Functionalization of polysulfide nanoparticles and their performance as circulating carriers.

We here present an evaluation of the carrier performance of nanoparticles that are biofunctional, i.e. derivatized to provide a controlled biological activity, and environmentally responsive, since they respond to the presence of oxidants. In particular, we focus on the possibilities (a) to make the nanoparticles detectable and (b) to control their uptake in phagocytic cells, which determines their lifetime in vivo. We first describe techniques for labeling selectively the nanoparticle surface or bulk with imaging moieties (fluorophores or gold). We then show how surface composition and size, which are both controlled through the use of PEG derivatives, influence uptake by macrophages in vitro and blood circulation in vivo: for example, in vitro uptake is negligible for small (40 nm) particles but not for larger (100 nm) ones and, correspondingly, in vivo blood circulation half-life time decreases from 6.0 to 2.9 h. However, upon decoration with RGD peptides also the small particles can be significantly internalized.

In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles.

Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100nm diameter poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-containing DCs (and other APCs) was seen at 96h vs. 24h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.

Oxidation-sensitive polymeric nanoparticles.

We have recently demonstrated the possible use of organic polysulfides for the design of oxidation-sensitive colloidal carriers in the form of polymeric vesicles, which are particularly suitable for the encapsulation of hydrosoluble drugs. In the present research we extend our efforts to carriers specifically suitable for hydrophobic molecules. Exploiting the living emulsion polymerization of episulfides, we have produced new cross-linked polysulfide nanoparticles. Here we demonstrate how this process allows the production of stable nanoparticles with a good control over their size and functionality. The nanoparticles showed negligible cytotoxicity on a fibroblast model; furthermore, they exhibited sensitivity to oxidative conditions, which first produce swelling and then solubilize the material.

Diffusion NMR spectroscopy for the characterization of the size and interactions of colloidal matter: the case of vesicles and nanoparticles.

We report the application of the pulse gradient spin-echo (PGSE) NMR technique (PGSE NMR) to the analysis of large colloidal materials, specifically vesicles formed from macromolecular amphiphiles and nanoparticles. Measurements of size and size distribution were demonstrated to be comparable to those obtained through dynamic light scattering or hydrodynamic chromatography. In comparison to these more common analytical methods, the use of PGSE NMR is particularly advantageous in that, as a spectroscopic technique, it adds chemical selectivity to the study of physical dimensions. In this way, chemically different species contemporarily present in a sample may be individually studied. In addition, we demonstrate the use of PGSE NMR to probe the existence of equilibria between macroamphiphiles present in solution and those present in vesicles or on the surface of nanoparticles. This feature in particular opens exciting possibilities for the characterization of the phase behavior and of the surface adsorption phenomena of colloids.

Intra-portal injection of 400- microm microcapsules in a large-animal model.

To date, encapsulated grafts have usually been implanted in the peritoneal cavity. This site is, however, not ideal, mainly because of its low blood supply. We have investigated the feasibility of intra-portal injection of (400 microm) microcapsules in the pig. Ten-thousand microcapsules per kilogram body weight were injected into six Large White pigs. Portal pressure, various biological tests, portographies and liver histology were recorded before and at various time points after injection. As a result, portal pressure increased after injection (15+/-2.3 vs 8.7+/-1.7 mmHg) but remained within an acceptable range (<20 mmHg) and returned to normal values at 3 months (8.5+/-3.7 mmHg). During the 3-month follow up, liver function and liver tests remained stable. Portographies showed a homogenous implantation of the capsule, with the portal flow always directed to the liver. At histological examination after 3 months the capsules demonstrated various degrees of fibrosis. We can thus conclude that these results demonstrate that intra-portal injection of microcapsules is feasible in a large-animal model. Hemodynamic, biological and radiological results are similar to those observed in clinical free-islet transplantation.

Improving cell encapsulation through size control.

Capsules based on the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride have previously shown important advantages for cell encapsulation. However, in vivo long-term applications require capsule features that are well suited for the functionality of encapsulated cells. These should be targeted to the site of implantation with an appropriate size, a relative stability, and suitable diffusion properties. This study shows the effect of capsule size reduction, from 1 mm to 400 microm, on capsule quality control, mechanical stability, diffusion properties, and in vitro activities of the encapsulated cells. Following a controlled preparation, it was determined that the capsule mechanical stability was largely dependent on the volume ratio of the capsule over the membrane. The molecule diffusion time was related to the surface/volume ratio of the capsule even for the capsules exhibiting an identical cut-off towards the proteins and the dextran molecules. Finally, the in vitro cellular activities, for both primary cultures of rat islets and murine hepatocytes, were improved for cells encapsulated into the 400 microm capsules compared with those in the 1 mm capsules. All of these findings suggest that the smaller capsules present better properties for future clinical applications, at the same time widening the choice of implantation site, and strengthen the notion that slight changes in the capsular morphological parameters can largely influence the graft function in vivo.