Huan Xu

Renal fibrosis plays a key role in the pathogenesis of chronic kidney disease (CKD), in which the persistent high expression of transforming growth factor beta 1 (TGF-beta 1) and alpha-smooth muscle actin (alpha-SMA) contributes to the progression of CKD to renal failure. In order to improve the solubility, bioavailability, and targeting of tanshinone IIA (Tan IIA), a novel targeting material, aminoethyl anisamide-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphate ethanolamine (AEAA-PEG-DSPE, APD) modified Tan IIA liposomes (APD-Tan IIA-L) was constructed. An animal model of glomerulonephritis induced by doxorubicin in BALB/c mice was established. APD-Tan IIA-L significantly decreased blood urea nitrogen and serum creatinine (SCr), and the consequences of renal tissue oxidative stress indicators showed that APD-Tan IIA-L downregulated malondialdehyde, upregulated superoxide dismutase, catalase, and glutathione peroxidase. Masson's trichrome staining showed that the deposition of collagen in the APD-Tan IIA-L group decreased significantly. The pro-fibrotic factors (fibronectin, collagen I, TGF-beta 1, and alpha-SMA) and epithelial-mesenchymal transition marker (N-cadherin) were significantly inhibited by APD-Tan IIA-L. By improving the microenvironment of fibrotic kidneys, APD-Tan IIA-L attenuated TGF-beta 1-induced excessive proliferation of fibroblasts and alleviated oxidative stress damage to the kidney, providing a new strategy for the clinical treatment of renal fibrosis.

To improve the solubility and targeting of Ginsenoside Rg3 (G-Rg3), in the current study, we constructed a novel targeting functional material folic acid -poly(2-ethyl-2-oxazoline)-cholesteryl meth...

In this study, we evaluated and screened the effects of the molecular weight (MW) and molar ratio of poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) on the pH sensitivity, stability, and antitumor efficacy of liposomes. The pH sensitivity of PEtOz-CHMC with different MWs and molar ratios was screened by drug release and cytotoxicity experiments at different pH levels. Results indicated that the liposomes coated with PEtOz1k-CHMC (7% molar ratio) and PEtOz2k-CHMC (5% molar ratio) exhibited the desirable pH responsiveness. When the MW of PEtOz was relatively low, 7% of the modified ratio obtained the strongest stability, but the turbidity of the liposomes did not obviously change when the molar ratio of PEtOz-CHMC was further increased. A375 cells were used as models to investigate the cellular uptake and intracellular localization of coumarin-6-loaded liposomes (C6-L), PEGylated liposomes (PEG-C6-L), and PEtOzylated liposomes. PEtOz1k-C6-L and PEtOz2k-C6-L presented remarkably stronger fluorescence intensity at low pH than at pH 7.4, whereas C6-L and PEG-C6-L did not achieve any obvious diversity at different pH conditions. Compared with C6-L and PEG-C6-L, PEtOz-C6-L showed efficient intracellular trafficking, including endosomal/lysosomal escape and cytoplasmic release. Pharmacokinetic experiments demonstrated that half-lives of PEG2k-C6-L, PEtOz2k-C6-L, and PEtOz1k-C6-L were 11.89-, 7.00-, and 5.29-fold times higher than those of C6-L, respectively. Among the liposomes, the DOX·HCl-loaded liposomes coated with PEtOz2k-CHMC demonstrated the strongest antitumor efficacy against B16 tumor xenograft models in vivo. These findings provide the feasibility of using PEtOz-CHMC with optimal pH sensitivity and long circulation to extend the application of liposomes to efficient anticancer drug delivery.

