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1.
Molecules ; 29(10)2024 May 16.
Article in English | MEDLINE | ID: mdl-38792206

ABSTRACT

Various conjugates with rhodamines were prepared by starting with betulinic acid (BA) and platanic acid (PA). The molecules homopiperazine and piperazine, which were identified in earlier research, served as linkers between the rhodamine and the triterpene. The pentacyclic triterpene's ring A was modified with two acetyloxy groups in order to possibly boost its cytotoxic activity. The SRB assays' cytotoxicity data showed that conjugates 13-22, derived from betulinic acid, had a significantly higher cytotoxicity. Of these hybrids, derivatives 19 (containing rhodamine B) and 22 (containing rhodamine 101) showed the best values with EC50 = 0.016 and 0.019 µM for A2780 ovarian carcinoma cells. Additionally, based on the ratio of EC50 values, these two compounds demonstrated the strongest selectivity between malignant A2780 cells and non-malignant NIH 3T3 fibroblasts. A375 melanoma cells were used in cell cycle investigations, which showed that the cells were halted in the G1/G0 phase. Annexin V/FITC/PI staining demonstrated that the tumor cells were affected by both necrosis and apoptosis.


Subject(s)
Apoptosis , Rhodamines , Triterpenes , Triterpenes/chemistry , Triterpenes/pharmacology , Triterpenes/chemical synthesis , Humans , Rhodamines/chemistry , Mice , Animals , Cell Line, Tumor , NIH 3T3 Cells , Apoptosis/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Betulinic Acid , Pentacyclic Triterpenes/pharmacology , Pentacyclic Triterpenes/chemistry , Pentacyclic Triterpenes/chemical synthesis , Cell Cycle/drug effects , Cell Survival/drug effects , Cell Proliferation/drug effects , Lupanes
2.
Viruses ; 16(5)2024 05 11.
Article in English | MEDLINE | ID: mdl-38793642

ABSTRACT

Mouse adenoviruses (MAdV) play important roles in studying host-adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.


Subject(s)
Genetic Vectors , Plasmids , Virus Replication , Animals , Mice , Genetic Vectors/genetics , Plasmids/genetics , Adenoviridae/genetics , NIH 3T3 Cells , Cloning, Molecular , Genes, Reporter
3.
Int J Mol Sci ; 25(8)2024 Apr 13.
Article in English | MEDLINE | ID: mdl-38673904

ABSTRACT

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.


Subject(s)
Benzopyrans , Glucokinase , Trypanocidal Agents , Trypanosoma cruzi , Animals , Humans , Mice , Benzopyrans/pharmacology , Benzopyrans/chemistry , Chagas Disease/drug therapy , Chagas Disease/parasitology , Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Glucokinase/metabolism , Glucokinase/antagonists & inhibitors , High-Throughput Screening Assays , Molecular Docking Simulation , NIH 3T3 Cells , Structure-Activity Relationship , Trypanocidal Agents/pharmacology , Trypanocidal Agents/chemistry , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/enzymology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology
4.
Bioorg Chem ; 147: 107374, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38636433

ABSTRACT

The incidence of idiopathic pulmonary fibrosis (IPF) has been steadily increasing each year, posing significant challenges in its treatment. In this study, we conducted the design and synthesis of 23 new inhibitors that specifically target the TGF-ß1/Smad3 pathway. Initially, we employed a cell model of TGF-ß-induced pulmonary fibrosis, using cell survival rate and HYP expression as indicators to identify the potent ingredient 5aa, which demonstrated significant anti-pulmonary fibrosis activity. Subsequently, we induced mice with bleomycin (BLM) to establish an experimental animal model of pulmonary fibrosis, and evaluated the pharmacodynamics of 5aa in vivo against pulmonary fibrosis. The alterations in HYP and collagen levels in BLM-induced pulmonary fibrosis mice were analyzed using ELISA and immunohistochemistry techniques. The results indicated that compound 5aa effectively suppressed the fibrotic response induced by TGF-ß1, inhibited the expression of the fibrotic marker α-SMA, and hindered the EMT process in NIH3T3 cells. Additionally, oral administration of 5aa demonstrated significant therapeutic effects in a mouse model of IPF, comparable to the established drug Nintedanib. Moreover, compound 5aa exhibited higher bioavailability in vivo compared to Nintedanib. These collective outcomes suggest that 5aa holds promise as a potential inhibitor of TGF-ß1/Smad3 signaling for the treatment of IPF.


