Description

During the past five decades, various types of chemistries have been used for conjugation of molecules such as antibodies, peptides, proteins or other reactive ligands to the surface of liposomes. In general, the conjugation can be achieved through the N-terminus, the C-terminus or the available sulfur (e.g. Fab’ fraction or thiolated antibodies). Not all chemistries have the same yield and efficiency of conjugation and often reproducing biocompatible batches can be a challenge.

Copper-free click chemistry is a fairly new chemistry that has been commercialized during the past few years. More and more click chemistry-based reagents are becoming available commercially which makes the formulation development much easier for scientists. The great advantage of this chemistry is biocompatibility since no cytotoxic copper catalyst is required. By far, click chemistry is the most efficient and easiest conjugation chemistry available for coupling of antibodies and other reactive ligands to the surface of the liposomes. The conjugation chemistry is based on the reaction of the dibenzocyclooctyne (DBCO) reagent with an azide linker to form a stable triazole. DBCO moiety can be on the antibody and azide moiety can be on liposomes and vice versa. Here, azide moiety is on the liposome and DBCO moiety is on the antibody, protein or peptide.

There are many commercialized reagents that can be used for DBCO modification of proteins, peptides and antibodies. To see the list of commercialized reagents for DBCO modification see here.

Immunodox®-Azide is a PEGylated product. For other Immunodox® products suitable for other types conjugation methods see here.

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Formulation Information

Immunodox®-Azide (PEGylated)

Lipid Composition	Concentration (mg/ml)	Concentration (mM)	Molar Ratio Percentage
Total	15.90 mg/ml	21.58 mM	100
Hydrogenated Soy PC	9.58	12.22	57
Cholesterol	3.19	8.25	38
DSPE-PEG(2000)	2.5	0.89	4
DSPE-PEG(2000)-Azide	0.63	0.22	1

Conjugation Protocol

Materials and Equipment

You need the following materials and equipment in order to use the kit.

1. Laboratory vortex mixer is recommended to have.
2. Laboratory magnetic stirrer is needed for dialysis.
3. Float-A-Lyzer® with a proper MWCO that easily allows the cleanup of your liposome conjugated ligand from free and non-conjugated protein/peptide/ligand. You need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore your liposomes can be dialyzed out. You cannot use dialysis cassettes blindly. Please understand the technique before using either spin column or dialysis cassette. If you do not use the correct MWCO, you can lose your entire prep. For this protocol, we recommend MWCO of 300,000 dalton.

Preparation Method

1. The total lipid concentration in Immunodox®-Azide is 21.58 mM. 1% mol of the lipid in liposomes contains azide group and only half of them are exposed to the outside of the liposomes, which is equal to 0.11 mM of reactive conjugable lipid. For 2 ml volume liposome, this is equal to 2.20×10^-7 mol, and for 5 ml volume liposome, this is equal to 5.50×10^-7 mol of azide. Add 1 mol equivalent of Azide-lipids in liposomes to 2.5 mol equivalents of DBCO containing protein. Incubate the mixture of liposome and antibody at room temperature for 4 h followed by overnight incubation at 4 °C in a refrigerator.
2. Remove the non-conjugated protein, peptide or antibody from the immunoliposomes by dialysis. We prefer dialysis to size exclusion columns. Dialysis is a much slower process but there will be minimum loss of immunoliposomes after the prep is cleaned from non-conjugated protein/peptide/ligand. Spin columns are much faster; however, you can easily lose over 50% of the liposomes on the spin column. We recommend using Float-A-Lyzer® dialysis cassette from Spectrum Labs. You will need to choose a cassette with proper MWCO depending on the MW of your protein, peptide, antibody or antibody fragment. NOTE: If you decide to use a dialysis cassette, you will need to make sure that the MWCO is below 1,000,000 dalton. At 1,000,000 dalton, the pore size on the dialysis membrane gets close to 100 nm and therefore, your liposomes can be dialyzed out. You cannot use dialysis cassettes and spin columns blindly. They come in various sizes and you need to choose the correct size wisely. Dialyze the immunoliposome solution in 1 liter of PBS at pH 7.4 for 8 hours. Change the dialysis buffer with a fresh 1 liter of PBS and let is dialyze for another 8 hours. After this step, your cleaned up immunoliposome is ready to be used.

