Exploring the ability of TAT, a cell-penetrating peptide, to deliver proteins in a non-fused form / 药学学报

Accumulating evidence has shown that the cell-penetrating peptide TAT can be applied to deliver different types of drug molecules, including nucleic acids, proteins and small molecule drugs. Usually TAT delivers cargoes on the basis of their covalent bonds or non-covalent interactions. However, there are few reports on the delivery of proteins by TAT in a non-covalent manner, and no quantitative comparisons have been made on the protein delivery ability of TAT in fusion and non-fusion manners. In order to explore the ability of TAT to deliver proteins in non-fusion manner, here we used fluorescence microscopy and flow cytometry to investigate the ability of TAT to deliver enhanced green fluorescent protein (EGFP) into non-small cell lung cancer cells A549 in a non-fusion manner. It was found that TAT could deliver EGFP into A549 cells, and its delivery ability was positively correlated with its concentration. In addition, the fusion protein TAT-EGFP was overexpressed and purified, and its permeability across cell membrane was also investigated. In this paper, based on quantitative comparison, we found that the delivery of EGFP by TAT in fusion manner is significantly efficient than that of TAT in non-fusion manner. This is the report that TAT can deliver EGFP in a non-fusion manner. Although its delivery efficiency remains to be improved as compared with the fusion manner, the non-fusion manner has shown incomparable advantages in ease of operation, suggesting that it is also a candidate for delivery strategy in the future.

Preparation of oxytocin liposomes modified by cell penetrating peptides and evaluation on brain targeting efficiency via intranasal delivery / 药学学报

The water-soluble polypeptide drug oxytocin was encapsulated in liposomes by reverse-phase evaporation vesicle method to obtain oxytocin loaded liposomes (OT@LPs) which was further modified with cationic cell penetrating peptide—arginine octamer (R8) to get R8 modified oxytocin loaded liposomes (OT@LPs-R8) which showed enhanced mucoadhesive. The brain targeting efficiency was evaluated preliminarily after nasal administration. OT@LPs-R8 showed a round shape with a particle size distribution of 110.2 ± 7.3 nm, a surface potential as high as +18 mV, a drug loading (62.17 ± 1.88) %, an encapsulation rate (5.85 ± 0.72) %, and stood stable in nasal mucus. After nasal administration, it could significantly prolong the retention and enhance the distribution in the brain with no irritation to the nasal mucosa. The animal experiment in line with the regulations of the Department of Laboratory Animal Science of Fudan University on the ethics of animal experiments had been carried out after passing the review of the Animal Ethics Committee of Fudan University. The results showed nasal administration of OT@LPs-R8 could promote oxytocin directly into the brain from the nose which expected to become a new carrier for delivery of oxytocin to the brain.

Research progress of histidine-based tumor-targeting drug delivery systems / 药学学报

As a basic amino acid, histidine has a pKa close to the acidity of the tumor microenvironment, thus the charge and solubility of histidine are able to vary as the pH changes. Under a neutral environment, histidine is not charged and exhibits hydrophobic properties, while it can be protonated and becomes hydrophilic when exposed to mildly acidic pH, such as tumor microenvironment. Therefore, histidine is widely used in the design of drug delivery systems to target the mildly acidic pH of tumor microenvironment. This article reviews the recent progresses of histidine-based tumor-targeting drug delivery systems, and summarizes the principles on promoting internalization and tuning drug release by taking advantage of histidine. Finally, we point out the common issues on histidine application and illustrate its future prospects.

Study on Preparation and in vitro Anticancer Activity of PFV-modified Paclitaxel/artesunate Co-loaded Targeting Micelles / 中国药房

