A near-infrared fluorescent probe with assembly/aggregation-induced retention effect for specific diagnosis of metastasis and image-guided surgery in breast cancer.

Image-guided surgery is crucial for achieving complete tumor resection, reducing postoperative recurrence and improving patient survival. However, current clinical near-infrared fluorescent probes, such as indocyanine green (ICG), face two main limitations: 1) lack of active tumor targeting, and 2) short retention time in tumors, which restricts real-time imaging during surgery. To address these issues, we developed a near-infrared fluorescent probe capable of in situ nanofiber formation within tumor lesions. This probe actively targets the integrin αvß3 receptors overexpressed on breast cancer cells and exhibits assembly/aggregation-induced retention effects at the tumor site, significantly extending the imaging time window. Additionally, we found that the probe's fluorescence intensity can be enhanced under receptor induction. Due to its excellent tumor specificity and sensitivity, 1FCG-FFGRGD not only identifies primary breast cancer but also precisely locates smaller lymph node metastases and detects sub-millimeter peritoneal metastases. In summary, this near-infrared probe, leveraging assembly/aggregation-induced retention effects, holds substantial potential for various biomedical applications.

Modular Design and Self-assembly of pH-Responsive Multidomain Peptides Incorporating Non-natural Ionic Amino Acids.

Stimuli-responsive peptides, particularly pH-responsive variants, hold significant promise in biomedical and technological applications by leveraging the broad pH spectrum inherent to biological environments. However, the limited number of natural pH-responsive amino acids within biologically relevant pH ranges presents challenges for designing rational pH-responsive peptide assemblies. In our study, we introduce a novel approach by incorporating a library of non-natural amino acids featuring chemically diverse tertiary amine side chains. Hydrophobic and ionic properties of these non-natural amino acids facilitate their incorporation into the assembly domain when uncharged, and electrostatic repulsion promotes disassembly under lower pH conditions. Furthermore, we observed a direct relationship between the number of substitutions and the hydrophobicity of these amino acids, influencing their pH-responsive properties and enabling rational design based on desired transitional pH ranges. The structure-activity relationship of these pH-responsive peptides was evaluated by assessing their antimicrobial properties, as their antimicrobial activity is triggered by the disassembly of peptides to release active monomers. This approach not only enhances the specificity and controllability of pH responsiveness but also broadens the scope of peptide materials in biomedical and technological applications.

A Membrane-Anchoring Self-Assembling Peptide Allows Bioorthogonal Coupling of Type-I AIEgens for Pyroptosis-Induced Cancer Therapy.

Enrichment of photosensitizers (PSs) on cancer cell membranes via bioorthogonal reactions is considered to be a very promising therapeutic modality. However, azide-modified sugars-based metabolic labeling processes usually lack targeting and the labeling speed is relatively slow. Moreover, it has been rarely reported that membrane-anchoring pure type-I PSs can induce cancer cell pyroptosis. Here, we report an alkaline phosphatase (ALP) and cholecystokinin-2 receptor (CCK2R) dual-targeting peptide named DBCO-pYCCK6, which can selectively and rapidly self-assemble on cancer cell membrane, and then bioorthogonal enrich type-I aggregation-induced emission luminogens (AIEgen) PSs (SAIE-N3) on the cell membrane. Upon light irradiation, the membrane-anchoring SAIE-N3 could effectively generate type-I reactive oxygen species (ROS) to induce gasdermin E (GSDME)-mediated pyroptosis. In vivo experiments demonstrated that the bioorthogonal combination strategy of peptide and AIEgen PSs could significantly inhibit tumor growth, which is accompanied by CD8+ cytotoxic T cell infiltration. This work provides a novel self-assembly peptide-mediated bioorthogonal reaction strategy to bridge the supramolecular self-assembly and AIE field through strain-promoted azide-alkyne cycloaddition (SPAAC) and elucidates that pure type-I membrane-anchoring PSs can be used for cancer therapy via GSDME-mediated pyroptosis.

Peptide-Amphiphilic Nanoassemblies as a Responsive Senolytic Navigator for Targeted Removal of Senescent Cardiomyocytes to Ameliorate Heart Failure.

