{"paper_id":"1fa9cbb5-98b6-42fb-a774-50d0a0b02ef4","body_text":"1 \nTackling Anticancer Drug Resistance and Endosomal Escape in Aggressive Brain \nTumors Using Bioelectronics \nAkhil Jain,1,5,‡,*Philippa Wade,2,‡ Snow Stolnik,3 Alistair N. Hume,4 Ian D. Kerr,4 Beth \nCoyle,2 Frankie Rawson5,* \n1Division of Pharmacy and Optometry, School of Health Sciences, University of Manchester, \nManchester, M13 9PL  \n2Children’s Brain Tumour Research Centre, School of Medicine, University of Nottingham, \nBiodiscovery Institute, Nottingham, NG7 2RD, UK \n3Division of Molecular Therapeutics and Formulation Division, School of Pharmacy, \nUniversity of Nottingham, Nottingham, Nottingham, NG7 2RD, UK \n4 School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 \n2UH, UK \n5Bioelectronics laboratory, Division of Regenerative Medicine and Cellular Therapies, School \nof Pharmacy, University of Nottingham, Biodiscovery Institute, University of Nottingham, \nNottingham, Nottingham, NG7 2RD, UK \n*Corresponding author email – Frankie.Rawson@nottingham.ac.uk and \nAkhil.Jain@manchester.ac.uk \n‡Authors contributed equally. \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 2 \nAbstract \nChemotherapy resistance and endosomal entrapment , controlled by intracellular trafficking \nprocesses, is a major factor in treatment failure . Here, we test the hypothesis that external \nelectrical stimulus can be used to modulate intracellular trafficking of chemotherapeutic drugs \nin most common malignant brain tumors in childhood ( medulloblastoma) and gold \nnanoparticles (GNPs)  in adulthood ( glioblastoma). We demonstrate that application of \nalternating current (AC) with frequencies ranging from KHz -MHz and low strength (1V/cm) \nlead to killing of cisplatin and vincristine resistant (mediated by extracellular vesicles) \nmedulloblastoma cell lines. On the other hand, in primary glioblastoma cells, high frequency \nAC (MHz) regulated the endosomal escape of GNPs. No s ignificant effect on the viability of \nthe control medulloblastoma cells (resistant cells cultured in drug free media and non-resistant \ncells) and glioblastoma cells after AC treatment confirmed targeting of intracellular trafficking \nprocess. This work supp orts future application of AC in drug delivery and brain cancer \ntherapy. \nKeywords: alternating current , extracellular vesicles, medulloblastoma, glioblastoma,  gold \nnanoparticles, endosomal escape. \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 3 \n1. Introduction  \nCells have honed their ability to maintain  homeostasis throug h delicately balancing their \ninternal environment by exerting precise control over what enters and exits the cell. This fine-\ntuned regulation is primarily orchestrated by cell and organelle membranes, the gateway to the \ncell and organelles, respectively.1 The implications of this cellular control mechanism extend \nfar beyond basic biological processes. They hold profound significance in the realm of disease \ntreatment, particularly in the context of combating cancer. Cancer cells are notorious for their \nability to evade therapeutic interventions and have evolved strategies to resist the effects of \nchemotherapy drugs through membrane bound organelles and particles such as endosomes and \nextracellular vesicles (EVs) , respectively effectively t hwarting their intended actions .2 This \nadaptation highlights the critical role played by cellular homeostasis in the development of \nresistance and underscores the need for novel approaches to tune these transport mechanisms.  \nEVs play a crucial role in the function of glioblastoma (GBM) and more broadly gliomas, \nwhich are difficult-to-treat cancers.3,4 Within this context, EVs have gained prominence due to \ntheir critical role in various cellular processes, including intercellular communication, cell \nsignaling, and immune regulation. 5 GBM cells secrete these small membranous vesicles, \ncontaining an array of cargo molecules such as proteins, RNA, and lipids. 6 EVs play a \nsignificant role in GBM biology and pathogenesis, where they contribute to tumor growth, \ninvasion, and metastasis by delivering oncogenic proteins and signaling molecules to other \ncells in the tumor microenvironment, suppressing the anti-tumor immune response, promoting \nangiogenesis and inducing resistance to chemotherapy and radiation therapy5.  \nEVs have also emerge d as crucial transport system in medulloblastoma.  