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Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination Yuanyuan Zhen a,1 , Ning Wang b,c,1 , Zibin Gao d , Xiaoyu Ma a , Biao Wei a , Yihui Deng c , Ting Wang a,∗ a
School of Pharmacy, Anhui Medical University, 81 Plum Hill Road, Hefei, Anhui Province 230032, China School of Medical Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui Province 230009, China c School of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, China d Department of Pharmacy, Hebei University of Science and Technology, 70 Yuhua East Road, Shijiazhuang 050018, China b
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Freeze-drying Immunization Toll-like receptor C-type lectin receptor ELISA Controlled temperature chain
a b s t r a c t To develop effective, convenient and stable mucosal vaccines, mannose-PEG-cholesterol (MPC)/lipid Aliposomes (MLLs) entrapping model antigen bovine serum albumin (BSA) were prepared by the procedure of emulsification–lyophilization and used to constitute microneedles, forming the proMLL-filled microneedle arrays (proMMAs). The proMMAs were rather stable and hard enough to pierce porcine skin and, upon rehydration, dissolved rapidly recovering the MLLs without size and entrapment change. The proMMAs given to mice via oral mucosal (o.m.) route, rather than routine intradermal administration, elicited robust systemic and mucosal immunoresponses against the loaded antigens as evidenced by high levels of BSA-specific IgG in the sera and IgA in the salivary, intestinal and vaginal secretions of mice. Enhanced levels of IgG2a and IFN-␥ in treated mice revealed that proMMAs induced a mixed Th1/Th2 immunoresponse. Moreover, a significant increase in CD8+ T cells confirmed that strong cellular immunity had also been established by the immunization of the proMMAs. Thus, the proMMAs can be immunized via o.m. route to set up an effective multiple defense against pathogen invasion and may be an effective vaccine adjuvant-delivery system (VADS) applicable in the controlled temperature chain. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Mucosal vaccines are inoculated at the site of cavity or lumen mucosas which are commonly rich in dendritic cells (DCs) and are, therefore, able to induce both systemic and mucosal immunoresponses not only at the site of antigen exposure but also at remote mucosas due to the existence of generalized mucosal
Abbreviations: MPC, mannose-PEG-cholesterol; MLL, MPC/lipid A-liposome; proMMA, proMLL-filled microneedle array; BMA, blank microneedle array; VADS, vaccine adjuvant-delivery system; CTC, controlled temperature chain; SPC, soy phosphatidylcholine; MAIM, microneedle array inverse mold; AE, association efficiency; MD, mean diameter; o.m., oral mucosal; i.d., intradermal; s.c., subcutaneous; a.e., anesthesia/anesthetization; BSA-Al, BSA-alum; MALT, mucosa-associated lymphoid tissue; DC, dendritic cell; CTL, cytotoxic T lymphocyte; PEL, procedure of emulsification–lyophilization; OCT, optimal cutting temperature; s.l., sublingual. ∗ Corresponding author. Tel.: +86 15395082913. E-mail addresses: [email protected], [email protected] (T. Wang). 1 Both authors contributed equally to this work.
immune network where numerous mucosa-associated lymphoid tissues (MALT) are located [1]. To function effectively, mucosal vaccines must approach the professional antigen-presenting cells (APCs) to induce potent immunoresponses to produce functional pathogen-specific antibodies and cytotoxic T lymphocytes (CTLs), neutralizing and lysing the invaded pathogens. Ideally, a mucosal vaccine would be able to elicit functional immunocytes to secrete mucosal immunoglobulins blocking the establishment of initial infection by the pathogens, such as HIV and HPV, which once enter the cells can rapidly integrate into the host genome to establish a latent reservoir that can hardly be eliminated by conventional antiretroviral agents [2]. Thus, the mucosal vaccines that can block the initial invasion of intractable pathogens are most desirably needed. However, the mammal mucosas suitable for vaccination are usually covered with a defending layer of mucus which is a continuously renewed viscous fluid and contains various categories of agents, such as antiseptic lysozyme, proteins and glycoprotein mucins [3]. The defending mucus and enzymes impose a potential
http://dx.doi.org/10.1016/j.vaccine.2015.03.081 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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damage to vaccine antigens and prevent mucosal vaccines from approaching and crossing the epithelial layer, under which the professional APCs are most likely sited. Moreover, the tightly-lined epithelial cells with intercellular spaces sealed by tight junctions also form a barrier to mucosal vaccines to reach the professional APCs which are the necessary sponsors for the immunoresponses to the vaccines. By comparison to other sites, oral mucosa confronts a mild environment and is easily accessible and relatively safe for vaccination [4]. The oral mucosa is also rich in APCs and can mediate innate and adaptive immunoresponses to block local and systemic infections [5]. However, oral mucosal immunization is further limited by its anatomic structure of stratified squamous epithelium (SSE) as well as by rapid clearance of the subjects from mucosal surfaces by flow of saliva, movement of tongue and jaws, and chewing and swallowing [6]. To conquer these barriers to oral mucosal delivery, numerous technologies have been developed, including supersaturation, eutectic formation, iontophoresis, electroporation, sonophoresis, laser radiation, photomechanical waves and needleless injection [6]. But a common strategy was to incorporate a bioadhesive component to the drug/carrier to achieve prolonged mucosal contact and higher drug concentration on the mucosal surface [7]. Unfortunately, the bioadhesive component may cause oral paraesthesia or foreign body sensation which accelerates in biofeedback salivation and swallow leading