Rationalization of a nanoparticle-based nicotine nanovaccine as an effective next-generation nicotine vaccine: A focus on hapten localization

Abstract

A lipid-polymeric hybrid nanoparticle-based next-generation nicotine nanovaccine was rationalized in this study to combat nicotine addiction. A series of nanovaccines, which had nicotine-haptens localized on carrier protein (LPKN), nanoparticle surface (LPNK), or both (LPNKN), were designed to study the impact of hapten localization on their immunological efficacy. All three nanovaccines were efficiently taken up and processed by dendritic cells. LPNKN induced a significantly higher immunogenicity against nicotine and a significantly lower anti-carrier protein antibody level compared to LPKN and LPNK. Meanwhile, it was found that the anti-nicotine antibodies elicited by LPKN and LPNKN bind nicotine stronger than those elicited by LPKN, and LPNK and LPNKN resulted in a more balanced Th1-Th2 immunity than LPKN. Moreover, LPNKN exhibited the best ability to block nicotine from entering the brain of mice. Collectively, the results demonstrated that the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine could be enhanced by modulating hapten localization, providing a promising strategy to combatting nicotine addiction.

Keywords: Anti-nicotine antibody; Hapten localization; Lipid-polymeric hybrid nanoparticle; Nicotine vaccine; Smoking cessation.

Figure 1

Verification of the hapten conjugate chemistry. (A) CLSM images showing the co-localization of model hapten dyes with hybrid NPs. Scale bars represent 10 μm. FT-IR spectra of Nic-hapten (Nic), Nic-KLH conjugate (KN), and KLH (B); Nic-hapten, hybrid NPs (LP), and Nic-hapten-conjugated LPN NPs (LPN) (C); LPKN, LPNK, and LPNKN (D).

Figure 2

Characterization of nanovaccine NPs. (A) TEM images showing the morphological characteristics of NPs. Scale bars represent 200 nm. (B) Size distribution of LPKN, LPNK, and LPNKN NPs. (C) and (D) show the stability of nanovaccines in PBS and DI water at 4 °C, respectively.

Figure 3

Flow cytometry assay of the uptake of nanovaccine NPs by dendritic cells. (A) Population distribution of cells treated with 20 μg of nanovaccine NPs for 15 min or 120 min. The percentage of NBD-positive cells (B) and NBD median intensity in cells (C) were analyzed.

Figure 4

Uptake of nanovaccine NPs by dendritic cells analyzed by CLSM. The lipid-layer of hybrid NPs was labeled by NBD. Nic-hapten on KLH was substituted with AF647 to provide fluorescence. Cells were treated with 20 μg of nanovaccine NPs for 15 min or 120 min. Scale bars represent 10 μm.

Figure 5

Anti-nicotine antibody titers (A) and anti-KLH antibody titers (B) determined by ELISA. Significantly different compared to the previous studied day: & p < 0.05, && p < 0.01, &&& p < 0.001. Significantly different compared to the other three groups on the same studied day: ## p < 0.01, ### p < 0.001. Significantly different: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Anti-nicotine antibody affinity estimated by competition ELISA. (A) Time-course affinity of anti-nicotine antibodies induced by various nicotine nanovaccines. (B) Endpoint comparison of antibody’s affinity among different hapten localization nanovaccine groups on day 40. Significantly different: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Anti-nicotine subclass antibody titers of (A) IgG 1, (B) IgG 2a, (C) IgG 2b, and (D) IgG 3. (E) Th1/Th2 index induced by immunization with nicotine nanovaccines. Th1/Th2 index= (IgG2a+IgG3)/2/IgG1. Significantly different: * p < 0.05, *** p < 0.001.

Figure 8

Pharmacokinetic efficacy of nanovaccines with different hapten localizations. Nicotine levels in the serum (A) and brain (B) of mice 3 min after challenged with 0.06 mg/kg nicotine. Data were reported as means ± standard error. Significantly different compared to the blank group: # p < 0.05, ### p < 0.001. Significantly different: * p < 0.05.

Figure 9

H&E staining of the sections of major organs including heart, kidney, lung, liver, and spleen harvested from the mice immunized with different nicotine vaccines.

Scheme

Schematic illustration of the structure of hybrid NP-based nicotine nanovaccines with different hapten localizations.