Human Foreskin Fibroblast Cell Line Research Articles

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report.

Stable Cas9 (CRISPR-associated protein 9)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas9 system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time. This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes. Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction. The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy. YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research.

Green synthesis of gold nanoparticles from Coleus scutellarioides (L.) Benth leaves and assessment of anticancer and antioxidant properties

Gold nanoparticles (AuNPs) have gained significant attention in the field of biomedicine due to their versatile potential and diverse applications. In this study, AuNPs were biologically synthesized using the leaf extract of Coleus scutellarioides (L.) Benth to explore the in vitro anticancer and antioxidant properties. The synthesized AuNPs underwent thorough characterization through various analyses, the UV–Visible Spectroscopy (UV–Vis) of the AuNPs displayed a peak at 532 nm, indicating the occurrence of surface plasmon resonance. The crystalline structure of the AuNPs was confirmed by analyzing the X-ray Diffraction pattern (XRD). Moreover, Transmission Electron Microscopy (TEM) analysis revealed a spherical morphology for the AuNPs, with an average particle size of 40.10 nm, consistent with the measurements obtained from Dynamic light scattering (DLS). After that, the biological performance of those biomolecules functionalized AuNPs was studied. This study investigated the potential cytotoxic effects of synthesized AuNPs using the MTT assay. The assay was employed to evaluate the cell viability against the human breast cancer cell line (MDA-MB-231) compared to the human foreskin fibroblast cell line (Hs-27), with concentrations ranging from 7.5 to 60 µg/ml over 24 h. The study findings reveal a concentration-dependent impact of synthesized AuNPs on MDA-MB-231 cells. As the AuNPs concentration increases, there is a corresponding increase in the inhibition of MDA-MB-231 cells, reaching an IC50 value of 36.10 µg/ml within a 24-hour period. Contrarily, the synthesized AuNPs exhibited no apparent toxicity against the noncancerous normal Hs-27 cells. Furthermore, the antioxidant efficacy of AuNPs was evaluated through a free radical scavenging DPPH assay. These findings suggest the potential of Coleus scutellarioides (L.) Benth leaf extract as an effective bio-reductant for synthesizing stable AuNPs with anticancer activity and antioxidant properties.

In vitro pro-apoptotic and anti-metastatic potentials of harmaline-silver containing folate-linked chitosan nano-drug delivery system.

Harmaline and green-synthesized silver nanoparticles were encapsulated by folate-linked chitosan molecules as a receptor-mediated drug delivery system to evaluate its pro-apoptotic and anti-metastatic potentials on human ovarian (A2780) and epithelioid (PANC) cancer cells. The Ag nanoparticles (AgNP) were synthesized utilizing an herbal bio-platform (Bistorta officinalis) and embedded with harmalin. The Harmaline-ag containing folate-linked chitosan nanoparticles (HA-fCNP) were synthesized utilizing the ionic gelation method. Both the AgNP and HA-fCNP nanoparticles were characterized by DLS, FESEM, and Zeta potential analysis. Moreover, the chemical properties of HA-fCNP and the crystallinity of AgNPs were determined by applying FTIR and XRD methods, respectively. The HA-fCNP cytotoxicity was analyzed on A2780, PANC, and HFF cell lines. Moreover, pro-apoptotic and anti-metastatic potentials of HA-fCNP were studied by analyzing the BAX-BCL2 and MMP2-MMP9 gene expression profiles, respectively. The A2780 cellular death was determined by AO/PI and flow cytometry methods. The HA-fCNP significantly exhibited a selective cytotoxic impact on A2780 and PANC cancerous cell lines compared with normal human foreskin fibroblast (HFF) cells. The increased SubG1-arrested A2780 cells and up-regulated BAX gene expression following the increased treatment concentrations of hA-fCNP indicated its selective pro-apoptotic activity on A2780 cells. Also, the notable down-regulated expressions of MMP2 and MMP9 metastatic genes following the increasing doses of HA-fCNP treatment on A2780 cells confirmed its anti-metastatic activity. The cancer-selective cytotoxicity, apoptotic, and anti-metastatic properties of HA-fCNP are considered the appropriate properties of an anticancer compound.

