Hydrophobic chain modified low molecular weight polyethylenimine for efficient antigen delivery

Abstract

The development of new therapeutic vaccines for the efficient induction of cellular immunity by antigen cross-presentation in antigen-presenting cells, especially dendritic cells, is regarded as a promising approach to prime antigen-specific T cell responses and to destroy tumor cells. We synthesized low molecular weight polyethylenimine (C12-PEI) with a modified hydrophobic lipid chain by the direct coupling of 1-bromododecane with low molecular weight polyethylenimine (MW 423 Da) in a 2 : 1 molar ratio. This was bound with model antigen ovalbumin (OVA) to form sphere-like polyplex nanoparticles (size 158–302 nm; zeta potential −18.4 to −13.1 mV) by electrostatic interaction at an C12-PEI/OVA weight ratio of 0.03–0.08. The in vitro cytotoxicity of the C12-PEI/OVA nanoparticles was strongly dependent on the weight ratio of the C12-PEI and OVA. The antigen cross-presentation effect (IL-2 secretion) of the C12-PEI/OVA polyplexes was also dependent on the weight ratio. Optimized cross-presentation was achieved on the C12-PEI/OVA-0.07/1 polyplexes, which was 5.3-fold higher than that of the free OVA solution and 1.9-fold higher than that of the PEI/OVA polyplexes. The intracellular distribution of the C12-PEI/rhodamine-labeled OVA polyplexes demonstrated the lysosome localization effect. These results indicated that the synthesized C12-PEI cationic polymer with a modified hydrophobic lipid chain could potentially be used as a therapeutic vaccine carrier for delivery of the OVA antigen.

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1. Introduction

Cancer immunotherapy, in which therapeutic vaccines are used to enhance the power and specificity of the immune system to target tumors, is considered to be a promising tool in cancer treatment.^1–4 The molecular identification of human cancer antigens as therapeutic vaccines has allowed the development of antigen-specific immunotherapy; in this vaccination approach, the provision of an antigen together with an adjuvant to prime therapeutic antigen-specific T cells in vivo⁵ is essential. However, the antigens used as cancer vaccines have a protein-based architecture and are easily degraded by proteases or absorbed by serum proteins in the blood circulation system. Antigen proteins could also induce a predominantly antibody response rather than an antigen-specific T lymphocyte response. These side-effects greatly decrease their immuno-therapeutic efficacy and limit their clinical applications against tumors.^6,7 Thus the key issues in the stimulation of cellular immunity against tumor cells is developing new carrier materials:⁸ (1) to protect the protein-based antigens/vaccines against rapid biodegradation or absorption in the bloodstream; and (2) to control the antigen processing of the vaccines inside antigen-presenting cells (APCs) through the major histocompatibility complex (MHC) I pathway.

The development of new synthetic polymeric materials for use as vaccine carriers has attracted increasing attention in cancer immunotherapy.^9,10 Positively charged cationic polymers/lipids have been used as polymeric carriers to absorb or load negatively charged therapeutic antigen/vaccine proteins^11,12 via electrostatic interactions. Polyethylenimines (PEIs) are a well-known series of commercially available, low cost and highly efficient polymeric gene vectors with a high positive charge density and unique “proton sponge” effect,^13,14 which facilitates disruption of the endosome/lysosome compartments and the release of DNA into the cytosol. This feature also makes them potential candidates to bind or load negatively charged antigen proteins and to promote endosome/lysosome escape for the efficient therapeutic delivery of antigens or vaccines. We previously reported the in vitro delivery of ovalbumin (OVA) antigen using commercially available PEI-25k as the polymeric vaccine carrier. We found that the PEI-25k/antigen polyplex nanoparticles improved the antigen cross-presentation efficiency in professional APCs.¹⁵ However, the design and synthesis of new PEI-based polymeric antigen/vaccine carriers that can form nano-scale antigen carriers or antigen complexes with good stability in solution and enhanced antigen cross-presentation efficiency in APCs are still challenging. There is a need to further elucidate the correlation between the polymer structures and their capability to deliver antigens and vaccines. Many modification approaches have been developed in the field of gene delivery to enhance the utility of PEI-based gene carriers/vectors. Hydrophobic modifications of lower molecular weight PEI have been shown to be promising approaches to achieve a high cell membrane penetration capability and highly efficient gene delivery. Bhattacharya and co-workers¹⁶ found that hydrophobic cholesterol-modified low molecular weight PEI enhanced the cellular uptake capability of the pDNA-loaded polyplexes and improved the efficiency of gene delivery. This could be explained by the hydrophobic interaction between the hydrophobic aliphatic chains and the lipophilic cell membrane, which resulted in enhanced cellular uptake of the polyplexes. There have been several successful studies of hydrophobic modifications to PEI polymers in gene delivery systems, demonstrating that modification of the hydrophobic lipid chains combined with low molecular weight PEI could improve the efficiency of gene delivery.^17,18 Thus it is expected that the development of new PEI-based therapeutic vaccine delivery systems by the hydrophobic modification of low molecular weight PEI might also give rise to a higher cell membrane penetration capability and higher efficiency of vaccine delivery.

