Hi Tarun,

I have prepared gelatin methacrylate using methacrylic anhydride (MAAh), but also with glycidyl methacrylate (GMA) which I think works slightly better. Here is a copy of my experimental protocol - hope it helps :)

1. Heat 250 mL of DI water in a 500 mL beaker to 50°C.

2. Add 25 g of gelatin to the beaker and stir through till dissolved fully (10 mins).

3. Roughly neutralise to pH 7.0 with 0.4 M NaOH solution (stable pH for 3 mins).

4. Dispense 10 g of gelatin solution into a 20 mL glass vial (on a top loading balance).

5. Volumetrically add required amount of GMA to the vial (density of GMA = 1.07 g.mL-1).

6. Leave to react at 40°C for 24 hours in the dark.

The temperature, pH and light are all fairly important parameters to control. Especially if you want to use MAAh because it will dramatically drop the pH - you want to keep the pH around 7-8.

Also, if you are wondering how much GMA is hypothetically needed to fully methacrylate the gelatin the hypothetical amount of groups in 1 g of gelatin that are able to react with GMA can be estimated as follows:

The residues in gelatin which are able to react with GMA are arginine, hydroxylysine, and lysine which comprise 7.8%, 1.0% and 3.5% of gelatin’s total amino acids by weight (ref http://www.gmap-gelatin.com/about_gelatin_AminoAcidComp.html), and equate to 448 µmol.g-1, 61.7 µmol.g-1, and 240 µmol.g-1, respectively, for a total of 750 µmol.g-1. The terminal amine of each gelatin molecule can also react with GMA, so for 87,500 g.mol-1 gelatin this would add 11.4 µmol.g-1 of reactable groups per gram of gelatin for a total of 761 µmol.g-1.

Seeing as the reaction stoichiometry between amines and epoxides is 1:1, the amount of GMA required to react with every available site in one gram of gelatin is 761 µmol which equates to 0.108 g.g-1 or 10.8 %(w/w).

Good luck!!!

You most probably formed a gelatin plug-like aggregate. What happens when you increase the temperature again at the end? Which pH does your reaction mixture has after the reaction? Did you avoid light? Maybe the Methacrylate groups are kind of crosslinked. You could try Acrylate instead of Methacrylate, which is less reactive.

You could try decreasing the gelatin solution concentration before adding the anhydride. The above mentioned question should be considered as well. There is evidence of undesired cyclization reactions involving carboxylic and hydroxyl groups in alcaline medium. 

Regards.

You could try increasing gelatin solution to 90 degree C, or decreasing the concentration of gelatin solution.

or you can add the methacrylic anhydride solution at a higher rate into the gelatin solution.

The acrylate would be more reactive and more prone to crosslinking than the methacrylate.  There are other ways to make gelMA.  You can use glycidyl methacrylate or use methacrylic acid with EDC and NHS.

http://onlinelibrary.wiley.com/doi/10.1002/mabi.201100232/full

http://polymer.zju.edu.cn/biomaterials/uploads/Publications/2012/210.pdf

Hi Tarun,

I have prepared gelatin methacrylate using methacrylic anhydride (MAAh), but also with glycidyl methacrylate (GMA) which I think works slightly better. Here is a copy of my experimental protocol - hope it helps :)

1. Heat 250 mL of DI water in a 500 mL beaker to 50°C.

2. Add 25 g of gelatin to the beaker and stir through till dissolved fully (10 mins).

3. Roughly neutralise to pH 7.0 with 0.4 M NaOH solution (stable pH for 3 mins).

4. Dispense 10 g of gelatin solution into a 20 mL glass vial (on a top loading balance).

5. Volumetrically add required amount of GMA to the vial (density of GMA = 1.07 g.mL-1).

6. Leave to react at 40°C for 24 hours in the dark.

The temperature, pH and light are all fairly important parameters to control. Especially if you want to use MAAh because it will dramatically drop the pH - you want to keep the pH around 7-8.

Also, if you are wondering how much GMA is hypothetically needed to fully methacrylate the gelatin the hypothetical amount of groups in 1 g of gelatin that are able to react with GMA can be estimated as follows:

The residues in gelatin which are able to react with GMA are arginine, hydroxylysine, and lysine which comprise 7.8%, 1.0% and 3.5% of gelatin’s total amino acids by weight (ref http://www.gmap-gelatin.com/about_gelatin_AminoAcidComp.html), and equate to 448 µmol.g-1, 61.7 µmol.g-1, and 240 µmol.g-1, respectively, for a total of 750 µmol.g-1. The terminal amine of each gelatin molecule can also react with GMA, so for 87,500 g.mol-1 gelatin this would add 11.4 µmol.g-1 of reactable groups per gram of gelatin for a total of 761 µmol.g-1.

