Dataset Information

Positively charged cholesterol-recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions.

ABSTRACT:

Purpose

Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.

Methods

Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.

Results

It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.

Conclusion

Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.

SUBMITTER: Kadengodlu PA

PROVIDER: S-EPMC3471541 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Positively charged cholesterol-recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions.

Kadengodlu Pallavi A PA Hebishima Takehisa T Takeshima Shin-Nosuke SN Ito Mika M Liu Mingzhe M Abe Hiroshi H Aida Yoko Y Aigaki Toshiro T Ito Yoshihiro Y

International journal of nanomedicine 20121011

<h4>Purpose</h4>Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.<h4>Methods</h4>Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and catio ...[more]

PMID: 23091385

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Project description:In this project, we combine two areas of research, experimental characterization and molecular docking studies of the interaction of positively charged oligopeptides with crucial blood plasma proteins. The investigated peptides are rich in NH2 groups of amino acid side chains from Dap, Orn, Lys, and Arg residues, which are relevant in protein interaction. The peptides are 9- and 11-mer with the following sequences: (Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt), (Lys-Dab-Ala-Gly-Orn-Pro-His-Lys-Arg), and (Lys-Dab-Dab-Gly-Orn-Pro-Phe(2-F)-Lys-Arg). The net charge of the compound strongly depends on the pH environment and it is an important aspect of protein binding. The studied oligopeptides exhibit therapeutic properties: anti-inflammatory activity and the capacity to diminish reactive oxygen species (ROS). Therefore, the mechanism of potential binding with blood plasma components is the next challenge. The binding interaction has been investigated under pseudo-physiological conditions with the main blood plasma proteins: albumin (BSA), α1-acid glycoprotein (AAG), and γ-globulin fraction (GGF). The biomolecular quenching constant (kq) and binding constant (Kb) were obtained by fluorescence spectroscopy at various temperatures. Simultaneously, the changes in the secondary structure of proteins were monitored by circular dichroism (CD) and infrared spectroscopy (IR) by quantity analysis. Moreover, molecular docking studies were conducted to estimate the binding affinity, the binding domain, and the chemical nature of these interactions. The results show that the investigated oligopeptides could be mainly transported by albumin, and the binding domain I is the most favored cavity. The BSA and GGF are able to form stable complexes with the studied compounds as opposed to AAG. The binding reactions are spontaneous processes. The highest binding constants were determined for Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt peptide, in which the values of the binding constants Kb to BSA and GGF were 10.1 × 104 dm3mol-1 and 3.39 × 103 dm3mol-1, respectively. The positively charged surface of peptides participated in salt bridge interaction with proteins; however, hydrogen bonds were also formed. The secondary structure of BSA and GGF after contact with peptides was changed. A reduction in the α-helix structure was observed with an increase in the β-sheet and β-turn and random coil structures.

Project description:Lacking an effective mechanism to safely and consistently enhance macromolecular uptake across the intestinal epithelium, prospects for successful development of oral therapeutic peptide drugs remain unlikely. We previously addressed this challenge by identifying an endogenous mechanism that controls intestinal paracellular permeability that can be activated by a peptide, termed PIP 640, which can increase cellular levels of phosphorylated myosin light chain at position S19 (MLC-pS19). Apical application in vitro or luminal application in vivo was shown to increase macromolecular solute transport within minutes that recovered completely within a few hours after removal. We now examine the nature of PIP 640-mediated permeability changes. Confluent Caco-2 cell monolayers treated with PIP 640 enhanced apical-to-basolateral (AB) transport of 4-kDa, but not 10-kDa, dextran. Expression and/or cellular distribution changes of tight junction (TJ) proteins were restricted to increased claudin-2 over a time course that correlated with an apparent shift in its distribution from the nucleus to the membrane fraction of the cell. PIP 640-mediated epithelial changes were distinct from the combined actions of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-?) and interferon gamma (IFN-?). While TNF-?/IFN-? treatment also increased MLC-pS19 levels, these cytokines enhanced AB transport for 70-kDa dextran and decreased occludin expression at TJs. Claudin-2-dependent changes induced by PIP 640 resulted in an AB transport bias for positively-charged macromolecules demonstrated in vitro using charge variants of 4-kDa dextrans and by comparing transport of salmon calcitonin to exenatide. Comparable outcomes of increased TJ localization of claudin-2 and enhanced transport of these therapeutic peptides that biased toward cationic characteristics was demonstrated in vivo following after intra-luminal injection into rat jejunum. Together, these data have shown a potential mechanism for PIP 640 to enhance paracellular permeability of solutes in the size range of small therapeutic peptides that is biased toward positively-charged solutes.

Dataset Information

Positively charged cholesterol-recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions.

Purpose

Methods

Results

Conclusion

Publications

Positively charged cholesterol-recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions.

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