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Introduction: Hyaluronic acid (HA) is an anionic biopolymer composed of alternating disaccharide units of D-glucuronic acid and Nacetyl-D-glucosamine with (14)interglycosidic linkage [1]. It is the only nonsulfated glycosaminoglycan (GAG) that is abundant in the synovial fluid and extracellular matrix (ECM) [1-2]. The over all goal of this study is to fabricate and characterize nanospheres based on hyaluronan using an ionic gelation method for therapeutic sustained and controlled gene delivery. Materials and Methods: Plasmid DNA loaded HA nanospheres were prepared by ionic gelation followed by cross-linking method. In the present study nanospheres were prepared by 20% w/v sodium sulphate and cross linked by various crosslinkers i.e. glutaraldehyde, EDC/NHS and 4 arm star PEG. Nanospheres were characterized for particle size, zeta potential, SEM, TEM and in vitro DNA release. FITC-dextran loaded Nanospheres were also prepared following the same method and used for cell uptake studies using adipose derived stem cells (ADSCs). In vitro cell viability of crosslinked nanospheres was determined using Picogreen assay.