Synthesis of PHP-Cho
Synthesis of PEI-Cho
[edit]9/22/14
300 mg PEI18 + 1 ml DCM + 10 ul trethtlamine (TEA)
100 mg CHol-Chl in 0.5 ml DCM (0.2 mg/ul)
| Sample # |
PEI18 mg |
PEI18 ul |
Chol-Chl mg |
CHol-Chl, ul |
DCM |
After reaction |
| E1 |
30 mg |
100 ul |
10 mg |
50 ul |
0 |
Not soluble in H2O |
| E2 |
30 mg |
100 ul |
7.5 |
37.5 |
12.5 |
Not soluble in H2O |
| E3 |
30 mg | 100 ul | 5 |
25 |
25 |
Not soluble in H2O |
| E4 |
30 mg | 100 ul | 2.5 |
12.5 |
37.5 |
Partial soluble in H2O |
| E5 |
30 mg | 100 ul | 1 |
5 |
45 |
soluble in H2O |
| E6 |
30 mg | 100 ul | 0 |
0 |
50 |
soluble in H2O |
React at 4~8 oC (cold room) o/n
+ 100 ul H2O, + 200 ul Ethyl acetate to extract, spin, remove top organic phase, + 10 ul H2O to bottom phase, repeat washing with EtoAc several time, dry in the fume hood
--> total vol ~=100 ul, 0.3 mg/ul
Conjugate to PHP
[edit]9/24/14
| Sample # |
PHP (9/21/14) |
PEI-Cho |
DSP 10 mg/ml |
| F1 |
25 ul |
E1 3 ul |
3.5 ul |
| F2 |
25 ul |
E2 3 ul |
3.5 |
| F3 |
25 ul | E3 3 ul | 3.5 |
| F4 |
25 ul | E4 3 ul | 3.5 |
| F5 |
25 ul | E5 3 ul | 3.5 |
| F6 |
25 ul | E6 3 ul | 3.5 |
conjugate at RT o/n
Transfection
[edit]Make 1:30, 1:50, 1:75 dilution, use 3 ul of diluted sol, + 1 ug DNA, 293T, 24 well plate
E1 to E6: use 3 ul undiluted, all cells died after o/n incubation
| E1 |
E2 |
E3 |
E4 |
E5 |
E6 |
| F1, 1:10 |
F2, 1:10 |
F3, 1:10 |
F4, 1:10 | F5 , 1:10 | F6 , 1:10 |
| F1, 1:30 |
F2, 1:30 |
F3, 1:30 |
F4, 1:30 | F5, 1:30 | F6, 1:30 |
| F1, 1:50 |
F2, 1:50 |
F3, 1:50 |
F4, 1:50 |
F5, 1:50 |
F6, 1:50 |
9/24/14
9/26/14 morning
9/26/14 afternoon
9/28/14
Result and Conclusion
very little of Chol conjuated to PHP is good, too much is no good
PHP-Cho, different degree of conjugation
[edit]9/26/14
Conjugate Cho to PHP
[edit]dissolve 450 mg PEI18 in 1 ml DCM, + 10 ul TEA
100 mg Chol-Chl in 0.5 ml DCM (0.2 mg/ul)
| PEI18 |
Cho-Chl in DCM | |
| G1 |
10 mg, 22 ul |
1 mg, 5ul |
| G2 |
20 mg, 44 ul |
1 mg, 5ul |
| G3 |
30 mg, 66 ul |
1 mg, 5ul |
| G4 |
40 mg, 88 ul | 1 mg, 5ul |
| G5 |
50 mg, 110 ul | 1 mg, 5ul |
| G6 |
60 mg, 132 ul |
1 mg, 5ul |
conjugate at RT o/n
ext with 100 ul ethyl ether, 2 times
+ 100 ul DCM, + 500 ul ethyl ether, spin,--> ppt
air dry, + H2O to reconsititute to 2 X original vol, i.e., G1: 44 ul; G2: 88 ul, etc
Conjugate PEI18-Cho to PHP
[edit]| PHP, 9/21/14 |
PEI18-Cho |
DSP 10 mg/ml in DMSO | |
| I-1 |
25 ul |
G1 6 ul |
3.5 ul |
| I-2 |
25 ul |
G2 6 ul |
3.5 ul |
| I-3 |
25 ul | G3 6 ul | 3.5 ul |
| I-4 |
25 ul |
G4 6 ul | 3.5 ul |
| I-5 |
25 ul | G5 6 ul | 3.5 ul |
| I-6 |
25 ul | G6 6 ul | 3.5 ul |
| I-7 |
25 ul | 0.3 mg/ul unconjugated PEI18 |
3.5 ul |
Conugate RT o/n
Conjugate PHP-TREN to Cho
[edit]Dilute 0.5 mg ul Chol-Chl to 5 ug/ul with DCM (10 ul of 500 ug/ul to 1 ml DCM)
| Cho-Chl |
PHP-TREN, A3 |
PHP-TREN, B1 | |
| H1 |
2.5 ul |
20 ul |
|
| H2 |
5 ul |
20 ul |
|
| H3 |
10 ul | 20 ul | |
| H4 |
2.5 ul | 20 ul | |
| H5 |
5 ul | 20 ul | |
| H6 |
10 ul | 20 ul |
PHP-TREN in water is not soluble in DCM, and Cho-Chl in DCM is not miscible with H2O.
