Article pubs.acs.org/molecularpharmaceutics
Development of Soluble Inulin Microparticles as a Potent and Safe Vaccine Adjuvant and Delivery System Sunny Kumar and Hemachand Tummala* College of Pharmacy, South Dakota State University, SAV # 255, Box 2202C, Brookings, South Dakota 57006, United States S Supporting Information *
ABSTRACT: The goal of the present study is to develop a potent and safe vaccine adjuvant that can also stabilize vaccine formulations during lyophilization and storage. Inulin is a safe plant polysaccharide, and in its water soluble isoform, it is known to stabilize protein formulations during storage. However, soluble inulins have never been shown to stimulate the immune system. In this study, for the first time, we showed that water soluble inulins could be developed into vaccine adjuvants by formulating as antigen encapsulated microparticles. A method was developed to prepare soluble inulin microparticles (sIMs) with high encapsulation efficiency (∼75%) and loading (∼75 μg/mg) of the antigen. When immunized in mice, sIMs have generated robust Th2-type antibody titers (IgG1: 500,000) compared to unadjuvanted antigens (IgG1: 17,500) or alum adjuvanted antigens (IgG1: 80,000). In vitro assays showed that a higher proportion of antigen presenting cells (APC’s) have taken up the antigen when presented in sIMs versus in solution (99 % vs 22 %). In addition, the amount of antigen taken up per cell has also been enhanced by more than 25 times when antigen was presented in sIMs. Efficient uptake of the antigen by APCs through sIMS was attributed to the observed enhancement in the immune response by antigen loaded sIMs. The sIMs neither caused any granuloma/tissue damage at the injection site in mice nor were they toxic to the APC’s in cell culture. In conclusion, the current study has developed a safe, soluble inulin based vaccine adjuvant and delivery system. KEYWORDS: vaccine, vaccine adjuvant, vaccine delivery, polymers, soluble inulin, ovalbumin, microparticles
1. INTRODUCTION The main goal of vaccination is to provide long lasting protection against infections by generating strong immune responses against the administered antigen.1 Although the new generation of vaccines based on recombinant proteins, peptides, and DNA are considered safe, they generate significantly lower immune responses compared to the conventional vaccines based on live/killed whole organisms.2,3 A majority of the subunit vaccines require the addition of helper agents called adjuvants to enhance the antigen specific immune responses.3,4 Adjuvants can act either as immunostimulatory agents and/or as vaccine delivery vehicles.3,4 Over the last few decades, a wide variety of materials including alum, MF59, AS03, complete Freund’s adjuvant (CFA), quil A, lipopolysaccharides (LPS), saponins, etc. have been investigated for their adjuvant effects.5 However, these adjuvants have limitations to be used in humans due to one or more of the following reasons: lack of clinical efficacy, difficult method of preparation, excessive cost, instability during storage, and most importantly undesirable local or systemic toxicities.5 At present, alum (aluminum salts) is used as a vaccine adjuvant in a majority of the vaccines in the United States. However, alum has several drawbacks such as pain, inflammation, lymphadenopathy, necrosis, and granulomas at the injection site.6,7 Moreover, alum adjuvanted vaccines lose © 2013 American Chemical Society
potency at elevated temperature and therefore require cold storage.8,9 This makes it difficult to deliver these vaccines in countries where cold storage is a limitation. Hence, there is an unmet need to develop a safe and potent vaccine adjuvant and/ or delivery system, which is also stable at room temperature. Several studies have reported the role of plant polysaccharides in preventing the degradation of proteins/vaccines during lyophillization and storage.10−13 Once lyophilized, these vaccine formulations could be stored at room temperature without affecting their stability. Inulin is a nontoxic and biocompatible plant polysaccharide used in the human diet for several years.14 Inulin exists as soluble (e.g., α and β) and insoluble isoforms (e.g., γ and δ). The insoluble isoforms of inulin (γ and δ) stimulate the immune system by the activating alternate complement pathway (ACP) and thus act as potent vaccine adjuvants.15,16 However, they cannot function as cryoprotectants or protein stabilizers. In addition, these insoluble isoforms of inulin are prepared by special methods from a soluble extract of inulin with little yield.17 However, the most abundant form of inulin from natural sources is the water-soluble polymorphic Received: Revised: Accepted: Published: 1845
November 7, 2012 March 4, 2013 March 18, 2013 March 18, 2013 dx.doi.org/10.1021/mp3006374 | Mol. Pharmaceutics 2013, 10, 1845−1853
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form, which is well known to stabilize proteins and vaccine formulations during freezing and drying,12,13,18 but it does not activate ACP16 and has never been shown to have an immune stimulatory effect. The main objective of this study was to test the hypothesis that the soluble inulin could stimulate the immune system when formulated as antigen encapsulated soluble inulin microparticles (sIMs). Therefore, in this study, sIMs were explored as a safe vaccine adjuvant and delivery system. The rationale for this study is that (i) particulate forms of antigens are more potent in stimulating the immune system compared to the soluble antigens, mainly due to the enhanced antigen uptake by immune cells,19−23 (ii) the backbone of inulin will provide the necessary costimulatory signals to the immune system for enhanced immune response,24 (iii) the safety profile for soluble inulin is well established, and that (iv) the vaccine formulation could be stored at room temperature as a lyophilized powder without any other cryoprotectant as soluble inulin itself acts as a cryoprotectant.
