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Oral gavage delivery of PR8 antigen with #-glucan conjugated GRGDS carrier to enhance M-cell targeting ability and induce immunity Dong-Yi Lee, Md Nurunnabi, Sung Hun Kang, Md Nafiujjaman, Kang Moo Huh, Yong-kyu Lee, and Yeu-Chun Kim Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01855 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017

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Oral gavage delivery of PR8 antigen with β-glucan conjugated GRGDS carrier to enhance M-cell targeting ability and induce immunity Dong-Yi Lee a,1, Md Nurunnabi b,c,1, Sung Hun Kangd, Md Nafiujjamane, Kang-Moo Huhb, Yong-Kyu Lee d,e*, Yeu-Chun Kim a,*

a

Department of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea b

Department of Polymer Science & Engineering, Chungnam National University, Daejeon, Korea

c

Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, USA 02129 d

Department of Chemical & Biological Engineering, Korea National University of Transportation, Chungju-380-702, Korea

e

Department of Green Bioengineering, Korea National University of Transportation, Chungju 380-702, Korea

Keywords: oral vaccine; β-glucan; GRGDS; PR8; in vivo; immunization.

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ABSTRACT Oral gavage is known as one of most convenient routes for therapeutic administration in comparison with other available routes such as intravenous, intra muscular, suppository, etc. An oral vaccine delivery system has additional potential as it may provide a convenient way to prevent infectious diseases by introducing optimum immunization in mucus. Although oral vaccine delivery has attracted tremendous interest in vaccine delivery research, various limitations have prevented its rate of progress up to the level that was initially expected. However, the major problems of oral vaccine delivery are vaccine instability and lack of absorbability, resulting from degradation of the sophisticated antigens in the acidic medium in the stomach. In order to obtain adequate microfold-cell (M-cell) targeting and uptake, the therapeutic material is required to pass through stomach and reach the small intestine without degradation. In this project, we have introduced a conjugate of β-glucan and glycine-arginineglycine-aspartic acid-serine (GRGDS) that is effective for simultaneous protection of the antigen (PR8) and M-cell targeting. According to the experimental results, the cationic β-glucan-GRGDS conjugate can encapsulate a certain amount of anionic PR8 through electrostatic interaction, which forms nanoparticles with a range of diameter of 200-250 nm. Also, the PR8 incorporated nanoparticles showed high cell viability and stability in diverse environments. Finally, excellent M-cell targeting ability was verified in an in vitro M-cell model. Most importantly, the in vivo test obviously demonstrated the superiority of this system, which significantly increases antibody concentration in serum, intestine, and mucus as measured 21 days after immunization.

Introduction


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Oral vaccination can be particularly useful because both systemic and mucosal immunities are induced1. For example, when vaccines are administered through the oral route, a certain portion of the antigen that survives degradation is absorbed in the small intestine. Here, some vaccines elicit mucosal immune responses because mucous membranes can be covered with IgA antibodies, as vaccines directly target immune cells that exist near the small intestine. Other vaccines induce systemic immunity when they arrive at blood vessels after passing through the liver. Compared to other routes such as injection routes (transdermal or subcutaneous delivery) and the nasal route, the oral route shows various advantages in terms of vaccine delivery. Specifically, the oral route has advantages such as low production cost, high patient-compliance, and easy handling2. Considering these issues, the oral route for vaccine delivery can be regarded as one of the most effective routes, even if some limitations and obstacles must be overcome. However, despite its advantages, oral vaccination has not been commercialized until now because of several existing issues. First, antigens themselves are not stable in the harsh and acidic environment of the gastrointestinal (GI) tract, so that orally administered antigens undergo degradation3. Second, there is always the necessity for a trusty carrier that can protect the antigens from gastrointestinal tract as well as carry the antigen to the intestinal side; this must be followed by active transportation through the membrane and mucus to reach the Payer’s Patch4. Therefore, it is necessary to design a carrier that has the potential to shield and carry the entrapped antigens, and actively transport them to the Payer’s Patch so that sufficient level of mucosal immunity may be achieved. In order to deal with these problems, particulate systems have been developed and introduced to oral delivery areas because nanoparticles can load drugs or vaccines through various methods, such as emulsion or charge interaction, and protect them from external environments5-8. In this


