RESUMEN
A multidimensional mass spectrometry (MS) methodology is introduced for the molecular level characterization of polymer-peptide (or polymer-protein) copolymers that cannot be crystallized or chromatographically purified. It encompasses electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) coupled with mass analysis, tandem mass spectrometry (MS(2)) and gas-phase separation by ion mobility mass spectrometry (IM-MS). The entire analysis is performed in the mass spectrometer ("top-down" approach) within milliseconds and with high sensitivity, as demonstrated for hybrid materials composed of hydrophobic poly(tert-butyl acrylate) (PtBA) or hydrophilic poly(acrylic acid) (PAA) blocks tethered to the hydrophobic decapeptide VPGVGVPGVG (VG2) via triazole linkages. The composition of the major products can be rapidly surveyed by MALDI-MS and MS(2). For a more comprehensive characterization, the ESI-IM-MS (and MS(2)) combination is more suitable, as it separates the hybrid materials based on their unique charges and shapes from unconjugated polymer and partially hydrolyzed products. Such separation is essential for reducing spectral congestion, deconvoluting overlapping compositions and enabling straightforward structural assignments, both for the hybrid copolymers as well as the polymer and peptide reactants. The IM dimension also permits the measurement of collision cross-sections (CCSs), which reveal molecular architecture. The MS and MS(2) spectra of the mobility separated ions conclusively showed that [PtBA-VG2]m and [PAA-VG2]m chains with the expected compositions and sequences were formed. Single and double copolymer blocks (m = 1-2) could be detected. Further, the CCSs of the hybrids, which were prepared via azide/alkyne cycloadditions, confirmed the formation of macrocyclic structures. The top-down methodology described would be particularly useful for the detection and identification of peptide/protein-polymer conjugates which are increasingly used in biomedical and pharmaceutical applications.
Asunto(s)
Acrilatos/química , Resinas Acrílicas/química , Oligopéptidos/química , Secuencia de Aminoácidos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Triazoles/químicaRESUMEN
Elastin-mimetic hybrid copolymers with an alternating molecular architecture were synthesized via the step growth polymerization of azide-functionalized, telechelic poly(tert-butyl acrylate) (PtBA) and an alkyne-terminated, valine and glycine-rich peptide with a sequence of (VPGVG)2 (VG2). The resultant hybrid copolymer, [PtBA-VG2]n, contains up to six constituent building blocks and has a polydispersity index (PDI) of ~1.9. Trifluoroacetic acid (TFA) treatment of [PtBA-VG2]n gave rise to an alternating copolymer of poly(acrylic acid) (PAA) and VG2 ([PAA-VG2]n). The modular design permits facile adjustment of the copolymer composition by varying the molecular weight of PAA (22 and 63 repeat units). Characterization by dynamic light scattering indicated that the multiblock copolymers formed discrete nanoparticles at room temperature in aqueous solution at pH 3.8, with an average diameter of 250-270 nm and a particle size distribution of 0.34 for multiblock copolymers containing PAA22 and 0.17 for those containing PAA63. Upon increasing the pH to 7.4, both types of particles were able to swell without being disintegrated, reaching an average diameter of 285-300 nm for [PAA22-VG2]n and 330-350 nm for [PAA63-VG2]n, respectively. The nanoparticles were not dissociated upon the addition of urea, further confirming their unusual stability. The nanoparticles were capable of sequestering a hydrophobic fluorescent dye (pyrene), and the critical aggregation concentration (CAC) was determined to be 1.09 × 10-2 or 1.05 × 10-2 mg/mL for [PAA22-VG2]n and [PAA63-VG2]n, respectively. We suggest that the multiblock copolymers form through collective H-bonding and hydrophobic interactions between the PAA and VG2 peptide units, and that the unusual stability of the multiblock nanoparticles is conferred by the multiblock architecture. These hybrid multiblock copolymers are potentially useful as pH-responsive drug delivery vehicles, with the possibility of drug loading through concerted H-bonds and hydrophobic interactions.
RESUMEN
We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.
Asunto(s)
Reactivos de Enlaces Cruzados/síntesis química , Elastina/síntesis química , Imitación Molecular , Reactivos de Enlaces Cruzados/química , Elastina/química , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Estructura Molecular , Tamaño de la Partícula , Péptidos/síntesis química , Péptidos/química , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polimerizacion , Propiedades de SuperficieRESUMEN
We are interested in developing peptide/polymer hybrid hydrogels that are chemically diverse and structurally complex. Towards this end, an alanine-based peptide doped with charged lysines with a sequence of (AKA(3)KA)(2) (AK2) was selected from the crosslinking regions of the natural elastin. Pluronic(®) F127, known to self-assemble into defined micellar structures, was employed as the synthetic building blocks. Fundamental investigations on the environmental effects on the secondary structure and assembly properties of AK2 peptide were carried out with or without the F-127 micelles. At a relatively low peptide concentration (~0.5 mg/mL), the F127 micelles are capable of not only increasing the peptide helicity but also stabilizing it against thermal denaturation. At a higher peptide concentration in basic media, the AK2 peptide developed a substantial amount of ß-sheet structure that is conducive to the formation of nanofibrils. The fibril formation was confirmed collectively by atomic force microscopy (AFM), small angle neutron scattering (SANS) and transmission electron microscopy (TEM). The assembly kinetics is strongly dependent on solution temperature and pH; an increased temperature and a more basic environment led to faster fibril assembly. The self-assembled nanoscale structures were covalently interlocked via the Michael-type addition reaction between vinyl sulfone-decorated F127 micelles and the lysine amines exposed at the surface of the nanofibers. The crosslinked hybrid hydrogels were viscoelastic, exhibiting an elastic modulus of approximately 17 kPa and a loss tangent of 0.2.
RESUMEN
We are interested in developing elastin-mimetic hybrid polymers (EMHPs) that capture the multiblock molecular architecture of tropoelastin as well as the remarkable elasticity of mature elastin. In this study, multiblock EMHPs containing flexible synthetic segments based on poly(ethylene glycol) (PEG) alternating with alanine-rich, lysine-containing peptides were synthesized by step-growth polymerization using α,ω-azido-PEG and alkyne-terminated AKA(3)KA (K = lysine, A = alanine) peptide, employing orthogonal click chemistry. The resulting EMHPs contain an estimated three to five repeats of PEG and AKA(3)KA and have an average molecular weight of 34 kDa. While the peptide alone exhibited α-helical structures at high pH, the fractional helicity for EMHPs was reduced. Covalent cross-linking of EMHPs with hexamethylene diisocyanate (HMDI) through the lysine residue in the peptide domain afforded an elastomeric hydrogel (xEMHP) with a compressive modulus of 0.12 MPa when hydrated. The mechanical properties of xEMHP are comparable to a commercial polyurethane elastomer (Tecoflex SG80A) under the same conditions. In vitro toxicity studies showed that while the soluble EMHPs inhibited the growth of primary porcine vocal fold fibroblasts (PVFFs) at concentrations ≥0.2 mg/mL, the cross-linked hybrid elastomers did not leach out any toxic reagents and allowed PVFFs to grow and proliferate normally. The hybrid and modular approach provides a new strategy for developing elastomeric scaffolds for tissue engineering.