Cyclization-enhanced poly(ß-amino ester)s vectors for efficient CRISPR gene editing therapy.

Among non-viral gene delivery vectors, poly(ß-amino ester)s (PAEs) are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. Over two decades, PAEs have evolved from linear to highly branched structures, significantly enhancing gene delivery efficacy. Building on the proven efficient sets of monomers in highly branched PAEs (HPAEs), this work introduced a new class of cyclic PAEs (CPAEs) constructed via an A2 + B4 + C2 cyclization synthesis strategy and identified their markedly improved gene transfection capabilities in gene delivery applications. Two sets of cyclic PAEs (CPAEs) with rings of different sizes and topologies were obtained. Their chemical structures were confirmed via two-dimensional nuclear magnetic resonance and the photoluminescence phenomena, and their DNA delivery behaviours were investigated and compared with the HPAE counterparts. In vitro assessments demonstrated that the CPAEs with a macrocyclic architecture (MCPAEs), significantly enhanced DNA intracellular uptake and facilitated efficient gene expression while maintaining perfect biocompatibility. The top-performance MCPAEs have been further employed to deliver a plasmid coding dual single guide RNA-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. In recessive dystrophic epidermolysis bullosa (RDEB) patient-derived epidermal keratinocytes, MCPAEs facilitated the CRISPR plasmid delivery and achieved efficient targeted gene editing in multiple colonies.

Backbone cationized highly branched poly(ß-amino ester)s as enhanced delivery vectors in non-viral gene therapy.

Gene therapy holds great potential for treating Lung Cystic Fibrosis (CF) which is a fatal hereditary condition arising from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in dysfunctional CFTR protein. However, the advancement and clinical application of CF gene therapy systems have been hindered due to the absence of a highly efficient delivery vector. In this work, we introduce a new generation of highly branched poly(ß-amino ester) (HPAE) gene delivery vectors for CF treatment. Building upon the classical chemical composition of HPAE, a novel backbone cationization strategy was developed to incorporate additional functional amine groups into HPAE without altering their branching degree. By carefully adjusting the type, proportion, and backbone distribution of the added cationic groups, a series of highly effective HPAE gene delivery vectors were successfully constructed for CF disease gene therapy. In vitro assessment results showed that the backbone cationized HPAEs with randomly distributed 10% proportion of 1-(3-aminopropyl)-4-methylpiperazine (E7) amine groups exhibited superior transfection performance than their counterparts. Furthermore, the top-performed backbone cationized HPAEs, when loaded with therapeutic plasmids, successfully reinstated CFTR protein expression in the CFBE41o- disease model, achieving levels 20-23 times higher than that of normal human bronchial epithelial (HBE) cells. Their therapeutic effectiveness significantly surpassed that of the currently advanced commercial vectors, Xfect and Lipofectamine 3000.

CRISPR-Cas9-based non-viral gene editing therapy for topical treatment of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal monogenic skin disease caused by mutations in COL7A1 gene and lack of functional type VII collagen (C7). Currently, there is no cure for RDEB, and most of the gene therapies under development have been designed as ex vivo strategies because of the shortage of efficient and safe carriers for gene delivery. Herein, we designed, synthesized, and screened a new group of highly branched poly(ß amino ester)s (HPAEs) as non-viral carriers for the delivery of plasmids encoding dual single-guide RNA (sgRNA)-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. The selected HPAEs (named PTTA-DATOD) showed robust transfection efficiency, comparable with or surpassing that of leading commercial gene transfection reagents such as Lipofectamine 3000, Xfect, and jetPEI, while maintaining negligible cytotoxicity. Furthermore, CRISPR-Cas9 plasmids delivered by PTTA-DATOD achieved efficient targeted deletion and restored bulk C7 production in RDEB patient keratinocyte polyclones. The non-viral CRISPR-Cas9-based COL7A1 exon deletion approach developed here has great potential to be used as a topical treatment for RDEB patients with mutations in COL7A1 exon 80. Besides, this therapeutic strategy can easily be adapted for mutations in other COL7A1 exons, other epidermolysis bullosa subtypes, and other genetic diseases.

