Intranasal delivery of rapamycin via brain-targeting polymeric micelles for Alzheimer’s disease treatment

Abstract

Alzheimer’s disease (AD) is a long-term neurological disorder associated with neuroinflammation and amyloid-beta (Aβ) aggregation, which leads to a decline in cognitive and behavioral changes. Rapamycin (Rapa) is an immunosuppressive drug effective in preventing organ rejection after a kidney transplant. In the last few years, orally delivered Rapa has emerged as a potential candidate for improving cognitive function in patients with AD. However, it is evident that long-term oral treatment of Rapa causes systemic toxicity, and although controversial, it may even trigger the aggregation of Aβ deposition. This study investigated the therapeutic potential of intranasally delivered brain-targeting polymeric micelles carrying Rapa. We successfully prepared Fibronectin CS1 peptide-conjugated poly(ethylene glycol)-block-poly(D, L-lactic acid) (FibCS1-PEG-b-PLA) micelles carrying Rapa, which were 98.08 ± 1.15 nm in particle size with a polydispersity index of 0.21 ± 0.01. FibCS1-PEG-b-PLA micelles showed a significant improvement for nasal permeation of Rapa across RPMI-2650 epithelial cells. Behavioral studies such as corner, novel object recognition and Morris Water Maze tests showed promising results towards the improvement of cognitive function in a 3xTg-AD mice model when treated with intranasal FibCS1-PEG-b-PLA micelles carrying RAPA at a dose of 0.2 mg/kg (q4dx5). The western blot and ELISA results of the brain tissues of 3xTg-AD mice treated with intranasal FibCS1-PEG-b-PLA micelles carrying Rapa showed significant reductions in Aβ and two pro-inflammation markers (e.g. interleukin (IL)-1β, tumor necrosis factor (TNF)-α). Here, we conclude that brain-targeting FibCS1-PEG-b-PLA micelles carrying Rapa were effective in reaching the brain via intranasal route, reduced pro-inflammatory markers and Aβ, and improved cognitive function in AD-induced mice.

Highlights

Brain-targeting FibCS1-PEG-b-PLA micelles carrying rapamycin enhanced the nasal permeability of rapamycin.

FibCS1-PEG-b-PLA micelles carrying rapamycin reduced Aβ protein levels and pro-inflammatory markers (TNF-α and IL-6) in vivo.

Intranasally delivered FibCS1-PEG-b-PLA micelles carrying rapamycin showed significant improvement in cognitive behavior.

Introduction

Alzheimer’s disease (AD) is a neurological disorder that impairs neurocognitive function. The main pathological features found in AD are the presence of amyloid β (Aβ) plaques and tau neurofibrillary tangles in the brain, resulting in neuronal loss and brain atrophy (Srivastava et al., 2021). The presence of Aβ plaque is considered the primary pathological condition of AD. It has been reported that the mechanistic target of rapamycin (mTOR) activity is closely related to Aβ deposition and clearance (Caccamo et al., 2010). Under normal circumstances, the brain rapidly clears insoluble Aβ. However, for patients suffering from AD, the imbalance between the production and clearance of Aβ in the brain causes an increased accumulation of Aβ (Jack et al., 2013). For these patients, mTOR is abnormally activated, resulting in the inhibition of Aβ clearance. Therefore, inhibiting mTOR promotes the Aβ clearance. Inversely, the accumulation of Aβ is known to alter the function of mTOR, which is directly linked to learning and memory.

