A novel and efficient nicotine vaccine using nano-lipoplex as a delivery vehicle

Abstract

A number of vaccines conjugating nicotine haptens with carrier proteins have been developed to combat nicotine caused tobacco dependence. Some vaccines, such as NicVAX, NicQb, advanced into clinical trials, but none of them were successful. Most of those vaccines have some innate disadvantages such as low nicotine loading capacity, easy degradation, and vulnerable to the clearance by reticulo-endothelial system (RES). Thus, there is undoubtedly an urgent need for developing novel vaccines against nicotine addiction. In this study, we assembled a liposome-protein based nanoparticle as a nicotine hapten delivery system. The nanoparticle (Scheme 1) was constructed by conjugating a model hapten carrier protein, bovine serum albumin (BSA), to cationic liposomes. This nano-sized complex, lipoplex, was characterized using zetasizer, transmission electron microscope (TEM), and flow cytometry. The efficacy of the lipoplex vaccine was evaluated in mice and compared with that of Nicotine-BSA conjugate (Nic-BSA). The lipoplex vaccine with Alum was able to elicit the highest NicAb titer of 11169 ± 2112, which was significantly higher than that induced by either the vaccine without Alum or Nic-BSA with Alum. The significant immunostimulatory effect of this nano-lipoplex may provide a novel strategy to improve the immunogenic ability of current nicotine vaccines or other vaccines using small molecules as immunogens.

Scheme 1. Illustration of lipoplex-based nicotine vaccine.

Introduction

Tobacco smoking is one of the most devastating habits that people have ever indulged. Just in 2011, the use of tobacco killed 6 million people and caused hundreds of billions of dollars of economic damage worldwide.¹ Unfortunately, very few of the smokers are able to quit smoking completely even with pharmaceutical aid,² but meanwhile, more and more people start their addiction to cigarettes. If such trends continue, more than 1 billion people will die from smoking related diseases in the 21st century. It is known that tobacco dependence is largely attributed to nicotine, which can stimulate brain mesolimbic dopamine neurons and induce rewarding behavior.³ Therefore, how to reduce the amount of nicotine that could be delivered to brain during smoking is the key question for smoking cessation. Currently, some pharmacological aids including nicotine replacement therapy, bupropion, and varenicline are available for smoking cessation, but they only impose short-term effect and majority of the quitters will relapse.^3,4 Inspired by the fact that human immune system is able to produce antibodies to clear foreign substances, such as virus, bacteria, and proteins, researchers have developed some innovative anti-nicotine vaccines to generate antibodies that are capable of specifically binding to nicotine molecules in peripheral circulation, and thus reducing their access to brain.^5,6 These vaccines share some common traits, e.g. nicotine haptens are conjugated to a variety of carrier proteins, and the vaccines are injected with appropriate adjuvants during vaccination. Many of the vaccines were reported to elicit high titers of anti-nicotine antibodies during animal studies; some of them even advanced to human clinical trials.⁷ However, most of the delivery systems used in previous nicotine vaccines contain solely carrier proteins, which may be too small to effectively present the nicotine haptens to the antigen presenting cells (APC) to elicit strong immune response.⁸ In addition, those carrier proteins may be easily cleared by RES before presenting nicotine haptens to immune cells.⁹ Moreover, the number of available ligands on carrier proteins for hapten conjugation is limited.

Therefore, it is necessary to develop new delivery systems that can both increase the antigen presenting efficiency and hapten loading capacity. Recently, cationic liposomes have been recognized as novel adjuvants and vaccine delivery systems due to some of their unique advantages over traditional carrier proteins on immunogenic stimulation:¹⁰ first, a non-immunogenic substance may be converted to an immunogenic one;¹¹ second, sizes, charges, and components of liposomes could be easily adjusted to cope with the need of different antigens;¹² third, liposomes increase the antigen presentation to APCs.¹³ In our previous work, lipoplex coupling cationic liposome with human liver fatty acid binding protein 1 was assembled and well characterized as a potential vaccine delivery platform.¹⁴ It is possible cationic liposomes combined with carrier proteins could be utilized to construct a delivery vehicle for small addictive compounds to achieve high titers of effective antibodies.

