in-vitro-permeation-fitc-loaded-ferritins-across-rat-blood-brain

<em>In Vitro</em> Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Università di Milano

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein which promotes the final liquefaction of infected host larvae. The potential of this viral molecule as a new tool for insect control is explored here. The ChiA gene was isolated from the AcMNPV genome by PCR and expressed in E. coli. The recombinant protein, purified by affinity chromatography, showed both exo- and endo-chitinase activities and produced perforations on the peritrophic membrane (PM) of Bombyx mori larvae which increased in number and in size, in a dose-dependent manner. This structural alteration resulted into a significant increase of PM permeability to methylene blue and to the small neuropeptide proctolin. When the fifth instar larvae of B. mori were fed on a artificial diet supplemented with the recombinant ChiA, 100% mortality was observed at a dose of 1 microg/g of larval body weight (LW), while at sub-lethal doses of 0.56 microg/g LW, a reduced larval growth was recorded. These results indicate that AcMNPV-ChiA may offer interesting new opportunities for pest control.

AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae.

In the last decade, the study of peptide and protein absorption by the insect gut has received increasing attention because of the considerable impact this information may have on the development of new delivery strategies for insecticide macromolecules targeting haemocoelic receptors. Available experimental evidence in vivo suggests that, in insects, peptides and proteins can cross the intestinal barrier reaching the haemocoel, but the functional bases of this absorption pathway have not yet been thoroughly investigated. The current knowledge of the mechanisms involved in protein and polypeptide absorption in animals derives from the extensive studies performed in mammalian polarised epithelial cells, where the transcellular transport of proteins by transcytosis has been demonstrated. In this process, proteins are internalised at one pole of the cell and transported by cytoplasmic vesicular traffic to the opposite plasma membrane domain, where they are released with unchanged biological activity. Here we report data on albumin translocation across the isolated midgut of Bombyx mori caterpillars perfused in vitro. The functional properties of the transepithelial transport of this protein are described and, since absorption prevails over secretion, its lumen-to-haemolymph flux is characterised. Low-temperature incubations nearly abolish the transepithelial transport, while the peculiar physiological features of the larval midgut, i.e. the high lumen positive transepithelial voltage and the luminal alkaline pH, do not affect the flux. The obtained results indicate that albumin crosses B. mori larval midgut by transcytosis.

Absorption of albumin by the midgut of a lepidopteran larva.

Increasing experimental evidence indicates that ingested proteins can in part reach the haemocoel undegraded, but information on the mechanisms involved in protein transport across the insect gut is very limited, in spite of the implications that this may have on the development of novel delivery strategies of insecticide proteins targeting haemocoelic receptors. Here we contribute to this field of study, by focusing on horseradish peroxidase (HRP) transport through Bombyx mori larval midgut, isolated and perfused in vitro. The protein crossed the intestinal barrier in a time-dependent manner and the influx was linearly related to time between 30 and 90 min of incubation. HRP absorption was strongly affected by temperature and inhibition of cell metabolism: protein influx at 4 degrees C was reduced to 27% of that measured at 25 degrees C and was similarly inhibited by the metabolic inhibitor DNP. Transmission electron microscopy analysis of midgut columnar cells exposed to HRP showed the presence of the protein both in vesicular structures inside the cytoplasm and in the space between two adjacent absorptive cells, indicating the occurrence of both a transcellular and a paracellular permeation route. The analysis of HRP influx as a function of increasing protein concentration in the lumen supported this morphological indication. The J(max) relative to the HRP transcellular transport component was 121+/-24 pmol/cm(2)/h and the K(d) of the passage through the paracellular route was 1.9+/-0.3 microl/cm(2)/h. The paracellular electrical resistance decreased in midguts exposed to HRP, indicating that its passage through this pathway was likely due to an alteration exerted on the junctional complex by the protein itself. The role of the cytoskeleton in HRP transport was investigated by assessing the impact of drugs affecting microtubules and actin filaments.

Absorption of horseradish peroxidase in Bombyx mori larval midgut.

Biotechnology has allowed the development of novel strategies to obtain plants that are more resistant to pests, fungal pathogens and other agents of biotic stress. The obvious advantages of having genotypes with multiple beneficial traits have recently fostered the development of gene pyramiding strategies, but less attention has been given to the study of genes that can increase resistance to different types of harmful organisms. Here we report that a recombinant Chitinase A protein of the Autographa californica nuclear polyhedrosis virus (AcMNPV) has both antifungal and insecticide properties in vitro. Transgenic tobacco plants expressing an active ChiA protein showed reduced damages caused by fungal pathogens and lepidopteran larvae, while did not have an effect on aphid populations. To our knowledge, this is the first report on the characterisation and expression in plants of a single gene that increases resistance against herbivorous pests and fungal pathogens and not affecting non-target insects. The implications and the potential of the ChiA gene for plant molecular breeding and biotechnology are discussed.