Heart failure (HF) represents the terminal stage of numerous cardiovascular disorders and lacks effective therapeutic strategies. The accumulation of senescent cardiomyocytes is a cardinal characteristic of HF, contributing to myocardial dysfunction and deteriorating the myocardial microenvironment through the development of senescence-associated secretory phenotypes (SASPs), ultimately culminating in pathological remodeling. Senolytics, a promising therapeutic strategy that selectively induces apoptosis in senescent cells, faces challenges due to nonspecific effects, raising concerns for clinical implementation. In this study, we developed peptide-amphiphilic nanoassemblies as responsive drug navigators for targeted delivery. The modular nanoassemblies comprise a hydrophilic domain containing a CD9-binding peptide, a hydrophobic domain incorporating a reactive oxygen species (ROS)-responsive motif, and an alkyl tail for encapsulation of the senolytic ABT263. The CD9-targeted and ROS-responsive nanoassemblies (AP@ABT263) specifically recognized senescent cardiomyocytes and modulated the release of ABT263 in the presence of elevated intracellular ROS levels. AP@ABT263 treatment significantly enhanced the targeted delivery of ABT263 to senescent cells in both in vitro and in vivo while showing minimal toxicity to normal cardiomyocytes and other tissues. Our findings provide compelling evidence that AP@ABT263 efficiently eradicated senescent cardiomyocytes, enhanced cardiac function, and attenuated the deleterious effects of SASP, thereby preventing adverse cardiac remodeling. In summary, AP@ABT263 represents a highly promising approach for responsive and controlled drug release in senescent cardiomyocytes, providing valuable insights into the development of intelligent pharmaceutical interventions for the management of HF.

Development of barley proteins into peptides nanomicelles for encapsulation of hydrophobic bioactive ingredient.

BACKGROUND: As natural polymer materials, barley proteins have been utilized to fabricate nanocarriers to encapsulate and delivery hydrophobic bioactive ingredients. However, as a result of the high proportion of hydrophobic amino acids and structural rigidity, barley protein-based nanocarriers tend to aggregate easily and have a low loading capacity, which greatly limits their application. In the present study, barley proteins were enzymolyzed to fabricate nanomicelles and then applied to encapsulate hydrophobic bioactive ingredient. RESULTS: Self-assembled barley peptides could be obtained by controllable enzymolysis of barley proteins. The obtained barley peptides could self-assemble into nanomicelles (BPNMs) with a diameter of approximately 90 nm when the concentration was > 2.1 µg mL-1. Hydrophobic interaction, disulfide bonds and hydrogen bonds were involved in maintaining the structure of BPNMs. Six self-assembled peptides (QQPFPQ, QTPLPQ, QLPQIPE, QPFPQQPQLPH, QPFPQQPPFGL and QPFPQQPPFWQQQ) were identified and they were characterized by alternating arrangement of hydrophobic amino acids and hydrophilic amino acids. Moreover, BPNMs were utilized to encapsulate hydrophobic bioactive ingredient quercetin. When quercetin was encapsulated by BPNMs, its water solubility was significantly increased, being approximately 30-fold higher than free quercetin. Meanwhile, encapsulation of BPNMs could greatly increase quercetin stability. The interaction between BPNMs and quercetin occurred spontaneously, mainly driven by van der Waals forces and hydrogen bonds. CONCLUSION: In the present study, BPNMs were successfully developed and could be used as a promising delivery system to improve the water solubility and stability of hydrophobic bioactive ingredients. © 2024 Society of Chemical Industry.

Clustering of the Membrane Protein by Molecular Self-Assembly Downregulates the Signaling Pathway for Cancer Cell Inhibition.

This work reports a cyclic peptide appended self-assembled scaffold that recognizes the membrane protein EGFR and arrests the EGFR signaling through multivalent interactions by assembly-induced aggregation. When incubated with cells, the oligomers of PAD-1 first recognize the overexpressed EGFR on cancer cell membranes for arresting EGFR, which then initiates cellular uptake through endocytosis. The accumulation of PAD-1 and EGFR in the lysosome results in the formation of nanofibers, leading to the lysosomal membrane permeabilization (LMP). These processes disrupt the homeostasis of EGFR and inhibit the downstream signaling transduction of EGFR for cancer cell survival. Moreover, LMP induced the release of protein aggregates that could generate endoplasmic reticulum (ER) stress, resulting in cancer cell death selectively. In vivo studies indicate the efficient antitumor efficiency of PAD-1 in tumor-bearing mice. As a first example, this work provides an alternative strategy for controlling protein behavior for tuning cellular events in living cells.

On-Demand Elongation of Peptide Nanofibrils at Cellular Interfaces to Modulate Cell-Cell Interactions.