OBJECTIVE：To prepare cell penetrating peptide PFV-modified paclitaxel （PTX）/artesunate（ART）co-loaded targeting micelles ，and to investigate in vitro anti-tumor activity. METHODS ：According to optimal technology ，PFV-modified PTX/ ART co-loaded targeting micelles were prepared by membrane hydration method ，and were characterized. Using blank micelle as blank control ，sulforhodamine B （SRB）method was used to evaluate the toxicity of PTX micelles ，ART micelles ，PTX/ART micelles and PFV-modified PTX/ART co-loaded targeting micelles to human gastric cancer BGC- 823 cells. The coumarin was used as fluorescent probe replacing PTX to prepare corresponding micelles. Then ，the uptake of BGC- 823 cells to corresponding micelles and targeting effect were observed and determined by flow cytometry and fluorescence microscope. The effects of PTX micelles ， ART micelles ，PTX/ART micelles and PFV-modified PTX/ART co-loaded targeting micelles on the invasion of BGC- 823 cells were investigated by Transwell chamber method. RESULTS ：Average particle size of PFV-modified PTX/ART co-loaded targeting micelles was （51.30±3.95）nm；PDI was 0.19±0.01，and Zeta potential was （0.21±0.02）mV. The encapsulation efficiency of PTX and ART were higher than 90％. The shape of micelles were spherical. The blank micelles had no obvious toxicity to BGC-823 cells. The IC 50 value of PTX micelles ，PTX/ART micelles and PFV-modified PTX/ART co-loaded targeting micelles to BGC-823 cells were （3.09±0.22），（1.93±0.24），（1.11±0.15）μmol/L，respectively. The distribution amount of different micelles in BGC- 823 cell nucleus in the descending order were PFV-modified coumarin/ART micelles ＞coumarin/ART micelles ＞coumarin micelles＞blank control. The order of inhibitory effect was PFV-modified PTX/ART co-loaded targeting micelles ＞PTX/ART micelles＞ART micelles ＞PTX micelles ＞blank control. CONCLUSIONS： Prepared PFV-modified PTX/ART No.81874347） co-loaded targeting micelles are in line with the quality of 1915286446@qq.com Chinese Pharmacopoeia . It shows strong cytotoxicity to BGC-823 cells，can improve the drug targeting and the cell uptake，and inhibit the inv asion and metastasis of BGC- 823 cells.

Preparation of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B and its effect on proliferation and migration of human skin fibroblasts / 中草药

Objective To prepare the liposomes of salvianolic acid B modified with cell penetrating peptide TAT (SAB-TAT-LIP), of which has effects on preventing and treating hypertrophic scars (HS), and establish the method of quality evaluation, as well as preliminarily investigate the effect on the proliferation and migration of human skin fibroblasts (HSF). Methods Liposomes were prepared by pH gradient reverse-phase evaporation method, and the entrapment efficiency was measured by ultrafiltration. Box-Behnken design was performed to optimize the formulation of liposomes by using encapsulation rate as evaluating index. The physicochemical properties of liposomes including morphology, entrapment efficiency, particle size, zeta potential, in vitro release and transdermal absorption, and stability were studied. In addition, the effect of liposomes on proliferation of HSF was examined by MTT assay, and the effect of liposomes on migration of HSF was investigated by scratching method and Transwell assay. Results Based on the optimal formulation of SAB-TAT-LIP, the entrapment efficiency of salvianolic acid B was (86.70 ± 0.85)%, the average particle size was (219.90 ± 5.09) nm, and the zeta potential was (-9.25 ± 0.92). The in vitro 24 h cumulative release was 62.49% of the total drug with no burst effect. The in vitro 32 h cumulated skin penetration rate was 17.21%, the permeance rate was (28.33 ± 4.9) μg/(cm2∙h), and the retention volume of dermis was (44.39 ± 6.87) μg/cm2. The stability was good when placed at 4 ℃ for 10 d. The in vitro cell studies showed that SAB-TAT-LIP can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts, compared with the control group (P < 0.01). Conclusion The optimized SAB-TAT-LIP have higher encapsulation efficiency, smaller particle size, good sustained release effect, and good dermal retention effect which all satisfy the in vitro release and transdermal regulation of local transdermal preparation, and it can significantly inhibit the proliferation, migration and invasion of human skin fibroblasts in vitro.

Effect of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B on proliferation,migration and cell cycle of human skin fibroblasts / 中国中药杂志

Hypertrophic scar( HS) is a very common skin fibrosis disorder after human skin injury and wound healing. The objective of this study was to investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B( SAB-TAT-LIP) on proliferation,migration and cell cycle of human skin fibroblasts( HSF),and preliminarily evaluate its effect on prevention and treatment of HS. HSF were cultured in vitro,and MTT assay was used to detect the inhibitory effect of SAB-TAT-LIP on cell proliferation. Cell migration was assessed by Transwell chamber method and scratch method; and cell cycle change was detected by flow cytometry. In vitro cell studies showed that blank liposome basically had no toxic effect on HSF. Different concentrations of SABTAT-LIP inhibited proliferation on HSF in varying degrees after intervention for different periods in a dose and time dependent manner;meanwhile,SAB-TAT-LIP significantly inhibited the migration and invasion of HSF. At the same time,SAB-TAT-LIP could block the cell cycle at G0/G1 phase after intervention for 48 h,P<0.01 as compared with the blank control group. Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP has significant inhibitory effect on cells proliferation,invasion and migration,with blocking effect on G0/G1 phase. This may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.

