Effects of Caryota mitis profilin-loaded PLGA nanoparticles in a murine model of allergic asthma

Animals and materials

Female BALB/c mice (specific pathogen-free grade, aged 5–6 weeks, weight 16–20 g) were purchased from the Animal Center of Guangdong Province and fed in a specific pathogen-free grade breeding room. rCmP, PLGA (lactide:glycolide 50:50, molecular weight 24,000–38,000) and polyvinyl alcohol (80% hydrolyzed) were obtained from Sigma-Aldrich (St Louis, MO, USA). Analytical grade dichloromethane obtained from J&K Chemical Co, Ltd (Beijing, People’s Republic of China) was used without any further purification. Al(OH)₃ and methacholine were also purchased from Sigma-Aldrich. Biotin-labeled goat anti-mouse IgE and horseradish peroxidase (HPR)-labeled goat anti-mouse IgG and IgG2a were from eBioscience (San Diego, CA, USA). Mouse interleukin (IL)-4, IL-10, and interferon gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kits were obtained from BioLegend (San Diego, CA, USA).

Expression and purification of rCmP

The strain was expressed by the gene sequences BL21 of rCmP at 37°C in a shaking water bath with a shaking speed of 120 rpm, which has been described previously18 The protein was Purified with a Ni²⁺ affinity chromatography column. The elution, which including resultant protein was dialyzed and lyophilized.

Formation of rCmP-loaded nanoparticles

rCmP-loaded PLGA nanoparticles were prepared by a w/o/w double emulsion solvent evaporation method as reported by Jain et al with slight modifications.19 Briefly, aqueous phase (0.5 mL) containing 10 mg of rCmP was added to organic phase (2 mL/dichloromethane and acetone at a volume ratio 9:1) including copolymer PLGA (5% w/v). The primary emulsion (w/o) was obtained by probe sonication in an ice bath for 120 seconds. The primary emulsion was then added to 100 mL of aqueous solution containing polyvinyl alcohol (1% w/v) as an emulsifier and sonicated in an ice bath for 60 seconds to form the secondary emulsion (w/o/w). The resulting emulsion was stirred vigorously overnight to evaporate the organic phase. The nanoparticles were collected by centrifugation (20,000 rpm, 15 minutes, 4°C), washed twice with distilled water to remove residual polyvinyl alcohol, and subjected to lyophilization. The final nanoparticles were stored at 4°C in a refrigerator.

Characterization of rCmP-loaded nanoparticles

SDS-PAGE and Western blotting of rCmP-loaded nanoparticles

The protein components in rCmP and in the rCmP-loaded PLGA nanoparticles (PLGA-rCmP) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) identification and electroblotted onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin (BSA) overnight at 4°C and then incubated in diluted (1:5) positive serum of allergic patients at 37°C for 2 hours.

Biotin-labeled goat anti-human IgE diluted (1:3,000) by TBST (TBS/0.05% Tween-20) was added to the resulting membranes, which were then incubated at 37°C for 2 hours. After adding HRP-labeled streptavidin diluted (1:1,000) by TBST, the membranes were incubated at 37°C for another 1.5 hours. The membranes were washed three times after each step (5 minutes). They were then visualized in freshly prepared diaminobenzidine substrate solution and observed after being washed in double-distilled water.

Animal sensitization and specific immunotherapy

The administrative committee on animal research at the Graduate School of Shenzhen, Tsinghua University, approved all the protocols used for the animal experiments. The animal sensitization model and specific immunotherapy were designed (Figure 1) by combining the previous literature and the requirements herein.21–24 The mice were randomly divided into five groups (n=8), ie, a normal group, a model group, a PLGA-rCmP group, a rCmP/Al(OH)₃ (ie, rCmP) vaccine group, and a blank PLGA nanoparticle control group. Mice in the normal group were sensitized and treated with normal saline, and the other groups were sensitized three times every 3 weeks by intraperitoneal injection of 50 μg Caryota mitis (Cm) pollen crude proteins adsorbed on 2 mg of Al(OH)₃. The treatment was initiated on day 36. The PLGA-rCmP group was subcutaneously injected with nanoparticles containing 50 μg rCmP, the rCmP group was injected with rCmP adsorbed on 2 mg of Al(OH)₃, and the PLGA group was injected with an equivalent amount of PLGA. The mice were treated three times every 4 weeks. The mice were intranasally challenged three times daily with 200 μg of crude proteins one week after the final treatment. The mice were subjected to an airway hyperresponsiveness (AHR) test 24 hours after the final challenge and were then euthanized.

