Lysosomal Permeabilization Research Articles

Nanoplatforms for Magnetic-Photo-Heating of Thermo-Resistant Tumor Cells: Singular Synergic Therapeutic Effects at Mild Temperature.

A self-assemble amphiphilic diblock copolymer that can incorporate iron oxide nanocubes (IONCs) in chain-like assemblies as heat mediators for magnetic hyperthermia (MHT) and tuneable amounts of IR780 dye as agent for photothermal therapy (PTT) is developed. MHT-heating performance of photobeads in viscous media have the same heat performances in water at magnetic field conditions of clinical use. Thanks to IR780, the photobeads are activated by infrared laser light within the first biological window (808nm) with a significant enhancement of photo-stability of IR780 enabling the raise of the temperature at therapeutic values during multiple PTT cycles and showing unchanged optical features up to 8 days. Moreover, the photobeads fluorescent signal is preserved once internalized by glioblastoma multiforme (GBM) cells. Peculiarly, the photobeads are used as toxic agents to eradicate thermo-resistant GBM cells at mild heat, as low as 41°C, with MHT and PTT both of clinical use. Indeed, a high U87 GBM cell mortality percentage is obtained only with dual MHT/PTT while each single treatment dose not provide the same cytotoxic effects. Only for the combined treatment, the cell death mechanism is assigned to clear sign of apoptosis as observed by structural/morphological cell studies and enhanced lysosome permeability.

Group A Streptococcus induces lysosomal dysfunction in THP-1 macrophages.

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Butylated hydroxyanisole induces vascular endothelial injury via TFEB-mediated degradation of GPX4 and FTH1

Butylated hydroxyanisole (BHA) is one of the most commonly used antioxidants and is widely used in food, but whether it causes vascular damage has not been clearly studied. The present study demonstrated for the first time that BHA reduced the viability of human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (BEND3) in a dose- and time-dependent manner. Moreover, BHA inhibited the migration and proliferation of vascular endothelial cells (ECs). Further analysis revealed that in ECs, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed the BHA-induced increase in Fe2+ and malonaldehyde (MDA) levels. Acridine orange staining demonstrated that BHA increased lysosomal permeability. At the protein level, BHA increased the expression of transcription factor EB (TFEB) and decreased the expression of glutathione peroxidase (GPX4), solute carrier family 7 member 11 (SLC7A11, xCT), and ferritin heavy chain 1 (FTH1). Moreover, these effects of BHA could be reversed by knocking down TFEB. In vivo experiments confirmed that BHA caused elevated pulse wave velocity (PWV) and reduced acetylcholine-dependent vascular endothelial diastole. In conclusion, BHA degrades GPX4, xCT, and FTH1 through activation of the TFEB-mediated lysosomal pathway and promotes ferroptosis, ultimately leading to vascular endothelial cell injury.

The Influence of Lysosomal Stress on Dental Pulp Stem Cell-Derived Schwann Cells.

Dysregulation of the endo-lysosomal-autophagy pathway has been identified as a critical factor in the pathology of various demyelinating neurodegenerative diseases, including peripheral neuropathies. This pathway plays a crucial role in transporting newly synthesized myelin proteins to the plasma membrane in myelinating Schwann cells, making these cells susceptible to lysosome-related dysfunctions. Nevertheless, the specific impact of lysosomal dysfunction in Schwann cells and its contribution to neurodegeneration remain poorly understood. We aim to mimic lysosomal dysfunction in Schwann cells using chloroquine, a lysosomal dysfunction inducer, and to monitor lysosomal leakiness, Schwann cell viability, and apoptosis over time. Additionally, due to the ethical and experimental issues associated with cell isolation and the culturing of human Schwann cells, we use human dental pulp stem cell-derived Schwann cells (DPSC-SCs) as a model in our study. Chloroquine incubation boosts lysosomal presence as demonstrated by an increased Lysotracker signal. Further in-depth lysosomal analysis demonstrated an increased lysosomal size and permeability as illustrated by a TEM analysis and GAL3-LAMP1 staining. Moreover, an Alamar blue assay and Caspase-3 staining demonstrates a reduced viability and increased apoptosis, respectively. Our data indicate that prolonged lysosomal dysfunction leads to lysosomal permeability, reduced viability, and eventually apoptosis in human DPSC-SCs.

