ligands, and the potential of such modified serum albumins for receptor-mediated
delivery of small lipophilic molecules has been investigated thoroughly. In this
context, modified BSA and HSA molecules have been described as accommodating
small drug molecules such as doxorubicin [34], camptothecin [35], and
methotrexate [36].
In order to assess whether polycationic albumins still bear the lipophilic binding
pockets that the native proteins offer, electron spin resonance experiments with
spin-labeled fatty acids have been carried out [37]. Native HSA and BSA are able to
bind about eight fatty acids within their scaffolds and their relative locations have
been determined by EPR analysis. After cationization, the flexibility and mobility
of the protein scaffold changed significantly, making cHSA considerably less
flexible than native HSA. In addition, many fatty acid ligands are bound tightly
to the surface of cHSA but none of these molecules has been found in the lipophilic
binding pockets (Fig. 5a). It was speculated that the decreased mobility of the
albumin scaffold might limit the capacity of cHSA to adapt to the shape of the guest
molecules and that a sufficient flexibility might be required for guest uptake.
However, it might be also plausible that the fatty acids are trapped on the surface
of cHSA due to electrostatic interactions.
In order to dissipate the positive charges within a larger volume, polyamidoamine
(PAMAM) dendrons have been attached to the HSA scaffold. PAMAM dendrimers
are highly branched, structurally well-defined macromolecules with many primary
amino groups within their outer shell. The dendritic branches that form a dendrimer
are called dendrons. It has been shown before that PAMAM dendrimers are able to
traffic into cells by clathrin-dependent endocytosis [38, 39]. The attachment of about
32 dendrons of the second and third generation to an azido-functionalized albumin by
1,3-dipolar cycloadditions yielded dendronized HSA core–shell macromolecules
(DHSA-G2 and DHSA-G3) with positive net charges. Interestingly, EPR experiments
indicate the accessibility of the binding pockets of albumin because five or six fatty
acids were bound to DHSA-G2 and even to DHSA-G3 (Fig. 5b). In addition, due to
the positive net charge of the dendrons, DHSA-G2 revealed membrane uptake, as
observed by confocal microscopy. Also, the cytotoxicity of both dendronized
core–shell HSA derivatives was considerably lower than that of PAMAM dendrimers
with similar numbers of positive charges. This suggests that protein-based
polycations and polymer polycations interact differently with cellular membranes,
e.g., the latter might have a higher tendency to induce cell leakage due to hole
formation in the membrane. The capacity of DHSA-G2 to accommodate and deliver
the lipophilic antitumor drug doxorubicin (DOX) has been further addressed
[40]. DHSA-G2 reveals a significantly higher loading compared to native HSA
since about 11 DOX molecules are bound tightly even in buffer or cell culture
medium. In vitro studies indicate a fast cellular uptake of the DHSA-G2–DOX
complexes, and DOX release into the cytosol has been substantiated by the observed
high cytotoxicity as well as activation of intracellular caspases 3 and 7, which
ultimately leads to apoptosis. Therefore, protein dendronization could potentially be
considered an attractive strategy for increasing the molecular weight of a protein to
Polymer Complexes in Biological Applications
221
delivery of small lipophilic molecules has been investigated thoroughly. In this
context, modified BSA and HSA molecules have been described as accommodating
small drug molecules such as doxorubicin [34], camptothecin [35], and
methotrexate [36].
In order to assess whether polycationic albumins still bear the lipophilic binding
pockets that the native proteins offer, electron spin resonance experiments with
spin-labeled fatty acids have been carried out [37]. Native HSA and BSA are able to
bind about eight fatty acids within their scaffolds and their relative locations have
been determined by EPR analysis. After cationization, the flexibility and mobility
of the protein scaffold changed significantly, making cHSA considerably less
flexible than native HSA. In addition, many fatty acid ligands are bound tightly
to the surface of cHSA but none of these molecules has been found in the lipophilic
binding pockets (Fig. 5a). It was speculated that the decreased mobility of the
albumin scaffold might limit the capacity of cHSA to adapt to the shape of the guest
molecules and that a sufficient flexibility might be required for guest uptake.
However, it might be also plausible that the fatty acids are trapped on the surface
of cHSA due to electrostatic interactions.
In order to dissipate the positive charges within a larger volume, polyamidoamine
(PAMAM) dendrons have been attached to the HSA scaffold. PAMAM dendrimers
are highly branched, structurally well-defined macromolecules with many primary
amino groups within their outer shell. The dendritic branches that form a dendrimer
are called dendrons. It has been shown before that PAMAM dendrimers are able to
traffic into cells by clathrin-dependent endocytosis [38, 39]. The attachment of about
32 dendrons of the second and third generation to an azido-functionalized albumin by
1,3-dipolar cycloadditions yielded dendronized HSA core–shell macromolecules
(DHSA-G2 and DHSA-G3) with positive net charges. Interestingly, EPR experiments
indicate the accessibility of the binding pockets of albumin because five or six fatty
acids were bound to DHSA-G2 and even to DHSA-G3 (Fig. 5b). In addition, due to
the positive net charge of the dendrons, DHSA-G2 revealed membrane uptake, as
observed by confocal microscopy. Also, the cytotoxicity of both dendronized
core–shell HSA derivatives was considerably lower than that of PAMAM dendrimers
with similar numbers of positive charges. This suggests that protein-based
polycations and polymer polycations interact differently with cellular membranes,
e.g., the latter might have a higher tendency to induce cell leakage due to hole
formation in the membrane. The capacity of DHSA-G2 to accommodate and deliver
the lipophilic antitumor drug doxorubicin (DOX) has been further addressed
[40]. DHSA-G2 reveals a significantly higher loading compared to native HSA
since about 11 DOX molecules are bound tightly even in buffer or cell culture
medium. In vitro studies indicate a fast cellular uptake of the DHSA-G2–DOX
complexes, and DOX release into the cytosol has been substantiated by the observed
high cytotoxicity as well as activation of intracellular caspases 3 and 7, which
ultimately leads to apoptosis. Therefore, protein dendronization could potentially be
considered an attractive strategy for increasing the molecular weight of a protein to
Polymer Complexes in Biological Applications
221