to ingredient loss as well as poor patient compliance; moreover, due to its adherence to the upper epithelia of SSE, bioadhesive component also limits vaccine uptake into the underlying professional APCs resulting in weakened efficacy. Another approach is to use permeation enhancers (e.g., a mucolytic agent of N-acetyl-l-cysteine) to enhance the bioavailability of protein-containing nanoparticles delivered via mucosal route, however, the mucus barrier properties cannot be undermined without compromising the mucosa [8]. Recently, researchers modified nanoparticles with PEG as socalled mucus-penetrating particles (MPPs) for topical delivery of antibiotics in vagina and proved that MPPs improved vaginal drug distribution and retention over the vaginal epithelium compared to conventional particles [9]. But no enhanced uptake of agents into cells of interest can be expected for such an MPP as PEG is a known barrier to the access by most kinds of cells [10]. Previously, we showed that the mannose-PEG-cholesterol MPC/lipid A-liposomes (MLLs) could efficiently carry, protect and present antigens and, therefore, proved an effective oral mucosal vaccine adjuvant-delivery system (VADS) [11]. However, the MLLs also confront the above multiple obstacles which are difficult to overcome with the abovementioned common methods. Recently, microneedle arrays (MAs) with sub-millimeter structures designed
to pierce the skin and deliver vaccines in the epidermis or dermis compartments without pain provide an especially attractive option for intradermal delivery [12–15]. Interestingly, some researchers primed mice with intradermal (i.d.) biodegradable microneedles and then boosted by intranasal inoculation with the aqueous formulation, and finally they elicited robust antigen-specific humoral as well as mucosal immunity [13]. Inspired by this, we proposed that microneedle vaccines might as well be administered at oral mucosal sites to penetrate the barriers encountered with conventional mucosal vaccines. In this report, the MPC/lipid A-liposomes (MLLs) (Fig. 1) entrapping a model antigen, BSA, were also prepared by the procedure of emulsification–lyophilization (PEL) but subsequently used to constitute the microneedles of a biodegradable microneedle array. The proMLL-constituted microneedle arrays (proMMAs) (Fig. 1) were rather stable due to lack of water. When given to mice at oral cavity mucosa, the proMMAs could induce robust systemic and wide mucosal immunoresponses against the loaded antigens. Such a design for the proMMA and its administration eliminate expectedly several substantial obstacles, such as the inability of intradermal microneedles to induce extensive mucosal immunoresponses, the inefficiency of conventional vaccines in penetrating the mucus and tight epithelium of mucosa, and the loss of a large fraction of active ingredients when vaccines gone with mucus fluids and saliva, which are confoundedly encountered in the development of conventional oral mucosal vaccines or microneedle vaccines.
2. Materials and methods 2.1. Materials Soy phosphatidylcholine (SPC, purity > 97%) was purchased from Lipoid (Ludwigshafen, Germany). Bovine serum albumin (BSA), ovalbumin (OVA), aluminum phosphate (−200 mesh), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytochalasin D, stearylamine (SA), monophosphoryl lipid A (LA), IFN-␥ and IL-4 assay kits were commercial products by Sigma (Shanghai, China). Goat anti-mouse IgG-horse radish peroxidase (HRP), IgG1-HRP, IgG2a-HRP and IgA-HRP were purchased with sales package of 200 g per 0.5 mL from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PE-conjugated antimouse CD8+ mAb (monoclonal antibody) and FITC-conjugated anti-mouse CD4+ mAb were biological products of eBioscience (San Diego, USA). The APC surface mannose receptor-binding molecule
Fig. 1. Structure of the proMMA and MLL.
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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mannose-PEG1000 -cholesterol conjugate (MPC) was synthesized according to a previous report [16]. 2.2. Preparation of the proMMAs The microneedle array inverse molds (MAIMs) made of PDMS (polydimethylsiloxane) from a stainless steel master mold were the generous gifts provided kindly by Gao’s group who described in detail the related preparation methods in a recent publication [17]. Liposomes were prepared by a procedure of emulsification–lyophilization (PEL) which was recently established to prepare liposomes or anhydrous reverse micelles (ARMs) [18–21]. Briefly, an appropriate amount of SPC/MPC/SA/LA (100/5/10/1, mole ratio) was dissolved in a cosolvent of chloroform/cyclohexane (1:3, v/v) which was used as oil phase (O). A PBS (pH 7.4, for immunization) containing 10% (w/v) sucrose was used as water phase (W). One aliquot of O was added into 3 aliquots of W, and then using an ice/water bath to keep the temperature under 25 ◦ C, the mixture was sonicated in pulse mode (pulse on, 3 s; pulse off, 5 s; 80 W) to form submicron O/W emulsions which were immediately lyophilized. The lyophilization was carried out in a freeze dryer (FDU-1100/DRC-1000, Eyela, Japan) and performed as follows: freezing at −80 ◦ C for 4 h in an ultra-low temperature refrigerator (MDF-382E, Sanyo, Japan); primary drying at −40 ◦ C for 4 h, −25 ◦ C for 2 h, −15 ◦ C for 2 h; and secondary drying 20 ◦ C for 4 h. Then the lyophilized emulsions were rehydrated with an aqueous solution containing BSA (with BSA/SPC of 10:1, mass ratio) and 20% PVPk30 (w/v) to form MPC/lipid A-liposomes (MLLs), which were subsequently put onto MAIMs and filled into the microholes by decreasing pressure and then, after collection of the redundancy of MLLs for recycling use, covered with an appropriate aliquot of W also containing 20% PVPk30 . The loaded MAIMs were then put into a dessicator containing anhydrous CaCl2 and dried. When the drying process of around 7 h was completed, a 6 × 6 microneedle array was carefully peeled off from the MAIM and put in a dish and filled with nitrogen gas, covered and sealed, and stored protected from light at 4, 25, or 40 ◦ C for stability investigation. Upon rehydration, the microneedles rapidly dissolved, and the MLLs recovered. The final dry products were regarded as the proMLLs-stacked microneedle arrays (proMMAs). All experiments were repeated in triplicate (n = 3). The residue solvents were detected by gas chromatography (GC2010; Shimadzu, Japan) according to a previous report [19]. 