Oleogels for the ocular delivery of epalrestat: formulation, in vitro, in ovo, ex vivo and in vivo evaluation.

The ocular administration of lipophilic and labile drugs such as epalrestat, an aldose reductase inhibitor with potential for diabetic retinopathy treatment, demands the development of topical delivery systems capable of providing sufficient ocular bioavailability. The aim of this work was to develop non-aqueous oleogels based on soybean oil and gelators from natural and sustainable sources (ethyl cellulose, beeswax and cocoa butter) and to assess their reproducibility, safety and efficiency in epalrestat release and permeation both ex vivo and in vivo. Binary combinations of gelators at 10% w/w resulted in solid oleogels (oleorods), while single gelator oleogels at 5% w/w remained liquid at room temperature, with most of the oleogels displaying shear thinning behavior. The oleorods released up to 4 µg epalrestat per mg of oleorod in a sustained or burst pattern depending on the gelator (approx. 10% dose in 24 h). The HET-CAM assay indicated that oleogel formulations did not induce ocular irritation and were safe for topical ocular administration. Corneal and scleral ex vivo assays evidenced the permeation of epalrestat from the oleorods up to 4 and 2.5 µg/cm2 after six hours, respectively. Finally, the capacity of the developed oleogels to sustain release and provide significant amounts of epalrestat to the ocular tissues was demonstrated in vivo against aqueous-based niosomes and micelles formulations loaded with the same drug concentration. Overall, the gathered information provides valuable insights into the development of oleogels for ocular drug delivery, emphasizing their safety and controlled release capabilities, which have implications for the treatment of diabetic neuropathy and other ocular conditions.

Editorial for the Special Issue Titled "Design of Polymeric Hydrogels Biomaterials".

Hydrogels have attracted great interest in the biomedical applications field in recent years [...].

A sterilizable platform based on crosslinked xanthan gum for controlled-release of polymeric micelles: Ocular application for the delivery of neuroprotective compounds to the posterior eye segment.

TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate) polymeric micelles show interesting properties for ocular administration thanks to their solubilization capability, nanometric size and tissue penetration ability. However, micelles formulations are generally characterized by low viscosity, poor adhesion and very short retention time at the administration site. Therefore, the idea behind this work is the preparation and characterization of a crosslinked film based on xanthan gum that contains TPGS micelles and is capable of controlling their release. The system was loaded with melatonin and cyclosporin A, neuroprotective compounds to be delivered to the posterior eye segment. Citric acid and heating at different times and temperatures were exploited as crosslinking approach, giving the possibility to tune swelling, micelles release and drug release. The biocompatibility of the platform was confirmed by HET-CAM assay. Ex vivo studies on isolated porcine ocular tissues, conducted using Franz cells and two-photon microscopy, demonstrated the potential of the xanthan gum-based platform and enlightened micelles penetration mechanism. Finally, the sterilization step was approached, and a process to simultaneously crosslink and sterilize the platform was developed.

Dexamethasone phosphate and penetratin co-eluting contact lenses: a strategy to enhance ocular drug permeability.

Contact lenses (CLs) have been suggested as drug delivery platforms capable of increasing the drug residence time on the cornea and therefore its bioavailability. However, when targeting the posterior segment of the eye, the drug released from CLs still encounters the barrier effect of the ocular tissues, which considerably reduces the efficacy of administration. This work aims at the development of CLs able to simultaneously deliver an anti-inflammatory drug (dexamethasone sodium phosphate) and a cell-penetrating peptide (penetratin), the latter acting as a drug carrier across the tissues. Hydroxyethyl methacrylate (HEMA)-based hydrogels were functionalized with acrylic acid (AAc) and/or aminopropyl methacrylamide (APMA) to serve as CL materials with increased affinity for the drug and peptide. APMA-functionalized hydrogels sustained the dual release for 8 h, which is compatible with the wearing time of daily CLs. Hydrogels demonstrated suitable light transmittance, swelling capacity and in vitro biocompatibility. The anti-inflammatory activity of the drug was not compromised by the presence of the peptide nor by sterilization. The ocular distribution of the drug after 6 h of CL wearing was evaluated in vivo in rabbits and revealed that the amount of drug in the cornea and aqueous humor significantly increased when the drug was co-delivered with penetratin.

