One of the major challenges in the hepatocellular carcinoma (HCC) treatment is its insensitivity to chemotherapeutic drugs. Here, we report the development of novel lipid-coated cisplatin nanoparticles co-loaded with microRNA-375 (NPC/miR-375) as a potential treatment for chemotherapy insensitive HCC. The NPC/miR-375 was fabricated by mixing two reverse microemulsions containing KCl solution and a highly soluble cis-diaminedihydroplatinum (II) coated with a cationic lipid layer. Subsequently, the miR-375 was incorporated into the lipid-coated cisplatin nanoparticles. The NPC/miR375 nanoparticles were expected to further decrease cell proliferation and to enhance the anti-tumor effect of cisplatin in chemotherapy resistant HCC cells. In vitro analysis of intracellular trafficking revealed that NPC/miR-375 were able to escape from the late endosomes instead of lysosomes thus avoiding degradation of the miR-375 in lysosomes. Importantly, NPC/miR-375 enhanced apoptosis and induced cell cycle arrest in HCC cells in vitro. In the double oncogenes Akt/Ras-induced primary HCC mouse model, multiple doses of NPC/miR-375 significantly inhibited tumor growth and delayed the tumor relapse. Our results indicate that cisplatin nanoparticles co-loaded with miR-375 represent a potential therapeutic agent for chemotherapy-insensitive HCC.

Cabazitaxel-loaded human serum albumin nanoparticles (Cbz-NPs) were synthesized to overcome vehicle-related toxicity of current clinical formulation of the drug based on Tween-80 (Cbz-Tween). A salting-out method was used for NP synthesis that avoids the use of chlorinated organic solvent and is simpler compared to the methods based on emulsion-solvent evaporation. Cbz-NPs had a narrow particle size distribution, suitable drug loading content (4.9%), and superior blood biocompatibility based on in vitro hemolysis assay. Blood circulation, tumor uptake, and antitumor activity of Cbz-NPs were assessed in prostatic cancer xenograft-bearing nude mice. Cbz-NPs exhibited prolonged blood circulation and greater accumulation of Cbz in tumors along with reduced toxicity compared to Cbz-Tween. Moreover, hematoxylin and eosin histopathological staining of organs revealed consistent results. The levels of blood urea nitrogen and serum creatinine in drug-treated mice showed that Cbz-NPs were less toxic than Cbz-Tween to the kidneys. In conclusion, Cbz-NPs provide a promising therapeutic for prostate cancer.

A series of thiophene derivatives (TPs) were synthesized and evaluated for cytotoxicity in HepG2 and SMMC-7721 cell lines by MTT assay. TP 5 was identified as a potential anticancer agent based on its ability to inhibit tumor cell growth. Drawbacks of TPs, including poor solubility and high toxicity, were overcome through delivery using self-assembling HSA nanoparticles (NPs). The optimum conditions for TP 5-NPs synthesis obtained by adjusting the temperature and concentration of TP 5. The NPs had an encapsulation efficiency of 99.59% and drug-loading capacity of 3.70%. TP 5 was slowly released from TP 5-NPs in vitro over 120 h. HepG2 and SMMC-7721 cell lines were employed to study cytotoxicity of TP 5-NPs, which exhibited high potency. ROS levels were elevated and mitochondrial membrane potentials reversed when the two cell lines were treated with TP 5-NPs for 12 h. Cellular uptake of fluorescence-labeled TP 5-NPs in vitro was analyzed by flow cytometry and laser confocal scanning microscopy. Fluorescence intensity increased over time, suggesting that TP 5-NPs were efficiently taken up by tumor cells. In conclusion, TP 5-NPs showed great promise as an anticancer therapeutic agent.

Mechanical stimulation of airway epithelial cells causes apical release of ATP, which increases ciliary beat frequency (CBF) and speeds up mucociliary clearance. The mechanisms responsible for this ATP release are poorly understood. CALHM1, a transmembrane protein with shared structural features to connexins and pannexins, has been implicated in ATP release from taste buds, but it has not been evaluated for a functional role in the airway. In the present study, Calhm1 knockout, Panx1 knockout, and wild-type mouse nasal septal epithelial cells were grown at an air-liquid interface (ALI) and subjected to light mechanical stimulation from an air puff. Apical ATP release was attenuated in Calhm1 knockout cultures following mechanical stimulation at a pressure of 55 mmHg for 50 milliseconds (p < 0.05). Addition of carbenoxolone, a PANX1 channel blocker, completely abolished ATP release in Calhm1 knockout cultures but not in wild type or Panx1 knockout cultures. An increase in CBF was observed in wild-type ALIs following mechanical stimulation, and this increase was significantly lower (p < 0.01) in Calhm1 knockout cultures. These results demonstrate that CALHM1 plays a newly defined role, complementary to PANX1, in ATP release and downstream CBF modulation following a mechanical stimulus in airway epithelial cells.

