Background:The efficient transdermal delivery of biologically active molecules remains a major challenge because of the structural barrier of the stratum corneum,which limits the penetration of large or hydrophilic mo...Background:The efficient transdermal delivery of biologically active molecules remains a major challenge because of the structural barrier of the stratum corneum,which limits the penetration of large or hydrophilic molecules.Low-molecular-weight protamine(LMWP)has a structure similar to that of the HIV TAT protein-derived peptide and is a representative cell-penetrating peptide(CPP)used to increase cell permeability.However,protamine has been reported to have many toxicities and side effects.Objectives:We developed human-derived low-molecular-weight protamine(hLMWP),which is based on fish-derived LMWP but designed using human protein sequences to improve safety and functionality.As it is derived from human proteins,it may reduce side effects and immune rejection during long-term or repeated administration.Additionally,we confirmed in our preliminary study that hLMWP enhances permeability compared to LMWP.In this study,we evaluated physiological activities and skin cell penetration of hLMWP conjugates to assess the potential applications of hLMWP.Methods:cDNA sequences for hLMWP-EGF(His)and hLMWP-SOD-1(His)were synthesized by connecting hLMWP(RSRRRRRRSCQTRRR)to the N-terminus of Epidermal growth factor(EGF)and Superoxide dismutase 1(SOD-1),respectively,with a 6 His-tag added to the C-terminus.The constructs were cloned into a expression vector and expressed in E.coli BL21-CodonPlus pET-41b(+)(DE3)-RIL cells.Expressed proteins were purified using a nickel column and eluted with imidazole buffer.Protein purity was confirmed by SDS-PAGE,and concentrations were quantified using a BCA assay.To evaluate the functional properties of these hLMWP-protein conjugates,a series of in vitro assays were conducted using keratinocyte and macrophage cell lines.These included assessments of permeability,proliferation,wound healing,and anti-inflammatory activity.Results:The results demonstrated that hLMWP-EGF and hLMWP-SOD-1 exhibited superior biological activities,including increased cell proliferation,wound healing,and anti-inflammatory effects,compared to EGF and SOD-1.Moreover,hLMWP-EGF and hLMWP-SOD-1 significantly enhanced the skin permeability of both EGF and SOD-1,as shown by Franz diffusion cell assay and immunofluorescence analysis.Conclusion:Our findings demonstrate that hLMWP significantly enhances skin permeability and biological activity of functional proteins such as EGF and SOD-1 while maintaining safety.This suggests its potential for application in transdermal drug delivery,regenerative medicine,and cosmeceutical formulations.展开更多
Chilled chicken has become the mainstream of chicken consumption.In order to explore the effect of post-mortem aging on water-soluble flavor precursors of chicken,pH,adenosine triphosphate(ATP)degradation,flavor nucle...Chilled chicken has become the mainstream of chicken consumption.In order to explore the effect of post-mortem aging on water-soluble flavor precursors of chicken,pH,adenosine triphosphate(ATP)degradation,flavor nucleosides,free amino acids and water-soluble low molecular weight peptides were determined using Qingyuan partridge yellow-feathered broilers as material during 0-4℃ post-mortem aging in 48 h.The results showed that the pH value fell to the limit pH 5.64(4 h)in chicken breast and 6.21(3 h)in thigh.Regardless of chicken breast or thigh,ATP dropped rapidly within 3 h.It was found that the K-value in chicken thigh was the lowest at 2 h indicating the freshness was the best.Considering the equivalent umami concentration(EUC),the value at 3 h and 4 h was relatively high,but the corresponding electronic tongue umami value was not high,which further showed that the water-soluble low molecular taste peptide played an important role on the post-mortem aging process.Combined with cluster analysis and partial least squares discriminant analysis(PLS-DA),it was preliminarily inferred that the optimal time for chilled chicken during 0-4℃ post-mortem aging was 2 h,which could provide a theoretical basis for the further processing of fresh chicken.展开更多
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 intracellula...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.展开更多
基金financially supported by the Ministry of Small and Mediumsized Enterprises(SMEs)and Startups(MSS),Republic of Korea,under the“Regional Specialized Industry Development Plus Program(R&D,S3363773)”supervised by the Korea Technology and Information Promotion Agency(TIPA)for SMEs.
