Master of Science
Dr. Qingguo Xu
Corneas are the most commonly transplanted tissue worldwide, with approximately 80,000 transplantations performed in the United States in 2018 alone. Corneal transplantation is used to restore the vision due to opaque corneas in various diseases such as fungal keratitis, keratoconus, Fuchs’s dystrophy, corneal ulceration, traumatic injury, abrasions or scarring, inflammation, and infection. Corneal transplant surgery could involve replacing the entire cornea (penetrating keratoplasty) or selective replacement of the corneal layers (e.g. Descemet’s membrane endothelial keratoplasty and deep anterior lamellar keratoplasty). Immunologic graft rejection is one of the main causes of graft failure in these corneal transplantations. The 2-year rejection rate for non-inflamed and avascular “low-risk” surgeries is approximately 10%, whereas it can be as high as 50% for “high-risk” corneal transplantations or patients who have a previous history of graft rejection or an inflamed bed. These eyes with rejected corneal transplants will require re-transplantation and become “high-risk” which has a much high rejection risk. Topical corticosteroids are the most commonly used immunosuppressive agents for postoperative management and treatment of corneal allograft rejections. These medications have a dosing frequency of up to 6-8 times per day for a period of time which even though tapered down further is associated with poor patient compliance that can lead to compromised efficacy. To address this issue, we developed biodegradable nanoparticles (NP) that can be administered by subconjunctival injection (SCT) at the time of surgery and provide sustained release of dexamethasone sodium phosphate (DSP), thus potentially removing the compliance burden on patients. We employed a zinc chelation method between the carboxylic acid group on poly(lactic-co-glycolic acid) (PLGA) and the phosphate group on DSP to encapsulate high content of water-soluble DSP into PLGA nanoparticles (DSP-NP), by nanoprecipitation-solvent diffusion method in the presence of Pluronic F127. My work focuses on the development and characterization of the optimized DSP-NP formulations that release DSP for up to 3 months suitable for long term in vivo corneal allograft rejection treatment.
In the first part, the polymers used to prepare NP were fully characterized for their terminal carboxyl group content. We successfully used the potentiometric titration method to measure the terminal carboxyl content in PLGA and poly(lactic acid) (PLA) polymers with different molecular weights ( e.g. Mn 3.4, 10, and 34 kDa) and different terminal carboxyl groups (single or dual carboxyl groups). The potentiometric titration experiments revealed that there is a decrease in the free terminal carboxylic group content in the polymer with an increase in the MW for the polymers with the same terminal structure. It would lead the low MW polymers with more terminal carboxyl groups to have a higher drug loading capacity due to their higher capacity to chelate with DSP and Zn. In the second part, we carried out a systemic experiment to prepare various DSP-NP formulations using different polymers and different initial target drug loading, and then fully characterized DSP-NP formulations including particle size and distribution, surface charge, drug loading, drug encapsulation efficiency, and drug release profiles in vitro. The DSP-NP were discrete spherical particles with a relatively uniform particle size distribution (PDI values < 0.2) and average particle size in 150 – 300 nm size range suitable for subconjunctival injection to the eye using fine gauge needles. They had a nearly neutral surface charge due to Pluronic F127 coating which were found to provide dense PEG coatings on biodegradable NP. Pluronic F127 is a triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO). The drug content in DSP-NP was found to increase with the increase of the initial target drug loading and reached a plateau of ~9% for PLGA-1COOH 3.4 kDa polymer when target drug loading is 30%. For PLA-1COOH3.2 kDa the drug content plateau occurred at a higher target drug loading > 30% in comparison to PLGA-1COOH 3.4 kDa at a similar molecular weight. It indicates that more hydrophobic PLA-1COOH3.2 kDa showed a better drug loading as compared to PLGA-1COOH 3.4 kDa in the DSP-NP formulations. PLA-2COOH, a novel PLA polymer with dual terminal carboxylic groups, showed an improved drug loading profile as compared to conventional PLA-1COOH3.2 kDa polymer single carboxylic terminal group. Then, the DSP-NP formulations with 20% and 30% target drug loading for both PLGA and PLA were used for further in vitro drug release studies. We found that the duration of drug release can be extended by increasing polymer MW or increasing the ratio of lactic acid: glycolic acid in the polymer. PLA-1COOH3.2 kDa had a more prolonged release profile as compared to PLGA-1COOH 3.4 kDa and showed release for twice the amount of time. All DSP-NP formulations exhibited minimal burst release. In the third part, ICP-OES was used to determine the zinc content in the DSP-NP formulations to understand the mechanism of encapsulation of DSP into carboxyl-terminated PLGA/PLA nanoparticles using zinc chelation method. Quantification of zinc using ICP-OES revealed an approximate molar ratio of 1:1:1 for the 3 components (DSP, zinc, carboxyl groups) in the DSP-NP formulation with the plateau drug content, suggesting that DSP may be encapsulated into carboxyl-terminated PLGA through ion bridging of both phosphate group from DSP and COOH group from polymer with zinc ions at 1:1:1 molar ratio. Further investigations will be warranted to fully understand the drug encapsulation mechanism.
In the overall conclusion, we efficiently encapsulated DSP in the polymeric nanoparticles using non-covalent zinc ion bridging between the carboxyl groups of the polymer and phosphate groups on DSP, along with dense PEG coating on the surface. DSP-NP were made with materials having a long history of safety which should facilitate translation to human use. Through the systemic formulation development and characterization, we optimized DSP-NP formulations and identified the lead formulation of PLA-2COOH6.5kDa DSP-NP that exhibited high drug content of ~11% and prolonged drug release up to 3 months in vitro, and it has been progressed to future in vivo efficacy study. The sustained drug release provided by DSP-NP may improve patient outcomes by enhancing compliance and improving efficacy for treatment of corneal graft rejection following subconjunctival (SCT ) administration.
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Available for download on Tuesday, May 20, 2025