In Vivo Efficacy and Toxicity of an Antimicrobial Peptide in a Model of Endotoxin-Induced Pulmonary Inflammation.

SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. Here we report two in vivo studies to assess the toxicity and efficacy of the peptide in a murine model of pulmonary inflammation. First, we present the toxicity study in which SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. We then report an efficacy study of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration.

Safety evaluations of a synthetic antimicrobial peptide administered intravenously in rats and dogs.

The antimicrobial peptide SET-M33 is under study for the development of a new antibiotic against major Gram-negative pathogens. Here we report the toxicological evaluation of SET-M33 administered intravenously to rats and dogs. Dose range finding experiments determined the doses to use in toxicokinetic evaluation, clinical biochemistry analysis, necroscopy and in neurological and respiratory measurements. Clinical laboratory investigations in dogs and rats showed a dose-related increase in creatinine and urea levels, indicating that the kidneys are the target organ. This was also confirmed by necroscopy studies of animal tissues, where signs of degeneration and regeneration were found in kidney when SET-M33 was administered at the highest doses in the two animal species. Neurological toxicity measurements by the Irwin method and respiratory function evaluation in rats did not reveal any toxic effect even at the highest dose. Finally, repeated administration of SET-M33 by short infusion in dogs revealed a no-observed-adverse-effect-level of 0.5 mg/kg/day.

Antimicrobial Peptides Active in In Vitro Models of Endodontic Bacterial Infections Modulate Inflammation in Human Cardiac Fibroblasts.

Endodontic and periodontal disease are conditions of infectious origin that can lead to tooth loss or develop into systemic hyperinflammation, which may be associated with a wide variety of diseases, including cardiovascular. Endodontic and periodontal treatment often relies on antibiotics. Since new antimicrobial resistances are a major threat, the use of standard antibiotics is not recommended when the infection is only local. Antimicrobial peptides were recently demonstrated to be valid alternatives for dental treatments. The antimicrobial peptide M33D is a tetrabranched peptide active against Gram-negative and Gram-positive bacteria. It has a long life, unusual for peptides, because its branched form provides resistance to proteases. Here the efficacy of M33D and of its analog M33i/l as antibiotics for local use in dentistry was evaluated. M33D and M33i/l were active against reference strains and multidrug-resistant clinical isolates of Gram-negative and Gram-positive species. Their minimum inhibitory concentration against different strains of dental interest was between 0.4 and 6.0 µM. Both peptides acted rapidly on bacteria, impairing membrane function. They also disrupted biofilm effectively. Disinfection of the root canal is crucial for endodontic treatments. M33D and M33i/l reduced E. faecalis colonies to one-twentieth in a dentin slices model reproducing root canal irrigation. They both captured and neutralized lipopolysaccharide (LPS), a bacterial toxin responsible for inflammation. The release of IL-1ß and TNFα by LPS-stimulated murine macrophages was reduced by both peptides. Human cardiac fibroblasts respond to different insults with the release of proinflammatory cytokines, and consequently, they are considered directly involved in atherogenic cardiovascular processes, including those triggered by infections. The presence of M33D and M33i/l at MIC concentration reduced IL6 release from LPS- stimulated human cardiac fibroblasts, hence proving to be promising in preventing bacteria-induced atherogenesis. The two peptides showed low toxicity to mammalian cells, with an EC50 one order of magnitude higher than the average MIC and low hemolytic activity. The development of antimicrobial peptides for dental irrigations and medication is a very promising new field of research that will provide tools to fight dental infections and their severe consequences, while at the same time protecting standard antibiotics from new outbreaks of antimicrobial resistance.

Inhalable Polymeric Nanoparticles for Pulmonary Delivery of Antimicrobial Peptide SET-M33: Antibacterial Activity and Toxicity In Vitro and In Vivo.

