Development of dual drug loaded-hydrogel scaffold combining microfluidics and coaxial 3D-printing for intravitreal implantation

Abstract

Treating diabetic retinopathy (DR) effectively is challenging, aiming for high efficacy with minimal discomfort. While intravitreal injection is the current standard, it has several disadvantages. Implantable systems offer an alternative, less invasive, with long-lasting effects drug delivery system (DDS). The current study aims to develop a soft, minimally invasive, biodegradable, and bioadhesive material-based hydrogel scaffold to prevent common issues with implants. A grid-shaped scaffold was created using coaxial 3D printing (3DP) to extrude two bioinks in a single filament. The scaffold comprises an inner core of curcumin-loaded liposomes (CUR-LPs) that prepared by microfluidics (MFs) embedded in a hydrogel of hydroxyethyl cellulose (HEC), and an outer layer of hyaluronic acid-chitosan matrix with free resveratrol (RSV), delivering two Sirt1 agonists synergistically activating Sirt1 downregulated in DR. Optimized liposomes, prepared via MFs, exhibit suitable properties for retinal delivery in terms of size (<200 nm), polydispersity index (PDI) (<0.3), neutral zeta potential (ZP), encapsulation efficiency (∼97 %), and stability up to 4 weeks. Mechanical studies confirm scaffold elasticity for easy implantation. The release profiles show sustained release of both molecules, with different patterns related to different localization of the molecules. RSV released initially after 30 min with a total release more than 90 % at 336 h. CUR release starts after 24 h with only 4.78 % of CUR released before and gradually released thanks to its internal localization in the scaffold. Liposomes and hydrogels can generate dual drug-loaded 3D structures with sustained release. Microscopic analysis confirms optimal distribution of liposomes within the hydrogel scaffold. The latter resulted compatible in vitro with human retinal microvascular endothelial cells up to 72 h of exposition. The hydrogel scaffold, composed of hyaluronic acid and chitosan, shows promise for prolonged treatment and minimally invasive surgery.

Introduction

Diabetes is a prevalent global problem, with an estimated 12 % of the adult population aged 20–79 years-old worldwide expected to be affected by 2045. Diabetic retinopathy (DR), a frequent complication of diabetes, is projected to affect an increased population of 642 million by 2050 (Abou Taha et al., 2024). DR is defined as a microvascular and neurodegenerative disease involving oxidative stress and inflammatory conditions. Metabolic dysfunction related to lipids and glucose leads to accumulation of reactive oxygen species (ROS), leukocyte activation, angiogenesis, and microvascular complications (Leley et al., 2021). The gold standard treatment for this pathology is the intravitreal injection of anti-VEGF agents. However, numerous drawbacks are associated with this administration, including non-adherence and lack of persistence to therapy, poor patient compliance, the need for repeated injections, sustained elevation of intraocular pressure after injections, corneal oedema, and a high risk of endophthalmitis (Cui et al., 2019, Forrester et al., 2020, Liu et al., 2023). Despite the safety and high compliance of non-invasive methods such as topical administration, the drug bioavailability in the aqueous vitreous humor is generally less than 3 % after instillation (Sigford et al., 2015). This is primarily due to poor bioavailability of the molecules. This is caused by the presence of highly selective barriers, such as the corneal epithelium, conjunctiva, sclera, and the choriocapillaris, as well as the rapid drainage of the formulation into the nasolacrimal system, due to dynamic barriers, such as lacrimation, blinking, conjunctival hyperaemia, and conjunctival absorption into the systemic circulation (Farkouh et al., 2016, Naageshwaran et al., 2022). These factors make targeting drugs to the posterior eye a challenging task.

Implants have received much attention as they allow for extended drug release, reduce the need for repeated administration, increase patient compliance, they are suitable for sterilization, and provide a defined and proper concentration of the drug directly at the site of administration. An increasing number of implants are now available in the market. Among them, Vitrasert® was the first and is a non-biodegradable implant. This was part of the first generation of implants, along with Illuvien®, Retisert®, and Durasert®. The second generation includes biodegradable implants such as Ozurdex® (Cao et al., 2019, Jervis, 2017).

Compared to biodegradable implants, non-biodegradable implants have some drawbacks, as they require surgical insertion and removal, often through invasive procedures. Additionally, they can cause anterior chamber migration, leading to corneal oedema and corneal decompensation, as well as an increased incidence of post-operative cataracts (Khurana et al., 2014).