The aim of the present study was to explore the possibility of building up an efficient delivery system based on lipoplex for nicotine vaccine. Here, we present a method of preparing such a new nano-lipoplex assembled by incorporating BSA to the surface of cationic liposomes composed by 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide[polyethylene glycol]-2000) (ammonium salt) (DSPE-PEG[2000]-maleimide), and demonstrate its potential value as a delivery system for nicotine haptens to elicit high titers of anti-nicotine antibodies in mice study.

Results

Analysis of Nic-BSA conjugates

Conjugates with various number of rac-trans 3′-hydroxymethylnicotine hemisuccinate (Nic) linked to BSA (0, 0, 15.1, 17.54, and 17.81) were constructed by coupling Nic to BSA at different initial molar ratio 1:10, 1:20, 1:50, 1:100, and 1:200 (Table 1). No Nic was detected on BSA with initial molar ratio of 1:10 and 1:20. The amount of coupled Nic could not be further increased by increasing the ratio to 1:200 compared to that at ratio of 1:100. The Nic-BSA conjugates with 15 Nic haptens were subsequently thiolated using 200- and 500-fold molar equivalents of Traut’s reagent (Table 2). The thiolated Nic-BSA bearing 6 sulfhydryl groups was prepared for loading to cationic liposomes.

Table 1. The coupling efficiency of Nic to BSA at various molar ratios.

Molar ratio of BSA/Nic	Amino groups on BSA consumed (%)	Number of Nic conjugated to BSA
1:10	0.0	0.0
1:20	0.0	0.0
1:50	26.0	15
1:100	30.0	18
1:200	30.0	18

Table 2. The degree of Nic-BSA thiolation at different molar ratio of Nic-BSA/Traut’s reagent.

BSA: Nic (molar ratio)	Nic-BSA: Traut’s reagent (molar ratio)	Thiol equivalents coupled to BSA
1:50	1:200	6
1:50	1:500	8

Analysis of physicochemical properties of Nic-BSA-liposomes (NBLs)

The size, charge, and polydisperisty of NBLs constituted by various molar ratio of DSPE-PEG(2000)- maleimide/DOTAP (0.5%, 1%, 2%, and 4%) and Nic-BSA were measured using Malvern Nano-ZS zetasizer (Table 3). For all formulations, lipid composition affects the size of liposomes. Nanoparticles with decreasing sizes (276.97 ± 6.66 nm, 271.33 ± 4.33 nm, 254.67 ± 2.21 nm, 178.67 ± 4.16 nm) were detected using dynamic light scattering (DLS) with the increasing DSPE-PEG(2000)-maleimide ratio (0.5%, 1%, 2%, and 4%).

Table 3. Comparison of size and surface-charge of Nic-BSA-liposmes with different ratios of DSPE-PEG(2000)-Maleimide and DOTAP (mean ± S.D.; n = 3).

DSPE-PEG(2000)-Maleimide: DOTAP (%)	Zeta potential (mV)	Size (nm)	Polydispersity
0.5	29.53 ± 1.29	276.97 ± 6.66	0.20 ±  0.02
1	22.20 ± 0.98	271.33 ± 4.33	0.23 ±  0.02
2	11.80 ± 0.75	254.67 ± 2.21	0.19 ±  0.02
4	8.88 ± 0.85	178.67 ± 4.16	0.14 ±  0.03

TEM investigation showed that DOTAP with addition of 4% DSPE-PEG (2000)-maleimide formed unilamellar vesicles with non-uniform diameters ranging from 100 to 300 nm (Fig. 1), and this result was consistent with the size distribution obtained by DLS (Table 3). Zeta potentials were reduced from 29.53 ± 1.29 mV to 8.88 ± 0.85 mV as the amount of neutral DSPE and charge shielding agent PEG increased.

Figure 1. TEM image of liposomes.

