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 bather (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. h order to overcome the bathers 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 proteinsare 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. (C) 2016 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.

Engineering a cell-penetrating hyperstable antibody scFv(Ras)-An extraordinary approach to cancer therapeutics

In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment.Additionally,it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system.As an alternative,this study modifies the hyperstable single-chain fragment variable(scFv)antibody to eliminate cancer using its linear shape.The scFv(F8)antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site.Furthermore,a cell-penetrating peptide(CPP)was attached to the scFv(Ras)antibody model to allow entrance to the cell,creating CPP-scFv(Ras).Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blotting,and the binding assay were performed to prove its effectiveness.As a result,CPP-scFv(Ras)was successfully engineered and bound to the antigen,HRas(G12V).

Cell-penetrating riboflavin conjugate for antitumor photodynamic therapy

Riboflavin(RF,vitamin B2)is an essential vitamin and has been considered as a promising natural photosensitizer for photodynamic therapy(PDT).However,further exploration of RF in antitumor application was limited by its poor cellular uptake.In this study,using cell-penetrating peptides Arg8,(Cha-Arg)3 and small molecule triphenylphosphine(TPP)as delivery compounds,three RF conjugates were prepared to increase the accumulation of RF in cells,termed as Arg8-RF,(Cha-Arg)3-RF and TPP-RF,respectively.Compared with TPP-RF and Arg8-RF,(Cha-Arg)3-RF exhibited better cell internalization and stronger cytotoxicity against HeLa cells upon exposure to blue light.Further researches proved that(Cha-Arg)3-RF generated reactive oxygen species(ROS)under irradiation,which could indiscriminately destroy endogenous proteins and mitochondria,ultimately inducing cell death.This work provides a new approach to explore RF as a natural photosensitizer for antitumor photodynamic therapy.

SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

Background The hairpin cell-penetrating peptides （hCPPs） demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 （MMP-2） positive human ovary adenocarcinoma cell lines, SKOV-3. Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N＇,N＂,N＂＇-tetraacetic acid gadolinium （111） （Gd-DOTA） and fluorescein isothiocyanate （FITC） by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction （RT-PCR） and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging （MRI） and transmission electron microscopy （TEM）. Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- （r=0.990, P 〈0.01） and concentration-dependent manner （r=0.964, P 〈0.001）, but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium. Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

Design of pH-responsive antimicrobial peptide melittin analog-camptothecin conjugates for tumor therapy

Melittin,a classical antimicrobial peptide,is a highly potent antitumor agent.However,its significant toxicity seriously hampers its application in tumor therapy.In this study,we developed novel melittin analogs with pH-responsive,cell-penetrating and membranelytic activities by replacing arginine and lysine with histidine.After conjugation with camptothecin(CPT),CPT-AAM-1 and CPT-AAM-2 were capable of killing tumor cells by releasing CPT at low concentrations and disrupting cell membranes at high concentrations under acidic conditions.Notably,we found that the C-terminus of the melittin analogs was more suitable for drug conjugation than the N-terminus.CPT-AAM-1 significantly suppressed melanoma growth in vivo with relatively low toxicity.Collectively,the present study demonstrates that the development of antitumor drugs based on pH-responsive antimicrobial peptide-drug conjugates is a promising strategy.

Peptides-based vaccine against SARS-nCoV-2 antigenic fragmented synthetic epitopes recognized by T cell and β-cell initiation of specific antibodies tofightthe infection

The World Health Organization has declared the rapidly spreading coronavirus to be a global pandemic.The FDA is yet to approve a vaccine for human novel coronavirus.Here,we developed a peptide-based vaccine and used high-throughput screening by molecular dynamics simulation to identify T-cell-and p-cell-recognized epitopes for producing specific antibod-ies against SARS-nCoV-2.We construct~12 P'antigenic epitope peptides to develop a more effective vaccine and identify specific antibodies.These epitope peptides selectively presented the best antigen presentation scores for both human pMHC class I and II alleles to develop a strong binding affinity.All antigens identified of SARS-nCoV-2 different proteins by each attached specific~1-7 L linker adaptor were used to construct a broad single peripheral peptide vaccine.It is expected to be highly antigenic with a minimum allergic effect.As a result of these exciting outcomes,expressing a vaccine using the intimated peptide was highly promising and positive to be highly proposed as epitope-based peptide vaccine of specific antibody against SARS-nCoV-2 by initiating T cells and β-cells.An in vitro study for the proposed peptide-based vaccine is.mostly recommended.Further clinical trials are required to check the efficacy of this vaccine.