Subject(s)
Idiopathic Pulmonary Fibrosis , Signal Transduction , Smad3 Protein , Transforming Growth Factor beta1 , Animals , Smad3 Protein/metabolism , Smad3 Protein/antagonists & inhibitors , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Idiopathic Pulmonary Fibrosis/chemically induced , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/antagonists & inhibitors , Mice , Signal Transduction/drug effects , Molecular Structure , Humans , Bleomycin , Structure-Activity Relationship , Mice, Inbred C57BL , NIH 3T3 Cells , Dose-Response Relationship, Drug , Male
5.
Int J Biol Macromol ; 267(Pt 2): 131389, 2024 May.
Article in English | MEDLINE | ID: mdl-38582461

ABSTRACT

This work developed Acer tegmentosum extract-mediated silver nanoparticles (AgNPs) loaded chitosan (CS)/alginic acid (AL) scaffolds (CS/AL-AgNPs) to enhance the healing of E. coli-infected wounds. The SEM-EDS and XRD results revealed the successful formation of the CS/AL-AgNPs. FTIR analysis evidenced that the anionic group of AL (-COO-) and cationic amine groups of CS (-NH3+) were ionically crosslinked to form scaffold (CS/AL). The CS/AL-AgNPs exhibited significant antimicrobial activity against both Gram-positive (G+) and Gram-negative (G-) bacterial pathogens, while being non-toxic to red blood cells (RBCs), the hen's egg chorioallantoic membrane (HET-CAM), and a non-cancerous cell line (NIH3T3). Treatment with CS/AL-AgNPs significantly accelerated the healing of E. coli-infected wounds by regulating the collagen deposition and blood parameters as evidenced by in vivo experiments. Overall, these findings suggest that CS/AL-AgNPs are promising for the treatment of infected wounds.


Subject(s)
Acer , Alginates , Anti-Bacterial Agents , Chitosan , Escherichia coli , Metal Nanoparticles , Plant Extracts , Silver , Wound Healing , Chitosan/chemistry , Chitosan/pharmacology , Metal Nanoparticles/chemistry , Silver/chemistry , Silver/pharmacology , Animals , Wound Healing/drug effects , Escherichia coli/drug effects , Mice , Acer/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , NIH 3T3 Cells , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Alginates/chemistry , Alginates/pharmacology , Escherichia coli Infections/drug therapy , Tissue Scaffolds/chemistry
6.
Int J Biol Macromol ; 267(Pt 2): 131375, 2024 May.
Article in English | MEDLINE | ID: mdl-38604424

ABSTRACT

Compostable zein-polycaprolactone (PZ) electrospun nanofiber integrated with different concentrations of Aster yomena extract loaded halloysite nanotubes (A. yomena-HNT) as bioactive nanofibrous food packaging is reported. SEM micrographs reveal heterogeneous nanofibers. A. yomena extract used in the study showed weak antioxidant activity with AAI and TEAC values of 0.229 and 0.346. In vitro, release profile over 7 days of A. yomena indicates a controlled, sustained, and prolonged release. The prepared nanofibers were effective against both gram-positive and gram-negative bacteria. The prepared composite nanofibers were rendered biocompatible and nontoxic when subjected to WST-1 and LDH assay after incubating with NIH 3T3 mouse fibroblast cell line. PZ-15 nanofiber packaging showed the best postharvest quality preservation in Black mulberry fruits after 4 days of storage at 25 °C and 85 % Rh. Moreover, the in vitro decomposition test reveals that the fabricated nanofibers decompose in the soil and do not pose as a threat to the environment.


