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. 2025 Aug 28;5(9):4346–4360. doi: 10.1021/jacsau.5c00721

A Metal-Peptide Framework as a Nanozyme for the Attenuation of Amyloid‑β Aggregation and Reactive Oxygen Species

Zhuo Zhang †, Mingchen Lv †, Jiaxi Xu †, Yaping Liu †, Jinlong Qin ‡,*, Zhen Fan †,‡,*, Jianzhong Du †,‡,*
PMCID: PMC12458022  PMID: 41001643

Abstract

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by abnormal performance in memory, cognition, and language, and it imposes a heavy economic burden worldwide. Amyloidosis and oxidative stress are highly associated with AD progression, yet limited clinical drugs are available at present. Nanozymes exhibit diverse enzyme-mimetic activities and have attracted widespread attention as a promising alternative candidate for scavenging reactive oxygen species to maintain the oxidation–antioxidation balance in cells. Neurotoxic amyloid-β (Aβ) aggregation is also a critical event in AD pathology. The development of dual-targeting nanomaterials with antiamyloidosis ability and enzyme-mimicking activity is expected to be a promising strategy for the treatment of amyloidosis and reactive oxygen species-mediated AD progression. Here, bimetallic-peptide framework nanozymes (CuZn-PEP NZs) with amyloid-β (Aβ) attenuating ability, multiple enzyme-mimicking properties, and broad-spectrum reactive oxygen species scavenging capacity were endowed to inhibit Aβ fibrillization, disaggregate Aβ fibrils, and scavenge Aβ fibril-induced reactive oxygen species. An obvious inhibitory effect on Aβ fibrillization and a disaggregation effect on Aβ fibrils were observed after treatment with CuZn-PEP NZs. Meanwhile, the cytotoxicity of Aβ fibrils toward PC12 cells was significantly reduced by CuZn-PEP NZs. Meanwhile, CuZn-PEP NZs with multiple redox pairs exhibit superoxide dismutase, catalase, and glutathione peroxidase-mimicking enzyme properties simultaneously, which further display cytoprotective effects against Aβ fibril-induced reactive oxygen species and mitochondrial damage. Besides, cellular studies verified that CuZn-PEP NZs possess excellent biocompatibility and blood–brain barrier penetration capacity. Overall, these bimetallic-peptide framework nanozymes represent a promising perspective for attenuation of amyloid-β aggregation and reactive oxygen species simultaneously, which highlights the potential of nanozymes for the treatment of amyloidosis and reactive oxygen species-mediated AD progression.

Keywords: peptide, nanozyme, amyloid-β, Alzheimer’s disease, oxidative stress

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Introduction

As one of the most common neurodegenerative diseases, Alzheimer’s disease (AD) triggers severe dysfunction of the brain and central nervous system, inducing abnormal performance in memory, cognition, and language. , Meanwhile, AD has become a heavy economic burden worldwide, affecting 150 million people globally by 2050. Extracellular senile plaques composed of amyloid-β (Aβ) peptide and intracellular neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein are the leading hallmarks of AD. The excessive production and accumulation of toxic Aβ species (oligomers, fibrils, and plaques) in the central nervous system are believed to initiate irreversible synaptic and neuronal dysfunction, according to the amyloid cascade hypothesis. Over the past few decades, numerous classes of therapeutic strategies focusing on toxic Aβ species have been developed to halt the progression of AD pathology. , Current approvals of the monoclonal antibodies aducanumab and lecanemab by the U.S. Food and Drug Administration (FDA) present potential effects in amyloidosis. Both monoclonal antibodies have shown efficacy in eliminating Aβ plaques and ameliorating cognitive decline; however, undesirable abnormal brain magnetic resonance imaging findings and enormous treatment costs still need to be optimized. Furthermore, more clinical drugs focusing on toxic Aβ species are in trials, demonstrating that amyloidosis pathology remains an effective target for AD treatment.

Meanwhile, oxidative stress is also considered one of the key pathological features of AD. Excessive reactive oxygen species (ROS) have been proven to damage neuronal membrane lipids, cellular organelles, as well as biological macromolecules such as proteins, enzymes, and DNA. − Most importantly, ROS can promote Aβ production and aggregation. In return, toxic Aβ species further exacerbate the generation of ROS, together inducing downstream cascade pathologies that promote irreversible cellular dysfunction and apoptosis. , Based on the oxidative stress pathology of AD, antioxidant therapy strategies have emerged as a promising target in AD treatment. Considering that Aβ and ROS are two correlative factors in AD progression, there is an urgent need to design an Aβ and ROS dual-targeted therapeutic system against AD.

With recent advancements in nanotechnology, nanozymes exhibit diverse enzyme-mimetic activities, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxidase (POD) activities. Inspired by copper/zinc superoxide dismutase (Cu/Zn-SOD), which exhibits significant antioxidative activity in living organisms, many researchers have designed artificial Cu/Zn-nanozymes and demonstrated their antioxidant enzyme-like activities in disease treatment. , In the structure of Cu/Zn-SOD, the copper ion is coordinated by histidine residues and is an essential active site in catalysis through cyclically oxidizing and reducing the negatively charged superoxide radical. , While zinc ion not only maintains the structural stability of SOD but also directly influences catalytic reactivity by adjusting the proper coordination and reduction potential of the active Cu site. Therefore, the synergistic effect of copper and zinc is crucial to the enzyme’s activity, and a rational design for a copper/zinc-bimetallic biomimetic site is expected to emerge with SOD-like activity. Moreover, L-carnosine is a natural dipeptide composed of β-alanine and L-histidine, and it possesses metal-ion chelation, anti-inflammatory property, and antioxidant capacity. Most importantly, the existence of L-histidine contributes to coordinating the metal active site, , showing potential for use in constructing metal-based nanozymes.

In this work, copper/zinc ion-peptide nanozymes (termed CuZn-PEP NZs) with diverse enzyme-mimetic activities were synthesized for the inhibition of Aβ fibrillization and disaggregation of Aβ fibrils, as well as ROS scavenging to save neuronal cells from the cytotoxicity of Aβ fibrils. As shown in Scheme , CuZn-PEP frameworks were designed and prepared based on a coordination-driven peptide biomimetic mineralization strategy. Then, CuZn-PEP NZs were obtained through liquid-phase exfoliation of CuZn-PEP frameworks. Within the structure of the biomimetic CuZn-PEP NZs, both Zn and Cu sites are coordinated to the imidazole rings, amino groups, and carboxyl groups of the peptide. More interestingly, thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectra, transmission electron microscopy (TEM) imaging, and isothermal titration calorimetry (ITC) assay demonstrated that CuZn-PEP NZs were able to inhibit Aβ fibrillization and disaggregate Aβ fibrils through hydrogen bonding and electrostatic interactions. In addition, CuZn-PEP NZs exhibit CAT/SOD/GPx-like antioxidant activities, which effectively eliminate Aβ fibril-induced ROS and mitochondrial damage in PC12 cells. Overall, the achievement of the Aβ fibrils and ROS dual-targeted strategy has successfully addressed the dilemma of the Aβ fibrils and ROS in neuronal cells, and thus might represent a promising candidate for AD treatment.

