Elastin is the primary structural component of elastic tissues, e.g., it accounts for
about 50 wt% of the total vascular extracellular matrix [12, 13]. Primarily it is
formed from tropoelastin molecules as a precursor that assemble to microfibrils and
are covalently interlinked by enzymatic oxidation of four lysine residues to
desomosine units. The primary structure comprises hydrophilic segments with the
lysine units and repeating hydrophobic polypenta- and polyhexapeptide sequences
[14] that act as an entropic spring [15, 16]. Furthermore, temperature- and
pH-dependent coacervation has been demonstrated for tropoleastin and ELPs
[17, 18]. This coacervation effect is thought to be related to the self-assembly
mechanism for the formation of the microfibrils [12, 19]. The controlled coacervation
of ELPs has been exploited for novel and intriguing drug delivery concepts,
e.g., injectable depots of an ELP fusion protein [10]. Another example is ELP
block copolymers fused to a protein, which assemble into thermoresponsive
micelles [11]. This concept has been extended to enable specific uptake into
tumor cells by temperature-controlled assembly only in the tumor, as is possible
in hyperthermia therapy. ELP block copolymers were conjugated to pentaarginine
blocks that were presented at the surface of the micelles and triggered cell penetration only when they were assembled by the micelle formation. The experiments
indicated that endocytosis plays an important role in internalization [20].
2 Experimental Details
2.1 Materials
Soluble elastin ES12 was purchased from Elastin Company (USA) as a salt-free
lyophilized powder. Elastin Company indicates a molecular weight of 60,000 Da,
but that the sample contains lower molecular weight peptides and smears on PAGE.
Coacervates are formed at pH 5 and 37
C. Samples were dissolved in borate buffer
(pH 9). Poly(ethylene glycol) diglycidyl ether (PEG-DGE) (M W ¼ 526 g/mol) and
bis(sulfosuccinimidyl) suberate (BS3) (M W ¼ 572.43 g/mol) were purchased from
Sigma Aldrich (Germany) and Piercenet Biotechnology Inc (USA) respectively.
N-Hexane (99%, VWR), Span 80 (Sigma), Tween 80 (Sigma), tetrahydrofuran
(99% Sigma Aldrich), Texas Red dextran (M W ¼ 70 KDa, Leunonostoc bacteria,
Invitrogen) were used as received. Dialysis membranes (molecular weight cut-off
3.5, 25, and 100 kDa) were purchased from Spectrum Laboratories.
2.2 Elastin-PEG DGE and Elastin-BS3 Microgels
Microgels were prepared by the inverse miniemulsion method with molar ratios of
elastin to crosslinker of 1:2, 1:1.5, 1:1 1:0.5, and 1:0.25. By agitation for 10 min,
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S. Singh et al.
about 50 wt% of the total vascular extracellular matrix [12, 13]. Primarily it is
formed from tropoelastin molecules as a precursor that assemble to microfibrils and
are covalently interlinked by enzymatic oxidation of four lysine residues to
desomosine units. The primary structure comprises hydrophilic segments with the
lysine units and repeating hydrophobic polypenta- and polyhexapeptide sequences
[14] that act as an entropic spring [15, 16]. Furthermore, temperature- and
pH-dependent coacervation has been demonstrated for tropoleastin and ELPs
[17, 18]. This coacervation effect is thought to be related to the self-assembly
mechanism for the formation of the microfibrils [12, 19]. The controlled coacervation
of ELPs has been exploited for novel and intriguing drug delivery concepts,
e.g., injectable depots of an ELP fusion protein [10]. Another example is ELP
block copolymers fused to a protein, which assemble into thermoresponsive
micelles [11]. This concept has been extended to enable specific uptake into
tumor cells by temperature-controlled assembly only in the tumor, as is possible
in hyperthermia therapy. ELP block copolymers were conjugated to pentaarginine
blocks that were presented at the surface of the micelles and triggered cell penetration only when they were assembled by the micelle formation. The experiments
indicated that endocytosis plays an important role in internalization [20].
2 Experimental Details
2.1 Materials
Soluble elastin ES12 was purchased from Elastin Company (USA) as a salt-free
lyophilized powder. Elastin Company indicates a molecular weight of 60,000 Da,
but that the sample contains lower molecular weight peptides and smears on PAGE.
Coacervates are formed at pH 5 and 37
C. Samples were dissolved in borate buffer
(pH 9). Poly(ethylene glycol) diglycidyl ether (PEG-DGE) (M W ¼ 526 g/mol) and
bis(sulfosuccinimidyl) suberate (BS3) (M W ¼ 572.43 g/mol) were purchased from
Sigma Aldrich (Germany) and Piercenet Biotechnology Inc (USA) respectively.
N-Hexane (99%, VWR), Span 80 (Sigma), Tween 80 (Sigma), tetrahydrofuran
(99% Sigma Aldrich), Texas Red dextran (M W ¼ 70 KDa, Leunonostoc bacteria,
Invitrogen) were used as received. Dialysis membranes (molecular weight cut-off
3.5, 25, and 100 kDa) were purchased from Spectrum Laboratories.
2.2 Elastin-PEG DGE and Elastin-BS3 Microgels
Microgels were prepared by the inverse miniemulsion method with molar ratios of
elastin to crosslinker of 1:2, 1:1.5, 1:1 1:0.5, and 1:0.25. By agitation for 10 min,
418
S. Singh et al.