Acidic Nanoparticles Restore Lysosomal Acidification and Rescue Metabolic Dysfunction in Pancreatic ß-Cells under Lipotoxic Conditions.

Type 2 diabetes (T2D), a prevalent metabolic disorder lacking effective treatments, is associated with lysosomal acidification dysfunction, as well as autophagic and mitochondrial impairments. Here, we report a series of biodegradable poly(butylene tetrafluorosuccinate-co-succinate) polyesters, comprising a 1,4-butanediol linker and varying ratios of tetrafluorosuccinic acid (TFSA) and succinic acid as components, to engineer lysosome-acidifying nanoparticles (NPs). The synthesized NPs are spherical with diameters of ≈100 nm and have low polydispersity and good stability. Notably, TFSA NPs, which are composed entirely of TFSA, exhibit the strongest degradation capability and superior acidifying properties. We further reveal significant downregulation of lysosomal vacuolar (H+)-ATPase subunits, which are responsible for maintaining lysosomal acidification, in human T2D pancreatic islets, INS-1 ß-cells under chronic lipotoxic conditions, and pancreatic tissues of high-fat-diet (HFD) mice. Treatment with TFSA NPs restores lysosomal acidification, autophagic function, and mitochondrial activity, thereby improving the pancreatic function in INS-1 cells and HFD mice with lipid overload. Importantly, the administration of TFSA NPs to HFD mice reduces insulin resistance and improves glucose clearance. These findings highlight the therapeutic potential of lysosome-acidifying TFSA NPs for T2D.

Blood-Brain Barrier-Targeting Nanoparticles: Biomaterial Properties and Biomedical Applications in Translational Neuroscience.

Overcoming the blood-brain barrier (BBB) remains a significant hurdle in effective drug delivery to the brain. While the BBB serves as a crucial protective barrier, it poses challenges in delivering therapeutic agents to their intended targets within the brain parenchyma. To enhance drug delivery for the treatment of neurological diseases, several delivery technologies to circumvent the BBB have been developed in the last few years. Among them, nanoparticles (NPs) are one of the most versatile and promising tools. Here, we summarize the characteristics of NPs that facilitate BBB penetration, including their size, shape, chemical composition, surface charge, and importantly, their conjugation with various biological or synthetic molecules such as glucose, transferrin, insulin, polyethylene glycol, peptides, and aptamers. Additionally, we discuss the coating of NPs with surfactants. A comprehensive overview of the common in vitro and in vivo models of the BBB for NP penetration studies is also provided. The discussion extends to discussing BBB impairment under pathological conditions and leveraging BBB alterations under pathological conditions to enhance drug delivery. Emphasizing the need for future studies to uncover the inherent therapeutic properties of NPs, the review advocates for their role beyond delivery systems and calls for efforts translating NPs to the clinic as therapeutics. Overall, NPs stand out as a highly promising therapeutic strategy for precise BBB targeting and drug delivery in neurological disorders.

Peptide-based allosteric inhibitor targets TNFR1 conformationally active region and disables receptor-ligand signaling complex.

Tumor necrosis factor (TNF) receptor 1 (TNFR1) plays a pivotal role in mediating TNF induced downstream signaling and regulating inflammatory response. Recent studies have suggested that TNFR1 activation involves conformational rearrangements of preligand assembled receptor dimers and targeting receptor conformational dynamics is a viable strategy to modulate TNFR1 signaling. Here, we used a combination of biophysical, biochemical, and cellular assays, as well as molecular dynamics simulation to show that an anti-inflammatory peptide (FKCRRWQWRMKK), which we termed FKC, inhibits TNFR1 activation allosterically by altering the conformational states of the receptor dimer without blocking receptor-ligand interaction or disrupting receptor dimerization. We also demonstrated the efficacy of FKC by showing that the peptide inhibits TNFR1 signaling in HEK293 cells and attenuates inflammation in mice with intraperitoneal TNF injection. Mechanistically, we found that FKC binds to TNFR1 cysteine-rich domains (CRD2/3) and perturbs the conformational dynamics required for receptor activation. Importantly, FKC increases the frequency in the opening of both CRD2/3 and CRD4 in the receptor dimer, as well as induces a conformational opening in the cytosolic regions of the receptor. This results in an inhibitory conformational state that impedes the recruitment of downstream signaling molecules. Together, these data provide evidence on the feasibility of targeting TNFR1 conformationally active region and open new avenues for receptor-specific inhibition of TNFR1 signaling.

