Editing Synthesis of PHP-Cho

== Synthesis of PEI-Cho ==

'''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)

{| width="492" cellspacing="1" cellpadding="1" border="1"
|-
| Sample #<br/>
| PEI18 mg<br/>
| PEI18 ul<br/>
| Chol-Chl mg<br/>
| CHol-Chl, ul<br/>
| DCM<br/>
| After reaction<br/>
|-
| E1<br/>
| 30 mg<br/>
| 100 ul<br/>
| 10 mg<br/>
| 50 ul<br/>
| 0<br/>
| Not soluble in H2O<br/>
|-
| E2<br/>
| 30 mg&nbsp;&nbsp;<br/>
| 100 ul<br/>
| 7.5<br/>
| 37.5<br/>
| 12.5<br/>
| Not soluble in H2O<br/>
|-
| E3<br/>
| 30 mg
| 100 ul
| 5<br/>
| 25<br/>
| 25<br/>
| Not soluble in H2O<br/>
|-
| E4<br/>
| 30 mg
| 100 ul
| 2.5<br/>
| 12.5<br/>
| 37.5<br/>
| Partial soluble in H2O<br/>
|-
| E5<br/>
| 30 mg
| 100 ul
| 1<br/>
| 5<br/>
| 45<br/>
| soluble in H2O<br/>
|-
| E6<br/>
| 30 mg
| 100 ul
| 0<br/>
| 0<br/>
| 50<br/>
| soluble in H2O<br/>
|}



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 ==

9/24/14

{| width="289" cellspacing="1" cellpadding="1" border="1"
|-
| Sample #<br/>
| PHP (9/21/14)<br/>
| PEI-Cho<br/>
| DSP 10 mg/ml<br/>
|-
| F1<br/>
| 25 ul<br/>
| E1 3 ul<br/>
| 3.5 ul<br/>
|-
| F2<br/>
| 25 ul&nbsp;<br/>
| E2 3 ul&nbsp;<br/>
| 3.5&nbsp;<br/>
|-
| F3<br/>
| 25 ul
| E3 3 ul
| 3.5
|-
| F4<br/>
| 25 ul
| E4 3 ul
| 3.5
|-
| F5<br/>
| 25 ul
| E5 3 ul
| 3.5
|-
| F6<br/>
| 25 ul
| E6 3 ul
| 3.5
|}

conjugate at RT o/n



== Transfection ==

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

{| width="471" cellspacing="1" cellpadding="1" border="1"
|-
| E1<br/>
| E2<br/>
| E3<br/>
| E4<br/>
| E5<br/>
| E6<br/>
|-
| F1, 1:10<br/>
| F2, 1:10<br/>
| F3, 1:10<br/>
| F4, 1:10
| F5 , 1:10
| F6 , 1:10
|-
| F1, 1:30<br/>
| F2, 1:30<br/>
| F3, 1:30&nbsp;<br/>
| F4, 1:30
| F5, 1:30
| F6, 1:30
|-
| F1, 1:50<br/>
| F2, 1:50<br/>
| F3, 1:50<br/>
| F4, 1:50<br/>
| F5, 1:50<br/>
| F6, 1:50<br/>
|}



9/24/14

[[File:Cho conjugation 2014 9 25.png|RTENOTITLE]]

9/26/14 morning

[[File:Cho conjugation 2014 9 26.png|RTENOTITLE]]

[[File:Cho conjugation 2014 9 26 1.JPG|RTENOTITLE]]

9/26/14 afternoon

[[File:Cho conjugation 2014 9 26 2.JPG|RTENOTITLE]]

9/28/14

[[File:Cho conjugation 2014 9 28.JPG|RTENOTITLE]]

Result and Conclusion

very little of Chol conjuated to PHP is good, too much is no good

== PHP-Cho, different degree of conjugation ==

9/26/14

=== Conjugate Cho to PHP ===

dissolve 450 mg PEI18 in 1 ml DCM, + 10 ul TEA

100 mg Chol-Chl in 0.5 ml DCM (0.2 mg/ul)

{| width="470" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| PEI18<br/>
| Cho-Chl in DCM<br/>
|-
| G1<br/>
| 10 mg, 22 ul<br/>
| 1 mg, 5ul<br/>
|-
| G2<br/>
| 20 mg, 44 ul<br/>
| 1 mg, 5ul&nbsp;<br/>
|-
| G3<br/>
| 30 mg, 66 ul<br/>
| 1 mg, 5ul
|-
| G4<br/>
| 40 mg, 88 ul
| 1 mg, 5ul<br/>
|-
| G5<br/>
| 50 mg, 110 ul
| 1 mg, 5ul
|-
| G6<br/>
| 60 mg, 132 ul<br/>
| 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 ===