Designing and fabrication of colloidal nano-phytosomes with gamma-oryzanol and phosphatidylcholine for encapsulation and delivery of polyphenol-rich extract from pomegranate peel

Nano-carriers are well-known delivery systems to encapsulate different bioactive compounds and extracts. Such nano-systems are used in various food and drug areas to protect active ingredients, increase bioavailability, control the release, and deliver bioactive substances. This study aimed to design and fabricate a stable colloidal nano-delivery system to better preserve the antioxidant properties of pomegranate peel extract (PPE) and protect its sustained release in a gastrointestinal model. To achieve this goal, a nano-phytosomal system was fabricated with plant-based, cost-effective, and food-grade compounds, i.e., phosphatidylcholine (PC) and gamma-oryzanol (GO) for encapsulation of PPE. To fabricate the nano-phytosomes, thin film hydration/sonication method was used. The parameters of particle size, zeta potential, polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) were investigated to evaluate the efficiency of the produced nano-system. In summary, the size, zeta potential, PDI, LC, and EE of homogenous spherical PC-GO-PPE nano-phytosomes (NPs) in the ratio of 8:2:2 % w/w were achieved as 60.61 ± 0.81 nm, −32.24 ± 0.84 mV, 0.19 ± 0.01, 19.13 ± 0.30 %, and 95.66 ± 1.52 %, respectively. Also, the structure of NPs was approved by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). The optimized NPs were stable during one month of storage at 4 °C, and changes in the size of particles and PPE retention rate were insignificant (p > 0.05). The nano-encapsulation of PPE significantly decreased the loss of its antioxidant activity during one month of storage at 4 °C. The optimized NPs exhibited prolonged and sustained release of PPE in a gastrointestinal model, so that after 2 h in simulated gastric fluid (SGF) and 4 h in simulated intestinal fluid (SIF), 22.66 ± 2.51 % and 69.33 ± 4.50 % of initially loaded PPE was released, respectively. Optimized NPs had considerable cytotoxicity against the Michigan Cancer Foundation-7 cell line (MCF7) (IC50 = 103 μg/ml), but not against Human Foreskin Fibroblast cell line (HFF-2) (IC50 = 453 μg/ml). In conclusion, spherical PC-GO-PPE NPs were identified as a promising delivery system to efficiently encapsulate PPE, as well as protect and preserve its bioactivity, including antioxidant and cytotoxicity against cancer cell line.

Green Synthesis and Bioactivity of Aliphatic N-Substituted Glycine Derivatives.

Standard amino acids have an asymmetric α-carbon atom to which -COOH, -NH2, -H, and -R groups are bonded. Among them, glycine is the simplest (R = -H) with no asymmetric carbon, and other natural amino acids are C-substituted of glycine. Here, we have designed and made a green synthesis of some new N-substituted glycine derivatives with R-(NH)CH2-COOH formula, where R is flexible and hydrophobic with different chain lengths and benches of the type propyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, 2-aminoheptyl, and octyl. These glycine derivatives were characterized by recording their melting points and FT-IR, mass, 1H NMR, and 13C NMR spectra. DFT studies revealed that 2-aminoheptyl glycine had the highest electronegativity value and can thus act as a good bidentate ligand for the metal centers. ADME comparative results and bioavailability radars indicated that both octyl- and 2-aminoheptyl glycine had the most lipophilicity, making them good agents in cell passing. Furthermore, lipophilicity determination showed that octyl glycine was the best and propylgly was more soluble than others. Based on solubility, lipophilicity, and dipole moment values, propyl- and 2-aminoheptyl-glycine were considered for bio-macromolecular interaction studies. Thus, the interaction of these two agents with DNA and HSA was studied using absorption spectroscopy and circular dichroism techniques. Due to the presence of the R-amine group, they can interact with the DNA by H-binding and hydrophobicity, while electrostatic mode could not be ruled out. Meanwhile, molecular docking studies revealed that octyl- and 2-aminoheptyl glycine had the highest negative docking energy, which reflects their higher tendency to interact with DNA. The DNA binding affinity of two candidate AAs was determined by viscosity measurement and fluorescence emission recording, which confirms that groove binding occurs. Also, the toxicity of these synthesized amino acid derivates was tested against the human foreskin fibroblast (HFF) cell line. They showed IC50 values within the range of 127-344 μM after 48 h with the highest toxicity for 2-aminoheptyl glycine.

Methanolic extract of Kigelia africana and wound healing: an in vitro study.

