A Nucleus-Targeting WT1 Antagonistic Peptide Encapsulated in Polymeric Nanomicelles Combats Refractory Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is recognized as a classic clonal myeloproliferative disorder. Given the limited treatment options for CML patients in the accelerated phase (AP) and blast phase (BP), there is an evident need to develop new therapeutic strategies. This has the potential to improve outcomes for individuals in the advanced stages of CML. A promising therapeutic target is Wilms' tumor 1 (WT1), which is highly expressed in BP-CML cells and plays a crucial role in CML progression. In this study, a chemically synthesized nucleus-targeting WT1 antagonistic peptide termed WIP2W was identified. The therapeutic implications of both the peptide and its micellar formulation, M-WIP2W, were evaluated in WT1+ BP-CML cell lines and in mice. The findings indicate that WIP2W can bind specifically to the WT1 protein, inducing cell cycle arrest and notable cytotoxicity in WT1+ BP-CML cells. Moreover, subcutaneous injections of M-WIP2W were observed to significantly enhance intra-tumoral accumulation and to effectively inhibit tumor growth. Thus, WIP2W stands out as a potent and selective WT1 inhibitor, and the M-WIP2W nanoformulation appears promising for the therapeutic treatment of refractory CML as well as other WT1-overexpressing malignant cancers.

Nanomicelles co-loading CXCR4 antagonist and doxorubicin combat the refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is featured with poor prognosis and high mortality, because chemo-resistance, nonspecific distribution and dose-limiting toxicity lead to a high rate of relapse and a very low 5-year survival percentage of less than 25%. CXCR4 is a highly expressed chemokine receptor in multiple types of AML cells and closely associated with the drug resistance and relapse. In this work, we integrate a chemically synthesized CXCR4 antagonistic peptide and doxorubicin using DSPE-mPEG2000 micelles (referred to as M-E5-Dox) that is applied to a very challenging refractory AML mouse model as well as human AML cell lines. Results showed that M-E5-Dox can effectively bind to the CXCR4-expressing AML cells, downregulating the signaling proteins mediated by CXCR4/CXCL12 axis and increasing the cellular uptake of Dox. Importantly, M-E5-Dox remarkably decreases the leukemic cells in the peripheral blood and bone marrow, as well as their infiltration in the spleen and liver of the AML mice, which in turn prolongs the survival significantly. Meanwhile, M-E5-Dox did not increase the cardiotoxicity of Dox. In conclusion, M-E5-Dox harnesses the functions of CXCR4 specific binding and CXCR4 antagonism of the peptide and the tumor cell killing capacity of Dox, which displays significant therapeutic effects and promising translational potentials for the treatment of refractory AML.

CD123 Antagonistic Peptides Assembled with Nanomicelles Act as Monotherapeutics to Combat Refractory Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Due to the development of drug resistance to traditional chemotherapies and high relapse rate, AML still has a low survival rate and there is in an urgent need for better treatment strategies. CD123 is widely expressed by AML cells, also associated with the poor prognosis of AML. In this study, we fabricated nanomicelles loaded with a lab-designed CD123 antagonistic peptide, which were referred to as mPO-6. The antagonistic and therapeutic effects were investigated with CD123+ AML cell lines and a refractory AML mouse (AE and CKITD816V) model. Results show that mPO-6 can specifically bind to the CD123+ AML cells and inhibit the cell viability effectively. Intravenous administration of mPO-6 significantly reduces the percentage of AML cells' infiltration and prolongs the median survival of AML mice. Further, the efficiency of mPO-6 is demonstrated to interfere with the axis of CD123/IL-3 via regulating the activation of STAT5, PI3K/AKT, and NF-κB signaling pathways related to cell proliferation or apoptosis at the level of mRNA and protein in vivo and in vitro. In conclusion, the novel CD123 antagonistic peptide micelle formulation mPO-6 can significantly enhance apoptosis and prolong the survival of AML mice by effectively interfering with the axis of CD123/IL-3 and therefore is a promising therapeutic candidate for the treatment of refractory AML.

Poroptosis: A form of cell death depending on plasma membrane nanopores formation.

