Sialic Acid Conjugated Cationic Nanostructured Lipid Carriers Loaded with Natural Bioactives for the Management of Alzheimer's Disease

Abstract

The present research aimed to design and evaluate Myricetin (MY) loaded nanostructured lipid carriers (MY-NLCs) to observe the enhancement of the bioavailability in the brain and cognitive function in Alzheimer’s. MY-NLCs were prepared and optimized using central composite design (CCD). In-vitro MTT assay and cellular accumulation were evaluated in SH-SY5Y neuroblastoma cells. Animals were i.p injected the MYS and MY-NLCs at a dose of 40 mg/kg body weight and MY concentration in brain and plasma were analysed (n=3). Further, the pharmacodynamics studies were evaluated in the (Aβ1-42) induced Alzheimer’s rat model (n=6) and cognitive performance was assessed using Morris water maze (MWM) test followed by histological and neurotransmitters analyses in rats’ brain. The optimized MY-NLCs showed 89.7±26.0 nm particle size, 80.81±10.39% entrapment efficiency, and 5.08±1.0% of drug loading capacity. The in-vitro release studies revealed a biphasic release pattern and also demonstrated distinct cellular internalization in SH-SY5Y cells. Developed MY-NLCs exhibited 2.77 folds higher AUC 0-24 in plasma and drug targeting efficiency for MY into the brain was found 127.05% as compared to MYS. The mitigating potential of MY-NLCs (10mg/kg) was also observed in behavioural parameters and neurotransmitter levels regulation in the rat brain. MY-NLCs would be investigated as a potentially promising drug delivery platform for a variety of neurodegenerative payloads. Based on preliminary successful findings, we further develop and evaluate cationic MY fabricated nanostructured lipid carrier (Cat-MY-NLC) and sialic acid (SA) grafted Sia-Cat-MY-NLC for Alzheimer’s disease (AD) management. After characterization studies, In-vitro cytotoxicity, cellular uptake, Aβ aggregation inhibition and mitochondrial membrane potential of prepared NLCs were observed in SH-SY5Y cells. The trans- endothelial electrical resistance (TEER) was measured through HCMEC/D3 cells. Finally, the pharmacokinetic and pharmacodynamic studies were also performed. The optimized formulations showed the particle sizes (142.26±24.16 nm and 236.3±15.26 nm), zeta potentials (36.5±2.43 mv and -2.4±1.30 mv) respectively for Cat-MY-NLC and Sia-Cat-MY-NLC. SA conjugation was supported with XPS spectroscopy analysis. NLCs revealed the in-vitro cellular accumulation and significant neuroprotective effects in SH-SY5Y cells. Moreover, NLCs were able to cross the in-vitro endothelial barrier of HCMEC/D3 cells. Results of pharmacokinetic studies manifested, the bioavailability was 5.3 and 5.88 folds enhanced with Cat-MY-NLC and Sia-Cat-MY-NLC respectively than MYS administration. In pharmacodynamic studies, the travelled path length, irregularly shaped neurons, Aβ plaque formation and reactive astrocytes in AD rats were markedly declined, revealing the better neuroprotective potential of prepared NLCs than MYS, MYN and Cat-MY-NLC treatment. So, Sia-Cat-MY-NLC would be explored as a promising ligand conjugated lipid-based drug delivery platform for neurodegenerative disorders. Another bioactive in our study is Asiatic acid (AA) which has been greatly acclaimed for its neuroprotective potential in AD. So we aimed to design and develop the AA encapsulated nanostructured lipid carriers (AAN) for the treatment of AD promoting its better bioavailability of the drug into the brain. The behavioural acquisition effects were evaluated in Aβ1-42 induced AD model followed by the evaluation of histology and quantification of neurotransmitters levels. The optimized AAN revealed desired PS (44.1 ± 12.4 nm), ZP (- 47.1 ± 0.017 mv) and EE (73.41 ± 2.53 %) for brain targeting delivery of AA. The results of pharmacodynamic studies manifested the AAN treatment significantly (p < 0.05) ameliorated the cognitive deficits. As a result, developed AAN has neuroprotective potential and should be considered as an unconventional platform in preclinical models for the management of Alzheimer's disease. Further, cationic nanostructured lipid carriers (NLCs) of AA (CA-AA-NLCs) and sialic acid (SA) surface modified CA-AA NLCs (SA-CA-AA-NLCs) were also prepared for investigating the neuroprotective potential in AD. The PS and ZP were shifted from 174.77±27.63 nm to 249.90± 4.23 nm and 23.2± 3.21 mv to 18.27±1.30 mv after the surface modification of CA-AA-NLCs to SA-CA-AA-NLCs. In-vitro, neuroprotective efficiency was evaluated in SH-SY5Y cells and HCMEC/D3 cells and found significant potential effects with treatments of developed NLCs. The pharmacokinetic data revealed the AA content with brain-plasma ratio >1, indicating their higher permeability through blood brain barrier than AAS. Pharmacodynamic studies were also assessed in Aβ1-42 intracerebroventricular injected rats. The results revealed significant (p < 0.05) refurbishment of cholinergic functions and reduction of immunoreactivity responses after 28 days treatment of these developed NLCs. Over all the present study developed a platform technology using sialic acid surface modification for enhanced bioavailability and pharmacological effects for two important neuroprotective bioactives myricetin and Asiatic acid. Prepared NLCs have shown better pharmacokinetic and pharmacodynamic behaviour than the free bioactives.