Medulloblastoma-\nderived EVs promote tumor growth, invasion, and metastasis, often by mechanisms akin to \nthose observed in GBM -derived EVs .7 The therapeutic potential of EVs in GBM and \nmedulloblastoma is under exploration. EVs could serve as vehicles for delivering therapeutic \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 4 \ndrugs or genes to tumor cells, modulating the tumor microenvironment to suppress tumor \ngrowth and progression, or developing vaccines targeting tumor -specific antigens .8 \nFurthermore, the role of EVs as biomarkers for monitoring disease progression and therapy \nresponse in patients with these aggressive brain tumors holds great promise. 9 Importantly for \nthis study, the reviews of the involvement of EVs in gliomas reveal evidence suggesting that \nglioma cells utilise the vesicles to expel therapeutics, thereby enhancing resistance .4 The \nchallenges encountered in advanced drug delivery further underscore the importance of \nunderstanding cellular homeostasis.10 A long standing challenge in advanced drug delivery is \nthat drugs and nanoparticles can be trafficked and siloed in endosomes and subsequently \ndegraded in lysosomes. To date, only a small fraction of these systems has advanced to clinical \nuse, mainly due to issues such as entrapment in endosomes and degradation in lysosomes. 11 \nFor instance: delivery of siRNA and other chemotherapeutics by lipid nanoparticles or EV \nbased therapeutic delivery is limited by endosomal entrapment. 12, 13 This unfortunate fate can \nrender potentially life-saving medications ineffective.12  \nConsequently, there is an urgent need to develop  widely applicable  technologies that can \nmodulate and precisely control the trafficking of drug, molecules, and nanoparticles within \ncells. By unraveling the intricate mechanisms and developing disruptive technological \napproaches to govern intracellular transport, we can begin to overcome the challenges posed \nto drug delivery and enhance the efficacy of treatments. Such advancements hold promise for \nrevolutionising the field of medicine and could one day improve patient outcomes. \nBioelectronic medicines are an emerging therapeutic approach 14 in which electrical input can \nbe used for the treatment of disease. There has been a recent shift to develop wireless electrical \nsystem for triggering release of drugs from their carriers, as reviewed by Mirvakili and Langer \n15, 16. Additionally, electroporation has been heavily studied for enhancing cell delivery of an \nanti-cancer drug with the most recent study investigating this in single cells .17 We have \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 5 \npreviously demonstrated that ultrasound can be used intracellularly release drug from \nliposomes.18 Our group has recently shown that AC could bypass the plasma membrane to exert \neffects across the cell membrane.16, 19 Moreover, it has been established that electrical input on \nlipid bilayers causes subtle structural perturbations in their structure.20Therefore, w e \nhypothesized that by exploiting electrical input intracellular fate of nanoparticle-based delivery \nsystems for anti-cancer drugs could be modulated.  This could influence two distinct processes \nin cancer treatment: (1) transport of anticancer drug out of cell via EVs. Moderating EVs \ntransport processes would lead to increase cytoplasmic exposure of the drug and cancer cells \nkilling. (2) R elease of GNPs from endosomal/lysosomal compartment into cytoplasm.  This \ncould lead to generation of new nanomedicine from labs to clinics for improved therapeutic \noutcomes. To the best of our knowledge there has been no demonstration of using AC to \nprevent transport of chemotherapies outside of cells via EVs. Furthermore, the added potential \nof using AC to innovate nanoparticle drug delivery systems by increasing their accumulation \nwithin the cytoplasm by facilitating endosomal escape.  \nIn this work, we conducted a study aiming to merge electronics for delivery of AC with chemo-\nresistant (Cisplatin - cis and Vincristine – vin) medulloblastoma and GBM cells. Our objective \nwas to investigate whether this delivery of HF-AC to cells could help overcome the problem \nof EV-mediated drug transport in cancer cells and modulate the intracellular escape of \nnanoparticles by influencing transport process . The results of our study successfully \ndemonstrate this concept, indicating that by combining electronics with cancer cells, we can \npotentially introduce a new bioelectronic technology that holds promise for the treatment of \nresistant cancers. Further dev elopment of  this approach could enhance the efficacy of \nchemotherapies and more broadly the field of cancer therapeutics. \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 6 \n2. Methodology  \n2.1. Cell lines and standard culture conditions : Sonic-hedgehog (SHH) medulloblastoma \nDAOY cell line was purchased from ATCC (ATCC® HTB-186™) and grown in DMEM with \n10% fetal bovine serum (FBS, HyClone (Logan, Utah, USA). Three vehicle and cis -tolerant \nMB Group3 cell lines (DT-D283-DMF, DT-D283-Cis, DT-HD-MB03-DMF, DT-HD-MB03-\nCis, DT -D458-DMF and DT -D458-Cis) were utilised, as previously published .21 The DT-\nD283 and DT-D458 lines were derived in-house whereas the DT-HD-MB03-Cis was obtained \nfrom Gianpiero Di Leva (Keele University, UK).  