Subject(s)
Clay , Food Packaging , Nanofibers , Nanotubes , Plant Extracts , Polyesters , Zein , Nanofibers/chemistry , Polyesters/chemistry , Zein/chemistry , Mice , Animals , Clay/chemistry , Food Packaging/methods , Nanotubes/chemistry , NIH 3T3 Cells , Plant Extracts/chemistry , Plant Extracts/pharmacology , Antioxidants/pharmacology , Antioxidants/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry
7.
Int J Biol Macromol ; 267(Pt 1): 131328, 2024 May.
Article in English | MEDLINE | ID: mdl-38574901

ABSTRACT

The management of invasive fungal infections in humans poses significant challenges due to the intricate nature of the treatment, which is both arduous and costly, necessitating routine diagnostic procedures. Consequently, this investigation aimed to formulate a chitosan-based nanoemulsion (CS NEMs) incorporating the antifungal agent undecanoic acid (UDA), characterizing these NEMs and assessing their antifungal efficacy against both filamentous and non-filamentous fungal pathogens. The CS-based UDA NEMs were synthesized by introducing the surfactant Triton X-100 and the stabilizer glycerol. Nanoparticle tracking analysis (NTA) and SEM demonstrated the CS-UDA NEMs with an average size of 145 nm and 164.5 ± 24 nm, respectively. The successful formation of CS-UDA NEMs was verified through FTIR and XRD. CS-UDA NEMs exhibited exceptional inhibition against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Candida albicans with MFC of 500, 500, 250 and 250 µg/mL, respectively. Additionally, CS-UDA NEMs displayed comparatively lower antioxidant activity as determined by DPPH and ABTS radical scavenging assays. Importantly, CS-UDA NEMs demonstrated no cytotoxic effects on NIH3T3 cells even at higher concentration (1000 µg/mL), as confirmed by cell viability and fluorescent staining assays. In conclusion, this study suggests that the developed CS-UDA NEMs hold promise as potent antifungal agents with diverse potential applications.


Subject(s)
Antifungal Agents , Chitosan , Emulsions , Fatty Acids , Chitosan/chemistry , Chitosan/pharmacology , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Emulsions/chemistry , Fatty Acids/chemistry , Mice , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Microbial Sensitivity Tests , Nanoparticles/chemistry , NIH 3T3 Cells , Antioxidants/pharmacology , Antioxidants/chemistry , Candida albicans/drug effects
8.
Cells ; 13(8)2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38667312

ABSTRACT

The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC50) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.


Subject(s)
Nanoparticles , Animals , Mice , NIH 3T3 Cells , Nanoparticles/toxicity , Nanoparticles/chemistry , RAW 264.7 Cells , Cell Survival/drug effects , Holography/methods , Quantitative Phase Imaging
9.
Biomed Phys Eng Express ; 10(3)2024 Apr 02.
Article in English | MEDLINE | ID: mdl-38564254

ABSTRACT

The high demand for bone grafts has motivated the development of implants with excellent osteogenic activity, whereas the risk of implant-associated infection, particularly given the rise of antimicrobial resistance, has compelled the development of implants with innovative antimicrobial strategies in which a small amount of bactericidal agent can effectively kill a wide range of bacteria. To induce antibacterial property, the surface of Grade-5 bone plate titanium implants used in clinical applications was modified using direct current (DC) sputter coating followed by thermal annealing. The 15 nm silver film-coated implants were thermally annealed in the furnace for 15 min at 750 °C. The modified implant surface's antibacterial efficacy againstEscherichia coli(E. coli),Staphylococcus aureus(S. aureus),Salmonella typhi, andMethicillin-resistant staphylococcus aureusbacteria has been assessed using a colony-forming assay. On the modified implant surface, the growth ofE. coliandS. aureusbacteria is reduced by 99.72%, while highly drug-resistant bacteria are inhibited by 96.59%. The MTT assay was used to assess the cytotoxicity of the modified bone-implant surface against NIH3T3 mouse fibroblast cells. The modified bone-implant surface promoted fibroblast growth and demonstrated good cytocompatibility. Furthermore, the mechanical properties of the implant were not harmed by this novel surface modification method. This method is simple and provides new insight into surface modification of commercial metallic implants to have effective antibacterial properties against various classes of bacteria.