Contact lenses that transform gold into nanoparticles for prophylaxis of light-related events and photothermal therapy.

This work describes for first time how anisotropic gold nanoparticles (AuNPs) can be spontaneously formed inside preformed contact lenses (CLs) avoiding the use of additional reductant agents (reagent-free) through a precise tunning of the monomeric composition, the saline concentration, and the application of steam heat sterilization. Protocols to generate AuNPs in solution using inorganic or small organic reductants are widely available. Differently, gold precursors interactions with polymer networks have been overlooked and, thus, the interest of chemically cross-linked hydrogels as organic reductants is still to be elucidated. In the ocular field, incorporation of AuNPs to CLs may expand their applications in prophylaxis, therapy and diagnosis. To carry out the work, a variety of hydrogels and commercially available CLs were incubated with gold salt solution without any other chemical reagent. AuNPs formation was monitored by changes in localized surface plasmon resonance (LSPR) bands and quantifying the gold sorbed. Only silicone hydrogels induced AuNPs formation at room temperature in few days; methacrylic acid red-shifted the LSPR band (550-600 nm), while monomers bearing F hindered the reduction. Storage of hydrogels in the gold precursor solution allowed a gradual formation of anisotropic AuNPs, which could be stopped at any time by washing the hydrogel with water. The developed CLs behave as efficient filters against highly penetrant light and also exhibit photoresponsiveness as demonstrated as rapid (10 s), focused mild hyperthermia when irradiated with green, red and NIR lasers.

Mucoadhesive gellan gum-based and carboxymethyl cellulose -based hydrogels containing gemcitabine and papain for bladder cancer treatment.

Local treatment of bladder cancer faces several limitations such as short residence time or low permeation through urothelium tissue. The aim of this work was to develop patient-friendly mucoadhesive gel formulations combining gemcitabine and the enzyme papain for improved intravesical chemotherapy delivery. Hydrogels based on two different polysaccharides, gellan gum and sodium carboxymethylcellulose (CMC), were prepared with either native papain or papain nanoparticles (nanopapain) to explore for the first time their use as permeability enhancers through bladder tissue. Gel formulations were characterized regarding enzyme stability, rheological behavior, retention on bladder tissue and bioadhesion, drug release properties, permeation capacity, and biocompatibility. After 90 days of storage, the enzyme loaded in the CMC gels retained up to 83.5 ± 4.9 % of its activity in the absence of the drug, and up to 78.1 ± 5.3 with gemcitabine. The gels were mucoadhesive and the enzyme papain showed mucolytic action, which resulted in resistance against washing off from the urothelium and enhanced permeability of gemcitabine in the ex vivo tissue diffusion tests. Native papain shortened lag-time tissue penetration to 0.6 h and enhanced 2-fold drug permeability All formulations demonstrated pseudoplastic behavior and no irritability. Overall, the developed formulations have potential as an upgraded alternative to intravesical therapy for bladder cancer treatment.

Nanostructured Lipid Carriers Aimed to the Ocular Delivery of Mangiferin: In Vitro Evidence.

Although mangiferin (MGN) is a natural antioxidant that could be a good candidate for the treatment of ocular diseases, its use in ophthalmology is strongly compromised due to its high lipophilicity. Its encapsulation in nanostructured lipid carriers (NLC) seems to be an interesting strategy for improving its ocular bioavailability. As reported in our previous work, MGN-NLC showed high ocular compatibility and fulfilled the nanotechnological requirements needed for ocular delivery. The aim of the present work was to investigate, in vitro and ex vivo, the capability of MGN-NLC to act as a potential drug delivery system for MGN ocular administration. The data obtained in vitro on arising retinal pigment epithelium cells (ARPE-19) did not show cytotoxic effects for blank NLC and MGN-NLC; likewise, MGN-NLC showed the maintenance of the antioxidant role of MGN by mitigating ROS (Reactive Oxygen Species) formation and GSH (glutathione) depletion induced by H2O2. In addition, the capacity of MGN-released to permeate through and accumulate into the ocular tissues was confirmed ex vivo using bovine corneas. Finally, the NLC suspension has been formulated as a freeze-dried powder using mannitol at a concentration of 3% (w/v) in order to optimize its storage for long periods of time. All this evidence suggests a potential application of MGN-NLC in the treatment of oxidative stress-related ocular diseases.

