Activation of CYP3A by Accelerated Blood Clearance Phenomenon Potentiates the Hepatocellular Carcinoma-Targeting Therapeutic Effects of PEGylated Anticancer Prodrug Liposomes.

Reduced enzyme activity in hepatocellular carcinoma (HCC) and poor targeting limit the application of enzyme-activating prodrugs, which is also detrimental to the effective treatment of HCC. Here, we investigated whether accelerated blood clearance (ABC) phenomenon occurs in HCC models following repeated injections of PEGylated liposomes (PEG-L), thus inducing prodrug accumulation and activation in the liver and exerting highly effective and low-toxicity therapeutic effects on HCC. First, PEGylated liposomal cyclophosphamide was prepared by solvent injection and characterized. Importantly, preinjection of PEG-L induced the ABC phenomenon and activation of CYP3A in both HCC rats and HCC mice by studying the effects of repeated injections of PEG-L on pharmacokinetics and tissue distribution. Next, the efficacy and toxicity of repeated injections of PEG-L in HCC mice were examined, and our data indicate that repeated injections are administered in a manner that significantly enhances the antitumor effect compared with controls, with little or no toxicity to other organs. To further reveal the pharmacokinetic mechanism of PEG-L repeated administration for the treatment of HCC, the protein expression of hepatic CYP3A and the concentration of cyclophosphamide in the liver and spleen of HCC mice by inhibiting CYP3A were analyzed. These results revealed that inducing CYP3A to accelerate the rapid conversion of prodrugs that accumulate significantly in the liver is a key mechanism for the treatment of HCC with repeated injections of PEG-L. Collectively, this work taps into the application potential of the ABC phenomenon and provides new insights into the clinical application of PEGylated nanoformulations. SIGNIFICANCE STATEMENT: This study revealed that repeated injections of PEGylated liposomes could induce the accelerated blood clearance (ABC) phenomenon characterized by hepatic accumulation and CYP3A activation based on hepatocellular carcinoma (HCC) rats and HCC mice. Furthermore, it was verified that induction of the ABC phenomenon dependent on hepatic accumulation and CYP3A activation could enhance the antihepatocellular carcinoma effects of PEGylated anticancer prodrugs in HCC mice. This elucidated the relevant pharmacokinetic mechanisms and unearthed new clues for solving the clinical application of PEGylated nanoparticles.

Activation of Pregnane X Receptor-Cytochrome P450s Axis: A Possible Reason for the Enhanced Accelerated Blood Clearance Phenomenon of PEGylated Liposomes In Vivo.

Recently, we reported that repeated injection of PEGylated liposomes (PEG-L) at certain intervals to the same rat lead to the disappearance of their long-circulation properties, referred to as the "accelerated blood clearance (ABC) phenomenon". Evidence from our recent studies suggested that cytochrome P450s (P450s) contribute to induction of the ABC phenomenon, a possibility that had been previously ignored. However, few details are known about the mechanism for induction of P450s. The present study was undertaken to investigate the roles in the ABC phenomenon of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), the major upstream transcriptional regulators of the P450 genes, including CYP3A1, CYP2C6, and CYP1A2. The results demonstrated that expression of rat PXR and CAR was significantly increased in the ABC phenomenon and was accompanied by elevated CYP3A1, CYP2C6, and CYP1A2 levels. Further findings revealed that PXR but not CAR protein was substantially upregulated in the hepatocyte nucleus, together with marked nuclear colocalization of the PXR-retinoid X receptor alpha (RXRα) transcriptionally active heterodimer, indicating that nuclear translocation of PXR was induced in the ABC phenomenon, whereas nuclear translocation of CAR was not observed. Notably, pretreatment with the specific PXR inducer dexamethasone significantly induced accelerated systemic clearance of the subsequent injection of PEG-L, associating with increased nuclear colocalization of PXR-RXRα These results revealed that the induction of P450s in the ABC phenomenon may be attributable largely to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of the PXR-P450s axis in promoting the ABC phenomenon. SIGNIFICANCE STATEMENT: The results of this study revealed that the induction of P450s in the ABC phenomenon may be largely attributable to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of the PXR-P450s axis in promoting the ABC phenomenon. The data may help to extend our insights into 1) the role of P450s, which are regulated by the liver-enriched nuclear receptor PXR, in the ABC phenomenon, and 2) the therapeutic potential of targeting the PXR-P450 axis for reducing the magnitude of the ABC phenomenon in clinical practice.

Induction of Cytochrome P450 Involved in the Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomes In Vivo.