Based on this hypothesis, we designed and synthesized a hydrophobic-modified low molecular weight PEI (C12-PEI) by direct coupling of 1-bromododecane with low molecular weight PEI (MW 423 Da) in a 2 : 1 molar ratio. The C12-PEI polymer was used as a vaccine carrier to deliver the model antigen OVA into dendritic cells (DCs) and to further prime the immune response of therapeutic antigen-specific T cells by antigen cross-presentation (Scheme 1). The average particle size, zeta potential, morphology and stability in solution of the C12-PEI/OVA complexes were measured by dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cytotoxicity of the C12-PEI/OVA polyplexes was evaluated by the CCK-8 assay. DCs derived from bone marrow and Kb-restricted OVA-specific T cells (RF33.70) were used as efficient antigen-presenting model cell lines to evaluate the cross-presentation efficiency using an IL-2 secretion assay. The intracellular localization of the C12-PEI/rhodamine-labeled OVA polyplexes was recorded by confocal fluorescent microscopy.

Scheme 1 Synthesis of the C12-PEI cationic polymer as a vaccine carrier to deliver the model antigen OVA into dendritic cells and to further prime the immune response of OVA antigen-specific T cells (RF33.70) by antigen cross-presentation.

2. Materials and methods

2.1. Materials

Low molecular weight polyethylenimine (PEI, branched, MW 423 Da), PEI-25k (branched, MW 25 kDa) and antigen OVA were purchased from Sigma-Aldrich (St Louis, MO, USA). 1-Bromododecane (99.0%) was purchased from Aladdin-Reagent (Shanghai, China). Mouse granulocyte-macrophage colony-stimulating factor and mouse interleukin (IL-4) were purchased from R&D Systems (Minneapolis, MN, USA). Rhodamine red succinimidyl ester was purchased from Life Technology (Shanghai, China). Cell culture media were obtained from Gibco Invitrogen (Carlsbad, CA, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Shanghai, China).

Kb-restricted OVA-specific hybridoma T cells RF33.70 (specific for the peptide of OVA257–264) were kindly provided by Professor Xuetao Cao from the Second Military Medical University (Shanghai, China). These cells were maintained in RPMI-1640 culture medium with 10% heat-inactivated fetal bovine serum and antibiotics. Male C57BL/6 mice (H-2b) (5–6 weeks old) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and were housed in a specific pathogen-free room at the School of Pharmacy, Shanghai Jiaotong University. All experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Pharmacy (Shanghai, China). DCs derived from bone marrow were prepared using a previously reported procedure.¹⁹ Bone marrow from C57BL/6 mice was collected and cultured in RPMI-1640 complete medium (10% heat-inactivated fetal bovine serum, 100 U mL⁻¹ penicillin, 100 U mL⁻¹ streptomycin) supplemented with 10 ng mL⁻¹ mouse granulocyte-macrophage colony-stimulating factor and 1 ng mL⁻¹ mouse IL-4. Non-adherent cells were removed after 3 days and the adherent cells were replanted in fresh RPMI-1640 complete medium supplemented with mouse granulocyte-macrophage colony-stimulating factor and IL-4. The non-adherent and loosely adherent cells were harvested as DCs after another 3 days.

