Construction and in vitro evaluation of pH-responsive and tumor-targeted PTEN/PLGA-(HE)10-MAP nanoparticles / 中国药科大学学报

@#To construct PTEN/PLGA-(HE)10-MAP nanoparticles, which encapsulated PTEN plasmid DNA and combined with the pH-responsive cell-penetrating peptides (CPPs), and to investigate their effects of gene delivery and anti-tumor targets in vitro. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with PTEN plasmid DNA were prepared by double emulsification-solvent evaporation method. PTEN/PLGA-(HE)10-MAP nanoparticles were prepared by coupling the histidine-glutamic acid-model amphipathic peptide nanocomplex [(HE)10-MAP] to the surface through amide condensation reaction. Particle size, Zeta potential, encapsulation rate and drug loading were tested to characterize the nanoparticles. By analyzing the cytotoxicity, cellular uptake, targeted transfection of eukaryotic expression plasmids and anti-tumor cell proliferation, the feasibility as a targeted gene delivery system were evaluated. The particle size of PTEN/PLGA-(HE)10-MAP nanoparticles was (266.5 ± 2.86) nm, with the encapsulation efficiency (80.6 ± 6.11)%. Zeta potentials were -(6.7 ± 0.26) mV, +(0.7 ± 0.22) mV and +(37.5 ± 0.85) mV at pH 7.4, 7.0 and 6.5, respectively. In the cytotoxicity test, the cell survival rates of tumor and normal cells were above 80%.Non-loading PLGA-(HE)10-MAP nanoparticles showed no obvious cytotoxicity. The results of cellular uptake experiments showed that PTEN/PLGA-(HE)10-MAP nanoparticles were more readily taken up by cells.The results of CCK-8 showed that the nanoparticles could pH-specifically inhibit proliferation of tumor cell in vitro.And PTEN/PLGA-(HE)10-MAP nanoparticles may be applied in tumor gene therapy.

Advances in the research and application of cell penetrating peptides / 生物工程学报

Cell-penetrating peptides (CPPs) are short peptides that can penetrate the cell membrane or tissue barrier. CPPs can deliver a variety of biomacromolecules, such as proteins, RNA and DNA, into cells to produce intracellular functional effects. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms for CPPs-mediated cargo delivery. Compared with other non-natural chemical molecules-based delivery reagents, the CPPs have better biocompatibility, lower cytotoxicity, are easily degraded after cargo delivery, and can be fused and recombined expressed with bioactive proteins. Because of these advantages, the CPPs have become an important potential tool for delivery of developing drugs which targets intracellular factors. As a novel delivery tool, the CPPs also show promising application prospects in biomedical researches. This review summarized recent advances regarding the classification characteristics, the cellular uptake mechanisms and therapeutic application potentials of CPPs.

Construction of artesunate nanoparticles modified by hyaluronic acid and cell-penetrating peptides and its inhibitory effect on cancer cells in vitro / 中国中药杂志

Hyaluronic acid (HA) and cell-penetrating peptide (CPP) R6H4-SA modified artesunate nanostructured lipid carrier (HA-R6H4-NLC/ART) for anti-tumor therapy was prepared. The physicochemical properties and in vitro drug release of HA-R6H4-NLC/ART were evaluated, and the uptake and cytotoxicity of liver cancer HepG2 cells were studied. The results showed that HA-R6H4-NLC/ART was spherical like in appearance, and the average particle size was about 160 nm. In vitro release experiments showed that the drug delivery system had sustained release characteristics. Cell results showed that, in slightly acidic environment, pH sensitive CPP R6H4-SA mediated cellular uptake of nanoparticles was significantly higher than that of non-sensitive peptide R8-SA. Meanwhile, HA-R6H4-NLC/ART had a targeting effect on HepG2 cells, and the HA receptor saturation experiment showed that the endocytosis of HA-R6H4-NLC/ART was mediated by the HA receptor on the cell surface. As compared with the unmodified or R6H4-SA single modified group, HA and R6H4-SA co-modified HA-R6H4-NLC/ART significantly improved the cell uptake and had a stronger anti-tumor effect under the conditions of the slightly acid environment and hyaluronidase degradation. The above results showed that hyaluronic acid and CPP R6H4-SA co-modified artesunate nanostructured lipid carrier, which can effectively identify and penetrate the tumor cell membrane into the cell, is a potentially efficient targeting delivery system for anti-tumor drugs.

