Self-assembly of a hydrophobic polypeptide containing a short hydrophilic middle segment: Vesicles to large compound micelles

This report describes a facile route to prepare the vesicles and large compound micelles (LCMs) from a series of poly(epsilon-benzyloxycarbonyl L-lysine)-block-poly[diethylene glycol bis(3-amino propyl) ether]-block-poly(epsilon-benzyloxycarbonyl L-lySine) (PZLL-DGBE-PZLL) in their water solution, depending on molecular weight of the polypeptides. A pyrene probe is used to demonstrate the aggregate formation of PZLL-DGBE-PZLL in solution, and also to measure their critical micelle concentration as a function of molecular weight of the polymer.

Synthesis and self-assembly of a novel Y-shaped copolymer with a helical polypeptide arm

novel biodegradable Y-shaped copolymer, poly(L-lactide)(2)-b-poly(gamma-benzyl-L-glutamic acid) (PLLA(2)-b-PBLG), was synthesized by the ring-opening polymerization (ROP) of N-carboxyanhydride of gamma-benzyl-L-glutamate (BLG-NCA) with centrally amino-functionalized poly(L-lactide), PLLA(2)-NH2, as a macroinitiator in a convenient way. The Y-shaped copolymer and its precursors were characterized by H-1 NMR, FT-IR, GPC, WAXD and DSC measurements. The self-assembly of the PLLA(2)-b-PBLG copolymer in toluene and benzyl alcohol was examined. It was found that the self-assembly of the copolymer was dependent on solvent and on relative length of the PBLG block. For a copolymer with PLLA blocks of 26 in total degree of polymerization (DP), if the PBLG block was long enough (e.g., DP = 54 or more), the copolymer/toluene solution became a transparent gel at room temperature. In benzyl alcohol Solution, only PLLA(2)-b-PBLG containing ca. 190 BLG residues could form a gel: those with shorter PBLG blocks (e.g., DP = 54) became nano-scale fibrous aggregates and these aggregates were dispersed in benzyl alcohol homogeneously.

Purification, sequencing and biological activity of polypeptide from velvet antler

The velvet antler polypeptide CNT14 was extracted and purified by gel filtration, ion exchange chromatography and RP C, which showed a single peak in HPLC chromatography and a single band in SDS-PAGE. The molecular weight measured by MALDI/TOF/MS spectrum was 1479. 9028. The polypeptide consisted mostly of Glu, Leu, Val, Pro. The amino acid sequence of the polypeptide was detected with ESI-MS/ MS, and the sequence was E-P-T-V-L-D-E-V-C-L-A-H-G-P. The experiments of biological activity of polypeptide CNT14 in vivo were carried out, and the results show that CNT14 has stimulant effects on the growth of rat HT22 cells. Then we produced the polypeptide CNT14 according the amino acid sequence by solid phase synthesis, confirmed the sequence of the polypeptide to be consistent with the amino acid sequence of polypeptide CNT14 which was separated from the velvet antler.

Self-assembly of polypeptide-containing ABC-type triblock copolymers in aqueous solution and its pH dependence

Self-assembling of novel biodegradable ABC-type triblock copolymer poly(ethylene glycol)-poly(L-lactide)-poly(L-glutamic acid) (PEG-PLLA-PLGA) is studied. In aqueous media, it self-assembles into a spherical micelle with the hydrophobic PLLA segment in the core and the two hydrophilic segments PEG and PLGA in the shell. With the lengths of PEG and PLLA blocks fixed, the diameter of the micelles depends on the length of the PLGA block and on the volume ratio of H2O/dimethylformamide (DMF) in the media. When the PLGA block is long enough, morphology of the self-assembly is pH-dependent. It assembles into the spherical micelle in aqueous media at pH 4.5 and into the connected rod at or below pH 3.2. The critical micelle concentration (cmc) of the copolymer changes accordingly with decreasing solution pH. Both aggregation states can convert to each other at the proper pH value. This reversibility is ascribed to the dissociation and neutralization of the COOH groups in the LGA residues. When the PLGA block is short compared to the PEG or PLLA block, it assembles only into the spherical micelle at various pH values.

Polypeptide modification of multiwalled carbon nanotubes by a graft-from approach

Covalent surface functionalization of carbon nanotubes with polypeptides is promising for possible medical applications. This work presents a graft-from approach to perform the polypeptide modification of multiwalled carbon nanotubes (MWTNs). The raw MWNTs are first amine-functionalized. The amine-functionalized MWNTs are then used as the initiator to initiate the ring-opening polymerization of gamma-benzyl-L-glutamate N-carboxyanhydride (BLG- NCA), to results in the polypeptide-grafted MWNTs. FT-IR, XPS, and TGA data demonstrate that the functionalization is successful. The TEM images of the products show that the thickness of the polypeptide shell of the PBLG-MWNT is about 4.5-22 nm. Using the facile route developed here, carbon nanotubes functionalized with other types of polypeptides can be easily fabricated using the corresponding NCAs.

