Yanhong Zhu

Role of cellular uptake in the reversal of multidrug resistance by PEG-b-PLA polymeric micelles.

Understanding the processes involved in the cellular uptake of nanoparticles is critical for developing effective nano drug delivery systems. In this paper we found that PEG-b-PLA polymeric micelles firstly interacted with cell membrane using atomic force microscopy (AFM) and then released their core-loaded agents into the cell membrane by fluorescence resonance energy transfer (FRET). The released agents were internalized into the cells via lipid raft/caveolae-mediated endocytosis using total internal reflection fluorescence microscopy (TIRFM) and endocytic inhibitors. Further studies revealed that paclitaxel (PTX)-loaded PEG-b-PLA micelles (M-PTX) increased the cellular accumulation of PTX in PTX-resistant human ovarian cell line A2780/T which resulted in more apoptosis as measured by flow cytometry and the cleavage of poly (ADP-ribose) polymerase (PARP) compared with free PTX. PEG-b-PLA micelles inhibited P-glycoprotein (Pgp) function and Pgp ATPase activity but had no effect on Pgp protein expression. The membrane microenvironment studies showed that PEG-b-PLA micelles induced cell membrane depolarization and enhanced membrane microviscosity. These results suggested that PEG-b-PLA micelles might inhibit Pgp function to reverse multidrug resistance (MDR) via interaction with cell membrane to affect the membrane microenvironment. This study provides a foundation for understanding the mechanism of reversing MDR by nanoparticles better and designing more effective nano drug carriers.

Lactoferrin-conjugated superparamagnetic iron oxide nanoparticles as a specific MRI contrast agent for detection of brain glioma in vivo.

A specific contrast agent for magnetic resonance imaging (MRI) is crucial to brain tumor patients for the surgical operation or the postoperative radiology. This study explored lactoferrin-conjugated superparamagnetic iron oxide nanoparticles (Lf-SPIONs) as an MRI contrast agent for the detection of brain gliomas in vivo. The hydrodynamic diameter of about 75 nm, saturation magnetization of 51 emu/g Fe and T(2) relaxivity of 75.6 mM(-1)S(-1) of the Lf-SPIONs suggested its applicability for MRI. Using a rat model of C6 glioma, Lf-SPIONs provided a better picture or more sensitivity to depict brain glioma on MR images than that of SPIONs. Significantly enhanced T(2)-weighted images of brain glioma were documented in vivo with Lf-SPIONs until 48 h after injection. Moreover, Lf-SPIONs were clearly observed around vascular region of the tumor slices after 48 h. High level expression of Lf receptors was confirmed in brain tumor tissues by RT-PCR and Western Blot compared to normal brain tissues. These findings suggested that Lf-SPIONs could be potentially employed as a sensitive and specific MRI contrast agent in the diagnosis of brain glioma.

pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin for MR and fluorescence imaging of glioma in rats

Glioma is the most common primary brain tumor and causes a disproportionate level of morbidity and mortality across a wide range of individuals. From previous clinical practices, definition of glioma margin is the key point for surgical resection. In order to outline the exact margin of glioma and provide a guide effect for the physicians both at pre-surgical planning stage and surgical resection stage, pH/temperature sensitive magnetic nanogels conjugated with Cy5.5-labled lactoferrin (Cy5.5-Lf-MPNA nanogels) were developed as a promising contrast agent. Due to its pH/te mperature sensitivity, Cy5.5-Lf-MPNA nanogels could change in its hydrophilic/hydrophobic properties and size at different pH and temperatures. Under physiological conditions (pH 7.4, 37 °C), Cy5.5-Lf-MPNA nanogels were hydrophilic and swollen, which could prolong the blood circulation time. In the acidic environment of tumor tissues (pH 6.8, 37 °C), Cy5.5-Lf-MPNA nanogels became hydrophobic and shrunken, which could be more easily accumulated in tumor tissue and internalized by tumor cells. In addition, lactoferrin, an effective targeting ligand for glioma, provides active tumor targeting ability. In vivo studies on rats bearing in situ glioma indicated that the MR/fluorescence imaging with high sensitivity and specificity could be acquired using Cy5.5-Lf-MPNA nanogels due to active targeting function of the Lf and enhancement of cellular uptake by tailoring the hydrophilic/hydrophobic properties of the nanogels. With good biocompatibility shown by cytotoxicity assay and histopathological analysis, Cy5.5-Lf-MPNA nanogels are hopeful to be developed as a specific and high-sensitive contrast agent for preoperative MRI and intraoperative fluorescence imaging of glioma.