Assessment of AHR to methacholine challenge

Twenty-four hours after the final challenge, AHR was measured using unrestrained whole-body plethysmography with a four-chamber system (Buxco Research Systems, Wilmington, NC, USA). The baseline response was recorded for 5 minutes in the event of stabilized respiration after 10 minutes of adaptation. The responses of the mice were then recorded every 5 minutes after inhaling different concentrations of atomized methacholine solutions (0, 6.25, 12.5, 25, 50, and 100 mg/mL PBS). Tests at two different concentrations were separated temporarily to allow the respiratory intensity to drop back to baseline. The percentage curves for Penh values at different methacholine concentrations were plotted based on that of PBS.

Determination of allergen-specific IgE, IgG, and IgG2a antibodies in serum

Blood was sampled from mouse eyes 24 hours after the AHR test, left undisturbed for 2 hours, and stored overnight at 4°C. The blood was then centrifuged at 4°C and 3,000 rpm for 10 minutes, and the supernatant serum was collected. Next, the responses of C. mitis pollen-specific IgE, IgG, and IgG2a were detected in serum using the indirect ELISA protocol. The antibodies were coated with 1 ng/μg rCmP and stored overnight at 4°C. The plates, which were washed with PBST and blocked with 3% BSA/PBS (200 μL/well), were incubated at 37°C for 2 hours. Mouse serum diluted (IgG, 1:2,000; IgG2a, 1:1,000; IgE, 1:5) by 1% BSA-PBST and 1% BSA-PBST was added to the test wells and the control well (100 μL/well) after the plates were washed with PBST, and incubated at 37°C for one hour. Next, 100 μL of biotin-labeled goat anti-mouse secondary antibodies diluted (HRP-IgG, 1:2,000; HRP-IgG2a, 1:2,000; biotin-IgE, 1:20,000) with 1% BSA/PBST were added after the plates were washed with PBST, and incubated at 37°C for a further hour. The plates were subjected to color development in the dark at 37°C for 10 minutes utilizing 3,3′,5,5′-tetramethylbenzidine solution after being washed with PBST, and the reactions were terminated by 1 M H₂SO₄. The absorbances at 450 nm were then measured on a microplate reader.

Bronchoalveolar lavage and inflammatory cell differential counting

Immediately after being euthanized the mice were subjected to bronchoalveolar lavage. The lung was immediately lavaged via the trachea cannula with 0.8 ml of PBS. The bronchoalveolar lavage fluid was collected after five bouts of pumping. Each mouse underwent bronchoalveolar lavage three times, yielding approximately 2 mL of bronchoalveolar lavage fluid each time (recovery rate >80%). The combined bronchoalveolar lavage fluid was centrifuged at 4°C for 10 minutes, and the supernatant was then sampled to determine cytokine levels. The precipitate was resuspended with 100 μL of PBS, which was utilized for cell counting (10 μL) and cell differential counting (90 μL). The stained cells were then classified and counted using Liu’s stain. At least 200 cells were counted and classified as neutrophils, eosinophils, lymphocytes, or macrophages according to their morphologic characteristics.

