Reduction of Protein Adsorption

Albumin is a model protein commonly used to study the protein-resistance properties of surfaces [53]. A 15fold reduction of albumin adsorption was found with

PEG-coated PEG-PLA nanospheres compared to PLA ones [54]. Similar albumin-rejecting abilities were observed in the case of PLGA nanospheres surface modified by adsorption of PEG-PLA diblock copolymers [55].

Fibrinogen was used as a model blood protein to test the efficacy of the PEG coating on PEG-PACA nanospheres [49]. After freeze-fracture, the adsorbed fibrinogen layer could only be visualized at the surface of PACA nanospheres but not at the surface of the PEG-coated ones, showing the protein-rejection properties of these last ones.

As it has been previously emphasized, complement is one of the main opsonins responsible for the nanoparticles uptake by MPS. Studies have been devoted to complement consumption by PEG-coated nanospheres [56-59].

In the case of PEG-PLA nanospheres, complement consumption was expressed as a function of the PEG surface density [56]. The nanospheres possessing an estimated average density exceeding 0.2 PEG molecules per square nanometer were found to consume very low amounts of complement, whereas PEG-PLA nanospheres possessing a lower PEG surface density consumed very high amounts. This threshold in complement consumption reduction corresponds to a distance between two terminally attached PEG chains of about 2.2 nm [56]. Moreover, contrary to the PLA nanospheres, due to their PEG coating, the PLA-PEG nanospheres were remarkably inert toward coagulation factors and calcium ions [57]. When prepared by another method (double emulsion), the PEG-PLA nano-spheres exhibited the same behavior with regard to complement consumption as the nanospheres previously described [56, 57], prepared by nanoprecipitation. Complement consumption was higher and faster in the case of PLA nano-spheres than in the case of PEG-coated ones and decreased with increasing the PEG surface density.

In the case of PEG-coated poly(isobutyl cyanoacrylate) nanoparticles, it was found that complement consumption was affected by the PEG-surface density, as well as by the PEG configuration at the surface ("loop" or "brush," see Fig. 2) [59].

After intravenous administration, the nanospheres simultaneously interact with a large number of proteins. Two-dimensional gel electrophoresis (2D-PAGE) provides an unique possibility to simultaneously detect hundreds of plasma proteins competitively adsorbing onto the nano-spheres' surfaces [60, 61]. Pioneer studies showed the interest of this technique in the case of PLA nanospheres and on PLA nanospheres surface modified by PEG [62]. Spots of the apolipoproteins A-IV and E were found on the PLA nanospheres but not on the PEG coated ones. It was, therefore, hypothesized that these specific apolipoproteins might play a role in the recognition process of the nanospheres by the MPS.

Two-dimensional gel electrophoresis was successfully used in the case of nanospheres made of PEG-PLGA diblock copolymers [63]. It was shown that the PEG coating effectively reduced the total amount of protein adsorbed onto the nanospheres' surface. Similar findings were reported in the case of PEG-PACA nanospheres [49].

The adsorption of plasma proteins was studied by 2D-PAGE onto nanospheres made by using a series of diblock copolymers: PEG-PLA, PEG-PLGA and PEG-PCL, where the molecular weight of both hydrophilic and hydrophobic blocks was varied [64]. This allowed varying of both the PEG surface density and the nature of the core. A threshold was found, in terms of PEG molecular weight, between 2000 and 5000 g/mol, resulting in maximum reduction of plasma protein adsorption. This result is in agreement with previous studies in the literature [65] suggesting a PEG molecular weight around 3500 g/mol to minimize albumin and fibrinogen adsorption on poly(ethylene terephthalate). However, even at maximal PEG-surface density, the adsorption of proteins could not be completely prevented. Furthermore, for each protein adsorbed, a threshold for the PEG chain length required for maximum reduction of adsorption was found. Finally, a distance D between two terminally attached PEG chains was determined to minimize protein adsorption. This distance (around 2 nm) was in agreement with the threshold found to avoid complement activation [56] and with theoretical predictions [27, 30]. Finally, it was found that the nature of the core plays a main role by determining the nature and amount of the adsorbed plasma proteins.

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