2.2. Methods

2.2.1. Synthesis of the C12-PEI cationic lipopolymer. A 0.43 g mass of low molecular polyethylenimine (PEI, 423 Da) was dissolved in 10 mL of dehydrated chloroform (CHCl₃) and then twice the molar ratio of 1-bromododecane (0.50 g) was added dropwise. The mixture was heated at 70 °C for 24 h under the protection of an N₂ atmosphere and the reaction was monitored by ¹H NMR until the starting material of 1-bromododecane had reacted completely. The organic solvent was then removed under reduced pressure and washed five times with 100 mL of diethyl ether and dried under vacuum at 55 °C to give the oil-like C12-PEI cationic lipopolymer as the final product. ¹H NMR spectra of the as-synthesized C12-PEI cationic lipopolymer were recorded on a Brucker Avance DPX 300 NMR spectrometer (Bruker, Germany); the chemical shifts (in ppm) of the proton NMR spectra were calibrated with tetramethylsilane as the internal reference.
C12-PEI. ¹H NMR (CDCl₃, δ ppm): 4.31–4.12 (br, NH, PEI), 3.17–2.18 (m, 34H, NHCH₂CH₂NH, PEI), 1.41–1.06 (m, 40H, CH₂, alkyl chain), 0.94–0.72 (t, 6H, CH₃, alkyl chain). FTIR (cm⁻¹): 3255, 2920, 2850, 2814, 1639, 1456, 1353, 1302, 1157, 1119, 1008, 938, 770, 720.

2.2.2. Preparation of the C12-PEI/OVA polyplexes. OVA was used as a model antigen and pre-dissolved in 10 mM HEPES buffer (pH 7.4) to prepare a stock solution (10 mg mL⁻¹). The synthesized C12-PEI cationic lipopolymer was dissolved in double-distilled water for preparation of the stock solution (2 mg mL⁻¹). The polyplex nanoparticles were prepared by mixing the OVA solution (1 mg mL⁻¹) with the C12-PEI polymer solution in different weight ratios and vortexing for 20 s. The mixture was then further incubated at room temperature for 10 min to form the C12-PEI/OVA polyplex nanoparticles.

2.2.3. Characterization of the C12-PEI/OVA polyplex nanoparticles by DLS. The average particle size and zeta potential of the C12-PEI/OVA nanoparticles were determined by DLS (Malvern Instruments Zetasizer Nano ZS90) at 25 °C with a scattering angle of 90°.

2.2.4. Morphology of the C12-PEI/OVA polyplex nanoparticles observed by TEM and AFM. C12-PEI/OVA-0.07/1 and C12-PEI/OVA-0.08/1 polyplexes were prepared by mixing the OVA solution (10 mg mL⁻¹, 50 μL, 500 μg OVA) with the C12-PEI polymer (2 mg mL⁻¹) at weight ratios of 0.07 : 1 and 0.08 : 1, respectively, and then incubating at room temperature for 20 min. For the TEM measurements, the as-prepared polyplex solution was dropped onto a carbon-coated copper grid and left to dry in air at room temperature for 24 h. The nanoparticle morphologies were then recorded using a transmission electron microscope (JEM-1230, JEOL, Japan) operated at an accelerating voltage of 80 kV. For the AFM measurements, the as-prepared C12-PEI/OVA-0.07/1 polyplexes were diluted 10× with ultrapure water and then deposited onto freshly cleaved mica. After evaporating the water to give a dry sample, the morphology of the C12-PEI/OVA-0.07/1 nanoparticles was observed by AFM (Nanonavi E-Sweep, Seiko Instruments).

2.2.5. Stability of the C12-PEI/OVA polyplex nanoparticles in solution measured by DLS. The as-prepared C12-PEI/OVA-0.07/1 polyplex nanoparticles (100 μL) were added to 900 μL of RPMI-1640 culture medium (or 0.9% NaCl and 10 mM HEPES) at room temperature. The samples were incubated for a predetermined time and then the average particle size and zeta potential of the C12-PEI/OVA nanoparticles were determined by DLS (Zetasizer Nano ZS90 instrument) at 25 °C with a scattering angle of 90°.

2.2.6. Cytotoxicity of the C12-PEI/OVA in DCs cells by CCK-8 assay. The cytotoxicity of the C12-PEI/OVA polyplex nanoparticles was examined by a CCK-8 assay. A predetermined amount of the C12-PEI/OVA polyplex nanoparticles was incubated with DC cells (5 × 10⁴ per well) overnight; 10 μL of CCK-8 solution were then added per well and incubated for 4 h in 96-well plates. The absorption of the CCK-8 was measured at 450 nm with a background correction of 620 nm using an ELISA reader (Bio-Rad). The relative cell growth (%) was calculated as (OD_sample − OD₀)/(OD_control − OD₀) × 100%. All data are presented as mean ± SD values for three experiments performed in triplicate.