Análogos sintéticos da cheferina I: interação com íons metálicos divalentes e o seu efeito na internalização celular e nas atividades anticandida e candidacida / Synthetic analogues of Shepherin I: interaction with metal divalent ions and their effect on cellular internalization and on anticandidal/candidacidal activitie

O desenvolvimento de resistência antimicrobiana e a consequente seleção de microrganismos multirresistentes consolidam-se como grandes ameaças à saúde global. Neste contexto, a busca por novas drogas antimicrobianas/microbicidas é fundamental e compostos como os peptídeos antimicrobianos (AMPs) tornaram-se alvos atraentes. Os AMPs são compostos químicos de massa molar média e grande diversidade estrutural, produzidos por todos os seres vivos e com capacidade de inibir o crescimento de e/ou matar microrganismos. O AMP Cheferina I (Chef I) foi isolado das raízes de Capsella bursa-pastoris e é resultado da proteólise de uma proteína da família das proteínas ricas em glicina, que em plantas estão relacionadas às funções de defesa e cicatrização. O nosso grupo de pesquisa foi pioneiro no desenvolvimento e estudo de análogos truncados amidados deste AMP atípico rico em glicina (67,9%) e histidina (28,6%), que se mostraram ativos frente às diferentes cepas de Candida e a S. cerevisiae pela internalização/ação celular acompanhada de manutenção da integridade da membrana plasmática; o análogo amidado (Chef Ia) e o análogo marcado com 5(6)-carboxifluoresceína/FAM (FAM-Chef Ia) tiveram as suas atividades antifúngicas potencializadas por íons Zn2+. Este trabalho deu continuidade ao estudo do efeito dos íons metálicos divalentes Zn2+, Cu2+, Ca2+ e Mg2+ nas atividades anticandida/fungistática e candidacida/fungicida a diferentes pHs e forças iônicas, estruturas e localizações intracelulares destes análogos. Os resultados na ausência de íons em pH 5,1 revelaram maior atividade do análogo fluorescente em relação à do não fluorescente. Neste mesmo pH, as atividades anticandida e candidacida de Chef Ia foram influenciadas negativamente pelos íons Ca2+ e Mg2+ (2-4 vezes) enquanto que, na presença de íons Zn2+ as atividades anticandida de ambos os análogos foram aumentadas (Chef Ia: 8-64 vezes; FAM-Chef Ia: 4-32 vezes). Os íons Cu2+ aumentaram a atividade anticandida de Chef Ia (2-4 vezes), mas não a do análogo fluorescente, mas as atividades candidacidas de ambos foram melhoradas (Chef Ia: 2-8 vezes; FAM-Chef Ia: 2 vezes). Em pH 5,1, os íons Zn2+ mantiveram a atividade anticandida de Chef Ia em alta força iônica, mas só FAM-Chef Ia exibiu atividade candidacida. Em pH 7,4 ambos análogos foram inativos em baixa e alta forças iônicas na ausência e presença de Zn2+ ou Cu2+. As maiores porcentagens de folhas-ß-antiparalelas e dobras foram observadas no espectro de DC de Chef Ia em pH 7,4, sendo que aqueles registrados em pH 5,1 e 7,4 em presença de íons Zn2 e Cu2+ indicaram a formação de quelatos estruturalmente distintos. Ambos os peptídeos são bioquelantes em potencial, sendo as proporções peptídeo: íon obtidas as seguintes: FAM-Chef Ia = 1:2 para Cu2+, 1:10 para Zn2+; Chef Ia = 1:1 para Cu2+. A análise da internalização celular de FAM-Chef Ia permitiu a suposição de dois mecanismos de internalização (translocação direta e endocitose), sendo que nas células vivas a presença de Zn2+ afetou negativamente a translocação direta (p 0,0343) e potencializou a endocitose (p 0,0002)