1. Schematic Illustration of (A) the Synthesis of CuZn-PEP NZs and (B) the Attenuation of Amyloid-β Aggregation and ROS .

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i Nerve cells were damaged after Aβ monomers aggregated into Aβ fibrils (red arrows). Aβ aggregation was monitored to maintain Aβ monomers with the treatment of CuZn-PEP NZs, and ROS were eliminated by CuZn-PEP NZs in cells (green arrows).

Results and Discussion

Preparation and Characterization of CuZn-PEP NZs

Metal–organic frameworks with peptides as linkers are particularly attractive because of their improved biocompatibility and enhanced structural and chemical diversity, which may further afford unique catalytic properties. , The framework can be formed through the coordination between zinc and L-carnosine (Figure S1, termed Zn-PEP NZs), showing therapeutic potential according to the literature. Herein, the novel design and composition of CuZn-PEP frameworks are shown in Scheme . Initially, CuZn-PEP frameworks were synthesized by a facile “one-pot” method, coordinating L-carnosine with Cu and Zn ions (Scheme A). CuZn-PEP frameworks exhibited a rod-like morphology with a hydrodynamic diameter of approximately 472.8 nm, as characterized by TEM and dynamic light scattering (DLS) (Figure A–C). Next, the size and dispersal of CuZn-PEP frameworks were optimized by the liquid-phase exfoliation method. As shown in Figure B, nanomorphology was acquired after the optimization process, which displayed a significantly decreased hydrodynamic diameter of approximately 145.9 nm, with only a slight change observed in the zeta potential results, as monitored by DLS (Figure C,D). In addition, as shown in Figure E, X-ray diffraction (XRD) spectra of CuZn-PEP NZs showed that the peaks were in good agreement with those of Zn-PEP NZs, indicating that the crystalline structure of CuZn-PEP NZs resembles that of Zn-PEP NZs. Furthermore, Fourier transform infrared (FTIR) spectroscopy studies were conducted to analyze the coordination between Cu/Zn ions and L-carnosine (Figures F and S2). In the FTIR spectra, the absorption band appearing at 3243 cm–1 was assigned to the N–H stretching vibration of the amino group derived from L-carnosine, which shifted to 3289 cm–1 in Zn-PEP NZs and CuZn-PEP NZs, demonstrating the coordination between Zn and N. Meanwhile, the absorption band appearing at 3060 cm–1 was assigned to the O–H stretching vibration of the carboxyl group from L-carnosine, which weakened in Zn-PEP NZs and CuZn-PEP NZs. The absorption band appearing at 1645 cm–1 was assigned to the CO stretching vibration from L-carnosine, which shifted to 1661 cm–1 in Zn-PEP NZs and CuZn-PEP NZs. These results indicate the coordination between Zn and O. , Additionally, high-resolution TEM and corresponding elemental mapping further verified the presence of Cu and Zn within CuZn-PEP NZs, which was consistent with the above results, suggesting the successful coordination of Cu and Zn ions with L-carnosine (Figure S3). Of note, according to the results of ICP-OES (Figure S4), the element content of Zn within Zn-PEP NZs is 21.2%. Meanwhile, the total metal content within CuZn-PEP NZs is 19.8%, among which the element content of Zn is 16.9%, and the element content of Cu is 2.9%. Taken together, these results confirmed that CuZn-PEP NZs with bimetallic-peptide coordination were successfully synthesized via a peptide coordination-driven strategy.

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Characterizations of the CuZn-PEP NZs. TEM images of (A) CuZn-PEP frameworks and (B) CuZn-PEP NZs. Nanomorphology of CuZn-PEP NZs was obtained with an average diameter of approximately 145.9 nm. (C) Hydrodynamic diameter of CuZn-PEP frameworks and CuZn-PEP NZs. (D) Zeta potential of CuZn-PEP frameworks and CuZn-PEP NZs. (E) XRD spectra of Zn-PEP NZs and CuZn-PEP NZs. (F) FT-IR spectra of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs. (G) XPS spectra of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs. High-resolution XPS spectra of (H) N 1s, (I) O 1s, (J) Zn 2p, and (K) Cu 2p in L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs. (L) High-resolution XPS spectra of Cu 2p in CuZn-PEP NZs. A stable crystal structure with redox pairs was obtained due to the coordination between copper/zinc ions and L-carnosine.

Afterward, X-ray photoelectron spectroscopy (XPS) was used to examine the elemental composition of the CuZn-PEP NZs in greater detail. As shown in Figure G–K, characteristic peaks for C, N, O, and Zn elements were present in Zn-PEP NZs, along with additional characteristic peaks of Cu observed in CuZn-PEP NZs. Moreover, there were slight shifts in the characteristic peaks of N 1s (from 400.34 eV to 399.24 eV) and O 1s (from 530.94 eV to 531.64 eV) in Zn-PEP NZs and CuZn-PEP NZs (Figure H,I), confirming that the construction of the metal-peptide frameworks was driven by the coordination between metal ions and N and O derived from L-carnosine, which was in line with the FTIR results. Furthermore, as shown in Figure L, the high-resolution spectra of Cu 2p show the main peaks of Cu 2p3/2 at 934.54 eV and Cu 2p1/2 at 954.04 eV with spin–orbit splitting of 19.5 eV, while other peaks appearing at 944.84, 942.04, and 962.74 eV are attributed to the satellite peaks of Cu2+. The peak of Cu 2p3/2 at 934.54 eV fits two peaks corresponding to Cu0 (932.64 eV) and Cu+ (934.64 eV), respectively. Meanwhile, the peak located at 940.04 eV is attributed to Cu2+ as well. , These results proved that three different valences were present in CuZn-PEP NZs, and thus a stable crystal structure was obtained when copper ions coordinated with L-carnosine. Most importantly, CuZn-PEP NZs, which possessed these redox pairs, implying excellent redox ability against ROS.

