Preparation of vincristine loaded low density lipoprotein nanoparticles modified by T7 peptide and evaluation of therapeutic effect on glioma / 药学学报

The purpose of this study was to prepare T7 peptide modified vincristine loaded low density lipoprotein (T7-LDL-VCR) nanoparticles to penetrate through blood brain barrier for targeting the brain tumor cells. Firstly, the low density lipoprotein (LDL) nanoparticles were extracted and separated from human serum by density gradient centrifugation method, and then was loaded into the nanoparticle's lipid core by the dry film method, T7 peptide was covalent modified on the surface of the nanoparticles. T7-LDL-VCR was characterized by particle size, entrapment efficiency and peptide attachment efficiency. The fluorescent probe DiR was used to track the brain biodistribution of T7-LDL-VCR in mice bearing intracranial C6 glioma by means of in vivo imaging. The therapeutic effect of nanoparticles was observed with magnetic resonance imaging (MRI). Finally, relative tumor volume and survival curve were determined in mice. The results showed that the mean size of the prepared T7-LDL-VCR nanoparticle was about 30 nm, encapsulation efficiency was 30.1%, and peptide attachment efficiency was 63.88%. As expected, the prepared preparation has good brain targeting and good effect on the treatment of glioma in mice:the relative tumor volumes of T7-VCR-LDL, LDL-VCR and VCR were 30%, 51.50% and 79.25%, respectively; the median survival time (36 days), which was 2, 1.85 and 1.38 fold higher than that of physiological saline, free VCR and LDL-VCR, respectively. This study suggests that dual modified hposomes possessed a better ability penetrating the blood brain barrier to target the brain tumor with significant antitumor activities.

A novel thermosensitive in-situ gel of gabexate mesilate for treatment of traumatic pancreatitis: An experimental study / 华中科技大学学报（医学）（英德文版）

Gabexate mesilate (GM) is a trypsin inhibitor, and mainly used for treatment of various acute pancreatitis, including traumatic pancreatitis (TP), edematous pancreatitis, and acute necrotizing pancreatitis. However, due to the characteristics of pharmacokinetics, the clinical application of GM still needs frequently intravenous administration to keep the blood drug concentration, which is difficult to manage. Specially, when the blood supply of pancreas is directly damaged, intravenous administration is difficult to exert the optimum therapy effect. To address it, a novel thermosensitive in-situ gel of gabexate mesilate (GMTI) was developed, and the optimum formulation of GMTI containing 20.6% (w/w) P-407 and 5.79% (w/w) P188 with different concentrations of GM was used as a gelling solvent. The effective drug concentration on trypsin inhibition was examined after treatment with different concentrations of GMTI in vitro, and GM served as a positive control. The security of GMTI was evaluated by hematoxylin-eosin (HE) staining, and its curative effect on grade II pancreas injury was also evaluated by testing amylase (AMS), C-reactive protein (CRP) and trypsinogen activation peptide (TAP), and pathological analysis of the pancreas. The trypsin activity was slightly inhibited at 1.0 and 5.0 mg/mL in GM group and GMTI group, respectively (P<0.05 vs. P-407), and completely inhibited at 10.0 and 20.0 mg/mL (P<0.01 vs. P-407). After local injection of 10 mg/mL GMTI to rat leg muscular tissue, muscle fiber texture was normal, and there were no obvious red blood cells and infiltration of inflammatory cells. Furthermore, the expression of AMS, CRP and TAP was significantly increased in TP group as compared with control group (P<0.01), and significantly decreased in GM group as compared with TP group (P<0.01), and also slightly inhibited after 1.0 and 5.0 mg/mL GMTI treatment as compared with TP group (P<0.05), and significantly inhibited after 10.0 and 20.0 mg/mL GMTI treatment as compared with TP group (P<0.01). HE staining results demonstrated that pancreas cells were uniformly distributed in control group, and they were loosely arranged, partially dissolved, with deeply stained nuclei in TP group. Expectedly, after gradient GMTI treatment, pancreas cells were gradually restored to tight distribution, with slightly stained nuclei. This preliminary study indicated that GMTI could effectively inhibit pancreatic enzymes, and alleviate the severity of trauma-induced pancreatitis, and had a potential drug developing and clinic application value.