To confirm that BSA with sulfhydryl groups could be incorporated to liposomes with exposed maleimides, BSA and liposome were labeled with Rhodamine B isothiocyanate (Rhod B) and fluorescein isothiocyanate-dextran (FITC), respectively, and measured using flow cytometer. Figure 2A shows that no fluorescent signal was detected in blank BSA-liposome nanoparticles for either Rhod B or FITC. In FITC labeled liposome, only FITC signal was detected (Fig. 2B). Only Rhod B signal was detected in BSA-liposome nanoparticles with BSA labeled with Rhod B (Fig. 2C). The above results showed that without labeling, BSA-liposome nanoparticles did not emit either Rhod B signal or FITC signal, and BSA and liposome could be labeled with Rhod B and FITC, respectively. Figure 2D shows that the nanoparticles emitted both Rhod B and FITC signals, indicating BSA was incorporated with liposome. As shown in Figure 3, the association efficiency of Nic-BSA to cationic liposomes increased (13.67%, 27.41%, 57.47%, 75.02%, and 87.45%) with the increasing molar ratio of maleimide/Nic-BSA (0, 1:1, 2:1, 4:1, and 8:1). Since Nic-BSA was negatively charged, the increased association efficiency led to reduction of charge on NBL (Fig. 4), which was indicated by the decreasing zeta potential of NBLs (41.56 ± 1.00 mV, 29.53 ± 1.29 mV, 22.20 ± 0.98 mV, 11.80 ± 0.75 mV, and 8.88 ± 0.85 mV). Interestingly, the increasing association rate displays a highly linear relationship with the decreasing zeta potential of NBL (Fig. 4), affirming that it was the association of Nic-BSA to liposome that led to the decrease in net charge on NBL. Furthermore, since high association efficiency will reduce the loss of Nic-BSA during NBL assembly and higher concentration of haptens on the vaccine complex is more likely to induce stronger immune response, NBL produced at 8:1 molar ratio of maleimide/Nic-BSA was chosen for animal studies.

Figure 2. Flow cytometric analysis of BSA-liposome complex. (A) BSA-Liposome without labeling; (B) FITC labeled liposome; (C) Rhod B-BSA-Liposome; (D) Rhod B-BSA-FITC-liposome.

Figure 3. Association efficiency of Nic-BSA to liposome at maleimide/ Nic-BSA molar ratio of 0, 1:1, 2:1, 4:1, 8:1, respectively.

Figure 4. The relationship between association efficiency of Nic-BSA to liposome and charge of Nic-BSA-liposome complex.

Animal tests of assembled Nic vaccines

Mice were immunized on day 0, and boosted on days 14, 28 with NBL and Nic-BSA with Alum and NBL without Alum to study the immunogenicity of respective vaccines containing 50 µg Nic-BSA. Sera were collected on days 0, 13, 27, 33, and 40, and anti-Nic antibodies (NicAb) were assayed on enzyme-linked immunosorbent assay (ELISA) plates coated with Nic-keyhole limpet hemocyanin (Nic-KLH). As shown in Figure 5, no NicAb was detected in sera collected on day 0, suggesting that there was no NicAb in the mice used in this study prior to Nic vaccine treatment. Thirteen days after the primary immunization, NicAb titers of 320 ± 208, 349 ± 210, 131 ± 27 were detected in samples injected respectively with NBL+Alum, NBL without Alum, and Nic-BSA+Alum. The NicAb titers achieved by NBL+Alum, NBL without Alum were significantly higher than that immunized with Nic-BSA+Alum. Titers for respective vaccines were further increased after first and second boost injection, and, in particular, the first booster (day 27) drastically increased titers of NBL+Alum, NBL without Alum, and Nic-BSA+Alum to 7932 ± 1047, 5720 ± 3952, 3953 ± 826, respectively. The maximal titers of 11169 ± 2112, 9876 ± 1932, and 7182 ± 314 for the three vaccines were achieved on day 40.

Figure 5. Time course of nicotine specific antibodies elicited by Nic-BSA-liposome + Alum, Nic-BSA-liposome without Alum, and Nic-BSA + Alum (* means P value < 0.05).

