Forest carbon removal potential and sustainable development in Japan.

Forests play a crucial role in mitigating climate change and reducing emissions as a major carbon sink. However, its value in removing carbon dioxide (CO2) from the atmosphere is always underestimated in natural capital (NC) accounting and sustainability assessments. This study predicted Japan's forest CO2 removal by afforestation and forest management and its monetary value until 2042 from national to gridded level, with statistical data and complementary satellite data products, and explored how that CO2 removal will contribute to sustainable development under the inclusive wealth (IW) framework. The results show that: (1) the annual CO2 removal by forests has the potential to offset 15.3% of the emission and increase NC by 6.8% in Japan, significantly contributing to carbon neutrality and IW growth; (2) the total CO2 removal in exiting forests will peak at around 2030 and then decrease, but expanding afforestation could offset that decrease in later years; (3) the spatial distribution patterns of IW and forest CO2 removal are opposite. This indicates a national carbon trading market could create new wealth for rural communities where vast forests exist, and then effectively balance the inequal urban-rural development in Japan. The explicit spatial information of this study could provide valuable information for differentiating policy priorities of forestry planning and sustainable development in different local communities.

Neurobiological Underpinnings of Hyperarousal in Depression: A Comprehensive Review.

Patients with major depressive disorder (MDD) exhibit an abnormal physiological arousal pattern known as hyperarousal, which may contribute to their depressive symptoms. However, the neurobiological mechanisms linking this abnormal arousal to depressive symptoms are not yet fully understood. In this review, we summarize the physiological and neural features of arousal, and review the literature indicating abnormal arousal in depressed patients. Evidence suggests that a hyperarousal state in depression is characterized by abnormalities in sleep behavior, physiological (e.g., heart rate, skin conductance, pupil diameter) and electroencephalography (EEG) features, and altered activity in subcortical (e.g., hypothalamus and locus coeruleus) and cortical regions. While recent studies highlight the importance of subcortical-cortical interactions in arousal, few have explored the relationship between subcortical-cortical interactions and hyperarousal in depressed patients. This gap limits our understanding of the neural mechanism through which hyperarousal affects depressive symptoms, which involves various cognitive processes and the cerebral cortex. Based on the current literature, we propose that the hyperconnectivity in the thalamocortical circuit may contribute to both the hyperarousal pattern and depressive symptoms. Future research should investigate the relationship between thalamocortical connections and abnormal arousal in depression, and explore its implications for non-invasive treatments for depression.

Homocysteine Combined with Apolipoprotein B as Serum Biomarkers for Predicting Carotid Atherosclerosis in the Oldest-Old.

Background: The measurement of serum biomarkers is a promising decision aid in the assessment of atherosclerosis. However, data on the levels and epidemiological distribution of serum biomarkers of carotid atherosclerosis (CAS) in the oldest-old are limited. This study aimed to investigate the characteristics of CAS serum biomarkers in the oldest-old over 80 and explore their predictive value for CAS. Methods: As part of the China Hainan Centenarian Cohort Study, a total of 1565 individuals over 80 years old were included. Atherosclerosis was assessed by carotid plaque and carotid intima-media thickness. Serum biomarker levels, demographic indicators, and physical examination indicators were detected. Prediction factors correlated to the CAS were explored by logistic regression and verified by receiver operating characteristic curve analysis. Multivariate regression models were fitted, along with subgroup analysis and robustness tests. Results: Among the oldest-old population, 83.5% (1306) had CAS. In a fully adjusted multivariate logistic regression model, systolic blood pressure (SBP), heart rhythm (HR), serum homocysteine (Hcy), and apolipoprotein B (ApoB) levels were significantly and positively associated with CAS in the oldest-old (PS < 0.001). ROC analysis indicated that the combination of serum Hcy, ApoB, SBP, and HR increased the predictive value for CAS in the oldest-old (area under the curve: 0.856, 95% CI: 0.803-0.879; sensitivity: 81.8%; specificity: 85.9%). Conclusion: SBP, HR, Hcy and ApoB are independent risk factors for CAS in the oldest-old. The specific set of biomarkers and their combination with other risk markers may be a promising strategy for assessing CAS in the elderly, especially in global aging.

Polysaccharide of Atractylodes macrocephala Koidz alleviate lipopolysaccharide-induced liver injury in goslings via the p53 and FOXO pathways.

