Search

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Drug delivery to the retina remains a challenge due to ocular barriers and fast clearing mechanisms. Nanocarrier drug delivery systems (NDDSs) hold the promise of prolonging intraocular retention times and increasing drug concentrations in the retina., Methods: Anionic and cationic PEGylated liposomes, loaded with oxaliplatin (OxPt) to be used as trace element, were prepared from dry lipid powders. The differently charged liposomes were intravitreally injected in C57BL/6JrJ mice; eyes were harvested 2 hours and 24 hours post-injection. To investigate active transport mechanisms in the eye, a subset of mice were pre-injected with chloroquine before injection with cationic liposomes. Eyes were dissected and the distribution of OxPt in different tissues were quantified by inductively coupled plasma mass spectrometry (ICP-MS)., Results: Both liposome formulations enhanced the retention time of OxPt in the vitreous over free OxPt. Surprisingly, when formulated in cationic liposomes, OxPt translocated through the retina and accumulated in the RPE-sclera. Pre-injection with chloroquine inhibited the transport of liposomal OxPt from the vitreous to the RPE-sclera., Conclusions: We show that liposomes can enhance the retention time of small molecular drugs in the vitreous and that active transport mechanisms are involved in the trans retinal transport of NDDS after intravitreal injections., Translational Relevance: These results highlight the need for understanding the dynamics of ocular transport mechanisms in living eyes when designing NDDS with the back of the eye as the target. Active transport of nanocarriers through the retina will limit the drug concentration in the neuronal retina but might be exploited for targeting the RPE.

Here, we describe a series of protocols detailing the steps for evaluating SARS-CoV-2 infection in models of the human eye. Included are protocols for whole eye organoid differentiation, SARS-CoV-2 infection, and processing organoids for single-cell RNA sequencing. Additional protocols describe how to dissect and culture adult human ocular cells from cadaver donor eyes and how to compare infection of SARS-CoV-2 and the presence of SARS-CoV-2 entry factors using qPCR, immunofluorescence, and plaque assays. For complete details on the use and execution of this protocol, please refer to Eriksen et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro , ex vivo , and in vivo platforms that currently are being developed to accelerate this emerging field of research., Competing Interests: None., (This journal is © The Royal Society of Chemistry.)

Targeting nanocarrier drug delivery systems, that deliver drug payloads to the site of disease action, are frequently viewed as the future of nanocarrier based therapies but have struggled to breakthrough to the clinic in comparison to non-targeting counterparts. Using unilamellar liposomes as model nanocarriers, we show that cell targeting strategy (electrostatic, ligand and antigen) influences both the intracellular fate of the liposomes and the corresponding efficacy of the loaded drug, doxorubicin, in endothelial cells. We show that increased liposome uptake by cells does not translate to improved efficacy in this scenario but that liposome intracellular trafficking, particularly distribution between recycling endosomes and lysosomes, influences in vitro efficacy. Choosing targeting strategies that promote desired nanocarrier intracellular trafficking may be a viable strategy to enhance the in vivo efficacy of drug delivery systems., (Copyright © 2020. Published by Elsevier B.V.)

