α-helical segments only in the hydrophobic domains. Specifically, the aqueous selfassembly of a series of poly(L-lysine)-b-poly(L-leucine) block copolypeptides was
studied (K x L y , where x ranged from 20 to 80, and y ranged from 10 to 30 residues)
as well as the poly(L-glutamatic acid)-b-poly(L-leucine) block copolypeptide,
E 60 L 20 [91]. In other work, it was found that samples with high K to L molar ratios
(e.g., K 180 L 20 ) could be dissolved directly in deionized water, yielding transparent
hydrogels composed of twisted fibrils (vide infra) [92]. It was reasoned that use of
shortened charged segments would relax repulsive polyelectrolyte interactions and
allow formation of charged polypeptide membranes. Samples were processed by
suspending the polymers in THF/water (1:1) followed by dialysis. Analysis of these
assemblies using DIC optical microscopy revealed the presence of large, sheet-like
membranes for K 20 L 20 and thin fibrils for K 40 L 20 . The K 60 L 20 sample was most
promising, as only large vesicular assemblies were observed by DIC [91].
The K 60 L 20 polypeptide vesicles obtained directly from dialysis are polydisperse
and range in diameter from ca. 5 μm down to 0.8 μm, as determined using DIC and
DLS (Fig. 4). For applications such as drug delivery via blood circulation, a vesicle
diameter of about 50–100 nm is desired. It was observed that aqueous suspensions
of K 60 L 20 vesicles could be extruded through nuclear track-etched polycarbonate
membranes with little loss of polypeptide material. After two passes through a filter,
reductions in vesicle diameter to values in close agreement to filter pore size were
observed. These results showed that the charged copolypeptide vesicles are readily
extruded, allowing good control over vesicle diameter in the tens to hundreds of
nanometers range (Fig. 4). DLS analysis revealed that the extruded vesicles were
also less polydisperse than before extrusion and contained no micellar contaminants. The vesicular morphology was also confirmed through TEM imaging of the
submicron K 60 L 20 suspensions. Thus, it appears that the membranes of the K 60 L 20
vesicles are more flexible and compliant than those of purely rod-like uncharged
polypeptides. The extruded vesicles were monitored for 6 weeks using DLS and
were found to be stable. The vesicles were also found to have high thermal stability.
An aqueous suspension of 1 μm vesicles was held at 80
C for 30 min, after which
no vesicle disruption could be detected [91]. Only after heating to 100
C for 30 min
were the vesicles disrupted, yielding large flat membrane sheets.
Stability of these highly charged polypeptide vesicles in ionic media is important
for use in most applications ranging from personal care products to drug delivery.
Although the K 60 L 20 vesicles are unstable at high salt concentrations (>1 M),
they are stable in 100 mM phosphate-buffered saline (PBS) buffer as well as in
serum-free Dulbecco’s modified Eagle’s medium (DMEM) cell culture media
[91]. Addition of serum, which contains anionic proteins, resulted in vesicle
disruption, most likely due to polyion complexation between the serum proteins
and the oppositely charged polylysine chains. Accordingly, it was observed that the
negatively charged polypeptide vesicles prepared using E 60 L 20 are stable in
DMEM containing 10% fetal bovine serum. Based on these results, these charged
polypeptide vesicles may have potential as encapsulants for water-soluble
therapeutics as an alternative to liposomes. These copolypeptides retain much of
the stability of the uncharged polypeptide vesicles described earlier, but allow
Synthesis and Self-Assembly of Well-Defined Block Copolypeptides via. . .
23
studied (K x L y , where x ranged from 20 to 80, and y ranged from 10 to 30 residues)
as well as the poly(L-glutamatic acid)-b-poly(L-leucine) block copolypeptide,
E 60 L 20 [91]. In other work, it was found that samples with high K to L molar ratios
(e.g., K 180 L 20 ) could be dissolved directly in deionized water, yielding transparent
hydrogels composed of twisted fibrils (vide infra) [92]. It was reasoned that use of
shortened charged segments would relax repulsive polyelectrolyte interactions and
allow formation of charged polypeptide membranes. Samples were processed by
suspending the polymers in THF/water (1:1) followed by dialysis. Analysis of these
assemblies using DIC optical microscopy revealed the presence of large, sheet-like
membranes for K 20 L 20 and thin fibrils for K 40 L 20 . The K 60 L 20 sample was most
promising, as only large vesicular assemblies were observed by DIC [91].
The K 60 L 20 polypeptide vesicles obtained directly from dialysis are polydisperse
and range in diameter from ca. 5 μm down to 0.8 μm, as determined using DIC and
DLS (Fig. 4). For applications such as drug delivery via blood circulation, a vesicle
diameter of about 50–100 nm is desired. It was observed that aqueous suspensions
of K 60 L 20 vesicles could be extruded through nuclear track-etched polycarbonate
membranes with little loss of polypeptide material. After two passes through a filter,
reductions in vesicle diameter to values in close agreement to filter pore size were
observed. These results showed that the charged copolypeptide vesicles are readily
extruded, allowing good control over vesicle diameter in the tens to hundreds of
nanometers range (Fig. 4). DLS analysis revealed that the extruded vesicles were
also less polydisperse than before extrusion and contained no micellar contaminants. The vesicular morphology was also confirmed through TEM imaging of the
submicron K 60 L 20 suspensions. Thus, it appears that the membranes of the K 60 L 20
vesicles are more flexible and compliant than those of purely rod-like uncharged
polypeptides. The extruded vesicles were monitored for 6 weeks using DLS and
were found to be stable. The vesicles were also found to have high thermal stability.
An aqueous suspension of 1 μm vesicles was held at 80
C for 30 min, after which
no vesicle disruption could be detected [91]. Only after heating to 100
C for 30 min
were the vesicles disrupted, yielding large flat membrane sheets.
Stability of these highly charged polypeptide vesicles in ionic media is important
for use in most applications ranging from personal care products to drug delivery.
Although the K 60 L 20 vesicles are unstable at high salt concentrations (>1 M),
they are stable in 100 mM phosphate-buffered saline (PBS) buffer as well as in
serum-free Dulbecco’s modified Eagle’s medium (DMEM) cell culture media
[91]. Addition of serum, which contains anionic proteins, resulted in vesicle
disruption, most likely due to polyion complexation between the serum proteins
and the oppositely charged polylysine chains. Accordingly, it was observed that the
negatively charged polypeptide vesicles prepared using E 60 L 20 are stable in
DMEM containing 10% fetal bovine serum. Based on these results, these charged
polypeptide vesicles may have potential as encapsulants for water-soluble
therapeutics as an alternative to liposomes. These copolypeptides retain much of
the stability of the uncharged polypeptide vesicles described earlier, but allow
Synthesis and Self-Assembly of Well-Defined Block Copolypeptides via. . .
23