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research, β-glucan was used as a shielding material to protect a sophisticated antigen from the harsh gastric environment. β-glucan is a type of polysaccharide; it is acid resistant and biocompatible extracted product of barley9-11. Previous studies have proved that β-glucan is capable of reacting to dendritic-cells through dectin-1 receptor10. Thus, if β-glucan is used to fabricate nanoparticle, loaded vaccines will be protected from stomach acid and enzymes, and immune responses can be elicited by β-glucan. However, although it was previously reported that β-glucan can target M-cells, that targeting ability is not sufficient to obtain active targeting efficiency. Therefore, we have introduced Glycine-Arginine-Glycine-Aspartic acid-Serine (GRGDS) in association with β-glucan to enhance the M-cell targeting ability of the carrier. GRGDS is a microfold-cell (M-cell) targeting material and M-cells exist in the mucous membrane of the small intestine12, 13. Therefore, the GRGDS conjugated β-glucan carrier will protect sophisticated vaccines from the harsh environment of the stomach and intestine, as well as facilitate vaccine penetration into the intestine via M-cells12. In this research, we have synthesized a conjugate of β-glucan and GRGDS and analyzed it to confirm the chemical conjugation; we have also measured the conjugation number of GRGDS to each β-glucan quantitatively. PR8 is an inactivated antigen of influenza A derived from Influenza A/PuertoRico/8/34 (PR8;H1N1). Studies reported earlier that PR8 induces IgG titer in the sera of mice as well as PR8-spefcific mucosal IgA antibody titers in the nasal washes of the treated mice.14 To optimize the antigen-polymer complex formation process, PR8 was wrapped in the cationic polymer-peptide conjugate and nanoparticles were formed through electrostatic interaction. Characterization was also conducted to measure size, morphology, and zeta potential of the nanoparticles. Next, excellent in vitro M-cell targeting ability was verified in Caco-2 and Raji cell lines. Excellent transportation ability was observed through the M-cell modeled


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monolayer formed by co-culturing of Caco-2 and Raji B cells. In vivo studies conducted in rats showed that the existence of GRGDS has an influence on the induction of antibodies. In the future, further study on specific disease models may be used to confirm our concept that an Mcell targeting carrier such as β-glucan conjugated GRGDS has the potential to enhance immunity. Materials and Methods Materials β-glucan extracted from barley (low viscosity) (MW: 166,000Da) was purchased from megazyme (Island). 4-nitrophenylchloroformate (4-NPC) (>98%), 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) (>98%), and triethylamine (TEA) (>99%) were purchased from TCI (Japan). Ethylenediamine (EDA) and N-Hydroxy-succinimide (>98%) (NHS) were purchased from Sigma-Aldrich (United States). Glycine-Arginine-Glycine-Aspartic Acid-Serine (GRGDS) was purchased from Anygen (Korea). PR8 influenza virus was obtained from Professor Bok-SilJeon’s laboratory at Kyung-Hee University. β-glucan-GRGDS conjugation GRGDS was introduced into β-glucan by forming an amide bond. First, 50 mg of β-glucan was stirred in DMSO solvent. After 30 min, the temperature was decreased to 4°C, followed by addition of 4-NPC to the beaker. After 4-NPC was conjugated to β-glucan, the temperature rose until arriving at room temperature. After that, TEA was added as reaction catalyst and excess EDA was also added in order to conjugate EDA into β-glucan and introduce amine groups. After roughly 5 hr, amine group conjugated β-glucan was dialyzed in deionized water and lyophilized.


