Enzyme-instructed morphology transformation of mitochondria-targeting peptide for the selective eradication of osteosarcoma.

The treatment of osteosarcoma involves an adjuvant therapy that combines surgery and chemotherapy. However, considering that children are the main victims of osteosarcoma, replacing such a harsh treatment with a soft but powerful method that ensures a complete cure while having no adverse effects is highly desirable. To achieve this aim, we have developed a supramolecular therapeutic strategy based on morphology-transformable mitochondria-targeting peptides for the eradication of osteosarcoma with enhanced selectivity and reduced side effects. A newly designed micelle-forming amphiphilic peptide, l-Mito-FFYp, consisting of a phosphate substrate for the biomarker enzyme of osteosarcoma alkaline phosphatase (ALP), disassembles in response to the ALP enzyme in the cell membrane to generate positively charged l-Mito-FFY molecules, which diffuse inside the targeted cell and self-assemble to form nanostructures specifically inside the mitochondria to induce cell apoptosis.

Application of self-assembly peptides targeting the mitochondria as a novel treatment for sorafenib-resistant hepatocellular carcinoma cells.

Currently, there is no appropriate treatment option for patients with sorafenib-resistant hepatocellular carcinoma (HCC). Meanwhile, pronounced anticancer activities of newly-developed mitochondria-accumulating self-assembly peptides (Mito-FF) have been demonstrated. This study intended to determine the anticancer effects of Mito-FF against sorafenib-resistant Huh7 (Huh7-R) cells. Compared to sorafenib, Mito-FF led to the generation of relatively higher amounts of mitochondrial reactive oxygen species (ROS) as well as the greater reduction in the expression of antioxidant enzymes (P < 0.05). Mito-FF was found to significantly promote cell apoptosis while inhibiting cell proliferation of Huh7-R cells. Mito-FF also reduces the expression of antioxidant enzymes while significantly increasing mitochondrial ROS in Huh7-R cells. The pro-apoptotic effect of Mito-FFs for Huh7-R cells is possibly caused by their up-regulation of mitochondrial ROS, which is caused by the destruction of the mitochondria of HCC cells.

Spatiotemporal Self-Assembly of Peptides Dictates Cancer-Selective Toxicity.

The intracellular or pericellular self-assembly of amphiphilic peptides is emerging as a potent cancer therapeutic strategy. Achieving the self-assembly of amphiphilic peptides inside a cell or cellular organelle is challenging due to the complex cellular environment, which consists of many amphiphilic biomolecules that may alter the self-assembling propensity of the synthetic peptides. Herein, we show that the hydrophobic-hydrophilic balance of the amphiphilic peptides determines the self-assembling propensity, thereby controlling the fate of the cell. A series of peptides were designed to target and self-assemble inside the mitochondria of cancer cells. The hydrophobicity of the peptides was tuned by varying their N-terminus capping. The analysis showed that the largest hydrophobic peptide was self-assembled before reaching the mitochondria and showed no selectivity toward cancer cells, whereas hydrophilic peptides could not self-assemble inside the mitochondria. Optimum balance between hydrophobicity and hydrophilicity is a critical factor for achieving self-assembly inside the mitochondria, thereby providing greater selectivity against cancer cells.

Novel Therapeutic Application of Self-Assembly Peptides Targeting the Mitochondria in In Vitro and In Vivo Experimental Models of Gastric Cancer.

Here, we provide the possibility of a novel chemotherapeutic agent against gastric cancer cells, comprising the combination of 5-fluorouracil (5-FU) and a mitochondria-targeting self-assembly peptide, which is a phenylalanine dipeptide with triphenyl phosphonium (Mito-FF). The anticancer effects and mechanisms of 5-FU and Mito-FF, individually or in combination, were compared through both in vitro and in vivo models of gastric cancer. Our experiments consistently demonstrated that the 5-FU and Mito-FF combination therapy was superior to monotherapy with either, as manifested by both higher reduction of proliferation as well as an induction of apoptotic cell death. Interestingly, we found that combining 5-FU with Mito-FF leads to a significant increase of reactive oxygen species (ROS) and reduction of antioxidant enzymes in gastric cancer cells. Moreover, the inhibition of ROS abrogated the pro-apoptotic effects of combination therapy, suggesting that enhanced oxidative stress could be the principal mechanism of the action of combination therapy. We conclude that the combination of 5-FU and Mito-FF exerts potent antineoplastic activity against gastric cancer cells, primarily by promoting ROS generation and suppressing the activities of antioxidant enzymes.

Intra-mitochondrial self-assembly to overcome the intracellular enzymatic degradation of l-peptides.

The design of peptide-based therapeutics is generally based on the replacement of l-amino acids with d-isomers to obtain improved therapeutic efficiency. However, d-isomers are expensive and frequently induce undesirable immune responses. In the present work, we demonstrate that an intra-mitochondrially self-assembling amphiphilic peptide exhibits analogous activity in both d- and l-isomeric forms. This outcome is in contrast to the general observation considering higher therapeutic efficiencies of d-isomers compared with l-analogues. This suggests that l-peptides overcome proteolytic degradation during intra-mitochondrial self-assembly both in vitro and in vivo.

Recent Progress in Mitochondria-Targeted Drug and Drug-Free Agents for Cancer Therapy.