2.2.7. Assay for antigen processing and presentation. C12-PEI/OVA polyplex nanoparticles were added to DCs with a final antigen concentration of 0.1 μM and the cells were incubated overnight (12 h). The DCs were then harvested and diluted with serum-free 1640 complete medium. A total of 5 × 10⁴ DCs and 2 × 10⁵ RF33.70 cells per well were mixed and incubated in 96-well plates. After 24 h of incubation, the IL-2 concentration in the culture supernatant was determined by ELISA. The IL-2 concentration was compared with that for cells treated with an OVA solution of the same concentration. All data are presented as mean ± SD values of three experiments performed in triplicate.

2.2.8. Intracellular distribution of the C12-PEI/rhodamine-labeled OVA polyplexes. To visualize the intracellular distribution of the C12-PEI/OVA polyplexes, the OVA antigen was labeled with rhodamine red succinimidyl ester to prepare rhodamine-labeled OVA (Rho-OVA) in pH 9.0 buffer and free dye was removed by Hitrap column on AKTA EXPLORER. Thereafter, the as-prepared Rho-OVA samples were mixed with C12-PEI to prepare the C12-PEI/Rho-OVA polyplexes, which were then added to the DCs (5 × 10⁴ per well), incubated for a preset period (15 min, 30 min or 1 h) and washed several times with serum-free RPMI-1640. Before fluorescence imaging, two fluorescent dyes, DAPI (10 μL, 5 mg mL⁻¹, for cell nuclei staining) and Lysotracker Green DND-26 (1 μL, for lysosome staining), were added to the DCs and incubated for 20 min and then washed several times with serum-free RPMI-1640. The intracellular distribution of the C12-PEI/Rho-OVA polyplexes was observed by confocal laser scanning microscopy (Leica TCS SP8 microscope).

3. Results and discussion

3.1. Synthesis of the C12-PEI cationic lipopolymer

It is known that the introduction of a hydrophobic block/moiety to cationic amphiphiles may facilitate the formation of nano-aggregates, improve the stability of the gene/drug payloads in solution, and enhance cell membrane fusion and permeation.^20,21 Cao and Feng²² found that doxorubicin conjugated with lipid containing vitamin E had a lower IC₅₀ value than free doxorubicin. In an effort to improve the performance of gene vectors, we previously found that the presence of a hydrophobic poly(L-lactic acid) block in some amphiphilic triblock dendritic poly(L-lysine)-b-poly(L-lactic acid)-b-dendritic poly(L-lysine) blocks could significantly enhance the DNA binding affinity and efficiency of transfection.²³ In the work reported here, to mimic the membrane fusion lipids bearing double alkyl chains (such as DOPE and DOTMA), we synthesized a low molecular weight PEI (C12-PEI) bearing a double alkyl chain by direct coupling of 1-bromododecane with low molecular weight PEI (MW 423 Da) in a 2 : 1 molar ratio.¹⁶ The reaction mixture was heated under comparatively mild conditions (70 °C) for 24 h until the 1-bromododecane reacted completely (monitored by ¹H NMR); the final product (C12-PEI) was obtained as yellowish oil without further purification. The synthetic routes for the C12-PEI cationic polymers from the 423 Da PEI and 1-bromododecane are shown in Scheme 1; the structural characterization of the C12-PEI cationic polymer by ¹H NMR and FTIR is described in detail in the experimental section (see Fig. S1†). In the ¹H NMR spectrum, the proton signals at δ 0.94–0.72 ppm (–CH₃) and δ 1.41–1.06 ppm (–CH₂–) represent the alkyl chains and the signal at δ 3.17–2.18 was identified as the –CH₂–CH₂– protons of the 423 Da PEI. Strong absorption bands at 2920, 2850 and 2814 cm⁻¹ (ν_CH3 and ν_CH2) and a comparably broad absorption band at 3500–3150 cm⁻¹ (ν_NH2 and ν_NH) were seen in the IR spectrum. These results confirmed the successful preparation of the C12-PEI lipopolymer.