Discussion

Much effort has been devoted to the development of vaccines against nicotine, but so far there are no nicotine vaccines for clinical use. Nicotine is non-immunogenic by itself; it has to be conjugated to carrier proteins in order to elicit immune response.¹⁵ The nicotine hapten used in this study is rac-trans 3′-hydroxymethylnicotine hemisuccinate, which possesses a carboxyl sidearm functional group enabling covalent link with an amino group on carrier proteins. In previous studies, rac-trans 3′-hydroxymethylnicotine hemisuccinate coupled to human serum albumin and KLH was found to generate highly effective antibodies in rabbits.¹⁶ Currently, most of the macromolecule carriers employed in nicotine vaccine are KLH,³ recombinant exotoxin A (rEPA),¹⁷ tetanus toxoid (TT),¹⁸ and some virus-like particles.¹⁹ However, none of them has yielded a clinically approved nicotine vaccine to date. The limited success might be attributed to the fast degradation of carrier proteins by enzymes and its rapid nonspecific clearance by human body. In this study, we attempt to demonstrate the feasibility of using nano-lipoplex particle as a delivery system for nicotine vaccine development. BSA was used as a model carrier protein, and it recently has been shown to be effective as a carrier protein in vaccines development. The anti-cancer vaccine, in which BSA was conjugated with 3′-fluoro-TF antigen-MUC1, was able to generate high titers of antibodies which could specifically bind to the tumor-associated glycopeptide antigen analog.²⁰ In another vaccine against malaria, Asn-Ala-Asn-Pro (NANP) repeats were bound to BSA, and the resulting immunogens were able to elicit high titers of antibodies against circumsporozoite protein.²¹

In this study, DOTAP was chosen as the major constituent of liposome, largely due to the fact that cationic liposomes promote a “depot effect” that facilitates antigen uptake.²² Cationic liposomes have long been proven to have immunostimulatory effect due to their active interaction with cells which usually possess negative charges, and such an interaction induces adsorptive endocytosis.²³ In addition, it was proven that cationic liposomes consisting of DOTAP and DOTMA could significantly enhance dendritic cell (DC) maturation by up regulating the expression of CD80 and CD86.²⁴ Furthermore, liposomes with diameters less than 500 nm were shown to efficiently enhance immunogenic performance of liposome vaccines over large liposomes (>500 nm).²⁵ Therefore, the size of liposomes produced in this study was designed to be around 200 nm. Polyethylene glycol (PEG), a biocompatible and hydrophilic polymer, was utilized to provide a hydrophilic protective layer outside drugs that can prevent nonspecific absorption of serum proteins and avoid clearance by RES, thereby prolonging the blood circulation time of drugs.¹⁰ PEG(2000) incorporated into cationic liposomes has two important roles: first, PEG moiety is able to prevent particle aggregation, extend the circulation time of liposomes by lowering adsorption of plasma proteins and reducing RES uptake, and improve the immunogenicity of cationic liposomes; second, it would be easier for maleimide, which is linked to the long chain of PEG(2000), to react with sulfhydryl-bearing BSA. Though some amine groups on BSA have been consumed during Nic-BSA conjugation, considerable amount of Nic-BSA was still conjugated to liposomes when the ratio between maleimide and Nic-BSA was increased, showing the high conjugation efficiency between maleimide and the -SH groups on the protein.

In mice immunization, NBL with Alum achieved higher NicAb titer compared with either NBL without Alum or Nic-BSA+Alum. NicAb titers are essential to the efficacy of Nic vaccine, because higher titers of antibody are expected to bind more nicotine molecules in the periphery, and thus reduce the amount of nicotine entering brain.²⁶ Alum has been commonly used as an adjuvant in many vaccines licensed by the US Food and Drug Administration.²⁷ Adding Alum adjuvant to NBL significantly enhanced the immunogenicity of the vaccine, which was shown by the higher titers of NBL + Alum compared to NBL without Alum after the first boost injection. The significant difference in titers of NicAb induced by NBL alone and Nic-BSA + Alum suggests the significant immune-stimulating effect of nano-lipoplex.