Mix occasionally, conjugate o/n at RT, DCM phase is gone after o/n conjugation
ppt with ethyl eter, + H2O to redissolve the pellet
H4 , H5, H6 (from B1) is quite soluble in isopropanol after conjugation, so need to add 1.5 ml ethyl ether to ppt conjugate
Transfection
[edit]9/28/14 Transfection
use 1 ug DNA, 3 ul diluted transfection reagent
| H1, 1:50 dilution |
H2, 1:50 |
H3, 1:50 | H4, 1:50 | H5, 1:50 | H6, 1:50 |
| F5, 1:30 |
F:5, 1:50 |
F5, 1:75 |
F5, 1:100 |
F6, 1:30 |
F6, 1:50 |
| I-1, 1:50 |
I-2, 1:50 |
I-3, 1:50 |
I-4, 1:50 |
I-5, 1:50 |
I-6, 1:50 |
| I-1, 1:100 |
I-2, 1:100 |
I-3, 1:100 |
I-4, 1:100 |
I-5, 1:100 |
I-6, 1:100 |
| I-7, 1:50 | I-7, 1:100 |
9/29/14
9/30/14 Morning
9/30/14 afternoon
10/1/14
Conjugate PHP-PEI and PHP-TREN to Cho-Chl
[edit]| PHP-PEI18, 9/21/14 |
PHP-TREN |
Cho-Chl (5ug/ul) | |
| K1 |
25 ul |
2.5 ul | |
| K2 |
25 ul |
5 ul | |
| K3 |
25 ul | 5 ul | |
| K4 |
25 ul | 2.5 ul | |
| K5 |
25 ul | 5 ul | |
| K6 |
25 ul | 10 ul | |
| K7 |
25 ul A3 |
2.5 ul | |
| K8 |
25 ul A3 |
5 ul | |
| K9 |
25 ul A3 |
10 ul | |
| K10 |
25 ul B1 | 2.5 ul | |
| K11 |
25 ul B1 | 5 ul | |
| K12 |
25 ul B1 | 10 ul | |
| K13 |
25 ul 9/6/14 A3-Pyro |
2.5 ul | |
| K14 |
25ul 9/6/14 A3-Pyro |
5 ul | |
| K15 |
25ul 9/6/14 A3-Pyro | 10 ul |
10/6/14
10/7/14
10/8/14
10/9/14
9/11/14
10/11/14
Repeated result
| PHP conjugated to Cho-Chl, then to PEI, Guanydinylated | 1:10 | 1:20 | 1:30 | 1:50 | 1:75 | 1:100 |
| 1:30 dilution of K1 to K15 |
K1 | K2 | K3 | K4 | K5 | K6 |
| K7 | K8 | K9 | K10 | K11 | K12 | |
| K13 | K14 | K15 | PHP-PEI18 | A3 | B1 |
Conjugate PHP-TREN-B1 to water soluble PEI-Cho
[edit]Prepare water soluble PEI18-Cho
[edit]10/5/14
4 g PEI18 in 14 ml DCM chill to 4oC on ice. Add ice cold 200 mg Cho-Chl in 1 ml DCM dropwise. React at 4~10 oC (cold room) o/n (15 Hours). Extract with 30 ml ethyl ether and 30 ml water, collect bottom phase. evaporate ethyl ether in fume hood.--> 27.5 ml water soluble PEI18-Cho.
Preparation of PHP-TREN-B1
[edit]10/9/14
1.5 g DAP
3.6 g DBP
1.5 ml MeOH
Add 196 mg TREN in 1.5 ml MeOH
50 oC react o/n
then 60 oC for 3 hours
Wash with 4X 6 ml MeOH.