Expression and Activities Experiment of DNA Transduction Motif Based on GAL4 in Pichia Pastoris

The genes encoding DNA-binding domain（BD） designed based on the yeast transcriptional activator GAL4 and protein transduction domain of HIV-1 Tat protein were fused via soft linker peptide sequence, and cloned into yeast expression vector pPIC9k. The resulted plasmid pTG was linearized and transfected into Pichia pastoris strains GS 115 by electroporation. High copies of transformants were obtained with Muts and HIS＋ phenotype identi- fication, PCR amplification and screening of G418. After flask culture and expression induced by methanol, the target protein named TG was well expressed and analyzed by SDS-PAGE and Western blot. Under optimized conditions, the yield of soluble recombinant protein was approximately 39.7 mg/L. DNA binding activity and cell transduction property of TG were analyzed by gel eleetrophoresis and fluorescent microscopy. The results show that the recombinant protein could bind strongly to the plasmid containing upstream activating sequence（UAS）. The cell experiments revealed that TG could deliver the binding plasmid into HEK-293 cells effectively. In summary, the work presented here suggests that TG is specific toward UAS containing plasmid and has the potential for use as nonviral DNA delivery agent.

The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro andin vivo application.Nevertheless,their intemalization pathway is obscure and conficting resultshave been obtained concerning their intracellular distribution.In this study,we demonstrate that octa-D-arginine(r:)undergoes diffuse localization throughout the cytoplasm and nucleus even atlow concentrations and that rs(r:D-arginine)enters the cells via direct membrane translocation,unlike R:(R:L-arginine),of which endocytosis is the major internalization pathway.The observation that Rs and rg enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry affects the uptake mechanism of oligoarginines.

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.

Study on the Penetrability of PEP-1-P27mt for Cell Membranes in Vitro

Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, pro- karyotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS trans- formed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentra- tions in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological pro- tocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

Charge-guided masking of a membrane-destabilizing peptide enables efficient endosomal escape for targeted intracellular delivery of proteins

Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules,largely limiting their therapeutic potential.Here,we converted a cell-penetrating peptide(CPP),low molecular weight protamine(LMWP),to endosomal escape peptides(EEPs)by masking LMWP with a pH-responsive counter-ionic peptide.The resulting masked CPPs(mLMWP and mLMWP2)effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm.Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage.Minimal reactive oxygen species(ROS)elevation or cell death was observed.Based on mLMWP2,we constructed an intracellular protein delivery system containing an antibody as a targeting module,mLMWP2 as an endosomal escape module,and the desired protein cargo.With the HER2-targeting delivery system,we efficiently translocated cyclization recombination enzyme(Cre)and BH3-interacting domain death agonist(BID)into the cytosol of HER2^(+)cells to exert their biological activity.Thereby,the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications.

Dual-targeting and microenvironment-responsive micelles as a gene delivery system to improve the sensitivity of glioma to radiotherapy

Dbait is a small double-stranded DNA molecule that has been utilized as a radiosensitizer to enhance the sensitivity of glioma to radiotherapy(RT). However, there is no effective drug delivery system to effectively overcome the blood–brain barrier(BBB). The aim of this study was to develop a gene delivery system by using the BBB and glioma dual-targeting and microenvironment-responsive micelles(ch-Kn(s-s)R8-An) to deliver Dbait into glioma for RT. Angiopep-2 can target the low-density lipoprotein receptor-related protein-1(LRP1) that is overexpressed on brain capillary endothelial cells(BCECs) and glioma cells. In particular, due to upregulated matrix metalloproteinase 2(MMP-2) in the tumor microenvironment, we utilized MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The results showed that ch-Kn(s-s)R8-An micelles maintained a reasonable size(80–160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration(CMC) with positive surface charge, ranging from 15 to40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency in vitro, improved uptake in glioma cells and good biocompatibility in vitro and in vivo. In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells in vitro. Thus, ch-K5(s-s)R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells.

Tauroursodeoxycholic acid(TUDCA) inhibits influenza A viral infection by disrupting viral proton channel M2

Influenza is a persistent threat to human health and there is a continuing requirement for updating antiinfluenza strategies. Initiated by observations of different endoplasmic reticulum(ER) responses of host to seasonal H1N1 and highly pathogenic avian influenza(HPAI) A H5N1 infections, we identified an alternative antiviral role of tauroursodeoxycholic acid(TUDCA), a clinically available ER stress inhibitor, both in vitro and in vivo. Rather than modulating ER stress in host cells, TUDCA abolished the proton conductivity of viral M2 by disrupting its oligomeric states, which induces inefficient viral infection. We also showed that M2 penetrated cells, whose intracellular uptake depended on its proton channel activity,an effect observed in both TUDCA and M2 inhibitor amantadine. The identification and application of TUDCA as an inhibitor of M2 proton channel will expand our understanding of IAV biology and complement current anti-IAV arsenals.