Kigelia africana (Lam.) Benth. (Bignoniaceae) syn. Kigelia pinnata (Jacq. DC) is a tropical plant that is native to tropical Africa. The aim of this study was to determine if a methanolic extract prepared from Kigelia africana (KAE) can promote wound healing in treated human normal epidermal keratinocyte (HaCaT) cells and human normal foreskin fibroblast cell line (BJ) cells compared with untreated cells. Experimental steps included: the methanolic extraction of the leaf and fruit of the Kigelia africana plant; the preparation of HaCaT and BJ cell lines; cell culture with a stable tetrazolium salt-based proliferation assay; and the evaluation of the wound healing effect of KAE (2μg/ml) in BJ and HaCaT cells. The phytochemical contents of KAE were determined using liquid chromatography quadrupole time-of-flight mass spectrometry. The following molecules were identified as being present in the KAE, among others: cholesterol sulfate; lignoceric acid; embelin; isostearic acid; linoleic acid; dioctyl phthalate; arg-pro-thr; 15-methyl-15(S)-PGE1; sucrose; benzododecinium (Ajatin); and 9-Octadecenamide (oleamide). KAE effected faster wound healing in treated cells compared with untreated cells for both cell lines. HaCaT cells that had been mechanically injured and treated with KAE healed completely in 48 hours compared with 72 hours for untreated HaCaT cells. Treated BJ cells healed completely in 72 hours compared with 96 hours for untreated BJ cells. Concentrations of KAE up to 300μg/ml had a very low cytotoxic effect on treated BJ and HaCaT cells. The experimental data in this study support the potential of KAE-based wound healing treatment to accelerate wound healing.

An Exploration of Osteosarcoma Metastasis Diagnostic Markers Based on Tumor-Associated Neutrophils.

The high rate of the recurrence and metastasis of osteosarcoma (OS) is the major cause of its poor prognosis. There is a strong correlation between tumor-associated neutrophils (TANs) and tumor progression, progression, and metastasis. This study aimed to identify potential markers that could predict OS metastasis based on analysis of TANs in the tissues of OS patients. A single-cell sequencing dataset (GSE152048), containing seven primary OS lesions, two recurrent OS lesions, and two lung metastatic OS lesions was used for TANs subset identification using the R software (version 4.1.0, R Project for Statistical Computing, Vienna, Austria; https://www.r-project.org/). Immune cell infiltration and immune score were analyzed using CIBERSORT algorithm and ESTIMATE database, respectively. The differentially expressed genes (DEGs) of TANs were used for weighted gene co-expression network analysis (WGCNA) to screen key genes associated with OS metastasis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were used to analyze the functions and signaling pathways involved in key genes. The mRNA levels of protein phosphatase 2 regulatory subunit B'gamma (PPP2R5C) were validated in human osteosarcoma cell lines U2-OS and MG63, human normal cervical endometrial cell line HUCEC, and human foreskin fibroblast (HFF-1) cell line by real-time qPCR (RT-qPCR). PPP2R5C-siRNA991 was transfected into U2-OS and MG63 for 48 h, then the expression levels of PPP2R5C, AKT serine/threonine kinase (AKT), and phospho-AKT (p-AKT) were determined by RT-qPCR and Western blotting. Cell proliferation, migration, and apoptosis were measured by cell counting kit-8 (CCK-8), Transwell, and flow cytometry, respectively. We identified TANs subsets in primary, metastatic, and recurrent OS. Immune infiltration analysis showed that TANs were expressed in OS. Compared with non-metastatic OS, metastatic OS had lower stromal score, immune score, ESTIMATE score, and higher tumor purity. WGCNA classified DEGs into five clusters, according to their function and identified PPP2R5C, protein phosphatase 2 regulatory subunit B'epsilon (PPP2R5E), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) and CREB binding protein (CREBBP), as potential markers that may affect TANs-induced OS metastasis via hypoxia inducible factor 1 (HIF-1), phosphatidylinositol 3-kinases (PI3K)-AKT and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathways. In vitro experiments demonstrated that the mRNA and protein expressions of PPP2R5C, PPP2R5E, YWHAG, and CREBBP were highly expressed in U2-OS and MG63 cells (p < 0.01). Furthermore, PPP2R5C reduced proliferation and migration (p < 0.01) and increased apoptosis and p-AKT protein levels in U2-OS and MG6 cells (p < 0.01). PPP2R5C affects OS metastasis via PI3K/AKT pathway, which may be a potential marker for OS metastasis and recurrence.

