Polymer nanoparticles.

Over the past few decades, interest in designing and developing polymeric nanoparticles has undergone considerable explosion. Indeed, these nanoparticulated polymer-based systems provide potential solution to improve therapeutic efficacy and diagnosis sensitivity. In this chapter, general properties, production, and characterization of polymer nanoparticles are introduced. Specifically, the development and application of polyhydroxyalkanoate (PHA)-based nanoparticles are emphasized because of the good biocompatible, biodegradable properties, as well as their mechanical flexibility. These PHAs nanoparticles can serve as targeting drug delivery carriers and protein purification and immobilization matrices. The perspective outlook in the last section highlights the future application of polymer nanoparticles in translational science.

Apogossypolone, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro.

Limited treatment options are available for aggressive prostate cancer. Gossypol has been reported to have a potent anticancer activity in many types of cancer. It can increase the sensitivity of cancer cells to alkylating agents, diminish multidrug resistance and decrease metastasis. Whether or not it can induce autophagy in cancer cells has not yet been determined. Here we investigated the antiproliferative activity of apogossypolone (ApoG2) and (-)-gossypol on the human prostate cancer cell line PC3 and LNCaP in vitro. Exposure of PC-3 and LNCaP cells to ApoG2 resulted in several specific features characteristic of autophagy, including the appearance of membranous vacuoles in the cytoplasm and formation of acidic vesicular organelles. Expression of autophagy-associated LC3-II and beclin-1 increased in both cell lines after treatment. Inhibition of autophagy with 3-methyladenine promoted apoptosis of both cell types. Taken together, these data demonstrated that induction of autophagy could represent a defense mechanism against apoptosis in human prostate cancer cells.

Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol.

We investigated the antiproliferative activity of (-)-gossypol on the human prostate cancer cell line PC3 in vitro and in vivo to elucidate its potential molecular mechanisms. Cell growth and viability were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was detected by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and electron microscopy. Expression of proliferating cell nuclear antigen (PCNA), Bcl-2, CD31, caspase-3 and caspase-8 in tumour tissue was determined by immunohistochemistry. The drug concentration that yielded 50% cell inhibition (IC(50) value) was 4.74 microg mL(-1). In the PC-3 tumour xenograft study, (-)-gossypol (> 5 mg kg(-1)) given once a day for 7 days significantly inhibited tumour growth in a dose-dependent manner. Immunohistochemical analysis revealed that (-)-gossypol enhanced caspase-3 and caspase-8 expression and decreased the expression of PCNA, Bcl-2 and CD31 in tumour tissues. It suggested that cell apoptosis and inhibition of angiogenesis might contribute to the anticancer action of (-)-gossypol.

[Oral immunization of Lugurus lugurus with the plasmid nanoemulsion of zona pellucida 3 to enhance infertility effects].

Immunocontraceptive technology is beginning to attract more attention for the control of wild mouse or other pest animals. Oral vaccination of animals and man, to provide effective mucosal and/or systemic immunity, is a good delivery system, but it remains poor bioavailablity, one of the most promising strategies to improve the oral delivery relies on polymeric nanoparticles carriers to encapsulate vaccine. Nanoemulsion was used as the carriers of oral immunization of Lugurus Lugurus zona pellucida 3, the immune response and antifertility potential of this vaccine is valuated. Nanoemulsion-encapsulated plasmid pcDNA3.0-Aat-COMP-lzp3-C3d3 (pACLC DNA vaccine) was prepared and evaluated the encapsulation efficiency by anion exchange chromatography. The mice were orally delivered with nanoemulsion-encapsulated plasmid, to detect specific IgG in serum and IgA in vaginal fluid by indirect ELISA, and to perform fertility experiment, to do histological analysis of immunized mice ovary. The results showed that the specific IgG and IgA antibody is elicited, the fertility rate is deceased at a certain extent. In conclusion, it is feasible that nanoemulsion is used as the carriers of oral immunization.

Visual recognition and efficient isolation of apoptotic cells with fluorescent-magnetic-biotargeting multifunctional nanospheres.

BACKGROUND: Fluorescent-magnetic-biotargeting multifunctional nanospheres are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. We have been developing such multifunctional nanospheres for biomedical applications. METHODS: We covalently coupled avidin onto the surfaces of fluorescent-magnetic bifunctional nanospheres to construct fluorescent-magnetic-biotargeting trifunctional nanospheres and analyzed the functionality and specificity of these trifunctional nanospheres for their ability to recognize and isolate apoptotic cells labeled with biotinylated annexin V, which recognizes phosphatidylserine exposed on the surfaces of apoptotic cells. RESULTS: The multifunctional nanospheres can be used in combination with propidium iodide staining of nuclear DNA to identify cells at different phases of the apoptotic process. Furthermore, we demonstrate that apoptotic cells induced by exposure to ultraviolet light can be isolated simply with a magnet from living cells at an efficiency of at least 80%; these cells can then be easily visualized with a fluorescence microscope. CONCLUSIONS: Our results show that fluorescent-magnetic-biotargeting trifunctional nanospheres can be a powerful tool for rapidly recognizing, magnetically enriching and sorting, and simultaneously identifying different kinds of cells.

