(T1430-06-35) Fibronectin CS1-Conjugated Polymeric Micelles for Rapamycin Delivery and Modulation of Pro-Inflammatory Cytokines in Alzheimer’s Disease

Purpose: Alzheimer's disease (AD) is marked by neuroinflammation that is triggered by elevated levels of pro-inflammatory cytokines such as amyloid-β (Aβ), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). This neuroinflammation is associated with cognitive impairment and further disease development. Current medications offer symptomatic alleviation, emphasizing the critical need for disease-modifying drugs. Rapamycin, an mTOR inhibitor, has emerged as a prospective contender because of its neuroprotective, autophagy-enhancing, and anti-inflammatory effects. However, rapamycin's systemic administration is limited due to weak brain penetration and severe off-target effects. This study aims to develop and evaluate fibronectin CS1-conjugated PEG-PLA micelles as a targeted nanocarrier system for rapamycin to enhance its anti-inflammatory effects and improve its distribution in AD, bypassing the blood-brain barrier (BBB) using the intranasal route of administration as depicted in Figure 1.

Methods: Polymeric micelles carrying rapamycin were synthesized using an amphiphilic polymer, namely N-hydroxysuccinimide ester-poly(ethylene glycol)- b-poly(D, L lactide) (NHS-PEG-PLA), by the solvent evaporation technique. These micelles were further conjugated with the fibronectin CS1 peptide for better targeted delivery. The micelles were evaluated for particle size, PDI, zeta potential, encapsulation efficiency (%EE), and drug loading (%DL). In vitro studies were performed utilizing SIM-A9 microglial cells to assess cytotoxicity, cellular uptake of polymeric micelles, and cytokine expression levels. In vivo evaluation of the formulation's ability to influence brain cytokine levels and reduce cytokine burden was conducted using a 3xTg AD animal model. The effect of rapamycin was further examined in AD animal models by intranasal administration of Fib CS1 conjugated polymeric micelles containing an imaging agent, before and after the treatment with Fib CS1 conjugated polymeric micelles containing rapamycin.

Results: Polymeric micelles were synthesized and evaluated for their physicochemical parameters, which are explained in the Table.1  In vitro studies employing SIM A9 microglial cells revealed that CS1-modified micelles significantly reduced inflammatory cytokine levels compared to non-targeted micelles (Figure 2A). Intranasal administration of Fib-CS1-conjugated micelles containing NIR (near-infrared) imaging agent has shown higher intensity in the brain of the AD-bearing mice before treatment with Fib-CS1-conjugated micelles containing rapamycin. After the treatment, the intensity was reduced as illustrated in Figures 2B and C, demonstrating significant reduction in neuroinflammation in the brain in vivo. Western blot analysis of brain tissue in vivo proved a substantial reduction in pro-inflammatory cytokines in mice administered CS1-rapamycin micelles (Figure 2D). Amyloid-β (Aβ) levels, normalized to GAPDH, showcased a significant decrease in Group 3 (targeted micelles) relative to Group 1 (control) (p < 0.01). In contrast, the difference between Groups 1 and 2 (non-targeted micelles) was not statistically significant. In Group 2, TNF-α expression decreased by more than 75% relative to the control (p < 0.001), while a moderate reduction was observed in Group 3 (p < 0.05). IL-6 levels were significantly higher in Group 2 compared to Group 1 (*p < 0.0001). In contrast, Group 3 (CS1-micelles) exhibited a notable reduction in IL-6 levels relative to Group 2 (p < 0.01), thereby confirming the immunomodulatory efficacy of the targeted system. These findings suggest that CS1-rapamycin micelles effectively reduce neuroinflammation by significantly decreasing TNF-α, and IL-6 levels in brain tissues, indicating their potential as a therapeutic strategy for AD.

Conclusion: The present study highlights that fibronectin CS1-conjugated PEG-PLA micelles serve as an efficient nanocarrier for the targeted delivery of rapamycin in the context of AD. The micelles demonstrated advantageous physicochemical characteristics and markedly improved rapamycin absorption in microglial cells. The CS1-functionalized system significantly reduced key pro-inflammatory markers—amyloid-β, TNF-α, and IL-6— both in vitro and in vivo, as demonstrated. The outcomes emphasize the capability of this targeted delivery platform to regulate neuroinflammation, presenting an attractive therapeutic approach for reducing the progression of AD over time.

References:

References:
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Acknowledgements:

Funding: Research reported in this work was supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R16GM154661. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.


Figure 1. Schematic representation of the preparation and intranasal administration of rapamycin-loaded polymeric micelles conjugated with Fibronectin CS1 peptide.


Table 1. Physicochemical parameters of polymeric micelles encapsulating rapamycin before and after FibCS1 conjugation. Data are presented as mean ± SD (n = 5).


Figure 2. (A) Western blot analysis of SIM-A9 microglia cells for the levels of proinflammatory cytokines. (B) In vivo biodistribution and anti-inflammatory effect of FibCS1-conjugated micelles encapsulating rapamycin in a 3xTg AD mouse model: Representative in vivo fluorescence images showing brain and nasal cavity signal intensities before treatment in untreated control mice, mice administered with non-targeted micelles, and mice administered with FibCS1-targeted micelles. (C) Fluorescence images post-treatment showing signal intensities in control mice, the non-targeted micelles group, and the targeted micelles group. (D) Densitometric analysis of western blot results from excised brain tissue showing expression of pro-inflammatory cytokines.