3.2. Average particle size and zeta potential of the C12-PEI/OVA polyplexes

It is known that the average particle size and the zeta potential of the drug/gene payloads influence their cytotoxicity, the delivery efficiency of the drug/gene and the intracellular uptake and trafficking behavior.^24,25 The average particle size and zeta potential of the C12-PEI/OVA polyplexes in solution were determined by DLS. The C12-PEI/OVA polyplexes were prepared by mixing OVA (1 mg mL⁻¹) with various amounts of C12-PEI (0.03–0.08 mg mL⁻¹) in HEPES buffer (10 mM, pH 7.4). Fig. 1A shows the dependence of the average particle sizes of the C12-PEI/OVA polyplexes in aqueous solution on the weight ratio. The C12-PEI cationic polymer could compact OVA into relatively small nanoparticles (diameter 158–302 nm) with a narrow polydispersity index (PDI 0.1–0.28) in the weight ratio range of 0.03 : 1 to 0.08 : 1 (see Fig. S2† for the DLS curve at 0.07 : 1). It was difficult for the C12-PEI and OVA to form complexes when the weight ratio was >0.08 : 1. At C12-PEI/OVA weight ratios of 0.03 : 1 to 0.08 : 1, the zeta potential of the C12-PEI/OVA particles was in the range −18.4 to −13.1 mV (Fig. 1B). The negative charge of the complexes showed that these nanoparticles were different from other PEI-based nano-delivery systems.¹⁵ It is suggested that, in aqueous solution, the C12-PEI polymer first assembled into micelles with a hydrophobic core containing an alkyl chain and a hydrophilic, positively charged shell containing 423 Da PEI. The negatively charged OVA proteins could be absorbed (loaded) onto the micelle surface to form the negatively charged C12-PEI/OVA nanoparticles.

Fig. 1 (A) Average particle size and polydispersity index (PDI) and (B) zeta potential of the C12-PEI/OVA complexes.

3.3. Morphology of the C12-PEI/OVA polyplexes

To further elucidate the assembly/aggregation features of the as-prepared C12-PEI/OVA polyplexes, the morphology of the C12-PEI/OVA-0.07/1 and C12-PEI/OVA-0.08/1 polyplexes was observed by TEM and AFM. The C12-PEI/OVA-0.07/1 and C12-PEI/OVA-0.08/1 polyplexes were prepared by mixing the OVA solution (1 mg mL⁻¹) with the C12-PEI polymer solution at C12-PEI/OVA weight ratios of 0.07 : 1 and 0.08 : 1, respectively, and were then incubated at 37 °C for 20 min. From the TEM images in Fig. 2, it can be seen that the C12-PEI/OVA-0.07/1 and C12-PEI/OVA-0.08/1 polyplexes were uniformly dispersed sphere-like nanoparticles with average particle sizes of around 40–70 nm; a similar sphere-like morphology and particle size (60–100 nm) were also observed by AFM (Fig. S3†). Larger particle sizes (∼290 nm) were measured by DLS than observed by TEM/AFM, which could be a result of shrinkage of the nanoparticles during drying of the C12-PEI/OVA polyplexes before observation by TEM/AFM.²⁶

Fig. 2 Transmission electron microscopy (TEM) images of the C12-PEI/OVA polyplex nanoparticles: (A–C) C12-PEI/OVA-0.07/1 at different magnifications; and (D) C12-PEI/OVA-0.08/1.

3.4. Stability of the C12-PEI/OVA polyplexes in solution

The stability of the antigen/vaccine payload in solution is an essential parameter for both the in vitro and in vivo delivery of therapeutic substances.^27,28 We evaluated the stability of the C12-PEI/OVA polyplexes by DLS under various conditions. Fig. 3 shows that the average particle size of the C12-PEI/OVA-0.07/1 polyplexes was still in the range 298–312 nm in the RPMI-1640 culture medium at 37 °C, indicating that the binding of C12-PEI with OVA could form stable C12-PEI/OVA-0.07/1 polyplexes. This binding could be a result of the induced self-aggregation of the hydrophobic dodecyl chain, which could subsequently lead to the formation of micelles with hydrophobic lipid cores.²⁹ This would decrease the dissociation of the C12-PEI/OVA-0.07/1 polyplexes in the RPMI-1640 culture solution. We have previously found that the stability of nanoparticles in a series of triblock polymer (DL_n-PLLA_m-DL_n)/DNA polyplexes could be enhanced by lengthening the hydrophobic PLLA block.²³ We further examined the stability of the C12-PEI/OVA polyplexes by DLS in 0.9% NaCl and 10 mM HEPES (Fig. S4†). The size of the polyplexes could be maintained at around 250–420 nm within 2 days. These results showed that the C12-PEI/OVA polyplexes could maintain their average particle size at a comparatively stable level under various solution conditions for up to 2 days. This short-term stability at room temperature might make them suitable for potential use in a vaccine formula in practical applications.

