Xiaoqian Shan

Long-circulating polymeric nanoparticles bearing a combinatorial coating of PEG and water-soluble chitosan

A major obstacle in the development of polymeric nanoparticles (NPs) as effective drug delivery vesicles is the rapid clearance from blood. In order to realize a significant prolongation in blood circulation, a combinatorial design, covalent attachment of polyethylene glycol (PEG) to polylactic acid (PLA) and physical adsorption of water-soluble chitosan (WSC) to particle surface, has been developed for surface modification of PLA NPs. Two types of WSC, cationic partially deacetylated chitin (PDC) and anionic N-carboxy propionyl chitosan sodium (CPCTS) were investigated. All the NPs formulated in the size range of 100-200nm were prepared by a modified w/o/w technique and physicochemically characterized. In vitro phagocytosis by mouse peritoneal macrophage (MPM), in vivo blood clearance and biodistribution following intravenous administration in mice, of these NPs labeled with 6-coumarin, were evaluated. The presence of WSC, whether alone or with PEG, highly improved the surface hydrophilicity as well as suspension stability of NPs. Their surface charge was greatly affected by the WSC coating, being close to neutrality for PEG/PDC NPs and highly negative in the case of PEG/CPCTS NPs. In comparison to NPs treated with PEG or WSC alone, the synergistic action of PEG and WSC strongly inhibited the macrophage uptake and extended the circulation half-life (t(1/2)) with concomitant reduced liver sequestration. Particularly, PEG/PDC NPs showed the most striking result with regard to their performance in vitro and in vivo. Calculated t(1/2) of PEG/PDC NPs and PEG/CPCTS NPs was 63.5h and 7.1h, respectively, much longer than that of control PEG/PVA NPs (1.1h). More WSC materials need to be evaluated, but the present data suggest that, a combinatorial coating of PEG and PDC greatly prolongs the systemic circulation of NPs and represents a significant step in the development of long-circulating drug delivery carriers.

In vitro macrophage uptake and in vivo biodistribution of long-circulation nanoparticles with poly(ethylene-glycol)-modified PLA (BAB type) triblock copolymer

The effect of the PEG-grafted degree in the range of 0-30% on the in vitro macrophage uptake and in vivo biodistribution of poly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol) (PELE) nanoparticles (NPs) were investigated in this paper. The prepared NPs were characterized in terms of size, zeta potential, hydrophilicity, poly(vinyl alcohol) (PVA) residual on nanoparticles surfaces as well as drug loading. The macrophage uptake and biodistribution including plasma clearance kinetics following intravenous administration in mice of the NPs labeled by 6-coumarin were evaluated. The results showed that, except for the particles size, the hydrophilicity, superficial charges and in vitro phagocytosis amount of NPs are dependent on the PEG content in the copolymers greatly. The higher of the PEG content, the more hydrophilicity and the nearer to neutral surface charge was observed. And the prolonged circulation half-life (t(1/2)) of the PELE NPs in plasma was also strongly depended on the PEG content with the similar trend. In particular for PELE30 (containing 30% of PEG content) NPs, with the lowest phagocytosis uptake accompanied the highest hydrophilicity and approximately neutral charge, it had the longest half-life in vivo with almost 12-fold longer and accumulation in the reticuloendothelial system organs close to 1/2-fold lower than those of reference PLA. These results demonstrated that the PELE30 NPs with neutral charge and suitable size has a promising potential as a long-circulating oxygen carrier system with desirable biocompatibility and biofunctionality.

Long-circulation of hemoglobin-loaded polymeric nanoparticles as oxygen carriers with modulated surface charges

The aim of this study was to investigate the effects of the surface charges on the in vitro macrophage cellular uptake and in vivo blood clearance and biodistribution of the hemoglobin-loaded polymeric nanoparticles (HbPNPs). The surface charges of the HbPNPs fabricated from mPEG-PLA-mPEG were modulated with cationized cetyltrimethylammonium bromide (CTAB) and anionized sodium dodecyl sulphate (SDS), respectively. In vitro macrophage cellular uptake and in vivo biodistribution of the coumarin 6-labeled HbPNPs with different electric charges were investigated, and the half-lives in the circulation were pharmacokinetically analyzed. The particle sizes of the HbPNPs were all below 200 nm with a narrow size distribution and high encapsulation efficiency (>84%). And the zeta-potentials of the untreated, cationized and anionized HbPNPs in phosphate buffered sodium chloride solution (PBS) were -12.3, +3.28 and -25.4 mV, respectively. The HbPNPs did not occur significant aggregation or sedimentation, even after 5 days. Compared with the untreated HbPNPs, 1-fold decrease/increase of the uptake percentage associated with the cationized/anionized HbPNPs was observed. In vivo experiment demonstrated that the calculated half-life of the cationized HbPNPs was 10.991 h, 8-fold longer than that of the untreated HbPNPs (1.198 h). But the anionized HbPNPs displayed opposite effect. Furthermore, the cationized HbPNPs mainly accumulated in the liver, lung and spleen after 48 h injection. MTT results showed that the HbPNPs with different surface charges all exhibited slight toxicity. These results demonstrated that the CTAB-modulated HbPNPs with low positive charge and suitable size have a promising potential as a long-circulating oxygen carrier system with desirable biocompatibility and biofunctionality.