Although not clearly elucidatedas of yet, it has been believed that inhibiting mTOR increases the expression of sirtunin 1 (SIRT1), a key regulator of α-secretase, which, as a result, inhibits the production of Aβ. Rapamycin (Rapa) is an mTOR inhibitor produced by Streptomyces hygroscopicus. Caccamo et al reported that AD-induced mice (3xTg) fed with Rapa-containing food (2.24 mg/kg) for 10 weeks showed a significant reduction in Aβ immunoreactivity of neurons in the CA1 region of the hippocampus (Hou et al., 2023). Chronic inflammation is another major factor in the progression of AD. Nuclear factor-κB (NF-κB) is a key mediator known for the anti-inflammatory effect of Rapa. Rapa exerts anti-inflammatory effects by down-regulating p65, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and other factors associated with NF-κB. IL-6 takes a complex role by stimulating the synthesis of Aβ precursor protein while exerting a neuroprotective effect by activating the phagocytic activity of microglia to degrade Aβ. Although controversial, it has been reported that Rapa upregulates IL-6 expression in astrocytes in a Parkinson’s disease mouse model, showing a neuroprotective effect (Zhang et al., 2017). It is evident that Rapa improves AD-induced cognitive dysfunction (Tramutola et al., 2018). The restoration of mTORC1 activity after Rapa treatment demonstrated a significant effect on cognitive performance in vivo (Tramutola et al., 2018). Rapa is reported to enhance synaptic plasticity, which plays a key role in the development of the nervous system, learning and memory, and cognitive function (Li and Selkoe, 2020, Roda et al., 2020, Caccamo et al., 2014). Rapa also increases synaptic protein expression by increasing mitochondrial autophagy and preventing cytochrome C-mediated apoptosis (Li and Selkoe, 2020). Rapa is known to exert a major effect on aging in rodents (Richardson et al., 2015, Ehninger et al., 2014). The NIA’s intervention testing program (ITP) identified Rapa as the compound that extends lifespan in rodents when given orally via the mouse chow (14 ppm) and the effect was noticeable across both males and females. The primary mechanism of extending lifespan appears to be the inhibition of lethal neoplastic disease (Miller et al., 2014).

Although Rapa has been proven effective in AD, several questions remain to be addressed (Hou et al., 2023). First, Rapa lacks specific targeted effects and as a result potential side effects may occur. There are a few instances regarding Rapa’s adverse effects associated with oral Rapa monotherapy. The most common side effects at the highest dose tested (20 mg/week for 6–16 weeks) were mouth ulcers, headache, fatigue, and neutropenia (Carosi and Sargeant, 2019). As per Shi et al, it was found that the long-term oral use of rapamycin for 2–3 months inhibited mTOR activity and reduced TREM2 expression in microglia of AD mice, reducing the uptake and clearance of Aβ by microglia and aggravating AD-like pathological changes in the brains of 5xFAD AD mice (Shi et al., 2022). The animals received Rapa via daily diet at 143 ppm encapsulated Rapa (14 ppm active Rapa; 2.24 mg/kg, 5 g/day). Additional side effects such as glucose intolerance, diabetes, and immunosuppression resulting from long-term use of Rapa are of concern to AD patients (Hou et al., 2023). In this study, to address the non-targeted effect of Rapa and its side effects owing to the long-term oral use, a low dose (0.2 mg/kg) of Rapa was delivered to AD-induced mice intranasally via brain-targeting polymeric micelles. We hypothesized that brain-targeting polymeric micelles carry Rapa effectively to the brain via nose-to-brain route at the lower dose of Rapa, and brain-targeting polymeric micelles carrying Rapa offer the greater therapeutic efficacy on short-term treatment regimen, thereby reducing systemic side effects. The current AD therapeutics are mainly delivered via oral and parenteral routes. The major drawback of these approaches is poor drug concentration, reduced therapeutic efficacy, and greater side effects of systemic toxicity (Alexander and Saraf, 2018).