Seeing as the reaction stoichiometry between amines and epoxides is 1:1, the amount of GMA required to react with every available site in one gram of gelatin is 761 µmol which equates to 0.108 g.g-1 or 10.8 %(w/w).

Good luck!!!

Hi Jaclyn,

the fact that you can not filtrate the dialyzed solution through a filter or that you can not dissolve the material after freeze-drying indicates that you formed a gel-like aggregate, possibily already before you perform dialysis. As Damian described in his answer: "The temperature, pH and light are all fairly important parameters to control. Especially if you want to use MAAh because it will dramatically drop the pH - you want to keep the pH around 7-8."

I suggest to use a strong buffer system (without any NH2 groups, this will react with the Methacrylic anhydride!), e.g. HEPES, PIPES or MOPS and always check the pH during the reaction. I attached a link giving an overview about different buffer systems.

Good luck!

http://en.wikipedia.org/wiki/Buffer_solution

Hi all, I have question, 

Instead of photo-crosslinking can I cross-linked the ethylene( c=c) group in GelMA  with primary amine or other functional groups.... can You suggest me the protocols if Any....

A carbonate–bicarbonate buffer system works much better for gelatin MA synthesis than PBS buffer. 

Please refer to the below article.

RSC Adv., 2015, 5, 106094

Efficient and controllable synthesis of highly

substituted gelatin methacrylamide for

mechanically stiff hydrogels

We report an effective and novel method to controllably produce highly substituted gelatin-MA with nearly 100% degree of substitution despite the use of a very low concentration of methacrylic anhydride (MAA). The method is based on sequential time-lapse loading of MAA after pH adjustment in a carbonate–bicarbonate buffer system. Rheological studies indicate that hydrogels formed from more highly substituted gelatin-MA exhibit significantly improved mechanical stiffness. 

RSC Adv., 2015, 5, 106094

Hi Bea Hoon, really interesting article you have! I was reading it carefully but didn't seen how do you actually adjust the pH of your Buffer. By adding NaOH? At which molarity?

many thanks in advance

MAA (Methacrylic anhydride) is added to gelatin solution for GelMA reaction right after the pH of gelatin solution in CB buffer is adjusted to 9 by using 5.0 N NaOH.

 Thank you very much

Full, peer-reviewed protocol here: http://www.nature.com/nprot/journal/v11/n4/full/nprot.2016.037.html

Precise Tuning of Facile One-Pot Gelatin Methacryloyl (GelMA) Synthesis

Scientific Reports 6, Article number: 31036 (2016)

Gelatin-methacryloyl (GelMA) is one of the most commonly used photopolymerizable biomaterials in bio-applications. However, GelMA synthesis remains suboptimal, as its reaction parameters have not been fully investigated. The goal of this study is to establish an optimal route for effective and controllable GelMA synthesis by systematically examining reaction parameters including carbonate-bicarbonate (CB) buffer molarity, initial pH adjustment, MAA concentration, gelatin concentration, reaction temperature, and reaction time. We employed several analytical techniques in order to determine the degree of substitution (DS) and conducted detailed structural analysis of the synthesized polymer. The results enabled us to optimize GelMA synthesis, showing the optimal conditions to balance the deprotonation of amino groups with minimizing MAA hydrolysis, which led to nearly complete substitution. The optimized conditions (low feed ratio of MAA to gelatin (0.1 mL/g), 0.25 M CB buffer at pH 9, and a gelatin concentration of 10–20%) enable a simplified reaction scheme that produces GelMA with high substitution with just one-step addition of MAA in one pot. Looking forward, these optimal conditions not only enable facile one-pot GelMA synthesis but can also guide researchers to explore the efficient, high methacrylation of other biomacromolecules.

i have met the same problem. i dissolved the gelatin(10% w/v) for more than two hours to make the clear solution as the article mentioned, but the solution seemed cloudy. After i filtered the solution with 0.22 filter, i got a yellow clear solution. in this case, i have problem that when you get the solution, it is yellow or without color? 

There are some good answers already. I'll add few points as per my few years of experience working with GelMA:

1) I have experienced that white thick mass during or after the addition of MA that only happened when I was preparing the GelMA the very first time. The reason was excessive evaporation of water from the solution. If a glass bottle is being used, try to cover the opening with paraffin film when it is uncapped and add MA by using using a needle through the paraffin film.

2) Even better, a two neck round bottom flask with a dropper can be used to add MA at desired rate by using the dropper knob. 

3) On a separate note, I found that the GelMA hydrogel depends a lot on the state of the GelMA prepolymer during UV crosslinking, For example, if you incubate the GelMA solution at 4C for sometime before you UV crosslink, the resulting hydrogel can be 5-8 fold stronger! We use his method routinely in lab to modulate the stiffness of GelMA hydrogel for different applications because it does not require addition of any other chemical component to increase the strength.

http://www.sciencedirect.com/science/article/pii/S0142961216307372