Data Mining of Microarray Datasets in Translational Neuroscience.

Data mining involves the computational analysis of a plethora of publicly available datasets to generate new hypotheses that can be further validated by experiments for the improved understanding of the pathogenesis of neurodegenerative diseases. Although the number of sequencing datasets is on the rise, microarray analysis conducted on diverse biological samples represent a large collection of datasets with multiple web-based programs that enable efficient and convenient data analysis. In this review, we first discuss the selection of biological samples associated with neurological disorders, and the possibility of a combination of datasets, from various types of samples, to conduct an integrated analysis in order to achieve a holistic understanding of the alterations in the examined biological system. We then summarize key approaches and studies that have made use of the data mining of microarray datasets to obtain insights into translational neuroscience applications, including biomarker discovery, therapeutic development, and the elucidation of the pathogenic mechanisms of neurodegenerative diseases. We further discuss the gap to be bridged between microarray and sequencing studies to improve the utilization and combination of different types of datasets, together with experimental validation, for more comprehensive analyses. We conclude by providing future perspectives on integrating multi-omics, to advance precision phenotyping and personalized medicine for neurodegenerative diseases.

Integrative multi-omics and systems bioinformatics in translational neuroscience: A data mining perspective.

Bioinformatic analysis of large and complex omics datasets has become increasingly useful in modern day biology by providing a great depth of information, with its application to neuroscience termed neuroinformatics. Data mining of omics datasets has enabled the generation of new hypotheses based on differentially regulated biological molecules associated with disease mechanisms, which can be tested experimentally for improved diagnostic and therapeutic targeting of neurodegenerative diseases. Importantly, integrating multi-omics data using a systems bioinformatics approach will advance the understanding of the layered and interactive network of biological regulation that exchanges systemic knowledge to facilitate the development of a comprehensive human brain profile. In this review, we first summarize data mining studies utilizing datasets from the individual type of omics analysis, including epigenetics/epigenomics, transcriptomics, proteomics, metabolomics, lipidomics, and spatial omics, pertaining to Alzheimer's disease, Parkinson's disease, and multiple sclerosis. We then discuss multi-omics integration approaches, including independent biological integration and unsupervised integration methods, for more intuitive and informative interpretation of the biological data obtained across different omics layers. We further assess studies that integrate multi-omics in data mining which provide convoluted biological insights and offer proof-of-concept proposition towards systems bioinformatics in the reconstruction of brain networks. Finally, we recommend a combination of high dimensional bioinformatics analysis with experimental validation to achieve translational neuroscience applications including biomarker discovery, therapeutic development, and elucidation of disease mechanisms. We conclude by providing future perspectives and opportunities in applying integrative multi-omics and systems bioinformatics to achieve precision phenotyping of neurodegenerative diseases and towards personalized medicine.

Lysosomal acidification dysfunction in microglia: an emerging pathogenic mechanism of neuroinflammation and neurodegeneration.