Guanidyl-Rich Poly(ß Amino Ester)s for Universal Functional Cytosolic Protein Delivery and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 Ribonucleoprotein Based Gene Editing.

Protein therapeutics are highly promising for complex disease treatment. However, the lack of ideal delivery vectors impedes their clinical use, especially the carriers for in vivo delivery of functional cytosolic protein. In this study, we modified poly(ß amino ester)s (PAEs) with a phenyl guanidine (PG) group to enhance their suitability for cytosolic protein delivery. The effects of the PG group on protein binding, cell internalization, protein function protection, and endo/lysosomal escape were systematically evaluated. Compared to the unmodified PAEs (L3), guanidyl rich PAEs (L3PG) presented superior efficiency of protein binding and protein internalization, mainly via clathrin-mediated endocytosis. In addition, both PAEs showed robust capabilities to deliver cytosolic proteins with different molecular weight (ranging from 30 to 464 kDa) and isoelectric points (ranging from 4.3 to 9), which were significantly improved in comparison with the commercial reagents of PULsin and Pierce Protein Transection Reagent. Moreover, L3PG successfully delivered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 ribonucleoprotein (RNP) into HeLa cells expressing green fluorescent protein (GFP) and achieved more than 80% GFP expression knockout. These results demonstrated that guanidyl modification on PAEs can enhance its capabilities for intracellular delivery of cytosolic functional proteins and CRISPR/Cas9 ribonucleoprotein. The guanidyl-rich PAEs are promising nonviral vectors for functional protein delivery and potential use in protein and nuclease-based gene editing therapies.

Artificial Intelligence (AI)-Aided Structure Optimization for Enhanced Gene Delivery: The Effect of the Polymer Component Distribution (PCD).

Gene therapy has emerged as a significant advancement in medicine in recent years. However, the development of effective gene delivery vectors, particularly polymer vectors, remains a significant challenge. Limited understanding of the internal structure of polymer vectors has hindered efforts to enhance their efficiency. This work focuses on investigating the impact of polymer structure on gene delivery, using the well-known polymeric vector poly(ß-amino ester) (PAE) as a case study. For the first time, we revealed the distinct characteristics of individual polymer components and their synergistic effects-the appropriate combination of different components within a polymer (high MW and low MW components) on gene delivery. Additionally, artificial intelligence (AI) analysis was employed to decipher the relationship between the polymer component distribution (PCD) and gene transfection performance. Guided by this analysis, a series of highly efficient polymer vectors that outperform current commercial reagents such as jetPEI and Lipo3000 were developed, among which the transfection efficiency of the PAE-B1-based polyplex was approximately 1.5 times that of Lipo3000 and 2 times that of jetPEI in U251 cells.

3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(ß-Amino Ester)s from Step Growth Polymerization.

The topological structures of polymers play a critical role in determining their gene delivery efficiency. Exploring novel polymeric structures as gene delivery vectors is thus of great interest. In this work, a new generation of multi-cyclic poly(ß-amino ester)s (CPAEs) with unique topology structure was synthesized for the first time via step growth polymerization. Through controlling the occurrence stage of cyclization, three types of CPAEs with rings of different sizes and topologies were obtained. In vitro experiments demonstrated that the CPAEs with macro rings (MCPAEs) significantly boosted the transgene expression comparing to their branched counterparts. Moreover, the MCPAE vector with optimized terminal group efficiently delivered the CRISPR plasmid coding both Staphylococcus aureus Cas9 nuclease and dual guide sgRNAs for gene editing therapy.

Efficacy evaluation of chimeric antigen receptor-modified human peritoneal macrophages in the treatment of gastric cancer.