Natural cells can achieve specific cell-cell interactions by enriching nonspecific binding molecules on demand at intercellular contact faces, a pathway currently beyond synthetic capabilities. We are inspired to construct responsive peptide fibrils on cell surfaces, which elongate upon encountering target cells while maintaining a short length when contacting competing cells, as directed by a strand-displacement reaction arranged on target cell surfaces. With the display of ligands that bind to both target and competing cells, the contact-induced, region-selective fibril elongation selectively promotes host-target cell interactions via the accumulation of nonspecific ligands between matched cells. This approach is effective in guiding natural killer cells, the broad-spectrum effector lymphocytes, to eliminate specific cancer cells. In contrast to conventional methods relying on target cell-specific binding molecules for the desired cellular interactions, this dynamic scaffold-based approach would broaden the scope of cell combinations for manipulation and enhance the adjustability of cell behaviors for future applications.

Design of parallel 𝛽-sheet nanofibrils using Monte Carlo search, coarse-grained simulations, and experimental testing.

Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel ß-sheets", that is, ß-sheets with N-terminus-to-C-terminus 𝛽-strand vectors oriented in parallel. To accomplish the desired ß-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-ß spine defined by Sawaya et al. This molecular configuration includes two layers of parallel ß-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent ß-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel 𝛽-sheet arrangement, two formed fibril-like structure with <50% in parallel 𝛽-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel ß-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.

Enzyme-Instructed Photoactivatable Supramolecular Antigens on Cancer Cell Membranes for Precision-Controlled T-Cell-Based Cancer Immunotherapy.

Cancer immunotherapies based on cytotoxic CD8+ T lymphocytes (CTLs) are highly promising for cancer treatment. The specific interaction between T-cell receptors and peptide-MHC-I complexes (pMHC-I) on cancer cell membranes critically determines their therapeutic outcomes. However, the lack of appropriate endogenous antigens for MHC-I presentation disables tumor recognition by CTLs. By devising three antigen-loaded self-assembling peptides of pY-K(Ag)-ERGD, pY-K(Ag)-E, and Y-K(Ag)-ERGD to noncovalently generate light-activatable supramolecular antigens at tumor sites in different manners, we report pY-K(Ag)-ERGD as a promising candidate to endow tumor cells with pMHC-I targets on demand. Specifically, pY-K(Ag)-ERGD first generates low-antigenic supramolecular antigens on cancer cell membranes, and a successive light pulse allows antigen payloads to efficiently release from the supramolecular scaffold, directly producing antigenic pMHC-I. Intravenous administration of pY-K(Ag)-ERGD enables light-controlled tumor inhibition when combined with adoptively transferred antigen-specific CTLs. Our strategy is feasible for broadening tumor antigen repertoires for T-cell immunotherapies and advancing precision-controlled T-cell immunotherapies.

Quest for New Generation Biocompatible Materials: Tailoring ß-Peptide Structure and Interactions via Synergy of Experiments and Modelling.

Peptide-based self-assembly has been used to produce a wide range of nanostructures. While most of these systems involve self-assembly of α-peptides, more recently ß-peptides have also been shown to undergo supramolecular self-assembly, and have been used to produce materials for applications in tissue engineering, cell culture and drug delivery. In order to engineer new materials with specific structure and function, theoretical molecular modelling can provide significant insights into the collective balance of non-covalent interactions that drive the self-assembly and determine the structure of the resultant supramolecular materials under different conditions. However, this approach has only recently become feasible for peptide-based self-assembled nanomaterials, particularly those that incorporate non α-amino acids. This perspective provides an overview of the challenges associated with computational modelling of the self-assembly of ß-peptides and the recent success using a combination of experimental and computational techniques to provide insights into the self-assembly mechanisms and fully atomistic models of these new biocompatible materials.

pH-responsive drug-loaded peptides enhance drug accumulation and promote apoptosis in tumor cells.

The efficacy of chemotherapeutic drugs in tumor treatment is limited by their toxicity and side effects due to their inability to selectively accumulate in tumor tissue. In addition, chemotherapeutic agents are easily pumped out of tumor cells, resulting in their inadequate accumulation. To overcome these challenges, a drug delivery system utilizing the amphiphilic peptide Pep1 was designed. Pep1 can self-assemble into spherical nanoparticles (PL/Pep1) and encapsulate paclitaxel (PTX) and lapatinib (LAP). PL/Pep1 transformed into nanofibers in an acidic environment, resulting in longer drug retention and higher drug concentrations within tumor cells. Ultimately, PL/Pep1 inhibited tumor angiogenesis and enhanced tumor cell apoptosis. The use of shape-changing peptides as drug carriers to enhance cancer cell apoptosis is promising.

A method for in situ self-assembly of the catalytic peptide in enzymatic compartments of glucan particles.

