Puyuan Wu

Intelligently Targeted Drug Delivery and Enhanced Antitumor Effect by Gelatinase-Responsive Nanoparticles

Aims The matrix metalloproteinase (MMP) 2/9, also known as collagenases IV and gelatinases A/B, play a key role in cancer invasion and metastasis. However, the clinical trials of the MMP inhibitors (MMPIs) ended up with disappointing results. In this paper, we synthesized a gelatinase-responsive copolymer (mPEG-PCL) by inserting a gelatinase cleavable peptide (PVGLIG) between mPEG and PCL blocks of mPEG-PCL for anticancer drug delivery to make use of MMP2/9 as an intelligent target for drug delivery. Materials and Methods mPEG-pep-PCL copolymer was synthesized via ring-opening copolymerization and double-amidation. To evaluate whether Nanoparticles (NPs) prepared from this copolymer are superior to NPs prepared from mPEG-PCL, NPs prepared from mPEG-PCL copolymer were used as positive control. Docetaxel-loading NPs using mPEG-pep-PCL and mPEG-PCL were prepared by nano-precipitation method, mentioned as Gel-NPs and Con-NPs, respectively. The morphologic changes of the NPs after treatment with gelatinases were observed macroscopically by spectrophotometer and microscopically by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cellular uptake amount and cytotoxicity of Gel-NPs and Con-NPs, respectively, in cell lines with different levels of gelatinase expression were studied. Moreover, the cytotoxicity study on the primary cancer cells isolated from pericardial fluids from a patient with late-stage lung cancer was conducted. Results The Gel-NPs aggregated in response to gelatinases, which was confirmed macroscopically and microscopically. The cellular uptake amount of Gel-NPs was correlated with the level of gelatinases. The in vitro antitumor effect of Gel-NPs was also correlated with the level of gelatinases and was superior to Taxotere (commercially available docetaxel) as well as the Con-NPs. The cytotoxicity study on the primary lung cancer cells also confirmed the effectiveness of Gel-NPs. Conclusion The results in this study preliminarily demonstrated the effectiveness of gelatinase-responsive targeting strategy and the prospect of this intelligent nano-drug delivery system though further studies are needed.

Enhancement of radiotherapy efficacy by mir-200c-loaded gelatinase-stimuli Peg-Pep-Pcl nanoparticles in gastric cancer cells

Radiotherapy is the main locoregional control modality for many types of unresectable tumors, including gastric cancer. However, many patients fail radiotherapy due to intrinsic radioresistance of cancer cells, which has been found to be strongly associated with cancer stem cell (CSC)-like properties. In this study, we developed a nanoparticle formulation to deliver miR-200c, which is reported to inhibit CSC-like properties, and then evaluated its potential activity as a radiosensitizer. miR-200c nanoparticles significantly augmented radiosensitivity in three gastric cancer cell lines (sensitization enhancement ratio 1.13–1.25), but only slightly in GES-1 cells (1.06). In addition to radioenhancement, miR-200c nanoparticles reduced the expression of CD44, a putative CSC marker, and the percentage of CD44+ BGC823 cells. Meanwhile, other CSC-like properties, including invasiveness and resistance to apoptosis, could be suppressed by miR-200c nanoparticles. CSC-associated radioresistance mechanisms, involving reactive oxygen species levels and DNA repair capacity, were also attenuated. We have demonstrated that miR-200c nanoparticles are an effective radiosensitizer in gastric cancer cells and induce little radiosensitization in normal cells, which suggests that they are as a promising candidate for further preclinical and clinical evaluation.