Rutin mitigates fluoride-induced nephrotoxicity by inhibiting ROS-mediated lysosomal membrane permeabilization and the GSDME-HMGB1 axis involved in pyroptosis and inflammation

Fluoride is known to induce nephrotoxicity; however, the underlying mechanisms remain incompletely understood. Therefore, this study aims to explore the roles and mechanisms of lysosomal membrane permeabilization (LMP) and the GSDME/HMGB1 axis in fluoride-induced nephrotoxicity and the protective effects of rutin. Rutin, a naturally occurring flavonoid compound known for its antioxidative and anti-inflammatory properties, is primarily mediated by inhibiting oxidative stress and reducing proinflammatory markers. To that end, we established in vivo and in vitro models. In the in vivo study, rats were exposed to sodium fluoride (NaF) throughout pregnancy and up until 2 months after birth. In parallel, we employed in vitro models using HK-2 cells treated with NaF, n-acetyl-L-cysteine (NAC), or rutin. We assessed lysosomal permeability through immunofluorescence and analyzed relevant protein expression via western blotting. Our findings showed that NaF exposure increased ROS levels, resulting in enhanced LMP and increased cathepsin B (CTSB) and D (CTSD) expression. Furthermore, the exposure to NaF resulted in the upregulation of cleaved PARP1, cleaved caspase-3, GSDME-N, and HMGB1 expressions, indicating cell death and inflammation-induced renal damage. Rutin mitigates fluoride-induced nephrotoxicity by suppressing ROS-mediated LMP and the GSDME/HMGB1 axis, ultimately preventing fluoride-induced renal toxicity occurrence and development. In conclusion, our findings suggest that NaF induces renal damage through ROS-mediated activation of LMP and the GSDME/HMGB1 axis, leading to pyroptosis and inflammation. Rutin, a natural antioxidative and anti-inflammatory dietary supplement, offers a novel approach to prevent and treat fluoride-induced nephrotoxicity.

Uncovering impaired mitochondrial and lysosomal function in adipose-derived stem cells from obese individuals with altered biological activity

BackgroundAdipose-derived stem cells (ADSCs) have been extensively used in preclinical and clinical trials for treating various diseases. However, the differences between ADSCs from lean individuals (L-ADSCs) and those from obese individuals (O-ADSCs) have not been thoroughly investigated, particularly regarding their mitochondrial and lysosomal functions. Therefore, this study aims to evaluate the differences between L-ADSCs and O-ADSCs in terms of cell biological activity, mitochondria, and lysosomes.MethodsWe first isolated and cultured L-ADSCs and O-ADSCs. We then compared the differences between the two groups in terms of biological activity, including cell proliferation, differentiation potential, and their effect on the polarization of macrophages. Additionally, we observed the mitochondrial and lysosomal morphology of ADSCs using an electronic microscope, MitoTracker Red, and lysotracker Red dyes. We assessed mitochondrial function by examining mitochondrial membrane potential and membrane fluidity, antioxidative ability, and cell energy metabolism. Lysosomal function was evaluated by measuring autophagy and phagocytosis. Finally, we performed transcriptome analysis of the ADSCs using RNA sequencing.ResultsThe biological activities of O-ADSCs were decreased, including cell immunophenotypic profiles, cell proliferation, and differentiation potential. Furthermore, compared to L-ADSCs, O-ADSCs promoted M1-type macrophage polarization and inhibited M2-type macrophage polarization. Additionally, the mitochondrial morphology of O-ADSCs was altered, with the size of the cells becoming smaller and mitochondrial fragments increasing. O-ADSCs also exhibited decreased mitochondrial membrane potential and membrane fluidity, antioxidative ability, and energy metabolism. With respect to lysosomes, O-ADSCs contained ungraded materials in their lysosomes, enhanced lysosomal permeability, and reduced autophagy and phagocytosis ability. RNA sequence analysis indicated that the signalling pathways related to cell senescence, cancer, and inflammation were upregulated, whereas the signalling pathways associated with stemness, cell differentiation, metabolism, and response to stress and stimuli were downregulated.ConclusionsThis study indicates that ADSCs from individuals (BMI > 30 kg/m2) exhibit impaired mitochondrial and lysosomal function with decreased biological activity.

Arsenic-Induced Thyroid Hormonal Alterations and Their Putative Influence on Ovarian Follicles in Balb/c Mice.