Lipopolysaccharide (LPS) can affect the immune system of geese by inducing liver injury. The polysaccharide of Atractylodes macrocephala Koidz (PAMK) have obvious immune-enhancing effects. Accordingly, this experiment investigated the effect of PAMK on LPS-induced liver injury in goslings. Two hundred 1-day-old goslings were randomly divided into the control group, LPS group, PAMK group, and PAMK+ LPS group, and the PAMK and PAMK+ LPS groups were fed the basal diet with 400 mg/kg PAMK, while the control and LPS groups were fed the basal diet. On D 21, 23, and 25 of the formal trial, the goslings in the LPS and PAMK+LPS groups were injected intraperitoneally with 2 mg/kg LPS, and goslings in the control and PAMK groups were injected intraperitoneally with the same amount of saline. Livers were collected on D 25. HE-stained sections showed that PAMK could effectively alleviate the LPS-induced indistinct hepatic cord structure, loss of cytoplasmic contents of hepatocytes, and dilatation of hepatic sinusoids. The biochemical parameters of liver tissues showed that PAMK could alleviate the LPS-induced upregulation of alanine aminotransferase and aspartate aminotransferase. To further investigate the mechanism of the mitigating effect of PAMK on LPS-induced injury, livers from the LPS and PAMK+LPS groups were selected for transcriptome sequencing. The sequencing results showed that there were 406 differentially expressed genes (DEGs) in the livers of LPS and PAMK+LPS goslings, of which 242 upregulated and 164 downregulated. The Kyoto Encyclopedia of Genes and Genome (KEGG) analysis showed that DEGs were significantly enriched in immune signal transduction, cell cycle, and cell metabolism. Besides, protein‒protein interaction analysis showed that 129 DEGs were associated with each other, including 7 DEGs enriched in the p53 and FOXO signaling pathway. In conclusion, PAMK may alleviate LPS-induced liver injury in gosling through the p53 and FOXO signaling pathway. These results provide a basis for further development of PAMK as an immunomodulator.

Measurement and Destruction of Porcine Endogenous Retrovirus in the Chinese Bama Minipig.

Porcine hepatocytes are widely used in bioartificial liver (BAL) systems for the treatment of liver failure, and Chinese Bama minipigs (BMPs) are extensively used for animal experiments in the field of medicine in China. The genome of porcine endogenous retroviruses (PERVs) has not yet been accurately quantified, posing a threat to their clinical application because they act as a source of cells. In this study, we used genome sequence data from BMPs to predict PERV copies and their distribution. We validated and quantified the identified PERV copies and subtypes across different BMP individuals and tissues using quantitative real-time polymerase chain reaction and droplet digital polymerase chain reaction, respectively, and found that the BMP genome contains only 11 to 21 PERV copies. Notably, they lack the C subtype, which is a relatively safe cell source. Moreover, we applied CRISPR/Cas9 technology to knock out the pol fragment of PERVs in primary renal fibroblasts (PRFs) from BMPs and obtain PERV-destructed cells. Overall, our results lay a foundation for obtaining PERV-destructed BMPs as a safe source of hepatocytes for BALs for future applications.

LPS-Induced Liver Injury of Magang Geese through Toll-like Receptor and MAPK Signaling Pathway.

Lipopolysaccharide (LPS) is one of the main virulence factors of Gram-negative bacteria. In the process of waterfowl breeding, an inflammatory reaction due to LPS infection is easily produced, which leads to a decline in waterfowl performance. The liver plays a vital role in the immune response and the removal of toxic components. Therefore, it is necessary to study the mechanism of liver injury induced by LPS in goose. In this study, a total of 100 1-day-old goslings were randomly divided into a control group and LPS group after 3 days of pre-feeding. On days 21, 23, and 25 of the formal experiment, the control group was intraperitoneally injected with 0.5 mL normal saline, and the LPS group was intraperitoneally injected with LPS 2 mg/(kg body weight) once a day. On day 25 of the experiment, liver samples were collected 3 h after the injection of saline and LPS. The results of histopathology and biochemical indexes showed that the livers of the LPS group had liver morphological structure destruction and inflammatory cell infiltration, and the levels of ALT and AST were increased. Next, RNA sequencing analysis was used to determine the abundances and characteristics of the transcripts, as well as the associated somatic mutations and alternative splicing. We screened 727 differentially expressed genes (DEGs) with p < 0.05 and |log2(Fold Change)| ≥ 1, as the thresholds; GO and KEGG enrichment analysis showed that LPS-induced liver injury may be involved in the Toll-like receptor signaling pathway, MAPK signaling pathway, NOD-like receptor signaling pathway, FoxO, and PPAR signaling pathway. Finally, we intersected the genes enriched in the key pathway of LPS-induced liver injury with the top 50 key genes in protein−protein interaction networks to obtain 28 more critical genes. Among them, 17 genes were enriched in Toll-like signaling pathway and MAPK signaling pathway. Therefore, these results suggest that LPS-induced liver injury in geese may be the result of the joint action of Toll-like receptor, MAPK, NOD-like receptor, FoxO, and PPAR signaling pathway. Among them, the TLR7-mediated MAPK signaling pathway plays a major role.

