Brain-neuron targeted nanoparticles for peptide synergy ...

Introduction

Alzheimer's disease (AD), characterized by the extracellular senile plaques constituted of amyloid-β (Aβ) peptides and the intraneuronal neurofibrillary tangles composed of hyperphosphorylated tau (p-tau), is a progressive neurodegenerative disorder with discontented therapeutic outcomes [[1], [2], [3]]. A series of drugs and vaccines based on the “amyloid hypothesis” and “tau pathophysiology” have been designed, however, although anti-amyloid monoclonal antibody, aducanumab, has been conditionally approved by the FDA, most Aβ-targeting therapies have failed in clinical trials and tau-targeting treatments have not yet produced positive findings [[4], [5], [6]]. This unsatisfactory status quo is in part because AD is a multifactorial disease with complex interactions of multiple pathophysiological pathways, which limits the therapeutic effects of monotherapies acting on only one pathologic element. In recent years, combination therapy has been considered as the most promising avenue to overcome the mechanism complexity-induced dilemma in AD treatment [[7], [8], [9]].

The efforts to develop combination therapy for AD treatment have yielded preliminary results. As shown on clinicaltrails.gov, compared with the monotherapy of cholinesterase inhibitor donepezil, the combination therapy of donepezil and the NMDA noncompetitive receptor antagonist memantine approved by the FDA in 2014 has been proved to provide clinical benefits to patients with moderate-to-severe AD [10]. CPC-201, a combination of donepezil and peripheral cholinergic antagonist monoclonal antibody solifenacin, is now in phase II clinical trials [11]. AMX0035, an oral combination of sodium phenylbutyrate and tauroursodeoxycholic acid, is presently in a 24-week, double-blind, placebo-controlled trial [12]. However, drug combination regimens for AD are still at the early stages of implementation and facing critical challenges, including target selection and combination scheme optimization, which are of great importance for the superiority of combination therapy.

Noteworthily, the identified interactions of Aβ and p-tau connect these two main pathological features of AD, forming a vicious circle that accelerates and complicates the disease progression [[13], [14], [15]]. Activating several kinases, such as tyrosine kinase Fyn, extracellular signal-regulated kinase 1 (ERK1), ERK2 and ribosomal protein S6, Aβ stimulates the development of tau pathology including tau phosphorylation, aggregation, mislocalization, and accumulation [16,17]. Additionally, extracellular Aβ plaques and intraneuronal Aβ oligomers (the main toxic species of Aβ) also participate in the inflammation-mediated induction of p-tau [18]. In turn, as a shuttle for Fyn, tau directs Fyn to dendrites and spines to mediate the formation of Aβ excitotoxic complex, through which Aβ triggers excitotoxicity on postsynapse [[19], [20], [21]]. Besides, Aβ and p-tau have also been shown to cooperate in mitochondrial dysfunction and synapse impairment [22,23]. Therefore, it is tenable to conjecture that the concurrent alleviation of Aβ and tau pathology can breach the pathological vicious circle and further construct a virtuous circle in treatment.

Based on this hypothesis, two peptides, H102 and NAP, which act on Aβ and tau respectively, are chosen as therapeutic agents. H102 (HKQLPFFEED) peptide is a β-sheet breaker that can interfere with β-sheet regions of Aβ to inhibit the accumulation of Aβ and decrease its neurotoxicity [24,25]. NAP (NAPVSIPQ) peptide, a neuronal microtubule-interacting agent, can preferentially interact with neuronal and glial tubulin, promote microtubules assembly, and reduce tau hyperphosphorylation [[26], [27], [28]]. Nevertheless, the application of H102 and NAP is constricted because of their poor blood-brain barrier (BBB) permeability and weak neuron-targetability, which undermine their effects on Aβ and p-tau that mainly exist in lesion neurons. In addition, for combination therapy, it is inevitable to consider the differences between these two peptides in pharmacokinetics, biodistribution and stability. The half-life of H102 is much shorter than that of NAP [29,30], making it difficult to design a suitable co-administration scheme of free peptides. Fortunately, nanocarrier shows its special merits in overcoming the aforementioned problems. Nanocarrier can not only facilitate the specific delivery of peptides but also partly uniformize the in vivo behaviors of peptides to reconcile the usage of several peptides with different properties [31].

