Enrico Mastrobattista

Chitosan-based delivery systems for protein therapeutics and antigens

Therapeutic peptides/proteins and protein-based antigens are chemically and structurally labile compounds, which are almost exclusively administered by parenteral injections. Recently, non-invasive mucosal routes have attracted interest for administration of these biotherapeutics. Chitosan-based delivery systems enhance the absorption and/or cellular uptake of peptides/proteins across mucosal sites and have immunoadjuvant properties. Chitosan is a mucoadhesive polysaccharide capable of opening the tight junctions between epithelial cells and it has functional groups for chemical modifications, which has resulted in a large variety of chitosan derivatives with tunable properties for the aimed applications. This review provides an overview of chitosan-based polymers for preparation of both therapeutic peptides/protein and antigen formulations. The physicochemical properties of these carrier systems as well as their applications in protein and antigen delivery through parenteral and mucosal (particularly nasal and pulmonary) administrations are summarized and discussed.

Artificial viruses: a nanotechnological approach to gene delivery

Nanotechnology is a rapidly expanding multidisciplinary field in which highly sophisticated nanoscale devices are constructed from atoms, molecules or (macro)molecular assemblies. In the field of gene medicine, systems for delivering nucleic acids are being developed that incorporate virus-like functions in a single nanoparticle. Although their development is still in its infancy, it is expected that such artificial viruses will have a great impact on the advancements of gene therapeutics.

Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells

PurposeKnowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake.Materials and MethodsThe effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.ResultsBoth pDMAEMA- and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrin- and caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake.ConclusionsThese results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

Biomedical Applications of Self-Assembling Peptides.

Self-assembling peptides have gained increasing attention as versatile molecules to generate diverse supramolecular structures with tunable functionality. Because of the possibility to integrate a wide range of functional domains into self-assembling peptides including cell attachment sequences, signaling domains, vaccine epitopes, and even therapeutic moieties, complex nanostructures can be obtained with a wide range of applications in the biomedical field. The first part of this Review provides a concise overview of how peptide primary and secondary structure dictate the way such self-assembling peptides organize into higher ordered, supramolecular structures. Next, an overview of the literature will be given on recent studies on peptide self-assembly for application in drug delivery, vaccination, and tissue engineering.

Peptide nanocarriers for intracellular delivery of photosensitizers

Previously we have shown that recombinantly produced amphiphilic oligopeptides with amino acid sequence Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu spontaneously assemble into nano-sized vesicles with an average diameter of 120 nm. Moreover, peptide vesicles could be stabilized by introducing multiple cysteine residues within the hydrophobic domain of these amphiphilic oligopeptides, allowing the formation of intermolecular disulfide bridges. In this study, the cellular association and internalization of peptide vesicles were assessed. Flow cytometry and confocal laser-scanning microscopy showed that peptide vesicles were internalized by cells predominantly via adsorptive macropinocytosis. Furthermore, the potential of these peptide vesicles as delivery system for photosensitizers was explored. Water-insoluble phthalocyanines could be quantitatively entrapped within the hydrophobic domains of these peptide vesicles. Confocal laser-scanning microscopy analysis showed that internalized peptides co-localized with the phthalocyanine, suggesting that peptide vesicles are internalized in their intact form. Upon illumination, the phthalocyanine-containing peptide vesicles showed an active photodynamic response towards the cells leading to effective cell killing. In contrast, the free phthalocyanine or empty peptide vesicles did not show any cytotoxicity. In conclusion, this is the first demonstration that peptide vesicles show promise as delivery systems for photosensitizers to be used in photodynamic therapy.

Immunoliposomes for the targeted delivery of antitumor drugs

This review presents an overview of the field of immunoliposome-mediated targeting of anticancer agents. First, problems that are encountered when immunoliposomes are used for systemic anticancer drug delivery and potential solutions are discussed. Second, an update is given of the in vivo results obtained with immunoliposomes in tumor models. Finally, new developments on the utilization of immunoliposomes for the treatment of cancer are highlighted.

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles

Abstract Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical Editor The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.

Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain.

In many different studies nanocarriers modified with targeting ligands have been used to target to the brain. Many ligands have been successful, but it is difficult to compare results from different studies to determine which targeting ligand is the best. Therefore, we selected five targeting ligands (transferrin, RI7217, COG133, angiopep-2, and CRM197) and compared their ability to target liposomes to the brain in vitro and in vivo. In vitro, only CRM197-modified liposomes were able to bind to murine endothelial cells (bEnd.3). Both CRM197 and RI7217-modified liposomes associated with human endothelial cells (hCMEC/D3). In vivo, uptake of targeted liposomes was tested at 12h after iv injection. For some of the ligands, additional time points of 1 and 6h were tested. Only the RI7217 was able to significantly enhance brain uptake in vivo at all time points. Uptake in the brain capillaries was up to 10 times higher compared to untargeted liposomes, and uptake in the brain parenchyma was up to 4.3 times higher. Additionally, these results show that many targeting ligands that have been described for brain targeting, do not target to the brain in vivo when coupled to a liposomal delivery vehicle.

The Nuclear Pore Complex: The Gateway to Successful Nonviral Gene Delivery

One of the limiting steps in the efficiency of nonviral gene delivery is transport of genetic material across the nuclear membrane. Trafficking of nuclear proteins from the cytoplasm into the nucleus occurs via the nuclear pore complex and is mediated by nuclear localization signals and their nuclear receptors. Several strategies employing this transport mechanism have been designed and explored to improve nonviral gene delivery. In this article, we review the mechanism of nuclear import through the nuclear pore complex and the strategies used to facilitate nuclear import of exogenous DNA and improve gene expression.

Targeting tumor antigens to dendritic cells using particulate carriers

Dendritic cells play a central role in antigen presentation and generation of cytotoxic T lymphocyte (CTL) response required for anticancer vaccination. The review focuses on use of particulate carriers like lipid and polymeric nanoparticles for targeting tumor antigens to the dendritic cells. The role of various physicochemical parameters of nanocarriers such as size, surface charge in passive targeting is detailed. Utilization of different ligands such as mannose, Fc receptor, CD11c/CD 18, DEC-205 and DC-SIGN on DC for active targeting is reviewed. Smart nanocarriers such as pH sensitive nanocarriers, pre forming liposomes, cell penetrating peptide containing systems and virosomes that can specifically increase the CTL response generating greater antitumor immunity have also been discussed.