Selective drug delivery to the retinal cells: Biological barriers and avenues.

Retinal drug delivery is a challenging, but important task, because most retinal diseases are still without any proper therapy. Drug delivery to the retina is hampered by the anatomical and physiological barriers resulting in minimal bioavailability after topical ocular and systemic administrations. Intravitreal injections are current method-of-choice in retinal delivery, but these injections show short duration of action for small molecules and low target bioavailability for many protein, gene based drugs and nanomedicines. State-of-art delivery systems are based on prolonged retention, controlled drug release and physical features (e.g. size and charge). However, drug delivery to the retina is not cell-specific and these approaches do not facilitate intracellular delivery of modern biological drugs (e.g. intracellular proteins, RNA based medicines, gene editing). In this focused review we highlight biological factors and mechanisms that form the basis for the selective retinal drug delivery systems in the future. Therefore, we are presenting current knowledge related to retinal membrane transporters, receptors and targeting ligands in relation to nanomedicines, conjugates, extracellular vesicles, and melanin binding. These issues are discussed in the light of retinal structure and cell types as well as future prospects in the field. Unlike in some other fields of targeted drug delivery (e.g. cancer research), selective delivery technologies have been rarely studied, even though cell targeted delivery may be even more feasible after local administration into the eye.

The Effect of Single Nucleotide Variations in the Transmembrane Domain of OATP1B1 on in vitro Functionality.

PURPOSE: Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. METHODS: Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. RESULTS: All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. CONCLUSIONS: Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.

Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor.

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, Cα-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.

Release of functional dexamethasone by intracellular enzymes: A modular peptide-based strategy for ocular drug delivery.

Tissue barriers limit drug delivery in the eye. Therefore, retinal diseases are treated with intravitreal injections. Delivery systems with reduced dosing frequency and/or cellular drug delivery properties are needed. We present here a modular peptide-based delivery system for cell targeted release of dexamethasone in the retinal pigment epithelial cells. The peptide-dexamethasone conjugates consist of cell penetrating peptide, enzyme cleavable linker and dexamethasone that is conjugated with hydrazone bond. The conjugates are chemically stable in the vitreous, internalize into the retinal pigment epithelial cells and release dexamethasone intracellularly by enzymatic action of cathepsin D. In vitro binding assay and molecular docking confirm binding of the released dexamethasone fragment to the human glucocorticoid receptor. In vivo rabbit studies show increased vitreal retention of dexamethasone with a peptide conjugate. Modular peptide conjugates are a promising approach for drug delivery into the retinal cells.

Hyaluronic Acid Graft Copolymers with Cleavable Arms as Potential Intravitreal Drug Delivery Vehicles.

Treatment of retinal diseases currently demands frequent intravitreal injections due to rapid clearance of the therapeutics. The use of high molecular weight polymers can extend the residence time in the vitreous and prolong the injection intervals. This study reports a water soluble graft copolymer as a potential vehicle for sustained intravitreal drug delivery. The copolymer features a high molecular weight hyaluronic acid (HA) backbone and poly(glyceryl glycerol) (PGG) side chains attached via hydrolysable ester linkers. PGG, a polyether with 1,2-diol groups in every repeating unit available for conjugation, serves as a detachable carrier. The influence of synthesis conditions and incubation in physiological media on the molecular weight of HA is studied. The cleavage of the PGG grafts from the HA backbone is quantified and polymer-from-polymer release kinetics are determined. The biocompatibility of the materials is tested in different cell cultures.

Differentially cleaving peptides as a strategy for controlled drug release in human retinal pigment epithelial cells.

Currently, drug delivery to the posterior eye segment relies on intravitreal injections of therapeutics. This approach requires frequent injections and does not guarantee drug delivery to intracellular targets. Controlled release systems and nanoparticles are being investigated to mitigate these challenges but most of these approaches lack translational success to the clinics. In our present study, we report a peptide-based delivery system that utilizes enzyme assisted cleavable linkers to release conjugated cargo within the retinal pigment epithelial (RPE) cells. Peptide linkers with differential cleavage rates were developed and tested in the vitreous humor, RPE cell homogenates and intact RPE cells. Selected peptide linkers were conjugated to cell penetrating peptides and d-peptide cargoes. The peptide-based delivery systems were non-toxic to the RPE cells, chemically stable in porcine vitreous and delivered cargo prototypes (hydrophobic & hydrophilic) to the RPE cells. Importantly, we show quantitatively with LC/MS analytics that the intracellular cargo release is controlled by the sequence of the peptide linker. The controlled cleavage of the peptide linkers is not only a useful strategy for intracellular drug delivery to the RPE targets but might also be useful in utilizing the RPE cells as mediators of drug delivery to intracellular targets and surrounding tissues (such as neural retina and choroid).

Pharmacokinetic aspects of retinal drug delivery.

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory.

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture.

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.

Recruitment pharmacophore for interleukin 5 receptor alpha antagonism.