Chitosan Nanocomposites as Wound Healing Materials: Advances in Processing Techniques and Mechanical Properties

This review discusses the increasing potential of chitosan nanocomposites as viable materials capable of targeting these debilitating factors. This review focuses on various techniques used to process chitosan nanocomposites and their mechanical properties. Chitosan nanocomposites are regarded as highly effective antimicrobials for the treatment of chronic wounds. Chitosan nanocomposites, such as chitosan/polyethylene and oxide/silica/ciprofloxacin, demonstrate efficient antibacterial activity and exhibit no cytotoxicity against Human Foreskin Fibroblast Cell Lines (HFF2). Other studies have also showcased the capacity of chitosan nanocomposites to accelerate and improve tissue regeneration through increment in the number of fibroblast cells and angiogenesis and reduction of the inflammation phase. The layer-by-layer technique has benefits, ensuring its suitability in preparing chitosan nanocomposites for drug delivery and wound dressing applications. While the co-precipitation route requires a cross-linker to achieve stability during processing, the solution-casting route can produce stable chitosan nanocomposites without a cross-linker. By using the solution casting method, fillers such as multi-walled carbon nanotubes (MWCNTs) and halloysite nanotubes (HTs) can be uniformly distributed in the chitosan, leading to improved mechanical properties. The antibacterial effects can be achieved with the introduction of AgNPs or ZnO. With the increasing understanding of the biological mechanisms that control these diseases, there is an influx in the introduction of novel materials into the mainstream wound care market.

Nanoliposomal peptides derived from Spirulina platensis protein accelerate full-thickness wound healing

Spirulina platensis is a type of blue-green algae that contains large amounts of protein with therapeutic effects. The present study was performed to investigate the effects of encapsulated Spirulina protein hydrolysates (SPH) with nanoliposomes (NLPs) in reducing wound healing period. SPH-loaded NLPs showed the size and zeta potential of 158 nm and −48 mV, respectively; as well as a uniform non-aggregated morphology. In-vitro MTT toxicity studies on the Human Foreskin Fibroblast (HFFF-2) cell line exhibited that the hydrolyzed peptides had no toxic effect and increased cell growth. The scratch test confirmed the MTT results. For in-vivo study, 162 mice were divided into nine groups, including the mice groups treated with blank gel, blank NLPs, and those treated with 2.5, 5, and 10 % SPH and SPH-loaded NLPs. The histopathological assessment was done to investigate rate of fibroblast proliferation and epithelialization. Immunofluorescence staining for bFGF, CD31, COL1A was conducted. The results showed that the mice group treated with SPH-NLPs showed higher wound contraction, epithelization, fibroblast proliferation, and higher expressions for bFGF, CD31, COL1A compared with blanks and other groups. In conclusion, the derived and encapsulated peptides showed significant effects in accelerating wound healing via angiogenesis and collagen production.

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report.

BackgroundFibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have wound-healing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells.ObjectiveGeneration and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs).Materials and MethodsIn this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flow-cytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45.ResultsThe YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics.ConclusionThis study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine.

Co-Functionalization of Gold Nanoparticles with C7H2 and HuAL1 Peptides: Enhanced Antimicrobial and Antitumoral Activities.

The functionalization of nanoparticles with therapeutic peptides has been pointed out as a promising strategy to improve the applications of these molecules in the field of health sciences. Peptides are highly bioactive but face several limitations such as low bioavailability due to the difficulty of overcoming the physiological barriers in the body and their degradation by enzymes. In this work, gold nanoparticles (AuNPs) were co-functionalized with two therapeutic peptides simultaneously. The peptides from the complementary determining region of monoclonal antibodies, composed of the amino acid sequences YISCYNGATSYNQKFK (C7H2) and RASQSVSSYLA (HuAL1) were chosen for having exhibited antitumor and antimicrobial activity before. The peptides-conjugated AuNPs were characterized regarding size, morphology, and metal concentration by using TEM, dynamic light scattering, and ICP-OES techniques. Then, peptides-conjugated AuNPs were evaluated regarding the antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. The antitumoral activity was evaluated in vitro by cell viability assays with metastatic melanoma cell line (B16F10-Nex2) and the cytotoxicity was evaluated against human foreskin fibroblast (Hs68) cell line. Finally, in vivo assays were performed by using a syngeneic animal model of metastatic melanoma. Our findings have highlighted the potential application of the dual-peptide AuNPs in order to enhance the antitumor and antimicrobial activity of peptides.

