1. Introduction
Infection by the high-risk human papillomavirus (HPV) is the most significant risk factor in the development of malignant tumors of the anogenital tract, including cervical cancer. Despite the increasing use of prophylactic vaccines against HPV infection, cervical cancer is still a primary health threat for women living in developing countries. Vaccine cost, inadequate healthcare infrastructure and socio-cultural issues are some of the main reasons for this apparent lack of effectivity. Hence, there is still a pressing need for new and efficient strategies for HPV transmission control, detection and treatment [
1,
2].
Genital dysplasia and cervical cancer are associated with a subset of HPVs referred to as high risk, consisting of HPV types 16, 18, 31, 33, 45, 52 and 58. HPVs are small (50 nm) icosahedral viruses with an 8 kb double-stranded circular DNA genome, encoding eight early and late genes. Early genes regulate cellular transformation, and early and late viral DNA replication. The late genes code for the L1 and L2 proteins, which self-assemble within the host cell nucleus to encapsidate the viral DNA and constitute infective virions. High-risk HPVs infect mucosal genital epithelia, where the key event is the binding of the positively charged viral capsid to heparan sulfate (HS), a negatively charged linear oligosaccharide attached to a protein core on the cell surface [
3].
Gold nanoparticles (GNPs) are biocompatible Au clusters from 1 to 100 nm in size. GNPs display distinctive physicochemical properties depending on their size and shape, including surface plasmon resonance (SPR) and fluorescence enhancement effects [
4]. Different forms of GNPs have been used as small-volume, ultra-sensitive label-free optical sensors, leading to the development of several biomedical applications, such as real-time imaging and biosensing, as well as the delivery of either genes or anti-tumor drugs for cancer diagnosis and therapy, respectively. An easily noticeable red to blue color shift results from particle–particle plasmonic coupling of GNPs. While in suspension in water, GNPs electrostatically repulse each other, maintaining a stable color. However, the presence of counterions (i.e., Na
++) neutralizes electrostatic repulsion and the distance between the GNPs decreases, producing near-field coupling that results in bigger particle size and a strong enhancement of the localized electric field within the interparticle spacing, leading to a pronounced color shift. The clearly distinguishable color shift facilitates detection readout to such a degree that it can be performed with the naked eye [
4].
GNP conjugation (functionalization) with biomolecules has been extensively reported in several biodetection methods. Non-functionalized GNPs (nfGNPs) in water suspension will eventually aggregate and precipitate as metallic gold powder. However, nfGNP stabilization by protective or capping agents, such as thiols or citrate, prevents uncontrolled aggregation, allowing long-term storage. The capping agent generally forms a monolayer coat (ligand shell) that plays a critical role against aggregation, thus improving solubility and electron-transfer efficiency while allowing controlled shape and assembly. The ability to integrate nfGNPs into biological systems relies on the basic understanding of their interactions with electrolytes, macromolecules and metabolites adsorbed onto their surface by electrostatic, hydrophobic, van der Waals and dispersive forces forming a dense coating known as the biocorona. Such a coating shields the original nfGNP surface properties acting as a “complex” surfactant interface modifying their size and composition, thus defining the physiological response and interaction with living systems including viruses [
5].