Protective effect of PEGylation against poly(amidoamine) dendrimer-induced hemolysis of human red blood cells.

Poly(amidoamine) (PAMAM) dendrimers are widely used in medical applications. However, dendrimers bearing positively charged surface groups are prone to destabilize cell membrane and cause cell lysis. The lytic effect of dendrimers on red blood cells (RBCs) namely hemolysis is extremely dangerous when administered in vivo. To diminish the hematologic toxicity, we modified PAMAM dendrimers with poly(ethylene glycol) (PEG) of three molecular weights (2k, 5k, and 20k). The protective effect of PEGylation against PAMAM dendrimer-induced hemolysis was studied. RBCs morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). The results indicated that PAMAM and PEG-2k modified dendrimers induced hemolysis at 0.1 and 0.5 mg/mL respectively, whereas PEG-5k and PEG-20k modified dendrimers showed no significant difference in hemolysis compared with control even at 5 mg/mL. OM and AFM investigation indicated PAMAM and PEG-2k modified dendrimers caused RBCs aggregation and lysis. However, no changes were observed in the overall shape of RBCs treated with PEG-5k and PEG-20k modified dendrimers. The surface roughness of RBCs treated with PEGylated dendrimers were far lower than that of RBCs treated with PAMAM dendrimers. This study demonstrated that hemocompatibility of PAMAM dendrimers could be greatly enhanced by PEGylation.

Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum

A specific and sensitive immunoassay based on magnetic microbead separation for schistosomiasis japonica screening is presented in this article. So far as we know, this is the first time that magnetic microbead-based enzyme-linked immunoassay (MEIA) has been used for the determination of Schistosoma japonicum (Sj) antibody in human serum. Fluorescein isothiocyanate (FITC)-labeled soluble egg antigen (SEA) and polymer-coated magnetic beads, to which anti-FITC monoclonal antibodies were immobilized, were used as separation support in MEIA. Immunoassay parameters were optimized based on a direct immunoreaction of SEA on the magnetic microbead and Sj antibody in serum samples. The laboratory experimental results showed that the MEIA method was more sensitive and more precise than traditional SEA-ELISA (enzyme-linked immunosorbent assay). In the field test, human sera collected from 513 infected humans and 2260 uninfected humans were tested with indirect hemagglutination assay (IHA), dipstick dye immunoassay (DDIA), and MEIA. IHA and DDIA were then compared with MEIA, and a lower false negative rate (0.97%) was obtained.

Water-soluble l-cysteine-coated FePt nanoparticles as dual MRI/CT imaging contrast agent for glioma

Nanoparticles (NPs) are advantageous for the delivery of diagnosis agents to brain tumors. In this study, we attempted to develop an l-cysteine coated FePt (FePt-Cys) NP as MRI/CT imaging contrast agent for the diagnosis of malignant gliomas. FePt-Cys NPs were synthesized through a co-reduction route, which was characterized by transmission electron microscopy, high-resolution transmission electron microscopy, powder X-ray diffraction, Fourier transform infrared spectroscopy, and dynamic light scattering. The MRI and CT imaging ability of FePt-Cys NPs was evaluated using different gliomas cells (C6, SGH44, U251) as the model. Furthermore, the biocompatibility of the as-synthesized FePt-Cys NPs was evaluated using three different cell lines (ECV304, L929, and HEK293) as the model. The results showed that FePt-Cys NPs displayed excellent biocompatibility and good MRI/CT imaging ability, thereby indicating promising potential as a dual MRI/CT contrast agent for the diagnosis of brain malignant gliomas.

Glioma-targeting micelles for optical/magnetic resonance dual-mode imaging.

Surgical resection is the primary mode for glioma treatment, while gross total resection is difficult to achieve, due to the invasiveness of the gliomas. Meanwhile, the tumor-resection region is closely related to survival rate and life quality. Therefore, we developed optical/magnetic resonance imaging (MRI) bifunctional targeted micelles for glioma so as to delineate the glioma location before and during operation. The micelles were constructed through encapsulation of hydrophobic superparamagnetic iron oxide nanoparticles (SPIONs) with polyethylene glycol-block-polycaprolactone (PEG-b-PCL) by using a solvent-evaporation method, and modified with a near-infrared fluorescent probe, Cy5.5, in addition to the glioma-targeting ligand lactoferrin (Lf). Being encapsulated by PEG-b-PCL, the hydrophobic SPIONs dispersed well in phosphate-buffered saline over 4 weeks, and the relaxivity (r2) of micelles was 215.4 mM−1·s−1, with sustained satisfactory fluorescent imaging ability, which might have been due to the interval formed by PEG-b-PCL for avoiding the fluorescence quenching caused by SPIONs. The in vivo results indicated that the nanoparticles with Lf accumulated efficiently in glioma cells and prolonged the duration of hypointensity at the tumor site over 48 hours in the MR image compared to the nontarget group. Corresponding with the MRI results, the margin of the glioma was clearly demarcated in the fluorescence image, wherein the average fluorescence intensity of the tumor was about fourfold higher than that of normal brain tissue. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay results showed that the micelles were biocompatible at Fe concentrations of 0–100 μg/mL. In general, these optical/MRI bifunctional micelles can specifically target the glioma and provide guidance for surgical resection of the glioma before and during operation.

