Ying-Shan Chen

Sustained intravitreal delivery of connexin43 mimetic peptide by poly(d,l-lactide-co-glycolide) acid micro- and nanoparticles – Closing the gap in retinal ischaemia

Recent research has shown that transient block of connexin43 (Cx43) hemichannels by mimetic peptides (MP) after retinal ischaemia inhibits uncontrolled hemichannel opening causing blood-brain barrier permeability and endothelial cell loss, and consequently provides improved retinal ganglion cell (RGC) survival. However, the highly hydrophilic character and potentially poor stability of native peptides can limit efficient delivery in a clinical setting. The present study investigated the ability of intravitreally injected Cx43 MP encapsulated into slow-release poly(lactic-co-glycolic) acid (PLGA) nano-(Nps) and microparticles (Mps) to promote RGC survival in a retinal ischaemia-reperfusion rat model. The particle size was around 113 nm (Nps) and 9 μm (Mps), respectively, with Cx43 MP entrapment efficiencies of 70% (Nps) and 97% (Mps). A triphasic in vitro release profile was observed with an initial burst of surface-bound Cx43 MP followed by slow release due to polymer erosion and further drug release at the point of complete particle breakdown, with 100% release achieved after 63 (Nps) and 112 (Mps) days, respectively. Nps showed the most promising results on both Cx43 down-regulation and RGC rescue in this acute injury model. Mps treatment, on the other hand, was unable to down regulate the initial inflammatory response possibly due to trapping of the bigger particles in the vitreous and the much slower release of Cx43 MP from these particles, but displayed a delayed effect on Cx43 regulation and RGC preservation due to the sustained release.

Intravitreal injection of lipoamino acid-modified connexin43 mimetic peptide enhances neuroprotection after retinal ischemia

Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Recent research has shown that transient block of connexin43 (Cx43) hemichannels by a Cx43 mimetic peptide (MP), Peptide5, delivered systemically or by intravitreal injection after retinal ischemia inhibits uncontrolled hemichannel opening to provide significantly reduced vessel leak and inflammation as well as significantly enhanced RGC survival. We have previously shown, in vitro, that a chemically modified C12-C12-Cx43 MP has a twofold greater half-life in bovine vitreous (ex vivo) than the native peptide. The present study investigated the ability of intravitreally injected, chemically modified C12-C12-Cx43 MP to further enhance RGC survival in a rat retinal ischemia-reperfusion model. Intravitreally injected native Cx43 MP or C12-C12-Cx43 MP both minimized vessel leak, reduced inflammation, and protected RGC after ischemic injury. However, the modified C12-C12-Cx43 MP, with its prolonged vitreous stability, showed significantly lower levels of Cx43 expression post-injury, with a trend towards a greater reduction in vessel leak and further RGC protection, suggesting that these molecules may be a clinically relevant neuroprotective tool in the treatment of optic neuropathy.

Cytotoxicity and vitreous stability of chemically modified connexin43 mimetic peptides for the treatment of optic neuropathy

Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Unregulated connexin (Cx) hemichannel opening plays a role in RGC loss. Thus, inhibition via Cx43-specific mimetic peptides (MP) may prevent further cell death. However, the highly hydrophilic character and poor stability of native peptides prevent their efficient delivery across biological membranes. The present study aimed to improve the stability of Cx43 MP by conjugation to C12-lipoamino acid (C12-Laa) or sugar groups. Unmodified and modified Cx43 MP were synthesized using solid-phase peptide synthesis. Their functionality was assessed by propidium iodide (PI) uptake into NT2 cells, a human testicular carcinoma progenitor cell line able to differentiate into astrocytes, whereas the stability in ocular vitreous was measured by reversed-phase high-performance liquid chromatography. PI uptake studies showed inhibition of hemichannel opening for unmodified and modified Cx43 MP. Stability measurements revealed improved stability of modified Cx43 MP, with two Laa groups increasing the peptide half-life in bovine vitreous more than twofold. Conjugation to C12 -Laa or sugar did not affect the functionality of Cx43 MP, but addition of two C12-Laa groups significantly improved peptide stability. Laa-modifications may therefore offer improved stability and retinal delivery of peptides in vivo.