2.3. Characterization 2.3.1. Observation of the proMMA A proMMA was imaged with a digital camera, and the microneedles of a proMMA were observed and imaged with a fluorescence microscope. The sizes of the products were measured with a calibrated scale. 2.3.2. Observation of the proMMAs by scanning electron microscopy (SEM) The microneedles of proMMAs were smashed and a small aliquot of the powders were fixed on an SEM-stub using conductive double-sided tape, coated in a vacuum with a thin layer of gold. And the samples were examined in a field emission scanning electron microscope (S-4800, Hitachi, Japan) with an accelerating voltage of 3 kV. 2.3.3. Verification of formation of liposomes by PEL and in microholes of MAIM by transmission electron microscopy (TEM) The microneedles (without base substrate) of proMMAs were dipped into water and snapped with a digital camera, and, subsequently, the aqueous suspensions were collected and observed in
3
a TEM. In addition, the microneedles (without basal substrate) still held in MAIMs were rehydrated in situ with an appropriate amount of water, and the suspensions were collected by squeezing the elastic MAIMs and observed in a TEM to verify the formation of liposomes (MLLs) in the microenvironment. The TEMs were obtained using a negative stain technique. Briefly, a small drop of the collected aqueous suspensions was placed on a collodion-coated grid and blotted with filter paper. A drop of 2% (w/v) phosphotungstic acid was applied to the grid and blotted, and the stained sample was allowed to dry. The sample-loaded grids were examined in a JEM100B (JEMO, Japan). 2.3.4. Detection of the size and zeta potential () of the MLLs The mean diameter (MD) and of the MLLs with or without BSA that were formed by PEL or recovered from proMMAs by rehydration were tested using a Malvern Zetasizer ZS90 (Malvern, Worcestershire, UK) at 25 ◦ C. The results were given as an average MD with PDI (polydispersity index) and ± SD (standard deviation). 2.3.5. Quantification of components of the MLLs The samples of MLLs recovered from proMMAs were collected, diluted with W and centrifuged at 100,000 × g for 30 min at 4 ◦ C in an ultracentrifuge (Micro Ultracentrifuge CS150NX, Hitachi, Japan). BSA in the supernatant was determined with the classical Bradford protein assay method [22]. Association efficiency (AE) of MLLs for BSA was determined as Eq. (1): AE =
total amount of BSA − unassociated BSA total amount of BSA
(1)
Unincorporated LA was quantified by Fiske and Subbarow method of phospho-molybdenum blue photometry after digestion of phosphorous [23]. Free SA in the supernatant was assayed by the fluorescamine labeling method [24]. Unincorporated MPC was determined by HPLC with an ODS column (4.6 mm × 150 mm, 5 m particle diameter) and the mobile phase of water (0.2% TEA)–methanol (25:75, v/v), and the detection was performed at a wavelength of 210 nm at 30 ◦ C. 2.3.6. BSA release feature The MLLs formed by PEL or recovered from proMMAs were transferred into a conical flasks diluted with PBS (pH7.4) and incubated at 37 ◦ C under stirring at 100 rpm. At different time intervals an appropriate amount of release medium was accurately taken out of the flask which was immediately supplemented with an identical amount of PBS. The released BSA in the medium was isolated by ultracentrifuge and subsequently determined with the Bradford method. 2.4. Penetration ability of microneedles Though the main immunization route of proMMAs was via mammal oral mucosa which is rather vulnerable, the ability of the microneedles to penetrate skins was also tested. The proMMA was patched onto porcine ear skins with an appropriate force and then removed. The microneedle-inserted area of skin was stained with trypan blue and subsequently blotted with paper towel and imaged using a digital camera. To observe whether MLLs were formed after skin insertion, a proMMA was patched onto the porcine ear skins for 2 min, and then the base substrate was removed. The microneedle-inserted area of skin was harvested and embedded in OCT (optimal cutting temperature) compound in a cryo-mold (Sakura Finetechnical Co., Ltd., USA), and then frozen at −80 ◦ C in an ultralow temperature refrigerator overnight. And the skin histological sections with 10 m thickness were made and imaged using florescence microscopy (BZ-8000; Keyence Corporation, Osaka, Japan).
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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2.5. Phagocytosis test
2.8. Sample collection and antibody titration
The ability of the MLLs recovered from proMMAs to facilitate the phagocytosis was evaluated as follows. The ether-anesthetized mice were sacrificed and the peritoneal cells were collected by flushing the peritoneal cavity with 10 mL of PBS. The mononuclear cells were isolated by centrifuge with a Ficoll gradient and suspended in RPMI 1640 complete medium and, subsequently, transferred into a 12-well plate with 1 mL of 103 mononuclear cells per well and incubated in a cell culture chamber containing 5% CO2 , 95% air (humidified), at 37 ◦ C for 12 h. Then 100 l of free calcein (10 M calcein-entrapping MLLs recovered from proMMAs) were added into the cells which were continuously incubated for 4 h. For control experiments, a phagocytosis inhibitor of cytochalasin D (10 g/mL) was added to the culture medium 1 h prior to the addition of MLLs and to the vesicles suspensions. Finally, mononuclear cells were isolated by repeated centrifuge (500 × g for 10 min) and suspension to get rid of unassociated calcein, and the cell samples were observed using a fluorescence microscope.