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Next, conjugated β-glucan was stirred in deionized water until β-glucan was totally dissolved. After this, EDC and NHS were added as catalysts to form amide bonds between the carboxylic acid of GRGDS and the amine of β-glucan. GRGDS was inserted and the reaction was allowed to continue for 12 hr. Final products were dialyzed in deionized water and freeze-dried15. The βglucan and β-glucan-GRGDS conjugates were characterized by 1H-NMR and confirmed their successful conjugation (figure S1). Antigen condensation into the β-glucan-GRGDS carrier

Nanoparticles were fabricated by electrostatic interaction. In brief, different ratios of β-glucanGRGDS and PR8 (0.5/1, 1/1, and 1/2) were mixed together and nanoparticles were produced by physical interaction. β-glucan-GRGDS and PR8 were dissolved in deionized water separately at a concentration of 0.1 mg/mL and mixed by gentle vortexing; this was followed by incubation for 30 min at room temperature. Particle sizes and morphology of the nanoparticles were measured by dynamic laser scattering (DLS) and SEM, respectively. Characterization and analysis The biocompatibilities of NH2 conjugated and GRGDS conjugated β-glucan were measured by 3-(4,5-Dimethylthiazol-2-yl) 2,5-Diphenyltetrazolium Bromide (MTT) assay with Caco-2 cells. Caco-2 cells were cultivated in Dulbecco’s modified Eagle’s Medium (DMEM) with 1% antibiotic solution, 10% nonessential amino acid, and 10% fetal bovine serum. Then, Caco-2 cells were seeded onto well plates at a density of 50000 cells/well. The cells were incubated with 100 µL of culture medium at 37 °C, with 5% CO2, for 48 hr. After this, the culture medium was completely removed and the aggregated cells were treated with 100 µL of the above two


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samples. After 20 hr incubation of the samples, 100 µL of MTT solution was put into each well. The cells were incubated for another 4 hr, and the entire medium was substituted with dimethyl sulfoxide (DMSO). Next, the absorbance of each well was measured using Multiskan GO equipment. In addition, cells treated with only culture medium were utilized as a control to calculate the cell viability16. Nanoparticle stabilities were examined at diverse values of pH and FaSSIF17 analysis was also conducted. After each particle was set to pH 2 and pH 7 conditions, nanoparticle sizes were analyzed with DLS. Additionally, nanoparticle sizes dissolved in FaSSIF were measured at each time using DLS. M-cell model and targeting ability Caco-2 cell or Caco-2+Raji B cell monolayers were formed on an 8 well Transwell® culture plate. Formation of cell monolayer was conducted according to our previously published method9, 18. In brief, Caco-2 cells were seeded on an apical layer of a trans-well and medium was changed every 2 days; cells were observed by microscope to confirm proper growth. After 3 weeks, we confirmed the growth of a proper cell monolayer by measuring the conductivity. For the monolayer of Caco-2 and Raji B cells, Raji B cells were seeded with the Caco-2 cell monolayer; this was followed by incubation for 3 days. The prepared formulation was dissolved in cell culture medium and added to the apical layer of the wells. For understanding of the transportation ability of the formulations, the solution from the basolateral layer was collected at the specified time and analyzed by ELISA reader to determine the amount of transported rhodamine-B conjugated formulation. Biodistribution


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In order to understand the absorption and biodistribution profile of the formulation, rhodamine-B was conjugated with β-glucan-GRGDS and orally administered to mice. After certain specified intervals, the mice were dissected to analyze the distribution to and deposition of the formulation in the specific organs. For qualitative analysis, the organs were isolated and washed with PBS to remove the blood; this was followed by slicing into sections with thickness of 15 µm, which were embedded on glass slides for imaging with an optical microscope19. Presence of Rhodamine-B was imaged by an optical microscope to determine the relative distribution of the formulations across the different organs. For quantitative analysis, the tissues were weighed, digested, and homogenized; this was followed by centrifuging to discard the precipitated tissues19. To determine the relative distribution of the rhodamine-B labeled formulations in the orally administered mice, the supernatant was taken from the micro well plate and analysis was conducted for quantitative measurement of the relative fluorescence (RFU) of rhodamine-B. Oral immunization Female Swiss albino rats were obtained from DehanShin Tech Bio (Cheongju, Korea), and used at 8 weeks of age 20. The animals were maintained under standard pathogen-free conditions and provided with free access to food and water during the experiments. All animals were maintained and used in accordance with the guidelines of the Korea National University of Transportation for the care and use of laboratory animals. The rats were divided into 4 groups (4 mice per group). Each rat was given an oral gavage of 200 µL PBS containing an amount equivalent to 100 mg of PR8 in either PR8 solution, β-glucan-GRGDS/PR8, β-glucan-NH2, or the control (PBS). All groups received a total of 5 doses of the vaccines (or control) on days 0, 1, 7, 8, and 14. To assess the immune responses, serum tail vein blood and feces samples were taken at day