Development of inhalable formulations for delivering peptides to the conductive airways and shielding their interactions with airway barriers, thus enhancing peptide/bacteria interactions, is an important part of peptide-based drug development for lung applications. Here, we report the construction of a biocompatible nanosystem where the antimicrobial peptide SET-M33 is encapsulated within polymeric nanoparticles of poly(lactide-co-glycolide) (PLGA) conjugated with polyethylene glycol (PEG). This system was conceived for better delivery of the peptide to the lungs by aerosol. The encapsulated peptide showed prolonged antibacterial activity, due to its controlled release, and much lower toxicity than the free molecule. The peptide-based nanosystem killed Pseudomonas aeruginosa in planktonic and sessile forms in a dose-dependent manner, remaining active up to 72 h after application. The encapsulated peptide showed no cytotoxicity when incubated with human bronchial epithelial cells from healthy individuals and from cystic fibrosis patients, unlike the free peptide, which showed an EC50 of about 22 µM. In vivo acute toxicity studies in experimental animals showed that the peptide nanosystem did not cause any appreciable side effects, and confirmed its ability to mitigate the toxic and lethal effects of free SET-M33.

Identification of a Neutralizing Epitope on TOSV Gn Glycoprotein.

Emerging and re-emerging viral infections have been an important public health problem in recent years. We focused our attention on Toscana virus (TOSV), an emergent neurotropic negative-strand RNA virus of the Phenuiviridae family. The mechanisms of protection against phlebovirus natural infection are not known; however, it is supposed that a virus-neutralizing antibody response against viral glycoproteins would be useful to block the first stages of infection. By using an improved memory B cell immortalization method, we obtained a panel of human mAbs which reacted with TOSV antigens. We identified three epitopes of TOSV Gn glycoproteins by neutralizing mAbs using synthetic peptide arrays on membrane support (SPOT synthesis). These epitopes, separated in primary structure, might be exposed near one another as a conformational epitope in their native structure. In vivo studies were conducted to evaluate the humoral response elicited in mice immunized with the identified peptides. The results underlined the hypothesis that the first two peptides located in the NH2 terminus could form a conformational epitope, while the third, located near the transmembrane sequence in the carboxyl terminus, was necessary to strengthen neutralizing activity. Our results emphasize the importance of identifying neutralizing epitopes shared among the various phleboviruses, which could be exploited for the development of a potential epitope-based diagnostic assay or a polyvalent protective vaccine against different phleboviruses.

Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

Heparan Sulfate Proteoglycans Can Promote Opposite Effects on Adhesion and Directional Migration of Different Cancer Cells.

Heparan sulfate proteoglycans take part in crucial events of cancer progression, such as epithelial-mesenchymal transition, cell migration, and cell invasion. Through sulfated groups on their glycosaminoglycan chains, heparan sulfate proteoglycans interact with growth factors, morphogens, chemokines, and extracellular matrix (ECM) proteins. The amount and position of sulfated groups are highly variable, thus allowing differentiated ligand binding and activity of heparan sulfate proteoglycans. This variability and the lack of specific ligands have delayed comprehension of the molecular basis of heparan sulfate proteoglycan functions. Exploiting a tumor-targeting peptide tool that specifically recognizes sulfated glycosaminoglycans, we analyzed the role of membrane heparan sulfate proteoglycans in the adhesion and migration of cancer cell lines. Starting from the observation that the sulfated glycosaminoglycan-specific peptide exerts a different effect on adhesion, migration, and invasiveness of different cancer cell lines, we identified and characterized three cell migration phenotypes, where different syndecans are associated with alternative signaling for directional cell migration.

Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids.

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., PLoS ONE, 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7-6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives Staphylococcus aureus, Staphylococcus saprophyticus, and Enterococcus faecalis, and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of S. aureus with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.

Endocytosis and Trafficking of Heparan Sulfate Proteoglycans in Triple-Negative Breast Cancer Cells Unraveled with a Polycationic Peptide.

