Correction to: Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells.

Following publication of our article [1], we became aware that Roberto Di Gesù had been omitted from the list of authors. The corrected author list and authors' contribution statement appear below. We apologize for any inconvenience this may have caused.

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery.

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

Nanoassemblies Based on Supramolecular Complexes of Nonionic Amphiphilic Cyclodextrin and Sorafenib as Effective Weapons to Kill Human HCC Cells.

Sorafenib (Sor), an effective chemiotherapeutic drug utilized against hepatocellular carcinoma (HCC), robustly interacts with nonionic amphiphilic cyclodextrin (aCD, SC6OH), forming, in aqueous solution, supramolecular complexes that behave as building blocks of highly water-dispersible colloidal nanoassemblies. SC6OH/Sor complex has been characterized by complementary spectroscopic techniques, such as UV-vis, steady-state fluorescence and anisotropy, resonance light scattering and (1)H NMR. The spectroscopic evidences and experiments carried out in the presence of an adamantane derivative, which competes with drug for CD cavity, agree with the entrapment of Sor in aCD, pointing out the role of the aCD cavity in the interaction between drug and amphiphile. Nanoassemblies based on SC6OH/Sor display size of ∼200 nm, negative zeta-potential (ζ = -11 mV), and both maximum loading capacity (LC ∼ 17%) and entrapment efficiency (EE ∼ 100%). Kinetic release profiles show a slower release of Sor from nanoassemblies with respect to the free drug. SC6OH/Sor nanoassemblies have very low hemolytic activity and high efficiency in vitro in decreasing cell growth and viability of HCC cell lines, such as HepG2, Hep3B, and PLC/PRF/5, opening promising chances to their in vivo applications.

Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells.

BACKGROUND: In hepatocellular carcinoma (HCC), different signaling pathways are de-regulated, and among them, the expression of the epidermal growth factor receptor (EGFR). Tyrphostin AG-1478 is a lipophilic low molecular weight inhibitor of EGFR, preferentially acting on liver tumor cells. In order to overcome its poor drug solubility and thus improving its anticancer activity, it was entrapped into nanostructured lipid carriers (NLC) by using safe ingredients for parenteral delivery. RESULTS: Nanostructured lipid carriers (NLC) carrying tyrphostin AG-1478 were prepared by using the nanoprecipitation method and different matrix compositions. The best system in terms of mean size, PDI, zeta potential, drug loading and release profile was chosen to evaluate the anti-proliferative effect of drug-loaded NLC versus free drug on human hepatocellular carcinoma HA22T/VGH cells. CONCLUSIONS: Thanks to the entrapment into NLC systems, tyrphostin AG-1478 shows an enhanced in vitro anti-tumor activity compared to free drug. These finding raises hope of future drug delivery strategy of tyrphostin AG-1478 -loaded NLC targeted to the liver for the HCC treatment.

Effects in cigarette smoke stimulated bronchial epithelial cells of a corticosteroid entrapped into nanostructured lipid carriers.

BACKGROUND: Nanomedicine studies have showed a great potential for drug delivery into the lung. In this manuscript nanostructured lipid carriers (NLC) containing Fluticasone propionate (FP) were prepared and their biocompatibility and effects in a human bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extracts (CSE) were tested. RESULTS: Biocompatibility studies showed that the NLC did not induce cell necrosis or apoptosis. Moreover, it was confirmed that CSE increased intracellular ROS production and TLR4 expression in bronchial epithelial cells and that FP-loaded NLC were more effective than free drug in modulating these processes. Finally, the nanoparticles increased GSH levels improving cell protection against oxidative stress. CONCLUSIONS: The present study shows that NLC may be considered a promising strategy to improve corticosteroid mediated effects in cellular models associated to corticosteroid resistance. The NLC containing FP can be considered good systems for dosage forms useful for increasing the effectiveness of fluticasone decreasing its side effects.

Supramolecular assemblies based on complexes of nonionic amphiphilic cyclodextrins and a meso-tetra(4-sulfonatophenyl)porphine tributyltin(IV) derivative: potential nanotherapeutics against melanoma.