Anti-EGFR-iRGD recombinant protein conjugated silk fibroin nanoparticles for enhanced tumor targeting and antitumor efficiency

In this study, we report a novel kind of targeting with paclitaxel (PTX)-loaded silk fibroin nanoparticles conjugated with iRGD–EGFR nanobody recombinant protein (anti-EGFR-iRGD). The new nanoparticles (called A-PTX-SF-NPs) were prepared using the carbodiimide-mediated coupling procedure and their characteristics were evaluated. The cellular cytotoxicity and cellular uptake of A-PTX-SF-NPs were also investigated. The results in vivo suggested that NPs conjugated with the recombinant protein exhibited more targeting and anti-neoplastic property in cells with high EGFR expression. In the in vivo antitumor efficacy assay, the A-PTX-SF-NPs group showed slower tumor growth and smaller tumor volumes than PTX-SF-NPs in a HeLa xenograft mouse model. A real-time near-infrared fluorescence imaging study showed that A-PTX-SF-NPs could target the tumor more effectively. These results suggest that the anticancer activity and tumor targeting of A-PTX-SF-NPs were superior to those of PTX-SF-NPs and may have the potential to be used for targeted delivery for tumor therapies.

Comparative studies of salinomycin-loaded nanoparticles prepared by nanoprecipitation and single emulsion method

To establish a satisfactory delivery system for the delivery of salinomycin (Sal), a novel, selective cancer stem cell inhibitor with prominent toxicity, gelatinase-responsive core-shell nanoparticles (NPs), were prepared by nanoprecipitation method (NR-NPs) and single emulsion method (SE-NPs). The gelatinase-responsive copolymer was prepared by carboxylation and double amination method. We studied the stability of NPs prepared by nanoprecipitation method with different proportions of F68 in aqueous phase to determine the best proportion used in our study. Then, the NPs were prepared by nanoprecipitation method with the best proportion of F68 and single emulsion method, and their physiochemical traits including morphology, particle size, zeta potential, drug loading content, stability, and in vitro release profiles were studied. The SE-NPs showed significant differences in particle size, drug loading content, stability, and in vitro release profiles compared to NR-NPs. The SE-NPs presented higher drug entrapment efficiency and superior stability than the NR-NPs. The drug release rate of SE-NPs was more sustainable than that of the NR-NPs, and in vivo experiment indicated that NPs could prominently reduce the toxicity of Sal. Our study demonstrates that the SE-NPs could be a satisfactory method for the preparation of gelatinase-responsive NPs for intelligent delivery of Sal.

Efficacy of decitabine-loaded gelatinases-stimuli nanoparticles in overcoming cancer drug resistance is mediated via its enhanced demethylating activity to transcription factor AP-2 epsilon

Hypermethylation of the transcription factor AP-2 epsilon (TFAP2E) gene affects 5-fluorouridine (5-FU) resistance in gastric cancer (GC) patients. The epigenetic inhibitor 5-Aza-2′-deoxycytidine (DAC), which reverses DNA methylation by targeting DNA methyltransferases (DNMTs), has potential to sensitize GC to 5-FU. Nevertheless, DNA demethylation only DAC transiently occurs since DAC is unstable in aqueous solutions, which limits its potential. Here we developed intelligent nanoparticles (NPs) comprising gelatinase with polyethylene glycol (PEG) and poly-ε-caprolactone) (PCL) to specifically deliver DAC (DAC-TNPs) to tumors. DAC-carrying PEG-PCL NPs (DAC-NPs) lacking gelatinase features served as controls. 72 hours after administration of DAC-TNPs or DAC-NPs, 5-FU was sequentially applied to GC cells and human GC xenografts in nude mice. Both in vitro and in vivo evaluations demonstrated that the combination treatment of DAC-TNPs and 5-FU greatly improved tumor suppression in GC cells and mouse xenograft models with hypermethylation TFAP2E (MKN45 cells). We thus propose that the sequential administration of DAC-TNPs and 5-FU could be significant in the development of novel targeted therapies.