Biodegradable scaffolds do not require surgical removal; thus, despite their shorter lifespan compared to non-biodegradable ones, they can ensure greater patient comfort and compliance. Within this category, hydrogel implants offer the advantages of being softer, usefully flexible for repositioning, and causing less discomfort. The main purpose of the current study is to develop a highly biocompatible hydrogel scaffold, made of biodegradable, highly biocompatible and bioadhesive materials to prevent anterior chamber migration (Barth et al., 2016, Rafael et al., 2023, Tan et al., 2022). The hydrogel scaffolds were prepared using hyaluronic acid (HA) and chitosan (CS) by a 3D bioprinting approach.

HA is widely used in the formation of bioinks, and often functionalized to improve its mechanical properties and slow its degradation. HA is endogenous to the body and is non-immunogenic. Specifically, in the eye, it constitutes a major component of the vitreous body (at concentrations of 140–340 μg/ml) along with collagen and it has ligands for receptors found in many types of retinal cells, such as CD-44 (Mishra et al., 2023, Zhang et al., 2021). Despite the aim of formulating a hydrogel based on HA to be as compatible as possible with the target site for intravitreal implantation, its poor mechanical properties and rapid degradation in vivo are significant disadvantages and must be considered. The use of chitosan to prepare a polyelectrolyte complex (PEC) with HA is a widely used strategy in the literature (Barroso et al., 2019, Drozdova et al., 2022, Khoonkari et al., 2023, Maiz-Fernández et al., 2021). It serves as a secondary material aimed at forming an oppositely charged PEC with HA, thereby enhancing its mechanical properties, rendering HA mouldable, and increasing the bioadhesion of the system (Amato et al., 2020, Li et al., 2014). The combination of two materials is used to form bioink, but mostly both materials, in particular HA is modified to allow the printing. Our work proposes a formation of bioink without modification of polymers that resulted a challenge for printing approach. Moreover, no studies in literature investigate the combination of HA and CS for preparation of 3D printing hydrogel scaffold addressed to the intravitreal implantation. A challenge is the printability of this mixture reported as a difficult step. The aim to use CS lies in adhesive properties with various mucous tissues and, owing to its positive charge, can bind the negatively charged polymer network of vitreous humor, primarily due to the presence of heparan sulfate. Polymers with cationic properties could enhance tissue adhesion, thereby prolonging permanence and preventing the typical disadvantage of anterior chamber system migration associated with many implants. The use of coaxial 3D printing allows to the constitution of system for delivery of two compound at a time with different localization: one in the inner core and one in the shell. This approach resulted a strategy not yet investigated in the ocular field.

Lipid-based nanoparticles (NPs) have been investigated as a compelling strategy to enhance the solubility and bioavailability of drugs with low water solubility, regulate release kinetics, increase apparent solubility in water, boost drug loading, and reduce ocular clearance (Chaw et al., 2021, Moiseev et al., 2022). Among these, liposomes have garnered significant attention for ocular administration due to their pronounced biocompatibility and low cytotoxicity. Particularly, pegylated liposomes have demonstrated success in overcoming vitreal barriers, improving retinal targeting, stability, and retention in the retinal tissue (Tavakoli et al., 2020). Given the advantages associated with liposomes, this study aims to deliver a Sirt1 agonist molecule via a biodegradable implant. By combining two techniques, microfluidics (MFs) and coaxial 3DP, it becomes feasible to deliver two molecules with Sirt1 agonist properties simultaneously, enhancing synergistic effects and exploring potential differences in behaviour between the internal and external matrices, as recently investigated by Fratini et al., for wound healing treatments (Fratini et al., 2023). Coaxial 3DP offers the advantage of creating an internal core capable of housing a hydrogel embedded with liposomes, thus delivering, and protecting, one molecule inside, while also forming an external shell composed of HA/CHI matrix embedded with a second molecule. Importantly, MFs and 3DP are both environmentally friendly and economically viable approaches (Weaver et al., 2022).