Hyaluronidase-triggered anticancer drug and siRNA delivery from cascaded targeting nanoparticles for drug- resistant breast cancer therapy

Drug resistance renders standard chemotherapy ineffective in the treatment of connective tissue growth factor （CTGF）-overexpressing breast cancer. By co-embedding the breast tumor cell-penetrating peptide （PEGA-pVEC） and hyaluronic acid （HA） as a targeting media, novel cascaded targeting nanoparficles （HACT NPs） were created on a rattle mesoporous silica （rmSiO2） scaffold for the pinpoint delivery of siRNAs along with an anticancer drug, aiming at overcoming the drug resistance of CTGF-overexpressing breast cancer in vivo. The targeting nanoparticles selectively accumulated in the vasculature under the guidance of the PEGA-pVEC peptide, cascaded by receptor-mediated endocytosis with the aid of another targeting agent, HA, presenting a greater in vivo tumor targeting ability than single targeting ligand vectors. In addition, an HA shell prevented the leakage of therapeutic drugs during the cargo transport process, until the hyaluronidase （HAase）-triggered degradation upon lysosomes entering, guaranteeing a controllable drug release inside the target cells. When the protective shell disintegrates, the released siRNA took charge to silence the gene associated with drug resistance, CTGF, thus facilitating doxorubicin-induced apoptosis. The cascaded targeting media （PEGA-pVEC and HA） advances precision-guided therapy in vivo, while the encapsulation of siRNAs into a chemotherapy drug delivery system provides an efficient strategy for the treatment of drug resistance cancers.

Oral delivery of superoxide dismutase by lipid polymer hybrid nanoparticles for the treatment of ulcerative colitis

Protein-based drugs have received extensive attention in the field of drug research in recent years.However,protein-based drug activity is difficult to maintain during oral delivery,which limits its application.This study developed bifunctional oral lipid polymer hybrid nanoparticles(R8-PEG-PPNPs)that deliver superoxide dismutase(SOD)for the treatment of ulcerative colitis(UC).R8-PEG-PPNPs was composed of PCADK,PLGA,lecithin,and co-modified with stearic acid-octa-arginine and polyethylene glycol.The nanoparticles(NPs)are uniformly dispersed with a complete spherical structure.In vitro stability and release studies showed that R8-PEG-PPNPs exhibited good stability and protection.In vitro cell culture experiments demonstrated that R8-PEG-PPNPs as carriers have no significant toxic effects on cells at concentration below 1000µg/mL and promote cellular uptake.In experiments with ulcerative colitis mice,R8-PEG-PPNPs were able to enhance drug absorption by intestinal epithelial cells and accumulate effectively at the site of inflammation.Its therapeutic effect further demonstrates that R8-PEG-PPNPs are a promising delivery system for oral delivery of protein-based drugs.

Macrocyclic peptides as regulators of protein-protein interactions

Protein-protein interactions(PPIs) are recognized as attractive therapeutic targets. However targeting PPIs especially intracellular ones has been proven extremely difficult for conventional drug-like small molecules, and biological drugs such as monoclonal antibodies have difficulty in reaching intracellular targets. Macrocyclic peptides are promising candidates of PPI regulators for their potential in combining high potency and biological stability together. Cell permeability of macrocyclic peptides may also be achieved by structural modifications or conjugation to a cell-penetrating sequence. Significant progress has been made in this research area in recent years. Important technology progress and recent examples of macrocyclic peptide PPI modulators are reviewed.

Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs

Background:Zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs)have been successfully used to knock out endogenous genes in stem cell research.However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases.A new delivery method that can improve the utility of these nucleases is needed.Results:In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery.Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively.However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture.Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine(C-C motif)receptor 5(CCR5,a co-receptor for HIV-1 entry into cells).Hypothermic treatment greatly enhanced the TAT-TALENmediated gene disruption efficiency.A 5%modification rate was observed in human induced pluripotent stem cells(hiPSCs)treated with TAT-TALEN as measured by the Surveyor assay.Conclusions:TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells.This new technique may advance the clinical application of TALEN technology.