Immunogenic cell death (ICD) in malignant cells can decrease tumor burden and activate antitumor immune response to obtain lasting antitumor immunity, leading to the elimination of distant metastases and prevention of recurrence. Here, we reveal that ppM1 peptide is capable of forming irreparable transmembrane pores on tumor cell membrane, leading to ICD which we name poroptosis. Poroptosis is directly dependent on cell membrane nanopores regardless of the upstream signaling of cell death. ppM1-induced poroptosis was characterized by the sustained release of intracellular LDH. This unique feature is distinct from other well-characterized types of acute necrosis induced by freezing-thawing (F/T) and detergents, which leads to the burst release of intracellular LDH. Our results suggested that steady transmembrane-nanopore-mediated subacute cell death played a vital role in subsequent activated immunity that transforms to an antitumor immune microenvironment. Selectively generating poroptosis in cancer cell could be a promise strategy for cancer therapy.

A nucleus-targeting peptide antagonist towards EZH2 displays therapeutic efficacy for lung cancer.

EZH2 is an overexpressed nuclear protein associated with relatively poor survival and chemoresistance in lung cancer. In this study, a nucleus-targeting peptide antagonist EIP103 capable of penetrating cell membrane and nuclear envelope was identified, and has high binding affinity towards EZH2 localized in the nucleus of lung cancer cells. To improve the stability and therapeutic efficacy of EIP103, PEG-PE micelle encapsulated EIP103 (M-EIP103) was successfully conducted. In vitro results indicated that M-EIP103 exhibited better stability, higher intracellular uptake and stronger cytotoxicity than free EIP103 in H446 and A549 cells. Mechanistic studies suggested that M-EIP103 inhibited proliferation by down-regulating the H3K27me3 expression level in cancer cells. In vivo assays further confirmed that both EIP103 and M-EIP103 significantly inhibited lung cancer progression. Notably, enhanced therapeutic efficacy of EIP103 by PEG-PE micelle encapsulation could be identified. The observed anti-tumor activity of EIP103 and M-EIP103 demonstrated a promising therapy to improve clinical treatment of lung cancers as well as other EZH2-overexpressing malignant cancers. This study also illustrates the feasibility of developing targeted delivery of therapeutic peptides to nucleus for cancer therapy.

Principles of Amino-Acid-Nucleotide Interactions Revealed by Binding Affinities between Homogeneous Oligopeptides and Single-Stranded DNA Molecules.

We have determined the binding strengths between nucleotides of adenine, thymine, guanine and cytosine in homogeneous single stranded DNAs and homo-octapeptides consisting of 20 common amino acids. We use a bead-based fluorescence assay for these measurements in which octapeptides are immobilized on the bead surface and ssDNAs are in solutions. Comparative analyses of the distribution of the binding energies reveal unique binding strength patterns assignable to each DNA nucleotide and amino acid originating from the chemical structures. Pronounced favorable (such as Arg-G, etc.) and unfavorable (such as Ile-T, etc.) binding interactions can be identified in selected groups of amino acid and nucleotide pairs that could provide basis to elucidate energetics of amino-acid-nucleotide interactions. Such interaction selectivity, specificity and polymorphism establish the contributions from DNA backbone, DNA bases, as well as main chain and side chain of the amino acids.

Peptide-based coacervates in therapeutic applications.

Coacervates are droplets formed by liquid‒liquid phase separation. An increasing number of studies have reported that coacervates play an important role in living cells, such as in the generation of membraneless organelles, and peptides contribute to condensate droplet formation. Peptides with versatile functional groups and special secondary structures, including α-helices, ß-sheets and intrinsically disordered regions, provide novel insights into coacervation, such as biomimetic protocells, neurodegenerative diseases, modulations of signal transmission, and drug delivery systems. In this review, we introduce different types of peptide-based coacervates and the principles of their interactions. Additionally, we summarize the thermodynamic and kinetic mechanisms of peptide-based coacervates and the associated factors, including salt, pH, and temperature, affecting the phase separation process. We illustrate recent studies on modulating the functions of peptide-based coacervates applied in biological diseases. Finally, we propose their promising broad applications and describe the challenges of peptide-based coacervates in the future.

