Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation.

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.

Brain Targeting Nanomedicines: Pitfalls and Promise.

Brain diseases are the most devastating problem among the world's increasingly aging population, and the number of patients with neurological diseases is expected to increase in the future. Although methods for delivering drugs to the brain have advanced significantly, none of these approaches provide satisfactory results for the treatment of brain diseases. This remains a challenge due to the unique anatomy and physiology of the brain, including tight regulation and limited access of substances across the blood-brain barrier. Nanoparticles are considered an ideal drug delivery system to hard-to-reach organs such as the brain. The development of new drugs and new nanomaterial-based brain treatments has opened various opportunities for scientists to develop brain-specific delivery systems that could improve treatment outcomes for patients with brain disorders such as Alzheimer's disease, Parkinson's disease, stroke and brain tumors. In this review, we discuss noteworthy literature that examines recent developments in brain-targeted nanomedicines used in the treatment of neurological diseases.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Targeting triple-negative breast cancer cells with a ß1-integrin binding aptamer.

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumor heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity-targeting ligands, aptamers show high selectivity toward target molecules. Compared with antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported discovery of the GreenB1 aptamer, which is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets ß1-integrin, a protein linked previously to breast cancer cell invasiveness and migration. Aptamer binds to ß1-integrin with low nanomolar affinity. Our findings suggest potential applications for GreenB1-guided precision agents for diagnosis and therapy of cancers overexpressing ß1-integrin.

Intravitreal CendR peptides target laser-induced choroidal neovascularization sites in mice.

Choroidal neovascularization (CNV) is a common ocular pathology that may be associated in a variety of eye diseases. Although intravitreal injection treatment of anti-vascular endothelial growth factor (anti-VEGF) drugs shows significant clinical benefits in CNV treatment, the limitations of the current therapy need to be addressed. The aim of our study was to investigate the potential utility of three C-end Rule (CendR) peptides (RPARPAR, PL3, iRGD) for CNV targeting and to evaluate the efficacy of peptides for treating experimental CNV in mice. We observed that the CendR peptides localize to the CNV lesion sites after intravitreal injection and were mainly found in the outer nuclear cell layer (ONL) of the mouse retina. Interestingly, experimental therapy with tenascin-C (TNC-C) and neuropilin-1 (NRP-1)-targeting PL3 peptide, reduced angiogenesis and decreased vascular leakage. The results suggest that PL3 and potentially other CendR peptides could serve as affinity targeting ligands and therapeutics for ocular diseases that involve pathological CNV.

Targeting of Tomato Bushy Stunt Virus with a Genetically Fused C-End Rule Peptide.

Homing peptides are widely used to improve the delivery of drugs, imaging agents, and nanoparticles (NPs) to their target sites. Plant virus-based particles represent an emerging class of structurally diverse nanocarriers that are biocompatible, biodegradable, safe, and cost-effective. Similar to synthetic NPs, these particles can be loaded with imaging agents and/or drugs and functionalized with affinity ligands for targeted delivery. Here we report the development of a peptide-guided Tomato Bushy Stunt Virus (TBSV)-based nanocarrier platform for affinity targeting with the C-terminal C-end rule (CendR) peptide, RPARPAR (RPAR). Flow cytometry and confocal microscopy demonstrated that the TBSV-RPAR NPs bind specifically to and internalize in cells positive for the peptide receptor neuropilin-1 (NRP-1). TBSV-RPAR particles loaded with a widely used anticancer anthracycline, doxorubicin, showed selective cytotoxicity on NRP-1-expressing cells. Following systemic administration in mice, RPAR functionalization conferred TBSV particles the ability to accumulate in the lung tissue. Collectively, these studies show the feasibility of the CendR-targeted TBSV platform for the precision delivery of payloads.

Paclitaxel-Loaded Cationic Fluid Lipid Nanodiscs and Liposomes with Brush-Conformation PEG Chains Penetrate Breast Tumors and Trigger Caspase-3 Activation.

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.

SARS-CoV-2 infects adipose tissue in a fat depot- and viral lineage-dependent manner.

Visceral adiposity is a risk factor for severe COVID-19, and a link between adipose tissue infection and disease progression has been proposed. Here we demonstrate that SARS-CoV-2 infects human adipose tissue and undergoes productive infection in fat cells. However, susceptibility to infection and the cellular response depends on the anatomical origin of the cells and the viral lineage. Visceral fat cells express more ACE2 and are more susceptible to SARS-CoV-2 infection than their subcutaneous counterparts. SARS-CoV-2 infection leads to inhibition of lipolysis in subcutaneous fat cells, while in visceral fat cells, it results in higher expression of pro-inflammatory cytokines. Viral load and cellular response are attenuated when visceral fat cells are infected with the SARS-CoV-2 gamma variant. A similar degree of cell death occurs 4-days after SARS-CoV-2 infection, regardless of the cell origin or viral lineage. Hence, SARS-CoV-2 infects human fat cells, replicating and altering cell function and viability in a depot- and viral lineage-dependent fashion.

ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Editorial on Special Issue "Precision Delivery of Drugs and Imaging Agents with Peptides".

Homing peptides and cell-penetrating peptides allow for systemic targeting of diseased tissues and/or efficient intracellular delivery of payloads [...].