Amphiphilic cyclodextrin (ACyD) provides water-soluble and adaptable nanovectors by modulating the balance between the hydrophobic and hydrophilic chains at both CyD sides. This work aimed to design nanoassemblies based on nonionic and hydrophilic ACyD (SC6OH) for the delivery of a poor-water-soluble organotin(IV)-porphyrin derivative [(Bu3Sn)4TPPS] to melanoma cancer cells. To characterize the porphyrin derivatives under simulated physiological conditions, a speciation was performed using complementary techniques. In aqueous solution (≤ 20 µM), (Bu3Sn)4TPPS primarily exists as a monomer (2 in Figure 1), as suggested by the low static anisotropy (ρ ≈ 0.02) with a negligible formation of porphyrin supramolecular aggregates. MALDI-TOF spectra indicate the presence of moieties (i.e., [(Bu3Sn)3TPPS](-)) that are derivatives of the monomeric species. Spectrofluorimetry coupled with potentiometric measurements primarily assesses the presence of the hydrolytic [(Bu3Sn)4TPPS (OH)4](4-) species under physiological conditions. Nanoassemblies of (Bu3Sn)4TPPS/SC6OH were prepared by dispersion of organic films in PBS at pH 7.4 and were investigated using a combination of spectroscopic and morphological techniques. The UV-vis and emission fluorescence spectra of the (Bu3Sn)4TPPS/SC6OH reveal shifts in the peculiar bands of the organotin(IV)-porphyrin derivative due to its interaction with the ACyD supramolecular assemblies in aqueous solution. The mean size was within the range of 100-120 nm. The ξ-potential was negative (-16 mV) for the (Bu3Sn)4TPPS/SC6OH nanoassemblies, with an entrapment efficiency of approximately 67%. The intracellular delivery, cytotoxicity, nuclear morphology and cell growth kinetics were evaluated via fluorescence microscopy on A375 human melanoma cells. The delivery of (Bu3Sn)4TPPS by ACyD with respect to free (Bu3Sn)4TPPS increases the internalization efficiency and cytotoxicity to induce apoptotic cell death and, at lower concentrations, changes the cellular morphology and prevents cell proliferation.

Minimalism in radiation synthesis of biomedical functional nanogels.

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.

Salmeterol Xinafoate (SX) loaded into mucoadhesive solid lipid microparticles for COPD treatment.

Chronic obstructive pulmonary disease (COPD) is one of the main health problems worldwide. It is characterised by chronic inflammation in the lungs that leads to progressive, chronic, largely irreversible airflow obstruction. The use of long-acting ß agonists remain today the frontline treatment for COPD with the aim of minimizing side effects and enhancing therapeutic usefulness. To this purpose, in this paper, mucoadhesive solid lipid microparticles (SLMs) containing a long-acting ß-2 agonist, Salmeterol Xinafoate (SX) were prepared, characterised (size, z-potential, aerodynamic diameter, turbidimetric evaluations, drug loading and entrapping efficiency) and tested in a model of bronchial epithelial cells. It was demonstrated that the incorporation of SX into SLMs led to the production of particles suitable for inhalation and more efficient than the free molecule at increasing the cAMP expression in bronchial epithelial cells. In conclusion, the prepared systems, due to their aerodynamic behaviour and mucoadhesive properties, could improve the retention time of SX in the lung epithelium and its therapeutic effect, thus representing a good strategy for the treatment of COPD patients.

Radiosensitizing effect of curcumin-loaded lipid nanoparticles in breast cancer cells.

