Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

Wei Fan1,2,*, Xin Wu1,*, Baoyue Ding3,*, Jing Gao4, Zhen Cai1, Wei Zhang1, Dongfeng Yin1, Xiang Wang1, Quangang Zhu1, Jiyong Liu1, Xueying Ding4, Shen Gao1 1Department of Pharmaceutics, Changhai Hospital, Second Military Medical University, Shanghai, 2Department of Pharmaceutics, The 425th Hospital of PLA, Sanya, 3Department of Pharmaceutics, Medical College of Jiaxing University, Jiaxing, 4Department of Pharmaceutics, School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China*These authors contributed equally to this workBackground: Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers' structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake.Methods: This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers.Results: Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus.Conclusion: This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future.Keywords: Pluronics, gene transfer, nonviral vectors, transfection efficiency, cellular uptak

Physical Association of PDK1 with AKT1 Is Sufficient for Pathway Activation Independent of Membrane Localization and Phosphatidylinositol 3 Kinase

Frequent activation of the AKT serine-threonine kinase in cancer confers resistance to therapy. AKT is activated by a multi-step process involving phosphatidylinositide (PtdIns) phosphate-mediated recruitment of AKT and its upstream kinases, including 3-Phosphoinositide-dependent kinase 1 (PDK1), to the inner surface of the cell membrane. PDK1 in the appropriate context phosphorylates AKT at threonine 308 (T308) to activate AKT. Whether PtdIns(3,4,5)Ps (PtdInsP3) binding and AKT membrane translocation mediate functions other than formation of a functional PDK1::AKT complex have not been fully elucidated. We fused complementary fragments of intensely fluorescent protein (IFP) to AKT1 and PDK1 to induce a stable complex to study the prerequisites of AKT1 phosphorylation and function. In the stabilized PDK1-IFPC::IFPN-AKT1 complex, AKT1 T308 phosphorylation was independent of PtdIns, as demonstrated by treatment with Phosphatidylinositol 3 Kinase (PI3K) inhibitors. Further when interaction with PtdIns and the cell membrane was prevented by creating PH-domain mutants of AKT1 (R25A) and PDK1 (R474A), AKT1 phosphorylation on T308 was maintained in the PDK1-IFPC::IFPN-AKT1 complex. The PDK1-IFPC::IFPN-AKT1 complex was sufficient for phosphorylation of known AKT substrates, and conferred resistance to inhibitors of PI3K (LY294002, PI103, GDC0941 and TGX286) but not inhibitors of the downstream TORC1 complex (rapamycin). Thus the locus of action of targeted therapeutics can be elucidated by the constitutively active AKT1 complex. Our data indicate that PtdIns and membrane localization are not required for AKT phosphorylation and activation, but rather serve to induce a functional physical interaction between PDK1 and AKT. The PDK1-IFPC::IFPN-AKT1 complex provides a cell-based platform to examine specificity of drugs targeting PI3K pathway components

MiR-770-5p inhibits cisplatin chemoresistance in human ovarian cancer by targeting ERCC2

In this study, we examined the role of the miRNA miR-770-5p in cisplatin chemotherapy resistance in ovarian cancer (OVC) patients. miR-770-5p expression was reduced in platinum-resistant patients. Using a 6.128-fold in expression as the cutoff value, miR-770-5p expression served as a prognostic biomarker and predicted the response to cisplatin treatment and survival among OVC patients. Overexpression of miR-770-5p in vitro reduced survival in chemoresistant cell lines after cisplatin treatment. ERCC2, a target gene of miR-770-5p that participates in the NER system, was negatively regulated by miR-770-5p. siRNA-mediated silencing of ERCC2 reversed the inhibition of apoptosis resulting from miR-770-5p downreglation in A2780S cells. A comet assay confirmed that this restoration of cisplatin chemosensitivity was due to the inhibition of DNA repair. These findings suggest that endogenous miR-770-5p may function as an anti-oncogene and promote chemosensitivity in OVC, at least in part by downregulating ERCC2. miR-770-5p may therefore be a useful biomarker for predicting chemosensitivity to cisplatin in OVC patients and improve the selection of effective, more personalized, treatment strategies

Smoking Dose Modifies the Association between C242T Polymorphism and Prevalence of Metabolic Syndrome in a Chinese Population