Subject(s)
Alloys , Methicillin-Resistant Staphylococcus aureus , Silver , Animals , Mice , Titanium , Bone Plates , Escherichia coli , NIH 3T3 Cells , Staphylococcus aureus , Anti-Bacterial Agents/pharmacology
10.
ACS Appl Mater Interfaces ; 16(15): 18327-18343, 2024 Apr 17.
Article in English | MEDLINE | ID: mdl-38588343

ABSTRACT

58S bioactive glass (BG) has effective biocompatibility and bioresorbable properties for bone tissue engineering; however, it has limitations regarding antibacterial, antioxidant, and mechanical properties. Therefore, we have developed BGAC biocomposites by reinforcing 58S BG with silver and ceria nanoparticles, which showed effective bactericidal properties by forming inhibited zones of 2.13 mm (against Escherichia coli) and 1.96 mm (against Staphylococcus aureus; evidenced by disc diffusion assay) and an increment in the antioxidant properties by 39.9%. Moreover, the elastic modulus, hardness, and fracture toughness were observed to be increased by ∼84.7% (∼51.9 GPa), ∼54.5% (∼3.4 GPa), and ∼160% (∼1.3 MPam1/2), whereas the specific wear rate was decreased by ∼55.2% (∼1.9 × 10-11 m3/Nm). X-ray diffraction, high-resolution transmission electron microscopy, and field emission scanning electron microscopy confirmed the fabrication of biocomposites and the uniform distribution of the nanomaterials in the BG matrix. The addition of silver nanoparticles in the 58S BG matrix (in BGA) increased mechanical properties by composite strengthening and bactericidal properties by damaging the cytoplasmic membrane of bacterial cells. The addition of nanoceria in 58S BG (BGC) increased the antioxidant properties by 44.5% (as evidenced by the 2,2-diphenyl-1-picrylhydrazyl assay). The resazurin reduction assay and MTT assay confirmed the effective cytocompatibility for BGAC biocomposites against mouse embryonic fibroblast cells (NIH3T3) and mouse bone marrow stromal cells. Overall, BGAC resulted in mechanical properties comparable to those of cancellous bone, and its effective antibacterial and cytocompatibility properties make it a good candidate for bone healing.


Subject(s)
Cerium , Metal Nanoparticles , Silver , Animals , Mice , Antioxidants , NIH 3T3 Cells , Fibroblasts , Anti-Bacterial Agents/pharmacology , Glass
11.
Biomed Mater ; 19(3)2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38574669

ABSTRACT

Recently,in vitromodels of intestinal mucosa have become important tools for drug screening and studying the physiology and pathology of the intestine. These models enable the examination of cellular behavior in diseased states or in reaction to alterations in the microenvironment, potentially serving as alternatives to animal models. One of the major challenges in constructing physiologically relevantin vitromodels of intestinal mucosa is the creation of three-dimensional microstructures that accurately mimic the integration of intestinal epithelium and vascularized stroma. Here, core-shell alginate (Alg) microspheres were generated to create the compartmentalized extracellular matrix microenvironment needed to simulate the epithelial and vascularized stromal compartments of the intestinal mucosa. We demonstrated that NIH-3T3 and human umbilical vein endothelial cells embedded in the core of the microspheres can proliferate and develop a vascular network, while human colorectal adenocarcinoma cells (Caco-2) can form an epithelial monolayer in the shell. Compared to Caco-2 monolayer encapsulated within the shell, the presence of the vascularized stroma enhances their proliferation and functionality. As such, our core-shell Alg microspheres provide a valuable method for generatingin vitromodels of vascularized intestinal mucosa with epithelial and vascularized stroma arranged in a spatially relevant manner and demonstrating near-physiological functionality.


