Giancarlo Morelli

Silver Nanoparticles as Potential Antibacterial Agents

Multi-drug resistance is a growing problem in the treatment of infectious diseases and the widespread use of broad-spectrum antibiotics has produced antibiotic resistance for many human bacterial pathogens. Advances in nanotechnology have opened new horizons in nanomedicine, allowing the synthesis of nanoparticles that can be assembled into complex architectures. Novel studies and technologies are devoted to understanding the mechanisms of disease for the design of new drugs, but unfortunately infectious diseases continue to be a major health burden worldwide. Since ancient times, silver was known for its anti-bacterial effects and for centuries it has been used for prevention and control of disparate infections. Currently nanotechnology and nanomaterials are fully integrated in common applications and objects that we use every day. In addition, the silver nanoparticles are attracting much interest because of their potent antibacterial activity. Many studies have also shown an important activity of silver nanoparticles against bacterial biofilms. This review aims to summarize the emerging efforts to address current challenges and solutions in the treatment of infectious diseases, particularly the use of nanosilver antimicrobials.

A New Nerve Growth Factor-Mimetic Peptide Active on Neuropathic Pain in Rats

Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 μm. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest “in vitro” activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.

Clickable Functionalization of Liposomes with the gH625 Peptide from Herpes simplex Virus Type I for Intracellular Drug Delivery

Liposomes externally modified with the nineteen residues gH625 peptide, previously identified as a membrane-perturbing domain in the gH glycoprotein of Herpes simplex virus type I, have been prepared in order to improve the intracellular uptake of an encapsulated drug. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of the hydrophobic gH625 peptide on the external surface of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPG)-based liposomes. Electron paramagnetic resonance studies, on liposomes derivatized with gH625 peptides, which are modified with the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label in several peptide positions, confirm the positioning of the coupled peptides on the liposome external surface, whereas dynamic light scattering measurements indicate an increase of the diameter of the liposomes of approximately 30% after peptide introduction. Liposomes have been loaded with the cytotoxic drug doxorubicin and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells.

Peptide-based targeting strategies for simultaneous imaging and therapy with nanovectors

Theranostic agents based on nanovectors (liposomes, naposomes, micelles, polymeric micelles and micelles built around a solid core) able to carry, simultaneously, a drug and a contrast agent and externally modified with targeting peptides are described.

Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs.

Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs); and G-protein coupled receptors (GPCRs). Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles) loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors.

Peptide-modified liposomes for selective targeting of bombesin receptors overexpressed by cancer cells: a potential theranostic agent

Objectives Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors. Methods A new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized. Results Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with peptide-free DSPC liposomes (1.4% ± 0.2% at 37°C). Incubation of cells with DSPC/ MonY-BN/Dox showed significantly lower cell survival compared with DSPC/Dox-treated cells, in the presence of 100 ng/mL and 300 ng/mL drug amounts, in cytotoxicity experiments. Intravenous treatment of PC-3 xenograft-bearing mice with DSPC/MonY-BN/Dox at 10 mg/kg Dox dose produced higher tumour growth inhibition (60%) compared with nonspecific DSPC/ Dox liposomes (36%) relative to control animals. Conclusion The structural and loading properties of DSPC/MonY-BN liposomes along with the observed in-vitro and in-vivo activity are encouraging for further development of this approach for target-specific cancer chemotherapy.

Micelles derivatized with octreotide as potential target-selective contrast agents in MRI†‡