Interleukin-5 receptor alpha is a therapeutic target for hypereosinophilic diseases including allergic inflammations and asthma. The cyclic peptide AF17121 (Ac-VDE[CWRIIASHTWFC]AEE-CONH(2)) has been identified as a submicromolar inhibitor of interleukin 5 (IL5)-interleukin 5 receptor alpha (IL5Ralpha) interaction from a random peptide screen. However, this inhibitor has limitations as a drug lead because of its relatively large size. We used chemical synthesis of peptides with natural and non-natural amino acids along with kinetic binding and cell proliferation competition assays to expand definition of structural elements in the peptide that are important for receptor antagonism and to elucidate the underlying pharmacophore. We found that the specific steric array of hydrogen bonding groups in the Arg 6 guanido side chain is critical for receptor inhibition. We also investigated noncharged structural elements in AF17121. Screening a set of five hydrophobic residues showed that peptide function is strongly sensitive to variations in several of these residues, most prominently Ile 7 and Trp 13. We postulate that presentation of charged, hydrogen bonding and hydrophobic structural elements within the disulfide-constrained peptide drives IL5Ralpha recruitment by AF17121. We hypothesize from these results and previous receptor mutagenesis studies that Arg 6 recruitment of IL5Ralpha occurs through hydrogen bonding as well as charge-charge interactions with Asp 55 in site one of domain 1 of IL5Ralpha, and that this interaction is complemented by additional charged and hydrophobic interactions around the Asp 55 locus. Scaffolding a limited set of structural elements in the inhibitor pharmacophore may be useful for small molecule antagonist design inspired by the peptide.

Recombinant-protein production in insect cells utilizing a hollow-fibre bioreactor.

Here, we demonstrate for the first time that the hollow-fibre bioreactor is an excellent tool for the production of Drosophila-expressed recombinant proteins. Using the example of the soluble extracellular portion of the human IL-5 (interleukin 5) receptor alpha expression in S2 (Schneider's Drosophila melanogaster cell line 2) cells, we found that it is possible to produce multi-milligram amounts of functional recombinant protein continuously for several months on a laboratory scale with minimal maintenance requirements. The insect cells grow to high density and express concentrated functional recombinant protein in a small volume, simplifying and economizing downstream purification.

Asymmetric usage of antagonist charged residues drives interleukin-5 receptor recruitment but is insufficient for receptor activation.

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.

Receptor epitope usage by an interleukin-5 mimetic peptide.

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.

Cyclic peptide interleukin 5 antagonists mimic CD turn recognition epitope for receptor alpha.

The cyclic peptide AF17121 (Ac-VDECWRIIASHTWFCAEE) that inhibits interleukin 5 (IL-5) function and IL-5 receptor alpha-chain (IL-5Ralpha) binding has been derived from recombinant random peptide library screening and follow-up synthetic variation. To better understand the structural basis of its antagonist activity, AF17121 and a series of analogs of the parent peptide were prepared by solid phase peptide synthesis. Sequence variation was focused on the charged residues Asp(2), Glu(3), Arg(6), Glu(17), and Glu(18). Two of those residues, Glu(3) and Arg(6), form an EXXR motif that was found to be common among library-derived IL-5 antagonists. The E and R in the EXXR motif have a proximity similar to charged residues in a previously identified receptor alpha binding region, the beta-strand between the C- and D-helices of human IL-5. Optical biosensor interaction kinetics and cell proliferation assays were used to evaluate the antagonist activities of the purified synthetic peptides, by measuring competition with the highly active single chain IL-5. Analogs in which acidic residues (Asp(2), Glu(3), Glu(17), and Glu(18)) were replaced individually by Ala retained substantial competition activity, with multiple replacements in these residues leading to fractional loss of potency at most. In contrast, R6A analogs had strongly reduced competition activity. The results reveal that the arginine residue is crucial for the IL-5Ralpha binding of AF17121, while the acidic residues are not essential though likely complex-stabilizing particularly in the Asp(2)-Glu(3) region. By CD, AF17121 exhibited mostly disordered structure with evidence for a small beta-sheet content, and replacement of the arginine had no influence on the observed secondary structure of the peptides. The dominance of Arg(6) in AF17121 activity corresponds to previous findings of dominance of the positive charge balance in the antiparallel beta-sheet of IL-5 composed of (88)EERRR(92) in one strand of the CD turn region of IL-5 and with Arg(32) in the neighboring beta-strand. These results argue that AF17121 and related library-derived peptides function by mimicking the CD turn receptor alpha recognition epitope in IL-5 and open the way to small molecule antagonist design.

Immunomodulation by peptide analogs of retroviral envelope protein.

The mechanism by which retroviral proteins exert their immunosuppressive influence has remained enigmatic. Early studies have demonstrated that retroviral infection suppresses cellular and humoral immune responses. A hydrophilic 26 amino acid region of the otherwise hydrophobic transmembrane envelope protein of murine and feline leukemia viruses, p15E, is conserved among the transmembrane envelope proteins of numerous animal retroviruses (e.g. murine, feline, bovine and simian) as well as in human T-cell leukemia virus, and to a lesser extent, in human immunodeficiency virus (HIV). We evaluated the immunomodulatory properties of various synthetic retroviral envelope peptides synthesized as overlapping fragments to this conserved sequence. We report that two small peptides inhibit human mixed lymphocyte reaction (MLR), interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) production. These peptides did not affect human natural killer (NK) cell cytotoxicity in vitro, and nitric oxide (NO) production in mouse macrophage cells, RAW264.7. Our observations suggests immunomodulatory potential of two retroviral peptide analogs.