Subject(s)
Alginates , Cell Proliferation , Human Umbilical Vein Endothelial Cells , Intestinal Mucosa , Microspheres , Tissue Engineering , Alginates/chemistry , Humans , Intestinal Mucosa/metabolism , Animals , Mice , Caco-2 Cells , Tissue Engineering/methods , NIH 3T3 Cells , Extracellular Matrix/metabolism , Tissue Scaffolds/chemistry , Hexuronic Acids/chemistry
12.
Cell Biol Int ; 48(5): 726-736, 2024 May.
Article in English | MEDLINE | ID: mdl-38439187

ABSTRACT

Cellular senescence is an irreversible cell-cycle arrest in response to a variety of cellular stresses, which contribute to the pathogenesis of a variety of age-related degenerative diseases. However, effective antisenescence strategies are still lacking. Drugs that selectively target senescent cells represent an intriguing therapeutic strategy to delay aging and age-related diseases. Thus, we thought to investigate the effects of dihydroartemisinin (DHA) on senescent cells and elucidated its mechanisms underlying aging. Stress-induced premature senescence (SIPS) model was built in NIH3T3 cells using H2O2 and evaluated by ß-galactosidase staining. Cells were exposed to DHA and subjected to cellular activity assays including viability, ferroptosis, and autophagy. The number of microtubule-associated protein light-chain 3 puncta was detected by immunofluorescence staining. The iron content was assessed by spectrophotometer and intracellular reactive oxygen species (ROS) was measured by fluorescent probe dichlorodihydrofluorescein diacetate. We found that DHA triggered senescent cell death via ferroptosis. DHA accelerated ferritin degradation via promoting autophagy, increasing the iron contents, promoting ROS accumulation, thus leading to ferroptotic cell death in SIPS cells. In addition, autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. Moreover, Atg5 silencing and autophagy inhibitor BafA1 preconditioning inhibited ferroptosis induced by DHA. We also revealed that the expression of p-AMP-activated protein kinase (AMPK) and p-mammalian target of rapamycin (mTOR) in senescent cells was downregulated. These results suggested that DHA may be a promising drug candidate for clearing senescent cells by inducing autophagy-dependent ferroptosis via AMPK/mTOR signaling pathway.


Subject(s)
AMP-Activated Protein Kinases , Artemisinins , Ferroptosis , Animals , Mice , AMP-Activated Protein Kinases/metabolism , Autophagy , Cellular Senescence , Hydrogen Peroxide/pharmacology , Iron , NIH 3T3 Cells , Reactive Oxygen Species/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
13.
Int J Biol Macromol ; 266(Pt 1): 131257, 2024 May.
Article in English | MEDLINE | ID: mdl-38554908

ABSTRACT

The infected wounds pose one of the major threats to human health today. To address this issue, it is necessary to develop innovative wound dressings with superior antibacterial activity and other properties. Due to its potent antibacterial, antioxidant, and immune-boosting properties, epigallocatechin gallate (EGCG) has been widely utilized. In this study, a multifunctional curdlan hydrogel loading EGCG (Cur-EGCGH3) was designed. Cur-EGCGH3 exhibited excellent physicochemical properties, good biocompatibility, hemostatic, antibacterial, and antioxidant activities. Also, ELISA data showed that Cur-EGCGH3 stimulated macrophages to secrete pro-inflammatory and pro-regenerative cytokines. Cell scratch results indicated that Cur-EGCGH3 promoted the migration of NIH3T3 and HUVECs. In vivo experiments confirmed that Cur-EGCGH3 could inhibit bacterial infection of the infected wounds, accelerate hemostasis, and promote epithelial regeneration and collagen deposition. These results demonstrated that Cur-EGCGH3 holds promise for promoting healing of the infected wounds.


Subject(s)
Anti-Bacterial Agents , Catechin , Catechin/analogs & derivatives , Hemostatics , Hydrogels , Wound Healing , beta-Glucans , Catechin/pharmacology , Catechin/chemistry , Animals , Wound Healing/drug effects , Mice , beta-Glucans/chemistry , beta-Glucans/pharmacology , Hydrogels/chemistry , Hydrogels/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Humans , NIH 3T3 Cells , Hemostatics/pharmacology , Hemostatics/chemistry , Wound Infection/drug therapy , Wound Infection/microbiology , Antioxidants/pharmacology , Antioxidants/chemistry , Human Umbilical Vein Endothelial Cells/drug effects
14.
Adv Sci (Weinh) ; 11(20): e2307487, 2024 May.
Article in English | MEDLINE | ID: mdl-38520715