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.

Atorvastatin Entrapped Noisome (Atrosome): Green Preparation Approach for Wound Healing

The present study aimed to formulate atorvastatin niosome (Atrosome) through an ultrasonic technique and to determine its contribution to the extent of wound healing in an animal model. The optimized Atrosome formulation (Atrosome-2) was stable at 4 °C for 3 months. Differential scanning calorimetry (DSC), ATR-Fourier transform infrared spectroscopy (ATR-FTIR), and powder X-ray diffraction (PXRD) analysis revealed that atorvastatin (ATR) was well encapsulated within the niosomes either in a stabilized amorphous form or a molecularly dispersed state. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscope (AFM) confirmed the spherical nature of the Atrosomes. The optimized formulation showed polydispersity index, particle size, drug encapsulation efficiency (EE%), and zeta potential of 0.457 ± 0.05, 196.33 ± 6.45 nm, 86.15 ± 0.58 %, and − 20.73 ± 0.98 mV, respectively. ATR release from the Atrosome gel followed the first-order kinetic model and showed no cytotoxicity in the in vitro cytotoxicity test. Cell viability (human foreskin fibroblast cell line) was nearly 99%. An excision wound model was also applied in male Wistar rats to examine the in vivo efficacy of the optimized formulation, followed by investigating malondialdehyde (MDA, an end-product of lipid peroxidation), superoxide dismutase (SOD, an endogenous antioxidant), hydroxyproline levels, and glutathione peroxidase (GPx) in skin tissue samples. MDA significantly decreased in the Atrosome gel group after 21 days, while GPx, SOD, and hydroxyproline levels demonstrated an increase. According to histological results, rats receiving Atrosomes were treated effectively faster when compared to the other formulation used.

Abstract PO-041: Systemic screening of gene delivery methods in pancreatic ductal adenocarcinoma cells

Abstract Deaths in the United States due to Pancreatic Ductal Adenocarcinoma (PDAC) has risen steadily since 1990, and PDAC is expected to be the second leading cause of cancer death by 2025. Such a dismal prognosis is mainly attributed to the fact that tumors are detected too late for an effective treatment. Additionally, even when detected early, PDAC is challenging to treat with existing treatment options. Thus, there is a need for improved treatment strategies for pancreatic cancer patients. Great efforts have been made to develop and test new targets in various model systems such as tumor cell lines, tumor organoid models, and patient-derived xenografts (PDXs). We previously demonstrated a proof-of-principle of cellular reprogramming to pluripotent state to model PDAC progression and utilized it as a discovery tool for early detection markers for PDAC. However, reprogramming efficiency was remarkably low and it is, at least in part, attributed to uneven gene delivery efficiencies across cell types in human pancreatic normal and cancer cells. Intriguingly, classical ductal cell types were more resistant to transduction with conventional VSV-G-pseudotyped lentivirus (LeV) than squamous subtype pancreatic cancer. To better understand the susceptibility of different types of pancreatic cancer cells to viral transduction and improve the gene delivery efficiency, we analyzed transduction of LeV, Sendai Virus (SeV), and episomal vector transfection efficiencies in classical ductal and squamous cell types. We used human pancreatic duct epithelial and foreskin fibroblast cell lines as controls. We found that transduction efficiency of LeV in both types of PDAC cell lines are significantly lower compared to control cell lines and considerably higher in squamous type compared to classical ductal type. In contrast, transduction efficiency of SeV was similar for both classical ductal and squamous types of PDAC cell lines and significantly higher compared to LeV efficiency (90% vs 5-25% at MOI 1). We also found that nucleofection transfection efficiency of episomal vector is significantly higher in squamous cell type compared to classical ductal cells (67% vs 55%). This is significantly higher than transduction efficiency of LeV (25 and 5% at MOI 1 correspondingly) but lower compared to SeV transduction efficiency. Thus, both SeV and nucleofection delivery methods show higher efficiency compared to LeV and can be successfully used as gene delivery methods in PDAC cell lines. By completing this study, we can provide a tailored gene delivery method for pancreatic cancer, and this information can be harnessed for cellular reprograming of human PDAC as well as testing newly developing targets in other model systems in vitro and ex vivo.This work is supported by OHSU/CEDAR project award 68182-939-000. Citation Format: Dmytro Grygoryev, Taelor Ekstrom, Jason M. Link, Rosalie C. Sears, Jungsun Kim. Systemic screening of gene delivery methods in pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-041.