The development of antimicrobial resistance and the consequent selection of multiresistant microorganisms have become major threats to global health. In this context, the search for new antimicrobial/microbicidal drugs is crucial and the antimicrobial peptides (AMPs) have been seen as attractive targets. AMPs are chemical compounds of medium molecular mass and high structural diversity produced by all living beings, capable of inhibiting the growth of microorganisms and killing them. The AMP Shepherin I (Shep I) was isolated from the roots of Capsella bursa-pastoris, being a bioactive peptide encrypted in a glycine-rich protein from a family that in plants are strictly related to defense and healing functions. Our research group has pioneered the development and study of amidated truncated analogues of this atypical glycine- (67.9%) and histidine-rich (28.6%) AMP, which has shown activity against different strains of Candida and S. cerevisiae through cellular internalization with maintenance of the plasma membrane integrity. The amide analogue (Chef Ia) and its fluorescent analog labeled with 5 (6) - carboxyfluorescein / FAM (FAM-Chef Ia) had their antifungal activities potentiated by Zn2+ ions, so the present work continued examining the effect of the divalent metallic ions Zn2+, Cu2+, Ca2+ and Mg2+ on the anticandidal/fungistatic and candidacidal/fungicide activities at different pHs and ionic forces, structures and intracellular locations of these analogues. The results in the absence of those ions at pH 5.1 revealed that the fluorescently labelled analog was more potent than the nonfluorescent. At the same pH, Shep Ia anticandidal and candidacidal activities were negatively influenced by Ca2+ and Mg2+ ions (2-4 fold), whereas in the presence of Zn2+ ions the anticandidal activities of both analogues were increased (Shep Ia: 8-64 fold, FAM- Shep Ia: 4-32 fold). Cu2+ ions increased Shep Ia anticandidal activity (2-4 fold) but not that of FAM-Shep Ia, nevertheless, the candidacidal activities of both analogues were increased (Shep Ia: 2-8 fold, FAM-Shep Ia: 2 fold). Also at pH 5.1, the Zn2+ ions helped retaining the anticandidal activity of Shep Ia at high ionic strength, although only FAM-Shep Ia exhibited candidacidal activity. At pH 7.4 both analogues were inactive at low and high ionic strengths in the absence or presence of Zn2+ or Cu2+. The highest percentages of antiparallel ß-sheet and turns were observed in Shep Ia CD spectrum at pH 7.4, while those recorded at pH 5.1 and 7.4 in the presence of Zn2+ or Cu2+ ions indicated the formation of structurally different chelates. Both peptides are potential biochelates, with the following peptide:ion ratios: FAM-Shep Ia = 1: 2 for Cu2+, 1:10 for Zn2+; Shep Ia = 1: 1 for Cu2+. The analysis of the cellular internalization of FAM-Chef Ia allowed the assumption of two mechanisms of internalization (direct translocation and endocytosis) and in the living cells the presence of Zn2+ negatively affected the direct translocation (p 0.0343) and potentiated endocytosis (p 0.0002)

Improved method for synthesis of low molecular weight protamine-siRNA conjugate

RNAi technology has aroused wide public interest due to its high efficiency and specificity to treat multiple types of diseases. However, the effective delivery of siRNA remains a challenge due to its large molecular weight and strong anionic charge. Considering their remarkable functions and features that are often desired in drug delivery carriers, biomimetic systems for siRNA delivery become an effective and promising strategy. Based on this, covalent attachment of synthetic cell penetrating peptides (CPP) to siRNA has become of great interest. We developed a monomeric covalent conjugate of low molecular weight protamine (LMWP, a well-established CPP) and siRNA a cytosol-cleavable disulfide linkage using PEG as a crosslinker. Results showed that the conjugates didn't generate coagulation, and exhibited much better RNAi potency and intracellular delivery compared with the conventional charge-complexed CPP/siRNA aggregates. Three different synthetic and purification methods were compared in order to optimize synthesis efficiency and product yield. The methodology using hetero-bifunctional NHS-PEG-OPSS as a crosslinker to synthesize LMWP-siRNA simplified the synthesis and purification process and produced the highest yield. These results pave the way towards siRNA biomimetic delivery and future clinical translation.