BACKGROUND: Gastric cancer is one of the most common cancers. Peritoneal carcinomatosis (PC) appears to be the most common pattern of recurrence, and more than half of the GC patients eventually die from PC. Novel strategies for the management of patients with PC are urgently needed. Recently, rapid progress has been made in adoptive transfer therapy by using macrophages as the effector cells due to their capabilities of phagocytosis, antigen presentation, and high penetration. Here, we generated a novel macrophage-based therapy and investigated anti-tumoral effects on GC and potential toxicity. METHODS: We developed a novel Chimeric Antigen Receptor-Macrophage (CAR-M) based on genetically modifying human peritoneal macrophages (PMs), expressing a HER2-FcεR1γ-CAR (HF-CAR). We tested HF-CAR macrophages in a variety of GC models in vitro and in vivo. RESULTS: HF-CAR-PMs specifically targeted HER2-expressed GC, and harboured the FcεR1γ moieties to trigger engulfment. Intraperitoneal administration of HF-CAR-PMs significantly facilitated the HER2-positive tumour regression in PC mouse model and prolonged the overall survival rate. In addition, the combined use of oxaliplatin and HF-CAR-PMs exhibited significantly augment anti-tumour activity and survival benefit. CONCLUSIONS: HF-CAR-PMs could represent an exciting therapeutic option for patients with HER2-positive GC cancer, which should be tested in carefully designed clinical trials.

Branch Unit Distribution Matters for Gene Delivery.

As a key nonviral gene therapy vector, poly(ß-amino ester) (PAE) has demonstrated great potential for clinical application after two decades of development. However, even after extensive efforts in structural optimizations, including screening chemical composition, molecular weight (MW), terminal groups, and topology, their DNA delivery efficiency still lags behind that of viral vectors. To break through this bottleneck, in this work, a thorough investigation of highly branched PAEs (HPAEs) was conducted to correlate their fundamental internal structure with their gene transfection performance. We show that an essential structural factor, branch unit distribution (BUD), plays an important role for HPAE transfection capability and that HPAEs with a more uniform distribution of branch units display better transfection efficacy. By optimizing BUD, a high-efficiency HPAE that surpasses well-known commercial reagents (e.g., Lipofectamine 3000 (Lipo3000), jetPEI, and Xfect) can be generated. This work opens an avenue for the structural control and molecular design of high-performance PAE gene delivery vectors.

A New Optimization Strategy of Highly Branched Poly(ß-Amino Ester) for Enhanced Gene Delivery: Removal of Small Molecular Weight Components.

Highly branched poly(ß-amino ester) (HPAE) has become one of the most promising non-viral gene delivery vector candidates. When compared to other gene delivery vectors, HPAE has a broad molecular weight distribution (MWD). Despite significant efforts to optimize HPAE targeting enhanced gene delivery, the effect of different molecular weight (MW) components on transfection has rarely been studied. In this work, a new structural optimization strategy was proposed targeting enhanced HPAE gene transfection. A series of HPAE with different MW components was obtained through a stepwise precipitation approach and applied to plasmid DNA delivery. It was demonstrated that the removal of small MW components from the original HPAE structure could significantly enhance its transfection performance (e.g., GFP expression increased 7 folds at w/w of 10/1). The universality of this strategy was proven by extending it to varying HPAE systems with different MWs and different branching degrees, where the transfection performance exhibited an even magnitude enhancement after removing small MW portions. This work opened a new avenue for developing high-efficiency HPAE gene delivery vectors and provided new insights into the understanding of the HPAE structure-property relationship, which would facilitate the translation of HPAEs in gene therapy clinical applications.

Hyperbranched Poly(ß-amino ester)s (HPAEs) Structure Optimisation for Enhanced Gene Delivery: Non-Ideal Termination Elimination.

Many polymeric gene delivery nano-vectors with hyperbranched structures have been demonstrated to be superior to their linear counterparts. The higher delivery efficacy is commonly attributed to the abundant terminal groups of branched polymers, which play critical roles in cargo entrapment, material-cell interaction, and endosome escape. Hyperbranched poly(ß-amino ester)s (HPAEs) have developed as a class of safe and efficient gene delivery vectors. Although numerous research has been conducted to optimise the HPAE structure for gene delivery, the effect of the secondary amine residue on its backbone monomer, which is considered the non-ideal termination, has never been optimised. In this work, the effect of the non-ideal termination was carefully evaluated. Moreover, a series of HPAEs with only ideal terminations were synthesised by adjusting the backbone synthesis strategy to further explore the merits of hyperbranched structures. The HPAE obtained from modified synthesis methods exhibited more than twice the amounts of the ideal terminal groups compared to the conventional ones, determined by NMR. Their transfection performance enhanced significantly, where the optimal HPAE candidates developed in this study outperformed leading commercial benchmarks for DNA delivery, including Lipofectamine 3000, jetPEI, and jetOPTIMUS.

