ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein system exerts genome editing effect through cleaving DNA double strands using RNA-guided endonuclease. Double-strand breaks were repaired via homology directed repair (HDR) or nonhomologous end joining (NHEJ), accompanied by insertions, deletions or replacements into the genome. As a powerful tool, CRISPR/Cas system has provided tremendous convenience for basic researches and may pave the path to treat genetic diseases and cancers. Genome editing could be achieved only when both CRISPR RNA and Cas protein are delivered into nucleus of target cell. Compared with physical and viral delivery, nonviral delivery of CRISPR/Cas system possesses unique advantages in terms of safety, loading capacity and preparation. Hence, many researchers have devoted themselves to the development of nonviral vectors with high delivery efficiency which is important for the application and translation of the promising technology. Advances on cationic liposomes, lipid like nanoparticles, cationic polymers, AuNPs, vesicles, polypeptides, proteins and so on have been made. We will give a brief introduction to the mechanism of CRISPR/Cas9, problems faced by nonviral delivery of CRISPR/Cas9 system in forms of plasmid, mRNA and protein; examples of non-viral vectors, hoping to give some hints on design of safe and efficient nonviral vectors for genome editing.
ABSTRACT
In this study a reduction-responsive nanoparticles (NPs) modified with hyaluronic acid (HA) was prepared for the co-delivery of doxorubicin (DOX) and siRNA and then evaluated as a lung cancer targeting delivery system in vitro. The amphiphilic polymer of poly-L-lysine-lipoic acid (PLA) based on poly-L-lysine (PLL) with lipoic acid (LA) was synthesized via amidation reaction and characterized by 1H NMR. The DOX loaded PLA NPs were prepared via dialysis method, and siRNA was loaded via electrostatic attraction to prepare the co-delivery NPs system (PLA/DOX-siRNA-NPs). Then PLA/DOX-siRNA-NPs were coated with HA to obtain HA-PLA/DOX-siRNA-NPs. The tumor microenvironment-responsive properties under different pH or reduction condition of HA-PLA/DOX-siRNA-NPs were evaluated by investigating the particle size and zeta potential. Cellular uptake of HA-PLA/DOX-siRNAFAM-NPs by A549 cells and endosomal escape of siRNA were studied using confocal laser scanning microscope (CLSM). 1H NMR spectrum demonstrated that PLA was successfully synthesized with LA grafting rate of 25.1%. The encapsulation efficiency (EE) and drug loading (DL) of HA-PLA/DOX-NPs was (86.93±8.91)% and (4.17±0.68)%, respectively, and siRNA was loaded at an N/P of 6:1 in carrier. HA-PLA/DOX-siRNA-NPs exhibited a suitable size of (167.3±9.9) nm and negative charge of (-15.5±1.4) mV with the optimal ratio of PLA and HA of 1:3. Additionally, the zeta potential of HA-PLA/DOX-siRNA-NPs significantly increased with charge reversal from negative to positive after the treatment with HAase, and the particle size of HA-PLA/DOX-siRNA-NPs changed significantly under the condition of 10 mmol·L-1 glutathione (GSH). The release profiles in vitro demonstrated that HA-PLA/DOX-NPs exhibited a maintained release behavior at pH 7.4 and the adding of GSH (10 mmol·L-1) led to rapid release of DOX from NPs. In vitro cellular uptake and subcellular distribution study demonstrated that themodification of HA enhanced the affinity of NPs to A549 cells and targeting ability, and the cellular uptake of HA-PLA/DOX-siRNAFAM-NPs significantly increased after the treatment with HAase. It was observed that HA-PLA/DOX-siRNAFAM-NPs could escape from endo-lysosomes followed by sharp payloads release to their relative targets. All these results demonstrated that the co-loaded NPs have a high entrapment efficiency of DOX and siRNA. And they also exhibited an active tumor targeting efficiency and tumor microenvironment-responsive properties, which were beneficial to cellular uptake and intracellular release of DOX and siRNA. In conclusion, these reduction-responsive NPs modified with HA have great potential as co-delivery systems for antitumor agents and siRNA.
