Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells

The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin 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; 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). gH625 liposomes showed 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. 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

Biophysical Characterization and Membrane Interaction of the Two Fusion Loops of Glycoprotein B from Herpes Simplex Type I Virus

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

Peptide based platforms for cancer drug delivery

Cancer remains one of main causes of death in humans, accounting for 8.2 milion deaths worldwide in 2012. Chemotherapy, the most widely used cancer therapy, is the most effective and potent strategy to treat malignant tumors, but has the disadvantage of not delivering the therapeutic agents only to tumor sites. Nanomedicine may allow the controlled release of drugs by biodegradation and self-regulation of nanomaterials in vitro and in vivo. The goal of this PhD project was to create a delivery tool, that transports the drug to the target cells not only with high efficacy, but also with minimal toxicity against normal cells and avoiding its degradation and entrapment in endosomes. The first part of this thesis was focused on the design of the strategies for the achievement of a toolbox easily to functionalize and on its obtainment. The second phase was the physico-chemical characterization of each selected nanosystem with particular attention to the size, zeta potential and drug loading and release. The third phase was to analyze in vitro the subcellular fate of the vectorized drug and the effect on the cells. Several nanosystems (liposomes, magnetic nanoparticles, polystyrene nanoparticles) were selected and functionalized with peptides both to enhance tumor targeting and facilitate intracellular uptake. In particular, we used a novel Cell Penetrating Peptide (namely, gH625) which is able to overcome the known limits of classic CPPs. In fact, gH625 is able to efficiently traverse biological membranes, promoting lipid-membrane reorganizing processes, such as fusion or pore formation and involving temporary membrane destabilization and subsequent reorganization; it is able to circumvent the endosomal entrapment either favouring the escape from the endosome or by directly translocating the drug across the membrane. In order to make the nanosystem cell and tissue specific, we have further functionalized the surface of the nanosystem with a targeting peptide. We exploited the EGB peptide, which recognizes the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor overexpressed in several solid tumors. The first nanoplatform is liposome based. The presence of gH625 on the surface of liposomes is favouring their uptake in both sensitive and drug-resistant tumor cell lines allowing an increase of cell growth inhibition: in fact, a greater quantity of Doxo from functionalized liposomes is accumulated into cells. Doxo encapsulated in functionalized liposomes was able to enter in the nuclei of Doxo-resistant cancer cells indicating that the peptide gH625 was probably inducing a greater and more rapid internalization also in resistant cells, which could contribute to overcome drug resistance. The second nanoplatform is based on multifunctional magnetic nanoparticles (SPIONs). Our goal was to verify if we could also enhance the cellular uptake of this kind of cargo. We focused on optimization of the gH625 conjugation strategy, in order to find the best compromise between the colloidal stability of nanosystems, their half-life in blood and their efficient translocation into cells. We optimized several important parameters such as the concentration of gH625 on the polymeric surface of SPIONs and characterized the obtained nanosystem by Circular Dichroism (CD). Data confirmed that gH625 retains its helical structure when bound to the nanoparticle surface, suggesting that the secondary structure of the peptide was not disturbed by attachment to SPIONs. The third nanoplatform is based on polystyrene nanoparticles (NPs). We explored the possibility of using NPs functionalized with gH625 to deliver a drug across the Blood Brain Barrier (BBB). The uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide. Moreover, gH625 plays a key role in controlling the uptake mechanism. In fact, gH625 is able to change the mechanism of uptake of the cargo and it is able to cross the BBB. In summary, these results establish that gH625 may represent a good choice for the design of promising carriers to deliver drugs for the treatment of human diseases and we have developed a nanoplatform for targeted drug delivery to be used for several pathologies

