Intra-Lysosomal Peptide Assembly for the High Selectivity Index against Cancer.

Lysosomes remain powerful organelles and important targets for cancer therapy because cancer cell proliferation is greatly dependent on effective lysosomal function. Recent studies have shown that lysosomal membrane permeabilization induces cell death and is an effective way to treat cancer by bypassing the classical caspase-dependent apoptotic pathway. However, most lysosome-targeted anticancer drugs have very low selectivity for cancer cells. Here, we show intra-lysosomal self-assembly of a peptide amphiphile as a powerful technique to overcome this problem. We designed a peptide amphiphile that localizes in the cancer lysosome and undergoes cathepsin B enzyme-instructed supramolecular assembly. This localized assembly induces lysosomal swelling, membrane permeabilization, and damage to the lysosome, which eventually causes caspase-independent apoptotic death of cancer cells without conventional chemotherapeutic drugs. It has specific anticancer effects and is effective against drug-resistant cancers. Moreover, this peptide amphiphile exhibits high tumor targeting when attached to a tumor-targeting ligand and causes significant inhibition of tumor growth both in cancer and drug-resistant cancer xenograft models.

Protein-Precoated Surface of Metal-Organic Framework Nanoparticles for Targeted Delivery.

Metal-organic framework (MOF) nanoparticles have recently emerged as a promising vehicle for drug delivery with high porosity and feasibility. However, employing a MOF-based drug delivery system remains a challenge due to the difficulty in controlling interfaces of particles in a biological environment. In this paper, protein corona-blocked Zr6 -based MOF (PCN-224) nanoparticles are presented for targeted cancer therapy with high efficiency. The unmodified PCN-224 surface is precoated with glutathione transferase (GST)-fused targetable affibody (GST-Afb) proteins via simple mixing conjugations instead of chemical modifications that can induce the impairment of proteins. GST-Afb proteins are shown to stably protect the surface of PCN-224 particles in a specific orientation with GST adsorbed onto the porous surface and the GST-linked Afb posed outward, minimizing the unwanted interfacial interactions of particles with external biological proteins. The Afb-directed cell-specific targeting ability of particles and consequent induction of cell death is demonstrated both in vitro and in vivo by using two kinds of Afb, which targets the surface membrane receptor, human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR). This study provides insight into the way of regulating the protein-adhesive surface of MOF nanoparticles and designing a more effective MOF-hosted targeted delivery system.

Self-Assembly of Mitochondria-Targeted Photosensitizer to Increase Photostability and Photodynamic Therapeutic Efficacy in Hypoxia.

The development of photosensitizers for cancer photodynamic therapy has been challenging due to their low photostability and therapeutic inefficacy in hypoxic tumor microenvironments. To overcome these issues, we have developed a mitochondria-targeted photosensitizer consisting of an indocyanine moiety with triphenylphosphonium arms, which can self-assemble into spherical micelles directed to mitochondria. Self-assembly of the photosensitizer resulted in a higher photostability by preventing free rotation of the indoline ring of the indocyanine moiety. The mitochondria targeting capability of the photosensitizer allowed it to utilize intramitochondrial oxygen. We found that the mitochondria-targeted photosensitizer localized to mitochondria and induced apoptosis of cancer cells both normoxic and hypoxic conditions through generation of ROS. The micellar self-assemblies of the photosensitizer were further confirmed to selectively localize to tumor tissues in a xenograft tumor mouse model through passive targeting and showed efficient tumor growth inhibition.

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.

Unveiling the Power of Cloaking Metal-Organic Framework Platforms via Supramolecular Antibody Conjugation.

Targeted drug delivery systems based on metal-organic frameworks (MOFs) have progressed tremendously since inception and are now widely applicable in diverse scientific fields. However, translating MOF agents directly to targeted drug delivery systems remains a challenge due to the biomolecular corona phenomenon. Here, we observed that supramolecular conjugation of antibodies to the surface of MOF particles (MOF-808) via electrostatic interactions and coordination bonding can reduce protein adhesion in biological environments and show stealth shields. Once antibodies are stably conjugated to particles, they were neither easily exchanged with nor covered by biomolecule proteins, which is indicative of the stealth effect. Moreover, upon conjugation of the MOF particle with specific targeted antibodies, namely, anti-CD44, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR), the resulting hybrid exhibits an augmented targeting efficacy toward cancer cells overexpressing these receptors, such as HeLa, SK-BR-3, and 4T1, as evidenced by flow cytometry. The therapeutic effectiveness of the antibody-conjugated MOF (anti-M808) was further evaluated through in vivo imaging and the assessment of tumor inhibition effects using IR-780-loaded EGFR-M808 in a 4T1 tumor xenograft model employing nude mice. This study therefore provides insight into the use of supramolecular antibody conjugation as a promising method for developing MOF-based drug delivery systems.

Surface protein-retractive and redox-degradable mesoporous organosilica nanoparticles for enhanced cancer therapy.