Novel silk fibroin nanoparticles incorporated silk fibroin hydrogel for inhibition of cancer stem cells and tumor growth

Background A multi-drug delivery platform is needed as the intra-tumoral heterogeneity of cancer leads to different drug susceptibility. Cancer stem cells (CSCs), a small population of tumor cells responsible for tumor seeding and recurrence, are considered chemotherapy-resistant and have been reported to be sensitive to salinomycin (Sal) instead of paclitaxel (Ptx). Here we report a novel silk fibroin (SF) hydrogel-loading Sal and Ptx by incorporating drug-loaded silk fibroin nanoparticles (SF-NPs) to simultaneously kill CSCs and non-CSCs. Methods Using the method we have previously reported to prepare Ptx-loaded SF-NPs (Ptx-SF-NPs), Sal-loaded SF-NPs (Sal-SF-NPs) were fabricated under mild and non-toxic conditions. The drug-loaded SF-NPs were dispersed in the ultrasound processed SF solution prior to gelation. Results The resulting SF hydrogel (Sal-Ptx-NP-Gel) retained its injectable properties, exhibited bio-degradability and demonstrated homogeneous drug distribution compared to the non-NP incorporated hydrogel. Sal-Ptx-NP-Gel showed superior inhibition of tumor growth compared to single drug-loaded hydrogel and systemic dual drug administration in the murine hepatic carcinoma H22 subcutaneous tumor model. Sal-Ptx-NP-Gel also significantly reduced CD44+CD133+ tumor cells and demonstrated the least tumor formation in the in vivo tumor seeding experiment, indicating superior inhibition of cancer stem cells. Conclusion These results suggest that SF-NPs incorporated SF hydrogel is a promising drug delivery platform, and Sal-Ptx-NP-Gel could be a novel and powerful locoregional tumor treatment regimen in the future.

Strengthening Gastric Cancer Therapy by Trastuzumab-Conjugated Nanoparticles with Simultaneous Encapsulation of Anti-MiR-21 and 5-Fluorouridine

Background/Aims: MicroRNA-21 is an oncogenic miR (oncomiR) frequently elevated in gastric cancer (GC). Overexpression of miR-21 decreases the sensitivity of GC cells to 5-fluorouridine (5-Fu) and trastuzumab, a humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2). Receptor-mediated endocytosis plays a crucial role in the delivery of biotherapeutics including anti-miRNA oligonucleotides (AMOs). This study is a continuation of earlier findings involving poly(ε-caprolactone) (PCL)-poly (ethylene glycol) (PEG) nanoparticles (PEG-PCL NPs), which were coated with trastuzumab to target GC with HER2 receptor over-expression using anti-miRNA-21 (AMO-21) and 5-Fu. Methods: HER-PEG-PCL NPs were prepared by one-step carbodiimide coupling using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAc) and Sulfo-NHS in aqueous phase. Covalent coupling of amino groups at the surface of PEG-PCL with the carboxyl groups of trastuzumab was analyzed by X-ray photoelectron spectroscopy (XPS). AMO-21/5-Fu NPs were formulated by a double-emulsion solvent evaporation technique. The cell line specificity, cellular uptake and AMO-21 delivery were investigated through the rhodamine-B-labeled 6-carboxyfluorescein (FAM)-AMO-21-PEG-PCL NPs coated with or without the antibody in both Her2-positive (NUGC4) and negative GC cells (SGC7901) visualized by fluorescence microscopy. The cytotoxicity of the HER-PEG-PCL NPs encapsulating AMO-21 was evaluated by MTT and apoptosis. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to examine miR-21 and phosphatase and tensin homolog (PTEN) and Sprouty2 expression in GC cell lines. The antitumor effects of AMO-21/5-Fu NPs were compared with other groups in xenograft gastric cancer mice. Results: The antibody conjugates significantly enhanced the cellular uptake of NPs. The AMO-21/5-Fu NPs effectively suppressed the target miRNA expression in GC cells, which further up-regulated PTEN and Sprouty2. As a result, the sensitivity of HER2-expressing gastric cancer to trastuzumab and 5-Fu were enhanced both in vitro and in vivo. The approach enhanced the targeting by trastuzumab as well as antibody-dependent cellular cytotoxicity (ADCC) of immune effector cells Conclusions: Taken together, the results provide insight into the biological and clinical potential of targeted AMO-21 and 5-Fu co-delivery using modified trastuzumab for GC treatment.