Polyethylene glycol (PEG) is recognized as an attractive excipient to modify liposomes due to its extended-circulation properties. Nevertheless, intravenous injection of polyethylene glycol-coated liposomes (PEG-L) usually triggers a rapid systemic clearance of the subsequent dose from blood circulation, which is referred to as an accelerated blood clearance (ABC) phenomenon. Therefore, since the induction of cytochrome P450 (P450) activity may lead to enhanced drug clearance, it motivated us to investigate the possibility of P450 involvement in the ABC phenomenon. In this study, polyethylene glycol-coated liposomal docetaxel was prepared and used to evaluate the magnitude of the ABC phenomenon in rats induced by repeated injection of PEG-modified liposomes. Notably, the ABC phenomenon was observed when the time interval between two doses was from 1 to 7 days, and its magnitude reached the maximum level at 3 days before gradually decreasing the time. Meanwhile, increased activity of CYP3A1, CYP2C6, and CYP1A2 was detected when PEG-L was repeatedly injected in male rats at a 3-day interval. Consistently, the expression levels of hepatic CYP3A1, CYP2C6, and CYP1A2 were also significantly increased in the repeated injection groups and their levels were highest in the 3-day interval group. P450 selective inhibitors confirmed the inhibition of hepatic CYP3A1 was accompanied by an attenuated magnitude of the ABC phenomenon, which strongly suggests that P450s may be induced by repeated injection of PEG-L, thus favoring metabolic clearance of the second dose. Collectively, herein, for the first time we demonstrate that the contribution of P450s should not be ignored in the ABC phenomenon.

Nano-delivery of salvianolic acid B induces the quiescence of tumor-associated fibroblasts via interfering with TGF-ß1/Smad signaling to facilitate chemo- and immunotherapy in desmoplastic tumor.

As the key stromal cells that mediate the desmoplastic reaction, tumor-associated fibroblasts (TAFs) play a critical role in the limited nanoparticle penetration and suppressive immune tumor microenvironment. Herein, we found that salvianolic acid B-loaded PEGylated liposomes (PEG-SAB-Lip) can interfere with the activation of TAFs by inhibiting the secretion of TGF-ß1. After inhibiting the activation of TAFs, collagen deposition in tumors was reduced, and the penetration of nanoparticles in tumors was enhanced. The results of RT-qPCR and immunofluorescence staining showed the high expression of Th1 cytokines and chemokines (CXCL9 and CXCL10) and the recruitment of CD4+, CD8+ T cells, and M1 macrophages in the tumor area. At the same time, the low expression of Th2 cytokine and chemokine CXCL13, as well as the decrease of MDSCs, Tregs, and M2 macrophages were also observed in the tumor area. These results were related to the inactivation of TAFs. The combined treatment of PEG-SAB-Lip and docetaxel-loaded PEG-modified liposomes (PEG-DTX-Lip) can significantly inhibit tumor growth. Moreover, PEG-SAB-Lip further inhibited tumor metastasis to the lung. Therefore, our results showed that PEG-SAB-Lip can remodel the tumor microenvironment and improve the efficacy of nanoparticles.

Expandable carboxymethyl chitosan/cellulose nanofiber composite sponge for traumatic hemostasis.

Uncontrolled hemorrhage poses a severe life-threatening situation. However, traditional hemostats still have various limitations. It is urgent to develop a material with excellent biocompatibility and hemostatic ability. Evidence has shown that carboxymethyl chitosan (CMCS) has hemostatic properties and good compatibility. Herein, we develop an expandable hemostatic sponge by modifying CMCS with cellulose nanofibrils (CNFs) through the CO-NH cross-linking method. We verified its potential as a hemostatic agent both in vivo and in vitro. The results demonstrated that the prepared carboxymethyl chitosan/cellulose nanofiber composite (CNF-CMCS) sponges could absorb blood, quickly expand to exert pressure in the wound, and exhibit an excellent coagulation ability. The CNF-CMCS sponges significantly decreased the bleeding time and blood loss in several hemorrhage models and possessed a significant advantage in treating the deep penetrating injury hemorrhage. Therefore, the sponges provide a unique application prospect and potential as a penetrating trauma hemostatic agent.

Seadragon genome analysis provides insights into its phenotype and sex determination locus.

The iconic phenotype of seadragons includes leaf-like appendages, a toothless tubular mouth, and male pregnancy involving incubation of fertilized eggs on an open "brood patch." We de novo-sequenced male and female genomes of the common seadragon (Phyllopteryx taeniolatus) and its closely related species, the alligator pipefish (Syngnathoides biaculeatus). Transcription profiles from an evolutionary novelty, the leaf-like appendages, show that a set of genes typically involved in fin development have been co-opted as well as an enrichment of transcripts for potential tissue repair and immune defense genes. The zebrafish mutants for scpp5, which is lost in all syngnathids, were found to lack or have deformed pharyngeal teeth, supporting the hypothesis that the loss of scpp5 has contributed to the loss of teeth in syngnathids. A putative sex-determining locus encoding a male-specific amhr2y gene shared by common seadragon and alligator pipefish was identified.

Enhanced nuclear delivery of H1-S6A, F8A peptide by NrTP6-modified polymeric platform.

