Biomaterial Synthesis and Characterisation

Standard Operation Procedures for Peptide Synthesis

Here, we report an example of a standard procedure for peptide synthesis in solid phase that we have optimised to functionalise the surface of biomaterials with anti-angiogenic peptides

APPROVALS

REVISIONS

Scope

To prepare QK peptide, produced from the helix region (17-25) of the VEGF binding interface (D’Andrea et al. PNAS (2005) 102; 40 p14215-14220). Sequence KLTWQELYQLKYKGI using the Biotage peptide synthesiser at the University of Brighton.

Purpose

To outline the assembly of a VEGF binding sequence KLTWQELYQLKYKGI. As peptide is assembled from the C-terminus, the assembly sequence will be IGKYKLQYLEQWTLK.

Materials

TITLE: ASSEMBLY OF GENERATION 3 POLYLYSINE SEMI-DENDRIMER             PABS: PEP-SYN-002

EFF. DATE: 17^thAPRIL 2007                                                                                                                          REVISION: 0

Equipment required:

 Pasteur Pipettes Unplugged (Long and short)

Clamp Stand

Boss Head

Clamp

50 cm³ Centrifuge Tubes

Centrifuge

Experimental method

Assembly of Peptide

• Remove all amino acids, Rink amide and Tenta Gel S resin from fridge or freezer and allow to reach ambient temperature (approximately 30 minutes) before opening containers.
• Weigh empty syringe and then add 0.5 g of TentaGel S NH₂ resin into the syringe.
• Add 5 mL DMF to syringe, gently shake and leave resin to swell for 15 minutes.
• Remove DMF from the syringe
• Add 0.4 mmoles of Rink amide linker to a glass vial, then add 0.4 mmole HBTU, 140 µL of DIPEA and add 3 mL DMF and sonicate for a few seconds to solubilise, and add to syringe.
• Gently shake syringe and leave for 30 minutes to react. Remove the solvent from the syringe.
• Wash resin with 3 x 5 mL DMF. Removing solvent from syringe after each addition of DMF.
• To deprotect add 5 mL of 20 % piperidine in DMF to syringe. Gently shake and leave for 2 minutes. Remove solvent.
• Repeat step 8 twice more.
• Next add 0.4 mmole amino acid to a glass vial and then add 0.4 mmole HBTU and 140 µL of DIPEA and add 3 mLDMF and sonicate for a few seconds to solubilise, and add to syringe.
• Gently shake syringe and leave for 30 minutes to react. Remove the solvent from the syringe.
• Wash resin with 3 x 5 mL DMF. Removing solvent from syringe after each addition of DMF.
• To deprotect add 5 mL of 20 % piperidine in DMF to syringe. Gently shake and leave for 2 minutes. Remove solvent.
• Repeat step 13 twice more.
• Repeat coupling and deprotection steps 10 to 14 for each amino acid. Once all amino acids in the sequence have been added, the resin should be washed to remove unreacted reagents.
• Add 5 mL of dichloromethane to syringe and transfer syringe contents to a pre-weighed 10 mL fritted syringe, repeat until all resin has been transferred to new syringe. Wash with 8 x 5 mL of dichloromethane
• Wash resin with 8 x 5 mL methanol.
• Wash resin with 8 x 5 mL diethylether and remove excess solvent allow to air dry for a few hours.
• The resin and syringe are then placed in a Christ Alpha 2-4 freeze dryer overnight to remove solvent and then the syringe and contents reweighed.

Cleavage of Peptide from Resin

1. To check that synthesis has been successful, remove resin from fridge and weigh 100 mg of resin into plastic weighing boat. For each 100 mg prepare 2 mL cleavage mixture. For composition of cleavage mixture see handout – Cleavage, Deprotection, etc of Peptides.
2. Add 2 mL cleavage mixture into a dropping vial, then add resin to vial taking care to add resin into the bottom of the vial. Add stopper to vial.
3. Leave for 3 hours, occasionally gently shaking vial.
4. Fill a polystyrene box with ice, then add 20 mL of diethylether into a unskirted 50 mL centrifuge tube and place in ice.
5. Assemble a short Pasteur pipette containing glass wool compacted down to a height of 0.5 cm.
6. Assemble a clamp stand over the box of ice and clamp in place the pipette containing glass wool above the centrifuge tube containing the diethylether.
7. Add the resin/peptide mixture in the pipette containing glass wool using a glass pipette. Once all the washings have passed down the glass wool containing pipette and collected in the diethylether, remove centrifuge tube from the ice and seal lid. Dry tube, then weigh it.
8. Into another centrifuge tube add an amount of water to match the mass of the tube containing the washings.
9. Place tubes in centrifuge directly opposite each other, making sure the lids are tightly sealed and the tubes are securely held in their holders.
10. Close lid and centrifuge at 3500 rpm at room temperature for 5 minutes.
11. After the cycle finishes remove tubes from centrifuge and decant off diethylether, taking care not to pour off pellet.
12. Add 20 mL diethylether, close lid then place centrifuge tube on mixer for 10 seconds to disperse the peptide pellet.
13. Repeat steps 8-13, three more times.
14. Decant off almost all the diethylether then dry the peptide pellet under a stream of helium gas, until constant weight.

