Nanoparticles with a Lipid Core Can Enhance the Infection of Epithelial Cells with an Enterovirus

Nanoparticles (NPs) are nano-sized materials (1–100 nm) used in a wide range of applications in the biomedical field, especially for the diagnosis, prevention, and treatment of diseases, due to their physicochemical and biological properties. Based on their composition, NPs are generally classified into the carbon-based, inorganic, and organic NPs [1] and can be internalized into mammalian cells via clathrin/caveolar-mediated endocytosis, phagocytosis, macropinocytosis, or pinocytosis pathways [2]. Organic NPs can be made from compounds such as carbohydrates, proteins, lipids, or polymers. Among organic NPs, lipid-based NPs (NPLs) are of particular interest due to their ability to encapsulate and deliver a wide range of molecules to cells, including drugs, peptides, proteins, and nucleic acids [3‒5]. NPLs are composed of bioavailable and biodegradable lipids, which provide stability and protect the therapeutic payload from degradation, to enhance cellular uptake and specificity [3, 6]. NPLs offer several advantages over other drug delivery systems, such as low toxicity, high biocompatibility, and ease of manufacturing [7].

NPLs can be used as a carrier for nucleic acid-based vaccines [3, 8], antiviral molecules [9, 10], or immune-stimulating antigens [11‒15]. Maltodextrin-based NPs lipidated with a phospholipid core can efficiently uptake and deliver proteins into epithelial [5, 16], immune [16], and neuronal cells [17]. It has been suggested that the porous structure of the NPLs allows the loading of proteins inside these particles [18]. An association of proteins with these NPLs or with other NPLs such as liposomes on their surface has also been suggested [18].

Carbon-based NPs or inorganic metallic NPs based on gold, silver, cerium oxide, or aluminum have demonstrated intrinsic antiviral properties in vitro against a variety of viruses such as human immunodeficiency virus [19, 20], herpes simplex virus [21, 22], enterovirus 71 [23], coronaviruses [20, 24, 25], and poliovirus (PV) [26]. In contrast, titanium-based metallic NPs exposure has been shown to enhance respiratory syncytial virus infection in human bronchial epithelial cells [27] and to exacerbate respiratory syncytial virus-induced airway inflammation [28].

Oncolytic virotherapy is a therapeutic approach for cancer since treatments such as chemotherapy and radiotherapy can result in acute and long-term toxic effects or sometimes in poor outcomes. Viruses, in particular PV, have been proposed as a candidate for oncolytic virotherapy [29, 30]. Attenuated or recombinant oncolytic PVs can selectively induce cancer cell death and have shown promising results in the treatment of malignant gliomas, ectodermal/neuroectodermal tumors, or breast and prostate cancers [31‒33].

PV is a small (around 30 nm), non-enveloped, single-strand RNA virus with an icosahedral capsid that belongs to the Enterovirus genus of the Picornaviridae family. The capsid is composed of 60 capsomers, each containing a single copy of the structural proteins VP1, VP2, VP3, and VP4 [34]. Following attachment of viral particles to specific PV receptor; the viral replication takes place in the cytoplasm which can result in a cytopathic effect (CPE) in cell layers [34]. The aim of this study was to determine the effect of NPLs on the infection of a human tumoral cell line with PV.

The human HEp-2 cell line (comprised of epithelial cervical adenocarcinoma cells derived via HeLa cell line contamination) [35] was provided by BioWhittaker (Vervier, Belgium). They were cultured in Eagles’s Minimum Essential Medium (Gibco^®, Invitrogen, UK) supplemented with 10% inactivated fetal bovine serum, 1% l-glutamine, 1% penicillin (100 U/mL), streptomycin (100 mg/mL), and fungizone (0.25 mg/mL; Invitrogen, Saint Aubin, France) in a 5% CO₂ atmosphere at 37°C. The PV type 1 Sabin strain, provided by the EUROVIR Hygiene-Institute (Luckenwalde, Germany), was propagated in HEp-2 cells. After three thaw-freeze cycles, the suspension was collected and clarified at 2,000 g for 10 min at 4°C. The resulting supernatant was aliquoted and stored at −80°C. The viral titer was assessed on HEp-2 cell monolayers using the end-point dilution assay, and the Spearman-Karber method was used to determine the tissue culture 50% infectious dose (TCID₅₀). The results were expressed as TCID₅₀/mL. The detection limit of this method is 10^1.5 TCID₅₀/mL. The virus titer was 10⁶ TCID₅₀/mL.

Virus preparations in sterile 6-well plates were exposed to 15W UV-C lamps (Philips^® Electronic Instruments) with an energy density of 20 mJ/cm² at room temperature. After UV irradiation, the loss of infectivity was ascertained by the lack of viral replication on HEp-2 cells. The inactivated viral particles in cell culture supernatant were then filtered by centrifugation at 3,000 g for 2 min at 15°C using a Vivaspin^® 20 Centrifugal Concentrator with a molecular weight cutoff of PES membranes of 100 kDa (Sartorius, Germany). The concentration of protein in the inactivated cell culture supernatant was then measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific).