Diseases of the retina cause vision loss and blindness, which have a profound impact on an individual's quality of life. The number of therapies available to treat retinal diseases is limited. Nanoparticle (NP)-based medicines represent one strategy to expand both the number of available therapies and the range of retinal diseases treated. Liposomes, phospholipid vesicles that frequently contain cholesterol and/or modified surface chemistries, have already had minor success in retinal disease treatment and hold significant promise. Here, we provide a snapshot of recent research developments in liposomal drug delivery systems for retinal diseases and discuss the challenges associated with liposomal systems in the context of recent developments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A common event in optic neuropathies is the loss of axons and death of retinal ganglion cells (RGCs) resulting in irreversible blindness. Mammalian target of rapamycin (mTOR) signaling pathway agonists have been shown to foster axon regeneration and RGC survival in animal models of optic nerve damage. However, many challenges remain in developing therapies that exploit cell growth and tissue remodeling including (i) activating/inhibiting cell pathways synergistically, (ii) avoiding tumorigenesis, and (iii) ensuring appropriate physiological tissue function. These challenges are further exacerbated by the need to overcome ocular physiological barriers and clearance mechanisms. Here we present liposomes loaded with multiple mTOR pathway stimulating biologics designed to enhance neuroprotection after retina damage. Liposomes were loaded with ciliary neurotrophic factor, insulin-like growth factor 1, a lipopeptide N-fragment osteopontin mimic, and lipopeptide phosphatase tension homologue inhibitors for either the ATP domain or the c-terminal tail. In a mouse model of N-methyl-d-aspartic acid induced RGC death, a single intravitreal administration of liposomes reduced both RGC death and loss of retina electrophysiological function. Furthermore, combining liposomes with transplantation of induced pluripotent stem cell derived RGCs led to an improved electrophysiological outcome in mice. The results presented here show that liposomes carrying multiple signaling pathway modulators can facilitate neuroprotection and transplant electrophysiological outcome.

Purpose: To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid-loaded functionalized liposomes that exhibit both enhanced uptake by HRECs and superior biologic activity compared to nontargeting liposomes and free drug., Methods: EPCR expression of HRECs was investigated through flow cytometry and Western blot assays. EPCR-targeting liposomes were developed by functionalizing EPCR-specific antibodies onto liposomes, and the uptake of liposomes was assessed with flow cytometry and confocal laser scanning microscopy. The therapeutic potential of EPCR-targeting liposomes was determined by loading them with prednisolone either through bilayer insertion and/or by remote loading into the aqueous core. The carrier efficacy was assessed in two ways through its ability to inhibit secretion of interleukins in cells stimulated with high glucose and angiogenesis in vitro by using an endothelial cell tube formation assay., Results: HRECs express EPCR at a similar level in both human aortic and umbilic vein endothelial cells. The EPCR-targeting liposomes displayed at least a 3-fold higher uptake compared to nontargeting liposomes. This enhanced uptake was translated into superior anti-inflammatory efficacy, as the corticosteroid-loaded EPCR-targeting liposomes significantly reduced the secretion of IL-8 and IL-6 and inhibited the development of cell tube formations in contrast to nontargeting liposomes., Conclusions: We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised.

The diffusion dynamics of nanocarriers in the vitreous and the influence of nanocarrier physicochemical properties on these dynamics is an important aspect of the efficacy of intravitreal administered nanomedicines for the treatment of posterior segment eye diseases. Here we use fluorescence correlation spectroscopy (FCS) to determine liposome diffusion coefficients in the intact vitreous (D Vit ) of ex vivo porcine eyes using a modified Miyake-Apple technique to minimize the disruption of the vitreous fine structure. We chose to investigate whether the zeta potential of polyethylene glycol functionalized (i.e. PEGylated) liposomes altered liposome in situ diffusion dynamics in the vitreous. Non-PEGylated cationic nanocarriers have previously shown little to no diffusion in the vitreous, whilst neutral and anionic have shown diffusion. The liposomes investigated had diameters below 150nm and zeta potentials ranging from -20 to +12mV. We observed that PEGylated cationic liposomes had significantly lower D Vit values (1.14μm 2 s -1 ) than PEGylated neutral and anionic liposomes (2.78 and 2.87μm 2 s -1 ). However, PEGylated cationic liposomes had a similar biodistribution profile across the vitreous to the other systems. These results show that PEGylated cationic liposomes with limited cationic charge can diffuse across the vitreous and indicate that the vitreous as a barrier to nanocarriers (Ø<500nm) is more complicated than simply an electrostatic barrier as previously suggested., (Copyright © 2017 Elsevier B.V. All rights reserved.)