A series of 1,2-dihydroquinoline derivatives were synthesized and evaluated for cytotoxicity in HeLa, Hep G2 and 6HEK-293T cell lines. EEDQ2 was identified as a promising anti-cancer agent with low IC50 in HeLa (18.55μg/ml) and Hep G2 (14.53μg/ml) cells. For improving the antitumor activity and tumor selectivity of EEDQ2, we prepared transferrin (Tf)-modified liposomes (LPs) to deliver EEDQ2. When HeLa and Hep G2 cells were treated with LP-delivered EEDQ2, the ROS level was significantly enhanced, and mitochondrial membrane potential was reversed. Tf-LPs improved cell uptake of EEDQ2 by about 3.7 times compared with non-targeted LPs. These data suggest that Tf-LPs delivering EEDQ2 is a promising strategy to treat cancer.

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small-cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b-treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.Molecular Therapy-Nucleic Acids (2013) 2, e84; doi:10.1038/mtna.2013.14; published online 16 April 2013.

A novel antisense oligonucleotide (ASO) carrier, polyethylenimine conjugated to linoleic acid (PEI-LA), was synthesized and evaluated for delivery of LOR-2501 to tumor cells. LOR-2501 is an ASO targeting ribonucleotide reductase R1 subunit (RRM1). In this study, PEI-LA was synthesized by reacting PEI (Mw ~ 800) with linoleoyl chloride. Gel retardation assay showed complete complexation between PEI-LA and LOR-2501 at N/P ratio above 8. No significant cytotoxicity was observed with these complexes at the tested dosage levels. Interestingly, at N/P ratio of >6, levels of cellular uptake of PEI-LA/LOR-2501 were double that of PEI/LOR-2501 complexes of the same N/P ratio. PEI-LA/LOR-2501 induced downregulation of 64% and 70% of RRM1 at mRNA and protein levels, respectively. The highest transfection activity was shown by PEI-LA/LOR-2501 complexes at N/P ratio of 10. Finally, using pathway specific inhibitors, clathrin-mediated endocytosis was shown to be the principle mechanism of cellular internalization of these complexes. In conclusion, PEI-LA is a promising agent for the delivery of ASOs and warrants further investigation.

Despite advances in chemo and immunotherapeutic agents for B chronic lymphocytic leukemia (B-CLL), the undesirable adverse side effects due to non-specific cellular uptake remain to be addressed. We identified anti-CD37 monoclonal antibody immunoliposomes (ILs) as vehicles for targeted delivery to B chronic lymphocytic leukemia cells. To achieve maximal benefits for all patients, a new strategy of dual-ligand immunoliposomes (dILs) was developed. A combinatorial antibody microarray technology was adapted to quickly identify optimal antibody combinations for individual patient cells. For proof-of-concept, a B-cell specific antibody, either anti-CD19 or anti-CD20, was combined with anti-CD37 to construct dILs with enhanced selectivity and efficacy. Consistent with data from the antibody microarray, these dILs provided highly specific targeting to both leukemia cell lines and B-CLL patient cells. Compared with the single antibody ILs, the anti-CD19/CD37 dILs clearly demonstrated superior delivery efficiency and apoptosis induction to B-CLL patient cells, whereas the anti-CD20/anti-CD37 dILs were found to be the most efficient for delivery to leukemia cell lines. In addition, it was observed that anti-CD37 ILs without payload drug mediated effective CD37 cross-linking and induced potent apoptosis induction. The anti-CD19/CD20 dILs showed the improved cell apoptosis induction compared to either anti-CD19 ILs or anti-CD20 ILs. Our findings suggest that the dual-ligand ILs may provide a preferred strategy of personalized nanomedicine for the treatment of B-cell malignancies.

