I had a gnome bard (the one in Sharn: CoT) write a play about the characters, spinning everything they did into a "comedic" piece. Then he dissed them in their own inn. And backed the fighter's opponent for a city council seat. Finally, he slipped, invisible, into the room of the little girl they were guarding, and cast an illusion of her laying in bed, with a dagger in her heart.
They paid the Tyrants (a group of doppelgangers) to set the bard up for murder, and then sneaked into the jail and slit his throat in his sleep. But they really hated that guy.
There was also a pair of Warforged who set first crossed them in the Forgotten Forge (the adventure in the back of the ECS). They cost them a pit fight by cheating, accosted the rogue in an alleyway, and (in my best plan yet) even bailed them out of jail, just to do the "evil monologue". The PC's killed them in a pit fight surrounded by web spells, with the help of a local guild leader.
They also hated Lucan Stellos from "Whisper of the Vampire's Blade", for his dodgy, never quite willing to stand up and fight attitude.
I think the main way to make your players hate your npcs is to set up a few encounters with the npcs where the pcs can't get a hand up on them. After two or three encounters, the players will be chomping at the bit to put them in their place.