a-simple-benchtop-filtration-method-to-isolate-small-extracellular

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this ...

Monash University Malaysia

Nanomedicine is an emerging area in the medical field, particularly in the treatment of cancers. Nanostructured lipid carrier (NLC) was shown to be a good nanoparticulated carrier for the delivery of tamoxifen (TAM). In this study, the tamoxifen-loaded erythropoietin-coated nanostructured lipid carriers (EPO-TAMNLC) were developed to enhance the anti-cancer properties and targetability of TAM, using EPO as the homing ligand for EPO receptors (EpoRs) on breast cancer tissue cells. Tamoxifen-loaded NLC (TAMNLC) was used for comparison. The LA7 cells and LA7 cell-induced rat mammary gland tumor were used as models in the study. Immunocytochemistry staining showed that LA7 cells express estrogen receptors (ERs) and EpoRs. EPO-TAMNLC and TAMNLC significantly (p<0.05) inhibited proliferation of LA7 in dose- and time-dependent manner. EPO-TAMNLC induced apoptosis and G0/G1 cell cycle arrest of LA7 cells. Both drug delivery systems showed anti-mammary gland tumor properties. At an intravenous dose of 5 mg kg-1 body weight, EPO-TAMNLC and TAMNLC were not toxic to rats, suggesting that both are safe therapeutic compounds. In conclusion, EPO-TAMNLC is not only a unique drug delivery system because of the dual drug-loading feature, but also potentially highly specific in the targeting of breast cancer tissues positive for ERs and EpoRs. The incorporation of TAM into NLC with and without EPO coat had significantly (p<0.05) improved specificity and safety of the drug carriers in the treatment of mammary gland tumors.

Enhanced anti-mammary gland cancer activities of tamoxifen-loaded erythropoietin-coated drug delivery system.

The use of fungal cultures have been well documented in human history. Although its used in healthcare, like penicillin and statins, have saved countless of lives, but there is still no fungal products that are specifically indicated for cancers. Research into fungal-derived materials to curb cancers in the recent decades have made a considerable progress in terms of drug delivery vehicles, anticancer active ingredients and cancer immunotherapy. Various parts of the organisms have successfully been exploited to achieve specific tasks. Apart from the identification of novel anticancer compound from fungi, its native capsular structure can also be used as drug cargo to achieve higher oral bioavailability. This review summarises the anticancer potential of fungal-derived materials, highlighting the role of capsular polysaccharides, proteins, and other structures in variety of innovative utilities to fit the current pharmaceutical technology. Many bioactive compounds isolated from fungi have also been formulated into nanoparticles to achieve greater anticancer activity. The progress of fungal compounds and their analogues in clinical trials is also highlighted. In addition, the potential of various fungal species to be developed for anticancer immunotherapy are also discussed.

How far have we explored fungi to fight cancer?

Recombinant human erythropoietin (rHuEPO) is the erythropoiesis-stimulating hormone that is being used concurrently with chemotherapeutic drugs in the treatment of anemia of cancer. The effect of rHuEPO on cancer cells in 3-dimensional (3D) cultures is not known. The objective of the study was to determine the effect of rHuEPO on the viability of MCF-7 breast cancer cells from 2-dimensional (2D) and 3D cell cultures. The monolayer MCF-7 cells from 2D culture and MCF-7 cell from 3D culture generated by ultra-low adhesive microplate technique, were treated with 0, 0.1, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The effects of rHuEPO on MCF-7 cell viability and proliferation were determined using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red retention time (NRRT), trypan blue exclusion assay (TBE), DNA fragmentation, acridine orange/propidium iodide staining (AO/PI) assays. The MCF-7 cells for 3D culture were also subjected to caspase assays and cell cycle analysis using flow cytometry. rHuEPO appeared to have greater effect at lowering the viability of MCF-7 cells from 3D than 2D cultures. rHuEPO significantly (p < 0.05) decreased viability and down-regulated the caspase activities of 3D MCF-7 cells in dose- and time-dependent manner. The cell cycle analysis showed that rHuEPO caused MCF-7 cells to enter the subG0/G1 phase. Thus, the study suggests that rHuEPO has a cytostatic effect on the MCF-7 breast cancer cells from 3D culture.