Liver Organoids: Formation Strategies and Biomedical Applications.

The liver is the most important digestive organ in the body. Several studies have explored liver biology and diseases related to the liver. However, most of these studies have only explored liver development, mechanism of liver regeneration and pathophysiology of liver diseases mainly based on two-dimensional (2D) cell lines and animal models. Traditional 2D cell lines do not represent the complex three-dimensional tissue architecture whereas animal models are limited by inter-species differences. These shortcomings limit understanding of liver biology and diseases. Liver organoid technology is effective in elucidating structural and physiological characteristics and basic tissue-level functions of liver tissue. In this review, formation strategies and a wide range of applications in biomedicine of liver organoid are summarized. Liver organoids are derived from single type cell culture, such as induced pluripotent stem cells (iPSCs), adult stem cells, primary hepatocytes, and primary cholangiocytes and multi-type cells co-culture, such as iPSC-derived hepatic endoderm cells co-cultured with mesenchymal stem cells and umbilical cord-derived endothelial cells. In vitro studies report that liver organoids are a promising model for regenerative medicine, organogenesis, liver regeneration, disease modelling, drug screening and personalized treatment. Liver organoids are a promising in vitro model for basic research and for development of clinical therapeutic interventions for hepatopathy.

[Rapid screening of single guide RNA targeting pig genome and the harvesting of monoclonal cells by microarray seal].

The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.

Creating rat hepatocyte organoid as an in vitro model for drug testing.

BACKGROUND: Liver organoids have recently been applied as models for liver disease and drug screening, especially when combined with liver-on-a-chip technologies. Compared to hepatocyte-like cells, primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro. Mesenchymal stem cells (MSCs) enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes. MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix (ECM) proteins. In this study, primary hepatocytes were co-cultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration. AIM: To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix (PLECM) gel. METHODS: Perfusion and enzymatic hydrolysis were used to form the PLECM gel. Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids. Generated organoids were evaluated through hematoxylin and eosin, periodic acid-Schiff, immuno-histological, and immunofluorescence staining, and quantitative PCR for alb, CYP450 gene markers, and urea cycle genes. Culture medium was collected to detect albumin (ALB) and urea production on days 2, 4, 6, 8, 14, and 20. RESULTS: The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel. The structural components and basement membrane composition of the ECM, such as collagen type I, collagen type IV, fibronectin, and laminin, were demonstrated to be retained. Through interaction of human MSCs with the liver-derived ECM, primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h. The mRNAs of the gene alb, the CYP450 gene markers cyp1a1, cyp1a2, and cyp3a2 as well as urea cycle genes arg-1, asl, ass-1, cps-1, nags were highly expressed in hepatocyte organoids. Long-term survival of the primary hepatocyte organoids, as well as stable functionality, was demonstrated via ALB and urea production in vitro. CONCLUSION: Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Using the Patterned Microarray Culture to Obtain Gene-Editing Monoclonal Cells.

Genetically modified pigs are the first choice for xenotransplantation research, but there have been problems with monoclonal screening of edited cells before nuclear transfer. Our objective was to get a novel strategy to quickly obtain monoclonal cells with low damage by microarray and to produce efficient gene-editing monoclonal cells in batches. Micropattern array printing technology was introduced to limit only a single cell was adhered on a micropattern substrate, and after 4 days of culture, the single cell grew into a monoclonal cell sphere and then came off from the bottom of the petri dish automatically. After sequencing, the results showed that a single cell is confined to a micropattern and grows into a sphere of monoclonal cells.

Design, synthesis, and biological activity of a novel series of benzofuran derivatives against oestrogen receptor-dependent breast cancer cell lines.

