Collagen is the structural framework for a wide range of animal connective tissues. It is known to induce cell proliferation and differentiation by direct epitope binding and by serving as a reservoir for growth factors and signaling molecules. [1] In the past, collagen mimetic peptide (CMP), a synthetic peptide composed of a collagen-like repetitive amino acid sequence, has played a central role in elucidating the triple-helical structure and thermal stability of natural collagens. [2][3][4][5][6][7] Herein, we show for the first time that a biochemically inert but highly helicogenic CMP, (Pro-HypGly) 7 (Hyp = hydroxyproline), binds preferentially to the gap regions within the surface of intact type I collagen fibers. This binding behavior was demonstrated by synthesizing CMPconjugated gold nanoparticles (NPs) that are colloidally stable under a wide range of aqueous conditions and by transmission electron microscopy (TEM) observation of their attraction to type I collagen fibers under physiological conditions. The results suggest that such binding affinity may exist in other natural proteins/peptides that contain collagen-like sequences.[8] The study also demonstrates the potential use of nanoparticle-labeling techniques in detecting structurally unstable domains in collagen fibers which are related to many debilitating human diseases. [9] The collagen triple helix is composed of three poly-(proline)-II strands that are held together by interchain hydrogen bonds. [4,10] This structure is similar to the doublehelical structure of DNA. Both collagen and DNA exhibit melting transitions that reflect the stability and strength of the multiplex assemblies. Strand invasion in DNA by short DNA strands or peptide nucleic acids is well documented. [11] Although collagens are known to incorporate thermally unstable domains where small segments of the triple helix are thought to be partially unraveled, [12] strand invasion by other collagen molecules or collagen analogues have not been reported to date.We previously presented a method of collagen modification that uses CMP as a collagen-specific molecular "hitchhiker".[13] Our results suggested that the CMP, (Pro-HypGly) x binds to a film composed of type I collagen molecules (in nonfibrous form) through a process that involves both strand invasion and assembly of a triple helix. However, the binding location and physical state of the collagen molecules that interact with CMP have not been studied in detail.In nature, type I collagen exists not as individual molecules but instead forms fibrous bundles of high tensile strength. Under TEM, collagen fibers exhibit characteristic banding patterns that indicate the structural integrity of the collagen molecules and the regularity of their assembly. The banding patterns also provide approximate position markers along the length of collagen molecules. As CMP is invisible under TEM, we prepared CMP-conjugated gold nanoparticles to use as an electron-dense TEM marker for the location of CMPs. We investigated binding between type I ...
Collagen is the structural framework for a wide range of animal connective tissues. It is known to induce cell proliferation and differentiation by direct epitope binding and by serving as a reservoir for growth factors and signaling molecules. [1] In the past, collagen mimetic peptide (CMP), a synthetic peptide composed of a collagen-like repetitive amino acid sequence, has played a central role in elucidating the triple-helical structure and thermal stability of natural collagens. [2][3][4][5][6][7] Herein, we show for the first time that a biochemically inert but highly helicogenic CMP, (Pro-HypGly) 7 (Hyp = hydroxyproline), binds preferentially to the gap regions within the surface of intact type I collagen fibers. This binding behavior was demonstrated by synthesizing CMPconjugated gold nanoparticles (NPs) that are colloidally stable under a wide range of aqueous conditions and by transmission electron microscopy (TEM) observation of their attraction to type I collagen fibers under physiological conditions. The results suggest that such binding affinity may exist in other natural proteins/peptides that contain collagen-like sequences.[8] The study also demonstrates the potential use of nanoparticle-labeling techniques in detecting structurally unstable domains in collagen fibers which are related to many debilitating human diseases. [9] The collagen triple helix is composed of three poly-(proline)-II strands that are held together by interchain hydrogen bonds. [4,10] This structure is similar to the doublehelical structure of DNA. Both collagen and DNA exhibit melting transitions that reflect the stability and strength of the multiplex assemblies. Strand invasion in DNA by short DNA strands or peptide nucleic acids is well documented. [11] Although collagens are known to incorporate thermally unstable domains where small segments of the triple helix are thought to be partially unraveled, [12] strand invasion by other collagen molecules or collagen analogues have not been reported to date.We previously presented a method of collagen modification that uses CMP as a collagen-specific molecular "hitchhiker".[13] Our results suggested that the CMP, (Pro-HypGly) x binds to a film composed of type I collagen molecules (in nonfibrous form) through a process that involves both strand invasion and assembly of a triple helix. However, the binding location and physical state of the collagen molecules that interact with CMP have not been studied in detail.In nature, type I collagen exists not as individual molecules but instead forms fibrous bundles of high tensile strength. Under TEM, collagen fibers exhibit characteristic banding patterns that indicate the structural integrity of the collagen molecules and the regularity of their assembly. The banding patterns also provide approximate position markers along the length of collagen molecules. As CMP is invisible under TEM, we prepared CMP-conjugated gold nanoparticles to use as an electron-dense TEM marker for the location of CMPs. We investigated binding between type I ...
Using live-cell confocal microscopy and particle tracking technology, the simultaneous transport of intracellular vesicles of the endo-lysosomal pathway and nonviral polyethylenimine (PEI)/DNA nanocomplexes was investigated. Due to potential problems associated with the use of acid-sensitive probes in combination with a gene vector that is hypothesized to buffer the pH of intracellular vesicles, the biological location of PEI/DNA gene vectors was revealed by probing their trafficking in cells expressing fluorescent versions of either early endosome antigen 1, a protein that localizes to early endosomes, or Niemann Pick C1, a protein that localizes to late endosomes and lysosomes. Studies directly show that PEI/DNA nanoparticles are actively transported within both early and late endosomes, and display similar overall transport rates in each. Additionally, gene vector transfer between endosomes is observed. Over time posttransfection, gene vectors accumulate in late endosomes/lysosomes; however, real-time escape of vectors from membrane-bound vesicles is not observed.
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