Recombinant Human erythropoietin reduces viability of MCF-7 breast cancer cells from 3D culture without caspase activation.

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells.

Copper complex has been gaining much attention in anticancer research as a targeted agent since cancer cells uptake more copper than non-cancerous cells. Our group synthesised a ternary copper complex, which is composed of 1,10-phenanthroline and tyrosine [Cu(phen)(L-tyr)Cl].3H0. These two payloads have been designed to cleave DNA and inhibit protein degradation system (proteasome) concurrently in cancer cells, making this copper complex a dual-target compound.

Induction of Apoptosis and Autophagy by Ternary Copper Complex Towards Breast Cancer Cells.

Cell therapy involves the transplantation of human cells to replace or repair the damaged tissues and modulate the mechanisms underlying disease initiation and progression in the body. Nowadays, many different types of cell-based therapy are developed and used to treat a variety of diseases. In the past decade, cell-free therapy has emerged as a novel approach in regenerative medicine after the discovery that the transplanted cells exerted their therapeutic effect mainly through the secretion of paracrine factors. More and more evidence showed that stem cell-derived secretome, i.e., growth factors, cytokines, and extracellular vesicles, can repair the injured tissues as effectively as the cells. This finding has spurred a new idea to employ secretome in regenerative medicine. Despite that, will cell-free therapy slowly replace cell therapy in the future? Or are these two modes of treatment still needed to address different diseases and conditions? This review provides an indepth discussion about the values of stem cells and secretome in regenerative medicine. In addition, the safety, efficacy, advantages, and disadvantages of using these two modes of treatment in regenerative medicine are also critically reviewed.

Comparing the Therapeutic Potential of Stem Cells and their Secretory Products in Regenerative Medicine.

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p < 0.05) increased the caspase-3 and -9 activities in the spheroid MCF-7 cells by 200 to 550% of the control. Combination rHuEPO and tamoxifen produced much lesser effect on the caspase-8 activity. The rHuEPO in the combination treatment had concentration-dependently caused decrease in the caspase activities. rHuEPO-tamoxifen combination markedly increased MCF-7 cells entering the SubG0/G1 phase of the cell cycle by more than 500% of the control, while decreasing those entering the G2 + M and S phases by 50%. After 72 h, the combination treatment produced greater (p < 0.05) change in the SubG0/G1 phase than tamoxifen treatment alone. Morphologically, spheroid MCF-7 cells subjected to combination rHuEPO-tamoxifen treatment showed nuclear condensation and margination, cytoplasmic blebbing, necrosis, and early and late apoptosis. Thus, the study showed that rHuEPO-tamoxifen combination induced apoptosis in the spheroid MCF-7 cells. The apoptotic effect of the rHuEPO-tamoxifen combination treatment on the MCF-7 cells was greater than that produced by tamoxifen alone. The rHuEPO-tamoxifen treatment enhanced the caspase-independent apoptotic effects of tamoxifen on the spheroid MCF-7 cells.

Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells.

Since the commercialization of morphine in 1826, numerous alkaloids have been isolated and exploited effectively for the betterment of mankind, including cancer treatment. However, the commercialization of alkaloids as anticancer agents has generally been limited by serious side effects due to their lack of specificity to cancer cells, indiscriminate tissue distribution and toxic formulation excipients. Lipid-based nanoparticles represent the most effective drug delivery system concerning clinical translation owing to their unique, appealing characteristics for drug delivery. To the extent of our knowledge, this is the first review to compile in vitro and in vivo evidence of encapsulating anticancer alkaloids in lipid-based nanoparticles. Alkaloids encapsulated in lipid-based nanoparticles have generally displayed enhanced in vitro cytotoxicity and an improved in vivo efficacy and toxicity profile than free alkaloids in various cancers. Encapsulated alkaloids also demonstrated the ability to overcome multidrug resistance in vitro and in vivo. These findings support the broad application of lipid-based nanoparticles to encapsulate anticancer alkaloids and facilitate their clinical translation. The review then discusses several limitations of the studies analyzed, particularly the discrepancies in reporting the pharmacokinetics, biodistribution and toxicity data. Finally, we conclude with examples of clinically successful encapsulated alkaloids that have received regulatory approval and are undergoing clinical evaluation.