In vitro-in vivo correlation of drug release profiles from medicated contact lenses using an in vitro eye blink model.

There is still a paucity of information on how in vitro release profiles from drug-loaded contact lenses (CLs) recorded in 3D printed eye models correlate with in vivo profiles. This work aims to evaluate the release profiles of two drug-loaded CLs in a 3D in vitro eye blink model and compare the obtained results with the release in a vial and the drug levels in tear fluid previously obtained from an animal in vivo study. In vitro release in the eye model was tested at two different flow rates (5 and 10 µL/min) and a blink speed of 1 blink/10 s. Model CLs were loaded with two different drugs, hydrophilic pravastatin and hydrophobic resveratrol. The release of both drugs was more sustained and lower in the 3D eye model compared to the in vitro release in vials. Interestingly, both drugs presented similar release patterns in the eye model and in vivo, although the total amount of drugs released in the eye model was significantly lower, especially for resveratrol. Strong correlations between percentages of pravastatin released in the eye model and in vivo were found. These findings suggest that the current 3D printed eye blink model could be a useful tool to measure the release of ophthalmic drugs from medicated CLs. Nevertheless, physiological parameters such as the composition of the tear fluid and eyeball surface, tear flow rates, and temperature should be optimized in further studies.

Phosphorylcholine-Based Contact Lenses for Sustained Release of Resveratrol: Design, Antioxidant and Antimicrobial Performances, and In Vivo Behavior.

Design of advanced contact lenses (CLs) demands materials that are safe and comfortable for the wearers and that preserve the normal eye microbiota, avoiding chronic inflammation and biofilm development. This work aimed to combine the natural antibiofouling phosphorylcholine and the antioxidant and prebiotic resveratrol as integral components of CLs that may have the additional performance of preventing oxidative-stress related eye diseases. Different from previous uses of 2-methacryloyloxyethyl phosphorylcholine (MPC) as coating, we explored the feasibility of adding MPC at high proportions as a comonomer of 2-hydroxyethyl methacrylate (HEMA)-based hydrogels while still allowing for the loading of the hydrophobic resveratrol. Homogeneous distribution of MPC along the hydrogel depth (confirmed by Raman spectroscopy) notably increased solvent uptake and the proportion of free water while it decreased Young's modulus. Relevantly, MPC did not hinder the uptake of resveratrol by CLs (>10 mg/g), which indeed showed network/water partition coefficients of >100. Protocols for CLs sterilization and loading of resveratrol under aseptic conditions were implemented, and the effects of tear proteins on resveratrol release rate were investigated. CLs sustained resveratrol release for more than 24 h in vitro, and sorption of albumin onto the hydrogel, although attenuated by MPC, slowed down the release. The combination of MPC and resveratrol reduced P. aeruginosa and S. aureus growth as tested in a novel hydrogel disk-agar interface biofilm growth setup. The developed CLs showed excellent anti-inflammatory properties and biocompatibility in in ovo and rabbit tests and provided higher and more prolonged levels of resveratrol in tear fluid, which favored resveratrol biodistribution in anterior and posterior eye segments compared to eye drops. Correlations between the release profiles of resveratrol in vitro and in vivo were assessed. Relevantly, the CLs preserved the antioxidant properties of resveratrol during the entire 8 h of wearing. In sum, CLs prepared with high proportion in MPC may help address safety and comfort requirements while having drug releasing capabilities.

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests.

Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to -17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9-27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10-15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.

Embryonic nutritional hyperglycemia decreases cell proliferation in the zebrafish retina.

Diabetic retinopathy (DR) is one of the leading causes of blindness in the world. While there is a major focus on the study of juvenile/adult DR, the effects of hyperglycemia during early retinal development are less well studied. Recent studies in embryonic zebrafish models of nutritional hyperglycemia (high-glucose exposure) have revealed that hyperglycemia leads to decreased cell numbers of mature retinal cell types, which has been related to a modest increase in apoptotic cell death and altered cell differentiation. However, how embryonic hyperglycemia impacts cell proliferation in developing retinas still remains unknown. Here, we exposed zebrafish embryos to 50 mM glucose from 10 h postfertilization (hpf) to 5 days postfertilization (dpf). First, we confirmed that hyperglycemia increases apoptotic death and decreases the rod and Müller glia population in the retina of 5-dpf zebrafish. Interestingly, the increase in cell death was mainly observed in the ciliary marginal zone (CMZ), where most of the proliferating cells are located. To analyze the impact of hyperglycemia in cell proliferation, mitotic activity was first quantified using pH3 immunolabeling, which revealed a significant decrease in mitotic cells in the retina (mainly in the CMZ) at 5 dpf. A significant decrease in cell proliferation in the outer nuclear and ganglion cell layers of the central retina in hyperglycemic animals was also detected using the proliferation marker PCNA. Overall, our results show that nutritional hyperglycemia decreases cellular proliferation in the developing retina, which could significantly contribute to the decline in the number of mature retinal cells.

Contact lenses for pravastatin delivery to eye segments: Design and in vitro-in vivo correlations.

Oral administration of cholesterol-lowering statins, HMG-CoA reductase inhibitors, is associated with beneficial effects on eye conditions. This work aims to design contact lenses (CLs) that can sustainedly deliver pravastatin and thus improve the ocular efficacy while avoiding systemic side effects of statins. Bioinspired hydrogels were prepared with monomers that resemble hydrophobic (ethylene glycol phenyl ether methacrylate) and amino (2-aminoethyl methacrylamide hydrochloride) functionalities of the active site of HMG-CoA. Best performing CLs loaded >6 mg/g, in vitro fulfilled the release demands for daily wearing, and showed anti-inflammatory activity (lowering TNF-α). High hydrostatic pressure sterilization preserved the stability of both the drug and the hydrogel network. Ex vivo tests revealed the ability of pravastatin to accumulate in cornea and sclera and to penetrate through transscleral route. In vivo tests (rabbits) confirmed that, compared to eye drops and for the same dose, CLs provided significantly higher pravastatin levels in tear fluid within 1 to 7 h of wearing. Moreover, after 8 h wearing pravastatin was present in cornea, sclera, aqueous humour and vitreous humour. Strong correlations between percentages of drug released in vitro and in vivo were found. Effects of volume and proteins on release rate and Levy plots were identified.

Design of Nanotechnological Carriers for Ocular Delivery of Mangiferin: Preformulation Study.

(1) Background: Mangiferin (MGN) is a natural compound, showing anti-inflammatory and antioxidant activities for the potential treatment of eye diseases. The poor physicochemical features of MGN (low solubility and high instability) justify its nanoencapsulation into nanostructured lipid carriers (NLC) to improve its ocular bioavailability. (2) Methods: Firstly, MGN-NLC were prepared by the high shear homogenization coupled with the ultrasound (HSH-US) method. Finally, unloaded and MGN-loaded NLC were analyzed in terms of ocular tolerance. (3) Results: MGN-NLC showed good technological parameters suitable for ocular administration (particle size below 200 nm). The ORAC assay was performed to quantify the antioxidant activity of MGN, showing that the antioxidant activity of MGN-NLC (6494 ± 186 µM TE/g) was higher than that of the free compound (3521 ± 271 µM TE/g). This confirmed that the encapsulation of the drug was able to preserve and increase its activity. In ovo studies (HET-CAM) revealed that the formulation can be considered nonirritant. (4) Conclusions: Therefore, NLC systems are a promising approach for the ocular delivery of MGN.

