Luisa Fiandra

HER2 Expression in Breast Cancer Cells Is Downregulated Upon Active Targeting by Antibody-Engineered Multifunctional Nanoparticles in Mice

Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.

Assessing the in vivo targeting efficiency of multifunctional nanoconstructs bearing antibody-derived ligands

A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their in vivo localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct.

Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect.

Ferritin nanocages: A biological platform for drug delivery, imaging and theranostics in cancer

Nowadays cancer represents a prominent challenge in clinics. Main achievements in cancer management would be the development of highly accurate and specific diagnostic tools for early detection of cancer onset, and the generation of smart drug delivery systems for targeted chemotherapy release in cancer cells. In this context, protein-based nanocages hold a tremendous potential as devices for theranostics purposes. In particular, ferritin has emerged as an excellent and promising protein-based nanocage thanks to its unique architecture, surface properties and high biocompatibility. By exploiting natural recognition of the Transferrin Receptor 1, which is overexpressed on tumor cells, ferritin nanocages may ensure a proper drug delivery and release. Moreover, researchers have applied surface functionalities on ferritin cages for further providing active tumor targeting. Encapsulation strategies of non metal-containing drugs within ferritin cages have been explored and successfully performed with encouraging results. Various preclinical studies have demonstrated that nanoformulation within ferritin nanocages significantly improved targeted therapy and accurate imaging of cancer cells. Aims of this review are to describe structure and functions of ferritin nanocages, and to provide an overview about the nanotechnological approaches implemented for applying them to cancer diagnosis and treatment.

Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice

UNLABELLED Eradication of virus by sanctuary sites is a main goal in HIV management. The central nervous system (CNS) is a classic model of sanctuary where viral replication occurs despite a complete viral suppression in peripheral blood. In recent years, nanotechnologies have provided a great promise in the eradication of HIV from the CNS. We hereby demonstrate for the first time that the structurally complex antiretroviral drug enfuvirtide (Enf), which normally is unable to penetrate the cerebrospinal fluid, is allowed to cross the blood brain barrier (BBB) in mice by conjugation with a nanoconstruct. Iron oxide nanoparticles coated with an amphiphilic polymer increase Enf translocation across the BBB in both in vitro and in vivo models. The mechanism involves the uptake of nanoconjugated-Enf in the endothelial cells, the nanocomplex dissociation and the release of the peptide, which is eventually excreted by the cells in the brain parenchyma. FROM THE CLINICAL EDITOR Despite the success of cocktail therapy of antiretroviral drugs, the complete eradication of HIV remains elusive, due to existence of viral sanctuary sites. The authors showed in this study that an antiretroviral drug complexed with iron oxide nanoparticles and coated with PMA amphiphilic polymer crosses the blood brain barrier. Furthermore, there was significant anti-viral activity. The results would aid further drug designs to eradicate HIV.

The intestinal barrier in lepidopteran larvae : Permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>>cysteine, lysine>>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

A viral chitinase enhances oral activity of TMOF.

In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors.

Nanometronomic treatment of 4T1 breast cancer with nanocaged doxorubicin prevents drug resistance and circumvents cardiotoxicity

Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach. However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, “metronomic” chemotherapy is based on frequent drug administrations at lower doses, resulting in neovascularization inhibition and induction of tumor dormancy. Here we show the potential of H-ferritin (HFn)-mediated targeted nanodelivery of metronomic doxorubicin (DOX) in the setting of a highly aggressive and metastatic 4T1 breast cancer mouse model with DOX-inducible expression of chemoresistance. We find that HFn-DOX administered at repeated doses of 1.24 mg kg−1 strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. We find that such a potent antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity. Our results suggest that HFn-DOX has tremendous potential for the development of “nanometronomic” chemotherapy toward safe and tailored oncological treatments.

Oral delivery of insulin via polyethylene imine-based nanoparticles for colonic release allows glycemic control in diabetic rats.

In this study, insulin-containing nanoparticles were loaded into pellet cores and orally administered to diabetic rats. Polyethylene imine-based nanoparticles, either placebo or loaded with insulin, were incorporated by extrusion and spheronization technology into cores that were subsequently coated with three overlapping layers and a gastroresistant film. The starting and coated systems were evaluated in vitro for their physico-technololgical characteristics, as well as disintegration and release performance. Nanoparticles-loaded cores showed homogeneous particle size distribution and shape. When a superdisintegrant and a soluble diluent were included in the composition enhanced disintegration and release performance were observed. The selected formulations, coated either with enteric or three-layer films, showed gastroresistant and release delayed behavior in vitro, respectively. The most promising formulations were finally tested for their hypoglycemic effect in diabetic rats. Only the nanoformulations loaded into the three-layer pellets were able to induce a significant hypoglycemic activity in diabetic rats. Our results suggest that this efficient activity could be attributed to a retarded release of insulin into the distal intestine, characterized by relatively low proteolytic activity and optimal absorption.