double-sided tape and sputter coated with a gold layer for analysis. 2.6. Estimation of Antigen Loading and Encapsulation Efficiency. To measure the amount of ova present per mg of the particles (ova loading), fluorescein isothiocyanate labeled ova (FITC-ova) were used instead of free ova during the preparation of sIMs. A known amount of FITC-ova loaded sIMs was dissolved in 1% w/v sodium dodecyl sulfate (SDS) solution. The mixture was centrifuged at 10,000g for 10 min. The protein content in the supernatant was determined by measuring the fluorescence values of FITC-ova at excitation and emission wavelengths of 490 and 530 nm, respectively. Ova loading is reported as μg of ova present per mg of sIMs (w/w). 2.7. Endotoxin Levels. The endotoxin level in the final formulation was determined by the LAL assay method with the commercially available Endotoxin Assay Kit (ToxinSensor Chromogenic LAL Endotoxin Assay Kit) from GenScript (Piscataway, NJ, USA)27 by following the manufacturer’s instructions. The endotoxin levels in the formulation are within the limits for parenteral injections as described in the United States of Pharmacopeia (5 EU/dose/kg of body weight). 2.8. In Vitro Release Studies. FITC-ova loaded sIMs (10 mg) were dispersed in 1 mL of 0.1 M phosphate buffer (pH 7.4) and incubated at 37 °C with 100 rpm shaking. Tubes (triplicates) were taken out at predetermined time intervals (0.5, 1, 2, 4, 8, 16, and 24 h) and centrifuged at 20,000g for 10 min at 4 °C. A 50 μL of supernatant was taken for the measurement of released FITC-ova and replaced with an equal volume of fresh phosphate buffer. The released ova concentration in the supernatant was measured using a fluorometer as described above. 2.9. Immunization Studies. Male Balb/C mice (n = 4 per group, 6−8 weeks old) purchased from Charles River laboratories (Wilmington, MA, USA) were used for the immunization study. The mice were immunized via an intradermal (i.d.) route with the following groups: (i) ova (100 μg per mouse), (ii) 100 μg of ova with 200 μg of alum (aluminum hydroxide) per mouse, (iii) physical mixture of ova (100 μg per mouse) and blank soluble inulin particles, and (iv) ova loaded sIMs (equivalent to 100 μg ova per mouse). The vaccine formulations in 100 μL of phosphate buffered saline (PBS, 10 mM phosphate buffer, and 150 mM sodium chloride, pH 7.4) were administered at two different sites (50 μL at each site) to the shaved back skin of the mouse using a standard disposable 27 1/2-gauge syringe. The mice were vaccinated on day 1 with the primary dose followed by a booster dose on day 21. Blood samples were collected in serum gel tubes (MicroHematocrit, Pittsburgh, PA, USA) from the retro orbital plexus at the first and third weeks after primary and booster doses. Samples were centrifuged at 3,200g for 30 min, and the sera were stored at −80 °C until further analyzed by ELISA. Animal experiments were conducted in full compliance with regulations of the Institutional Animal Care and Use Committees (IACUC) of South Dakota State University, Brookings, SD, USA. 2.10. Detection of Anti-Ova Antibodies Using the Enzyme Linked Immunosorbent Assay (ELISA). Sera from the immunized mice were tested for the presence of antibodies (IgG-total, IgG-1, and IgG-2a) generated against the ova by an indirect-ELISA assay method. Briefly, 96- well ELISA plates (Maxisorp, NUNC, Rochester, NY, USA) were coated with ova (1 μg/well) in 50 mM carbonate buffer (pH 9.6) and incubated overnight at 4 °C. The plates were washed three times with