Resveratrol-Loaded Hydrogel Contact Lenses with Antioxidant and Antibiofilm Performance.

Contact lenses (CLs) are prone to biofilm formation, which may cause severe ocular infections. Since the use of antibiotics is associated with resistance concerns, here, two alternative strategies were evaluated to endow CLs with antibiofilm features: copolymerization with the antifouling monomer 2-methacryloyloxyethyl phosphorylcholine (MPC) and loading of the antioxidant resveratrol with known antibacterial activity. MPC has, so far, been used to increase water retention on the CL surface (Proclear® 1 day CLs). Both poly(hydroxyethyl methacrylate) (HEMA) and silicone hydrogels were prepared with MPC covering a wide range of concentrations (from 0 to 101 mM). All hydrogels showed physical properties adequate for CLs and successfully passed the hen's egg-chorioallantoic membrane (HET-CAM) test. Silicone hydrogels had stronger affinity for resveratrol, with higher loading and a slower release rate. Ex vivo cornea and sclera permeability tests revealed that resveratrol released from the hydrogels readily accumulated in both tissues but did not cross through. The antibiofilm tests against Pseudomonas aeruginosa and Staphylococcus aureus evidenced that, in general, resveratrol decreased biofilm formation, which correlated with its concentration-dependent antibacterial capability. Preferential adsorption of lysozyme, compared to albumin, might also contribute to the antimicrobial activity. In addition, importantly, the loading of resveratrol in the hydrogels preserved the antioxidant activity, even against photodegradation. Overall, the designed hydrogels can host therapeutically relevant amounts of resveratrol to be sustainedly released on the eye, providing antibiofilm and antioxidant performance.

Atorvastatin-Eluting Contact Lenses: Effects of Molecular Imprinting and Sterilization on Drug Loading and Release.

Statins are receiving increasing attention in the ophthalmic field. Their activity as 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors is clinically used to regulate cholesterol levels and leads to pleiotropic effects, which may help in the management of diabetes-related ocular pathologies. This work aims to design bioinspired contact lenses (CLs) with an affinity for atorvastatin by mimicking the active site of HMG-CoA reductase. Sets of imprinted and nonimprinted 2-hydroxyethyl methacrylate (HEMA) hydrogels were synthesized, varying the contents in functional monomers that bear chemical groups that resemble those present in HMG-CoA reductase, namely, ethylene glycol phenyl ether methacrylate (EGPEM), 2-aminoethyl methacrylate hydrochloride (AEMA), and N-(3-aminopropyl) methacrylamide hydrochloride (APMA). The hydrogels were characterized in terms of suitability as CLs (solvent uptake, light transmission, mechanical properties, and biocompatibility) and capability to load and release atorvastatin. Three sterilization protocols (steam heat, gamma radiation, and high hydrostatic pressure) were implemented and their effects on hydrogel properties were evaluated. Copolymerization of AEMA and, particularly, APMA endowed the hydrogels with a high affinity for atorvastatin (up to 11 mg/g; KN/W > 200). Only high hydrostatic pressure sterilization preserved atorvastatin stability and hydrogel performance. Permeability studies through the porcine cornea and sclera tissues revealed that the amount of atorvastatin accumulated in the cornea and sclera could be effective to treat ocular surface diseases.

Anti-biofilm multi drug-loaded 3D printed hearing aids.