DT-D283 and DT-D458 cells were cultured \nin DMEM with 10% fetal bovine serum and the DT -D283-CisDT and DT -D458-CisDT \nsupplemented with 1.6 and 0.6 µ M cis (Selleckchem (Houston, TX, USA), S1166) \nrespectively. DT-HD-MB03 were grown in RPMI 1640 with 10% FBS and the DT-HD-MB03-\nCisDT cells were supplemented with 0.5 µm cis. The equivalent volume of vehicle (DMF) was \nadded to the matched vehicle line. All cell lines were mycoplasma tested monthly and grown \nin antibiotic-free culture conditions at 5% CO2 and 37 °C. EV-depleted FBS was generated by \nultracentrifugation at 100,000 × g at 4°C for 18 hours. Filter sterilization using a 0.22 µm filter \n(Millipore) was carried out prior to its addition to culture medium, resulting in EV -depleted \nmedium. \nGBM cells - Glioma INvasive Marginal 31 (GIN) cells from the infiltrative tumour margin and \nGlioma Core Enhanced 31 (GCE) from the core of the tumor were isolated previously 22 16 . \nBoth GIN and GCE cells were cultured in DMEM (Gibco) supplemented with 10% FBS, 1% \nPenicillin/Streptomycin and 1% L-Glutamine. Cells were maintained at 37°C in an incubator, \ncontaining 5% CO2. Cells were tested for mycoplasma every month, where they were grown \nin an antibiotic -free medium for one week before mycoplasma testing. All cells used were \nmycoplasma-free. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 7 \n2.2 Generation of vincristine- and cisplatin-resistant cell lines \nA continuous model of selection was used to generate drug-tolerant MB cell lines resistant to \nvin and cis. SHH DAOY cells were cultured continuously in the presence of vin (Selleckchem, \nS1241) and the concentration dose was escalated upon cell proliferation. Cells were passaged \nin T-25 flasks and initially treated with 1/10th of their vin  EC50 and the dose increased upon \ncell proliferation. A matched DMSO vehicle cell line was generated alongside to account for \nmorphological and genetic changes resulting from vehicle exposure and long -term culture. \nCells were considered resistant when the EC50 against vincristine for the treated cells had \nexceeded the treatment dose and was significantly increased in comparison to the EC50 of the \nvehicle line. DT-DAOY cells were grown in DMEM with 10% FBS, and the DT-DAOY-VinDT \nline supplemented with 2.8 nM vincristine. Akin to the DT-DAOY cells, cis-resistant cell lines \nDT-D458, DT-HD-MB03 and DT -D283 were also generated in a similar manner.21 In brief, \ncells were treated with increasing doses of cis until their EC50 exceeded the treatment dose or \nwas significantly increased in comparison to the vehicle cell line (DMF).  \n2.3 Drug cytotoxicity assay \nCells were seeded into clear bottomed, black -walled 96 -well plates (Greiner; 655096) at a \ndensity of either 1 ×103 (DT-DAOY), 5 × 103 (DT-HD-MB03) or 1 × 104 (DT-D283 and DT-\nD458) cells per well and left overnight. Cells were then challenged with varying concentrations \nof vin or cis prior to being left for 72  hours at 37°C and 5% CO 2. After 72 hours, metabolic \nactivity was assessed using PrestoBlue (Thermo Fisher; A13262) and fluorescence was \nmeasured using the FLUOstar Omega microplate reader at 560/590 nm . Cell viability was \ncalculated as a percentage relative to the vehicle control and EC50 were calculated in GraphPad \nPRISM 9 using nonlinear regression with three parameters. Significant differences between \nEC50 were calculated using one -way ANOVA with Sidak’s multiple comparisons. The data \nrepresents the SEM of three independent experiments. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 8 \n2.4 Isolation of extracellular vesicles  \nTo isolate extracellular vesicles from cell cultures, cells were grown in 1 × T-225 flask up to \n30% confluence, washed twice with Hanks’ Balanced Salt Solution (HBSS, Gibco \n(Loughborough, UK)) and incubated in EV -depleted medium for 48h. Cell culture medium \nwas collected and centrifuged at 300 × g for 5 minutes to pellet cells. The supernatant was then \ncentrifuged again at 1,500 × g for 10 minutes, followed by a final centrifugation at 10,000 x g \nand 4°C for 10 minutes to remove any debris and large particles. The supernatant was filtered \nthrough a 0.22 µm filter prior to ultrafiltration using a 100K MWCO protein