Journal of Nanobiotechnology

In the past years, we have designed biodegradable poly(benzyl malate) (PMLABe73) homopolymer and amphiphilic poly(ethylene glycol)-b-PMLABe (PEG42-b-PMLABe73) copolymer and several modified (co)polymers to produce biocompatible polymeric nanoparticles (NPs) capable of targeting hepatic cells in vitro with the goal to develop applications in the treatment of liver diseases. The current study aimed at comparing the uptake of PMLABe73 PEG42-b-PMLABe73-based NPs in human hepatic HepaRG cells, primary macrophages and peripheral blood mononuclear cells (PBMC). The uptake of NPs prepared from PEG42-b-PMLABe73 was significantly lower than that of PMLABe73 in both hepatic cells and macrophages. In addition, the NPs uptake by HepaRG cells was inversely correlated to the density of PEG present on their surface. In contrast, the internalization of with PMLABe-based NPs by human macrophages was not affected by low PEG densities, only uptake of fully pegylated PEG42-b-PMLABe73based-NPs was significantly decreased. Herein, we also showed that PMLABe-based NPs did not strongly accumulated in PBMC, T lymphocytes and neutrophils while monocytes showed slightly higher uptake of these NPs. Moreover, we further demonstrated that PMLABe-derived NPs by did not trigger inflammasome activation and secretion of pro-inflammatory cytokines neither in macrophages nor HepaRG cells. Then, we demonstrated that peptide GBVA10-9 derived from George Baker (GB) Virus A, known to exhibit a good hepatotropism did not significantly affect the uptake of PMLABe73-based NPs in HepaRG cells and macrophages, when grafted onto these NPs. The present results demonstrate that PMLABe-derived NPs are very efficiently internalized in both macrophages and hepatocytes but not in PBMC and reinforce our previous reports regarding their biocompatibility.

Cellular accumulation of biomedical nanoparticles could be affected by cellular biological properties. However, little is known about the influence of cell cycle and autophagy on nanoparticle accumulation. Whats even more tough is that several long-lasting methodological barriers have hampered the experimental performance and restricted related research progress. Herein, a multi-functional platform was constructed for simultaneously overcoming existing obstacles by integrating several technical approaches, particularly mitotic shake-off, for thorough cell cycle phase separation. Strikingly, application of this platform revealed that G2-phase and M-phase cells, two cell populations previously muddled up together as G2/M-phase cells, respectively exhibited the maximum and minimum accumulation of lipid-based nanoparticles. Moreover, although further verification is needed, we have provided a novel line of evidence for enhanced nanoparticle accumulation by autophagy blockade. Besides providing a technical solution, this study discovered characteristic cell cycle- and autophagy-associated nanoparticle accumulations that may offer new insights for optimization and application of nanomedicines.

BackgroundTriple-negative breast cancer is an aggressive breast cancer subtype characterized by the absence of human epidermal growth factor receptor 2, estrogen and progesterone receptors, limiting targeted therapy options. Cisplatin, a chemotherapeutic agent, induces DNA damage and exhibits some efficacy against triple-negative breast cancer, but its effectiveness is often reduced by chemoresistance and systemic toxicity. A very promising strategy to augment cisplatin treatment can be based on combining it with the biologic Cetuximab, an epidermal growth factor receptor inhibitor, which boosts cisplatin efficacy by inducing ferroptosis. ResultsTo optimize this strategy in a biocompatible and precise manner, we developed a nanoplatform based on red blood cell-derived extracellular vesicles for the combined delivery of Cetuximab and cisplatin, enabling immune evasion, and the possibility of autologous personalization and GMP-compliant production. Owing to their DNA-free lumen and lack of EGFR, RBC-EVs preserve cisplatin activity and prevent interference with cetuximab. This formulation enhances cisplatins cytotoxicity by up to 50%, as shown in vitro and in patient-derived organoids. It effectively reduces chemoresistance by downregulating hypoxia-related genes and promoting ferroptosis, additionally, it improves cisplatins cytotoxic effects while reducing hemotoxicity compared to the administration of free cisplatin. ConclusionsThese findings highlight the potential of red blood cell-derived extracellular vesicles as a biocompatible delivery system enabling combined therapy and offering a promising strategy to overcome current limitations in TNBC treatment.