The process of heparan sulfate proteoglycan (HSPG) internalization has been described as following different pathways. The tumor-specific branched NT4 peptide has been demonstrated to bind HSPGs on the plasma membrane and to be internalized in tumor cell lines. The polycationic peptide has been also shown to impair migration of different cancer cell lines in 2D and 3D models. Our hypothesis was that HSPG endocytosis could affect two important phenomena of cancer development: cell migration and nourishment. Using NT4 as an experimental tool mimicking heparin-binding ligands, we studied endocytosis and trafficking of HSPGs in a triple-negative human breast cancer cell line, MDA-MB-231. The peptide entered cells employing caveolin- or clathrin-dependent endocytosis and macropinocytosis, in line with what is already known about HSPGs. NT4 then localized in early and late endosomes in a time-dependent manner. The peptide had a negative effect on CDC42-activation triggered by EGF. The effect can be explained if we consider NT4 a competitive inhibitor of EGF on HS that impairs the co-receptor activity of the proteoglycan, reducing EGFR activation. Reduction of the invasive migratory phenotype of MDA-MB-231 induced by NT4 can be ascribed to this effect. RhoA activation was damped by EGF in MDA-MB-231. Indeed, EGF reduced RhoA-GTP and NT4 did not interfere with this receptor-mediated signaling. On the other hand, the peptide alone determined a small but solid reduction in active RhoA in breast cancer cells. This result supports the observation of few other studies, showing direct activation of the GTPase through HSPG, not mediated by EGF/EGFR.

Molecular definition of the interaction between a tumor-specific tetrabranched peptide and LRP6 receptor.

The tumor-specific tetrabranched peptide NT4 binds membrane sulfate glycosaminoglycans and receptors belonging to the low density lipoprotein receptor-related protein (LRP) family, like LRP6, which are overexpressed in cancer. The binding occurs through a multimeric positively-charged motif of NT4 that interacts with negatively charged motives in both glycosaminoglycans and LRP receptors. LRP6 has an essential function in canonical Wnt signaling, acting together with receptors of the Frizzled family as coreceptor for Wnt ligands. The extracellular domain of LRP6 contains four YWTD ß-propellers, which are fundamental for interactions with ligands, such as Wnt and Wnt inhibitors. To investigate the molecular interactions between the NT4 peptide and LRP6 receptor, we synthesized a library of epitope mapping peptides reproducing the YWTD ß-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of ß-propeller 3 and contained negatively charged residues, including glutamic acid-708 which is known to be involved in Wnt3a interaction. The results pave the way for a possible development of peptide inhibitors of Wnt3a pathway to be used as drugs in oncology.

A New NT4 Peptide-Based Drug Delivery System for Cancer Treatment.

The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.

Antimicrobial Peptide-Loaded Nanoparticles as Inhalation Therapy for Pseudomonas aeruginosa Infections.

INTRODUCTION: Antibiotic-resistant bacteria kill 25,000 people every year in the EU. Patients subject to recurrent lung infections are the most vulnerable to severe or even lethal infections. For these patients, pulmonary delivery of antibiotics would be advantageous, since inhalation can achieve higher concentration in the lungs than iv administration and can provide a faster onset of action. This would allow for the delivery of higher doses and hence reduce the number of treatments required. We report here about a new nanosystem (M33-NS) obtained by capturing SET-M33 peptide on single-chain dextran nanoparticles. SET-M33 is a non-natural antimicrobial peptide synthesized in branched form. This form gives the peptide resistance to degradation in biological fluids. SET-M33 has previously shown efficacy in vitro against about one hundred of Gram-negative multidrug and extensively drug-resistant clinical isolates and was also active in preclinical infection models of pneumonia, sepsis and skin infections. METHODS: The new nanosystem was evaluated for its efficacy in bacteria cells and in a mouse model of pneumonia. Toxicity and genotoxicity were also tested in vitro. Biodistribution and pharmacokinetic studies in healthy rats were carried out using a radiolabeled derivative of the nanosystem. RESULTS: The M33-nanosystem, studied here, showed to be effective against Pseudomonas aeruginosa in time-kill kinetic experiments. Cytotoxicity towards different animal cell lines was acceptable. Lung residence time of the antimicrobial peptide, administered via aerosol in healthy rats, was markedly improved by capturing SET-M33 on dextran nanoparticles. M33-NS was also efficient in eradicating pulmonary infection in a BALB/c mouse model of pneumonia caused by P. aeruginosa. DISCUSSION: This study revealed that the encapsulation of the antimicrobial peptide in dextran nanoparticles markedly improved lung residence time of the peptide administered via aerosol. The result has to be considered among the aims of the development of a new therapeutic option for patients suffering recurrent infections, that will benefit from high local doses of persistent antimicrobials.

An antimicrobial molecule mitigates signs of sepsis in vivo and eradicates infections from lung tissue.