Preclinical Validation of Tumor-Penetrating and Interfering Peptides against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults. The disease is characterized by the accumulation of tumoral B cells resulting from a defect of apoptosis. We have in vitro and in vivo preclinically validated a tumor-penetrating peptide (named TT1) coupled to an interfering peptide (IP) that dissociates the interaction between the serine/threonine protein phosphatase 2A (PP2A) from its physiological inhibitor, the oncoprotein SET. This TT1-IP peptide has an antitumoral effect on CLL, as shown by the increased survival of mice bearing xenograft models of CLL, compared to control mice. The peptide did not show toxicity, as indicated by the mouse body weight and the biochemical parameters, such as renal and hepatic enzymes. In addition, the peptide-induced apoptosis in vitro of primary tumoral B cells isolated from CLL patients but not of those isolated from healthy patients. Finally, the peptide had approximately 5 h half-life in human serum and showed pharmacokinetic parameters compatible with clinical development as a therapeutic peptide against CLL.

Depletion of Mannose Receptor-Positive Tumor-associated Macrophages via a Peptide-targeted Star-shaped Polyglutamate Inhibits Breast Cancer Progression in Mice.

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Significance: A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

New Tools for Streamlined In Vivo Homing Peptide Identification.

In vivo peptide-phage display is an unbiased technique for mapping of the vascular diversity and identification of homing peptides. This chapter is intended to serve as a structured practical guide to execute in vivo T7 phage biopanning and data analysis experiments. We discuss experimental designs and protocols with emphasis on application of high-throughput sequencing-based technologies for streamlined in vivo biopanning and validation of homing peptides.

Homing Peptide-Based Targeting of Tenascin-C and Fibronectin in Endometriosis.

The current diagnostic and therapeutic strategies for endometriosis are limited. Although endometriosis is a benign condition, some of its traits, such as increased cell invasion, migration, tissue inflammation, and angiogenesis are similar to cancer. Here we explored the application of homing peptides for precision delivery of diagnostic and therapeutic compounds to endometriotic lesions. First, we audited a panel of peptide phages for the binding to the cultured immortalized endometriotic epithelial 12Z and eutopic stromal HESC cell lines. The bacteriophages displaying PL1 peptide that engages with angiogenic extracellular matrix overexpressed in solid tumors showed the strongest binding to both cell lines. The receptors of PL1 peptide, tenascin C domain C (TNC-C) and fibronectin Extra Domain-B (Fn-EDB), were expressed in both cells. Silver nanoparticles functionalized with synthetic PL1 peptide showed specific internalization in 12Z and HESC cells. Treatment with PL1-nanoparticles loaded with the potent antimitotic drug monomethyl auristatin E decreased the viability of endometriotic cells in 2D and 3D cultures. Finally, PL1-nanoparticless bound to the cryosections of clinical peritoneal endometriotic lesions in the areas positive for TNC-C and Fn-EDB immunoreactivities and not to sections of normal endometrium. Our findings suggest potential applications for PL1-guided nanoparticles in precision diagnosis and therapy of endometriosis.

PL1 Peptide Engages Acidic Surfaces on Tumor-Associated Fibronectin and Tenascin Isoforms to Trigger Cellular Uptake.

Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.

A widespread viral entry mechanism: The C-end Rule motif-neuropilin receptor interaction.

Many phylogenetically distant animal viruses, including the new coronavirus severe acute respiratory syndrome coronavirus 2, have surface proteins with polybasic sites that are cleaved by host furin and furin-like proteases. Other than priming certain viral surface proteins for fusion, cleavage generates a carboxy-terminal RXXR sequence. This C-end Rule (CendR) motif is known to bind to neuropilin (NRP) receptors on the cell surface. NRPs are ubiquitously expressed, pleiotropic cell surface receptors with important roles in growth factor signaling, vascular biology, and neurobiology, as well as immune homeostasis and activation. The CendR-NRP receptor interaction promotes endocytic internalization and tissue spreading of different cargo, including viral particles. We propose that the interaction between viral surface proteins and NRPs plays an underappreciated and prevalent role in the transmission and pathogenesis of diverse viruses and represents a promising broad-spectrum antiviral target.

Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50-100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

Bi-Functional Peptides as a New Therapeutic Tool for Hepatocellular Carcinoma.

BACKGROUND: The interfering peptides that block protein-protein interactions have been receiving increasing attention as potential therapeutic tools. METHODS: We measured the internalization and biological effect of four bi-functional tumor-penetrating and interfering peptides into primary hepatocytes isolated from three non-malignant and 11 hepatocellular carcinomas. RESULTS: These peptides are internalized in malignant hepatocytes but not in non-malignant cells. Furthermore, the degree of peptide internalization correlated with receptor expression level and tumor aggressiveness levels. Importantly, penetration of the peptides iRGD-IP, LinTT1-IP, TT1-IP, and RPARPAR-IP induced apoptosis of the malignant hepatocytes without effect on non-malignant cells. CONCLUSION: Receptor expression levels correlated with the level of peptide internalization and aggressiveness of the tumor. This study highlights the potential to exploit the expression of tumor-penetrating peptide receptors as a predictive marker of liver tumor aggressiveness. These bi-functional peptides could be developed for personalized tumor treatment.

Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics.

Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure-activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL-NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL-NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases.

P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas.

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.