Nucleus is the central regulator of cell metabolism, growth and differentiation, which is considered as an effective target for the treatment of many diseases. To efficiently deliver drugs into nucleus, delivery systems have to bypass a number of barriers especially crossing the cell membrane and nuclear envelope. Here we report a nucleolar targeting peptide (NrTP6) modified polymeric conjugate platform based on N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymers for enhanced nuclear delivery of H1-S6A, F8A peptide to hinder c-Myc from binding to DNA. On one hand, the modification of NrTP6 would promote cellular uptake and nuclear accumulation of the conjugates, and on the other hand, the conjugates can release smaller molecular weight subunits (H1-NrTP6) via cleavage of lysosomally enzyme-sensitive spacer for facilitating nucleus transport. It was found that NrTP6 modified HPMA copolymer-H1 peptide conjugates could improve internalization and nuclear accumulation of H1 peptide by 2.2 and 37.1-fold, respectively, compared to the non-NrTP6 modified ones, in HeLa cells. Moreover, the same trend was found in MDA-MB-231 cells and 4T1 cells. In addition, we found that the nuclear targeting mechanism of NrTP6 peptide mediation may be associated with the importin α/ß pathway. Furthermore, the in vivo investigation revealed that NrTP6-modified polymeric platform exhibited the best therapeutic efficacy with a tumor growth inhibition rate of 77.0%. These results indicated that NrTP6 modification was a promising strategy for simultaneously realizing cellular internalization and nuclear targeting, which might provide a new path for intranuclear drug delivery.

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM.

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as "accelerated blood clearance (ABC) phenomenon." Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose-dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.

A novel αVß3 ligand-modified HPMA copolymers for anticancer drug delivery.

The integrin αVß3 receptor emerged as one of the most promising targets owing to its high expression on the surface of various malignant tumour cells and tumour angiogenesis endothelial cells, but with little expression in mature endothelial cells and the majority of normal cells. Here, we report a new targeting ligand FQSIYPpIK (FQS) with high affinity to integrin αVß3 receptor. To take the advantage of the particular interaction between FQS and integrin αVß3 receptor, FQS was linked to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. A model drug doxorubicin (DOX) was simultaneously conjugated to the same HPMA copolymers via pH-sensitive hydrazone linkages (FQS-HPMA-DOX). In in vitro study, FQS-HPMA-DOX could be internalised into αVß3 receptor-overexpressed B16F10 cells via a highly specific ligand - receptor pathway (5.0 times and 4.5 times higher cellular internalisation than HPMA-DOX and a scrambled peptide (s)-FQS (sequence: SYFIPKQIp)-modified copolymers ((s)-FQS-HPMA-DOX)). It is worth noting that compared with the classical αVß3 ligand cRGDfK-modified HPMA copolymers (cRGDfK-HPMA-DOX), FQS-HPMA-DOX also showed superior targeting efficiency. In in vivo study in the B16F10 melanoma bearing mice model showed the antitumour efficiency of FQS-HPMA-DOX (83.9%) were significantly higher than HPMA-DOX (44.9%) and cRGDfK-HPMA-DOX (77.5%). These results suggest that FQS peptide can act as an effective targeting ligand for the delivery of therapeutic agents.

Charge-Reversible Multifunctional HPMA Copolymers for Mitochondrial Targeting.

Mitochondrial-oriented delivery of anticancer drugs has been considered as a promising strategy to improve the antitumor efficiency of chemotherapeutics. However, the physiological and biological barriers from the injection site to the final mitochondrial action site remain great challenges. Herein, a novel mitochondrial-targeted multifunctional nanocomplex based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers (MPC) is designed to enhance drug accumulation in mitochondria. MPC possesses various functions such as extracellular pH response, superior cellular uptake, lysosomal escape, and mitochondrial targeting. In detail, MPC was formed by two oppositely charged HPMA copolymers, that is, positively charged mitochondrial-targeting guanidine group-modified copolymers and charge-reversible 2,3-dimethylmaleic anhydride (DMA)-modified copolymers (P-DMA). It was validated that MPC could remain stable in the blood circulation (pH 7.4) but could be cleaved to expose the positive charge of the guanidine group immediately in response to the mild acidity of tumor tissues (pH 6.5). The gradual exposure of positively charged guanidine will simultaneously facilitate endocytosis, endosomal/lysosomal escape, and mitochondrial targeting. The in vitro experiments showed that compared with copolymers without guanidine modification, the cellular uptake and mitochondrial-targeting ability of MPC in the simulated tumor environment (MPC@pH6.5) separately increased 4.3- and 23.8-fold, respectively. The in vivo experiments were processed on B16F10 tumor-bearing C57 mice, and MPC showed the highest accumulation in the tumor site and a peak tumor inhibition rate of 82.9%. In conclusion, multifunctional mitochondrial-targeting HPMA copolymers provide a novel and versatile approach for cancer therapy.