Curcumin is a natural polyphenol with anti-oxidative, anti-inflammatory and anti-cancer properties but its therapeutic potential is substantially hindered by the rather low water solubility and bioavailability. Thus, in this work, a new soluble inclusion complex of curcumin with sulfobutylether--cyclodextrin (SBE-β-CD) was prepared in solution and at the solid state using different preparation techniques and characterized by FT-IR, NMR, DSC, SEM, phase solubility studies and Job’s plot method. Results clearly indicate that curcumin reacts with SBE-β-CD to form a host-guest complex with an apparent formation constant of 1455 M-1. Moreover, SBE-β-CD strongly increases water solubility of curcumin (from 0.56 to 102.78 g/ml, at 25◦C) and lyophilization method seems to be the best preparation technique to obtain the complex at the solid state. Finally, an in vitro test on a human hepatic cancer cell line (HepG-2) shows that complexation positively influences curcumin anticancer and antioxidant activity

Nanoparticles (NPs) emitting in the second biological near infrared (NIR) window of the electromagnetic spectrum have been successfully synthesized by growing a silica shell on the hydrophobic surface of OLEA/TOP PbS nanocrystals (NCs), by means of a reverse microemulsion approach, and subsequently decorated with biotin molecules. The fabrication of very uniform and monodisperse NPs, formed of SiO2 shell coated single core PbS NCs, has been demonstrated by means of a set of complementary optical and structural techniques (Vis-NIR absorption and photoluminescence spectroscopy, transmission electron microscopy) that have highlighted how experimental parameters, such as PbS NC and silica precursor concentration, are crucial to direct the morphology and optical properties of silica coated PbS NPs. Subsequently, the silica surface of the core-shell NPs has been grafted with amino groups, in order to achieve covalent binding of biotin to NIR emitting silica coated NPs. Finally the successful reaction with a green-fluorescent labelled streptavidin has verified the molecular recognition response of the biotin molecules decorating the PbS@SiO2 NP surface. Dynamic light scattering (DLS) and zeta-potential techniques have been used to monitor the hydrodynamic diameter and colloidal stability of both PbS@SiO2 and biotin decorated NPs, showing their high colloidal stability in physiological media, as needed for biomedical applications. Remarkably the obtained biotinylated PbS@SiO2 NPs have been found to retain emission properties in the 'second optical window' of the NIR region of the electromagnetic spectrum, thus representing attractive receptor-targeted NIR fluorescent probes for in vivo tumour imaging.

Solid inclusion complex between hydroxypropyl-β-cyclodextrin (HP-β-CD) and minoxidil (MXD) was prepared by freeze-drying and characterized by yield, drug loading and dissolution rate. Moreover, the complex was formulated as alginate gel (GEL HP-β-CD)/MXD 3.5% w/w). The efficacy of the novel GEL HP-β-CD)/MXD 3.5% w/w and of MXD 3.5% w/w ethanolic/propylene-glycol solution (MXD solution) were evaluated by monitoring the hair growth of dorsal skin 1-4 weeks after depilation followed by histological analysis and gene expression in skin biopsies in male rat. Patch-clamp experiments and cell-dehydrogenase activity (CDA) were performed to evaluate the capability of the formulations to activate "in vitro" the ATP-sensitive K(+)-channels (KATP) and their effects on cell viability in skin fibroblasts. After 3 weeks, the MXD solution and MXD/HP-β-CD GEL enhanced the hair growth, respectively, of 80.1±2% and 84.3±4% vs controls. After 4 weeks, the MXD/HP-β-CD GEL significantly enhanced the hair length and bulb diameter vs others groups. The MXD/HP-β-CD GEL significantly enhanced the mRNA levels of the SUR2 and Kir6.1 subunits of the KATP channels and AKT2 vs other groups. The AR gene was down-regulated vs controls following the treatment with either MXD formulations. Either MXD (10(-4)M) formulations were effective in potentiating the KATP currents. The MXD solution and its vehicle after 9 h of incubation time, but not MXD/HP-β-CD, reduced CDA in fibroblasts. In sum, the MXD/HP-β-CD GEL shows a favorable profile following topical long-term use.