Over 5% of the world's population has disabling hearing loss, which affects approximately one third of individuals over 65 years. Hearing aids are commonly used in this population group, but prolonged use of these devices may cause ear infections. We describe for the first time, the use of 3D printing to fabricate hearing aids loaded with two antibiotics, ciprofloxacin and fluocinolone acetonide. Digital light processing 3D printing was employed to manufacture hearing aids from two polymer resins, ENG hard and Flexible. The inclusion of the antibiotics did not affect the mechanical properties of the hearing aids. All multi-drug-loaded devices exhibited a hydrophilic surface, excellent blood compatibility and anti-biofilm activity against P. aeruginosa and S. aureus. Hearing aids loaded with ciprofloxacin (6% w/w) and fluocinolone acetonide (0.5% w/w) sustained drug release for more than two weeks and inhibited biofilm formation on the surface of the devices and bacteria growth in the surrounding medium. In summary, this work highlights the potential of vat photopolymerization 3D printing as a versatile manufacturing approach to fabricate high-fidelity patient-specific medical devices with anti-bacterial properties.

Anandamide-nanoformulation obtained by electrospraying for cardiovascular therapy.

Anandamide (AEA), an endogenous cannabinoid, has a relevant antihypertensive effect. However, its cardioprotective role has been barely explored due to unfavorable physico-chemical properties and, sometimes, undesirable psychoactive effects. In this context, drug encapsulation in nanocarriers could overcome the limitations associated with the administration of AEA in free form. The aim of the present study was to encapsulate AEA in poly-ε-caprolactone/Pluronic® F127 nanoparticles (AEA/PCL/PF127 NPs) by means of electrospraying, to characterize their physico-chemical properties and cytocompatibility and to evaluate their effect in an in vivo model of cardiovascular remodeling caused by hypertension. AEA/PCL/PF127 NPs were characterized in terms of morphology, size, polydispersity, Z-potential, hydrophilicity, thermal and spectroscopic properties. Also, the encapsulation and loading efficiencies and in vitro release of AEA were analyzed. AEA/PCL/PF127 NPs (700-1000 nm) showed adequate cytocompatibility. For the cardiovascular remodeling studies, normotensive (WKY) and hypertensive (SHR) male rats were treated or not with AEA/PCL/PF127 NPs (5 mg/Kg, intraperitoneal injection) weekly for 1 month. Inflammatory markers and hemodynamic, structural and cardiac functional parameters were monitored. In SHR, the treatment with AEA/PCL/PF127 NPs reversed all altered cardiovascular markers and parameters (p < 0.05). Overall, nanoformulated AEA obtained by electrospraying proved to be effective for the treatment of hypertension and its comorbidities, especially cardiovascular remodeling.

Bioinspired hydrogels for drug-eluting contact lenses.

Efficient ocular drug delivery that can overcome the challenges of topical application has been largely pursued. Contact lenses (CLs) may act as light-transparent cornea/sclera bandages for prolonged drug release towards the post-lens tear fluid, if their composition and inner architecture are fitted to the features of the drug molecules. In this review, first the foundations and advantages of using CLs as ocular drug depots are revisited. Then, pros and cons of common strategies to prepare drug-loaded CLs are analyzed on the basis of recent examples, and finally the main section focuses on bioinspired strategies that can overcome some limitations of current designs. Most bioinspired strategies resemble a reverse engineering process to create artificial receptors for the drug inside the CL network by mimicking the human natural binding site of the drug. Related bioinspired strategies are being also tested for designing CLs that elute comfort ingredients mimicking the blinking-associated renewal of eye mucins. Other bioinspired approaches exploit the natural eye variables as stimuli to trigger drug release or take benefit of bio-glues to specifically bind active components to the CL surface. Overall, biomimicking approaches are being revealed as valuable tools to fit the amounts loaded and the release profiles to the therapeutic demands of each pathology. STATEMENT OF SIGNIFICANCE: Biomimetic and bioinspired strategies are remarkable tools for the optimization of drug delivery systems. Translation of the knowledge about how drugs interact with the natural pharmacological receptor and about components and dynamics of anterior eye segment may shed light on the design criteria for obtaining efficient drug-eluting CLs. Current strategies for endowing CLs with controlled drug release performance still require optimization regarding amount loaded, drug retained in the CL structure during storage, regulation of drug release once applied onto the eye, and maintenance of CL physical properties. All these limitations may be addressed through a variety of recently growing bioinspired approaches, which are expected to pave the way of medicated CLs towards the clinics.