Normal Dermal Fibroblasts Research Articles

Characterization and Physiological Differences of Two Primary Cultures of Human Normal and Hypertrophic Scar Dermal Fibroblasts: A Pilot Study.

Background/Objectives: Dermal fibroblasts (DFs) are key participants in skin hypertrophic scarring, and their properties are being studied to identify the molecular and cellular mechanisms underlying the pathogenesis of skin scarring. Methods: In the present work, we performed a comparative analysis of DFs isolated from normal skin (normal dermal fibroblasts, NDFs), and hypertrophic scar skin (hypertrophic scar fibroblasts, HTSFs). The fibroblasts were karyotyped and phenotyped, and experiments on growth rate, wound healing, and single-cell motility were conducted. Results: Comparative analysis revealed a minor karyotype difference between cells. However, HTSFs are characterized by higher proliferation level and motility compared to NDFs. These significant differences may be associated with quantitative and qualitative differences in the cell secretome. A proteomic comparison of NDF and HTSF found that differences were associated with metabolic proteins reflecting physiological differences between the two cells lines. Numerous unique proteins were found only in the vesicular phase of vHTSFs. Some proteins involved in cell proliferation (protein-glutamine gamma-glutamyltransferase K) and cell motility (catenin delta-1), which regulate gene transcription and the activity of Rho family GTPases and downstream cytoskeletal dynamics, were identified. A number of proteins which potentially play a role in fibrosis and inflammation (mucin-5B, CD97, adhesion G protein-coupled receptor E2, antileukoproteinase, protein S100-A8 and S100-A9, protein caspase recruitment domain-containing protein 14) were detected in vHTSFs. Conclusions: A comparative analysis of primary cell cultures revealed their various properties, especially in the cell secretome. These proteins may be considered promising target molecules for developing treatment or prevention strategies for pathological skin scarring.

Development of morphine analgesia using biocompatible synthesized molecular imprinted hydrogel based on tragacanth gum

AbstractA novel molecular imprinted hydrogel using tragacanth gum (MITMIH) was synthesized for extending morphine analgesic efficacy by controlled release. The characterized MITMIH was used as a new morphine carrier that can increase analgesic time window after its single injection in mice. During in vitro optimization, the MITMIH showed maximum adsorption capacity (~113 mg g−1) at pH = 6.5 after 20 min immersion (400 μM initial morphine concentration, 10 mg MITMIH). During in vivo test, we examined the analgesic effect in mice by dissolving morphine in physiologic saline or by loading on MITMIH. The results of a single injection revealed that morphine‐loaded MITMIH shifted the time profile of morphine analgesia from 123.4 to 257.6 min. This indicates almost 100% efficacy in the controlled release of the drug during 5 h after the injection. Furthermore, cytotoxicity and mitochondrial membrane potential test was evaluated against normal rat kidney epithelial (NRK) and Rat Dermal Fibroblasts (RDF). The results confirmed that the MITMIH could be considered as a potential carrier increasing length of morphine analgesia after the first single injection without any cytotoxicity and negative effect on cell tissues. This may in turn reduce repeated use of the drug to achieve satisfactory analgesia in patients with chronic pain.

Discovery of Novel Fluorescent Azaindoles with Cytotoxic Action in A2780 Ovarian Carcinoma Cells.

Azaindole scaffold is a privileged structure in medicinal chemistry and some derivatives have demonstrated to be potential anticancer drugs. Herein, a set of novel azaindoles, comprising the four regioisomers, bearing a morpholine (azaindoles 3 a-d) and N-methyl-N-benzylamine (azaindoles 4 a-d) groups were prepared. Among these compounds, azaindoles 4 exhibited higher cytotoxicity against the ovarian cancer cell line A2780 and normal dermal fibroblasts compared to azaindoles 3. Furthermore, azaindoles 4 b and 4 c promoted a delay in the cell cycle of the cancer cell line, inspiring an investigation into the intracellular localization of these derivatives.

Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN.

In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.

