Bemiparin (fractionated low molecular weight heparin)-loaded nanoparticles were prepared by two consecutive w/o emulsions and an inversion to an o/w emulsion for various polymer systems as controlled release formulations. New synthetic block copolymers, poly(methyl methacrylate-b-trimethyl aminoethyl methacrylate) (PMMA-b-PMAETMA), with controlled microstructure and molecular weight, were prepared by RAFT (Reversible Addition-Fragmentation chain-Transfer) polymerization creating a set of polymers with different amounts of cationic charges. For comparison, a non-biodegradable positively charged polymer, Eudragit® RS PO, and a biodegradable polymer poly(lactic-co-glycolic acid), PLGA, were used. The microstructural arrangement of MMA and MAETMA sequences in PMMA-b-PMAETMA results in self-assembled core-shell nanoparticles in water with a positively charged surface, which interacts with bemiparin. The formulations were evaluated in terms of particle size, zeta potential and morphology by scanning electron microscopy (SEM). The entrapment of bemiparin molecules was confirmed by a negatively increased zeta potential value and the detection of a sulfur signal by energy dispersive X-ray spectroscopy (EDAX). High encapsulation efficiency was reached with all the polymeric matrices, ranging from 89 to 98%. Systems prepared with synthetic block copolymers PMMA-b-PMAETMA and PLGA showed higher in vitro bemiparin release than Eudragit® RS PO systems. For each formulation, bemiparin released from nanoparticles preserved its biological activity as shown by the BaF32 cell proliferation assay in the presence of fibroblast growth factor (FGF2).