Samples of blood, saliva, vaginal and intestinal flush were collected 3 weeks after immunization in treated mice. In addition, collection of blood, saliva, and vaginal flush samples was continued at 9, 18, 27, 36 and 45 weeks after immunization in all groups except the one received the 40 ◦ C-stored proMMAs. For sample collection, the treated mice were deprived of food, but not water, overnight and anesthetized by i.p. injection of chloral hydrate. Saliva was collected from mice 5 min after s.c. injection of pilocarpine injection (20 g per animal). The vaginal samples were collected by two successive washes with 100 l of PBS introduced into the vaginal tract using a pipette, and the solution was withdrawn and again reintroduced ten times, and the two washes were pooled. Then blood was collected from the retro-orbital plexus and allowed to stand at room temperature for 20 min and then centrifuged for 10 min at 10,000 rpm. The supernatant serum was collected and stored at −20 ◦ C until further assay. Finally, intestinal lavages were collected after rinsing repeatedly the small intestinal lumen with 2 mL of PBS. All mucosal fluids were clarified by centrifugation for 10 min at 10,000 rpm, and the supernatant was collected and stored at −20 ◦ C until further assay. The conventional indirect ELISA method was employed for assaying the BSA-specific antibodies, and a microplate reader (QuantTM , BioTek Instruments, Inc., Vermont, USA) was used to determine the optical absorbance (OA) at 450 nm of the samples with an appropriate dilution [21].
2.6. Stability investigation The proMMAs had been stored at 4 ◦ C for up to 3 months, at for up to 2 weeks, and at 40 ◦ C for up to 4 days. At different time intervals, the integrity of BSA in the stored proMMAs was tested using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions [17]. Briefly, BSA was released from the MLLs with 1% Triton-X, and SDS-PAGE experiments were performed on 5% stacking gel and 10% separation (resolving) gel. The BSA samples were heated to 100 ◦ C for 10 min to denature with strong reducing agent, sodium dodecyl sulfate (SDS) and heat. Subsequently, the samples were loaded onto the vertical slab gel and subjected to electrophoresis at 80 mV for 30 min and then at 120 mV until the indicator bromophenol blue reached the bottom of the plate using a Bio-Rad mini protean II dual slab cell (Bio-Rad, Hemel Hempstead, UK). Gels were subsequently fixed and stained with Coomassie brilliant blue. Also, the size, potential and AE of the MLLs that were recovered from the stored proMMAs were tested to evaluate the stability of the products. 25 ◦ C
2.7. Vaccination of mice Animal protocols were approved by the Animal Ethic Committee at Anhui Medical University and carried out in compliance with the Declaration of Helsinki for Care and Use of Laboratory Animals. Female Kunming mice aged 4 weeks were from the Experimental Animal Center of Anhui Medical University and divided into eight groups (9 mice per group). Four groups of mice were immunized by oral mucosal (o.m.) administration of blank microneedle arrays (BMAs, containing sucrose and PVP), proMMAs that had been stored at room temperature for 2 weeks or at 40 ◦ C for 3 days, or MLLs which were freshly constructed by rehydration of the proMMAs. Another two groups of mice were immunized by intradermal (i.d.) administration of proMMAs or by s.c. injection of an aqueous suspension of BSA-Al (BSA/alum, 10:1, w/w). In order to comment on the loss of ingredients with oral mucus fluids and saliva, two additional groups of mice were immunized with MLLs or proMMAs through oral mucosal administration under anesthesia (labeled as a.e./o.m. group) which was achieved by intraperitoneal (i.p.) injection of chloral hydrate at a dose of 0.5 mg/g body weight. For all formulations, except BMA, the dose of BSA was kept at 4 g per mouse.
2.9. Splenocyte proliferation test and cytokine assay The spleens of treated mice were removed after anesthetization under an aseptic operation, and then the spleen lymphocytes were isolated and incubated in a 96-well microplate at a concentration of 100 l of 5 × 105 cells per well in the presence of 2.5 mg/mL BSA for 72 h at 37 ◦ C in a cell chamber. The splenocyte proliferation was evaluated with MTT method and expressed with the stimulation index (SI), which was calculated with Eq. (2): SI =
OA (treated mice splenocytes) OA (negative control mice splenocytes)
(2)
where OA was the optical absorbance at 540 nm of the samples treated according to MTT protocol. To analyze the type of T helper (Th) 1 and Th2 immune response induced by different formulations, the sandwich enzyme immunoassay was employed to determine the concentrations of IFN-␥ and IL-4 in the sera of the immunized mice and in the culture supernatants of the splenocytes that had been isolated from the immunized mice and subsequently incubated in the presence of BSA. Cytokine concentrations were calculated from the curves established with standard IFN-␥ or IL-4 dilutions. The samples were assayed using Quant microplate reader with OA tested at 450 nm. 2.10. Flow cytometry (FCM) of T lymphocyte subset Three weeks after immunization, the spleen cells were isolated from immunized mice after anesthetization with RMPI 1640 medium without serum. Cells adjusted to 1 × 106 with PBS were stained with 0.5 g of PE-conjugated anti-mouse CD4+ mAb and FITC-conjugated anti-mouse CD8+ mAb (eBioscience, CA, USA), T lymphocyte subsets were measured using a flow cytometer (BD FACSVerseTM , San Jose, CA, USA). 2.11. Statistical analysis Results were presented as mean ± SD (standard deviation). Statistical differences among multiple groups were analyzed with
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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ANOVA, followed by Dunnett’s post hoc t test to estimate the statistical differences between groups using the SPSS software. A p value
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JVAC-16309; No. of Pages 11
