Ultrasound-Responsive Nrf2-Targeting siRNA-Loaded Nanobubbles for Enhancing the Treatment of Melanoma.

The siRNA-mediated inhibition of nuclear factor E2-related factor 2 (Nrf2) can be an attractive approach to overcome chemoresistance in various malignant tumors, including melanoma. This work aims at designing a new type of chitosan-shelled nanobubble for the delivery of siRNA against Nrf2 in combination with an ultrasound. A new preparation method based on a water-oil-water (W/O/W) double-emulsion was purposely developed for siRNA encapsulation in aqueous droplets within a nanobubble core. Stable, very small NB formulations were obtained, with sizes of about 100 nm and a positive surface charge. siRNA was efficiently loaded in NBs, reaching an encapsulation efficiency of about 90%. siNrf2-NBs downregulated the target gene in M14 cells, sensitizing the resistant melanoma cells to the cisplatin treatment. The combination with US favored NB cell uptake and transfection efficiency. Based on the results, nanobubbles have shown to be a promising US responsive tool for siRNA delivery, able to overcome chemoresistance in melanoma cancer cells.

Enhanced Antimicrobial and Antibiofilm Effect of New Colistin-Loaded Human Albumin Nanoparticles.

Multidrug-resistant (MDR) Gram-negative bacteria (GNB), such as Acinetobacter and Klebsiella, are responsible for severe hospital-acquired infections. Colistin, despite its toxicity and low tissue penetration, is considered the last resort antibiotic against these microorganisms. Of concern, the use of Colistin has recently been compromised by the emergence of Colistin resistance. Herein, we developed a new formulation consisting of multifunctional chitosan-coated human albumin nanoparticles for the delivery of Colistin (Col/haNPs). Col/haNPs were in vitro characterized for encapsulation efficiency, drug release, stability and cytotoxicity and were evaluated for antibacterial activity against MDR GNB (Acinetobacter baumannii and Klebsiella pneumoniae). Col/haNPs showed sizes lower than 200 nm, high encapsulation efficiency (98.65%) and prolonged in vitro release of Colistin. The safety of the nanoformulation was demonstrated by a negligible cytotoxicity on human fibroblasts and hemolytic activity. Col/haNPs evidenced a high antibacterial effect with a significant decrease in MIC values compared to free Colistin, in particular against Col-resistant strains with a pronounced decline of bacterial growth over time. Moreover, Col/haNPs exhibited an inhibitory effect on biofilm formation that was 4 and 60 fold higher compared to free Colistin, respectively for Colistin susceptible and resistant A. baumannii. Our findings suggest that Col/haNPs could represent a promising Colistin nanocarrier with high antimicrobial activity on MDR GNB.

Albumin nanoformulations as an innovative solution to overcome doxorubicin chemoresistance.

Aim: Resistance to chemotherapy is a major limiting factor that hamper the effectiveness of anticancer therapies. Doxorubicin is an antineoplastic agent used in the treatment of a wide range of cancers. However, it presents several limitations such as dose-dependent cardiotoxicity, lack of selectivity for tumor cells, and induced cell resistance. Nanotechnology represents a promising strategy to avoid these drawbacks. In this work, new albumin-based nanoparticles were formulated for the intracellular delivery of doxorubicin with the aim to overcome cancer drug resistance. Methods: Glycol chitosan-coated and uncoated albumin nanoparticles were prepared with a tuned coacervation method. The nanoformulations were in vitro characterized evaluating the physicochemical parameters, morphology, and in vitro release kinetics. Biological assays were performed on A2780res and EMT6 cells from human ovarian carcinoma and mouse mammary cell lines resistant for doxorubicin, respectively. Results: Cell viability assays showed that nanoparticles have higher cytotoxicity than the free drug. Moreover, at low concentrations, both doxorubicin-loaded nanoparticles inhibited the cell colony formation in a greater extent than drug solution. In addition, the cell uptake of the different formulations was investigated by confocal microscopy and by the HPLC determination of doxorubicin intracellular accumulation. The nanoparticles were rapidly internalized in greater extent compared to the free drug. Conclusion: Based on these results, doxorubicin-loaded albumin nanoparticles might represent a novel platform to overcome the mechanism of drug resistance in cancer cell lines and improve the drug efficacy.

Improvement in the Anti-Tumor Efficacy of Doxorubicin Nanosponges in In Vitro and in Mice Bearing Breast Tumor Models.