New amphiphilic monomers (OCA‐DTPAGlu and OCA‐DOTA) containing, in the same molecule, three different functions: (i) the chelating agent (DTPAGlu or DOTA) able to coordinate gadolinium ion, (ii) the octreotide bioactive peptide able to target somatostatin receptors, and (iii) a hydrophobic moiety with two 18‐carbon atoms alkyl chains have been designed and synthesized by solid‐phase methods. The novel amphiphilic monomers aggregate, in water solution, giving stable micelles at very low concentration (cmc values of 2.3 × 10−6 mol kg−1 and 2.5 × 10−6 mol kg−1 for OCA‐DTPAGlu and OCA‐DOTA, respectively) as confirmed by fluorescence spectroscopy. Fluorescence studies and circular dichroism experiments indicate, for the two compounds as well as for their gadolinium complexes (OCA‐DOTA(Gd) and OCA‐DTPAGlu(Gd)), the complete exposure of octreotide on the micelle surface, and the predominant presence of an antiparallel β‐sheet peptide conformation characterized by a β‐like turn. The high relaxivity value (r1p = 13.9 mM−1 s−1 at 20 MHz and 25 °C), measured for micelles obtained by the gadolinium complex OCA‐DTPAGlu(Gd), indicates these aggregates as promising target‐selective magnetic resonance imaging (MRI) contrast agents. Copyright

Peptide-Lipid Interactions: Experiments and Applications

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.

Target-Selective Drug Delivery through Liposomes Labeled with Oligobranched Neurotensin Peptides

The structure and the in vitro behavior of liposomes filled with the cytotoxic drug doxorubicin (Doxo) and functionalized on the external surface with a branched moiety containing four copies of the 8–13 neurotensin (NT) peptide is reported. The new functionalized liposomes, DOPC‐NT4Lys(C18)2, are obtained by co‐aggregation of the DOPC phospholipid with a new synthetic amphiphilic molecule, NT4Lys(C18)2, which contains a lysine scaffold derivatized with a lipophilic moiety and a tetrabranched hydrophilic peptide, NT8–13, a neurotensin peptide fragment well known for its ability to mimic the neurotensin peptide in receptor binding ability. Dynamic light scattering measurements indicate a value for the hydrodynamic radius (RH) of 88.3±4.4 nm. The selective internalization and cytotoxicity of DOPC‐NT4Lys(C18)2 liposomes containing Doxo, as compared to pure DOPC liposomes, were tested in HT29 human colon adenocarcinoma and TE671 human rhabdomyosarcoma cells, both of which express neurotensin receptors. Peptide‐functionalized liposomes show a clear advantage in comparison to pure DOPC liposomes with regard to drug internalization in both HT29 and TE671 tumor cells: FACS analysis indicates an increase in fluorescence signal of the NT4‐liposomes, compared to the DOPC pure analogues, in both cell lines; cytotoxicity of DOPC‐NT4Lys(C18)2‐Doxo liposomes is increased four‐fold with respect to DOPC‐Doxo liposomes in both HT29 and TE671 cell lines. These effects could to be ascribed to the higher rate of internalization for DOPC‐NT4Lys(C18)2‐Doxo liposomes, due to stronger binding driven by a lower dissociation constant of the NT4‐liposomes that bind the membrane onto a specific protein, in contrast to DOPC liposomes, which approach the plasma membrane unselectively.

Peptide inhibitors against herpes simplex virus infections

Herpes simplex virus (HSV) is a significant human pathogen causing mucocutaneous lesions primarily in the oral or genital mucosa. Although acyclovir (ACV) and related nucleoside analogs provide successful treatment, HSV remains highly prevalent worldwide and is a major cofactor for the spread of human immunodeficiency virus. Encephalitis, meningitis, and blinding keratitis are among the most severe diseases caused by HSV. ACV resistance poses an important problem for immunocompromised patients and highlights the need for new safe and effective agents; therefore, the development of novel strategies to eradicate HSV is a global public health priority. Despite the continued global epidemic of HSV and extensive research, there have been few major breakthroughs in the treatment or prevention of the virus since the introduction of ACV in the 1980s. A therapeutic strategy at the moment not fully addressed is the use of small peptide molecules. These can be either modeled on viral proteins or derived from antimicrobial peptides. Any peptide that interrupts protein–protein or viral protein–host cell membrane interactions is potentially a novel antiviral drug and may be a useful tool for elucidating the mechanisms of viral entry. This review summarizes current knowledge and strategies in the development of synthetic and natural peptides to inhibit HSV infectivity. Copyright