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28 (prior to the first immunization). The blood samples were centrifuged for 10 min at 5000 rpm; the serum was collected and stored at 70oC until analysis could be performed for anti-PR8 IgG titer. Rat intestines were isolated and washed with PBS. Mucus from every intestinal membrane were collected by gentle scratching with a spatula and PBS was added. The mucus samples containing PBS were centrifuged at 6000 rpm for 10 min and the supernatant was separated in order to measure the mucus anti-PR8 IgA. The remaining intestines (~3 cm) were added to PBS and digestive solution; this was followed by homogenizing in ice cold PBS. The homogenized extract was centrifuged at 10,000 rpm for10 min to separate the supernatant and precipitate. The supernatants were collected and analyzed for the presence of antigen-specific IgA. All the samples were stored at -70 °C until ELISA reader analysis for the presence of IgA titers 21-23. Evaluation of antibody production 96-well ELISA plates were coated overnight at 4 °C with 2 µg of Pr8 influenza per well with a coating buffer (0.05 m CBS, pH 9.6). Wells were washed with PBST (0.01 m PBS containing 0.05% [m/v] Tween 20, pH 7.4) and were blocked by incubation with 2% (m/v) BSA in PBST for 60 min at 37 °C. After washing with PBST, 100 µL per well of appropriate sera dilutions were added to the plates. Each sample was diluted two-fold with the addition of buffer (PBST containing 0.1% [m/v] BSA), followed by incubation for 30 min at 37 °C. Plates were then washed and incubated with 100 µL horseradish peroxidase-conjugated goat antibody against either mouse IgG (Sigma–Aldrich, St. Louis, MO, USA), IgG1, or IgG2a (Santa Cruz, CA, USA) (IgG diluted 1:20,000; IgG1 and IgG2a diluted 1:2000) for 30 min at 37 °C. Thereafter, the plates were washed again with PBST. 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added to each well and samples were incubated for 20 min at room temperature.


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After stopping the reaction by adding 50 µL of 2 M H2SO4 to each well, the optical density (OD, 450 nm) was measured by ELISA reader 22,23. Statistical analysis All the data were expressed as mean ± SEM. Data were statistically analyzed by the Origin pro 8.0. One-way analysis of variance (ANOVA) was used to analyze the data for comparison of treatment groups with the negative control group.

Results and Discussion Regarding the ability to orally deliver the PR8 antigen with high efficiency 23, targeting of microfold-cells (M-cells) is important because this pathway assures delivery of vaccines to the Peyer’s Patch, where many immune cells are located. Our hypothesis is that the polysaccharide β-glucan will act as a shield through the other surfaces of the particles to protect the internal acid sensitive PR8 antigen from the harsh gastric acid and enzymes and from association with GRGDS, which is a well-known and widely used peptide that can target M-cells10. The synthesis processes of β-glucan and GRGDS are shown in Scheme 1; the β-glucan was modified to introduce primary amine groups, which facilitate conjugation of the β-glucan through the formation of amide bonds. The introduction of primary amine groups to β-glucan was characterized by elementary analysis; the results are shown in Table 1. Elemental analysis shows an increase of nitrogen content, which is solid evidence of the conjugation of ethylenediamine, which is a source of primary amine. The results indicate that the amine groups were successfully introduced to β-glucan. Furthermore, elementary analysis results shown in Table 1 confirm the increase of nitrogen percentage that resulted from the conjugation of GRGDS and β-glucan15. To calculate the number of GRGDS molecules conjugated to each β-glucan, GRGDS was labeled


