The potential use of H102 peptide-loaded dual-functional ...

Abstract

Alzheimer's disease (AD) is a complex neurodegenerative disease with few effective treatments. The non-targeted distribution of drugs decreases drug efficiency and cause side effects. The cascade targeting strategy has been suggested for precise drug delivery. We developed a dual-functional nanoparticle drug delivery system loaded with β-sheet breaker peptide H102 (TQNP/H102). Two targeting peptides, TGN and QSH, were conjugated to the surface of the nanoparticles for blood–brain barrier transport and Aβ42 targeting, respectively. The prepared nanoparticles were spherical and uniform. The brain distribution study of H102 was conducted with the HPLC–mass spectrometry method to evaluate whether this nano-carrier could achieve increased AD-lesion delivery. The highest uptake of H102 was observed in the hippocampi of the TQNP/H102 group mice 1 h after administration, which was 2.62 and 1.86 times the level of non-modified nanoparticles (NP/H102) and TGN modified nanoparticles (TNP/H102), respectively. The neuroprotective effects of H102 preparations were evaluated using Morris water maze experiment, biochemical indexes assay and tissue histology. The spatial learning and memory of the AD model mice in the TQNP/H102 group were significantly improved compared with the AD control group, and were also better than other preparations at the same dosage, even the TNP/H102 group. These results were consistent with the values of biochemical indexes in mouse hippocampi as well as the histological observations. The results demonstrate that TQNP is a promising carrier for peptide or protein drugs, such as H102, for entry into the central nervous system (CNS) and subsequent location of brain AD lesions, thus offering a highly-specific method for AD therapy.

Introduction

Alzheimer's disease (AD) is a complex neurodegenerative disease and the most common form of dementia in elderly people [1]. The disease affects 26.6 million people worldwide, and it is predicted that over 100 million patients will develop AD by 2050 [2], [3]. The annual cost per patient with dementia is estimated to be nearly $20,000, which exceeds the assumed costs for patients with cancer or cardiovascular diseases [4]. Thus AD is producing severe epidemiologic and economic impacts on the whole society [5], and definitive diagnoses, resource-intensive care and effective therapeutic measures are urgently needed to enhance patients' well-being [6].

The identified neuropathological hallmarks of AD are the presence of Aβ peptide deposition in senile plaques and neurofibrillary tangles of hyperphosphorylated tau protein in the brains of patients. The “amyloid cascade hypothesis”, which proposes that AD is driven by the accumulation and deposition of synaptotoxic and neurotoxic Aβ oligomers [7] and the elevated levels of brain Aβ correlate with cognitive impairment [8], remains the leading hypothesis to explain the pathophysiology of AD. In this hypothesis, Aβ40 and Aβ42 are two major forms of Aβ peptides generated from amyloid precursor protein (APP). Although Aβ40 is produced at higher levels, Aβ42 is more neurotoxic as its higher hydrophobicity leads to faster oligomerization and aggregation, representing the majority of deposited parenchymal Aβ [9]. Accordingly, targeting brain Aβ42 to reduce its formation, prevent its aggregation or facilitate its removal has potential as an important strategy in AD therapy [1].

For this strategy, a cascade targeting delivery system has been developed for the precise delivery of drugs to AD brain lesions [10]. This system comprises a poly(ethylene glycol)-poly(lactic acid) (PEG–PLA) nanoparticle system modified with TGN and QSH peptides, both of which are screened by phage display. TGN is a brain-targeting peptide, and the nanoparticles modified with TGN are able to achieve 3.6-times greater accumulation in the brain than unmodified nanoparticles [11]. In addition, QSH has good affinity with Aβ42 and binds Aβ42 in the sub-micromolar range [10]. These dual-functional targeted nanoparticles can achieve enhanced and precise delivery to amyloid plaques in the brains of AD model mice after intravenous injection and thus present great potential in AD treatment [10]. However, no drug was loaded onto this delivery system in the previous study to evaluate whether this dual-functional TQNP could achieve better AD therapeutic effects compared with NP or TNP.

A large number of drugs are now available or under development for AD treatment. However, clinical drugs such as cholinesterase inhibitors or glutamate receptor antagonists can only ameliorate the symptoms instead of curing the disease, and an actual therapeutic strategy for AD is still lacking. β-sheet breaker peptides are a new class of oligopeptide drugs that are designed to specifically interfere with β-sheets within Aβ, preventing the misfolding and deposition of Aβ and decreasing its neurotoxicity [12], [13]. H102 (HKQLPFFEED) peptide is a recently found β-sheet breaker [14], [15], that can improve the spatial memory impairment of APP transgenic mice, and reduce the quantity of senile plaques and the level of APP and Aβ after intracerebroventricular injection and thus it presents a great potential for AD treatment. However, free H102 is barely transported across the BBB and can be rapidly cleared after intravenous injection, with a plasma half-life of 0.8 min, which limits its clinical application.

In this study, the H102 peptide was encapsulated into the dual-functional targeted PEG–PLA nanoparticle system modified with both TGN and QSH peptides. The brain distribution study of H102 was conducted to determine whether this nano-carrier can enhance drug delivery to the AD lesions compared with non-modified or single-peptide modified nano-carriers. Subsequently the neuroprotective effects of the preparations were evaluated using Morris water maze experiments, biochemical index assays and tissue histology.

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Materials

Maleimide-poly(ethylene glycol)3000-poly(lactic acid)70,000 (Mal-PEG–PLA) and methoxy poly(ethylene glycol)3000-poly(lactic acid)50,000 (MePEG-PLA) were synthesized by the East China University of Science. TGN (TGNYKALHPHNGC), QSH (QSHYRHISPAQVC) and Aβ42 were obtained from the Chinese Peptide Company (Hangzhou, China). H102 peptide (HKQLPFFEED) was purchased from GL Biochem (Shanghai) Ltd. (purity 97%). AChE and ChAT activity assay kits were purchased from Jiancheng Bioengineering Institute

Preparation and characterization of H102-loaded nanoparticles

The mean particle sizes of the H102-loaded nanoparticles were approximately 120 nm with a narrow particle distribution (Table 2). The zeta potentials were approximately − 28 mV. Nanoparticles presented as spherical and uniform under transmission electron microscopy (Fig. 1). Conjugation with peptides on the surface slightly increased the diameter of nanoparticles. The loading capacities of these four H102-loaded nanoparticles were similar, approximately 0.54–0.61% with an encapsulation efficiency

Discussion

The brain–blood barrier (BBB) is a formidable obstacle for most drugs entering the brain. Negotiating the BBB is essential for successful treatment of the central nervous system (CNS) diseases, such as AD. In the last 20 years, with the development of brain-targeting delivery systems, some drugs encapsulated by nano-scale particles conjugated with BBB targeting ligands have been able to be transported directly into the brain [5]. However, non-specific drug distribution in normal brain tissues

Conclusions

In conclusion, TGN- and QSH-conjugated PEG–PLA nanoparticles were prepared for the precise delivery of the β-sheet breaker peptide H102 to the brain lesions of AD model mice. This dual-functional drug delivery system effectively increased the H102 accumulation at brain Aβ42 concentrated in the hippocampal region and provided better neuroprotective effects in the AD model mice compared with non-modified or TGN-modified nanoparticles. These results demonstrate that TQNP may be a promising carrier

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81273461 and No. 81473150) and the National Basic Research Program of China (2013CB932500).

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