Our group first reported the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and nfGNPs, which resulted in the stabilization of purified VLPs and the inhibition of the nfGNPs’ salt-induced aggregation. Ionic-competition and genetic data showed that the nature of an nfGNP–VLP interaction was mainly hydrophobic [
6]. Furthermore, nfGNPs covered substantial areas on the VLPs’ surface, suggesting that nfGNPs may be used as a steric inhibitor of the HPV-16 virion interaction with the host cell surface receptor, thus blocking infection. The present work showed, for the first time, the interaction of nfGNPs with functional HPV16 PsVs and characterized the effect of the PsVs in the nfGNPs’ aggregation process and the impact of the nfGNPs in the HPV virion cell entrance process in vitro. Aggregation assays showed that PsVs can inhibit the nfGNPs’ color shift in the presence of NaCl in a dose-dependent manner. High-resolution TEM showed that nfGNPs can directly interact with HPV16 PsVs. The nfGNP–PsV complex prevented the characteristic red to blue color shift in aggregation assays and, in the presence of ethanol, revealed that the interaction is essentially hydrophobic. The PsVs’ aggregate size in solution decreased rapidly in the presence of nfGNPs, as previously determined with VLPs. The nfGNPs were capable of inhibiting virus entry in a pseudovirus model with no toxic effects. This is not unique to nanometric gold, as non-functionalized silver nanoparticles (nfSNPs) can also inhibit the viral cell entry process. We compared the inhibition effect of non-functionalized metallic nanoparticles (nfMNPs) with heparin, the canonical inhibitor of HPV, and showed that nfGNPs can inhibit the infection process more efficiently than heparin.
Non-functionalized metallic nanoparticles (nfMNPs) can be useful to efficiently inhibit the HPV16 infection process and have the potential to be incorporated in prophylactic formulations as a functional component.
3. Discussion
Pseudo-infection inhibition by nfGNPs was better than by heparin, a molecule structurally related to HS and widely accepted as an efficient inhibitor of the HPV infection [
3]. However, the antiviral application of heparin is contraindicated due to its anticoagulant activity and inherent toxicity. On the other hand, nfGNPs have high specificity, even in a complex mixture of proteins, and have a hydrophobic surface that mediates nfGNP–PsV interaction, as recently reported for an influenza virus-sensing platform [
11].
Metal nanoparticles (MNPs) have been widely used to inhibit viral infection, and this has been extensively reviewed by Kaminee Maduray and Raveen Parboosing [
12]. Functionalized gold nanoparticles with polyethylene glycol (GNP-PEG) of 10 nm in size have been used to inhibit the HIV cell fusion process, supporting the idea of inhibiting the HIV infection process by gp120-CD4 recognition by raising 60% of inhibition at 0.8 mg/mL [
13]. Polysulfated gold nanoparticles (GNP-SO
4Na) from 110 nm were used to efficiently inhibit the binding of vesicular stomatitis virus (VSV) by a three-dimensional network formation that prevents VSV from cell binding; conversely, 19 nm GNP-SO
4Na only decorate the virion at the concentration tested, probably inhibiting virus-cell binding by a complete steric shielding [
14]. nfGNPs from 9.5 nm in size (initially 24 h after infection and every 24 h thereafter, for a total of three times at concentrations of 5 mg/kg and 20 mg/kg) were applied intranasally in 8–10-week-old female BALB/c mice against H2N2 influenza virus, obtaining 100% survival at eight days compared with zero survival for the control [
15].
In perspective, nfGNPs could be a component of a novel topical application product as suggested for carrageenan (a polysaccharide such as heparin and a potent inhibitor of HPV infection) and the positively charged polymer AGMA1 [
11]. Thus, incorporating nfGNPs into a mixture with carrageenan and AGM1 in a single treatment could completely inhibit HPV entry in different ways, resulting in an effective prophylactic tool. The major concern with nanoparticle application in vivo is its potential toxicity; this concern has been widely reviewed by Sani et al. [
16]. nfGNPs have a great potential to be incorporated as a functional component in a lubricant, where the nanoparticles would be in contact with the mucus-coated vaginal tissue—a non-keratinized stratified squamous epithelia. Vaginal epithelium is structured by basal membrane, stratum granulosum, stratum spinosum, intermediate cells, superficial squamous flat cells and stratum corneum cells. Stratum corneum cells, where nfGNP toxicity could occur, slough off by a natural process [
17]. Ensign et al. exposed the difficulty of going through the mucous layer coating the vagina epithelium. Mucus traps particles efficiently by adhesive and steric interactions, preventing cell penetration. Particles and pathogens trapped in the upper mucus layer would be expected to rapidly clear [
18].