The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco.

The Autographa californica nucleopolyhedrovirus chitinase A (AcMNPV ChiA) is a chitinolytic enzyme with fungicidal and insecticidal properties. Its expression in transgenic plants enhances resistance against pests and fungal pathogens. We exploited tobacco for the production of a biologically active recombinant AcMNPV ChiA (rChiA), as such species is an alternative to traditional biological systems for large-scale enzyme production. The protein was purified from leaves using ammonium sulfate precipitation followed by anion exchange and gel-filtration chromatography. Transgenic plants produced an estimated 14 mg kg(-1) fresh leaf weight, which represents 0.2% of total soluble proteins. The yield of the purification was about 14% (2 mg kg(-1) fresh leaf weight). The comparison between the biochemical and kinetic properties of the rChiA with those of a commercial Serratia marcescens chitinase A indicated that the rChiA was thermostable and more resistant at basic pH, two positive features for agricultural and industrial applications. Finally, we showed that the purified rChiA enhanced the permeability of the peritrophic membrane of larvae of two Lepidoptera (Bombyx mori and Heliothis virescens) and inhibited spore germination and growth of the phytopatogenic fungus Alternaria alternata. The data indicated that tobacco represents a suitable platform for the production of rChiA, an enzyme with interesting features for future applications as "eco-friendly" control agent in agriculture.

Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco.

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.

A viral chitinase enhances oral activity of TMOF.

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.

HER2 expression in breast cancer cells is downregulated upon active targeting by antibody-engineered multifunctional nanoparticles in mice.

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.

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

A genetically engineered apoferritin variant consisting of 24 heavy-chain subunits (HFn) was produced to achieve a cumulative delivery of an antitumor drug, which exerts its cytotoxic action by targeting the DNA at the nucleus of human cancer cells with subcellular precision. The rationale of our approach is based on exploiting the natural arsenal of defense of cancer cells to stimulate them to recruit large amounts of HFn nanoparticles loaded with doxorubicin inside their nucleus in response to a DNA damage, which leads to a programmed cell death. After demonstrating the selectivity of HFn for representative cancer cells compared to healthy fibroblasts, doxorubicin-loaded HFn was used to treat the cancer cells. The results from confocal microscopy and DNA damage assays proved that loading of doxorubicin in HFn nanoparticles increased the nuclear delivery of the drug, thus enhancing doxorubicin efficacy. Doxorubicin-loaded HFn acts as a "Trojan Horse": HFn was internalized in cancer cells faster and more efficiently compared to free doxorubicin, then promptly translocated into the nucleus following the DNA damage caused by the partial release in the cytoplasm of encapsulated doxorubicin. This self-triggered translocation mechanism allowed the drug to be directly released in the nuclear compartment, where it exerted its toxic action. This approach was reliable and straightforward providing an antiproliferative effect with high reproducibility. The particular self-assembling nature of HFn nanocage makes it a versatile and tunable nanovector for a broad range of molecules suitable both for detection and treatment of cancer cells.

Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.

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.

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

New perspectives on nanotechnology and antiretroviral drugs: a 'small' solution for a big promise in HIV treatment?

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.

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

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.

Article	Year
A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut. Journal of insect physiology\| PubMed ID: 12770086	2002
AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae. Insect biochemistry and molecular biology\| PubMed ID: 15522616	2004
Absorption of albumin by the midgut of a lepidopteran larva. Journal of insect physiology\| PubMed ID: 15935372	2005
Absorption of horseradish peroxidase in Bombyx mori larval midgut. Journal of insect physiology\| PubMed ID: 17391693	2007
The Chitinase A from the baculovirus AcMNPV enhances resistance to both fungi and herbivorous pests in tobacco. Transgenic research\| PubMed ID: 17851776	2008
Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco. Journal of biotechnology\| PubMed ID: 20302895	2010
A viral chitinase enhances oral activity of TMOF. Insect biochemistry and molecular biology\| PubMed ID: 20457253	2010
HER2 expression in breast cancer cells is downregulated upon active targeting by antibody-engineered multifunctional nanoparticles in mice. ACS nano\| PubMed ID: 21790185	2011
Assessing the in vivo targeting efficiency of multifunctional nanoconstructs bearing antibody-derived ligands. ACS nano\| PubMed ID: 23758591	2013
Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 25312541	2014
Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 25839392	2015
New perspectives on nanotechnology and antiretroviral drugs: a 'small' solution for a big promise in HIV treatment? AIDS (London, England)\| PubMed ID: 26807964	2016
Ferritin nanocages: A biological platform for drug delivery, imaging and theranostics in cancer. Pharmacological research\| PubMed ID: 26968122	2016
Oral delivery of insulin via polyethylene imine-based nanoparticles for colonic release allows glycemic control in diabetic rats. Pharmacological research\| PubMed ID: 27181095	2016

Luisa Fiandra

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