Neuroprotection in the treatment of glaucoma – A focus on connexin43 gap junction channel blockers

Glaucoma is a form of optic neuropathy and a common cause of blindness, affecting over 60 million people worldwide with an expected rise to 80 million by 2020. Successful treatment is challenging due to the various causes of glaucoma, undetectable symptoms at an early stage and inefficient delivery of drugs to the back of the eye. Conventional glaucoma treatments focus on the reduction of elevated intraocular pressure (IOP) using topical eye drops. However, their efficacy is limited to patients who suffer from high IOP glaucoma and do not address the underlying susceptibility of retinal ganglion cells (RGC) to degeneration. Glaucoma is known as a neurodegenerative disease which starts with RGC death and eventually results in damage of the optic nerve. Neuroprotective strategies therefore offer a novel treatment option for glaucoma by not only preventing neuronal loss but also disease progression. This review firstly gives an overview of the pathophysiology of glaucoma as well as current treatment options including conventional and novel delivery strategies. It then summarizes the rational for neuroprotection as a novel therapy for glaucomatous neuropathies and reviews current potential neuroprotective strategies to preserve RGC, with a focus on connexin43 (Cx43) gap junction channel blockers.

Hyaluronic Acid Coated Albumin Nanoparticles for Targeted Peptide Delivery to the Retina

Recent studies have shown that Connexin43 mimetic peptide (Cx43 MP) can prevent secondary damage following several retinal ischemic and inflammatory disorders by blocking the pathological opening of gap junction hemichannels. However, the poor stability of peptides and the presence of various intraocular barriers limit efficient retinal delivery in the clinical setting. The present study aimed to prolong the bioactivity of Cx43 MP and achieve targeted delivery to the retina by loading the peptide into hyaluronic acid (HA) coated human serum albumin nanoparticles (HSA NPs). Two different loading methods, adsorption and incorporation, were used with the peptide released slowly over a period of up to four months. Compared to uncoated particles, HA coated HSA NPs exhibited enhanced in vitro cellular uptake and ex vivo retinal penetration via HA-CD44 receptor mediated interactions. Furthermore, cell viability and Cx43 MP functionality assays showed that NPs protected Cx43 MP from degradation, sustained its release, and thus prolonged its bioactivity without reducing cell viability at concentrations used for Cx43 hemichannel blocking. Therefore, HA coated HSA NPs could have great potential for sustained and targeted delivery of Cx43 MP to treat various retinal inflammatory conditions.

In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides

The efficacy of antisense oligodeoxynucleotides (AsODNs) is compromised by their poor stability in biological fluids and the inefficient cellular uptake due to their size and negative charge. Since chemical modifications of these molecules have resulted in a number of non-antisense activities, incorporation into particulate delivery systems has offered a promising alternative. The aim of this study was to evaluate various poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for AsODN entrapment and delivery. PLGA nanoparticles were prepared using the double emulsion solvent evaporation method. The influence of formulation parameters such as PLGA concentration and volume ratio of internal aqueous phase volume (Va1) to organic phase volume (Vo) to external aqueous phase volume (Va2) on particle size, polydispersity index (PDI) and zeta potential (ZP) was investigated using a full factorial study. The particle size increased with increasing PLGA concentrations and volume ratios, with an interaction detectable between the two factors. AsODN entrapment efficiencies ranged between 49.97% and 54.95% with no significant difference between various formulations. By fitting the in vitro release profiles to a dual first order release model it was shown that the AsODN release occurred via two processes: a diffusion controlled process in the early phase (25 to 32% within one day) and a PLGA degradation process in the latter (39 to 70% after 14 days). Cellular uptake studies using primary corneal epithelial cells suggested active transport of nanoparticles via endocytosis. PLGA nanoparticles therefore show potential to successfully entrap AsODNs, transport them into cells and release them over time due to polymer erosion.

Overcoming ocular drug delivery barriers through the use of physical forces

Abstract Overcoming the physiological barriers in the eye remains a key obstacle in the field of ocular drug delivery. While ocular barriers naturally have a protective function, they also limit drug entry into the eye. Various pharmaceutical strategies, such as novel formulations and physical force‐based techniques, have been investigated to weaken these barriers and transport therapeutic agents effectively to both the anterior and the posterior segments of the eye. This review summarizes and discusses the recent research progress in the field of ocular drug delivery with a focus on the application of physical methods, including electrical fields, sonophoresis, and microneedles, which can enhance penetration efficiency by transiently disrupting the ocular barriers in a minimally or non‐invasive manner. Graphical abstract Figure. No Caption available.

