Raloxifene-loaded and aptamer-bonded exosomes induce autophagic and apoptotic death in HeLa cells by enhancing the lysosomotropic effect

Background:Raloxifene,a selective estrogen receptor modulator,is also known to be a lysosomotropic agent.The bioavailability of raloxifene is around 2%due to extensive hepatic transport.Exosomes are nanosized vesicles that are naturally released from cells.Method:In this study,exosomes released from HeLa cervical cancer cells were loaded with raloxifene to increase its bioavailability,and an aptamer was attached to the exosome membrane for targeting only HeLa cells.Characterization of exosomes isolated from HeLa cells was performed by transmission electron microscopy,zeta sizer,and western blotting.In addition,the cytotoxic,apoptotic,autophagic,and lysosomotropic effects of the prepared Exo-Apt-Ral formulation on HeLa cervical cancer cells were investigated.Results:According to zeta analysis,the sizes of the empty exosome and Exo-Apt-Ral formulation were measured as 66±12 and 120±21 nm,respectively.There was a rise in the lysosomal permeability of HeLa cells after the Exo-Apt-Ral application.In addition,both apoptotic and autophagic death mechanisms were triggered in HeLa cells after the Exo-Apt-Ral application.Conclusion:This study showed that raloxifene functionalized by loading into aptamer-bound exosomes can be a new targeted drug carrier system for cervical cancer.

Microfluidic methods used in exosome isolation

Exosomes are important biomarkers for clinical diagnosis.It is critical to isolate secreted exosomes from bodily fluids such as blood,saliva,breast milk,and urine for liquid biopsy applications.The field of microfluidics provides numerous benefits for biosample processing,diagnostics,and prognostics.Several microfluidics-based methods have been employed for the isolation and purification of exosomes in the last ten years.These microfluidic methods can be grouped into two categories based on passive and active isolation mechanisms.In the first group,inertial and hydrodynamic forces are employed to separate exosomes based on their size differences.In the second group,external forcefields are integrated into microfluidic platforms to actively isolate exosomes from other bioparticles.In this paper,the application of microfluidic methods in exosome isolation is discussed,and future perspectives on this field are highlighted.

Bio-fabrication of liver cancer stem cells and testing of gemcitabine

Aim:Cancer stem cells are cell populations that are essential in drug resistance and cancer metastasis.Some liver cancer cells exhibit the characteristics of cancer stem cells,and it is crucial to study the activities and interactions of drugs in these cells.Huh7 is a human liver cancer cell with stem cell biomarkers and is used with induced pluripotent stem cells to form various cancer organoids through encapsulation methods.Due to their ease of use without animal testing,bio-fabrication studies of cell-encapsulated models have gained importance in the pharmaceutical industry in recent years.This study aimed to biofabricate Huh7 human liver cancer stem cells by alginate encapsulation and test gemcitabine’s efficacy.Methods:Huh7 cells were encapsulated with alginate(0.8%w/v)and fibronectin,and their viability was evaluated with 3.2μM gemcitabine on days 1,3,6,9,and 12.Furthermore,gene expressions of stem cell markers CD90 and AFP were evaluated in encapsulated Huh7 cells by qPCR.In addition,IL-6 secretion in the cell medium was measured by ELISA for the tumor microenvironment.Results:Encapsulation of Huh7 cells was found to maintain their viability and stem cell properties for up to 12 days.In addition,alginate-encapsulated Huh7 cells were bio-fabricated to demonstrate long-term gemcitabine response.While the effect of the gemcitabine was evaluated in alginate-encapsulated Huh7 cells,CD90 and AFP mRNA levels were significantly reduced in the cells and IL-6 secretion was decreased in the tumor microenvironment.Conclusion:This study demonstrated that bio-fabrication of alginate-encapsulated Huh7 cells is a novel approach for long-term drug testing in liver cancer models.Bio-fabricated alginate-encapsulated cancer stem cells may be a cheaper and faster method for the testing of many drugs.