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1 Available anticancer drugs do not seem to modify the prognosis of metastatic melanoma. Salicylate and acetyl salicylic acid (aspirin) were found to suppress growth in a number of transformed cells, that is, prostate and colon. Therefore, we studied the direct effects of aspirin on metastatic B16 melanoma cells. 2 Aspirin at a plasma-attainable and nontoxic level suppressed the proliferation of B16 cells. 3 Aspirin induced the activation of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. 4 Inhibition of JNK, but not p38, decreased the suppressive effect of aspirin upon the proliferation of B16 cells. 5 The aspirin-induced reduction in B16 proliferation was cumulative over time. 6 Aspirin and the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) induced B16 cell death synergistically. 7 In addition to the murine B16 cell line, the proliferation of SK-28 human melanoma cells was also suppressed by aspirin. 8 In conclusion, aspirin suppresses the proliferation of metastatic B16 cells in a JNK-dependent mechanism.

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

The design and development of contrast agents (CAs) for magnetic resonance imaging (MRI) in clinical analysis is expected to improve the image spatial resolution and to increase the detection sensitivity, especially regarding neurological disorders and cancer disease. In particular, advanced CAs for T1-weighted images are investigated to achieve the sensitive detection of early-stage primary tumors or brain metastases. In this study, we present a strategy toward diagnostic T1 CAs for MRI, based on polymer-modified MnO nanoparticles (NPs). Two different nanosystems were synthesized, consisting of (1) colloidal MnO nanocrystals wrapped by a multidentate amphiphilic polymer and (2) MnO nanocrystals embedded within poly(lactic-co-glycolic acid) (PLGA) NPs. These nanosystems were compared in terms of their MRI contrast power and biological safety. The latter system combines the excellent biocompatibility of PLGA with the unique magnetic properties of MnO NPs and allows sustained contrast enhancement over time. Longitudinal relaxivities of both MnO composite nanomaterials proved to be higher than those of commercial Gd-based CAs and Teslascan, both in phosphate-buffered saline and in plasma, also exhibiting low cytotoxicity. The high relaxation rates achieved with these contrast enhancers are promising toward future application in in vivo imaging.

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Magnetic resonance imaging (MRI) is one of the most sophisticated diagnostic tools that is routinely used in clinical practice. Contrast agents (CAs) are commonly exploited to afford much clearer images of detectable organs and to reduce the risk of misdiagnosis caused by limited MRI sensitivity. Currently, only a few gadolinium-based CAs are approved for clinical use. Concerns about their toxicity remain, and their administration is approved only under strict controls. Here, we report the synthesis and validation of a manganese-based CA, namely, Mn@HFn-RT. Manganese is an endogenous paramagnetic metal able to produce a positive contrast like gadolinium, but it is thought to result in less toxicity for the human body. Mn ions were efficiently loaded inside the shell of a recombinant H-ferritin (HFn), which is selectively recognized by the majority of human cancer cells through their transferrin receptor 1. Mn@HFn-RT was characterized, showing excellent colloidal stability, superior relaxivity, and a good safety profile. In vitro experiments confirmed the ability of Mn@HFn-RT to efficiently and selectively target breast cancer cells. In vivo, Mn@HFn-RT allowed the direct detection of tumors by positive contrast enhancement in a breast cancer murine model, using very low metal dosages and exhibiting rapid clearance after diagnosis. Hence, Mn@HFn-RT is proposed as a promising CA candidate to be developed for MRI.

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in vivo. Nanoparticles could be the solution to this problem, as they are the right size to infiltrate and interact with biological systems (i.e., size is similar to proteins). They could allow the division of this big problem into two smaller problems: in vivo delivery, achieved by the nanoparticle, and target site interaction, achieved by the drug. This chapter focuses on the first smaller problem, summarizing the nanoparticle properties governing their biodistribution, i.e., size, surface charge, nonspecific interactions, elasticity, geometry, molecular recognition, and triggered response. It discusses current research focused on controlling and characterizing these parameters and the creation of new drug delivery platforms for specific applications.

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method might indeed be useful for this purpose in the future. The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method might indeed be useful for this purpose in the future.

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

Successful application of nanoconjugates either in vitro or in vivo does depend on the design strategy used for their development. In this chapter, we focus on the most important factors that need to be taken into account to optimize nanoparticle biofunctionalization and provide an overview of recent approaches yielding the efficient and orientation-controlled immobilization of complex molecules, including peptides and proteins. In addition, we discuss on the elements that cooperate with ligand orientation in affecting the biological activity of nanoconjugates. Indeed, the control of ligand orientation on the nanoparticle surface is of crucial importance in determining the affinity of the immobilized biomolecules toward the biological target. However, the ability of a nanoconjugate to recognize its target is even strongly affected by other important items, including (1) multivalency as a result of ligand density and organization; (2) conformational changes potentially occurring on binding of ligands to the nanoparticle; (3) thermodynamic and kinetic factors related to the interaction of the ligand with the nanoparticle surface; and (4) interaction between nanoparticle and its surrounding environment to form the so-called “protein corona.” Altogether, these five issues are the driving force in the specificity and selectivity of designed nanoconjugates toward their target, and, as a consequence, define their potential utility for biomedical application. Therefore, a careful control on the abovementioned “magic five” contributions is needed to improve the quality standard of the next-generation nanomedicines. In addition to the illustrated nanobioconjugation strategies, we emphasize that all these features need to be carefully optimized when assessing the biological effects of synthetic nanoparticles designed for biomedical purposes.

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., A common strategy to target nanoparticles to tumours is conjugation with specific antibodies, targeting protein expressed preferentially by cancer cells. Here the authors show that the number of antibodies bound to the nanoparticle influences the targeting ability in vitro and in vivo.