Liposome Particle Calculator

Immunodox® liposomes are unilamellar and sized to 100 nm. The molar concentration of liposome is 21.58 mM. By having liposome diameter (nm) and lipid concentration (µM), you can calculate the total number of the lipids in one liposome and the number of the liposomes in one milliliter of the liposome solution. To use the calculator click here.

Technical Notes

• Doxorubicin is a fluorescent molecule with λ_ex 470 nm and λ_em 585 nm. If you are using a fluorescent tag on your antibody or ligand, you need to make sure that they will not interfere with each other.
• Before starting the conjugation process please make sure to avoid buffers that contain azides, which can react with DBCO.
• Reactions of DBCO and azides are more efficient at high concentrations and temperatures (i.e., up to 37°C). In order to avoid denaturation of proteins, peptides and antibodies it is recommended to incubate molecules with liposomes at room temperature followed by refrigeration (See step 2).
• Typical reaction times are less than 12 h; however, incubating for longer can improve efficiency.
• Spin columns can be used for the immunoliposome separation, and they are very fast method for purification. However, a large quantity of the samples are lost on the column. Dialysis is a slower process with minimal sample loss and therefore, we recommend dialysis over spin columns.
• If you are using a ligand or peptide that is hydrophobic then it is recommended to solubilize it in DMSO or DMF and then add the buffer to it. It is recommended not to use more than 5% volume of DMSO or DMF in the solution. DMF and DMSO are both compatible with liposomes and they are also miscible in water. Other organic solvent such as ethanol and chloroform are not compatible with liposomes and will cause the liposomes to lyse. If you end up using DMSO or DMF then after the conjugation reaction is done, you need to remove DMSO and DMF from the liposomes. In order to do that you need to use a dialysis cassette that is made from REGENERATED CELLULOSE MEMBRANE. NOTE: Not all membranes are compatible with DMF and DMSO. We recommend using a Slide-A-Lyzer™ MINI Dialysis Device with MWCO of 2K made from regenerated cellulose membrane manufactured by ThermoFisher. After DMSO or DMF is removed, you can use Float-A-Lyzer® dialysis device for the final step of cleaning up the prep.
• Liposomes should be kept at 4°C and NEVER be frozen.

Database

Direct link to the database page for easy navigation: Immunoliposomes Conjugation Database

Appearance

Immunodox®-Azide is a red translucent liquid made of nano size unilamellar liposomes. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. The liposomes are packaged in an amber vial.

Educational Video

Ordering/Shipping Information

• All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
• Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
• Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
• We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
• Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
• If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
• Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.

Storage and Shelf Life

Storage

Immunodox® products should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. If the suspension is frozen, the encapsulated drug can be released from the liposomes thus limiting its effectiveness. In addition, the size of the liposomes will also change upon freezing and thawing.

Shelf Life

Immunodox®-Azide is made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 4 months of the manufacturing date.

References and background reading

1. Wang Y, Xie Y, Li J, Peng ZH, Sheinin Y, Zhou J, Oupický D. Tumor-Penetrating Nanoparticles for Enhanced Anticancer Activity of Combined Photodynamic and Hypoxia-Activated Therapy. ACS nano. 2017 Feb 6;11(2):2227-38.

2. Kunjachan S, Ehling J, Storm G, Kiessling F, Lammers T. Noninvasive imaging of nanomedicines and nanotheranostics: principles, progress, and prospects. Chemical reviews. 2015 Jul 13;115(19):10907-37.

3. Wang, Hua, Marianne Gauthier, Jamie R. Kelly, Rita J. Miller, Ming Xu, William D. O’Brien, and Jianjun Cheng. “Targeted Ultrasound‐Assisted Cancer‐Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry.” Angewandte Chemie 128, no. 18 (2016): 5542-5546.

4. D’souza AA, Shegokar R. Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications. Expert opinion on drug delivery. 2016 Sep 1;13(9):1257-75.

5. Oude Blenke E, Sleszynska M, Evers MJ, Storm G, Martin NI, Mastrobattista E. Strategies for the activation and release of the membranolytic peptide melittin from liposomes using endosomal pH as a trigger. Bioconjugate chemistry. 2017 Jan 13;28(2):574-82.

6. Zhang H, Weingart J, Jiang R, Peng J, Wu Q, Sun XL. Bio-inspired liposomal thrombomodulin conjugate through bio-orthogonal chemistry. Bioconjugate chemistry. 2013 Mar 15;24(4):550-9.

7. Marqués-Gallego P, de Kroon AI. Ligation strategies for targeting liposomal nanocarriers. BioMed research international. 2014 Jul 14;2014.