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination Yuanyuan Zhen a,1 , Ning Wang b,c,1 , Zibin Gao d , Xiaoyu Ma a , Biao Wei a , Yihui Deng c , Ting Wang a,∗ a
School of Pharmacy, Anhui Medical University, 81 Plum Hill Road, Hefei, Anhui Province 230032, China School of Medical Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui Province 230009, China c School of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning Province 110016, China d Department of Pharmacy, Hebei University of Science and Technology, 70 Yuhua East Road, Shijiazhuang 050018, China b
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Freeze-drying Immunization Toll-like receptor C-type lectin receptor ELISA Controlled temperature chain
a b s t r a c t To develop effective, convenient and stable mucosal vaccines, mannose-PEG-cholesterol (MPC)/lipid Aliposomes (MLLs) entrapping model antigen bovine serum albumin (BSA) were prepared by the procedure of emulsification–lyophilization and used to constitute microneedles, forming the proMLL-filled microneedle arrays (proMMAs). The proMMAs were rather stable and hard enough to pierce porcine skin and, upon rehydration, dissolved rapidly recovering the MLLs without size and entrapment change. The proMMAs given to mice via oral mucosal (o.m.) route, rather than routine intradermal administration, elicited robust systemic and mucosal immunoresponses against the loaded antigens as evidenced by high levels of BSA-specific IgG in the sera and IgA in the salivary, intestinal and vaginal secretions of mice. Enhanced levels of IgG2a and IFN-␥ in treated mice revealed that proMMAs induced a mixed Th1/Th2 immunoresponse. Moreover, a significant increase in CD8+ T cells confirmed that strong cellular immunity had also been established by the immunization of the proMMAs. Thus, the proMMAs can be immunized via o.m. route to set up an effective multiple defense against pathogen invasion and may be an effective vaccine adjuvant-delivery system (VADS) applicable in the controlled temperature chain. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Mucosal vaccines are inoculated at the site of cavity or lumen mucosas which are commonly rich in dendritic cells (DCs) and are, therefore, able to induce both systemic and mucosal immunoresponses not only at the site of antigen exposure but also at remote mucosas due to the existence of generalized mucosal
Abbreviations: MPC, mannose-PEG-cholesterol; MLL, MPC/lipid A-liposome; proMMA, proMLL-filled microneedle array; BMA, blank microneedle array; VADS, vaccine adjuvant-delivery system; CTC, controlled temperature chain; SPC, soy phosphatidylcholine; MAIM, microneedle array inverse mold; AE, association efficiency; MD, mean diameter; o.m., oral mucosal; i.d., intradermal; s.c., subcutaneous; a.e., anesthesia/anesthetization; BSA-Al, BSA-alum; MALT, mucosa-associated lymphoid tissue; DC, dendritic cell; CTL, cytotoxic T lymphocyte; PEL, procedure of emulsification–lyophilization; OCT, optimal cutting temperature; s.l., sublingual. ∗ Corresponding author. Tel.: +86 15395082913. E-mail addresses: [email protected], [email protected] (T. Wang). 1 Both authors contributed equally to this work.
immune network where numerous mucosa-associated lymphoid tissues (MALT) are located [1]. To function effectively, mucosal vaccines must approach the professional antigen-presenting cells (APCs) to induce potent immunoresponses to produce functional pathogen-specific antibodies and cytotoxic T lymphocytes (CTLs), neutralizing and lysing the invaded pathogens. Ideally, a mucosal vaccine would be able to elicit functional immunocytes to secrete mucosal immunoglobulins blocking the establishment of initial infection by the pathogens, such as HIV and HPV, which once enter the cells can rapidly integrate into the host genome to establish a latent reservoir that can hardly be eliminated by conventional antiretroviral agents [2]. Thus, the mucosal vaccines that can block the initial invasion of intractable pathogens are most desirably needed. However, the mammal mucosas suitable for vaccination are usually covered with a defending layer of mucus which is a continuously renewed viscous fluid and contains various categories of agents, such as antiseptic lysozyme, proteins and glycoprotein mucins [3]. The defending mucus and enzymes impose a potential
http://dx.doi.org/10.1016/j.vaccine.2015.03.081 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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damage to vaccine antigens and prevent mucosal vaccines from approaching and crossing the epithelial layer, under which the professional APCs are most likely sited. Moreover, the tightly-lined epithelial cells with intercellular spaces sealed by tight junctions also form a barrier to mucosal vaccines to reach the professional APCs which are the necessary sponsors for the immunoresponses to the vaccines. By comparison to other sites, oral mucosa confronts a mild environment and is easily accessible and relatively safe for vaccination [4]. The oral mucosa is also rich in APCs and can mediate innate and adaptive immunoresponses to block local and systemic infections [5]. However, oral mucosal immunization is further limited by its anatomic structure of stratified squamous epithelium (SSE) as well as by rapid clearance of the subjects from mucosal surfaces by flow of saliva, movement of tongue and jaws, and chewing and swallowing [6]. To conquer these barriers to oral mucosal delivery, numerous technologies have been developed, including supersaturation, eutectic formation, iontophoresis, electroporation, sonophoresis, laser radiation, photomechanical waves and needleless injection [6]. But a common strategy was to incorporate a bioadhesive component to the drug/carrier to achieve prolonged mucosal contact and higher drug concentration on the mucosal surface [7]. Unfortunately, the bioadhesive component may cause oral paraesthesia or foreign body sensation which accelerates in biofeedback salivation and swallow leading to ingredient