NPLs were synthesized as previously described [5]. Maltodextrin (100 g; Roquette, France) was dissolved in 2 N sodium hydroxide with magnetic stirring at 25°C, and then epichlorohydrin (Merck, France) and GTMA (Merck, France) were added to obtain a cationic polysaccharide gel. The gel was then neutralized with acetic acid and crushed using a high-pressure homogenizer (LM20, Microfluidics, France). The particles were then purified by tangential flow ultrafiltration (AKTA flux 6, GE Healthcare, France) to obtain purified NP + particles. These NP + particles were then lipidated with dipalmitoylphosphatidyl glycerol (Lipoid, Germany) to produce NPLs. The concentration of NPLs used in the present study was 10 μg/mL. It was shown by our team that NPLs at 10 μg/mL were not cytotoxic toward epithelial cell lines [36]. In our preliminary studies, NPLs at this concentration was not cytotoxic toward HEp-2 cells as shown by UptiBlue assay.

The association of NPL with inactivated PV was performed by mixing both components in PBS at 37°C for 1 h 30 min with a PV/NPL ratio of 1:3 or 1:10 (w/w). The size, the polydispersity index (PDI), the zeta potential of NPLs, and inactivated PV and PV/NPL formulations were measured at 25°C by DLS and by electrophoretic light scattering, respectively, using a Zetasizer Nano ZS (Malvern Instruments, Orsay, France).

PV was filtered by centrifugation at 3,000 g for 2 min at 15°C using a Vivaspin^® 20 Centrifugal Concentrator with a molecular weight cutoff of the PES membrane of 100 kDa (Sartorius, Germany). The virus was then diluted in PBS at infectious doses 1, 10, and 100 TCID₅₀/mL, mixed with NPLs, and incubated for 1 h 30 min at 37°C and 5% CO₂ in a humidified atmosphere. HEp-2 cells were harvested and seeded at 10⁵ cells per well in 24-well plates and were inoculated with PV or PV/NPL formulations. NPLs have been shown to deliver proteins to dendritic or epithelial cell lines more efficiently after 24 h of incubation [16]; therefore, the incubation time of PV and NPL mixtures with HEp-2 cells in the present study was 24 h. Thus, after 24 h of incubation at 37°C and 5% CO₂ in the humidified atmosphere, the medium was removed, cells were then washed once with PBS, and fresh medium was added. The cell layers were daily observed by using an inverted microscope (Olympus CKX41) to assess the appearance of a CPE.

Mock-infected, PV-infected HEp-2 cells and PV/NPL-inoculated HEp-2 cells were cultured for 18 h at 37°C and 5% CO₂ on sterile coverslips. Cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol/acetone. The presence of the intracellular viral capsid protein VP1 in HEp-2 cells was investigated by immunofluorescence as previously described [37] using a primary anti-enterovirus VP1 monoclonal antibody (clone 5D8/1, Dako^®), and a secondary anti-mouse IgG rabbit polyclonal antibody conjugated with Alexa Fluor 488 (Molecular probes^®). Cell nuclei were revealed by Hoechst dye solution (Sigma-Aldrich), and stained cells were visualized with a fluorescence microscope (Lietz, Diaplan).

Mock-infected, PV-infected HEp-2 cells and PV/NPL-inoculated HEp-2 cells were washed twice with PBS and collected at 18 h postinoculation. The total viral RNA was extracted with TRI Reagent^® RNA isolation reagent/Chloroform (Sigma Aldrich) following manufacturer’s instructions and quantified by a reading absorbance at 260 nm and 280 nm with a µDrop^® plate and spectrophotometer (Thermo Fischer Scientific). Then, the RNA retro-transcription step was performed with the AffinityScript^® QPCR cDNA Synthesis Kit (Agilent, Lees Ulis, France) on a Perkin Elmer 2400 thermocycler. The Brilliant II^® QPCR Kit (Agilent technology, France) was used for the PCR step on the MX3000p^® instrument (Stratagene). Enterovirus 71 RNA (Vircell) was used as the standard for quantification. Primers used for the enterovirus genome and RT-PCR cycling conditions were previously described [38].

Data are presented as mean ± standard deviation. Comparisons were performed using Student’s t test when appropriate. p values <0.05 were considered statistically significant. Graphs and data analyses were performed with GraphPad Prism^® V6.0 Software.

The association of NPL with inactivated PV was performed with a PV/NPL ratio of 1:3 or 1:10 (w/w). The size and zeta potential of NPLs, inactivated PV, and of the association PV to NPLs were determined. NPLs and inactivated PV had a cationic and anionic zeta potential, respectively. When PV and NPLs were associated, the zeta potential values were cationic regardless of the PV/NPL formulation ratio (shown in Table 1). The diameter of PV/NPL formulation (1:3) was larger than that of NPLs. The PDI is used to determine whether samples are homogeneous based on low PDI values [39]. When the ratio of PV/NPL formulation was 1:3, the PDI was lower compared with the ratio 1:10. Therefore, in the rest of the study, the ratio of PV/NPL formulation was 1:3.

