Comparison between Nanoparticle Encapsulation and Surface Loading for Lysosomal Enzyme Replacement Therapy

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) enhance the delivery of therapeutic enzymes for replacement therapy of lysosomal storage disorders. Previous studies examined NPs encapsulating or coated with enzymes, but these formulations have never been compared. We examined this using hyaluronidase (HAse), deficient in mucopolysaccharidosis IX, and acid sphingomyelinase (ASM), deficient in types A–B Niemann–Pick disease. Initial screening of size, PDI, ζ potential, and loading resulted in the selection of the Lactel II co-polymer vs. Lactel I or Resomer, and Pluronic F68 surfactant vs. PVA or DMAB. Enzyme input and addition of carrier protein were evaluated, rendering NPs having, e.g., 181 nm diameter, 0.15 PDI, −36 mV ζ potential, and 538 HAse molecules encapsulated per NP. Similar NPs were coated with enzyme, which reduced loading (e.g., 292 HAse molecules/NP). NPs were coated with targeting antibodies (> 122 molecules/NP), lyophilized for storage without alterations, and acceptably stable at physiological conditions. NPs were internalized, trafficked to lysosomes, released active enzyme at lysosomal conditions, and targeted both peripheral organs and the brain after i.v. administration in mice. While both formulations enhanced enzyme delivery compared to free enzyme, encapsulating NPs surpassed coated counterparts (18.4- vs. 4.3-fold enhancement in cells and 6.2- vs. 3-fold enhancement in brains), providing guidance for future applications

DNA-Methylome based Tumor Hypoxia Classifier Identifies HPV-negative Head & Neck Cancer Patients at Risk for Locoregional Recurrence After Primary Radiochemotherapy

BACKGROUND Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in Head and Neck Squamous Cell Carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia. METHODS A DNA methylome-based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA-HNSCC cohort based on matched assignments using gene expression-based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of Human Papilloma Virus (HPV)-negative HNSCC patients treated with primary radiochemotherapy (RCHT). RESULTS While hypoxia-GSEs failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (LR, HR=4.3, p=0.001) and overall survival (OS, HR=2.34, p=0.03) but not distant metastasis (DM) after RCHT in the both cohorts. Hypoxia-M status was inversely associated with CD8 T-cells infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR=1.83, p=0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status. CONCLUSIONS Our findings highlight an unexplored avenue for DNA Methylation-based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors. TRIAL REGISTRATION Retrospective observational study from the German Cancer Consortium (DKTK-ROG), not interventional

Comparative study of nanocarriers targeted to different transport pathways into and across the endothelium for brain delivery of therapeutic enzymes

[eng] The blood-brain barrier (BBB) is a major obstacle for the treatment of neurological diseases such as common Parkinson’s disease or rare lysosomal storage disorders (LSDs). LSDs are characterised by deficiency of lysosomal components, mainly enzymes, resulting in lysosomes accumulation of macromolecules affecting the CNS and peripheral organs. Examples are Niemann-Pick disease (NPD) and Gaucher disease (GD), characterised by deficiency in acid sphingomyelinase (ASM) and glucocerebrosidase (GBA), respectively. Additionally, GBA alterations are involved in Parkinson’s. Currently, enzyme replacement therapy (ERT) fails to treat neuropathic symptoms due to the BBB. Targeting enzymes across the BBB using nanocarriers capable to transcytosis in this interface offers an interesting approach to improve ERT. This project studied the transport of differently targeted therapeutic nanocarriers across the BBB to identify the most suitable formulations, using polymer nanocarriers targeted to different routes, BBB cell models and in vivo animal models, as well as physicochemical measurements of colloidal properties, radioactive tracing, and confocal fluorescence microscopy. Four aims were addressed. First, biodegradable PLGA nanocarriers and non-degradable polystyrene models were produced, which had similar physicochemical properties, were functionalized for targeting, loaded with a enzyme and increased enzyme delivery in cell cultures, also providing in vivo targeting. Non degradable nanocarriers were thereafter used to focus on transport solely avoiding degradation artifacts. Second, transport across a cellular BBB was examined for formulations targeting intercellular adhesion molecule 1 (ICAM-1), identifying a dual simultaneous transport to endothelial lysosomes and BBB transcytosis with additional basolateral re-uptake of nanocarriers, intertwined and dependent on the targeting valency of nanocarriers. Third, the effects of NPD on this transport was examined for ICAM-1 targeting nanocarriers along with formulations targeting the transferrin receptor (TfR) or plasmalemma vesicle associated protein 1 (PV1). Disease altered all pathways in different ways, with ICAM-1 targeting nanocarriers being the most efficient formulation for BBB transport in this disease model. Fourth, a similar study was conducted in GD, where different alterations were also found, yet again ICAM-1 targeting nanocarrier were the most suitable candidate for trans BBB delivery. Thus, this study, encompassing multidisciplinary basic and applicable research, found relevant findings for a better understanding of the pathological alterations associated to these neuropathies and the practical design of therapeutic nanocarriers

