusion SCI using a single dose of brain-derived neurotrophic factor (BDNF) mRNA nanomicelles prepared with polyethylene glycol polyamino acid block copolymer directly injected into the injured tissue. BDNF levels in the injured spinal cord tissue were approximately doubled by mRNA treatment. Motor function was monitored using the Basso Mouse Scale and Noldus CatWalk Automated Gait Analysis System for 6 weeks post-injury. BDNF-treated mice showed improved motor function recovery, demonstrating the feasibility of mRNA delivery to treat SCI.

Intervertebral disk (IVD) degeneration is often associated with severity of lower back pain. IVD core is an avascular, highly hydrated tissue composed of type II collagen, glycosaminoglycans, and proteoglycans. The disk degeneration is not only a destruction of IVD structure but also is related to a disorder of the turnover of the disk matrix, leading the jelly-like IVD core to be replaced by fibrous components. Here we present a disease-modifying strategy for IVD degenerative diseases by direct regulation of the cells in the IVD using mRNA medicine, to alter the misbalanced homeostasis during disk degeneration. When mRNA encoding a cartilage-anabolic transcription factor, runt-related transcription factor-1, was administered to a rat model of coccygeal disk degeneration using a polyplex nanomicelle composed of polyethylene glycol-polyamino acid block copolymers and mRNA, the disk height was maintained to a significantly higher extent (≈81%) compared to saline control (69%), with prevention of fibrosis in the disk tissue. In addition, the use of nanomicelles effectively prevented inflammation, which was observed by injection of naked mRNA into the disk. This proof-of-concept study revealed that mRNA medicine has a potential for treating IVD degenerative diseases by introducing a cartilage-anabolic factor into the host cells, proposing a new therapeutic strategy using mRNA medicine. Keywords: mRNA, mRNA medicine, runt-related transcription factor-1, RUNX1, transcription factor, intervertebral disk disease, polyplex nanomicelle

We developed a novel chemically modified miR-143 (miR-143#12), and with it we investigated the contribution of miR-143 to the pathogenesis of bladder cancer (BC), in which miR-143 is extremely downregulated. Since miR-143 silenced K-RAS and RAS effector-signaling molecules Erk and Akt, we performed the ectopic expression of miR-143 in human BC 253J-BV cells, and we examined the growth inhibition and the mechanism of it in vitro and in orthotopic model mice. As a result, miR-143#12 induced a marked growth inhibition with apoptosis through impairing RAS-signaling networks, including SOS1, which exchanges guanosine diphosphate (GDP)/RAS for active guanosine triphosphate (GTP)/RAS. In the in vivo study, miR-143#12 exhibited a marked anti-tumor activity by either systemic or intravesical administration with polyionic copolymer (PIC) as the carrier, compared with the activity obtained by use of lipofection. These findings raised the possibility that the chemically modified miR-143#12 would be a candidate of microRNA (miRNA) medicine for BC delivered by intravesical infusion. Keywords: miR-143, K-RAS, SOS1, bladder cancer, PI3K/Akt, MEK/Erk signaling, intravesical infusion

Nanoparticle delivery of siRNA has problems with penetration and off target accumulation. Here, the authors report on the development of Y-shaped block catiomers which dynamically wrap around siRNA; demonstrate increased circulation times and delivery into hard to reach brain and pancreas tumour models.

Abstract Nano‐immunotherapy regimens have high potential to improve patient outcomes, as already demonstrated in advanced triple negative breast cancer with nanoparticle albumin‐bound paclitaxel and the immune checkpoint blocker (ICB) atezolizumab. This regimen, however, does not lead to cures with median survival lasting less than two years. Thus, understanding the mechanisms of resistance to and development of strategies to enhance nano‐immunotherapy in breast cancer are urgently needed. Here, in human tissue it is shown that blood vessels in breast cancer lung metastases are compressed leading to hypoxia. This pathophysiology exists in murine spontaneous models of triple negative breast cancer lung metastases, along with low levels of perfusion. Because this pathophysiology is consistent with elevated levels of solid stress, the mechanotherapeutic tranilast, which decompressed lung metastasis vessels, is administered to mice bearing metastases, thereby restoring perfusion and alleviating hypoxia. As a result, the nanomedicine Doxil causes cytotoxic effects into metastases more efficiently, stimulating anti‐tumor immunity. Indeed, when combining tranilast with Doxil and ICBs, synergistic effects on efficacy, with all mice cured in one of the two ICB‐insensitive tumor models investigated is resulted. These results suggest that strategies to treat breast cancer with nano‐immunotherapy should also include a mechanotherapeutic to decompress vessels.