Intranasal “nose-to-brain” drug delivery route has been discovered as an alternative approach that addresses these issues and treats the neurological disorders such as migraine (e.g. Onzetra Xsail®) and Parkinson’s disease at greater efficacy and improved patient compliance (Agrawal et al., 2018). Intranasally delivered therapeutics can bypass the blood brain barrier (BBB) and reach the central nervous system (CNS) via the olfactory and trigeminal neural pathways (Hanson and Frey, 2008). Several pathways for nose-to-brain delivery have been proposed based on pre-clinical studies including the olfactory pathway. To date, olfactory pathway, transporting drugs directly to the brain from the nasal cavity along the olfactory and trigeminal nerves, is considered to be the most direct “nose-to-brain” pathway. Once inhaled via nasal pathway, the formulation enters the nasal vestibule where vibrissae, turbulence, and mucosal contact filter particles >12 μm in size (Trevino et al., 2021). It then arrives in a respiratory region which is lined with ciliated pseudostratified columnar epithelium and contains the highly vascularized nasal turbinates. The formulation then reaches the olfactory region located on the roof of the nasal cavity. The olfactory nerve (the maxillary branch of the trigeminal nerve) entering the CNS through the pons provides the formulation with direct CNS access by evading the BBB. Such direct CNS access can be achieved via intracellular and extracellular pathways (Crowe et al., 2018).

The intracellular pathway begins with endocytosis by olfactory sensory cells, followed by axonal transport to the synaptic clefts in the olfactory bulb. The drug undergoes exocytosis onto the olfactory bulb, where neurons projecting to brain regions repeat this process. In the extracellular pathway, drugs are transported directly into the cerebral spinal fluid across the paracellular space in the nasal epithelium, then pass through the perineural space to reach the subarachnoid space of the brain.

To further improve the efficacy of drug delivery, nanoparticles have been utilized as the emerging vehicles. As the axonal transport of rodents is < 100 nm, nanoparticles in average particle size < 100 nm were detected inside olfactory epithelial cells (Rabiee et al., 2021). As the nasal mucous membrane is negative in charge, zeta potential of the nanoparticle at > 30 mV appears to stabilize the nanoparticles, avoiding agglomeration due to the electrostatic repulsion within the nasal cavity and facilitating the interaction between the nanoparticles and the mucosal cells. It is noteworthy that higher positive zeta potential may cause greater toxicity in nasal mucous membrane.

Polymeric micelles are spherically shaped nanoparticles composed of amphiphilic block copolymers, featuring a hydrophobic core and a hydrophilic shell (Micelles, 2024). The most widely adopted hydrophilic block is PEG and the dense brush of PEG on the surface of the micelles ensure that the micellar network embedded with hydrophobic compounds are soluble in water. The modified PEG such as N-Hydroxysuccinimide-PEG (NHS-PEG), permits the versatile conjugation of targeting moieties on the surface of the PEG-based polymeric micelles. The hydrophobic blocks impart unique features and predictive properties of polymeric micelles for drug delivery. For example, poly(ε-caprolactone) (PCL) is a semicrystalline polymer with a melting temperature (Tm) of 55 °C, whereas PLA is amorphous with a glass transition temperature (Tg) of 50 °C. PEG-b-PCL is expected to offer a greater stability for the loaded drugs in a liquid form due to the semicrystalline core of the micelles, however, lyophilization of PEG-b-PCL micelles is rather challenging due to the fragile semicrystalline core. PEG-b-PLA is expected to change the average particle size depending on the drug loading; however, it is easier to prepare lyophilized PEG-b-PLA micelles with greater stability.

In this study, we prepared fibronectin CS1 peptide-conjugated poly(ethylene glycol)-block-poly(D,L-lactic acid) (FibCS1-PEG-b-PLA) micelles carrying Rapa. The primary focus of the study was to demonstrate the effectiveness of the tumor-targeting micellar formulation in reaching the brain via intranasal route, reducing pro-inflammatory markers and Aβ and improving cognitive function in AD-induced mice. Most of the studies evaluating the efficacy of Rapa on improving cognitive functions and extending lifespan have been conducted with the long-term oral administration of Rapa. In this study, we highlighted the feasibility of short-term intranasal administration of brain-targeting micelles carrying Rapa for improving cognitive behavior including motor coordination, recognition memory, and special learning and memory in an AD-bearing mouse model.