Viral Peptide Targeted Delivery of Nano-Therapeutics

Over the past decade, an increasing number of potential drugs have been suggested for therapeutic applications. Often bio-macromolecules show a limited ability to cross the plasma membrane resulting in poor cellular access, which largely prevents them from reaching intracellular targets and from crossing epithelial or endothelial barriers. Moreover, neurological disorders contribute significantly to the global burden of disease and are likely to increase in the coming years due to an aging population; however, significant efforts have been devoted towards the development of improved therapies for central nervous system (CNS) diseases, treatments remain limited due to the inability of therapeutic agents to effectively cross the blood-brain-barrier (BBB).[1] The development of nanotechnology provides powerful tools to deliver therapeutics to target sites. This presentation targets major advances in the design and realization of nano-scaffolds for theranostics. A fundamental limitation of current diagnostics and therapeutics is the lack of a single delivery system that has the potential to not only deliver therapeutics to the disease site of interest with high fidelity, i.e. target delivery, but also allows for diagnostics and cell delivery, i.e. cell penetration and uptake and thus the obtainment of a toolbox that combines targeting and delivery with imaging and targeted cell uptake. The discovery of several peptides with the ability to cross the plasma membrane of eukaryotic cells by a possibly receptor- and endocytosis-independent mechanism, has opened a new avenue in biomedical research. Among the so-called cell penetrating peptides (CPPs), Tat, penetratin and VP22 have been widely used and have shown to be entrapped in intracellular organelles. Thus, one of the main goals of recent research is the obtainment of novel delivery systems that are able to cross membranes without being entrapped in intracellular organelles[2]. Viral derived peptides, and in particular those derived by viral entry proteins, may be useful as delivery vehicles due to their intrinsic properties of inducing membrane perturbation. The present talk will describe results obtained with the use of a peptide derived from Herpes simplex virus type 1 for the delivery of bioactive molecules or fluorescent dyes inside the host cell. In particular, its use for the intracellular delivery of quantum dots (QDs), liposomes, dendrimers and nanoparticles will be addressed

Synthesis and in vitro evaluation of fluorescent and magnetic nanoparticles functionalized with a cell penetrating peptide for cancer theranosis

International audienceWe 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

Enhanced uptake of gH625 by blood brain barrier compared to liver in vivo: characterization of the mechanism by an in vitro model and implications for delivery

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

Protein-and Peptide-functionalized nanovectors for anticancer theranosis

Among biomedical applications of nanotechnology, cancer theranosis (therapy and diagnosis) is one of the most promising domains. In order to improve the nanosystems biocompatibility, they are usually coated with neutral biocompatible polymers like polethylene glycol (PEG). For cancer cell enhanced and specific targeting with injectable nanosystems, the common strategy consists in engeneering the nanosystem surface with various biologically relevant ligands (from small molecules to antibodies). Peptides have several interesting properties as ligands for anticancer nanomedicine. In the present talk, we will expose our recently developed PEGylated and peptide-functionalized injectable nanovectors based on nanoliposomes or on superparamagnetic iron oxides (SPIONs). Their PEG polymeric coating was covalently coupled to (i) membranotropic cell-penetrating peptides gH625[1] or to (ii) scFv fragments of antibody trastuzumab, specific of HER2 positive breast cancers[2]. Once synthetized, the novel nanovectors were carefully characterized in order to establish their physicochemical properties (size and zeta potential in a wide range of pH, chemical composition and structure). Then, the nanovectors interaction with cancer cells has been studied in vitro, on various cancer cell lines, overexpressing or not specific receptors, thus playing a role of positive or negative controls. By comparing ligand-free PEGylated (control) and ligand carrying PEGylated nanovectors we attempt to establish a possible role of the ligands. As we will show, the ligands are able to affect nanosystemcell interactions both quantitatively (intracellular accumulation) and qualitatively (nanosystem internalisation, subcellular localisation/interaction and drug delivery). We will also present some methodological developments we made to shed a light on the nanosystem-cell interactions, in particular on the intracellular fate of the nanosystems and of the drugs they deliver

Nanocarriers Conjugated with Cell Penetrating Peptides: New Trojan Horses by Modern Ulysses

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

Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells.

International audienceThe 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