Do Lipid-based Nanoparticles Hold Promise for Advancing the Clinical Translation of Anticancer Alkaloids?

Exosomes are the small extracellular vesicles secreted by cells for intercellular communication. Exosomes are rich in therapeutic cargos such as microRNA (miRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA), DNA, protein, and lipids. Recently, many studies have focused on miRNAs as a promising therapeutic factor to support cartilage regeneration. Exosomes are known to contain a substantial amount of a variety of miRNAs. miRNAs regulate the post-transcriptional gene expression by base-pairing with the target messenger RNA (mRNA), leading to gene silencing. Several exosomal miRNAs have been found to play a role in cartilage regeneration by promoting chondrocyte proliferation and matrix secretion, reducing scar tissue formation, and subsiding inflammation. The exosomal miRNA cargo can be modulated using techniques such as cell transfection and priming as well as post-secretion modifications to upregulate specific miRNAs to enhance the therapeutic effect. Exosomes are delivered to the joints through direct injection or via encapsulation within a scaffold for sustained release. To date, exosome therapy for cartilage injuries has yet to be optimized as the ideal cell source for exosomes, and the dose and method of delivery have yet to be identified. More importantly, a deeper understanding of the role of exosomal miRNAs in cartilage repair is paramount for the development of more effective exosome therapy.

Mesenchymal Stem Cell-Derived Exosomes and MicroRNAs in Cartilage Regeneration: Biogenesis, Efficacy, miRNA Enrichment and Delivery.

Human umbilical cord mesenchymal stem cell-derived small extracellular vesicle (hUC-MSCs-sEVs) therapy has shown promising results to treat diabetes mellitus in preclinical studies. However, the dosage of MSCs-sEVs in animal studies, up to 10 mg/kg, was considered high and may be impractical for future clinical application. This study aims to investigate the efficacy of low-dose hUC-MSCs-sEVs treatment on human skeletal muscle cells (HSkMCs) and type 2 diabetes mellitus (T2DM) rats. Treatment with hUC-MSCs-sEVs up to 100 μg/mL for 48 h showed no significant cytotoxicity. Interestingly, 20 μg/mL of hUC-MSCs-sEVs-treated HSkMCs increased glucose uptake by 80-90% compared to untreated cells. The hUC-MSCs-sEVs treatment at 1 mg/kg improved glucose tolerance in T2DM rats and showed a protective effect on complete blood count. Moreover, an improvement in serum HbA1c was observed in diabetic rats treated with 0.5 and 1 mg/kg of hUC-MSCs-sEVs, and hUC-MSCs. The biochemical tests of hUC-MSCs-sEVs treatment groups showed no significant creatinine changes, elevated alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels compared to the normal group. Histological analysis revealed that hUC-MSCs-sEVs relieved the structural damage to the pancreas, kidney and liver. The findings suggest that hUC-MSCs-sEVs could ameliorate insulin resistance and exert protective effects on T2DM rats. Therefore, hUC-MSCs-sEVs could serve as a potential therapy for diabetes mellitus.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorated Insulin Resistance in Type 2 Diabetes Mellitus Rats.

The significant growth in type 2 diabetes mellitus (T2DM) prevalence strikes a common threat to the healthcare and economic systems globally. Despite the availability of several anti-hyperglycaemic agents in the market, none can offer T2DM remission. These agents include the prominent incretin-based therapy such as glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 inhibitors that are designed primarily to promote GLP-1R activation. Recent interest in various therapeutically useful gastrointestinal hormones in T2DM and obesity has surged with the realisation that enteroendocrine L-cells modulate the different incretins secretion and glucose homeostasis, reflecting the original incretin definition. Targeting L-cells offers promising opportunities to mimic the benefits of bariatric surgery on glucose homeostasis, bodyweight management, and T2DM remission. Revising the fundamental incretin theory is an essential step for therapeutic development in this area. Therefore, the present review explores enteroendocrine L-cell hormone expression, the associated nutrient-sensing mechanisms, and other physiological characteristics. Subsequently, enteroendocrine L-cell line models and the latest L-cell targeted therapies are reviewed critically in this paper. Bariatric surgery, pharmacotherapy and new paradigm of L-cell targeted pharmaceutical formulation are discussed here, offering both clinician and scientist communities a new common interest to push the scientific boundary in T2DM therapy.