{| width="377" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| PHP, 9/21/14<br/>
| PEI18-Cho<br/>
| DSP 10 mg/ml in DMSO<br/>
|-
| I-1<br/>
| 25 ul<br/>
| G1 6 ul<br/>
| 3.5 ul<br/>
|-
| I-2<br/>
| 25 ul<br/>
| G2 6 ul<br/>
| 3.5 ul<br/>
|-
| I-3<br/>
| 25 ul
| G3 6 ul
| 3.5 ul&nbsp;
|-
| I-4<br/>
| 25 ul<br/>
| G4 6 ul
| 3.5 ul<br/>
|-
| I-5<br/>
| 25 ul
| G5 6 ul
| 3.5 ul
|-
| I-6<br/>
| 25 ul
| G6 6 ul
| 3.5 ul
|-
| I-7<br/>
| 25 ul
| 0.3 mg/ul unconjugated PEI18<br/>
| 3.5 ul<br/>
|}

Conugate RT o/n



=== Conjugate PHP-TREN to Cho ===

Dilute 0.5 mg ul Chol-Chl to 5 ug/ul with DCM (10 ul of 500 ug/ul to 1 ml DCM)

{| width="419" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| Cho-Chl<br/>
| PHP-TREN, A3<br/>
| PHP-TREN, B1<br/>
|-
| H1<br/>
| 2.5 ul<br/>
| 20 ul<br/>
| <br/>
|-
| H2<br/>
| 5 ul<br/>
| 20 ul<br/>
| <br/>
|-
| H3<br/>
| 10 ul
| 20 ul
| <br/>
|-
| H4<br/>
| 2.5 ul
| <br/>
| 20 ul
|-
| H5<br/>
| 5 ul
| <br/>
| 20 ul
|-
| H6<br/>
| 10 ul
| <br/>
| 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 ===

9/28/14 Transfection

use 1 ug DNA, 3 ul diluted transfection reagent

{| width="653" cellspacing="1" cellpadding="1" border="1"
|-
| H1, 1:50 dilution<br/>
| H2, 1:50<br/>
| H3, 1:50
| H4, 1:50
| H5, 1:50
| H6, 1:50
|-
| F5, 1:30<br/>
| F:5, 1:50<br/>
| F5, 1:75<br/>
| F5, 1:100<br/>
| F6, 1:30<br/>
| F6, 1:50<br/>
|-
| I-1, 1:50<br/>
| I-2, 1:50<br/>
| I-3, 1:50<br/>
| I-4, 1:50<br/>
| I-5, 1:50<br/>
| I-6, 1:50<br/>
|-
| I-1, 1:100<br/>
| I-2, 1:100<br/>
| I-3, 1:100<br/>
| I-4, 1:100<br/>
| I-5, 1:100<br/>
| I-6, 1:100<br/>
|-
| I-7, 1:50
| I-7, 1:100
| <br/>
| <br/>
| <br/>
| <br/>
|}





9/29/14

[[File:Cho conjugation 2014 9 29.JPG|RTENOTITLE]]

[[File:Cho conjugation 2014 9 29 2.JPG|RTENOTITLE]]



9/30/14 Morning

[[File:Cho conjugation 2014 9 30.JPG|RTENOTITLE]]

9/30/14 afternoon

[[File:Cho conjugation 2014 9 30 2.JPG|RTENOTITLE]]

10/1/14

[[File:Cho conjugation 2014 10 1.JPG|RTENOTITLE]]



== Conjugate PHP-PEI and PHP-TREN to Cho-Chl ==

{| width="498" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| PHP-PEI18, 9/21/14<br/>
| PHP-TREN<br/>
| Cho-Chl (5ug/ul)<br/>
|-
| K1<br/>
| 25 ul<br/>
| <br/>
| 2.5 ul<br/>
|-
| K2<br/>
| 25 ul<br/>
| <br/>
| 5 ul<br/>
|-
| K3<br/>
| 25 ul
| <br/>
| 5 ul<br/>
|-
| K4<br/>
| 25 ul
| <br/>
| 2.5 ul<br/>
|-
| K5<br/>
| 25 ul
| <br/>
| 5 ul<br/>
|-
| K6<br/>
| 25 ul
| <br/>
| 10 ul<br/>
|-
| K7<br/>
| <br/>
| 25 ul A3<br/>
| 2.5 ul
|-
| K8<br/>
| <br/>
| 25 ul A3&nbsp;&nbsp;<br/>
| 5 ul<br/>
|-
| K9<br/>
| <br/>
| 25 ul A3<br/>
| 10 ul<br/>
|-
| K10<br/>
| <br/>
| 25 ul B1
| 2.5 ul
|-
| K11<br/>
| <br/>
| 25 ul B1
| 5 ul<br/>
|-
| K12<br/>
| <br/>
| 25 ul B1
| 10 ul<br/>
|-
| K13<br/>
| <br/>
| 25 ul 9/6/14 A3-Pyro<br/>
| 2.5 ul
|-
| K14<br/>
| <br/>
| 25ul 9/6/14 A3-Pyro<br/>
| 5 ul<br/>
|-
| K15<br/>
| <br/>
| 25ul 9/6/14 A3-Pyro
| 10 ul<br/>
|}

