solutions of ha and of edc were mixed to yield a preparation system of ha and edc each at final concentrations of 1 7 mg ml the ph of the preparation system was adjusted to 3 by titration with 1n hcl the preparation system was incubated for a time period at 37 u00b0 c with stirring table 3 shows an example of variation in the pre incubation time period for reacting ha with edc a pre incubation period of 3 hours is preferred to activate the carboxylic residues of ha table 3______________________________________effects of pre incubationha liposome binding a pre incubation mg haperiod hours bound mmole lipid______________________________________0 01 03 22 8 u00b1 0 924 20 9 u00b1 2 8______________________________________ a liposomes are luvet incubation was at 37 u00b0 c incubation of complete reaction mixture at ph 3 with the addition of borate buffer for 24 hours after the pre incubation period pe liposome samples were added and followed by the addition of a 0 1m borate buffer at ph 8 5 the ha pe liposome mixture was incubated at 37 u00b0 c in a shaker bath for 24 hours removal of excess unbound ha and reagents was by ultracentrifugation and washings initial and final concentrations of ha lipid are reported in table 5 various parameters affect the successful binding of ha to pe liposomes when using edc as the crosslinking reagent these parameters include a pre incubation procedure ph of the reaction mixture use of buffer solution in the incubation system and the contact area between liposomes and ha tables 4 and 5 provide data on variations of these parameters.