[Preparation of plasmid nanoemulsion and its character determination by anion exchange chromatography method].

Nanoemulsion-encapsulated lzp3 DNA vaccine (pCDNA3-Aat-COMP-lzp3-C3d3, lzp3) was prepared, and its quality was evaluated. The interfacial emulsification method was employed to make the nanoemulsion-encapsulated plasmid, and its sizes and distribution were detected by electron microscope. Anion exchange chromatography was used to separate the nanoemulsion from the plasmid based on the charge characteristics of plasmid. The determination method for encapsulation efficiency of the plasmid nanoemulsion was established by anion exchange chromatography. The results indicated that the average size of the plasmid nanoemulsion is (23 +/- 10) nm, and the encapsulation efficiency is 80.5%. The separation and determination conditions of nanoemulsion-encapsulated plasmid was as follows: eluent buffer was 0.05 mol/L Tris-HCl solution at a flow rate of 0.7 mL/min, while detection wavelength for plasmid was 260 nm, the temperature of column was maintained at 30 degrees C, and injection volume was 2mL. On this condition, nanoemulsion and plasmid can be separated on the column of anion exchange chromatography significantly. This method is simple, sensitive and reproducible with respect to good linearity in the range of 0.05-0.80 mg/mL (r = 0.9983), which can be applied to determine the entrapment efficiency of nanoemulsion-encapsulated plasmid.

[Preparation of gelatin nanogel modified with fluoride anion and its ultrasonic triggered release characteristics].

AIM: To investigate the preparation, shape and ultrasound triggered release characteristics of gelatin nanogel modified with fluoride anion. METHODS: Adriamycin gelatin nanogel modified with fluoride anion (ADM-FMNG) was prepared by co-precipitation with fluoride anion. The content and encapsulation rate of adriamycin were measured by HPLC method. The size and shape of ADM-FMNG were determined by electron microscope. The size and distribution of ADM-FDNG before and after sonication were measured by laser size analysis device. RESULTS: The average diameter of ADM-FMNG was (46 +/- 12) nm. Adriamycin encapsulated rate and loading were 87.2% and 0.091 g x L(-1), respectively. 48.5% of adriamycin was released within 50 h while in vitro at 37 degrees C. Under the action of ultrasound that has the frequency of 20 kHz, 0.4 W x cm(-2) of power density and 7-8 min duration, 51. 5% of adriamycin in ADM-FMNG was released that was significantly higher than control group, the size of ADM-FMNG was changed from (46 +/- 12) nm to (1,212 +/- 35) nm and restored after ultrasound stopped for 3-4 min. CONCLUSION: ADM-FDNG system has the sensitive ultrasound triggered release characteristics.

Biofunctionalization of fluorescent-magnetic-bifunctional nanospheres and their applications.

Hydrazide-containing bifunctional nanospheres were covalently coupled on the surface with IgG, avidin, and biotin, to generate novel fluorescent-magnetic-biotargeting trifunctional nanospheres, which can be used in a number of biomedical applications, including visual sorting and manipulation of apoptotic cells as demonstrated here.

[Preparation and characterization of nanoemulsion].

AIM: To prepare nanoemulsion-encapsulated BSA-FITC (NEBSA-FITC), study its characteristics, and measure its uptake by dendritic cells (DCs) and peritoneal macrophages. METHODS: NEBSA-FITC was prepared by a method of interfacial polymerization.The encapsulation rate, drug-carrying capacity and stability of the nanoemulsion were determined by Sephadex-G100 chromatography. The shape and size of NEBSA-FITC were observed under electron microscope. The uptake of NEBSA-FITC by DCs and macrophage cells was detected by FACS and laser confocal microscopy. RESULTS: The mean size of NEBSA-FITC was (25+/-10) nm. The encapsulation rate was 91%, the drug-carrying capacity was 0.091 g/L and NEBSA-FITC had a good stability. The FACS analysis showed that DCs and macrophage cells could take in more NEBSA-FITC than free BSA. The observation under laser confocal microscope found that NEBSA-FITC was located in the cytoplasm of DCs. CONCLUSION: Nanoemulsion can be efficiently taken by DCs and macrophage cells, and therefore may be promising efficient carrier of APCs-targeted antitumor vaccine.