2. MATERIALS AND METHODS 2.1. Materials. Inulin from dahlia tubers, ovalbumin (ova), aluminum hydroxide gel, and all other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). Goat antimouse IgG/IgG1/IgG2a-HRP conjugates were purchased from Southern Biotech (Birmingham, Alabama, USA). 2.2. Cell Lines. The mouse dendritic cell line DC2.4 was a generous gift from Dr. K. L. Rock (University of Massachusetts Medical Center, Worcester, MA, USA). DC2.4 cells are shown to display dendritic morphology, express dendritic cell-specific markers, MHC molecules, and costimulatory molecules, and have phagocytic properties with antigen presenting capability.25 DC2.4 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, and 2 mM Lglutamine and penicillin/streptomycin antibiotics. 2.3. Preparation of Soluble Inulin. Soluble inulin was prepared from raw inulin by the ethanol precipitation method.24,26 Commercially available raw inulin obtained from dahlia tubers (Fisher Scientific) was dispersed in 100% ethanol and allowed to precipitate overnight at 4 °C. The next day, precipitated inulin was separated after centrifugation and lyophilized. The soluble inulin was characterized by its free solubility in water at 37 °C.17 The dried soluble inulin was used for all further studies. 2.4. Preparation of Ova Loaded Soluble Inulin Microparticles (sIMs). sIMs were prepared by the water in oil (w/o) emulsion−precipitation technique. Soluble inulin (100 mg) and ova (10 mg) were dissolved in 10 mL of 10 mM phosphate buffer at pH 7.4 (aqueous phase). This aqueous phase was added dropwise into 30 mL of light mineral oil containing 1% w/v of Tween-80 as surfactant with continuous stirring to obtain a stable w/o emulsion. The emulsion was stirred for 4 h, and then 30 mL of acetone was added dropwise to precipitate soluble inulin particles. The suspension was left for overnight stirring, and sIMs were collected by centrifugation at 3,200g for 30 min at 4 °C. sIMs were washed twice with nhexane, stored at −80 °C for 1 h, and lyophilized for 48 h. The final microparticles were completely water-soluble at 37 °C. 2.5. Scanning Electron Microscopy (SEM). The size and morphology of the sIMs were investigated using scanning electron microscopy (SEM, Model S-3400N, Hitachi, Japan). The particles were mounted on metal holders using conductive 1846
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the ova loaded sIMs was 1.5 ± 0.12 μm (Table 1). SEM pictures of the microparticles showed that ova loaded sIMs
wash solution (50 mM Tris, 140 mM NaCl, and 0.05% Tween 20, pH 8.0) and then blocked with 200 μL of blocking solution (50 mM Tris, 140 mM NaCl, and 1% BSA, pH 8.0) for 30 min. Subsequently, the plates were washed three times with wash solution, and the test sera were added (100 μL/well at different dilutions) and incubated for 1 h. After washing the plates three times, 100 μL of peroxidase-conjugated goat antimouse antibodies (1:6000) was added and incubated for 1 h. After incubation, the plates were washed; 100 μL of substrate (ABTS, Becton Dickinson, Franklin Lakes, NJ) was added and further incubated for 5 min for color development. All of the incubations in ELISA were performed at room temperature. The reaction was stopped using 2 M H2SO4, and absorbance (450 nm) was measured. Results were expressed as serum immunoglobulin G (IgG) titers defined as the reciprocal of the highest dilution of a sera from immunized mice that yielded an optical density (OD) value more than two standard deviations above the average OD generated from nonimmunized control sera samples. The titers are plotted on a log10 scale. 2.11. In Vitro Uptake of Antigen by Dendritic Cells. Antigen uptake by dendritic cells (DC2.4) was evaluated by flow cytometric and fluorescence microscopic analysis. DC2.4 cells (1 × 106 cells/well) in a 6-well plate were incubated with the following groups: no treatment (only media), FITC-ova (25 μg/mL), and FITC-ova loaded inside sIMs (equivalent to 25 μg/mL of FITC-ova) for 1 h at 37 °C. After incubation, attached cells were washed and trypsinized to make a single cell suspension. The suspended cells were fixed with 4% w/v paraformaldehyde in PBS, and the percent of cells containing FITC-ova and the relative amount of FITC-ova per cell were analyzed using flow cytometry. To evaluate the cellular uptake of antigen by DC2.4 cells using fluorescence microscopy, DC2.4 cells were cultured on glass coverslips. The cells were incubated with only media, FITC-ova (25 μg/mL), or FITCova (equivalent to 25 μg/mL of FITC-ova) loaded in sIMs for 1 h at 37 °C. Subsequently, cells were washed and fixed with 4% w/v paraformaldehyde, and cellular uptake of FITC-ova was observed under a fluorescence microscope. 