Isolation and identification of hyperin and naringenin from guava cultivated in Iraq and evaluation of cytotoxic activity of hyperin

The guava plant, Psidium guajava L., serves as proof of the abundant donations of nature, providing a delicious guava fruit; this plant is rich in groups of medicinal and nutritional benefits. Guava belonging to the Myrtaceae family, many previous studies reported many phytochemical constituents in its leaves that have many pharmacological activities and medicinal properties; this study focuses on the isolation, structural elucidation and calculation concentration of flavonoids, assessment of the cytotoxic activityof hyperin from Psidium guajava leaves newly cultivated in Iraq. The isolation process involved the use of thin-layer chromatography (TLC) and preparative high-performance liquid chromatography (PHPLC) and structural elucidation involved NMR (nuclear magnetic resonance spectroscopy) and FTIR (Fourier transform infrared spectrometer) for detailed structural insights into 2 isolated flavonoids. The isolation techniques proved effective in obtaining pure samples of hyperin and naringenin from the ethyl acetate and n.butanol fractions and structural elucidation techniques gives a good explanation for 2 isolated flavonoids. Evaluate cytotoxic activity of hyperin flavonoid against prostate cancer cell line (PC-3), human breast cancer cell line (MCF-7) and normal dermal fibroblast neonatal (HdFn), hyperin flavonoid exhibited a decrease in cell viability (%) with IC50 58.9 ?g /mL against the prostate cancer cell and IC50 90.58 ?g /mL against human breast cancer and noncytotoxic to HDFn, with an IC50 value substantially surpassing concentrations of 100 ?g/mL. These analytical approaches provided a comprehensive understanding of the chemical composition of the isolated flavonoids and interpreted that guava has cytotoxic activity against some cancers, depending on a concentration-dependent mode.

Biomechanical Activation of Keloid Fibroblasts Promotes Lysosomal Remodeling and Exocytosis

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM deposition and conferred resistance myosin inhibitors. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

Superoxide Dismutase Mimetic Avasopasem Manganese Enhances Radiation Therapy Effectiveness in Soft Tissue Sarcomas and Accelerates Wound Healing.

Soft tissue sarcomas (STSs) are mesenchymal malignant lesions that develop in soft tissues. Despite current treatments, including radiation therapy (RT) and surgery, STSs can be associated with poor patient outcomes and metastatic recurrences. Neoadjuvant radiation therapy (nRT), while effective, is often accompanied by severe postoperative wound healing complications due to damage to the surrounding normal tissues. Thus, there is a need to develop therapeutic approaches to reduce nRT toxicities. Avasopasem manganese (AVA) is a selective superoxide dismutase mimetic that protects against IR-induced oral mucositis and lung fibrosis. We tested the efficacy of AVA in enhancing RT in STSs and in promoting wound healing. Using colony formation assays and alkaline comet assays, we report that AVA selectively enhanced the STS (liposarcoma, fibrosarcoma, leiomyosarcoma, and MPNST) cellular response to radiation compared to normal dermal fibroblasts (NDFs). AVA is believed to selectively enhance radiation therapy by targeting differential hydrogen peroxide clearance in tumor cells compared to non-malignant cells. STS cells demonstrated increased catalase protein levels and activity compared to normal fibroblasts. Additionally, NDFs showed significantly higher levels of GPx1 activity compared to STSs. The depletion of glutathione using buthionine sulfoximine (BSO) sensitized the NDF cells to AVA, suggesting that GPx1 may, in part, facilitate the selective toxicity of AVA. Finally, AVA significantly accelerated wound closure in a murine model of wound healing post RT. Our data suggest that AVA may be a promising combination strategy for nRT therapy in STSs.

The Conjugates of Indolo[2,3-b]quinoline as Anti-Pancreatic Cancer Agents: Design, Synthesis, Molecular Docking and Biological Evaluations.