Cell death induced in glioblastoma cells by Plasma-Activated-Liquids (PAL) is primarily mediated by membrane lipid peroxidation and not ROS influx.

Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects.

Crosstalk between IL-15Rα+ tumor-associated macrophages and breast cancer cells reduces CD8+ T cell recruitment.

BACKGROUND: Interleukin-15 (IL-15) is a promising immunotherapeutic agent owing to its powerful immune-activating effects. However, the clinical benefits of these treatments are limited. Crosstalk between tumor cells and immune cells plays an important role in immune escape and immunotherapy drug resistance. Herein, this study aimed to obtain in-depth understanding of crosstalk in the tumor microenvironment for providing potential therapeutic strategies to prevent tumor progression. METHODS: T-cell killing assays and co-culture models were developed to determine the role of crosstalk between macrophages and tumor cells in breast cancer resistant to IL-15. Western blotting, histological analysis, CRISPR-Cas9 knockout, multi-parameter flow cytometry, and tumor cell-macrophage co-injection mouse models were developed to examine the mechanism by which IL-15Rα+ tumor-associated macrophages (TAMs) regulate breast cancer cell resistance to IL-15. RESULTS: We found that macrophages contributed to the resistance of tumor cells to IL-15, and tumor cells induced macrophages to express high levels of the α subunit of the IL-15 receptor (IL-15Rα). Further investigation showed that IL-15Rα+ TAMs reduced the protein levels of chemokine CX3C chemokine ligand 1 (CX3CL1) in tumor cells to inhibit the recruitment of CD8+ T cells by releasing the IL-15/IL-15Rα complex (IL-15Rc). Administration of an IL-15Rc blocking peptide markedly suppressed breast tumor growth and overcame the resistance of cancer cells to anti- programmed cell death protein 1 (PD-1) antibody immunotherapy. Interestingly, Granulocyte-macrophage colony-stimulating factor (GMCSF) induced γ chain (γc) expression to promote tumor cell-macrophage crosstalk, which facilitated tumor resistance to IL-15. Additionally, we observed that the non-transcriptional regulatory function of hypoxia inducible factor-1alpha (HIF-1α) was essential for IL-15Rc to regulate CX3CL1 expression in tumor cells. CONCLUSIONS: The IL-15Rc-HIF-1α-CX3CL1 signaling pathway serves as a crosstalk between macrophages and tumor cells in the tumor microenvironment of breast cancer. Targeting this pathway may provide a potential therapeutic strategy for enhancing the efficacy of cancer immunotherapy.

Pretreatment of umbilical cord derived MSCs with IFN-γ and TNF-α enhances the tumor-suppressive effect on acute myeloid leukemia.

Currently, the standard therapeutic approach of AML consists of chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). However, these strategies are usually associated with adverse side effects and high risk of relapse following HSCT. Thus, it is imperative to find an alternative way against AML progression. Here, we showed that treatment with umbilical cord-derived mesenchymal stem cells (UC-MSCs) could efficiently induce apoptosis in both primary AML patient-derived leukemic cells and AML cell lines. Mechanistically, tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in UC-MSCs mediated the proapoptotic effect in AML cells. Besides, indoleamine 2,3-dioxygenase (IDO) secreted by UC-MSCs blocked the cell cycle progression and inhibited the proliferation of AML cells. Importantly, we found that incubation of UC-MSCs with IFN-γ and TNF-α could upregulate the expression of TRAIL and IDO, resulting in an intensive pro-apoptotic efficacy. UC-MSCs pre-treatment could not only relieve the AML burden but also eliminate AML cells in a xenograft AML model. Our findings have shed light on an effective pre-activated approach to aggravating the anti-leukemia effect of MSC. Furthermore, a novel and safe stem cell-based therapy approach for AML treatment.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

An Injectable Chitosan-Based Self-Healable Hydrogel System as an Antibacterial Wound Dressing.