The main aim of the present study was to estimate the carrier characteristics affecting the dissolution efficiency of Griseofulvin (Gris) containing blends (BLs) using partial least squares (PLS) regression analysis. These systems were prepared at three different drug/carrier weight ratios (1/5, 1/10, and 1/20) by the solvent evaporation method, a well-established method for preparing solid dispersions (SDs). The carriers used were structurally different including polymers, a polyol, acids, bases and sugars. The BLs were characterised at the solid-state by spectroscopic (Fourier transform infrared spectroscopy), thermoanalytical (differential scanning calorimetry) and X-ray diffraction studies and their dissolution behaviours were quantified in terms of dissolution efficiencies (log DE/DEGris). The correlation between the selected descriptors, including parameters for size, lipophilicity, cohesive energy density, and hydrogen bonding capacity and log DE/DEGris (i.e., DE and DEGris are the dissolution efficiencies of the BLs and the pure drug, respectively) was established by PLS regression analysis. Thus two models characterised by satisfactory coefficient of determination were derived. The generated equations point out that aqueous solubility, density, lipophilic/hydrophilic character, dispersive/polar forces and hydrogen bonding acceptor/donor ability of the carrier are important features for dissolution efficiency enhancement. Finally, it could be concluded that the correlations developed may be used to predict at a semiquantitative level the dissolution behaviour of BLs of other essentially neutral drugs possessing hydrogen bonding acceptor groups only.

Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a] pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Subcellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents.

Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy.

Binary asymmetric nanocrystals (BNCs), composed of a photoactive TiO2 nanorod joined with a superparamagnetic gamma-Fe2O3 spherical domain, were embedded in polyethylene glycol modified phospholipid micelle and successfully bioconjugated to a suitably designed peptide containing an RGD motif. BNCs represent a relevant multifunctional nanomaterial, owing to the coexistence of two distinct domains in one particle, characterized by high photoactivity and magnetic properties, that is particularly suited for use as a phototherapy and hyperthermia agent as well as a magnetic probe in biological imaging. We selected the RGD motif in order to target integrin expressed on activated endothelial cells and several types of cancer cells. The prepared RGD-peptide/BNC conjugates, comprehensively monitored by using complementary optical and structural techniques, demonstrated a high stability and uniform dispersibility in biological media. The cytotoxicity of the RGD-peptide/BNC conjugates was studied in vitro. The cellular uptake of RGD-peptide conjugates in the cells, assessed by means of two distinct approaches, namely confocal microscopy analysis and emission spectroscopy determination in cell lysates, displayed selectivity of the RGD-peptide-BNC conjugate for the alpha v beta 3 integrin. These RGD-peptide-BNC conjugates have a high potential for theranostic treatment of cancer.

To evaluate the utility of new Translocator protein 18 kDa (TSPO)-targeted fluorescent probes for in vivo molecular imaging of activated microglia. Compounds 2-4 were synthesized; their stability and affinity for TSPO were determined. Compounds 2-4 were incubated both with Ra2 cells in the presence of LPS, a potent activator of microglia, and with tissue sections of normal and chemically injured brains. Compounds 2-4 were injected into carotid artery or directly in striatum of mice. Cells and tissue sections from these in vitro and in vivo studies were observed by fluorescence microscopy after histochemical treatments. Compounds 2-4 are stable in both buffer and physiological medium and showed high affinity for TSPO and were found to stain live Ra2 microglial cells effectively. Double staining with Mito Tracker Red suggested that binding sites of compounds 2 and 3 may exist on mitochondria. In vivo studies showed that compounds 2-4 may penetrate in part into brain; moreover, cells in mouse striatum were stained with compounds 2-4 and microglial marker CD11b. Compounds 2-4 can fluorescently label activated microglia in vitro and in vivo.