ObjectiveThe aim of our study was to integrate bone marrow-derived stem cells (BMSCs) with macrophages and to investigate the performance of this hybrid in IgG clearance, immunosuppression, and the mitigation of inflammatory injury. MethodsBMSCs and RAW264.7 cells were fused through transient transfection using the COVID-19 spike glycoprotein and human angiotensin-converting enzyme 2 (hACE2), respectively. The resulting hybrids were purified using magnetic sorting, and their characteristics were evaluated by examining morphology, glucose uptake rates, phagocytic capacity, cytoskeletal morphology, and the levels of Fc{gamma} receptors, as well as anti-inflammatory, antioxidant, adhesion, and complement inhibitory factors. The IgG clearance capability of the hybrids was assessed by measuring internalization efficiency and infiltration into renal organoids. Additionally, the reparative effects of the hybrids were evaluated using doxorubicin-treated MPC5 damage models. Live cell imaging techniques were employed to investigate the interactions between the hybrids and immature dendritic cells (iDCs). Hybrid-derived nanovesicles were isolated, and their abilities to target IgG and clear IL-6 were evaluated using immunofluorescence and ELISA. Furthermore, chloroplasts and C-dots were co-incubated with the hybrids, and their benefits were assessed by measuring cell viability, oxygen content, ROS levels, mitochondrial function, and the levels of anti-inflammatory, antioxidant, and pro-repair factors. A scratch assay was conducted to determine the impact of chloroplast transplantation on the migration ability of 3T3 cells. ResultsHigh-purity hybrids were successfully produced that cleared IgG while maintaining their anti-inflammatory and antioxidant effects. They promoted the recovery of podocyte injury through vesicular and mitochondrial delivery, as well as efferocytosis. Furthermore, the hybrids inhibited iDCs primarily through migrasomes, intercellular nanomicrotubule connections, and phagocytosis. The derived nanovesicles were capable of residing in IgG-enriched regions and adsorbing IL-6. Interestingly, the transplanted chloroplasts enabled the hybrids to utilize light energy to enhance their antioxidant capacity and promote the migration of 3T3 cells, which contributes to wound repair. Subsequently, the loading of C-dots was beneficial for enhancing resistance to oxidative damage. ConclusionsOur results suggest that hybrids-mediated therapy is a new and creative therapeutic approach for managing immune-mediated nephropathies.Hybrids demonstrated effective immunomodulatory and promoted injury recovery. The derived nanovesicles have the potential to alleviate the inflammatory burden at sites of antibody immune complex deposition. Additionally, the incorporation of chloroplasts and C-dots enhanced the hybrids adaptability to ischemic-hypoxic microenvironments.

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

Current extracellular vesicle (EV) isolation methods depend on large expensive equipment like ultracentrifuges and are laborious and time consuming. There is also currently no method available for high throughput isolation to meet clinical demands. Here, we present a method that combines our previous published ExtraPEG method and magnetic beads. Western blot and nanoparticle tracking analysis (NTA) of the purified EVs revealed higher or equivalent recovery and purity with this method compared to ExtraPEG or size exclusion chromatography (SEC) methods. With this newly developed workflow and automated liquid handling instrument, we have successfully isolated up to 96 EV samples from 5 {micro}L pre-cleared serum in 45 minutes. Moreover, DNA / small RNA / protein purification and profiling steps could be seamlessly integrated into the isolation workflow. To profile EV protein markers, EVs were lysed from the binding step and covalently bound to the surface of the beads. TotalSeq or ELISA antibody can be applied with under a standard protocol. With this extended protocol, researchers can easily complete EV isolation and protein profiling experiment within 8 hours. Taken together, we provide a high throughput method for EV isolation and molecular analyses that may be used for sensitive biomarker detection from biological fluids.