Multienzyme-Like Activity of CuZn-PEP NZs

In view of the crucial role of ROS in the progression of AD, the potential of CuZn-PEP NZs to possess multiple antioxidant enzyme-mimetic activities for ROS scavenging, including superoxide anion (O2 •−) and hydrogen peroxide (H2O2), was further investigated using SOD, CAT, and GPx-like activity assay kits. At first, the oxidation of xanthine by xanthine oxidase produces O2 •−, which then reduces the nitro-blue tetrazolium to formazan with a characteristic absorption peak at 560 nm. SOD can eliminate O2 •−, thus reducing the formation of formazan and affecting the corresponding absorption at 560 nm. Thereby, the quantization of SOD-like activity can be achieved through counting O2 •− scavenging capacity. As shown in Figure A, CuZn-PEP NZs exhibited superior SOD-like O2 •− scavenging activity over both L-carnosine and Zn-PEP NZs. At the same concentration, approximately 94.7% of the O2 •− was eliminated in reaction with CuZn-PEP NZs. On the contrary, only 9.8% and 1.0% of O2 •− was eliminated in reactions with L-carnosine and Zn-PEP NZs, respectively. Meanwhile, the SOD-like activity of CuZn-PEP NZs enhanced with the increase of the concentration, and only 25 μg/mL of CuZn-PEP NZs were required to eliminate more than 90% of the concentration of O2 •− (Figure B). Second, H2O2 possesses a characteristic absorption peak at 240 nm.35 The introduction of CAT can decompose H2O2 and decrease the absorption intensity at 240 nm, thereby enabling the quantification of CAT-like activity. As illustrated in Figure C, CuZn-PEP NZs displayed better CAT-like activity for H2O2 scavenging capacity than both L-carnosine and Zn-PEP NZs. More than half of H2O2 was decomposed by CuZn-PEP NZs. Correspondingly, only 3.6% and 5.0% of H2O2 was decomposed by L-carnosine and Zn-PEP NZs, respectively. In the meantime, CuZn-PEP NZs demonstrated effective CAT-like H2O2 scavenging activity in a concentration-dependent manner (Figure D); about 50% of H2O2 was decomposed when the concentration of CuZn-PEP NZs was above 100 μg/mL. Eventually, reduced glutathione (GSH) can be oxidized to oxidized glutathione (GSSG) in the presence of GPx and H2O2. Meanwhile, the remaining GSH can react with the chromogenic substrate 5,5′-dithio-bis-nitrobenzoic acid (DTNB) to produce GSSG and 2-nitro-5-thiobenzoic acid (TNB), with a characteristic absorption at 412 nm. Therefore, the GPx-like activity for H2O2 scavenging capacity can be calculated based on the amount of TNB, as characterized by the absorbance at 412 nm. As shown in Figure E, CuZn-PEP NZs exhibited preferable GPx-like H2O2 scavenging activities over both L-carnosine and Zn-PEP NZs. Approximately 80% of H2O2 was scavenged when treated with CuZn-PEP NZs. On the contrary, only a slight GPx-like activity for H2O2 scavenging was observed for L-carnosine and Zn-PEP NZs. Meanwhile, CuZn-PEP NZs demonstrated efficient GPx-like activity in a concentration-dependent manner as well (Figure F), and approximately 90% of H2O2 was scavenged by 200 μg/mL of CuZn-PEP NZs.

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Characterization of the multienzyme-like activities of CuZn-PEP NZs. (A) SOD-like activity of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs (200 μg/mL). (B) SOD-like activity of CuZn-PEP NZs with various concentrations. (C) CAT-like activity of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs (200 μg/mL). (D) CAT-like activity of CuZn-PEP NZs with various concentrations. (E) GPx-like activity of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs (200 μg/mL). (F) GPx-like activity of CuZn-PEP NZs with various concentrations. (G) •OH scavenging ability of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs (200 μg/mL). (H) •OH scavenging ability of CuZn-PEP NZs with various concentrations. (I) Schematic illustration of the enzyme-like activities of CuZn-PEP NZs. Multiple antioxidant enzyme-mimetic activities for ROS scavenging were confirmed in CuZn-PEP NZs.

On the additional antioxidant capacity of CuZn-PEP NZs was evaluated using an •OH scavenging rate detection kit. The •OH was generated by the Fenton reaction between H2O2 and Fe2+, which then convert 1,10-phenanthroline-Fe2+ to 1,10-phenanthroline-Fe3+ with a characteristic absorption peak at 536 nm. Hence, the •OH scavenging capacity is positively correlated with the absorbance intensity of 1,10-phenanthroline-Fe2+ at 536 nm. As displayed in Figure G, CuZn-PEP NZs showed excellent •OH scavenging activity compared to L-carnosine and Zn-PEP NZs. Almost 100% of the •OH was eliminated after treatment with CuZn-PEP NZs, whereas only 15.0% and 14.6% of the •OH were eliminated after treatment with L-carnosine and Zn-PEP NZs, respectively. Moreover, as shown in Figure H, the results indicated that CuZn-PEP NZs also exhibit •OH scavenging ability in a concentration-dependent manner, and 6.25 μg/mL CuZn-PEP NZs were able to eliminate more than 50% of the •OH.

As evidenced in Figures L and , the emergence of three different valences (Cu0, Cu+, and Cu2+) in CuZn-PEP NZs is contributed to the highest multienzyme-like activities than L-carnosine and Zn-PEP NZs due to the presence of Cu2+/Cu+/Cu0 with more favorable oxidation–reduction active sites capable of catalyzing negatively charged ROS. Of note, more Cu+ sites corresponds to a higher oxidation–reduction potential, which exhibits higher catalytic reactivity than Cu2+. Specifically, the asymmetric coordination between Cu, N, and O atoms effectively improves the stability of Cu–N or Cu–O bonds, thereby improving the electrochemical stability and further enhancing the multienzyme-like activities of CuZn-PEP NZs. On the contrary, Zn2+ contributes to maintaining structural stability and catalytic reactivity, demonstrating the indispensable role of Zn2+ in these CuZn-PEP NZs. All in all, our studies collectively demonstrate that CuZn-PEP NZs possess SOD/CAT/GPx-like activities (Figure I), enabling them to act as robust antioxidants capable of scavenging a broad spectrum of ROS, thereby highlighting their potential for AD treatment.

Inhibition and Disaggregation of Aβ Fibrils by CuZn-PEP NZs

According to the amyloid cascade hypothesis, toxic Aβ fibrils play a vital role in the amyloidosis of AD. As indicated in the above section, CuZn-PEP NZs possess multiple antioxidant enzyme-mimetic activities for ROS scavenging (Figure ). We then investigated the inhibition and disaggregation effects of CuZn-PEP NZs on Aβ fibrils. First, Aβ aggregation kinetics were monitored by using a ThT fluorescence assay in the presence and absence of various concentrations of CuZn-PEP NZs. As shown in Figure A, the ThT fluorescence intensity of Aβ monomers alone continued to increase within 72 h of incubation, suggesting the formation of Aβ fibrils. Meanwhile, the Aβ fibrillization process was delayed when Aβ monomers were incubated together with CuZn-PEP NZs, which exhibited a concentration-dependent manner in inhibiting Aβ fibrillization. Around 75–100% was achieved when the concentration of CuZn-PEP NZs ranged from 25 to 200 μg/mL (Figures A and S5). On the contrary, the disaggregation effect of CuZn-PEP NZs on Aβ fibrils was also monitored via the ThT fluorescence assay. As shown in Figure B, the ThT fluorescence intensity of Aβ fibrils was maintained within 72 h of incubation, indicating the stable structure of Aβ fibrils. Meanwhile, the ThT fluorescence intensity continued to decrease within 72 h after coincubating Aβ fibrils with CuZn-PEP NZs, showing a concentration-dependent manner (Figure S6). The ThT fluorescence intensity was reduced by 35.9–72.9% after coculturing Aβ fibrils with 25–200 μg/mL of CuZn-PEP NZs for 72 h.