A novel CD123-targeted therapeutic peptide loaded by micellar delivery system combats refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival rate due to the refractory feature and high rate of relapse. CD123 is highly expressed on multiple types of AML cells, especially leukemia stem cells, and closely associated with the poor prognosis of AML. Aiming to meet the urgent demand to targeted therapeutics for the refractory AML patients, herein we synthesize a CD123 antagonistic peptide (PO-6) loaded in nanomicelles (mPO-6), and investigated its therapeutic effect and pharmacokinetics on a lab-established refractory AML mice model (AE & CKITD816V). It is shown that the PO-6 can effectively bind to the CD123+ AML cells and the micellar formulation mPO-6 increases the dissolution stability and the specific binding capacity. When injected intravenously, mPO-6 significantly prolongs the survival of the refractory AML mice by interfering CD123/IL-3 axis, evidenced by the down regulation of phosphorylation of STAT5 and PI3K/AKT and the inhibition of activated NF-κB in the nucleus, as well as by the analysis results of next generation RNA-sequencing (RNA-seq) with the bone marrow of the AML mice. The antagonistic effect leads to the significantly reduction of AML cells infiltration in the bone marrow of the AML mice. In conclusion, mPO-6 could provide a potent antagonistic therapeutic approach for targeted treatment of AML.

Synthetic Neutralizing Peptides Inhibit the Host Cell Binding of Spike Protein and Block Infection of SARS-CoV-2.

Antiviral treatments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been extensively pursued to conquer the pandemic. To inhibit the viral entry to the host cell, we designed and obtained three peptide sequences via quartz crystal microbalance measurement screening, which showed high affinity at nanomole to the S1 subunit of the spike protein and wild-type SARS-CoV-2 pseudovirus. Circular dichroism spectroscopy measurements revealed significant conformation changes of the S1 protein upon encounter with the three peptides. The peptides were able to effectively block the infection of a pseudovirus to 50% by inhibiting the host cell lines binding with the S1 protein, evidenced by the results from Western blotting and pseudovirus luciferase assay. Moreover, the combination of the three peptides could increase the inhibitory rate to 75%. In conclusion, the three chemically synthetic neutralizing peptides and their combinations hold promising potential as effective therapeutics in the prevention and treatment of COVID-19.

Peptide-Enabled Targeted Delivery Systems for Therapeutic Applications.

Receptor-targeting peptides have been extensively pursued for improving binding specificity and effective accumulation of drugs at the site of interest, and have remained challenging for extensive research efforts relating to chemotherapy in cancer treatments. By chemically linking a ligand of interest to drug-loaded nanocarriers, active targeting systems could be constructed. Peptide-functionalized nanostructures have been extensively pursued for biomedical applications, including drug delivery, biological imaging, liquid biopsy, and targeted therapies, and widely recognized as candidates of novel therapeutics due to their high specificity, well biocompatibility, and easy availability. We will endeavor to review a variety of strategies that have been demonstrated for improving receptor-specificity of the drug-loaded nanoscale structures using peptide ligands targeting tumor-related receptors. The effort could illustrate that the synergism of nano-sized structures with receptor-targeting peptides could lead to enrichment of biofunctions of nanostructures.

Enhancement of gold-nanocluster-mediated chemotherapeutic efficiency of cisplatin in lung cancer.

A novel delivery system for cisplatin was constructed based on electrostatics-mediated assemblies of gold nanoclusters and PEGylated cationic peptide (cisplatin@GC-pKs). Encapsulated cisplatin in the as-formed micelle like assemblies was observed to demonstrate improved cellar uptake and enhanced chemotherapeutic efficiency in the cisplatin-resistant lung cancer cells. In vivo assays further confirmed that cisplatin@GC-pKs had profound anti-tumor efficiency due to deep penetration and accumulation of nanoscale cisplatin@GC-pKs via the enhanced permeability and retention (EPR) effect at tumor tissues. The constructed cisplatin@GC-pKs in this work demonstrated enhanced anti-tumor activity for lung cancer therapy, as well as a potential treatment strategy for a variety of cisplatin-resistance related malignancies.