NPLs were formulated with PV at 1, 10, and 100 TCID₅₀/mL and then added to HEp-2 cells. When HEp-2 cells were inoculated with PV or PV/NPL formulations (10 and 100 TCID₅₀/mL), the CPE was around 100% as soon as 2 days postinfection (p.i). There was no CPE on day 6 p.i when the cells were inoculated with PV at 1 TCID₅₀/mL, but CPE was 100% when HEp-2 cells were inoculated with 1 TCID₅₀/mL PV associated with NPLs (shown in Fig. 1a).

When HEp-2 cells were infected with the virus (1 TCID₅₀/mL), the infectious titer of the culture supernatant was below the detection limit (10^1.5 TCID₅₀/mL) up to 6 days p.i. When HEp-2 cells were infected with PV (1 TCID₅₀/mL)/NPL formulation, the infectious titer of culture supernatants increased during the follow-up, reaching 2 × 10⁶ TCID₅₀/mL as soon as day 4 p.i (shown in Fig. 1b).

The proportion of VP1-positive cells in HEp-2 cells infected with PV or PV/NPL formulation was evaluated by immunofluorescence at 18 h p.i. When the cells were inoculated with the virus, VP1 was only detected in cells infected with PV at 10 TCID₅₀/mL (0.4% ± 0.6). In contrast, when the cells were inoculated with PV/NPL formulations, the percentages of VP1-positive cells were 6.8% ± 1 and 16.7% ± 0.4 for infectious doses at 1 and 10 TCID₅₀/mL, respectively (shown in Fig. 1c).

The level of intracellular viral RNA in HEp-2 cells inoculated with PV and PV/NPL formulation was evaluated by RT-qPCR. The level of viral RNA was significantly lower in PV-infected cells than in cells inoculated with the PV/NPL formulation (100 ± 30 vs. 965 ± 154 copies of viral RNA/ng of total RNA, p < 0.05) (shown in Fig. 1d).

It was previously reported that maltodextrin-based NPLs are able to efficiently deliver proteins into epithelial cell line [16, 40]. In the present study, the effect of these NPLs on the viral infection of epithelial cells was investigated in vitro. NPLs were shown to enhance PV infection of HEp-2 cells. Indeed, our results show that in HEp-2 cell cultures inoculated with the PV/NPL formulation, intracellular viral RNA was significantly higher than in HEp-2 cells inoculated with the virus alone. Interestingly, the viral titer of culture supernatants, the percentages of CPE, and the proportion of VP1-positive cells were higher when HEp-2 cells were inoculated with the PV/NPL formulation. Taken together, these results show that PV infection of HEp-2 cells can be enhanced by NPLs.

NPLs are porous vectors able to encapsulate and deliver large amounts of proteins into cells via endocytosis mediated by a combination of pathways including those of clathrin, caveolae, and dynamin [5, 18]. The presence of an anionic phospholipid core inside maltodextrin NPs markedly improve the interactions with the cell membrane as well as cytoplasmic delivery of vectorized proteins/antigens compared to other organic NPs [16, 18, 40, 41]. The lowered cationic surface charge and the greater diameter of PV/NPL formulation with a ratio of 1:3 compared to empty NPLs suggest that viral particles are adsorbed to their surface and not loaded within the core of NPLs. Further studies are needed to clarify the mechanisms of the infection when cells are inoculated with PV/NPL formulation.

Viruses, and PV in particular, are oncolytic agents that can be administered to the patient, by systemic or intratumoral injection [29, 30]. However, several parameters can reduce the delivery of oncolytic viruses to cancer cells [30, 33, 42]. This study shows that NPLs can enhance the infection of a tumor cell line with a virus, especially PV. NPLs have shown satisfactory bio-distribution in vivo [16, 41]. Whether NPLs can be used in virotherapy to increase the virus-induced lysis of tumor cells deserves further investigation.

In conclusion, NPLs have previously been shown to be a promising and safe delivery system for drugs and vaccines. The present study shows that NPLs can enhance the infection of a cell line with a virus and provides arguments in favor of the potential value of these NPs as vectors of oncolytic viruses.

The authors thank the team of the Laboratoire de Virologie ULR3610 and all their collaborators.

This article does not contain any studies with human participants or animals. This study did not require ethical approval or written informed consent in accordance with local/national guidelines.

The authors have no conflicts of interest to declare.

This work was supported by Ministère de l’Education Nationale de la Recherche et de la Technologie, Université de Lille (ULR3610), and Centre Hospitalier et Universitaire de Lille. Inès Vergez was supported by a scholarship of Région Hauts de France, France, and Centre Hospitalier et Universitaire de Lille.

Methodology and writing – original draft preparation: Inès Vergez and Cédric Rubrecht; writing – review and editing: Magloire Pandoua Nekoua, François Fasquelle, Angelo Scuotto, and Enagnon Kazali Alidjinou; conceptualization, supervision, funding acquisition, and writing – review and editing: Didier Betbeder and Didier Hober. All the authors have read and approved the published version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.