2.12. Safety of Vaccine Formulation. The safety and the toxicity of vaccine formulations were analyzed by two different methods: evaluating the gross structural damage at the injection site in mouse skin and in vitro cytotoxicity to the dendritic cells (DC2.4 cells). Structural damage to the mouse skin at the injection site was evaluated 21 days after the injection of different vaccine formulations (ova only, ova loaded sIMs, and ova-alum) by histological observation of skin sections after hemotoxylin and eosin (H&E) staining. The cellular toxicity of the sIMs was tested on dendritic (DC2.4) cells by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The DC2.4 cells (3 × 104 cells/well) were incubated with different concentrations (10, 50, 100, 500, and 1000 μg/mL) of sIMs for 24 h at 37 °C. After incubation, cells were washed 2−3 times with PBS, and the cells were incubated with 50 μL of MTT solution (0.5 mg/mL) for 4 h. Subsequently, 150 μL of DMSO was added to each well, and the OD of the well was measured at 540 nm by using a UV−visible spectrophotometer.
Table 1. Physiochemical Characterization of Ova Loaded sIMsa type of particles ova loaded soluble inulin particles
ova loading (μg/mg)
size (μm)
encapsulation efficiency (%)
75.87 ± 2.74
1.5 ± 0.12
75.31 ± 42
a
Particle size was measured using scanning electron microscopy (SEM). Data represent mean ± standard deviation (n = 3). Ova loading represents μg of ova present per mg of microparticles. Encapsulation efficiency defined as the mass ratio of the entrapped ova in microparticles to the starting amount of ova used in the preparation.
Figure 1. Scanning electron microscopy (SEM) images of soluble inulin microparticle formulation. SEM image shows the size and shape of soluble inulin microparticles. Inset of the figure shows the enlarged picture of a single particle representing the average size and shape of sIMs.
were spherical in shape (Figure 1). The encapsulation efficiency and ova loading in inulin particles could not be performed with the commonly used method of protein estimation such as the BCA protein assay as the soluble inulin was interfering with the BCA assay. Therefore, to determine the encapsulation efficiency and loading, FITC-ova was loaded into the sIMs instead of the ova. The encapsulation efficiency was around 75% with 75.87 ± 2.74 μg of FITC-ova loaded per mg of sIMs (Table 1). Furthermore, the in vitro release study in 10 mM phosphate buffer (pH 7.4) at 37 °C showed that around 90% of the encapsulated ova was released from the sIMs within 16 h of incubation. However, around 20% of the encapsulated ova was released into the medium within the first 30 min of the incubation (burst release) (Figure 2). 3.2. Immunization Study. Immunization studies were performed in Balb/C mice via the intradermal route. The ova specific antibody titers (IgG-total, IgG-1, and IgG-2a) in the immunized mice sera at the first and third week after primary and booster immunizations are shown in Figure 3. The ova loaded sIMs induced significantly higher IgG-total and IgG-1 antibody titers (titers: 200,000, and 500,000) compared to the unadjuvanted ova (titers: 10,000, and 17,500) or alum
3. RESULTS 3.1. Preparation and Analysis of Antigen Loaded Soluble Inulin Microparticles (sIMs). Ova loaded soluble inulin microparticles were prepared by the w/o single emulsion−precipitation technique. The size and shape of sIMs were analyzed by SEM microscopy. The average size of 1847
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a high number of immune infiltrating cells at the site of injection. In addition to being safe at the injection site, sIMs did not cause any substantial cellular toxicity to the dendritic cells, even at concentrations of up to 1 mg/mL, as estimated by the MTT assay (Figure 5B).