A docking study of a novel series of benzofuran derivatives with ERα was conducted. In this study, we report the synthesis of a novel series of benzofuran derivatives and evaluation of their anticancer activity in vitro against MCF-7 human breast cancer cells, as well as their potential toxicity to ER-independent MDA-MB-231 breast cancer cells, human renal epithelial HEK-293 cells, and human immortal keratinocytes (HaCaT cells) by using the MTT colorimetric assay. The screening results indicated that the target compounds exhibited anti-breast cancer activity. The target compound 2-benzoyl-3-methyl-6-[2-(morpholin-4-yl)ethoxy]benzofuran hydrochloride (4e) exhibited excellent activity against anti-oestrogen receptor-dependent breast cancer cells and low toxicity. The preliminary structure-activity relationships of the target benzofuran derivatives have been summarised. In conclusion, the novel benzofuran scaffold may be a promising lead for the development of potential oestrogen receptor inhibitors.

Heterotopic vascularization and functionalization of implantable bio engineered hepatic tissue alleviates liver injury in rats.

BACKGROUND: The challenge of using bioengineered liver lies in sustaining the quantity of high-quality hepatocytes and the vasculature for blood perfusion. We characterized the heparinization of a porcine decellularized liver scaffold (DLS) as a carrier to support hepatocyte angiogenesis, thereby developing functional and vascularized hepatic tissue useful to treat liver injury. METHOD: The porcine DLS was obtained by the removal of cellular components and then subjected to heparinization by the end-point attachment technique. The heparinized DLSs were recellularized with rat hepatocyte spheroids to construct engineered hepatic tissue. The hepatic tissue was heterotopically implanted in the omentum majus of a rat model with liver injury induced by carbon tetrachloride (CCl4 ). RESULTS: Hepatocyte spheroids in the heparinized DLS remained viable for at least 10 weeks in vivo. The entire scaffold was populated with hepatocytes and arranged well. The volume of the heparinized DLS group was expanded over 400-fold. Liver-specific functions such as albumin synthesis, glycogen storage and cytochrome P 3A4 activity were highly expressed in the hepatic tissue. In addition, endothelial cells were recruited, as shown by CD31 staining, and new blood vessels formed, as visualized by fluorescein isothiocyanate-labelled dextran intravital confocal microscopy. The heparinized bioengineered hepatic tissue alleviated CCl4 -induced liver injury by regulating the deposition and degradation of the extracellular matrix. CONCLUSION: Primary hepatocyte spheroids survived for an extended time on the heparinized DLS and expanded to generate vascularized and functional bioengineered hepatic tissue that can alleviate liver injury in rats.

Bioartificial liver support systems for acute liver failure: A systematic review and meta-analysis of the clinical and preclinical literature.

BACKGROUND: Acute liver failure (ALF) has a high mortality varying from 80% to 85% with rapid progress in multi-organ system failure. Bioartificial liver (BAL) support systems have the potential to provide temporary support to bridge patients with ALF to liver transplantation or spontaneous recovery. In the past decades, several BAL support systems have been conducted in clinical trials. More recently, concerns have been raised on the renovation of high-quality cell sources and configuration of BAL support systems to provide more benefits to ALF models in preclinical experiments. AIM: To investigate the characteristics of studies about BAL support systems for ALF, and to evaluate their effects on mortality. METHODS: Eligible clinical trials and preclinical experiments on large animals were identified on Cochrane Library, PubMed, and EMbase up to March 6, 2019. Two reviewers independently extracted the necessary information, including key BAL indicators, survival and indicating outcomes, and adverse events during treatment. Descriptive analysis was used to identify the characteristics of the included studies, and a meta-analysis including only randomized controlled trial (RCT) studies was done to calculate the overall effect of BAL on mortality among humans and large animals, respectively. RESULTS: Of the 30 selected studies, 18 were clinical trials and 12 were preclinical experiments. The meta-analysis result suggested that BAL might reduce mortality in ALF in large animals, probably due to the recent improvement of BAL, including the type, cell source, cell mass, and bioreactor, but seemed ineffective for humans [BAL vs control: relative risk (95% confidence interval), 0.27 (0.12-0.62) for animals and 0.72 (0.48-1.08) for humans]. Liver and renal functions, hematologic and coagulative parameters, encephalopathy index, and neurological indicators seemed to improve after BAL, with neither meaningful adverse events nor porcine endogenous retrovirus infection. CONCLUSION: BAL may reduce the mortality of ALF by bridging the gap between preclinical experiments and clinical trials. Clinical trials using improved BAL must be designed scientifically and conducted in the future to provide evidence for transformation.

