Paul.xu at 14:07, 1 November 2014

2014-11-01T14:07:08Z

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== PHP-TREN co-polymer synthesis ==

8/6/14

{| width="372" cellspacing="1" cellpadding="1" border="1"
|-
| monomer 
| TREN-1 
| TREN-2 
| TREN-3 
|-
| DBP 
| 184 mg 
| 184 mg 
| 184 mg 
|-
| DAP 
| 38 mg 
| 38 mg 
| 0 
|-
| TREN 
| 61 mg 
| 30 mg 
| 61 mg 
|-
| MeOH
| 150 ul
| 150 ul
| 150 ul
|-
| 
| Form solid rx product after adding TREN 
| yellowish liquid 
| form clear crystal, it sticks on the wall, gel-like polymer, not soluble in water 
|-
| 
| DNA binding using Sybe-Gold: good 
| DNA binding using Sybe-Gold: good 
| DNA binding using Sybe-Gold: MeOH soluble, ok, water phase is gel-like suspension, not binding to DNA 
|}

 react at 50 oC for 24 hours 

'''DAP is required for  polymer synthesis''' 

Purify polymer by adding isopropanol to ppt TREN-1 and TREN2. Add water to dissolve pellet 

== Transfection ==

O(ornithine)+DBP+AG(agmatine); see page 000726 for synthesis 

 

Before Isopropanol Purification

{| width="600" cellspacing="1" cellpadding="1" border="1"
|-
|
O(ornithine)+DBP+AG(agmatine) 

 

| O+CBA+AG 
| TREN-1, cells are dead 
| TREN-2, cells are dead 
| TREN-3, MeOH 
| TREN-3, H2O 
|-
| O+DBP+AG, 1:10 dilution 
| O+CBA+AG, 1:10 dilution 
| TREN-1, 1:10 dilution 
| TREN-2, 1:10 dilution  
| TREN-3, 1:10 dilution
| TREN-3, 1:10 dilution
|-
| GuP-1 
| GuP-2 
| GuP-3 
| Stability-1 
| 
| GFP 
|}

Picture of 8-10-14 

[[Image:TREN before purification.JPG]] 

 

After Purification 

{| width="503" cellspacing="1" cellpadding="1" border="1"
|-
| QC: 8-15-14, 1:10 
| QC: 8-15-14-P, 1:10 
| TREN-1-P, 1:10, 90% cells are dead 
| TREN-1-P, 1:30 
| TREN-2-P, 1:10 
| TREN-2-P, 1:30 
|-
| 8-15-14, 1:30  
| 8-15-14-P, 1:30
| L3K 
| L2K 
| TurboFect 
| ViaFect 
|-
| 8-15-14, 1:50
| 8-15-14-P, 1:50
| GuP-1 
| 
| 
| 
|}

Picture of 8-10-14 

[[Image:QC 8 15 14 TREN 8 10 14.JPG]] 

8/11/14

[[Image:QC 8 15 14 TREN 8 11 14.JPG]] 

8/12/14 

[[Image:QC 8 15 14 TREN 8 11 14.JPG]] 

 

== Synthesis of PHP-TREN ==

8/16/14 

Mix A: 184 mg DBP, 38 mg DAP, 75 ul MeOH 

Divide into 3 vials 

Mix B: 184 mg DBP, 75 mg DAP, 75 ul MeOH 

Divide into 3 vials 

Reactions 

{| width="379" cellspacing="1" cellpadding="1" border="1"
|-
| 
| A1 
| '''A2''' 
| '''A3''' 
| B1 
| B2 
| B3 
|-
| DBP 
| 61 mg 
| 61 mg 
| 61 mg
| 61 mg
| 61 mg
| 61 mg
|-
| DAP 
| 12.5 mg 
| 12.5 mg    
| 12.5 mg
| 25 mg
| 25 mg
| 25 mg
|-
| TREN 
| 10 mg in 75 ul MeOH 
| 20 mg in 75 ul MeOH 
| 30 mg in 75 ul MeOH 
| 10 mg in 75 ul MeOH 
| 20 mg in 75 ul MeOH 
| 30 mg in 75 ul MeOH 
|-
| Total MeOH 
| 150 ul 
| 150 ul 
| 150 ul 
| 150 ul 
| 150 ul 
| 150 ul 
|-
| Appearance 
| yellowish oil 
| white ppt + yellowish oil 
| white ppt + yellowish oil 
| yellow/white gel, white gel is not soluble in H2O 
| white polymer 
| white polymer 
|}

50 oC react o/n (24 hours) 

Reaction Products 

[[Image:PHP TREN reaction products 8 2014.JPG]] 