Current status and prospect of translational medicine in nanotechnology / 药学学报

Nowadays, nanotechnologies have shown wide application foreground in the biomedical field of medicine laboratory tests, drug delivery, gene therapy and bioremediation. However, in recent years, nanomaterials have been labeled poisonous, because of the disputes and misunderstandings of mainstream views on their safety. Besides, for the barriers of technical issues in preparation like: (1) low efficacy (poor PK & PD and low drug loading), (2) high cost (irreproducibility and difficulty in scale up), little of that research has been successfully translated into commercial products. Currently, along with the new theory of "physical damage is the origin of nanotoxicity", biodegradability and biocompatibility of nanomaterials are listed as the basic principle of safe application of nanomaterials. Combining scientific design based on molecular level with precision control of process engineering will provide a new strategy to overcome the core technical challenges. New turning point of translational medicine in nanotechnology may emerge.

A review on the influences of size and surface charge of liposome on its targeted drug delivery in vivo / 药学学报

Liposomes can be cleared by the reticuloendothelial system (RES) when it is in the blood circulation in the body. And they can accumulate in the organs rich in RES in the body by passive targeting. Targeting of the liposomes is an important factor for its use as a drug carrier, and particle size as well as surface charge are important for its in vivo targeting. In this paper, studies on the influences of particle size and surface charge of the liposomes on cell binding and phagocytosis mechanism were reviewed. A comprehensive review on passive targeting effect of the particle size and surface charge of liposomes on blood, liver, spleen as well as tumor tissue was made. At last, an outlook for future research directions was made.

Taking the advantages of new drug delivery system to accelerate the development of Chinese pharmaceutic industry / 国际药学研究杂志

New drug research and development has entered a new era of preparation innovation, and new drug delivery system (DDS) has become a new drug development hot spot. Chinese pharmaceutical industry should make new molecular entities (NME) and new formulation products as the two pillars of support to ensure a sustained and healthy development. Based on the current status of the Chinese pharmaceutical industry, NME development is more suitable to serve as long term strategy of the Chinese pharmaceutical industry, while the nnovation in DDS should be adopted as a phasic development strategy. DDS can form the competitive strength, industrial superiority and scale more quickly and will become a catalyst and driving force in a short time for the Chinese pharmaceutical industry. Chinese pharmaceutical enterprises need to attach importance to and grasp the new opportunities brought by innovation preparation.

Pharmacokinetics of epirubicin hydrochloride long-circulating thermosensitive liposomes in rat plasma / 药学学报

To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of epirubicin hydrochloride (EPI) in rat plasma, daunorubicin hydrochloride was used as internal standard. The plasma samples were deproteinated with methanol, and separation was performed on a reversed-phase CAPCELL PAK C18 column (3.0 mm x 50 mm, 3 microm). The mobile phase contained methanol-0.1% formic acid (80:20). Detection was carried out by multiple reaction monitoring on a HP1200-6410 QQQ LC/MS system. Different preparations of EPI solution, EPI-LIP (EPI-liposome) and EPI-LTSL (EPI-thermosensitive liposome) was administered in rats by i.v with the same dosage (12 mg kg(-1)). The pharmacokinetic model and parameters were fitted and calculated by the DAS ver2.0 software. The calibration curve was linear in the range of 0.01-50 microg mL(-1). The limit of quantification was 0.01 microg mL(-1). RSDs of intra- and interbatch precisions were all less than 11.9%. The average extract recovery was 89.3% and 92.1%, respectively. The pharmacokinetics of EPI in rats with all preparations were fitted to three compartments, which all fast distributed and slowly eliminated. The t1/2 alpha, t1/2 beta, t1/2 gamma, AUC(0-infinity), and MRT(0-infinity) of EPI-LTSL group were 7.5, 1.3, 12.6, 12.9, 3.7 times those of EPI solution group; and 1.6, 1.4, 12.3, 2.9, 2.6 times those of EPI-LIP group. Moreover, the CL of the latter two groups was about 13.4 times of the former EPI-LTSL group. EPI-LTSL can significantly improve AUC and prolong the circulation time of EPI in rat plasma.

Optimizing the operating variables that affect the transfection experiment of antisense oligodeoxyribonucleotide by gas-filled microbubbles / 药学学报

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.