Revisiting the concept of incretin and enteroendocrine L-cells as type 2 diabetes mellitus treatment.

The conventional concept of using nanocarriers to deliver chemotherapeutic drugs has advanced to accommodate additional diagnostic capability. Nanotheranostic agents (NTA), combining both treatment and diagnostic tools, are an ideal example of engineering-health integration for cancer management. Owing to the diverse materials used to construct NTAs, their safety, effectiveness, and diagnostic accuracy could vary substantially. This systematic review consolidated current NTAs incorporating 5-fluorouracil and elucidated their toxicity, anticancer efficacy, and imaging capability. Medline and Embase databases were searched up to March 18, 2022. The search, selection, and extraction were performed by the preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines to ensure completeness and reproducibility. Original research papers involving 5-fluorouracil in the preparation of nanoparticles which reported their efficacy, toxicity, and diagnostic capability in animal cancer models were recruited. The quality of included studies was assessed using the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) checklist. Nine studies were eligible for the systematic review. There was no significant toxicity reported based on animal weight and organ histology. Complete tumor remission was observed in several animal models using chemo-thermal ablation with NTAs, proving the enhancement of 5-fluorouracil efficacy. In terms of imaging performance, the time to achieve maximum tumor image intensity correlates with the presence of targeting ligand on NTAs. The NTAs, which are composed of tumor-targeting ligands, hold promises for further development. Based on the input of current NTA research on cancer, this review proposed a checklist of parameters to recommend researchers for their future NTA testing, especially in animal cancer studies. : website, identifier registration number.

Article	Year
Enhanced anti-mammary gland cancer activities of tamoxifen-loaded erythropoietin-coated drug delivery system. PloS one\| PubMed ID: 31291309	2019
How far have we explored fungi to fight cancer? Seminars in cancer biology\| PubMed ID: 33737109	2021
Recombinant Human erythropoietin reduces viability of MCF-7 breast cancer cells from 3D culture without caspase activation. Saudi journal of biological sciences\| PubMed ID: 33935571	2021
Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells. Molecular biotechnology\| PubMed ID: 34061307	2021
Induction of Apoptosis and Autophagy by Ternary Copper Complex Towards Breast Cancer Cells. Anti-cancer agents in medicinal chemistry\| PubMed ID: 34315396	2022
Comparing the Therapeutic Potential of Stem Cells and their Secretory Products in Regenerative Medicine. Stem cells international\| PubMed ID: 34422061	2021
Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells. Saudi journal of biological sciences\| PubMed ID: 34466099	2021
Do Lipid-based Nanoparticles Hold Promise for Advancing the Clinical Translation of Anticancer Alkaloids? Cancers\| PubMed ID: 34771511	2021
Mesenchymal Stem Cell-Derived Exosomes and MicroRNAs in Cartilage Regeneration: Biogenesis, Efficacy, miRNA Enrichment and Delivery. Pharmaceuticals (Basel, Switzerland)\| PubMed ID: 34832875	2021
Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorated Insulin Resistance in Type 2 Diabetes Mellitus Rats. Pharmaceutics\| PubMed ID: 35336023	2022
Revisiting the concept of incretin and enteroendocrine L-cells as type 2 diabetes mellitus treatment. Pharmacological research\| PubMed ID: 35487405	2022
Emerging Nanotheranostics for 5-Fluorouracil in Cancer Therapy: A Systematic Review on Efficacy, Safety, and Diagnostic Capability. Frontiers in pharmacology\| PubMed ID: 35662688	2022

Chee Wun How

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