Hence, we develop a brain neuron-targeting nano-combination system to co-regulate Aβ and tau pathology. Poly(ethylene glycol)-poly(lactic acid) (PEG-PLA), a widely used material in peptide delivery with high biocompatibility [32], is chosen for nanoparticle preparation. The targeting strategy, dual-decoration with the BBB targeting ligand, CGN peptide, and the neuron-targeting ligand, Tet1 peptide, whose specific brain-neuron targetability has been demonstrated on gene carriers in our previous study [33], is employed to realize the efficient and specific peptide delivery to lesion neurons. Furthermore, in order to take full advantage of combination therapy, it is a must to investigate the combination scheme. For nano-combination systems, combination ratio and combination mode are two variables closely related to therapeutic effects in the combination scheme design. Generally, combination modes of nano-combination systems mainly include co-loading and separated-loading, both of which have their advantages. Co-loading possesses more accurate drug delivery to target sites or target cells at the same time in proportion, while separated-loading exhibits flexible adjustment of combination ratios. Therefore, in addition to the combination ratio conventionally screened, the combination mode is also sifted for the optimization of the nano-combination system. Besides, proper screening criteria are essential for the correct analysis of the pros and cons of various combination schemes. Since synergism is the highlight of combination therapy, the synergy precisely evaluated via combination index (CI) and U test value is identified as the core screening indicator in this study.

Through screening, separated-loading nanoparticles with the molar ratio of 2:1 (H102:NAP), CT-NP/H102 + CT-NP/NAP(2:1), is determined as the optimal nano-combination system, which has synergistically-enhanced therapeutic effects on 3 × Tg-AD transgenic mice, reducing toxic Aβ and p-tau, breaking copathogenic Aβ-tau interactions, finally alleviating memory deficits (Scheme 1). Overall, we construct an effective nano-combination system that achieves synergistic effects by simultaneous interference with Aβ and tau pathology, providing deep insights into combination therapy for AD.

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Section snippets

Materials

CGN (d-CGNHPHLAKYNGTC), Tet1 (HLNILSTLWKYRC), NAP (NAPVSIPQ, Mw = 824.9), Aβ25–35, and Aβ1–42 were obtained from the Chinese Peptide Company (Hangzhou, China). H102 (HKQLPFFEED, Mw = 1289.4) was purchased from GL Biochem Ltd. (Shanghai, China). Maleimide-poly(ethylene glycol)3000-poly (lactic acid)70,000 (Mal-PEG-PLA) and methoxy-poly(ethylene glycol)3000-poly(lactic acid)50,000 (MPEG-PLA) were synthesized by Dai gang Biomaterial Company (Jinan, China). Coumarin-6 (Cou-6),

Characterization of H102 or/and NAP loaded nanoparticles

CGN and Tet1 peptides were covalently conjugated to the surface of the nanoparticles with conjugation efficiencies of 100% through a maleimide (Mal)-thiol coupling reaction and the densities of CGN and Tet1 peptides per nanoparticle were 7.9% and 11.85% (mol/mol), respectively. The mean particle sizes of separated-loading nanoparticles (CT-NP/H102 and CT-NP/NAP) were around 140 nm. The co-loading nanoparticles (CT-NP/(H102 + NAP)) possessed particle sizes that slightly decreased with the

Discussion

Considering that the complex etiologies of AD impede the therapeutic efficacy of monotherapy, combination therapy targeting different pathological pathways in parallel has been becoming an appealing research direction in the current landscape of AD treatment. The Aβ and tau pathologies are two interconnected central mechanisms of AD, suggesting that the logical combination of agents acting on these two pathological targets may achieve an intriguing therapeutic improvement. Besides, another

Conclusion

In this study, we developed a nanoparticle-based combination therapeutic strategy to precisely deliver two peptide drugs to neurons in AD lesions and intervene in Aβ and tau pathology simultaneously. The systemic screening of the combination ratio and mode maximized the synergy of combination therapy. The nano-combination at the molar ratio of 2:1 (H102:NAP) achieved synergistic therapeutic effects at Aβ and tau targets and efficiently restored the cognitive performance in 3 × Tg-AD mice.

CRediT authorship contribution statement

Qian Guo: Conceptualization, Methodology, Investigation, Visualization, Software, Formal analysis, Data curation, Writing – original draft. Yixian Li: Investigation, Visualization, Software, Formal analysis, Writing – original draft, Writing – review & editing. Shuting Xu: Investigation, Visualization, Validation, Software, Formal analysis. Pengzhen Wang: Investigation, Validation. Kang Qian: Investigation, Validation. Peng Yang: Investigation, Validation. Dongyu Sheng: Investigation. Liuchang

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 81473150, 82073780, 81690263, 82003702), and Shanghai Municipal Natural Science Foundation (No. 19ZR140620).

1

These authors made equal contributions to this work.

© 2023 Published by Elsevier B.V.