Phylogenetic and population structural inference from genomic ancestry maintained in present-day common wheat Chinese landraces.

Hexaploid common wheat is one of the most important food crops worldwide. Common wheat domestication began in the Fertile Crescent of the Near East approximately 10 000 years ago and then spread west into Europe and eastward into East Asia and China. However, the possible spreading route into and within China is still unclear. In this study, we successfully extracted DNA from single ancient wheat seeds and sequenced the whole genome of seven ancient samples from Xiaohe and Gumugou cemeteries in Xinjiang, China. Genomic inference and morphological observation confirmed their identity as hexaploid common wheat grown in prehistoric China at least 3200 years before present (BP). Phylogenetic and admixture analyses with RNA-seq data of modern hexaploid wheat cultivars from both China and Western countries demonstrated a close kinship of the ancient wheat to extant common wheat landraces in southwestern China. The highly similar allelic frequencies in modern landraces of the Qinghai-Tibetan plateau with the ancient wheat support the previously suggested southwestern spreading route into highland China. A subsequent dispersal route from the Qinghai-Tibetan plateau margins to the Yangtze valley was proposed in this study. Furthermore, the common wheat populations grown in the Middle and Lower Yangtze valley wheat zones were also proposed to be established by population admixture with the wheat grown in the Upper Yangtze valley. Our study reports ancient common wheat sequences at a genome-wide scale, providing important information on the origin, dispersal, and genetic improvement under cultivation of present-day wheat landraces grown in China.

Efficient Generation of an Fah/Rag2 Dual-Gene Knockout Porcine Cell Line Using CRISPR/Cas9 and Adenovirus.

The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 108 human hepatocytes per mouse. We hypothesized that 2-10 × 1010 human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.

Fabrication and Characterization of In Situ Synthesized SiC/Al Composites by Combustion Synthesis and Hot Press Consolidation Method.

The in situ SiC/Al composites were fabricated in Al-Si-C systems with different Si/C mass ratios and holding time by the method of combustion synthesis and hot press consolidation. The influences of Si/C mass ratio and holding time on the phase constitution, microstructure, and hardness of the composites were investigated. The results indicate that the increase of Si/C mass ratio leads to more uniform size distribution of the SiC particles in the Al matrix. Moreover, by improving the Si/C mass ratio from 4 : 1 to 5 : 1, the maximum size of SiC particle was reduced from 4.1 µm to 2.0 µm. Meanwhile, the percentage of submicroparticles was increased from 22% to 63%, and the average hardness value of the composites was increased by 13%. In addition, when the holding time is set to be fifteen minutes, the Al4C3 phase did not exist in the composites because of its total reactions with Si atoms to form SiC particles, and the average hardness value was 73.8 HB.

Injectable self-assembled block copolymers for sustained gene and drug co-delivery: an in vitro study.

A temperature-responsive pentablock copolymer (PB) was designed to deliver DNA and provide prolonged gene expression by forming a thermogelling release depot after subcutaneous or intratumoral injection. A synthetic barrier gel was developed based on poly(ethylene glycol) diacrylate to enable the released vectors to instantly and continuously transfect cultured cells. The aim of this setup was to provide a simple and controlled in vitro system to mimic tumor matrix to optimize the release system and to study the influence of the continuous and sustained release of the polyplexes on the in vitro transfection. The porosity of the barrier gel was adjusted by addition and removal of Pluronic F127 (PL), and the properties of the gel were characterized by visual inspection and scanning electron microscopy (SEM). Concentrated PB-based vector was administered to the barrier gel and allowed to be released to the buffer. We found that most of feed vector could be released from the barrier gel without unpacking the polymer-DNA complexes. Based on the specific construction of the PB-based vectors, an anti-cancer drug paclitaxel (PTX) was further loaded into the same vector designed for DNA delivery, thus formulating a gene and drug co-delivery system. This system was tested for sustained delivery to human ovarian carcinoma cells SKOV3 using the barrier gel in vitro as a tumor mimic. Transfection efficiency was found to be significantly enhanced by co-delivering PTX, while PTX also showed its effect as an anti-cancer drug to induce cell death. Yet both of the two effects of PTX shown in SKOV3 cells turned out to be dramatically weaker in ARPE-19 cells, a human non-cancerous cell line, which might be related to the nature of the vector itself. Instant release of vectors showed the ability to maintain transfection up to the fourth day, making the gene expression stable at least for the first 5 days. Further study is still needed to improve the duration of effective release.