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Inhibition and disaggregation of Aβ fibrils by CuZn-PEP NZs. The relative ThT fluorescence intensity of (A) Aβ monomers and (B) Aβ fibrils in the absence or presence of various CuZn-PEP NZs at different incubation times. Data are presented as mean ± SD, n = 3. The ThT fluorescence intensity of each sample at 0 h was normalized to 1.0. (C) Circular dichroism spectra of Aβ monomers or Aβ fibrils with or without incubation with CuZn-PEP NZs. (D) Secondary structure of Aβ monomers or Aβ fibrils with or without incubation with CuZn-PEP NZs. TEM images of (E) Aβ monomers, (F) Aβ fibrils, (G) Aβ monomers cocultured with CuZn-PEP NZs for 72 h, and (H) Aβ fibrils cocultured with CuZn-PEP NZs for 72 h. Obvious inhibition and disaggregation effects of CuZn-PEP NZs toward Aβ fibrils were obtained according to the ThT fluorescence assay, CD spectra, and TEM images. (I) Typical ITC data for the titration of CuZn-PEP NZs into a solution of Aβ. (J) Summary of binding stoichiometry N, binding constant K, enthalpy changes ΔH, entropy changes ΔS, and Gibbs free energy changes ΔG of ITC data. Hydrogen bonding and electrostatic interactions between Aβ and CuZn-PEP NZs are the main driving forces for inhibiting and disaggregating Aβ fibrils via CuZn-PEP NZs.

To further evaluate the inhibition and disaggregation effects of CuZn-PEP NZs toward Aβ fibrils, CD and TEM were employed to investigate the secondary structural transitions and morphological changes of Aβ fibrils. As shown in Figure C,D, the secondary structure of Aβ monomers alone was predominantly random coils that transitioned into β-sheets after incubation for 24 h. The content of β-sheets in Aβ monomers increased from 26.5% to 62.9% in Aβ fibrils, while the content of random coils in Aβ monomers decreased from 51.0% to 21.3% in Aβ fibrils. Meanwhile, when Aβ monomers were coincubated with CuZn-PEP NZs for 24 h, the content of β-sheets only increased to 45.2%, which was significantly lower than that in Aβ fibrils alone (62.9%). Furthermore, after coincubation of Aβ fibrils with CuZn-PEP NZs for 24 h, the content of β-sheets decreased to 38.5%, and the content of random coils increased to 39.7%. These results provide a potent demonstration that CuZn-PEP NZs can inhibit the secondary structure transition of Aβ monomers to toxic β-sheets and induce pretransformed β-sheets into nontoxic random coils. In addition, upon incubation of Aβ monomers for 72 h, typical fibril morphology was observed (Figure E,F). Meanwhile, only scarce fibrils appeared after coincubation of Aβ monomers with CuZn-PEP NZs for 72 h (Figure G). Dense fibril aggregation was broken into segments when Aβ fibrils were coincubated with CuZn-PEP NZs for 72 h (Figure H). In short, these results clearly indicate that CuZn-PEP NZs possess excellent inhibition and disaggregation effects toward Aβ fibrils.

To uncover the mechanism of CuZn-PEP NZs against Aβ fibrils, ITC measurement was employed further (Figure I). , As summarized in Figure J, the binding stoichiometry (N) and the binding constant (K) indicate the existing binding affinity between Aβ and CuZn-PEP NZs. Meanwhile, the favorable enthalpy change (ΔH< 0) and unfavorable entropy loss (ΔS < 0) suggest that the binding affinity between Aβ and CuZn-PEP NZs is mainly due to hydrogen bond and electrostatic interactions. Moreover, the Gibbs free energy change (ΔG) between Aβ and CuZn-PEP NZs implies an energetically favorable binding process between Aβ and CuZn-PEP NZs. Overall, the above ITC results demonstrate that hydrogen bonding and electrostatic interactions can be formed between Aβ and CuZn-PEP NZs, which contribute to the excellent inhibition and disaggregation effects of CuZn-PEP NZs toward Aβ fibrils.

CuZn-PEP NZs Penetrate Blood–Brain Barrier and Rescue Cytotoxicity Induced by Aβ Fibrils In Vitro

Aβ aggregation process can result in neuronal cell apoptosis by triggering various deleterious cellular responses, including membrane disruption and oxidative stress. , We further evaluated whether CuZn-PEP NZs could rescue neuronal cells from Aβ-induced cytotoxicity using a standard CCK-8 (Cell Counting Kit-8) assay and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. First, we monitored the cytotoxicity of CuZn-PEP NZs. As human umbilical vein endothelial cells (HUVEC) and bEND.3 are commonly applied vascular endothelial cell lines in the study of brain-related diseases, and PC12 cells are capable of differentiating to resemble sympathetic neurons in morphology and function, all of these cell lines could be employed for the research of neurodegenerative diseases. As illustrated in Figure A–C, no significant toxicities were observed in various cell lines, including HUVEC, bEND.3, and PC12 cells, suggesting that CuZn-PEP NZs possess excellent biocompatibility for biomedical applications. Afterward, the uptake of CuZn-PEP NZs by PC12 cells was examined using a confocal laser scanning microscope and quantified by the optical density in the images. The results indicated that CuZn-PEP NZs could be absorbed by PC12 cells within 12 h (Figures D and S7). Then, the cell internalization mechanism of CuZn-PEP NZs was investigated. PC12 cells were treated with various endocytosis inhibitors, including the caveolae-mediated inhibitor methyl-β-cyclodextrin, the dynamin-mediated inhibitor dynasore (which inhibits both clathrin-mediated and caveolae-mediated endocytosis), the pinocytosis inhibitor amiloride, and the clathrin-mediated inhibitor chlorpromazine. , After treatment with the above inhibitors, the cells pretreated with methyl-β-cyclodextrin and dynasore showed almost no uptake of CuZn-PEP NZs. However, the uptake of CuZn-PEP NZs was not inhibited by amiloride or chlorpromazine (Figure E). These results demonstrate that CuZn-PEP NZs enter PC12 cells through caveolae-mediated endocytosis pathways, which depend on cholesterol-dependent lipid rafting. However, methyl-β-cyclodextrin and low temperatures, which deplete cholesterol, highly blocked the internalization of CuZn-PEP NZs. In addition, the in vitro BBB penetration of CuZn-PEP NZs in bEND.3 cells via a Transwell model was tested. As shown in Figure F, bright red fluorescence was observed in the PC cells, implying significant BBB penetration of CuZn-PEP NZs. On the contrary, as Aβ aggregates can induce neurotoxicity through membrane disruption and oxidative stress, a reduced cell viability of 45.1% was observed when PC12 cells were cocultured with Aβ fibrils for 24 h (Figure G). Meanwhile, after Aβ monomers and Aβ fibrils were treated with CuZn-PEP NZs and then cocultured with PC12 cells for 24 h, the cell viability of PC12 cells increased to 76.2% and 73.3%, respectively. The presence of CuZn-PEP NZs could rescue PC12 cells from Aβ-induced cytotoxicity.

