Monica Argenziano

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

Background The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

Nanobubbles: a promising efficienft tool for therapeutic delivery

In recent decades ultrasound-guided delivery of drugs loaded on nanocarriers has been the focus of increasing attention to improve therapeutic treatments. Ultrasound has often been used in combination with microbubbles, micron-sized spherical gas-filled structures stabilized by a shell, to amplify the biophysical effects of the ultrasonic field. Nanometer size bubbles are defined nanobubbles. They were designed to obtain more efficient drug delivery systems. Indeed, their small sizes allow extravasation from blood vessels into surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. Additionally, nanobubbles might be endowed with improved stability and longer residence time in systemic circulation. This review will describe the physico-chemical properties of nanobubbles, the formulation parameters and the drug loading approaches, besides potential applications as a therapeutic tool.

GSH-targeted nanosponges increase doxorubicin-induced toxicity "in vitro" and "in vivo" in cancer cells with high antioxidant defenses.

Several reports indicate that chemo-resistant cancer cells become highly adapted to intrinsic oxidative stress by up-regulating their antioxidant systems, which causes an increase of intracellular GSH content. Doxorubicin is one of the most widely used drugs for tumor treatment, able to kill cancer cells through several mechanisms. However, doxorubicin use is limited by its toxicity and cancer resistance. Therefore, new therapeutic strategies able to reduce doses and to overcome chemo-resistance are needed. A new class of glutathione-responsive cyclodextrin nanosponges (GSH-NS), is able to release anticancer drugs preferentially in cells having high GSH content. Doxorubicin-loaded GSH-NS, in the cancer cells with high GSH content, inhibited clonogenic growth, cell viability, topoisomerase II activity and induced DNA damage with higher effectiveness than free drug. Moreover, GSH-NS reduced the development of human tumor in xenograft models more than free drug. These characteristics indicate that GSH-NS can be a suitable drug delivery carrier for future applications in cancer therapy.

2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues.

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.

Antimicrobial chitosan nanodroplets: new insights for ultrasound-mediated adjuvant treatment of skin infection

BACKGROUND Chronic wounds, characterized by hypoxia, inflammation and impaired tissue remodeling, are often worsened by bacterial/fungal infections. Intriguingly, chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLNs) have proven effective in delivering oxygen to hypoxic tissues. AIM The present work aimed at investigating nanodroplet antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans, toxicity on human keratinocytes (HaCaT) and ultrasound (US)-triggered transdermal delivery. MATERIALS & METHODS Nanodroplet antibacterial/antifungal properties, human cytotoxicity, and US-triggered transdermal delivery were measured through microbiological, biochemical, and sonophoresis assays, respectively. RESULTS OLNs and oxygen-free nanodroplets (OFNs) displayed short- or long-term cytostatic activity against MRSA or Candida albicans, respectively. OLNs were not toxic to keratinocytes, whereas OFNs slightly affected cell viability. Complementary US treatment promoted OLN transdermal delivery. CONCLUSION As such, US-activated chitosan-shelled OLNs appear as promising, nonconventional and innovative tools for adjuvant treatment of infected chronic wounds.

Drug Delivery Nanoparticles in Treating Chemoresistant Tumor Cells

Intrinsic or acquired chemoresistance represents the main obstacle to the successful treatment of cancer patients. Several mechanisms are involved in multidrug resistance: decreased uptake of hydrophilic drugs, increase of energy dependent efflux, alteration of the redox state, alteration of apoptotic pathways, and modification of the tumor microenvironment. In recent years, several types of nanoparticles have been developed to overcome these obstacles and improve the accumulation and release of drugs at the pathological site. In this review, we describe the main mechanisms involved in multidrug resistance and the nanovehicles which have been proposed to target specific aspects of this phenomenon.

Enhanced cytotoxic effect of camptothecin nanosponges in anaplastic thyroid cancer cells in vitro and in vivo on orthotopic xenograft tumors

Abstract Anaplastic carcinoma of the thyroid (ATC) is a lethal human malignant cancer with median survival of 6 months. To date, no treatment has substantially changed its course, which makes urgent need for the development of novel drugs or novel formulations for drug delivery. Nanomedicine has enormous potential to improve the accuracy of cancer therapy by enhancing availability and stability, decreasing effective doses and reducing side effects of drugs. Camptothecin (CPT) is an inhibitor of DNA topoisomerase-I with several anticancer properties but has poor solubility and a high degradation rate. Previously, we reported that CPT encapsulated in β-cyclodextrin-nanosponges (CN-CPT) increased solubility, was protected from degradation and inhibited the growth of prostate tumor cells both in vitro and in vivo. The aim of this study was to extend that work by assessing the CN-CPT effectiveness on ATC both in vitro and in vivo. Results showed that CN-CPT significantly inhibited viability, clonogenic capacity and cell-cycle progression of ATC cell lines showing a faster and enhanced effect compared to free CPT. Moreover, CN-CPT inhibited tumor cell adhesion to vascular endothelial cells, migration, secretion of pro-angiogenic factors (IL-8 and VEGF-α), expression of β-PIX, belonging to the Rho family activators, and phosphorylation of the Erk1/2 MAPK. Finally, CN-CPT significantly inhibited the growth, the metastatization and the vascularization of orthotopic ATC xenografts in SCID/beige mice without apparent toxic effects in vivo. This work extends the previous insight showing that β-cyclodextrin-nanosponges are a promising tool for the treatment of ATC.

Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

BACKGROUND In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. OBJECTIVE To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. METHODS HaCaT cells were treated for 24h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. RESULTS Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. CONCLUSION Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds.

Nanomedicine formulations for the delivery of antiviral drugs: a promising solution for the treatment of viral infections

ABSTRACT Introduction: Viral infections represent a public health problem and one of the leading causes of global mortality. Nanomedicine strategies can be considered a powerful tool to enhance the effectiveness of antiviral drugs, often associated with solubility and bioavailability issues. Consequently, high doses and frequent administrations are required, resulting in adverse side effects. To overcome these limitations, various nanomedicine platforms have been designed. Areas covered: This review focuses on the state of the art of organic-based nanoparticles for the delivery of approved antivirals. A brief description of the main characteristics of nanocarriers is followed by an overview of the most promising research addressing the treatment of most important viral infections. Expert opinion: The activity of antiviral drugs could be improved with nanomedicine formulations. Indeed, nanoparticles can affect the fate of the encapsulated drugs, allowing controlled release kinetics, enhanced bioavailability, modified pharmacokinetics, and reduced side effects. In addition, the physicochemical properties of nanocarriers can enable their capability to target specific sites and to interact with virus structures. In this regard, nanomedicines can be considered an opportunity to enhance the therapeutic index of antivirals. Efficacy, safety, and manufacturing issues need to be carefully assessed to bring this promising approach to the clinic.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.