Sensing polymer/DNA polyplex dissociation using quantum dot fluorophores.

We characterized the dissociation of polymer/DNA polyplexes designed for gene delivery using water-soluble quantum dots (QDs). A pH-responsive pentablock copolymer was designed to form stable complexes with plasmid DNA via tertiary amine segments. Dissociation of the polyplex was induced using chloroquine where the efficiency of this process was sensed through changes in QD fluorescence. We found that increasing concentrations of pentablock copolymer and DNA led to quenching of QD fluorescence, while chloroquine alone had no measurable effect. The mechanism of quenching was elucidated by modeling the process as the combination of static and dynamic quenching from the pentablock copolymer and DNA, as well as self-quenching due the bridging of QDs. Tertiary amine homopolymers were also used to study the effect of chain length on quenching. Overall, these QDs were found to be highly effective at monitoring the dissociation of pentablock copolymer/DNA polyplexes in vitro and may have potential for studying the release of DNA within cells.

The mechanism of selective transfection mediated by pentablock copolymers; part II: nuclear entry and endosomal escape.

Transfection efficiencies of non-viral gene delivery vectors commonly vary with cell type, owing to differences in proliferation rates and intracellular characteristics. Previous work demonstrated that the poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers exhibit transfection in vitro selectively in cancer cell lines as opposed to non-cancerous cell lines. This study continues the investigation of intracellular barriers to transfection using this vector in "normal" and cancer cell lines to understand the underlying mechanisms of the selectivity. Results from Part I of this investigation showed, using conjugated epidermal growth factor, that cellular uptake of these polyplexes is not a major barrier in these systems. Part II of this work continues the investigation into the other potential intracellular barriers, endosomal escape and nuclear entry, using a lysosomotropic agent chloroquine (CLQ), and a nuclear localization signal (NLS) SV40, respectively. Lack of effectiveness of NLS peptide in improving the transfection efficiency suggests that nuclear uptake might not be the major intracellular barrier using the pentablock copolymer vectors, or that the nuclear transport might not be primarily achieved through nuclear pores. However, inclusion of CLQ led to a dramatic enhancement in the level of gene expression, with an almost two orders of magnitude increase in expression seen in normal cell lines, compared with that the increase observed in cancer cell lines. The different lysosomal pH values in normal vs cancer cells was believed to cause the pentablock copolymer vectors to behave distinctly during transport through endocytic pathways, with greater loss of functional DNA occurring in normal cells containing more acidic endocytic vesicles in contrast to cancer cells with less acidic vesicles. Interestingly, CLQ introduced almost no enhancement in the transfection with the control vector ExGen which lacked selectivity of transfection. Exploiting intracellular differences between normal and cancer cells for gene delivery vector design offers a new paradigm to achieve transfection selectivity based on intracellular differences rather than conventional approaches involving vector modification using specific ligands for targeted delivery.

The mechanism of selective transfection mediated by pentablock copolymers; part I: investigation of cellular uptake.

Poly(diethylaminoethylmethacrylate) (PDEAEM) and Pluronic F127 based pentablock copolymer vectors with the ability to transfect cancer cells selectively over normal cells in in vitro cultures were developed, as described in a previous report. Understanding the mechanism of this selectivity will enable better polymeric vectors to be designed, with inherent selectivity for specific cell types based on intracellular differences and not on the use of targeting ligands, which have shown variable success, depending on the system. It is assumed that the selectivity was due to different intracellular barriers to transfection in the different cell types. Part I focuses on investigating whether cellular entry is one of the barriers to transfection, through conjugation of epidermal growth factor (EGF) to the pentablock copolymer vector. Results indicate that EGF conjugation increased transfection efficiency the most when conjugated to the outer surface of polyplexes, with minimal disruption to DNA packaging and maximal accessibility to receptors. The overall resulting enhancement in transfection, however, was a moderate three- to five-fold increase compared with the condition with no EGF involved, implying that the addition of EGF fails to overcome the intracellular barrier to transfection, which probably involves some step other than cellular uptake in pentablock copolymer system. Therefore, the differences observed in the selectivity of transfection between cancer and normal cell lines is probably not controlled by differences in cellular entry, and the intracellular barriers to transfection in this system are likely to be endosomal escape or nuclear entry, as investigated in Part II, the companion paper to this work.