Though the results of this current work prove the ability of nano-lipoplex to enhance the immunogenicity of nicotine hapten, numerous questions still need to be answered in order to improve this hapten delivery system. First, the carrier protein used here is a model protein, BSA, which is not clinically approved. Therefore, it is necessary to search for substituting carrier proteins which are both medically acceptable and immunologically effective. Second, the main purpose of this study was to demonstrate the immune-potentiating effect of nano-lipoplex, and some important variables such as nicotine epitope density of NBL nanoparticles were not sufficiently optimized. Theoretically, the more nicotine epitope each NBL nanoparticle contains, the higher probability that the vaccine can be recognized by B-cell receptors, and the higher chance that nicotine-specific naïve B cell will be activated. In this study, epitope density was dictated by the number of Nic that was conjugated to BSA and the incorporation efficiency of BSA to liposome. Therefore, further work needs to be done in these two aspects to increase nicotine epitope density. Third, only titers of antibody were evaluated in this study. The affinity of NicAb to nicotine and the specificity of those antibodies to nicotine need to be tested in the future. Fourth, this lipoplex could be further modified to enhance antibody response; for example targeting molecule can be linked to nano-lipoplex to specifically target the delivery system to immune cells.

In summary, in this study, a novel nicotine vaccine using lipoplex nanoparticle as a delivery system was developed. The formation of this vaccine was realized through two major steps: first, nicotine haptens were covalently linked to BSA using EDC; second, the Nic-BSA was conjugated to cationic liposomes via a reaction between the maleimide group in liposome and the sulfhydryl group in BSA. By adjusting the molar ratio between Nic and BSA or between Nic-BSA and maleimide, we could readily control the amount of Nic loaded onto this vaccine nanoparticle. The physiochemical properties of this vaccine were characterized using TEM, zetasizer, and flow cytometer. As a proof of concept study, the results from animal test showed that the nicotine vaccine using lipoplex as the delivery vehicle achieved significantly higher titers of anti-nicotine antibody than those from the vaccine delivered by carrier protein alone. In future work, BSA will be replaced with some clinically approved carrier proteins such as rEPA, TT, and the specificity and affinity of generated antibodies to nicotine molecule will be evaluated.

Materials and Methods

Materials

DOTAP (890890C) in chloroform and DSPE-PEG(2000)-maleimide (880126) in chloroform were purchased from Avanti Polar Lipids, Inc. 2,4,6-trinitrobenzene-1-sulfonic acid (TNBS) (P2297), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (E6383), BSA (A2153), 2-iminothiolane hydrochloride (Traut’s reagent) (I6256), Rhodamine B isothiocyanate (83689), and fluorescein isothiocyanate-dextran (F7250) were obtained from Sigma-Aldrich. Rac-trans3′-hydroxymethylnicotine hemisuccinate (H948190) was purchased from Toronto Research Chemicals. The micro BCA assay kit (23235) was purchased from Thermo Scientific. All other chemicals and reagents were purchased from Fisher Scientific and are of analytical grade.

Preparation of cationic liposome

Cationic liposomes were prepared according to the method described by Huang and Zhang.²⁸ In brief, lipid film consisting of 1.96 mg DOTAP and DSPE-PEG(2000)-maleimide ranging from 0.04 mg to 0.32 mg was hydrated with buffer containing 0.9% NaCl, 5% dextrose, and 10% sucrose in Tris-HCl buffer (0.05 M Tris Base, pH 7.4). The resulting suspension was incubated at 65 °C for 1 hour and extruded through polycarbonate membrane with pore sizes of 100 nm for 14 times. For FITC labeled liposomes, FITC and lipids at molar ratio of 1:10 was extruded according to the above method.

Synthesis of nicotine-BSA conjugates (Scheme 2 Step I)

Scheme 2. Illustration of NBL vaccine synthesis.

