Ennio Tasciotti

Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications.

Many nanosized particulate systems are being developed as intravascular carriers to increase the levels of therapeutic agents delivered to targets, with the fewest side effects. The surface of these carriers is often functionalized with biological recognition molecules for specific, targeted delivery. However, there are a series of biological barriers in the body that prevent these carriers from localizing at their targets at sufficiently high therapeutic concentrations. Here we show a multistage delivery system that can carry, release over time and deliver two types of nanoparticles into primary endothelial cells. The multistage delivery system is based on biodegradable and biocompatible mesoporous silicon particles that have well-controlled shapes, sizes and pores. The use of this system is envisioned to open new avenues for avoiding biological barriers and delivering more than one therapeutic agent to the target at a time, in a time-controlled fashion.

Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions

The therapeutic efficacy of systemic drug-delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. White blood cells of the immune system--known as leukocytes--possess all of these properties and exert their targeting ability through cellular membrane interactions. Here, we show that nanoporous silicon particles can successfully perform all these actions when they are coated with cellular membranes purified from leukocytes. These hybrid particles, called leukolike vectors, can avoid being cleared by the immune system. Furthermore, they can communicate with endothelial cells through receptor-ligand interactions, and transport and release a payload across an inflamed reconstructed endothelium. Moreover, leukolike vectors retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in a tumour.

Tailored Porous Silicon Microparticles: Fabrication and Properties

The use of mesoporous silicon particles for drug delivery has been widely explored thanks to their biodegradability and biocompatibility. The ability to tailor the physicochemical properties of porous silicon at the micro- and nanoscale confers versatility to this material. A method for the fabrication of highly reproducible, monodisperse, mesoporous silicon particles with controlled physical characteristics through electrochemical etching of patterned silicon trenches is presented. The particle size is tailored in the micrometer range and pore size in the nanometer range, the shape from tubular to discoidal to hemispherical, and the porosity from 46 to over 80%. In addition, the properties of the porous matrix are correlated with the loading of model nanoparticles (quantum dots) and their three-dimensional arrangement within the matrix is observed by transmission electron microscopy tomography. The methods developed in this study provide effective means to fabricate mesoporous silicon particles according to the principles of rational design for therapeutic vectors and to characterize the distribution of nanoparticles within the porous matrix.

Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

Nanotechnology for breast cancer therapy

Breast cancer is the field of medicine with the greatest presence of nanotechnological therapeutic agents in the clinic. A pegylated form of liposomally encapsulated doxorubicin is routinely used for treatment against metastatic cancer, and albumin nanoparticulate chaperones of paclitaxel were approved for locally recurrent and metastatic disease in 2005. These drugs have yielded substantial clinical benefit, and are steadily gathering greater beneficial impact. Clinical trials currently employing these drugs in combination with chemo and biological therapeutics exceed 150 worldwide. Despite these advancements, breast cancer morbidity and mortality is unacceptably high. Nanotechnology offers potential solutions to the historical challenge that has rendered breast cancer so difficult to contain and eradicate: the extreme biological diversity of the disease presentation in the patient population and in the evolutionary changes of any individual disease, the multiple pathways that drive disease progression, the onset of ‘resistance’ to established therapeutic cocktails, and the gravity of the side effects to treatment, which result from generally very poor distribution of the injected therapeutic agents in the body. A fundamental requirement for success in the development of new therapeutic strategies is that breast cancer specialists—in the clinic, the pharmaceutical and the basic biological laboratory—and nanotechnologists—engineers, physicists, chemists and mathematicians—optimize their ability to work in close collaboration. This further requires a mutual openness across cultural and language barriers, academic reward systems, and many other ‘environmental’ divides. This paper is respectfully submitted to the community to help foster the mutual interactions of the breast cancer world with micro- and nano-technology, and in particular to encourage the latter community to direct ever increasing attention to breast cancer, where an extraordinary beneficial impact may result. The paper initiates with an introductory overview of breast cancer, its current treatment modalities, and the current role of nanotechnology in the clinic. Our perspectives are then presented on what the greatest opportunities for nanotechnology are; this follows from an analysis of the role of biological barriers that adversely determine the biological distribution of intravascularly injected therapeutic agents. Different generations of nanotechnology tools for drug delivery are reviewed, and our current strategy for addressing the sequential bio-barriers is also presented, and is accompanied by an encouragement to the community to develop even more effective ones.

The impact of nanoparticle protein corona on cytotoxicity, immunotoxicity and target drug delivery

In a perfect sequence of events, nanoparticles (NPs) are injected into the bloodstream where they circulate until they reach the target tissue. The ligand on the NP surface recognizes its specific receptor expressed on the target tissue and the drug is released in a controlled manner. However, once injected in a physiological environment, NPs interact with biological components and are surrounded by a protein corona (PC). This can trigger an immune response and affect NP toxicity and targeting capabilities. In this review, we provide a survey of recent findings on the NP-PC interactions and discuss how the PC can be used to modulate both cytotoxicity and the immune response as well as to improve the efficacy of targeted delivery of nanocarriers.

Tailoring the degradation kinetics of mesoporous silicon structures through PEGylation

Injectable and implantable porosified silicon (pSi) carriers and devices for prolonged and controlled delivery of biotherapeutics offer great promise for treatment of various chronic ailments and acute conditions. Polyethylene glycols (PEGs) are important surface modifiers currently used in clinic mostly to avoid uptake of particulates by reticulo-endothelial system (RES). In this work we show for the first time that covalent attachment of PEGs to the pSi surface can be used as a means to tune degradation kinetics of silicon structures. Seven PEGs with varying molecular weights (245, 333, 509, 686, 1214, 3400, and 5000 Da) were employed and the degradation of PEGylated pSi hemispherical microparticles in simulated physiological conditions was monitored by means of ICP-AES, SEM, and fluorimetry. Biocompatibility of the systems with human macrophages in vitro was also evaluated. The results clearly indicate that controlled PEGylation of silicon microparticles can offer a sensitive tool to finely tune their degradation kinetics and that the systems do not induce release of proinflammatory cytokines IL-6 and IL-8 in THP1 human macrophages.

Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

Tailoring of the nanotexture of mesoporous silica films and their functionalized derivatives for selectively harvesting low molecular weight protein.

We present a fast, efficient, and reliable system based on mesoporous silica chips to specifically fractionate and enrich the low molecular weight proteome. Mesoporous silica thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different templates and concentrations in the precursor solution, various pore size distributions, pore structures, and connectivity were obtained and applied for selective recovery of low mass proteins. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. In addition, to overcome the limitations of the prefunctionalization method in polymer selection, plasma ashing was used for the first time for the treatment of the mesoporous silica surface prior to chemical modification. Surface charge modifications by different functional groups resulted in a selective capture of the low molecular weight proteins from serum sample. In conclusion, our study demonstrates that the ability to tune the physicochemical properties of mesoporous silica surfaces, for a selective enrichment of the low molecular weight proteome from complex biological fluids, has the potential to promote proteomic biomarker discovery.

Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation.

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.