concentrator \n(Thermo Scientific™ Pierce™; 88533) where the supernatant was spun at 3,000 × g until a ~ \n1 mL concentrate was left. The 1 mL concentrate was loaded directly onto size -exclusion \nchromatography columns obtained from Izon (qEV 1 / 70 nm ; IC1-70) and EV fractions \ncollected according to manufacturer’s instructions. Concentration of EVs was determi ned \nusing ZetaView® Nanoparticle Tracking Analysis relative to 1 × 106 cells. \n2.4. AC Stimulation: Electrical Stimulation (ES) with AC was carried out by inserting t wo \nsteel electrodes (0.5 mm ´ 25 mm) at the opposite end (fixed at 10 mm from each other) of \neach well in a 24-well plate and dipped in cell culture media. These electrodes were connected \nto an Arbitrary Function Generator (AFG-21225, RS PRO, UK) which delivered the AC sine-\nwave signals, frequency, and amplitude. The cells were stimulated with AC with a desired \nfrequency and peak voltage amplitude of 1V/cm for 30 minutes.  The strength of AC between \nthe electrodes was measured using a digital oscilloscope (TDS 210, Tektronix).  \n2.3. Metabolic activity assay: The medulloblastoma vehicle cell lines DT-D283-DMF, DT-\nD458-DMF, DT-HD-MB03-DMF and DT-DAOY-DMSO and the cell lines resistant to cis  - \ndrug treated (DT) and non-drug treated (NDT) (DT-D283-Cis, DT-D458-Cis, and DT-HD-MB03-\nCis) and vincristine  - drug treated ( DT) and non -drug treated ( NDT) (DT-DAOY-Vin) were \nseeded at density of 1.0 × 105 per well in a 24-well plate. The cells were stimulated using the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 9 \nprotocol mentioned above in the AC stimulation section. Immediately after stimulation with \nAC the cells were incubated at 37°C and 5% CO2 for 24 h before carrying out metabolic activity \nassay. Next, the media containing cells was replaced with fresh media containing 1% \nPrestoBlueTM HS cell viability reagent (ThermoFisher Scientific, UK) and incubated for 1 hour \nbefore reading the fluorescence at 590 nm/610 nm (excitation/ emission) in a Tecan microplate \nreader (Infinite M Plex and Spark 10M). Cells grown in culture media without AC t reatment \nwere used as the negative control. Values are presented in the results relative to negative \ncontrol. The data is represented as an average of triplicate experiment with 3 independent \nrepeats.  \n2.4. Live/Dead assay:  After stimulation with AC the cells were incubated for 24 h in an \nincubator at 37°C and 5% CO 2.  Next, cis resistant and vehicle cells were centrifuged (due to \ntheir semi-adherent nature) , and the pellet was dispersed in fresh media containing 1 mM \nCalcein AM and 1mg/mL Propidium iodide (ThermoFisher, UK), and incubated for 30 min at \n37°C and 5% CO2 in a 24 well plate. The cells were then centrifuged (300g for 5 minutes), and \nthe pellet was washed with PBS. Finally, the cells were placed in a 24 -well plate (m-plate 24 \nwell black, ibiTreat, Thistle Scientific, UK)  in phenol red free medium  and imaged using a \nLeica TCS SPE Confocal Microscope. The proportions of live and dead cells were quantified \nusing ImageJ software. \n2.5. Biocompatibility of gold nanoparticles: The GIN and GCE cells were seeded in a 96-well \nplate at a density of 5 × 103 cells/well and allowed to adhere for 24 h. The media was replaced \nwith fresh media containing Texas red conjugated 100 nm spherical gold nanoparticle (GNP, \nNanopartz, Inc, USA) conjugates at different concentrations (25, 50, and 100 mg/mL) and the \ncells were incubated for 8h, then cells were stimulated with AC of various frequencies at 1V/cm \nfor 30 min. The cells were then incubated for 24 h at 37°C and 5% CO 2. Next, the media was \nreplaced with complete media containing 10% PrestoBlue TM HS cell viability reagent \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 10 \n(ThermoFisher Scientific, UK) and incubated for an hour before reading the fluorescence at \n590 nm / 610 nm (excitation/ emission) in a Tecan microplate reader (Infinite M Plex and Spark \n10M). The  data is represented as an average of triplicate experiment with 3 independent \nrepeats.  \n2.6. Endo/lysosomal escape: GIN 31 and GCE 31 cells were seeded at a density of 4 ´ 104 \ncells/ well in a 24-well plate and incubated at 37°C and 5% CO2. After 24 h, the culture medium \nwas replaced with fresh medium containing CellLight™ Late Endosomes-GFP, BacMam 2.0 \n(ThermoFisher Scientific, UK) and incubated overnight at 37°C and 5% CO2. Later, the media \nwas replaced with fresh media containing 25 µg/mL of GNP and incubated for 8 h. Immediately \nafter the ES, the cells were washed with PBS and imaged using a Leica TCS SPE Confocal \nMicroscope. \n3. Results and Discussions:  \n3.1. Drug Resistance in medulloblastomas: Cis and Vin  are two of the standard of care \nchemotherapies for medulloblastoma. Previously we have described 3 cis  resistant \nmedulloblastoma cell lines;21 in that work, compared to matched vehicle controls, DT -D458-\nCisDT showed 8-fold resistance, DT-HB-MB03-CisDT showed 2-fold resistance and DT-D283-\nCisDT showed 5-fold resistance. In the current work (Fig. 1) we established vin tolerant DAOY \ncell line (DT -DAOY-VinDT) by continuous treatment with escalating concentrations of \nvincristine. DT -DAOY-VinDT has a 4 -fold increase in the EC50 compared to the matched \nvehicle cell line (p<0.001, Fig. 1). \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 11 \n \nFigure 1. Continuous long-term vincristine treatment promotes increased cell resistance \nto vincristine in medulloblastoma SHH DAOY cells. The cells were initially treated with vin \nat 1/10th of their EC50 and upon cell proliferation, the cells were subsequently challenged with \nan increasing dose of vin. Cell viability was assessed via drug -response assays and the EC 50 \ncalculated using nonlinear regression analysis with three parameters.  DAOY-WT - Wild-type \nDAOY cell line, DT-DAOY-Vin - Vin tolerant cell line, DT-DAOY-DMSO – Vehicle cell \nline. Significance was assessed using one -way ANOVA with Šídák ’s multiple comparisons \ntest.  ****p ≤ 0.0001. \nIntriguingly, comparison of the number of E Vs released by our cis (Fig. 2 a -c) tolerant lines \nrevealed a significant increase relative to their vehicle treated pairs (Fig. 2). On the other hand, \nin vin tolerant lines no significant difference was observed compared vehicles cells (Fig. 2d). \nNevertheless, an increase in average number E Vs in drug treated vin tolerant lines was \nobserved, which can be further supported with  literature, where it has also indicated that EVs \ncan act as transporters for efflux of drugs.23 Thus it can be concluded that number of EVs are \nenhanced in drug tolerant lines which together with EC50 data suggest EV mediated drug \ntolerance in medulloblastoma cell lines.  \n0.001 0.01 0.1 1 10 100\n0\n25\n50\n75\n100\n125\nConcentration Vincristine (nM)\nCell Viability (%)\nDAOY-WT\nDT-DAOY-DMSO\nDT-DAOY-Vin\nEC50: 1.5\nEC50: 1\nEC50: 4.1\n****\n****\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 12 \n \nFigure 2. Quantification of E Vs released in drug tolerant cell lines. (a -c) Cis-tolerant \nMBGroup3 cells release significantly more EVs in comparison to their matched vehicle cell lines. \ncis resistant & cis treated cell lines are denoted as DT-D458-CisDT, DT-D283-CisDT, DT-HD-\nMB03-CisDT and vehicle cell lines are denoted as DT-D458-DMF, DT-D283-DMF, DT-HD-\nMB03-DMF cell line (d) EVs release from Vin -tolerant DAOY cells.  Vin resistant & vin \ntreated cell lines (DT-DAOY-VinDT) and vehicle (DT-DAOY-DMSO) cell lines. EVs in both \ncis and vin tolerant/ vehicle cell lines were isolated using size-exclusion chromatography and \nparticle concentration from the pooled EV -containing fractions was quantified using \nZetaView® Nanoparticle Tracking Analysis. The concentration of EVs was calculated relative \nto 1 × 10 6 cells, where cells were counted at the point of harvest. Significant differences \nbetween vehicles and cis /vin-tolerant cells were calculated using unpaired t -test. The data \nrepresents the SEM of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ \n0.0001. \n \n0\n1×1010\n2×1010\n3×1010\n4×1010\n5×1010\n****\nConcentration of EVs / 106 cells\nDT-D458\n   DMF\nDT-D458\n   CisDT\n0\n2×109\n4×109\n6×109\n8×109\n1×1010\n*\nConcentration of EVs/ 106 cells\nDT-D283\n   DMF\nDT-D283\n   CisDT\n0.0\n5.0×109\n1.0×1010\n1.5×1010\n**\nDT-HD-MB-03\n       DMF\nDT-HD-MB-03\n       CisDT\nConcentration of EVs / 106 cells\n0\n2×109\n4×109\n6×109\n8×109\nConcentration of EVs / 106 cells\nns\nDT-DAOY\n  DMSO\nDT-DAOY\n    VinDT\n(b)(a)\n(c) (d)\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 13 \n3.2. Overcoming EVs mediated cisplatin and vincristine resistance in medulloblastoma: \nAfter establishing EV mediated drug resistance in medulloblastoma cell lines , we sought to \nprovide evidence to this effect and investigate whether bioelectronics systems can be used to \ndeliver electrical input to modulate efflux through these pathways . To study the response to \nexternal electrical input on the manipulation of intracellular trafficking modulated by sub -\ncellular entities such as EVs, we used AC (Fig. 3). AC with frequency range of 1 KHz – 5 MHz \nat a constant potential of 1V/cm were utilised to study the response. It is worth emphasising \nthat the AC used in this work are not tumour treating fields as the electrodes were not shielded \nby