The aim of this study is to assess if an adhesive biopolymer, sodium hyaluronate (NaHA), has synergistic effects with s-PRGF (a serum derived from plasma rich in growth factors and a blood derivative that has already shown efficacy in corneal epithelial wound healing), to reduce time of healing or posology. In vitro proliferation and migration studies, both in human corneal epithelial (HCE) cells and in rabbit primary corneal epithelial (RPCE) cultures, were carried out. In addition, we performed studies of corneal wound healing in vivo in rabbits treated with s-PRGF, NaHA, or the combination of both. We performed immunohistochemistry techniques (CK3, CK15, Ki67, ß4 integrin, ZO-1, α-SMA) in rabbit corneas 7 and 30 days after a surgically induced epithelial defect. In vitro results show that the combination of NaHA and s-PRGF offers the worst proliferation rates in both HCE and RPCE cells. Addition of NaHA to s-PRGF diminishes the re-epithelializing capability of s-PRGF. In vivo, all treatments, given twice a day, showed equivalent efficacy in corneal epithelial healing. We conclude that the combined use of s-PRGF and HaNA as an adhesive biopolymer does not improve the efficacy of s-PRGF alone in the wound healing of corneal epithelial defects.

This paper discusses the development and validation of a rapid method for the reversed phase HPLC-UV quantification of biodegradable poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres co-loaded with two neuroprotective agents (dexamethasone and melatonin) (DX-MEL-MSs) to be intravitreally administered as a promising glaucoma treatment. The study was performed to validate two procedures that quantify the content of the two active substances entrapped into the polymer matrix during an encapsulation efficiency assay and the amount of drugs liberated over time during the in vitro release assay. The reversed-phase method allowed for the simultaneous determination of dexamethasone and melatonin, which were respectively detected at 240.5 and 222.7 nm. Chromatographic separation was performed using an Ascentis^® C18 HPLC Column (25 cm × 4.6 mm, 5 µm) with an isocratic mobile phase composed of methanol-water (70:30, v/v) with 1.0 mL min⁻¹ flow rate. The two procedures were validated analytically in terms of system suitability testing, specificity, linearity, precision, accuracy, sensitivity, and robustness. Both the validated procedures were applied to characterize DX-MEL-MSs and were found appropriate to quantify the drug quantities encapsulated and estimate their release profile over 10 days. The validation study designed in this work can be helpful for planning any other protocols that refer to the quantification of PLGA based drug delivery systems.

Glaucoma is a group of chronic irreversible neuropathies that affect the retina and the optic nerve. It is considered one of the leading causes of blindness in the world. Although it can be due to various causes, the most important modifiable risk factor is the elevated intraocular pressure (IOP). In this case, the treatment of choice consists of instilling antihypertensive formulations on the ocular surface. The chronicity of the pathology, together with the low bioavailability of the drugs that are applied on the ocular surface, make it necessary to instill the formulations very frequently, which is associated, in many cases, with the appearance of dry eye disease (DED). The objective of this work is the design of topical ocular formulations capable of treating glaucoma and, at the same time, preventing DED. For this, two liposome formulations, loaded with brimonidine or with travoprost, were Tadeveloped using synthetic phospholipids and enriched by the addition of compounds with osmoprotective activity. The proposed formulations not only presented physicochemical characteristics (size, pH, osmolarity, surface tension, and viscosity) and encapsulation efficiency values (EE% of 24.78% and ≥99.01% for brimonidine and travoprost, respectively) suitable for ocular surface administration, but also showed good tolerance in human corneal and conjunctival cell cultures, as well as an in vitro osmoprotective activity. The hypotensive effect of both liposomal formulations was evaluated in normotensive albino New Zealand rabbits, showing a faster and longer lasting reduction of intraocular pressure in comparison to the corresponding commercialized products used as control. According to these results, the hypotensive liposomal formulations combined with osmoprotective agents would result in a very promising platform for the treatment of glaucoma and the simultaneous protection of the ocular surface.

