Biomedical Engineering Reference
In-Depth Information
proteins when ammonium sulfate was used in the precipita-
tion procedure [34]. Furthermore, ELP-mediated affinity
capture (EMAC) procedures, which avoid the need of
ELPylation, have been developed (Figure 14.2E). Here,
ELP is genetically or chemically fused to a caption partner
that binds specifically but reversibly to the target compound,
which could be proteins, plasmid DNA, glycopeptides, or
heavy metals [35-45]. This procedure also eliminates the
need for postpurification enzymatic or chemical cleavage of
the ELP, and allows a reuse of the ELP capture molecule.
The introduction of ELPs in biotechnology has not just
added one more way for the purification of recombinant
proteins but has revealed a generally new principle for
protein purification. ITC-based purification could make
conventional affinity chromatography-based protein purifi-
cation methods almost dispensable. The following section is
about the present status of how ELPs could aid drug
application in medicine or gave rise to new applications
in nanobiotechnology.
In addition, ELPs represent homing devices for pharma-
ceuticals in vivo and carrier for thermally targeted delivery
of therapeutics [51]. The effectiveness of pharmaceutical
treatments as well as the severity of the corresponding side
effects is largely dependent on the precise targeting of the
applied medicament. Especially. the application of toxic
substances, for example, in chemotherapy, can cause severe
ailments in nonafflicted tissues. By fusion to ELPs, the
active compound was precipitated in the targeted tissue
with a locally induced hyperthermia [52,53]. Micro-sized
ELP aggregates adhere to tumor vasculature only when
tumors are locally heated. Upon return to normothermia
and dissolving of the vascular ELP particles into the plasma,
the local vascular concentration was increased and drove
more ELPs across the tumor blood vessel thus enhancing its
extravascular accumulation [54]. Due to the hydrophobic
folding and the assembly into nano- and/or microparticles to
form a phase separated state above T t (Figure 14.3A) [55].
Microspheres are small spherical particles, with diameters in
the nano- to micrometer range, that might serve as drug
depot delivery devices in inflammation and tumor therapy.
Self-assembled particles of poly(Val-Pro-Ala-Val-Gly) as
vehicles for controlled release of dexamethasone phosphate
(DMP) were generated and once formed; they remain stable
either at ambient or body temperature. Significant amounts
of the model drug were encapsulated after self-assembly and
sustained DMP release for about 30 days was reported [56].
The temperature-dependent release of trapped particles from
cross-linked ELP-made microspheres was also demon-
strated for prednisone acetate, an immunosuppressant
drug. Pore sizes increased above T t , leading to an increased
particle release from ELP microspheres [57]. Some of the
recent studies focused on controlling size and homogeneity of
ELP microspheres, which are critical factors in improving
therapeutic efficacy [58-62]. To generate nanomeso scale
bioresponsive peptide-based particles of defined morphology,
which can encapsulate drugs, electrospraying was used for
preparing ELP particles containing doxorubicin [63]. Func-
tionalized ELP-fusion proteins might benefit from thermally
induced local multivalent ELP biopolymer formation by
triggering avidity resulting in simultaneous interaction of
multiple ELP-conjugated ligands with multiple receptors.
The strategy to use ELPs for the assembly of low-affinity
into high-avidity states was named dynamic affinity modula-
tion (DAM). Since the switch is thermally and locally induc-
ible, avidity effects can be targeted to the desired site of action,
thereby limiting unwanted interaction in nontarget sites [64].
Since ELPs are biopolymers capable of in vivo drug depot
formation through thermally stimulated complexes at phys-
iological temperatures, fusion of such polymers with soluble
tumor necrosis factor receptors (sTNFR) have been designed
and used as in situ anti-TNF drug depots. TNF is a major
inflammatory cytokine and blocking of TNF with monoclo-
nal antibodies or sTNFR-fragments could lead to remission
14.3 ELPYLATED PROTEINS IN MEDICINE
AND NANOBIOTECHNOLOGY
Besides the aforementioned property of ELPs, namely, the
inverse temperature transition, it is the extraordinary bio-
compatibility that makes this type of polymer usable for
medical applications. This was initially demonstrated for
poly(Gly-Val-Gly-Val-Pro) by generic biological tests for
materials and devices recommended by the American Soci-
ety for Testing and Materials (ASTM) in contact with
tissues, tissue fluids, and blood [17]. In vitro and in vivo
studies of poly(Val-Pro-Ala-Val-Gly) revealed no induction
of cytotoxicity, no nonspecific depression of cellular respi-
ration on macrophages and no inflammatory response
caused by ELPs [18]. This unmatched biocompatibility
could be based on the similarity of ELPs to native elastin,
making them undistinguishable for the immune system. Due
to the reported biocompatibility and the fact that the simple
polypeptide nature of ELPs prevented the generation of
monoclonal antibodies, ELP-containing proteins have
already found their way into numerous biomedical applica-
tions [11,46,47].
One application area of ELPs is to form scaffolds for the
growth of tissue, which can be used for tissue repair or de
novo tissue engineering. In vitro, genetically engineered
ELPs, resembling the ocular surface ECM, were useful as
a substratum to culture cells from the ocular surface [48].
Furthermore, ELP-containing polymers were used to con-
struct an ECM for thermally reversible cell sheet recovery of
cultured cells [49], and an ELP-hydrogel was used as a
growing matrix for human adipose-derived adult stem
(hADAS) cells, which showed signs of chondrogenesis
without addition of differentiation supplements [50].
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