New amide conjugates of hydroxycinnamic acids (HCAs) and the known antineoplastic 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ), an analog of the natural alkaloid neocryptolepine, were synthesized and tested in vitro for anticancer activity. The compound 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline (2), which contains the ortho-coumaric acid fragment, demonstrated dose-dependent effectiveness against both normal BxPC-3 and metastatic AsPC-1 pancreatic cancer cells. The IC50 values for AsPC-1 and BxPC-3 were 336.5 nM and 347.5 nM, respectively, with a selectivity index of approximately 5 for both pancreatic cancer cells compared to normal dermal fibroblasts. Conjugate 2 did not exhibit any hemolytic activity against human erythrocytes at the tested concentration. Computational studies were performed to predict the pharmacokinetic profile and potential mechanism of action of the synthesized conjugates. These studies focused on the ADME properties of the conjugates and their interactions with DNA, as well as DNA-topoisomerase alpha and beta complexes. All of the conjugates studied showed approximately one order of magnitude stronger binding to DNA compared to the reference DiMIQ, and approximately two orders of magnitude stronger binding to the topoisomerase II-DNA complex compared to DiMIQ. Conjugate 2 was predicted to have the strongest binding to the enzyme-DNA complex, with a Ki value of 2.8 nM.

Differential Photosensitivity of Fibroblasts Obtained from Normal Skin and Hypertrophic Scar Tissues.

It is unclear whether normal human skin tissue or abnormal scarring are photoreceptive. Therefore, this study investigated photosensitivity in normal skin tissue and hypertrophic scars. The expression of opsins, which are photoreceptor proteins, in normal dermal fibroblasts (NDFs) and hypertrophic scar fibroblasts (HSFs) was examined. After exposure to blue light (BL), changes in the expression levels of αSMA and clock-related genes, specifically PER2 and BMAL1, were examined in both fibroblast types. Opsins were expressed in both fibroblast types, with OPN3 exhibiting the highest expression levels. After peripheral circadian rhythm disruption, BL induced rhythm formation in NDFs. In contrast, although HSFs showed changes in clock-related gene expression levels, no distinct rhythm formation was observed. The expression level of αSMA was significantly higher in HSFs and decreased to the same level as that in NDFs upon BL exposure. When OPN3 knocked-down HSFs were exposed to BL, the reduction in αSMA expression was inhibited. This study showed that BL exposure directly triggers peripheral circadian synchronization in NDFs but not in HSFs. OPN3-mediated BL exposure inhibited HSFs. Although the current results did not elucidate the relationship between peripheral circadian rhythms and hypertrophic scars, they show that BL can be applied for the prevention and treatment of hypertrophic scars and keloids.

The conjugates of 5'-deoxy-5-fluorocytidine and hydroxycinnamic acids - synthesis, anti-pancreatic cancer activity and molecular docking studies.

New amide conjugates 1-6 of hydroxycinnamic acids (HCA) and 5'-deoxy-5-fluorocytidine (5-dFCR), the prodrug of 5-fluorouracil (5-FU), were synthesized and tested in vitro against pancreatic cancer lines (PDAC). The compounds showed slightly higher efficacy against primary BxPC-3 cells (IC50 values of 14-45 μM) than against metastatic AsPC-1 (IC50 values of 37-133 μM), and similar to that of 5-FU for both PDAC lines. Compound 1, which has a para-(acetyloxy)coumaroyl substituent, was found to be the most potent (IC50 = 14 μM) with a selectivity index of approximately 7 to normal dermal fibroblasts (IC50 = 96 μM). The potential pharmacological profiles were discussed on the basis of the ADME data. Docking to the carboxylesterase CES2 showed that the synthesized compounds have the ability to bind via hydrogen bonding between a specific acetate group of the sugar moiety and Ser228, which belongs to the catalytic triad that causes hydrolysis. Docking to albumin, a major transport protein in the circulatory system, revealed a strong interaction of the conjugates at the binding site which is native to warfarin and responsible for its transport in the body.