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with Rhodamine-B. The coupling ratio of GRGDS was around 4 to each mole of β-glucan, as measured by UV-vis spectrum analysis. In the next step, the PR8 antigen was incorporated with β-glucan-GRGDS resulting from formation of nanoparticles by electrostatic interaction between the anionic PR8 and the cationic β-glucan-GRGDS. The results show that at a 1/1 w/w ratio, β-glucan-GRGDS and PR8 form comparatively smaller and more uniform particles, assuming that the maximum amount of PR8 condenses with equivalent amounts of β-glucan-GRGDS and PR8. Figures 1 (A) and (C) show that the particle size ranged from 200 to 300 nm in diameter and that the zeta potential was -13 mV, as determined by DLS and a zeta analyzer, respectively. Also, Figure 1 (B) shows the size and morphology of the particles measured by scanning electron microscopy (SEM) which was also used to determine particle shapes. The SEM image shows that the particle shapes are spherical and that the particles are distributed uniformly because the sizes of most of the particles are smaller than 300 nm. In order to confirm the cytotoxicity and biocompatibility of the nanoparticles and their individual derivatives such as β-glucan-NH2, β-glucan-GRGDS, and PR8 incorporated nanoparticles, we tested the in vitro toxicity levels in Caco-2 cells16. The Caco-2 cell is a model human intestinal epithelial cell line used for feasibility studies of oral delivery formulations 25. In Figure 2, in vitro toxicity results reveal that none of these derivatives are toxic for the cell, as even after 24 h of incubation around 100% of the cells were viable compared to the saline treated group. Besides this, compatibility profile MTT colorimetric assay was employed and also confirmed the cells’ potential for oral vaccine delivery. The cell survival profile reveals that βglucan-GRGDS could be considered a safe vaccine delivery carrier overcoming the hazardous related issue.


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The stability of the formulations was monitored to determine their survival profile in vivo when they were administered orally. To monitor the physical stability, the formulations were not only dissolved in diverse pH buffers (pH=1,2,7, and 9) but also dissolved in Fasted State Simulated Intestinal Fluid (FaSSIF), which mimics the gastric solution 5. Upon reaching the stability state in the FsSSIF solution, we may be able to assume a stability profile of the formulation in the gastro-intestinal tract, more specifically in the stomach, for orally administered solutions. As can be seen in Figure 3 (A), particle size was kept constant even as the pH value changed from pH 9 to pH 1. Particles retain their shapes and sizes irrespective of pH value, so that it can be believed that these materials are not affected by gastric acid in the stomach. Furthermore, sizes of the particles rarely varied, even after they were dissolved in FaSSIF, as shown in Figure 3 (B). These results indicate that the enzymes in intestinal condition did not influence any of the samples to change in terms of morphology. To summarize, these nanoparticles are stable in the gastrointestinal tract and can protect vaccines from external environments until they arrive at the M-cells in the small intestine. The M-cell targeting ability was tested in an in vitro M-cell model in which Caco-2 cells and Raji B cells were co-cultured to form a monolayer on a Transwell® cell culture plate18. Caco-2 cell monolayer was also prepared as a negative control. Rho-GRGDS-BG and Rho-GRGDSBG/PR8 were co-incubated for 12 h with both the monolayers. Medium collection from the basolateral layer (bottom layer) was conducted at 0, 2, 4, 6, 8, 10, and 12 h of post addition; we measured the content of nanoparticles based on the absorbance of Rhodamine-B for those samples that passed through the monolayer conjugated particles. In Figure 4, our observations show that our designed nanoparticles have more targeting affinity for M-cells than they do forCaco-2 cells because the calculated absorbance of Basolateral layer of the M-cell was almost