10/6/14

[[File:Cho 10 6 14.JPG|850px|Cho 10 6 14.JPG]]

[[File:Cho 10 6 14 2.JPG|850px|Cho 10 6 14 2.JPG]]

10/7/14

[[File:Cho 10 7 14 1.JPG|RTENOTITLE]]



[[File:Cho 10 7 14 2.JPG|RTENOTITLE]]

10/8/14

[[File:Cho 10 8 14.JPG|RTENOTITLE]]



[[File:Cho 10 8 14 2.JPG|RTENOTITLE]]



10/9/14

[[File:Cho 10 8 14 1.JPG|850px|Cho 10 8 14 1.JPG]]

[[File:Cho 10 9 14 3.JPG|RTENOTITLE]]

[[File:Cho 10 9 14 2.JPG|RTENOTITLE]]

9/11/14

[[File:Cho 10 11 14 4.JPG|RTENOTITLE]]



[[File:Cho 10 11 14 3.JPG|RTENOTITLE]]

10/11/14

Repeated result

{| width="456" cellspacing="1" cellpadding="1" border="1"
|-
| PHP conjugated to Cho-Chl, then to PEI, Guanydinylated
| 1:10
| 1:20
| 1:30
| 1:50
| 1:75
| 1:100
|-
| rowspan="3" | 1:30 dilution of K1 to K15<br/>
| K1
| K2
| K3
| K4
| K5
| K6
|-
| K7
| K8
| K9
| K10
| K11
| K12
|-
| K13
| K14
| K15
| PHP-PEI18
| A3
| B1
|}



[[File:Cho 10 11 14 2.JPG|RTENOTITLE]]







== Conjugate PHP-TREN-B1 to water soluble PEI-Cho ==

=== Prepare water soluble PEI18-Cho ===

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 ===

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 ===

10/12/14

{| width="491" cellspacing="1" cellpadding="1" border="1"
|-
| <br/>
| PEI18 0.4g/ml in water, ul<br/>
| water soluble PEI-Cho, ul<br/>
| PHP-TREN-B1, ul<br/>
| DSP 100 mg/ml, ul<br/>
|-
| L1<br/>
| 2.5<br/>
| 1<br/>
| 25<br/>
| 3.5<br/>
|-
| L2<br/>
| 2.5<br/>
| 2<br/>
| 25&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br/>
| 3.5&nbsp;&nbsp;&nbsp;&nbsp;<br/>
|-
| L3<br/>
| 2.5
| 3<br/>
| 25
| 3.5
|-
| L4<br/>
| 2.5
| 4<br/>
| 25
| 3.5
|-
| L5<br/>
| 2.5
| 5<br/>
| 25
| 3.5<br/>
|-
| L6<br/>
| 0<br/>
| 1<br/>
| 25
| 3.5
|-
| L7<br/>
| 0<br/>
| 2<br/>
| 25
| 3.5
|-
| L8<br/>
| 0<br/>
| 3<br/>
| 25
| 3.5
|-
| L9<br/>
| 0<br/>
| 4<br/>
| 25
| 3.5<br/>
|-
| L10<br/>
| 0<br/>
| 5<br/>
| 25<br/>
| 3.5
|}



=== Transfection ===

10/13/14

293T,&nbsp; 24 wells

Plate 1

{| width="484" cellspacing="1" cellpadding="1" border="1"
|-
| 
1:30 L1

| L2<br/>
| L3<br/>
| L4<br/>
| L5<br/>
| L6<br/>
|-
| L7<br/>
| L8<br/>
| L9<br/>
| L10<br/>
| Unconjugated PHP-TREN-B1<br/>
| PHP-Cho-Gu<br/>
|-
| 1:50 L1<br/>
| L2<br/>
| L3<br/>
| L4<br/>
| L5<br/>
| L6<br/>
|-
| L7<br/>
| L8<br/>
| L9<br/>
| L10<br/>
| Unconjugated PHP-TREN-B1<br/>
| PHP-Cho-Gu<br/>
|}



