Precise delivery of multi-stimulus-responsive nanocarriers based on interchangeable visual guidance.

Cancer treatment is imminent, and controlled drug carriers are an important development direction for future clinical chemotherapy. Visual guidance is a feasible means to achieve precise treatment, reduce toxicity and increase drug efficacy. However, the existing visual control methods are limited by imaging time-consuming, sensitivity and side effects. In addition, the ability of the carrier to respond to environmental stimuli in vivo is another difficulty that limits its application. Here, we propose a highly stimulus-responsive GC liposome with precise tracing and sensitive feedback capabilities. It combines magnetic resonance imaging and fluorescence imaging, and addresses the need for precise visualization by alternating imaging modalities. More importantly, GC liposomes are a carrier that can accumulate stimuli. In this paper, by tracking the fragmentation process of empty GC and drug-loaded D-GC liposomes, we confirm the synergistic effect between multiple stimuli, which can result in a more efficient drug release performance. Finally, in mice models we examined the GC liposome imaging approach and the D-GC + UV group guided by this visualization exhibited the highest tumor inhibition efficiency (6.85-fold). This study highlights the advantages of alternate visualization-guided and co-stimulation treatment strategies, and provides design ideas and potential materials for efficient and less toxic cancer treatments.

Association between SCN1A polymorphism rs3812718 and valproic acid resistance in epilepsy children: a case-control study and meta-analysis.

Resistance to valproic acid (VPA), a first-line antiepileptic drug (AED), is occurring at an alarming rate, particularly in children. Signal nucleotide polymorphisms are considered crucial in this process. Therefore, we investigated whether the SCN1A polymorphism rs3812718 could be associated with VPA resistance. A total of 231 children with epilepsy who were solely administered VPA were enrolled. DNA was extracted from the peripheral blood samples and was genotyped by the Mass Array method. Furthermore, a meta-analysis was conducted between the drug responsive and resistant patients who were exposed to voltage-gated sodium channels. Results revealed that the TT genotype was associated with a higher risk of developing drug resistance (OR = 2.636, 95% CI 1.08-6.433, P = 0.033). After adjusting for the risk factors, a significant difference was still observed between the responsive and resistant groups (OR = 2.861, 95% CI 1.141-7.174, P = 0.025). Moreover, the recessive model was associated with a decreased drug resistance (OR = 0.402, 95% CI 0.167-0.968, P = 0.042) after correcting the risk factors. Meta-analysis of nine studies revealed similar results. In conclusion, our results proved that the rs3812718 TT genotype was associated with a high risk of developing drug resistance, and the recessive model could decrease the risk of VPA resistance.

[Luminescence Characteristics of Scintillator Y2SiO5∶Ce].

Cerium doped Y2SiO5 (YSO) is an important scintillator material due to its high density, non-hygroscopic, excellent light output and fast decay time nature. in the paper, Y2SiO5∶Ce3+0.2%(YSO∶Ce) was grown with high-temperature solid-phase method. The time-resolved excitation and emission spectra and fluorescent decay curves at low temperature and room temperature (RT) were measured and discussed. There were two types of luminescence, one was the crystal defect emission, the center at 320 nm; the other one was doped Ce3+ ions 5d→4f emission, the center at 440 nm. Only when the excitation energy (Ex) was greater than the band gap width (Eg), the crystal defect emission can be observed corresponding to slow process, and the emission intensity was higher at low temperature. The crystals defect emission was hardly observed in the time-resolved emission spectra when the temperature rose to room temperature because of temperature quenching. Regions from 60~300 nm corresponding to emission due to 5d→4f transitions in the activator Ce3+ ions peaks at 440 nm, a plurality of excitation peaks were observed. Among them, the excitation with energy less than 6.1 eV(Ex

[Nonlalocalized interference in multiple-beam interferometer].

The authors studied the nonlocalized interference in multiple-beam interferometer. The light intensity distribution function was obtained. The result shows that the function in the circle center has the same form with localized interference. Numerical simulation method was used to analyse the light intensity distribution function. As the reflection coefficient increases, the stripe becomes sharp, Resolution ratio was improved, while the noise occured around the lower interference index. The noise becomes obviously as the reflection coefficient increases. While changing the receiving screen distance, the simulation result shows that linear relationship exists between inteference index and cosine value of interference stripe dip angle. The mirror spacing can be obtained through the straight line slope. With changing the mirror spacing, the numerical simulation result shows that linear relationship exists between interference index and stripe radii square. The straight slope shows a linear relationship with the mirror spacing.

[Advances in the study of organelles targeting nanocarriers].

