Sequential assembled chimeric peptide for precise synergistic phototherapy and photoacoustic imaging of tumor apoptosis

doi:10.1016/j.cej.2021.130775

Chemical Engineering Journal

Volume 427, 1 January 2022, 130775

https://doi.org/10.1016/j.cej.2021.130775 Get rights and content

Highlights

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Continuous assembly strategy gives more functions.
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Proper initiation of self-assembly maximizes the therapeutic potential of the drug.
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Accurate photoacoustic apoptosis monitoring optimized the therapeutic protocol.
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A simpler versatile nanoagent.

Abstract

Although considerable progress has been made in nanomedicine, it is still of great significance for nanomedicine machines with natural targeting, diverse functions, maximum drug utilization, and outstanding performance. In this work, a simple small molecule chimeric peptide with the capability of sequential two-step self-assembly was developed. This chimeric peptide could be self-assembled into larger nanoparticles from smaller nanoparticles at tumor extracellular acidic microenvironment and further aggregated in apoptotic tumor cells mediated by photodynamic therapy. In addition to the nanostructure changes, the increasing of size could accelerate cell internalization and tumor accumulation of chimeric peptide for better photodynamic therapy. And the aggregation in the apoptotic cell could enhance the photothermal effect and photoacoustic signal of chimeric peptide for further photothermal therapy and photoacoustic imaging of tumor apoptosis. Both in vitro and in vivo studies demonstrated that these self-assembly behaviors endowed chimeric peptide with well tumor accumulation, preferable tumor suppression, and precise therapeutic evaluation, which should provide a new way for the construction of efficient theranostic agent based on chimeric peptides.

Graphical abstract

Introduction

Recently, the intelligent molecular self-assembly strategy in pathological condition has attracted tremendous attention due to its hierarchical nanostructures as well as fascinating biological effect [1], [2], [3], [4], [5], [6]. By reassembling the small molecules in specific biological events, many desirable physical and chemical properties different from those of its precursor can be found, which have been provided many new ideas for the treatment and diagnosis of tumor, such as, enhanced MRI [3], induced cell death [7], sensitization chemotherapy [8], enhanced retention and bioimaging [9], [10]. However, due to the irreversibility of the supramolecular assembly process, these works were usually accomplished through a single assembly process. If multiple assembly processes could be realized in an individual nanomaterial, a more versatile nanoagent may be expected to handle cumbersome theranostics [11].

On the other hand, due to the irreversibility of the assembly, some valuable properties of precursors may also be neglected. For example, Zheng et al. constructed a novel porphysome nanovesicles comparable to gold nanorods though supramolecular assembly [12]; Yan et al. developed a series of ultra-high biocompatibility photothermal nanodots based on the assembly in vitro [13], [14], [15]; Wang et al. also reported a pioneering fiber-like theranostic nanoagent based on MMP-2 responsive assembly [16]. However, these assembly processes usually occurred in vitro or triggered by overexpressed markers that already exist in the tumor and the compromise of the precursors' photodynamic therapeutic properties due to premature assembly might not be noticed. Consequently, if the assembly process can be adjusted appropriately, both the two therapeutic properties may be harnessed simultaneously.

Moreover, the evaluation of treatment response is of great importance for the control of disease. A quick and sensitive therapeutic feedback can contribute to the further improvement of drug and optimization of treatment protocol. Previously, some work has achieved a real-time therapeutic feedback capability by incorporating additional apoptotic-responsive reporter [18], [19], [20]. However, most of these works are based on fluorescence emission [21]. In addition to the complex synthesis and modification process, the low penetration depth and poor spatial resolution also have limited its application in deeper biological tissues. In recent years, photoacoustic imaging (PAI) has been widely used in biomedicine due to its higher tissue penetration and spatial resolution [22], such as, detection of disease [23], [24], tracing of the drug [25], and so on. However, despite the great progress, the system with photoacoustic self-monitoring of treatment efficiency was still rare [26]. Therefore, it is desirable to develop a simple system with higher penetration and spatial resolution for real-time treatment feedback.