This is the first report of nfGNPs blocking HPV infection. Although a full understanding of the molecular mechanisms governing the nfGNP–HPV interaction remains to be fully understood, the strong inhibitory effect of the nfGNPs on the PsV pseudo-infection process could provide a novel and affordable prophylactic method against HPV infection, thus preventing cervical cancer.
4. Materials and Methods
4.1. Synthesis of Non-Funtionalized GNPs (nfGNPs)
Non-functionalized GNPs (nfGNPs) were prepared by a modified Turkevich method, as described [
19,
20]. Briefly, 45 mL of deionized water was added to a reaction flask and refluxed. A mixture of 5 mL of 0.1% tetrachloroauric acid, 2 mL of 1% trisodium citrate and 42.5 μL of 0.1% silver nitrate was added dropwise. Synthesis reactions were carried out for 60 min and allowed to cool to room temperature. Colloidal nfGNPs were stored at 4 °C until used. The nfGNP characterization was made in the synthesis final reaction conditions (pH 5.5), nfGNP shape was determined by HAADF-STEM in a JEM-ARM200F transmission electron microscope (JEOL Ltd., Tokyo, Japan). nfGNP size was determined using dynamic light scattering (DLS) in a Zetasizer Nano-ZS90 (Malvern Panalytical Ltd., Cambridge, UK) with detection at 173°, and nfGNP concentration was determined by UV-visible spectra, using the equation c = A450/Ɛ450, where c is the concentration in mol per liter, A450 is the absorbance at 450 nm and Ɛ450 is the molar extinction coefficient for GNPs of 18 nm (3.87 × 108 M
−1 cm
−1) [
21]. The nfGNP suspension pH was changed to pH 7.2–7.7 through 1:100 dilution in DPBS buffer before incubation with the PsVs.
4.2. Production of HPV16 L1 Virus-Like Particles (VLPs)
HPV16 L1 virus-like particles (VLPs) were produced as we previously reported [
1,
22]. Briefly,
Spodoptera frugiperda Sf21 cells (Life Technologies Corporation, Carlsbad, CA, USA) in Sf900TMII medium (Life Technologies) were infected with L1-producing recombinant baculovirus for 72 h at 26 °C. Infected cells were pelleted by centrifugation and resuspended in 1X Dulbecco’s phosphate-buffered saline (D-PBS) (2.67 mM KCl, 1.47 mM KH
2PO
4, 137.93 NaCl mM and 8.06 mM Na
2HPO
4-7H
2O pH 7.2–7.7) complemented with cOmplete
TM protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MI, USA) in siliconized microtubes and lysed by sonication in a GEX 130 PB ultrasonic processor (Cole-Parmer Instrument Co., Vernon Hills, IL, USA) at 60% cycle duty 3 times (5 s each). Total lysates were incubated overnight at 37 °C for VLP maturation; digested at 37 °C for 1 h with
Serratia marcescens nuclease (Sigma-Aldrich) at 0.1% final concentration; and immediately chilled on ice, mixed with a 0.17 volumes of 5 M NaCl and clarified by centrifugation at 2000×
g for 15 min. The resulting supernatant was purified through CsCl isopycnic centrifugation and extensively dialyzed against 1X D-PBS at 4 °C. VLP production was verified by negative stain in TEM and immunoblotting, and VLP stock was stored in 1X D-PBS at 4 °C until use.
4.3. GNP Aggregation Assays
Non-functionalized GNP aggregation was carried out in 96-well microplates by adding nfGNPs (100 μL), VLPs or PsV and NaCl for a 250 μL final volume. Absorbance at 520 and 650 nm was measured immediately using an Epoch™ microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). The mean and standard deviation of the color-shift 650/520 ratio from each sample were plotted by triplicate.
4.4. Dynamic Light Scattering (DLS)
Sample measurements were performed at 25 °C using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK) with detection at 90°. The hydrodynamic size of the VLPs was recorded as Z-average hydrodynamic diameter (Dh). The plotted data represent the average size of five measurements from the same sample.