Dissolve in 12 ml H2O
Conjugation
[edit]10/12/14
| PEI18 0.4g/ml in water, ul |
water soluble PEI-Cho, ul |
PHP-TREN-B1, ul |
DSP 100 mg/ml, ul | |
| L1 |
2.5 |
1 |
25 |
3.5 |
| L2 |
2.5 |
2 |
25 |
3.5 |
| L3 |
2.5 | 3 |
25 | 3.5 |
| L4 |
2.5 | 4 |
25 | 3.5 |
| L5 |
2.5 | 5 |
25 | 3.5 |
| L6 |
0 |
1 |
25 | 3.5 |
| L7 |
0 |
2 |
25 | 3.5 |
| L8 |
0 |
3 |
25 | 3.5 |
| L9 |
0 |
4 |
25 | 3.5 |
| L10 |
0 |
5 |
25 |
3.5 |
Transfection
[edit]10/13/14
293T, 24 wells
Plate 1
|
1:30 L1 |
L2 |
L3 |
L4 |
L5 |
L6 |
| L7 |
L8 |
L9 |
L10 |
Unconjugated PHP-TREN-B1 |
PHP-Cho-Gu |
| 1:50 L1 |
L2 |
L3 |
L4 |
L5 |
L6 |
| L7 |
L8 |
L9 |
L10 |
Unconjugated PHP-TREN-B1 |
PHP-Cho-Gu |
Plate 2
|
1:75 L1 |
L2 |
L3 |
L4 |
L5 |
L6 |
| L7 |
L8 |
L9 |
L10 |
Unconjugated PHP-TREN-B1 |
PHP-Cho-Gu |
| 1:100 L1 |
L2 |
L3 |
L4 |
L5 |
L6 |
| L7 |
L8 |
L9 |
L10 |
Unconjugated PHP-TREN-B1 |
PHP-Cho-Gu |
Result
[edit]10/14/14
10/15/14
10/17/14
Comparison
[edit]Comparion 1
[edit]10/11/14
Experiment done by Shanyi
| 1:30 | A3 | B1 | K10 | K11 | K12 | L3K |
| 1:50 | A3 | B1 | K10 | K11 | K12 | L3K |
Comparison 2
[edit]
10/17/14
Comparison 3
[edit]L: PHP-TREN-B1 large scale
1.5 g DAP, 3.6 g DBP in 1.5 ml MeOH
TREN 200 ul (196 mg) in 1.5 ml MeOH
50 oC react o/n, then 3 hours at 60 oC
purify with 5 X 5 ml MeOH, spin
dissolve H2O to 12 ml
store at 4 oC--> turbid, + 600 ul water soluble PEI18-Cho (become clear), + 80 mg DSP react o/n
LP: Purified L y isopropanol precipitation
| 1:30 | L | LP | B1 | K10 | L6 | |
| 1:50 | L | LP | B1 | K10 | L6 | L2K |
| 1:75 | L | LP | B1 | K10 | L6 | L3K |
10/17/14
10/18/14
Comparison 4
[edit]to make M1
10/16/14
1.5 g DAP, 3.6 g DBP + 1.5 ml MeOH
+ 196 mg TREN (200 ul) in 1.5 ml MeOH
50 oC react o/n, purify using EtOH 20 ml , 2 X
+ H2O to 12 ml
| M1 | 25 ul Purify by 300 ul isopropanol ppt, reconsitited to 25 ul |
| M2 | 25 ul purify by 300 ul isopropanol ppt, reconsitited to 25 ul, add 1 ul soluble PEI-CHO |
| N0 |
PHP-TREN-A3-CHo-Gu |
| N1 |
PHP-TREN-A3-CHO-Gu, 25 ul + 1 ul soluble PEI18-Cho |
| N2 |
PHP-TREN-B1-Cho-Gu, 10-9-14, unpurified |
| N3 |
PHP-TREN-B1-Cho-Gu, 10-9-14, purified using isopropanol |
| N4 |
PHP-TREN-B1-Cho |
| B1 |
10/19/14
10/20/14
Comparison 5
[edit]To Make PHP-TREN-B1-Cho and PHP-TREN-B1-PEI18-Cho
10/16/14
| B1 |
PHP-TREN-B1 |
| K10 |
PHP-TREN-B1-Cho |
| M1 | 25 ul Purify by 300 ul isopropanol ppt, reconsitited to 25 ul |
| M2 | 25 ul purify by 300 ul isopropanol ppt, reconsitited to 25 ul, add 1 ul soluble PEI-CHO |
| M3 |
25 ul M1 + 2.5 ul Cho-Chl in DCM, 5 ug/ul---->to make PHP-TREN-B1-Cho |
| M4 |
25 ul M2 + 2.5 ul 100 mg/ml DSP---> to make PHP-TREN-B1-PEI18-Cho |
10/20/14: Transfection
10/21/14
10/22/14
10/23/14
