Engineering of Bone- and CD44-Dual-Targeting Redox-Sensitive Liposomes for the Treatment of Orthotopic Osteosarcoma.

This study aimed to develop an efficient step-by-step osteosarcoma (OS)-targeting liposome system functionalized with a redox-cleavable, bone- and cluster of differentiation 44 (CD44)-dual-targeting polymer. Furthermore, the effect of coadministration of a tumor-penetrating peptide, internalizing RGD (iRGD), was investigated. First, a bone-targeting moiety, alendronate (ALN), was conjugated with hyaluronic acid (HA), a ligand for CD44. This ALN-HA conjugate was coupled with DSPE-PEG2000-COOH through a bioreducible disulfide linker (-SS-) to obtain a functionalized lipid, ALN-HA-SS-L, to be postinserted into preformed liposomes loaded with doxorubicin (DOX). The roles of ALN, HA, and the redox sensitivity of the ALN-HA-SS-L liposomes (ALN-HA-SS-L-L) in the anti-OS effect were critically evaluated against various reference liposomal formulations (with only ALN, HA, or redox sensitivity). ALN-HA-SS-L-L displayed a zeta potential of -26.07 ± 0.32 mV and selectively disassembled in the presence of a reducing agent, 10 mM glutathione, which can be found in cancer cells. Compared to various reference liposomes, ALN-HA-SS-L-L/DOX had significantly higher cytotoxicity to human OS MG-63 cells alongside high and rapid cellular uptake. In the orthotopic OS nude mouse models, ALN-HA-SS-L-L/DOX showed remarkable tumor growth suppression and prolonged survival time. This result was further improved by the coadministration of iRGD. The antitumor effects of various liposomes were ranked in the same order as the degree of tumor biodistribution shown by in vivo/ex vivo imaging: ALN-HA-SS-L-L coadministered with iRGD > ALN-HA-SS-L-L > HA-SS-L-L > HA-L-L > PEG-L> free drug. ALN-HA-SS-L-L/DOX also reduced the cardiotoxicity of DOX and lung metastases. Overall, this study demonstrated that ALN-HA-SS-L-L/DOX, equipped with bone- and CD44-dual-targeting abilities and redox sensitivity, could be a promising OS-targeted therapy. The efficacy could also be augmented by coadministration of iRGD.

Estrogen-functionalized liposomes grafted with glutathione-responsive sheddable chotooligosaccharides for the therapy of osteosarcoma.

An estrogen (ES)-functionalized cationic liposomal system was developed and exploited for targeted delivery to osteosarcoma. Natural biocompatible chotooligosaccharides (COS, MW2-5 KDa) were covalently tethered to the liposomal surface through a disulfate bond (-SS-) to confer reduction-responsive COS detachment, whereas estrogen was grafted via polyethylene glycol (PEG 2 K) chain to achieve estrogen receptor-targeting. The liposomal carriers were prepared by the ethanol injection method and fluorescent anticancer drug doxorubicin (DOX) was loaded with ammonium sulfate gradient. The physicochemical properties, reduction-sensitivity, and the roles of estrogen on cellular uptake and tumor-targeting were studied. The Chol-SS-COS/ES/DOX liposomes were spherical with an average size about 110 nm, and high encapsulation efficiency. The liposomes were stable in physiological condition but rapidly release the payload in response to tumoral intracellular glutathione (20 mM). MTT cytotoxicity assay confirmed that Chol-SS-COS/ES/DOX liposomes exhibited higher cytotoxicity to MG63 osteosarcoma cells than to liver cells (LO2). Flow cytometry (FCM) and confocal laser scanning microscopy revealed that cellular uptake of Chol-SS-COS/ES/DOX liposomes by MG63, than the free DOX or Chol-SS-COS/DOX. Ex vivo fluorescence distribution study showed that the multifunctional liposomes selectively accumulated in the MG63 xenografts versus the organs. Chol-SS-COS/ES/DOX liposomes strongly inhibited the tumor growth and enhanced the animal survival rate. Overall, the COS grafted estrogen-functionalized cationic liposomes, fortified with glutathione-responsiveness, showed great potential for specific intracellular drug delivery to estrogen receptor-expressing tumors such as osteosarcoma.

Chitooligosaccharides Modified Reduction-Sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models.