loss as well as poor patient compliance; moreover, due to its adherence to the upper epithelia of SSE, bioadhesive component also limits vaccine uptake into the underlying professional APCs resulting in weakened efficacy. Another approach is to use permeation enhancers (e.g., a mucolytic agent of N-acetyl-l-cysteine) to enhance the bioavailability of protein-containing nanoparticles delivered via mucosal route, however, the mucus barrier properties cannot be undermined without compromising the mucosa [8]. Recently, researchers modified nanoparticles with PEG as socalled mucus-penetrating particles (MPPs) for topical delivery of antibiotics in vagina and proved that MPPs improved vaginal drug distribution and retention over the vaginal epithelium compared to conventional particles [9]. But no enhanced uptake of agents into cells of interest can be expected for such an MPP as PEG is a known barrier to the access by most kinds of cells [10]. Previously, we showed that the mannose-PEG-cholesterol MPC/lipid A-liposomes (MLLs) could efficiently carry, protect and present antigens and, therefore, proved an effective oral mucosal vaccine adjuvant-delivery system (VADS) [11]. However, the MLLs also confront the above multiple obstacles which are difficult to overcome with the abovementioned common methods. Recently, microneedle arrays (MAs) with sub-millimeter structures designed
to pierce the skin and deliver vaccines in the epidermis or dermis compartments without pain provide an especially attractive option for intradermal delivery [12–15]. Interestingly, some researchers primed mice with intradermal (i.d.) biodegradable microneedles and then boosted by intranasal inoculation with the aqueous formulation, and finally they elicited robust antigen-specific humoral as well as mucosal immunity [13]. Inspired by this, we proposed that microneedle vaccines might as well be administered at oral mucosal sites to penetrate the barriers encountered with conventional mucosal vaccines. In this report, the MPC/lipid A-liposomes (MLLs) (Fig. 1) entrapping a model antigen, BSA, were also prepared by the procedure of emulsification–lyophilization (PEL) but subsequently used to constitute the microneedles of a biodegradable microneedle array. The proMLL-constituted microneedle arrays (proMMAs) (Fig. 1) were rather stable due to lack of water. When given to mice at oral cavity mucosa, the proMMAs could induce robust systemic and wide mucosal immunoresponses against the loaded antigens. Such a design for the proMMA and its administration eliminate expectedly several substantial obstacles, such as the inability of intradermal microneedles to induce extensive mucosal immunoresponses, the inefficiency of conventional vaccines in penetrating the mucus and tight epithelium of mucosa, and the loss of a large fraction of active ingredients when vaccines gone with mucus fluids and saliva, which are confoundedly encountered in the development of conventional oral mucosal vaccines or microneedle vaccines.
2. Materials and methods 2.1. Materials Soy phosphatidylcholine (SPC, purity > 97%) was purchased from Lipoid (Ludwigshafen, Germany). Bovine serum albumin (BSA), ovalbumin (OVA), aluminum phosphate (−200 mesh), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytochalasin D, stearylamine (SA), monophosphoryl lipid A (LA), IFN-␥ and IL-4 assay kits were commercial products by Sigma (Shanghai, China). Goat anti-mouse IgG-horse radish peroxidase (HRP), IgG1-HRP, IgG2a-HRP and IgA-HRP were purchased with sales package of 200 g per 0.5 mL from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PE-conjugated antimouse CD8+ mAb (monoclonal antibody) and FITC-conjugated anti-mouse CD4+ mAb were biological products of eBioscience (San Diego, USA). The APC surface mannose receptor-binding molecule
Fig. 1. Structure of the proMMA and MLL.
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mannose-PEG1000 -cholesterol conjugate (MPC) was synthesized according to a previous report [16]. 2.2. Preparation of the proMMAs The microneedle array inverse molds (MAIMs) made of PDMS (polydimethylsiloxane) from a stainless steel master mold were the generous gifts provided kindly by Gao’s group who described in detail the related preparation methods in a recent publication [17]. Liposomes were prepared by a procedure of emulsification–lyophilization (PEL) which was recently established to prepare liposomes or anhydrous reverse micelles (ARMs) [18–21]. Briefly, an appropriate amount of SPC/MPC/SA/LA (100/5/10/1, mole ratio) was dissolved in a cosolvent of chloroform/cyclohexane (1:3, v/v) which was used as oil phase (O). A PBS (pH 7.4, for immunization) containing 10% (w/v) sucrose was used as water phase (W). One aliquot of O was added into 3 aliquots of W, and then using an ice/water bath to keep the temperature under 25 ◦ C, the mixture was sonicated in pulse mode (pulse on, 3 s; pulse off, 5 s; 80 W) to form submicron O/W emulsions which were immediately lyophilized. The lyophilization was carried out in a freeze dryer (FDU-1100/DRC-1000, Eyela, Japan) and performed as follows: freezing at −80 ◦ C for 4 h in an ultra-low temperature refrigerator (MDF-382E, Sanyo, Japan); primary drying at −40 ◦ C for 4 h, −25 ◦ C for 2 h, −15 ◦ C for 2 h; and secondary drying 20 ◦ C for 4 h. Then the lyophilized emulsions were rehydrated with an aqueous solution containing BSA (with BSA/SPC of 10:1, mass ratio) and 20% PVPk30 (w/v) to form MPC/lipid A-liposomes (MLLs), which were subsequently put onto MAIMs and filled into the microholes by decreasing pressure and then, after collection of the redundancy of MLLs for recycling use, covered with an appropriate aliquot of W also containing 20% PVPk30 . The loaded MAIMs were then put into a dessicator containing anhydrous CaCl2 and dried. When the drying process of around 7 h was completed, a 6 × 6 microneedle array was carefully peeled off from the MAIM and put in a dish and filled with nitrogen gas, covered and sealed, and stored protected from light at 4, 25, or 40 ◦ C for stability investigation. Upon rehydration, the microneedles rapidly dissolved, and the MLLs recovered. The final dry products were regarded as the proMLLs-stacked microneedle arrays (proMMAs). All experiments were repeated in triplicate (n = 3). The residue solvents were detected by gas chromatography (GC2010; Shimadzu, Japan) according to a previous report [19]. 