Modern drug delivery system demands high therapeutic efficacy and low toxicity which depends on efficient intracellular transportation of therapeutics to specific organisms, cells, even targeted organelles such as cytosol, nucleus, mitochondria, lysosome and endoplasmic reticulum. Intracellular barriers which prevent drug molecules accessing to their targets mainly include cell membrane, lysosomal degradation and the endomembrane system. Nanocarriers can preserve the bioactivities of protein, enzyme and DNA, and also they are easy to be modified and functionalized. In this paper, we summarized the intracellular fate of nanocarriers, especially how to bypass intracellular barriers and then target cytosol, nucleus, mitochondria, lysosome and endoplasmic reticulum by pharmaceutical modifications.

Telomerase-targeting antisense oligonucleotides carried by polycation liposomes enhance the growth inhibition effect on tumor cells.

In this study, a novel nonviral gene delivery system, which could enhance the inhibition effect of antisense oligonucleotides (ASODN) against the tumor cells, was developed. The polycation liposomes (PCLs) were prepared using the film hydration method with dioleoylphosphatidylethanolamine (DOPE) and amphipathic compound polyethylenimine-cholesterol (PEI 800-Chol), synthesized by low-molecular-weight polyethylenimine (PEI, MW 800) covalent conjugation with cholesterol. The formation of PEI 800-Chol was confirmed by IR and critical micelle concentration detection. The transfection efficiency of PCLs mediating Green Fluorescence Protein plasmid (pEGFP) in HeLa cells was evaluated and the highest gene expression was obtained by PCLs containing DOPE, which was 1.6-fold of that induced by commercial Lipofectamine 2000, and the gene expression efficiency was influenced in the present of serum. Subsequently, human telomerase reverse transcriptase gene antisense oligonucleotides (hTERT-ASODN) were used as therapeutic gene, and the results showed that PCLs, which demonstrated very low cytotoxicity itself, could significantly enhance the inhibition efficiency of hTERT-ASODN in the growth of tumor cells. These results suggested that the PCLs could be widely applied for ASODN delivery.

In vitro and in vivo tumor suppressive activity induced by human telomerase transcriptase-targeting antisense oligonucleotides mediated by cationic liposomes.

The objective of this study was to investigate the in vitro and in vivo influence of cationic liposomes on the tumor suppressive effect of antisense telomerase oligodeoxynucleotides to human cervical adenocarcinoma cells (HeLa). Antisense oligodeoxynucleotides (ASODN) against the human telomerase transcriptase (hTERT) served as telomerase inhibitors. The cholesterol derivative, 3beta [N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), was synthesized, and cationic liposomes (CL) were prepared using DC-Chol and dioleoylphosphatidylethanolamine (DOPE). The in vitro IC50 of the CL-ASODN complex was 1.88 mumol/l, while the IC50 of the cells treated with free ASODNs or CL alone was 25.24 mumol/l and 55.18 mumol/l, respectively. The CL-ASODN complex inhibited HeLa cell growth for at least 120 h. In vivo, the CL-ASODN complex inhibited the tumor growth rate by 55.11%, which increased to 89.47% when CL-ASODN was combined with 5-fluorouracil treatment. ASODNs alone failed to induce tumor-suppressive activity, suggesting that CL prepared from DOPE and DC-Chol can significantly enhance the growth inhibitory effect of ASODN on tumor cells both in vitro and in vivo.

Liposomes modified with polycation used for gene delivery: preparation, characterization and transfection in vitro.

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer. The development of efficient and safe gene transfer systems could be one of the most important factors for successful gene therapy. In the present study, an amphiphilic compound, polyethylenimine (PEI, MW 800)-cholesterol (PEI 800-Chol), firstly designed to modify the surface of liposomes, was synthesized. Polycation liposomes (PCLs) composed of soybean phospholipids (SPL), cholesterol (Chol) and PEI 800-Chol were prepared using film hydration method. The mean particle size of the PCLs was 133.0 nm and the zeta potential was 50.1+/-2.6 mV. Due to the PEI anchored onto the surface of liposomes, higher buffering capacity of PCLs was observed, indicating the potential for buffering in the acidic pH environment of the endosomes. Compared to Lipofectamine 2000, PCLs have equivalent transfection efficiency with significantly low cytotoxicity. Interestingly, the transfection activity of PCLs was not influenced in the presence of serum. Furthermore, we constructed another PCL composed of PEI 800-Chol and DOPE, and transfection efficiency increased notably. In conclusion, the PCLs described in this study have high transfection efficiency with low cytotoxicity, as well as the protection ability from serum, which suggests PCLs would be a potential non-viral gene delivery system.

[Preparation and quality evaluation of quercetin self-emulsifyied drug delivery systems].