PURPOSE: To investigate the potential of a reduction-sensitive and fusogenic liposomes, enabled by surface-coating with chotooligosaccharides (COS) via a disulfide linker, for tumor-targeted cytoplasmic drug delivery. METHODS: COS (MW2000-5000) were chemically tethered onto the liposomes through a disulfide linker (-SS-) to cholesterol (Chol). Doxorubicin (DOX) was actively loaded in the liposomes. Their reduction-sensitivities, cellular uptake, cytotoxicity, pharmacokinetics and antitumor efficacy were investigated. RESULTS: The Chol-SS-COS/DOX liposomes (100 nm) had zeta potential of 33.9 mV and high drug loading (13% w/w). The liposomes were stable with minimal drug leakage under physiological conditions but destabilized in the presence of reducing agents, dithiothreitol (DTT) or glutathione (GSH) at 10 mM, the cytosolic level. MTT assay revealed that the cationic Chol-SS-COS/DOX liposomes had higher cytotoxicity to MG63-osteosarcoma cells than non-reduction sensitive liposome (Chol-COS/DOX). Flow cytometry and confocal microscopy revealed that Chol-SS-COS/DOX internalized more efficiently than Chol-COS/DOX with more content to cytoplasm whereas Chol-COS/DOX located around the cell membrane. Chol-SS-COS/DOX preferentially internalized into MG63 cancer cell over LO2 normal liver cells. In rats both liposomes produced a prolonged half-life of DOX by 4 - 5.5 fold (p < 0.001) compared with the DOX solution. Chol-SS-COS/DOX exhibited strong inhibitory effect on tumor growth in MG63 cell-bearing nude mice (n = 6), and extended animal survival rate. CONCLUSIONS: Reduction-responsive Chol-SS-COS liposomes may be an excellent platform for cytoplasmic delivery of anticancer drugs. Conjugation of liposomes with COS enhanced tumor cell uptake, antitumor effect and survival rate in animal models.

Redox-sensitive and hyaluronic acid functionalized liposomes for cytoplasmic drug delivery to osteosarcoma in animal models.

This study aimed to develop redox-sensitive and CD44-targeted liposomes to improve chemotherapy of osteosarcoma. Cationic liposomes were prepared and stabilized with a novel detachable polyethylene glycol (PEG2000) conjugated with cholesterol through a bio-reducible disulfide linker (Chol-SS-mPEG). Hyaluronic acid (HA, MW 20-40kDa), a ligand to CD44, was non-covalently coated on the cationic liposomes. Doxorubicin (DOX) was actively loaded in the liposomes as a model drug. The roles of HA and Chol-SS-mPEG on intracellular drug delivery efficiency, and antitumor efficacy were studied. The structure of Chol-SS-mPEG was confirmed with Fourier-transform infrared and nuclear magnetic resonance (1H NMR). The liposomes, Chol-SS-mPEG/HA-L had a mean diameter of 165nm, zeta potential -28.9mV, and destabilized in reducing or acidic (pH5-6) conditions. In vitro release of DOX was well-controlled at physiological conditions, but a burst release of 60% was observed in the presence of 10mM glutathione (GSH), in contrast to non-redox sensitive liposomes (Chol-mPEG/HA-L and Chol-mPEG-L). MTT cell viability assay showed that the dual-functional Chol-SS-mPEG/HA-L with a drug loading of 15.0% (w/w) had significantly higher cytotoxicity to MG63 osteosarcoma cells compared with non-reduction sensitive or non-HA coated liposomes (p<0.01), consistent with the cellular uptake and intracellular trafficking studies using confocal microscopy and flow cytometry. Furthermore, the HA-coated GSH-responsive liposomes preferentially internalized to MG63 over human liver cells LO2. In rats, liposomes stabilized with either Chol-SS-mPEG or Chol-mPEG, with or without HA, increased the half-life of DOX by >10-fold. In a MG63 xenograft mouse model, Chol-SS-mPEG/HA-L showed the most effective tumor suppression with minimal uptake by the liver compared with other liposomes. All animals treated with liposomal formulations survived, in contrast to those free-DOX treated. In conclusion, the easily prepared Chol-SS-mPEG/HA-L was demonstrated as an excellent CD44-mediated intracellular delivery system capable of long-circulation and GSH-triggered cytoplasmic drug release. Further translational and multidisciplinary research is required to make it real clinical benefits to cancer patients.

The altered tight junctions: an important gateway of bacterial translocation in cachexia patients with advanced gastric cancer.

UNLABELLED: Tight junctions (TJs) are the structural basis for the intestinal epithelium barrier. Increased intestinal permeability caused by variations in TJ proteins may result in bacterial translocation (BT). There is increasing evidence that BT might contribute to the occurrence and development of cancer cachexia, but the details are not known. Aims, we undertook further investigations into the pathway of BT in cancer cachexia. RESULTS: BT-positive patients had a higher level of claudins-2 (CL-2, P=0.035) and a lower level of occludin (P=0.038) and Zonula occluden-1 (P=0.01) than BT-negative patients. Moreover, the levels of IL-6, TNF-α, and IFN-γ in BT-positive cachexia patients were higher compared with BT-negative cachexia patients (P<0.001, P=0.01, P<0.001) and BT-positive noncachexia patients (P<0.001, P=0.025, P<0.001). In the BT-positive cachexia patients, the local concentration of IL-6, TNF-α, and IFN-γ, in the middle colic vein, was higher than in the peripheral venous (P=0.04, P=0.03, P=0.038). In addition, endotoxin was detected within the small intestinal wall, and the concentration of endotoxin decreased from the mucosal side to the serosal side gradually in BT-positive patients. This study suggests that the altered TJs could be an important gateway of BT in gastric cancer cachexia and local cytokines could play a more important role than systemic cytokines in the process.

