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1.
Sci Rep ; 8(1): 13836, 2018 09 14.
Article in English | MEDLINE | ID: mdl-30218088

ABSTRACT

We have investigated the crossing of the blood brain barrier (BBB) by the peptide gH625 and compared to the uptake by liver in vivo. We clearly observed that in vivo administration of gH625 allows the crossing of the BBB, although part of the peptide is sequestered by the liver. Furthermore, we used a combination of biophysical techniques to gain insight into the mechanism of interaction with model membranes mimicking the BBB and the liver. We observed a stronger interaction for membranes mimicking the BBB where gH625 clearly undergoes a change in secondary structure, indicating the key role of the structural change in the uptake mechanism. We report model studies on liposomes which can be exploited for the optimization of delivery tools.


Subject(s)
Blood-Brain Barrier/metabolism , Peptides/metabolism , Viral Envelope Proteins/metabolism , Animals , Biological Transport , Blood-Brain Barrier/physiology , Liposomes/chemistry , Liver/metabolism , Liver/physiology , Male , Nanoparticles/chemistry , Peptides/chemistry , Rats , Rats, Wistar , Viral Envelope Proteins/chemistry
2.
J Colloid Interface Sci ; 499: 209-217, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28388503

ABSTRACT

We synthesized rationally designed multifunctional nanoparticles (NPs) composed of a superparamagnetic iron oxide nanoparticle (SPION) core, cyanine fluorescent dye emitting in far red, polyethylene glycol (PEG5000) coating, and the membranotropic peptide gH625, from the cell-penetrating peptides (CPP) family. The peptide sequence was enriched with an additional cysteine so it can be involved as a reactive moiety in a certain orientation- and sequence-specific coupling of the CPP to the PEG shell of the NPs. Our data indicate that the presence of approximately 23 peptide molecules per SPION coated with approximately 137 PEG chains minimally changes the overall NP characteristics. The final CPP-capped NP hydrodynamic diameter was 98nm, the polydispersity index was 0.192, and the zeta potential was 4.08mV. The in vitro evaluation, performed using an original technique fluorescence confocal spectral imaging, showed that after a short incubation duration (maximum 30min), SPIONs-PEG-CPP uptake was 3-fold higher than that for SPIONs-PEG. The CPP also drives the subcellular distribution of a higher NP fraction towards low polarity cytosolic locations. Therefore, the major cellular uptake mechanism for the peptide-conjugated NPs should be endocytosis. Enhancement/acceleration of this mechanism by gH625 appears promising because of potential applications of SPIONs-PEG-gH625 as a multifunctional nanoplatform for cancer theranosis involving magnetic resonance imaging, optical imaging in far red, drug delivery, and hyperthermia.

3.
Curr Pharm Biotechnol ; 17(8): 700-22, 2016.
Article in English | MEDLINE | ID: mdl-27087493

ABSTRACT

Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems.


Subject(s)
Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/pharmacokinetics , Drug Delivery Systems/methods , Nanoparticles/chemistry , Endosomes/metabolism , Humans
4.
Oncotarget ; 7(4): 4077-92, 2016 Jan 26.
Article in English | MEDLINE | ID: mdl-26554306

ABSTRACT

New delivery systems including liposomes have been developed to circumvent drug resistance. To enhance the antitumor efficacy of liposomes encapsulating anti-cancer agents, we used liposomes externally conjugated to the 20 residue peptide gH625. Physicochemical characterization of the liposome system showed a size of 140 nm with uniform distribution and high doxorubicin encapsulation efficiency. We evaluated the effects of increasing concentrations of liposomes encapsulating Doxo (LipoDoxo), liposomes encapsulating Doxo conjugated to gH625 (LipoDoxo-gH625), empty liposomes (Lipo) or free Doxo on growth inhibition of either wild type (A549) or doxorubicin-resistant (A549 Dx) human lung adenocarcinoma. After 72 h, we found that the growth inhibition induced by LipoDoxo-gH625 was higher than that caused by LipoDoxo with an IC50 of 1 and 0.3 µM in A549 and A549 Dx cells, respectively. The data on cell growth inhibition were paralleled by an higher oxidative stress and an increased uptake of Doxo induced by LipoDoxo-gH625 compared to LipoDoxo, above all in A549 Dx cells. Cytometric analysis showed that the antiproliferative effects of each drug treatment were mainly due to the induction of apoptosis. In conclusion, liposomes armed with gH625 are able to overcome doxorubicin resistance in lung adenocarcinoma cell lines.


Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Resistance, Neoplasm/drug effects , Liposomes/chemistry , Lung Neoplasms/drug therapy , Peptides/administration & dosage , Viral Envelope Proteins/administration & dosage , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Antibiotics, Antineoplastic/chemistry , Apoptosis/drug effects , Blotting, Western , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Doxorubicin/chemistry , Flow Cytometry , Humans , Liposomes/administration & dosage , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Microscopy, Confocal , Oxidative Stress/drug effects , Peptides/chemistry , Tumor Cells, Cultured , Viral Envelope Proteins/chemistry
5.
Int J Pharm ; 488(1-2): 59-66, 2015 Jul 05.
Article in English | MEDLINE | ID: mdl-25891256

ABSTRACT

The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of polyethylene glycol (PEG) liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin (Doxo) to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; functionalized and not liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). FACS data confirmed that gH625 increases the total intracellular MTX content. CSI data indicated that when liposomes are decorated with gH625 an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. The cytotoxicity on HeLa cell line was higher for functionalized liposomes within 4-6h of treatment. To summarise, the MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug at the delay of 4-6h.


Subject(s)
Antineoplastic Agents/administration & dosage , Liposomes/chemistry , Mitoxantrone/administration & dosage , Nanoparticles/chemistry , Peptides/chemistry , Viral Envelope Proteins/chemistry , Antineoplastic Agents/pharmacology , Cell Survival , Drug Liberation , Flow Cytometry , HeLa Cells , Humans , Microscopy, Confocal , Mitoxantrone/pharmacology , Polyethylene Glycols/chemistry
6.
PLoS One ; 7(2): e32186, 2012.
Article in English | MEDLINE | ID: mdl-22384173

ABSTRACT

The molecular mechanism of entry of herpesviruses requires a multicomponent fusion system. Cell invasion by Herpes simplex virus (HSV) requires four virally encoded glycoproteins: namely gD, gB and gH/gL. The role of gB has remained elusive until recently when the crystal structure of HSV-1 gB became available and the fusion potential of gB was clearly demonstrated. Although much information on gB structure/function relationship has been gathered in recent years, the elucidation of the nature of the fine interactions between gB fusion loops and the membrane bilayer may help to understand the precise molecular mechanism behind herpesvirus-host cell membrane fusion. Here, we report the first biophysical study on the two fusion peptides of gB, with a particular focus on the effects determined by both peptides on lipid bilayers of various compositions. The two fusion loops constitute a structural subdomain wherein key hydrophobic amino acids form a ridge that is supported on both sides by charged residues. When used together the two fusion loops have the ability to significantly destabilize the target membrane bilayer, notwithstanding their low bilayer penetration when used separately. These data support the model of gB fusion loops insertion into cholesterol enriched membranes.


Subject(s)
Biophysics/methods , Herpesvirus 1, Human/chemistry , Herpesvirus 1, Human/metabolism , Viral Envelope Proteins/chemistry , Acrylamide/chemistry , Amino Acid Motifs , Crystallography, X-Ray/methods , Lipids/chemistry , Peptides/chemistry , Protein Structure, Secondary , Spectrometry, Fluorescence/methods , Tryptophan/chemistry , Tyrosine/chemistry
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