Ten milligrams of EDC dissolved in 700 µL DI water was mixed with appropriate volume of 100 mg/mL Nic, also dissolved in DI water. After incubation at 0 °C for 10 min, the mixture was added with 10 mg BSA (40 mg/mL) and appropriate amount of DI water to a total volume of 1 mL, and stirred at room temperature for 12 h. The pH of the DI water used in this step was adjusted to 6.76 with 0.01 M sodium hydroxide. The resulting Nic-BSA was purified by size exclusion chromatography with a Sephadex G-25 column using an AKTA FPLC system (Amersham Biosciences). Briefly, 0.5 mL sample was loaded onto the column, followed by elution using phosphate buffered saline (PBS) (0.1 M, pH 7.4) as the mobile phase at 1 mL/min. Nic-BSA was collected and concentrated to 1 mg/mL using Microcon centrifugal filter units (50 000 MWCO) from EMD Millipore. The Rhod B labeled BSA was synthesized using the same method as Nic-BSA conjugation, except the molecular ratio between BSA and Rhod B was 50:1.

Quantifying the number of Nic haptens on Nic-BSA

The number of Nic haptens on Nic-BSA was determined by measuring the difference in the number of unreacted lysine groups on the surface of BSA before and after conjugation using TNBS. In brief, 200 μL of Nic-BSA mixed with 200 μL of 4% NaHCO₃ solution was added with 200 μL of 0.1% TNBS solution, and the resulting mixture was incubated for 1 h at 37 °C. The color of the solution was read at 335 nm. The amount of −NH₂ groups consumed during conjugation of Nic to BSA was calculated from the difference between the O.D. of the control and that of the conjugate.²⁹

Loading Nic-BSA onto liposomes

Thiol groups were introduced to Nic-BSA by incubating Nic-BSA obtained in the previous step with 1 mg/mL of Traut's reagent for one hour in darkness under continuous stirring (Scheme 2, Step II). The thiolated Nic-BSA was purified using FPLC as described for Nic-BSA purification and concentrated to 2 mg/mL in PBS (0.1 M, pH 7.4) by Microcon centrifugal filter units (50 000 MWCO). The amount of thiol groups on BSA was quantified through a colorimetric sulfhydryl assay using Ellman's reagent (5,5′-dithiobis[2-nitrobenzoic acid]).³⁰ Briefly, 80 μL of 4 mg/mL Ellman's reagent was added to 800 μL of thiolated Nic-BSA, and the mixture was incubated at room temperature for 15 minutes. Free sulfhydryl levels were determined from the absorbance at 412 nm using the following formula generated from a set of cysteine standards: SH = 1.1A₄₁₂ / 14398C_Nic-BSA, where A₄₁₂ is the absorbance at 412 nm, C_Nic-BSA is the protein concentration and SH is the number of thiol equivalents. One hundred and twenty-five μL Mal-PEG-liposome, 450 µL thiolated Nic-BSA conjugates, and 425 µL 0.15 M NaCl-0.2 mM EDTA (pH 7.4) were mixed to form Nic-BSA-Liposome (Scheme 2, Step III). The resulting Nic-BSA-Liposome was purified by dialysis using dialysis membrane (MWCO 1000 kD) from Spectrum Laboratories in 10 mM NaCl.

Measuring the amount of Nic-BSA loaded to liposomes

Nic-BSA associated with liposomes was assayed using a modified protocol described by Ansell et al.³¹ In detail, 20 μL of prepared liposomes (before/after purification) were withdrawn and diluted with 1 ml 0.15 M pH 7.4 NaCl solution (working-dispersion). To assess the total and conjugated protein concentration, 500 μL of working-dispersion was mixed with 100 μL of 5% (v/v) Triton X-100 and this dispersion was maintained at 65 °C for 5 min to disrupt all the vesicles. Both total and conjugated Nic-BSA concentrations were assessed using micro BCA protein assay kit from Pierce. The association efficiency of Nic-BSA to liposomes was calculated according to the following equation: Association efficiency (%) = BSA_conjugated / BSA_Total × 100%

DLS analysis

The sizes of NBL nanoparticles were evaluated using DLS. DLS measurements were performed using a Malvern Nano-ZS zetasizer (Malvern Instruments Ltd.). Newly prepared samples were diluted by 10-fold with 0.9% sodium chloride saline (pH 7.4) and each measurement was done in triplicate.