Fig. 3 Change with time of the average particle size and PDI of the C12-PEI/OVA-0.07/1 polyplexes during incubation at 37 °C in RPMI-1640 culture medium.

3.5. In vitro cytotoxicity of the C12-PEI/OVA polyplexes by CCK-8 assay

The development of carriers with low cytotoxicity is considered to an essential prerequisite for safe therapeutic applications. The in vitro cytotoxicity of the C12-PEI/OVA polyplexes was evaluated in DCs by a CCK-8 assay³⁰ and the polyplexes formed by 25 kDa PEI and OVA (PEI/OVA) was used as the control. Fig. 4 shows that PEI/OVA had a high cytotoxicity (about 54% survival) at a weight ratio of 0.04 : 1, whereas C12-PEI/OVA had a very low cytotoxicity (about 99% survival) at a weight ratio of 0.04 : 1 to 0.05 : 1, which might be due to the lower molecular weight of the cationic PEI decreasing the disruption of the cell membrane.³¹ However, as the mass ratio increased to 0.06 : 1 and higher, the cytotoxicity significantly increased. As the C12-PEI/OVA polyplexes were negatively charged nanoparticles, the cytotoxicity could be attributed to their intracellular metabolism instead of the disruption of the cell membrane induced by a cationic charge.³² The results also suggested that the in vitro cytotoxicity largely depended on the weight ratio of C12-PEI/OVA in the polyplex nanoparticles.

Fig. 4 In vitro cytotoxicity of the C12-PEI/OVA polyplexes in DCs by the CCK-8 assay; PEI/OVA-0.04/1 was used as a control.

3.6. Antigen cross-presenting efficiency by the C12-PEI/OVA polyplexes

The antigen cross-presenting efficiency of the C12-PEI/OVA polyplex nanoparticles was further evaluated with the co-culture of antigen-pulsed DCs (pre-incubated with the C12-PEI/OVA polyplexes) with RF33.70 hybridoma cells specific for the MHCI/OVA257–264 complex on the cell surface.³³ Once the antigens from the DCs had been cross-presented by the MHC I complexes, they could interact with specifically with these T cells and up-regulate the secretion of IL-2. We have previously reported the delivery of OVA antigen using PEI-25k as the polymer carrier, which gave rise to enhanced IL-2 secretion and improved the antigen cross-presentation efficiency of the DCs.¹⁵ Here, we evaluated the secretion of IL-2 induced by C12-PEI/OVA; the polyplex formed by PEI-25k and OVA (PEI/OVA) was used as a reference. Fig. 5 shows that the secretion of IL-2 by RF33.70 cells in the C12-PEI/OVA polyplex nanoparticles at weight ratios of 0.04 : 1 to 0.09 : 1 was about 1.2–5.3-fold higher than that of the free OVA solution. The highest secretion of IL-2 from the RF33.70 cells was observed when they were incubated with the C12-PEI/OVA polyplex nanoparticles at a weight ratio of 0.07; the secretion of IL-2 was 1.9-fold higher than that seen with the PEI/OVA-0.04/1 polyplex nanoparticles. These results suggest that the weight ratio of the C12-PEI/OVA polyplex nanoparticles was a critical factor in antigen presentation and the comparatively high IL-2 secretion indicated that the C12-PEI/OVA polyplex might be a promising tool for the delivery of OVA antigen.

Fig. 5 Secretion of IL-2 by RF33.70 cells after incubation with different amounts of the C12-PEI/OVA polyplexes to stimulate the DCs.