Evaluation of the effectiveness of low-level laser therapy on proliferation of fibroblasts isolated from chronic wounds in human in vitro

Chronic wounds pose significant challenges in healthcare due to impaired healing mechanisms. Fibroblast cells play a crucial role in wound healing by orchestrating proliferation and migration. This study aimed to assess fibroblast cells derived from chronic wounds and explore the impact of low-level laser therapy (LLLT) on their growth and migration. Dermal samples from chronic pressure ulcers and diabetic ulcers were obtained from 20 patients at three sites. Fibroblasts from wound base, margins, and adjacent healing skin were isolated and characterized. Proliferation and migration capabilities of these cells were evaluated. LLLT was applied at various energy levels (2.5, 3, 3.5, 4, and 5 J/cm²) to assess its effect on cell count. Fibroblasts from chronic wounds exhibited slower proliferation and migration rates compared to normal dermal fibroblasts. Notably, LLLT intervention at different energy levels led to a significant increase in cell count, with the most pronounced effect observed at 3 J/cm². LLLT at an energy level of 3 J/cm² demonstrated a notable enhancement in fibroblast migration. These findings underscore the potential of LLLT as a therapeutic approach for chronic wounds, offering insights into its efficacy in augmenting fibroblast functions crucial for wound healing.

Ex vivo identification of transcriptional regulation landscape and potential therapeutic targets against oxidative stress using Alzheimer’s disease patient‐derived dermal fibroblast: findings from BICWALZS cohort

AbstractBackgroundReactive oxygen species (ROS) is considered as a central pathophysiology for neurodegeneration in Alzheimer’s disease (AD). Many studies using in vitro cell line or in vivo mouse model have shown the possible association between ROS defense mechanisms and cellular damage. However, that connection was not elucidated well in AD patient‐derived cell or brain organoids. In this study, we aimed to identify transcriptional regulation landscape and potential therapeutic targets against oxidative stress using AD dementia patient‐derived dermal fibroblastMethodsFor the purposes of ex vivo research, we performed skin biopsy and stabilized dermal fibroblasts in vitro. Total of 20 AD dementia patients and 22 cognitively normal older adults were assessed. All of AD dementia patients were amyloid positive on PET imaging, APOE e4 carrier, and cognition was imparied. On the contrary, all of cognitively normal older adults were amyloid negative on PET imaging, and APOE e4 non‐carrier. We applied H2O2 (600 uM) to dermal fibroblast for 24 hours as an oxidant agent to promote oxidative stress. After that, to identify transcriptional regulation landscape against oxidative stress, RNA sequencing was performed.ResultsTotal of 406 transcripts (up = 61, down = 345) were commonly differentially expressed in both of AD dementia and cognitively normal older adults derived dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to cell division, cell cycle, and DNA repair. Interestingly, 133 transcripts (up = 41, down = 92) were only differentially expressed in AD dementia dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to DNA duplex unwinding, cell division, and DNA replication. Total of 147 transcripts (up = 67, down = 80) were only differentially expressed in cognitively normal older adults dermal fibroblast by ROS‐H2O2. These transcripts were enriched in GO categories related to negative regulation of cell proliferation, notch signaling pathway, and NMDA glutamate receptor signaling pathway.ConclusionWe identified transcriptional regulation landscape by ROS‐H2O2 in AD dementia and cognitively normal older adults dermal fibroblast. Further bioinformatic analysis with computational tools and validation experiments will be performed to find potential therapeutic targets against oxidative stress.

Ex vivo identification of transcriptional regulation landscape and potential therapeutic targets against ER stress using Alzheimer’s disease patient‐derived dermal fibroblast: findings from BICWALZS cohort

