Abstract:
Background: Neurological disorders affect hundreds of millions of people worldwide and rapidly rising. One of the biggest hurdles for treating neurological disorders is the limited scope of drugs that can be delivered to the brain via non-invasive routes. Peptide drugs like Glycine-Proline-Glutamate (GPE) and cyclic Glycine Proline (cGP) have been found to be very effective at treating a wide range of neurodegenerative conditions like stroke and Parkinson’s Disease (PD). The main issue with these drugs is that they are susceptible to degradation and/or elimination when either orally or parenterally administered to patients. By incorporating these peptide drugs into a formulation, it is possible to both protect and deliver the drugs directly to the brain. Niosomes were chosen as the preferred vesicle of choice as they are biocompatible and able to entrap both hydrophilic and lipophilic drugs. Notably, niosomes are also able to incorporate ligands into its structure to improve both the efficacy of transport and specificity targeting towards the blood-brain-barrier (BBB). Two different ligands were chosen to be incorporated into the formulation to help deliver the drugs to the brain. The two ligands are the non-specific cell-penetrating peptide Poly-L-arginine (PLR) and the monoclonal antibody RI7, which is a non-invasive targeting ligand specific towards the BBB to improve oral drug delivery to the brain. These two ligands can be used to improve the cellular uptake and transport of GPE and cGP to the brain across the BBB.
Aim: The aim of this project is to design an optimised a bi-ligand niosomal delivery system to orally deliver GPE and cGP across the BBB.
Methods: An HPLC method was developed and validated for the determination of both peptide drugs, GPE and cGP. The niosomes were prepared via a thin-film hydration technique and a factorial design was used to efficiently explore and optimise the different formulation parameters. Chemical and physical properties of the niosomes such as particle size, entrapment efficiency, zeta potential and in vitro release profiles were characterised. The cytotoxicity of the bi-ligand niosomes and both free drugs were evaluated in both human colorectal adenocarcinoma cells (Caco-2)/HT29-MTX-E12 (E12) and Rat brain microvascular endothelial cell (RBMVEC). Cellular uptake was determined on both Caco-2 and RBMVEC for various niosome formulations and parameters such as time, concentration, temperature, and the use of transport inhibitors. Both a gastrointestinal (Caco-2/E12) and BBB (RBMVEC/Astrocytes) model were used to determine the transport of the free drug and niosomal formulations through Transwell® inserts.
Results and Discussion: The optimum formulation was obtained with the following conditions: 0.5 mg of drug, 1:1 ratio between cholesterol and Span 80 surfactant (total 150 μmol), 5 μmol of dicetyl phosphate, 10 ml hydration volume, and 30 min hydration time. This resulted in entrapment efficiency of 28.3% for GPE niosomes and 68.1% for cGP niosomes. The size of the bi-ligand niosome after sonication did not exceed 300 nm with a PDI of less than 0.2 and zeta potential of more than -50 mV. The small size means that the niosomes are suitable for uptake into the cells and are the overall formulation is stable due to the negative zeta potential causing repulsion between vesicles. The in vitro release studies indicated release of drug from the niosome follows an initial burst release of 50% followed by a slow and sustained release of drug over 48 hours. This release profile fits the Korsmeyer-Peppas model and shows that the release of drug from the niosome is via more than 1 phase. The cytotoxicity studies with Caco-2 and RBMVEC is used to determine the dose range to use for subsequent uptake and transport experiments. Uptake results of the niosomal formulation into Caco-2 cells showed that it was time-dependent, concentration-dependent, temperature dependent and the mechanism of uptake is partially via adsorptive-mediated endocytosis pathway. Uptake of the niosomal formulation into RBMVEC showed it was time-dependent, concentration-dependent, temperature dependent and the mechanism of uptake is partially via active transport, adsorptive mediated transport and clathrin mediated transport. Importantly, when free drug uptake is compared to bi-ligand niosome uptake there was 2.5 times more GPE and 3 times more cGP found in the cells when delivered using niosomes. Also, the RI7 ligand only improved uptake into RBMVECs but not Caco-2 cells, whereas the PLR ligand significantly improved uptake into both cell types. Transport experiments utilising a Caco2/E12 gastrointestinal model and RBMVEC/Astrocyte BBB model were carried out, but no detectable amount of drug was found in the basolateral compartment in both models. This suggests that even though uptake of drug into cells was successful, transport of drugs across the cell was not, which could be due the significant degradation of the drug.
Conclusion: This project was meant to develop an optimised novel niosomal delivery system to entrap a wide range of hydrophilic or lipophilic drugs for delivery across the BBB. Successful optimisation, characterisation, and fabrication of the novel bi-ligand niosomes that can entrap both GPE and cGP was achieved. Niosome with both ligands significantly improved GPE or cGP cellular uptake into both Caco-2 and RBMVEC cells, however no detectable amounts of drugs was observed through the cells during transport studies. Further investigation
and correlation with an animal model is required to prove the BBB transport ability of this novel delivery system.