Purpose: Diabetic retinopathy (DR) affects about one third of the estimated 422 million people with diabetes mellitus and is a leading cause of vision-loss worldwide. It is associated with retinal neurovascular degeneration and studies indicated that systemic, subconjunctival or intravitreal injection of insulin may reduce the risk of DR onset and progression. However, insulin has a short half-life and the risk of hypoglycemia limits patients’ ability to take enough insulin systemically. Administration of drugs to the eye using eye drops and eye ointments is associated with patient incompliance and precorneal elimination, which results in less than 5% - usually less than 1% - of an applied dose reaches the intraocular tissue. Subconjunctival administration has shown promises in delivering drugs including insulin to the retina via sclera; however, the existences of the blood retinal barrier (BRB), and conjunctival and choroidal vasculature clearances limit sufficient passage of drugs from sclera to retina. Subconjunctival administration of drug loaded nanocarriers may enhance the retention time of the drug in the eye, help to bypass the BRB, and release the loaded drug in a controlled manner. Therefore, the purpose of this project is to develop thermoresponsive and biodegradable nanogels for aqueous loading and sustained release of insulin after subconjunctival injection. Successful completion of the project will have a significant impact in the prevention and treatment of DR.
Methods: Insulin loaded surfactant free, thermoresponsive, biodegradable nanogels, based on Dextran-lactate-HEMA macromer and N-isopropylacrylamide (NIPAAM) monomer were synthesized at 45 °C using Irgacure® 2959 as an UV initiator. To render the nanogels negatively charged, 10 mol% of acrylic acid with respect to NIPAAM was added to the reaction mixture. The nanoparticles were characterized with respect to size, zeta-potential, yield, and insulin loading efficiency. The effect of initiator concentration and UV exposure time on nanoparticle size and yield was thoroughly investigated. The in vitro release kinetics of insulin from the nanogels was optimized by varying the chain length of the lactate spacer on the macromer (DP), the degree of substitution of lactate-HEMA on the dextran segment of the macromer (DS), and the macromer to NIPAAM ratio. The released insulin was quantified by a developed UPLC method. The toxicity of the nanoparticles synthesized at different macromer to crosslinker ratios was assessed in ARPE-19 cells by an MTT assay, after incubating the nanogels in cell culture media without FBS at 37 °C for 0, 1, 3, 7, 14, 21 and 28 days. Currently, we are quantifying the amount insulin in the different ocular tissues, including aqueous humor, vitreous humor, lens, cornea, sclera, ciliary body and iris, choroid and retina, which were collected 1, 3 and 7 days after subconjunctival injection of the insulin-loaded nanogels to the left eyes of SD rats (5/group). The right eyes were injected with vehicle (PBS, pH 7.4) to serve as internal sham controls. Rats injected with equivalent amounts of free insulin and rats without any injection served as positive and naïve controls, respectively. A sensitive LC-MS/MS method is developed for insulin quantification using bovine insulin as an internal standard.
Results: Negatively charged, insulin loaded, thermoresponsive, and biodegradable nanogels with insulin loading efficiency of > 98% and yield > 80% were obtained. The sizes of the nanoparticles are 70 – 200 nm depending on the nanogel composition and temperature. When the initiator concentration was increased from 0.1% to 1%, the yield increased from about 60% to 90% and the nanoparticle size increased from about 70 nm to 160 nm. At 1% initiator concentration, the reaction reached into completion in less than 15 min of UV exposure. Insulin release from the nanogels was highly dependent on the DP/DS of the macromer as well as the macromer to monomer ratio and drug release for more than 7 days was attained in vitro. The nanogels as well as their degradation products, obtained by incubating the nanogels in cell culture media without FBS at 37 °C for 28 days, showed no sign of toxicity in ARPE-19 cells. The nanogels may release the loaded insulin in a controlled fashion in vivo.
Conclusion: The obtained thermoresponsive, biodegradable nanogels showed no sign of toxicity and released the loaded insulin for over 7 days, in vitro. Provided that the expected in vivo insulin pharmacokinetic are obtained, the nanogels will have a significant impact in the prevention and treatment of DR.
Cameron V. Fili– University of Tennessee Health Science Center
Sangyoon Kim– Graduate Research Assistant, University of Tennessee Health Science Center, Memphis, Tennessee
Jingzhao Tang– University of Tennessee Health Science Center
David J. Hamilton– University of Tennessee Health Science Center
Tao L. Lowe– University of Tennessee Health Science Center