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1.5 times higher than that of Caco-2 cells. No significant difference was observed between PR8loaded and PR8-free nanoparticles. The results clearly show that the GRGDS conjugated carrier and nanoparticles actively targeted the M-cells, thus potentially enhancing the permeability. The biodistribution results show the accumulation of the orally administered formulation in different organs; this accumulation was observed both quantitatively and qualitatively. Figure 5shows fluorescence images and ELISA analysis results of the fluorescence for respective organs. We have observed that the highest fluorescence appeared in the liver and intestine, indicating the highest deposition of rhodamine-B labeled particles in those organs. However, limited amounts of the orally administered formulations were non-specifically transported to the kidney, lung, and spleen. Observation of a certain amount of the formulations in the intestine indicates that the formulation was viable in the harsh conditions of stomach in vivo and was able to bind with the M-cells, which led to its deposit in the intestinal membrane. The absolute objective of this project was the rational design of a non-viral carrier that could potentially enhance M-cell targeting; we also wanted to investigate the immune responses of PR8 release from β-glucan-GRGDS/PR8 nanoparticles after M-cell targeting and accumulation. Production of secretory IgA was found to locally provide adequate evidence and establish that the formulation is sufficiently feasible to produce a first line of defense at the mucosal surface, which leads to blocking of pathogen adhesion. Figures 6 (A) and (B) indicate the titers of IgA in blood serum and mucus, respectively, where we observed the highest amount of IgA antibody for the mice treated with β-glucan-GRGDS/PR8 formulation. Interestingly, the level of IgA measured in mucus was 1.8 times higher than that of the IgA antibody in the intestine. These results clearly indicate that the carrier has significant binding ability to the M-cells and potentially enhances immunity in not only the mucus layer but also the intestinal membrane of


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the gastro intestinal tract. Secretory antibody IgG was also measured in blood serum, and was found to have a level almost 6 fold higher than that level in the intestinal tract and 3 folds higher than the level of mucus antibody IgA. The overall immunization results demonstrate that the βglucan-GRGDS carrier has the potential for active targeting of the M-cells and releases entrapped PR8 to the specific sites; this process leads to enhanced immunization in the mucus and intestine.

Conclusion In the present work, GRGDS and β-glucan were synthesized via the formation of biocompatible amide bonds. Various characterization methods were applied to confirm the success of the chemical conjugation and formulation of nanoparticles through electrostatic interaction. Also, MTT assay results show that these particles are not significantly toxic to the cell and are stable in different acidic buffers for a certain period of time. Furthermore, the M-cell targeting ability of the M-cell model is very high compared to that of the Caco-2 cell mono-culture system; this is clear evidence of the potential and efficiency of the newly designed and simple method for delivery of an oral vaccine. We believe that this non-viral gene delivery approach will be applied further for oral delivery of therapeutic genes and for vaccine delivery according to the disease model through further optimization if required.

Figures


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Figure 1. Nanoparticle characterization.(A)Nanoparticle size of amine conjugated β-glucan and GRGDS conjugated β-glucan. (B) SEM image of the GRGDS conjugated β-glucan nanoparticles, which show equal distribution (scale bar is 1um). (C) Zeta potential value of the GRGDS conjugated β-glucan nanoparticles (-13.4mV). Data are expressed as mean ± SEM (n = 4).


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Figure 2. MTT assay of amine conjugated β-glucan and GRGDS conjugated β-glucan. Data are expressed as mean ± SEM (n = 5).


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Figure 3. Nanoparticle stabilities: (A) Size of GRGDS conjugated β-glucan nanoparticles according to different pH values;(B) Size of GRGDS conjugated β-glucan nanoparticles according to different time values. Data are expressed as mean ± SEM (n = 5).


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*

Figure 4.Absorbance value of orally administered rhodamine B dye labeled nanoparticles at different time points of post-administration. Data are expressed as mean ± SEM (n = 5). (*: P

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