The Optimisation of Novel, Hyaluronic Acid and Poly(L-lysine) Polyelectrolyte Complex Formulations for the Intraocular Delivery of NAD+

Background:
Aging is a primary risk factor of visual impairment. In 2020, 295 million people worldwide are suffering with a moderate to severe vision impairment (MSVI) . This equates to a 36% increase in MSVI cases between 2015 and 2020. This figure is set to increase exponentially by 2050, resulting in over 460 million people being diagnosed with an MSVI. Ocular diseases, such as age related macular degeneration, glaucoma and diabetic retinopathy, are the primary causes of MSVIs. At a cellular level, the onset of such diseases are strikingly similar, predominantly caused by a progressive loss of cellular homeostasis. These diseases are typically treated in a reactive manner, using intravitreal injections and laser photocoagulation surgery. Although effective, these treatments can be invasive and can potentially cause secondary ocular abnormalities, particularly if administered frequently. So, what if we could pre-treat ocular disease by replenishing the concentration of endogenous co-factors essential for cellular longevity and minimising the deleterious effects of aging. This research study aims to optimise a natural, biocompatible nanoparticulate system, composed of hyaluronic acid and poly(L-lysine) polyelectrolyte complexes (PEC). These PEC formulations will be used for the topical, intraocular delivery of NAD+, co-factor that plays a vital role in the intracellular metabolism of glucose to cellular energy.

Methods:
Hyaluronic acid and poly(L-lysine) complexes were prepared via polyelectrolyte complexation at ambient conditions using both one-shot addition and colloidal titration methods. The resulting formulations were subsequently purified using a three-cycle centrifugation programme. To optimise the intrinsic parameters required for PEC formation, complexes were prepared using various polyelectrolyte concentrations and charge ratios (n-/n+). The optimal pH for complex formation was determined using potentiometric titrations. PEC size, zeta potential and polydispersity index (PDI) were analysed using dynamic light scattering (DLS) analysis. Fourier transform infrared spectroscopy was also used to monitor PEC complexation at various timepoints over a period of 24 hours.

Results:
A pH of 6 was identified as an optimal pH for complexation. Colloidal titration has shown greater efficacy in comparison to one-shot addition regarding the minimisation of polyplex gels at higher polyelectrolyte concentrations. Two optimal formulations have been identified, based on size, zeta potential and PDI. The particle size of the 2% (n-/n+ = 0.96) and 2% (w/ v) (n-/n+ = 1.4) PECs were 222 ± 50.3 nm and 412 ± 69.3 nm (n = 2), respectively. This suggests that PEC size is dependent on the proportion of hyaluronic acid within the complex formulation. The zeta potential and PDIs of these formulations were -79.0 ± 22.4 mV, 0.23 ± 0.098 and – 41.3 ± 5.9 mV, 0.12 ± 0.036, respectively, thus suggesting the formulations exhibit sufficient colloidal stability via electrostatic repulsion.



Conclusion and Future Work:
Colloidal PEC dispersions have been successfully prepared via polyelectrolyte complexation. To enhance PEC stability, in accordance with DLVO theory, further studies involving the use of various ionic strengths and complexation temperatures will be conducted. NAD+ loading and encapsulation efficiency studies are also required. Phase solubility studies will be used to determine the optimal NAD+ to complex stoichiometric ratio.