Compounded insulin eye drops were prepared at 1 IU/mL from commercially available subcutaneous insulin by dilution in saline solution or artificial tears. Physicochemical characterization and in vitro tolerance testing in human and conjunctival cells were followed by a 28-day short-term stability study under various conditions. The formulations were isotonic (280–300 mOsm/L), had a pH close to neutral (7–8), medium surface-tension values (<56 MN/m⁻¹), and low (≈1 mPa·s) and medium (≈5 mPa·s) viscosities (compounded normal saline solution and artificial tear-based preparation, respectively). These values remained stable for 28 days under refrigeration. Microbiological stability was also excellent. Insulin potency remained in the 90–110% range in the compounded formulations containing normal saline solution when stored at 2–8 °C for 28 days, while it decreased in those based on artificial tears. Although both formulations were well tolerated in vitro, the compounded insulin diluted in a normal saline solution exhibited better cell tolerance. Preliminary data in humans showed that insulin in saline solution was an effective and safe treatment for persistent corneal epithelial defects. Compounded insulin eye drops diluted in normal saline solution could, therefore, constitute an emergent therapy for the treatment of persistent corneal epithelial defects.

The prevalence of dry eye disease (DED) is rising globally, increasingly affecting young adults. Although artificial tears are commonly used, unfortunately they do not address the inflammatory component of the disease. For this reason, in patients whose symptoms persist despite the use of artificial tears, anti-inflammatory agents such as the immunosuppressant cyclosporine A (CyA) are clinically indicated to reduce the underlying inflammation in DED. In this study, a CyA-loaded liposomal formulations composed of analogous components to the lipid and aqueous layers of the natural tears (CyA-lipo) and dispersed in an aqueous solution of sodium hyaluronate (CyA-lipo-NaHa) has been developed. Formulations were analysed for physicochemical properties, and in vitro tolerance using human corneal and conjunctival cell lines over a short-term stability study at 25 °C and 2–8 °C. In vivo tolerance was assessed in rabbits, and therapeutic efficacy was evaluated in dogs diagnosed with DED. Average vesicles’ size resulted in 204.2 ± 4.3 nm and 198.7 ± 6.0 nm for CyA-lipo and CyA-lipo-NaHa respectively. Both formulations rendered osmolarity values close to 200 mOsm and surface tension values below 31 mN/m. The addition of sodium hyaluronate produced an increase of viscosity with values of 6.030 ± 0.316 mPa·s for CyA-lipo-NaHa and 0.950 ± 0.073mPa·s for CyA-lipo. Cell viability was above 80% in corneal and conjunctival cells, and no signs of ocular surface damage in rabbit were observed. In dogs, ocular surface parameters, Schirmer’s test values in particular, improved significantly after two months of treatment (< 13 mm/min before and higher than15 mm/min in all animals after treatment). This liposomal formulation demonstrates optimal physicochemical properties, biocompatibility, and therapeutic efficacy, supporting its potential as an optimised treatment for DED. Graphical Abstract

Background: Biodegradable microspheres (MSs) are good candidates for intravitreal administration in the posterior segment of the eye. MSs can be formulated as injectable preparations and administered using syringes with small-gauge needles. To allow proper injectability and avoid patients’ discomfort, the MS size should be as small as possible. However, the relationships between size, encapsulation efficiency, and drug-release behavior in these systems are widely recognized. Methods: In an attempt to optimize this requirement, we present here interesting dexamethasone (Dxm), vitamin E and human serum albumin (HSA) biodegradable MSs in the 2–20 µm range, elaborated by employing a version of the single-emulsion extraction–evaporation method. The method employed here renders MSs in the 2–40 µm range. The present work focuses on evaluating the smaller fraction, the 2–20 µm range, including physicochemical characterization, image analysis, dexamethasone release assays, cytotoxicity, and injectability evaluations. Results: The 2–20 µm fraction represents 25–30% of the entire population, has an average particle size of 18 µm, is spherical in shape, and has a Dxm loading between 38 and 50 µm per mg of MSs. In addition, a comparative evaluation between both fractions was carried out to assess whether relevant therapeutic differences can be expected. The in vitro release studies showed that MSs sized 2–20 µm, when vitamin E and HSA were included, have a low burst effect and, after that, a biphasic release profile during the first 4 weeks, with a long sustained Dxm release phase. This suggests that they might be better suited to maintain therapeutically anti-inflammatory concentrations of Dxm in the vitreous than the 20–40 µm MSs fraction, which also had a controlled Dxm release but in very reduced amounts after the first week. Conclusions: This 2–20 µm MS system might represent a success for its advantages over the existing alternatives in the marketplace, such as dexamethasone implants. Among others, due to the minimized risk associated with their administration that does not need ocular surgery, and consequently, the better the patient’s adherence is expected, there can be a medical preference for this system for its ease of use and possibility of personalizing the dose.