2.3. Characterization 2.3.1. Observation of the proMMA A proMMA was imaged with a digital camera, and the microneedles of a proMMA were observed and imaged with a fluorescence microscope. The sizes of the products were measured with a calibrated scale. 2.3.2. Observation of the proMMAs by scanning electron microscopy (SEM) The microneedles of proMMAs were smashed and a small aliquot of the powders were fixed on an SEM-stub using conductive double-sided tape, coated in a vacuum with a thin layer of gold. And the samples were examined in a field emission scanning electron microscope (S-4800, Hitachi, Japan) with an accelerating voltage of 3 kV. 2.3.3. Verification of formation of liposomes by PEL and in microholes of MAIM by transmission electron microscopy (TEM) The microneedles (without base substrate) of proMMAs were dipped into water and snapped with a digital camera, and, subsequently, the aqueous suspensions were collected and observed in
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a TEM. In addition, the microneedles (without basal substrate) still held in MAIMs were rehydrated in situ with an appropriate amount of water, and the suspensions were collected by squeezing the elastic MAIMs and observed in a TEM to verify the formation of liposomes (MLLs) in the microenvironment. The TEMs were obtained using a negative stain technique. Briefly, a small drop of the collected aqueous suspensions was placed on a collodion-coated grid and blotted with filter paper. A drop of 2% (w/v) phosphotungstic acid was applied to the grid and blotted, and the stained sample was allowed to dry. The sample-loaded grids were examined in a JEM100B (JEMO, Japan). 2.3.4. Detection of the size and zeta potential () of the MLLs The mean diameter (MD) and of the MLLs with or without BSA that were formed by PEL or recovered from proMMAs by rehydration were tested using a Malvern Zetasizer ZS90 (Malvern, Worcestershire, UK) at 25 ◦ C. The results were given as an average MD with PDI (polydispersity index) and ± SD (standard deviation). 2.3.5. Quantification of components of the MLLs The samples of MLLs recovered from proMMAs were collected, diluted with W and centrifuged at 100,000 × g for 30 min at 4 ◦ C in an ultracentrifuge (Micro Ultracentrifuge CS150NX, Hitachi, Japan). BSA in the supernatant was determined with the classical Bradford protein assay method [22]. Association efficiency (AE) of MLLs for BSA was determined as Eq. (1): AE =
total amount of BSA − unassociated BSA total amount of BSA
(1)
Unincorporated LA was quantified by Fiske and Subbarow method of phospho-molybdenum blue photometry after digestion of phosphorous [23]. Free SA in the supernatant was assayed by the fluorescamine labeling method [24]. Unincorporated MPC was determined by HPLC with an ODS column (4.6 mm × 150 mm, 5 m particle diameter) and the mobile phase of water (0.2% TEA)–methanol (25:75, v/v), and the detection was performed at a wavelength of 210 nm at 30 ◦ C. 2.3.6. BSA release feature The MLLs formed by PEL or recovered from proMMAs were transferred into a conical flasks diluted with PBS (pH7.4) and incubated at 37 ◦ C under stirring at 100 rpm. At different time intervals an appropriate amount of release medium was accurately taken out of the flask which was immediately supplemented with an identical amount of PBS. The released BSA in the medium was isolated by ultracentrifuge and subsequently determined with the Bradford method. 2.4. Penetration ability of microneedles Though the main immunization route of proMMAs was via mammal oral mucosa which is rather vulnerable, the ability of the microneedles to penetrate skins was also tested. The proMMA was patched onto porcine ear skins with an appropriate force and then removed. The microneedle-inserted area of skin was stained with trypan blue and subsequently blotted with paper towel and imaged using a digital camera. To observe whether MLLs were formed after skin insertion, a proMMA was patched onto the porcine ear skins for 2 min, and then the base substrate was removed. The microneedle-inserted area of skin was harvested and embedded in OCT (optimal cutting temperature) compound in a cryo-mold (Sakura Finetechnical Co., Ltd., USA), and then frozen at −80 ◦ C in an ultralow temperature refrigerator overnight. And the skin histological sections with 10 m thickness were made and imaged using florescence microscopy (BZ-8000; Keyence Corporation, Osaka, Japan).
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2.5. Phagocytosis test
2.8. Sample collection and antibody titration
The ability of the MLLs recovered from proMMAs to facilitate the phagocytosis was evaluated as follows. The ether-anesthetized mice were sacrificed and the peritoneal cells were collected by flushing the peritoneal cavity with 10 mL of PBS. The mononuclear cells were isolated by centrifuge with a Ficoll gradient and suspended in RPMI 1640 complete medium and, subsequently, transferred into a 12-well plate with 1 mL of 103 mononuclear cells per well and incubated in a cell culture chamber containing 5% CO2 , 95% air (humidified), at 37 ◦ C for 12 h. Then 100 l of free calcein (10 M calcein-entrapping MLLs recovered from proMMAs) were added into the cells which were continuously incubated for 4 h. For control experiments, a phagocytosis inhibitor of cytochalasin D (10 g/mL) was added to the culture medium 1 h prior to the addition of MLLs and to the vesicles suspensions. Finally, mononuclear cells were isolated by repeated centrifuge (500 × g for 10 min) and suspension to get rid of unassociated calcein, and the cell samples were observed using a fluorescence microscope.