Effect of targeting ligand designation of self-assembly chitosan-poloxamer nanogels loaded Paclitacel on inhibiting MCF-7 cancer cell growth

In this study, we investigated two formulations of chitosan-Pluronic P123 with different folate ligand designation for targeted delivery of Paclitaxel (PTX), in which folic acid (FA) was directly conjugated to chitosan (FA-Cs-P123) or substituted onto P123 (Cs-P123-FA). The results showed that the FA content of Cs-P123-FA was determined at 0.71 wt/wt% which was significantly higher than that of FA-Cs-P123 (0.31 wt/wt%). Two copolymers were low critical gel concentrations (CGC). FA-Cs-P123 and Cs-P123-FA nanogels performed high PTX encapsulation efficiency reaching 95.57 ± 5.51 and 92.51 ± 6.68 wt/wt%, respectively. Transmission electron microscopy (TEM) and zeta potential analysis indicated that the PTX-loaded nanogels were spherically formed around 60 nm in diameter along with positive charge. Furthermore, the PTX release profile was slow and it was controlled by the pH of the medium. In particular, in vitro biocompatibility assays indicated that both FA-Cs-P123 and Cs-P123-FA exhibited good biological compatibility with a human foreskin fibroblast cell line and well uptake efficiency into MCF-7 cancer cells. Cs-P123-FA nanogel significantly enhanced the cytotoxicity of PTX in comparison with FA-Cs-P123. The result indicates that Cs-P123-FA nanogels with a higher decorated FA content perform a better targeting efficiency; therefore, they could have great potential application towards breast cancer treatment.

Investigation of 3-sulfamoyl coumarins against cancer-related IX and XII isoforms of human carbonic anhydrase as well as cancer cells leads to the discovery of 2-oxo-2H-benzo[h]chromene-3-sulfonamide - A new caspase-activating proapoptotic agent.

Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives. Prepared compounds were tested invitro for inhibition of four physiologically relevant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed low nanomolar KI values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of A431cell growth with low nanomolar IC50 values. We demonstrated that 10q possessed a concentration-dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable lead for further development of new-generation anticancer agents.

An in vitro investigation into targeted paclitaxel delivery nanomaterials based on chitosan-Pluronic P123-biotin copolymer for inhibiting human breast cancer cells

Targeted chemotherapy is a current research trend in cancer treatment that aims to improve the effectiveness of drug access to cancer cells. Among the vitamins, biotin has been identified as an effective targeting agent in drug delivery to cancer cells. In this work, novel biotin-conjugated chitosan-Pluronic P123, termed Cs–P123-Bio, was developed as a carrier vehicle for delivering paclitaxel (PTX) to the tumour target. The results show that Cs–P123-Bio was stable with a low critical micelle concentration (31.83 ± 4.25 μg/mL), and the encapsulation efficacy and drug loading of Cs–P123-Bio were around 94.67 ± 3.67% and 1.89 ± 0.07%, respectively. Transmission electron microscopy (TEM), dynamic light scattering (DLS) and electrophoretic light scattering (ELS) analysis indicated that the Cs–P123-Bio nanoparticles loaded with PTX formed as a spherical shape with an average diameter of 63.4 ± 0.6 nm and a positive charge. Furthermore, the PTX release profile was slow and controlled by the pH of the medium. In particular, in vitro biocompatibility assays indicated that Cs–P123-Bio exhibited good biological compatibility with a human foreskin fibroblast cell line and Cs–P123-Bio manifested good internalization efficiency in MCF-7 cells. Cs–P123-Bio also enhanced the cytotoxicity of PTX in comparison with Cs–P123. Therefore, our approach could suggest excellent potential for tumour targeting in cancer therapy.