In breast cancer (BC) care, radiotherapy is considered an efficient treatment, prescribed both for controlling localized tumors or as a therapeutic option in case of inoperable, incompletely resected or recurrent tumors. However, approximately 90% of BC-related deaths are due to the metastatic tumor progression. Then, it is strongly desirable to improve tumor radiosensitivity using molecules with synergistic action. The main aim of this study is to develop curcumin-loaded solid nanoparticles (Cur-SLN) in order to increase curcumin bioavailability and to evaluate their radiosensitizing ability in comparison to free curcumin (free-Cur), by using an in vitro approach on BC cell lines. In addition, transcriptomic and metabolomic profiles, induced by Cur-SLN treatments, highlighted networks involved in this radiosensitization ability. The non tumorigenic MCF10A and the tumorigenic MCF7 and MDA-MB-231 BC cell lines were used. Curcumin-loaded solid nanoparticles were prepared using ethanolic precipitation and the loading capacity was evaluated by UV spectrophotometer analysis. Cell survival after treatments was evaluated by clonogenic assay. Dose-response curves were generated testing three concentrations of free-Cur and Cur-SLN in combination with increasing doses of IR (2-9 Gy). IC50 value and Dose Modifying Factor (DMF) was measured to quantify the sensitivity to curcumin and to combined treatments. A multi-"omic" approach was used to explain the Cur-SLN radiosensitizer effect by microarray and metobolomic analysis. We have shown the efficacy of the Cur-SLN formulation as radiosensitizer on three BC cell lines. The DMFs values, calculated at the isoeffect of SF = 50%, showed that the Luminal A MCF7 resulted sensitive to the combined treatments using increasing concentration of vehicled curcumin Cur-SLN (DMF: 1,78 with 10 µM Cur-SLN.) Instead, triple negative MDA-MB-231 cells were more sensitive to free-Cur, although these cells also receive a radiosensitization effect by combination with Cur-SLN (DMF: 1.38 with 10 µM Cur-SLN). The Cur-SLN radiosensitizing function, evaluated by transcriptomic and metabolomic approach, revealed anti-oxidant and anti-tumor effects. Curcumin loaded- SLN can be suggested in future preclinical and clinical studies to test its concomitant use during radiotherapy treatments with the double implications of being a radiosensitizing molecule against cancer cells, with a protective role against IR side effects.

Cationic Solid Lipid Nanoparticles as Non Viral Vectors for the Inhibition of Hepatocellular Carcinoma Growth by RNA Interference.

Hepatocellular carcinoma (HCC) is one of the most important causes of cancer deaths worldwide. Gene therapy is a novel approach for treating HCC. A safe and efficient gene delivery method, using viral or non-viral vectors, is a crucial factor for developing a successful HCC gene therapy. Among non-viral vectors, cationic solid lipid nanoparticles (cSLN) have advantages such as biocompatibility and transfection efficiency. In this study, novel cSLN were prepared, characterized and complexed with a plasmid (shNUPR1) capable of inhibiting the expression of the NUPR1 gene, which is involved in HCC growth and chemoresistance. The particles resulted biocompatible, as confirmed by haemolysis and cytotoxicity assays, and was able to protect the shNUPR1 plasmid from degradation by DNase I. We also demonstrated, by carrying out transfection and immunofluorescence studies, that the particles efficiently delivered the shNUPR1 plasmid into HCC cells, causing the downregulation of NUPR1-regulated genes and NUPR1 protein expression. These results suggest that the cSLN obtained could be proposed for further in vivo studies as novel transfection vectors for HCC gene therapy, having shown excellent in vitro transfection efficiency and biocompatibility.

Novel cationic solid-lipid nanoparticles as non-viral vectors for gene delivery.

In this paper, the suitability of novel cationic solid-lipid nanoparticles (SLN) as a nonviral transfection agent for gene delivery was investigated. SLN were produced by using the microemulsion method and Compritol ATO 888 as matrix lipid, dimethyldioctadecylammonium bromide as charge carrier and Pluronic F68 as surfactant. Obtained nanoparticles were approximately 120 nm in size and positively charged, with a zeta potential value equal to +45 mV in twice-distilled water. Cationic SLN were able to form stable complexes with DNA and to protect DNA against DNase I digestion. The SLN-DNA complexes were characterized by mean diameter and zeta potential measurements. In vitro studies on human liver cancer cells demonstrated a very low degree of toxicity of both SLN and SLN-DNA complexes. Further, SLN-DNA complexes were able to promote transfection of liver cancer cells. These data suggest that our cationic SLN may be potentially useful for gene therapy.

Nanostructured lipid carriers-containing anticancer compounds: preparation, characterization, and cytotoxicity studies.

This article describes the development of nanostructured lipid carriers (NLC) as colloidal carriers for two antitumor compounds that possess a remarkable antineoplastic activity. But their limited stability and low solubility in water could give a very low parenteral bioavailability. Results revealed an enhancement of the cytotoxicity effect of drug-loaded NLC on human prostate cancer (PC-3) and human hepatocellular carcinoma (HuH-6, HuH-7) cell lines with respect to that of both free drugs. Results of characterization studies strongly support the potential application of these drugs-loaded NLC as prolonged delivery systems for lipophilic drugs by several administration routes, in particular for intravenous administration.

Mucoadhesive solid lipid microparticles for controlled release of a corticosteroid in the chronic obstructive pulmonary disease treatment.