ABSTRACT

Collective cells, a typical active matter system, exhibit complex coordinated behaviors fundamental for various developmental and physiological processes. The present work discovers a collective radial ordered migration behavior of NIH3T3 fibroblasts that depends on persistent top-down regulation with 2D spatial confinement. Remarkably, individual cells move in a weak-oriented, diffusive-like rather than strong-oriented ballistic manner. Despite this, the collective movement is spatiotemporal heterogeneous and radial ordering at supracellular scale, manifesting as a radial ordered wavefront originated from the boundary and propagated toward the center of pattern. Combining bottom-up cell-to-extracellular matrix (ECM) interaction strategy, numerical simulations based on a developed mechanical model well reproduce and explain above observations. The model further predicts the independence of geometric features on this ordering behavior, which is validated by experiments. These results together indicate such radial ordered collective migration is ascribed to the couple of top-down regulation with spatial restriction and bottom-up cellular endogenous nature.


Subject(s)
Cell Movement , Animals , Mice , Cell Movement/physiology , NIH 3T3 Cells , Extracellular Matrix/physiology , Extracellular Matrix/metabolism , Fibroblasts/cytology , Fibroblasts/physiology
15.
J Vis Exp ; (204)2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38436407

ABSTRACT

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.


Subject(s)
Cell Communication , Organoids , Humans , Mice , Animals , NIH 3T3 Cells , Circadian Rhythm , Fibroblasts
16.
Int J Pharm ; 654: 123994, 2024 Apr 10.
Article in English | MEDLINE | ID: mdl-38484859

ABSTRACT

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.


Subject(s)
Fatty Acids, Monounsaturated , Glioblastoma , Liposomes , Quaternary Ammonium Compounds , Mice , Animals , Humans , Liposomes/chemistry , Polymers/chemistry , RNA, Small Interfering , Glioblastoma/genetics , Glioblastoma/therapy , NIH 3T3 Cells , Nanomedicine , Lipids/chemistry
17.
Int J Nanomedicine ; 19: 1749-1766, 2024.
Article in English | MEDLINE | ID: mdl-38414527

ABSTRACT

Purpose: Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer. However, the effect of current treatment strategies by inducing tumor cell apoptosis alone is not satisfactory. The growth, metastasis and treatment sensitivity of tumors can be strongly influenced by cancer-associated fibroblasts (CAFs) in the microenvironment. Effective cancer therapies may need to target not only the tumor cells directly but also the CAFs that protect them. Methods: Celastrol and small-sized micelles containing betulinic acid were co-encapsulated into liposomes using the thin-film hydration method (CL@BM). Folic acid was further introduced to modify liposomes as the targeting moiety (F/CL@BM). We established a novel NIH3T3+4T1 co-culture model to mimic the tumor microenvironment and assessed the nanocarrier's inhibitory effects on CAFs-induced drug resistance and migration in the co-culture model. The in vivo biological distribution, fluorescence imaging, biological safety evaluation, and combined therapeutic effect evaluation of the nanocarrier were carried out based on a triple-negative breast cancer model. Results: In the present study, a novel multifunctional nano-formulation was designed by combining the advantages of sequential release, co-loading of tretinoin and betulinic acid, and folic acid-mediated active targeting. As expected, the nano-formulation exhibited enhanced cytotoxicity in different cellular models and effectively increased drug accumulation at the tumor site by disrupting the cellular barrier composed of CAFs by tretinoin. Notably, the co-loaded nano-formulations proved to be more potent in inhibiting tumor growth in mice and also showed better anti-metastatic effects in lung metastasis models compared to the formulations with either drug alone. This novel drug delivery system has the potential to be used to develop more effective cancer therapies. Conclusion: Targeting CAFs with celastrol sensitizes tumor cells to chemotherapy, increasing the efficacy of betulinic acid. The combination of drugs targeting tumor cells and CAFs may lead to more effective therapies against various cancers.


