Development of injectable, resorbable drug-releasing copolymer scaffolds for minimally invasive sustained ophthalmic therapeutics

Abstract

Copolymers based on N-isopropylacrylamide (NIPAAm), acrylic acid N-hydroxysuccinimide (NAS) and varying concentrations of acrylic acid (AA) and acryloyloxy dimethyl-γ-butyrolactone (DBA) were synthesized to create thermoresponsive, resorbable copolymers for minimally invasive drug and/or cell delivery to the posterior segment of the eye to combat retinal degenerative diseases. Increasing DBA content was found to decrease both copolymer water content and lower critical solution temperature. The incorporation of NAS provided an amine-reactive site, which can be exploited for facile conjugation of bioactive agents. Proton nuclear magnetic resonance analysis revealed the onset of hydrolysis-dependent opening of the DBA lactone ring, which successfully eradicated copolymer phase transition properties and should allow the gelled polymer to re-hydrate, enter systemic circulation and be cleared from the body without the production of degradation byproducts. Hydrolytic ring opening occurs slowly, with over 85% copolymer mass remaining after 130 days of incubation in 37 °C phosphate buffered saline. These slow-degrading copolymers are hypothesized to be ideal delivery vehicles to provide minimally invasive, sustained, localized release of pharmaceuticals within the posterior segment of the eye to combat retinal degenerative diseases.

Introduction

Efficient delivery of pharmaceuticals to the back of the eye is one of the most significant unmet needs of visual health care. Numerous pharmaceuticals show promise for the treatment of posterior segment ocular conditions, including vascular endothelial growth factor (VEGF) antagonists capable of minimizing ocular neovascularization, corticosteroids to combat retinal edema and other promising compounds such as antioxidants and anti-hypertensive drugs [1]. However, conventional drug delivery modalities are inefficient for delivering therapeutically relevant doses of pharmaceuticals to affected tissues in the back of the eye. Delivery of drugs to the posterior segment is made difficult by the isolated nature of the eye, which is separated from systemic circulation by blood ocular barriers, including the blood retinal barrier (BRB) and blood aqueous barrier (BAB) [2]. Furthermore, the eye is a segmented structure with numerous barriers to delivery and effective clearance mechanisms efficiently eliminating pharmaceuticals that successfully reach the posterior segment [2]. Topically applied drugs can enter the anterior chamber by crossing the cornea, or through the conjunctiva and sclera [2] or via the systemic circulation, but must cross the BAB. Clearance from the anterior chamber occurs via aqueous turnover, or by re-absorption into systemic circulation [2]. The half-life of a typical drug within the anterior chamber is ∼1 h; however, this varies depending on the properties of the pharmaceutical [3]. Drugs can be introduced into the posterior segment, which houses the light-sensitive retina, through systemic circulation by crossing the BRB, through non-corneal permeation into the uvea, or by direct injection into the vitreous [2], with the latter being the most efficient method of delivering drugs to the posterior eye. Drug clearance from the posterior segment occurs through either the anterior or posterior route; the anterior route involves diffusion across the vitreous and elimination via uveal blood flow and aqueous turnover, whereas elimination via the posterior route requires permeation through the BRB [2].

As a result of the numerous barriers, the effective clearance routes and the segmented nature of the eye, delivery of drugs to the posterior segment is particularly challenging. Topically applied eye drops typically result in less than 5% uptake into the anterior chamber and negligible amounts entering the back of the eye [2]. Furthermore, only an estimated 1–2% of a systemically applied dose crosses the restrictive ocular barriers [4]. Therefore, high systemic doses are required to achieve therapeutic concentrations of drug within the posterior segment of the eye [5]. Additionally, many new pharmaceuticals are protein-based and are therefore not suitable for oral delivery as they are rapidly broken down and denatured in the digestive system [6]. Direct injection into the vitreous cavity is a highly efficient technique to introduce therapeutically relevant doses of drug into the vitreous body and retinal tissues while minimizing off-target exposure [5]. However, due to the previously mentioned clearance mechanisms, frequent injections (often every 4–6 weeks) are required to maintain therapeutically relevant concentrations [7]. While intravitreal injections are an acceptable means of delivery, in addition to being inconvenient for the physician and patient, frequent injections are associated with increased risk of complications such as endophthalmitis, cataract formation, vitreous hemorrhage, retinal detachment and patient discomfort [5], [8]. Therefore, approaches that safely utilize the intravitreal route to provide sustained localized delivery of therapeutic concentrations of drug and do not require frequent perforation of the eye wall represent an exciting potential to treat numerous debilitating ocular conditions.

