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Scheme of chitosan nanoparticle and lipochitoplex formation.Chitosan as a polycationic non-viral vector for gene delivery has the advantage of being a biocompatible and biodegradable polymer. However, without laborious chemical modifications to its structure, it is of limited use as a gene delivery vehicle due to its low ability to efficiently transfect under physiological conditions. To address this problem, we developed novel liposome encapsulated chitosan nanoparticles; lipochitoplexes (LCPs). Chitosan nanoparticles (CsNPs) were obtained using the ionic gelation technique. For this purpose, an ultrapure low molecular weight chitosan with a high degree of deacetylation was cross-linked using polyanionic tripolyphosphate resulting in efficient entrapment of plasmid DNA (pDNA) inside the nanoparticles. LCPs were prepared by incubating chitosan nanoparticles together with anionic liposomes (DPPC/Cholesterol). The LCPs offered better pDNA protection, reduced cytotoxicity and at least twofold increase in the transfection efficiency under physiological conditions. The efficiency of our delivery vehicle was also proved in vivo in the chorioallantoic membrane model (CAM). LCPs were able to transfect the CAM without traumatising the surrounding blood vessels. This new biocompatible composite system devoid of chemical modifications, organic solvents and harsh production conditions makes it an optimal gene delivery vehicle for in vivo applications offering new insights into the field of non-viral gene therapy.