Add 1 ml isopropanol to ppt. Dissolve pellet in 500 ul water 

dilute in 10 mM HEPES, pH 7.0: 1:30 and 1:10 dilutions 

'''8/18/14 Transfection'''

use 3 ul 1:10 and 1:30 diluted polymer, 1 ug DNA, 24 well plate 

{| width="363" cellspacing="1" cellpadding="1" border="1"
|-
| 1:10 A1 
| 1:10 A2 
| 1:10 A3 
| 1:10 B1 
| 1:10 B2 
| 1:10 B3 
|-
| 1:30 A1 
| 1:30 A2 
| 1:30 A3 
| 1:30 B1 
| 1:30 B2 
| 1:30 B3 
|-
| 8/6/14 TREN1 1:10 
| 8/6/14 TREN1 1:30 
| 8/6/14 TREN2 1:10 
| 8/6/14 TREN2 1:30 
| ProteinOne liposome #1 DOTAP-DOPE 
| ProteinOne liposome #2 DOTAP-DOPE-PEG 
|-
| L2K 
| 
| 
| 
| 
| 
|}

8/19/14

[[Image:PHP-TREN 8 19 14 2.JPG]] 

8/20/14

[[Image:PHP-TREN 8 20 14 1.JPG]]

8/22/14

[[Image:PHP-TREN 8 22 14 1.JPG]]

{| width="592" cellspacing="1" cellpadding="1" border="1"
|-
| A1, 1:10, 2 ug DNA 
| A2, 1:10, 2 ug DNA 
| A3, 1:10, 2 ug DNA 
| B1, 1:10, 2 ug DNA 
| B2, 1:10, 2 ug DNA 
| B3, 1:10, 2 ug DNA 
|-
| L2K, 2ugDNA 
| 
| 
| 
| 
| 
|}

8/19/14 

[[Image:PHP-TREN 8 19 14 1.JPG]] 

8/20/14 

[[Image:PHP-TREN 8 20 14 2.JPG]]

 

8/22/14

[[Image:PHP-TREN 8 22 14 2.JPG]]

 

=== Transfection of Diluted PHP-TREN ===

8/22/2014: 

Transfection, 1ug DNA, 293T 

{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Dilution 
| 1:30 
| 1:50 
| 1:75 
| 1:30 
| 1:50 
| 1:75 
|-
| rowspan="4" | 
| A1 
| A1 
| A1 
| A2 
| A2 
| A2 
|-
| A3 
| A3 
| A3 
| B1 
| B1 
| B1 
|-
| B2 
| B2 
| B2 
| B3 
| B3 
| B3 
|-
| TREN1 
| TREN1 
| TREN1 
| TREN2 
| TREN2 
| TREN2 
|}

Conclusion: A2 and '''A3 '''are the best

Picture 8/23/2014 

[[Image:PHP TREN 8 23 2014.jpg]]

8/24/14

[[Image:PHP-TREN 8 24 14.JPG]]

8/25/14

[[Image:PHP-TREN 8 245 14.JPG]]

8/26/14

[[Image:PHP-TREN 8 26 14.JPG]]

8/27/14

[[Image:PHP-TREN 8 27 14.JPG]]

 

== Scale up synthesis of PHP-TREN ==

'''Synthesis''' 

9/6/14 

369 mg DAP 

1.8 g DBP 

+ 1 ml MeOH to dissolve 

+ 950 mg TREN (should be 885 mg, 6.5 mmole,  but added too much) + 2.0 ml MeOH 

Mix and react at 50 oC o/n, 24 hours 

+ 30 mg cystamine --> 60 0C react 3 hours 

+ 7 ml isopropanol, mix, spin 5 min-->brownish oil-like bottom phase, remove top phase (isopropanol) 

+ 5ml H2O--> 7.5 ml total vol, take out 10 ul for transfection assay 

Add 200 mg pyrazole-1-carboxyamidine hydrochloride, react at 25 oC for 2 hours---> take 10 ul for transfection assay 

Add 10 ml isopropanol, spin--remove top phase (isopropano). 

'''Transfection''' 

9/8/14 

1:30, 1:50, 1:75, 1:100 dilution 

3 ul diluted + 1 ug DNA 

{| width="404" cellspacing="1" cellpadding="1" border="1"
|-
| PHP-TREN, 1:30 
| GU-PHP-TREN, 1:30 
| PHP-DSP, 1:30 
| PHP-TREN, A2, 1:50 
| L3K 
| 
|-
| PHP-TREN, 1:50 
| GU-PHP-TREN, 1:50
| PHP-DSP, 1:50  
| PHP-TREN, A3, 1:50 
| L2K 
| 
|-
| PHP-TREN, 1:75
| GU-PHP-TREN, 1:75
| PHP-DSP, 1:75
| PHP-TREN, TREN1, 1:100 
| 
| 
|-
| PHP-TREN, 1:100
| GU-PHP-TREN, 1:100
| PHP-DSP, 1:100
| PHP-TREN, B1, 1:50 
| 
| 
|}

