Topical vaccination with novel imiquimod ultradeformable nanovesicles

CAIMI, AT; DE FARIAS, MARCELO; VILLARES PORTUGAL, RODRIGO; PEREZ, AP; ROMERO, EL; MORILLA, MJ

Congreso; XVII Brazil MRS Meeting - SBPMat; 2018

IntroductionThe development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies. Topical vaccination however, is challenged by the barrier that the stratum corneum (SC) interposed between antigens-adjuvants and the skin-associated lymphoid tissue (SALT) lying few hundred micrometers depth from skin surface. Topical vaccination with soft matter is a new attractive way to induce immune protection without disrupting the SC. In this scenario, we previously showed that ultradeformable archaeosomes (UDA: made of total polar archaeolipids (TPA) extracted from the archaea Halorubrum tebenquichense plus soybean phosphatidylcholine (SPC) and sodium cholate (NaChol) (3:3:1 w/w) topically applied can penetrate the SC and induces antigen specific IgG serum titers [1]. TPA, key components of UDA, is a mixture of sn2,3 glycerol ether linked fully saturated polyisoprenoid chains. On one hand, TPA are ligands for scavenger receptor class A express on phagocytic cells and immature antigen presenting cells (APC) [2]. APC much more pronouncedly captures UDA than TPA-lacking nanovesicles. On the other hand, TPA confers UDA stability to heat sterilization and storage under cold-free conditions [3]. The immune response elicited by topical UDA, however, is lower than the obtained after parenteral administration and the IgG2a component is lower than the IgG1 component, suggesting that the response is more humoral (Th2) than cellular (Th1). Imiquimod (IMQ), on the other hand, is a synthetic ligand of the Toll Like Receptor 7 (TLR7), that induces the production of several cytokines as IFN- α, TNF-α, IL-1a, IL-6, IL-8 and IL-12 in immune cells like monocytes/macrophages, dendritic cells, B cells, keratinocytes and granulocytes. Topically administered IMQ induces functional maturation of epidermal Langerhans cells and stimulates migration of these APC to regional lymph nodes where they promote a specific T cell response. A 5 % IMQ creme called Aldara ® is approved to treat human dermatologic skin conditions including external genital and perianal warts, small superficial basal cell carcinoma and actinic keratoses on the face and scalp. Aldara is an oil-in-water emulsion consisting of isostearic acid as the oil phase in which IMQ is solubilized at 5%. Clinical trials have shown that topical application of Aldara to patients with melanoma and prostate cancer vaccinated with peptides, increase the cytotoxic response. However, the responses induced by Aldara rapidly fade away, resulting in poor memory formation and only partial tumor protection. Moreover, isostearic acid cause frequent cutaneous adverse reactions in 75% of the patients. Besides, this emulsion is not stable, Aldara cream is marketed in single-use packaging and it is recommended that packets unused should be discarded. All these data suggest that Aldara is not an ideal preparation for topical vaccination. The hypothesis of this work is that combining the penetration into skin and targeting to APC of UDA with immunomodulation activity of IMQ will improve the immunological response after topical application as compared with UDA or IMQ alone. The aim of this work was to prepared UDA loaded with IMQ and to test their adjuvant properties in vivo. MethodsUDA made of SPC:TPA:NaChol 3:3:1: (w/w) were prepared by the film hydration method. Briefly, mixtures of lipids were dissolved in chloroform:methanol 1:1 v/v and 3.6%w/v of linoleic acid and 4 mg/ml IMQ were added. Then solvents were rotary evaporated at 40°C until elimination. The lipid films were flushed with N2 and hydrated with 10 mM Tris-HCl buffer pH 7.4 with 0.9 % w/w NaCl up to a final concentration of 40 mg/ml total lipids. The resultant suspensions were sonicated and extruded 10 times through a 0.4 μm pore size polycarbonate filters. UDA-IMQ was characterized in terms of particle size, zeta potential and IMQ content. The viability of keratinocytes (HaCaT cells) and macrophages (J774 cells) upon 24 and 48 h of incubation with free IMQ and UDA-IMQ was measured by the MTT method. The in vitro skin penetration of free IMQ and UDA-IMQ was measured upon 1 h incubation on human skin explants using the Saarbrücken method, followed by tape stripping and IMQ extraction and quantification. Finally, we screened ability of free IMQ and UDA-IMQ to stimulate immune responses toward the model antigen OVA upon topically application on Balb-c mice.ResultsIMQ is a small molecule (molecular weight 240.3) that is practically insoluble in water, sparingly soluble in other common pharmaceutical solvents and shows very poor skin permeability. To incorporated IMQ into UDA matrix, we have solubilized IMQ in linoleic acid and mixed with lipids in the film preparation step. The UDA-IMQ obtained showed a mean size of 190 nm, polydispersity index of 0.3 and Z potential of -35 mV. IMQ concentration resulted 2.1 mg/ml of IMQ with a IMQ/phospholipid ratio of 62 g/mg and an encapsulation efficiency of 55 %. Transmission electron microscopy images showed unilamellar and bilamellar vesicles of around 100-200 nm. The colloidal stability and IMQ content of UDA-IMQ remained unchanged up to 6 months of storage at 4°CThe viability of J774 cells decreased in a dose and time dependent form for free IMQ. Viability decreased 25 % at concentrations higher than 20 g/ml and 80 % at 5 g/ml of free IMQ after 24 h and 48 h of incubation, respectively. The viability of J774 cells did not significantly decrease after 24 h of incubation with UDA-IMQ; but the viability decreased 50 % after 48 h of incubation at the higher concentration tested. The viability of HaCaT cells decreased only for free IMQ in a dose and time dependent form, reducing 25 and 50 % the viability at 40 g/ml after 24 h and 48 h of incubation, respectively. Afterwards, we measured the capacity of free IMQ and UDA-IMQ at non-cytotoxic concentrations to induce the release of the pro-inflammatory cytokines IL-6 and TNF- on J774 and HaCaT cells. We found that on J774 cells, both free IMQ as well as UDA-IMQ promoted the release of IL-6, but only UDA-IMQ generated TNF-. On HaCaT cells, UDA-IMQ promoted more IL-6 release than free IMQ, however neither free IMQ nor UDA-IMQ induced TNF- The IMQ accumulation in human skin was 1.5 times higher when applied as UDA-IMQ (30 g/cm2) than as free IMQ (20 g/cm2).Finally, we found that topically applied on mice IMQ plus OVA induced low IgG serum titers that decreased post boost and only the IgG1 isotype was induced with no production of IgG2a antibody response. In contrast, UDA plus OVA and UDA-IMQ plus OVA induced higher IgG titers; but only for UDA-IMQ the IgG2a component was increased. UDA alone and free IMQ induced more IgG1 isotype, that is related to a humoral response. Conclusions.Our results demonstrate that UDA-IMQ was more efficient at generating cellular response, as measured by an increase in the IgG2a antibody response compared with free IMQ and UDA alone. The higher skin IMQ accumulation plus the higher induction of pro-inflammatory cytokines produced by UDA-IMQ could contributed to the increase cellular response found after topical application of UDA-IMQ.