PLGA nanoparticle-loaded light-responsive insitu forming injectable implants for effective drug delivery to the back of the eye

Abstract

Background and Aim: Currently, frequent intravitreal (IVT) drug solution injections are used to treat posterior eye diseases and may be associated with pain and serious side effects. Commercially available site specific implants, on the other hand, require surgical implantation and removal after complete drug release. To overcome these limitations, this thesis aimed to develop nanoparticle-loaded light-responsive in-situ forming injectable implants (ISFIs) as a promising platform for minimally invasive and effective drug delivery to the posterior eye segment. Methods: A high performance liquid chromatography (HPLC) method was developed and validated to determine stability and degradation kinetics of connexin43 mimetic peptide (Cx43MP) under various experimental conditions, and in biological fluids. A validated liquid chromatography tandem mass spectroscopy (LC-MS/MS) method was used along with intravitreal microdialysis to determine the ex-vivo tissue distribution of Cx43MP following IVT administration. During the first phase of formulation development, poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) were prepared using the one-step nanoprecipitation technique and statistically optimized using Box-Behnken design, with their biocompatibility also evaluated. Due to the low peptide entrapment achieved using the one-step method, the two-step nanoprecipitation technique was subsequently used and the developed Cx43MP-loaded PLGA NPs characterized for various physicochemical properties including peptide-excipients compatibility, size, zetapotential (ZP), polydispersity index (PDI), entrapment efficiency (%EE) and cell uptake. In the second part of formulation development, methacrylated alginate (MA) was synthesized and developed formulations were characterized for various physicochemical properties including gelling time, clarity, morphology, syringeability, hardness, rate of swelling and degradation. Ex-vivo studies were performed to investigate the formation of ISFIs inside bovine vitreous upon photoirridiation through the cornea. The biocompatibility of developed NPs and ISFIs was evaluated using the sulforhodamine B (SRB) assay on ARPE-19 cells and the zebrafish embryo toxicity (ZET) model. Finally, Cx43MP-loaded NPs were incorporated into light-responsive ISFIs and in-vitro drug release from NPs, ISFIs and NP-loaded ISFIs was studied. Findings: The HPLC method showed excellent linearity over the concentration range of 0.9 to 250 μg/ml (R2 ≥ 0.998), with the limits of detection (LOD) and quantification (LOQ) found to be 0.90 and 2.98 μg/mL, respectively. Accelerated stability studies revealed that Cx43MP was more sensitive to basic conditions, completely degrading within 24 h at 37 ºC. Cx43MP was found to be more stable in bovine vitreous (t1/2 slow= 171.8 min) compared to human plasma (t1/2 slow = 39.3 min) at 37 ºC, according to the two phase degradation kinetics model. During ex-vivo microdialysis studies, Cx43MP concentration was found to increase slowly in the vitreous body for up to 16 h, after which it declined. After 48 h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humour. NPs developed using the two-step nanoprecipitation technique ranged between 149.3 to 235.4 nm in size, with a PDI between 0.24 and 0.46, a ZP of -32.4 to -27.0 mV and an EE% of 34.3 to 55.3%. The successful synthesis of MA and its subsequent photocrosslinking upon photoirridiation was confirmed by 1H NMR. Photocrosslinked MA exhibited the required clarity and its gelling time, morphology, syringeability, hardness, rate of swelling and degradation were found suitable for prolonging its residence in the posterior eye segment. The SRB assay on ARPE-19 cells and the ZET model confirmed the biocompatibility of NPs and ISFIs. NP entrapment within the gel network was confirmed via morphological studies. Finally, in-vitro peptide release studies revealed peptide instability in phosphate buffer saline over prolonged periods at 37 ºC. Conclusion: This project reported, for the first time, a validated stability indicating HPLC method and degradation kinetics of Cx43MP in biological fluids. Compared to drug-free human plasma, Cx43MP was found relatively stable in bovine vitreous which assures its clinical applicability. Moreover, the developed ex-vivo microdialysis model may be a useful tool for investigating short-term ocular disposition and intravitreal kinetics of drugs after IVT injection. Both NPs and light-responsive ISFIs exhibited suitable physicochemical properties and a high degree of biocompatibility, rendering them suitable for ocular drug delivery applications. However, no conclusions could be drawn with regard to the improved in-vitro release profile of the combined system as the peptide seemed to degrade rapidly in the release medium at physiological temperature. Nevertheless, the information obtained from the present in-vitro release studies with regard to Cx43MP stability under various experimental conditions will be highly valuable in designing a suitable peptide delivery system in the future.