Thyroid issues are common among women in their reproductive years, and women with thyroid dysfunction often encounter challenges with fertility. Arsenic is known for its toxic effects on the thyroid and ovaries, investigated independently. However, there is no known study directly or indirectly addressing the association between arsenic, thyroid function, and ovarian reserve. This study aims to investigate the effect of arsenic on thyroid function and its possible implications on ovarian follicular reserve. Female Balb/c mice were given sodium arsenite (0.2ppm, 2ppm, and 20ppm) via drinking water for 30days. Findings showed that arsenic decreased thyroid hormone levels (fT3 and fT4) while increasing TSH levels, which might have led to elevated levels of FSH and LH. Furthermore, arsenic treatment not only decreased thyroid follicle sizes but also altered the ovarian follicular count. The finding demonstrates that arsenic significantly reduced the expression of LAMP1, a lysosomal marker protein. This reduction leads to increased lysosomal permeability in the thyroid, resulting in a significant release of cathepsin B. These changes led to hypothyroidism, which might indirectly affect the ovaries. Also, the elevated levels of growth differentiation factor-8 in arsenic-treated ovaries indicate impaired folliculogenesis and ovulation. Furthermore, arsenic significantly increased the expressions of pAkt and pFoxo3a, implying that arsenic accelerated the activation of the primordial follicular pools. In conclusion, arsenic disrupts lysosomal stabilization, potentially leading to a decline in circulating fT3 and fT4 levels. This disturbance could, in turn, affect the estrous cycle and may alter the pattern of follicular development.

Lysosomal membrane stabilization by imipramine attenuates gentamicin-induced renal injury: Enhanced LAMP2 expression, down-regulation of cytoplasmic cathepsin D and tBid/cytochrome c/cleaved caspase-3 apoptotic signaling

Nephrotoxicity is a serious complication commonly encountered with gentamicin (GTM) treatment. Permeabilization of lysosomes with subsequent cytoplasmic release of GTM and cathepsins is considered a crucial issue in progression of GTM toxicity. This study was designed to evaluate the prospective defensive effect of lysosomal membrane stabilization by imipramine (IMP) against GTM nephrotoxicity in rats. GTM (30 mg/kg/h) was intraperitoneally administered over 4 h daily (120 mg/kg/day) for 7 days. IMP (30 mg/kg/day) was orally administered for 14 days; starting 7 days before and then concurrently with GTM. On 15th day, samples (urine, blood, kidney) were collected to estimate biomarkers of kidney function, lysosomal stability, apoptosis, and inflammation. IMP administration to GTM-treated rats ameliorated the disruption in lysosomal membrane stability induced by GTM. That was evidenced by enhanced renal protein expressions of LAMP2 and PI3K, but reduced cathepsin D cytoplasmic expression in kidney sections. Besides, IMP guarded against apoptosis in GTM-treated rats by down-regulation of the pro-apoptotic (tBid, Bax, cytochrome c) and the effector cleaved caspase-3 expressions, while the anti-apoptotic Bcl-2 expression was enhanced. Additionally, the inflammatory cascade p38 MAPK/NF-κB/TNF-α was attenuated in GTM + IMP group along with marked improvement in kidney function biomarkers, compared to GTM group. These findings were supported by the obvious improvement in histological architecture. Furthermore, in vitro enhancement of the antibacterial activity of GTM by IMP confers an additional benefit to their combination. Conclusively, lysosomal membrane stabilization by IMP with subsequent suppression of tBid/cytochrome c/cleaved caspase-3 apoptotic signaling could be a promising protective strategy against GTM nephrotoxicity.

Hyaluronan nanogel co-loaded with chloroquine to enhance intracellular cisplatin delivery through lysosomal permeabilization and lysophagy inhibition

Hyaluronan (HA) has been widely used to construct nanocarriers for cancer-targeted drug delivery, due to its excellent biocompatibility and intrinsic affinity towards CD44 that is overexpressed in most cancer types. However, the HA-based nanocarriers are prone to trapping in lysosomes following the HA-mediated endocytosis, which limited the delivered drug to access its pharmacological action sites and subsequently compromised the therapeutic efficacy. To overcome this intracellular obstacle, here we demonstrated the co-loading of chloroquine (CQ) in HA nanogel could efficiently promote the intracellular delivery of cisplatin. The cisplatin coordination with HA generated the nanogel that could also co-encapsulate CQ (HA/Cis/CQ nanogel). Compared with cisplatin-loaded HA nanogel (HA/Cis), HA/Cis/CQ significantly promoted the lysosomal escape of cisplatin as well as enhanced tumor inhibition in the triple-negative breast cancer model. Mechanism studies suggested that co-delivery of CQ not only induced the lysosomal membrane permeabilization but also inhibited the lysophagy, which collectively contributed to the lysosomal instability and cisplatin escape. This HA/Cis/CQ nanogel elicited less toxicity compared with the combination of free Cis and CQ, thus suggesting a promising HA nanocarrier to boost the cisplatin delivery towards cancer-targeted therapy.