文摘Background:The efficient transdermal delivery of biologically active molecules remains a major challenge because of the structural barrier of the stratum corneum,which limits the penetration of large or hydrophilic molecules.Low-molecular-weight protamine(LMWP)has a structure similar to that of the HIV TAT protein-derived peptide and is a representative cell-penetrating peptide(CPP)used to increase cell permeability.However,protamine has been reported to have many toxicities and side effects.Objectives:We developed human-derived low-molecular-weight protamine(hLMWP),which is based on fish-derived LMWP but designed using human protein sequences to improve safety and functionality.As it is derived from human proteins,it may reduce side effects and immune rejection during long-term or repeated administration.Additionally,we confirmed in our preliminary study that hLMWP enhances permeability compared to LMWP.In this study,we evaluated physiological activities and skin cell penetration of hLMWP conjugates to assess the potential applications of hLMWP.Methods:cDNA sequences for hLMWP-EGF(His)and hLMWP-SOD-1(His)were synthesized by connecting hLMWP(RSRRRRRRSCQTRRR)to the N-terminus of Epidermal growth factor(EGF)and Superoxide dismutase 1(SOD-1),respectively,with a 6 His-tag added to the C-terminus.The constructs were cloned into a expression vector and expressed in E.coli BL21-CodonPlus pET-41b(+)(DE3)-RIL cells.Expressed proteins were purified using a nickel column and eluted with imidazole buffer.Protein purity was confirmed by SDS-PAGE,and concentrations were quantified using a BCA assay.To evaluate the functional properties of these hLMWP-protein conjugates,a series of in vitro assays were conducted using keratinocyte and macrophage cell lines.These included assessments of permeability,proliferation,wound healing,and anti-inflammatory activity.Results:The results demonstrated that hLMWP-EGF and hLMWP-SOD-1 exhibited superior biological activities,including increased cell proliferation,wound healing,and anti-inflammatory effects,compared to EGF and SOD-1.Moreover,hLMWP-EGF and hLMWP-SOD-1 significantly enhanced the skin permeability of both EGF and SOD-1,as shown by Franz diffusion cell assay and immunofluorescence analysis.Conclusion:Our findings demonstrate that hLMWP significantly enhances skin permeability and biological activity of functional proteins such as EGF and SOD-1 while maintaining safety.This suggests its potential for application in transdermal drug delivery,regenerative medicine,and cosmeceutical formulations.
基金supported by China Agriculture Research System of MOF and MARA (CARS-41)Wens Fifth Five R&D Major Project (WENS-2020-1-ZDZX-007)
文摘Chilled chicken has become the mainstream of chicken consumption.In order to explore the effect of post-mortem aging on water-soluble flavor precursors of chicken,pH,adenosine triphosphate(ATP)degradation,flavor nucleosides,free amino acids and water-soluble low molecular weight peptides were determined using Qingyuan partridge yellow-feathered broilers as material during 0-4℃ post-mortem aging in 48 h.The results showed that the pH value fell to the limit pH 5.64(4 h)in chicken breast and 6.21(3 h)in thigh.Regardless of chicken breast or thigh,ATP dropped rapidly within 3 h.It was found that the K-value in chicken thigh was the lowest at 2 h indicating the freshness was the best.Considering the equivalent umami concentration(EUC),the value at 3 h and 4 h was relatively high,but the corresponding electronic tongue umami value was not high,which further showed that the water-soluble low molecular taste peptide played an important role on the post-mortem aging process.Combined with cluster analysis and partial least squares discriminant analysis(PLS-DA),it was preliminarily inferred that the optimal time for chilled chicken during 0-4℃ post-mortem aging was 2 h,which could provide a theoretical basis for the further processing of fresh chicken.
基金supported by supported by Beijing Municipal Science&Technology Commission(Z231100007223008,China)the National Key R&D Program of China(2017YFA0207900,China)+1 种基金Tsinghua University Initiative Scientific Research Program(2023Z11DSZ001,China)the Tsinghua-Peking Joint Center for Life Sciences.
文摘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.