New conjugates between inulin (INU) and vitamin E (VITE) able to form amphiphilic systems self-assembling in nanostructures thought for biomedical applications such as the therapy of urinary tract affections were prepared. This work, shows the syntheses, characterization and self-assembling properties of the obtained polymeric conjugates (INVITE). The reaction between VITE and INU has been carried out in bulk, without isolation of intermediate products leading to a convenient one-step reaction. To tailor the physical-chemical characteristics of the INVITE bioconjugate, six different INVITE conjugates were obtained by varying the relative amount of bonded VITE respect to INU repeating units. The obtained products were characterized by 1H-NMR, 13C-NMR, FT-IR and DSC. Furthermore, to verify that VITE does not undergo any oxidation during the reactions, UV-VIS analyses have been performed and the ability of the new conjugates to form nanoparticulate systems in water or 0.9 % saline was performed by dynamic light scattering. Furthermore, in the same media, was evaluated the stability of the INVITE nanosystems after incubation at 25 °C up to 12 days, by measuring at different time points their size variation. The degree of derivatization was found not influenced by the temperature, but it mostly depend from the different molar ratios. Interestingly, the introduction of VITE in the inulin backbone resulted almost quantitative. All conjugates resulted self-assembling in water forming nanosystems sized below 100 nm. The nanosystems are stable and do not aggregate after 12 days incubation. These results strongly encourage to prove these systems for drug delivery applications.

Aim of this project is the synthesis and characterization of nanostructured drug delivery systems (DDS) to be used in the therapy of urinary tract affections. The main idea is based on the synthesis of a bioconjugate between inulin and vitamin E (INVITE) able to produce amphiphilic systems self-assembling in nanostructured DDS even at low concentrations. A so ideated system should solubilize, incorporate and protect lipophilic drugs while targeting them in the urinary tract [1,2].

This study investigates the use of codrugs of the GABAergic agent 2-phenyl-imidazo[1,2-a]pyridinacetamide and dopamine (DA) or ethyl ester L-Dopa (LD) as a strategy to deliver DA and simultaneously activate GABA-receptors in the brain. For this purpose, both DA and LD ethyl ester were linked by carbamate bond to imidazo[1,2-a]pyridine acetamide moieties to yield two DA- and two LD-imidazopyridine derivatives. These compounds were evaluated in vitro to assess their stability, binding affinities and cell membrane transport, and in vivo to assess their bio-availability via microdialysis studies. The two DA derivatives were adequately stable in buffered solution, but underwent cleavage in diluted human serum. By contrast, the LD derivatives were unstable in buffered solution. Receptor binding studies showed that the DA-imidazopyridine carbamates had binding affinity for benzodiazepine receptors in the nanomolar range. Brain microdialysis experiments indicated that intraperitoneal administration of the DA derivatives sustained DA levels in rat striatum over a 4-h period. These results suggest that DA-imidazopyridine carbamates are new DA codrugs with potential application for DA replacement therapy.

Peripheral benzodiazepine receptors (PBRs, also named TSPO) are overexpressed in many tumor types, with the grade of TSPO overexpression correlating with the malignancy of the tumor. For this reason, TSPO-binding ligands have been widely explored as carriers for receptor-mediated drug delivery. In this paper we have selected a ligand with nanomolar affinity for TSPO, [2-(4-chlorophenyl)-8-aminoimidazo[1,2-a]pyridin-3-yl]]-N,N-di-n-propyla cetamide (3), for preparing platinum adducts that are structural analogues to picoplatin, cis-[PtCl(2)(NH(3))(2-picoline)] (AMD0473, 6), a platinum analogue currently in advanced clinical investigation. In vitro studies assessing receptor binding and cytotoxicity against human and rat glioma cells have shown that the new compounds cis-[PtX(2)(NH(3)){[2-(4-chlorophenyl)-8-aminoimidazo[1,2-a]pyridin-3-yl ]-N,N-di-n-propylacetamidel}] (X = I,4; X = Cl, 5) keep high affinity and selectivity for TSPO (nanomolar concentration) and are as cytotoxic as cisplatin. Moreover, they appear to be equally active against sensitive and cisplatin-resistant A2780 cells. Similar to cisplatin, these compounds induce apoptosis but show a favorable 10- to 100-fold enhanced accumulation in the glioma cells.

