The latest results of Wang Huaimin's team from the School of Science of Westlake University appeared on the cover of Nano Letters to control the dynamics of peptide assembly, causing a "butterfly effect" in cells

Figure 1.
? Nano Letters cover image

Cancer is one of the major public health problems worldwide, clinically mainly through surgical removal of cancerous tissue, radiotherapy, chemotherapy and immunotherapy to alleviate cancer symptoms, but the solubility of radiotherapy and chemotherapy drugs is poor, toxic side effects are large, will be indiscriminate killing of healthy cells, frequent use of drugs will also cause tumor cells drug resistance
.
However, the response rate of immunotherapy is low and the individual variation is large
.
Therefore, the development of new strategies is of great significance
for tumor treatment.
With the development of biomaterials, enzyme-induced peptide self-assembly has been applied to cancer treatment, however, the rate of peptide assembly has a significant impact
on its biological activity.
So, how to control the self-assembly of peptide molecules within specific organelles? How to regulate enzymatic assembly kinetics within cells?

Wang Huaimin's research group at Westlake University has conducted research in the field of peptide assembly for many years, and Professor Huang Feihe of Zhejiang University has long been engaged in the research of
subject-guest interaction.
After long-term discussion and research, they introduced host-guest interaction into the peptide self-assembly system to construct a small molecule peptide self-assembly system with controllable enzymatic assembly kinetics in tumor cells to achieve the purpose of
selectively killing cancer cells.
On the one hand, the formation of host-guest complexes can control the speed of enzyme reactions and enhance cell uptake without reducing the ability of peptide assembly; On the other hand, its pH response characteristics can make enzymatic self-assembly "slowly" and "quickly" in cell membranes and mitochondria, increase the local assembly concentration of peptides in mitochondria, and increase the targeting and efficacy
of tumor therapy.

As shown in the figure below, the research team synthesized Fc-TPP1, a peptide molecule with targeting tumor cell mitochondria, which consists of four parts: self-assembly hydrophobic polypeptide fragment, tumor cell selective functional group, mitochondrial targeting group, and capping functional group that can occur host and guest recognition, in order to regulate the enzymatic assembly kinetics of polypeptide molecules, the authors introduced water-soluble column [6] aromatic hydrocarbons (WP6) into the
assembly system 。 The synthesized small molecule polypeptide can interact with hydrophilic WP6 to form a complex through host-guest interaction under in vitro neutral conditions, which greatly reduces the enzymatic hydrolysis rate of polypeptide
molecules.
When the complex enters the cell, under the acidic environment of lysosomes, the host-guest complex is destroyed, and under the action of targeting mitochondrial groups, peptide self-assembly precursor molecules escape from lysosomes, target mitochondria for in-situ self-assembly, destroy cell mitochondria, cause cell iron death, and thus achieve the purpose of killing cancer cells (Figure 2).

Figure 2.
? A) Chemical structure of peptide molecules Fc-TPP1 and WP6; Fc-TPP1 and WP6 undergo controlled enzymatic self-assembly in B) in vitro and C) cancer cells, causing iron death in cancer cells

First, the authors verified the host-guest interaction between Fc-TPP1 and WP6 in vitro, and determined that the binding constant between Fc-TPP1 and WP6 was 39.
9 μM and the binding ratio was 1:1
.
When alkaline phosphatase (ALP) is added to the system, the host-guest complex transforms from aggregated nanoparticles to a homogeneous nanosphere structure
.
By adjusting the ratio between Fc-TPP1 and WP6, the particle size of the nanospheres can be precisely tuned
.

Fc-TPP1/WP6 complex and Fc-TPP1 were co-incubated with human osteosarcoma cells (Saos-2) and cervical cancer cells (HeLa) overexpressing ALP, respectively, and cytotoxicity experiments showed that Fc-TPP1 after host-guest interaction treatment exhibited greater cytotoxicity
.
The analysis of cell lysate showed that Saos-2 cells had a greatly improved uptake of Fc-TPP1 protected by WP6
.
Laser confocal microscopy (CLSM) has shown that Fc-TPP1/WP6 accumulates in cellular mitochondria and disrupts mitochondria (Figure 2A).

Further analysis showed that after incubation with host-guest complexes, the membrane potential of mitochondria decreased (Figure 2B), cells produced excess ROS (Figure 2C), and tumor cells developed ferrozoosis
.
Subsequently, bioelectron microscopy (Bio-TEM) results showed that after 0.
5 h treatment of Saos-2 cells with Fc-TPP1/WP6 host-guest complex, dense assembly appeared in the mitochondria, and after 24 h, the mitochondria were destroyed, the number of organelles in the cell decreased, and other organelles were damaged (Figure 2D).

。 Photoelectron spectroscopy (XPS) analysis of cell sections showed that cells treated with Fc-TPP1/WP6 had the highest Fe content, indicating that Fc-TPP1 protected by host-guest interaction could aggregate, assemble, and eventually cause cancer cell death
in tumor cells.
Further, injection of Fc-TPP1/WP6 complex into tumor-bearing mice can successfully achieve tumor suppression
.

Figure 3.
? A) CLSM images of Saos-2 cells incubated with Fc-TPP1/WP6 for 0.
5h, 6h, and 24h; B) changes in cell membrane potential and C) ROS production in Saos-2 cells treated with Fc-TPP1 and Fc-TPP1/WP6; D) Bio-TEM images of Saos-2 cells incubated with Fc-TPP1/WP6 for 0.
5 h and 24 h

This work proposes to regulate the assembly behavior of peptide molecules in cells by controlling the kinetics of intracellular enzymatic assembly, and precisely control their enzymatic self-assembly behavior to be carried out "slowly" and "quickly" on the cell membrane surface and mitochondria of tumor cells, respectively, which improves the water solubility, cell uptake ability, and targeting of tumor treatment of peptide molecules, and provides a new strategy
for controllable programming of peptides in cells 。 This work was supported
by grants from the National Natural Science Foundation of China (82022038, 22035006), the Westlake Education Foundation, and the Natural Science Foundation of Zhejiang Province (LD21B020001).
At the same time, the work is grateful to the Westlake University Molecular Science Public Experimental Platform, Material Science Public Experimental Platform and Biomedical Experimental Technology Center for assistance
in testing.

Dissertation information

Controlling Intracellular Enzymatic Self-Assembly of Peptide by Host-Guest Complexation for Programming Cancer Cell Death?

Xuejiao Yang, Bihan Wu, Jiong Zhou, Honglei Lu, Hongyue Zhang, Feihe Huang, and Huaimin Wang*

Nano Letters

https://doi.
org/10.
1021/acs.
nanolett.
2c02612

Introduction to the Smart Biomaterials Laboratory