[Influence of nanoparticle-encapsulated SEA on T-cell subgroups and its antitumor effect on hepatoma].

BACKGROUND & OBJECTIVE: Staphylococcal enterotoxin A (SEA) is one of the widely researched superantigens, which is prospective in antitumor therapy. However, its application is limited due to the toxicity. This study was conducted to prepare the nanoparticle-encapsulated SEA (NSEA) and to observe its influences on the T-cell subgroups and the antitumor effects in vivo. METHODS: NSEA was prepared by the interfacial polymerization method.BALB/c mice were divided into 3 groups (each group had 12 mice). After injection of 0.1 ml normal saline (NS I group), 0.1 ml 2 mg/L free SEA (SEA I group) and 0.1 ml 2 mg/L NSEA(NSEA I group), the changes of T-cell subgroups (CD4(+) and CD8(+)) were observed. The mice model bearing hepatocellular carcinoma H22 were injected with 0.1 ml NS(NS II group), 0.1 ml 2 mg/L free SEA(SEA II group), 0.1 ml 2 mg/L NSEA (NESA II group), then the tumor volume and the survival time were recorded. RESULTS: SEA and NSEA significantly improved the absolute number of CD4(+) and CD8(+) T cells (P< 0.01); while the proportion of CD4(+) to CD8(+) did not change (P >0.05). The numbers of CD4(+) and CD8(+) T cells in NSEA I group reached the peaks [(8.26+/-1.46) x 10(9)/L and (5.53+/-0.91) x 10(9)/L] at 72 hours. The absolute number of CD4(+) T cells in SEA I group reached the peak of (8.61+/-1.59) x 10(9)/L at 48 hours,and the absolute number of CD8(+) T cells reached the peak of (6.05+/-1.31) x 10(9)/L at 72 hours; both of them descended to normal level at 96 hours. The inhibition rates of SEA II group and NSEA II group were 58.9% and 50% and the percentages of life span increase were 167% and 169%, respectively. CONCLUSION: NSEA and SEA could induce the activation and proliferation of T cells in vivo but could not influence the proportion of CD4(+) and CD8(+) cells in the mice. The effects of NSEA were weaker but longer than that of SEA. This study demonstrated that NSEA has the sustained release effects and prolongs the effective time.

[The determination of konjac glucomannan in konjac refined powder and monosaccharide compositions by HPLC].

OBJECTIVE: To establish a quantitative method for the content determination and monosaccharide composition analysis of Konjac glucomannan (KGM) in Konjac refined powder by pre-column derivatization high performance liquid chromatographic method (HPLC). METHOD: The two derivatives combined reducing monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) were separated by reverse-phase HPLC using a developed fragment gradient elution process, and monitored by ultraviolet detector at 250 nm. The broad reagent peak of PMP was separated very well from all the PMP-sugars, and good separation was achieved for derivatives of mannose and glucose. The quantitative methods of two reducing monosaccharides were studied by the method combined internal and external standard; while the KGM content in Konjac refined powder was determined. RESULT: Linearity of glucose was good (r = 0.9990) in range of 1.002-8.016 nmol; while mannose (r = 0.9994) in range of 1.001-8.008 nmol. The average recovery of this method was 98.1%, RSD of repeatability was 1.72%. KGM content in Konjac refined powder was 79.5%, ratio of glucose to mannose in KGM was 1:1.51. CONCLUSION: This method is a sample, convenient and rapid method that can determine KGM content and analyze monosaccharide compositions in KGM, which will be helpful to quality assessment of Konjac refined powder.

[Studies on ultrasonic wave extracting method determining konjac glucomannanin konjac refined powder].

OBJECTIVE: To investigate the effect of ultrasonic wave on extracting Konjac Glucomannan(KGM) in Konjac refined powder. METHOD: Free reduced sugar in Konjac refined powder was removed and Konjac refined powder in the aqueous solution was processed by ultrasonic wave and KGM content was measured by spectrophotometry. RESULT: KGM content in the Konjac refined powder aqueous solution by ultrasonic process at fixed 40 kHz, 100 W, 30-45 min was equal to that by routine method at 4 h; whereas, by 1 h of ultrasonic process, KGM content was significantly enhanced than that by 4 h of routine method(P < 0.01), enhancement rate was 6.5%. Linearity of standard glucose was good (r = 0.9996) in range of 0.2-1.6 mg. The average recovery was 97.8%, RSD of repeatability was 1.27%. CONCLUSION: Ultrasonic extraction in aqueous solution is a reliable and rapid method that can enhance extraction efficiency of KGM in Konjac refined powder.