135
2. Perform the purification of tetrahydrofuran using standard
high-vacuum techniques. THF is refluxed over Na under N 2
atmosphere in the presence of benzophenone, until a bright
deep purple color is attained, before being collected in a
round-bottom flask containing fresh finely grounded CaH 2 .
Connect this flask to the vacuum line; degas the solvent and
distill in a flask containing a Na/K (1/3 by weight) alloy. The
bright blue color, which develops after stirring for some time,
indicates that the solvent is free from impurities.
3. Ethyl acetate is fractionally distilled over phosphorus
pentoxide.
4. Hexane is fractionally distilled over sodium.
5. Purify dimethylformamide by short-path fractional distillation
under vacuum in a custom-made apparatus. Use always the
middle fraction.
6. Distill fractionally 1,6-diaminohexane under high vacuum and
treat it with sodium at room temperature for 1 day. Then subsequently distill it into precalibrated ampoules with break
seals. Dilute it then with DMF to the appropriate concentration in a sealed apparatus equipped with precalibrated
ampoules and keep away from light.
7. Synthesize the polypeptides using high-vacuum techniques.
Design the polymerization reactors to have volumes at least
three times larger than that of the CO 2 generated by each
polymerization.
Carry
out
polymerizations
with
1,6- diaminohexane as the initiator.
8. The selective cleavage of trityl and tert-butoxycarbonyl groups
from poly (l-histidine) and poly(l-lysine) segments, respectively, is achieved by treating with trifluoroacetic acid (TFA),
followed by addition of triethyl silane. The solution is precipitated in diethyl ether, and the white solid is filtered and dried.
9. These terpolypeptides are composed of one middle hydrophilic block (PLys), two hydrophobic blocks poly(l-histidineco-γ-benzyl-l-glutamate (or l-leucine)), followed by two
hydrophilic blocks of PLys, each one connected at the outer
sides of the hydrophobic blocks. The middle hydrophilic block
of poly(l-lysine) should have a high molecular weight to form
strong hydrogels at very low concentrations, that is, 3.33%
(w/w) in water.
10. A similar procedure is followed for the synthesis of the terpolypeptides exhibiting poly(l-leucine) (PLEU) instead of PBLG.
11. To ensure the formation of a homogeneous mixture, the
hydrogel is inserted into a syringe and passed between two
syringes multiple times using a syringe connector, prior to the
in vivo tests.
Nanostructured Hydrogels for Controlled Drug Delivery
2. Perform the purification of tetrahydrofuran using standard
high-vacuum techniques. THF is refluxed over Na under N 2
atmosphere in the presence of benzophenone, until a bright
deep purple color is attained, before being collected in a
round-bottom flask containing fresh finely grounded CaH 2 .
Connect this flask to the vacuum line; degas the solvent and
distill in a flask containing a Na/K (1/3 by weight) alloy. The
bright blue color, which develops after stirring for some time,
indicates that the solvent is free from impurities.
3. Ethyl acetate is fractionally distilled over phosphorus
pentoxide.
4. Hexane is fractionally distilled over sodium.
5. Purify dimethylformamide by short-path fractional distillation
under vacuum in a custom-made apparatus. Use always the
middle fraction.
6. Distill fractionally 1,6-diaminohexane under high vacuum and
treat it with sodium at room temperature for 1 day. Then subsequently distill it into precalibrated ampoules with break
seals. Dilute it then with DMF to the appropriate concentration in a sealed apparatus equipped with precalibrated
ampoules and keep away from light.
7. Synthesize the polypeptides using high-vacuum techniques.
Design the polymerization reactors to have volumes at least
three times larger than that of the CO 2 generated by each
polymerization.
Carry
out
polymerizations
with
1,6- diaminohexane as the initiator.
8. The selective cleavage of trityl and tert-butoxycarbonyl groups
from poly (l-histidine) and poly(l-lysine) segments, respectively, is achieved by treating with trifluoroacetic acid (TFA),
followed by addition of triethyl silane. The solution is precipitated in diethyl ether, and the white solid is filtered and dried.
9. These terpolypeptides are composed of one middle hydrophilic block (PLys), two hydrophobic blocks poly(l-histidineco-γ-benzyl-l-glutamate (or l-leucine)), followed by two
hydrophilic blocks of PLys, each one connected at the outer
sides of the hydrophobic blocks. The middle hydrophilic block
of poly(l-lysine) should have a high molecular weight to form
strong hydrogels at very low concentrations, that is, 3.33%
(w/w) in water.
10. A similar procedure is followed for the synthesis of the terpolypeptides exhibiting poly(l-leucine) (PLEU) instead of PBLG.
11. To ensure the formation of a homogeneous mixture, the
hydrogel is inserted into a syringe and passed between two
syringes multiple times using a syringe connector, prior to the
in vivo tests.
Nanostructured Hydrogels for Controlled Drug Delivery