Doxorubicin (DOX) is an anthracycline widely used in cancer therapy and in particular in breast cancer treatment. The treatment with DOX appears successful, but it is limited by a severe cardiotoxicity. This work evaluated the in vitro and in vivo anticancer effect of a new formulation of ß-cyclodextrin nanosponges containing DOX (BNS-DOX). The BNS-DOX effectiveness was evaluated in human and mouse breast cancer cell lines in vitro in terms of effect on cell growth, cell cycle distribution, and apoptosis induction; and in vivo in BALB-neuT mice developing spontaneous breast cancer in terms of biodistribution, cancer growth inhibition, and heart toxicity. BNS-DOX significantly inhibited cancer cell proliferation, through the induction of apoptosis, with higher efficiency than free DOX. The breast cancer growth in BALB-neuT mice was inhibited by 60% by a BNS-DOX dose five times lower than the DOX therapeutic dose, with substantial reduction of tumor neoangiogenesis and lymphangiogenesis. Biodistribution after BNS-DOX treatment revealed a high accumulation of DOX in the tumor site and a low accumulation in the hearts of mice. Results indicated that use of BNS may be an efficient strategy to deliver DOX in the treatment of breast cancer, since it improves the anti-cancer effectiveness and reduces cardiotoxicity.

Study of oxyresveratrol complexes with insoluble cyclodextrin based nanosponges: Developing a novel way to obtain their complexation constants and application in an anticancer study.

The complexation of the bioactive compound oxyresveratrol (OXY) with a polymer called cyclodextrin-based nanosponge (CD-NS) and its application was studied.A new methodology is used to calculate, an apparent inclusion complex constant (KFapp) between a ligand and CD-NSs. Moreover, the KFapp of resveratrol was also evaluated and compared. The complex of OXY with the nanosponge ß-CDI 1:4, was studied in vitro using DSC, TGA and FTIR techniques and its drug loading and release behavior were studied. An in vitro digestion showed higher protection of OXY complexed than free OXY. The bioactivity enhancing capacity of OXY was also studied against prostate (PC-3) and colon (HT-29 and HCT-116) cancer cell lines, where it showed stronger cell viability inhibition than the free drug. The findings as a whole represent a new opportunity for studying the complexation of drugs in CD-NSs and the use of oxyresveratrol as an ingredient in nutraceutical products.

Evaluation of solubility enhancement, antioxidant activity, and cytotoxicity studies of kynurenic acid loaded cyclodextrin nanosponge.

Kynurenic acid demonstrates antioxidant, neuroprotective and free radical scavenging properties. However, low aqueous solubility of kynurenic acid limits its therapeutic activity. In the present study, cyclodextrin nanosponges were used to improve the solubility and therapeutic activity of kynurenic acid. The formation of kynurenic acid loaded nanosponge was confirmed by different characterization techniques. The solubility of kynurenic acid was significantly increased with nanosponge (111.1 µg/ml) compared to free kynurenic acid (16.4 µg/ml) and ß-cyclodextrin (28.6 µg/ml). High drug loading (19.06%) and encapsulation efficiency (95.31%) were achieved with NS. The particle size and zeta potential of kynurenic acid loaded nanosponge was around 255.8 nm and -23 mV respectively. Moreover, higher solubilization of kynurenic acid loaded nanosponge produced better antioxidant activity compared to free kynurenic acid. The kynurenic acid loaded nanosponge and blank nanosponge were found nontoxic in the cytotoxicity assay. Thus, these studies demonstrated that nanosponges can be used as a carrier for the delivery of kynurenic acid.

'In Vitro', 'In Vivo' and 'In Silico' Investigation of the Anticancer Effectiveness of Oxygen-Loaded Chitosan-Shelled Nanodroplets as Potential Drug Vector.

PURPOSE: Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects 'per se', using a mathematical model validated on experimental data. METHODS: OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section. In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN. RESULTS: OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results. CONCLUSIONS: Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

α-Cyclodextrin and α-Cyclodextrin Polymers as Oxygen Nanocarriers to Limit Hypoxia/Reoxygenation Injury: Implications from an In Vitro Model.

The incidence of heart failure (HF) is increasing worldwide and myocardial infarction (MI), which follows ischemia and reperfusion (I/R), is often at the basis of HF development. Nanocarriers are interesting particles for their potential application in cardiovascular disease. Impaired drug delivery in ischemic disease is challenging. Cyclodextrin nanosponges (NS) can be considered innovative tools for improving oxygen delivery in a controlled manner. This study has developed new α-cyclodextrin-based formulations as oxygen nanocarriers such as native α-cyclodextrin (α-CD), branched α-cyclodextrin polymer (α-CD POLY), and α-cyclodextrin nanosponges (α-CD NS). The three different α-CD-based formulations were tested at 0.2, 2, and 20 µg/mL to ascertain their capability to reduce cell mortality during hypoxia and reoxygenation (H/R) in vitro protocols. H9c2, a cardiomyoblast cell line, was exposed to normoxia (20% oxygen) or hypoxia (5% CO2 and 95% N2). The different formulations, applied before hypoxia, induced a significant reduction in cell mortality (in a range of 15% to 30%) when compared to samples devoid of oxygen. Moreover, their application at the beginning of reoxygenation induced a considerable reduction in cell death (12% to 20%). α-CD NS showed a marked efficacy in controlled oxygenation, which suggests an interesting potential for future medical application of polymer systems for MI treatment.