A novel polypeptide from shark cartilage with potent anti-angiogenic activity

Using guanidine-HCl extraction, acetone precipitation, ultra-filtration and chromatography, a novel polypeptide with potent anti-angiogenic activity was purified from cartilage of the shark, Prionace glauca. N-terminal amino acid sequence analysis and SDS-PAGE revealed that the substance is a novel polypeptide with MW 15500 (PG155). The anti-angiogenic effects of PG155 were evaluated using zebrafish embryos model in vivo. Treatment of the embryos with 20 mu g/ml PG155 resulted in a significant reduction in the growth of subintestinal vessels (SIVs). A higher dose resulted in almost complete inhibition of SIV growth, as observed by endogenous alkaline phosphatase (EAP) staining assay. An in vitro transwell experiment revealed that the polypeptide inhibited vascular endothelial growth factor (VEGF) induced migration and tubulogenesis of human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs in 20 mu g/ml PG155 significantly decreased the density of migrated cells. Almost complete inhibition of cell migration was found when HUVECs were treated with 40-80 mu g/ml PG155. PG155 (20 mu g/ml) markedly inhibited the tube formation of HUVECs and a dose-dependent effect was also found when treatment of HUVECs with PG155 at the concentration from 20 to 160 mu g/ml.

Neural network analysis identifies scaffold properties necessary for in vitro chondrogenesis in elastin-like polypeptide biopolymer scaffolds.

The successful design of biomaterial scaffolds for articular cartilage tissue engineering requires an understanding of the impact of combinations of material formulation parameters on diverse and competing functional outcomes of biomaterial performance. This study sought to explore the use of a type of unsupervised artificial network, a self-organizing map, to identify relationships between scaffold formulation parameters (crosslink density, molecular weight, and concentration) and 11 such outcomes (including mechanical properties, matrix accumulation, metabolite usage and production, and histological appearance) for scaffolds formed from crosslinked elastin-like polypeptide (ELP) hydrogels. The artificial neural network recognized patterns in functional outcomes and provided a set of relationships between ELP formulation parameters and measured outcomes. Mapping resulted in the best mean separation amongst neurons for mechanical properties and pointed to crosslink density as the strongest predictor of most outcomes, followed by ELP concentration. The map also grouped formulations together that simultaneously resulted in the highest values for matrix production, greatest changes in metabolite consumption or production, and highest histological scores, indicating that the network was able to recognize patterns amongst diverse measurement outcomes. These results demonstrated the utility of artificial neural network tools for recognizing relationships in systems with competing parameters, toward the goal of optimizing and accelerating the design of biomaterial scaffolds for articular cartilage tissue engineering.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted. © 2003 Elsevier Science Ltd. All rights reserved.

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(LyS(37)PAL) and N-AcGIP(LyS(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25 nmol kg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(LyS(37)PAL) or N-AcGIP(LyS37PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP. (c) 2006 Elsevier Inc. All rights reserved.

GIP(Lys16PAL) and GIP(Lys37PAL): novel long-acting acylated analogues of glucose-dependent insulinotropic polypeptide with improved antidiabetic potential.

Glucose-dependent insulinotropic polypeptide (GIP) is a physiological insulin releasing peptide. We have developed two novel fatty acid derivatized GIP analogues, which bind to serum albumin and demonstrate enhanced duration of action in vivo. GIP(Lys(16)PAL) and GIP(Lys(37)PAL) were resistant to dipeptidyl peptidase IV (DPP IV) degradation. In vitro studies demonstrated that GIP analogues retained their ability to activate the GIP receptor through production of cAMP and to stimulate insulin secretion. Intraperitoneal administration of GIP analogues to obese diabetic (ob/ob) mice significantly decreased the glycemic excursion and elicited increased and prolonged insulin responses compared to native GIP. A protracted glucose-lowering effect was observed 24 h following GIP(LyS(37)PAL) administration. Once a day injection for 14 days decreased nonfasting glucose, improved glucose tolerance, and enhanced the insulin response to glucose. These data demonstrate that fatty acid derivatized GIP peptides represent a novel class of long-acting stable GIP analogues for therapy of type 2 diabetes.