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Cytotoxicity analysis of CuZn-PEP NZs against (A) HUVEC cells, (B) bEND.3 cells, and (C) PC12 cells. Data are presented as mean ± SD, n = 6. No obvious cytotoxicities were observed in HUVEC, bEND.3, and PC12 cells at various concentrations of CuZn-PEP NZs. (D) Confocal images of PC12 cells cocultured with RBITC-labeled CuZn-PEP NZs at different incubation times. (E) Confocal images of PC12 cells treated with various inhibitors before incubation with RBITC-labeled CuZn-PEP NZs for 4 h. (F) Confocal images of in vitro BBB penetration of CuZn-PEP NZs by PC12 and bEND.3 cells via a Transwell model. CuZn-PEP NZs exhibited significant BBB penetration ability in vitro. (G) Cytotoxicity analysis of Aβ fibrils, Aβ monomers/CuZn-PEP NZs, and Aβ fibrils/CuZn-PEP NZs against PC12 cells. Data are presented as mean ± SD, n = 6.

Subsequently, we further explored the alleviation effect of CuZn-PEP NZs on Aβ fibril-induced cytotoxicity through the Calcein/PI dye staining assay. The nuclei of late apoptotic/dead cells were stained with PI (red), and healthy cells were stained with Calcein (green) simultaneously. As illustrated in Figure A, an obvious PI fluorescence (red) was observed when PC12 cells were cocultured with Aβ fibrils, proving the severe cytotoxicity of Aβ fibrils toward PC12 cells. Meanwhile, when Aβ monomers and Aβ fibrils were pretreated with CuZn-PEP NZs, only negligible PI fluorescence (red) was observed after incubation. This result is consistent with the above study (Figure F), and all of these findings demonstrate the efficiency of CuZn-PEP NZs in alleviating Aβ-induced cytotoxicity. Afterward, the neuroprotective effect of CuZn-PEP NZs was explored in detail. Membrane disruption is one of the main manifestations of Aβ-induced cytotoxicity. Here, DIO was used as a fluorescent probe to stain the cell membrane and Aβ fibrils, and 4’,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. As illustrated in Figure B, compared to PC12 cells alone with an intact membrane structure, membrane disruption accompanied by Aβ fibrils was observed after the incubation of PC12 cells with Aβ fibrils. The colocalization of Aβ fibrils around the membrane implies a potential threat of Aβ fibrils toward the membrane structure, ultimately leading to PC12 cell apoptosis. Meanwhile, only negligible Aβ fibrils were observed after Aβ monomers and Aβ fibrils were treated with CuZn-PEP NZs and then cocultured with PC12 cells, respectively. Overall, these results indicate that not only CuZn-PEP NZs possess excellent biocompatibility and BBB penetration capacity, but they also effectively alleviate the cytotoxicity of Aβ fibrils toward PC12 cells by attenuating Aβ aggregation.

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Fluorescence images of Calcein/PI dye staining of PC12 cells treated with Aβ fibrils, Aβ monomers/CuZn-PEP NZs, and Aβ fibrils/CuZn-PEP NZs. (B) Confocal images of PC12 cells after treatment with Aβ fibrils, Aβ monomers/CuZn-PEP NZs, and Aβ fibrils/CuZn-PEP NZs, stained with DIO. After being treated with CuZn-PEP NZs, the cytotoxicity induced by Aβ fibrils was obviously reduced, which contributed to maintaining the integral membrane structure of PC12 cells.

CuZn-PEP NZs Alleviate Reactive Oxygen Species and Rescue Mitochondrial Damage Induced by Aβ Fibrils In Vitro

When it comes to AD, oxidative stress is one of the main factors involved in the pathological progression, accompanied by the accumulation of ROS. In light of the excellent ROS scavenging capacity (Figure ), we further validated whether CuZn-PEP NZs could rescue neuronal cells under Aβ-induced ROS. Owing to the fact that 2,7-dichlorofluorescein with green fluorescence can be obtained when 2,7-dichlorofluorescein diacetate (DCFH-DA) reacts with ROS, DCFH-DA is normally employed to visualize intracellular ROS levels. As shown in Figure A, the DCFH-DA staining assay revealed a significant increase in ROS (strong green fluorescence) in PC12 cells upon the addition of Aβ fibrils, indicating a disruption of intracellular ROS homeostasis. Meanwhile, when Aβ monomers and Aβ fibrils were treated with CuZn-PEP NZs and then cocultured with PC12 cells, an inhibited ROS level with weak green fluorescence intensity was obtained (Figure S8), which is attributed to the attenuation of Aβ aggregation and ROS by CuZn-PEP NZs, verifying the strong antioxidant capability of CuZn-PEP NZs at the cellular level. On the contrary, ROS produced in the mitochondria appear to be extremely prominent among ROS sources. Mitochondrial damage and apoptosis have been reported to be strongly associated with oxidative stress and ROS. For further assessment of the antioxidant function of CuZn-PEP NZs, Aβ fibril-induced mitochondrial damage was detected by using a mitochondrial membrane potential kit (JC-1 probe). JC-1 can aggregate in the healthy mitochondrial matrix and produce red fluorescence, while it remains as a monomer when mitochondria are damaged, emitting green fluorescence. As illustrated in Figure B–D, enhanced green fluorescence intensity and attenuated red fluorescence intensity were observed in PC12 cells upon the addition of Aβ fibrils, confirming that Aβ fibrils induce the destruction of the mitochondrial membrane and improve mitochondrial-mediated apoptosis in PC12 cells. Meanwhile, suppressive green fluorescence intensity and recovered red fluorescence intensity were obtained after Aβ monomers and Aβ fibrils were treated with CuZn-PEP NZs, implying the neuroprotective effect of CuZn-PEP NZs. Taken together, these results indicate that CuZn-PEP NZs can significantly alleviate Aβ fibril-induced ROS and mitochondrial damage.

6.