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AIM To study the anti-inflammatory effects of compound Cervi Cornu Degelatinatum extract on adjuvant arthritic rats.METHODS The rats,except for those assigned into a blank group,were induced to be the adjuvant arthritis models by Freund's complete adjuvant and randomly divided into model group,Prednisone Acetate Tablets group,Tripterygium wilfordii polyglycoside tablets group,and compound Cervi Cornu Degelatinatum extract groups (1 000 mg/kg high-dose group,500 mg/kg medium-dose group,and 250 mg/kg low-dose group) to observe changes in body weight,mental state,and extent of joints injury.Post-treatment levels of TNF-α,IL-6,IL-10 and PGE-2 were determined by ELISA,and the pathological changes of ankle joints were assessed by HE staining.RESULTS Significantly lower body weight and IL-10 level,markedly higher levels of TNF-α,IL-6,PGE-2,and joints injury in the model group than those in the blank group were observed (P ＜0.01).Such indices also revealed the significant superiority in the high-dose,medium-dose groups of compound Cervi Cornu Degelatinatum extract and the positive control group as compared with the model group.CONCLUSION Compound Cervi Cornu Degelatinatum extract highlights the rheumatoid arthritis management through proinflammatory cytokines secretion reduction,the antiinflammatory factors improvement,and the inflammatory reaction and tissue damage alleviation.
ABSTRACT
cRGD-carboxymethyl chitosan-palmitic acid (cRGD-CMCh-PA) was synthesized and a pHsensitive paclitaxel-loaded cRGD-CMCh-PA micelles (PTX-cRGD-CMCh-PA) was prepared with the film dispersion method; related substances were characterized by FT-IR and 1H NMR. PTX-cRGD-CMCh-PA micelles were studied with the particle size distribution, zeta potential, morphology and release behavior in vitro was investigated by the method of equilibrium dialysis. In vitro cytotoxicity of different formulations on A549 cells was tested by MTT assay. The uptake process of micelles was explored using confocal microscopy and a live cell station was used to observe the dynamic phagocytosis. The subcutaneous and orthotropic tumor models were built to study the distribution of DiR-labeled micelles by near-infrared fluorescence (NIR) imaging system. The FT-IR spectra and 1H NMR spectra confirmed the successful conjugation of cRGD-CMCh-PA polymer and the degree of carboxymethyl and the palmitic acid grafted on chitosan were 45.0% and 15.0%. PTX-cRGD-CMCh-PA micelles were prepared with particle size of (162.9±1.5) nm, zeta potential of +26.3 mV and encapsulation efficiency and the drug loading of 99.67% and 28.5%, respectively. The micelles released slowly in pH 7.4 whose release curves were accorded with the Higuchi equation; they had an initial burst effect in second hours and showed a pH sensitive release behavior in pH 5.3. The IC50 of PXT-CMCh-PA and PTX-cRGD-CMCh-PA were 2.077 μg·mL-1 and 0.876 μg·mL-1, respectively. The cells uptake process of micelles in A549 cells revealed that the micelles were mainly co-located with lysosome and PTX-cRGD-CMCh-PA showed much better targeting effect. The NIR fluorescence imaging results showed that the micelles had a good targeting effect on both subcutaneous and orthotropic tumors. In this study, a novel copolymer cRGDCMCh-PA was synthesized with a sustained and pH-dependent drug release activity which would potentially become a new carrier for hydrophobic drugs.
ABSTRACT
Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.
Subject(s)
Humans , Male , Apoptosis , Blotting, Western , Caspase 3 , Metabolism , Cell Line, Tumor , Cell Survival , Diosgenin , Chemistry , Pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Flow Cytometry , Molecular Structure , Prostatic Neoplasms , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Time Factors , bcl-2-Associated X Protein , MetabolismABSTRACT
Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.