Plate 2

{| width="484" cellspacing="1" cellpadding="1" border="1"
|-
| 
1:75 L1

| L2<br/>
| L3<br/>
| L4<br/>
| L5<br/>
| L6<br/>
|-
| L7<br/>
| L8<br/>
| L9<br/>
| L10<br/>
| Unconjugated PHP-TREN-B1<br/>
| PHP-Cho-Gu<br/>
|-
| 1:100 L1<br/>
| L2<br/>
| L3<br/>
| L4<br/>
| L5<br/>
| L6<br/>
|-
| L7<br/>
| L8<br/>
| L9<br/>
| L10<br/>
| Unconjugated PHP-TREN-B1<br/>
| PHP-Cho-Gu<br/>
|}

=== <br/>Result ===

10/14/14

[[File:PHP Cho 2014 10 14.jpg|850px|PHP Cho 2014 10 14.jpg]]



[[File:PHP Cho 2014 10 14 2.jpg|850px|PHP Cho 2014 10 14 2.jpg]]

10/15/14

[[File:PHP TREN B1 PEI18 Cho 10 15 14 1.JPG|RTENOTITLE]]

[[File:PHP TREN B1 PEI18 Cho 10 15 14 2.JPG|RTENOTITLE]]



10/17/14

[[File:PHP TREN B1 PEI18 Cho 10 17 14 1.JPG|RTENOTITLE]]

[[File:PHP TREN B1 PEI18 Cho 10 17 14 2.JPG|RTENOTITLE]]

== Comparison ==

=== Comparion 1 ===

10/11/14

Experiment done by Shanyi

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| 1:30
| A3
| B1
| K10
| K11
| K12
| L3K
|-
| 1:50
| A3
| B1
| K10
| K11
| K12
| L3K
|}



[[File:Cho 10 11 14 1.JPG|RTENOTITLE]]



=== Comparison 2 ===

[[File:Comparison 204 10 15.png|850px|Comparison 204 10 15.png]]

10/17/14

[[File:Comparison 204 10 17.JPG|RTENOTITLE]]



=== Comparison 3 ===

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

{| width="526" cellspacing="1" cellpadding="1" border="1"
|-
| 1:30
| L
| LP
| B1
| K10
| L6
| <br/>
|-
| 1:50
| L
| LP
| B1
| K10
| L6
| L2K
|-
| 1:75
| L
| LP
| B1
| K10
| L6
| L3K
|}



10/17/14

[[File:Comparison 204 10 17 2.JPG|RTENOTITLE]]

10/18/14

[[File:Comparison 204 10 18.JPG|RTENOTITLE]]



=== Comparison 4 ===

'''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





{| width="624" cellspacing="1" cellpadding="1" border="1"
|-
| 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<br/>
| PHP-TREN-A3-CHo-Gu<br/>
|-
| N1<br/>
| PHP-TREN-A3-CHO-Gu, 25 ul + 1 ul soluble PEI18-Cho<br/>
|-
| N2<br/>
| PHP-TREN-B1-Cho-Gu, 10-9-14, unpurified<br/>
|-
| N3<br/>
| PHP-TREN-B1-Cho-Gu, 10-9-14, purified using isopropanol<br/>
|-
| N4<br/>
| PHP-TREN-B1-Cho<br/>
|-
| B1<br/>
| <br/>
|}



[[File:Comparison-10-18-2014 s.jpg|850px|Comparison-10-18-2014 s.jpg]]

10/19/14

[[File:Comparison 204 10 19.JPG|RTENOTITLE]]

10/20/14

[[File:Comparison 204 10 20.JPG|RTENOTITLE]]



=== Comparison 5 ===

'''To Make PHP-TREN-B1-Cho and PHP-TREN-B1-PEI18-Cho'''

10/16/14

{| width="624" cellspacing="1" cellpadding="1" border="1"
|-
| B1<br/>
| PHP-TREN-B1<br/>
|-
| K10<br/>
| PHP-TREN-B1-Cho<br/>
|-
| 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<br/>
| 25 ul M1 + 2.5 ul Cho-Chl in DCM, 5 ug/ul---->to make PHP-TREN-B1-Cho<br/>
|-
| M4<br/>
| 25 ul M2 + 2.5 ul 100 mg/ml DSP---> to make PHP-TREN-B1-PEI18-Cho<br/>
|}

10/20/14: Transfection



10/21/14

[[File:Comparison 2014 10 21.png|RTENOTITLE]]

10/22/14

[[File:Comparison 2014 10 22.JPG|RTENOTITLE]]



10/23/14

[[File:Comparison 2014 10 23.JPG|RTENOTITLE]]
[[Category:Paul]] [[Category:Transfection]]