6

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Confocal images of cellular ROS in PC12 cells after treatments with Aβ fibrils, Aβ monomers/CuZn-PEP NZs, and Aβ fibrils/CuZn-PEP NZs, stained with DCFH-DA (green). (B) Confocal images, (C) corresponding mean fluorescence intensity, and (D) red/green fluorescence intensity ratio of mitochondrial membrane potential in PC12 cells after treatment with Aβ fibrils, Aβ monomers/CuZn-PEP NZs, and Aβ fibrils/CuZn-PEP NZs, stained with JC-1 probes. Aβ fibril-induced ROS and mitochondrial damage were significantly alleviated after treatment with CuZn-PEP NZs.

Conclusions

In summary, we have presented a bimetallic-peptide framework as nanozymes that mimic an intracellular antioxidant enzyme-based defense system. The introduction of copper ions with multiple redox pairs endows CuZn-PEP NZs with excellent multienzyme-like activities, which were verified through various ROS scavenging capacity tests. Besides, owing to strong electrostatic interactions and hydrogen bond interactions with Aβ fibrils, enhanced inhibition and disaggregation effects of CuZn-PEP NZs were achieved. The concentration-dependent manner in inhibiting Aβ fibrillization and disaggregating Aβ fibrils was obtained with the introduction of CuZn-PEP NZs, as characterized by the ThT fluorescence intensity. Meanwhile, an obvious secondary structure transition was observed according to the CD assay; the content of β-sheet only reached 45.2% and 38.5% when Aβ monomers and Aβ fibrils were treated with CuZn-PEP NZs, respectively, which was significantly lower than that in Aβ fibrils alone (62.9%). In addition, based on the TEM images, only scarce fibrils appeared in the presence of CuZn-PEP NZs, rather than the morphology of dense fibril aggregation seen in Aβ fibrils alone. Of note, ITC results demonstrated that the formation of hydrogen bonds and electrostatic interactions between Aβ and CuZn-PEP NZs plays a major role in the inhibition and disaggregation effects. Furthermore, in vitro experiments demonstrated excellent biocompatibility on various cell lines and BBB penetration capacity of CuZn-PEP NZs. Meanwhile, the presence of CuZn-PEP NZs could rescue PC12 cells from Aβ-induced cytotoxicity, and cell viability increased to above 70% by the MTT assay. Significantly, CuZn-PEP NZs exhibited excellent antioxidant capability and neuroprotective effects, which were verified by maintaining intracellular ROS homeostasis and alleviating Aβ fibril-induced mitochondrial damage in PC12 cells. Our study represents a promising antioxidant nanomaterial with multiple enzyme-mimicking activities for the treatment of amyloidosis and ROS-mediated AD progression simultaneously.

During the last decades, nanoparticles (typically 50–200 nm in diameter) have been investigated as therapeutic platforms for in vivo studies in the context of stroke, Alzheimer’s disease, or Parkinson’s disease, which usually exploit clathrin-mediated endocytosis for translocation across the plasma membrane. However, blood–brain barrier penetration effectiveness and therapeutic efficacy are still controversial because of distinct gaps between dosage and availability. Currently, there have been certain studies on nanoparticle-based blood–brain barrier penetration. For example, Jiang et al. designed a nanozyme-integrated MOF with antioxidant activity and chiral-dependent blood–brain barrier penetration as antineuroinflammatory agents for the treatment of Parkinson’s disease. These chiral nanozymes show higher accumulation with more pathways to traverse the blood–brain barrier in vivo, including clathrin-mediated and caveolae-mediated endocytosis. The Tang group designed near-infrared-II aggregation-induced emission (AIE) nanocomposites for Alzheimer’s disease therapy. NIR-II imaging verified that the Ang-2 modification assisted the nanocomposites in efficiently crossing the blood–brain barrier. In addition, physical disruption of tight junctions via osmotic pressure or focused ultrasound has been investigated to overcome this blood–brain barrier. Kim et al. prepared piezoelectric nanoparticles, which could produce nitric oxide and generate direct current under high-intensity focused ultrasound and can be used to stimulate deep tissue in brain. The symptoms of Parkinson’s disease were alleviated through the ultrasound-responsive nanoparticles in vivo without causing overt toxicity. Besides, cell-membrane-modified nanoparticles have also emerged as one of the most promising techniques for central nervous system drug delivery. By fusing natural cell membranes, the nanoparticles inherently mimic the properties of the source cells from which their membrane is derived, thereby achieving efficient blood-brain barrier penetration. Lu et al. encapsulated self-assembled nanoparticles by membranes derived from glioma cells, which can readily penetrate the blood–brain barrier and target glioblastoma multiforme through homotypic recognition. Strong therapeutic effects against cancer cells in vivo are achieved through this cellmembrane-modified blood–brain barrier penetration strategy. It will be beneficial to further encapsulate CuZn-PEP NZs with certain cell membranes for enhanced blood–brain barrier penetration. In addition, by conjugating ultrasound-responsive moieties with CuZn-PEP NZs, improved delivery efficiency toward the brain could also be achieved with the assistance of focused ultrasound.

Materials and Methods

Materials

Zinc nitrate hexahydrate (AR, purity 99%) was purchased from Sinopharm Chemical Reagent Co., Ltd. Amyloid-β42 was purchased from Hangzhou ALL PEPTIDE Biology Co., Ltd. with a purity of 95%. Copper­(II) nitrate trihydrate (purity 99%), L-carnosine (Ala-His, purity 98%), sodium hydroxide (NaOH, purity 98%), 4,6-diamidino-2-phenylindole (DAPI, purity 98%), and methyl-β-cyclodextrin (purity 98%) were purchased from Shanghai Adamas Reagents Co., Ltd. N,N-dimethylformamide (DMF, purity 99.5%) was purchased from Beijing Innochem Science and Technology Co., Ltd. Rhodamine B isothiocyanate (RBITC), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, purity 99.5%), phosphotungstic acid, chlorpromazine hydrochloride, amiloride hydrochloride hydrate, dynasore, dimethyl sulfoxide (DMSO, purity 99.8%), and thioflavin-T (ThT) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Superoxide dismutase (SOD) activity assay kit, catalase (CAT) activity assay kit, CCK-8 cell proliferation and cytotoxicity assay kit, mitochondrial membrane potential kit (JC-1 assay), phosphate-buffered saline (pH 7.2–7.4, 0.01 M), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 Medium (RPMI-1640), fetal bovine serum (FBS), and penicillin–streptomycin solution were purchased from Beijing Solarbio Science and Technology Co., Ltd. Total glutathione peroxidase activity assay kit (GPx), 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO), 4,6-diamidino-2-phenylindole (DAPI), Calcein/PI cell viability and cytotoxicity assay kit, and reactive oxygen species assay kit were purchased from Beyotime Biotechnology Company (Shanghai, China). The hydroxyl radical scavenging rate detection kit was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

Measurements and Characterization

Transmission electron microscopy (TEM) images were measured using a JEOL JEM-F200 instrument at 200 kV. Dynamic light scattering (DLS) measurements were performed with a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK). The XRD spectra were obtained using an X-ray powder diffractometer (SmartLab SE, Japan). The XPS spectra were recorded using X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific). Fourier-Transform Infrared (FT-IR) spectroscopy data were collected with a Thermo-Nicolet iS5. The elemental composition assay was conducted using an inductively coupled plasma optical emission spectrometer (720ES, Agilent Technologies). Fluorescence signals were recorded using a fluorospectrophotometer (Hitachi, FL4700). The confocal images were captured using a Nikon Ti2 confocal laser scanning microscope (Nikon, Japan). The CCK-8 and MTT assays were analyzed using a microplate reader (Multiskan FC, Thermo Fisher Scientific). The CD spectra were obtained using a circular dichroism spectrometer (J-1500, Japan). The isothermal titration calorimetry (ITC) measurements were recorded by using a Malvern MicroCal VP-ITC.