AIM: Therapeutic efficacy of pulmonary diseases is often limited and drug delivery systems offer new solutions to clinical problems. Solid lipid microparticles (SLMs) are suggested as systems for the delivery of therapeutics to the lung as, because of their size, they are able to deposit into secondary bronchi. MATERIALS & METHODS: Here, we describe two novel different SLMs using chitosan and alginate such as mucoadhesive polymers and we also studied their biocompatibility and their effectiveness compared with the free drug in controlling senescence and inflammatory processes in cigarette smoke extracts. RESULTS: Data reported show that fluticasone propionate (FP)-loaded SLMs are more effective than FP alone in controlling oxidative stress. CONCLUSION: The therapeutic approach using FP-loaded microparticles could be a promising strategy for the treatment of the chronic inflammatory pulmonary diseases.

Polyaspartamide-Based Nanoparticles Loaded with Fluticasone Propionate and the In Vitro Evaluation towards Cigarette Smoke Effects.

This paper describes the evaluation of polymeric nanoparticles (NPs) as a potential carrier for lung administration of fluticasone propionate (FP). The chosen polymeric material to produce NPs was a copolymer based on α,ß-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) whose backbone was derivatised with different molecules, such as poly(lactic acid) (PLA) and polyethylenglycol (PEG). The chosen method to produce NPs from PHEA-PLA-PEG2000 was the method based on high-pressure homogenization and subsequent solvent evaporation by adding Pluronic F68 during the process and trehalose before lyophilisation. Obtained colloidal FP-loaded NPs showed a slightly negative surface charge and nanometric dimensions that are maintained after storage for one year at -20 °C and 5 °C. The FP loading was about 2.9 wt % and the drug was slowly released in simulated lung fluid. Moreover, the obtained NPs, containing the drug or not, were biocompatible and did not induce cell necrosis and cell apoptosis on bronchial epithelial cells (16-HBE). Further in vitro testing on cigarette smoke extract (CSE)-stimulated 16-HBE revealed that FP-loaded NPs were able to reduce the survivin expression, while either free FP or empty NPs were not able to significantly reduce this effect.

Surfactant effect on the physicochemical characteristics of cationic solid lipid nanoparticles.

Solid lipid nanoparticles (SLNs) may be considered as a new approach for therapeutics for many diseases. In addition to drug delivery, their use as non-viral vectors for gene delivery can be obtained by including cationic lipids, which provide a positive surface potential that favors binding to the nucleic acids as DNA, siRNA, miRNA, etc. In fact, the addition of cationic surfactants is indispensable for obtaining nanoparticles with surface positive charge. In this study, three different cationic lipids (dioctadecyl dimethyl ammonium bromide, cetyltrimethyl ammonium bromide, cetylpyridinium chloride) and Brij 76 as nonionic surfactant were employed to formulate Precirol ATO 5 based cSLN using pEGFP-LUC as model plasmid. The physicochemical properties of cSLN were influenced by both type and amount of surfactants. Thermal analyses of bulk cSLN showed endothermic peaks significantly different from the ones of the single pure components, hinting a complete entanglement of the lipid matrix with the surfactants and justifying the different behavior of the cSLN in the ability to interact with the plasmid DNA. Finally, the biocompatibility of cSLN was demonstrated by hemolytic assays. These results may give an insight into the choice of surfactants in order to obtain non-toxic and highly effective delivery systems for gene therapy.

Biocompatible Lipid Nanoparticles as Carriers To Improve Curcumin Efficacy in Ovarian Cancer Treatment.

Curcumin is a natural molecule with proved anticancer efficacy on several human cancer cell lines. However, its clinical application has been limited due to its poor bioavailability. Nanocarrier-based drug delivery approaches could make curcumin dispersible in aqueous media, thus overtaking the limits of its low solubility. The aim of this study was to increase the bioavailability and the antitumoral activity of curcumin, by entrapping it into nanostructured lipid carriers (NLCs). For this purpose here we describe the preparation and characterization of three kinds of curcumin-loaded NLCs. The nanosystems allowed the achievement of a controlled release of curcumin, the amounts of curcumin released after 24 h from Compritol-Captex, Compritol-Miglyol, and Compritol NLCs being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. Considering the slower curcumin release profile, Compritol NLCs were chosen to perform successive in vitro studies on ovarian cancer cell lines. The results show that curcumin-loaded NLCs maintain anticancer activity, and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was decreased after treatment with 5 µM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLC significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore potentiating the activity of curcumin to inhibit A2780S cell growth. The obtained results clearly suggest that the entrapment of curcumin into NLCs increases curcumin efficacy in vitro, indicating the potential use of NLCs as curcumin delivery systems.