4. DISCUSSION An ideal vaccine adjuvant or delivery system should stimulate the immune system robustly against the administered antigen. In addition, it should be safe to administer, economical to produce, and also protect the formulated antigen from degradation during storage.28 In the current study, we have successfully explored antigen encapsulated soluble inulin microparticles (sIMs) as a safe vaccine adjuvant and delivery system, which generated a strong immune response with little toxicity or inflammation at the injection site, was economical, and may protect the antigen against physical stability during lyophilization. Inulin is a safe plant polysaccharide, which exists in different isoforms (α, β, γ, δ, and ε) based on their solubility in water at different temperatures.16,17 Soluble inulin (α and β isoforms) has been shown to protect protein and vaccine formulations from degradation during storage.12,13,18 However, soluble isoforms of inulin cannot activate the alternate complement pathway (ACP)17 and have never been shown to stimulate the immune system or act as vaccine adjuvants. However, water insoluble isoforms (γ and δ) of inulin stimulate ACP and thereby, act as vaccine adjuvants when coinjected with an antigen,17 but they cannot function as protein stabilizers. In this article, we have shown for the first time that soluble isoforms of inulin could be made into vaccine adjuvants when formulated as antigen encapsulated microparticles. The following logical observations completely rule out the presence of significant amounts of water insoluble γ-inulin in the soluble inulin microparticle preparation tested in this study and its role in the immunostimulatory effect observed: (a) γ-Inulin is completely insoluble at 37 °C. However, the sIMs are completely water soluble at 37 °C. No significant residue was left at the bottom after 24 h of incubation and centrifugation. (b) γ-Inulin is known to stimulate the immune system when coinjected with an antigen.15 On the contrary, sIMs did not stimulate the immune system when coinjected with the antigen (Figure 3). The antigen needs to be encapsulated inside the sIMs to act as an adjuvant. (c) γ-Inulin activates the alternate complement pathway (ACP),17 whereas neither the starting material that we have used nor the final sIMs stimulated ACP (Supporting Infotmation, Figure 1). In this assay, we have used γ-inulin as a positive control. (d) γ-Inulin is known to stimulate both Th1 and Th2 types of immune responses.15 However, sIMs in this study have stimulated only the Th2 type immune response. Taken together, the above observations clearly indicate that soluble inulin is a safe biomaterial that could function as a vaccine adjuvant when formulated as antigen loaded microparticles. It also suggests that sIMs enhance the immune response by a different mechanism than insoluble isoforms of inulin. Although additional mechanisms may not be ruled out for sIMs as vaccine adjuvants, we hypothesize that enhanced internalization of the antigen in particulate form could have contributed to an increased immune response observed with antigen encapsulated sIMs (Figure 4 and Table 2). Similarly, previous studies have demonstrated that the immune stimulating effect of some other carbohydrates is due to the
Figure 2. In vitro release kinetics of ova from sIMs. A release study was performed with 10 mg of FITC-ova loaded sIMs dispersed in 1 mL of 0.1 M phosphate buffer (pH 7.4) with ∼100 rpm shaking at 37 °C. At different time intervals, samples were taken and replaced with equal volumes of fresh phosphate buffer. The amount of FITC-ova in the supernatant of the collected sample was measured by fluorescence readings of at λEx at 490 nm and λEmat 520 nm. n = 3 in triplicate.
