Macrophages-aPKCɩ-CCL5 Feedback Loop Modulates the Progression and Chemoresistance in Cholangiocarcinoma

Background Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. Methods 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKCɩ in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKCɩ-siRNA and detect the antitumor effects in CCA. Results M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKCɩ expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKCɩ expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGFβ1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKCɩ-mediated NF-κB signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKCɩ-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKCɩ-siRNA significantly inhibited macrophages infiltration and CCA progression. Conclusion our study demonstrates the role of Macrophages-aPKCɩ-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKCɩ-siRNA and GEM co-delivered by liposomes for CCA.

Gene Therapy Approach for Treating DeltaF508, G542X and R553X Cystic Fibrosis Mutations Through the Usage Of CRISPR Prime Editing

Cystic Fibrosis is a rare genetic disease that affects the transmission of chloride ions due to mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Even though there are nearly 2000 mutations identified to be related to the condition, the most common mutation is F508del; deletion of a phenylalanine residue at 508. On the other hand, G542X which is a Class I mutation is also found very commonly and there are no modulator treatments available for it. Furthermore, it was investigated that R553X mutation can as well be corrected simultaneously with G542X mutation. Therefore, the main focus is on designing a gene therapy project that can correct all these three mutations at once by utilizing the prime editing technique via lipid-based delivery. In this way, the mutations can be edited through plasmids that were designed containing 2 pegRNAs and the Cas enzyme. To implement such an approach efficiently, both ex vivo, an animal model, and in vivo steps are to be designed. For the cell line, fibroblasts are selected due to their simplicity and low cost. The animal model of the experiment is determined to be a ferret concerning the high similarity to the human's CFTR protein and finally, the procedure will follow on a direct application in human Cystic Fibrosis patients. The plasmids are thought to be delivered through a cationic liposome that will reach the lungs with the aid of a nebulizer. At the last stage of the experimental procedure, Sanger Sequencing will be done to see if the desired edit within the CFTR has been performed successfully, and Next Generation Sequencing will be executed to see if there has been an off-target mutation in the remainder of the genome. Whereas for detecting the presence and expression of CFTR protein in humans, immunodetection with flow cytometry will be conducted.

Cationic Lipid-Based Formulations for Encapsulation and Delivery of Anti-EFG1 2’OMethylRNA Oligomer

Background: The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2’OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. Results: To accomplish this, cationic 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoylphosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2’OMe oligomers and consequently inhibit C. albicans filamentation up to 60 % after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg=3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection.Conclusions: In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2’OMe oligomers.

Macrophages-aPKCι-CCL5 Feedback Loop Modulates the Progression and Chemoresistance in Cholangiocarcinoma

Background: Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. Methods: 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKCi in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKCi-siRNA and detect the antitumor effects in CCA. Results: M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKCi expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKCi expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGFb1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKCi-mediated NF-kB signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKCi-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKCi-siRNA significantly inhibited macrophages infiltration and CCA progression. Conclusion: our study demonstrates the role of Macrophages-aPKCi-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKCi-siRNA and GEM co-delivered by liposomes for CCA.

Preparation and Characterization of Hypoxia Sensitive Cationic Liposomal Doxorubicin and Evaluation Its Anti-tumor Activity in Mice Bearing C26 Tumors

The goal of this study was to prepare cationic nanoliposomal doxorubicin in which PEG molecule attached to the liposome via a hypoxia-sensitive azo linker. The cost-effective hypoxia-sensitive molecule (HSM) was synthesized composing of C18H37 lipophilic tail, azo-linker, and PEG2000 hydrophilic molecule. The NMR and FTIR were employed to characterize the HSM. Then, this compound was post-inserted into the cationic liposome (Cat-lip), and PEG-Azo-Cat-lip was prepared and characterized using DLS. In vitro release and cytotoxicity studies were performed in normoxic and hypoxic conditions. In vivo biodistribution and anti-tumor activities of the formulations were studied on mice bearing C-26 colon carcinoma tumor model and compared with PEGylated liposomal doxorubicin (Caelyx®). Besides, the histological test confirmed the formulation biosafety on healthy mice. The results of NMR and FTIR indicated the synthesis of HSM. The decrease in the zeta-potential of formulation from +18.4 mV for Cat-lip to +6.1 mV along with the increase in the size of the PEG-Azo-Cat-lip indicated the successful post-insertion of HSM. The release study showed that PEGylation results in the more stable PEG-Azo-Cat-lip compared to the Cat-lip. The increased cytotoxicity of the PEG-Azo-Cat-lip in the hypoxic condition also indicated the cleavage of the azo-linker in the hypoxic environment. In vivo biodistribution using animal imaging has shown higher tumor accumulation of the PEG-Azo-Cat-lip than Cat-lip during the 120 h of the study. The results of anti-tumor activities and biosafety of the formulations also showed the higher efficiency of the PEG-Azo-Cat-lip compared with the Cat-lip. The results of this study, indicated the anti-tumor efficacy of this hypoxia-sensitive which merits further investigation.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2–3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.