Comparative study of nanocarriers targeted to different transport pathways into and across the endothelium for brain delivery of therapeutic enzymes

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Bioenginyeria de Catalunya (IBEC)[eng] The blood-brain barrier (BBB) is a major obstacle for the treatment of neurological diseases such as common Parkinson’s disease or rare lysosomal storage disorders (LSDs). LSDs are characterised by deficiency of lysosomal components, mainly enzymes, resulting in lysosomes accumulation of macromolecules affecting the CNS and peripheral organs. Examples are Niemann-Pick disease (NPD) and Gaucher disease (GD), characterised by deficiency in acid sphingomyelinase (ASM) and glucocerebrosidase (GBA), respectively. Additionally, GBA alterations are involved in Parkinson’s. Currently, enzyme replacement therapy (ERT) fails to treat neuropathic symptoms due to the BBB. Targeting enzymes across the BBB using nanocarriers capable to transcytosis in this interface offers an interesting approach to improve ERT. This project studied the transport of differently targeted therapeutic nanocarriers across the BBB to identify the most suitable formulations, using polymer nanocarriers targeted to different routes, BBB cell models and in vivo animal models, as well as physicochemical measurements of colloidal properties, radioactive tracing, and confocal fluorescence microscopy. Four aims were addressed. First, biodegradable PLGA nanocarriers and non-degradable polystyrene models were produced, which had similar physicochemical properties, were functionalized for targeting, loaded with a enzyme and increased enzyme delivery in cell cultures, also providing in vivo targeting. Non degradable nanocarriers were thereafter used to focus on transport solely avoiding degradation artifacts. Second, transport across a cellular BBB was examined for formulations targeting intercellular adhesion molecule 1 (ICAM-1), identifying a dual simultaneous transport to endothelial lysosomes and BBB transcytosis with additional basolateral re-uptake of nanocarriers, intertwined and dependent on the targeting valency of nanocarriers. Third, the effects of NPD on this transport was examined for ICAM-1 targeting nanocarriers along with formulations targeting the transferrin receptor (TfR) or plasmalemma vesicle associated protein 1 (PV1). Disease altered all pathways in different ways, with ICAM-1 targeting nanocarriers being the most efficient formulation for BBB transport in this disease model. Fourth, a similar study was conducted in GD, where different alterations were also found, yet again ICAM-1 targeting nanocarrier were the most suitable candidate for trans BBB delivery. Thus, this study, encompassing multidisciplinary basic and applicable research, found relevant findings for a better understanding of the pathological alterations associated to these neuropathies and the practical design of therapeutic nanocarriers

Role of the Lactide:Glycolide Ratio in PLGA Nanoparticle Stability and Release under Lysosomal Conditions for Enzyme Replacement Therapy of Lysosomal Storage Disorders

Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤−35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56–78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs

Value of PET imaging for radiation therapy *

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