Lipid nanocarriers containing sorafenib inhibit colonies formation in human hepatocarcinoma cells.

Here, the potential of two nanostructured lipid carriers (NLC) for controlled release of sorafenib was evaluated. The obtained systems showed characteristics suitable as drug delivery systems for the treatment of hepatocellular carcinoma (HCC) through parenteral administration. The use of a mixture between a solid lipid (tripalmitin) with a liquid lipid (Captex 355 EP/NF or Miglyol 812) to prepare NLC systems could give a higher drug loading capacity and a longer term stability during storage than that obtained by using only solid lipids. The obtained nanoparticles showed a nanometer size and high negative zeta potential values. Scansion electron microscopy (SEM) of the sorafenib loaded NLC revealed a spherical shape with a diameter <300 nm. In vitro biological studies demonstrated that sorafenib loaded into NLC had enhanced anti-tumor activity compared to that of free drug. This finding raises hope in terms of future drug delivery strategy of sorafenib loaded NLC, that can be useful for therapeutic application in HCC.

Nanotechnology applications for the therapy of liver fibrosis.

Chronic liver diseases represent a major global health problem both for their high prevalence worldwide and, in the more advanced stages, for the limited available curative treatment options. In fact, when lesions of different etiologies chronically affect the liver, triggering the fibrogenesis mechanisms, damage has already occurred and the progression of fibrosis will have a major clinical impact entailing severe complications, expensive treatments and death in end-stage liver disease. Despite significant advances in the understanding of the mechanisms of liver fibrinogenesis, the drugs used in liver fibrosis treatment still have a limited therapeutic effect. Many drugs showing potent antifibrotic activities in vitro often exhibit only minor effects in vivo because insufficient concentrations accumulate around the target cell and adverse effects result as other non-target cells are affected. Hepatic stellate cells play a critical role in liver fibrogenesis , thus they are the target cells of antifibrotic therapy. The application of nanoparticles has emerged as a rapidly evolving area for the safe delivery of various therapeutic agents (including drugs and nucleic acid) in the treatment of various pathologies, including liver disease. In this review, we give an overview of the various nanotechnology approaches used in the treatment of liver fibrosis.

An allergen-polymeric nanoaggregate as a new tool for allergy vaccination.

A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E. coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, zeta potential, electrophoretic mobility and atom force microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity suggesting its employment as potential vaccine against Parietaria-induced allergies.

Novel composed galactosylated nanodevices containing a ribavirin prodrug as hepatic cell-targeted carriers for HCV treatment.

In this paper, we describe the preparation of liver-targeted nanoparticles potentially able to carry to hepatocytes a ribavirin (RBV) prodrug, exploiting the presence of carbohydrate receptors in the liver (i.e., ASGPR in hepatocytes). These particles were obtained starting from a galactosylated phospholipid-polyaminoacid conjugate. This latter was obtained by chemical reaction of alpha,beta-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-DL-aspartamide (PHEA-EDA) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) sodium salt (DPPE), and subsequent reaction with lactose, obtaining PHEA-EDA-DPPE-GAL copolymer. To enhance the entrapment into obtained nanostructures, a hydrophobic RBV prodrug, i.e., RBV tripalmitate, was synthesized and its capability to release RBV in the presence of an adequate enzymatic activity was demonstrated. RBV tripalmitate-loaded nanoparticles were obtained starting from PHEA-EDA-DPPE-GAL copolymer by using the dialysis method. These particles showed spherical shape and nanometric size. By in vitro experiments the absence of haemolytic activity of RBV tripalmitate-loaded PHEA-EDA-DPPE-GAL nanoparticles and their specificity toward HepG2 were demonstrated by using a competitive inhibition assay in the presence of free GAL and assessing nanoparticle uptake in the presence of free GAL and/or non-galactosylated nanoparticles. This finding raises hope in terms of future nanoparticle-based liver-targeted drug delivery strategy for the hepatitis C treatment.