Preparation of Metal-Peptide Frameworks

For Zn-PEP frameworks, zinc nitrate hexahydrate aqueous solution (21.04 mL, 0.336 M), L-carnosine aqueous solution (0.442 M), 8.0 mL of DI water, and 80 mL of DMF were added into a pressure flask initially. Then, the flask was heated at 100 °C for 12 h with a heating rate of 1 °C/min. After the completion of the reaction, the flask was cooled to room temperature at a cooling rate of 1 °C/min. Subsequently, the reactant was filtered and washed three times with methanol to remove impurities. The Zn-PEP frameworks were obtained after the reactant was vacuum dehydrated for 24 h. The preparation of CuZn-PEP frameworks is the same as that of Zn-PEP frameworks, except that copper (II) nitrate trihydrate aqueous solution (2.104 mL, 0.336 M) and zinc nitrate hexahydrate aqueous solution (18.936 mL, 0.336 M) were added into the reaction system. Finally, the framework powder was dispersed in DI water and subjected to sonication for 24 h. The solution was then centrifuged (500 g, 15 min) to collect the supernatant. At last, Zn-PEP NZs or CuZn-PEP NZs were obtained by using ultrafiltration.

Preparation of Aβ Monomers and Fibrils

At first, Aβ42 peptide powder (1.0 mg/mL) was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) for 2 h at room temperature. Then, a peptide film was obtained after HFIP was removed under a gentle nitrogen stream. The film was stored at −20 °C until use. To prepare Aβ fibrils, the peptide film was dissolved in NaOH aqueous solution (20 mM) initially. After undergoing ultrasound treatment for 10 min, the solution was diluted to 20 μM in a PBS.

ThT Fluorescence Assay

The concentration of the ThT stock solution was 20 μM in PBS. For the inhibition of Aβ fibrillation, various concentrations of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs were introduced into the Aβ solution, and then, the mixture was incubated at 37 °C for 3 days; the Aβ solution alone was used as the control. At different incubation times, 100 μL of the different Aβ solutions and 900 μL of the ThT solution were mixed together, and the ThT fluorescence was measured (λex = 440 nm, λem = 484 nm).

For the disaggregation of Aβ fibrils, different concentrations of L-carnosine, Zn-PEP NZs, and CuZn-PEP NZs were added to the preformed Aβ fibril solution, and then, the mixture was incubated at 37 °C for 3 days. The ThT fluorescence results were recorded as described above.

CD Spectroscopy

The secondary structure contents of various Aβ solutions were determined using a JASCO J-1500 circular dichroism spectrophotometer. The final concentration of Aβ was 40 μM, and 200 μL of each solution was transferred to a quartz cuvette with a path length of 1 mm. The temperature of the cuvette holder was set to 25 °C. CD spectra were recorded from 190 to 400 nm at a scan speed of 100 nm/min. Percentage secondary structure contents were determined by analyzing the CD data via Beta Structure Selection.

TEM

To prepare the Aβ-related TEM sample, 5 μL of sample solution was dropped onto a carbon-coated copper grid for 90 s, and then, the solution was removed using filter paper. Afterward, the sample was negatively stained with 1% phosphotungstic acid for 90 s, then the solution was removed, and the grid was air-dried at room temperature.

ITC Measurement

ITC experiments were performed at 25 °C, and the stirring speed was set to 437 rpm. In the ITC experiment, 270 μL of CuZn-PEP NZs solution (2 mM) was injected in steps of 10 μL into 1.44 mL of an Aβ solution (20 μM).

SOD-Like Activity of Nanozymes

SOD-like activity of nanozymes was assayed using superoxide dismutase (SOD) activity assay kit according to the manufacturer’s instructions. Briefly, O2 •− was produced through the oxidation of xanthine by xanthine oxidase, which then reduced nitro-blue tetrazolium (NBT) to formazan with a characteristic absorption at 560 nm. After the addition of various nanozymes to the reaction solution at 37 °C for 30 min, the absorbance at 560 nm was measured using UV–vis spectroscopy.

CAT-Like Activity of Nanozymes

CAT-like activity of nanozymes was assessed using a catalase (CAT) activity assay kit according to the manufacturer’s instructions. Briefly, H2O2 possess characteristic absorption peak at 240 nm; the introduction of CAT can decompose H2O2 and decrease the absorption intensity at 240 nm. The reaction solution with various nanozymes was prepared, and the absorbance at 240 nm was measured using UV–vis spectroscopy.

GPx-Like Activity of Nanozymes

GPx-like activity of nanozymes was estimated using a total glutathione peroxidase activity assay kit according to the manufacturer’s instructions. Briefly, reduced glutathione (GSH) is oxidized to oxidized glutathione (GSSG) in the presence of GPx and organic peroxides. Meanwhile, the remaining GSH reacts with the chromogenic substrate 5,5′-dithio-bis-nitrobenzoic acid (DTNB) to produce GSSG and 2-nitro-5-thiobenzoic acid (TNB), which has a characteristic absorption at 412 nm. Various nanozymes were added to the reaction solution at ambient temperature for 15 min. After the introduction of the peroxide reagent, the absorbance at 412 nm was tested using UV–vis spectroscopy immediately.

•OH Scavenging Activity of Nanozymes

OH scavenging activity of nanozymes was determined using a hydroxyl radical scavenging rate detection kit according to the manufacturer’s instructions. Briefly, •OH was generated by the Fenton reaction between H2O2 and Fe2+, which then convert Fe2+ to Fe3+ with a characteristic absorption at 536 nm. The reaction solution with various nanozymes was reacted at 37 °C for 1 h. Then, the absorbance peak at 536 nm of the reaction solution was measured using UV–vis spectroscopy.