Several studies demonstrate a correlation between the downregulation of Sirt1 and DR. This protein plays a cytoprotective role in ocular tissues such as the retina and the optic nerve. Downregulation leads to increased susceptibility of these tissues to degenerative disorders (Hammer et al., 2021). Polyphenolic compounds function as activators of Sirt1, enhancing its activity by reducing the Michaelis constant (Km) of its substrates, i.e. the substrate concentration at which an enzyme is half saturated, which leads to a small amount of substrate being required to saturate the enzyme. Recent studies indicate that compounds such as resveratrol, curcumin, berberine, quercetin, and others act as agonists of this protein (Wiciński et al., 2023). However, their therapeutic efficacy is hindered by low solubility in aqueous environments, limited bioavailability, degradation in various environments, and instability over time (Wiciński et al., 2023). Resveratrol (RSV) stands out as the most potent activator of Sirt 1in retinal cells, as it allosterically binds to Sirt1 and reduces the Michaelis constant of Sirt1 for acetylated substrate (Cao et al., 2020). Therefore, RSV has been designated for incorporation into the outer hydrogel matrix. Curcumin (CUR), a polyphenolic compound with Sirt1 activator properties, is extensively studied in the field of eye treatment and theranostics. A water-soluble fraction of CUR at a low concentration (5 μM) induces a cytoprotective effect in retinal cells exposed to oxidative stress (Nedzvetsky et al., 2021). CUR also serves as an anti-VEGF agent, beneficial for treating neovascularization in proliferative diabetic retinopathy (Jiang et al., 2023). Encapsulating CUR inside liposomes enhances its protection and stability (Ballacchino et al., 2021). When RSV and CUR are used together, they exhibit increased antidiabetic and antioxidant effects. The combination of RSV and CUR leads to a greater reduction in plasma glucose levels compared to either molecule alone. Additionally, as hyperglycaemia exacerbates oxidative stress by increasing reactive oxygen species (ROS) production, the combination of RSV and CURC elevates levels of glutathione (GSH) and superoxide dismutase (SOD) compared to individual molecules (Hussein and El-Maksoud, 2013). The development of a hydrogel scaffold prepared through a combination of microfluidics and coaxial 3DP, which simultaneously loads two molecules, represents a novel approach in the ocular field since no studies with the same aim are present in the literature until now. MFs was utilized to produce DMPC:DSPE-PEG:Chol liposomes encapsulating curcumin for sustained and controlled release in retinal tissues. Coaxial 3D bioprinting, on the other hand, was chosen to combine CUR-LPs with hydroxyethyl cellulose (HEC) and a hybrid hydrogel composed of HA and CS enriched with free RSV, achieving a burst release of the active pharmaceutical ingredient (API) and prompt control of the inflammatory state. The resulting scaffolds were characterized and analysed to confirm proper loading of the liposomes, efficient release of both curcumin and RSV from the implant. Ultimately, cytocompatibility in vitro was assessed by exposing the HS/CS hydrogel scaffold with human retinal microvascular endothelial cells (HREMCs) for 72 h in culture.

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Materials

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Sigma Aldrich (Germany). Cholesterol was purchased from TCI (Japan). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) was purchased from Lipoid GmBH (Germany). Ethanol (≥99,8 %), Methanol (≥99,9 %), acetonitrile (for HPLC, gradient grade, ≥ 99,9 %), Acetic acid (for HPLC, gradient grade, 99 %) and phosphate-buffered saline tablets (PBS, pH 7.4) were purchased from Sigma-Aldrich (Germany). Chitosan (CS) high molecular weight and hydroxyethyl-cellulose (HEC) were purchased from Sigma-Aldrich (Germany). Sodium Hyaluronate from Bacteria (HA) was purchased from TCI (Japan). CUR [hydroalcoholic extract from Curcuma Longa L., rhizome; purity 95.0 % by HPLC] and RSV [trans-3,4′,5-Trihydroxystilbene; hydroalcoholic extract from Polygonum cuspidatum, Siebold et Zucc., roots; purity 99.0 % by HPLC] was produced by Giellepi SpA (Seregno, Italy) and kindly gifted by Labomar SpA (Istrana, Italy).

Elide Zingale, Edward Weaver, Pietro Maria Bertelli, Imre Lengyel, Rosario Pignatello, Dimitrios A. Lamprou, Development of dual drug loaded-hydrogel scaffold combining microfluidics and coaxial 3D-printing for intravitreal implantation, International Journal of Pharmaceutics, Volume 665, 2024, 124700, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124700.