Study on cell-penetrating peptides carrying foreign proteins into eyeball / 眼科新进展

Objective To investigate whether poly arginine as the carrier can carry foreign proteins to penetrate the cell membrane and even penetrate the eyeball barrier.Methods Poly-Args (R9) was used as a CPP in this study.R9-green fluorescent protein (GFP) and GFP were constructed.In vitro,human lens epithelial cells were treated with these two proteins.Then,MTT assay were used to detect whether the protein could affect the proliferation of the cells.Flow cytometry and laser confocal microscopy were used to detect the penetrability of CPPs on the cells.In vivo,eyes of mice were treated with protein in eye drops way for 7 days.Then total protein were extracted,ELISA were used to detect the penetrability of CPPs.Results The results of MTT,flow cytometry and laser confocal microscopy showed that CPPs could carry protein into cells in a dose dependent manner without affecting cell proliferation.In vivo,slit lamp showed that the mice eyeballs had no any abnormal after treated by GFP,R9-GFP,and ELISA results also showed that R9 could effectively get foreign protein into the eyeball.Conelusion R9 can carry foreign protein into the cell membrane and eyeball barrier.This study provides the basis for the eye medication and dosing mode improvement.

Research progress of cell-penetrating peptides in tumor-targeted therapy / 第二军医大学学报

Advances in biotechnology give much importance to the therapeutic biomacromolecules in the therapy of diseases, such as proteins, oligonucleotides, and peptides. But their effects are limited in practical application because of cell membrane barrier. Cell-penetrating peptides (CPPs) are promising oligopeptides with a remarkable capacity for membrane translocation, which can carry various macromolecules into cells. In this paper, we reviewed the classification and transmembrane mechanism of CPPs as nanoparticles, with particular focus on their recent progress in tumor-targeted therapy.

CADY-based cell penetrating peptides as siRNA delivery vector:research progress / 国际药学研究杂志

How to deliver small interfering RNA(siRNA)molecules into target cells efficiently and safely to silence target genes is a bottleneck in the clinical application of RNA interference. In recent years,siRNA delivery technology based on cell pene-trating peptides has developed rapidly. Among them,the secondary amphipathic peptide,called CADY,has little cytotoxicity and no immunogenicity,forms stable siRNA complexes and improves their delivery into a wide variety of cell lines. Therefore,it has become a very promising delivery carrier for siRNA applications. This review summarizes the design idea,structural characteristic,spatial con-figuration,internalization mechanism,modification and application of CADY.

Screening and identification of cell-penetrating peptides by display technology / 国际药学研究杂志

Screening and identification of cell-penetrating peptides, CPP which have different functions by display technology are hot topics in recent years. Currently the most widely used display technology is phage display and mRNA display technology. This paper describes the principle and application of display technology such as phage display and mRNA display and reviews in detail the advance in study on the screening and identification of CPP by display technology in recent years.

Tat-LK15-mediated delivery of nNOS siRNA in rats with chronic inflammatory pain / 实用医学杂志

Objective To investigate the potential application of a non-viral gene carrier , TAT-LK15 , for delivering nNOSsiRNAin vivo and to study whether TAT-LK15/siRNA can be a new treatment method for chronic inflammatory pain. Method TAT-LK15 was complexed with nNOSsiRNA or scrambled control siRNA. The expression of nNOS was determined in SCDH of chronic inflammatory pain rats by western-blot assay. Pain control efficacy was evaluated by mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD) assays. Results nNOS protein expression was efficiently inhibited by intrathecal injection of TAT-LK15/siRNA complexes , with the reduction of nNOS protein by 52%. Moreover , injection of TAT-LK15/siRNA com-plexes significantly could decrease MWT , but increase TWD in rats with chronic inflammatory pain. Conclusions TAT-LK15 can efficiently deliver nNOSsiRNAin vivo and nNOSsiRNA can relieve chronic inflammatory pain in rats.

Screening and identification of cell-penetrating peptides by display technology / 国际药学研究杂志

Screening and identification of cell-penetrating peptides(CPP)which have different functions by display technolo?gy are hot topics in recent years. Currently the most widely used display technology is phage display and mRNA display technology. This paper describes the principle and application of display technology such as phage display and mRNA display and reviews in detail the advance in study on the screening and identification of CPP by display technology in recent years.