Cell Viability of CuZn-PEP Nanozymes

HUVEC cells, bEND.3 cells, and PC12 cells, with a density of 1 × 105 cells were seeded into 96-well plates. After incubation for 24 h, different concentrations of CuZn-PEP NZs were introduced and incubated for an additional 24 h. Then, cell viability was measured using the CCK-8 assay.

In Vitro BBB Penetration Assay

bEND.3 cells with a density of 1 × 105 cells were seeded into the upper chamber, and the medium was changed every other day for 5 days. Afterward, PC12 cells with a density of 1 × 105 cells were seeded into the lower chamber for 24 h. Then, the medium in the upper chamber was changed to medium containing RBITC-labeled CuZn-PEP NZs. After incubation for 24 h, the cells were washed with PBS three times . The fluorescence images were obtained using a confocal fluorescence microscope, and fluorescence intensity was analyzed by using NIS-Elements software. PC12 cells alone were used as the control.

Cellular Uptake Assay

PC12 cells, at a density of 1 × 105 cells, were seeded into 6-well plates. After incubation for 24 h, the medium was replaced with a medium containing RBITC-labeled CuZn-PEP NZs. At various incubation times, the cells were washed with PBS three times . Then, DAPI dye was added, and the plates were placed in a cell culture incubator for 10 min. After washing with PBS, fluorescence images were acquired using a confocal fluorescence microscope, and fluorescence intensity was analyzed by using NIS-Elements software. PC12 cells alone were used as the control.

For the internalization mechanism study, PC12 cells, at a density of 1 × 105 cells, were seeded into 35 mm confocal dishes. After incubation for 24 h, PC12 cells were treated with methyl-β-cyclodextrin, chlorpromazine hydrochloride, amiloride hydrochloride hydrate, and dynasore separately for 1 h. Then, RBITC-labeled CuZn-PEP NZs were added and incubated for another 4 h. After that, the cells were washed with PBS twice. Then, Hoechst 33342 was added, and the plates were placed in a cell culture incubator for 10 min. After washing with PBS twice, fluorescence images were acquired. PC12 cells alone and cells treated with RBITC-labeled CuZn-PEP NZs without any inhibitors were used as the control.

Aβ-Induced Cytotoxicity

PC12 cells, at a density of 5 × 103 cells, were seeded into 96-well plates. After incubation for 24 h, Aβ fibrils that had preinteracted with CuZn-PEP NZs (after the process of inhibition and disaggregation) were added to the PC12 cells and allowed to incubate for another 24 h. PC12 cells alone and Aβ fibrils alone were used as control groups. Then, cell viability was measured using the MTT assay.

Confocal Fluorescence Imaging

For Calcein/PI staining assay, PC12 cells, at a density of 5 × 103 cells, were seeded into 96-well plates. After incubation for 24 h, a certain concentration of Aβ fibrils, with or without CuZn-PEP NZs, was added and coincubated for another 12 hours, followed by washing with PBS three times. Then, the Calcein/PI buffer solution was introduced. After incubation at 37 °C for 30 min, fluorescence images were obtained using a confocal fluorescence microscope. PC12 cells alone were used as the control.

For the DIO staining assay, PC12 cells, at a density of 1 × 105 cells, were seeded onto glass slides within 6-well plates and incubated for 24 h. Afterward, a certain concentration of Aβ fibrils, with or without CuZn-PEP NZs, was added and cocultured for an additional 8 h. After being washed once with PBS, DIO buffer solution was added and incubated at 37 °C for 20 min. Subsequently, after being washed twice with PBS, DAPI dye was added and incubated at 37 °C for 10 min. Fluorescence images were acquired using a confocal fluorescence microscope after the solution was removed, and the cells were washed with PBS. PC12 cells alone were used as the control.

For the DCFH-DA staining assay, the cell culture process is the same as that for the Calcein/PI staining assay. After being treated with various Aβ fibrils/CuZn-PEP NZs, the cells were stained with DCFH-DA at 37 °C for 20 min and then imaged using a confocal fluorescence microscope. PC12 cells alone were used as the control.

For the JC-1 staining assay, the cell culture process is the same as that of the DIO staining assay. After treating the cells with various Aβ fibrils/CuZn-PEP NZs, the cells were stained with JC-1 at 37 °C for 20 min. After washing twice with PBS, fluorescence images were acquired using a confocal fluorescence microscope. PC12 cells alone were used as the control.

Statistical Analysis

The ThT fluorescence intensity of each sample at 0 h was normalized to 1, and the relative ThT intensity was calculated based on the normalized ThT intensity of each sample at 0 h. Results in the figures are presented as the mean ± SD unless otherwise stated. Sample size (n) for cytotoxicity analysis was n = 6, and sample size (n) for other statistical analyses was n = 3 unless otherwise stated. One-way analysis of variance was used to assess significant differences. Statistical significance was calculated as * p < 0.05. OriginPro 2021 (OriginLab Corp., USA) was used for statistical analysis and to draw plots.

Supplementary Material

au5c00721_si_001.pdf (1.9MB, pdf)

Acknowledgments

This research was supported by the National Natural Science Foundation of China (52222306, 22475154, 22335005), Innovation Program of Shanghai Municipal Education Commission (2023ZKZD28), the Fundamental Research Funds for the Central Universities, and the International Scientific Collaboration Fund of the Science and Technology Commission of Shanghai Municipality (23520710900).

Glossary

Abbreviations

AD

Alzheimer’s disease

Aβ

amyloid-β

NFTs

intracellular neurofibrillary tangles

FDA

Food and Drug Administration

ROS

reactive oxygen species

RNS

reactive nitrogen species

SOD

superoxide dismutase

GSH

glutathione

GPx

glutathione peroxidase

CAT

catalase

H2O2

hydrogen peroxide

•OH

hydroxyl radical

O2 •−

superoxide anion

POD

peroxidase

ThT

thioflavin T

CD

circular dichroism

TEM

transmission electron microscope

ITC

isothermal titration calorimetry

DLS

dynamic light scattering

XRD

X-ray diffraction

FTIR

Fourier transform infrared

XPS

X-ray photoelectron spectroscopy.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c00721.

  • TEM images, hydrodynamic diameter, and zeta potential of Zn-PEP frameworks and Zn-PEP NZs; FT-IR spectra of L-carnosine, Zn-PEP, and CuZn-PEP NZs; elemental mapping of CuZn-PEP NZs and the elemental composition of Zn-PEP NZs and CuZn-PEP NZs; and mean fluorescence intensity according to various results (PDF)

All authors have given approval to the final version of the manuscript. Z.Z. and M.L.: data curation, formal analysis, investigation, methodology, visualization, writingoriginal draft, writingreview and editing; J.X.: investigation, software; Y.L.: investigation and visualization; J.Q., Z. F., and J.D.: conceptualization, data curation, formal analysis, funding acquisition, project administration, resources, supervision, validation, writingoriginal draft, and writingreview and editing.

The authors declare no competing financial interest.

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