Drawing inspiration from cellular compartmentalization, enzymatic compartments play a pivotal role in bringing enzymes and substrates into confined environments, offering heightened catalytic efficiency and prolonged enzyme lifespan. Previously, we engineered bioinspired enzymatic compartments, denoted as TPE-Q18H@GPs, achieved through the spatiotemporally controllable self-assembly of the catalytic peptide TPE-Q18H within hollow porous glucan particles (GPs). This design strategy allows substrates and products to freely traverse, while retaining enzymatic aggregations. The confined environment led to the formation of catalytic nanofibers, resulting in enhanced substrate binding affinity and a more than two-fold increase in the second-order kinetic constant (kcat/Km) compared to TPE-Q18H nanofibers in a dispersed system. In this work, we will introduce how to synthesize the above-mentioned enzymatic compartments using salt-responsive catalytic peptides and GPs.

Metal co-factors to enhance catalytic activity of short prion-derived peptide sequences.

Development of biomolecular enzyme mimics to efficiently catalyse biochemical reactions are of prime relevance for the bulk scale production of industrially relevant biocatalyst. In this regard, amyloidogenic peptides act as suitable self-assembling scaffolds, providing stable nanostructures with high surface area facilitating biocatalysis. Herein, we rationally design two positional amyloidogenic peptide isomers, "Fmoc-VYYAHH (1)" and "Fmoc-VHHAYY (2)" considering catalytic and metal binding affinity of histidine and tyrosine when placed in periphery vs. inner core of the peptide sequence. With an ultimate objective of designing metalloenzyme mimic, we choose Co2+ and Cu2+ as divalent transition metal cations for peptide complexation to aid in catalysis. After optimizing self-assembly of innate peptides, we investigate metal-peptide binding ratio and co-ordination, finally selecting 1:1 peptide metal complex suitable for biocatalysis. Metallopeptides act as better catalysts than the innate peptides as acyl esterase when tyrosines were present at the periphery. Kinetic parameters for assessing hydrolysis rate were calculated by fitting data into Michaelis-Menten and Lineweaver Burk plots. Catalytic activity is altered depending on the stability of peptide metal complexes. 2-Cu acting as the best biocatalyst with a kcat/KM = 0.08 M/s. The protocols mentioned in this chapter meticulously cover the design, synthesis, self-assembly and enzyme kinetics.

Redesign of a thioflavin-T-binding protein with a flat ß-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.

Effects of amyloid-ß-mimicking peptide hydrogel matrix on neuronal progenitor cell phenotype.

Self-assembling peptide-based hydrogels have become a highly attractive scaffold for three-dimensional (3D) in vitro disease modeling as they provide a way to create tunable matrices that can resemble the extracellular matrix (ECM) of various microenvironments. Alzheimer's disease (AD) is an exceptionally complex neurodegenerative condition; however, our understanding has advanced due to the transition from two-dimensional (2D) to 3D in vitro modeling. Nonetheless, there is a current gap in knowledge regarding the role of amyloid structures, and previously developed models found long-term difficulty in creating an appropriate model involving the ECM and amyloid aggregates. In this report, we propose a multi-component self-assembling peptide-based hydrogel scaffold to mimic the amyloid-beta (ß) containing microenvironment. Characterization of the amyloid-ß-mimicking hydrogel (Col-HAMA-FF) reveals the formation of ß-sheet structures as a result of the self-assembling properties of phenylalanine (Phe, F) through π-π stacking of the residues, thus mimicking the amyloid-ß protein nanostructures. We investigated the effect of the amyloid-ß-mimicking microenvironment on healthy neuronal progenitor cells (NPCs) compared to a natural-mimicking matrix (Col-HAMA). Our results demonstrated higher levels of neuroinflammation and apoptosis markers when NPCs were cultured in the amyloid-like matrix compared to a natural brain matrix. Here, we provided insights into the impact of amyloid-like structures on NPC phenotypes and behaviors. This foundational work, before progressing to more complex plaque models, provides a promising scaffold for future investigations on AD mechanisms and drug testing. STATEMENT OF SIGNIFICANCE: In this study, we engineered two multi-component hydrogels: one to mimic the natural extracellular matrix (ECM) of the brain and one to resemble an amyloid-like microenvironment using a self-assembling peptide hydrogel. The self-assembling peptide mimics ß-amyloid fibrils seen in amyloid-ß protein aggregates. We report on the culture of neuronal progenitor cells within the amyloid-mimicking ECM scaffold to study the impact through marker expressions related to inflammation and DNA damage. This foundational work, before progressing to more complex plaque models, offers a promising scaffold for future investigations on AD mechanisms and drug testing.

Impact of glycan nature on structure and viscoelastic properties of glycopeptide hydrogels.

Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics.