VP22 enhances the expression and distribution of the tumor suppressor protein PTEN in BT549 / 中国药学杂志

OBJECTIVE: To prepare a eukaryotic expression vector for cell penetrating peptide fusion protein PTEN-VP22, and to evaluate whether the cell penetrating peptide VP22 increase the expression and distribution of the tumor suppressor protein PTEN in BT549. METHODS: The eukaryotic expression vectors pcDNA3-PTEN and pcDNA3-PTEN-VP22 were constructed and then transferred to BT549. The expression and distribution of PTEN protein and PTEN-VP22 fusion protein were identified by Western blot and immunofluorescence. RESULTS: PTEN protein and PTEN-VP22 fusion protein were successfully expressed in BT549. The expression and distribution of PTEN-VP22 were significantly increased compared with those of PTEN. The distribution of PTEN-VP22 was in a typical VP22 pattern and mainly distributed in nucleus. CONCLUSION: The cell penetrating peptide VP22 enhances the expression and distribution of the tumor suppressor protein PTEN in BT549.

Advances in non-viral vector research of siRNA / 中国药理学通报

siRNA drug research and development is becoming one of the main objectives in the future. However, due to the in-stability of siRNA and the complex environment in vivo, the safe and effective delivery of siRNA is limited in vivo. Thus, special vectors are used to assist siRNA to express biological effects. This paper reviews the advances in non-viral vector for delivery of siRNA in vivo.

Brain targeting effect of PEGylated liposomes modified with RDP peptide / 中国药理学通报

Aim Targeted drug delivery in the brain is the necessary way for the treatment of brain diseases. In our study, a new peptide derived from the rabies vi-rus glycoprotein ( RVG-derived peptide, RDP ) was used as a targeted carrier to modify the curcumin stealth liposomes, and their characteristics and brain targeting effect were studied. Methods The curcumin liposomes were prepared by thin film dispersion. The release test in vitro was conducted to investigate their drug release. Curcumin distribution in several organs of mice was investigated by caudal vein injection of curcumin suspension liquid ( CUR ) , curcumin lipo-somes ( CUR-L) , RDP modified curcumin stealth lipo-somes ( RDP-CUR-L) via HPLC assay at different time points. Results The prepared stealth nano liposomes had a size of around 100 nm, and also had a good dis-persion and reproducibility. The entrapment efficiency was larger than 85%. After caudal vein injection of CUR, CUR-L and RDP-CUR-L in mice respectively, no curcumin was detected in brain of CUR group, and only a little was detected in CUR-L group. Neverthe-less, high concentration of curcumin was detected in RDP-CUR-L group. Conclusion RDP can deliver li-posome into the brain, which may provide a new meth-od for the treatment of brain diseases.

Delivery of therapeutic siRNA by Tat-LK15 targeting nNOS fusion gene in RGC-5 cells / 中国药理学通报

Aim To investigate the potential applica-tion of a non-viral gene carrier Tat-LK15 for delivering siRNA targeting nNOS in vitro, which provides evi-dence of Tat-LK15 mediating siRNA targeting nNOS in vivo for treatment of neuropathic pain. Methods 1. Tat-LK15 was mixed with siRNA, then the mixture was analyzed the best ratio by Gel retardation. The trans-fection efficiency of FAM-siRNA mediated by Tat-LK15 on RGC-5 cells was examined by Flow Cytome-try. The apoptosis ratio of RGC-5 was identified by Flow Cytometry 24 h after treated with the different do-ses of Tat-LK15 (1, 2. 5, 5, 10 and 20 μg). 2. The model of RGC-5 cell overexpression of nNOS protein was prepared. 3. RGC-5 cells were randomly divided into 5 groups:control group,model group, Tat-S group ( Tat-LK15 mediate nNOS/siRNA transfection model cell) , Lipo-S group ( LipofectamineTM RNAiMAX me-diate nNOS/siRNA transfection model cell) and Tat-N group ( Tat-LK15 mediate NCsiRNA transfection model cell) . Real-time Quantitative polymerase chain reac-tion( Q-PCR) and Western blot were used to evaluate nNOS expression level assay. Results It indicated that the Tat-LK15/siRNA complex completely formed at the weight ratio of 2∶ 1 (μg/μg) , and the transfec-tion efficiency was (84. 4 ± 3. 9)%. It caused cotytox-icity when Tat-LK15 dose was 20 μg ( 6. 1 μmol · L-1 ) , and the apoptosis rate more than control group [(10. 3 ± 1. 1)% vs (7. 4 ± 0. 9)%, P<0. 05]. The nNOS protein level of RGC-5 cells was significantly el-evated after modeling. Compared with that of model group, Tat-LK15/siRNA efficiently inhibited the ex-pression of nNOS at transcriptional level or protein leve1 of Tat-S group ( P <0. 05 ) , and there was no significant difference of the efficiency inhibited between Tat-S group and Lipo-S group. Conclusions Tat-LK15’ advantage is with high efficiency, low cytotox-icity. The Tat-LK15 can deliver siRNA targeting nNOS in vitro efficiently and safely.

Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity / 吉林大学学报(医学版)

Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Preparation and evaluation of flutide-loaded PLGA nanoparticles modified with cell-penetrating peptides / 军事医学

Objective To prepare and evaluate flutide-loaded PLGA nanoparticles modified with cell-penetrating peptide-TAT.Methods The sequence of TAT was synthesized with florenl methyoxycarbonyl amino acids .The purity and molecular weight of TAT were determined using RP-HPLC and MALDI-TOF-MS.PLGA was modified with the TAT peptide and then prepared into flutide-loaded nanoparticles ( TAT-PLGA NPs) with the double emulsion method .The physical and chemical properties were evaluated , including size distribution, Zeta potential, SEM of nanoparticles , loading ratio of drug content and release profiles of TAT-PLGA NPs in vitro.The cytotoxicity of TAT-PLGA NPs was evaluated by CCK-8 methods.Results The purity of synthesized TAT was 95.6%, and molecular weight was 1495.8.The mean diameter,Zeta potential, drug loading ratio of TAT-PLGA nanoparticals were (159.5 ±2.1) nm, -(1.87 ±0.6) mV, and (5.75 ±0.17)μg/mg, respectively.The nanoparticles observed by transmission electron microscopy (TEM) had a spherical shape and uniform size without aggregation .In vitro release test showed sustained release of flutide from TAT-PLGA nanoparticles .Cell proliferation assay revealed that the TAT-PLGA nanoparticles did not damage the cell growth in vitro and showed good compatibility.Conclusion TAT-PLGA nanoparticles are prepared successfully by double emulsion method,and have sustained-release effect and good compatibility in vitro.They have potential application prospect in prevention and treatment of influenza .

Construction of RGD-TAT modified liposomes and evaluation of its targeting on glioma / 中国生化药物杂志

Objective To construct RGD-TAT modified liposomes(RGD-TAT-LPs)and evaluate its glioma targeting efficiency.Methods RGD-TAT-LPs was constructed by film-ultrasonic method,its appearance,particle size and Zeta potential were mearsured. Cellular uptake of LPs,TAT-LPs, RGD-LPs and RGD-TAT-LPs was used to evaluate the affinity to C6 cells.C6 cells were xenografted in athymic mice to establish the animal model,which were used to evaluate the distribution of liposomes in vivo. Results The particle diameter of RGD-TAT-LPs was (1 16.5 ±1 1.3 )nm,and its Zeta potential was (23.2 ±3.5 )mV. Cellular uptake experiments demonstrated the cell uptake efficiency of RGD-TAT-LPs by C6 cells were 2.9-fold,2.3-fold and 4.7-fold than that of RGD-LPs,TAT-LPs and LPs respectively. The in vivo imaging showed that RGD-TAT-LPs had the strongest fluorescence intensity in brain. Conclusion The RGD-TAT-LPs might serve as a promising delivery system of antitumor drugs.