dielectric material  and does not cause any significant change in the temperature of cell \nculture medium.24 To avoid potential of electrolysis potentials above 1V/cm were avoided. For \nall 4 lines, metabolic activity only decreased in the drug tolerant line when an AC was applied \nin the presence of drugs (Fig. 3 a-d and Fig. S1). This effect increased at high frequencies (Fig. \n3 a-d) and was confirmed to be cell death caused enhanced concentration of drug (cis and vin) \nwithin the cytoplasm (Fig. 3 e -h). This  was in -fitting wit h previous findings that outer \nmembranes are capacitively coupled to AC at high frequency (KHz -MHz). This  leads to \nmembrane electro-permeabilization which further allow the interaction of HF-AC with the sub-\ncellular structures.25 Furthermore, no change in the viability of control cells (not drug treated) \nafter the treatment with HF-AC suggests their interaction with membrane bound EVs carrying \nanticancer drugs  (Fig. S2 ). This could be supported by previous observations reported in \nliterature where low voltage electric fields lead to disruption of EVs and release of its content. \n26 Therefore, based on the obtained data it can be concluded that HF-AC could manipulate \nintracellular trafficking by targeting membrane bound EVs which leads increased vulnerability \nof resistant cells towards cis and vin.  \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 14 \nFigure 3. AC-EFs overcomes cisplatin and vincristine resistance in medulloblastoma cells \nin vitro. The cells were stimulated with s inewave AC (1 MHz, 3 MHz, and 5 MHz) using a \nfrequency generator at a potential of 1V/cm for 30 min  followed by a PrestoBlue assay 24 h \npost electrical stimulation. (a-c) The metabolic activity of cis resistant & cis treated cell lines \n(DT-D458-CisDT, DT-D283-CisDT, DT-HD-MB03-CisDT), cis resistant but non-cis treated cell \nlines (DT-D458-CisNDT, DT-D283-CisNDT, DT-HD-MB03-CisNDT), and vehicle cell lines (DT-\nD458-DMF, DT-D283-DMF, DT-HD-MB03-DMF) cell line . (d)Vin resistant & vin treated \ncell lines (DT-DAOY-VinDT), vin resistant but non-vin treated cell lines (DT-DAOY-VinNDT), \nand vehicle (DT-DAOY-DMSO) cell lines . The error bars represent S.E.M of three \nindependent experiment in triplicates. (e-h) Live/dead staining of ci s and vin  resistant \nmedulloblastoma cell line s. The cells were stained with calcein AM (green, live cells) and \npropidium iodide (red, dead cells) 24 h after stimulation with AC-EFs and imaged using GFP \nand Texas red filter in a Leica TCS SPE Confocal Microscope. Scale bars = 100 µm. NDT – no \ndrug treatment, DT – drug treated. \n0\n1 KHz10 KHz100 KHz500 KHz1 MHz3 MHz5 MHz\n0\n25\n50\n75\n100Metabolic Activity (%) \nDT-D283-DMF\nDT-D283-CisDT\nAC frequency at 1 V/cm\nDT-D283-CisNDT\n0\n1 KHz10 KHz100 KHz500 KHz1 MHz3 MHz5 MHz\n0\n25\n50\n75\n100Metabolic activity %\nDT-D458-DMF\nDT-D458-CisDT\nAC frequency at 1 V/cm\nDT-D458-CisNDT\n(a) (b)\n0\n1 KHz10 KHz100 KHz500 KHz1 MHz3 MHz5 MHz\n0\n25\n50\n75\n100Metabolic activity %\nDT-HD-MB03-DMF\nDT-HD-MB03-CisDT\nAC frequency at 1 V/cm\nDT-HD-MB03-CisNDT\n0\n1 KHz10 KHz100 KHz500 KHz1 MHz3 MHz5 MHz\n0\n20\n40\n60\n80\n100Metabolic Activity (%) \nDT-DAOY-DMSO\nDT-DAOY-VINDT\nAC frequency at 1 V/cm\nDT-DAOY-VinNDT\n(c) (d)\nDT-D283-CisDT cells\nDT-D458-CisDT cells\nDT-HD-MB-03-CisDT cells\nDT-DAOY-VinDT cells\nControl 1 MHz 3 MHz 5 MHz\nControl 1 MHz 3 MHz 5 MHz\nControl 1 MHz 3 MHz 5 MHz\nControl 1 MHz 3 MHz 5 MHz\n(e)\n(f)\n(g)\n(h)\nControl\nControl 3 MHz1 MHz\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 15 \n3.3. Endo/lysosomal escape of gold nanoparticles: We next tested our hypothesis that HF-AC \ntargets subcellular structure involved in intracellular trafficking by studying the endosomal \nescape of GNPs in primary heterogenous brain tumour cell  cultures after the treatment with \nHF-AC (Fig. 4). HF-AC (1-5 MHz) were able to induce endosomal escape of GNPs from \nprimary glioma cells isolated from the invasive edge viz. GIN cells (Fig 4. a) and the tumour \ncore viz. GCE cells (Fig. 4 b ). No significant toxicity was observed after GNP and AC \ntreatment (Fig. S3). This was further validated by obtaining Pearson’s correlation coefficient  \n(PCC) which predicted the degree of overlap between green channel (late endosomes) and \nGNPs (red).27 The PCC obtained from confocal microscopy images  shown in Fig 4 a and b,  \nconfirmed that application of HF -AC leads to  endosomal escape  of GNPs.  