Receptor-targeted delivery of imaging and therapeutic agents can lead to enhanced efficacy for both. Multimodality imaging offers unique advantages over traditional single modality imaging. Tumor marker folate receptor (FR)-targeted fluorescent paramagnetic bimodal liposomes were synthesized to co-deliver paramagnetic and fluorescence agents for magnetic resonance (MR) and optical bimodal imaging contrast enhancement.

Development of antibody-drug conjugates (ADCs) is a promising therapeutic strategy for cancer therapy. In this study, folate was conjugated via a polyethyleneglycol (PEG) linker to immunoglobulin G (IgG), which was linked to doxorubicin (DOX), to form a novel ADC folate-PEG-IgG-DOX (FA-PEG-IgG-DOX). The FA-PEG-IgG-DOX showed high targeting efficiency in HeLa and KB cells and significantly improved the uptake and retention of DOX compared with IgG-DOX about 10-fold. Subsequently, FA-PEG-IgG-DOX was shown to have at least 8 times higher antitumor activity than IgG-DOX both in HeLa and KB cells and also induced more apoptosis in those cells than IgG-DOX. Moreover, FA-PEG-IgG-DOX had a 2 times longer circulating time than FA-IgG-DOX, but did not increase the DOX distribution in mouse hearts. Importantly, FA-PEG-IgG-DOX treatment significantly inhibited tumor growth in xenograft mice. Together, our results indicate that FA-PEG-IgG is an effective ADC carrier for delivery of chemotherapeutic agents and that conjugating tumor targeting ligands to antibodies is a promising strategy for producing ADC drugs.

The POP Trial was a phase 1, open-label, rising-dose, randomised study that explored the safety and tolerability of calmangafodipir (superoxide dismutase mimetic) co-treatment with n-acetylcysteine (NAC) for paracetamol overdose.

Proliposomes were used to improve the solubility and oral bioavailability of nifedipine. Nifedipine proliposomes were prepared by methanol injection-spray drying method. The response surface method was used to optimize formulation to enhance the encapsulation efficiency (EE%) of nifedipine. The particle size of nifedipine proliposomes after rehydration was 114 nm. Surface morphology of nifedipine proliposomes was observed by a scanning electron microscope (SEM) and interaction of formulation ingredients was assessed by differential scanning calorimetry (DSC). The solubility of nifedipine is improved 24.8 times after forming proliposomes. In vitro release experiment, nifedipine proliposomes had a control release effect, especially in simulated gastric fluid. In vivo, nifedipine proliposomes significantly improved the bioavailability of nifedipine. The area under the concentration-time curve (AUC0-∞) of nifedipine proliposomes was about 10 times than nifedipine after oral administration. The elimination half-life (T1/2β) of nifedipine was increased from 1.6 h to 6.6 h. In conclusion, proliposomes was a promising system to deliver nifedipine through oral route and warranted further investigation.

Bitter taste receptors (T2Rs) localize to airway motile cilia and initiate innate immune responses in retaliation to bacterial quorum sensing molecules. Activation of cilia T2Rs leads to calcium-driven NO production that increases cilia beating and directly kills bacteria. Several diseases, including chronic rhinosinusitis, COPD, and cystic fibrosis, are characterized by loss of motile cilia and/or squamous metaplasia. To understand T2R function within the altered landscape of airway disease, we studied T2Rs in non-ciliated airway cell lines and primary cells. Several T2Rs localize to the nucleus in de-differentiated cells that typically localize to cilia in differentiated cells. As cilia and nuclear import utilize shared proteins, some T2Rs may target to the nucleus in the absence of motile cilia. T2R agonists selectively elevated nuclear and mitochondrial calcium through a G-protein-coupled receptor phospholipase C mechanism. Additionally, T2R agonists decreased nuclear cAMP, increased nitric oxide, and increased cGMP, consistent with T2R signaling. Furthermore, exposure to T2R agonists led to nuclear calcium-induced mitochondrial depolarization and caspase activation. T2R agonists induced apoptosis in primary bronchial and nasal cells differentiated at air-liquid interface but then induced to a squamous phenotype by apical submersion. Air-exposed well-differentiated cells did not die. This may be a last-resort defense against bacterial infection. However, it may also increase susceptibility of de-differentiated or remodeled epithelia to damage by bacterial metabolites. Moreover, the T2R-activated apoptosis pathway occurs in airway cancer cells. T2Rs may thus contribute to microbiome-tumor cell crosstalk in airway cancers. Targeting T2Rs may be useful for activating cancer cell apoptosis while sparing surrounding tissue.

