Polyethylene glycolated (PEGylated)curcumin-grafted-chitosan (PCC) conjugates were synthesized with three PEG/chitosan feed molar ratios (1/5, 1/7.5, and 1/10), namely PCC1, PCC2 and PCC3. Chemical structures of these conjugates were characterized by Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR). The degrees of substitution (DS) of PEG were 0.75%, 0.45% and 0.33%, respectively, for PCC1, PCC2 and PCC3by 1H NMR analysis. Self-assembled PCC nanoparticles (NPs) were spherical as observed in transmission electron microscope images. Mitoxantrone (MTO)-loaded PCC NPs were prepared to analyze the particle size, zeta potential, drug loading, drug release and in vitro cytotoxicity. The MTO-loaded PCC3 NP (DS = 0.33%) possessed the smallest size (~183.1 nm), highest zeta potential (~+34.0 mV) and the largest loading capacity of curcumin (CUR, ~16.1%) and MTO (~8.30%). The release results showed that MTO-loaded PCC3 NP demonstrated the lowest percentage of MTO release and increased as pH decreased, but the CUR release could only be detected at pH 4.0. In the cytotoxicity study, MTO-loaded PCC3 NP displayed the highest cytotoxicity in HepG2 cell line and the best synergistic effect among the tested NPs. Our results suggest that the DS of PEG has impacts on the structures and functions of PCC NPs: the smaller DS of PEG was associated with the smaller size, the higher zeta potential, the slower drug release, and the higher cytotoxicity of NPs.

BACKGROUND: Identification of molecular markers that reflect the characteristics of the tumor microenvironment (TME) may be beneficial to predict the prognosis of post-operative hepatocellular carcinoma (HCC) patients. OBJECTIVE AND METHODS: A total of 100 tissue samples from HCC patients were separately stained by immunohistochemistry to examine the expression levels of CD56, CD8α, CD68, FoxP3, CD31 and pan-Keratin. The prognostic values were analyzed by Cox regression and the Kaplan-Meier method. RESULTS: Univariate and multivariate logistic analysis showed that FoxP3 was the independent factor associated with microvascular invasion (MVI), tumor size and envelop invasion; CD68 was associated with envelope invasion and AFP. Kaplan-Meier survival curves revealed that CD68 and FoxP3 expression were significantly associated with relapse free survival (RFS) of HCC patients (P< 0.05). The ROC curve indicated that the combination of tumor number, MVI present and CD68 expression yielded a ROC curve area of 82.3% (86.36% specificity, 68.75% sensitivity) to evaluate the prognosis of HCC patients, which was higher than the classifier established by the combination of tumor number and MVI (78.8% probability, 63.64% specificity and 85.42% sensitivity). CONCLUSIONS: Our study indicated that CD68 and FoxP3 are associated with prognosis of HCC patients, and CD68 can be considered as a potential prognostic and predictive biomarker.

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Background: This study attempts to detect the expression of FoxP3, CD68, CD8α, and PD-L1 in the tumor microenvironment (TME) of intrahepatic cholangiocarcinoma (ICC), and analyze the relationship between the corresponding cells and clinicopathological characteristics as well as prognosis of ICC.Methods: RNA sequencing (RNA-seq) provided the general landscape of the TME in ICC. A total of 99 ICC patients and the corresponding specimens were used for multiplex immunofluorescence and relapse-free survival (RFS) was analyzed. Flow cytometry further validated the effect of regulatory T (Treg) cells on ICC relapse.Results: RNA-seq data showed that the infiltration of Treg cells, CD8+ T cells, and macrophages were likely associated with ICC relapse. The survival analysis based on multiplex immunofluorescence showed that the high FoxP3(+) Treg cells ratio and low CD68(+) macrophages ratio in mesenchyme were associated with higher RFS rate, respectively. Low FoxP3(+) Τreg cells ratio was associated with more perineural invasion, and high CD68(+) macrophages ratio was correlated with more lymph node metastasis. Cox regression analysis revealed that FoxP3(+) Treg cells ratio was an independent predictive factor for ICC relapse. Flow cytometry showed that TregIII was the predominant Treg cell subtype in both tumor tissue and peripheral blood of ICC patients, and high TregIII abundance in peripheral blood was significantly associated with longer RFS of ICC patients. Conclusion:High FoxP3(+) Treg cells ratio in the mesenchyme of ICC tumor tissue predicted longer RFS and was an independent favorable prognostic factor for ICC The first two authors contributed equally to this article.