ABSTRACT
The study is to observe the effect of racemic TJ0711 on blood pressure and heart rate as well as protection of cardiovascular system of renal hypertensive rats after long-term administration. The renal hypertensive models were established by the two-kidney, one-clip (2K1C) method in Wistar rats. Four weeks later, assigned the rats whose SBP had increased at least 4 kPa randomly into 5 groups: racemic TJ0711 10, 20 and 40 mg x kg(-1) groups, carvedilol control group, model group and sham group (n=10), ig administration once daily. The changes of BP (blood press) and HR (heart rate) before and after administration were measured by tail-cuff method weekly. Plasma samples of all animals were taken in 6-8 weeks, and plasma MDA as well as renin, angiotensin II (Ang II) and endothelin-1 (ET-1) levels were measured. Left ventricle was cut off after 9 weeks, and left ventricular weight index (LVWI) and hydroxyproline were measured. The significant decrease of the BP of TJ0711 40 mg x kg(-1) group was observed after TJ0711 ig administration for 4 weeks, and this effect remained till the end of the study. In 8th week, the systolic blood pressure values were: TJ0711 40 mg x kg(-1) group 18.93 +/- 1.82 kPa (vs 21.30 +/- 2.30 kPa, P < 0.05); 20 mg x kg(-1) group 20.68 +/- 3.29 kPa (vs 22.19 +/- 2.88 kPa). The plasma MDA level of all treated groups was significantly lower than that of model group, so were the plasma renin, Ang II and ET-1 levels (P < 0.05). LVWI and hydroxyproline content of myocardial tissue decreased to some extent, but was not significant as compared with that of model group. The study showed that TJ0711 repeated dosing could reduce BP level beginning from drug administration; besides block adrenal alpha and beta receptors to play an antihypertensive role. The sustained antihypertensive effect also related to reduce plasma vasoconstrictor substances and oxidation product MDA. These effects benefited cardiovascular protection.
Subject(s)
Animals , Female , Male , Rats , Angiotensin II , Blood , Antihypertensive Agents , Pharmacology , Blood Pressure , Endothelin-1 , Blood , Heart Rate , Heart Ventricles , Metabolism , Pathology , Hydroxyproline , Metabolism , Hypertension, Renal , Blood , Longitudinal Studies , Malondialdehyde , Blood , Organ Size , Phenoxypropanolamines , Pharmacology , Random Allocation , Rats, Wistar , Renin , BloodABSTRACT
<p><b>AIM</b>To perpare and identify irisquinone hydroxypropyl-beta-cyclodextrin inclusion complex (irisquinone-HP-beta-CD), as well as to study the inclusion mechanism and molecule stoichiometry between irisquinone and hydroxypropyl-beta-cyclodextrin.</p><p><b>METHODS</b>Irisquinone-HP-beta-CD was prepared by freeze-drying technique. The ratio of host and guest was also studied in inclusion process by mol gradient and continuing variational methods. At the same time, the inclusion complex was identified by X-ray diffraction (XRD) and differential scanning calorimetry (DSC).</p><p><b>RESULTS</b>It was demonstrated that the solubility of irisquinone was enhanced markedly by inclusion with HP-beta-CD when stoichiometry was 2:1 of host and guest at 25 degrees C, 35 degrees C and 45 degrees C.</p><p><b>CONCLUSION</b>The solubility and stability of irisquinone could be increased by preparing the inclusion complex with hydroxypropyl-beta-cyclodextrin.</p>
Subject(s)
2-Hydroxypropyl-beta-cyclodextrin , Benzoquinones , Chemistry , Calorimetry, Differential Scanning , Drug Compounding , Methods , Drug Stability , Freeze Drying , Solubility , X-Ray Diffraction , beta-Cyclodextrins , ChemistryABSTRACT