This could be \nexplained based on previous studies that suggest that HF-AC can induce transmembrane \npotentials which cause transient disruption in cell membrane structures , without causing any \ntoxicity Importantly, it has been reported that low MHz frequencies can penetrate deep into the \ncytoplasm to  manipulate sub -cellular structures .28  Based on these literature studies the \npossibility of GNPs from endosomes cannot be ruled out however leaking of 100 nm GNPs \ndue to the electro -permeabilization of plasma membrane requires further studies. Another \npossible mechanism that could be considered is the behavior of GNPs as electric field \ntransducers.29 In this case, the  polarization of GNPs in presence of AC could allow them to \ninteract with plasma membrane in a way that facilitates their movements outwards. However, \nit is unclear how HF -AC could manipulate endosomal membrane, thus highlighting the need \nfor new investigations on understanding the underlying mechanism  such as AC mediated \nproton sponge effect . In literature there are various reports about the use of external stimuli \nsuch as light and ultrasound to enhance the cytoplasmic concentration of drugs from polymeric \nor metallic nanoparticles outside the endosomal compartment. 30, 31  However, it must be \nemphasized that this effect relies on the properties conjugating ligand or peptides that facilitate \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 16 \nendosomal escape but not on the external stimuli. Although conjugation of ligands that \nenhances endosomal escape has shown great potential, they have been criticized for causing \noff-set toxicity and reducing the surface coverage for the attachment of targeting moieties.31, 32 \nOn the other hand, this work highlights the importance of using external electrical stimuli such \nas AC to enhance cytoplasmic concentration of not only drugs but also metallic nanoparticles.  \n \nFigure 4. AC mediated endosomal escape of AuNPs in patient derived glioblastoma cells \n(GIN 31 and GCE 31) . Confocal microscopy image s to demonstrate endosomal escape of \nGIN 31\nGCE 31\nControl 5 MHz\nGNP + 3 MHz GNP+ 5 MHz\nGNP\nGNP+ 1 MHz\nControl 5 MHz\nGNP + 3 MHz GNP+ 5 MHz\nGNP\nGNP+ 1 MHz\n(a)\n(b)\n(c)\nGIN 31 GCE 31\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0Pearson’s coefficient (a.u.)  \nGNP \nGNP + 1 MHz\nGNP + 3 MHz\nGNP + 5 MHz\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 17 \nAuNPs (8-hour incubation with cells) in (a) GIN 31 and (b) GCE 31 cells immediately after \ntreatment with sine wave HF-AC (3 MHz and 5 MHz) at a potential of 1V/cm for 30 min. Cells \nwere stained with late endosome dye (green) and imaged using a Leica confocal microscope \nwith GFP (late endosomes) and Texas-red (AuNPs) filter settings. Scale bar = 100 µm. To \nconfirm the co-localisation of AuNPs at least 50 cells were analysed. (c) Violin plot depicting \nPearson’s correlation coefficient (obtained using colocalization plugin in ImageJ) to quantify \nco-localisation of AuNPs (red) with late endosome (green) upon application of EFs.  A value \nof 1 represents perfect co -localisation of Texas red (AuNPs) with green channel (late -\nendosome).  \n4. Conclusions: Our findings  support the hypothesis that chemotherapeutic resistance in \naggressive brain tumors may be mediated via intracellular trafficking of increased numbers of \nEVs. Importantly, we have shown that AC can disrupt this  EVs mediated trafficking of \nanticancer drugs to enhance their vulnerability in drug treated medulloblastoma cells . \nFurthermore, we showed that HF-AC could enhance the endosomal escape of GNPs in patient-\nderived GBM cells. Overall, together with ease of HF -AC delivery with no toxic effects on \ncells by itself potentiates the future application of AC in drug delivery  to achieve enhanced \ntherapeutic efficacy for better treatment outcomes.   \n5. Acknowledgements: This work was supported by the Engineering and Physical Sciences \nResearch Council Grant number [EP/R004072/1].  \n6. Authors Contribution: Akhil Jain:  Conceptualization, Methodology, Validation, \nInvestigation, Writing - original draft, Visualization, Writing - review & editing, Formal \nanalysis, Supervision, Funding acquisition. Philippa Wade: Investigation, Writing - original \ndraft, Visualization, Writing - review & editing, Formal analysis. Snow Stolnik: Supervision, \nWriting - review & editing. Alistair N Hume: Supervision, Writing - review & editing. Ian D. \nKerr: Supervision, Writing - review & editing. Beth Coyle: Conceptualization, Methodology, \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 18 \nValidation, Writing - original draft, Writing - review & editing, Supervision, Funding \nacquisition. Frankie Rawson: Conceptualization, Methodology, Validation, Writing - original \ndraft, Writing - review & editing, Supervision, Funding acquisition.  \n7. Conflict of Interest: The authors declare no conflict of interest. All the authors read and \nreviewed the manuscript and agreed for journal submission. \n8. Supporting Information: The data related to live dead staining of medulloblastoma cells at \nfrequencies below <1 MHz (Fig. S1), live dead images of vehicle cell lines (Fig. S2), and \nbiocompatibility of GNPs in primary GBM cells (Fig. S3). \n9. Data Availability: All the data will be available to the readers at free of cost at \nhttps://nottingham.rdmc.ac.uk. \n10. References  \n(1) Mogre, S. S.; Brown, A. I.; Koslover, E. F. Getting around the cell: physical transport in \nthe intracellular world. Physical Biology 2020, 17 (6), 061003. \n(2) Yang, Q.; Xu, J.; Gu, J.; Shi, H.; Zhang, J.; Zhang, J.; Chen, Z.-S.; Fang, X.; Zhu, T.; Zhang, \nX. Extracellular Vesicles in Cancer Drug Resistance: Roles, Mechanisms, and Implications. \nAdvanced Science 2022, 9 (34), 2201609. DOI: https://doi.org/10.1002/advs.202201609. \nFontana, F.; Carollo, E.; Melling, G. E.; Carter, D. R. Extracellular vesicles: Emerging \nmodulators of cancer drug resistance. 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It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 23 \nSupporting Information \n \nTackling Anticancer Drug Resistance and Endosomal Escape in Aggressive Brain \nTumors Using Bioelectronics \nAkhil Jain,1,5,‡,*Philippa Wade,2,‡ Snow Stolnik,3 Alistair N. Hume,4 Ian D. Kerr,4 Beth \nCoyle,2 Frankie Rawson5,* \n1Division of Pharmacy and Optometry, School of Health Sciences, University of Manchester, \nManchester, M13 9PL  \n2Children’s Brain Tumour Research Centre, School of Medicine, University of Nottingham, \nBiodiscovery Institute, Nottingham, NG7 2RD, UK \n3Division of Molecular Therapeutics and Formulation Division, School of Pharmacy, \nUniversity of Nottingham, Nottingham, Nottingham, NG7 2RD, UK \n4 School of Life Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 \n2UH, UK \n5Bioelectronics laboratory, Division of Regenerative Medicine and Cellular Therapies, School \nof Pharmacy, University of Nottingham, Biodiscovery Institute, University of Nottingham, \nNottingham, Nottingham, NG7 2RD, UK \n*Corresponding author email – Frankie.Rawson@nottingham.ac.uk and \nAkhil.Jain@manchester.ac.uk \n‡Authors contributed equally. \n \n \n \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 24 \n \nFigure S1. – AC-EFs overcomes cis and vin resistance in medulloblastoma cells in vitro. The \ncells were stimulated with square wave AC -EFs (1 KHz, 10 KHz, 100 KHz, and 500 KHz) \nusing a frequency generator at a potential of 1V/cm for 30 min. Live/dead staining of ci s and \nvin resistant medulloblastoma cell lines. The cells were stained with calcein AM (green, live \ncells) and propidium iodide (red, dead cells) 24 h after stimulation with AC -EFs and imaged \nusing GFP and Texas red filter in a Leica TCS SPE Confocal Micros cope. Scale bars = 100 \nµm. \n1 KHz 10 KHz 100 KHz 500 KHz\n(a)\n(b)\n(c)\n(d)\n1 KHz 10 KHz 100 KHz 500 KHz\n1 KHz 10 KHz 100 KHz 500 KHz\n1 KHz 10 KHz 100 KHz 500 KHz\nDT-D283-CisDT cells\nDT-D458-CisDT cells\nDT-HD-MB-03-CisDT cells\nDT-DAOY-VinDT cells\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 25 \n \nFigure S2. Live/dead staining of vehicle medulloblastoma cell lines. The cells were stained \nwith calcein AM (green, live cells) and propidium iodide (red, dead cells) 24 h after stimulation \nwith AC -EFs and imaged using GFP and Texas red filter in a Leica TCS SPE Confocal \nMicroscope. Scale bars = 100 µm. \n \nControl 1 KHz 10 KHz 100 KHz\n500 KHz 1 MHz 3 MHz 5 MHz\nControl 1 KHz 10 KHz 100 KHz\n500 KHz 1 MHz 3 MHz 5 MHz\nDT-HD-MB-03-CisNDT cells\nDT-DAOY-VinNDT cells\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint \n\n 26 \n \nFigure S3. Biocompatibility of AuNPs on GIN 31 and GCE 31 in presence of AC-EFs. The \ncells were treated with AuNPs for 8 h before stimulation with square wave AC -EFs (1 KHz, \n10 KHz, 100 KHz, and 500 KHz) using a frequency generator at a potential of 1V/cm for 30 \nmin. The metabolic activity of cells was determined 24 hours after stimulation with AC -EFs \nusing PrestoBlue assay. The error bars represent the S.E.M. from a triplicate experiment \nrepeated thrice. \n \n \n(a)\n(b)\n0 5 10 20 40 80\n0\n20\n40\n60\n80\n100\nAuNP concentration (µg/ mL)\nMetabolic Activity (%) \nControl\n500 KHz\n 1 MHz\n 3 MHz\n 5 MHz\nGIN 31\nEF at 1V/cm\n0 5 10 20 40 80\n0\n20\n40\n60\n80\n100\nAuNP concentration (µg/ mL)\nMetabolic Activity (%) \nControl\n500 KHz\n1 MHz\n3 MHz\n5 MHz\nGCE 31\nEF at 1V/cm\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted June 3, 2024. ; https://doi.org/10.1101/2024.06.03.597127doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}