The increasing importance of sigma-2 receptor as target for the diagnosis and therapy of tumors paves the way for the development of innovative optically traceable fluorescent probes as tumor cell contrast and therapeutic agents. Here, a novel hybrid organic-inorganic nanostructure is developed by combining the superior fluorescent properties of inorganic quantum dots (QDs), coated with a hydrophilic silica shell (QD@SiO2 NPs), the versatility of the silica shell, and the high selectivity for sigma-2 receptor of the two synthetic ligands, namely, the 6-[(6-aminohexyl)oxy]-2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroisoquinolin-1(2H)-one (MLP66) and 6-[1-[3-(4-cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy]hexylamine (TA6). The proposed nanostructures represent a challenging alternative to all previously studied organic small fluorescent molecules, based on the same sigma-2 receptor affinity moieties. Flow cytometry and confocal fluorescence microscopy experiments, respectively, on fixed and living cancerous MCF7 cells, which overexpress the sigma-2 receptor, prove the ability of functionalized (QD@SiO2-TA6 and QD@SiO2-MLP66) NPs to be internalized and demonstrate their affinity to the sigma-2 receptor, ultimately validating the targeting properties conveyed to the NPs by sigma-2 ligand conjugation. The presented QD-based nanoprobes possess a great potential as in vitro selective sigma-2 receptor imaging agent and, consequently, could provide a significant impact to future theranostic applications.

Background: Ligand targeted therapy (LTT) is a powerful pharmaceutical strategy to achieve selective drug delivery to pathological cells, for both therapeutic and diagnostic purposes, with the advantage of limited side effects and toxicity. This active drug targeting approach is based on the discovery that there are receptors overexpressed on pathological cells, compared to their expression in normal tissues. Purpose: The purpose of this article is to review recently published data on LTT with applications, both in the field of cancer therapy and other diseases. Moreover, data on LTT exploiting receptors overexpressed at cytoplasmatic level are also reviewed. Methods: Data were deduced from Medline (PubMed) and SciFinder and their selections were made with preference to papers where the most relevant receptors were involved. Results: Several groups have reported improved delivery of targeted nanocarriers, as compared to nontargeted ones, to pathological cells. LTT offers several advantages, but there are also limitations in the development of this strategy. Moreover, LTT have shown encouraging results in in vitro and in animal models in vivo; hence their clinical potential awaits investigation. Conclusion: Recent studies highlight that the ligand density plays an important role in targeting efficacy. Furthermore, LTT applications in diseases different from cancer and those exploiting receptors overexpressed at cytoplasmatic level are growing.

A new tridentate 2-phenyl-imidazopyridin-dipropylacetamide ligand (CB239-H) with high (nanomolar) affinity for the TSPO protein was synthesized and its coordination compound with rhenium tricarbonyl, fac-[Re(CO)3(CB239-N,N,O)] was investigated. The procedure established for the synthesis of the 187/185Re complex can be also used for the synthesis of 99mTc and 188/186Re analogues, which find application in SPECT diagnosis and in therapy. Because of the tridentate coordination of CB239-H and the kinetic inertness of the carbonyl ligands, the new complex was expected to exhibit low reactivity towards plasma proteins and hence greater resistance to deactivation. Being TSPO overexpressed in numerous types of cancers and in activated microglial cells occurring in inflammatory neurodegenerative diseases, TSPO ligands can be exploited as carriers for receptor-mediated drug targeting and hence can be used in diagnosis as well as in therapy. Very suprisingly, fac-[Re(CO)3(CB239-N,N,O)] resulted to be not very stable in diluted human serum but maintained a good affinity towards TSPO.

The transport of dopamine across the blood brain barrier represents a challenge for the management of Parkinson's disease. The employment of central nervous system targeted ligands functionalized nanocarriers could be a valid tactic to overcome this obstacle and avoid undesirable side effects. In this work, transferrin functionalized dopamine-loaded liposomes were made by a modified dehydration-rehydration technique from hydrogenated soy phosphatidylcoline, cholesterol and 1,2-stearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(poly(ethylene glycol)-2000)]. The physical features of the prepared liposomes were established with successive determination of their endothelial permeability across anin vitromodel of the blood-brain barrier, constituted by human cerebral microvascular endothelial cells (hCMEC/D3). Functionalized dopamine-loaded liposomes with encapsulation efficiency more than 35% were made with sizes in a range around 180 nm, polydispersity indices of 0.2, and positive zeta potential values (+7.5 mV). Their stability and drug release kinetics were also evaluated. The apparent permeability (Pe) values of encapsulated dopamine in functionalized and unfunctionalized liposomes showed that transferrin functionalized nanocarriers could represent appealing non-toxic candidates for brain delivery, thus improving benefits and decreasing complications to patients subjected to L-dopa chronical treatment.