3.7. Intracellular distribution of the C12-PEI/rhodamine-labeled OVA polyplexes

To further understand the intracellular distribution of the C12-PEI/OVA polyplexes in DCs, rhodamine-labeled OVA (Rho-OVA) was prepared and mixed with C12-PEI to prepare the C12-PEI/Rho-OVA polyplexes, which were then incubated with DCs for 30 min and then observed by fluorescence microscopy. Fig. 6 shows the fluorescence images (400×) obtained after the incubation of C12-PEI/Rho-OVA polyplexes for 30 min with DCs. The red fluorescence of the rhodamine dye showed the successful and efficient cellular internalization of the C12-PEI/Rho-OVA polyplexes. Fluorescent images for the polyplexes after incubation periods of 15 min, 30 min and 1 h were recorded (Fig. S5†) and showed that the C12-PEI/Rho-OVA polyplexes could be taken up into the DC cells within a relatively short time of 30 min. Most of the red fluorescent Rho-OVA co-localized with the green fluorescent dye Lysotracker Green, indicating that a lysosome localization effect³⁴ was involved in the cellular trafficking process for the C12-PEI/Rho-OVA polyplexes. This may lead to efficient intracellular OVA release by the well-known “proton sponge” effect of low molecular weight 423 Da PEI. The rapid uptake might be caused by the relatively small nanoparticle size and the membrane fusion capability of the OVA-loaded nanoparticles. It has been reported that some cationic lipids bearing alkyl chains are inclined to be taken up via a pathway mediated by lipid rafts or caveolae.³⁵ We therefore suggest that the C12-PEI/Rho-OVA polyplexes might be internalized through a lipid raft or caveolae dependent pathway to finally give rise to efficient delivery of the OVA antigen. Further investigation of the intracellular trafficking mechanisms of the antigen/polymer carrier polyplexes has been carried out in our laboratory.

Fig. 6 Fluorescence images of the intracellular distribution of the C12-PEI/Rho-OVA polyplexes in DCs after incubation for 30 min.

4. Conclusion

We have designed and synthesized a hydrophobic-modified low molecular weight PEI (C12-PEI) by direct coupling of 1-bromododecane with low molecular weight PEI (MW 423 Da) in a 2 : 1 molar ratio. The synthesized C12-PEI could bind or load OVA antigen at weight ratios of 0.03 : 1 to 0.08 : 1 to form C12-PEI/OVA polyplexes with an average particle size range of 158–302 nm and a zeta potential range of −18.4 to −13.1 mV. Stability studies showed that the C12-PEI/OVA-0.07/1 polyplexes had short-term storage stability (up to 2 days) in RPMI-1640 culture medium, 0.9% NaCl and 10 mM HEPES at room temperature. The CCK-8 assay results indicated that the cytotoxicity of the C12-PEI/OVA polyplexes greatly depended on the weight ratio of C12-PEI/OVA and the antigen cross-presentation (IL-2 secretion) effect of the C12-PEI/OVA polyplexes was also dependent on the weight ratio. Optimized IL-2 secretion was achieved on the C12-PEI/OVA-0.07/1 polyplexes and was 5.3-fold higher than with free OVA and 1.9-fold higher than on the PEI/OVA-0.04/1 polyplexes. The intracellular distribution of the C12-PEI/Rho-OVA polyplexes showed the obvious lysosome localization effect of the polyplexes, suggesting that the “lysosomal escape” effect of the C12-PEI/OVA polyplexes may play an essential role in subsequent OVA antigen release and cross-presentation.

This study has demonstrated that the synthesized hydrophobic C12-PEI lipopolymer with modified with an alkyl chain could potentially be used as a therapeutic vaccine carrier for the delivery of the OVA antigen. The hydrophobic chain number and length, the degree of unsaturation of the hydrophobic chain, the charge density, and the linkers and linkage bonds may play important roles in determining the cytotoxicity and delivery efficiency of drugs, genes or vaccines.³⁶ The correlation between the chemical structural factors of the nanocarriers³⁷ and the vaccine delivery properties are under further investigation in our laboratory.

Acknowledgements

The authors are grateful for partial funding from the National Science Foundation of China (NSFC 81001406 and 21372251) and the Funds for Scientific Research by Minhang Committee of Science and Technology (No. 2015MH138). We also acknowledge the generous help from the employees of Instrumental Analysis Centre of Shanghai Jiao Tong University. Dr Ruilong Sheng thanks the Chinese Academy of Sciences (CAS) for the CAS–Canada Young Scientist Visiting Scholarship and Youth Innovation Promotion Association for sponsorship.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25919c

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