Huanjun Zhou

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.

Hierarchically microporous/macroporous scaffold of magnesium-calcium phosphate for bone tissue regeneration.

Hierarchically 3D microporous/macroporous magnesium-calcium phosphate (micro/ma-MCP) scaffolds containing magnesium ammonium phosphate hexahydrate [NH(4)MgPO(4).6H(2)O] and hydroxyapatite [Ca(10)(PO(4))(6)(OH)(2)] were fabricated from cement utilizing leaching method in the presence of sodium chloride (NaCl) particles and NaCl saturated water solution. NaCl particles produced macroporosity, and NaCl solution acted as both cement liquid and porogens, inducing the formation of microporosity. The micro/ma-MCP scaffolds with porosities varied from 52 to 78% showed well interconnected and open macropores with the sizes of 400-500 microm, and degradation of the scaffolds was significantly enhanced in Tris-HCl solution compared with macroporous MCP (ma-MCP) and corresponding calcium phosphate cement (CPC) scaffolds. Cell attachment and proliferation of MG(63) on micro/ma-MCP were significantly better than ma-MCP and CPC scaffolds because of the presence of microporosity, which enhanced the surface area of the scaffolds. Moreover, the alkaline phosphatase (ALP) activity of the MG(63) cells on micro/ma-MCP was significantly higher than ma-MCP and CPC scaffolds at 7 days, and the MG(63) cells with normal phenotype spread well and formed confluent layers across the macroporous walls of the micro/ma-MCP scaffolds. Histological evaluation confirmed that the micro/ma-MCP scaffolds improved the efficiency of new bone regeneration, and exhibited excellent biocompatibility, biodegradability and faster and more effective osteogenesis in vivo.

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration.

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200-500 μm and micropores of 2-5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.

In vitro cytotoxicity and induction of apoptosis by silica nanoparticles in human HepG2 hepatoma cells

Background Silica nanoparticles have been discovered to exert cytotoxicity and induce apoptosis in normal human cells. However, until now, few studies have investigated the cytotoxicity of silica nanoparticles in tumor cells. Methods This study investigated the cytotoxicity of 7–50 nm silica nanoparticles in human HepG2 hepatoma cells, using normal human L-02 hepatocytes as a control. Cell nucleus morphology changes, cellular uptake, and expression of procaspase-9, p53, Bcl-2, and Bax, as well as the activity of caspase-3, and intracellular reactive oxygen species and glutathione levels in the silica nanoparticle-treated cells, were analyzed. Results The antitumor activity of the silica nanoparticles was closely related to particle size, and the antiproliferation activity decreased in the order of 20 nm > 7 nm > 50 nm. The silica nanoparticles were also cytotoxic in a dose- and time-dependent manner. However, the silica nanoparticles showed only slight toxicity in the L-02 control cells, Moreover, in HepG2 cells, oxidative stress and apoptosis were induced after exposure to 7–20 nm silica nanoparticles. Expression of p53 and caspase-3 increased, and expression of Bcl-2 and procaspase-9 decreased in a dose-dependent manner, whereas the expression of Bax was not significantly changed. Conclusion A mitochondrial-dependent pathway triggered by oxidative stress mediated by reactive oxygen species may be involved in apoptosis induced by silica nanoparticles, and hence cytotoxicity in human HepG2 hepatic cancer cells.

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.

Development of magnesium calcium phosphate biocement for bone regeneration

Magnesium calcium phosphate biocement (MCPB) with rapid-setting characteristics was fabricated by using the mixed powders of magnesium oxide (MgO) and calcium dihydrogen phosphate (Ca(H2PO4)2·H2O). The results revealed that the MCPB hardened after mixing the powders with water for about 7 min, and the compressive strength reached 43 MPa after setting for 1 h, indicating that the MCPB had a short setting time and high initial mechanical strength. After the acid–base reaction of MCPB containing MgO and Ca(H2PO4)2·H2O in a molar ratio of 2 : 1, the final hydrated products were Mg3(PO4)2 and Ca3(PO4)2. The MCPB was degradable in Tris–HCl solution and the degradation ratio was obviously higher than calcium phosphate biocement (CPB) because of its fast dissolution. The attachment and proliferation of the MG63 cells on the MCPB were significantly enhanced in comparison with CPB, and the alkaline phosphatase activity of MG63 cells on the MCPB was significantly higher than on the CPB at 7 and 14 days. The MG63 cells with normal phenotype spread well on the MCPB surfaces, and were attached in close proximity to the substrate, as seen by scanning electron microscopy (SEM). The results demonstrated that the MCPB had a good ability to support cell attachment, proliferation and differentiation, and exhibited good cytocompatibility.

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.