Objectives: To improve stability and shelf life, lyophilized formulations of 20(R)-Ginsenoside Rg3 liposomes (G-Rg3-Ls) were prepared. Methods: Glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-β-cyclodextrin and polyethylene glycol were used as single lyoprotectant and then compared in terms of their ability to protect lyophilized G-Rg3-Ls. Further, a glucose–mannitol complex was used to determine the optimal lyophilized preparation. The analysis of lyophilized liposomes or lyoprotectant was further investigated by scanning electron microscopy, thermogravimetry-differential thermal analysis, X-ray diffractometry and Fourier transform infrared spectroscopy. Cytotoxicity assay was used to assess the cyto-inhibition of freshly prepared and lyophilized liposomes. Key findings: When the ratio of glucose–mannitol to phospholipids was 4 : 2 : 1 (w/w) the lyophilized G-Rg3-Ls exhibited good appearance, high DRR (86.52% ± 5.02%), small change in particle size (45.83 ± 0.50%) and short rehydration reconstruction time (8.3 ± 1.5 s). All indices were considerably better than those of each single protective agent. Results indicated that when the two lyoprotectants were combined, the stabilizing effect of glucose and shaping effect of mannitol were well maintained. The cyto-inhibition of freshly prepared and lyophilized G-Rg3 liposomes showed that lyophilization did not affect the bioactivity of G-Rg3. Conclusions: The application of glucose–mannitol composite lyoprotectants can obtain a good G-Rg3 lyophilized preparation.

In this study, the buffering capacity of amphiphilic pH-sensitivity copolymer poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (PEOZ-CHMC) was evaluated. The ammonium sulfate gradient method was used to prepare doxorubicin hydrochloride (DOX x HCl)-loaded liposomes (DOX-L), and then the post-insertion method was used to prepare PEOZ-CHMC and polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE) modified DOX x HCl-loaded liposomes (PEOZ-DOX-L and PEG-DOX-L). The physico-chemical properties, in vitro drugs release behavior, cellular toxicity and intracellular delivery of liposomes were evaluated, separately. The results showed that PEOZ-CHMC has a satisfactory buffering capacity. The sephadex G-50 column centrifugation method and dynamic light scattering were used to determine the encapsulation efficiency (EE) and particle size of liposomes. The EE and particle size of DOX-L were (97.3 ± 1.4) % and 120 nm, respectively, and the addition of PEOZ-CHMC or PEG-DSPE had no influence on EE and particle size. The zeta potentials of three kinds of liposomes were negative. The release behavior of various DOX liposomes in vitro was investigated by dialysis method. In phosphate buffer solution (PBS) at pH 7.4, DOX x HCl was released from PEOZ-DOX-L in a sustained manner. While in PBS at pH 5.0, the release rate of DOX x HCl from PEOZ-DOX-L increased significantly, which suggested DOX x HCl was released from PEOZ-DOX-L in a pH-dependent manner. The intracellular delivery of liposomes was investigated by confocal laser scanning microscopy (CLSM). The CLSM images indicated that PEOZ-DOX-L showed efficient intracellular trafficking including endosomal escape and release DOX x HCl into nucleus, as well as the DOX-L and PEG-DOX-L had no this effect. The cytotoxicity of liposomes against MCF-7 cells was detected by using MTT assay. The results showed that antiproliferative effects of PEOZ-DOX-L enhanced with pH value decreased, whereas DOX-L and PEG-DOX-L did not have any significant difference in inhibitions at different pH conditions. Therefore, the problems of the inhibition of cellular uptake of liposomes and the failed endosomal escape of pH-sensitive liposomes by PEG chain can be overcome by the pH-sensitive liposomes constructed by PEOZ-CHMC.

Polyethylene glycol (PEG) is extensively used to increasing the in vivo and in vitro stability of liposomes. However, PEGylated liposomes also produce some negative effects with further research, such as low cellular uptake, poor "endosomal escape" of pH sensitive liposome (PSL) and accelerated blood clearance (ABC) phenomenon, and this situation is referred as the "PEG dilemma". "PEG dilemma" posed severe challenges for the targeted delivery of PEGylated liposomes-loaded anticancer drugs, effective intracellular release of PEGylated PSL-encapsulated gene and protein drugs, and repeated administration of PEGylated liposomes. Therefore, it is urgent to solve the "PEG dilemma". This review focused on the definition, classification of "PEG dilemma", and discussed several possible approaches to overcome "PEG dilemma".

A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study. PEtOz-CHMC was synthesized and characterized by TLC, IR and ¹H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified. PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t _1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL. PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t _1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.