Effect of a CrossMab cotargeting CD20 and HLA-DR in non-Hodgkin lymphoma

Abstract Objectives To evaluate the anti-tumor activities of CD20/HLA-DR CrossmabCH1-CL through cell and animal models. Methods Based on “knobs-into-holes” and “crossover” technology, CrossMab, targeting CD20 and HLA-DR, was constructed. A binding assay and a competitive inhibition assay were performed to confirm its specificity. The effects of CrossMab on antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were measured. Cell apoptosis, lysosome-mediated cell death, and lysosomal permeability were quantified. In addition, the effects of CrossMab on peripheral blood leukocytes were tested. The pharmacokinetics were determined with a noncompartmental analysis model. Human malignant lymphoma xenograft models in CB17-SCID mice were established for an in-vivo efficacy study. Results The antitumor activities of CrossMab were shown both in vitro and in vivo. CrossMab exhibited strong binding to CD20 and HLA-DR at the same time in Raji cells. CrossMab also demonstrated antilymphoma effects by inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Furthermore, CrossMab altered the lysosomal membrane permeability. The toxicity of CrossMab on normal peripheral blood lymphocytes (PBLs) was specific to B cells. A mouse xenograft model demonstrated the antitumor activities of CrossMab in vivo. Conclusions CrossMab exhibited an enhanced antigen recognition ability and antitumor activities in lymphoma without evident toxicity. CrossMab could be an effective immunotherapeutic strategy for non-Hodgkin lymphoma.

Externally Applied Electromagnetic Fields and Hyperthermia Irreversibly Damage Cancer Cells

At present, the applications and efficacy of non-ionizing radiations (NIR) in oncotherapy are limited. In terms of potential combinations, the use of biocompatible magnetic nanoparticles as heat mediators has been extensively investigated. Nevertheless, developing more efficient heat nanomediators that may exhibit high specific absorption rates is still an unsolved problem. Our aim was to investigate if externally applied magnetic fields and a heat-inducing NIR affect tumor cell viability. To this end, under in vitro conditions, different human cancer cells (A2058 melanoma, AsPC1 pancreas carcinoma, MDA-MB-231 breast carcinoma) were treated with the combination of electromagnetic fields (EMFs, using solenoids) and hyperthermia (HT, using a thermostated bath). The effect of NIR was also studied in combination with standard chemotherapy and targeted therapy. An experimental device combining EMFs and high-intensity focused ultrasounds (HIFU)-induced HT was tested in vivo. EMFs (25 µT, 4 h) or HT (52 °C, 40 min) showed a limited effect on cancer cell viability in vitro. However, their combination decreased viability to approximately 16%, 50%, and 21% of control values in A2058, AsPC1, and MDA-MB-231 cells, respectively. Increased lysosomal permeability, release of cathepsins into the cytosol, and mitochondria-dependent activation of cell death are the underlying mechanisms. Cancer cells could be completely eliminated by combining EMFs, HT, and standard chemotherapy or EMFs, HT, and anti-Hsp70-targeted therapy. As a proof of concept, in vivo experiments performed in AsPC1 xenografts showed that a combination of EMFs, HIFU-induced HT, standard chemotherapy, and a lysosomal permeabilizer induces a complete cancer regression.

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

Abstract 2826: Electromagnetic fields and hyperthermia induce lysosomal permeabilization and death in different cancer cell models: in vitro and in vivo studies