Guiding Transient Peptide Assemblies with Structural Elements Embedded in Abiotic Phosphate Fuels.

Despite great progress in the construction of non-equilibrium systems, most approaches do not consider the structure of the fuel as a critical element to control the processes. Herein, we show that the amino acid side chains (A, F, Nal) in the structure of abiotic phosphates can direct assembly and reactivity during transient structure formation. The fuels bind covalently to substrates and subsequently influence the structures in the assembly process. We focus on the ways in which the phosphate esters guide structure formation and how structures and reactivity cross regulate when constructing assemblies. Through the chemical functionalization of energy-rich aminoacyl phosphate esters, we are able to control the yield of esters and thioesters upon adding dipeptides containing tyrosine or cysteine residues. The structural elements around the phosphate esters guide the lifetime of the structures formed and their supramolecular assemblies. These properties can be further influenced by the peptide sequence of substrates, incorporating anionic, aliphatic and aromatic residues. Furthermore, we illustrate that oligomerization of esters can be initiated from a single aminoacyl phosphate ester incorporating a tyrosine residue (Y). These findings suggest that activated amino acids with varying reactivity and energy contents can pave the way for designing and fabricating structured fuels.

Self-Assembled Food Peptides: Recent Advances and Perspectives in Food and Health Applications.

Self-assembling peptides are rapidly gaining attention as novel biomaterials for food and biomedical applications. Peptides self-assemble when triggered by physical or chemical factors due to their versatile physicochemical characteristics. Peptide self-assembly, when combined with the health-promoting bioactivity of peptides, can also result in a plethora of biofunctionalities of the biomaterials. This perspective highlights current developments in the use of food-derived self-assembling peptides as biomaterials, bioactive nutraceuticals, and potential dual functioning bioactive biomaterials. Also discussed are the challenges and opportunities in the use of self-assembling bioactive peptides in designing biocompatible, biostable, and bioavailable multipurpose biomaterials.

Two-Dimensional Layered Nanomaterials Steering Self-Assembly of Dodecapeptides with Three Building Blocks.

Self-assembly of peptides on layered nanomaterials such as graphite and MoS2 in the formation of long-range ordered two-dimensional nanocrystal patterns leading to its potential applications for biosensing and bioelectronics has attracted significant interest in nanoscience and nanotechnology. However, controlling the self-assembly of peptides on nanomaterials is still challenging due to the unclear role of nanomaterials in steering self-assembly. Here, we used the in-situ AFM technique to capture different changes of peptide coverage as well as lengthening and widening rates depending on peptide concentrations, show the distinct boundary dynamics of two stabilized peptide domains, and resolve the molecular resolution structural differences and specific orientation of peptide on both nanomaterials. Moreover, ex-situ results showed that the nanomaterial layers tuned the opposite changes of nanowire heights and densities and displayed the different water-resistance stabilities on both nanomaterials. This work provides a basis for understanding nanomaterials steering peptide self-assembly and using hybrid bionanomaterials as a scaffold, enabling for potential biosensing and bioelectronics applications.

Co-Assembly of Cancer Drugs with Cyclo-HH Peptides: Insights from Simulations and Experiments.

Peptide-based nanomaterials can serve as promising drug delivery agents, facilitating the release of active pharmaceutical ingredients while reducing the risk of adverse reactions. We previously demonstrated that Cyclo-Histidine-Histidine (Cyclo-HH), co-assembled with cancer drug Epirubicin, zinc, and nitrate ions, can constitute an attractive drug delivery system, combining drug self-encapsulation, enhanced fluorescence, and the ability to transport the drug into cells. Here, we investigated both computationally and experimentally whether Cyclo-HH could co-assemble, in the presence of zinc and nitrate ions, with other cancer drugs with different physicochemical properties. Our studies indicated that Methotrexate, in addition to Epirubicin and its epimer Doxorubicin, and to a lesser extent Mitomycin-C and 5-Fluorouracil, have the capacity to co-assemble with Cyclo-HH, zinc, and nitrate ions, while a significantly lower propensity was observed for Cisplatin. Epirubicin, Doxorubicin, and Methorexate showed improved drug encapsulation and drug release properties, compared to Mitomycin-C and 5-Fluorouracil. We demonstrated the biocompatibility of the co-assembled systems, as well as their ability to intracellularly release the drugs, particularly for Epirubicin, Doxorubicin, and Methorexate. Zinc and nitrate were shown to be important in the co-assembly, coordinating with drugs and/or Cyclo-HH, thereby enabling drug-peptide as well as drug-drug interactions in successfully formed nanocarriers. The insights could be used in the future design of advanced cancer therapeutic systems with improved properties.