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Late-stage dry age-related macular degeneration (AMD) or geographic atrophy (GA) is an irreversible blinding condition characterized by degeneration of retinal pigment epithelium (RPE) and the associated photoreceptors. Clinical and genetic evidence supports a role for dysfunctional lipid processing and accumulation of harmful oxidized lipids in the pathogenesis of GA. Using an oxidized low-density lipoprotein (ox-LDL)-induced RPE death assay, we screened and identified sterically-hindered phenol compounds with potent protective activities for RPE. The phenol-containing PPARγ agonist, troglitazone, protected against ox-LDL–induced RPE cell death, whereas other more potent PPARγ agonists did not protect RPE cells. Knockdown of PPARγ did not affect the protective activity of troglitazone in RPE, confirming the protective function is not due to the thiazolidine (TZD) group of troglitazone. Prototypical hindered phenol trolox and its analogs potently protected against ox-LDL–induced RPE cell death whereas potent antioxidants without the phenol group failed to protect RPE. Hindered phenols preserved lysosomal integrity against ox-LDL–induced damage and FITC-labeled trolox was localized to the lysosomes in RPE cells. Analogs of trolox inhibited reactive oxygen species (ROS) formation induced by ox-LDL uptake in a dose-dependent fashion and were effective at sub-micromolar concentrations. Treatment with trolox analog 2,2,5,7,8-pentamethyl-6-chromanol (PMC) significantly induced the expression of the lysosomal protein NPC-1 and reduced intracellular cholesterol level upon ox-LDL uptake. Our data indicate that the lysosomal-localized hindered phenols are uniquely potent in protecting the RPE against the toxic effects of ox-LDL, and may represent a novel pharmacotherapy to preserve the vision in patients with GA.

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.

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

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.

To improve therapeutic efficacy of nanocarrier drug delivery systems, it is essential to improve their uptake and penetration in tumour tissue, enhance cellular uptake and ensure efficient drug release at the tumour site. Here we introduce a tumour targeting drug delivery system based on the ultrasound-mediated delivery of enzyme sensitive liposomes. These enzyme sensitive liposomes are coated with cleavable poly(ethylene glycol) (PEG) which will be cleaved by two members of the enzyme matrix metalloproteinase family (MMP-2 and MMP-9). Cleavage of the PEG coat can increase cellular uptake and will destabilize the liposomal membrane which can result in accelerated drug release. The main aim of the work was to study the effect of focused ultrasound and microbubbles on the delivery and therapeutic efficacy of the MMP sensitive liposome. The performance of the MMP sensitive liposome was compared to a non-MMP sensitive version and Doxil-like liposomes. In vitro, the cellular uptake and cytotoxicity of the liposomes were studied, while in vivo the effect of ultrasound and microbubbles on the tumour accumulation, biodistribution, microdistribution, and therapeutic efficacy were investigated. For all tested liposomes, ultrasound and microbubble treatment resulted in an improved tumour accumulation, increased extravasation, and increased penetration of the liposomes from blood vessels into the extracellular matrix. Surprisingly, penetration depth was independent of the ultrasound intensity used. Ultrasound-mediated delivery of free doxorubicin and the Doxil-like and MMP sensitive liposome resulted in a significant reduction in tumour volume 28 days post the first treatment and increased median survival. The MMP sensitive liposome showed better therapeutic efficacy than the non-MMP sensitive version indicating that cleaving the PEG-layer is important. However, the Doxil-like liposome outcompeted the MMP and non-MMP sensitive liposome, both with and without the use of ultrasound and microbubbles. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 antiinflammatory 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.

Amorphous silica nanoparticles (a-SiNPs) with high surface area and small pore size have been synthesized using a zwitterionic surfactant in acid media. The produced nanoparticles displayed large specific surface area (-850 m2/g) with an average particles size of 45 nm. The loading efficiency was assessed by incorporating three model water insoluble active substances Carbamazepine (CBZ), Ibuprofen (IBU) and Cyclosporin (CyA) via passive loading and it was found to varying from 15-40%. The loaded silica nanoparticles were analyzed using X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) to investigate the state of the adsorbed active agents. CyA was found to be in amorphous state, IBU was partially crystalline while CBZ was transformed into form I. The dissolution profiles showed rapid release for CBZ while IBU and CyA demonstrated prolonged release for 24 hr. The viability of Caco-2 cells was not affected in the presence of a-SiNPs showing negligible cytotoxicity (85%) at high concentrations up to 15 mg/ml.

Solid lipid nanoparticles (SLNs) loaded with ibuprofen (IBU) were prepared by solvent-free high-pressure homogenization (HPH). The produced SLNs consisted of stearic acid, triluarin or tripalmitin as lipid matrixes and various stabilizers. The produced empty and IBU-loaded SLNs were characterized for particle size stability over 8 months. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) were implemented to characterize the IBU state of freeze-dried SLNs. IBU was found to be in both amorphous and crystalline form within the lipid matrix. The lyophilized powders showed increased dissolution rates for IBU depending on the lipid nature. SLNs were incubated in Caco-2 cells for 24 h showing negligible cell cytotoxicity up to 15 mg/mL.

High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterisation (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), x-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates the ability of identifying surface moieties that control protein adsorption and subsequent cell adhesion using ToF SIMS and multivariate analysis.

Cyclosporine (CyA) solid lipid nanoparticles were prepared by using a solvent free high pressure homogenization process. CyA was incorporated into SLNs that consisted of stearic acid, trilaurin or tripalmitin lipid solid cores in order to enhance drug solubility. The process was conducted by varying lipid compositions, drug initial loading and applied homogenization pressure. The processing temperatures were above the lipid melting points for all formulations. The empty and CyA loaded SLN particles made were characterized for particle size stability over six months. Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) showed particle sizes ranging from 112-177 nm for empty and 181-215 nm for loaded SLNs each with narrow particle size distributions. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques were used to characterize the CyA state, which was found to be amorphous, within the lipid cores of freeze-dried SLNs. The CyA metastable form showed a profound effect on the drug dissolution rates. SLNs were incubated in Caco-2 cells for 24 hr showing negligible cell cytotoxicity up to 15 mg/ml.

The application of high throughput surface characterization (HTSC) to the analysis of polymeric biomaterial libraries is an important advancement for the discovery and development of new biomedical materials and is the focus of this review. The potential for HTSC to identify structure/activity relationships for large libraries of materials can be utilized to accelerate materials discovery as well as providing insight into the underlying biological-material interactions. Furthermore, the correlations identified between surface chemical structure and cellular behavior could not have been predicted by a rational design approach based simply on review of bulk structure, which demonstrates the importance of HTSC in the assessment of cell-material and cell-biomolecular interactions that are dependent on surface properties.