adjuvanted ova (titers: 80,000, and 80,000) after the third week of booster immunization suggesting that sIMs are more efficient in stimulating antigen specific antibody response than the commercially available adjuvant, alum (Figure 3A and B). Interestingly, when ova was not encapsulated inside microparticles but coinjected with the blank sIMs, it failed to stimulate the immune system (titer: 20,000, and 30,000) as much as the ova encapsulated inulin particles. This suggests that the encapsulation of antigen is important for the adjuvant effect of sIMs. Although the IgG2a antibody titers in mice immunized with ova encapsulated sIMs (titer: 170) were significantly higher than the alum adjuvanted (titer: 50) group (Figure 3C), they may not be clinically sufficient to prevent or treat a disease which requires IgG2a immune response. 3.3. Cellular Uptake of Ova by Antigen Presenting Cells (APC’s). To understand the possible mechanism of ova loaded sIM mediated immune stimulation, antigen uptake in antigen presenting cells (dendritic cells) was evaluated. The incorporation of FITC-ova into the sIMs allowed quantitative estimation of the cellular uptake of the antigen in dendritic cells. FITC-ova either in solution or encapsulated inside sIMs was incubated with the dendritic cell line (DC2.4 cells) for 1 h, and the relative amount of ova taken up by the dendritic cells was quantified by flow cytometry and fluorescence microscopy. Flow cytometric data reveals that a significantly higher number of dendritic cells have taken up FITC-ova when presented inside sIMs (Table 2) in comparison to FITC-ova in solution (99% vs 22%). More cells have engulfed ova, and the amount of antigen (FITC-ova) taken up by each cell was around 25 times higher when ova was delivered inside sIMs (Figure 4A; Table 2, fluorescence intensity). The more efficient uptake of the antigen (ova) by dendritic cells when ova was presented inside sIMs was further confirmed by fluorescence microscopy (Figure 4B). This data clearly suggests that encapsulating with sIMs is a more efficient way of presenting the antigen to the APCs. 3.4. Safety and Toxicity of sIMs. Furthermore, the safety of sIMs was evaluated by histological observation of the mouse skin at the injection site after 21 days of booster immunization using hemotoxylin and eosin (H&E) staining. As shown in Figure 5A, no gross structural damage or inflammation was observed at the injection site of sIMs. The particles were cleared by immune cells, and there were no remnants of sIMs at the injection site. However, mice immunized with alum adjuvanted ova showed depot formation and the presence of 1848
dx.doi.org/10.1021/mp3006374 | Mol. Pharmaceutics 2013, 10, 1845−1853
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Figure 3. Ova specific antibody titers in the serum. Mice (n = 3−4 per group) were injected intradermally with ova (100 μg) alone or ova with alum (200 μg) or ova with blank sIMs or ova loaded sIMs on days 1 and 21 as primary and booster doses, respectively. Serum samples were collected at the 1st and 3rd week after the primary and booster immunizations for analysis of antibody titers (IgG-total, IgG-1, and IgG-2a) using indirect ELISA. The titer is the reciprocal of the highest dilution of sera from immunized mice that yielded an optical density (OD) value more than two standard deviations above the average OD generated from nonimmunized control sera samples. The titers are depicted as mean ± SD. * indicates significant results (P < 0.05 using Student’s t test) as compared to the alum adjuvanted vaccine group; n = 4−6 with triplicates. 1849
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There are several advantages for using sIMs compared to water insoluble isoforms of inulin as vaccine adjuvants: (i) soluble inulin is the most abundant form of raw inulin, and therefore, it was prepared26 with significantly higher yield (>90%) in this study as compared to the reported yields of water insoluble isoforms (γ) of inulin (∼40−50%);17 (ii) the method of preparing soluble inulin is much simpler compared to the preparation of water insoluble forms of inulin;17 and (iii) there are no local toxicities observed at the site of injection (Figure 5). We do not anticipate any systemic toxicities with sIMS once solubilized. This is based on the known safety of soluble inulins in humans as it has been used in parenterals (in kidney function tests) and orally (as a fiber diet). Although, particulate systems have advantages as vaccine adjuvants, it is challenging to encapsulate hydrophilic molecules in nano/microparticles with high encapsulation efficiency.31−33 In the present study, we have developed a method to encapsulate a model hydrophilic protein/antigen (ova) in inulin microparticles. Using this technique, high encapsulation efficiency (∼75%) and loading (75 μg of ova/mg of particles) of ova was achieved in sIMs (Table 1). This is significantly higher compared to the previously reported method (∼45%) for encapsulating a model hydrophilic material (BABCH) inside inulin microparticles. Higher encapsulation efficiency and loading not only reduces the loss of antigen during preparation that leads to lower cost of preparation (cost-effective) but also
Table 2. Flow Cytometric Analysis of Antigen Uptake by Dendritic Cellsa S.No.