OBJECTIVE: To prepare the quercetin self-emulsified formulation and evaluate its quality. METHOD: The quercetin self-emulsified formulation was optimized based on the quercetin solubility in different oils, and the self-microemulsified efficiency of various combinations of emulsifier and co-emulsifier evaluated using the pseudo-ternary phase diagram. The microemulsion of morphology, size and zeta potential were examined. The quercetin of solubility in self-emulsified system was tested and the formulation stability was investigated by accelerated experiment. RESULT: The blank self-emulsified system was composed of ethyl oleate/Cremophor EUL/butanol with weight ratio of 10: 54: 36. After being dilutied with water, the morphology of microemulsion was homogeneous small spherical drops observed under the electro-microscopy. The particle size and the zeta potential were 16.3 +/- 4.6 nm and 2.1 +/- 0.8 mV, respectively. The solubility of quercetin in self-emulsifing system was (62.42 +/- 0.11) mg x mL(-1), increased 2 229 folds compared with that of in water. The quality of quercetin self-emulsified formulation was stable during the 3 months storage at 40 degrees C. CONCLUSION: The solubility of quercetin is significantly increased in self-emulsified system and the formulation is stable and easy to prepare.

[Study on alpha-asarone reservoir-type patch].

OBJECTIVE: To prepare the alpha-asarone reservoir patch and investigate its release and transdermal absorption characteristics in vitro. The efficient enhancers were chosen to improve the drug's permeation rate. METHOD: The alpha-asarone reservoir patch was prepared using 1% hydroxypropyl methylcellulose (HPMC) of ethanol solution as medium and ethylene vinyl acetate (EVA) membrane to control the release of drug. The Franz diffusion cells were used and several permeation enhancers were evaluated. High performance liquid chromatorgraphy (HPLC) was used to determine alpha-asarone's content and permeation rate. RESULT: The release mechanisms of alpha K-asarone patch in vitro coincided with zero-order kinetic. 30% ethanol cooperates with 1% Isopropyl Myristate (IPM) have the best effect on permeation of the patch. The permeation rate reaches (20.67 +/- 1.33) microg x cm(-2) h(-1). CONCLUSION: Ethanol combined with IPM is good permeation enhancer, which facilitated the permeation of alpha K-asarone to fit the clinical requirements. However, the further studies of the skin's stimulation and bioavailability are needed.

[Preparation and release behavior of chitosan scaffolds encapsulating proteins loaded in PLGA microspheres].

AIM: To prepare cells scaffolds with the characteristics of sustained release of proteins. METHODS: Chitosan scaffolds was prepared by freeze-drying. Porosity and water content of scaffolds were determined. Bovine serum album (BSA) was selected as a model protein. Poly (lactic-co-glycolic acid) (PLGA) microspheres were prepared by double emulsion solvent evaporation and encapsulated into chitosan scaffolds. The morphology of PLGA microspheres and various scaffolds were observed using scanning electron microscope. Release behavior of BSA from various chitosan scaffolds was investigated. RESULTS: The chitosan scaffold represents porous. At the -70 degrees C of quenching temperature, the porosity and water content of chitosan scaffolds were 78.6% +/- 1.5% and 85.1% +/- 6.2%, respectively. PLGA microspheres can be uniformly encapsulated into scaffolds without any morphology change. Significant sustained release of BSA from PLGA microspheres encapsulated into scaffolds was obtained. The cumulative release at 168 h was only 33.5%, while that of BSA from chitosan scaffolds at 24 h was above 90%. The release behavior can be controlled by adjusting the amount of chitosan in scaffolds and the type of PLGA. CONCLUSION: The novel chitosan scaffolds encapsulating PLGA microspheres proved to be a promising cells scaffolds with controlling the release of growth factors in tissue engineering.

Development and validation of a sensitive reversed-phase HPLC method to determine intracellular accumulation of hydroxycamptothecin.

The intracellular accumulation of anti-cancer agents strongly influences the efficiency of chemotherapy for cancer. In the present study, a simple, rapid, sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated to determine hydroxycamptothecin (HCPT) in Eca109 cells. HCPT in cellular lysis solution were measured by RP-HPLC with a C18 column after extraction with ethyl acetate. The mobile phase contained 0.1% triethylamine-phosphoric acid buffer (pH 3.0) and acetonitrile (75:25, v/v). Fluorescence detector with excitation and emission wavelengths of 382 and 528 nm was used for determination of HCPT. The calibration curve was linear from 2 to 100 ng/ml with correlation coefficient of 0.9999, while the limit of quantification is 2 ng/ml. The recovery of assay was between 86.5 and 105.2%. The intra- and inter-day coefficients of variation were less than 10% (R.S.D.). Furthermore, the validated method was used to determine the accumulation of HCPT after incubating the liposomal formulation of HCPT and HCPT for injection with the intact cells. HCPT liposomes showed higher intracellular accumulation of HCPT at different incubation times compared with that of conventional HCPT injection.

[Gene expression of tumor cells both in vitro and in vivo enhanced by integrin-targeting adenovirus vector].