Comparison of the PLA-mPEG and mPEG-PLA-mPEG copolymers nanoparticles on the plasma protein adsorption and in vivo biodistribution

In order to address the chemical constitution-effect associated with the PEGylated amphiphilic block copolymer, the adsorption of the plasma proteins, in vitromacrophage uptake and in vivo biodistribution of the poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PLA, BA) nanoparticles (NPs) and the poly(ethylene glycol)-poly(D,L-lactic acid)-poly(ethylene glycol) (mPEG-PLA-mPEG, BAB) NPs, with the same molar ratio of PEG to PLA segment of 30% and the same EG chain length of 5 kDa were investigated and compared in the present study. PLA without PEGylation was also studied as control. All the NPs formulated with the diameter of about 180 nm were prepared by a modified w/o/w technique. The BAB NPs exhibited excellent hydrophilicity and an approximately neutralized surface charge of −2.23 mV, whereas the BA NPs presented the negative charge of −13.8 mV. X-Ray photoelectron spectroscopy (XPS) revealed that the 63.73% coverage of PEG chains was achieved on the BAB NPs surface, whereas the PEG amount on the surface of BA NPs was only 21.11%. BAB polymer could suppress the adsorption of both the big protein fibrinogen (inhibition ratio of 85%) and small protein albumin (inhibition ratio of 71%) significantly. But the mPEG-PLA could only repel the adsorption of big protein with inhibition ratio of 65%. In vitro and in vivo studies indicated that compared with the BA NPs, the BAB NPs strongly prohibited the macrophage uptake and extended the circulation half-life (t1/2) with concomitant reduced liver sequestration. Calculated t1/2 of the BAB NPs and the BA NPs was 600 ± 50 min and 160 ± 15 min, respectively. These results indicated that compared with the BA NPs, even with the same molar ratio of PEG to PLA segment and the same EG chain length, BAB NPs could effectively inhibit the adsorption of protein and prolong the systemic circulation of NPs.

Reduction and suppression of methemoglobin loaded in the polymeric nanoparticles intended for blood substitutes

Oxidation of hemoglobin (Hb) to nonfunctional methemoglobin (metHb) is a main challenge for the fabrication of an ideal Hb-based blood substitute. In this study, a novel nonenzymatic reduction and suppression route, combined with a fast prereduction, optimized double emulsion preparation, and a second sustaining postreduction, was developed to control the metHb in Hb-loaded nanoparticles (HbP) with porous microstructure to a desirable level. In the prereduction, the metHb in the raw Hb was effectively reduced from over 90% to 1.2% using sodium dithionite following gel filtration separation. During the preparation, higher the emulsion strength performed, higher was the extent of Hb oxidized. PEGylated polymer and addition of miscible solvent, such as acetonitrile, into the oil phase could pronouncedly suppress metHb formation. The resultant metHb level in HbP under the optimal fabrication was about 5.6%, which could be further reduced to 1.4% by the model reducing agents in human plasma with the help of superoxide dismutatse and catalase system, which are capable of sustaining postreduction. The oxygen dissociation curve of the HbP was close to that of native Hb, indicating that the oxygen-carrying ability of the Hb, despite initially losing this function due to the severe oxidation, recovered and retained well. The results achieved are promising for the fabrication of blood substitutes with controlled metHb level, which can fulfill the binding/delivering oxygen to tissues in vivo for future trials.

Porosity and semipermeability of hemoglobin‐loaded polymeric nanoparticles as potential blood substitutes

Porosity and semipermeability, allowing life-sustaining small molecules to penetrate, but hemoglobin (Hb) and other enzymes to cut off, predominantly affect the functionalities of the Hb-loaded polymeric nanoparticles (HbPNPs) as blood substitutes. In this article, HbPNPs formulated in the size range of 110-122 nm were prepared by a modified double-emulsion method with poly(lactic acid) (PLA)-based polymers. The influences of the main preparation conditions, including solvent composition, stirring speed, Hb concentration and polymer matrix, on the porosity were investigated in details. To evaluate the porosity of HbPNPs, a novel nondestructive testing method based on molecular weight cut-off (MWCO) was developed, and an effusion approach was applied to investigate the pore size in the particle shells with poly(ethylene glycol)s (PEGs) of different molecular weights (PEG200, PEG400, PEG600) as probes. Moreover, in vitro diffusion behaviors of ascorbic acid and reduced glutathione from HbPNPs fabricated with various polymer matrices were studied. The MWCO of HbPNPs by changing solvent composition, stirring speed, Hb concentration, and polymer composition varied from 200 to 600, especially the PEGylation of the polymer, which exhibited obvious influence on the MWCO of HbPNPs. Ascorbic acid with molecular weight 176.1 could diffuse into PEGylated nanoparticles with mPEG content of 5-30 wt % freely, while reduced glutathione with molecular weight 307.3 could not penetrate when mPEG content reached 30 wt %. These results suggest that the HbPNPs optimized with MWCO between 400 and 600 can facilitate the transport of all those life-sustaining small molecules.