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 m). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

Cancer remains the leading cause of death, with chemotherapy, radiotherapy, and surgical resection being the primary treatment methods. However, chemotherapys side effects, surgical limitations, and drug resistance present significant challenges. Small interfering RNA (siRNA) has emerged as a promising tool in cancer therapy due to its ability to silence disease-related genes selectively. Recent advancements in non-viral delivery systems, particularly cell-penetrating peptides (CPPs), have enhanced the efficacy of siRNA delivery. The use of siRNA as a therapeutic tool in cancer treatment has been reported. However, silencing only one target protein has only minor effects on tumor cell proliferation as previously shown for WRAP-based nanoparticles targeting cyclin-dependent kinase 4 (CDK4) in human glioblastoma cells. Here, we designed a more sophisticated approach to enhance therapeutic efficacy, encapsulating multiple siRNAs targeting CDK4, cyclin D1 (CD1), and Mcl-1 proteins. The siRNA cocktail, delivered via WRAP5 nanoparticles, effectively silenced these targets and reduced cell proliferation in human glioblastoma cells. Furthermore, the nanoparticles also demonstrated potential therapeutic impact in gastrointestinal stromal tumors (GIST), a rare cancer characterized by its tendency to resist standard treatments. This study highlights the versatility of WRAP5 nanoparticles as a platform for personalized cancer therapy, suggesting that siRNA delivery systems may be tailored to specific cancer types for more effective treatment strategies.

Micro and nanoplastics are pollutants which concentration in different biotopes increases continuously over time, which poses the question of their potential effects on health. In animals, these micro and nanoplastics are recognized as particulate materials and thus handled by macrophages, either directly in the case of lung exposure, or after the particles have crossed the epithelial barriers in case of oral or dermal exposure. It is thus important to study the potential effects of micro and nanoplastics on macrophages. Most studies have used an experimental scheme in which the cells of interest are exposed to a single dose of plastics, and where the readout of the studied parameters is made immediately after exposure. However, this classical experimental scheme does not take into account the impact of biopersistence, nor the potential cellular adaptation that may take place when cells are exposed repeatedly to a low dose of plastics. We thus used a repeated exposure scheme, in order to better take into account these phenomena. Within this frame, we compared the macrophages responses to a persistent nanoplastic, i.e. true-to-life polyethylene terephthalate nanoparticles and to a biodegradable nanoplastic, i.e. polycaprolactone, by a combination of proteomic and targeted experiments. Our results show that under this repeated exposure scheme, the proteome changes were of a lesser extent than under the acute exposure mode, indicating cell adaptation. However, polyethylene terephthalate nanoparticles induced oxidative stress and a pro-inflammatory response, while polycaprolactone nanoparticles induced a depression of macrophages functions, indicating harmful effects even in the repeated exposure scheme.

Nanoparticle delivery to solid tumors is known to be an inefficient process and various studies have tried to increase efficacy, but mechanistic and comparative studies remain scarce. Here, we use pharmacological agents to study the effect of vessel normalization or vessel disintegration on nanoparticle delivery to solid tumors. Using a multiparametric approach, we find that vessel disintegration fails to improve nanoparticle delivery and instead seems to have a limiting effect. Vessel normalization, however, improves delivery efficacy for nanoparticles ranging from 20 to 60 nm diameter. The normalization of the tumor blood vessels results in reduced hypoxia, reduced necrosis and an increase in Plvap+ CD276+ endothelial cells, which have been linked with nanoparticle delivery. Interestingly, where vessel disintegration stimulated cancer cell intravasation and associated metastases, vessel normalization impeded these processes. Together, these data reveal that, vessel normalization may be a safer and more suited approach for improving nanoparticle delivery to solid tumors, but its efficacy is limited by nanoparticle diameter and tumor parameters. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/538559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e5e08org.highwire.dtl.DTLVardef@14f82dcorg.highwire.dtl.DTLVardef@118708eorg.highwire.dtl.DTLVardef@18593d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods, have drawbacks such as the requirement of high concentration in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/ml) of several target proteins into outer membrane vesicles (OMVs) of E. coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency arranging from 90-97% for different cell lines. Moreover, the free penetration of molecules less than 600 Dalton across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