Subject(s)
Cancer-Associated Fibroblasts , Pentacyclic Triterpenes , Triple Negative Breast Neoplasms , Humans , Animals , Mice , Liposomes/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , NIH 3T3 Cells , Betulinic Acid , Tretinoin/pharmacology , Folic Acid/pharmacology , Cell Line, Tumor , Tumor Microenvironment
18.
Carbohydr Polym ; 331: 121878, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38388061

ABSTRACT

Pectins are a class of soluble polysaccharides that can have anticancer properties through several mechanisms. This study aimed to characterize the molecular structure of water-soluble fractions (WSF) derived from ripe and unripe papayas and assess their biological effects in two models: the 3D colon cancer spheroids to measure cell viability and cytotoxicity, and the in vivo model to investigate the inhibition of preneoplastic lesions in rats. WSF yield was slightly higher in ripe papaya, and both samples mainly consisted of pectin. Both pectins inhibited the growth of colon cancer HT29 and HCT116 spheroids. Unripe pectin disturbed HT29/NIH3T3 spheroid formation, decreased HCT116 spheroid viability, and increased spheroid cytotoxicity. Ripe pectin had a more substantial effect on the reduction of spheroid viability for HT29 spheroids. Furthermore, in vivo experiments on a rat model revealed a decrease in aberrant crypt foci (ACF) formation for both pectins and increased apoptosis in colonocytes for ripe papaya pectins. The results suggest potential anticancer properties of papaya pectin, with ripe pectin showing a higher potency.


Subject(s)
Carica , Colonic Neoplasms , Rats , Animals , Mice , Pectins/pharmacology , Pectins/chemistry , Carica/chemistry , NIH 3T3 Cells , Colonic Neoplasms/chemically induced , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Cell Proliferation , Colon
19.
Biomed Mater ; 19(3)2024 Mar 20.
Article in English | MEDLINE | ID: mdl-38417147

ABSTRACT

The present work focuses on developing 5% w/v oxidized alginate (alginate di aldehyde, ADA)-7.5% w/v gelatin (GEL) hydrogels incorporating 0.25% w/v silk fibroin (SF) and loaded with 0.3% w/v Cu-Ag doped mesoporous bioactive glass nanoparticles (Cu-Ag MBGNs). The microstructural, mechanical, and biological properties of the composite hydrogels were characterized in detail. The porous microstructure of the developed ADA-GEL based hydrogels was confirmed by scanning electron microscopy, while the presence of Cu-Ag MBGNs in the synthesized hydrogels was determined using energy dispersive x-ray spectroscopy. The incorporation of 0.3% w/v Cu-Ag MBGNs reduced the mechanical properties of the synthesized hydrogels, as investigated using micro-tensile testing. The synthesized ADA-GEL loaded with 0.25% w/v SF and 0.3% w/v Cu-Ag MBGNs showed a potent antibacterial effect againstEscherichia coliandStaphylococcus aureus. Cellular studies using the NIH3T3-E1 fibroblast cell line confirmed that ADA-GEL films incorporated with 0.3% w/v Cu-Ag MBGNs exhibited promising cellular viability as compared to pure ADA-GEL (determined by WST-8 assay). The addition of SF improved the biocompatibility, degradation rate, moisturizing effects, and stretchability of the developed hydrogels, as determinedin vitro. Such multimaterial hydrogels can stimulate angiogenesis and exhibit desirable antibacterial properties. Therefore further (in vivo) tests are justified to assess the hydrogels' potential for wound dressing and skin tissue healing applications.


Subject(s)
Fibroins , Nanoparticles , Mice , Animals , Hydrogels/chemistry , Fibroins/chemistry , NIH 3T3 Cells , Alginates/chemistry , Gelatin/chemistry , Anti-Bacterial Agents , Nanoparticles/chemistry
20.
Oncol Res ; 32(3): 477-487, 2024.
Article in English | MEDLINE | ID: mdl-38361760

ABSTRACT

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFß, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFß in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFß secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer.


Subject(s)
Breast Neoplasms , Metformin , Animals , Mice , Humans , Female , Metformin/pharmacology , Metformin/metabolism , Coculture Techniques , Interleukin-6/metabolism , Interleukin-6/pharmacology , NIH 3T3 Cells , Oxidative Stress , Breast Neoplasms/pathology , Fibroblasts/metabolism , Phenotype , Lactic Acid/metabolism , Lactic Acid/pharmacology , Transforming Growth Factor beta/metabolism , Cell Line, Tumor
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