Background: The C242T polymorphism of the CYBA gene that encodes p22phox, a component of NADPH oxidase, has been found to modulate superoxide production. Oxidase is a major source of the superoxide anion that contributes to individual components of metabolic syndrome. We examined the relationship of the C242T polymorphism with the prevalence of metabolic syndrome in a Chinese population, taking account of consumed cigarette amounts. Methodology/Principal Findings: In 870 participants, we collected biomarkers related to metabolic syndrome and detailed history of smoking and genotyped the C242T polymorphisms. After adjustment for covariates, the CT/TT genotypes were associated with a lower risk of metabolic syndrome (P = 0.0008). The odds of having metabolic syndrome in the CT/TT participants were 0.439 (95%CI: 0.265, 0.726), while for CC participants the odds were 1.110 (95%CI: 0.904, 1.362). There was significant (P = 0.014) interaction between the C242T polymorphism and smoking status in relation to the prevalence of metabolic syndrome. For smokers who smoke no less than 25 pack-years, those with CT/TT genotypes had lower risk of metabolic syndrome as compared with CC polymorphism carriers (P = 0.015). In the multiple regression analysis, the CT/TT genotypes were significantly associated with lower serum concentration of triglycerides both in all subjects and smokers; furthermore, the CT/TT genotypes were also related to smaller waist circumference in smokers. Conclusions: Our study suggests that the C242T gene polymorphism is indeed related to the prevalence of metaboli

Governing the Moral Hazard in China’s Sponge City Projects: A Managerial Analysis of the Construction in the Non-Public Land

As a new development mode for solving urban water control problems, a sponge city has been widely concerned and steadily promoted in China. The source engineering in the non-public land is called a low-impact development system. Although it is an important part of a sponge city, the effect of its construction and operation is not ideal at present. The main reason for this lies in the moral hazard behavior of the developer who is the responsible party and the agent. In this research, governance mechanisms for preventing the moral hazard behavior are put forward. Additionally, corresponding models are constructed and designed based on incentive theory and project governance theory. Furthermore, the existence constraint conditions of different governance mechanisms are obtained, as well as the optimal reward and punishment of the government. Meanwhile, this paper calculated the expected returns of the government and the developer. The result shows that project completion probability and supervision cost are two key factors affecting the choice of the governance mechanisms. According to different value of project completion probability and supervision cost, this paper evaluated the selection conditions of different governance mechanisms. Moreover, this research puts forward some governing tactics and suggestions for preventing the developer’s moral hazard behavior, in order to improve the effect of project construction, and promote sponge cities to develop in a more efficient and sustainable manner

Framework for Designing Project Delivery and Contract Strategy in Chinese Construction Industry Based on Value-Added Analysis

A project delivery method (PDM) and project contract strategy (PCS) are two critical factors for project success in construction industry. Currently, there are several options available for them in the construction industry, with several innovative PDMs and PCSs emerging along with the development of the industry. This paper maintains that the PDM should be designed in combination with the PCS given their close relationship. This paper focuses on the design of project delivery and contract strategy (PDCS), with the aim of developing a conceptual framework for the PDCS design. The design process is divided into two stages: preliminary design and detailed design, taking engineering design as a frame of reference. In the design process, the value-added analysis of project objectives is adopted as a tool to make decisions at different stages. The framework proposed in this paper is intended to provide a more distinct and direct path to improving PDCS selection or design, as well as enhancing the reliability of decision-making

Megaproject Governance Research in China: A Review and Visual Analysis from the Whole Life Cycle Perspective

At present, the number of megaprojects in China is rapidly increasing, with multiple projects ranked as the world’s highest. To showcase the theoretical and practical achievements of China’s megaprojects to the world, a visual analysis was carried out using CiteSpace software based on data from 520 pieces of core literature related to megaproject governance in the CNKI database from 2001 to 2023. Our findings indicate that: the number of publications has significantly increased since 2016, with a core group of researchers significantly contributing to this field, while team collaboration needs to be strengthened; the research hotspots include social stability risks, transaction governance, and innovation in megaprojects; the research can be divided into three stages, starting with early macro-studies, gradually forming clear and hierarchical research branches, and beginning to develop in the direction of Chinese characteristics and innovative deepening in recent years; research on the megaproject governance system is becoming gradually more enriched, but there is a lack of research at the levels of top-level governance and operational governance. Finally, in order to advance the study of megaproject governance in China, future research directions are suggested