Microglia are the resident innate immune cells in the brain with a major role in orchestrating immune responses. They also provide a frontline of host defense in the central nervous system (CNS) through their active phagocytic capability. Being a professional phagocyte, microglia participate in phagocytic and autophagic clearance of cellular waste and debris as well as toxic protein aggregates, which relies on optimal lysosomal acidification and function. Defective microglial lysosomal acidification leads to impaired phagocytic and autophagic functions which result in the perpetuation of neuroinflammation and progression of neurodegeneration. Reacidification of impaired lysosomes in microglia has been shown to reverse neurodegenerative pathology in Alzheimer's disease. In this review, we summarize key factors and mechanisms contributing to lysosomal acidification impairment and the associated phagocytic and autophagic dysfunction in microglia, and how these defects contribute to neuroinflammation and neurodegeneration. We further discuss techniques to monitor lysosomal pH and therapeutic agents that can reacidify impaired lysosomes in microglia under disease conditions. Finally, we propose future directions to investigate the role of microglial lysosomal acidification in lysosome-mitochondria crosstalk and in neuron-glia interaction for more comprehensive understanding of its broader CNS physiological and pathological implications.

Defective lysosomal acidification: a new prognostic marker and therapeutic target for neurodegenerative diseases.

Lysosomal acidification dysfunction has been implicated as a key driving factor in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Multiple genetic factors have been linked to lysosomal de-acidification through impairing the vacuolar-type ATPase and ion channels on the organelle membrane. Similar lysosomal abnormalities are also present in sporadic forms of neurodegeneration, although the underlying pathogenic mechanisms are unclear and remain to be investigated. Importantly, recent studies have revealed early occurrence of lysosomal acidification impairment before the onset of neurodegeneration and late-stage pathology. However, there is a lack of methods for organelle pH monitoring in vivo and a dearth of lysosome-acidifying therapeutic agents. Here, we summarize and present evidence for the notion of defective lysosomal acidification as an early indicator of neurodegeneration and urge the critical need for technological advancement in developing tools for lysosomal pH monitoring and detection both in vivo and for clinical applications. We further discuss current preclinical pharmacological agents that modulate lysosomal acidification, including small molecules and nanomedicine, and their potential clinical translation into lysosome-targeting therapies. Both timely detection of lysosomal dysfunction and development of therapeutics that restore lysosomal function represent paradigm shifts in targeting neurodegenerative diseases.

Restoration of lysosomal acidification rescues autophagy and metabolic dysfunction in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. High levels of free fatty acids in the liver impair hepatic lysosomal acidification and reduce autophagic flux. We investigate whether restoration of lysosomal function in NAFLD recovers autophagic flux, mitochondrial function, and insulin sensitivity. Here, we report the synthesis of novel biodegradable acid-activated acidifying nanoparticles (acNPs) as a lysosome targeting treatment to restore lysosomal acidity and autophagy. The acNPs, composed of fluorinated polyesters, remain inactive at plasma pH, and only become activated in lysosomes after endocytosis. Specifically, they degrade at pH of ~6 characteristic of dysfunctional lysosomes, to further acidify and enhance the function of lysosomes. In established in vivo high fat diet mouse models of NAFLD, re-acidification of lysosomes via acNP treatment restores autophagy and mitochondria function to lean, healthy levels. This restoration, concurrent with reversal of fasting hyperglycemia and hepatic steatosis, indicates the potential use of acNPs as a first-in-kind therapeutic for NAFLD.

Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives.

Membrane proteins (MPs) play key roles in cellular signaling pathways and are responsible for intercellular and intracellular interactions. Dysfunctional MPs are directly related to the pathogenesis of various diseases, and they have been exploited as one of the most sought-after targets in the pharmaceutical industry. However, working with MPs is difficult given that their amphiphilic nature requires protection from biological membrane or membrane mimetics. Polymersomes are bilayered nano-vesicles made of self-assembled block copolymers that have been widely used as cell membrane mimetics for MP reconstitution and in engineering of artificial cells. This review highlights the prevailing trend in the application of polymersomes in MP study and drug discovery. We begin with a review on the techniques for synthesis and characterization of polymersomes as well as methods of MP insertion to form proteopolymersomes. Next, we review the structural and functional analysis of the different types of MPs reconstituted in polymersomes, including membrane transport proteins, MP complexes, and membrane receptors. We then summarize the factors affecting reconstitution efficiency and the quality of reconstituted MPs for structural and functional studies. Additionally, we discuss the potential in using proteopolymersomes as platforms for high-throughput screening (HTS) in drug discovery to identify modulators of MPs. We conclude by providing future perspectives and recommendations on advancing the study of MPs and drug development using proteopolymersomes.