Abstract Introduction: Non-ionizing radiations (NIR), e.g. tumor-treating fields, high intensity focused ultrasounds (HIFU) or high frequency microwaves, have potential applications in Oncotherapy. Nevertheless, as today, their efficacy is limited. We investigated whether externally applied magnetic fields and non-ablative hyperthermia affect cancer cell viability and growth. Methods: Under in vitro conditions, human A2058 melanoma, AsPC1 pancreas carcinoma, and MDA-MB-231 breast carcinoma cells were treated with electromagnetic fields (EMFs, using solenoids) and/or hyperthermia (HT, using a thermostated water bath). The effect of both energies was also studied in combination with standard chemotherapy and targeted therapy. An experimental system combining EMFs and HIFU-induced HT was developed and tested in vivo. Results: EMFs (25 μT, 4h) or HT (52ºC, 40 min) quite moderately affect cancer cell viability. However, their combination decreased viability to approx. 16%, 50%, and 21% of controls values in A2058, AsPC1, and MDA-MB-231 cells, respectively). Increased lysosomal permeability, release of cathepsins into the cytosol and activation of the mitochondria-dependent cell death are the underlying mechanisms. Cancer cells were completely eliminated combining EMFs, HT and standard chemotherapy or EMFs, HT and anti-Hsp70-targeted therapy. In vivo experiments, using pancreatic cancer xenografts growing in nude mice, show that combination of EMFs, HIFU-induced HT and gemcitabine induces a drastic cancer regression (to approx. 3-4% of control values). Conclusion: Non-invasive NIR (EMFs and HIFU-induced HT) can complement current Oncotherapies. This strategy, associated to current chemotherapy or anti-Hsp70-targeted therapy, may facilitate the elimination of tumors that can be previously located using standard imaging techniques (CT scan and NMR). These results may help to overcome the limitations in the use of different NIR in Oncotherapy. Citation Format: Elena Obrador, Ali Jihad-Jebbar, Rosario Salvador-Palmer, Rafael López-Blanch, María Oriol-Caballo, María Paz Moreno-Murciano, Enrique Navarro, Rosa Cibrián, Jose M. Estrela. Electromagnetic fields and hyperthermia induce lysosomal permeabilization and death in different cancer cell models: in vitro and in vivo studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2826.

The Pd-catalyzed precise synthesis of self-assembled fluorescent nanoprobes with different molecular weights for enhancing the permeability of cell membrane and targeting lysosomes

As a common polymer, poly (dimethylaminoethyl methacrylate) (PDMAEMA) is mainly used to synthesize medical materials. However, there are few reports about the related polymer fluorescent probes. Moreover, as far as we know, no one has studied the effect of its molecular weight on the performance of polymer probes. In this work, polymer probes (NapNH2(1)-PDM(4), NapNH2(1)-PDM(9), NapNH2(1)-PDM(23), NapNH2(1)-PDM(34)) with different molecular weights were accurately constructed based on 1,8-naphthalimide fluorophore and poly(N,N-dimethylaminoethyl methacrylate) and their photophysical properties were systematically studied. The test results showed that they all showed good photophysical properties. Moreover, the polymer probe NapNH2(1)-PDM(23), which had the best self-assembly effect and good cell permeability, was successfully screened by confocal fluorescence imaging technology. Not only that, the probe NapNH2(1)-PDM(23) could also label lysosomes in cells quickly and accurately. Therefore, this work provided important reference to construct relevant polymer probes with good cell permeability in the future.

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways through permeabilization of IV membranes and the formation of endosomes and lysosomes. A method named chromophore-assisted laser inactivation (CALI) is applied to study the formation of IVs and the materials in controlling IV regulation. CALI is an imaging-based photodynamic methodology to study the signaling pathway induced by membrane permeabilization. The method allows spatiotemporal manipulation of the selected organelle to be permeabilized in a cell. The CALI method has been applied to observe and monitor specific molecules through the permeabilization of endosomes and lysosomes. The membrane rupture of IVs is known to selectively recruit glycan-binding proteins, such as galectin-3. Here, the protocol describes the induction of IV rupture by AlPcS2a and the use of galectin-3 as a marker to label impaired lysosomes, which is useful in studying the downstream effects of IV membrane rupture and their downstream effects under various situations.

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

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

Differential cascading cellular and subcellular toxicity induced by two sizes of nanoplastics