<p><b>AIM</b>To investigate the permeation mechanism of paclitaxel by enhancers and preparation factors.</p><p><b>METHODS</b>The fluidity of mucous membrane and membrane protein conformation changes were determined by using electron spin resonance (ESR) when mucous membrane was treated by several enhancers. At the same time, the factors of penetration of lower dissolution drug across the intestinal mucous membrane were studied in three formulas inclusion complex, microemulsion and injection.</p><p><b>RESULTS</b>Polyethylene glycol (PEG) 1500, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and phospholipid as enhancers could reinforce the permeation of paclitaxle because of loosening of protein conformation in intestinal mucous membrane. Paclitaxel-HP-beta-CD inclusion complex and paclitaxel microemulsion as vehicle could significantly increased permeation kinetic rate of paclitaxel with fluid diffuse method.</p><p><b>CONCLUSION</b>Characteristics of enhancing intestinal absorption of poor dissolution drug had been provided with enhancer the change of membrane fluid.</p>
Subject(s)
Animals , Rats , 2-Hydroxypropyl-beta-cyclodextrin , Antineoplastic Agents, Phytogenic , Pharmacokinetics , Drug Synergism , Electron Spin Resonance Spectroscopy , Emulsions , Injections , Intestinal Absorption , Intestinal Mucosa , Membrane Fluidity , Paclitaxel , Pharmacokinetics , Pharmaceutical Vehicles , Pharmacology , Phospholipids , Pharmacology , Polyethylene Glycols , Pharmacology , Rats, Sprague-Dawley , beta-Cyclodextrins , PharmacologyABSTRACT
<p><b>AIM</b>To investigate the absorption and distribution of recombinant hirudin-2 (rHV2) in the GI tract in rats after oral administration.</p><p><b>METHODS</b>Using HPLC, fluorescence spectrophotometry and confocal laser scanning microscopy to measure the amount of intact rHV2 absorbed into gastrointestinal mucosa and blood.</p><p><b>RESULTS</b>HPLC spectrum showed that there were intact rHV2 molecules in plasma after 1 h of oral administration. After oral administration for 3 h, 1.2%-26.8% of fluorescence was found in the GI tract in rats. Chromatographic analysis showed that 2.27%-38.75% of fluorescence recovered from the GI tract luminal contents and mucosa was eluted at the void volume of a Sephadex G-25 column. Microscopic examination showed that FITC-rHV2 was taken up throughout the whole small intestine but the ileum appeared to be a preferred site for FITC-rHV2 transport in rats.</p><p><b>CONCLUSION</b>rHV2 may partially survive in the GI lumen and subsequently absorbed in active form by gastrointestinal tract.</p>
Subject(s)
Animals , Male , Rats , Administration, Oral , Digestive System , Metabolism , Fibrinolytic Agents , Pharmacokinetics , Fluorescein-5-isothiocyanate , Hirudins , Pharmacokinetics , Ileum , Metabolism , Intestinal Absorption , Intestinal Mucosa , Metabolism , Random Allocation , Rats, Sprague-Dawley , Recombinant Proteins , Pharmacokinetics , Tissue DistributionABSTRACT
<p><b>AIM</b>To prepare the target drug delivery systems(TDDS), albendazole polybutycyanocrylate nanoparticles (ABZ-PBCA-NP), its pharmaceutical characters and tissue distributions were simultaneously investigated.</p><p><b>METHODS</b>Albendazole nanoparticles were prepared with the emulsification-polymerization method and the drug-load mechanism of polybutycyanocrylate nanoparticles was studied with the equal-tempaerature adsorption principle. The dialyse dynamic of albendazole from ABZ-PBCA-NP was investigated in four formulations in vitro. The tissue distribution of albendazole in different drug vehicles was studied with isotope labelling experiment.</p><p><b>RESULTS</b>ABZ-PBCA-NP and ABZ-PVP-PBCA-NP fit to the Higuchi and bi-exponent function in vitro respectively. The drug loaded in nanoparticles was abide by the Langmuir adsorption equation. Targeting index of albendazole in liver and spleen in mice are 11.4 and 3.9 after ig 3H-ABZ-PBCA-NP. The bioavailability of albendazole nanoparticle and suspension are 76.0% and 36.9% respectively.</p><p><b>CONCLUSION</b>The absorptive capability of drug was enhance when 4% PVP was added into the nanoparticle, and its release time was lengthen. At the same time, the nanoparticles vehicles increase the albendazole bioavailability.</p>