4.5. Cell Culture
The human embryonic kidney 293TT (ATCC CRL-3467) cells were cultured in Gibco® Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 5% Gibco® fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 IU/mL penicillin, 100 µg/mL streptomycin (PAA Laboratories Inc., Pasching, Austria) and 200 μg/mL Gibco® hygromycin (Thermo Fisher Scientific) at 37 °C and 5% CO2.
4.6. HPV16 Pseudovirus (PsVs) Production
HPV16 PsVs were produced as previously described [
23]. Briefly, 293TT cells were co-transfected with the p16sheLL expression plasmid [
11] and the reporter plasmid pSVLYFP using the Lipofectin
® transfection reagent (Thermo Fisher Scientific). Cells were harvested 72 h post-transfection, lysed in lysis buffer (DPBS-9.5 mM MgCl
2 and 0.25% Brij
® 58) and incubated at 37 °C for 24 h for maturation. The mature mixture was digested with Benzonase
® (Merck KGaA, Darmstadt, Germany) and clarified by centrifugation. Supernatant was placed on an OptiPrep™ gradient (Sigma-Aldrich) for ultracentrifugation at 170,000×
g for 20 h. The fraction containing PsVs was recovered and filtered through a Sephadex
® G25 column (Cytvia, Marlborough, MA, USA) in DPBS-0.5M NaCl. PsV production was verified by immunoblotting and transmission electron microscopy (TEM). Infectivity was a measure of the number of 293TT fluorescent cells generated by 1 µL of viral stock after 72 h post infection. Each fluorescent cell was considered an infectious unit (InU). PsVs were stored at 4 °C until use.
4.7. Pseudovirus Infection Inhibition
293TT cells were seeded into 24-well plates (1 × 104/well) and incubated overnight at 37 °C. Infection mixes were prepared with 5000 InU PsVs and increasing concentrations of nfGNPs (Thermo Fisher Scientific) until a final volume of 80 µL of DPBS. Infection mixes were maintained on gentle agitation at room temperature for 20 min and then added to 293TT in 500 μL of DMEM. PsV adsorption was allowed for 20 min before removal of the infection mix followed by addition of 1 mL of pre-warmed DMEM supplemented with 5% FBS. After 72 h, cells were harvested, washed with DPBS and resuspended in 0.5 mL of DPBS for analysis in a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with a band-pass filter at 530/30 nm (FL1). Excitation was performed with a 488 nm argon laser (1 × 104 cells/read).
4.8. Cellular Viability Assay
293TT cells were treated as described in the
Section 4.7. Seventy-two hours post treatment, the cells were harvested and an aliquot of the suspension cell was mixed with an equal volume of 0.4% blue trypan (Bio-Rad Laboratories, Hercules, CA, USA). The number of viable cells was evaluated in a TC10 automated cell counter (Bio-Rad Laboratories).
5. Conclusions
Nanomaterials used to integrate theranostic tools have become an essential part of health care research and development. In the present work, nfGNPs were tested as inhibitors for the infection of HPV16 (the main etiological agent of cervical cancer) in an in vitro PsV model that recapitulates the HPV16 L1 interaction with HS as an essential step for viral entry to the host cell. As with HPV16 VLPs, nfGNP NaCl-induced aggregation was inhibited by an interaction with HPV16 PsV, suggesting that nfGNPs can inhibit the viral entry process. Moreover, the nfGNPs stably interacted, disaggregating PsV–PsV complexes through direct hydrophobic contacts with the surface of the virions. Pseudo-infection experiments showed that the nfGNPs can significantly inhibit the HPV16 entry process in a dose-dependent manner without affecting cell viability and more efficiently than heparin, a classic inhibitor of HPV infection. A similar effect was observed with silver nanoparticles, suggesting a general mechanism for metallic nanoparticles.
Therefore, nfGNPs could be used as a part of topical formulations to prevent HPV infection on genital mucosa, thus providing a novel prophylactic alternative for the fight against cervical cancer.