Preparation of PLGA nanoparticles to enhance intracellular uptake in dendritic cell

SUMMARY Nanoparticles based approaches for antigen delivery in dendritic cells (DCs) have been studied in order to enhance intracellular uptake. In this study, we successfully prepared poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with chicken egg ovalbumin (OVA) and poly(inosinic : polycytidylic) acid (poly I:C) by self microemulsifying double emulsion method, which demonstrate highly safe and enhance DC uptake. INTRODUCTION Development of nanoparticles (NPs) has been attracted in biomedical applications for disease control. Recently, many multifunctional delivery systems have been designed for codelivery of effective payloads such as micelles, liposomes, and nanoparticles. In addition, NPs can enhance intracellular delivery efficiency to immune cell for cell based cancer therapies. PLGA exhibits many of the ideal properties of a nanoscale delivery system, providing long term release of the encapsulated agent and degrading into the biocompatible products of lactic and glycolic acid. PLGA has long been a popular use for drug delivery applications, particularly since it is already FDA-approved for use in human. Dendritic cells (DCs), which is a representative antigen presenting cell (APC), are the most effective mechanism that are able to present the antigens to T lymphatic cells, leading that activated T cells can contribute antitumor immunotherapy. Proteins as a model antigen presentation is a potential route for immunotherapy. However, these are poorly immunogenic for T cells when administered alone. To overcome this limitation, NPs based delivery platform particularly attractive. Moreover, manipulation of the functional DCs by using NPs has been attempted in clinical trial. Here, we developed novel nanocarriers loaded with OVA and Poly I:C as model protein and adjuvant to induce intracellular delivery efficiency in DCs. The PLGA NPs could provide a new strategy for antigen delivery in DCs, which have potential for DC based immunotherapy. EXPERIMENTAL METHODS PLGA NPs were prepared by “water-in-oilin-water” double emulsion-solvent evaporation method, which loaded with OVA (model antigen) and/or Poly I:C (model adjuvant) Briefly, 2 mg of OVA and 0.5 mg of Poly I:C were dissolved in 0.1 ml of de-ionized water, and then mixed with 1 ml of chloroform containing 30 mg of PLGA using microtip probe sonicator at 4°C for 1 min. The primary emulsion was further emulsified with a secondary aqueous phase (10 ml of 2% w/v polyvinyl alcohol (PVA) solution) at 4°C for 2 min to form a secondary emulsion. The emulsion was agitated using a magnetic stirrer at room temperature overnight until the chloroform was completely evaporated. After 18 h, the suspension of PLGA NPs was washed three times with de-ionized water at 4°C by centrifugation (13,000 rpm, 20 min). The size and zeta potential of PLGA NPs were analyzed by dynamic light scattering using an electrophoretic light scattering photometer (ELS-Z, Otsuka Electronics, Osaka, Japan). Encapsulated OVA and poly I:C were measured by SMARTTMmicro BCA kit (Intron Biotechnology, KOREA). Morphology of PLGA NPs was evaluated by scanning electron microscope (SEM). Cell viability of DCs for PLGA NPs was assessed thiazoyl blue tetrazolium bromide (MTT) assay. Bone marrow-derived dendritic cells (BMDCs) were incubated with PLGA NPs for 24 hr in 96-well plates at a density of 1 x 10. Absorbance was determined at 570 nm with an ELISA reader (SpectraMAX, Molecular Devices, Sunnyvale, CA, USA). Intracellular uptake of PLGA NPs in DCs was monitored by FACS Calibur flow cytometer (BD Biosciences). RESULTS AND DISCUSSION The physical properties of PLGA NPs and loading efficiency of drugs are shown in Figure 1. The mean diameter of PLGA NPs ranged from 160 nm to 230 nm and zeta potential was from -30 mV to -10 mV, respectively. In addition, loading efficiency of drugs in PLGA NPs was OVA (66.53 ± 6.4 %), Poly I:C (57.57 ± 7.8 %), and OVA/Poly I:C (53.39±6.2 %), respectively. Figure 1. Physical properties of PLGA nanoparticles. (A) mean diameter, (B) surface charge, (C) loading efficiency of drugs, and (D) morphology by SEM (bar: 5 μm). Error bars represent S.D. (n=3) *p < 0.05. We next assessed cytotoxicity of PLGA NPs against BMDCs (Figure 2). PLGA NPs resulted in no cytotoxicity on BMDCs with different concentration of PLGA NPs. Intracellular delivery of PLGA NPs in BMDC was confirmed by FACS (Figure 3). Intracellular delivery efficiency showed the enhancement based on PLGA NPs loaded with OVA. Figure 2. Cell viability of PLGA NPs in vitro. Cell viability was assessed by MTT assay at 24 hr. Error bars represent S.D. (n=3) *p < 0.05. Figure 3. Intracellular delivery efficiency of PLGA NPs into DCs. (A) FACS image and (B) bar graph for intracellular uptake. Error bars represent S.D. (n=3) *p < 0.05. CONCLUSION In summary, we have developed PLGA NPs to enhance intracellular uptake for DCs, which has broad potential as a delivery platform in human disease for DC based immunotherapy and could be adapted for other protein delivery methods. REFERENCES 1. Kirtane, AR.; Kalschever, SM.; Panyam, J. Adv. Drug Deliv. Rev. 2013, (13), 17311747. 2. Look, M.; Saltzman, WM.; Craft, J.; Fahmy, TM. Biomaterials. 2014, 35, (3), 1089-1095. ACKNOWLEDGMENTS This work was supported by Basic Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A4A1069575)