142
6. Measure the electrophoretic mobility of the empty and drugloaded vesicle dispersion. All the measurements are at least the
average of three runs performed at 25 °C. Carry out all measurements in isotonic (0.150 M NaCl) Tris buffer (0.010 M) at
pH = 7.4.
7. Cell cultures: Human pancreatic adenocarcinoma cell lines
AsPC1 and BxPC-3, obtained from the American Type Culture
Collection. Propagate the cells in medium containing 5% fetal
calf serum and antibiotics (penicillin, streptomycin) in 5% CO 2
atmosphere in a 37 °C incubator.
Perform the synthesis using high-vacuum techniques [2]. The
purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and is confirmed by FTIR along with
1
H NMR spectroscopy analysis [3].
1. Add N
α
,N
ε
-Di-(tert-butoxycarbonyl)-l-lysine into a flask, place
it on the vacuum line, and pump overnight.
2. Distill purified ethyl acetate, followed by argon insertion in
order to reach atmospheric pressure and by the addition of triphosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by
addition of triphosgene.
2. Heat the mixture at 70 °C until the solution becomes clear,
indicating the formation of the NCA.
3. Distill off the solvent in the vacuum line, and distill fresh dry
ethyl acetate in the flask, to dissolve the crude NCA, followed
by removal of the solvent by distillation.
4. Repeat twice this procedure in order to remove the excess phosgene that sublimes under high vacuum.
5. Remove the unreacted species, such as free amino acids along
with the HCl salts of the amino acids produced during the synthesis, by extraction with an alkali solution in water.
3.2 Synthesis
of Monomers
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-lLysine N-Carboxy
Anhydride, (Ν
ε
BOC-l-LYS
NCA)
3.2.2 Synthesis
of γ-Benzyl-l-Glutamate
N-Carboxy Anhydride
(BLG-NCA)
Hermis Iatrou et al.
6. Measure the electrophoretic mobility of the empty and drugloaded vesicle dispersion. All the measurements are at least the
average of three runs performed at 25 °C. Carry out all measurements in isotonic (0.150 M NaCl) Tris buffer (0.010 M) at
pH = 7.4.
7. Cell cultures: Human pancreatic adenocarcinoma cell lines
AsPC1 and BxPC-3, obtained from the American Type Culture
Collection. Propagate the cells in medium containing 5% fetal
calf serum and antibiotics (penicillin, streptomycin) in 5% CO 2
atmosphere in a 37 °C incubator.
Perform the synthesis using high-vacuum techniques [2]. The
purity of the NCA monomers is crucial for their successful living
polymerization utilizing ROP through primary amine difunctional
initiator and is confirmed by FTIR along with
1
H NMR spectroscopy analysis [3].
1. Add N
α
,N
ε
-Di-(tert-butoxycarbonyl)-l-lysine into a flask, place
it on the vacuum line, and pump overnight.
2. Distill purified ethyl acetate, followed by argon insertion in
order to reach atmospheric pressure and by the addition of triphosgene. Leave the mixture to react for 10 min.
3. Dilute triethylamine in dry ethyl acetate, add it dropwise, and
immerse the solution in an ice-water bath for 6 h.
4. Filter the precipitate, in order to remove the HCl salt of triethylamine, and immerse the clear solution in an ice bath.
5. Extract the NCA repeatedly with Milli-Q water, until neutral
pH of the aqueous phase is achieved.
6. Recrystallize the purified NCA three times under high vacuum
in a custom-made apparatus, with ethyl acetate/hexane
(1/5 v/v) pair at −20 °C. The yield is 60%.
1. Suspend γ-benzyl-l-glutamate in dry ethyl acetate followed by
addition of triphosgene.
2. Heat the mixture at 70 °C until the solution becomes clear,
indicating the formation of the NCA.
3. Distill off the solvent in the vacuum line, and distill fresh dry
ethyl acetate in the flask, to dissolve the crude NCA, followed
by removal of the solvent by distillation.
4. Repeat twice this procedure in order to remove the excess phosgene that sublimes under high vacuum.
5. Remove the unreacted species, such as free amino acids along
with the HCl salts of the amino acids produced during the synthesis, by extraction with an alkali solution in water.
3.2 Synthesis
of Monomers
3.2.1 Synthesis
of ε-tert-Butoxycarbonyl-lLysine N-Carboxy
Anhydride, (Ν
ε
BOC-l-LYS
NCA)
3.2.2 Synthesis
of γ-Benzyl-l-Glutamate
N-Carboxy Anhydride
(BLG-NCA)
Hermis Iatrou et al.