• • None

Preparation of VEGF Blocker-Functionalised Gellan Gum Hydrogel Discs

Gellan gum (iGG-MA) hydrogel discs were prepared in according an established protocol recently published [Silva-Correira 2011] where iGG-MA powder was initially dissolved with VEGF blocker formulation in distilled water at the final concentration of 2 % w/v. Under continuous agitation at 42°C, a stable functionalised gellan gum solution was formed and deposited as droplets of 100 µL on the bottom of dry 24-well tissue culture plates (Fisher Scientific). The samples were left at 4°C overnight to allow the hydrogel setting. Non-functionalised iGG-MA hydrogel discs were made with the same procedure and used as control.

Functionalisation and characterisaiton of super paramagnetic nanoparticles coated by poly(acrylic acid) by hyperbranched peptides

Fe₃O₄@PAA (13 mg) were washed three times with 20 ml of ethanol. A 20 ml volume of 0.1 M MES buffer containing 38 mg EDC and 6 mg NHS was added, the solution was vortexed for 30 seconds and then placed in the shaking incubation for 30 minutes.  L-cysteine (25 mg, 0.2 mmole) was added and the mixture was shaken for 1 h. The MES buffer was removed and the beads washed three times with ethanol. The beads were then split into two tubes. To conjugate the dendrimers to the cysteine via a disulfide bridge, 10 ml 8 M urea containing 3 % w/v hydrogen peroxide were added along with (i) 5.3 mg, 1 µmole dendrimer and (ii) 3 mg, 1 µmole of dendrimer. The tubes were then returned to the shaking incubator for 30 minutes. Scanning electron microscopy analysis was performed by a FEG-SEM Zeiss SIGMA at 5 keV on palladium-coated specimens. DSC was performed twice at heating rate of 10 ^oC/minute, over the range -50 to 50 ^oC using a Mettler Toledo DSC 1 and STAR^eThermal analysis software. Thermal transitions were studied in the 25 ^oC to 50 ^oC.

Synthesis of Calcium Phosphate gels

CaP gels were synthesized at room temperature using calcium nitrate tetrahydrated [Ca(NO3)2$4H2O, 99.0%; Sigma-Aldrich] and diphosphorus pentoxide (P2O5 99.99%; Sigma-Aldrich) as precursors and using ethanol as the solvent.

In particular, the procedure (Fig. 1A) consists of adding phosphorus pentoxide (P2O5) to ethanol, and after 30 min, to obtain different degrees of crystallinity, the medium alkalinity was adjusted by drop-wise addition of NH4OH up to pH of 9 and 11 (P solution). Calcium nitrate tetrahydrated was dissolved in ethanol at room temperature (Ca solution). After mixing for 20 min, P solution was added drop wise to Ca solution in their required amounts to achieve a Ca/P ratio of 1.67. CaP solutions were placed in a shaking incubator at 100 rpm, at 37oC until gelling occurred. After gelling, the gels were dialyzed in 0.01M phosphate-buffered saline (PBS), pH 7.4, until equilibrated to the buffer pH.

Synthesis of Chitosan nanoparticles (CNPs)

Chitosan 1 % (w/v) in 1 % (v/v) acetic acid was filtered using a pore membrane (0.8 µm, Millipore) and diluted (1:4) with methanol. Succinic anhydride (≥ 99 % GC) was dissolved in 5 mL of acetone at 4 % (w/v) and dropwise added under magnetic stirring at 20 °C. The solution was left overnight³³. The formed gel was removed, properly diluted in methanol and dialysed against ultrapure water for 3 days. The precipitate was collected by centrifugation and lyophilised (Suc-chi). Suc-chi (aq) (1 % w/v) was mixed with liquid paraffin (x10eq), homogenised for 30 minutes at 24000 rpm. Successively, Span 85 solution was added (0.1 volume eq.) and homogenised. 1 M NaOH solution (0.3 volume eq.) was dropwise added and the solution was homogenised, properly diluted in toluene and washed by centrifugation (3000 rpm). The above reported procedure was repeated using ethanol and collected in water and lyophilised, obtaining CNPs. Chitosan, succinic anhydride and all the reagents to prepare CNPs were purchased from Sigma Aldrich.