AbstractBackgroundEndoplasmic reticulum (ER) stress is considered as a central pathophysiology for neurodegeneration in Alzheimer’s disease (AD). Many studies using in vitro cell line or in vivo mouse model have shown the possible association between ER stress and neurodegeneration. However, that connection was not elucidated well in AD patient‐derived cell or brain organoids. In this study, we aimed to identify transcriptional regulation landscape and potential therapeutic targets against ER stress using AD dementia patient‐derived dermal fibroblast.MethodFor the purposes of ex vivo research, we performed skin biopsy and stabilized dermal fibroblasts in vitro. Total of 20 AD dementia patients and 22 cognitively normal older adults were assessed. All of AD dementia patients were amyloid positive on PET imaging, APOE e4 carrier, and cognition was imparied. On the contrary, all of cognitively normal older adults were amyloid negative on PET imaging, and APOE e4 non‐carrier. We applied thabsigargin (10 nM) to dermal fibroblast for 24 hours to promote ER stress. After that, to identify transcriptional regulation landscape against ER stress, RNA sequencing was performed.ResultTotal of 252 transcripts (up = 162, down = 90) were commonly differentially expressed in both of AD dementia and cognitively normal older adults derived dermal fibroblast by ER stress‐thabsigargin. These transcripts were enriched in GO categories related to protein folding in ER, response to ER stress, and ERAD pathway. Interestingly, 80 transcripts (up = 33, down = 47) were only differentially expressed in AD dementia dermal fibroblast by ER stress‐thabsigargin. These transcripts were enriched in GO categories related to cell division, miotic cell cycle, and microtubule‐based movement. Total of 60 transcripts (up = 41, down = 19) were only differentially expressed in cognitively normal older adults dermal fibroblast by ER stress‐thabsigargin. These transcripts were enriched in GO categories related to cholesterol biosynthesis process, DNA replication checkpoint, and cAMP‐mediated signaling.ConclusionWe identified transcriptional regulation landscape by ER stress‐thabsigargin in AD dementia and cognitively normal older adults dermal fibroblast. Further bioinformatic analysis with computational tools and validation experiments will be performed to find potential therapeutic targets against ER stress.

Highly cyto- and immune compatible new synthetic fluorapatite nanomaterials co-doped with rubidium(I) and europium(III) ions

In the present study, biocompatible luminescent of nanosized fluorapatite doped with rubidium(I) (Rb+ ion) and europium(III) (Eu3+ ion) ions were synthesized via hydrothermal method. It was investigated the influence of co-doped Rb+ and Eu3+ ions on the structural, and morphological characteristics of the obtained fluorapatite materials. The characterization techniques utilized included: X-ray powder diffraction (XRPD), infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). Moreover, to establish the influence of the co-doped Rb+ and Eu3+ ions on the luminescence properties of the lanthanide ion, emission excitation, emission spectrum and luminescence decays were measured. This confirmed a distinct red emission originating from Eu3+ ions and an increased emission lifetime. To determine the biocompatibility of the obtained fluorapatite compounds, in vitro studies using normal dermal human fibroblasts were performed. The results of these studies clearly demonstrate the remarkable biocompatibility of our compounds. This discovery opens exciting prospects for the use of synthetic fluorapatites doped with Eu3+ and Rb+ ions in various biomedical contexts. In particular, these materials hold great promise for potential applications in regenerative engineering, but also serve as innovative and practical solutions as bone scaffolds and dental implants containing nano-fluorapatite. Further discussion of these properties can be found in this article, along with a discussion of their importance and potential in the field of biomedical applications. However, according to our pervious study and based on our current investigations but also based on available scientific records, it was proposed potential molecular mechanism of Rb+ ions in the process of osteoclastogenesis.

Placental mesenchymal stem cells-secreted proenkephalin suppresses the p38 MAPK signaling to block hyperproliferation of keloid fibroblasts

BackgroundThanks to their multi-potency and secretory functions, mesenchymal stem cells (MSCs) have long been established as an ideal cell type for skin wound healing and a candidate therapeutic strategy for excessive pathological scarring in the meantime. This study focuses on the effect of placental MSCs (PMSCs) on the activity of keloid fibroblasts (KFs) and the potential involvement of proenkephalin (PENK). MethodsSecretory protein of PMSC that are lowly expressed in KFs were predicted by bioinformatics analyses. The expression of PENK in KFs was detected by RT-qPCR and western blot analysis. PMSCs were co-cultured with KFs and dermal fibroblasts (DFs) to examine their effect on proliferation, migration, invasion, and apoptosis of the distinct cell types. PENK secretion by PMSCs and its uptake by KFs were examined by ELISA, WB, and immunofluorescence staining. Loss-of-functions of PENK and p38-MAPK were induced to examine the activity of KFs in vitro and in mice. ResultsPENK, a secretory protein of PMSCs, was conspicuously downregulated in KFs compared to normal DFs. PMSC stimulation suppressed proliferation, migration, invasion, and resistance to apoptosis of the co-cultured KFs but not DFs, which was ascribed to the upregulation of PENK protein in KFs. PMSCs-secreted PENK suppressed p38 phosphorylation in KFs. The proliferative and aggressive properties of KFs in vitro and the nodule-forming capacity of KFs in vivo were promoted upon PENK downregulation but suppressed by the p38 MAPK inhibitor SB202190. ConclusionThis work unravels that PMSCs-secreted PENK suppresses the p38 MAPK signaling to block hyperproliferation of KFs.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes.