Tat-functionalized Ag-FeO nano-composites as tissue-penetrating vehicles for tumor magnetic targeting and drug delivery

In this paper, we prepared a dual functional system based on dextrin-coated silver nanoparticles which were further attached with iron oxide nanoparticles and cell penetrating peptide (Tat), producing Tat-modified Ag-FeO nanocomposites (Tat-FeAgNPs). To load drugs, an -SH containing linker, 3-mercaptopropanohydrazide, was designed and synthesized. It enabled the silver carriers to load and release doxorubicin (Dox) in a pH-sensitive pattern. The delivery efficiency of this system was assessed using MCF-7 cells, and using null BalB/c mice bearing MCF-7 xenograft tumors. Our results demonstrated that both Tat and externally applied magnetic field could promote cellular uptake and consequently the cytotoxicity of doxorubicin-loaded nanoparticles, with the IC of Tat-FeAgNP-Dox to be 0.63 µmol/L. The delivery efficiency of Tat-FeAgNP carrying Cy5 to the mouse tumor was analyzed using the optical imaging tests, in which Tat-FeAgNP-Cy5 yielded the most efficient accumulation in the tumor (6.7±2.4% ID of Tat-FeAgNPs). Anti-tumor assessment also demonstrated that Tat-FeAgNP-Dox displayed the most significant tumor-inhibiting effects and reduced the specific growth rate of tumor by 29.6% ( = 0.009), which could be attributed to its superior performance in tumor drug delivery in comparison with the control nanovehicles.

Melanoma cells apoptosis induced by novel cell-penetrating peptide and apoptin fusion protein / 中国药学杂志

OBJECTIVE: To investigate apoptosis induction ability of hPP10-Apoptin fusion protein to mouse B16 melanoma cell. METHODS: pET15b-hPP10-Apoptin expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded hPP10-Apoptin fusion protein was purified by Ni-NTA His-Bind Resin and confirmed by Western blotting assay. Melanoma cell apoptosis induced by hPP10-Apoptin fusion protein was analyzed by TUNEL assay, and the antitumor effect was examined in melanoma cell-bear mouse model. RESULTS: hPP10-Apoptin fusion protein was highly expressed in BL21 cells, Western blotting analysis result showed that fusion protein was expressed correctly. The fusion protein can induce melanoma cell apoptosis in vivo and in vitro. CONCLUSION: The results confirm that hPP10-Apoptin fusion protein could penetrate into melanoma cell and also has antitumor effect.

Comparison of three methods for preparation of bacterial ghosts from avian pathogenic Escherichia coli / 生物工程学报

Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD₆₀₀=0.1) by 10 μmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

Preliminary study on immune mechanisms of pH-sensitive transmembrane peptide modified liposome loaded with α-galactosylceramides / 药学学报

In this study, we aim to develop a pH-sensitive transmembrane peptide TH (AGYLLGHINLHH LAHL(Aib)HHIL-Cys) modified liposome loaded with immunoadjuvant α-galactosylceramides (αGC-TH- Lip) and then investigate its effect on the immune function in tumor-bearing mice and its immune mechanism of action. The liposomes were prepared by membrane dispersion-probe ultrasound method and the size and zeta potential of αGC-TH-Lip were also characterized. The uptake of TH modified liposomes (TH-Lip) and polyethylene glycols modified liposomes (PEG-Lip) in DC2.4 cells in vitro were analyzed and the activation of natural killer T (NKT), natural killer (NK) and macrophages in tumor-bearing mice were also measured after systemic administrations of samples. Besides, the degree of maturation of dendritic cell (DC), the number of cytotoxic T lymphocyte (CTL) and the differentiation of helper T cell (Th) were determined. The results showed the particle size of αGC-TH-Lip was about 117.9 nm and the zeta potential was about -8.37 mV under the neutral condition (pH 7.4) and the αGC-TH-Lip had high serum stability in 50% fetal bovine serum. The uptake of TH-Lip in DC2.4 cells in vitro was 1.48 times higher than that of PEG-Lip. After systemic administrations of the samples, the numbers of NKT cells, NK cells and macrophages in tumor-bearing mice were (0.43±0.048)%, (12.80±0.50)% and (3.13±0.26)%, respectively, and the number of mature DCs and CTLs reached (2.30±0.22)% and (32.30±0.80)% separately, which was significantly different from the con-trol group. Finally, we discovered the αGC-TH-Lip had the strongest induction effect on the differentiation of Th1 cells, while barely promote the differentiation of Th2 cells. All the above results demonstrated that the αGC-TH-Lip can improve the immune the activity of mice, enhance the effect of α-galactosylceramide and promote the differentiation of lymphocytes toward the direction of cellular immunity, which consequently achieve a better anti-cancer immune activity.