Composition-dependent multivalency of peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We have examined in this contribution the composition dependence of binding characteristics in peptide-peptide interactions between an oligopeptide octa-glycine and a series of tryptophan-containing octapeptides. The binding energy associated with tryptophan-glycine interactions manifests pronounced stepwise binding characteristics as the number of tryptophan increases from 0 to 8 in the octapeptides consisting only of glycine and can be attributed to mono-, di-, and tri-valent peptide-peptide interactions. At the same time, only weak fluctuations in binding energy were observed as the number of tryptophan increases from 2 to 7. Such distinctive nonlinearity of composition-dependent tryptophan-glycine binding energy characteristics due to continuously varying tryptophan compositions in the octapeptides could be considered as a reflection of combinatorial contributions due to the hydrogen bonds originated from the indole moieties of tryptophan with the main chains of octapeptide of glycine containing N-H and C=O moieties and the van der Waals interactions (including π-π and π-CH interactions) between peptides.

Enhanced lymphatic delivery of nanomicelles encapsulating CXCR4-recognizing peptide and doxorubicin for the treatment of breast cancer.

Lymph node metastases in cancer patients are associated with high aggressiveness, poor prognosis, and short survival time. The chemokine receptor 4 (CXCR4)/stroma derived factor 1α (CXCL12) biological axis plays a critical role in the spread of cancer cells. Designing effective delivery systems that can successfully deliver CXCR4 antagonists to lymph nodes, which are rich in CXCR4-overexpressing cancer cells, for controlling cancer metastasis remain challenging. In this study, we demonstrated that such a challenge may be alleviated by developing nanometer-sized polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles for the co-delivery of the CXCR4 antagonistic peptide E5 and doxorubicin (M-E5-Dox). This nanomicelle platform enables the preferential accumulation of cargos into lymph nodes and thus can better inhibit cancer metastasis and enhance antitumor efficacy than either free drugs or single drug-loaded micelles in breast cancer-bearing mouse models. Hence, M-E5-Dox is expected to be a potential therapeutic agent that would improve the clinical benefits of breast cancer therapy and treatment of various CXCR4-overexpressing malignancies.

Synthetic CXCR4 Antagonistic Peptide Assembling with Nanoscaled Micelles Combat Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common adult acute leukemia with very low survival rate due to drug resistance and high relapse rate. The C-X-C chemokine receptor 4 (CXCR4) is highly expressed by AML cells, actively mediating chemoresistance and reoccurrence. Herein, a chemically synthesized CXCR4 antagonistic peptide E5 is fabricated to micelle formulation (M-E5) and applied to refractory AML mice, and its therapeutic effects and pharmacokinetics are investigated. Results show that M-E5 can effectively block the surface CXCR4 in leukemic cells separated from bone marrow (BM) and spleen, and inhibit the C-X-C chemokine ligand 12-mediated migration. Subcutaneous administration of M-E5 significantly inhibits the engraftment of leukemic cells in spleen and BM, and mobilizes residue leukemic cells into peripheral blood, reducing organs' burden and significantly prolonging the survival of AML mice. M-E5 can also increase the efficacy of combining regime of homoharringtonine and doxorubicin. Ribonucleic acid sequencing demonstrates that the therapeutic effect is contributed by inhibiting proliferation and enhancing apoptosis and differentiation, all related to the CXCR4 signaling blockade. M-E5 reaches the concentration peak at 2 h after administration with a half-life of 14.5 h in blood. In conclusion, M-E5 is a novel promising therapeutic candidate for refractory AML treatment.

Position-coded multivalent peptide-peptide interactions revealed by tryptophan-scanning mutagenesis.

We demonstrate in this contribution the evidence that significant cooperative binding effect can be identified for the amino acid sites that are determinant to the binding characteristics in peptide-peptide interactions. The analysis of tryptophan-scanning mutagenesis of the 14-mer peptide consisting only of glycine provides a mapping of position-dependent contributions to the binding energy. The pronounced tryptophan-associated peptide-peptide interactions are originated from the indole moieties with the main chains of 14-mer glycines containing N-H and CO moieties. Specifically, with the presence of two tryptophans as determinant amino acids, cooperative binding can be observed, which are dependent on relative positions of the two tryptophans with a "volcano"-like characteristics. An optimal separation of 6-10 amino acids between two adjacent binding sites can be identified to achieve maximal binding interactions.

Peptide-enabled receptor-binding-quantum dots for enhanced detection and migration inhibition of cancer cells.

