Cyclodextrin-based nanosponges (NS) are solid nanoparticles, obtained from the cross-linking of cyclodextrins that have been proposed as delivery systems for many types of drugs. Various NS derivatives are currently under investigation in order that their properties might be tuned for different applications. In this work, new carboxylated cyclodextrin-based nanosponges (Carb-NS) carrying carboxylic groups within their structure were purposely designed as novel Acyclovir carriers. TEM measurements revealed their spherical shape and size of about 400 nm. The behaviour of Carb-NS, with respect to the incorporation and delivery of Acyclovir, was compared to that of NS, previously investigated as a drug carrier. DSC, XRPD and FTIR analyses were used to investigate the two NS formulations. The results confirm the incorporation of the drug into the NS structure and NS-Acyclovir interactions. The Acyclovir loading into Carb-NS was higher than that obtained using NS, reaching about 70% (w/w). In vitro release studies showed the release kinetics of Acyclovir from Carb-NS to be prolonged in comparison with those observed with NS, with no initial burst effect. The NS uptake into cells was evaluated using fluorescent Carb-NS and revealed the nanoparticle internalisation. Enhanced antiviral activity against a clinical isolate of HSV-1 was obtained using Acyclovir loaded in Carb-NS. © 2012 Elsevier B.V. All rights reserved.