Detection of micro- and nanoplastics (MNPs) in human tissues has raised growing concern about their biological effects on tissue and cell function. While previous studies have examined MNP-cell interaction, most focused on limited cell and plastic types. Here, we present a comprehensive, quantitative investigation into how different types of nanoplastics (NPs) associate with and affect diverse cell types under physiologically relevant conditions. Using microfluidic-calibrated fluorescence microscopy, we quantify NP accumulation in cells in vitro and match cellular NP concentrations to levels reported in human tissues. While cell-associated NPs could be gradually released in vitro, they persist in vivo for over one month without detectable reduction in a mouse model. We discover that NP exposure at these levels broadly impairs cell proliferation across epithelial, endothelial, fibroblast, and immune cells, with cell type-dependent sensitivity. NP exposure also reduces motility in T cells and fibroblasts, with more complex effects observed in macrophages. Mechanistically, NP-cell association and trans-epithelial transport involved not only classical endocytic regulators but also pathways related to ion and water transport. Notably, NP association and release were highly sensitive to the extracellular fluid environment within the physiological range. By testing inhibitors of these pathways, we identified molecules that reduce NP-cell association and promote release. We further compared common NPs found in human samples and widely used in research: polystyrene (PS), polyethylene (PE), and polypropylene (PP). Although these NPs similarly impaired proliferation and motility, they showed markedly different cellular association and release dynamics. These findings reveal the impact of NPs on tissue cell functions and uncover novel regulatory pathways, establishing a quantitative framework for studying NP-cell interactions in biologically relevant conditions.

High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, namely the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF). Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 114 and 155 nm) by using light-scatter based detection thresholds (SSC, FSC, VSSC) across a concentration range from 106 to 109 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding for their qualitative and quantitative analysis. We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.

Building on the success of COVID-19 vaccine development, lipid nanoparticles (LNPs) have emerged as leading vehicles for mRNA delivery in a range of therapeutic applications. Naturally-occurring extracellular vesicles (EVs), which share similar physical properties with LNPs, present a promising alternative platform because of their relative stability and lower immunogenicity. A key challenge common to both EVs and LNPs is enabling efficient vesicle - cell interactions and establishing a polarized permeability pathway required for effective cargo transfer. Membrane recognition and intercalation are essential for the function and delivery capacity of both systems, regardless of their complexity. In this study, we leveraged recent advances to create hybrid extracellular vesicles (HEVs) by using LNPs to load mRNA into EVs. We characterized HEV formation using Forster resonance energy transfer (FRET), cryo-electron microscopy (Cryo-EM), and super-resolution microscopy, and demonstrated their ability to deliver mRNA to recipient cells. In both, in vitro and in vivo models, HEVs exhibited superior transfection efficiency compared to conventional LNPs composed of synthetic lipids, while significantly reducing LNPs cytotoxicity - a not-well-recognized limitation of synthetic lipid-based systems. These results highlight HEVs as a safer and more effective alternative for mRNA and small molecule delivery. Future therapeutic strategies could involve isolating EVs from patients, hybridizing them with synthetic lipid carriers loaded with therapeutic cargo, and reintroducing them for personalized treatment.

The midbodysome (MBsome), a secreted remnant of midbody during cell division, is now known to play a key role in cytokinesis. It was thought that the MBsome is either released into the extracellular space or autophagically degraded by one of its daughter cells. However, recent studies have revealed that MBsomes can be maintained by cells even after cell division is complete, and that they accumulate in the cytoplasm and regulate cell proliferation and survival through integrin and epidermal growth factor receptor-dependent pathways. Here, we examined the ability of MBsomes to act as carriers of mRNAs, a novel function that has not been studied. We found that MBsomes isolated from human lung cancer and stem cells via sucrose cushion ultracentrifugation were 300-400 nm in size and stable for up to 4 days when stored at 4{degrees}C. In addition, we confirmed successful expression of the EGFP protein following incubation of the isolated MBsomes with the EGFP mRNA at room temperature. These results suggest that MBsomes have the potential to serve as mRNA carriers and therapeutic agents capable of delivering a gene-of-interest.

