Hyaluronic acid coating of gold nanoparticles for intraocular drug delivery: Evaluation of the surface properties and effect on their distribution.

Due to the unique anatomical structure of the eye, ocular drug delivery is a promising delivery route for the treatment of several ocular diseases, such as the ocular neovascularization that contributes to diabetic retinopathy. This disease is triggered by inflammation, retinal ischemia, and/or deposits of advanced-glycation end-products (AGEs), as well as increased levels of vascular endothelial growth factor (VEGF), interleukins, or reactive oxygen species (ROS). Gold has unique antioxidant and antiangiogenic properties and can inhibit angiogenic molecules. Furthermore, gold nanoparticles (GNPs) are not only biocompatible, they are easy to synthesize, they absorb and scatter visible light, and they can be made with precise control over size and shape. GNPs are an excellent candidate for ocular drug delivery because they can be conjugated to an extraordinarily diverse array of different biomolecules, and surface functionalization can improve the mobility of GNPs across the physiological barriers of the eye, such as the vitreous humour or the inner limiting membrane. For this purpose, we employed low molecular weight hyaluronan (HA) to increase the mobility of the nanoparticles as well as target them to HA receptors that are expressed in different cells of the eye. In this study, the combination of gold and HA enhanced the stability of the whole carrier and promoted their distribution across ocular tissues and barriers to reach the retina. Moreover, analysis in vitro, ex vivo, and in ovo revealed the protective and antiangiogenic effect of GNPs as inhibitors of AGEs-mediated- retinal pigment epithelial cell death and neovascularization. We demonstrated that conjugation with HA enhances GNP stability and distribution due to a specific CD44 receptor interaction. The capacity of HA-GNPs to distribute through the vitreous humour and their avidity for the deeper retinal layers ex vivo, suggest that HA-GNPs are a promising delivery system for the treatment of ocular neovascularization and related disorders.

Fertility preservation for trans men: frozen-thawed in vitro matured oocytes collected at the time of ovarian tissue processing exhibit normal meiotic spindles.

PURPOSES: At the moment of sex reassignment surgery (SRS), the ovarian tissue is sometimes cryopreserved as fertility preservation option for female-to-male trans men, also called trans men. During this preparation, cumulus-oocyte-complexes (COCs) can be found and in vitro matured. It is not known if these oocytes are developmentally competent. In order to use these oocytes for fertility preservation and subsequent fertilization, a normal spindle structure before and after vitrification is necessary. METHODS: A total of 680 COCs were collected from trans men (n = 16) at the time of SRS and after testosterone treatment. The COCs were subjected to in vitro maturation and those that reached the metaphase II stage (MII) were collected and split into two groups; group 1 was immediately fixed for spindle staining and group 2 was first vitrified and warmed followed by spindle staining. Statistical analysis was performed by Fisher's exact test. RESULTS: After 48 h in vitro maturation, 38.1% of COCs were at MII stage. Those oocytes were split in two groups: (1) 126 MII oocytes in the noncryopreservation group and (2) 133 MII oocytes underwent cryopreservation through vitrification. The oocyte survival rate, after 2 h warming, was 67.7%. Both the noncryopreserved and the vitrified group showed comparable results concerning normal spindle structure and chromosomes alignment, 85.7% vs. 92.2% (P = 0.27). CONCLUSIONS: Spindle structure analysis and chromosomal alignment after vitrification seem normal in in vitro matured COCs collected during the tissue processing of ovaries in trans men at the time of SRS. The MII oocytes do not seem to be morphologically affected by prolonged testosterone treatment.

siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat.

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.