treatment groups
mean fluorescence intensity (counts)
% of green cells
1 2 3
no treatment ova in solution ova loaded sIMs
4.60 ± 0.57 13.18 ± 1.06 324.16 ± 22.33*b
3.31 ± 1.61 22.0 ± 2.68 98.82 ± 0.71*b
Dendritic cells were analyzed by flow cytometry after incubating with FITC-ova either in solution or inside sIMs. Mean fluorescence intensity of FITC-ova in the green channel was represented as arbitrary fluorescence units. Cells were gated to remove autofluorescence observed in blank dendritic cells. Data represent the mean ± standard deviation (n = 3). bAsterisks indicate that the variance is significant (p < 0.0001) as compared to ova in the solution group. a
enhanced uptake of the antigen into the APC’s, possibly by binding to the carbohydrate-binding receptors such as the Ctype lectin receptors (CLR’s) present on the APC’s.29,30 For example, plant polysaccharides, such as those from Plantago asiatica, enhance the endocytosis of antigens into the APC’s, improve the processing and presentation of antigen to the Tcells and hence stimulate the immune system.29,30 Therefore, sIMs, which cannot activate ACP, may act as vaccine adjuvants by enhancing the delivery of antigen to the APC’s.
Figure 4. Uptake of antigen (ova) by dendritic cells. The dendritic (DC2.4) cells were incubated for 1 h with FITC-ova in solution or loaded inside sIMs at 37 °C. After 1 h of incubation, (A) cells were extensively washed, fixed in 4% (w/v) paraformaldehyde, and analyzed using a flow cytometer to determine the uptake of FITC-ova by DC2.4 cells. The quantification of this data is presented in Table 2. (B) Cells were extensively washed, fixed in 4% (w/v) paraformaldehyde, and observed under fluorescent microscope. The nucleus was stained with DAPI. Scale bars are equal to 50 μm. 1850
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Figure 5. Safety of the sIMs. (A) Histological images of tissue sections at the site of injection from the mice immunized with (i) ova solution, (ii) ova loaded sIMs, and (iii) ova with alum. Tissue sections were stained with H&E. The skin structure was found intact in mice immunized with ova solution and ova loaded sIMs. Alum adjuvanted ova immunized mice showed the formation of a depot at the injection site along with the presence of high number of immune infiltrating cells. The arrows indicate immune infiltrating cells. Scale bars are equal to 200 μm. (B) Cytotoxic effects of sIMs on DC2.4 cells. DC2.4 cells were incubated with different concentrations of sIMs for 24 h. The cells were washed and analyzed for cell death by the MTT assay. The percent cell viability was calculated with respect to untreated cells. The data represent the mean ± SD; n = 3 with triplicates.
generating a significant Th1 type immune response could be a limitation of this technology when it is planned to be used as an adjuvant in viral vaccines or vaccines against cancer. However, further optimization of physiochemical properties of soluble inulin or encapsulation of immune potentiating agents such as CpG, MPLA, etc. inside inulin particles could be utilized to improve the cellular immune response (Th1 type). Strong adjuvant activity is often associated with toxicity. For example, the FDA approved vaccine adjuvant, alum, also causes pain, inflammation, lymphadenopathy, necrosis, and granuloma at the injection site.6,7 As predicted, alum formed a depot at the injection site with inflammatory cells around it (Figure 5A). In contrast, sIMs were completely cleared by immune cells, and there was no cellular or tissue damage at the injection site (Figure 5A). There is no granuloma or depot, unlike that with alum, that could cause pain and inflammation at the injection site. In addition, sIMs did not cause any cellular toxicity to the APC’s (dendritic cells) even at the 1 mg/mL concentration in a cell culture experiment (Figure 5B). This suggests that sIMs are not only more potent than alum but also safe to administer as a vaccine adjuvant. In this article, we have reported a novel formulation as a vaccine adjuvant and delivery system that can be effectively used for existing vaccines where protection is provided by the generation of neutralizing antibodies (humoral immune
decreases the amount of vaccine formulation that needs to be injected (better patient compliance). The in vitro release study showed that around 20% of ova was released within the first 30 min of incubation from sIMs (Figure 2). However, more than 50% of the encapsulated BABCH was released within the first 5 min from inulin microparticles.34 This immediate release is called burst release, which is mainly coming from the surface adsorbed drug/antigen. The drug/antigen encapsulated inside the polymer matrix will be released slowly after the burst release. This is evident from sIMs because after burst release (20% in
