295 Research Article Received: 23 August 2010 Revised: 10 January 2011 Accepted: 18 January 2011 Published online in Wiley Online Library: 31 March 2011 (wileyonlinelibrary.com) DOI 10.1002/mrc.2740 Prilocaine – cyclodextrin – liposome: effect of pH variations on the encapsulation and topology of a ternary complex using 1 H NMR Luís Fernando Cabec ¸a, a Isis Martins Figueiredo, a Eneida de Paula b and Anita Jocelyne Marsaioli a* A better comprehension of the prilocaine (PLC)– β -cyclodextrin (β -CD) complex liberation to membranes was provided by studying the architectural supramolecular arrangements of PLC, β -CD and egg phosphatidylcholine (EPC) liposomes, a membrane model. The topologies and possible interactions of mixtures of PLC, β -CD and EPC liposomes were investigated by nuclear magnetic resonances combining experimental 1 H-NMR (1D ROESY, STD and DOSY) at different pHs. The results indicate that in the mixture PLC/β -CD/EPC at pH 10 the PLC molecules are almost totally embedded into the liposomes and little interaction was observed between PLC and β -CD. However, at pH 5.5 not only was PLC imbedded in the EPC bilayer, but PLC was also interacting with β -CD. These results were rationalized as a spontaneous PLC release from β -CD to liposomes vesicles, whereas the PLC/EPC complex formation was higher at pH 10 than pH 5.5. Copyright c  2011 John Wiley & Sons, Ltd. Keywords: NMR; 1 H; 1D ROESY; DOSY; STD; prilocaine; cyclodextrins; liposome Introduction Prilocaine (PLC) is an aminoamide local anesthetic (LA) widely used in dentistry. It is pharmacologically similar to lidocaine the LA of greatest use in the world, with the advantage of lower toxicity. [1,2] PLC is usually associated with a vasoconstrictor to increase the anesthetic duration by keeping it at the site of action longer. [2] Adverse effects of the use of PLC are associated with the development of methemoglobinemia when used at high doses. [3] This is due to the action of ortho-toluidine, one of the breakdown products of PLC metabolism that is able to convert ferrous iron to ferric iron in hemoglobin. [4] Thus, the development of new LA formulations – intended to prolong the anesthetic action without increasing the systemic toxicity – is a current pharmaceutical challenge. [5] Consequently, pharmaceutical research targets new drug delivery systems that enhance the anesthetic action and decrease the toxicity, based on drug-liposome encapsulations or drug- cyclodextrin (CD) inclusion complexes. Liposomes are vesicles that consist of one or more lipid layers separated by aqueous compartments and are widely used in the pharmaceutical field as drug delivery systems due to their versatility and clinical efficacy. [6,7] They can encapsulate a hydrophilic drug in the aqueous compartment and a lipophilic drug within the lipid bilayer. [8] However, most organic compounds are either poorly water soluble or subject to some hydrolysis, making their encapsulation difficult. [8,9] This can be bypassed by adding CDs to the drug – liposome mixture. CDs [9] form inclusion complexes with many drugs, improving their aqueous solubility, chemical stability and bioavailability; however, these inclusion complexes can have the drug displaced by other molecules with greater affinity for the cavity and can be rapidly removed from blood circulation into urine, along with some CD, inducing toxicity to the kidneys, especially after chronic use. [10 – 12] Therefore, encapsulating therapeutic agents using multicomponent systems such as drug-in-CD-in- liposome is a strategy to overcome these problems by increasing aqueous solubility and therapeutic activity with an increasing drug-to-lipid mass ratio. [8,9,13] Notwithstanding, the successful physical – chemical changes of the drug properties little have been mentioned about the architecture and topology of the mixture PLC–CD–liposome. Aiming at a better comprehension of the PLC-β -CD complex lib- eration to membranes, we studied the architectural supramolecu- lar arrangements of PLC, β -CD (β -CD) and egg phosphatidylcholine (EPC) liposomes, a membrane model, at different pHs (10.0, 7.0 and 5.5). [14] To probe these topologies, we have used 1 H NMR spec- troscopy (NOE, nuclear Overhauser effect), [15,16] diffusion-ordered spectroscopy (DOSY) [17 – 19] and saturation transfer difference (STD) technique. [20 – 25] Results and Discussion Investigating the topologies of PLC in β -CD in EPC liposomes at different pHs by NMR required full characterization of simple binary mixtures in aqueous solutions of PLC, β -CD and EPC. The ∗ Correspondence to: Anita Jocelyne Marsaioli, Institute of Chemistry, UNICAMP, PO Box 6154, 13083-970 Campinas, SP, Brazil. E-mail: anita@iqm.unicamp.br a Institute of Chemistry, UNICAMP, PO Box 6154, 13083-970 Campinas, SP, Brazil b Institute of Biology, UNICAMP, PO Box 6109, 13083-970 Campinas, SP, Brazil Magn. Reson. Chem. 2011, 49, 295–300 Copyright c  2011 John Wiley & Sons, Ltd.