Cytokine assay

Mouse spleen tissue was ground and filtered through a 200-mesh screen. The splenocytes, which were then resuspended by Roswell Park Memorial Institute 1640 medium (100 mg/L fetal bovine serum, 100 U/L penicillin, and 100 mg/L streptomycin) and erythrocyte lysate were collected by centrifugation. The cells were cultured in Roswell Park Memorial Institute 1640 medium in 24-well plates at a density of 5 × 10⁶/well, stimulated with C. mitis pollen crude proteins (200 μg/well), and incubated at 37°C for 72 hours. The culture medium was then collected and centrifuged at 4°C and 1,500 rpm. The supernatant was then collected and stored at −80°C for cytokine determination. The IL-4, IL-10, and INF-γ levels in the supernatants of the splenocyte culture medium and bronchoalveolar lavage fluid were measured strictly according to the instructions of the ELISA kit (Biolegend San Diego, CA, USA).

Lung histology and inflammation scoring

The mouse lungs were immediately removed and fixed in cold formalin solution for 24 hours, and then dehydrated by different concentrations of ethanol. The lung tissues were embedded in paraffin and cut into approximately 5 mm × 5 mm blocks, which were sliced using a slicing machine (4 μm thick). The slices were then stained with hematoxylin and eosin and sealed to observe any histologic changes under an optical microscope.

Statistical analysis

The data are expressed as the mean ± standard deviation. Statistical comparisons between two groups were made using the two-tailed, unpaired Student’s t-test with Statistical Package for the Social Sciences version 16 software (SPSS Inc, Chicago, IL, USA). P<0.05 was considered to be statistically significant.

Characterization of nanoparticles

The size and size distribution of the rCmP-loaded PLGA nanoparticles were investigated by dynamic light scattering. Physicochemical characteristics, such as particle size and surface properties, play an important role in vitro and in vivo. Therefore, they affect the therapeutic efficacy of specific immunotherapy.25 The mean diameter of the rCmP-loaded PLGA nanoparticles was approximately 180 nm, which is in the excellent size range for cellular uptake of nanoparticles.26 The nanoparticles exhibited a relatively narrow polydispersity index (≈ 0.135), which is useful for treatment effects. The zeta potential of nanoparticles is a crucial factor for stability in a suspension through the electrostatic repulsion between the nanoparticles, interaction with the cell membrane in vivo, and judgment of component onto the nanoparticle surface.27 The rCmP-loaded PLGA nanoparticles showed a negative surface charge of around −10 mV which was stable in vivo. The protein LC and EE of the rCmP-loaded PLGA nanoparticles was 9.79% and 63.56%, respectively.

The surface morphology of the rCmP-loaded PLGA nanoparticles was investigated by FESEM. Figure 2A shows an FESEM image for rCmP-loaded PLGA nanoparticles. The nanoparticles seemed to be about 180 nm in diameter and have a smooth surface within the resolution level. Further, the FESEM images demonstrated the particle size detected using a Mastersizer based on dynamic light scattering (Figure 2B).

The in vitro release research was carried out in PBS (pH 7.4). The release profile of rCmP-loaded PLGA nanoparticles showed a typical biphasic release pattern (Figure 2C). The first phase was a rapid burst release in the initial 24 hours (approximately 28.3%). This could be attributed to the amount of rCmP adherent to or just beneath the surface of the nanoparticles. This type of release is advantageous in terms of vaccine delivery, because it offers a primary immunization effect. The second phase consisted of constant continuous release of rCmP driven by diffusion through the rigid PLGA core. Release of rCmP reached a plateau in the following 192 hours (8 days), with a cumulative release of 65.2%. Further, we found that the cumulative protein release was 69.3% within 336 hours (14 days) and no further obvious release was observed. This may be due to degradation of the encapsulated protein because of the harsh conditions formed by PLGA. A similar result was reported by Jain et al.19

SDS-PAGE and Western blotting of rCmP-loaded PLGA nanoparticles

We used SDS-PAGE to detect changes in rCmP after coating with nanoparticles. The bands of PLGA-rCmP and rCmP are almost identical, indicating that rCmP and PLGA were bound without alterations (Figure 3). In addition, the Western blotting results (Figure 4) show that PLGA-rCmP binds to specific IgE in serum, suggesting that the activity of profilin in rCmP-loaded PLGA nanoparticles maintained intact after being bound physically to PLGA.