In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze-thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin-BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

From Kiwi Peels’ “End-of-Life” to Gold Nanoparticles: the Upcycling of a Waste

Following a green approach, kiwi peels (a waste) were washed in hot water to obtain a water-based polyphenolic extract (KPWW) used to reduce Au3+ (coming from a HAuCl4 water-based solution) for forming gold nanoparticles (AuNPs). Indeed, KPWW, as shown after performing high-performance liquid chromatography-mass spectrometry (HPLC/MS-MS) analysis, is mainly composed by different polyphenols acting as reductant agents, accomplishing a red-ox reaction and decorating the AuNPs-KPWW surface. Spectroscopic and morphologic techniques were used in synergy for investigating the AuNPs-KPWW main features. Polyhedral-shaped plasmonic nanoparticles with a mean size of 30±10 nm and a negative charge of −40 mV were thus obtained. The AuNPs’ stability was assessed under different working conditions, investigating the role of ionic strength, pH, and temperature. The photostability was also assessed by irradiating AuNPs-KPWW with a solar simulator lamp. Both temperature and solar light did not perturb AuNPs-KPWW. Thanks to the presence of polyphenols, the antioxidant and skin-lightening properties were positively demonstrated. Moreover, the protective role of AuNPs in scavenging H2O2 and ·OH was also investigated by inhibiting the oxidation of a biomolecule. The sunscreen ability of AuNPs-KPWW was also estimated, and the theoretical calculation of the sun protection factor (SPF) was determined. Finally, the AuNPs-KPWW biocompatibility was tested on endothelial colony-forming cells and normal dermal fibroblasts as human cell lines, revealing that AuNPs-KPWW did not affect cell viability and did not alter cell morphology, demonstrating their safety and their potential application in nanomedicine.

Syringin as TGF-βR1, HER2, EGFR, FGFR4 Kinase, and MMP-2 Inhibitor and Potential Cytotoxic Agent against ER+ Breast Cancer Cells

Background: Breast cancer is currently the most diagnosed cancer worldwide. Neoplastic cells and components of the tumor microenvironment trigger enzymes and receptors to facilitate cancer advancement. Syringin, a natural phenylpropanoid glycoside, has been reported to possess anti-cancer activity and affinity with numerous druggable targets of breast carcinoma. Objectives: This work aims to evaluate the effects of syringin on the growth of breast cancer cells (MCF-7) and normal dermal fibroblast cells (HDFn) and its ability to inhibit the protein targets of breast cancer. Methods: Syringin was investigated on cell lines in vitro via MTT assay. Using non-cell-based activity assay kits, its influence on the activity of transforming growth factor-beta receptor type 1 (TGF-βR1), human epidermal growth factor receptor (HER2), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 4 (FGFR4), and matrix metalloproteinase-2 (MMP-2) was evaluated. Results: Syringin exhibited significant cytotoxicity against MCF-7 cells (IC50: 32.11 μM for 24 hours and 21.35 μM for 48 hours) and was non-toxic on healthy HDFn cells (IC50: >100 μM for 24 and 48 hours). It significantly suppressed the activity of cancer and angiogenesis regulating enzymes in vitro with commendable IC50 values on TGF-βR1 kinase (IC50: 6.48 μM), HER2 kinase (IC50: 7.18 μM), EGFR kinase (IC50: 12.38 μM), FGFR4 kinase (IC50: 16.03 μM), and MMP-2 (IC50: 16.07 μM). Conclusion: Findings showed the selective toxicity of syringin on breast cancer cells and its potential against pro-angiogenic enzymes. These discoveries strongly indicate the significance and therapeutic potential of syringin in targeted cancer therapy.

Cell Uptake of Steroid-BODIPY Conjugates and Their Internalization Mechanisms: Cancer Theranostic Dyes.

Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11β-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.