Zeta potential measurements

Freshly made NBLs were diluted by 10 fold with deionized water (pH 7.0) and zeta potential of these samples was measured with the Malvern Nano ZS using the technique of Laser Doppler Velocimetry (LDV). Zeta potential was measured six times for each sample.³²

TEM analysis of liposome

Sample grids (carbon coated copper) were put into one drop of liposome for 30 s, then in distilled water drop for washing (10 s) and finally in a phosphotungstic acid drop for staining (10 s).³² The excessive stain on the grids was removed using filter paper. The prepared grids were analyzed by Morgagni™ Transmission Electron Microscope.

Active immunization of mice with nicotine vaccines

All animal studies were carried out following the National Institutes of Health guidelines for animal care and use. Animal protocols were approved by the Institutional Animal Care and Use Committee at Virginia Polytechnic Institute and State University. Groups of n = 8 female Balb/c mice (6−7 weeks, 16−20 g) were immunized by subcutaneous (s.c.) injection on days 0, 14, and 28 with Nic-BSA conjugates, Nic-BSA-liposome conjugates (50 µg for Nic-BSA) in 0.9% sodium chloride saline with Imject Alum Adjuvant (Pierce Biotechnology Inc.) or Nic-BSA-liposome conjugates (50 µg for Nic-BSA) without Alum adjuvant (total volume was 100 µl). Following vaccine administration, blood samples (~200 µl) were collected on days 0, 13, 27, 33, 40 via retroorbital puncture from each mouse. Sera (100 µl for each sample) centrifuged from blood were stored at −80 °C.

ELISA measurement

Mice sera were analyzed according to the ELISA procedure described by de Villiers et al. with appropriate modification.³ Nic was conjugated to KLH using the same method for Nic-BSA conjugate. Nic-KLH was used as the coating material to prevent nonspecific binding of BSA and liposome derived antibodies. ELISA-plates were coated with 100 µL of 10 µg/mL Nic-KLH conjugate at 25 °C for 5 h. The plates were washed with washing buffer for 4 times and DI water for 2 times, and were blocked with 300 µL non-protein blocking buffer from Pierce for 12 h. After washing, 100 µL of each dilution (1:25, 1:125, 1:625, 1:3125, 1:15 625, and 1:78 125) of serum from each mouse was incubated in plates at 25 °C for 2 h. The plates were washed again, and incubated with 100 µL anti-mouse IgG HRP (1:10 000) from Sigma-Aldrich for 1 h. After washing as before, 100 µL of TMB One Component Microwell Substrate was added into each well and incubated for 10 min, and the reaction was stopped by adding 100 µL of 0.5% (v/v) H₂SO₄. The absorbance for each well at 450 nm was recorded. Titer was defined as the dilution factor at which OD₄₅₀ falls to half of the maximal.

Data analysis

Serum NicAb concentrations were compared among groups using one way ANOVA and comparisons among paired groups were analyzed with Tukey’s HSD. The difference is considered as significant when P value is less than 0.05. Each measurement was carried out at least three times, and the results were expressed as mean ± standard deviation.

Glossary

Abbreviations:

APC

antigen presenting cells

BSA

bovine serum albumin

DC

dendritic cell

DLS

dynamic light scattering

DOTAP

1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)

DSPE-PEG(2000)-maleimide

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide[polyethylene glycol]-2000) (ammonium salt)

ELISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate–dextran

rEPA

recombinant exotoxin A

NBL

Nic-BSA-Liposome

Nic

rac-trans 3′-hydroxymethylnicotine hemisuccinate

NicAb

anti-Nic antibodies

Nic-BSA

nicotine-BSA conjugate

RES

reticulo-endothelial system

Rhod B

Rhodamine B isothiocyanate

TEM

transmission electron microscope

TT

tetanus toxoid

10.4161/hv.26635