The objective of this study was to develop long-circulating solid lipid nanoparticles (LSLN) containing a novel curcumin (CU) derivative (CU1), to improve CU1's pharmacokinetic behavior and its anti-cancer effects in MHCC-97H liver cancer cells.

Better management of head and neck squamous cell carcinomas (HNSCCs) requires a clearer understanding of tumor biology and disease risk. Bitter taste receptors (T2Rs) have been studied in several cancers, including thyroid, salivary, and GI, but their role in HNSCC has not been explored. We found that HNSCC patient samples and cell lines expressed functional T2Rs on both the cell and nuclear membranes. Bitter compounds, including bacterial metabolites, activated T2R-mediated nuclear Ca2+ responses leading to mitochondrial depolarization, caspase activation, and ultimately apoptosis. Buffering nuclear Ca2+ elevation blocked caspase activation. Furthermore, increased expression of T2Rs in HNSCCs from The Cancer Genome Atlas is associated with improved overall survival. This work suggests that T2Rs are potential biomarkers to predict outcomes and guide treatment selection, may be leveraged as therapeutic targets to stimulate tumor apoptosis, and may mediate tumor-microbiome crosstalk in HNSCC.

A series of thiophene derivatives were synthesized by functionalization of 2,3-fused thiophene scaffolds. Their cytotoxicity was assessed against HeLa and Hep G2 cells. Compound 480 was identified as a promising candidate because of its low IC50 in HeLa (12.61 μg/mL) and Hep G2 (33.42 μg/mL) cells. The drug was loaded into folic acid (FA)-coated nanoparticles (NPs) to address its poor water solubility and to improve its selectivity for cancer cells. Compound 480 was shown to induce apoptosis by changes in mitochondrial membrane potential (ΔΨm) and the reactive oxygen species level. Furthermore, FA-modified NPs enhanced uptake capacity compared to unmodified controls by flow cytometry. This drug delivered in folate nanocarriers is promising for the treatment of cancers.

Background: Vincristine is a potent therapeutic agent with well-defined activity against hematologic malignancies and solid tumors. It is a cell-cycle specific drug with concentration and exposure duration dependent activity. When used by liposomal delivery, it exhibits enhanced anti-tumor activity. However, vincristine liposome formulation in the clinic is supplied as a 3-vial-kit due to lacking sufficient stability. So it has to be prepared in situ prior to use through a multi-step process. Purpose: The purpose here is to develop a more stable and ready-to-use liposomal formulation for vincritstine in one vial. Patients and methods: A series of preparations were investigated based on sphingomyelin/cholesterol/PEG2000-DSPE lipid composition, with different drug/lipid (D/L) ratios (1/10, 1/5, 1/2), using an active sucrose octasulfate triethylamine salt gradient loading method. In this work, compared to generic vincristine sulfate liposome injection (GVM), the stability both in vivo and in vitro and efficacy in vivo of novel vincristine liposomes were investigated. Results: It was shown that the degradation of vincristine during 2-8°C storage was significantly decreased from 8.2% in 1 month (GVM) to 2.9% in 12 months (D/L ratio 1/5). The half-time for sphingomyelin/cholesterol/PEG2000-DSPE liposomes in vivo could be adjusted from 17.4 h (D/L ratio 1/10) to 22.7 h (D/L ratio 1/2) in rats, while the half-time for GVM was only 11.1 h. The increase in drug retention contributed to the lower in vivo toxicity. The antitumor efficacy was evaluated using a human melanoma tumor model and showed remarkable improvement compared to GVM. Conclusion: The study demonstrates that the new formulation with the drug/lipid ratio of 1/5 owns a higher encapsulation efficiency, better stability, lower toxicity and superior antitumor efficacy, which is screened out for further development.