Anethum Graveolens Essential Oil Nanoemulsions (AGEO-NE) as an Exclusive Apoptotic Inducer in Human Lung Adenocarcinoma (A549) Cells

Anethum graveolens essential oil (AGEO) is used as an anticancer compound that can be made more efficacious by improving its biosolubility and biocompatibility. In the current study, we aimed to improve AGEO bioactivity using the nanoemulsion-based encapsulation technique. To formulate stable AGEO-NE, an ultrasonication method was utilized. The size, stability, and morphology of the AGEO-NE was measured, and then its cytotoxic impacts were evaluated on adenocarcinomic human alveolar basal epithelial cells (A549). The ferric reducing antioxidant power (FRAP) test was done to measure the antioxidant activity of AGEO-NE. Its cytotoxic property was analyzed by measuring the viability percentage of cancer (A549) and normal human foreskin fibroblast (HFF) cell lines after increasing AGEO-NE treatment doses. AGEO-NE apoptotic activity was evaluated by studying the flow cytometry of the cells and measuring the apoptotic gene expression profile (Cas-3 and Cas-8) in the A549 cell line. The results showed a significant correlation between the increase of AGEO-NE concentrations and decrease of cancer (A549) cell viability (p < 0.05) when AGEO-NE was compared with normal HFF cells. Thus, AGEO-NE can be an efficient novel apoptosis and antioxidant inducer for human lung cancer cells without any undesirable side effects. However, further In Vitro and In Vivo studies are needed to confirm the results.

Fabrication of ciprofloxacin-loaded chitosan/polyethylene oxide/silica nanofibers for wound dressing application: In vitro and in vivo evaluations

Silica plays an effective role in collagen creation; hence, the degradation products of silica-based materials accelerate wound healing. In this regard, chitosan/polyethylene oxide/silica hybrid nanofibers were prepared by the combining the sol–gel method with electrospinning technique to accelerate the wound healing process. Ciprofloxacin, as an antibacterial drug, was then added to the electrospinning mixture. The nanofibers were characterized by SEM, EDX, X-ray mapping, TEM, TGA, FTIR, and XRD analysis. The degradation, swelling ratio, and release of ciprofloxacin were investigated in PBS. The prepared nanofiber could absorb water, maintain its morphological integrity during the degradation process, and gradually release ciprofloxacin. The nanofibers revealed an efficient antibacterial activity against Escherichia coli and Staphylococcus aureus. Cell viability assays showed that the nanofibers had no cytotoxicity against L929 mouse fibroblast and HFFF2 human foreskin fibroblast cell lines. The potential of the chitosan/polyethylene oxide/silica/ciprofloxacin nanofiber for healing full-thickness wound was assessed by applying the scaffold in the dorsal cutaneous wounds of the Balb/C mice. The white blood cell counts of the animals indicated the nanofiber-treated mice compared with the untreated ones had less infection and inflammation. According to the histopathologic data, the prepared nanofiber accelerated and enhanced tissue regeneration by increasing fibroblast cells and angiogenesis as well as decreasing the inflammation phase. The findings suggest that the prepared antibacterial scaffold with drug delivery properties could be an appropriate candidate for many medical and hygienic applications, especially as a bio-compatible and bio-degradable wound dressing.

In vitro and in vivo growth inhibitory activities of cryptolepine hydrate against several Babesia species and Theileria equi.

Piroplasmosis treatment has been based on the use of imidocarb dipropionate or diminazene aceturate (DA), however, their toxic effects. Therefore, the discovery of new drug molecules and targets is urgently needed. Cryptolepine (CRY) is a pharmacologically active plant alkaloid; it has significant potential as an antiprotozoal and antibacterial under different in vitro and in vivo conditions. The fluorescence assay was used for evaluating the inhibitory effect of CRY on four Babesia species and Theileria equi in vitro, and on the multiplication of B. microti in mice. The toxicity assay was evaluated on Madin–Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half-maximal inhibitory concentration (IC50) values of CRY on Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 1740 ± 0.377, 1400 ± 0.6, 790 ± 0.32, 600 ± 0.53, and 730 ± 0.025 nM, respectively. The toxicity assay on MDBK, NIH/3T3, and HFF cell lines showed that CRY affected the viability of cells with a half-maximum effective concentration (EC50) of 86.67 ± 4.43, 95.29 ± 2.7, and higher than 100 μM, respectively. In mice experiments, CRY at a concentration of 5 mg/kg effectively inhibited the growth of B. microti, while CRY–atovaquone (AQ) and CRY–DA combinations showed higher chemotherapeutic effects than CRY alone. Our results showed that CRY has the potential to be an alternative remedy for treating piroplasmosis.