Combined effect of cytokine gene polymorphisms on end-stage knee osteoarthritis from Chinese Han population.

The mechanism of osteoarthritis (OA) is not well understood. Cytokines have been implicated in the episode, and there is increasing evidence that the host's cytokine response is genetically determined. We determined the predictive value of IL-634G./C, ICAM-1 469 K/E and IL-10-1082A/G, -819T/C and -592A/C gene polymorphisms on knee OA. The study included 1007 patients with end-stage knee OA and 910 healthy controls. Genomic DNA was prepared from peripheral blood leukocytes. Genotypes and allele frequencies were determined using restriction fragment length polymorphism analysis of polymerase chain reaction products. No significant difference in the IL-10 promoter allele or haplotype frequencies between end-stage knee OA and controls was found. Patients with end-stage knee OA showed a significantly higher prevalence of IL-6-634G/ICAM-1 469E carrier than that in controls (P = 0.017). Results indicate that IL-6-634G/ICAM-1 469E carrier could be associated with increased susceptibility to end-stage knee OA.

[The value of erythrocyte sedimentation rate and C-reactive protein in evaluating disease activity in ankylosing spondylitis].

OBJECTIVE: To determine whether erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) is more appropriate in measuring the disease activity in ankylosing spondylitis (AS). METHODS: We studied 126 consecutive patients with AS. The external criteria for disease activity were Cowling clinical assessment of disease activity and the Bath AS disease activity index (BASDAI). In each measure we defined 3 levels of disease activity i.e. no activity, ambiguous activity and definite activity. The patients with AS were divided into 2 groups: those with spinal involvement only and those with peripheral arthritis as well. For each criterion of disease activity, the patients without activity and with definite activity were included in receiver operating characteristic curve, which was used to determine cutoff values with the highest sensitivity and specificity. We also calculated Spearman correlation. RESULTS: The median ESR and CRP were 25.3 mm/1 h and 11.1 mg/L in the spinal group and 30.0 mm/1 h and 15.0 mg/L in the peripheral group. In both groups the Spearman correlation coefficients between ESR and CRP were around 0.30. There was no correlation between ESR, CRP, and the 2 disease activity variables (0.027-0.282). Sensitivity for both ESR and CRP was between 39.4 %and 81.3% for Cowling assessment of disease activity and the BASDAI, while specificity was between 40.0% and 86.7% for all disease activity measures. CONCLUSION: It is concluded that neither ESR nor CRP is superior for assessing disease activity in ankylosing spondylitis.

[Experimental study of immunological tolerance induced by cytotoxic T lymphocyte-associated antigen 4 immunoglobulin in fresh bone allografts].

OBJECTIVE: To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyte-associated antigen 4 immuno globulin (CTLA4-Ig). METHODS: Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and 18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection of L6 (as a control), was named AL group. The second group, also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group, homologous BALB/C with injection of PBS buffer solution, was named AB group. The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6 (BC group) and C3H (BH group) mice respectively after first 12 mice being transplanted with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. RESULTS: Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets (P<0.05), the serum antibody (P<0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor (P<0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4 T cell subsets (P>0.05), the serum antibody (P>0.05), and lymphocyte proliferation of the second stimulation (P>0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared, cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (P<0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor (P<0.05), BC group was significantly lower than those of BH group. So the immunological tolerance induced by CTLA4-Ig was of donor-specificity. CONCLUSION: The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.

In vitro study of immune tolerance induced by CTLA4-Ig in bone transplantation: The effect on cell proliferation stimulated by lymphocytes and bone supernatant.

To clarify the effect of CTLA4-lg on immune rejection of bone grafts, we observed the effect of CTLA4-lg on lymphocyte proliferation of BALB/C mice stimulated by lymphocytes and bone supernatant of C57BL/6 mice. The splenic lymphocytes and bone supernatant of C57BL/6 mice, as the stimulator cells and stimulator antigens, were cultured in vitro with the splenic lymphocyte of BALB/C mice. At the same time, CTLA4-lg at a dose of 5,10 or 20 &mgr;g/ml and L6 (as control) at 20 &mgr;g/ml were added. Six days later, the incorporation of 3 H-TdR was determined. Results indicated that CTLA4-Ig at a dose of 5, 10 or 20 &mgr;g/ml significantly inhibited the cell proliferation stimulated by lymphocytes and bone supernatant of C57BL/6 mice. The effect was non-cytotoxic. L6 showed no significant inhibition of cell proliferation. CTLA4-Ig can efficiently block the proliferation of alloresponsive T cell stimulated by lymphocytes and bone supernatant of C57BL/6 mice. This study provides a basis for further study of CTLA4-induced immune tolerance of bone grafts.