A hand-targeted auxiliary personal protective equipment for intervention of fomite transmission of viruses.

In COVID-19, fomite transmission has been shown to be a major route for the spreading of the SARS-CoV-2 virus due to its ability to remain on surfaces for extended durations. Although glove wearing can mitigate the risk of viral transmission especially on high touch points, it is not prevalent due to concerns on diversion of frontline medical resources, cross-contamination, social stigma, as well as discomfort and skin reactions derived from prolonged wearing. In this study, we developed FlexiPalm, a hand-targeted auxiliary personal protective equipment (PPE) against fomite transmission of viruses. FlexiPalm is a unique palmar-side hand protector designed to be skin-conforming and transparent, fabricated from medical-grade polyurethane transparent film material as a base substrate. It serves primarily as a barrier to microbial contamination like conventional gloves, but with augmented comfort and inconspicuousness to encourage a higher public adoption rate. Compared to conventional glove materials, FlexiPalm demonstrated enhanced mechanical durability and breathability, comparable hydrophobicity, and displayed a minimal adsorption of SARS-CoV-2 spike protein and virus-like particles (VLP). Importantly, FlexiPalm was found to bind significantly less viral protein and VLP than artificial human skin, confirming its ability to reduce viral contamination. A pilot study involving participants completing activities of daily living showed a high level of comfort and task completion, illustrating the usability and functionality of FlexiPalm. Moreover, we have demonstrated that surface modification of FlexiPalm with microtextures enables further reduction in viral adsorption, thereby enhancing its functionality. An effective implementation of FlexiPalm will bolster PPE sustainability and lead to a paradigm shift in the global management of COVID-19 and other infectious diseases in general.

Synthesis of bioactive (1→6)-ß-glucose branched poly-amido-saccharides that stimulate and induce M1 polarization in macrophages.

ß-Glucans are of significant interest due to their potent antitumor and immunomodulatory activities. Nevertheless, the difficulty in purification, structural heterogenicity, and limited solubility impede the development of structure-property relationships and translation to therapeutic applications. Here, we report the synthesis of a new class of (1→6)-ß-glucose-branched poly-amido-saccharides (PASs) as ß-glucan mimetics by ring-opening polymerization of a gentiobiose-based disaccharide ß-lactam and its copolymerization with a glucose-based ß-lactam, followed by post-polymerization deprotection. The molecular weight (Mn) and frequency of branching (FB) of PASs is readily tuned by adjusting monomer-to-initiator ratio and mole fraction of gentiobiose-lactam in copolymerization. Branched PASs stimulate mouse macrophages, and enhance production of pro-inflammatory cytokines in a FB-, dose-, and Mn-dependent manner. The stimulation proceeds via the activation of NF-κB/AP-1 pathway in a Dectin-1-dependent manner, similar to natural ß-glucans. The lead PAS significantly polarizes primary human macrophages towards M1 phenotype compared to other ß-glucans such as lentinan, laminarin, and curdlan.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Modulating lysosomal pH: a molecular and nanoscale materials design perspective.

Lysosomes, membrane-bound organelles, play important roles in cellular processes including endocytosis, phagocytosis, and autophagy. Lysosomes maintain cellular homeostasis by generating a highly acidic environment of pH 4.5 - 5.0 and by housing hydrolytic enzymes that degrade engulfed biomolecules. Impairment of lysosomal function, especially in its acidification, is a driving force in the pathogenesis of diseases including neurodegeneration, cancer, metabolic disorders, and infectious diseases. Therefore, lysosomal pH is an attractive and targetable site for therapeutic intervention. Currently, there is a dearth of strategies or materials available to specifically modulate lysosomal acidification. This review focuses on the key aspects of how lysosomal pH is implicated in various diseases and discusses design strategies and molecular or nanoscale agents for lysosomal pH modulation, with the ultimate goal of developing novel therapeutic solutions.