The mitochondrion is a dynamic eukaryotic organelle that controls lethal and vital functions of the cell. Being a critical center of metabolic activities and involved in many diseases, mitochondria have been attracting attention as a potential target for therapeutics, especially for cancer treatment. Structural and functional differences between healthy and cancerous mitochondria, such as membrane potential, respiratory rate, energy production pathway, and gene mutations, could be employed for the design of selective targeting systems for cancer mitochondria. A number of mitochondria-targeting compounds, including mitochondria-directed conventional drugs, mitochondrial proteins/metabolism-inhibiting agents, and mitochondria-targeted photosensitizers, have been discussed. Recently, certain drug-free approaches have been introduced as an alternative to induce selective cancer mitochondria dysfunction, such as intramitochondrial aggregation, self-assembly, and biomineralization. In this review, we discuss the recent progress in mitochondria-targeted cancer therapy from the conventional approach of drug/cytotoxic agent conjugates to advanced drug-free approaches.

Heterochiral Assembly of Amphiphilic Peptides Inside the Mitochondria for Supramolecular Cancer Therapeutics.

Self-assembly of peptides containing both l- and d-isomers often results in nanostructures with enhanced properties compared to their enantiomeric analogues, such as faster kinetics of formation, higher mechanical strength, and enzymatic stability. However, occurrence and consequences of the heterochiral assembly in the cellular microenvironment are unknown. In this study, we monitored heterochiral assembly of amphiphilic peptides inside the cell, specifically mitochondria of cancer cells, resulting in nanostructures with refined morphological and biological properties owing to the superior interaction between the backbones of opposite chirality. We have designed a mitochondria penetrating tripeptide containing a diphenyl alanine building unit, named as Mito-FF due to their mitochondria targeting ability. The short peptide amphiphile, Mito-FF co-assembled with its mirror pair, Mito-ff, induced superfibrils of around 100 nm in diameter and 0.5-1 µm in length, while enantiomers formed only narrow fibers of 10 nm in diameter. The co-administration of Mito-FF and Mito-ff in the cell induced drastic mitochondrial disruption both in vitro and in vivo. The experimental and theoretical analyses revealed that pyrene capping played a major role in inducing superfibril morphology upon the co-assembly of racemic peptides. This work shows the impact of chirality control over the peptide self-assembly inside the biological system, thus showing a potent strategy for fabricating promising peptide biomaterials by considering chirality as a design modality.

Intra-mitochondrial biomineralization for inducing apoptosis of cancer cells.

The use of biomineralization that regulates cellular functions has emerged as a potential therapeutic tool. However, the lack of selectivity still limits its therapeutic efficacy. Here, we report a subcellular-targeting biomineralization system featuring a triphenylphosphonium cation (TPP) (the mitochondria-targeting moiety) and trialkoxysilane (the biomineralization moiety via silicification). The TPP-containing trialkoxysilane exhibited approximately seven times greater cellular uptake into cancer cells (SCC7) than into normal cells (HEK293T) due to the more negative mitochondrial membrane potentials of the cancer cells. In turn, its accumulation inside mitochondria (pH 8) induces specific silicification, leading to the formation of silica particles in the mitochondrial matrix and further activation of apoptosis. In vivo assessment confirmed that the biomineralization system efficiently inhibits tumor growth in a mouse xenograft cancer model. Exploiting both the subcellular specificity and the targeting strategy provides new insight into the use of intracellular biomineralization for targeted cancer therapy.

Reduction-Triggered Self-Cross-Linked Hyperbranched Polyglycerol Nanogels for Intracellular Delivery of Drugs and Proteins.

Owing to the unique advantages of combining the characteristics of hydrogels and nanoparticles, nanogels are actively investigated as a promising platform for advanced biomedical applications. In this work, a self-cross-linked hyperbranched polyglycerol nanogel is synthesized using the thiol-disulfide exchange reaction based on a novel disulfide-containing polymer. A series of structural analyses confirm the tunable size and cross-linking density depending on the type of polymer (homo- or copolymer) and the amount of reducing agent, dithiothreitol, used in the preparation of the nanogels. The nanogels retain not only small molecular therapeutics irrespective of hydrophilic and hydrophobic nature but also large enzymes such as ß-galactosidase by exploiting the self-cross-linking chemistry. Their superior biocompatibility together with the controllable release of active therapeutic agents suggests the applicability of nanogels in smart drug delivery systems.

Mitochondria localization induced self-assembly of peptide amphiphiles for cellular dysfunction.

Achieving spatiotemporal control of molecular self-assembly associated with actuation of biological functions inside living cells remains a challenge owing to the complexity of the cellular environments and the lack of characterization tools. We present, for the first time, the organelle-localized self-assembly of a peptide amphiphile as a powerful strategy for controlling cellular fate. A phenylalanine dipeptide (FF) with a mitochondria-targeting moiety, triphenyl phosphonium (Mito-FF), preferentially accumulates inside mitochondria and reaches the critical aggregation concentration to form a fibrous nanostructure, which is monitored by confocal laser scanning microscopy and transmission electron microscopy. The Mito-FF fibrils induce mitochondrial dysfunction via membrane disruption to cause apoptosis. The organelle-specific supramolecular system provides a new opportunity for therapeutics and in-depth investigations of cellular functions.Spatiotemporal control of intracellular molecular self-assembly holds promise for therapeutic applications. Here the authors develop a peptide consisting of a phenylalanine dipeptide with a mitochondrial targeting moiety to form self-assembling fibrous nanostructures within mitochondria, leading to apoptosis.