AIM: To construct an efficient recombinant viral vector for gene therapy. METHODS: First-generation adenovirus (Ad) vector was modified with the RGD peptide inserted into the fiber. Both in vitro and in vivo experiments of gene expression in different tumor cells with conventional and recombinant vectors were conducted. RT-PCR was used for detecting the expression of coxackievirus and adenovirus receptor and integrin at the surface of Meth-A cells. RESULTS: Fiber-mutant adenovirus vector showed a notably enhanced gene expression in A2058, B16BL6, OV-HM, and Meth-A tumor cells compared with that of conventional ones. In vivo study carried out using Meth-A tumor-bearing mice also demonstrated that the intra-tumoral injection of recombinant adenovirus induced strong gene expression in these CAR-deficient tumor cells. CONCLUSION: The recombinant vector can be a promising one for effective cancer gene therapy.

[Cellular uptake behavior of antisense oligodeoxynucleotides polymethacrylate submicroparticles].

AIM: To survey the uptake behavior and subcellular distribution of antisense oligodeoxynucleotide polymethacrylate submicroparticles (AS-ODN-SMP) and infer its mechanism in MGC cell lines. METHODS: MGC cells were incubated at certain concentration of AS-ODN-SMP or AS-ODN for 8 h at 4 degrees C or 37 degrees C. Then the fluorescence oligodeoxynucleotide- labeled cells were counted by flow cytometer and the intracellular fluorescence intensity was determined after incubated with chloroquine for 2 h. RESULTS: Cellular uptake of oligodeoxynucleotides was significantly increased following application of AS-ODN-SMP and total intracellular fluorescence intensity was enhanced by 683 folds with the vehicle concentration of 20 microg x mL(-1). AS-ODN-SMP entranced to cells profoundly with temperature-dependent manner. Rare cells took on fluorescence when incubated at 4 degrees C, while 37 degrees C they were significantly increased. But the intracellular fluorescence intensity appeared same level in present or absent of chloroquine. CONCLUSION: With the help of polyacrylate submicroparticles, oligonucleotides efficiently entranced the cells via endocytosis and could successfully escape the degradation in lysosome.

[Preparation of cationic liposomes and its role in enhancing cellular uptake of antisense oligonucleotides].

AIM: To prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently. METHODS: A cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON. RESULTS: The mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected. CONCLUSION: The cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.

[Study on polymethacrylate nanoparticles as delivery system of antisense oligodeoxynucleotides].

AIM: To investigate the possibility of polymethacrylate nanoparticles (NP) for antisense oligodeoxynucleotides delivery system. METHODS: The nanoparticles were prepared by evaporating ethenol solution containing Eudragit RL100 or RS100, and then mixtured with oligonucleotides. The morphology and size were investigated by a transmission electron microscope and Mastersizer particle characterization systems, and the cytotoxicity was evaluated by Trypan Blue staining and hemolysis test. The flow cytometer was used to determine the uptake of fluorescence-labelled oligodeoxynucleotides. RESULTS: The morphology of nanoparticles showed spherical and orderly, the average diameter was about 127 nm, and almost the antisense oligodeoxynucleotides (ODN) were loaded when NP: ODN was 6.6. The uptake of ODN was significantly increased when loaded by nanoparticles, which well depended on the nanoparticles concentration. Meanwhile, slightly cytotoxicity was observed when high dose of nanoparticles was used. CONCLUSION: The polymethacrylate nanoparticles appeared to be a promising vehicle for gene delivery.

[Study on the characteristics of antisense oligodeoxy-neucleotides-liposomes complex and cellular uptake].

AIM: To investigate factors affecting the properties of antisense oligodeoxy nucleotides (ASON)-liposomes complex and their cellular uptake. METHODS: Three types of blank liposomes were prepared by reverse-phase evaporation vesicles, and the complex were obtained through physical absorption. The light microscope was used to observe morphology characteristics of the complex. Drug loading capacity was analyzed by agarose gel electrophoresis. The transfected cell percentage and means fluorescence intensity were determined by flow cytometric analysis using M3 myeloma cell as a model. RESULTS: The neutral liposome showed no aggregation while the cationic liposomes appeared some different extent aggregation in different medium when associated antisense oligodeoxynucleotides. The drug loading capacity depended on the ratio of +/- and the cationic charge density on the lipid membrane. The two kinds of cationic liposomes appeared different principles of loading ASON. As far as cellular uptake, The neutral liposomes showed no improvement of cellular uptake of ASON. However, the cationic liposomes were shown to enhance the cellular uptake of ASON if the appropriate +/- charge ratio was used. The optimal cellular uptake was achieved when +/- charge ratio was at 0.5:1 and 1:1 for SA-I liposome and SA-II liposomes, respectively. CONCLUSION: The cationic liposomes improved the loading capacity and cell uptake of antisense oligodeoxynucleotides, which was determined by +/- charge ratio and charge density.