Degradable Nanoparticles Restore Lysosomal pH and Autophagic Flux in Lipotoxic Pancreatic Beta Cells.

Chronic exposure to high levels of fatty acids (lipotoxicity) in pancreatic beta cells (ß-cells) decreases lysosomal acidity and inhibits autophagic flux. Today, there are a lack of approaches to modify lysosomal acidity to determine whether impaired lysosomal acidification is causally inhibiting autophagic flux and cellular functions. Biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) with diameters of ≈100 nm localize to lysosomes and serve as an ideal method to deliver lactic and glycolic acid to lysosomes upon NP polymer degradation. In this study, the ability of PLGA NPs to lower lysosomal pH and restore autophagic flux is investigated in pancreatic insulin secreting (INS1) ß-cells. PLGA NPs display a concentration dependent performance with higher concentrations of PLGA NPs, lowering lysosomal pH, as well as restoring autophagic flux and insulin secretion in pancreatic ß-cells. These results document that acidifying the lysosome, via an external perturbation, in lipotoxic pancreatic ß-cells affords a specific biological outcome of improved cellular degradative activity.

Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Chronic exposure of pancreatic ß cells to high concentrations of free fatty acids leads to lipotoxicity (LT)-mediated suppression of glucose-stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to ß-cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose-dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome-targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re-acidification restores citrate synthase activity and ATP content in an insulin secreting ß-cell line (INS-1). Furthermore, nanoparticle-mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS-1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.-Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Biodegradable PLGA nanoparticles restore lysosomal acidity and protect neural PC-12 cells against mitochondrial toxicity.

Exposure of mitochondrial parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) to PC-12 cells results in significant cell death, decreases lysosomal acidity, and inhibits autophagic flux. Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) of ≈100 nm diameter localize to the lysosome, degrade, and subsequently release their acidic components to acidify the local lysosomal environment. The performance of PLGA NPs with different lysosomal pH modulating capabilities is investigated in PC-12 cells under MPP+ induced mitochondrial toxicity. PLGA NPs perform in a compositional dependent manner, where NPs with a higher glycolic acid to lactic acid ratio content degrade faster, and yield greater degrees of lysosomal pH modulation as well as autophagic flux modulation in PC-12 cells under MPP+ insult. These results show that slight compositional changes of the polymeric NP give rise to differing degrees of lysosomal acidification in PC-12 cells and afford improved cellular degradative activity.

Biologically Active Branched Polysaccharide Mimetics: Synthesis via Ring-Opening Polymerization of a Maltose-Based ß-Lactam.

Stereoregular poly-amido-saccharides bearing α-glucopyranose branches (Mal-PASs) are synthesized by anionic ring-opening polymerization of a maltose-based ß-lactam monomer followed by debenzylation. The polymerization affords high molecular weight polymers (up to 31500 g/mol) with narrow dispersities (D < 1.1). Deprotected Mal-PASs are highly soluble in water and adopt a left-handed helical conformation in solution. Turbidimetric assay shows that Mal-PASs are multivalent ligands to lectin Concanavalin A.

Synthesis of Altrose Poly-amido-saccharides with ß-N-(1→2)-d-amide Linkages: A Right-Handed Helical Conformation Engineered in at the Monomer Level.