AHR assessment

AHR changes were assessed by methacholine challenge 24 hours after the last allergen challenge. The results (Figure 5) show that AHR in the model group was significantly greater (P<0.01) than in the normal group, and that in animals challenged by 25 mg/mL, 50 mg/mL, and 100 mg/mL methacholine was particularly high. AHR in the PLGA-rCmP group was significantly lower than in the model group (P<0.01), whereas the Penh value did not differ significantly from that of the normal group. The similar AHR of the Al(OH)₃-containing rCmP group and the model group indicates that the rCmP-loaded PLGA nanoparticles performed better than the individual rCmP. The blank PLGA nanoparticle control group performed similarly to the model group.

Determination of serum IgE, IgG, and IgG2a

Serum IgE, IgG, and IgG2a levels were measured by ELISA to evaluate the efficacy of the vaccine. The results (Figure 6) show that serum IgE levels in the model group were significantly higher than those in the normal group (P<0.001). Compared with the model group, PLGA-rCmP inhibited the immune response of serum IgE to a greater extent than did rCmP, given the significantly lower serum IgE levels of the two groups (P<0.05). Serum IgE levels in the PLGA and model groups did not differ significantly. PLGA-rCmP and rCmP both elevated serum IgG and IgG2a levels significantly compared with the model group, and PLGA-rCmP induced serum IgG2a more obviously than did rCmP. Serum IgG and IgG2a levels in the PLGA and model groups did not differ significantly. In general, PLGA-rCmP facilitated the Th1 immune response while suppressing the Th2 response.

Bronchoalveolar lavage and inflammatory cell differential counting

Figure 7 shows that the total counts of cells and inflammatory cells in the bronchoalveolar lavage fluid sampled from the model group were significantly higher than those in the normal group (P<0.01). The inflammatory cells mainly comprised neutrophils and eosinophils. The total counts for cells and inflammatory cells were significantly lower after being treated with rCmP-loaded PLGA nanoparticles and rCmP compared with the model group. In particular, considerably fewer cells, eosinophils, and lymphocytes were observed in the PLGA-rCmP group. In contrast, the total counts of cells and inflammatory cells in the PLGA control group do not differ from those in the model group.

Cytokine assay

IL-4, IL-10, and IFN-γ concentrations in the splenocyte culture medium supernatant and bronchoalveolar lavage fluid detected by ELISA (Figure 8A and B) show that the model group had allergen-specific Th2 immune responses, ie, IL-4 and IFN-γ levels, that were evidently upregulated and downregulated, respectively, compared with the normal group. After being treated with rCmP-loaded PLGA nanoparticles and rCmP, IL-4 and IFN-γ levels were dramatically downregulated and upregulated, respectively, suggesting that treatment with the nanoparticles promoted the Th1 immune response and inhibited the Th2 immune response by excreting more IFN-γ. In addition, IL-10 levels in the PLGA-rCmP and rCmP groups were higher than in the model group, which boosted anti-inflammatory and immunosuppressive functioning more effectively. The Th1/Th2 imbalance in the PLGA group was not restored, based on the concentrations of IL-4, IL-10, and IFN-γ in the splenocyte culture medium supernatant and bronchoalveolar lavage fluid that were almost identical to those in the model group.

Lung histologic analysis

As shown in Figure 9, lung tissue structures in the normal group were well defined without discernible damage or edema, and the trachea and blood vessels were not infiltrated peripherally by inflammatory cells. However, lung tissue and bronchial structures in the model group were disturbed by internal hemorrhage and edema. The bronchial and vascular walls were thickened and infiltrated by a considerable number of inflammatory cells. Lung tissue in the PLGA-rCmP group showed histologic changes with occasional inflammatory cell infiltration similar to that seen in the normal group. Although lung tissue in the rCmP group was free from edema and damage, there was markedly irregular architecture and inflammatory cell infiltration. Regardless, the histologic results of the rCmP group generally improved relative to those of the model group.