We hypothesized that poly(N-isopropylacrylamide) (PNIPAAm), a thermally sensitive intelligent polymer, which undergoes a rapid, reversible phase transition from liquid to gel when heated above a lower critical solution temperature (LCST) of ∼32 °C, would serve as an ideal delivery scaffold for posterior segment ocular therapy [9]. PNIPAAm’s sub-physiologic LCST allows a liquid polymer/drug solution to be injected directly into the vitreal cavity, wherein a thermally induced phase transition drives the formation of a solid drug depot capable of providing sustained, localized therapy. However, as PNIPAAm is a non-degrading polymer, its introduction into the isolated vitreous would result in its persistence within the eye for the lifetime of the patient, unless surgically removed. Therefore, in an attempt to design clinically relevant materials, there has been an emphasis in recent years on developing degradable or resorbable formulations of PNIPAAm that maintain thermal sensitivity but promote the eventual clearance from the body. Neradovic et al. designed NIPAAm-based copolymers containing hydrolyzable lactate ester groups [10], [11]. Hydrolysis of hydrophobic side groups results in an increase in copolymer LCST, which, if raised above body temperature, allows the copolymer to undergo a reverse phase transition and revert to a hydrated liquid state, allowing uptake into the systemic circulation and clearance from the body via the kidneys. Ma et al. developed bioabsorbable NIPAAm copolymers possessing strong mechanical properties through copolymerization with methacrylate-polylactide (MAPLA) and hydroxyethyl methacrylate (HEMA) [12]. Yoshida et al. reported the synthesis of NIPAAm-based copolymers crosslinked with degradable poly(amino acids) [13]. Cui et al. demonstrated that through incorporation of dimethyl-γ-butyrolactone acrylate (DBA), a hydrolysis-dependent ring opening of the DBA lactone side group could result in an increase in the LCST above body temperature, allowing the polymer to re-solubilize and be cleared from the body without the formation of degradation byproducts [14], [15]. Furthermore, it was found that copolymers of NIPAAm and DBA yielded slow degrading scaffolds with degradation periods of roughly one year required to increase copolymer LCST above body temperature [14]. Based on these results, we propose that copolymers consisting of NIPAAm and DBA would serve as ideal delivery scaffolds for posterior segment therapeutics capable of providing minimally invasive, long-term localized drug release that will increase the time between injections, and upon exhaustion of the drug, hydrolytic degradation will promote clearance from the eye without the need for surgical intervention. However, these polymers lack synthetic flexibility and have limited potential for bioconjugation and cellular adhesion, which may be useful for the subsequent incorporation of cell adhesion peptides for the development of minimally invasive cell carriers capable of delivering cell-based payloads to the subretinal tissues to combat retinal degeneration. Therefore, in this work, we have synthesized a series of synthetically flexible copolymers based on NIPAAm, DBA, acrylic acid (AA) and acrylic acid N-hydroxysuccinimide (NAS). AA was incorporated to balance the hydrophilic and hydrophobic content of the copolymers and control the LCST while the inclusion of NAS was designed to provide a site capable of facile conjugation with biologically relevant molecules such as drugs or cell adhesive peptides. NAS functionality provides a site for copolymer/drug conjugation, which can theoretically be exploited to obtain controlled release through targeted destruction of copolymer/drug linkage, although this was not utilized in this work. Copolymer synthesis as well as physical, chemical and biological characterization are described herein.