The design and synthesis of amide-linked saccharide oligomers and polymers, which are predisposed to fold into specific ordered secondary structures, is of significant interest. Herein, right-handed helical poly amido-saccharides (PASs) with ß-N-(1→2)-d-amide linkages are synthesized by the anionic ring-opening polymerization of an altrose ß-lactam monomer (alt-lactam). The right-handed helical conformation is engineered into the polymers by preinstalling the ß configuration of the lactam ring in the monomer via the stereospecific [2+2] cycloaddition of trichloroacetyl isocyanate with a d-glycal possessing a 3-benzyloxy group oriented to the α-face of the pyranose. The tert-butylacetyl chloride initiated polymerization of the alt-lactam proceeds smoothly to afford stereoregular polymers with narrow dispersities. Birch reduction of the benzylated polymers gives water-soluble altrose PASs (alt-PASs) in high yields without degradation of the polymer backbone. Circular dichroism analysis shows the alt-PASs adopt a right-handed helical conformation in aqueous solutions. This secondary conformation is stable over a wide range of different conditions, such as pH (2.0 to 12.0), temperature (5 to 75 °C), ionic salts (2.0 M LiCl, NaCl, and KCl), as well as in the presence of protein denaturants (4.0 M urea and guanidinium chloride). Cytotoxicity studies reveal that the alt-PASs are nontoxic to HEK, HeLa, and NIH3T3 cells. The results showcase the ability to direct solution conformation of polymers through monomer design. This approach is especially well-suited and straightforward for PASs as the helical conformations formed result from constraints imposed by the relatively rigid and sterically bulky repeating units.

Lysosome acidification by photoactivated nanoparticles restores autophagy under lipotoxicity.

In pancreatic ß-cells, liver hepatocytes, and cardiomyocytes, chronic exposure to high levels of fatty acids (lipotoxicity) inhibits autophagic flux and concomitantly decreases lysosomal acidity. Whether impaired lysosomal acidification is causally inhibiting autophagic flux and cellular functions could not, up to the present, be determined because of the lack of an approach to modify lysosomal acidity. To address this question, lysosome-localizing nanoparticles are described that, upon UV photoactivation, enable controlled acidification of impaired lysosomes. The photoactivatable, acidifying nanoparticles (paNPs) demonstrate lysosomal uptake in INS1 and mouse ß-cells. Photoactivation of paNPs in fatty acid-treated INS1 cells enhances lysosomal acidity and function while decreasing p62 and LC3-II levels, indicating rescue of autophagic flux upon acute lysosomal acidification. Furthermore, paNPs improve glucose-stimulated insulin secretion that is reduced under lipotoxicity in INS1 cells and mouse islets. These results establish a causative role for impaired lysosomal acidification in the deregulation of autophagy and ß-cell function under lipotoxicity.

Nanoparticle tumor localization, disruption of autophagosomal trafficking, and prolonged drug delivery improve survival in peritoneal mesothelioma.

The treatment outcomes for malignant peritoneal mesothelioma are poor and associated with high co-morbidities due to suboptimal drug delivery. Thus, there is an unmet need for new approaches that concentrate drug at the tumor for a prolonged period of time yielding enhanced antitumor efficacy and improved metrics of treatment success. A paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) system is described that addresses two unique challenges to improve the outcomes for peritoneal mesothelioma. First, following intraperitoneal administration, eNPs rapidly and specifically localize to tumors. The rate of eNP uptake by tumors is an order of magnitude faster than the rate of uptake in non-malignant cells; and, subsequent accumulation in autophagosomes and disruption of autophagosomal trafficking leads to prolonged intracellular retention of eNPs. The net effect of these combined mechanisms manifests as rapid localization to intraperitoneal tumors within 4 h of injection and persistent intratumoral retention for >14 days. Second, the high tumor-specificity of PTX-eNPs leads to delivery of greater than 100 times higher concentrations of drug in tumors compared to PTX alone and this is maintained for at least seven days following administration. As a result, overall survival of animals with established mesothelioma more than doubled when animals were treated with multiple doses of PTX-eNPs compared to equivalent dosing with PTX or non-responsive PTX-loaded nanoparticles.