Radiation enhancement is a promising anti-cancer approach based on a local radiation dose increase due to the presence of metallic nanoparticles (NPs) within cancer cells. Depending on their composition, size and cellular properties, NPs can follow multiple cellular pathways and entry routes. We observed that gold, platinum and TiO2 NPs are internalized at higher levels in mesenchymal cells compared to epithelial cells in breast cancer models. A global survey of gene expression between epithelial and mesenchymal cells exposed to 4 different NP types revealed an involvement of membrane structure, and further experiments confirmed that the hyaluronic acid (HA) and its receptor CD44 are mediators of metallic NP uptake into cells. We extended our results to a larger panel of breast cancer cell lines and again showed a preferential uptake of all NPs tested in mesenchymal cells and relying on the HA/CD44 axis. These data provide considerations for the design of NP-based therapies targeting mesenchymal cancer cells, which are often resistant to treatment and correlate with poor prognosis and tumor recurrence.

Environmental degradation and accumulation of plastics results in micro- and nanoplastics (MNPLs) that are small enough to cross biological barriers, including the blood-brain barrier. Microglia, resident immune cells of brain, are critical regulators of neuroimmune homeostasis and represent a cellular target of nanoplastic exposure. In this study, we assessed the neurotoxic effects of two sizes of polystyrene nanoplastics (PS-NPs; 100 nm and 500 nm) using integrated in vivo and in vitro exposure and washout paradigms. In vivo exposure in mice (60 days; 0.15 or 1.5 mg/day) showed the accumulation of both PS-NP sizes in the cerebral cortex without histopathological damage. However, cortical microglia showed pronounced morphological remodeling, observed as increased expression of Iba1 and GFAP. Transcriptomic profiling of cortical tissue revealed a strong size-dependent response. The 100 nm PS-NP group revealed 18 DEGs (|log2FC| [&ge;] 2, padj < 0.05), whereas the 500 nm PS-NPs showed more than 4,000 DEGs, including upregulation of immune- and microglia-associated genes (CCL5, CXCL10, LCN2, LYZ2) and downregulation of synaptic and neuronal signaling genes (GRIN2B, SYN1, STX1B, MAP1B, ITPR1/2). In vitro assessment, using BV2 microglia cells, showed internalization of PS-NPs via the endolysosomal pathway, with strong co-localization to Rab7- and LAMP2-positive compartments and prolonged intracellular retention following exposure washout. Also, microglial activation markers (Iba1, CD68) exhibited a transient, size- and concentration-dependent increase, correlated with intracellular particle burden rather than cumulative exposure. Overall, these findings demonstrate that PS-NPs accumulate in brain, driving size-dependent microglia activation and transcriptomic reprogramming, even after cessation of exposure to PS-NPs. HighlightsO_LIPS-NPs (100 nm and 500 nm) reach mouse cerebral cortex following 60-day oral exposure. C_LIO_LIPS-NPs were internalized by microglia; accumulated in endolysosomal compartments. C_LIO_LIPS-NP exposure induced transient microglial activation without sustained cytotoxicity. C_LIO_LIMicroglial activation was correlated with intracellular PS-NPs burden. C_LIO_LITranscriptomics revealed disruption of neuroimmune and microglial regulatory pathways. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/712727v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1aba3eaorg.highwire.dtl.DTLVardef@1967641org.highwire.dtl.DTLVardef@12da637org.highwire.dtl.DTLVardef@1fb8441_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extracellular vesicles (EVs) hold potential as effective carriers for drug delivery, providing a promising approach to resolving challenges in lung cancer treatment. Traditional treatments, such as with the chemotherapy drug cisplatin, encounter resistance in standard cell death pathways like apoptosis, prompting the need to explore alternative approaches. This study investigates