Light-responsive in situ forming injectable implants for effective drug delivery to the posterior segment of the eye

ABSTRACT Introduction: Frequent intravitreal injections are currently the preferred treatment method for diseases affecting the posterior segment of the eye. However, these repeated injections have been associated with pain, risk of infection, hemorrhages, retinal detachment and high treatment costs. To overcome these limitations, light-responsive in situ forming injectable implants (ISFIs) may emerge as novel systems providing site-specific controlled drug delivery to the retinal tissues with great accuracy, safety, minimal invasiveness and high cost efficiency. Area covered: Complex ocular barriers, routes for drug delivery, types of injectable implants, ocular application of light and benefits of light-responsive systems are discussed with regards to challenges and strategies employed for effective drug delivery to the posterior segment of the eye. In particular, we have highlighted photoresponsive moieties, photopolymerization mechanisms and different development strategies with their limitations as well as recent advancements in the field. Expert opinion: Biodegradable light-responsive ISFIs are promising drug delivery systems that have shown a high degree of biocompatibility with sustained drug release in a number of applications. However, their use in intravitreal drug delivery is still in the very early stages. Issues related to the biocompatibility of the photoinitiator and the elimination of photo-degraded by-products from the ocular tissues need careful consideration, not only from a chemistry standpoint, but also from a biological perspective to improve the suitability of these systems for clinical applications.

Ultrasound-mediated nanoparticle delivery across ex vivo bovine retina after intravitreal injection

Graphical abstract Figure. No Caption available. HighlightsUltrasound enhanced nanoparticle penetration across the retina.Ultrasound improved the diffusive mobility of nanoparticles in the vitreous.Ultrasound did not cause any detectable damage to the ocular tissues. Abstract Intravitreal injection is the most common administration route for the treatment of retinal diseases. However, the vitreous and some of the retinal layers themselves act as significant barriers to efficient delivery of drugs administered intravitreally. This study aimed to improve the diffusive mobility of nanoparticles (NPs) in the vitreous and enhance their permeation across the retina after intravitreal injection by application of ultrasound (US). Ex vivo posterior bovine eye cups were used and the vitreous was either left intact or removed gently from the neural retina. Hyaluronic acid coated human serum albumin NPs were administered into the eye cups and continuous US with a frequency of 1 MHz, an intensity of 0.5 W/cm2, and a duration of 30 s was applied once or repeatedly via the transscleral route. After pre‐determined time points, fluorescence intensities in the vitreous and the retina were analyzed. Short pulses of US significantly improved the diffusive mobility of NPs through the vitreous as well as their penetration across the neural retina into the retinal pigment epithelium and choroid without causing any detectable damage to the ocular tissues. Therefore, transscleral US could be a powerful and safe tool to enhance retinal delivery of intravitreally injected NPs.

Hyaluronic acid coated albumin nanoparticles for targeted peptide delivery in the treatment of retinal ischaemia

Recent studies have shown that Connexin43 mimetic peptide (Cx43 MP) can prevent secondary damage following retinal ischaemic and inflammatory disorders by blocking uncontrolled Cx43 hemichannel opening. However, limitations in peptide stability and the presence of various intraocular barriers limit efficient retinal delivery in the clinical setting. The present study aimed to achieve targeted and sustained peptide delivery to the retina by encapsulating Cx43 MP into hyaluronic acid (HA) coated albumin nanoparticles (NPs). Intraocular biodistribution, particle retention, retinal targeting, and therapeutic efficacy of intravitreally injected NPs encapsulating Cx43 MP were evaluated in a rat model of retinal ischaemia-reperfusion injury. NPs rapidly diffused through the vitreous and specifically targeted CD44-expressing retinal cells. NPs remained at the target site for extended periods enabling sustained peptide release and thus prolonged therapeutic action. Compared to free Cx43 MP, Cx43 MP loaded NPs enabled enhanced therapeutic efficacy preventing thinning of retinal layers and disruption of retinal blood vessels. Immunohistochemical results confirm that Cx43 MP loaded NPs efficiently reduced Cx43 expression, thereby suppressing ongoing inflammation and preventing the loss of retinal ganglion cells. Overall, HA coated NPs could have great potential as a peptide delivery platform in the treatment of chronic retinal degenerative and inflammatory disorders.