The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET-M33DIM) and evaluated as a candidate drug for infections due to multidrug-resistant (MDR) Gram-negative pathogens. SET-M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration [MICs], 1.5-11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11-22 µM). It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC50 was as much as 22 times better than that of SET-M33, a peptide with the same amino-acid sequence, but synthesized in tetra-branched form (638 vs 28 µM). In in vivo and ex vivo experiments, SET-M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge. It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.

NMR Study of the Secondary Structure and Biopharmaceutical Formulation of an Active Branched Antimicrobial Peptide.

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. The secondary structure of this 40 amino acid peptide was investigated by NMR to fully characterize the product in the framework of preclinical studies. The possible presence of helixes or ß-sheets in the structure had to be explored to predict the behavior of the branched peptide in solution, with a view to designing a formulation for parenteral administration. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, we also report the studies on a new salt form, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient from a process development point of view and did not increase the toxicity of the antimicrobial drug.

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antagonist binding of HSPG. The binding affinity of NT4 with recombinant HSPG showed that NT4 bound glypican-3 and -4 and, with lower affinity, syndecan-4. NT4 binding to the cancer cell membrane was inversely correlated with sulfatase expression. NT4 binding was higher in cell lines with lower expression of SULF-1 and SULF-2, which confirms the determinant role of sulfate groups for recognition by NT4. Using 8-mer and 9-mer heparan sulfate (HS) oligosaccharides with analog disaccharide composition and different sulfation sites, a possible recognition motif was identified that includes repeated 6-O-sulfates alternating with N- and/or 2-O-sulfates. Molecular modeling provided a fully descriptive picture of binding architecture, showing that sulfate groups on opposite sides of the oligosaccharide can interact with positive residues on two peptide sequences of the branched structure, thus favoring multivalent binding and explaining the high affinity and selectivity of NT4 for highly sulfated GAGs. NT4 and possibly newly selected branched peptides will be essential probes for reconstructing and unraveling binding sites for cancer-involved ligands on GAGs and will pave the way for new cancer detection and treatment options.

Peptides and small molecules blocking the CXCR4/CXCL12 axis overcome bone marrow­induced chemoresistance in acute leukemias.

Notable advances in treatment have been made and increases in the cure rates of pediatric leukemia have been achieved. However, the majority of children with relapsed disease are not expected to survive, with chemotherapy resistance acting as the principal cause of treatment failure. Interaction between leukemic cells and the bone marrow microenvironment is the primary cause of relapse. It was identified that a multi­protein membrane complex, formed by potassium voltage­gated channel subfamily H member 2 (hERG1) channels, the ß1 integrin subunit and the stromal cell­derived factor 12 (CXCL12) receptor, C­X­C chemokine receptor type 4 (CXCR4), exerts a role in mesenchymal stromal cell (MSC)­mediated chemoresistance in pediatric leukemias. hERG1 blockade was able to overcome chemoresistance in vitro and in vivo. As an alternative strategy to overcome chemoresistance, the present study evaluated the effects of novel tools targeting the CXCR4/CXCL12 axis. The analysis of CXCL12 structural dynamics was used for the selection of a peptide (4­1­17) and a small molecule (8673), which interact with a transient hot spot, identified by a dynamic drug design approach. The present findings indicated that peptide 4­1­17 and small molecule 8673 inhibited leukemia cell proliferation and induced a pro­apoptotic effect, which was not reduced by the presence of MSCs. The combined treatment with 4­1­17 and 8673 had a stronger pro­apoptotic effect, particularly on cells cultured on MSCs in normoxic and hypoxic conditions, and was able to overcome MSC­induced resistance to cytarabine. Overall, the targeting of CXCL12 and the ensuing inhibition of the CXCR4/CXCL12 axis may be proposed as an alternative strategy to overcome chemoresistance in leukemia.

Heterologous Prime-Boost Combinations Highlight the Crucial Role of Adjuvant in Priming the Immune System.

The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4+ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone.

The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells.

Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in in vivo models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.

Near-infrared quantum dots labelled with a tumor selective tetrabranched peptide for in vivo imaging.

BACKGROUND: Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells. RESULTS: In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model. CONCLUSIONS: NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.

Coupling to a cancer-selective heparan-sulfate-targeted branched peptide can by-pass breast cancer cell resistance to methotrexate.

Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.