The aim of this work was to evaluate TSPO ligand-Ara-C conjugation as an approach for the selective delivery of the antineoplastic agent to brain tumors as well as for overcome P-gp resistance induction observed for the majority of cytotoxic agents, enhancing the drug clinical potential. To this end, the novel N-imidazopyridinacetyl-Ara-C conjugates 3a-c, 10 and 15 have been prepared and evaluated for their cytotoxicity against glioma cell lines. In contrast to that observed for 3a-c and 10, the conjugate 15 resulted stable in both phosphate buffer and physiological medium. In all cases, the release of free Ara-C from hydrolyzed conjugates was checked by HPLC and ESI-MS analysis. Conjugates 10 and 15 displayed very high in vitro TSPO affinity and selectivity, and, hence, they may possess potential for targeted brain delivery. Due to the favorable features displayed by the conjugate 15, it was further evaluated on glioma cell lines, expressing high levels of TSPO, in the presence and in the absence of specific nucleoside transport (NT) inhibitors. In contrast to that observed for the free Ara-C, the presence of NT inhibitors did not reduce the cytotoxic activity of 15. Moreover, conjugate 15, as N(4)-acyl derivative of Ara-C, should be resistant to inactivation by cytidine deaminase, and it may possess enhanced propensity to target brain tumor cells characterized by a reduced expression of NTs. In addition, this conjugate behaves as a clear P-gp modulator and thereby may be useful to reverse MDR. Transport studies across the MDCKII-MDR1 monolayer indicated that conjugate 15 should overcome the BBB by transcellular pathway. All these features may be useful for enhancing the clinical potential of the nucleoside drug Ara-C.

Translocator protein 18 kDa (TSPO) is a promising target for molecular imaging and for targeted drug delivery to tumors overexpressing TSPO. In our previous work, new macromolecular conjugates with a high affinity and selectivity for TSPO were prepared by conjugating the biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) polymer with two potent and selective TSPO ligands, namely, compounds 1 and 2. Based on this, nanoparticle delivery systems (NPs), employing TSPO ligand-PLGA conjugated (PLGA-TSPO) polymers, were prepared. Furthermore, to evaluate the ability of the new NPs to be used as a drug delivery systems for anticancer therapy, PLGA-TSPO NPs were loaded with 5-fluorouracil (5-FU), chosen as a model hydrophilic anticancer drug. The main goal of this work was to investigate the synergistic potential of using NP conjugates PLGA-TSPO, TSPO ligands being pro-apoptotic agents, to simultaneously deliver a cytotoxic anticancer drug. To better highlight the occurrence of synergistic effects, dual drug loaded PLGA NPs (PLGA NPs/5-FU/1) and dual drug loaded PLGA-TSPO NPs (PLGA-TSPO NPs/5-FU/1), with 5-FU and TSPO ligand 1 physically incorporated together, were also prepared and characterized. The particle size and size distribution, surface morphology, and drug encapsulation efficiency, as well as the drug release kinetics, were investigated. In vitro cytotoxicity studies were carried out on C6 glioma cells overexpressing TSPO, and to evaluate the potential uptake of these nanoparticulate systems, the internalization of fluorescent labeled PLGA-TSPO NPs (FITC-PLGA-TSPO NPs) was also investigated by fluorescence microscopy. Results demonstrated that PLGA-TSPO NPs/5-FU and dual drug loaded PLGA NPs/5-FU/1 and PLGA-TSPO NPs/5-FU/1 could significantly enhance toxicity against human cancer cells due to the synergistic effect of the TSPO ligand 1 with the anticancer drug 5-FU.