Samples of blood, saliva, vaginal and intestinal flush were collected 3 weeks after immunization in treated mice. In addition, collection of blood, saliva, and vaginal flush samples was continued at 9, 18, 27, 36 and 45 weeks after immunization in all groups except the one received the 40 ◦ C-stored proMMAs. For sample collection, the treated mice were deprived of food, but not water, overnight and anesthetized by i.p. injection of chloral hydrate. Saliva was collected from mice 5 min after s.c. injection of pilocarpine injection (20 g per animal). The vaginal samples were collected by two successive washes with 100 l of PBS introduced into the vaginal tract using a pipette, and the solution was withdrawn and again reintroduced ten times, and the two washes were pooled. Then blood was collected from the retro-orbital plexus and allowed to stand at room temperature for 20 min and then centrifuged for 10 min at 10,000 rpm. The supernatant serum was collected and stored at −20 ◦ C until further assay. Finally, intestinal lavages were collected after rinsing repeatedly the small intestinal lumen with 2 mL of PBS. All mucosal fluids were clarified by centrifugation for 10 min at 10,000 rpm, and the supernatant was collected and stored at −20 ◦ C until further assay. The conventional indirect ELISA method was employed for assaying the BSA-specific antibodies, and a microplate reader (QuantTM , BioTek Instruments, Inc., Vermont, USA) was used to determine the optical absorbance (OA) at 450 nm of the samples with an appropriate dilution [21].
2.6. Stability investigation The proMMAs had been stored at 4 ◦ C for up to 3 months, at for up to 2 weeks, and at 40 ◦ C for up to 4 days. At different time intervals, the integrity of BSA in the stored proMMAs was tested using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions [17]. Briefly, BSA was released from the MLLs with 1% Triton-X, and SDS-PAGE experiments were performed on 5% stacking gel and 10% separation (resolving) gel. The BSA samples were heated to 100 ◦ C for 10 min to denature with strong reducing agent, sodium dodecyl sulfate (SDS) and heat. Subsequently, the samples were loaded onto the vertical slab gel and subjected to electrophoresis at 80 mV for 30 min and then at 120 mV until the indicator bromophenol blue reached the bottom of the plate using a Bio-Rad mini protean II dual slab cell (Bio-Rad, Hemel Hempstead, UK). Gels were subsequently fixed and stained with Coomassie brilliant blue. Also, the size, potential and AE of the MLLs that were recovered from the stored proMMAs were tested to evaluate the stability of the products. 25 ◦ C
2.7. Vaccination of mice Animal protocols were approved by the Animal Ethic Committee at Anhui Medical University and carried out in compliance with the Declaration of Helsinki for Care and Use of Laboratory Animals. Female Kunming mice aged 4 weeks were from the Experimental Animal Center of Anhui Medical University and divided into eight groups (9 mice per group). Four groups of mice were immunized by oral mucosal (o.m.) administration of blank microneedle arrays (BMAs, containing sucrose and PVP), proMMAs that had been stored at room temperature for 2 weeks or at 40 ◦ C for 3 days, or MLLs which were freshly constructed by rehydration of the proMMAs. Another two groups of mice were immunized by intradermal (i.d.) administration of proMMAs or by s.c. injection of an aqueous suspension of BSA-Al (BSA/alum, 10:1, w/w). In order to comment on the loss of ingredients with oral mucus fluids and saliva, two additional groups of mice were immunized with MLLs or proMMAs through oral mucosal administration under anesthesia (labeled as a.e./o.m. group) which was achieved by intraperitoneal (i.p.) injection of chloral hydrate at a dose of 0.5 mg/g body weight. For all formulations, except BMA, the dose of BSA was kept at 4 g per mouse.
2.9. Splenocyte proliferation test and cytokine assay The spleens of treated mice were removed after anesthetization under an aseptic operation, and then the spleen lymphocytes were isolated and incubated in a 96-well microplate at a concentration of 100 l of 5 × 105 cells per well in the presence of 2.5 mg/mL BSA for 72 h at 37 ◦ C in a cell chamber. The splenocyte proliferation was evaluated with MTT method and expressed with the stimulation index (SI), which was calculated with Eq. (2): SI =
OA (treated mice splenocytes) OA (negative control mice splenocytes)
(2)
where OA was the optical absorbance at 540 nm of the samples treated according to MTT protocol. To analyze the type of T helper (Th) 1 and Th2 immune response induced by different formulations, the sandwich enzyme immunoassay was employed to determine the concentrations of IFN-␥ and IL-4 in the sera of the immunized mice and in the culture supernatants of the splenocytes that had been isolated from the immunized mice and subsequently incubated in the presence of BSA. Cytokine concentrations were calculated from the curves established with standard IFN-␥ or IL-4 dilutions. The samples were assayed using Quant microplate reader with OA tested at 450 nm. 2.10. Flow cytometry (FCM) of T lymphocyte subset Three weeks after immunization, the spleen cells were isolated from immunized mice after anesthetization with RMPI 1640 medium without serum. Cells adjusted to 1 × 106 with PBS were stained with 0.5 g of PE-conjugated anti-mouse CD4+ mAb and FITC-conjugated anti-mouse CD8+ mAb (eBioscience, CA, USA), T lymphocyte subsets were measured using a flow cytometer (BD FACSVerseTM , San Jose, CA, USA). 2.11. Statistical analysis Results were presented as mean ± SD (standard deviation). Statistical differences among multiple groups were analyzed with
Please cite this article in press as: Zhen Y, et al. Multifunctional liposomes constituting microneedles induced robust systemic and mucosal immunoresponses against the loaded antigens via oral mucosal vaccination. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.081
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ANOVA, followed by Dunnett’s post hoc t test to estimate the statistical differences between groups using the SPSS software. A p value