Nanoplastics (NPs) can be potentially accumulated by living organisms, but how they interact with cells at the cellular or subcellular level in the physiological environment is still largely unknown. In this study, time-resolved flow cytometry coupled with confocal imaging as well as other biomolecular approaches were used to investigate the cellular and subcellular responses to amine-modified polystyrene NPs of two different sizes (100 nm and 1000 nm). We first demonstrated that the two sizes of NPs displayed contrasting cytotoxicity to embryonic zebrafish fibroblast cell lines ZF4. Using the fluorescent-labeled NPs, the differentially internalized patterns between the two-sized NPs in a time-resolved manner were observed. Confocal images showed that the two sizes of NPs were deposited in lysosomes but could escape through lysosomal rupture, as evidenced by the induction of lysosomal acidification (for 1000 nm) and alkalization (for 100 nm) as well as permeabilization. Subsequent deposition of 100-NPs in the cytosol induced loss of mitochondrial membrane potential and significant reactive oxygen species production, and finally stimulated the activation of caspases, disrupted the mitochondrial mitophagy, leading to irreversible cell death. In contrast, 1000-NPs toxicity in ZF4 cells did not involve lysosomal permeabilization and loss of mitochondrial membrane potential. Lysosomal deposition of such larger sized nanoplastics mainly induced lysosome acidification, activated the autophagy as well as disrupted the integrity of cell membrane, but at the same time provoked the activation of caspases and finally triggered the apoptosis. Our study demonstrated a complicated relationship among lysosome damage, autophagy activation, and apoptosis, leading to contrasting toxicity of NPs of different sizes.

Lysosomal permeabilization as a mechanism of amyloid degradation toxicity in Alzheimer's disease.

The amyloid channel theory readily explains primary molecular damage induced by beta-amyloid (Aβ) but cannot interpret multiple major phenomena associated with Alzheimer's Disease such as autophagy failure and decreased metabolism. To explain them, the amyloid degradation toxicity hypothesis suggests that the cytotoxicity is initiated by the channel formation in lysosomal membranes by amyloid fragments (such as Aβ25-35 ) produced by the digestion of endocytosed Aβ. One amyloid channel is sufficient to neutralize lysosomal content and inactivate proteases; therefore, undigested amyloid accumulates, and autophagy stalls. Inadequate mitophagy results in an increased generation of reactive oxygen species and decreased energy production. Huge electrical conductance of amyloid channels prompted us to estimate the maximal size of the macromolecules which can leak from permeabilized lysosomes through amyloid channels. To estimate the channels' ability to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in the non-conductive membranes passing spheres with a density of average globular proteins. The histograms of single channel conductance at various pH were constructed from the previously published experimental dataset. An approximation with a power function was used to extrapolate the probabilities of channels with high molecular weight cut-offs (MWCO). A variability of channel sizes suggests that channel-forming barrel-shaped aggregates can have various number of monomers. Extrapolation of the channel size distribution predicts that a significant number of channels has MWCO exceeding 10 kDa, while acidic environment disproportionally promotes their formation. This range of MWCO is sufficient for the leakage of cathepsins (20-30 kDa) implicated in the induction of necrosis and/or apoptosis. Channels with MWCO above 1000 Da can release into the cytoplasm the amyloid fragments which can form membrane channels in the plasma membrane by entering from the internal leaflet. The time needed for endocytosis and proteolytic digestion explains why cellular responses to Aβ exposure are taking significant time (at least several minutes). While dissipation of the proton gradient by small amyloid channel readily explains lysosomal failure, relatively rare events of lysosomal permeabilization to large macromolecules by amyloid channels can be an alternative mechanism of cellular death induced by the exposure to Aβ.

Inhibiting autophagy increases the efficacy of low-dose photodynamic therapy

Rupture and permeabilization of endocytic vesicles can be triggered by various causes, such as pathogenic invasions, amyloid proteins, and silica crystals leading to cell death and degeneration. A cellular quality control process, called lysophagy was recently described to target damaged lysosomes for autophagic sequestration within isolation membranes in order to protect the cell from the consequences of lysosomal leakage. This protective process, however, might interfere with treatment conditions, such as photodynamic therapy (PDT) and the intracellular drug delivery method photochemical internalization (PCI). PCI-induced permeabilization of endosomes and lysosomes is purposely triggered to release drugs that are sequestered in these organelles into the cytosol in order to synergistically kill cancer cells. Here, we show that photochemical treatment with the PCI-photosensitizer TPCS2a/fimaporfin results in both induction of autophagy and inhibition of the autophagic flux. The autophagic response is accompanied by recruitment of ubiquitin (Ubq), p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3) to damaged vesicles, marked by Galectin 3 (Gal3). Furthermore, ultrastructural analysis revealed a homogenously thick p62-positive layer surrounding these permeabilized vesicles. Although p62 seems to be important during the selective autophagic sequestration, we show that its presence is not essential for the effective removal of damaged vesicles or the recovery of the lysosomal content. An active autophagic response and the presence of p62, however, is important for cancer cells to survive low-dose TPCS2a-PDT. Thus, targeting both p62 and autophagy together and independently, in a light-controlled/PCI based delivery of cancer therapeutics could increase the effectiveness of the treatment regime.