We report the efforts to construct active targeting quantum dots using receptor-binding peptide for enhanced detection and migration inhibition of cancer cells. Peptide E5 has specific binding with chemokine receptor 4 (CXCR4), which is a transmembrane G-coupled receptor involved in the metastasis of various types of cancers. E5 was introduced to the surface of CdSe/ZnS quantum dots via biotin-streptavidin interactions. The constructed CXCR4-targeting quantum dots (E5@QDs) was observed to display improved detection sensitivity and significantly enhanced binding affinity for CXCR4 over-expressed cancer cells, and the ability to inhibit cancer cells migration induced by CXCL12.

Modulation of ß-amyloid aggregation by graphene quantum dots.

Misfolding and abnormal aggregation of ß-amyloid peptide is associated with the onset and progress of Alzheimer's disease (AD). Therefore, modulating ß-amyloid aggregation is critical for the treatment of AD. Herein, we studied the regulatory effects and mechanism of graphene quantum dots (GQDs) on 1-42 ß-amyloid (Aß1-42) aggregation. GQDs displayed significant regulatory effects on the aggregation of Aß1-42 peptide as detected by thioflavin T (ThT) assay. Then, the changes of confirmations and structures induced by GQDs on the Aß1-42 aggregation were monitored by circular dichroism (CD), dynamic light scattering (DLS) and transmission electron microscope (TEM). The in vitro cytotoxicity experiments further demonstrated the feasibility of GQDs on the regulation of Aß1-42 aggregation. Meanwhile, the structural changes of a Aß1-42/GQDs mixture in different pH revealed that electrostatic interaction was the major driving force in the co-assembly process of Aß1-42 and GQDs. The proposed mechanism of the regulatory effects of GQDs on the Aß1-42 aggregation was also deduced reasonably. This work not only demonstrated the potential feasibility of GQDs as therapeutic drug for AD but also clarified the regulatory mechanism of GQDs on the Aß1-42 aggregation.

Principles of Inter-Amino-Acid Recognition Revealed by Binding Energies between Homogeneous Oligopeptides.

We have determined the interaction strengths of the common naturally occurring amino acids using a complete binding affinity matrix of 20 × 20 pairs of homo-octapeptides consisting of the 20 common amino acids between stationary and mobile states. We used a bead-based fluorescence assay for these measurements. The results provide a basis for analyzing specificity, polymorphisms, and selectivity of inter-amino-acid interactions. Comparative analyses of the binding energies, i.e., the free energies of association (ΔG A), reveal contributions assignable to both main-chain-related and side-chain-related interactions originating from the chemical structures of these 20 common amino acids. Side-chain-side-chain and side-chain-main-chain interactions are found to be pronounced in an identified set of amino acid pairs that determine the basis of inter-amino-acid recognition.

Porous Eleocharis@MnPE Layered Hybrid for Synergistic Adsorption and Catalytic Biodegradation of Toxic Azo Dyes from Industrial Wastewater.

The effective treatment of industrial wastewater to protect freshwater reserves for the survival of life is a primary focus of current research. Herein, a multicomponent Eleocharis-manganese peroxidase enzyme (Eleocharis@MnPE) layered hybrid with high surface area (1200 m2/m3), with a strong synergistic adsorption and catalytic biodegradation (SACB), has been developed through a facile method. A combination of outer porous (Eleocharis) and inner catalytically active (MnPE) components of the hybrid resulted in highly efficient SACB system, evidenced by high removal rate of 15 kg m-3 day-1 (100%) and complete degradation of toxic Orange II (OR) azo dye into nontoxic products (gases and weak acids). The Eleocharis@MnPE layered hybrid efficiently degraded both OR in synthetic wastewater and also other azo dyes (red, pink, and yellow dyes) present in three different textile industrial effluents. For the industrial effluents, these were evidenced by the color disappearance and reduction in biological oxygen demand (BOD), chemical oxygen demand (COD), and total organic carbon (TOC) of up to 97%, 92%, and 76%, respectively. Furthermore, reduced toxicity of treated wastewater was confirmed by decreased cell toxicity to 0.1%-1% and increased cell viability to 90%. We believe that designing a hybrid system with strong ability of SACB could be highly effective for industrial-scale treatment of wastewater.

Correction: Improving the inhibitory effect of CXCR4 peptide antagonist in tumor metastasis with an acetylated PAMAM dendrimer.

[This corrects the article DOI: 10.1039/C8RA08526A.].