Cell transplantation is promising for regenerative medicine. A combination of a three-dimensional spheroid culture system with gene transfection was developed to enhance the therapeutic effects of mesenchymal stem cell (MSC) transplantation. The spheroid cell culture system is based on micropatterned substrates composed of a regular array of 100-μm-diameter cell-adhesion areas coated with a temperature-responsive polymer, poly (N-isopropylacrylamide-co-methacrylic acid), which allows for spheroid detachment by simply cooling the plates. In this study, MSC spheroids were transfected with plasmid DNA encoding runt-related transcription factor 2 (Runx2) and tested for their ability to enhance bone regeneration. In vitro analyses revealed that osteogenic differentiation of the MSCs was enhanced by forming spheroids and was further promoted by Runx2 expression. The enhanced osteogenic differentiation was maintained under pathological conditions, such as hypoxia and inflammation. Transplanting Runx2-transfected MSC spheroids into bone defects on rat femurs induced significantly faster bone regeneration compared with nontransfected MSC spheroids or genetically modified MSCs from conventional monolayer culture. MSC migration into the bone defect area was enhanced by upregulation of cell-migration-related genes. In conclusion, genetically modified MSC spheroids are effective for enhancing bone regeneration, providing a promising option for cell transplantation therapy in the fields of regenerative medicine. Keywords: mesenchymal stem cell, spheroid, bone regeneration, cell transplantation, genetic modification, regenerative medicine

Abstract Objective Measuring motor function in mice is important for studying models of spinal cord injury (SCI) or other diseases. Several methods exist based on visual observation of mice moving in an open field. Though these methods require very little equipment, observers must be trained, and the possibility of human error or subjectivity cannot be eliminated. The Noldus CatWalk XT Automated Gait Analysis system assesses mouse motor function by taking high-resolution videos of the mice, with specialized software to measure several aspects of the animal’s gait. This instrument reduces the possibility of human error, but it is not always clear what data is important for assessing motor function. This study used data collected during mouse SCI experiments to create a simple mathematical model that combines the data collected by the CatWalk system into a single score, the Combined CatWalk Index or CCI. Results The CCI system produces similar results to the Basso Mouse Scale or the CatWalk’s Step Sequence Regularity Index. However, the CCI has a significantly smaller coefficient of variation than either other method. Additionally, CCI scoring shows slightly better correlation with impact force. The CCI system is likely to be a useful tool for SCI research.

Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.

Self-renewal of cancer stem cells can contribute to glioma progression. Here, the authors show that Notch1 activation in glioma stem cells induces expression of the lncRNATUG1, which promotes self-renewal through the repression of differentiation genes, and that targeting TUG1 represses glioma growth in vivo.

Sonodynamic therapy (SDT) is a minimally invasive anticancer therapy involving a chemical sonosensitizer and high-intensity focused ultrasound (HIFU). SDT enables the reduction of drug dose and HIFU irradiation power compared to those of conventional monotherapies. In our previous study, mouse models of colon and pancreatic cancer were used to confirm the effectiveness of SDT vs. drug-only or HIFU-only therapy. To validate its usefulness, we performed a clinical trial of SDT using an anticancer micelle (NC-6300) and our HIFU system in four pet dogs with spontaneous tumors, including chondrosarcoma, osteosarcoma, hepatocellular cancer, and prostate cancer. The fact that no adverse events were observed, suggests the usefulness of SDT.

Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.

BACKGROUND: We set out to examine the activity of γ-glutamyltranspeptidase (GGT) in lung cancer and the validity of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) for intraoperative imaging of primary lung cancer. METHODS: GGT activities and mRNA expression levels of GGT1 (one of the GGT subtypes) in five human lung cancer cell lines were examined by fluorescence imaging and quantitative reverse transcription polymerase chain reaction. In vivo imaging of an orthotopic A549 xenograft model in nude mice was performed to confirm its applicability to intraoperative imaging. Furthermore, ex vivo imaging of 73 specimens from lung cancer patients were performed and analyzed to calculate the sensitivity/specificity of gGlu-HMRG for lung cancer diagnosis. RESULTS: GGT activities and mRNA expression levels of GGT1 are diverse depending on cell type; A549, H441, and H460 showed relatively high GGT activities and expression levels, whereas H82 and H226 showed lower values. In the in vivo mouse model study, tiny pleural dissemination and hilar/mediastinal lymph node metastasis (less than 1 mm in diameter) were clearly detected 15 minutes after topical application of gGlu-HMRG. In the ex vivo study of specimens from patients, the sensitivity and specificity of gGlu-HMRG were calculated to be 43.8% (32/73) and 84.9% (62/73), respectively. When limited to female patients, never smokers, and adenocarcinomas, these values were 78.9% (15/19) and 73.7% (14/19), respectively. CONCLUSIONS: Although GGT activity of lung cancer cells vary, gGlu-HMRG can serve as an intraoperative imaging tool to detect small foci of lung cancer when such cells have sufficient GGT activity.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