Ginseng fruit saponins (GFS) extracted from the ginseng fruit are the bioactive triterpenoid saponin components. The aim of the present study was to develop a drug delivery system called proliposome using sodium deoxycholate (NaDC) as a bile salt to improve the oral bioavailability of GFS in rats. The liposomes of GFS were prepared by a conventional ethanol injection and formed the solid proliposomes (P-GFS) using spray drying method on mannitol carriers. The formulation of P-GFS was optimized using the response surface methodology. The physicochemical properties of liposome suspensions including encapsulation efficiency, in vitro drug release studies, particle size of the reconstituted liposome were tested. The solid state characterization studies using the method of Field emission-scanning electron microscope (FE-SEM), Fourier transform infrared (FT-IR) and Differential scanning colorimetric (DSC) were tested to study the molecular state of P-GFS and to indicate the interactions among the formulation ingredients. In vitro studies showed a delayed release of ginsenoside Re (GRe). In vivo studies were carried out in rats. The concentrations of GRe in plasma of rats and its pharmacokinetic behaviors after oral administration of GFS, Zhenyuan tablets (commercial dosage form of GFS) and P-GFS were studied using ultra performance liquid chromatography tandem mass spectrometry. It was founded that the GRe concentration time curves of GFS, Zhenyuan tablets and P-GFS were much more different in rats. Pharmacokinetic behaviors of P-GFS showed a second absorption peak on the concentration time curve. The pharmacokinetic parameters of GFS, Zhenyuan tablets, P-GFS in rats were separately listed as follows: T max 0.25 h, C max 474.96 ± 66.06 ng/ml and AUC0-∞ 733.32 ± 113.82 ng/ml h for GFS; T max 0.31 ± 0.043 h, C max 533.94 ± 106.54 ng/ml and AUC0-∞ 1151.38 ± 198.29 ng/ml h for Zhenyuan tablets; T max 0.5 h, C max 680.62 ± 138.051 ng/ml and AUC0-∞ 2082.49 ± 408.33 ng/ml h for the P-GFS. The bioavailability of P-GFS was nearly 284% and 181% of the GFS and Zhengyuan tablets respectively. In conclusion, the proliposomes significantly enhanced the drug bioavailability, absorption in the gastrointestinal tract and decreased its elimination time of GRe in rats and could be selectively applied for oral delivery of GFS.

Multidrug resistance (MDR) due to overexpression of P-glycoprotein (P-gp) is a major obstacle that hinders the treatment of hepatocellular carcinoma (HCC). It has been shown that miR-375 inhibits P-gp expression via inhibition of astrocyte elevated gene-1 (AEG-1) expression in HCC, and induces apoptosis in HCC cells by targeting AEG-1 and YAP1. In this study, we prepared lipid-coated hollow mesoporous silica nanoparticles (LH) containing doxorubicin hydrochloride (DOX) and miR-375 (LHD/miR-375) to deliver the two agents into MDR HCC cells in vitro and in vivo. We found that LHD/miR-375 overcame drug efflux and delivered miR-375 and DOX into MDR HepG2/ADR cells or HCC tissues. MiR-375 delivered by LHD/miR-375 was taken up through phagocytosis and clathrin- and caveolae-mediated endocytosis. Following release from late endosomes, it repressed the expression of P-gp in HepG2/ADR cells. The synergistic effects of miR-375 and hollow mesoporous silica nanoparticles (HMSN) resulted in a profound increase in the uptake of DOX by the HCC cells and prevented HCC cell growth. Enhanced antitumor effects of LHD/miR-375 were also validated in HCC xenografts and primary tumors; however, no significant toxicity was observed. Mechanistic studies also revealed that miR-375 and DOX exerted a synergistic antitumor effect by promoting apoptosis. Our study illustrates that delivery of miR-375 using HMSN is a feasible approach to circumvent MDR in the management of HCC. It, therefore, merits further development for potential clinical application.

The surface properties of an object, such as texture, glossiness or colour, provide important cues to its identity. However, the actual visual stimulus received by the eye is determined by both the properties of the object and the illumination. We tested whether operational colour constancy for glossy objects (the ability to distinguish changes in spectral reflectance of the object, from changes in the spectrum of the illumination) was affected by rotational motion of either the object or the light source. The different chromatic and geometric properties of the specular and diffuse reflections provide the basis for this discrimination, and we systematically varied specularity to control the available information. Observers viewed animations of isolated objects undergoing either lighting or surface-based spectral transformations accompanied by motion. By varying the axis of rotation, and surface patterning or geometry, we manipulated: (i) motion-related information about the scene, (ii) relative motion between the surface patterning and the specular reflection of the lighting, and (iii) image disruption caused by this motion. Despite large individual differences in performance with static stimuli, motion manipulations neither improved nor degraded performance. As motion significantly disrupts frame-by-frame low-level image statistics, we infer that operational constancy depends on a high-level scene interpretation, which is maintained in all conditions.