Targeted delivery along with controlled drug release is considered crucial in development of a drug delivery system (DDS) for efficient cancer treatment. In this paper, we present a strategy to obtain such a DDS by utilizing disulfide-incorporated mesoporous organosilica nanoparticles (MONs), which were engineered to minimize the surface interactions with proteins for better targeting and therapeutic performance. That is, after MONs were loaded with a chemodrug doxorubicin (DOX) through the inner pores, their outer surface was treated for conjugation to the glutathione-S-transferase (GST)-fused cell-specific affibody (Afb) (GST-Afb). These particles exhibited prompt responsivity to the SS bond-dissociating glutathione (GSH), which resulted in considerable degradation of the initial particle morphology and DOX release. As the protein adsorption to the MON surface appeared largely reduced, their targeting ability with GSH-stimulated therapeutic activities was demonstrated in vitro by employing two kinds of the GST-Afb protein, which target human cancer cells with the surface membrane receptor, HER2 or EGFR. Compared with unmodified control particles, the presented results show that our system can significantly enhance cancer-therapeutic outcomes of the loaded drug, offering a promising way of designing a more efficacious DDS.

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.

Intramitochondrial co-assembly between ATP and nucleopeptides induces cancer cell apoptosis.

Mitochondria are essential intracellular organelles involved in many cellular processes, especially adenosine triphosphate (ATP) production. Since cancer cells require high ATP levels for proliferation, ATP elimination can be a unique target for cancer growth inhibition. We describe a newly developed mitochondria-targeting nucleopeptide (MNP) that sequesters ATP by self-assembling with ATP inside mitochondria. MNP interacts strongly with ATP through electrostatic and hydrogen bonding interactions. MNP exhibits higher binding affinity for ATP (-637.5 kJ mol-1) than for adenosine diphosphate (ADP) (-578.2 kJ mol-1). To improve anticancer efficacy, the small-sized MNP/ADP complex formed large assemblies with ATP inside cancer cell mitochondria. ATP sequestration and formation of large assemblies of the MNP/ADP-ATP complex inside mitochondria caused physical stress by large structures and metabolic disorders in cancer cells, leading to apoptosis. This work illustrates a facile approach to developing cancer therapeutics that relies on molecular assemblies.

Targeting senescent retinal pigment epithelial cells facilitates retinal regeneration in mouse models of age-related macular degeneration.

Although age-related macular degeneration (AMD) is a multifactorial disorder with angiogenic, immune, and inflammatory components, the most common clinical treatment strategies are antiangiogenic therapies. However, these strategies are only applicable to neovascular AMD, which accounts for less than 20% of all AMD cases, and there are no FDA-approved drugs for the treatment of dry AMD, which accounts for ~ 80% of AMD cases. Here, we report that the elimination of senescent cells is a potential novel therapeutic approach for the treatment of all types of AMD. We identified senescent retinal pigment epithelium (RPE) cells in animal models of AMD and determined their contributions to retinal degeneration. We further confirmed that the clearance of senescent RPE cells with the MDM2-p53 inhibitor Nutlin-3a ameliorated retinal degeneration. These findings provide new insights into the use of senescent cells as a therapeutic target for the treatment of AMD.

Intramitochondrial Disulfide Polymerization Controls Cancer Cell Fate.

Recent advances in supramolecular chemistry research have led to the development of artificial chemical systems that can form self-assembled structures that imitate proteins involved in the regulation of cellular function. However, intracellular polymerization systems that operate inside living cells have been seldom reported. In this study, we developed an intramitochondrial polymerization-induced self-assembly system for regulating the cellular fate of cancer cells. It showed that polymeric disulfide formation inside cells occurred due to the high reactive oxygen species (ROS) concentration of cancer mitochondria. This polymerization barely occurs elsewhere in the cell owing to the reductive intracellular environment. The polymerization of the thiol-containing monomers further increases the ROS level inside the mitochondria, thereby autocatalyzing the polymerization process and creating fibrous polymeric structures. This process induces dysfunction of the mitochondria, which in turn activates cell necroptosis. Thus, this in situ polymerization system shows great potential for cancer treatment, including that of drug-resistant cancers.

Drug resistance-free cytotoxic nanodrugs in composites for cancer therapy.

Drug resistance is a major cause of treatment failure for small-molecule cancer chemotherapies, despite the advances in combination therapies, drug delivery systems, epigenetic drugs, and proteolysis-targeting chimeras. Herein, we report the use of a drug resistance-free cytotoxic nanodrug as an alternative to small-molecule drugs. The present nanodrugs comprise 2 nm core gold nanoparticles (AuNPs) covered completely with multivalent hydrocarbon chains to a final diameter of ∼10 nm as single drug molecules. This hydrophobic drug-platform was delivered in composite form (∼35 nm) with block-copolymer like other small-molecular drugs. Upon uptake by cells, the nanodrugs enhanced the intracellular levels of reactive oxygen species and induced apoptosis, presumably reflecting multivalent interactions between aliphatic chains and intracellular biomolecules. No resistance to our novel nanodrug was observed following multiple treatment passages and the potential for use in cancer therapy was verified in a breast cancer patient-derived xenograft mouse model. These findings provide insight into the use of nano-scaled compounds as agents that evade drug resistance to cancer therapy.

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.