Detection of IgG in sera of patients with schistosomiasis japonica by developing magnetic affinity enzyme-linked immunoassay based on recombinant 14-3-3 protein.

BACKGROUNDnLow intensity of Schistosoma infection is the current status in China after long time treatment with praziquantel, therefore more sensitive diagnostic methods are required now. In this study, a magnetic affinity enzyme-linked immunoassay (MEIA) based on the signal transduction protein 14-3-3 of Schistosoma japonicum (Sj14-3-3), was developed for detecting schistosomiasis.nnnMETHODSnSera of infected BALB/c mice were collected and analyzed with MEIA and ELISA. Both MEIA and ELISA based on Sj14-3-3 were further used to detect serum IgG in patients. Sera from 58 schistosomiasis-related patients with low-intensity infection, and 30 non-endemic negative controls, were collected to assess the assay. Six sera from paragonimiasis patients were used to analysis cross-reactions.nnnRESULTSnCompared with ELISA, MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio in infected mice (3.71 vs 2.45). Similar results were observed in humans, higher P/N of MEIA compared to ELISA (3.57 vs 2.68). There was no cross-reaction with the sera of paragonimiasis patients detected by both MEIA and ELISA.nnnCONCLUSIONSnOur studies suggested that MEIA based on recombinant Sj14-3-3 protein (rSj14-3-3) had the potential for the diagnosis of schistosomiasis.

Magnetic affinity enzyme-linked immunoassay based on recombinant 26 kDa glutathione-S-transferase for serological diagnosis of schistosomiasis japonica.

Schistosomiasis remains a serious worldwide public health problem. Improving the diagnostic assay for surveillance and monitoring will contribute to hastening the possible elimination of the disease in endemic regions. Therefore, this study aims to develop magnetic affinity enzyme-linked immunoassay (MEIA) for serological diagnosis of schistosomiasis based on recombinant 26kDa glutathione-S-transferase of Schistosoma japonicum (rSj26GST). BALB/c mice infected with S. japonicum cercariae (40 per mouse) were used. After infecting for 6 weeks, the antibody was detected by MEIA. All of the infected mouse sera were effectively determined by MEIA. Compared with the enzyme-linked immunosorbent assay (ELISA), MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio (3.92 versus 2.66). MEIA was further applied for diagnosis of human schistosomiasis. Sera from 28 schistosomiasis-confirmed patients with low-intensity infection, 15 treated patients, and 20 non-endemic negative controls, were used to assess the assay. The results showed that MEIA and ELISA had similarity in positive detection rates. However, the higher P/N of MEIA was observed at the same dilution ratio. MEIA had high negative rate in detection of specific IgG in the treated patients. Moreover, there was no cross reaction with the sera of paragonimiasis patients. These results suggested that MEIA based on rSj26GST is a simple, rapid, convenient assay for the diagnosis of schistosomiasis.

Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK(Ca)) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H(2)O(2)) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hbeta1 were cotransfected with the PTEN(wt), PTEN(C124S), PTEN(G129E) in HEK 293 cells. Typical BK(Ca) channel currents could be recorded in cell-attached configurations. We found that PTEN(wt) reduced the H(2)O(2)-induced BK(Ca) channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN(C124S)/PTEN(G129E) mutants that lack lipid phosphatase activity produced no regulation on the H(2)O(2)-induced BK(Ca) channel activation. These results demonstrated that PTEN regulated the H(2)O(2)-induced BK(Ca) channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H(2)O(2)-induced BK(Ca) channel activation was attenuated when cells were treated with H(2)O(2) at concentrations higher than 100 microM or at 100 microM for long-term treatment. In addition, the p-AKT expression level in PTEN(wt) overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca(2+)](i)) induced by H(2)O(2) was also inhibited. These findings may elucidate a new mechanism for H(2)O(2)-induced BK(Ca) channel activation and provide some evidences for the role of PTEN on vasodilation induced by H(2)O(2).