Due to their biodegradability and biocompatibility, chitosan-based hydrogels have great potential in regenerative medicine, with applications such as bacteriostasis, hemostasis, and wound healing. However, toxicity and high cost are problems that must be solved for chitosan-based hydrogel crosslinking agents such as formaldehyde, glutaraldehyde, and genipin. Therefore, we developed a biocompatible yet cost-effective chitosan-based hydrogel system as a candidate biomaterial to prevent infection during wound healing. The hydrogel was fabricated by crosslinking chitosan with dialdehyde chitosan (CTS-CHO) via dynamic Schiff-base reactions, resulting in a self-healable and injectable system. The rheological properties, degradation profile, and self-healable properties of the chitosan-based hydrogel were evaluated. The excellent antibacterial activity of the hydrogel was validated by a spread plate experiment. The use of Live/Dead assay on HEK 293 cells showed that the hydrogel exhibited excellent biocompatibility. The results demonstrate that the newly designed chitosan-based hydrogel is an excellent antibacterial wound dressing candidate with good biocompatibility.

Enhanced pyrazolopyrimidinones cytotoxicity against glioblastoma cells activated by ROS-Generating cold atmospheric plasma.

Pyrazolopyrimidinones are fused nitrogen-containing heterocyclic systems, which act as a core scaffold in many pharmaceutically relevant compounds. Pyrazolopyrimidinones have been demonstrated to be efficient in treating several diseases, including cystic fibrosis, obesity, viral infection and cancer. In this study using glioblastoma U-251MG cell line, we tested the cytotoxic effects of 15 pyrazolopyrimidinones, synthesised via a two-step process, in combination with cold atmospheric plasma (CAP). CAP is an adjustable source of reactive oxygen and nitrogen species as well as other unique chemical and physical effects which has been successfully tested as an innovative cancer therapy in clinical trials. Significantly variable cytotoxicity was observed with IC50 values ranging from around 11 µM to negligible toxicity among tested compounds. Interestingly, two pyrazolopyrimidinones were identified that act in a prodrug fashion and display around 5-15 times enhanced reactive-species dependent cytotoxicity when combined with cold atmospheric plasma. Activation was evident for direct CAP treatment on U-251MG cells loaded with the pyrazolopyrimidinone and indirect CAP treatment of the pyrazolopyrimidinone in media before adding to cells. Our results demonstrated the potential of CAP combined with pyrazolopyrimidinones as a programmable cytotoxic therapy and provide screened scaffolds that can be used for further development of pyrazolopyrimidinone prodrug derivatives.

Reactive oxygen species (ROS): utilizing injectable antioxidative hydrogels and ROS-producing therapies to manage the double-edged sword.

Reactive oxygen species (ROS) are generated in cellular metabolism and are essential for cellular signalling networks and physiological functions. However, the functions of ROS are 'double-edged swords' to living systems that have a fragile redox balance between ROS generation and elimination. A modest increase of ROS leads to enhanced cell proliferation, survival and benign immune responses, whereas ROS stress that overwhelms the cellular antioxidant capacity can damage nucleic acids, proteins and lipids, resulting in oncogenic mutations and cell death. ROS are therefore involved in many pathological conditions. On the other hand, ROS present selective toxicity and have been utilised against cancer and pathogens, thus also acting as a double-edged sword in the healthcare field. Injectable antioxidative hydrogels are gel precursors that form hydrogel constructs in situ upon delivery in vivo to maintain an antioxidative capacity. These hydrogels have been developed to counter ROS-induced pathological conditions, with significant advantages of biocompatibility, excellent moldability, and minimally invasive delivery. The intrinsic, readily controllable ROS-scavenging ability of the functionalised hydrogels overcomes many drawbacks of small molecule antioxidants. This review summarises the roles of ROS under pathological conditions and describes the state-of-the-art of injectable antioxidative hydrogels. A particular emphasis is also given to current ROS-producing therapeutic interventions, enabling potential application of injectable antioxidant hydrogels to prevent the adverse effects of many cancer and infection treatments.