In this work, we have reported an efficient “generalist” based on a simple chimeric peptide molecule (PDP). This chimeric peptide could sequential two-step assembly in response to tumor mildly-acidic environment and a delayed trigger (caspase-3) for precise synergistic phototherapy and PA imaging of tumor apoptosis. Scheme 1A presents the chemical formula of the chimeric peptide, containing i) a photosensitizer, pheophorbide-a (Pha), for photodynamic therapy (PDT) and ii) a hydrophilic PEGylated Asp-Glu-Val-Asp (DEVD) peptide sequence for pH and caspase-3 (casp-3) recognition and the active self-assembly process. When under physiological pH (7.4) condition, the PEG and carboxylate radical [COO–] on the glutamate and aspartic acid side chain could significantly increase the hydrophilicity of PDP, which helps PDP exist as a smaller nanoparticle. While at tumor extracellular acidic microenvironment, the protonated of carboxylate radical could increased the hydrophobic of PDP and driven a weak assembly of amphiphilic PDP nanoparticles into large size nanoparticles. The larger size nanoparticles accelerated cell internalization of PDP, resulting in higher tumor accumulation (Scheme 1B). Subsequently, when the PDT mediates tumor apoptosis, the activated caspase-3 in apoptotic cells could specifically cleave the hydrophilic PEGylated DEVD peptide sequence, resulting in a strong aggregation of PDP by intermolecular π-interaction. Instead of both fluorescence emission and singlet oxygen generation (SOG), the aggregated PDP could obtain higher photothermal conversion efficiency [27], which could realize the conversion from PDT properties to photothermal therapy (PTT) properties. Meanwhile, since the amplification of the photothermal effect was triggered by apoptotic enzyme, we could also make photoacoustic (PA) imaging of the apoptotic cell, which might facilitate the evaluation of therapeutic response.

Section snippets

Reagents and materials

Rink Amide resin, N-fluorenyl-9-methoxycarbonyl (Fmoc)-protected L-amino acids, diisopropylethylamine (DIEA), o-benzotriazole-N,N,N′,N'-tetramethyluronium hexafluorophosphate (HBTU) and piperidine were purchased from GL Biochem Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum (FBS), MTT were purchased from GIBCO Invitrogen Corp. Triisopropylsilane (TIS) and trifluoroacetic acid (TFA) were obtained from Shanghai Reagent Chemical Co. (China), and

Synthesis and characterization of PDP

The PDP (Ac-PEG₈-DEVDGK(Pha)) was synthesized on Amie Rink through the standard Fmoc solid-phase peptide synthesis (SPPS) method (Scheme S1) and the peak at 1699.82 ([M + 2Na]²⁺) in high-resolution mass spectrometry (HRMS) confirmed the validity of PDP (Fig. S1). The high-performance liquid chromatography (HPLC) indicated that the purity of PDP was about 90% (Fig. S2). The molecular state of the PDP was studied by the UV–vis spectrum. A broadened Qy-band at 685 nm (Fig. S4), which is

Conclusions

In summary, we developed a simple chimeric peptide (PDP) that could undergo two different degrees of self-assembly in response to the tumor microenvironment and cell apoptosis. Importantly, these continuous self-assembly not only change the nanostructure, but also provided multiple capabilities, enabling precise synergistic phototherapy and photoacoustic imaging of tumor apoptosis. Compared to some nanoagents that incorporated various functional units together or constructed by single

Author statement

The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. J. Zhang and Y. L. Mu contributed equally to this work.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Science and Technology Major Project of Guangxi (Gui Ke AA18118046), National Key R&D Program of China (2016YFD0500706), National Natural Science Foundation of China (21778020, 31750110464 and 31950410755) and Sci-tech Innovation Foundation of Huazhong Agriculture University (2662017PY042, 2662018PY024).

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¹: These authors contributed equally to this work.

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Sequential assembled chimeric peptide for precise synergistic phototherapy and photoacoustic imaging of tumor apoptosis

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents and materials

Synthesis and characterization of PDP

Conclusions

Author statement

Declaration of Competing Interest

Acknowledgments

J. Photochem. Photobiol. B: Biol.

Biomaterials

J. Colloid Interface Sci.

Biomaterials

Crit. Rev. Oncol./Hematol.

Multicomponent self-assembly as a tool to harness new properties from peptides and proteins in material design

Chem. Soc. Rev.

Supramolecular biomaterials

Nat Mater

Furin-mediated intracellular self-assembly of olsalazine nanoparticles for enhanced magnetic resonance imaging and tumour therapy

Nat Mater

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Advances

Supramolecular peptide constructed by molecular Lego allowing programmable self-assembly for photodynamic therapy

Nat. Commun.

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J. Am. Chem. Soc.