9/9/2014 morning 

[[Image:PHP TREN vs PHP DSP 2014 9 9.JPG]] 

9/9/2014 afternoon 

[[Image:PHP TREN vs PHP DSP 2014 9 9 2.JPG]]

9/10/14

[[Image:PHP TREN vs PHP DSP 2014 9 10 1.JPG]]

[[Image:PHP TREN vs PHP DSP 2014 9 10 2.JPG]]

9/11/14 

[[Image:PHP TREN vs PHP DSP 2014 9 11 1.JPG]][[Image:PHP TREN vs PHP DSP 2014 9 11 2.JPG]]

9/12/14 

[[Image:PHP TREN vs PHP DSP 2014 9 12 2.JPG]] 

[[Image:PHP TREN vs PHP DSP 2014 9 12 1.JPG]] 

9/13/14

[[Image:PHP TREN vs PHP DSP 2014 9 13 3.JPG]]

[[Image:PHP TREN vs PHP DSP 2014 9 13 2.JPG]]

[[Image:PHP TREN vs PHP DSP 2014 9 13 1.JPG]]

9/13/14 afternon

[[Image:PHP TREN vs PHP DSP 2014 9 13 4.JPG]]

9/14/14

[[Image:PHP TREN vs PHP DSP 2014 9 13 5.JPG]]

9/14/14

{| width="660" cellspacing="1" cellpadding="1" border="1"
|-
| GU-PHP_DSP,1:30 
| GU-PHP-TREN, B1, 1:30 
| GU-PHP-TREN, A3, 1:30 
| PHP-TREN, B1, 1:30 
| L3K 
| GU-PHP-TDSP, 1:30 
|-
| GU-PHP_DSP, 1:50 
| GU-PHP-TREN, B1,   1:50
| GU-PHP-TREN, A3,  1:50  
| PHP-TREN, B1, 1:50 
| L2K 
| GU-PHP-TDSP, 1:50 
|-
| GU-PHP_DSP, 1:75
| GU-PHP-TREN, B1, 1:75
| GU-PHP-TREN, A3, 1:75
| PHP-TREN, B1, 1:75 
| GU-PHP-TREN, A3, 1:30 
| GU-PHP-TREN, B1, 1:30 
|-
| GU-PHP_DSP, 1:100 
| GU-PHP-TREN, B1, 1:100
| GU-PHP-TREN, A3, 1:100
| Mix of Gu_PHP-DSP and Gu_PHP-TREN, A3, at 3:1 ratio, 1:30 
| GU-PHP-TREN, A3, 1:50
| GU-PHP-TREN, B1, 1:50 
|}

[[Image:PHP TREN vs PHP DSP 2014 9 14.JPG]]

 

== PHP-TREN-B1 Large Scale ==

=== Synthesis ===

10/25/14 

1.5 g DAP + 3.6 g DBP + 1.5 ml MeOH, dissolve, feel heat 

+ (200 ul TREN + 0.5 ml MeOH) 

mix 50 oC react o/n 

add 1 ml MeOH--> 60 oC for 3 hours 

=== Purification ===

10/26/14 

+ H2O to 10 ml, + 40 ml isopropanol, spin, repeat once 

dissolve in H2O and adjust to 10 ml-->P 

 

Derivatization 

{| width="494" cellspacing="1" cellpadding="1" border="1"
|-
| P1 
| PHP-TREN-B1-Cho 
| 25 ul P + 2.5 ul 0.5 mg/ul Cho-Chl
|-
| P2 
| PHP-TREN-B1-PEI 
| 25 ul P + 2.5 ul 0.4 mg/ml PEI + 3.5 ul DSP 
|-
| P3 
| PHP-TREN-B1--PEI-Cho 
| 25 ul P + 2.5 ul PEI-Cho + 3.5 ul DSP 
|}

=== Transfection ===

10/27/14 

R: PHP-TREN-A3-Cho-Gu (oil-like) 

RP: purify R using isopropanol 

Q: PHP-TREN-B1-Cho-Gu, 10-9-14, white pellet 

QP: purified Q using isopropanol 

=== Result ===

10/29/14 

[[Image:PHP-TREN-B1 2014 10 29.JPG]] 

 

10/30/14 

cell dead RP, P2, P3

[[Image:PHP-TREN-B1 2014 10 30.JPG]]

 

10/31/14

[[Image:PHP-TREN-B1 2014 10 31.JPG]]

11/1/14

[[Image:PHP-TREN-B1_2014_11_1.JPG]]

[[Category:Transfection]] [[Category:Paul]]

Synthesis of PHP-TREN - Revision history

Paul.xu at 14:07, 1 November 2014