Development of novel cell-penetrating peptide Cys-CPT2,9 penetratin with high translocation ability and antitumor activity / 药学学报

To develop a cell-penetrating peptide with high membrane penetrating ability and effective antitumor activity, we designed and synthesized an analogue of penetratin, [Cys-CPT2,9] penetratin, by substitution of Gln2 and Asn9 with Cys-CPT. We investigated the transmembrane activity and antitumor activity of [Cys-CPT2,9] penetratin. The fluorescence intensity of [Cys-CPT2,9] penetratin in HeLa cells was observed by laser confocal microscopy and flow cytometry, and the cell uptake mechanism of [Cys-CPT2,9] penetratin was evaluated by using different endocytic inhibitors, finally the anti-tumor activity of [Cys-CPT2,9] penetratin was tested by MTT assay. The results showed that the membrane activity of [Cys-CPT2,9] penetratin was significantly enhanced in laser confocal microscopy and flow cytometry assay, and the intracellular fluorescence intensity was 5 times higher than penetratin. The cell uptake mechanism study of [Cys-CPT2,9] penetratin indicated that it mainly entered the cell through the clathrin and endocytosis. Moreover, [Cys-CPT2,9] penetratin exhibited anti-tumor activity against HeLa cells, and its inhibitory effect on cancer cells was stronger than CPT. [Cys-CPT2,9] penetratin is a new cell-penetrating peptide with high translocation ability, and has anti-tumor activity against HeLa cells.

Effects of Cu/Zn SOD mediated by cell penetrating peptide 4 on hypoxia/reoxygenation injury in myocardial cells / 重庆医学

Objective To evaluate the effect of cell-penetrating peptide (protein transduction domain 4,PTD4) mediated copper-zinc superoxide dismutase (Cu/Zn SOD) on hypoxia/reoxygenation injury (HRI) in rat myocardial cells.Methods Rat myocardial cell H9C2 HRI model was prepared by using the anaerobic incubator (85% N2,10% H2,5% CO2).The HRI group (without adding any treating factor in HRI cell culture fluid),HRI+Cu/Zn SOD group (adding 10 μmol/L Cu/Zn SOD) and HRI+PTD4-Cu/Zn SOD group (10 μmol/L PTD4-Cu/Zn SOD) were set up.In addition,normally cultured myocardial cells served as the normal control group.After incubating for 30 min,the ultra microstructure of mitochondria was observed under transmission electron microscope.The mitochondrial membrane potential was detected by JC-1 kit.The myocardial cell apoptosis was detected by TdT mediated dUTP nick end labeling TUNEL technique.Results The mitochondria injury degree after 30 min incubation in the PTD4-Cu/Zn SOD group was significantly improved compared with the HRI group.Compared with the normal control group,the mitochondrial membrane potential in the HRI group was significantly decreased,while the mitochondrial membrane potential in the PTD4-Cu/Zn SOD group was lower than that in the normal control group,but compared with the HRI group,which was obviously recovered.The cardiomyocyte apoptosis in the HRI+PTD4-Cu/Zn SOD group was (10.20±2.77)%,which was significantly decreased compared with (28.40±2.41)% in the HRI group,the difference was statistically significant (P<0.05).Conclusion PTD4 mediated Cu/Zn SOD can attenuate HRI in rat myocardial cells.

Use of Cell-Penetrating Peptides in Dendritic Cell-Based Vaccination

Cell-penetrating peptides (CPPs) are short amino acids that have been widely used to deliver macromolecules such as proteins, peptides, DNA, or RNA, to control cellular behavior for therapeutic purposes. CPPs have been used to treat immunological diseases through the delivery of immune modulatory molecules in vivo. Their intracellular delivery efficiency is highly synergistic with the cellular characteristics of the dendritic cells (DCs), which actively uptake foreign antigens. DC-based vaccines are primarily generated by pulsing DCs ex vivo with various immunomodulatory antigens. CPP conjugation to antigens would increase DC uptake as well as antigen processing and presentation on both MHC class II and MHC class I molecules, leading to antigen specific CD4+ and CD8+ T cell responses. CPP-antigen based DC vaccination is considered a promising tool for cancer immunotherapy due to the enhanced CTL response. In this review, we discuss the various applications of CPPs in immune modulation and DC vaccination, and highlight the advantages and limitations of the current CPP-based DC vaccination.

Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood-brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP-proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

Construction and expression of cell-penetrating peptide CCL fusion protein expression vector / 中国感染控制杂志

Objective To evaluate the construction of expression vector for fusion protein of cell-penetrating pep-tide CCL (PEP-CCL).Methods CCL6-PEP-6XHis was inserted into plasmid pABP,pABP-CCL6-PEP plasmid was extracted and then transfected into HEK293 cells,CCL6-PEP-6XHis was expressed and purified by chromatog-raphy and detected with Western Blot.Results PEP-CCL express vector was successfully constructed and purified. PCR product of CCL6-PEP-6XHis Tag was ligated with T vector,recombinant was transferred into the host cells, then host cells were cultured,plasmid was extracted and sequenced,the sequence was identical to targeted gene. CCL6-PEP-6XHis was successfully inserted into the eukaryotic expression vector pABP,plasmid was extracted and digested,electrophoresis results revealed that a fragment with 430bp was digested by Hind Ⅲ+XbaⅠ,which was identical to the expected value.Western Blot revealed that CCL6-PEP fusion protein could be recognized by His monoclonal antibody.Conclusion PEP-CCL express vector can be constructed and expressed in eukaryotic cells.

Expression and functional study of green fluorescent protein-hemagglutinin A-cervical cancer fusion protein in vitro / 中国药学杂志

OBJECTIVE: To prepare green fluorescent protein-hemagglutinin A-cervical cancer (GFP-HA-PTP) fusion protein trageting HPV transformed cervical cancer cells with endosome escape capability, and further investigate its penetrating ability for cervical cancer cell lines. METHODS: pET15b-GFP-HA-PTP expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded GFP-HA-PTP fusion protein was purified by Ni-NTA His-Bind resin and conformed by Western blotting assay. Specific penetrating analysis of GFP-HA-PTP was performed under fluorescence microscopy. RESULTS: GFP-HA-PTP fusion protein was highly expressed in BL21 cells. Western blotting analysis result showed that the fusion protein was expressed correctly. The fusion protein can selectively penetrate into cervical cancer cell lines Siha and Caski with endosome escape efficiently. CONCLUSION: GFP-HA-PTP can specificly penetrate into cervical cancer cell lines after endosome escaping with high efficiency.

The development of cell-penetrating peptides in drug delivery system / 药学学报

Cell membrane serves as the natural barrier. Cell-penetrating peptides (CPPs) have been a powerful vehicle for the intracellular delivery of a large variety of cargoes cross the cell membrane. The efficiency of intracellular delivery of drugs, proteins, peptides and nucleic acid, as well as various nanoparticu-late pharmaceutical carriers (e.g., liposomes, polymeric micelles and inorganic nanoparticles) has been demon-strated both in vitro and in vivo. This review focuses on the CPPs-based strategy for intracellular delivery of small molecule drugs, proteins, peptides, nucleic acid and CPP-modified nanocarriers.

Activatable cell-penetrating peptides: a potential activatable modality for diseases diagnosis and therapy / 药学学报

Cell-penetrating peptides are composed of positively-charged amino acids that can mediate molecules or nano-carriers across cell membranes. However, most of the known cell-penetrating peptides have no cell-or tissue-specificity, with affinity to almost all types of cells in internalization. The non-specificity of cell-penetrating peptides is a significant obstacle in the application to targeted delivery of imaging probes and therapeutic agents. Accordingly, many studies focused on selective switching of systemically-delivered inert cell-penetrating peptides into active forms in diseased tissues. Tsien groups introduced the concept of activatable cell-penetrating peptides in 2004. Subsequently, a growing number of similar delivery systems (molecular or nano-sized) have been documented, and the sensitive factors have included enzyme, lower pH, light and exogenous component. In this paper, we make an overview of the development of activatable delivery system in recent years.