In this study, two types of biodegradable polycation (PAsp(DET) homopolymer and PEG-PAsp(DET) copolymer) were applied as vectors for inhalable dry gene powders prepared by spray freeze drying (SFD). The prepared dry gene powders had spherical and porous structures with a 5~10-μm diameter, and the integrity of plasmid DNA could be maintained during powder production. Furthermore, it was clarified that PEG-PAsp(DET)-based dry gene powder could more sufficiently maintain both the physicochemical properties and in vitro gene transfection efficiencies of polyplexes reconstituted after powder production than PAsp(DET)-based dry gene powder. From an in vitro inhalation study using an Andersen cascade impactor, it was demonstrated that the addition of l-leucine could markedly improve the inhalation performance of dry powders prepared by SFD. Following pulmonary delivery to mice, both PAsp(DET)- and PEG-PAsp(DET)-based dry gene powders could achieve higher gene transfection efficiencies in the lungs compared with a chitosan-based dry gene powder previously reported by us.

Chemical modification of nucleosides in mRNA is an important technology to regulate the immunogenicity of mRNA. In this study, various previously reported mRNA formulations were evaluated by analyzing in vitro protein expression and immunogenicity in multiple cell lines. For the macrophage-derived cell line, RAW 264.7, modified mRNA tended to have reduced immunogenicity and increased protein expression compared to the unmodified mRNA. In contrast, in some cell types, such as hepatocellular carcinoma cells (HuH-7) and mouse embryonic fibroblasts (MEFs), protein expression was decreased by mRNA modification. Further analyses revealed that mRNA modifications decreased translation efficiency but increased nuclease stability. Thus, mRNA modification is likely to exert both positive and negative effects on the efficiency of protein expression in transfected cells and optimal mRNA formulation should be determined based on target cell types and transfection purposes.

Several drugs targeting the pathogenesis of aortic aneurysm have shown efficacy in model systems but not in clinical trials, potentially owing to the lack of targeted drug delivery. Here, we designed a novel drug delivery system using nanoparticles to target the disrupted aortic aneurysm micro-structure. We generated poly(ethylene glycol)-shelled nanoparticles incorporating rapamycin that exhibited uniform diameter and long-term stability. When injected intravenously into a rat model in which abdominal aortic aneurysm (AAA) had been induced by infusing elastase, labeled rapamycin nanoparticles specifically accumulated in the AAA. Microscopic analysis revealed that rapamycin nanoparticles were mainly distributed in the media and adventitia where the wall structures were damaged. Co-localization of rapamycin nanoparticles with macrophages was also noted. Rapamycin nanoparticles injected during the process of AAA formation evinced significant suppression of AAA formation and mural inflammation at 7 days after elastase infusion, as compared with rapamycin treatment alone. Correspondingly, the activities of matrix metalloproteinases and the expression of inflammatory cytokines were significantly suppressed by rapamycin nanoparticle treatment. Our findings suggest that the nanoparticle-based delivery system achieves specific delivery of rapamycin to the rat AAA and might contribute to establishing a drug therapy approach targeting aortic aneurysm.

Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.

Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. In vitro analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of in vivo mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.

PurposeAge-related macular degeneration caused by choroidal neovascularization (CNV) remains difficult to be treated despite the recent advent of several treatment options. In this study, we investigated the in vivo angiogenic control by intravenous injection of polyion complex (PIC) micelle encapsulating plasmid DNA (pDNA) using a mice CNV model.MethodsThe transfection efficiency of the PIC micelle was investigated using the laser-induced CNV in eight-week-old male C57 BJ/6 mice. Firstly, each mouse received intravenous injection of micelle encapsulating pDNA of Yellow Fluorescent Protein (pYFP) on days 1,3 and 5. The expression of YFP was analyzed using fluorescein microscopy and western blotting analysis. In the next experiments, each mouse received intravenous injection of micelle encapsulating pDNA of soluble Fms-like tyrosine kinase-1 (psFlt-1) 1,3 and 5 days after the induction of CNV and the CNV lesion was analyzed by choroidal flatmounts on day 7.ResultsFluorescein microscopy and western blotting analysis revealed that the expression of YFP was confirmed in the CNV area after injection of the PIC micelle, but the expression was not detected neither in mice that received naked pDNA nor those without CNV. Furthermore, the CNV area in the mice that received intravenous injection of the psFlt-1-encapsulated PIC micelle was significantly reduced by 65% compared to that in control mice (pConclusionsTransfection of sFlt-1 with the PIC micelle by intravenous injection to mice CNV models showed significant inhibition of CNV. The current results revealed the significant potential of nonviral gene therapy for regulation of CNV using the PIC micelle encapsulating pDNA.