Echogenic phospholipids-based gas-filled microbubbles as delivery system of antisense oligodeoxynucleotides / 药学学报

<p><b>AIM</b>To investigate the feasibility of transfer antisense oligodeoxynucleotides (AS-ODNs) by the phospholipids-based gas-filled microbubbles (PGM) under ultrasound activation.</p><p><b>METHODS</b>An antisense oligodeoxynucleotides sequence ZL combined with luciferase reporter plasmid was used. A breast cancer cell line SK-BR-3 was exposed to different conditions to investigate the effects of such factors as ZL concentration, PGM concentration, mechanical index (MI) and ultrasound exposure duration on transfection efficiency and cell viability. The transfection efficiency and cell viability by other lipid vectors such as lipofectamine and liposome were also tested, whose results were comparied with that of PGM. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by PI (propidium iodide) assay.</p><p><b>RESULTS</b>Among the factors tested, ultrasound exposure duration, MI and PGM concentration had obvious impacts on transfection efficiency and cell viability. The results showed that the optimal ultrasound condition was the exposure to ultrasound at MI 1.0 for 30 s with 2% PGM concentration, which gave an overall transfection efficiency of 78% +/- 10%, increased nearly 18 folds over the transfection by PGM (4.0%) or lipofectamine (4.3%) without ultrasound. Under same ultrasound conditions, different vectors showed significant difference in transfection efficiency while there are similar results in cell viability.</p><p><b>CONCLUSION</b>Under proper ultrasound conditions, PGM can markedly enhance AS-ODNs transfection efficiency.</p>

Comparison of sonoporation effect of liposomes and phospholipids-based microbubbles on cultured cell membrane / 药学学报

<p><b>AIM</b>To compare sonoporation effect of two phospholipids-based vectors-liposomes and microbubbles on cultured cell membrane.</p><p><b>METHODS</b>A breast cancer cell line SK-BR-3 was exposed to ultrasound alone, 2% or 5% liposome + ultrasound and 2% or 5% microbubble + ultrasound, separately. Immediately after the experiment and 24 h after ultrasound exposure, atomic-force microscopy (AFM) scanning was used to observe the membrane change of SK-BR-3 cells.</p><p><b>RESULTS</b>After ultrasound exposure, normal SK-BR-3 cells more or less lost their natural shape, showing elliptic outline with obtuse curved boundary. In groups added with phospholipids-based microbubbles, more obtuse curved boundary of cells was observed. The membrane pores of SK-BR-3 cells had apparent changes after ultrasound exposure. With AFM technique, membrane pores under ultrasound alone or ultrasound with liposomes conditions were enlarged, the diameter of some pores exceeding 1 microm. But all the membrane pores in these conditions returned to normal appearance after 24 hours. In ultrasound with 2% microbubble condition, most membrane pores were about 1 - 3 microm in size and returned to normal appearance after 24 h. In ultrasound with 5% microbubble condition, however, pores of most cell membrane porosity was about 2 - 4 pm and did not totally return to normal appearance after 24 h.</p><p><b>CONCLUSION</b>At 2% concentration, phospholipids-based microbubble could enhance ultrasonic sonoporation effect and produce reparable membrane pores on SK-BR-3 cells, which appeared to be a promising vehicle for drug and gene delivery.</p>

Plasmid construction, expression, immunogenicity and protective efficacy of recombinant protein candidate vaccine of respiratory syncytial virus / 生物工程学报

To construct plasmid of recombinant protein candidate vaccine of respiratory syncytial virus, express it in E. coli, and to investigate its immunogenicity and protective efficacy. A CD8+ T cell epitope from respiratory syncytial virus (RSV) M2 protein F/M2:81 - 95 and the G:125-225 (G1) gene fragments from RSV-G protein containing B cell epitopes were amplified by PCR method and then inserted into the prokaryotic expression vector pET-DsbA after bonding to a linker. The fusion protein DsbA-G1-Linker-F/M2:81-95 (D-G1LF/M2) was expressed successfully in E. coli BL21 (DE3). The product was proved to be RSV-specific by Western-blot. After purified by affinity chromatography on Ni+ Sepharose and renatured by gradient dialysis. D-G1LF/M2 was used to immune BALB/c mice. D-G1LF/M2 induced high anti-D-G1LF/M2 IgG, anti-RSV IgG and neutralizing antibody titers in serum and lung of BALB/c mice, and elicied RSV-specific CTL responses. The IgG subclass distribution revealed that IgG1/IgG2a ratio was 2.66. Viral titration indicated that D-G1LF/M2 could protect BALB/c mice against RSV challenge in lung.

Differences in gene expression between Taxus chinensis cells during Taxol-synthesis phase and those during non-Taxol-synthesis phase / 生物工程学报

In plant, evocation of secondary metabolism is associated with complex biochemical and molecular events that are regulated by developmental and environmental factors. In order to get more information about Taxol biosynthesis, comparison of mRNA populations from Taxus chinensis cells during Taxol-synthesis phase and those during non-Taxol-synthesis phase were performed by mRNA differential display. The results suggested that genes specifically expressed in the Taxol-synthesis phase might be involved in Taxol biosynthesis.