RGD and cell penetrating peptides co-modiifed paclitaxel loaded liposome inhibit the A549 cells / 中国生化药物杂志

Objective To prepare RGD and TAT co-modified paclitaxel loaded liposome(RGD/TAT-LP-PTX)for A 549 cells targeting.Method The co-modified liposome was prepared by film-ultrasonic method. The appearance,particle size,Zeta potential were evaluated. The cellular uptake by A 549 cells in vitro was used to evaluate the targeting efficiency. The anti-proliferation efficiency of RGD/TAT-LP-PTX was evaluated by MTT assay. Results The particle diameter of the co-modified liposome was (118.5±11.4) nm with the Zeta potential of (21.58±2.42 )mV. The entrapment efficiency of PTX was 86.5%. The result demonstrated that the co-modified liposome uptaken by A 549 were 2.1, 2.8 times higher than that of TAT-LP and RGD-LP, respectively. The RGD/TAT-LP-PTX shows the highest anti-proliferation efficiency. Conclusion The co-modified liposome might serve as a promising tumor delivery system of antitumor drugs.

Development and Application of Cell-penetrating Peptides

Intracellular transduction of hydrophilic macromolecules has been problematic owing to the biochemical restriction imposed by lipid bilayer of the cytoplasmic membrane. Several technologies have been developed to improve the intracellular delivery of the large molecules for therapeutic purpose, including cell penetrating peptide. Cell penetrating peptides or cell permeable peptides (CPPs) were initially discovered based on the potency of certain full-length proteins or proteins to translocate across the plasma membrane. Currently, CPPs are broadly applied for intracellular delivery of biologically functional molecules in vivo and vitro, varying from small molecules, peptides, proteins, liposomes and nucleic acids. With introducing the history and characteristics of CPPs, this review will focus on the intracellular transduction mechanism and application of CPPs.

Expression of human Oct4 and cell penetrating peptide fusion protein / 第二军医大学学报

Objective: To express the fusion protein of hOct4 and cell penetrating peptides using prokaryotic expression systems, and to optimize its expression methods and observe the membrane penetrating ability of the fusion proteins. Methods: The pET-based prokaryotic expression system was constructed by genetic engineering, and the fusion plasmid was transferred into E. coli BL21(DE3) and Rosetta2(DE3). The protein was purified by Ni affinity chromatography and identified by Western blotting analysis. The penetrating ability of the Rhodamine-labelled fusion protein was investigated using BJ cells. Results: We successfully constructed pET21a(+)-hOct4-11R-His and pET21a(+)-EGFP-11R-His vectors. Fusion proteins hOct4-11-His and EGFP-11R-His were generated by transfering the plasmids into E. coli. The fusion protein was verified by Western blotting analysis and was detected in BJ cells. Conclusion: We have successfully generated EGFP-11R-His and hOct4-11R-His fusion proteins, and the proteins can effectively enter the BJ cells and locate around the nuclei.

Effects of heme oxygenase-1 mediated by cell penetrating peptide PEP-1 on myocardial ischemia/reperfusion injury in isolated rat hearts / 中华麻醉学杂志

Objective To investigate the protective effects of heme oxygenase-1 (HO-1) mediated by cell penetrating peptide PEP-1 on myocardium against ischemia/reperfusion (IR) injury in isolated rat hearts. Methods Healthy male SD rats weighing 220-280 g were anesthetized with intraperitoneal pentobarbital. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O2-5% CO2 at 37 ℃. Eighteen isolated rat hearts were randomly divided into 3 groups ( n = 6 each): Ⅰ group sham operation (group S);Ⅱ group IR and Ⅲ group PEP-1/HO-1 + IR (group HO-1). The isolated rat hearts were perfused with an oxygena-ted (95% O2-5% CO2 ) K-H solution at 37 ℃ in a Langendorff apparatus and were subjected to 40 min of global ischemia followed by 50 min of reperfusion after 30 min of stabilization. In group Ⅲ (group HO- 1 ) the isolated hearts were perfused with 50 μmol/L PEP-1/HO-1 for 15 min before ischemia. After 50 min of reperfusion, HO-1expression, MDA content and SOD activity in myocardial tissues were determined. The activities of creatine kinase (CK) and lactic dehydrogenase (LDH) in coronary effluent fluid were measured. Results The HO- 1 expression was significanfly higher in HO-1 group than in group IR. IR induced significant increase in MDA content and decrease in SOD activity in myocardium and CK and LDH activities in coronary effluent in group Ⅱ compared with group S. PEP-1/HO-1 significantly attenuated IR-induced changes. Conciusion HO-1 mediated by PEP-1 has protective effects on myocardium ngainst IR injury in rats.