Platinum nanoparticles inhibit intracellular ROS generation and protect against cold atmospheric plasma-induced cytotoxicity.

Platinum nanoparticles (PtNPs) have been investigated for their antioxidant abilities in a range of biological and other applications. The ability to reduce off-target cold atmospheric plasma (CAP) cytotoxicity would be useful in Plasma Medicine; however, little has been published to date about the ability of PtNPs to reduce or inhibit the effects of CAP. Here we investigate whether PtNPs can protect against CAP-induced cytotoxicity in cancerous and non-cancerous cell lines. PtNPs were shown to dramatically reduce intracellular reactive species (RONS) production in U-251 MG cells. However, RONS generation was unaffected by PtNPs in medium without cells. PtNPs protect against CAP induced mitochondrial membrane depolarization, but not cell membrane permeabilization which is a CAP-induced RONS-independent event. PtNPs act as potent intracellular scavengers of reactive species and can protect against CAP induced cytotoxicity. PtNPs, showing no significant biocorrosion, may be useful as a catalytic antioxidant for healthy tissue and for protecting against CAP-induced tissue damage.

Ursolic Acid Inhibits Collective Cell Migration and Promotes JNK-Dependent Lysosomal Associated Cell Death in Glioblastoma Multiforme Cells.

Ursolic acid (UA) is a bioactive compound which has demonstrated therapeutic efficacy in a variety of cancer cell lines. UA activates various signalling pathways in Glioblastoma multiforme (GBM) and offers a promising starting point in drug discovery; however, understanding the relationship between cell death and migration has yet to be elucidated. UA induces a dose dependent cytotoxic response demonstrated by flow cytometry and biochemical cytotoxicity assays. Inhibitor and fluorescent probe studies demonstrate that UA induces a caspase independent, JNK dependent, mechanism of cell death. Migration studies established that UA inhibits GBM collective cell migration in a time dependent manner that is independent of the JNK signalling pathway. Cytotoxicity induced by UA results in the formation of acidic vesicle organelles (AVOs), speculating the activation of autophagy. However, inhibitor and spectrophotometric analysis demonstrated that autophagy was not responsible for the formation of the AVOs. Confocal microscopy and isosurface visualisation determined co-localisation of lysosomes with the previously identified AVOs, thus providing evidence that lysosomes are likely to be playing a role in UA induced cell death. Collectively, our data identify that UA rapidly induces a lysosomal associated mechanism of cell death in addition to UA acting as an inhibitor of GBM collective cell migration.

Cold atmospheric plasma induces silver nanoparticle uptake, oxidative dissolution and enhanced cytotoxicity in glioblastoma multiforme cells.

Silver nanoparticles (AgNP) emerged as a promising reagent for cancer therapy with oxidative stress implicated in the toxicity. Meanwhile, studies reported cold atmospheric plasma (CAP) generation of reactive oxygen and nitrogen species has selectivity towards cancer cells. Gold nanoparticles display synergistic cytotoxicity when combined with CAP against cancer cells but there is a paucity of information using AgNP, prompting to investigate the combined effects of CAP using dielectric barrier discharge system (voltage of 75 kV, current is 62.5 mA, duty cycle of 7.5kVA and input frequency of 50-60Hz) and 10 nm PVA-coated AgNP using U373MG Glioblastoma Multiforme cells. Cytotoxicity in U373MG cells was >100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC50 of 4.30 µg/mL with PVA-AgNP alone compared with 0.07 µg/mL after 25s CAP and 0.01 µg/mL 40s CAP). Combined cytotoxicity was ROS-dependent and was prevented using N-Acetyl Cysteine. A novel darkfield spectral imaging method investigated and quantified AgNP uptake in cells determining significantly enhanced uptake, aggregation and subcellular accumulation following CAP treatment, which was confirmed and quantified using atomic absorption spectroscopy. The results indicate that CAP decreases nanoparticle size, decreases surface charge distribution of AgNP and induces uptake, aggregation and enhanced cytotoxicity in vitro.