the potential of iron oxide nanoparticles (IONP) and EVs to induce ferroptosis--a regulated cell death mechanism--in lung cancer cells. We formulated a novel EV and IONP-based system, namely ExoFeR, and observed that ExoFeR demonstrated efficient ferroptosis induction, evidenced by downregulation of ferroptosis markers (xCT/SLC7A11 and GPX4), increased intracellular and mitochondrial ferrous iron levels, and morphological changes in mitochondria. To enhance efficacy, tumor-targeting transferrin (TF)-conjugated ExoFeR (ExoFeRTF) was developed. ExoFeRTF outperformed ExoFeR, exhibiting higher uptake and cell death in lung cancer cells. Mechanistically, nuclear factor erythroid 2-related factor 2 (Nrf2)--a key regulator of genes involved in glutathione biosynthesis, antioxidant responses, lipid metabolism, and iron metabolism--was found downregulated in the ferroptotic cells. Inhibition of Nrf2 intracellular translocation in ExoFeRTF-treated cells was also observed, emphasizing the role of Nrf2 in modulating ferroptosis-dependent cell death. Furthermore, ExoFeR and ExoFeRTF demonstrated the ability to sensitize chemo-resistant cancer cells, including cisplatin-resistant lung cancer patient-derived tumoroid organoids. In summary, ExoFeRTF presents a promising and multifaceted therapeutic approach for combating lung cancer by intrinsically inducing ferroptosis and sensitizing chemo-resistant cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies and is frequently associated with chemotherapy resistance and immunosuppression. Inducible nitric oxide synthase (iNOS) is overexpressed in breast cancer and has been strongly correlated with poor clinical outcomes, owing to its role in promoting tumor progression, invasiveness, and resistance to therapy. Although highly selective iNOS inhibitors such as N-(3-(Aminomethyl)benzyl)acetamidine (1400W) exhibit considerable therapeutic promise, their clinical translation has been hindered by unfavorable pharmacokinetic properties. To overcome these limitations, we developed a pH-responsive nanoscale formulation based on Schiff base conjugation between 1400W and oxidized PEGylated alginate (OPA), in combination with ionic interactions. The resulting nanoparticles (NPs) exhibited efficient release of 1400W under acidic conditions and effectively suppressed nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. While the NPs alone did not induce significant cytotoxicity, they synergistically enhanced the anticancer efficacy of paclitaxel (PTX) in MDA-MB-231 TNBC cells, significantly inhibiting cell viability and migration. In addition, the NP-PTX combination markedly reduced endothelial tube formation in HUVECs, compared to PTX alone indicating potentiation of the anti-angiogenic activity of PTX. In conclusion, the pH-responsive NPs enables effective modulation of NO signaling and enhances the therapeutic activity of PTX in TNBC cells. These findings support the potential of iNOS-targeted nanomedicine as an adjuvant strategy for TNBC treatment and warrant further investigation using in vivo models to evaluate pharmacokinetics, tumor accumulation, and antitumor efficacy of the NPs.

1.Cell motility, shape supporting, and intracellular signaling are followed by changes in cell morphology and cytoskeleton. The cell reaction and the reorganization of the cytoskeleton occurs in a single volume of the cytoplasm and affects all components of the cytoskeleton: intermediate filaments, microtubules and microfilaments. A promising way to manipulate cells is magnetic nanoparticles that control cellular physiology. This approach is called magnetogenetics and has found application in various fields of cell and molecular biology. Using a magnetic field, it is possible to non-invasively regulate biochemical processes, migration and changes in the morphology of cells with magnetic nanoparticles. Our work opens up new possibilities for spatial manipulation of individual cytoskeletal components in vitro and operates biochemical pathways associated with individual cytoskeletal components.